Reduced Noradrenergic Excitability in the Locus Coeruleus Compromises Nociceptive Inhibition in a Diabetic Mouse Model

Reduced Noradrenergic Excitability in the Locus Coeruleus Compromises Nociceptive Inhibition in a Diabetic Mouse Model | 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 Article Reduced Noradrenergic Excitability in the Locus Coeruleus Compromises Nociceptive Inhibition in a Diabetic Mouse Model Alberto Mesa-Lombardo, Nuria García-Magro, Guillermo Cerrillo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9072982/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The locus coeruleus (LC) play an essential role in the regulation of nociceptive transmission by a widespread descending pathway to the spinal cord and the spinal trigeminal nucleus. We examined the effect of formalin injection in the vibrissal pad (nociceptive stimulus) on LC activity in isoflurane anesthetized control and in streptozotocin-induced diabetic (STZ-diabetic) mice which display neuropathic pain. Using unit recordings, we observed that formalin induced a sustained increase in the firing rate of LC neurons. In contrast, STZ-diabetic mice only showed an initial response, suggesting a reduced neuronal excitability. This finding was supported by a reduced c-Fos expression in comparison with control mice. We suggest that the reduction of LC excitability was due to a reduction of the insulin-like growth factor I (IGF-I) levels that occur in diabetes. In fact, immunohistochemistry studies showed that IGF-I receptors were diminished in STZ-diabetic mice. Treatment of STZ-diabetic mice with the IGF-I receptor sensitizer AIK3a305 restored LC activity. In conclusion, the lack of IGF-I in the brain of diabetic animals may be responsible for the reduced activity of LC neurons and, consequently, for the altered control of nociceptive transmission, facilitating chronic pain development in neuropathy diseases. Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Neuroscience Biological sciences/Physiology noradrenergic transmission formalin insulin-like growth factor I pain diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The locus coeruleus (LC) is a small, bilateral nucleus in the pontine tegmentum that is characterized by its rich concentration of noradrenergic (NA) neurons. NA neurons play an essential role in the regulation of cognitive function, sleep/wake state, arousal, attention, mood, and stress reactions 1 – 4 . According to the behavioral state, LC neurons display two different discharge patterns: tonic and phasic modes 5 , 6 . Spontaneous tonic activity consisted in a sustained regular discharge pattern of single spikes at 2–8 Hz. The phasic discharge pattern was characterized by brief 10–20 Hz bursts of two to three action potentials. In addition, LC also control nociceptive transmission by a widespread descending pathway which includes the spinal cord and spinal trigeminal nucleus 1 , 7 – 11 . The caudalis division of the spinal trigeminal nucleus (Sp5C) is the first central relay station in the circuitry involved in processing pain and non-noxious stimuli from the craniofacial region 12 – 18 . Neuroanatomical findings have demonstrated reciprocal connections between the LC and trigeminal sensory nuclei, suggesting that the LC may exert modulatory control over nociceptive processing in the orofacial region 19 – 21 . Consistent with this notion, acute noxious orofacial stimulation induces increased c-Fos immunoreactivity within the LC 22, 23 and stimulation of the LC neurons inhibits the sensory transmission in the Sp5C neurons through activation of α2-NA receptors 24 . Recently, we have published that LC also inhibit sensory responses in the Sp5C nucleus by activation of postsynaptic α2-NA receptors on glutamatergic Sp5C neurons and by excitation of GABAergic neurons via α1-NA receptors 25 , 26 . Neuropathic pain is one of the most common complications of diabetes. Although one of the most important functions of the LC is to control the nociceptive transmission, little is known about the changes in the LC activity in diabetes. Results from our laboratory have suggested that a reduction in NA neuronal activity may contribute to the generation and/or maintenance of neuropathic pain in these patients. The inhibition elicited by LC stimulation in Sp5C nucleus was reduced in streptozotocin (STZ)-induced diabetic mice, suggesting that an impairment of LC activity may contribute to the generation of chronic pain in diabetic patients 25 , 26 . For this reason, the goal of the present study was to investigate the changes in LC activity in STZ-diabetic mice and the mechanisms underlying the reduction of noradrenergic neuronal activity. We suggest that the decrease in insulin-like growth factor I (IGF-I) levels that occur in diabetes may be responsible for the reduced activity of LC neurons and, consequently, for the altered control of nociceptive transmission. Results Effect of formalin injection on locus coeruleus activity in control and STZ-diabetic mice To evaluate the impact of peripheral nociceptive stimulation on the activity of the LC neurons in control and STZ-diabetic mice, the spontaneous firing rate was recorded before and after formalin application to the vibrissal pad of anesthetized mice (20 µl; 2%). In control animals, formalin injections evoked a progressive increase of the firing rate of LC neurons from 6.5 ± 0.74 spikes/s under baseline conditions to 12.3 ± 1.0 spikes/s at 25 minutes after formalin injection (n = 17 neurons; p < 0.0001, paired t-test; Fig. 1 A, B). In contrast, STZ-diabetic animals exhibited a progressive decrease of the firing rate after formalin injection from 7.1 ± 0.57 spikes/s to 4.0 ± 0.49 spikes/s at 25 minutes after formalin injection (n = 20 neurons; p = 0.0001). A repeated measures ANOVA test indicated that both curves were statistically different (p < 0.0001). These results suggest an altered LC response to nociceptive stimuli in the context of diabetes. We studied the presence of burst in LC neurons recorded in basal conditions or after formalin injection because they occur in association with salient or new stimuli 27 . Control animals showed the presence of burst of action potentials that consisted in two-three spikes at a frequency of 10–20 Hz with a mean frequency of 55.9 ± 7.3 bursts measured in 5 minutes of basal activity (Fig. 1 C; see inset). Their frequency increased after formalin injection when they were recorded at 5 or 25 minutes after formalin injection to 92.4 ± 14.6 or 112.1 ± 12.7 bursts per 5 minutes, respectively (n = 17 neurons; p = 0.0162 or p = 0.0012). STZ-diabetic mice also showed burst of spikes in basal conditions 40.2 ± 6.8 bursts per 5 minutes. However, formalin injection did not modify the frequency of these bursts at 5 or 25 minutes after formalin injection (41.9 ± 8.6 or 37.1 ± 9.1 bursts per 5 minutes, respectively; n = 20 neurons; p > 0.05). The fact that formalin increased the spontaneous activity of LC neurons in control mice suggested that there was also an increase in neuronal excitability. However, this effect was not observed in STZ-diabetic mice. We studied the response of LC neurons to electrical stimulation at the Sp5C nucleus in basal conditions and at 5 and 25 minutes after the injection of formalin. Electrical stimulation ( 0.05). In control animals, LC responses increased from 1.6 ± 0.17 spikes/stimulus in basal conditions to 2.2 ± 0.17 spikes/stimulus and 2.5 ± 0.24 spikes/stimulus at 5 and 25 minutes after formalin injection (p = 0.0111, p = 0.0017, respectively; Fig. 1 D), indicating that LC increased their excitability after formalin injection. STZ-diabetic mice showed a response in basal conditions of 1.8 ± 0.14 spikes/stimulus however, the LC response did not increase 5 min. after formalin injection (1.9 ± 0.17 spikes/stimulus) and only increased to 2.2 ± 0.18 spikes/stimulus at 25 min. (p > 0.05 and p = 0.0272, respectively). These data suggested that the excitability of LC neurons in STZ-diabetic animals was reduced in comparison to control animals. Formalin increased LC activity assessed by c-Fos expression Expression of c-Fos is an indirect marker of neuronal activity because c-Fos is expressed when neurons fire action potentials. To corroborate that formalin induced an increase of LC neuronal excitability, we analyzed the expression of c-Fos in non-injected or formalin injected (2%) control or STZ-diabetic animals (Fig. 2 A). Formalin injection in control mice (n = 8 animals) led to a higher c-Fos expression in the LC than in non-injected control animals (23.7 ± 2.9 to 33.7 ± 1.7 IF intensity; n = 8, p = 0.011), indicating enhanced neuronal activation in response to the nociceptive stimulus (Fig. 2 B). Notably, even in the absence of formalin, STZ-diabetic mice showed reduced c-Fos expression in the LC compared to control mice (16.3 ± 1.6 IF intensity; n = 8, p = 0.0417), suggesting a basal reduction in neuronal activity associated with the diabetic condition. Additionally, within the STZ-diabetic group, animals injected with formalin displayed significantly higher c-Fos expression than non-injected animals (22.1 ± 1.5 IF intensity; n = 8; p = 0.0216); however, these values did not reach the c-Fos expression in formalin-injected control mice (p = 0.0004), indicating an impaired activation of the LC in response to nociceptive inputs. LC-evoked inhibition in Sp5C increased in control mice by formalin but not in STZ-diabetic mice It has been published that electrical stimulation of the LC induces an inhibition of vibrissal responses in the Sp5C nucleus through activation of NA receptors that may contribute to control sensory transmission of non-nociceptive and nociceptive inputs 24 – 26 , 28 . In our experiments, electrical stimulation of the LC induced a clear inhibition of the vibrissal responses in basal conditions in control mice (-11.2 ± 2.3%, n = 12 neurons) that increased when measured 5 min. after formalin injection (-23.8 ± 3.9%, n = 12 neurons; p = 0.0228, respect to basal values). This inhibition persisted up to 20 minutes after formalin injection (-25.2 ± 4.5%, n = 12 neurons; p = 0.0467, respect to basal values; Fig. 3 A). LC-evoked inhibition was smaller in STZ-diabetic animals in comparison with control animals (-0.9 ± 3.3%, n = 14 neurons; p = 0.0357 respect to basal values in control animals), as has been previously described. LC-evoked inhibition increased when measured 5 min. after formalin injection (-13.8 ± 3.3%, n = 14 neurons; p = 0.0430, respect to basal values) and returned to basal values at 20 min after formalin injection (-5.5 ± 4.1%, n = 14 neurons; p > 0.05). Therefore, the data indicated that the increase in LC activity by formalin injection resulted in an increase in the LC-evoked inhibition exerted on Sp5C neurons in control animals. However, in STZ-diabetic animals, this inhibitory effect was only observed in the first 5 min. after the injection of formalin. The IGF-I effect was reduced in the LC of diabetic mice IGF-I plays an essential role in the modulation of synaptic plasticity and in learning and memory processes 29 , 30 . Reduced levels of IGF-I and insulin have been proposed as potential causes of neurological disorders 31 – 34 . Since insulin and IGF-I levels were strongly reduced in diabetic animals 35 , 36 , we tested the effect of IGF-I on LC neurons in control and STZ-diabetic mice. Systemic injection of IGF-I in control mice (1 