Electrophysiological evidence about sexual dimorphism in the clonidine-induced inhibition of trigeminal wide dynamic range cell activity

Electrophysiological evidence about sexual dimorphism in the clonidine-induced inhibition of trigeminal wide dynamic range cell activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrophysiological evidence about sexual dimorphism in the clonidine-induced inhibition of trigeminal wide dynamic range cell activity Gustavo López-Córdoba, Guadalupe Martínez-Lorenzana, Miguel Condés-Lara, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7546267/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted 10 You are reading this latest preprint version Abstract Background The trigeminal system plays a key role in headaches pathophysiology. In this regard, nociceptive experiments in male rodents have shown that at the trigeminal level, clonidine induces antinociception through α 2A - but not α 2B/2C -adrenoceptors. Interestingly, although behavioural experiments suggest that males are more sensitive to clonidine-induced antinociception than female rats (an effect linked to hormonal factors), little is known about how neuronal nociceptive processing is affected in females (including the role of the α 2A/2B/2C -adrenoceptors subtypes involved). Since trigeminal second-order wide dynamic range (WDR) cells are one of the main gatekeepers involved in decoding and processing nociceptive inputs from peripheral facial structures, this study was designed to test the effect of clonidine on trigeminal WDR cell activity in male and female rats. Methods Extracellular unitary recordings of trigeminal WDR cells were performed in anaesthetised Wistar rats and analysed as Aδ-, and C-fibres associated discharge. Under these conditions, the effect of local clonidine (3.1–31 nmol) on electrical periorbital-evoked firing of WDR cells was recorded in both sexes. Furthermore, considering that at least three α 2A adrenoceptor subtypes exist (α 2A -, α 2B - and α 2C -adrenoceptors), pharmacological blockade of these receptor subtypes was performed using BRL 44408 (α 2A ), imiloxan (α 2B ), and JP-1302 (α 2C ). Results Clonidine inhibited the activity of Aδ-, and C-fibres in male and female rats. This inhibition was observed in 50–60% of cells recorded. Furthermore, the dose necessary to induce similar electrophysiological antinociception was higher in females (31 nmol vs. 10 nmol). In males, but not females, clonidine-induced inhibition of Aδ- and C-fibres activity was reversed using BRL 44408. Whereas in females, imiloxan and JP-1302 reversed the clonidineinduced inhibition of Aδ- but not C-fibres. Conclusion Male rodents are more sensitive than females to clonidine-induced WDR antinociception. In males but not females, clonidine-induced inhibition of Aδ- and C-fibres discharge relies on α 2A -adrenoceptor activation. However, in females, the activation of α 2B/2C adrenoceptors seems to be relevant to Aδ- but not C-fibre discharge inhibition by clonidine, implying that other mechanism/receptors may be involved in females. Adrenoceptors Headache Migraine Nociception Pain Sex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Since the mid-1980s, it has been shown that spinal administration of clonidine (an α 2 -adrenoceptor agonist) has analgesic effects in humans and antinociception in rodents [ 1 – 6 ]. At the spinal dorsal horn (SDH) level, many electrophysiological studies in male rodents have consistently reported that clonidine via the α 2 -adrenoceptor induces antinociception by presynaptic inhibition of the neuronal evoked activity associated with activation of Aδ- and C-fibres in second-order wide dynamic range (WDR) cells [ 7 – 11 ]. Similarly, although scarce, experiments at the trigeminal level, particularly in the medullary dorsal horn (MDH), have shown that clonidine also exerts antinociception by activating the α 2 -adrenoceptor. Interestingly, at MDH, using behavioural approaches (in male and female rodents), it was shown that females are less sensitive to clonidineinduced antinociception; hence, sexual dimorphism linked to the hormonal milieu has been implicated[ 12 , 13 ]. Beyond hormonal component, how sex impact the clonidine-induced antinociception at the neural level has not been explored; thus, the present study was designed to analyse the influence of sex on clonidine-induced neuronal inhibition of trigeminal nociception. Furthermore, we must keep in mind that α 2 -adrenoceptors can be pharmacologically subdivided into three functional subtypes, namely α 2A -, α 2B -, and α 2C -adrenoceptors [ 14 , 15 ]. Indeed, current data strongly support the notion that α 2A -adrenoceptors play a key role in spinal and trigeminal clonidineinduced antinociception [ 16 – 18 ]. Admittedly, these findings have been reported in male rats; however, the contribution of these receptor subtypes to clonidine-induced antinociception has not been explored in females. Certainly, the search for precise mechanisms involved in the modulation of nociception in female and male will provide basic evidence with potential clinical relevance. Hence, by using a pharmacological approach coupled with an in vivo analysis of the WDR cell response at the MDH, the effect of clonidine (administered at the trigeminal level) on periorbital-evoked nociceptive responses were analysed in male and female rats. The data showed that, although WDR firing was inhibited in both sexes by clonidine, females required a higher dose. Furthermore, the α 2A -adrenoceptor subtype elicits robust inhibition of Aδ- and C-fibres discharge in males, but not in females. In contrast, in females, the α 2B/2C -adrenoceptors subtypes seem to play a key role mediating inhibition of Aδ- discharge, but not upon C-fibres activity. MATERIAL AND METHODS 2.1. Experimental animals and ethical standards A total of 108 male and female rats (313 ± 4 and 288 ± 8 g, respectively; Fig. 1 ) from the bioterium of our institute were used in this study. The animals were housed in pairs in acrylic cages (18" L × 9" W × 8 H) with woodbased bedding and controlled temperature (23 ± 2°C) and humidity (50%) on a 12:12 h light/dark cycle (light beginning at 7:00 h), with food (LabDiet 5001) and tap water ad libitum. The rodents were allowed to acclimatise to their new environment for at least 72 h before handling for the experiments. All animal protocols were approved by our Institutional Ethics Committee, following the local animal welfare committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (80 − 23, revised in 1996). 2.2. General methods 2.2.1. Surgical procedures for electrophysiological recordings of trigeminal WDR cells The animals were anaesthetised with urethane (2 g/kg, i.p.). Adequacy of anaesthesia before surgery was judged by the absence of ocular reflexes, a negative tail flick test, and corporal relaxation. Under these conditions, an intratracheal cannula was inserted and the animals were mounted onto a stereotaxic frame (Narishige Group, Japan). The animals were immediately connected to a rodent ventilator (65–75 strokes/min) coupled to a homeothermic control module (RoVent®, Kent Scientific Corp., USA) to maintain ventilation and body temperature (~ 37°C). Next, the muscles of the dorsal neck were separated, cervical (C1) laminectomy was performed, and the overlying dura was carefully removed to allow access to the MDH, as previously reported [ 19 – 20 ]. The animals were not paralysed, and no withdrawal reactions were observed during the experiments. End-tidal CO 2 was monitored (Passport 2 Monitor, Datascope Corp., USA) and maintained within the physiological range (2.5-3.0%) throughout the experiment. At the end of the experiment, the animals were halted with an intracardiac overdose of KCl. 2.2.2. Extracellular recordings of trigeminal WDR cells Three quartz-platinum/tungsten microelectrodes (4–8 MΩ) mounted in a multichannel microdrive Mini Matrix System (Thomas Recording GmbH, Germany) were used to record neuronal activity of second-order wide dynamic range (WDR) cells with input from the periorbital receptive field (RF). Although this device allows the insertion of three microelectrodes into neural tissue (inter-electrode distance: 300 µm), and each microelectrode was inserted independently using the Eckhorn Matrix multiuser software (Thomas Recording GmbH, Giessen, Germany), we recorded only one extracellular unit per animal. The microelectrodes were positioned in the dorsolateral segment of the MDH and lowered (500–700 µm from the surface) in small steps (2–5 µm/s) to search for single-unit discharges evoked by innocuous tactile stimulation of the periorbital dermatome (mainly innervated by the ophthalmic branch of the trigeminal nerve, V1). Then, RF was assessed for non-noxious (brushing) and noxious (pinching) inputs. To verify the peripheral input relays in a WDR cell, electrical stimulation was applied using two electrodes inserted into the RF. In this case, two needles (27 G) attached to an electrical stimulator (2100 Isolated Pulse Stimulator; A-M Systems, LLC, USA) were inserted subcutaneously into the RF. Electrical test stimulation was then conducted. This test consisted of 20 square electrical stimuli (0.5 Hz) with a 1-ms pulse duration at 5–9 mA to evoke Aδ- and C-fibre responses. The evoked multiunit extracellular cluster signals were amplified ×100 (1700 Differential AC amplifier; A-M Systems, USA), digitised, and discriminated using Micro1401-4 CED hardware and Spike2 v10.19 software (Cambridge Electronic Design, UK). To identify the responding cells, the action potentials of the recorded cells were analysed using peri-stimulus time histograms (PSTH) and raster displays (100 ms duration; bin time = 1 ms). Raw data were fed through an audio monitor (Model 3300; A-M Systems LLC, USA) and displayed on a digital oscilloscope (TBS1064; Tektronix, Inc., USA). Furthermore, the waveforms and recorded spike trains were stored on a computer disk for offline analysis. Based on the distance between the RF and recording electrode, the spike latencies observed correspond to peripheral conduction velocities within the Aδ- (3–25 ms) and C-fibres (26–100 ms) [ 21 ]. On this basis, the threshold to evoke action potentials and their frequency of occurrence resulting from the stimulation of the periorbital region were attributed to the recruitment of Aδ- and C-fibres. In the process of identifying trigeminal WDR cells that respond to periorbital tactile and electrical RF stimulation, some neurones were categorised as solely tactile-sensitive and a smaller proportion as nociceptive-specific; these cell types were not subjected to further analysis. Thus, the number of action potentials in response to 20 RF stimuli (0.5 Hz, 1-ms pulse, 5–9 mA) was compared before (baseline) and after the treatment. The baseline response was established after an identified neuron had a variation of ≤ 10% in the evoked neural responses in at least three consecutive tests. In all cases, pharmacological treatments were delivered at the MDH level (topical) near the site of microelectrode insertion at a volume of 20 µL using a Hamilton® syringe. 2.3. Pharmacological treatments and electrophysiological study design As illustrated in Fig. 1 , the animals were allocated into two main sets: male (n = 50) and female (n = 58). In all cases, evoked trigeminal WDR cell firing was evaluated before (baseline) and after vehicle or clonidine administration at 10, 20, 30, 40, 50, and 60-min post-treatment (see Fig. 1 for details), by applying 20 RF electrical stimuli each time. Vehicle (n = 5 for each sex) or clonidine (3.1, 10, and 31 nmol; n = 10 for each dose) were administered immediately after the first test (i.e. baseline). A control group without treatment was used for both sexes (n = 5 for each group). At this point, we must clarify that the following experiments involving antagonists were designed with the following considerations: (i) females exhibit less sensitivity to the antinociceptive effects of clonidine than males, thus requiring a higher dose to achieve a similar degree of trigeminal antinociception (10 nmol in males vs. 31 nmol in females); and (ii) regardless of the clonidine dose administered, not all WDR cells were inhibited (~ 58% of the WDR cells were susceptible to inhibition). Therefore, before antagonist treatment, it was necessary to determine whether clonidine exerted an inhibitory effect on WDR cell firing. If 10 min after clonidine administration, the WDR cell activity was inhibited (> 15% inhibition), then 20 µl of the following antagonists was locally administered: (i) BRL 44408 (10 nmol; α 2A -adrenoceptor antagonist); (ii) imiloxan (10 nmol; α 2B -adrenoceptor antagonist); and (iii) JP-1302 (10 nmol; α 2C adrenoceptor antagonist). The per se effects of the antagonists used also was tested. A detailed description of the groups and doses used is shown in Fig. 1 . 2.4. Compounds In addition to urethane, we used the following chemicals from Sigma Chemical Co.: (i) clonidine hydrochloride (CAS number: 4205 91 8), (ii) 2-[2H-(1-methyl-1,3-dihydroisoindole) methyl]-4,5-dihydroimidazole maleate (BRL 44408; CAS number: 118343-19-4), (iii) 2-(1-ethyl-2-indazoyl) methyl-1,4-benzodioxan hydrochloride (imiloxan; CAS number: 81167-22-8); and (iv) N-[4-(4-methyl-1-piperazinyl)phenyl]-9-acridinamine dihydrochloride (JP-1302; CAS number: 1259314-65-2) acquired from Tocris Ltd. (USA). The doses of clonidine, BRL 44408, imiloxan, and JP-1302 were based on their free bases. All drugs were dissolved in saline solution (0. % NaCl). 