Hyperprolactinemia prevents short- and long-term memory deficits in ovariectomized rats and modifies the neuronal morphology of hippocampal CA1 neurons.

Hyperprolactinemia prevents short- and long-term memory deficits in ovariectomized rats and modifies the neuronal morphology of hippocampal CA1 neurons. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyperprolactinemia prevents short- and long-term memory deficits in ovariectomized rats and modifies the neuronal morphology of hippocampal CA1 neurons. Verónica Paniagua-Alegría, José Eduardo Suárez-Santiago, Marco Antonio Cerbón, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4774359/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The pituitary hormone prolactin (PRL) has been traditionally associated to reproductive functions, specifically to lactogenesis. Currently, this hormone has been linked to a great diversity of brain processes such as neurogenesis, neuroprotection, learning and memory formation. Regarding this later, literature is controversial since some studies indicate that PRL, like estrogens, improve some types of memory, while other report the opposite. Implanting pituitary homografts under the renal capsule induces a 10-fold increase of plasmatic PRL in male rats, but its effect on females is unknown. On the other hand, long-term (15 weeks) removal of ovaries produces a clear deficit in memory function by avoiding the action of estrogens and progesterone on this process. The present study was aimed at exploring the putative pro-cognitive actions of endogenous PRL and its role on the morphology of pyramidal neurons from hippocampus. The novel object recognition test, a hippocampal and cortical-dependent memory task was used for evaluating short- and long-term memory, while bromocriptine, a dopaminergic agonist, was assayed to block the actions of PRL on behavioral and morphological parameters. Main results indicate that PRL prevents the cognitive deficit observed in ovariectomized rats and increases the mushroom spines in CA1 pyramidal neurons; an effect that was partially impeded by bromocriptine. CA1 learning prolactin dendritic morphology ovariectomy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Prolactin (PRL) is a polypeptidic hormone commonly associated with lactogenesis and galactopoiesis and mainly synthesized by the anterior pituitary gland, whose synthesis and receptors location have also been reported in extra-pituitary tissue including various brain regions [ 1 , 2 ]. Nowadays, PRL is recognized as a pleiotropic hormone related to several brain processes including neurogenesis [ 3 ], neuroplasticity [ 4 ], neuroprotection[ 5 ], learning and memory, among others (see Cabrera-Reyes[ 6 ] for a review). Regarding this latter, studies analyzing the actions of PRL over cognition are scarce and controversial; in this line, one pioneer study reported that hyperprolactinemia (HPRL) in male rats, induced by pituitary homografts under the kidney capsule, facilitated learning acquisition of an active avoidance behavior [ 7 ]. More recently, it has been found that PRL injected in female mice improved the acquisition of new information during learning of a spatial memory task [ 8 ]. By contrast, Torner et al. [ 9 ], found that homografts-induced HPRL impaired performance of male rats in the novel object recognition test (NORT) without altering spatial learning evaluated in the Morris water maze. Interestingly, female patients suffering from prolactinomas, i.e., having high PRL levels, show poor performance in verbal memory and executive functions [ 10 ]. As mentioned above, one useful approach to investigate the role of PRL in the brain is to induce HPRL by implanting pituitary homografts beneath the renal capsule [ 11 ]; this procedure produces approximately a 10-fold increase of circulating PRL[ 9 ] and therefore it was used in the current study. Similar to PRL, the endogenous 17 beta-estradiol (E2) and some synthetic estrogens regulate multiple actions on neural tissues and in consequence have been identified as neuroprotective and pro-cognitive agents [ 12 , 13 ]. In this sense, estrogens improve several types of learning and memory including social learning, social discrimination, spatial and fear memory; all of them mediated by the amygdala, hippocampus, perirhinal cortex, prefrontal cortex, among other brain regions [ 14 ]. In line with this evidence, pregnancy, and lactation have been considered neuroprotective processes because, during these reproductive stages, the mother´s brain is exposed to several neurotrophic factors including progesterone, E2 and prolactin [ 15 ]. Indeed, it is well known that the high estrogenic levels found during proestrus and/or lower ones during the diestrous can modulate the dendritic arborization of some hippocampal subfields [ 16 , 17 ] The present study was aimed at elucidating the possible pro-cognitive action of PRL in female rats, by using the NORT. However, to avoid the influence of both progesterone and E2 on this process, it was necessary to remove the ovaries (OVX). This surgery unravels the effects of sex steroids on neural tissues and produces at long-term (15 weeks) a clear deficit in memory function, which is detectable in both the auto-shaping learning task and the NORT [ 18 – 20 ]. Thus, we explore if high levels of PRL induced by a double pituitary homograft, modify the cognitive performance of intact rats (in proestrus) and long-term OVX rats in the NORT. Bromocriptine (BRC) was assayed to block these actions. Moreover, these data were correlated to changes in dendritic branching pattern and number of dendritic spines of CA1 pyramidal neurons. Materials and methods Animals Eighty adult virgin Wistar female rats (180–200 g) were housed under standard laboratory conditions (12-h light/dark cycle, lights on at 7:00 a.m.) with water and food ad libitum . Donor female rats (forty) were previously used in other experiments and had already been scheduled to be sacrificed. The estrous cycle stage was determined by vaginal smears taken daily at 8 am for 10 days. As known, the proestrus phase is characterized by round and nucleated cells together with high estrogenic levels. All experimental animals were assigned to 4 groups: proestrus, OVX, OVX + HPRL, OVX + HPRL + BRC. All animals care procedures meet the official Mexican regulations for the use and care of laboratory animals (NOM-062-ZOO-1999). The current protocol was approved by the Animal Ethics Committee from the “Escuela Superior de Medicina, Instituto Politécnico Nacional” (México, protocol no. CICUAL-01/19-04-2019). Care was taken to minimize animal suffering and to reduce the number of animals used. Surgery Once the animals arrived to the laboratory, thirty of them were immediately OVX through a dorsal incision, under 2,2,2-tribromoethanol anesthesia (0.2 g/kg i.p.). The complete extraction of the ovaries was corroborated by visual inspection. Immediately after, each rat was placed on a warm platform until its complete post-surgery recovery and administered the non-steroidal anti-inflammatory drug meloxicam (2 mg/kg, s.c.) and topical anesthetic (Lidocaine / Epinephrine 2%, 0.3 mL, s.c.) to ease pain, and finally housed in a standard home cage for 2 weeks. Five weeks after this surgery, twenty of these animals received two adenopituitaries. For this surgery, animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p., Sigma-Aldrich, USA). After the kidney exposition through a small incision on the peritoneum, two adenopituitary glands from rat donors were placed under the kidney capsules of the host rats. Like to OVX surgery, rats were treated to easy pain and later housed in individual cages until they were 15 weeks post-OVX. Novel object recognition test The NORT, a hippocampal and cortical-dependent memory test, has been proposed as a useful tool for evaluating recognition memory [ 21 ]. This animal model considers the natural tendency of rodents to explore new objects instead of familiar ones. It consists of an open-field arena (61X42X20 cm) in which two identical objects (A + A ́) are located in opposite and symmetrical corners. During the first trial (acquisition phase) each rat was placed in the arena and allowed to freely explore for 5 min. Rats were tested again 1 h (short-term memory; STM) and 24 h (long-term memory; LTM) later, but in these two sessions, one