µg/g body weight, i.p.) induced an increase of the firing rate of LC neurons from 5.4 ± 0.5 spikes/s to 9.3 ± 0.17 spikes/s 30 minutes after IGF-I injection (n = 12 neurons; p = 0.0025; Fig. 4 A). Also, the response of LC neurons to Sp5C stimulation increased from 1.7 ± 0.23 spikes/stimulus to 2.4 ± 0.36 spikes/stimulus after IGF-I injection (n = 12 neurons; p = 0.0201; Fig. 4 B), indicating that IGF-I increased LC neuronal excitability. IGF-I also increased the firing rate of LC neurons in STZ-diabetic mice from 4.8 ± 0.39 spikes/s to 5.9 ± 0.49 spikes/s when was injected systemically (n = 12: p = 0.003; Fig. 4 A) although the increment was lower than in the control mice. Equally, the response of LC neurons to Sp5C stimulation increased from 2.1 ± 0.21 spikes/stimulus to 2.8 ± 0.32 spikes/stimulus after IGF-I injection (n = 12 neurons; p = 0.0182; Fig. 4 B), which indicates that LC neurons could increase their excitability if IGF-I levels were to rise. This reduced response of LC neurons in STZ-diabetic mice in comparison with control mice may be due to a reduction of IGF-I receptor (IGF-IR) in LC neurons. Immunohistochemical studies showed an ample distribution of the IGF-IR in the brainstem, as is shown in the LC (Fig. 5 A). The optical density of normalized fluorescence intensity indicated a statistically significant reduction of IGF-IR in STZ-diabetic animals respect to control ones (26.6 ± 1.0 to 12.3 ± 1.1 IF intensity; n = 14 and n = 10, respectively; p < 0.001; Fig. 5 B), indicating an impaired IGF-I signaling in this nucleus under diabetic conditions that may explain the reduced excitability of LC neurons in STZ-diabetic mice. AIK3a305 restore LC neuronal activity in STZ-diabetic mice To determine that dysregulated IGF-IR expression in LC neurons of STZ-diabetic mice may be responsible for the reduction of nociceptive responses in LC, we treated STZ-diabetic mice with AIK3a305, a novel IGF-IR sensitizer. We observed that the LC response to formalin injection in STZ-diabetic mice treated with AIK3a305 resembles control animals (Fig. 6 A, B). Also, the LC-evoked inhibition of vibrissal responses in Sp5C was recorded in treated animals. In control animals LC-evoked inhibition in basal condition was − 14.1 ± 1.9% (n = 22 neurons) while in STZ-diabetic animals was − 0.6 ± 1.6% (n = 18 neurons; p < 0.001). LC-evoked inhibition in STZ-diabetic mice treated with AIK3a305 reached the same values as in control animals (-16.7 ± 2.1% (n = 22 neurons; p < 0.001; Fig. 6 B). We next evaluated if TH and IGF-IR expression were recovered with the AIK3a305 treatment (Fig. 7 A). TH expression was reduced in STZ-diabetic mice respect to control mice (38.2 ± 1.1 to 23.3 ± 1.5 IF intensity; n = 10 and n = 11, respectively; p < 0.001). However, the expression of TH increased after the treatment with AIK3a305 respect to STZ-diabetic mice (30.5 ± 1.3 IF intensity; n = 8; p = 0.0022), although it did not reach the values in control mice (p < 0.001; Fig. 7 B). The same occurred with the expression of IGF-IR in the LC; it was significantly reduced in diabetic mice compared to control animals (26.6 ± 1.0 to 12.3 ± 1.1 IF intensity; n = 14 and n = 10, respectively; p < 0.001). Treatment of STZ-diabetic mice with AIK3a305 resulted in a marked increase in IGF-1R expression, surpassing the levels observed in diabetic animals (18.5 ± 1.2 IF intensity; n = 8; p = 0.0016). AIK3a305 treatment restores the response to mechanical stimulation in STZ-diabetic mice Mechanical stimulation response was evaluated during the second and third weeks following STZ administration and compared to baseline values recorded prior to the injection. STZ-diabetic mice exhibited a significant reduction in the mechanical withdrawal threshold in the vibrissal pad at 21 days, which became more pronounced by day 28 (Fig. 8 A). In contrast, control mice did not display any time-dependent changes in their response threshold. Notably, STZ-diabetic mice treated with AIK3a305 did not exhibit any alterations in mechanical sensitivity at 28 days, displaying thresholds comparable to those observed in control animals. Regarding response scores, diabetic mice showed a significant increase in their response to mechanical stimulation of the vibrissal pad at both 21 and 28 days compared to control animals and diabetic mice treated with AIK3a305. Although diabetic mice treated with AIK3a305 exhibited a slight increase in response scores, this change was not statistically significant when compared to controls (Fig. 8 B). Discussion The present results demonstrate that the responsiveness of LC neurons to nociceptive stimuli was altered in STZ-diabetic mice. Consequently, the LC-evoked inhibition of vibrissal responses in Sp5C was markedly reduced. Such a decrease in NA activity may contribute to the facilitation of chronic pain development. This reduction of LC activity could be due to the reduction in the brain IGF-I signaling, as its receptor expression in the LC was decreased in STZ-diabetic mice. In fact, treatment of STZ-diabetic mice with the IGF-IR sensitizer AIK3a305 restored LC activity. The mouse orofacial formalin test is a valid and reliable model for evaluation of orofacial nociceptive processing and modulation 37 . Formalin has been shown to produce a long-lasting nociceptive stimulus with a biphasic behavioral response; a direct action of formalin on nociceptive receptors is responsible for the first phase, whereas inflammation seemed to play a more important role in the second phase of formalin response 38 , 39 . Electrophysiological data present here reveal that LC neurons in control mice increased the firing rate after formalin injection and remained at least 25 minutes after the injection. Previous findings have also shown that neuropathic pain increases spontaneous and noxious-evoked activity of LC neurons 40 , 41 . Furthermore, the expression of c-Fos is a common marker for recent cellular activity, reflecting cell activation induced by acute events such as nociceptive stimulus which increases c-Fos expression in the LC 24, 42 . In agreement with previous findings, formalin injection in the vibrissal pad increased the firing rate of LC neurons as well as increased the c-Fos expression in the LC of control mice. However, LC neurons from STZ-diabetic mice increased firing rate immediately after formalin injection, but after that it decreased progressively even below the basal activity. STZ-diabetic mice also showed a reduced c-Fos expression in the LC compared to control mice even in the absence of formalin, suggesting a basal reduction in neuronal activity associated with the diabetic condition. Formalin injections in STZ-diabetic mice increased the c-Fos expression but values were lower than the c-Fos expression in formalin-injected control mice. Moreover, the reduction of LC activity in STZ-diabetic mice was accompanied by a reduction in the excitability of these neurons. Electrical stimulation of Sp5C evoked a short latency orthodromic response in LC neurons that increased after formalin injection in control animals, indicating that the nociceptive stimulation increased the excitability of LC neurons. Under baseline conditions, the evoked response was similar in STZ-diabetic mice than in control mice. However, after the formalin injection, the evoked response increase was lower than in control animals, indicating that nociceptive stimulus did not modify substantially the excitability of LC neurons. The increase in excitability caused by the formalin injection was also noted by the increase in the action potential firing rate of LC neurons and by a change in the discharge pattern. It is well known that LC neurons show tonic or bursting firing patterns 5 , 6 . Spontaneous tonic activity consisted in a sustained regular discharge pattern of single spikes at 2–8 Hz that may change to a phasic, bursting discharge pattern characterized by brief 10–20 Hz bursts when salient or new stimuli occur 27 . In control mice, the burst frequency increased after formalin injection but not in STZ-diabetic mice, suggesting that the excitability of LC neurons in STZ-diabetic mice was decreased and consequently, the response to nociceptive stimulation was also reduced. The descending NA inhibitory system from the LC to the dorsal horn and trigeminal nucleus is one of the main pathways involved in the endogenous pain modulation system 8 , 43 . Previous studies from our laboratory have demonstrated that LC evokes an inhibition of vibrissal responses in the Sp5C via activation of α2-NA on glutamatergic neurons and by α1-NA receptors on GABAergic neurons 25 , 26 . This inhibitory effect may be the modulatory mechanism for which LC controls the transmission of non-noxious and noxious stimuli. In agreement with this hypothesis, LC-evoked inhibition in the Sp5C increased after formalin injection respect to the basal condition in control mice and remained higher along the recording time, suggesting that the increase of excitability in LC neurons provoked by formalin induced an increase in the LC-evoked inhibition of nociceptive transmission in the Sp5C. However, this effect did not occur in STZ-diabetic mice in which LC-evoked inhibition only increment in the first 5 minutes after formalin injection, facilitating probably the appearance of chronic neuropathic pain. Taken together, the results suggest that in diabetic animals, the LC is capable of responding to an acute nociceptive stimulus but cannot maintain its response to a stimulus that is prolonged over time. IGF-I enhances neuronal and glia activity, controlling numerous brain processes 44 – 50 . IGF-I level is significantly reduced by 40% to 50% in type I as well as type II diabetes 35 , 36 . Reduced levels of IGF-I and insulin have been proposed as potential causes of neurological disorders because IGF-I application reduce cognitive deficits 31 – 33 , 51 . In the light of these findings, we propose that the reduction of IGF-I may be responsible for the reduction of excitability of LC neurons since IGF-I increases neuronal excitability in many brain regions 52 – 56 . We have just published that the reduction of IGF-I levels in STZ-diabetic mice is responsible for synaptic plasticity impairment in the somatosensory cortex 57 . The lack of IGF-I could explain the limited response that the LC exerts when the stimulus is prolonged over time. We observed that the expression of IGF-IR was significantly reduced in the LC of diabetic mice, suggesting the low levels of IGF-I circulating may reduce the number of IGF-IR on the membrane and, therefore, the excitability of neurons. In fact, systemic injection of IGF-I in control mice increased the spontaneous firing rate of LC neurons as well as the response to Sp5C electrical stimulation. However, these effects were reduced in STZ-diabetic mice probably due to reduction in the number of IGF-IR on the LC neurons. Furthermore, the expression of TH was also reduced in STZ-diabetic mice, which could indicate that there might be a neuronal reduction in the LC that decreases its activity. Previously, it has been demonstrated that the AIK3a305, a IGF-IR sensitizer, restored responses to IGF-I and sleep patterns in old mice 58 . In these experiments, treatment with AIK3a305 in STZ-diabetic mice recovered LC responses to formalin injection as well as the mechanical threshold sensitivity suggesting that the impairment of LC activity in these animals may be