2.5. Data presentation and statistical analysis One trigeminal WDR cell was studied per animal, and as previously reported [ 7 ], data were presented as mean ± standard error of the mean (SEM). Before performing a parametric statistical analysis, we checked for normality using the Shapiro-Wilk test (p > 0.05). Thus, the number of basal (baseline) evoked potentials (total spikes and number of Aδ- and C-fibres) in the different experimental groups was analysed. Because the normality test failed, a Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks was performed (see Table 1 ). Consequently, the data were normalised and the triggered potentials induced by electrical stimulation of the periorbital RF were expressed as a percentage change from the respective baseline. To assess the stability of the recorded neurones over the 60-minute period in the control and vehicle groups, we employed a one-way repeated measures ANOVA. Differences in neuronal activity evoked within one group of animals before and after treatment were compared using two-way repeated-measures ANOVA. Moreover, the temporal course was adjusted to obtain global neuronal activity due to the treatment (box and whisker plots), and an ordinary one-way ANOVA was performed. ANOVAs were followed (if applicable) by the Dunnett post-hoc test. In the case of two-way ANOVAs, sphericity was not assumed and corrections to degrees of freedom were made following the GreenhouseGeisser method. Differences were considered statistically significant at p < 0.05. Graphs and statistical analyses were performed using GraphPad Prism V6.0 software (USA). Table 1 Mean action potentials (± SEM) elicited by 20 electrical stimuli at baseline in the experimental groups for both sexes. A KruskalWallis one-way ANOVA on ranks was performed to compare the action potential elicited by the different treatments. MALES Control (n = 5) Vehicle 20 µl (n = 5) Clonidine (nmol) χ 2 , p Sensitive Non-sensitive +BRL44408 10 (n = 5) 3.1 (n = 5) 10 (n = 7) 31 (n = 6) 3.1 (n = 5) 10 (n = 3) 31 (n = 4) Aδ-fibres 64 ± 6 51 ± 6 83 ± 11 63 ± 13 73 ± 8 85 ± 10 67 ± 15 78 ± 6 89 ± 17 10.02, 0.26 C-fibres 166 ± 11 145 ± 12 182 ± 35 104 ± 23 205 ± 39 157 ± 30 151 ± 27 175 ± 26 222 ± 24 12.97, 0.11 FEMALES Control (n = 5) Vehicle 20 µl (n = 5) Clonidine (nmol) χ 2 , p Sensitive Non-sensitive +BRL44408 +Imiloxan +JP1302 10 (n = 4) 31 (n = 7) 10 (n = 6) 31 (n = 3) 31 (n = 5) 31 (n = 4) 31 (n = 5) Aδ-fibres 78 ± 6 76 ± 8 91 ± 6 79 ± 12 92 ± 18 86 ± 13 76 ± 13 64 ± 13 83 ± 9 4.66, 0.79 C-fibres 190 ± 35 190 ± 41 223 ± 45 187 ± 34 307 ± 38 151 ± 61 128 ± 34 111 ± 21 116 ± 14 17.27, 0.03 RESULTS 3.1. Effects of periorbital electrical stimulation on the trigeminal WDR cell firing Figure 2 illustrates the electrophysiological setup used to perform unitary electrophysiological recordings of the trigeminal WDR cells in vivo . Using Eckhorn Matrix software, the trigeminal WDR cells recorded were found at an average of 634 ± 9 µm from the MDH surface. In all cases, evoked action potentials were analysed using PSTH to depict the impact of different treatments on the Aδ- and C-fibre components. Considering that at baseline the mean evoked WDR potentials associated to activation of Aδ- and C-fibres in male and female rats did not follow a normal distribution, a Mann-Whitney U-test was performed. Statistical analysis showed that neuronal spikes at baseline (234 ± 34 in males vs. 267 ± 34 in females) did not differ between sexes ( U = 1239, n 1 = 50 n 2 = 58, p = 0.1939 two-tailed). However, as detailed in Table 1 , unlike males, the baseline means of evoked WDR potentials in females showed a statistically significant difference in C-fibres activity in the different treatments (Kruskal-Wallis test; χ 2 :17.27, p = 0.03). Thus, to homogenise the variability in the WDR firing responses, the following data were normalised. Taking into account that trigeminal WDR cells were recorded for 60 min, excluding a timedependent effect was crucial. One-way ANOVA showed that no time-dependent changes in neuronal responses occurred during our experimental protocols in either sex. Specifically, in the control group, time had no effect on Aδ- (males: F (4, 20) = 2.56, p = 0.07; females: F (4, 20) = 0.11, p = 0.98) or C-fibres (males: F (4, 20) = 0.15, p = 0.96; females: F (4, 20) = 0.76, p = 0.57), and similar results were obtained with vehicle on Aδ- (males: F (4, 20) = 2.152, p = 0.11; females: F (4, 20) = 0.60, p = 0.67) and C-fibres (males: F (4, 20) = 0.153, p = 0.96; females: F (4, 20) = 0.12, p = 0.97). 3.2. Effect of clonidine on the evoked trigeminal WDR cell discharge in male rats Since no time-dependent changes in neuronal responses occurred during the 60 min period, the following experiments were designed to test the effect of clonidine on trigeminal WDR cell firing. As shown in Fig. 3 A, 40 WDR cells were recorded in males: (i) control (n = 5); (ii) vehicle (n = 5); and (iii) clonidine (n = 30). The WDR cells were treated with clonidine (3.1, 10, and 31 nmol; n = 10 cells for each dose), and some cells were inhibited (i.e., sensitive, n = 18), whereas others were unaffected (non-sensitive, n = 12) by this compound. This dichotomy is illustrated in Fig. 3 B, using a representative PSTHs of two WDR cells. Furthermore, as illustrated in the upper panel of Fig. 3 B, the inhibition of WDR cell activity was associated with a decrease in the spike frequency associated with the activation of Aδ- and C-fibres and not due to a decrease in the amplitude of the processed signal. Upon quantifying the data from the PSTHs, the WDR cell firing inhibition by clonidine seems no to be dependent on the dose tested considering that: (i) 5/10 WDR cells recorded were inhibited with 3.1 nmol; (ii) 7/10 WDR cells recorded were inhibited with 10 nmol; and (iii) 6/10 WDR cells recorded were inhibited with 31 nmol. Specifically, the effect on the activity associated with Aδ- and C-fibre activation is illustrated in Figs. 3 C to 3 H. In this regard, in sensitive WDR cells, 10 nmol of clonidine consistently inhibited Aδ- and C-fibres discharge. Note that the antinociceptive effects of clonidine (10 and 31 nmol) was recorded in some WDR cells (Fig. 3 C – 3 E; sensitive WDR cells), whereas the other WDR cells were unaffected by this compound, irrespective of the dose tested (Fig. 3 F to 3 H; non-sensitive WDR cells). See Table 2 for details of the statistical analyses. Table 2 Two-way repeated-measures analysis of variance (ANOVA) data for Figures. Two-way RM ANOVA Figure Interaction Time Treatment 3C F (24, 138) = 3.06; p < 0.0001 F (3.040, 69.92) = 12.29; p < 0.0001 F (4, 23) = 6.48; p = 0.0012 3D F (24, 138) = 5.38; p < 0.0001 F (1.987, 45.71) = 19.43; p < 0.0001 F (4, 23) = 16.58; p < 0.0001 3F F (24, 102) = 2.90; p = 0.0001 F (4.219, 71.73) = 4.94; p = 0.0012 F (4, 17) = 4.14; p = 0.0160 3G F (24, 102) = 0.87; p = 0.6461 F (3.346, 56.89) = 1.87; p = 0.1393 F (4, 17) = 1.74; p = 0.1867 4C F (18, 102) = 3.81; p < 0.0001 F (3.518, 59.81) = 59.81; p < 0.0001 F (3, 17) = 13.93; p < 0.0001 4D F (18, 102) = 8.08; p < 0.0001 F (2.167, 36.83) = 16.92; p < 0.0001 F (3, 17) = 38.03; p < 0.0001 4F F (18, 90) = 0.88; p = 0.6035 F (3.955, 59.32) = 1.00; p = 0.4121 F (3, 15) = 1.09; p = 0.3850 4G F (18, 90) = 1.17; p = 0.3004 F (2.350, 35.26) = 0.56; p = 0.6052 F (3, 15) = 1.13; p = 0.3691 5A F (24, 132) = 3.33; p < 0.0001 F (3.028, 66.61) = 6.06; p = 0.0010 F (4, 22) = 5.23; p = 0.0041 5C F (24, 132) = 4.88; p < 0.0001 F (1.809, 39.81) = 4.91; p = 0.0147 F (4, 22) = 17.22; p < 0.0001 6A F (24, 114) = 1.43; p = 0.1093 F (2.706, 51.41) = 18.89; p < 0.0001 F (4, 19) = 2.83; p = 0.0537 6B F (24, 114) = 3.08; p < 0.0001 F (2.425, 46.08) = 47.22; p < 0.0001 F (4, 19) = 13.28; p < 0.0001 6D F (18, 90) = 1.09; p = 0.3738 F (4.071, 61.06) = 0.69; p = 0.6041 F (4, 15) = 1.23; p = 0.3342 6E F (18, 90) = 1.33; p = 0.1871 F (2.994, 44.90) = 0.66; p = 0.5790 F (4, 15) = 2.33; p = 0.1156 3.3. Effect of clonidine on the evoked trigeminal WDR cell discharge in female rats As mentioned above, no time-dependent changes in the neuronal response were observed during the 60 min period; therefore, the following experiments were designed to test the effect of clonidine on trigeminal WDR cell firing. Current evidence suggests that females are less sensitive to clonidine-induced antinociception; consequently, to reduce the number of animals tested, the dose of 3.1 nmol clonidine was not tested in females. As shown in Fig. 4 A, 30 WDR cells were recorded in females: (i) control (n = 5); (ii) vehicle (n = 5); and (iii) clonidine (n = 20). These cells were treated with clonidine (10 and 31 nmol; n = 10 for each dose), and similar to males, some cells were inhibited (i.e. sensitive, n = 11) or unaffected (non-sensitive n = 9) by this drug. As depicted above for males, these data are illustrated using representative PSTHs of two WDR cells (Fig. 4 B). Note that, as shown in the upper panel, the inhibition of WDR cell activity is associated with a decrease in the spike frequency of Aδ- and C-fibres and not due to a decrease in the amplitude of the processed signal. Upon quantifying the data from the PSTHs, we found that, independent of the dose tested, not all WDR cells were inhibited by clonidine. Specifically, (i) 4/10 cells recorded were inhibited by 10 nmol, and (ii) 7/10 cells recorded were inhibited by 31 nmol. As illustrated in Figs. 4 C and 4 D, although 10 nmol clonidine tended to diminish neuronal activity (inhibition > 20%, particularly of the C-fibres), statistical analysis of the temporal course data in these sensitive WDR cells showed that 31 nmol, but not 10 nmol clonidine, inhibited neuronal firing of the Aδ- and C-fibres. However, an analysis of global effects (Fig. 4 E), suggest that both doses of clonidine seemed to block the neuronal activity of the Aδ- and C-fibres. In this regard, we consider that 31 nmol of clonidine consistently inhibits Aδ- and C-fibres discharge over time. Figures 4 F to 4 H illustrates that some WDR cells were completely unaffected by this drug at all doses tested. See Table 2 for details about statistical analysis. 3.4. Effect of BRL 44408 on the clonidine-induced trigeminal electrophysiological antinociception in male rats Taking into account that data from the SDH and MDH consistently suggest that α 2A -adrenoceptor subtype activation mediates clonidine-induced antinociception in male rats [ 7 , 18 , 22 ], the following experiments were performed to corroborate previous findings under our experimental conditions. As shown in Fig. 5 A to 5 D, the 10 nmol clonidine-induced inhibition of WDR cell discharge was prevented (Aδ-fibres) or reversed (C-fibres) when sensitive to clonidine WDR cells were posttreated with an α 2A -adrenoceptor agonist (BRL 44408). Note that BRL 44408 per se had no effect on the WDR activity. The impact of BRL 44408 on clonidine-induced antinociception is illustrated using PSTHs (Fig. 5 E); note that the inhibition of WDR cell discharge by clonidine (baseline vs. 10 min) was reversed when the WDR cells were treated with BRL 44408 (30 min, lower panel). 3.5 Effect of BRL 44408, imiloxan and JP-1302 on the clonidine-induced trigeminal electrophysiological antinociception in female rats Current behavioural experiments suggest that α 2 -adrenoceptors play a key role in clonidineinduced trigeminal antinociception in both the sexes. However, no evidence regarding the receptor subtype and how the neural WDR code is modified has been tested in females. As shown in Fig. 6 A to 6 C, the experiments showed that unlike in males, blocking α 2A adrenoceptors with BRL 44408 (10 nmol) did not reverse clonidine-induced antinociception. In contrast, treatment with imiloxan (10 nmol α 2B -adrenoceptor subtype antagonist) or JP-1302 (10 nmol; α 2C -adrenoceptor subtype antagonist) reversed clonidine-induced antinociception, particularly during the temporal course of the Aδ-fibres (Fig. 6 A), but not on the C-fibres (Fig. 6 B), where the inhibitory effect remained. Similar data were obtained when the neuronal response was analysed as a global response (Fig. 6 C). It is important to mention that the antagonists used had no effect per se on the evoked WDR trigeminal responses (Fig. 6 D to 6 F). DISCUSSION 4.1. General Apart from the implications discussed below, electrophysiological experiments showed that clonidine inhibited trigeminal neuronal activity associated with periorbital electrical activation of Aδ- and C-fibres in both sexes. In addition, we found the following: i. Although male and female trigeminal WDR cells were inhibited by clonidine, not all WDR cells were inhibited, independent of the dose tested. Thus, the WDR cell population can be divided into sensitive and non-sensitive to clonidine (Figs. 3 and 4). ii. Females were less prone to clonidine-induced antinociception (i.e. the dose required to induce a similar antinociception was higher in females (10 vs. 31 nmol) (Figs. 3 and 4). iii. In males, as previously shown [11, 18], α 2A -adrenoceptor subtype activation is involved in clonidine-induced electrophysiological trigeminal antinociception (Fig. 5). iv. In contrast, in females, the α 2A -adrenoceptor subtype does not seem to be relevant to the inhibitory effect of clonidine on WDR cell discharge. Remarkably, trigeminal antinociception induced by clonidine seems to depend on α 2B - and α 2C - adrenoceptor subtypes activation in Ad-, but not in C-fibres (Fig. 6). 