of the familiar objects (A) was replaced by a new one (B for STM and C for LTM). One hour before the acquisition phase and the LTM session, one group of animals was injected with BRC (2.5 mg/kg; i.p). Both the route of administration and dose were selected taking into account previous studies [22–24]. All sessions were videotaped and then offline analyzed by a single observer, who was blind to the treatment conditions. Data are expressed as the discrimination index (DI), according to the equation DI = (B or C/A + B or C)*100 [ 25 ]. Here, a DI value above 50 indicates a novel object preference and is interpreted as normal cognitive performance. All experiments were run between 9–11 am. Open field test To discard the possibility that unspecific effects of PRL or BRC on motor skills could mask data from NORT, immediately after this evaluation all rats were submitted to an ambulatory activity test, which was performed in a black acrylic box (100X100X33 cm). During the test, each rodent was introduced into the box and its performance was automatically videotaped for 5 min employing a digital camera (Digital USB 2.0 CMOS camera, Stoelting Co., USA). In this model, the total distance traveled together with the time spent near to walls or at the center of the box was registered. The cage was always carefully cleaned with 70% ethanol and dried between each trial of the session. Enzyme-linked immunosorbent assay for PRL Seven to ten females from each experimental group were deeply anesthetized with an overdose of pentobarbital (200 mg/kg, i.p). Blood was collected from the right auricula before the beginning of the perfusion process. These samples were centrifuged at 1500 RPM for 10 min and serum was stored at − 80 o C. PRL concentrations were quantified by a prolactin Mouse/Rat ELISA Kit (Sigma-Aldrich SE120088), following the manufacturer’s instructions. Briefly; 1) 50 µL of each standard and sample was added into wells; 2) 100 µL of Biotinylated Antibody reagent was added to all wells and gently shaking the plate for 10 seconds to mix; 3). The plate was covered and incubated for 1 h at RT; 4) the solution was discarded and washed 3 times with 1x Wash Solution; 5) 100 µL of Streptavidin Enzyme Conjugate was added to all wells and incubated for 30 min at RT; 6). The solution was discarded and washed 3 times; 7) 100 µL of TMB Substrate solution was added, and the plate was covered and incubated for 15 min at RT; 8) Finally, Stop Solution was added into all wells and then absorbance values were read at 450 nm in a plate reader (Bio-Rad). Here it is important to clarify that data from pituitary-transplanted rats presenting PRL concentrations within the range of the proestrus group were discarded. Golgi method After the intracardial perfusion of five rats from each experimental group through the left cardiac ventricle with PBS followed by 4% paraformaldehyde, brains were removed and processed according to the FD Rapid GolgiStain™ Kit instructions (FD Neurotechnologies Inc, USA) that begin preparing the impregnation solution mixing equal volumes of solution A and B. This solution was changed after the first 24 hours of incubation. Later, the brains were transferred into solution C and incubated at 4°C for 1 week in the dark. Solution C was also changed after 24 h. At the end of the incubation in solution C, the brains were sliced (200 µm) by using a cryostat (Leica CM1520, Germany) at -22°C. The slices were collected into Superfrost Plus glass slides (Thermo Scientific, USA) with solution C and dried at RT. Further, the slides were rinsed with distilled water and then placed in a mixture of equal parts of solution D and solution E and 2 parts of water for 10 min. The slides were rinsed again with water and then dehydrated with ethanol (50%, 70%, 95%, and absolute) before clearing them with xylene. After coverslipped, slides were dried at RT until their analysis under a light microscope (Axiophot Zeiss, equipped with a video camera Olympus Q5). Neuronal morphology analysis In this study, 5 randomly selected rats per experimental group were used, and 30 neurons per group were chosen to determine both dendritic spine density, arborization, and spine morphology analysis. To determine these parameters, multiple images of pyramidal cells in the CA1 region of the dorsal hippocampus [Bregma − 2.80 to -3.80 mm; The Rat Brain in Stereotaxic Coordinates, 1982)] were obtained using a Nikon camera (200M optics) attached to a Nikon Eclipse E600 microscope. These neurons were identified by the triangular shape of their soma and the main apical dendrite oriented toward the pial surface. Only neurons that met the following criteria were analyzed: [ 1 ] specifically located within the area of interest, isolated from neighboring stained cells, [ 2 ] each cell needed to be completely stained and impregnated and with clearly visible spines (Fig. 3 ). The density of dendritic spines was determined by analyzing dendritic segments of 50 µm, while, for the study of the effects of hormonal treatments on the neuronal branching pattern (Sholl analysis), were taken into account two lengths from the soma, 50 and 100 µm. On the other hand, to determine the percentage of dendritic spines according to their morphology, 10 dendritic segments of 50 µm per animal were used (see Fig. 3 ). The dendritic spine density together with Sholl analysis and dendritic spine morphology were determined by the method described by Risher et al. [ 26 ], using ImageJ software [ 27 ]. The quantifications obtained using the Golgi staining method probably represent conservative data and not the actual values for all cells in the dorsal hippocampal CA1 region. However, histological procedures and measurements were performed in the same manner in all experimental groups. Statistics All data were analyzed using a one-way analysis of variance followed by Tukey's multiple comparisons. Results are expressed as mean \(\:\pm\:\:\) standard error. A p \(\:\le\:\) 0.05 was considered significant. Results Novel object recognition test Figure 1 shows the DI during the acquisition phase in the NORT of intact rats in the proestrus phase compared to OVX, OVX implanted with two pituitary homografts (OVX + HPRL) and OVX + HPRL treated with BRC. As expected, all animals spent approximately the same time, around 50%, exploring each of the two identical objects [F(3,32) = 1.196; p = 0.326)]. On the contrary, during the short- and long-term memory tests, the groups had dissimilar performance, with the OVX rats showing a low DI compared to the proestrus and the OVX + HPRL rats. The OVX + HPRL + BC group showed a DI just above the chance level. The corresponding ANOVA tests for the animals tested 1 and 24 hrs after the training phase in the NORT resulted as follows: for STM [F(3,32) = 8.140, p < 0.004], for LTM [F(3,32) = 8.140, p = 0.004]. Tukey’s post hoc test showed that at STM and LTM, the proestrus group (for STM, p = 0.012; for LTM, p = 0.006) and the OVX + HPRL group (for STM, p = 0.009; for LTM, p = 0.002) were statistically different with respect to OVX animals. Open field test In this test (Fig. 2 A) only the OVX and the OVX + HPRL groups displayed changes in comparison to all other groups. Thus, OVX rats explored the center of the arena for less time, staying more in the periphery. By contrast, HPRL rats explored the center longer time, remining less in the periphery and traveled more in comparison to OVX or OVX + HPRL treated with BRC rats. One-way ANOVA for the ambulatory activity was as follows: for time in the center of the arena [F(3,32) = 5.085, p = 0.005], for time in the periphery [F(3, 32) = 5.012, p = 0.005] and for total distance traveled [F(3,36) = 7.510, p = 0.005], while Tukey post hoc tests showed the difference of time spent in the center among OVX group vs. proestrus (p < 0.042) and OVX + HPRL groups (p < 0.004). Regarding distance traveled, differences were as follows: OVX vs . OVX + HPRL (p < 0.002) and vs . OVX + HPRL + BRC groups (p < 0.001). Panel B of this figure shows representative trajectories of the corresponding groups. Enzyme-linked immunosorbent assay (ELISA) for PRL Table I shows that the implantation of two adenopituitary glands in OVX animals produced a significant increase in plasmatic prolactin levels [F(3, 50) = 127.0; p < 0.001)]. This action was partially blocked by the BRC treatment since