due to a reduction of IGF-IR signaling and that could be partially recovered with the treatment. In agreement with that, the LC-evoked inhibition on Sp5C neurons was increased in STZ-diabetic mice treated with AIK3a305, reaching similar values as in control mice, and IGF-IR and TH expression were also increased. In conclusion, chronic pain in patients with diabetes may be due to the reduction in the inhibitory effect that the LC has on the transmission of nociceptive stimuli, as we have observed in an animal model of diabetes. This reduction in LC activity may be due to a decrease in IGF-I signaling, therefore the use of IGF-IR sensitizers such as AIK3a305 may represent a promising therapeutic strategy to restore impaired IGF-I signaling and improve pain modulation in diabetic patients. Methods Animals Young adult male C57BL/6J mice (2–3 months old; weight 22–28 g; Harlan Laboratories, Spain) were used in this study. The animals were assigned to two groups: STZ-induced diabetic mice (n = 48) and control mice receiving vehicle injections (n = 39). All mice were housed under standardized conditions, with a 12:12 h light/dark cycle at 22 ± 2°C and had free access to food and water. All experimental procedures complied with European legislation (Directive 2010/63/EU) and were approved by the Ethics Committee of the Autonomous University of Madrid and the Regional Government of Madrid (PROEX: 181.6/21). All efforts were made to minimize both animal suffering and the number of animals used. A double-blind design was implemented throughout the study: a technician was responsible for glucose measurements and animal care, while the investigator performing behavioral, anatomical, or electrophysiological assessments remained blind to the group assignments. STZ-Dependent Diabetes STZ is an antibiotic that selectively destroys pancreatic islet β-cells and is widely used to generate experimental models of type 1 diabetes mellitus 59 . This animal model enables the study of diabetes-related alterations and the evaluation of potential therapeutic strategies. According to the previously protocol by Perez-Taboada et al. (2020) 60 , STZ (50 mg/kg, intraperitoneally, i.p.; Sigma-Aldrich, St. Louis, MO) was administered intraperitoneally once daily for five consecutive days. The mice were considered diabetic when blood glucose levels exceeded 300 mg/dl measured from tail using a glucometer (Glucoleader- Yasee GLM-76, Nessler, Spain). Control animals received equivalent injections of the vehicle solution (10 mM sodium citrate, 0.9% NaCl; pH 4.5, i.p.). Glucose levels were assessed after a 4-hour fasting period prior to STZ administration, throughout the three-week period of diabetes development, and before experimental recordings or behavioral testing. Behavioral test Mechanical allodynia was evaluated using a series of six calibrated nylon von Frey monofilaments (0.008 to 0.4 g) to confirm the presence of orofacial hypersensitivity in diabetic mice. Testing was conducted according to the method described by Mesa-Lombardo et al. 2023 25 . Von Frey filaments were applied in ascending order to five distinct points on each vibrissal pad, with five repetitions per filament. Each filament was applied until it bent slightly, with a minimum interval of 5 seconds between stimulations. The response threshold was defined as the lowest filament force eliciting a brisk head withdrawal in more than 50% of trials (3/5). Testing was performed at three and four weeks after STZ or vehicle administration. In addition, behavioral responses were also scored based on observed reactions: eye blink (2.5 points), face scratching (5 points), and face withdrawal (10 points). Points were summed and averaged for the 0.07 g filament to obtain and overall response score. Electrophysiological recordings and electrical stimulation As described previously 25 , 26 , animals were recorded after three weeks of diabetes development in STZ-injected animals or three weeks of receiving vehicle in control animals. Animals were anesthetized with isoflurane (2% induction; 1–1.5% maintenance doses) and placed in a David Kopf stereotaxic device (Tujunga, CA, USA). Body temperature was maintained at 37 ºC through a heating pad (Gaymar T/Pump, NY, USA). A small incision was performed in the skin over the scalp and an unilateral craniotomy over the LC nucleus was made according to the atlas of Paxinos and Franklin (coordinates from Bregma: AP: -5.4 mm, ML: 0.9 mm, DV: 3.5 mm) 61 . Single-unit recordings were performed in the LC through tungsten microelectrodes (2 MΩ; AM-System, Sequim, USA), using a DAM50 preamplifier (World Precision Instruments, Friedberg, Germany; filter setting, 0.3-3 kHz). The signals were sampled at 10 kHz through an analog-to-digital converter (Power 1401 data acquisition unit, Cambridge Electronic Design, Cambridge, UK) and a personal computer for off-line analysis with Spike 2 software (Cambridge Electronic Design). Electrical stimulation of Sp5C (coordinates from Bregma: AP: -7.6 mm, ML: 2 mm; DV: 0.5–1.5 mm from the surface of the nucleus) was performed by a bipolar stimulation electrode (World Precision Instruments, Friedberg, Germany) as described previously 61 . Pulses of 0.3 ms duration were applied. For comparison, the stimulation intensity was fixed two times higher than the threshold to elicit spike firing in the LC neurons (10–100 µA intensity). Nociceptive and non-nociceptive stimulation Non-nociceptive stimulation was applied by vibrissal deflections that were evoked by brief air-pulses using a pneumatic pressure pump (Picospritzer, Hollis, NH, USA; 1–2 kg/cm2, 20 ms duration). They were applied to test the inhibition evoked by LC stimulation on vibrissal responses in Sp5C. The experimental protocol consisted of air pulses delivered to the vibrissae at 0.5 Hz for 1 minute (30 stimuli; basal condition) before electrical pulses in the LC nucleus followed by vibrissal stimuli at 50 ms delays for 1 minute (30 pair of stimuli; pair pulses). After the paired-pulse protocol, the vibrissal was stimulated for 1 minute at 0.5 Hz (30 stimuli) to test if the vibrissal response recovered from the effect of LC stimulation. Nociceptive stimulation was induced by subcutaneously injected formalin that produces a long-lasting nociceptive response and is commonly used as a model for pain studies 62 – 64 . Mice were injected subcutaneously with 20 µl of 2% formalin solution or saline, using a sterile insulin syringe (U-100 insulin 30G X 1/2- 0.33 X 8mm). Administration of IGF-I or AIK3a305 To test the effect of IGF-I on LC neuronal activity, it was systemically injected (1 µg/g body weight). AIK3a305 (Allinky Biopharma, Spain) or the vehicle (DMSO + saline) were injected i.p. at a dose of 20 mg/kg/day daily for 28 consecutive days (4 weeks) 58 . To induce diabetes, STZ was injected for 5 days at the beginning of treatment with AIK3a305. Immunohistochemistry This protocol was performed as previously described 25 , 26 . Briefly, animals were given an overdose of sodium pentobarbital (Dolethal; Vétoquinol, Madrid, Spain; 50 mg/kg) and perfused through the heart with with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brainstem was dissected. All extracted tissues were post-fixed overnight in the same fixative and subsequently cryoprotected in 30% sucrose in PB for 48 hours. Serial 30-µm-thick coronal sections of the brainstem were obtained using a sliding microtome (Leica SM2400, Leica Biosystems, Nussloch, Germany) and collected in 0.1 M PB for free-floating immunohistochemical processing. Sections were first incubated for 2 hours at room temperature in a blocking solution containing 0.1 M PB, 1% Triton X-100, and 10% normal donkey serum. They were then incubated at 4°C for 24 hours with primary antibodies. The following antibodies were used: rabbit monoclonal anti-tyrosine hydroxylase (TH) antibody (1:2000; ab137869, Abcam), mouse monoclonal anti-IGF-I receptor antibody (1: 500; sc-271606, Santa Cruz), and mouse polyclonal c-Fos (1:400; Abcam, UK, ab190289). After three washes with 0.1 M PB, sections were incubated for 2 hours at room temperature, in the dark, with an AlexaFluor 488-conjugated donkey anti-rabbit (1:200; A21206, ThermoFisher) and AlexaFluor 546-conjugated donkey anti-mouse (1:200; A10036, ThermoFisher) secondary antibodies. Finally, sections were washed twice in PB and incubated for 5 minutes with Hoechst stain (1:3000 dilution in PB; Thermo Fisher Scientific, Waltham, MA, USA) to visualize cell nuclei. Samples were then mounted on glass slides and coverslipped using ProLong™ mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). Microscopy and image analysis As previously described 25 , 26 , confocal microscopy 3D images of the Sp5C in both sides were obtained by means of a Leica Stellaris 8 Tau STED confocal microscope (Leica Mcrosystems AG; Wetzlar, Germany) using a 20X objective or 100X oil immersion objective to study the morphology and location of LC neurons or for densitometry analysis. Image stacks were acquired at 1024 x 1024 pixels using Leica software. The densitometric analysis was performed with a ImageJ image analysis software for Windows (Microsoft; Albuquerque, NM, USA). Images obtained by confocal microscopy were processed to create TIFF files with maximum intensity projections, using a consistent final tissue thickness of 10 µm for all series. Sections were processed together under identical conditions to ensure comparable immunostaining analysis. The region of interest (ROI) was outlined to include the LC. Optical density measurements were obtained by the ImageJ 'Set Measurement' routine. These values were measured to perform statistical analysis. Data analysis The mean firing rate was calculated every minute as the number of spikes/s in basal conditions and after formalin injection. Bursts were defined as two or three spikes firing at 10–20 Hz 5, 6 . The instantaneous frequency was calculated as the time interval between two consecutive spikes. It was used to identify the burst firing pattern of LC neurons. The frequency of the bursts was calculated in periods of 5 minutes in basal conditions or 5 and 25 minutes after formalin injection. The spike response was calculated by means of the peristimulus time histograms (PSTHs; 1 ms bin-width). The spikes that occur in a 50 ms time window following the stimulus onset were counted. The vibrissal response during the baseline recording was considered 100%, and the effect of LC stimulation was calculated during the paired-pulse (LC-vibrissa) stimulation protocol. To measure the feedback inhibition, paired-pulse stimulation of the vibrissa was applied; the mean response to the first stimuli was considered 100% and the reduction of the second response was considered as a measure of the inhibition. The statistical analysis was conducted using GraphPad Prism version 10 (San Diego, CA, USA). Depending on the number of independent variables, the distribution of the data (assessed via the Kolmogorov–Smirnov test), and the nature of the experimental comparisons, we used either Student’s t-test or a two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. When the data did not follow a normal distribution, comparisons between two groups were made using the Mann–Whitney U test. Data are presented as mean ± standard error of the mean (SEM). Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Declarations Competing interests The authors declare no competing interests. Funding: This work was supported by Grants from Spanish Ministerio de Ciencia, Innovación y Universidades (PID2024-158246OB-I00) and Convocatoria de Proyectos de Investigación 2025 (Evaluación del control inhibitorio del locus coeruleus en la neuralgia del trigémino: identificación de nuevas vías terapéuticas; UFV2025-07) from Universidad Francisco de Vitoria de Madrid. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Alberto Mesa-Lombardo, Nuria García-Magro and Guillermo Cerrillo. The first draft of the manuscript was written by Alberto Mesa-Lombardo and Nuria García-Magro and all authors commented on previous versions of the manuscript. Yasmina B. Martin and Angel Nuñez have written and review the final version. All authors have read and approved the final manuscript. Acknowledgement We like to thank R.M. Sánchez Lozano for technical support. 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Nociceptive stimuli induce changes in somatosensory responses of rat dorsal column nuclei neurons. Brain Res. 1025 , 169–176 (2004). Borghi, V. et al. Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonists in mice. Brain Res. 956 , 339–348 (2002). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Editor invited by journal 07 Apr, 2026 Submission checks completed at journal 22 Mar, 2026 First submitted to journal 22 Mar, 2026 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. 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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-9072982","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":621764896,"identity":"fb48ab1b-9dc7-4c7b-8bac-2ce96f166533","order_by":0,"name":"Alberto Mesa-Lombardo","email":"","orcid":"","institution":"Autonomous University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Mesa-Lombardo","suffix":""},{"id":621764897,"identity":"62188e69-7560-4918-beb2-da1aa844c2e1","order_by":1,"name":"Nuria García-Magro","email":"","orcid":"","institution":"Universidad Francisco de Vitoria","correspondingAuthor":false,"prefix":"","firstName":"Nuria","middleName":"","lastName":"García-Magro","suffix":""},{"id":621764898,"identity":"d049505b-1dbf-4e1e-a53c-ac3a43c54054","order_by":2,"name":"Guillermo Cerrillo","email":"","orcid":"","institution":"Autonomous University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Guillermo","middleName":"","lastName":"Cerrillo","suffix":""},{"id":621764899,"identity":"3b48515b-6c4f-4823-9b15-09cf78e27db2","order_by":3,"name":"Yasmina B. Martin","email":"","orcid":"","institution":"Universidad Francisco de Vitoria","correspondingAuthor":false,"prefix":"","firstName":"Yasmina","middleName":"B.","lastName":"Martin","suffix":""},{"id":621764900,"identity":"3a6cd08a-60ab-453e-85a1-720f5534943d","order_by":4,"name":"Ángel Nuñez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYLCCBxCK8QHxWhIgFLMByVrYJIhSzT+7/eKDBIY7idvZ259V89RsY+DnP4Bfi8SdM8UGCQzPEnf2HEi7zXPsNoPkjAQC1tzISZNIYDicuOFGwrHbPGy3GQxuENAhfyMn/QdYy/2HbcU8/24z2J8n4DCDG+nHGCC2MLMx87YBbYGFBi5geCOHWSLB4LDxhjNpzJJz+27zSNwgoEXuRvrDDx8qDstuOH784Yc3327L8fcTcBgDAw8wApHikIeQeiBgf0CEolEwCkbBKBjRAACUK0kmUYJEnQAAAABJRU5ErkJggg==","orcid":"","institution":"Autonomous University of Madrid","correspondingAuthor":true,"prefix":"","firstName":"Ángel","middleName":"","lastName":"Nuñez","suffix":""}],"badges":[],"createdAt":"2026-03-09 12:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9072982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9072982/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106879440,"identity":"530e9389-a3c3-482f-9d0f-c190157ca2e6","added_by":"auto","created_at":"2026-04-14 11:03:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":121196,"visible":true,"origin":"","legend":"\u003cp\u003eFormalin induces an increase in the excitability of LC neurons that is reduced in STZ-diabetic mice. \u003cstrong\u003eA,\u003c/strong\u003e formalin injection at the vibrissal pad (20 ml; 2%; vertical arrow) induces a progressive increase of the firing rate of LC neurons in control animals while decrease it in STZ-diabetic animals. Two-way repeated measure ANOVA, interaction group F (2, 50)= 15.20 p \u0026lt; 0,0001. \u003cstrong\u003eB,\u003c/strong\u003e Plot of the firing rate measure in basal conditions and 25 min. after formalin injection. \u003cstrong\u003eC,\u003c/strong\u003eNumber of burst measure in 5 min. in basal condition, 5 min. and 25 min. after formalin injection. Formalin only increases the burst firing in control animals. \u003cstrong\u003eD,\u003c/strong\u003e electrical pulses at Sp5C (\u0026lt;100 mA; 0.3 ms) evoke an orthodromic response in LC. Formalin increases the orthodromic response in control mice, indicating an increase of excitability, but only 25 min. after formalin in STZ-diabetic mice. In this and in the following figures *, p\u0026lt; 0.05; **, p\u0026lt; 0.01; *** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/8569f6ca9c50050dcb66a145.jpeg"},{"id":106960512,"identity":"6198071d-3d41-474b-b2c6-8c3377cf6557","added_by":"auto","created_at":"2026-04-15 09:21:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169536,"visible":true,"origin":"","legend":"\u003cp\u003eFormalin increases c-Fos expression in the LC nucleus. A, representative photomicrographs of c-Fos labeled cells in the LC of control and STZ-diabetic mice (Scale 50 µm except in the right column 10 µm). Formalin injection in the vibrissal pad increases the expression of c-Fos in both control and STZ-diabetic mice. B, optical density (IF) of c-Fos expression in non-injected or formalin injected (2%) control or STZ-diabetic mice. Both animal groups increase the optical density of c-Fos by formalin.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/c0db2bea91d927cd0c6ace73.jpeg"},{"id":106961245,"identity":"c0086bab-9daf-403d-b9ec-8935ecea3b9a","added_by":"auto","created_at":"2026-04-15 09:24:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38790,"visible":true,"origin":"","legend":"\u003cp\u003eFormalin increases the LC-evoked inhibition of vibrissal responses in Sp5C. A and B, plots show the mean LC-evoked inhibition in control mice (blue bars) and in STZ-diabetic mice (red bars) in basal conditions and after formalin injection. In control, the nociceptive stimulus of formalin increases the inhibition of vibrissal responses in Sp5C by LC. In STZ-diabetic mice, this increase is only observed at 5 min. after formalin injection.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/b529575635c40fa4dd3a5e63.jpeg"},{"id":106961140,"identity":"3a73f14c-57d5-4e85-94ab-655d8a6c02dc","added_by":"auto","created_at":"2026-04-15 09:24:26","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45589,"visible":true,"origin":"","legend":"\u003cp\u003eIGF-I increases the excitability of LC neurons. A, plot shows the mean firing rate of LC neurons in basal condition and 30 min. after systemic injection of IGF-I (1 μg/g body weight, i.p.) in control animals (blue bars) and in STZ-diabetic mice (red bars). IGF-I increases the firing rate in both animal groups although the effect is larger in control animals. B, plot of the orthodromic response in LC neurons by Sp5C electrical stimulation. IGF-I increases the orthodromic response in both animal groups.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/37038d0aafb5bc826b6389e3.jpeg"},{"id":106961184,"identity":"336407cf-6580-46c7-b1f8-6ec3e3a94ac4","added_by":"auto","created_at":"2026-04-15 09:24:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114660,"visible":true,"origin":"","legend":"\u003cp\u003eTH and IGF-IR Immunohistochemistry in the LC of control and STZ-diabetic mice. A, representative photomicrographs of TH labeled cells and IGF-IR in the LC of control and STZ-diabetic mice (Scale 50 µm except in the right column 10 µm). B, optical density (IF) of IGF-I expression in control and STZ-diabetic mice. A clear reduction of IGF-IR is shown.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/b9257f2dc358a9be190c4df4.jpeg"},{"id":106879447,"identity":"aaf07d8e-2143-4166-b813-408801b11ee0","added_by":"auto","created_at":"2026-04-14 11:03:48","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82053,"visible":true,"origin":"","legend":"\u003cp\u003eThe novel IGF-IR sensitizer AIK3a305 increases the response to formalin in STZ-diabetic mice. STZ was injected for 5 days to induce diabetes; at the same time AIKa305 was systemically injected for 28 days (1 μg/g body weight). A, same plot as in Figure 1A with the addition of the effect of formalin injection at the vibrissal pad (20 l; 2%; vertical arrow) in treated STZ-diabetic mice with AIK3a305 (beige points). Formalin induces a progressive increase of the firing rate of LC neurons in treated animals, as in control animals. Two-way repeated measure ANOVA, interaction group F (2, 50)= 15.17, p\u0026lt; 0,0001. B, plot of the LC-evoked inhibition of vibrissal responses in Sp5C. The inhibition of Sp5C responses by LC stimulation was the same in treated animals as in control animals.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/d16282c9eff26b91b095cd12.jpeg"},{"id":106961223,"identity":"60a50de8-801b-4238-96e7-3bea6baf248e","added_by":"auto","created_at":"2026-04-15 09:24:45","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":168572,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AIK3a305 on TH and IGF-IR Immunohistochemistry. A, representative photomicrographs of TH labeled cells and IGF-IR in the LC of control, STZ-diabetic mice and in STZ-diabetic mice treated with AIK3a305 (Scale 50 µm except in the right column 10 µm). B, optical density (IF) of TH+ expression and IGF-IR expression in control, STZ-diabetic mice and in STZ-diabetic mice treated with AIK3a305. A clear reduction of TH neurons is shown. Treatment of STZ-diabetic mice with AIK3a305 results in an increase in TH expression. C, optical density (IF) of IGF-IR expression in control, STZ-diabetic and STZ-diabetic mice treated with AIK3a305. The reduction of IGF-IR expression in STZ-diabetic mice is partially reverted by the treatment with AIK3a305.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/0aafb34d901b61cc4e98d881.jpeg"},{"id":106960513,"identity":"b689bab7-5539-4b38-9f66-eda26c2f6867","added_by":"auto","created_at":"2026-04-15 09:21:34","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":69517,"visible":true,"origin":"","legend":"\u003cp\u003eAIK3a305 treatment restores the response to mechanical stimulation in STZ-diabetic mice. A, STZ-diabetic animals show a decrease in the withdrawal threshold to mechanical stimulation in vibrissal pad the third and four weeks after STZ injection in comparison with basal values. Mice with AIK3a305 treatment almost reach control values. Two-way repeated measure ANOVA, interaction group F (2, 51)= 11.54, p\u0026lt; 0,0001. B, similar results are shown regarding response scores. Two-way repeated measure ANOVA, interaction group F (2, 30)= 3.571, p= 0.0406. # indicates the differences with the basal values and * indicate differences with respect to the values obtained in the control mice p\u0026lt; 0.05*, p\u0026lt; 0.01**, p\u0026lt; 0.001***.