4.2. Clonidine inhibited the evoked activity of male and female trigeminal WDR cells Although in vivo electrophysiological experiments have shown that in males, trigeminal administration of clonidine induces antinociception through the activation of α 2 -adrenoceptors [11, 23-26], this effect has not been analysed in females. To the best of our knowledge, only behavioural experiments have been made suggesting that similar than males, the α 2 -adrenoceptors play a role in the clonidine induced antinociception [13, 27]. However, beyond the a 2A/2B/2C -adrenoceptor subtypes involved (currently unknown in females), experiments in females have not been designed to analyse the impact of clonidine upon neural transmission. In this regard, the second-order WDR cells at MDH modulate the nociceptive input from the periphery to supraspinal centres, encoding the neural influx from non-nociceptive and nociceptive signals transmitted by primary Aβ-, Aδ-, and C-fibres. Hence, WDR cells are among the first relays in which the incoming sensorial input is computed and modulated [28]. Considering that under our experimental conditions, the electrically induced neuronal WDR response in both sexes was stable, a reduction in WDR cell firing by a given pharmacological treatment could be interpreted as a compound with potential analgesic action. Indeed, trigeminal application of analgesics (e.g. naratriptan, olcegepant, or morphine) suppresses the responsiveness of trigeminal WDR cells by inhibiting neuronal discharge associated with the activation of Ad- and C-fibres [28-31]. As expected, electrophysiological data showed that trigeminal clonidine induced antinociception in both sexes (Figs. 3 and 4). However, as previously reported in behavioural experiments [32, 33], females require a higher dose (10 vs. 31 nmol) of clonidine to obtain an inhibitory effect upon Ad- and C-fibres, similar to that observed in males. Certainly, in behavioural trigeminal pain models using ovariectomized or castrated rats with hormonal replacement therapy, sexual hormones have been shown to play a role in the degree of antinociception induced by clonidine [12, 13, 27]. Together, these data indicate the relevance of sex in sensitivity to analgesic treatments, as discussed extensively by Mogil et al. [34, 35]. In addition, it is interesting to note that in both sexes, the trigeminal WDR cells were clustered into two populations: (i) sensitive and (ii) non-sensitive to the antinociceptive effects elicited by clonidine. In this regard, at MDH of male rodents, and using in vitro and in vivo electrophysiological recordings consistently showed that not all cells recorded (WDR, nociceptive specific and low threshold cells) could be inhibited by clonidine or noradrenaline [11, 24, 25, 36, 37]. Thus, the most parsimonious interpretation could be that there is a heterogeneous distribution of clonidine-sensitive receptors on primary afferent fibres (PAF). Furthermore, given that our electrophysiological data in both sexes (see Figs. 3 and 4) showed that clonidine inhibited neuronal evoked activity associated with the activation of Aδ- and C-fibres, a presynaptic effect on nociceptive PAFs is supported. However, the degree of antinociception elicited by clonidine seems to be more relevant for C-fibres than Aδ-fibres. Therefore, at first glance, if we consider that clonidine exerts its antinociception by activation of the α 2 -adrenoceptor, we could propose that at the trigeminal level, sensitive receptors to clonidine must be predominantly expressed on C-fibres. In this regard, α 2A/2B/2C -adrenoceptors have been reported to be expressed at the spinal and trigeminal level [38-41], but the extent of expression of these receptor subtypes upon PAF has not been explored. 4.3. The role of α 2A/2B/2C -adrenoceptor subtypes in the clonidine-induced antinociception in males and females In males, it is well known that activation of trigeminal α 2 -adrenoceptors induces antinociception predominantly via the α 2A -adrenoceptor subtype [11, 23-27, 39]. Therefore, we decided to pharmacologically dissect under our experimental conditions whether this adrenoceptor subtype was involved in clonidine-induced electrophysiological antinociception in male and female trigeminal WDR cells. As expected, in males, pharmacological blockade of α 2A -adrenoceptor with BRL 44408 fully reversed clonidine-induced electrophysiological antinociception in Ad- and C‑fibres (Fig. 5). As above referenced, under acute nociception, α 2A - rather than α 2B/2C ‑adrenoceptors, plays a key role inhibiting nociception [42, 43]. In contrast, the inhibitory effect of clonidine persisted in females when the α 2A ‑adrenoceptor subtype was blocked (Fig. 6A to 6C). These data implied that in females α 2A -adrenoceptor do not play a pivotal role in clonidine-induced antinociception. We must keep in mind that males and females differ in the degree of antinociception induced by clonidine, where males are more sensitive to clonidine effects, at least in behavioural experiments [12, 13, 27, 36]. Based on this evidence, it is not surprising that the receptor subtypes involved could differ between the sexes. For example, in a neuropathic pain behavioural model, tizanidine (an α 2 -adrenoceptor agonist) induced antinociception in females by α 2B - rather than α 2A -adrenoceptor activation [44]. As shown in Figure 6A to 6C, when the α 2B -adrenoceptor in females was blocked with imiloxan, we found that the electrophysiological antinociception elicited by clonidine was particularly reversed on neuronal firing associated with the activation of Ad-fibres, without any effect on C-fibres. A similar outcome was obtained when α 2C -adrenoceptor was blocked with JP‑1302. It is relevant to mention that the antagonists used (i.e., BRL 44408, imiloxan and JP‑1302) devoid any effect per se on the evoked WDR cell activity (see Figs. 6D to 6F). At this point, beyond the finding that the receptors subtypes involved in clonidine-induced inhibition were different between males and females, it was also interesting that in females, the Ad- but not C‑fibres recovered their baseline neuronal firing after the blockade of α 2B - or α 2C -adrenoceptors. These results imply that in females: i. The specific expression of these α 2 -adrenoceptors subtypes on A d - and C-fibres differs. Indeed, although it is well known that in males, the global expression of the α 2 -adrenoceptor subtypes at the trigeminal level differs (i.e. α 2A >α 2C >>>α 2B ) [18, 25, 40, 45-50], currently no data about the distribution of these α 2 -adrenoceptor subtypes expression on Ad- and C-fibres exist. Certainly, transcriptomic profiling at single-cell resolution could be necessary to explore these sexual dimorphisms. ii. Clonidine-induced antinociception in C-fibres may be mediated by different mechanisms or receptors. For example, it has been reported that clonidine can activate imidazoline receptors [15, 51, 52]. Indeed, the role of imidazoline receptors exerting antinociception has been suggested, based on the fact that yohimbine (a preferent α 2 -adrenoceptor antagonist) did not reverse clonidine-induced spinal antinociception in the formalin test [53, 54]. However, several reports have shown that clonidine-induced spinal antinociception is resistant to treatment with selective I 1 /I 2 imidazoline receptor antagonists [55-58]. In agreement with these data, pretreatment with more selective α 2 -adrenoceptor antagonists (e.g., rauwolscine) is enough to abolish the effect of clonidine. Therefore, the current analysis of the receptors implicated in the antinociception induced by clonidine seems to exclude the role of imidazoline receptors. However, we must admit that our experimental design was not designed to analyse the role of imidazoline receptors in clonidine-induced antinociception. 4.4. Conclusion The data collectively indicate that α 2A -adrenoceptor activation induces significant antinociceptive effects on trigeminal Ad- and C-fibres in males, but not in females. In contrast, in females, the activation of α 2B/2C -adrenoceptors inhibits neuronal firing associated with the recruitment of Ad‑fibres, but not C-fibres. This suggests that in female rats, other mechanisms or receptors may mediate the inhibition of C-fibre discharge induced by clonidine. Abbreviations ANOVA Analysis of variance C1 C1 cervical vertebra CO 2 Carbon dioxide i.p. Intraperitoneal i.v. Intravenous KCl Potassium chloride MDH Medullary dorsal horn PAF Primary afferent fibres PSTH Peri-stimulus time histograms RF Receptive field SDH Spinal dorsal horn SEM Standard error of the mean V1 Ophthalmic branch of the trigeminal nerve WDR Wide dynamic range Declarations Acknowledgements. Gustavo López Córdoba is a doctoral student (ID number: 518010700) from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and has received a CONAHCYT (currently named SECIHTI) fellowship (no. 859394). This research article is part of his doctoral thesis, and the authors thank the Posgrado de Doctorado en Ciencias Biomédicas UNAM for their academic support. Nombre del Alumno is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and has received CONAHCYT fellowship (no. 859394). Authors' contributions. Conceptualisation (G.L-C., M.C-L., A.G-H.), investigation (G.L-C., G.M‑L., A.G-H.), data curation (G.L-C.), formal analysis (G.L-C., A.G-H.), supervision (G.M-L., M.C-L., A.G-H.), resources (M.C-L., A.G-H.), wrote the main manuscript text (G.L-C., A.G-H.), All authors have read and approved the final manuscript. Funding. This work on rodents was sponsored by Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM Mexico) under Grant agreement no. IN227225 (A.G-H.). Data availability. All the relevant data have been included in the manuscript. Ethics approval. This study was conducted in accordance with the principles of the Basel Declaration and recommendations of the (i) of Health Guidelines for the Care and Use of Laboratory Animals, (ii) ARRIVE Guidelines, and (iii) Standards for the Investigation of Experimental Pain in Animals [59]. The study protocol was approved by the Institutional Animal Care and Use Committee of the Instituto de Neurobiología, UNAM. Consent for publication. Not applicable. Competing interests . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Author information . Departamento de Neurobiología del Desarrollo y Neurofisiología, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus UNAM-Juriquilla. ORCiD / e-mail G.L-C. 0009-0006-3072-8010 / [email protected] G.M-L. 0009-0001-5184-6431 / [email protected] M.C-L. 0000-0003-1651-8931 / [email protected] A.G-H. 0000-0002-6472-1419 / [email protected] References Eisenach JC, Dewan DM, Rose JC et al (1987) Epidural clonidine produces antinociception, but not hypotension, in sheep. 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Cite Share Download PDF Status: Published Journal Publication published 25 Feb, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted Editorial decision: Revision requested 24 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 08 Sep, 2025 Editor assigned by journal 08 Sep, 2025 Submission checks completed at journal 08 Sep, 2025 First submitted to journal 05 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7546267","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512978654,"identity":"a699330c-f598-4459-82c8-34f7e9afc1d6","order_by":0,"name":"Gustavo López-Córdoba","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"López-Córdoba","suffix":""},{"id":512978656,"identity":"369b272e-5a9d-4ca6-bee0-512501cdb95e","order_by":1,"name":"Guadalupe Martínez-Lorenzana","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Guadalupe","middleName":"","lastName":"Martínez-Lorenzana","suffix":""},{"id":512978657,"identity":"ceeac9ff-dad2-487c-8bcc-692c8a19b05c","order_by":2,"name":"Miguel Condés-Lara","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Condés-Lara","suffix":""},{"id":512978660,"identity":"75c70cc4-12b3-4944-92f1-d87aa1e29576","order_by":3,"name":"Abimael González-Hernández","email":"data:image/png;base64,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","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":true,"prefix":"","firstName":"Abimael","middleName":"","lastName":"González-Hernández","suffix":""}],"badges":[],"createdAt":"2025-09-05 17:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7546267/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7546267/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10194-025-02221-x","type":"published","date":"2026-02-25T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91385205,"identity":"202913a9-fdf3-47b1-8d68-8ad8cf23f4cf","added_by":"auto","created_at":"2025-09-16 02:21:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284966,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of animals and the allocation of the experimental groups used in this study. The animals were divided into two main sets: male (set 1) and female (set 2).