the values from this group did not reach those obtained in intact (during proestrus) or OVX animals. Post-hoc comparisons were as follows: proestrus or OVX vs . HPRL, (p < 0.001); HPRL vs . BRC (p < 0.001), and BRC vs . proestrus or OVX, (p < 0.001). Sholl analysis Figure 4 shows the dendritic arborization of pyramidal neurons in the CA1 region at 50 and 100 µm from the soma. The ANOVAs were as follows: for 50 µm [F(3,49) = 17.66; p < 0.001], for 100 µm [F(3,40) = 27.42; p < 0.001]. Tukey's post hoc test showed a dendritic arborization decrease in the OVX, OVX + HPRL and OVX + HPRL + BRC versus proestrus cells at both distances analyzed (50 µm = p < 0.001; 100 µm = p < 0.001). In the group receiving adenohypophysis implantation, an increase in neuronal arborization was observed at 50 µm compared to the OVX group (p < 0.002), however, this phenomenon did not occur at a distance of 100 µm from the neuronal soma. Regarding dendritic length, ANOVA analysis showed a significant difference between the groups [F(3,80) = 16.53; p < 0.001]. The post hoc test showed a decrease in dendritic length in all the groups vs proestrus group (p < 0.001). Dendritic spines density The dendritic spine density of pyramidal cells of the CA1 region together with representative images of dendrites from each group are depicted in Fig. 5 . The ANOVA tests showed a difference between the groups [F(3,80) = 186.1; p < 0.001]. Tukey's post hoc test showed that there was a significant decrease in the number of dendritic spines in all the groups vs proestrus (p < 0.001). However, this reduction of spine density was less pronounced in OVX + HPRL rats. Interestingly, this last effect on spiny was inhibited by BRC (p < 0.001). Dendritic spine morphology analysis The ANOVA analyses of the percentage of spines (Fig. 6 ) according to their morphology were as follows: for filipodia [F(3,45) = 4.021, p = 0.012], for thin [(F(3,42) = 16.51, p < 0.001], for long thin [F(3,44) = 5.230, p = 0.003], for stubby [F(3,44) = 1.363, p = 0.266] for mushroom [F(3,44) = 14.78, p < 0.001] and for branched [F(3,44) = 5.227, p = 0.003]. Post hoc analysis showed a decrease of "filipodia" in OVX + HPRL + BRC vs. OVX (p < 0.007). Also, an increase of "thin" type spines was observed in OVX and OVX + HPRL + BRC groups versus proestrus (p < 0.001) and OVX + HPRL groups respectively (p < 0.004). Figure 6 also shows that long thin type spines increased in OVX vs proestrus (p = 0.003); an action blocked by BRC treatment (p = 0.002). The HPRL condition increased the mushroom spines without reaching the proestrus values. this effect was prevented by the BRC administration, reducing the expression of this type of spine at similar values to OVX rats. Finally, a decrease in branched spines was observed in both, OVX and OVX + HPRL + BRC when compared to the proestrus group (p = 0.011). Discussion The current study shows that: a) elevated levels of PRL induced by 2 pituitary homografts prevent the cognitive deficit induced by long-term OVX; b) these behavioral actions were clearly correlated to an increase of the mushroom spines percentage in CA1 pyramidal neurons, an effect blocked by BRC and c), the removal of ovaries induces changes in both dendrite complexity and dendritic spine morphology of hippocampal neurons. The pituitary homografts surgery introduced by Adler et al. [ 11 ] generates approximately a tenfold increase in the circulating levels of PRL (an observation confirmed by the current study) and can prevent the cognition deficit observed at long-term in rats without ovaries [ 28 ]. This finding suggest that the removal of ovaries represents a useful approximation for the study of extra-ovarian hormones like PRL on cognition. Regarding this point, some authors have reported that high doses of PRL in female mice for three days improve the learning of a spatial memory task [ 8 ], while spatial and recognition memory impairment caused by 1,2-Diacetylbenzene or kainic acid, can be reversed by PRL treatment [ 29 , 30 ]. In line with this, the low levels of this hormone found in PRL-null mice have been associated with a spatial learning deficit; an effect reverted by the chronic intrahippocampal infusion of PRL [ 3 ]. On the contrary, some authors have reported negative actions of PRL on cognition. For instance, induction of HPRL by subcutaneous osmotic pumps can impair memory in tests such as the Barnes maze [ 31 ] and, as above mentioned, HPRL male rats show a poor performance in the NORT but not in the Morris water maze, i.e., elevated concentrations of PRL impair recognition but not spatial learning [ 9 ]. As evident, the role of PRL on cognition is still controversial, however, data are showing that lactation is directly associated with neuroprotection and cognitive improvement in rats, effects that even appear to be long-lasting [ 32 ]. In this sense, it has been reported that nulliparous females, tested in the radial-arm maze show a poor performance in comparison to age-matched multiparous rats [ 33 , 34 ]. Accordingly, our group reported that pregnancy improves the performance in the Morris water maze [ 35 ] and increases the number of dendritic spines in the CA1 hippocampal subfield [ 15 ]. A possible explanation regarding these discrepancies could be the different plasmatic concentrations of PRL present during memory testing. In support of this idea is the finding that patients with elevated PRL levels show verbal and working memory deficits, which can be alleviated by administering dopaminergic agonists [ 36 , 37 ]. Therefore, it would be important to know the PRL levels achieved during the behavioral tests to clarify the true role of PRL on cognition. Several mechanisms have been proposed to explain the PRL actions on learning. For instance, the addition of exogenous PRL to primary adult hippocampal cells produces an approximate 50% increase in the number of neurospheres [ 3 ], which is in line with the PRL-induced hippocampal neurogenesis previously reported [ 38 , 39 ] in adult rodents. Thus, the pro-cognitive actions of PRL reported here could be partially explained by its capacity to promote neurogenesis. On the other hand, it has been described that PRL decreases the expression of both the parvalbumin-positive cells and the β2/3 subunit of the GABAA receptor in the hippocampal CA1 region, which is in line with the hypothesis that the attenuation of GABAergic activity at this level could be responsible for the improvement of cognitive performance [ 31 ]. The neuroprotective effects of PRL have already been studied in silico and associated with an increased expression of various transcription factors and genes involved in learning and memory [ 29 , 40 ]. Besides this later finding, other studies trying to explain the role of PRL on cognition include the PRL-releasing peptide, which seems to reduce the expression of inflammatory markers in the microglia by decreasing the activity of the NADPH oxidase-regulated NLRP3 inflammasome in rats [ 41 ], the reduction of Ca2 + input under conditions of Glu-induced excitotoxicity, the overexpression of anti-apoptotic proteins, etc. [ 42 ]. Taken together, all this evidence suggests that PRL possesses beneficial effects on learning and cognition, but its underlaying mechanisms remain to be elucidated. The cellular mechanisms involved in the pro-cognitive effects of PRL and its association with changes in the structural plasticity of regions related to learning such as the prefrontal cortex and hippocampus are still poorly understood. Nevertheless, some evidence has emerged suggesting a close relationship between these variables. For instance, Leuner and Gould [ 43 ] reported in mother rats an increase of dendritic spines density in pyramidal cells of the medial prefrontal cortex and hippocampal neurons from CA1 and linked this finding to an improvement of cognitive flexibility. More recently, Wang et al. [ 44 ] studying voles during the parental care stage found similar morphological changes in the same brain regions, which was close related to the PRL levels reached during this parental care stage in these animals. Such an increase in dendritic spines had already been observed during the lactation period, in tuberoinfundibular dopaminergic cells; a phenomenon associated with an intensification in excitatory inputs into this neuronal group [ 45 ]. In the piriform cortex