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/88d294a4adbb5fcf41034480.jpeg"},{"id":106964653,"identity":"ea124167-98bf-4473-90d5-287f4d0c6452","added_by":"auto","created_at":"2026-04-15 09:50:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1695143,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9072982/v1/c18c4e1b-ab9f-49eb-875e-df6e69b31f2b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reduced Noradrenergic Excitability in the Locus Coeruleus Compromises Nociceptive Inhibition in a Diabetic Mouse Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe locus coeruleus (LC) is a small, bilateral nucleus in the pontine tegmentum that is characterized by its rich concentration of noradrenergic (NA) neurons. NA neurons play an essential role in the regulation of cognitive function, sleep/wake state, arousal, attention, mood, and stress reactions \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. According to the behavioral state, LC neurons display two different discharge patterns: tonic and phasic modes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Spontaneous tonic activity consisted in a sustained regular discharge pattern of single spikes at 2\u0026ndash;8 Hz. The phasic discharge pattern was characterized by brief 10\u0026ndash;20 Hz bursts of two to three action potentials. In addition, LC also control nociceptive transmission by a widespread descending pathway which includes the spinal cord and spinal trigeminal nucleus \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe caudalis division of the spinal trigeminal nucleus (Sp5C) is the first central relay station in the circuitry involved in processing pain and non-noxious stimuli from the craniofacial region \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Neuroanatomical findings have demonstrated reciprocal connections between the LC and trigeminal sensory nuclei, suggesting that the LC may exert modulatory control over nociceptive processing in the orofacial region \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Consistent with this notion, acute noxious orofacial stimulation induces increased c-Fos immunoreactivity within the LC \u003csup\u003e22, 23\u003c/sup\u003e and stimulation of the LC neurons inhibits the sensory transmission in the Sp5C neurons through activation of α2-NA receptors \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Recently, we have published that LC also inhibit sensory responses in the Sp5C nucleus by activation of postsynaptic α2-NA receptors on glutamatergic Sp5C neurons and by excitation of GABAergic neurons via α1-NA receptors \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNeuropathic pain is one of the most common complications of diabetes. Although one of the most important functions of the LC is to control the nociceptive transmission, little is known about the changes in the LC activity in diabetes. Results from our laboratory have suggested that a reduction in NA neuronal activity may contribute to the generation and/or maintenance of neuropathic pain in these patients. The inhibition elicited by LC stimulation in Sp5C nucleus was reduced in streptozotocin (STZ)-induced diabetic mice, suggesting that an impairment of LC activity may contribute to the generation of chronic pain in diabetic patients \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For this reason, the goal of the present study was to investigate the changes in LC activity in STZ-diabetic mice and the mechanisms underlying the reduction of noradrenergic neuronal activity. We suggest that the decrease in insulin-like growth factor I (IGF-I) levels that occur in diabetes may be responsible for the reduced activity of LC neurons and, consequently, for the altered control of nociceptive transmission.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffect of formalin injection on locus coeruleus activity in control and STZ-diabetic mice\u003c/h2\u003e \u003cp\u003eTo evaluate the impact of peripheral nociceptive stimulation on the activity of the LC neurons in control and STZ-diabetic mice, the spontaneous firing rate was recorded before and after formalin application to the vibrissal pad of anesthetized mice (20 \u0026micro;l; 2%). In control animals, formalin injections evoked a progressive increase of the firing rate of LC neurons from 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 spikes/s under baseline conditions to 12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 spikes/s at 25 minutes after formalin injection (n\u0026thinsp;=\u0026thinsp;17 neurons; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, paired t-test; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In contrast, STZ-diabetic animals exhibited a progressive decrease of the firing rate after formalin injection from 7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 spikes/s to 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 spikes/s at 25 minutes after formalin injection (n\u0026thinsp;=\u0026thinsp;20 neurons; p\u0026thinsp;=\u0026thinsp;0.0001). A repeated measures ANOVA test indicated that both curves were statistically different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These results suggest an altered LC response to nociceptive stimuli in the context of diabetes.\u003c/p\u003e \u003cp\u003eWe studied the presence of burst in LC neurons recorded in basal conditions or after formalin injection because they occur in association with salient or new stimuli \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Control animals showed the presence of burst of action potentials that consisted in two-three spikes at a frequency of 10\u0026ndash;20 Hz with a mean frequency of 55.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 bursts measured in 5 minutes of basal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; see inset). Their frequency increased after formalin injection when they were recorded at 5 or 25 minutes after formalin injection to 92.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6 or 112.1\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 bursts per 5 minutes, respectively (n\u0026thinsp;=\u0026thinsp;17 neurons; p\u0026thinsp;=\u0026thinsp;0.0162 or p\u0026thinsp;=\u0026thinsp;0.0012). STZ-diabetic mice also showed burst of spikes in basal conditions 40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 bursts per 5 minutes. However, formalin injection did not modify the frequency of these bursts at 5 or 25 minutes after formalin injection (41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6 or 37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1 bursts per 5 minutes, respectively; n\u0026thinsp;=\u0026thinsp;20 neurons; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fact that formalin increased the spontaneous activity of LC neurons in control mice suggested that there was also an increase in neuronal excitability. However, this effect was not observed in STZ-diabetic mice. We studied the response of LC neurons to electrical stimulation at the Sp5C nucleus in basal conditions and at 5 and 25 minutes after the injection of formalin. Electrical stimulation (\u0026lt;\u0026thinsp;100 \u0026micro;A; 0.3 ms) elicited a short latency orthodromic response in LC neurons in both control (5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ms, n\u0026thinsp;=\u0026thinsp;13 neurons) and STZ-diabetic mice (6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ms, n\u0026thinsp;=\u0026thinsp;19 neurons; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In control animals, LC responses increased from 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 spikes/stimulus in basal conditions to 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 spikes/stimulus and 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 spikes/stimulus at 5 and 25 minutes after formalin injection (p\u0026thinsp;=\u0026thinsp;0.0111, p\u0026thinsp;=\u0026thinsp;0.0017, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating that LC increased their excitability after formalin injection. STZ-diabetic mice showed a response in basal conditions of 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 spikes/stimulus however, the LC response did not increase 5 min. after formalin injection (1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 spikes/stimulus) and only increased to 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 spikes/stimulus at 25 min. (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and p\u0026thinsp;=\u0026thinsp;0.0272, respectively). These data suggested that the excitability of LC neurons in STZ-diabetic animals was reduced in comparison to control animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFormalin increased LC activity assessed by c-Fos expression\u003c/h3\u003e\n\u003cp\u003eExpression of c-Fos is an indirect marker of neuronal activity because c-Fos is expressed when neurons fire action potentials. To corroborate that formalin induced an increase of LC neuronal excitability, we analyzed the expression of c-Fos in non-injected or formalin injected (2%) control or STZ-diabetic animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Formalin injection in control mice (n\u0026thinsp;=\u0026thinsp;8 animals) led to a higher c-Fos expression in the LC than in non-injected control animals (23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 to 33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 IF intensity; n\u0026thinsp;=\u0026thinsp;8, p\u0026thinsp;=\u0026thinsp;0.011), indicating enhanced neuronal activation in response to the nociceptive stimulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, even in the absence of formalin, STZ-diabetic mice showed reduced c-Fos expression in the LC compared to control mice (16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 IF intensity; n\u0026thinsp;=\u0026thinsp;8, p\u0026thinsp;=\u0026thinsp;0.0417), suggesting a basal reduction in neuronal activity associated with the diabetic condition. Additionally, within the STZ-diabetic group, animals injected with formalin displayed significantly higher c-Fos expression than non-injected animals (22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 IF intensity; n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;=\u0026thinsp;0.0216); however, these values did not reach the c-Fos expression in formalin-injected control mice (p\u0026thinsp;=\u0026thinsp;0.0004), indicating an impaired activation of the LC in response to nociceptive inputs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLC-evoked inhibition in Sp5C increased in control mice by formalin but not in STZ-diabetic mice\u003c/h3\u003e\n\u003cp\u003eIt has been published that electrical stimulation of the LC induces an inhibition of vibrissal responses in the Sp5C nucleus through activation of NA receptors that may contribute to control sensory transmission of non-nociceptive and nociceptive inputs \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In our experiments, electrical stimulation of the LC induced a clear inhibition of the vibrissal responses in basal conditions in control mice (-11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, n\u0026thinsp;=\u0026thinsp;12 neurons) that increased when measured 5 min. after formalin injection (-23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9%, n\u0026thinsp;=\u0026thinsp;12 neurons; p\u0026thinsp;=\u0026thinsp;0.0228, respect to basal values). This inhibition persisted up to 20 minutes after formalin injection (-25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5%, n\u0026thinsp;=\u0026thinsp;12 neurons; p\u0026thinsp;=\u0026thinsp;0.0467, respect to basal values; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). LC-evoked inhibition was smaller in STZ-diabetic animals in comparison with control animals (-0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, n\u0026thinsp;=\u0026thinsp;14 neurons; p\u0026thinsp;=\u0026thinsp;0.0357 respect to basal values in control animals), as has been previously described. LC-evoked inhibition increased when measured 5 min. after formalin injection (-13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, n\u0026thinsp;=\u0026thinsp;14 neurons; p\u0026thinsp;=\u0026thinsp;0.0430, respect to basal values) and returned to basal values at 20 min after formalin injection (-5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1%, n\u0026thinsp;=\u0026thinsp;14 neurons; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Therefore, the data indicated that the increase in LC activity by formalin injection resulted in an increase in the LC-evoked inhibition exerted on Sp5C neurons in control animals. However, in STZ-diabetic animals, this inhibitory effect was only observed in the first 5 min. after the injection of formalin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe IGF-I effect was reduced in the LC of diabetic mice\u003c/h3\u003e\n\u003cp\u003eIGF-I plays an essential role in the modulation of synaptic plasticity and in learning and memory processes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Reduced levels of IGF-I and insulin have been proposed as potential causes of neurological disorders \u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Since insulin and IGF-I levels were strongly reduced in diabetic animals \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, we tested the effect of IGF-I on LC neurons in control and STZ-diabetic mice.