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/a46debf83a806439bdb85527.jpeg"},{"id":91385078,"identity":"f879bd2d-5716-4a18-8990-4b6cd73777dc","added_by":"auto","created_at":"2025-09-16 02:13:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":374055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrophysiological setup. (A)\u003c/strong\u003e Raw data from a single second-order trigeminal WDR cell responding to 20 consecutive electrical stimuli delivered in the periorbital receptive field (RF). \u003cstrong\u003e(B)\u003c/strong\u003e Locations of the two stimulating electrodes in the RF are illustrated. The recording electrodes were inserted into the dorsolateral segment of the medullary dorsal horn (MDH), ipsilateral to the RF. \u003cstrong\u003e(C)\u003c/strong\u003e The upper panel illustrates the raw data of the WDR firing (upper panel) elicited by a single electrical stimulus, whereas in the middle and lower panels, a raster display and peri-stimulus time histogram (PSTH) constructed from 20 electrical pulses is shown. Note that, depending on the latency time, the Aδ- and C-fibre components of the WDR cell responses can be discriminated.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/21a70900b845ec6dee93c7b3.jpeg"},{"id":91385080,"identity":"b572d20c-9c82-49d8-b20c-3a3bb9e821b6","added_by":"auto","created_at":"2025-09-16 02:13:08","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":510813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResponse of trigeminal second-order WDR cells to clonidine in male rats. (A) \u003c/strong\u003eForty WDR cells were analysed: (i) control (n = 5); (ii) vehicle (n = 5); and (iii) clonidine (n = 30). Note that some WDR cells were non-sensitive to the antinociception induced by clonidine. \u003cstrong\u003e(B)\u003c/strong\u003e In the upper panels, raw data (100 ms) of WDR cell firing elicited by a single electrical stimulus are shown before (baseline) and after 10 nmol clonidine (10 and 30 min); after which the spike frequency, but not the amplitude of the signal, was diminished. In the middle and lower panels, the peri-stimulus time histograms (PSTH) of a sensitive and non-sensitive WDR cell to clonidine are illustrated; note that the Aδ- and C-fibre components of the WDR cells are depicted. Based on these data, sensitive and non-sensitive WDR cells to the clonidine antinociception were found. Panels (\u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e) show the time course of the percentage change in the Ad- and C-fibres activity in WDR cells inhibited by clonidine; such inhibition was preferential upon C-fibres discharge at all doses tested (3.1, 10, and 31 nmol). Analysis of these data as a global neuronal effect (\u003cstrong\u003eE\u003c/strong\u003e) showed that 10 nmol (but not 31 nmol) of clonidine consistently inhibited the activity of both fibres. In contrast, as shown in panels (\u003cstrong\u003eF \u003c/strong\u003eto\u003cstrong\u003e H\u003c/strong\u003e), some WDR cells were unaffected by clonidine at all doses tested.\u003cstrong\u003e \u003c/strong\u003eEmpty symbols (in the temporal courses) or * represent p \u0026lt; 0.05 vs control.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/7823b2b7a162ffea53315ee2.jpeg"},{"id":91385084,"identity":"566bccfb-082a-41fe-a14b-1e648d9084b5","added_by":"auto","created_at":"2025-09-16 02:13:08","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":498110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResponse of trigeminal second-order WDR cells in females.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eThirty WDR cells were analysed: (i) control (n = 5); (ii) vehicle (n = 5); and (iii) clonidine (n = 20). Note that some WDR cells were non-sensitive to the antinociception induced by clonidine. \u003cstrong\u003e(B) \u003c/strong\u003eIn the upper panels, raw data (100 ms) of WDR cell firing elicited by a single electrical stimulus is shown before (baseline) and after 31 nmol clonidine (10 and 30 min), after which the spike frequency, but not the amplitude of the signal, was diminished. In the middle and lower panels, the peri-stimulus time histograms (PSTH) of a sensitive and non-sensitive WDR cell to clonidine are illustrated; note that the Aδ- and C-fibre components of the WDR cells are depicted considering the latencies of each fibre. Based on these data, sensitive and non-sensitive WDR cells to the clonidine antinociception were found. Panels\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) show the time-course of the percentage change in the Ad- and C-fibres activity of WDR cells inhibited by clonidine; such inhibition was observed in Ad-, and C-fibres discharge at 31 nmol of clonidine; note that the inhibition was more pronounced upon C-fibres. Analysis of these data as a global neuronal effect (\u003cstrong\u003eE\u003c/strong\u003e) showed that both doses of clonidine inhibited the global activity of the Ad- and C-fibres. In contrast, as shown in panels (\u003cstrong\u003eF \u003c/strong\u003eto\u003cstrong\u003eH\u003c/strong\u003e), some WDR cells were unaffected by clonidine at all doses tested.\u003cstrong\u003e \u003c/strong\u003eEmpty symbols (in the temporal courses) or * represent p \u0026lt; 0.05 vs control.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/12ea8c4d04dc411e3647b1fc.jpeg"},{"id":91385210,"identity":"e001cc47-f049-448b-b9b9-e906a4a8afd2","added_by":"auto","created_at":"2025-09-16 02:21:08","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":447011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of trigeminal α\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-adrenoceptor subtypes in clonidine-induced antinociception in males.\u003c/strong\u003e (\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) Time-course of percentage of change and the global percentage of change (respectively) of Ad-fibre activity in sensitive WDR cells post-treated (10 min after clonidine) with 10 nmol BRL 44408. The blockade of α\u003csub\u003e2A\u003c/sub\u003e-adrenoceptors prevented the onset of the inhibitory effects of clonidine. Furthermore, as shown in panels (\u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e), 10 min after administration of BRL 44408, clonidine-induced inhibition of C-fibres discharge was reversed. Note that BRL 44408 administered alone did not have any effect on evoked neuronal firing in the Ad- and C-fibres. \u003cstrong\u003e(E)\u003c/strong\u003e The peri-stimulus time histograms (PSTH) of sensitive WDR cells (red) treated with clonidine and another sensitive WDR cell treated concomitantly with clonidine + BRL 44408 (black); notably, in the case of the WDR cell treated with clonidine alone (red), the number of events was clearly diminished, whereas in the WDR cell post-treated with BRL 44408 at min 10, the number of events measured at min 30 was similar to the baseline. Unfilled symbols (in the temporal courses) or * represent p \u0026lt; 0.05 vs control.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/7714365c88655943da59026c.jpeg"},{"id":91385082,"identity":"b8f1a45e-cb55-46f9-8454-6598a5b23bc8","added_by":"auto","created_at":"2025-09-16 02:13:08","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":519921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of trigeminal α\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A/2B/2C\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-adrenoceptors subtypes in clonidine-induced antinociception in females. \u003c/strong\u003eWDR cells were post-treated (10 min after clonidine) with BRL 44408, imiloxan, or JP-1302. Panels (\u003cstrong\u003eA\u003c/strong\u003e to \u003cstrong\u003eC\u003c/strong\u003e) show the time-course of the percentage of change and the global neuronal activity of the Ad- and C-fibres in the sensitive WDR cells post-treated with the different antagonists; note that in Ad-fibres, but not in C-fibres the blockade of the α\u003csub\u003e2B\u003c/sub\u003e- and α\u003csub\u003e2C\u003c/sub\u003e-adrenoceptors reversed the inhibitory effects of clonidine. Panels (\u003cstrong\u003eD \u003c/strong\u003eto\u003cstrong\u003e F\u003c/strong\u003e), illustrates that antagonists did not have a \u003cem\u003eper se\u003c/em\u003e effect. Empty symbols (in the temporal courses) or * represent p \u0026lt; 0.05 vs control.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/7a1f734119ad0d8925f73305.jpeg"},{"id":103765864,"identity":"9ccb9e78-d8b7-4d1a-b445-f15d0d7aa2e6","added_by":"auto","created_at":"2026-03-02 16:10:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4105543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7546267/v1/d74fc6c9-a177-4755-b933-9a73bedc1c79.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electrophysiological evidence about sexual dimorphism in the clonidine-induced inhibition of trigeminal wide dynamic range cell activity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSince the mid-1980s, it has been shown that spinal administration of clonidine (an α\u003csub\u003e2\u003c/sub\u003e-adrenoceptor agonist) has analgesic effects in humans and antinociception in rodents [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At the spinal dorsal horn (SDH) level, many electrophysiological studies in male rodents have consistently reported that clonidine via the α\u003csub\u003e2\u003c/sub\u003e-adrenoceptor induces antinociception by presynaptic inhibition of the neuronal evoked activity associated with activation of Aδ- and C-fibres in second-order wide dynamic range (WDR) cells [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, although scarce, experiments at the trigeminal level, particularly in the medullary dorsal horn (MDH), have shown that clonidine also exerts antinociception by activating the α\u003csub\u003e2\u003c/sub\u003e-adrenoceptor. Interestingly, at MDH, using behavioural approaches (in male and female rodents), it was shown that females are less sensitive to clonidineinduced antinociception; hence, sexual dimorphism linked to the hormonal milieu has been implicated[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond hormonal component, how sex impact the clonidine-induced antinociception at the neural level has not been explored; thus, the present study was designed to analyse the influence of sex on clonidine-induced neuronal inhibition of trigeminal nociception. Furthermore, we must keep in mind that α\u003csub\u003e2\u003c/sub\u003e-adrenoceptors can be pharmacologically subdivided into three functional subtypes, namely α\u003csub\u003e2A\u003c/sub\u003e-, α\u003csub\u003e2B\u003c/sub\u003e-, and α\u003csub\u003e2C\u003c/sub\u003e-adrenoceptors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Indeed, current data strongly support the notion that α\u003csub\u003e2A\u003c/sub\u003e-adrenoceptors play a key role in spinal and trigeminal clonidineinduced antinociception [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Admittedly, these findings have been reported in male rats; however, the contribution of these receptor subtypes to clonidine-induced antinociception has not been explored in females. Certainly, the search for precise mechanisms involved in the modulation of nociception in female and male will provide basic evidence with potential clinical relevance.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHence, by using a pharmacological approach coupled with an \u003cem\u003ein vivo\u003c/em\u003e analysis of the WDR cell response at the MDH, the effect of clonidine (administered at the trigeminal level) on periorbital-evoked nociceptive responses were analysed in male and female rats. The data showed that, although WDR firing was inhibited in both sexes by clonidine, females required a higher dose. Furthermore, the α\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor subtype elicits robust inhibition of Aδ- and C-fibres discharge in males, but not in females. In contrast, in females, the α\u003csub\u003e2B/2C\u003c/sub\u003e-adrenoceptors subtypes seem to play a key role mediating inhibition of Aδ- discharge, but not upon C-fibres activity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Experimental animals and ethical standards\u003c/h2\u003e\n \u003cp\u003eA total of 108 male and female rats (313\u0026thinsp;\u0026plusmn;\u0026thinsp;4 and 288\u0026thinsp;\u0026plusmn;\u0026thinsp;8 g, respectively; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) from the bioterium of our institute were used in this study. The animals were housed in pairs in acrylic cages (18\u0026quot; L \u0026times; 9\u0026quot; W \u0026times; 8 H) with woodbased bedding and controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (50%) on a 12:12 h light/dark cycle (light beginning at 7:00 h), with food (LabDiet 5001) and tap water ad libitum. The rodents were allowed to acclimatise to their new environment for at least 72 h before handling for the experiments. All animal protocols were approved by our Institutional Ethics Committee, following the local animal welfare committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (80\u0026thinsp;\u0026minus;\u0026thinsp;23, revised in 1996).