and posterolateral cortical amygdala, an increased density of mushroom and stubby dendritic spines has also been found during early and late gestation [ 46 ]. At the same stage, PRL has been reported to promote increased progenitor cell expression in the subventricular region in mice [ 47 ]. The findings of the present research and data reported in the literature suggest that PRL not only promotes the appearance of plastic changes in different neuronal groups but also facilitates the process of neurogenesis. It seems that the molecular events associated with changes in neuronal morphology after PRL exposure are related to its ability to induce, in addition to cell differentiation, neuronal and dendritic axonal growth through the expression of proteins associated with cytoskeleton modeling such as nestin and MAP2 at CA1 hippocampal level [ 48 , 49 ]. Thus, it is likely that the high arborization together with the high density of dendritic spines, especially of the mushroom type, found in the dorsal hippocampal CA1 of HPRL rats may be due to PRL’s ability to induce the expression of cytoskeletal proteins together with neurotrophic factors such as GDNF and BDNF, as demonstrated by Arnold et al. [ 50 ], who used the same method of the current study (implanting two pituitary homografts), to induce HPRL rodents. In general, these morphological changes were blocked by BRC, which implies the important role of PRL in promoting plastic changes at the hippocampus and their close relationship with the improvement of both STM and LTM. Our finding that the procognitive actions of PRL were not completely blocked by BRC may be due to the fact that the dose of BRC tested was not sufficient to fully impair the production of the hormone or only did it for a short period. Given that the dose used here had no impact on the rat´s motor skills (see Fig. 2 ), further experiments increasing the dose of BRC and blocking other neurotransmitter systems traditionally involved in the regulation of recognition memory are being carried out to clarify this point. Declarations The authors have no conflicts of interest to declare Acknowledgments This study was partially supported by COFAA and SIP-IPN (Mexico). We thank Histotechnologist Carmen Baltazar for her invaluable support with Golgi techniques. 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PLoS ONE 9. https://doi.org/10.1371/journal.pone.0107591 Rasband W (2015) ImageJ [Software]. U S National Institutes of Health, Bethesda, Maryland, USA Barreto-Cordero LM, Ríos-Carrillo J, Roldán-Roldán G et al (2020) Cyclic changes and actions of progesterone and allopregnanolone on cognition and hippocampal basal (stratum oriens) dendritic spines of female rats. Behav Brain Res. https://doi.org/10.1016/j.bbr.2019.112355 . 379: Nguyen NT, Le TL, Vo TKN et al (2023) Effect of a Polymorphism in Prolactin Gene on Some Reproductive Traits in TB Crossbred Ducks in Southern Vietnam. Adv Anim Vet Sci 11. https://doi.org/10.17582/journal.aavs/2023/11.6.886.892 Reyes-Mendoza J, Morales T (2016) Post-treatment with prolactin protects hippocampal CA1 neurons of the ovariectomized female rat against kainic acid-induced neurodegeneration. Neuroscience 328. https://doi.org/10.1016/j.neuroscience.2016.04.030 Mellado S, Moreno-Ruiz B, Expósito S et al (2022) Prolactin Reduces Hippocampal Parvalbumin and GABAAReceptor Expression in Female Mice. Neuroendocrinology 112. https://doi.org/10.1159/000520279 Burgos H, Hernández A, Constandil L et al (2019) Early postnatal environmental enrichment restores neurochemical and functional plasticities of the cerebral cortex and improves learning performance in hidden-prenatally-malnourished young-adult rats. Behav Brain Res. https://doi.org/10.1016/j.bbr.2019.02.001 . 363: Kinsley CH, Madonia L, Gifford GW et al (1999) Motherhood improves learning and memory. Nature 402. https://doi.org/10.1038/45957 Pawluski JL, Vanderbyl BL, Ragan K, Galea LAM (2006) First reproductive experience persistently affects spatial reference and working memory in the mother and these effects are not due to pregnancy or mothering alone. Behav Brain Res. https://doi.org/10.1016/j.bbr.2006.08.017 . 175: Cabrera-Pedraza VR, de Jesús Gómez-Villalobos M, de la Cruz F et al (2017) Pregnancy improves cognitive deficit and neuronal morphology atrophy in the prefrontal cortex and hippocampus of aging spontaneously hypertensive rats. Synapse 71. https://doi.org/10.1002/syn.21991 Castanho TC, Moreira PS, Portugal-Nunes C et al (2014) The role of sex and sex-related hormones in cognition, mood and well-being in older men and women. Biol Psychol 103. https://doi.org/10.1016/j.biopsycho.2014.08.015 Montalvo I, Llorens M, Caparrós L et al (2018) Improvement in cognitive abilities following cabergoline treatment in patients with a prolactin-secreting pituitary adenoma. Int Clin Psychopharmacol 33. https://doi.org/10.1097/YIC.0000000000000199 Larsen CM, Grattan DR (2012) Prolactin, neurogenesis, and maternal behaviors. Brain Behav Immun 26 Bridges RS, Grattan DR (2003) Prolactin-induced neurogenesis in the maternal brain. Trends Endocrinol Metabolism 14 Duc Nguyen H, Hee Jo W, Hong Minh Hoang N, Kim MS (2022) Anti-inflammatory effects of B vitamins protect against tau hyperphosphorylation and cognitive impairment induced by 1,2 diacetyl benzene: An in vitro and in silico study. Int Immunopharmacol 108. https://doi.org/10.1016/j.intimp.2022.108736 Hoang NMH, Jo W, Kim MS (2023) Protective effect of Prolactin releasing peptide against 1,2-diacetylbenzene -induced neuroinflammation. https://doi.org/10.1016/j.npep.2023.102349 . Neuropeptides 100: Rivero-Segura NA, Flores-Soto E, De La Cadena SG et al (2017) Prolactin-induced neuroprotection against glutamate excitotoxicity is mediated by the reduction of [Ca2+]i overload and NF-κB activation. PLoS ONE 12. https://doi.org/10.1371/journal.pone.0176910 Leuner B, Glasper ER, Gould E (2010) Sexual experience promotes adult neurogenesis in the hippocampus despite an initial elevation in stress hormones. PLoS ONE 5. https://doi.org/10.1371/journal.pone.0011597 Wang B, Laifu LI, Zhixiong HE et al (2018) Effects of reproductive experience on paternal behavior, levels of testosterone, prolactin in serum and dendritic spines in medial prefrontal cortex of mandarin voles. Integr Zool 13. https://doi.org/10.1111/1749-4877.12354 Yip SH, Araujo-Lopes R, Szawka RE et al (2020) Morphological plasticity of the tuberoinfundibular dopaminergic neurones in the rat during the oestrous cycle and lactation. J Neuroendocrinol 32. https://doi.org/10.1111/jne.12884 Matsuda KI, Takahashi T, Morishita S, Tanaka M (2024) Histological analysis of neuronal changes in the olfactory cortex during pregnancy. https://doi.org/10.1016/j.heliyon.2024.e26780 . Heliyon 10: Larsen CM, Grattan DR (2010) Prolactin-induced mitogenesis in the subventricular zone of the maternal brain during early pregnancy is essential for normal postpartum behavioral responses in the mother. Endocrinology 151. https://doi.org/10.1210/en.2009-1385 Havránek T, Bačová Z, Štrbák V et al (2014) Prolactin increases expression of cytoskeletal proteins in SK-N-SH cells. Folia Biologica (Czech Republic) 60 Leem YH, Park JS, Chang H et al (2019) Exercise Prevents Memory Consolidation Defects Via Enhancing Prolactin Responsiveness of CA1 Neurons in Mice Under Chronic Stress. Mol Neurobiol 56. https://doi.org/10.1007/s12035-019-1560-z Arnold E, Thebault S, Baeza-Cruz G et al (2014) The hormone prolactin is a novel, endogenous trophic factor able to regulate reactive glia and to limit retinal degeneration. J Neurosci 34. https://doi.org/10.1523/JNEUROSCI.2452-13.2014 Supplementary Files CompliancewithEthicalStandard.docx Cite Share Download PDF Status: Posted Version 1 posted 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-4774359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336630089,"identity":"88db80e9-7ab4-408f-81f2-96f77190623f","order_by":0,"name":"Verónica Paniagua-Alegría","email":"","orcid":"","institution":"Instituto Politécnico Nacional: Instituto Politecnico Nacional","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Verónica","middleName":"","lastName":"Paniagua-Alegría","suffix":""},{"id":336630090,"identity":"58a042b1-dac1-4bb0-8222-d97c8f51dfbd","order_by":1,"name":"José Eduardo Suárez-Santiago","email":"","orcid":"","institution":"Universidad Autónoma