\u003c/p\u003e \u003cp\u003eSystemic injection of IGF-I in control mice (1 \u0026micro;g/g body weight, i.p.) induced an increase of the firing rate of LC neurons from 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 spikes/s to 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 spikes/s 30 minutes after IGF-I injection (n\u0026thinsp;=\u0026thinsp;12 neurons; p\u0026thinsp;=\u0026thinsp;0.0025; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Also, the response of LC neurons to Sp5C stimulation increased from 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 spikes/stimulus to 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 spikes/stimulus after IGF-I injection (n\u0026thinsp;=\u0026thinsp;12 neurons; p\u0026thinsp;=\u0026thinsp;0.0201; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that IGF-I increased LC neuronal excitability. IGF-I also increased the firing rate of LC neurons in STZ-diabetic mice from 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 spikes/s to 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 spikes/s when was injected systemically (n\u0026thinsp;=\u0026thinsp;12: p\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) although the increment was lower than in the control mice. Equally, the response of LC neurons to Sp5C stimulation increased from 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 spikes/stimulus to 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 spikes/stimulus after IGF-I injection (n\u0026thinsp;=\u0026thinsp;12 neurons; p\u0026thinsp;=\u0026thinsp;0.0182; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which indicates that LC neurons could increase their excitability if IGF-I levels were to rise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis reduced response of LC neurons in STZ-diabetic mice in comparison with control mice may be due to a reduction of IGF-I receptor (IGF-IR) in LC neurons. Immunohistochemical studies showed an ample distribution of the IGF-IR in the brainstem, as is shown in the LC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The optical density of normalized fluorescence intensity indicated a statistically significant reduction of IGF-IR in STZ-diabetic animals respect to control ones (26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 to 12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 IF intensity; n\u0026thinsp;=\u0026thinsp;14 and n\u0026thinsp;=\u0026thinsp;10, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating an impaired IGF-I signaling in this nucleus under diabetic conditions that may explain the reduced excitability of LC neurons in STZ-diabetic mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAIK3a305 restore LC neuronal activity in STZ-diabetic mice\u003c/h3\u003e\n\u003cp\u003eTo determine that dysregulated IGF-IR expression in LC neurons of STZ-diabetic mice may be responsible for the reduction of nociceptive responses in LC, we treated STZ-diabetic mice with AIK3a305, a novel IGF-IR sensitizer. We observed that the LC response to formalin injection in STZ-diabetic mice treated with AIK3a305 resembles control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Also, the LC-evoked inhibition of vibrissal responses in Sp5C was recorded in treated animals. In control animals LC-evoked inhibition in basal condition was \u0026minus;\u0026thinsp;14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9% (n\u0026thinsp;=\u0026thinsp;22 neurons) while in STZ-diabetic animals was \u0026minus;\u0026thinsp;0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% (n\u0026thinsp;=\u0026thinsp;18 neurons; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). LC-evoked inhibition in STZ-diabetic mice treated with AIK3a305 reached the same values as in control animals (-16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% (n\u0026thinsp;=\u0026thinsp;22 neurons; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated if TH and IGF-IR expression were recovered with the AIK3a305 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). TH expression was reduced in STZ-diabetic mice respect to control mice (38.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 to 23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 IF intensity; n\u0026thinsp;=\u0026thinsp;10 and n\u0026thinsp;=\u0026thinsp;11, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, the expression of TH increased after the treatment with AIK3a305 respect to STZ-diabetic mice (30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 IF intensity; n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;=\u0026thinsp;0.0022), although it did not reach the values in control mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The same occurred with the expression of IGF-IR in the LC; it was significantly reduced in diabetic mice compared to control animals (26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 to 12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 IF intensity; n\u0026thinsp;=\u0026thinsp;14 and n\u0026thinsp;=\u0026thinsp;10, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment of STZ-diabetic mice with AIK3a305 resulted in a marked increase in IGF-1R expression, surpassing the levels observed in diabetic animals (18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 IF intensity; n\u0026thinsp;=\u0026thinsp;8; p\u0026thinsp;=\u0026thinsp;0.0016).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAIK3a305 treatment restores the response to mechanical stimulation in STZ-diabetic mice\u003c/h2\u003e \u003cp\u003eMechanical stimulation response was evaluated during the second and third weeks following STZ administration and compared to baseline values recorded prior to the injection. STZ-diabetic mice exhibited a significant reduction in the mechanical withdrawal threshold in the vibrissal pad at 21 days, which became more pronounced by day 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). In contrast, control mice did not display any time-dependent changes in their response threshold. Notably, STZ-diabetic mice treated with AIK3a305 did not exhibit any alterations in mechanical sensitivity at 28 days, displaying thresholds comparable to those observed in control animals.\u003c/p\u003e \u003cp\u003eRegarding response scores, diabetic mice showed a significant increase in their response to mechanical stimulation of the vibrissal pad at both 21 and 28 days compared to control animals and diabetic mice treated with AIK3a305. Although diabetic mice treated with AIK3a305 exhibited a slight increase in response scores, this change was not statistically significant when compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present results demonstrate that the responsiveness of LC neurons to nociceptive stimuli was altered in STZ-diabetic mice. Consequently, the LC-evoked inhibition of vibrissal responses in Sp5C was markedly reduced. Such a decrease in NA activity may contribute to the facilitation of chronic pain development. This reduction of LC activity could be due to the reduction in the brain IGF-I signaling, as its receptor expression in the LC was decreased in STZ-diabetic mice. In fact, treatment of STZ-diabetic mice with the IGF-IR sensitizer AIK3a305 restored LC activity.\u003c/p\u003e \u003cp\u003eThe mouse orofacial formalin test is a valid and reliable model for evaluation of orofacial nociceptive processing and modulation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Formalin has been shown to produce a long-lasting nociceptive stimulus with a biphasic behavioral response; a direct action of formalin on nociceptive receptors is responsible for the first phase, whereas inflammation seemed to play a more important role in the second phase of formalin response \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eElectrophysiological data present here reveal that LC neurons in control mice increased the firing rate after formalin injection and remained at least 25 minutes after the injection. Previous findings have also shown that neuropathic pain increases spontaneous and noxious-evoked activity of LC neurons \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Furthermore, the expression of c-Fos is a common marker for recent cellular activity, reflecting cell activation induced by acute events such as nociceptive stimulus which increases c-Fos expression in the LC \u003csup\u003e24, 42\u003c/sup\u003e. In agreement with previous findings, formalin injection in the vibrissal pad increased the firing rate of LC neurons as well as increased the c-Fos expression in the LC of control mice. However, LC neurons from STZ-diabetic mice increased firing rate immediately after formalin injection, but after that it decreased progressively even below the basal activity. STZ-diabetic mice also showed a reduced c-Fos expression in the LC compared to control mice even in the absence of formalin, suggesting a basal reduction in neuronal activity associated with the diabetic condition. Formalin injections in STZ-diabetic mice increased the c-Fos expression but values were lower than the c-Fos expression in formalin-injected control mice.\u003c/p\u003e \u003cp\u003eMoreover, the reduction of LC activity in STZ-diabetic mice was accompanied by a reduction in the excitability of these neurons. Electrical stimulation of Sp5C evoked a short latency orthodromic response in LC neurons that increased after formalin injection in control animals, indicating that the nociceptive stimulation increased the excitability of LC neurons. Under baseline conditions, the evoked response was similar in STZ-diabetic mice than in control mice. However, after the formalin injection, the evoked response increase was lower than in control animals, indicating that nociceptive stimulus did not modify substantially the excitability of LC neurons.