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. General methods\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1. Surgical procedures for electrophysiological recordings of trigeminal WDR cells\u003c/h2\u003e\n \u003cp\u003eThe animals were anaesthetised with urethane (2 g/kg, i.p.). Adequacy of anaesthesia before surgery was judged by the absence of ocular reflexes, a negative tail flick test, and corporal relaxation. Under these conditions, an intratracheal cannula was inserted and the animals were mounted onto a stereotaxic frame (Narishige Group, Japan). The animals were immediately connected to a rodent ventilator (65\u0026ndash;75 strokes/min) coupled to a homeothermic control module (RoVent\u0026reg;, Kent Scientific Corp., USA) to maintain ventilation and body temperature (~\u0026thinsp;37\u0026deg;C). Next, the muscles of the dorsal neck were separated, cervical (C1) laminectomy was performed, and the overlying dura was carefully removed to allow access to the MDH, as previously reported [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The animals were not paralysed, and no withdrawal reactions were observed during the experiments. End-tidal CO\u003csub\u003e2\u003c/sub\u003e was monitored (Passport 2 Monitor, Datascope Corp., USA) and maintained within the physiological range (2.5-3.0%) throughout the experiment. At the end of the experiment, the animals were halted with an intracardiac overdose of KCl.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2. Extracellular recordings of trigeminal WDR cells\u003c/h2\u003e\n \u003cp\u003eThree quartz-platinum/tungsten microelectrodes (4\u0026ndash;8 MΩ) mounted in a multichannel microdrive Mini Matrix System (Thomas Recording GmbH, Germany) were used to record neuronal activity of second-order wide dynamic range (WDR) cells with input from the periorbital receptive field (RF). Although this device allows the insertion of three microelectrodes into neural tissue (inter-electrode distance: 300 \u0026micro;m), and each microelectrode was inserted independently using the Eckhorn Matrix multiuser software (Thomas Recording GmbH, Giessen, Germany), we recorded only one extracellular unit per animal.\u003c/p\u003e\n \u003cp\u003eThe microelectrodes were positioned in the dorsolateral segment of the MDH and lowered (500\u0026ndash;700 \u0026micro;m from the surface) in small steps (2\u0026ndash;5 \u0026micro;m/s) to search for single-unit discharges evoked by innocuous tactile stimulation of the periorbital dermatome (mainly innervated by the ophthalmic branch of the trigeminal nerve, V1). Then, RF was assessed for non-noxious (brushing) and noxious (pinching) inputs. To verify the peripheral input relays in a WDR cell, electrical stimulation was applied using two electrodes inserted into the RF. In this case, two needles (27 G) attached to an electrical stimulator (2100 Isolated Pulse Stimulator; A-M Systems, LLC, USA) were inserted subcutaneously into the RF. Electrical test stimulation was then conducted. This test consisted of 20 square electrical stimuli (0.5 Hz) with a 1-ms pulse duration at 5\u0026ndash;9 mA to evoke A\u0026delta;- and C-fibre responses.\u003c/p\u003e\n \u003cp\u003eThe evoked multiunit extracellular cluster signals were amplified \u0026times;100 (1700 Differential AC amplifier; A-M Systems, USA), digitised, and discriminated using Micro1401-4 CED hardware and Spike2 v10.19 software (Cambridge Electronic Design, UK). To identify the responding cells, the action potentials of the recorded cells were analysed using peri-stimulus time histograms (PSTH) and raster displays (100 ms duration; bin time\u0026thinsp;=\u0026thinsp;1 ms). Raw data were fed through an audio monitor (Model 3300; A-M Systems LLC, USA) and displayed on a digital oscilloscope (TBS1064; Tektronix, Inc., USA). Furthermore, the waveforms and recorded spike trains were stored on a computer disk for offline analysis.\u003c/p\u003e\n \u003cp\u003eBased on the distance between the RF and recording electrode, the spike latencies observed correspond to peripheral conduction velocities within the A\u0026delta;- (3\u0026ndash;25 ms) and C-fibres (26\u0026ndash;100 ms) [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. On this basis, the threshold to evoke action potentials and their frequency of occurrence resulting from the stimulation of the periorbital region were attributed to the recruitment of A\u0026delta;- and C-fibres. In the process of identifying trigeminal WDR cells that respond to periorbital tactile and electrical RF stimulation, some neurones were categorised as solely tactile-sensitive and a smaller proportion as nociceptive-specific; these cell types were not subjected to further analysis. Thus, the number of action potentials in response to 20 RF stimuli (0.5 Hz, 1-ms pulse, 5\u0026ndash;9 mA) was compared before (baseline) and after the treatment. The baseline response was established after an identified neuron had a variation of \u0026le;\u0026thinsp;10% in the evoked neural responses in at least three consecutive tests. In all cases, pharmacological treatments were delivered at the MDH level (topical) near the site of microelectrode insertion at a volume of 20 \u0026micro;L using a Hamilton\u0026reg; syringe.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Pharmacological treatments and electrophysiological study design\u003c/h2\u003e\n \u003cp\u003eAs illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the animals were allocated into two main sets: male (n\u0026thinsp;=\u0026thinsp;50) and female (n\u0026thinsp;=\u0026thinsp;58). In all cases, evoked trigeminal WDR cell firing was evaluated before (baseline) and after vehicle or clonidine administration at 10, 20, 30, 40, 50, and 60-min post-treatment (see Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e for details), by applying 20 RF electrical stimuli each time. Vehicle (n\u0026thinsp;=\u0026thinsp;5 for each sex) or clonidine (3.1, 10, and 31 nmol; n\u0026thinsp;=\u0026thinsp;10 for each dose) were administered immediately after the first test (i.e. baseline). A control group without treatment was used for both sexes (n\u0026thinsp;=\u0026thinsp;5 for each group).\u003c/p\u003e\n \u003cp\u003eAt this point, we must clarify that the following experiments involving antagonists were designed with the following considerations: (i) females exhibit less sensitivity to the antinociceptive effects of clonidine than males, thus requiring a higher dose to achieve a similar degree of trigeminal antinociception (10 nmol in males vs. 31 nmol in females); and (ii) regardless of the clonidine dose administered, not all WDR cells were inhibited (~\u0026thinsp;58% of the WDR cells were susceptible to inhibition). Therefore, before antagonist treatment, it was necessary to determine whether clonidine exerted an inhibitory effect on WDR cell firing. If 10 min after clonidine administration, the WDR cell activity was inhibited (\u0026gt;\u0026thinsp;15% inhibition), then 20 \u0026micro;l of the following antagonists was locally administered: (i) BRL 44408 (10 nmol; \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor antagonist); (ii) imiloxan (10 nmol; \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e-adrenoceptor antagonist); and (iii) JP-1302 (10 nmol; \u0026alpha;\u003csub\u003e2C\u003c/sub\u003eadrenoceptor antagonist). The \u003cem\u003eper se\u003c/em\u003e effects of the antagonists used also was tested. A detailed description of the groups and doses used is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Compounds\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eIn addition to urethane, we used the following chemicals from Sigma Chemical Co.: (i) clonidine hydrochloride (CAS number: 4205 91 8), (ii) 2-[2H-(1-methyl-1,3-dihydroisoindole) methyl]-4,5-dihydroimidazole maleate (BRL 44408; CAS number: 118343-19-4), (iii) 2-(1-ethyl-2-indazoyl) methyl-1,4-benzodioxan hydrochloride (imiloxan; CAS number: 81167-22-8); and (iv) N-[4-(4-methyl-1-piperazinyl)phenyl]-9-acridinamine dihydrochloride (JP-1302; CAS number: 1259314-65-2) acquired from Tocris Ltd. (USA). The doses of clonidine, BRL 44408, imiloxan, and JP-1302 were based on their free bases. All drugs were dissolved in saline solution (0. % NaCl).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Data presentation and statistical analysis\u003c/h2\u003e\n \u003cp\u003eOne trigeminal WDR cell was studied per animal, and as previously reported [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e], data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Before performing a parametric statistical analysis, we checked for normality using the Shapiro-Wilk test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Thus, the number of basal (baseline) evoked potentials (total spikes and number of A\u0026delta;- and C-fibres) in the different experimental groups was analysed. Because the normality test failed, a Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks was performed (see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Consequently, the data were normalised and the triggered potentials induced by electrical stimulation of the periorbital RF were expressed as a percentage change from the respective baseline. To assess the stability of the recorded neurones over the 60-minute period in the control and vehicle groups, we employed a one-way repeated measures ANOVA. Differences in neuronal activity evoked within one group of animals before and after treatment were compared using two-way repeated-measures ANOVA. Moreover, the temporal course was adjusted to obtain global neuronal activity due to the treatment (box and whisker plots), and an ordinary one-way ANOVA was performed. ANOVAs were followed (if applicable) by the Dunnett \u003cem\u003epost-hoc\u003c/em\u003e test. In the case of two-way ANOVAs, sphericity was not assumed and corrections to degrees of freedom were made following the GreenhouseGeisser method. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Graphs and statistical analyses were performed using GraphPad Prism V6.0 software (USA).\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean action potentials (\u0026plusmn; SEM) elicited by 20 electrical stimuli at baseline in the experimental groups for both sexes. A KruskalWallis one-way ANOVA on ranks was performed to compare the action potential elicited by the different treatments.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eMALES\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eVehicle\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e20 \u0026micro;l\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eClonidine (nmol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e, p\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSensitive\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eNon-sensitive\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e+BRL44408\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e10\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e3.1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e10\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e31\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e3.1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e10\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e31\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eA\u0026delta;-fibres\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u0026thinsp;\u0026plusmn;\u0026thinsp;17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.02, 0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eC-fibres\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e166\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e145\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182\u0026thinsp;\u0026plusmn;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e205\u0026thinsp;\u0026plusmn;\u0026thinsp;39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e151\u0026thinsp;\u0026plusmn;\u0026thinsp;27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.97, 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eFEMALES\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eVehicle\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e20 \u0026micro;l\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eClonidine (nmol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026chi;\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e, \u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNon-sensitive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+BRL44408\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+Imiloxan\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+JP1302\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eA\u0026delta;-fibres\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e78\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.66, 0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC-fibres\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e190\u0026thinsp;\u0026plusmn;\u0026thinsp;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e190\u0026thinsp;\u0026plusmn;\u0026thinsp;41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e223\u0026thinsp;\u0026plusmn;\u0026thinsp;45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e187\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e307\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e151\u0026thinsp;\u0026plusmn;\u0026thinsp;61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.27, 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Effects of periorbital electrical stimulation on the trigeminal WDR cell firing\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the electrophysiological setup used to perform unitary electrophysiological recordings of the trigeminal WDR cells \u003cem\u003ein vivo\u003c/em\u003e. Using Eckhorn Matrix software, the trigeminal WDR cells recorded were found at an average of 634\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u0026micro;m from the MDH surface. In all cases, evoked action potentials were analysed using PSTH to depict the impact of different treatments on the A\u0026delta;- and C-fibre components.