de Chiapas: Universidad Autonoma de Chiapas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"Eduardo","lastName":"Suárez-Santiago","suffix":""},{"id":336630091,"identity":"1083e373-fc21-4ae5-8fc9-817b6cc4f2d8","order_by":2,"name":"Marco Antonio Cerbón","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"Antonio","lastName":"Cerbón","suffix":""},{"id":336630092,"identity":"993bceb4-003e-40a5-b986-3aec389bcf8c","order_by":3,"name":"Carlos Vera-Arzave","email":"","orcid":"","institution":"Instituto Politécnico Nacional: Instituto Politecnico Nacional","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Vera-Arzave","suffix":""},{"id":336630093,"identity":"09959d4d-d747-47b7-8d31-2ea250949244","order_by":4,"name":"Gabriel Roldán-Roldán","email":"","orcid":"","institution":"Universidad Nacional Autonoma de Mexico Biblioteca Conjunta de Ciencias de la Tierra: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Roldán-Roldán","suffix":""},{"id":336630094,"identity":"20b60f2f-b72c-495f-82ec-e8db423596e8","order_by":5,"name":"Sandra Orozco-Suárez","email":"","orcid":"","institution":"Instituto Mexicano del Seguro Social","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Orozco-Suárez","suffix":""},{"id":336630095,"identity":"f8fdf6bb-6762-48e2-a313-88cd9e679a04","order_by":6,"name":"Ofir Picazo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIie3PsQrCMBCA4QsHnVqySUTRV0gpqIMPk76Bk3R0qpN0regbuAiBzgXByepayVJwVejoqHFysnETzL8d3MclADbbL4akgjoCoHoQEyOCnKQHgPZME252hqMXA/BcDyZkOEdy8eIslKdNWFcc+rSVfybdHaK/PqpgUN4kez7MX67EZ8KQ7tl1qnqDsthqIrhqJOgw11FukBbybkzabqx6nC4y4yvopwcVsNLLRoIzg7/QHanqSIVJUsjzPRr3aaeBvOHXJjNd19H8m22bzWb7px7G9UEKX/Lw0gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4320-4997","institution":"Instituto Politecnico Nacional","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ofir","middleName":"","lastName":"Picazo","suffix":""}],"badges":[],"createdAt":"2024-07-20 18:56:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4774359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4774359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63814387,"identity":"55a7fcda-c856-4dbe-9247-45324d310e02","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83045,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ovariectomy (OVX), pituitary implant (OVX+HPRL) and bromocriptine administration (OVX+HPRL+BRC) on rats tested in the NOR during the acquisition (sample), 1 h (STM) and 24 h (LTM) after. Each column represents the mean ± SE (n=10) of the discrimination index percentage. The asterisk indicates a p\u0026lt;0.05 in comparison to proestrus, OVX and HPRL groups. The symbol # denotes a p\u0026lt;0.05 between\u003cem\u003e \u003c/em\u003ethe groups indicated by the bracket.\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/3034b79161dc994eb4ec20c0.png"},{"id":63814392,"identity":"4491ae48-5793-4de8-9a1e-307dd2fc3cd2","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":824933,"visible":true,"origin":"","legend":"\u003cp\u003eAmbulatory activity of rats in proestrus, without ovaries (OVX), OVX with pituitary homografts (OVX+HPRL), and after bromocriptine administration (OVX+HPRL+BRC). Panel A shows the time spent in the center or periphery of the arena, besides the distance traveled for each rat for 5 min. Columns represent the mean ± SE (n=10). The asterisk indicates a p\u0026lt;0.001 when compared to proestrus or OXV+HPRL; other comparisons are indicated by brackets where the symbol # denotes a p\u0026lt;0.05. Panel B shows representative images of ambulation traces under different experimental conditions.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/018f39bb34278dc46854491d.png"},{"id":63814391,"identity":"d77199f6-1c21-4930-9799-bcd6f34e79f3","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2521382,"visible":true,"origin":"","legend":"\u003cp\u003eImages of both the Sholl and the Golgi analysis (at right side) of a representative pyramidal neuron from the CA1 region of the hippocampus.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/82f2bb3f78943cde08cf20a0.png"},{"id":63814390,"identity":"0c1d2fb4-dbcf-4725-8289-8078f45b8161","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":235413,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the arborization at two distances (50 and 100 mm from the neuronal head) and the total dendritic length of pyramidal CA1 neurons from intact (proestrus), ovariectomized (OVX), hyperprolactinemic (HPRL) and bromocriptine (BRC) treated rats. Each asterisk indicates at least a p\u0026lt;0.05 in comparison to proestrus group, while the pound symbol implies a p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/95c20ec46887d5f0387b581e.png"},{"id":63814389,"identity":"c62a8ef8-8a45-45e9-a6b9-9ade69e0dbac","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1377448,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of apical dendritic spines in neurons from CA1 hippocampus of rats in proestrus, rats without ovaries (OVX), hyperprolactinemic rats without (OVX+HPRL) or with bromocriptine treatment (OVX+HPRL+BRC). The asterisk indicates a statistical difference vs. the proestrus group (p\u0026lt;0.001). The symbol # denotes a difference vs. OVX and OVX+HPRL+BRC groups (p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/90ede22683ba0be288703f70.png"},{"id":63814388,"identity":"8b96c766-2b75-4e82-8f05-a13200e5c915","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":281405,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent types of spines were observed in CA1 pyramidal neurons from rats in proestrus, rats without ovaries (OVX) and hyperprolactinemic rats without (OVX+HPRL) or with bromocriptine treatment (OVX+HPRL+BRC). The asterisks denote a statistical difference vs. the proestrus group (p\u0026lt;0.001). Other differences are indicated by brackets where the symbol # = p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"FIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/9577b91ae4d2c77febb8fd97.png"},{"id":66409100,"identity":"f1e9f1f2-71f1-4ecb-8729-42d692c86f87","added_by":"auto","created_at":"2024-10-11 13:23:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7100806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/f01d035c-554a-4f83-b613-9b8bf20c12be.pdf"},{"id":63814393,"identity":"b69a62dc-8b2b-46f3-bc37-30a1deb6c2f1","added_by":"auto","created_at":"2024-09-02 14:33:28","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":14680,"visible":true,"origin":"","legend":"","description":"","filename":"CompliancewithEthicalStandard.docx","url":"https://assets-eu.researchsquare.com/files/rs-4774359/v1/a15518681e7ec2b167c092f0.docx"}],"financialInterests":"","formattedTitle":"Hyperprolactinemia prevents short- and long-term memory deficits in ovariectomized rats and modifies the neuronal morphology of hippocampal CA1 neurons.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProlactin (PRL) is a polypeptidic hormone commonly associated with lactogenesis and galactopoiesis and mainly synthesized by the anterior pituitary gland, whose synthesis and receptors location have also been reported in extra-pituitary tissue including various brain regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Nowadays, PRL is recognized as a pleiotropic hormone related to several brain processes including neurogenesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], neuroplasticity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], neuroprotection[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], learning and memory, among others (see Cabrera-Reyes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] for a review). Regarding this latter, studies analyzing the actions of PRL over cognition are scarce and controversial; in this line, one pioneer study reported that hyperprolactinemia (HPRL) in male rats, induced by pituitary homografts under the kidney capsule, facilitated learning acquisition of an active avoidance behavior [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. More recently, it has been found that PRL injected in female mice improved the acquisition of new information during learning of a spatial memory task [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. By contrast, Torner et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], found that homografts-induced HPRL impaired performance of male rats in the novel object recognition test (NORT) without altering spatial learning evaluated in the Morris water maze. Interestingly, female patients suffering from prolactinomas, i.e., having high PRL levels, show poor performance in verbal memory and executive functions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As mentioned above, one useful approach to investigate the role of PRL in the brain is to induce HPRL by implanting pituitary homografts beneath the renal capsule [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]; this procedure produces approximately a 10-fold increase of circulating PRL[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and therefore it was used in the current study.