\u003c/p\u003e \u003cp\u003eThe increase in excitability caused by the formalin injection was also noted by the increase in the action potential firing rate of LC neurons and by a change in the discharge pattern. It is well known that LC neurons show tonic or bursting firing patterns \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Spontaneous tonic activity consisted in a sustained regular discharge pattern of single spikes at 2\u0026ndash;8 Hz that may change to a phasic, bursting discharge pattern characterized by brief 10\u0026ndash;20 Hz bursts when salient or new stimuli occur \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In control mice, the burst frequency increased after formalin injection but not in STZ-diabetic mice, suggesting that the excitability of LC neurons in STZ-diabetic mice was decreased and consequently, the response to nociceptive stimulation was also reduced.\u003c/p\u003e \u003cp\u003eThe descending NA inhibitory system from the LC to the dorsal horn and trigeminal nucleus is one of the main pathways involved in the endogenous pain modulation system \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Previous studies from our laboratory have demonstrated that LC evokes an inhibition of vibrissal responses in the Sp5C via activation of α2-NA on glutamatergic neurons and by α1-NA receptors on GABAergic neurons \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This inhibitory effect may be the modulatory mechanism for which LC controls the transmission of non-noxious and noxious stimuli. In agreement with this hypothesis, LC-evoked inhibition in the Sp5C increased after formalin injection respect to the basal condition in control mice and remained higher along the recording time, suggesting that the increase of excitability in LC neurons provoked by formalin induced an increase in the LC-evoked inhibition of nociceptive transmission in the Sp5C. However, this effect did not occur in STZ-diabetic mice in which LC-evoked inhibition only increment in the first 5 minutes after formalin injection, facilitating probably the appearance of chronic neuropathic pain. Taken together, the results suggest that in diabetic animals, the LC is capable of responding to an acute nociceptive stimulus but cannot maintain its response to a stimulus that is prolonged over time.\u003c/p\u003e \u003cp\u003eIGF-I enhances neuronal and glia activity, controlling numerous brain processes \u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48 CR49\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. IGF-I level is significantly reduced by 40% to 50% in type I as well as type II diabetes \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Reduced levels of IGF-I and insulin have been proposed as potential causes of neurological disorders because IGF-I application reduce cognitive deficits \u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In the light of these findings, we propose that the reduction of IGF-I may be responsible for the reduction of excitability of LC neurons since IGF-I increases neuronal excitability in many brain regions \u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We have just published that the reduction of IGF-I levels in STZ-diabetic mice is responsible for synaptic plasticity impairment in the somatosensory cortex \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The lack of IGF-I could explain the limited response that the LC exerts when the stimulus is prolonged over time.\u003c/p\u003e \u003cp\u003eWe observed that the expression of IGF-IR was significantly reduced in the LC of diabetic mice, suggesting the low levels of IGF-I circulating may reduce the number of IGF-IR on the membrane and, therefore, the excitability of neurons. In fact, systemic injection of IGF-I in control mice increased the spontaneous firing rate of LC neurons as well as the response to Sp5C electrical stimulation. However, these effects were reduced in STZ-diabetic mice probably due to reduction in the number of IGF-IR on the LC neurons. Furthermore, the expression of TH was also reduced in STZ-diabetic mice, which could indicate that there might be a neuronal reduction in the LC that decreases its activity.\u003c/p\u003e \u003cp\u003ePreviously, it has been demonstrated that the AIK3a305, a IGF-IR sensitizer, restored responses to IGF-I and sleep patterns in old mice \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In these experiments, treatment with AIK3a305 in STZ-diabetic mice recovered LC responses to formalin injection as well as the mechanical threshold sensitivity suggesting that the impairment of LC activity in these animals may be due to a reduction of IGF-IR signaling and that could be partially recovered with the treatment. In agreement with that, the LC-evoked inhibition on Sp5C neurons was increased in STZ-diabetic mice treated with AIK3a305, reaching similar values as in control mice, and IGF-IR and TH expression were also increased.\u003c/p\u003e \u003cp\u003eIn conclusion, chronic pain in patients with diabetes may be due to the reduction in the inhibitory effect that the LC has on the transmission of nociceptive stimuli, as we have observed in an animal model of diabetes. This reduction in LC activity may be due to a decrease in IGF-I signaling, therefore the use of IGF-IR sensitizers such as AIK3a305 may represent a promising therapeutic strategy to restore impaired IGF-I signaling and improve pain modulation in diabetic patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eYoung adult male C57BL/6J mice (2\u0026ndash;3 months old; weight 22\u0026ndash;28 g; Harlan Laboratories, Spain) were used in this study. The animals were assigned to two groups: STZ-induced diabetic mice (n\u0026thinsp;=\u0026thinsp;48) and control mice receiving vehicle injections (n\u0026thinsp;=\u0026thinsp;39). All mice were housed under standardized conditions, with a 12:12 h light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and had free access to food and water. All experimental procedures complied with European legislation (Directive 2010/63/EU) and were approved by the Ethics Committee of the Autonomous University of Madrid and the Regional Government of Madrid (PROEX: 181.6/21). All efforts were made to minimize both animal suffering and the number of animals used. A double-blind design was implemented throughout the study: a technician was responsible for glucose measurements and animal care, while the investigator performing behavioral, anatomical, or electrophysiological assessments remained blind to the group assignments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSTZ-Dependent Diabetes\u003c/h2\u003e \u003cp\u003eSTZ is an antibiotic that selectively destroys pancreatic islet β-cells and is widely used to generate experimental models of type 1 diabetes mellitus \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. This animal model enables the study of diabetes-related alterations and the evaluation of potential therapeutic strategies. According to the previously protocol by Perez-Taboada et al. (2020)\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, STZ (50 mg/kg, intraperitoneally, i.p.; Sigma-Aldrich, St. Louis, MO) was administered intraperitoneally once daily for five consecutive days. The mice were considered diabetic when blood glucose levels exceeded 300 mg/dl measured from tail using a glucometer (Glucoleader- Yasee GLM-76, Nessler, Spain). Control animals received equivalent injections of the vehicle solution (10 mM sodium citrate, 0.9% NaCl; pH 4.5, i.p.). Glucose levels were assessed after a 4-hour fasting period prior to STZ administration, throughout the three-week period of diabetes development, and before experimental recordings or behavioral testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral test\u003c/h2\u003e \u003cp\u003eMechanical allodynia was evaluated using a series of six calibrated nylon von Frey monofilaments (0.008 to 0.4 g) to confirm the presence of orofacial hypersensitivity in diabetic mice. Testing was conducted according to the method described by Mesa-Lombardo et al. 2023\u003csup\u003e25\u003c/sup\u003e. Von Frey filaments were applied in ascending order to five distinct points on each vibrissal pad, with five repetitions per filament. Each filament was applied until it bent slightly, with a minimum interval of 5 seconds between stimulations. The response threshold was defined as the lowest filament force eliciting a brisk head withdrawal in more than 50% of trials (3/5). Testing was performed at three and four weeks after STZ or vehicle administration.\u003c/p\u003e \u003cp\u003eIn addition, behavioral responses were also scored based on observed reactions: eye blink (2.5 points), face scratching (5 points), and face withdrawal (10 points). Points were summed and averaged for the 0.07 g filament to obtain and overall response score.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiological recordings and electrical stimulation\u003c/h2\u003e \u003cp\u003eAs described previously\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, animals were recorded after three weeks of diabetes development in STZ-injected animals or three weeks of receiving vehicle in control animals. Animals were anesthetized with isoflurane (2% induction; 1\u0026ndash;1.5% maintenance doses) and placed in a David Kopf stereotaxic device (Tujunga, CA, USA). Body temperature was maintained at 37 \u0026ordm;C through a heating pad (Gaymar T/Pump, NY, USA). A small incision was performed in the skin over the scalp and an unilateral craniotomy over the LC nucleus was made according to the atlas of Paxinos and Franklin (coordinates from Bregma: AP: -5.4 mm, ML: 0.9 mm, DV: 3.5 mm) \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSingle-unit recordings were performed in the LC through tungsten microelectrodes (2 MΩ; AM-System, Sequim, USA), using a DAM50 preamplifier (World Precision Instruments, Friedberg, Germany; filter setting, 0.3-3 kHz). The signals were sampled at 10 kHz through an analog-to-digital converter (Power 1401 data acquisition unit, Cambridge Electronic Design, Cambridge, UK) and a personal computer for off-line analysis with Spike 2 software (Cambridge Electronic Design).\u003c/p\u003e \u003cp\u003eElectrical stimulation of Sp5C (coordinates from Bregma: AP: -7.6 mm, ML: 2 mm; DV: 0.5\u0026ndash;1.5 mm from the surface of the nucleus) was performed by a bipolar stimulation electrode (World Precision Instruments, Friedberg, Germany) as described previously\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Pulses of 0.3 ms duration were applied. For comparison, the stimulation intensity was fixed two times higher than the threshold to elicit spike firing in the LC neurons (10\u0026ndash;100 \u0026micro;A intensity).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNociceptive and non-nociceptive stimulation\u003c/h2\u003e \u003cp\u003eNon-nociceptive stimulation was applied by vibrissal deflections that were evoked by brief air-pulses using a pneumatic pressure pump (Picospritzer, Hollis, NH, USA; 1\u0026ndash;2 kg/cm2, 20 ms duration). They were applied to test the inhibition evoked by LC stimulation on vibrissal responses in Sp5C. The experimental protocol consisted of air pulses delivered to the vibrissae at 0.5 Hz for 1 minute (30 stimuli; basal condition) before electrical pulses in the LC nucleus followed by vibrissal stimuli at 50 ms delays for 1 minute (30 pair of stimuli; pair pulses). After the paired-pulse protocol, the vibrissal was stimulated for 1 minute at 0.5 Hz (30 stimuli) to test if the vibrissal response recovered from the effect of LC stimulation.