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eConsidering that at baseline the mean evoked WDR potentials associated to activation of A\u0026delta;- and C-fibres in male and female rats did not follow a normal distribution, a Mann-Whitney U-test was performed. Statistical analysis showed that neuronal spikes at baseline (234\u0026thinsp;\u0026plusmn;\u0026thinsp;34 in males vs. 267\u0026thinsp;\u0026plusmn;\u0026thinsp;34 in females) did not differ between sexes (\u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1239, \u003cem\u003en\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50 \u003cem\u003en\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;58, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1939 two-tailed). However, as detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, unlike males, the baseline means of evoked WDR potentials in females showed a statistically significant difference in C-fibres activity in the different treatments (Kruskal-Wallis test; \u0026chi;\u003csup\u003e2\u003c/sup\u003e:17.27, p\u0026thinsp;=\u0026thinsp;0.03). Thus, to homogenise the variability in the WDR firing responses, the following data were normalised.\u003c/p\u003e\n \u003cp\u003eTaking into account that trigeminal WDR cells were recorded for 60 min, excluding a timedependent effect was crucial. One-way ANOVA showed that no time-dependent changes in neuronal responses occurred during our experimental protocols in either sex. Specifically, in the control group, time had no effect on A\u0026delta;- (males: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.56, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07; females: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.11, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.98) or C-fibres (males: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.96; females: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.57), and similar results were obtained with vehicle on A\u0026delta;- (males: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.152, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11; females: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.60, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.67) and C-fibres (males: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.153, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.96; females: F\u003csub\u003e(4, 20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.12, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.97).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Effect of clonidine on the evoked trigeminal WDR cell discharge in male rats\u003c/h2\u003e\n \u003cp\u003eSince no time-dependent changes in neuronal responses occurred during the 60 min period, the following experiments were designed to test the effect of clonidine on trigeminal WDR cell firing. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, 40 WDR cells were recorded in males: (i) control (n\u0026thinsp;=\u0026thinsp;5); (ii) vehicle (n\u0026thinsp;=\u0026thinsp;5); and (iii) clonidine (n\u0026thinsp;=\u0026thinsp;30). The WDR cells were treated with clonidine (3.1, 10, and 31 nmol; n\u0026thinsp;=\u0026thinsp;10 cells for each dose), and some cells were inhibited (i.e., sensitive, n\u0026thinsp;=\u0026thinsp;18), whereas others were unaffected (non-sensitive, n\u0026thinsp;=\u0026thinsp;12) by this compound. This dichotomy is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, using a representative PSTHs of two WDR cells. Furthermore, as illustrated in the upper panel of Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, the inhibition of WDR cell activity was associated with a decrease in the spike frequency associated with the activation of A\u0026delta;- and C-fibres and not due to a decrease in the amplitude of the processed signal.\u003c/p\u003e\n \u003cp\u003eUpon quantifying the data from the PSTHs, the WDR cell firing inhibition by clonidine seems no to be dependent on the dose tested considering that: (i) 5/10 WDR cells recorded were inhibited with 3.1 nmol; (ii) 7/10 WDR cells recorded were inhibited with 10 nmol; and (iii) 6/10 WDR cells recorded were inhibited with 31 nmol. Specifically, the effect on the activity associated with A\u0026delta;- and C-fibre activation is illustrated in Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC to \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH. In this regard, in sensitive WDR cells, 10 nmol of clonidine consistently inhibited A\u0026delta;- and C-fibres discharge. Note that the antinociceptive effects of clonidine (10 and 31 nmol) was recorded in some WDR cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026ndash; \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE; sensitive WDR cells), whereas the other WDR cells were unaffected by this compound, irrespective of the dose tested (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF to \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH; non-sensitive WDR cells). See Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e for details of the statistical analyses.\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTwo-way repeated-measures analysis of variance (ANOVA) data for Figures.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTwo-way RM ANOVA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFigure\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInteraction\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.06; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3.040, 69.92)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12.29; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 23)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.48; p\u0026thinsp;=\u0026thinsp;0.0012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.38; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(1.987, 45.71)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;19.43; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 23)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.58; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.90; p\u0026thinsp;=\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4.219, 71.73)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.94; p\u0026thinsp;=\u0026thinsp;0.0012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 17)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.14; p\u0026thinsp;=\u0026thinsp;0.0160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.87; p\u0026thinsp;=\u0026thinsp;0.6461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3.346, 56.89)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.87; p\u0026thinsp;=\u0026thinsp;0.1393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 17)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.74; p\u0026thinsp;=\u0026thinsp;0.1867\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.81; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3.518, 59.81)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;59.81; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3, 17)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.93; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.08; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(2.167, 36.83)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.92; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3, 17)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;38.03; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.88; p\u0026thinsp;=\u0026thinsp;0.6035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3.955, 59.32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.00; p\u0026thinsp;=\u0026thinsp;0.4121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3, 15)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.09; p\u0026thinsp;=\u0026thinsp;0.3850\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.17; p\u0026thinsp;=\u0026thinsp;0.3004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(2.350, 35.26)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.56; p\u0026thinsp;=\u0026thinsp;0.6052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3, 15)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.13; p\u0026thinsp;=\u0026thinsp;0.3691\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 132)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.33; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(3.028, 66.61)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.06; p\u0026thinsp;=\u0026thinsp;0.0010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 22)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.23; p\u0026thinsp;=\u0026thinsp;0.0041\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 132)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.88; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(1.809, 39.81)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.91; p\u0026thinsp;=\u0026thinsp;0.0147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 22)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.22; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 114)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.43; p\u0026thinsp;=\u0026thinsp;0.1093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(2.706, 51.41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;18.89; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 19)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.83; p\u0026thinsp;=\u0026thinsp;0.0537\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(24, 114)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.08; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(2.425, 46.08)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;47.22; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 19)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.28; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.09; p\u0026thinsp;=\u0026thinsp;0.3738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4.071, 61.06)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.69; p\u0026thinsp;=\u0026thinsp;0.6041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 15)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.23; p\u0026thinsp;=\u0026thinsp;0.3342\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(18, 90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.33; p\u0026thinsp;=\u0026thinsp;0.1871\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(2.994, 44.90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.66; p\u0026thinsp;=\u0026thinsp;0.5790\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF \u003csub\u003e(4, 15)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.33; p\u0026thinsp;=\u0026thinsp;0.1156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Effect of clonidine on the evoked trigeminal WDR cell discharge in female rats\u003c/h2\u003e\n \u003cp\u003eAs mentioned above, no time-dependent changes in the neuronal response were observed during the 60 min period; therefore, the following experiments were designed to test the effect of clonidine on trigeminal WDR cell firing. Current evidence suggests that females are less sensitive to clonidine-induced antinociception; consequently, to reduce the number of animals tested, the dose of 3.1 nmol clonidine was not tested in females. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, 30 WDR cells were recorded in females: (i) control (n\u0026thinsp;=\u0026thinsp;5); (ii) vehicle (n\u0026thinsp;=\u0026thinsp;5); and (iii) clonidine (n\u0026thinsp;=\u0026thinsp;20). These cells were treated with clonidine (10 and 31 nmol; n\u0026thinsp;=\u0026thinsp;10 for each dose), and similar to males, some cells were inhibited (i.e. sensitive, n\u0026thinsp;=\u0026thinsp;11) or unaffected (non-sensitive n\u0026thinsp;=\u0026thinsp;9) by this drug. As depicted above for males, these data are illustrated using representative PSTHs of two WDR cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Note that, as shown in the upper panel, the inhibition of WDR cell activity is associated with a decrease in the spike frequency of A\u0026delta;- and C-fibres and not due to a decrease in the amplitude of the processed signal.