\u003c/p\u003e \u003cp\u003eSimilar to PRL, the endogenous 17 beta-estradiol (E2) and some synthetic estrogens regulate multiple actions on neural tissues and in consequence have been identified as neuroprotective and pro-cognitive agents [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this sense, estrogens improve several types of learning and memory including social learning, social discrimination, spatial and fear memory; all of them mediated by the amygdala, hippocampus, perirhinal cortex, prefrontal cortex, among other brain regions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In line with this evidence, pregnancy, and lactation have been considered neuroprotective processes because, during these reproductive stages, the mother\u0026acute;s brain is exposed to several neurotrophic factors including progesterone, E2 and prolactin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Indeed, it is well known that the high estrogenic levels found during proestrus and/or lower ones during the diestrous can modulate the dendritic arborization of some hippocampal subfields [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe present study was aimed at elucidating the possible pro-cognitive action of PRL in female rats, by using the NORT. However, to avoid the influence of both progesterone and E2 on this process, it was necessary to remove the ovaries (OVX). This surgery unravels the effects of sex steroids on neural tissues and produces at long-term (15 weeks) a clear deficit in memory function, which is detectable in both the auto-shaping learning task and the NORT [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, we explore if high levels of PRL induced by a double pituitary homograft, modify the cognitive performance of intact rats (in proestrus) and long-term OVX rats in the NORT. Bromocriptine (BRC) was assayed to block these actions. Moreover, these data were correlated to changes in dendritic branching pattern and number of dendritic spines of CA1 pyramidal neurons.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eEighty adult virgin Wistar female rats (180\u0026ndash;200 g) were housed under standard laboratory conditions (12-h light/dark cycle, lights on at 7:00 a.m.) with water and food \u003cem\u003ead libitum\u003c/em\u003e. Donor female rats (forty) were previously used in other experiments and had already been scheduled to be sacrificed. The estrous cycle stage was determined by vaginal smears taken daily at 8 am for 10 days. As known, the proestrus phase is characterized by round and nucleated cells together with high estrogenic levels. All experimental animals were assigned to 4 groups: proestrus, OVX, OVX\u0026thinsp;+\u0026thinsp;HPRL, OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC. All animals care procedures meet the official Mexican regulations for the use and care of laboratory animals (NOM-062-ZOO-1999). The current protocol was approved by the Animal Ethics Committee from the \u0026ldquo;Escuela Superior de Medicina, Instituto Polit\u0026eacute;cnico Nacional\u0026rdquo; (M\u0026eacute;xico, protocol no. CICUAL-01/19-04-2019). Care was taken to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSurgery\u003c/h2\u003e \u003cp\u003eOnce the animals arrived to the laboratory, thirty of them were immediately OVX through a dorsal incision, under 2,2,2-tribromoethanol anesthesia (0.2 g/kg i.p.). The complete extraction of the ovaries was corroborated by visual inspection. Immediately after, each rat was placed on a warm platform until its complete post-surgery recovery and administered the non-steroidal anti-inflammatory drug meloxicam (2 mg/kg, s.c.) and topical\u003c/p\u003e \u003cp\u003eanesthetic (Lidocaine / Epinephrine 2%, 0.3 mL, s.c.) to ease pain, and finally housed in a standard home cage for 2 weeks.\u003c/p\u003e \u003cp\u003eFive weeks after this surgery, twenty of these animals received two adenopituitaries. For this surgery, animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p., Sigma-Aldrich, USA). After the kidney exposition through a small incision on the peritoneum, two adenopituitary glands from rat donors were placed under the kidney capsules of the host rats. Like to OVX surgery, rats were treated to easy pain and later housed in individual cages until they were 15 weeks post-OVX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNovel object recognition test\u003c/h2\u003e \u003cp\u003eThe NORT, a hippocampal and cortical-dependent memory test, has been proposed as a useful tool for evaluating recognition memory [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This animal model considers the natural tendency of rodents to explore new objects instead of familiar ones. It consists of an open-field arena (61X42X20 cm) in which two identical objects (A\u0026thinsp;+\u0026thinsp;A ́) are located in opposite and symmetrical corners. During the first trial (acquisition phase) each rat was placed in the arena and allowed to freely explore for 5 min. Rats were tested again 1 h (short-term memory; STM) and 24 h (long-term memory; LTM) later, but in these two sessions, one of the familiar objects (A) was replaced by a new one (B for STM and C for LTM). One hour before the acquisition phase and the LTM session, one group of animals was injected with BRC (2.5 mg/kg; i.p). Both the route of administration and dose were selected taking into account previous studies [22\u0026ndash;24].\u003c/p\u003e \u003cp\u003eAll sessions were videotaped and then offline analyzed by a single observer, who was blind to the treatment conditions. Data are expressed as the discrimination index (DI), according to the equation DI = (B or C/A\u0026thinsp;+\u0026thinsp;B or C)*100 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Here, a DI value above 50 indicates a novel object preference and is interpreted as normal cognitive performance. All experiments were run between 9\u0026ndash;11 am.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eTo discard the possibility that unspecific effects of PRL or BRC on motor skills could mask data from NORT, immediately after this evaluation all rats were submitted to an ambulatory activity test, which was performed in a black acrylic box (100X100X33 cm). During the test, each rodent was introduced into the box and its performance was automatically videotaped for 5 min employing a digital camera (Digital USB 2.0 CMOS camera, Stoelting Co., USA). In this model, the total distance traveled together with the time spent near to walls or at the center of the box was registered. The cage was always carefully cleaned with 70% ethanol and dried between each trial of the session.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay for PRL\u003c/h2\u003e \u003cp\u003eSeven to ten females from each experimental group were deeply anesthetized with an overdose of pentobarbital (200 mg/kg, i.p). Blood was collected from the right auricula before the beginning of the perfusion process. These samples were centrifuged at 1500 RPM for 10 min and serum was stored at \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003e C. PRL concentrations were quantified by a prolactin Mouse/Rat ELISA Kit (Sigma-Aldrich SE120088), following the manufacturer\u0026rsquo;s instructions. Briefly; 1) 50 \u0026micro;L of each standard and sample was added into wells; 2) 100 \u0026micro;L of Biotinylated Antibody reagent was added to all wells and gently shaking the plate for 10 seconds to mix; 3).