\u003c/p\u003e \u003cp\u003eNociceptive stimulation was induced by subcutaneously injected formalin that produces a long-lasting nociceptive response and is commonly used as a model for pain studies \u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Mice were injected subcutaneously with 20 \u0026micro;l of 2% formalin solution or saline, using a sterile insulin syringe (U-100 insulin 30G X 1/2- 0.33 X 8mm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAdministration of IGF-I or AIK3a305\u003c/h2\u003e \u003cp\u003eTo test the effect of IGF-I on LC neuronal activity, it was systemically injected (1 \u0026micro;g/g body weight). AIK3a305 (Allinky Biopharma, Spain) or the vehicle (DMSO\u0026thinsp;+\u0026thinsp;saline) were injected i.p. at a dose of 20 mg/kg/day daily for 28 consecutive days (4 weeks)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. To induce diabetes, STZ was injected for 5 days at the beginning of treatment with AIK3a305.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eThis protocol was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, animals were given an overdose of sodium pentobarbital (Dolethal; V\u0026eacute;toquinol, Madrid, Spain; 50 mg/kg) and perfused through the heart with with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brainstem was dissected. All extracted tissues were post-fixed overnight in the same fixative and subsequently cryoprotected in 30% sucrose in PB for 48 hours. Serial 30-\u0026micro;m-thick coronal sections of the brainstem were obtained using a sliding microtome (Leica SM2400, Leica Biosystems, Nussloch, Germany) and collected in 0.1 M PB for free-floating immunohistochemical processing. Sections were first incubated for 2 hours at room temperature in a blocking solution containing 0.1 M PB, 1% Triton X-100, and 10% normal donkey serum. They were then incubated at 4\u0026deg;C for 24 hours with primary antibodies. The following antibodies were used: rabbit monoclonal anti-tyrosine hydroxylase (TH) antibody (1:2000; ab137869, Abcam), mouse monoclonal anti-IGF-I receptor antibody (1: 500; sc-271606, Santa Cruz), and mouse polyclonal c-Fos (1:400; Abcam, UK, ab190289). After three washes with 0.1 M PB, sections were incubated for 2 hours at room temperature, in the dark, with an AlexaFluor 488-conjugated donkey anti-rabbit (1:200; A21206, ThermoFisher) and AlexaFluor 546-conjugated donkey anti-mouse (1:200; A10036, ThermoFisher) secondary antibodies. Finally, sections were washed twice in PB and incubated for 5 minutes with Hoechst stain (1:3000 dilution in PB; Thermo Fisher Scientific, Waltham, MA, USA) to visualize cell nuclei. Samples were then mounted on glass slides and coverslipped using ProLong\u0026trade; mounting medium (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy and image analysis\u003c/h2\u003e \u003cp\u003eAs previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, confocal microscopy 3D images of the Sp5C in both sides were obtained by means of a Leica Stellaris 8 Tau STED confocal microscope (Leica Mcrosystems AG; Wetzlar, Germany) using a 20X objective or 100X oil immersion objective to study the morphology and location of LC neurons or for densitometry analysis. Image stacks were acquired at 1024 x 1024 pixels using Leica software.\u003c/p\u003e \u003cp\u003eThe densitometric analysis was performed with a ImageJ image analysis software for Windows (Microsoft; Albuquerque, NM, USA). Images obtained by confocal microscopy were processed to create TIFF files with maximum intensity projections, using a consistent final tissue thickness of 10 \u0026micro;m for all series. Sections were processed together under identical conditions to ensure comparable immunostaining analysis. The region of interest (ROI) was outlined to include the LC. Optical density measurements were obtained by the ImageJ 'Set Measurement' routine. These values were measured to perform statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe mean firing rate was calculated every minute as the number of spikes/s in basal conditions and after formalin injection. Bursts were defined as two or three spikes firing at 10\u0026ndash;20 Hz \u003csup\u003e5, 6\u003c/sup\u003e. The instantaneous frequency was calculated as the time interval between two consecutive spikes. It was used to identify the burst firing pattern of LC neurons. The frequency of the bursts was calculated in periods of 5 minutes in basal conditions or 5 and 25 minutes after formalin injection. The spike response was calculated by means of the peristimulus time histograms (PSTHs; 1 ms bin-width). The spikes that occur in a 50 ms time window following the stimulus onset were counted. The vibrissal response during the baseline recording was considered 100%, and the effect of LC stimulation was calculated during the paired-pulse (LC-vibrissa) stimulation protocol. To measure the feedback inhibition, paired-pulse stimulation of the vibrissa was applied; the mean response to the first stimuli was considered 100% and the reduction of the second response was considered as a measure of the inhibition.\u003c/p\u003e \u003cp\u003eThe statistical analysis was conducted using GraphPad Prism version 10 (San Diego, CA, USA). Depending on the number of independent variables, the distribution of the data (assessed via the Kolmogorov\u0026ndash;Smirnov test), and the nature of the experimental comparisons, we used either Student\u0026rsquo;s t-test or a two-way ANOVA followed by Sidak\u0026rsquo;s post hoc test for multiple comparisons. When the data did not follow a normal distribution, comparisons between two groups were made using the Mann\u0026ndash;Whitney U test. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical significance is indicated as follows: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by Grants from Spanish Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades (PID2024-158246OB-I00) and Convocatoria de Proyectos de Investigaci\u0026oacute;n 2025 (Evaluaci\u0026oacute;n del control inhibitorio del locus coeruleus en la neuralgia del trig\u0026eacute;mino: identificaci\u0026oacute;n de nuevas v\u0026iacute;as terap\u0026eacute;uticas; UFV2025-07) from Universidad Francisco de Vitoria de Madrid.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Alberto Mesa-Lombardo, Nuria Garc\u0026iacute;a-Magro and Guillermo Cerrillo. The first draft of the manuscript was written by Alberto Mesa-Lombardo and Nuria Garc\u0026iacute;a-Magro and all authors commented on previous versions of the manuscript. Yasmina B. Martin and Angel Nu\u0026ntilde;ez have written and review the final version. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe like to thank R.M. S\u0026aacute;nchez Lozano for technical support. We want to thank Allinky BioPharma, manufacturer of AIK3a305, for providing us with the drug.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePertovaara, A. 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The formalin test: a quantitative study of the analgesic effects of morphine. meperidine, and brain stem stimulation in rats and cats. \u003cem\u003ePain\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 161\u0026ndash;174 (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta-Garcia, M. \u0026amp; Nu\u0026ntilde;ez, A. Nociceptive stimuli induce changes in somatosensory responses of rat dorsal column nuclei neurons. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cb\u003e1025\u003c/b\u003e, 169\u0026ndash;176 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorghi, V. et al. Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonists in mice. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cb\u003e956\u003c/b\u003e, 339\u0026ndash;348 (2002).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"noradrenergic transmission, formalin, insulin-like growth factor I, pain, diabetes","lastPublishedDoi":"10.21203/rs.3.rs-9072982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9072982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe locus coeruleus (LC) play an essential role in the regulation of nociceptive transmission by a widespread descending pathway to the spinal cord and the spinal trigeminal nucleus. We examined the effect of formalin injection in the vibrissal pad (nociceptive stimulus) on LC activity in isoflurane anesthetized control and in streptozotocin-induced diabetic (STZ-diabetic) mice which display neuropathic pain. Using unit recordings, we observed that formalin induced a sustained increase in the firing rate of LC neurons. In contrast, STZ-diabetic mice only showed an initial response, suggesting a reduced neuronal excitability. This finding was supported by a reduced c-Fos expression in comparison with control mice. We suggest that the reduction of LC excitability was due to a reduction of the insulin-like growth factor I (IGF-I) levels that occur in diabetes. In fact, immunohistochemistry studies showed that IGF-I receptors were diminished in STZ-diabetic mice. Treatment of STZ-diabetic mice with the IGF-I receptor sensitizer AIK3a305 restored LC activity. In conclusion, the lack of IGF-I in the brain of diabetic animals may be responsible for the reduced activity of LC neurons and, consequently, for the altered control of nociceptive transmission, facilitating chronic pain development in neuropathy diseases.\u003c/p\u003e","manuscriptTitle":"Reduced Noradrenergic Excitability in the Locus Coeruleus Compromises Nociceptive Inhibition in a Diabetic Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 11:03:40","doi":"10.21203/rs.3.rs-9072982/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"329091776478126230079253840641456477287","date":"2026-05-18T14:53:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T05:38:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178754458865429584852801526482864182969","date":"2026-05-02T03:59:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T15:05:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T15:01:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-07T12:43:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-22T18:13:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-22T09:18:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"feb90b69-b311-4d4f-b2cd-d7f8444dbd15","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"329091776478126230079253840641456477287","date":"2026-05-18T14:53:58+00:00","index":65,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T05:38:37+00:00","index":63,"fulltext":""},{"type":"reviewerAgreed","content":"178754458865429584852801526482864182969","date":"2026-05-02T03:59:56+00:00","index":54,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66163960,"name":"Health sciences/Diseases"},{"id":66163961,"name":"Health sciences/Endocrinology"},{"id":66163962,"name":"Biological sciences/Neuroscience"},{"id":66163963,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-14T11:03:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 11:03:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9072982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9072982","identity":"rs-9072982","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}