\u003c/p\u003e\n \u003cp\u003eUpon quantifying the data from the PSTHs, we found that, independent of the dose tested, not all WDR cells were inhibited by clonidine. Specifically, (i) 4/10 cells recorded were inhibited by 10 nmol, and (ii) 7/10 cells recorded were inhibited by 31 nmol. As illustrated in Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, although 10 nmol clonidine tended to diminish neuronal activity (inhibition\u0026thinsp;\u0026gt;\u0026thinsp;20%, particularly of the C-fibres), statistical analysis of the temporal course data in these sensitive WDR cells showed that 31 nmol, but not 10 nmol clonidine, inhibited neuronal firing of the A\u0026delta;- and C-fibres. However, an analysis of global effects (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggest that both doses of clonidine seemed to block the neuronal activity of the A\u0026delta;- and C-fibres. In this regard, we consider that 31 nmol of clonidine consistently inhibits A\u0026delta;- and C-fibres discharge over time. Figures \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF to \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH illustrates that some WDR cells were completely unaffected by this drug at all doses tested. See Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e for details about statistical analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Effect of BRL 44408 on the clonidine-induced trigeminal electrophysiological antinociception in male rats\u003c/h2\u003e\n \u003cp\u003eTaking into account that data from the SDH and MDH consistently suggest that \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor subtype activation mediates clonidine-induced antinociception in male rats [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], the following experiments were performed to corroborate previous findings under our experimental conditions. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA to \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, the 10 nmol clonidine-induced inhibition of WDR cell discharge was prevented (A\u0026delta;-fibres) or reversed (C-fibres) when sensitive to clonidine WDR cells were posttreated with an \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor agonist (BRL 44408). Note that BRL 44408 \u003cem\u003eper se\u003c/em\u003e had no effect on the WDR activity. The impact of BRL 44408 on clonidine-induced antinociception is illustrated using PSTHs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE); note that the inhibition of WDR cell discharge by clonidine (baseline vs. 10 min) was reversed when the WDR cells were treated with BRL 44408 (30 min, lower panel).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.5 Effect of BRL 44408, imiloxan and JP-1302 on the clonidine-induced trigeminal electrophysiological antinociception in female rats\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eCurrent behavioural experiments suggest that \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptors play a key role in clonidineinduced trigeminal antinociception in both the sexes. However, no evidence regarding the receptor subtype and how the neural WDR code is modified has been tested in females. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA to \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, the experiments showed that unlike in males, blocking \u0026alpha;\u003csub\u003e2A\u003c/sub\u003eadrenoceptors with BRL 44408 (10 nmol) did not reverse clonidine-induced antinociception. In contrast, treatment with imiloxan (10 nmol \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e-adrenoceptor subtype antagonist) or JP-1302 (10 nmol; \u0026alpha;\u003csub\u003e2C\u003c/sub\u003e-adrenoceptor subtype antagonist) reversed clonidine-induced antinociception, particularly during the temporal course of the A\u0026delta;-fibres (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), but not on the C-fibres (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB), where the inhibitory effect remained. Similar data were obtained when the neuronal response was analysed as a global response (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). It is important to mention that the antagonists used had no effect \u003cem\u003eper se\u003c/em\u003e on the evoked WDR trigeminal responses (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD to \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e4.1.\u0026nbsp; \u0026nbsp;\u0026nbsp;General\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApart from the implications discussed below, electrophysiological experiments showed that clonidine inhibited trigeminal neuronal activity associated with periorbital electrical activation of A\u0026delta;- and C-fibres in both sexes. In addition, we found the following:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ei.\u0026nbsp; \u0026nbsp;Although male and female trigeminal WDR cells were inhibited by clonidine, not all WDR cells were inhibited, independent of the dose tested. Thus, the WDR cell population can be divided into sensitive and non-sensitive to clonidine (Figs. 3 and 4).\u003c/p\u003e\n\u003cp\u003eii.\u0026nbsp;\u0026nbsp;Females were less prone to clonidine-induced antinociception (i.e. the dose required to induce a similar antinociception was higher in females (10 vs. 31 nmol) (Figs. 3 and 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiii.\u0026nbsp;In males, as previously shown [11, 18], \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor subtype activation is involved in clonidine-induced electrophysiological trigeminal antinociception (Fig. 5).\u003c/p\u003e\n\u003cp\u003eiv. In contrast, in females, the \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor subtype does not seem to be relevant to the inhibitory effect of clonidine on WDR cell discharge. Remarkably, trigeminal antinociception induced by clonidine seems to depend on \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e- and \u0026alpha;\u003csub\u003e2C\u003c/sub\u003e- adrenoceptor subtypes activation in Ad-, but not in C-fibres (Fig. 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.\u0026nbsp; \u0026nbsp;\u0026nbsp;Clonidine inhibited the evoked activity of male and female trigeminal WDR cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough \u003cem\u003ein vivo\u003c/em\u003e electrophysiological experiments have shown that in males, trigeminal administration of clonidine induces antinociception through the activation of \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptors [11, 23-26], this effect has not been analysed in females. To the best of our knowledge, only behavioural experiments have been made suggesting that similar than males, the \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptors play a role in the clonidine induced antinociception [13, 27]. However, beyond the\u0026nbsp;a\u003csub\u003e2A/2B/2C\u003c/sub\u003e-adrenoceptor subtypes involved (currently unknown in females), experiments in females have not been designed to analyse the impact of clonidine upon neural transmission.\u003c/p\u003e\n\u003cp\u003eIn this regard, the second-order WDR cells at MDH modulate the nociceptive input from the periphery to supraspinal centres, encoding the neural influx from non-nociceptive and nociceptive signals transmitted by primary A\u0026beta;-, A\u0026delta;-, and C-fibres. Hence, WDR cells are among the first relays in which the incoming sensorial input is computed and modulated [28]. Considering that under our experimental conditions, the electrically induced neuronal WDR response in both sexes was stable, a reduction in WDR cell firing by a given pharmacological treatment could be interpreted as a compound with potential analgesic action. Indeed, trigeminal application of analgesics (e.g. naratriptan, olcegepant, or morphine) suppresses the responsiveness of trigeminal WDR cells by inhibiting neuronal discharge associated with the activation of Ad- and C-fibres [28-31].\u003c/p\u003e\n\u003cp\u003eAs expected, electrophysiological data showed that trigeminal clonidine induced antinociception in both sexes (Figs. 3 and 4). However, as previously reported in behavioural experiments [32, 33], females require a higher dose (10 vs. 31 nmol) of clonidine to obtain an inhibitory effect upon Ad- and C-fibres, similar to that observed in males. Certainly, in behavioural trigeminal pain models using ovariectomized or castrated rats with hormonal replacement therapy, sexual hormones have been shown to play a role in the degree of antinociception induced by clonidine [12, 13, 27]. Together, these data indicate the relevance of sex in sensitivity to analgesic treatments, as discussed extensively by Mogil et al. [34, 35].\u003c/p\u003e\n\u003cp\u003eIn addition, it is interesting to note that in both sexes, the trigeminal WDR cells were clustered into two populations: (i) sensitive and (ii) non-sensitive to the antinociceptive effects elicited by clonidine. In this regard, at MDH of male rodents, and using \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e electrophysiological recordings consistently showed that not all cells recorded (WDR, nociceptive specific and low threshold cells) could be inhibited by clonidine or noradrenaline [11, 24, 25, 36, 37]. Thus, the most parsimonious interpretation could be that there is a heterogeneous distribution of clonidine-sensitive receptors on primary afferent fibres (PAF).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, given that our electrophysiological data in both sexes (see Figs. 3 and 4) showed that clonidine inhibited neuronal evoked activity associated with the activation of A\u0026delta;- and C-fibres, a presynaptic effect on nociceptive PAFs is supported. However, the degree of antinociception elicited by clonidine seems to be more relevant for C-fibres than A\u0026delta;-fibres. Therefore, at first glance, if we consider that clonidine exerts its antinociception by activation of the \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor, we could propose that at the trigeminal level, sensitive receptors to clonidine must be predominantly expressed on C-fibres. In this regard, \u0026alpha;\u003csub\u003e2A/2B/2C\u003c/sub\u003e-adrenoceptors have been reported to be expressed at the spinal and trigeminal level [38-41], but the extent of expression of these receptor subtypes upon PAF has not been explored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.\u0026nbsp; \u0026nbsp;\u0026nbsp;The role of \u0026alpha;\u003csub\u003e2A/2B/2C\u003c/sub\u003e-adrenoceptor subtypes in the clonidine-induced antinociception in males and females\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn males, it is well known that activation of trigeminal \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptors induces antinociception predominantly via the \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor subtype [11, 23-27, 39]. Therefore, we decided to pharmacologically dissect under our experimental conditions whether this adrenoceptor subtype was involved in clonidine-induced electrophysiological antinociception in male and female trigeminal WDR cells. As expected, in males, pharmacological blockade of \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor with BRL 44408 fully reversed clonidine-induced electrophysiological antinociception in Ad- and C‑fibres (Fig. 5). \u0026nbsp;As above referenced, under acute nociception, \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e- rather than \u0026alpha;\u003csub\u003e2B/2C\u003c/sub\u003e‑adrenoceptors, plays a key role inhibiting nociception [42, 43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, the inhibitory effect of clonidine persisted in females when the \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e‑adrenoceptor subtype was blocked (Fig. 6A to 6C). These data implied that in females \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor do not play a pivotal role in clonidine-induced antinociception. We must keep in mind that males and females differ in the degree of antinociception induced by clonidine, where males are more sensitive to clonidine effects, at least in behavioural experiments [12, 13, 27, 36]. Based on this evidence, it is not surprising that the receptor subtypes involved could differ between the sexes. For example, in a neuropathic pain behavioural model, tizanidine (an \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor agonist) induced antinociception in females by \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e- rather than \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor activation [44].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 6A to 6C, when the \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e-adrenoceptor in females was blocked with imiloxan, we found that the electrophysiological antinociception elicited by clonidine was particularly reversed on neuronal firing associated with the activation of Ad-fibres, without any effect on C-fibres. A similar outcome was obtained when \u0026alpha;\u003csub\u003e2C\u003c/sub\u003e-adrenoceptor was blocked with JP‑1302. It is relevant to mention that the antagonists used (i.e., BRL 44408, imiloxan and JP‑1302) devoid any effect \u003cem\u003eper se\u0026nbsp;\u003c/em\u003eon the evoked WDR cell activity (see Figs. 6D to 6F). At this point, beyond the finding that the receptors subtypes involved in clonidine-induced inhibition were different between males and females, it was also interesting that in females, the Ad- but not C‑fibres recovered their baseline neuronal firing after the blockade of \u0026alpha;\u003csub\u003e2B\u003c/sub\u003e- or \u0026alpha;\u003csub\u003e2C\u003c/sub\u003e-adrenoceptors. These results imply that in females:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ei. \u003cem\u003eThe specific expression of these \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptors subtypes on A\u003c/em\u003e\u003cem\u003ed\u003c/em\u003e\u003cem\u003e- and C-fibres differs.\u003c/em\u003e Indeed, although it is well known that in males, the global expression of the \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor subtypes at the trigeminal level differs (i.e. \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e\u0026gt;\u0026alpha;\u003csub\u003e2C\u003c/sub\u003e\u0026gt;\u0026gt;\u0026gt;\u0026alpha;\u003csub\u003e2B\u003c/sub\u003e) [18, 25, 40, 45-50], currently no data about the distribution of these \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor subtypes expression on Ad- and C-fibres exist. Certainly, transcriptomic profiling at single-cell resolution could be necessary to explore these sexual dimorphisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eii. \u003cem\u003eClonidine-induced antinociception in C-fibres may be mediated by different mechanisms or receptors.\u003c/em\u003e For example, it has been reported that clonidine can activate imidazoline receptors [15, 51, 52]. Indeed, the role of imidazoline receptors exerting antinociception has been suggested, based on the fact that yohimbine (a preferent \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor antagonist) did not reverse clonidine-induced spinal antinociception in the formalin test [53, 54]. However, several reports have shown that clonidine-induced spinal antinociception is resistant to treatment with selective I\u003csub\u003e1\u003c/sub\u003e/I\u003csub\u003e2\u003c/sub\u003e imidazoline receptor antagonists [55-58]. In agreement with these data, pretreatment with more selective \u0026alpha;\u003csub\u003e2\u003c/sub\u003e-adrenoceptor antagonists (e.g., rauwolscine) is enough to abolish the effect of clonidine. Therefore, the current analysis of the receptors implicated in the antinociception induced by clonidine seems to exclude the role of imidazoline receptors. However, we must admit that our experimental design was not designed to analyse the role of imidazoline receptors in clonidine-induced antinociception.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4.\u0026nbsp; \u0026nbsp;\u0026nbsp;Conclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data collectively indicate that \u0026alpha;\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor activation induces significant antinociceptive effects on trigeminal Ad- and C-fibres\u0026nbsp;in males, but not in females.\u0026nbsp;In contrast, in females,\u0026nbsp;the\u0026nbsp;activation of \u0026alpha;\u003csub\u003e2B/2C\u003c/sub\u003e-adrenoceptors inhibits neuronal firing associated with the recruitment of Ad‑fibres, but not C-fibres. This suggests that in female rats, other mechanisms or receptors may mediate the inhibition of C-fibre discharge induced by clonidine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Analysis of variance\u003c/p\u003e\n\u003cp\u003eC1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;C1 cervical vertebra\u003c/p\u003e\n\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Carbon dioxide\u003c/p\u003e\n\u003cp\u003ei.p.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intraperitoneal\u003c/p\u003e\n\u003cp\u003ei.v.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Intravenous\u003c/p\u003e\n\u003cp\u003eKCl\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Potassium chloride\u003c/p\u003e\n\u003cp\u003eMDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medullary dorsal horn\u003c/p\u003e\n\u003cp\u003ePAF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Primary afferent fibres\u003c/p\u003e\n\u003cp\u003ePSTH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Peri-stimulus time histograms\u003c/p\u003e\n\u003cp\u003eRF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Receptive field\u003c/p\u003e\n\u003cp\u003eSDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Spinal dorsal horn\u003c/p\u003e\n\u003cp\u003eSEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Standard error of the mean\u003c/p\u003e\n\u003cp\u003eV1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ophthalmic branch of the trigeminal nerve\u003c/p\u003e\n\u003cp\u003eWDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Wide dynamic range\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements.\u003c/em\u003e\u003c/strong\u003e Gustavo L\u0026oacute;pez C\u0026oacute;rdoba is a doctoral student (ID number: 518010700) from the Programa de Doctorado en Ciencias Biom\u0026eacute;dicas, Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico (UNAM) and has received a CONAHCYT (currently named SECIHTI) fellowship (no. 859394). This research article is part of his doctoral thesis, and the authors thank the Posgrado de Doctorado en Ciencias Biom\u0026eacute;dicas UNAM for their academic support.\u003c/p\u003e\n\u003cp\u003eNombre del Alumno is a doctoral student from the Programa de Doctorado en Ciencias Biom\u0026eacute;dicas, Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico (UNAM) and has received CONAHCYT fellowship (no. 859394).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026apos; contributions.\u003c/em\u003e\u003c/strong\u003e Conceptualisation (G.L-C., M.C-L., A.G-H.), investigation (G.L-C., G.M‑L., A.G-H.), data curation (G.L-C.), formal analysis (G.L-C., A.G-H.), supervision (G.M-L., M.C-L., A.G-H.), resources (M.C-L., A.G-H.), wrote the main manuscript text (G.L-C., A.G-H.), All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding.\u003c/em\u003e\u003c/strong\u003e\u0026nbsp; This work on rodents was sponsored by Programa de Apoyo a Proyectos de Investigaci\u0026oacute;n e Innovaci\u0026oacute;n Tecnol\u0026oacute;gica (PAPIIT-UNAM Mexico) under Grant agreement no. IN227225 (A.G-H.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability.\u003c/em\u003e\u003c/strong\u003e All the relevant data have been included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval.\u003c/em\u003e\u003c/strong\u003e This study was conducted in accordance with the principles of the Basel Declaration and recommendations of the (i) of Health Guidelines for the Care and Use of Laboratory Animals, (ii) ARRIVE Guidelines, and (iii) Standards for the Investigation of Experimental Pain in Animals [59]. The study protocol was approved by the Institutional Animal Care and Use Committee of the Instituto de Neurobiolog\u0026iacute;a, UNAM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication.\u003c/em\u003e\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor information\u003c/em\u003e\u003c/strong\u003e. Departamento de Neurobiolog\u0026iacute;a del Desarrollo y Neurofisiolog\u0026iacute;a, Instituto de Neurobiolog\u0026iacute;a, Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico, Campus UNAM-Juriquilla.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eORCiD / e-mail\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.L-C. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 0009-0006-3072-8010 / [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG.M-L. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0009-0001-5184-6431 / [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.C-L. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0000-0003-1651-8931 / [email protected] \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA.G-H. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0000-0002-6472-1419 / [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEisenach JC, Dewan DM, Rose JC et al (1987) Epidural clonidine produces antinociception, but not hypotension, in sheep. 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J Comp Neurol 372:111\u0026ndash;134\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUhlen S, Wikberg JES (1991) Rat spinal cord α\u003csub\u003e2\u003c/sub\u003e-adrenoceptors are of α\u003csub\u003e2A\u003c/sub\u003e-subtype: comparison with α\u003csub\u003e2A\u003c/sub\u003e- and α\u003csub\u003e2B\u003c/sub\u003e-adrenoceptors in rat spleen, cerebral cortex and kidney using \u003csup\u003e3\u003c/sup\u003eH-RX821002 ligand binding. Pharmacol Toxicol 69:341\u0026ndash;350\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Y, Stamer WD, Anthony TL et al (2002) Expression of α\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor subtypes in prenatal rat spinal cord. Brain Res Dev Brain Res 133:93\u0026ndash;104\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHieble JP, Bondinell WE, Ruffolo RR Jr (1995) α- and β-adrenoceptors: from the gene to the clinic. 1. Molecular biology and adrenoceptor subclassification. 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Pain 16(2):109\u0026ndash;110\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adrenoceptors, Headache, Migraine, Nociception, Pain, Sex","lastPublishedDoi":"10.21203/rs.3.rs-7546267/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7546267/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe trigeminal system plays a key role in headaches pathophysiology. In this regard, nociceptive experiments in male rodents have shown that at the trigeminal level, clonidine induces antinociception through α\u003csub\u003e2A\u003c/sub\u003e- but not α\u003csub\u003e2B/2C\u003c/sub\u003e-adrenoceptors. Interestingly, although behavioural experiments suggest that males are more sensitive to clonidine-induced antinociception than female rats (an effect linked to hormonal factors), little is known about how neuronal nociceptive processing is affected in females (including the role of the α\u003csub\u003e2A/2B/2C\u003c/sub\u003e-adrenoceptors subtypes involved). Since trigeminal second-order wide dynamic range (WDR) cells are one of the main gatekeepers involved in decoding and processing nociceptive inputs from peripheral facial structures, this study was designed to test the effect of clonidine on trigeminal WDR cell activity in male and female rats.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eExtracellular unitary recordings of trigeminal WDR cells were performed in anaesthetised Wistar rats and analysed as Aδ-, and C-fibres associated discharge. Under these conditions, the effect of local clonidine (3.1\u0026ndash;31 nmol) on electrical periorbital-evoked firing of WDR cells was recorded in both sexes. Furthermore, considering that at least three α\u003csub\u003e2A\u003c/sub\u003eadrenoceptor subtypes exist (α\u003csub\u003e2A\u003c/sub\u003e-, α\u003csub\u003e2B\u003c/sub\u003e- and α\u003csub\u003e2C\u003c/sub\u003e-adrenoceptors), pharmacological blockade of these receptor subtypes was performed using BRL 44408 (α\u003csub\u003e2A\u003c/sub\u003e), imiloxan (α\u003csub\u003e2B\u003c/sub\u003e), and JP-1302 (α\u003csub\u003e2C\u003c/sub\u003e).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eClonidine inhibited the activity of Aδ-, and C-fibres in male and female rats. This inhibition was observed in 50\u0026ndash;60% of cells recorded. Furthermore, the dose necessary to induce similar electrophysiological antinociception was higher in females (31 nmol vs. 10 nmol). In males, but not females, clonidine-induced inhibition of Aδ- and C-fibres activity was reversed using BRL 44408. Whereas in females, imiloxan and JP-1302 reversed the clonidineinduced inhibition of Aδ- but not C-fibres.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eMale rodents are more sensitive than females to clonidine-induced WDR antinociception. In males but not females, clonidine-induced inhibition of Aδ- and C-fibres discharge relies on α\u003csub\u003e2A\u003c/sub\u003e-adrenoceptor activation. However, in females, the activation of α\u003csub\u003e2B/2C\u003c/sub\u003eadrenoceptors seems to be relevant to Aδ- but not C-fibre discharge inhibition by clonidine, implying that other mechanism/receptors may be involved in females.\u003c/p\u003e","manuscriptTitle":"Electrophysiological evidence about sexual dimorphism in the clonidine-induced inhibition of trigeminal wide dynamic range cell activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 02:13:03","doi":"10.21203/rs.3.rs-7546267/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T03:39:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T00:24:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74214399695431742468137714738186969508","date":"2025-09-24T12:39:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267831942129360996206059566081234639096","date":"2025-09-10T09:01:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T08:07:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261303281815792387506324605099591878368","date":"2025-09-08T09:14:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T08:29:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T06:52:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T06:51:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Headache and Pain","date":"2025-09-05T17:18:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8ae39c2-08ad-494e-85f3-beb1a3744068","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-7546267","link":"https://doi.org/10.1186/s10194-025-02221-x","journal":{"identity":"the-journal-of-headache-and-pain","isVorOnly":false,"title":"The Journal of Headache and Pain"},"publishedOn":"2026-02-25 15:58:13","publishedOnDateReadable":"February 25th, 2026"},"versionCreatedAt":"2025-09-16 02:13:03","video":"","vorDoi":"10.1186/s10194-025-02221-x","vorDoiUrl":"https://doi.org/10.1186/s10194-025-02221-x","workflowStages":[]},"version":"v1","identity":"rs-7546267","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7546267","identity":"rs-7546267","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}