\u003c/p\u003e \u003cp\u003eThe plate was covered and incubated for 1 h at RT; 4) the solution was discarded and washed 3 times with 1x Wash Solution; 5) 100 \u0026micro;L of Streptavidin Enzyme Conjugate was added to all wells and incubated for 30 min at RT; 6). The solution was discarded and washed 3 times; 7) 100 \u0026micro;L of TMB Substrate solution was added, and the plate was covered and incubated for 15 min at RT; 8) Finally, Stop Solution was added into all wells and then absorbance values were read at 450 nm in a plate reader (Bio-Rad). Here it is important to clarify that data from pituitary-transplanted rats presenting PRL concentrations within the range of the proestrus group were discarded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eGolgi method\u003c/h2\u003e \u003cp\u003eAfter the intracardial perfusion of five rats from each experimental group through the left cardiac ventricle with PBS followed by 4% paraformaldehyde, brains were removed and processed according to the FD Rapid GolgiStain\u0026trade; Kit instructions (FD Neurotechnologies Inc, USA) that begin preparing the impregnation solution mixing equal volumes of solution A and B. This solution was changed after the first 24 hours of incubation. Later, the brains were transferred into solution C and incubated at 4\u0026deg;C for 1 week in the dark. Solution C was also changed after 24 h. At the end of the incubation in solution C, the brains were sliced (200 \u0026micro;m) by using a cryostat (Leica CM1520, Germany) at -22\u0026deg;C. The slices were collected into Superfrost Plus glass slides (Thermo Scientific, USA) with solution C and dried at RT.\u003c/p\u003e \u003cp\u003eFurther, the slides were rinsed with distilled water and then placed in a mixture of equal parts of solution D and solution E and 2 parts of water for 10 min. The slides were rinsed again with water and then dehydrated with ethanol (50%, 70%, 95%, and absolute) before clearing them with xylene. After coverslipped, slides were dried at RT until their analysis under a light microscope (Axiophot Zeiss, equipped with a video camera Olympus Q5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eNeuronal morphology analysis\u003c/h2\u003e \u003cp\u003eIn this study, 5 randomly selected rats per experimental group were used, and 30 neurons per group were chosen to determine both dendritic spine density, arborization, and spine morphology analysis. To determine these parameters, multiple images of pyramidal cells in the CA1 region of the dorsal hippocampus [Bregma \u0026minus;\u0026thinsp;2.80 to -3.80 mm; The Rat Brain in Stereotaxic Coordinates, 1982)] were obtained using a Nikon camera (200M optics) attached to a Nikon Eclipse E600 microscope. These neurons were identified by the triangular shape of their soma and the main apical dendrite oriented toward the pial surface. Only neurons that met the following criteria were analyzed: [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] specifically located within the area of interest, isolated from neighboring stained cells, [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] each cell needed to be completely stained and impregnated and with clearly visible spines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe density of dendritic spines was determined by analyzing dendritic segments of 50 \u0026micro;m, while, for the study of the effects of hormonal treatments on the neuronal branching pattern (Sholl analysis), were taken into account two lengths from the soma, 50 and 100 \u0026micro;m. On the other hand, to determine the percentage of dendritic spines according to their morphology, 10 dendritic segments of 50 \u0026micro;m per animal were used (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The dendritic spine density together with Sholl analysis and dendritic spine morphology were determined by the method described by Risher et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], using ImageJ software [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe quantifications obtained using the Golgi staining method probably represent conservative data and not the actual values for all cells in the dorsal hippocampal CA1 region. However, histological procedures and measurements were performed in the same manner in all experimental groups.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll data were analyzed using a one-way analysis of variance followed by Tukey's multiple comparisons. Results are expressed as mean \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003estandard error. A p\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNovel object recognition test\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the DI during the acquisition phase in the NORT of intact rats in the proestrus phase compared to OVX, OVX implanted with two pituitary homografts (OVX\u0026thinsp;+\u0026thinsp;HPRL) and OVX\u0026thinsp;+\u0026thinsp;HPRL treated with BRC. As expected, all animals spent approximately the same time, around 50%, exploring each of the two identical objects [F(3,32)\u0026thinsp;=\u0026thinsp;1.196; p\u0026thinsp;=\u0026thinsp;0.326)]. On the contrary, during the short- and long-term memory tests, the groups had dissimilar performance, with the OVX rats showing a low DI compared to the proestrus and the OVX\u0026thinsp;+\u0026thinsp;HPRL rats. The OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BC group showed a DI just above the chance level. The corresponding ANOVA tests for the animals tested 1 and 24 hrs after the training phase in the NORT resulted as follows: for STM [F(3,32)\u0026thinsp;=\u0026thinsp;8.140, p\u0026thinsp;\u0026lt;\u0026thinsp;0.004], for LTM [F(3,32)\u0026thinsp;=\u0026thinsp;8.140, p\u0026thinsp;=\u0026thinsp;0.004]. Tukey\u0026rsquo;s post hoc test showed that at STM and LTM, the proestrus group (for STM, p\u0026thinsp;=\u0026thinsp;0.012; for LTM, p\u0026thinsp;=\u0026thinsp;0.006) and the OVX\u0026thinsp;+\u0026thinsp;HPRL group (for STM, p\u0026thinsp;=\u0026thinsp;0.009; for LTM, p\u0026thinsp;=\u0026thinsp;0.002) were statistically different with respect to OVX animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eIn this test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) only the OVX and the OVX\u0026thinsp;+\u0026thinsp;HPRL groups displayed changes in comparison to all other groups. Thus, OVX rats explored the center of the arena for less time, staying more in the periphery. By contrast, HPRL rats explored the center longer time, remining less in the periphery and traveled more in comparison to OVX or OVX\u0026thinsp;+\u0026thinsp;HPRL treated with BRC rats. One-way ANOVA for the ambulatory activity was as follows: for time in the center of the arena [F(3,32)\u0026thinsp;=\u0026thinsp;5.085, p\u0026thinsp;=\u0026thinsp;0.005], for time in the periphery [F(3, 32)\u0026thinsp;=\u0026thinsp;5.012, p\u0026thinsp;=\u0026thinsp;0.005] and for total distance traveled [F(3,36)\u0026thinsp;=\u0026thinsp;7.510, p\u0026thinsp;=\u0026thinsp;0.005], while Tukey \u003cem\u003epost hoc\u003c/em\u003e tests showed the difference of time spent in the center among OVX group \u003cem\u003evs.\u003c/em\u003e proestrus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.042) and OVX\u0026thinsp;+\u0026thinsp;HPRL groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.004). Regarding distance traveled, differences were as follows: OVX \u003cem\u003evs\u003c/em\u003e. OVX\u0026thinsp;+\u0026thinsp;HPRL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002) and \u003cem\u003evs\u003c/em\u003e. OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Panel B of this figure shows representative trajectories of the corresponding groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA) for PRL\u003c/h2\u003e \u003cp\u003eTable I shows that the implantation of two adenopituitary glands in OVX animals produced a significant increase in plasmatic prolactin levels [F(3, 50)\u0026thinsp;=\u0026thinsp;127.0; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)]. This action was partially blocked by the BRC treatment since the values from this group did not reach those obtained in intact (during proestrus) or OVX animals. Post-hoc comparisons were as follows: proestrus or OVX \u003cem\u003evs\u003c/em\u003e. HPRL, (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001); HPRL \u003cem\u003evs\u003c/em\u003e. BRC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and BRC \u003cem\u003evs\u003c/em\u003e. proestrus or OVX, (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSholl analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the dendritic arborization of pyramidal neurons in the CA1 region at 50 and 100 \u0026micro;m from the soma. The ANOVAs were as follows: for 50 \u0026micro;m [F(3,49)\u0026thinsp;=\u0026thinsp;17.66; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001], for 100 \u0026micro;m [F(3,40)\u0026thinsp;=\u0026thinsp;27.42; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. Tukey's \u003cem\u003epost hoc\u003c/em\u003e test showed a dendritic arborization decrease in the OVX, OVX\u0026thinsp;+\u0026thinsp;HPRL and OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC \u003cem\u003eversus\u003c/em\u003e proestrus cells at both distances analyzed (50 \u0026micro;m\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 100 \u0026micro;m\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the group receiving adenohypophysis implantation, an increase in neuronal arborization was observed at 50 \u0026micro;m compared to the OVX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002), however, this phenomenon did not occur at a distance of 100 \u0026micro;m from the neuronal soma. Regarding dendritic length, ANOVA analysis showed a significant difference between the groups [F(3,80)\u0026thinsp;=\u0026thinsp;16.53; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. The \u003cem\u003epost hoc\u003c/em\u003e test showed a decrease in dendritic length in all the groups vs proestrus group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDendritic spines density\u003c/h2\u003e \u003cp\u003eThe dendritic spine density of pyramidal cells of the CA1 region together with representative images of dendrites from each group are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The ANOVA tests showed a difference between the groups [F(3,80)\u0026thinsp;=\u0026thinsp;186.1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. Tukey's post hoc test showed that there was a significant decrease in the number of dendritic spines in all the groups vs proestrus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, this reduction of spine density was less pronounced in OVX\u0026thinsp;+\u0026thinsp;HPRL rats. Interestingly, this last effect on spiny was inhibited by BRC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDendritic spine morphology analysis\u003c/h2\u003e \u003cp\u003eThe ANOVA analyses of the percentage of spines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) according to their morphology were as follows: for filipodia [F(3,45)\u0026thinsp;=\u0026thinsp;4.021, p\u0026thinsp;=\u0026thinsp;0.012], for thin [(F(3,42)\u0026thinsp;=\u0026thinsp;16.51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001], for long thin [F(3,44)\u0026thinsp;=\u0026thinsp;5.230, p\u0026thinsp;=\u0026thinsp;0.003], for stubby [F(3,44)\u0026thinsp;=\u0026thinsp;1.363, p\u0026thinsp;=\u0026thinsp;0.266] for mushroom [F(3,44)\u0026thinsp;=\u0026thinsp;14.78, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001] and for branched [F(3,44)\u0026thinsp;=\u0026thinsp;5.227, p\u0026thinsp;=\u0026thinsp;0.003]. \u003cem\u003ePost hoc\u003c/em\u003e analysis showed a decrease of \"filipodia\" in OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC vs. OVX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.007). Also, an increase of \"thin\" type spines was observed in OVX and OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC groups versus proestrus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and OVX\u0026thinsp;+\u0026thinsp;HPRL groups respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.004).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e also shows that long thin type spines increased in OVX vs proestrus (p\u0026thinsp;=\u0026thinsp;0.003); an action blocked by BRC treatment (p\u0026thinsp;=\u0026thinsp;0.002). The HPRL condition increased the mushroom spines without reaching the proestrus values. this effect was prevented by the BRC administration, reducing the expression of this type of spine at similar values to OVX rats. Finally, a decrease in branched spines was observed in both, OVX and OVX\u0026thinsp;+\u0026thinsp;HPRL\u0026thinsp;+\u0026thinsp;BRC when compared to the proestrus group (p\u0026thinsp;=\u0026thinsp;0.011).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study shows that: a) elevated levels of PRL induced by 2 pituitary homografts prevent the cognitive deficit induced by long-term OVX; b) these behavioral actions were clearly correlated to an increase of the mushroom spines percentage in CA1 pyramidal neurons, an effect blocked by BRC and c), the removal of ovaries induces changes in both dendrite complexity and dendritic spine morphology of hippocampal neurons.\u003c/p\u003e \u003cp\u003eThe pituitary homografts surgery introduced by Adler et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] generates approximately a tenfold increase in the circulating levels of PRL (an observation confirmed by the current study) and can prevent the cognition deficit observed at long-term in rats without ovaries [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This finding suggest that the removal of ovaries represents a useful approximation for the study of extra-ovarian hormones like PRL on cognition. Regarding this point, some authors have reported that high doses of PRL in female mice for three days improve the learning of a spatial memory task [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while spatial and recognition memory impairment caused by 1,2-Diacetylbenzene or kainic acid, can be reversed by PRL treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In line with this, the low levels of this hormone found in PRL-null mice have been associated with a spatial learning deficit; an effect reverted by the chronic intrahippocampal infusion of PRL [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. On the contrary, some authors have reported negative actions of PRL on cognition. For instance, induction of HPRL by subcutaneous osmotic pumps can impair memory in tests such as the Barnes maze [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and, as above mentioned, HPRL male rats show a poor performance in the NORT but not in the Morris water maze, i.e., elevated concentrations of PRL impair recognition but not spatial learning [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs evident, the role of PRL on cognition is still controversial, however, data are showing that lactation is directly associated with neuroprotection and cognitive improvement in rats, effects that even appear to be long-lasting [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this sense, it has been reported that nulliparous females, tested in the radial-arm maze show a poor performance in comparison to age-matched multiparous rats [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Accordingly, our group reported that pregnancy improves the performance in the Morris water maze [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and increases the number of dendritic spines in the CA1 hippocampal subfield [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A possible explanation regarding these discrepancies could be the different plasmatic concentrations of PRL present during memory testing. In support of this idea is the finding that patients with elevated PRL levels show verbal and working memory deficits, which can be alleviated by administering dopaminergic agonists [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, it would be important to know the PRL levels achieved during the behavioral tests to clarify the true role of PRL on cognition.\u003c/p\u003e \u003cp\u003eSeveral mechanisms have been proposed to explain the PRL actions on learning. For instance, the addition of exogenous PRL to primary adult hippocampal cells produces an approximate 50% increase in the number of neurospheres [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is in line with the PRL-induced hippocampal neurogenesis previously reported [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] in adult rodents. Thus, the pro-cognitive actions of PRL reported here could be partially explained by its capacity to promote neurogenesis.\u003c/p\u003e \u003cp\u003eOn the other hand, it has been described that PRL decreases the expression of both the parvalbumin-positive cells and the β2/3 subunit of the GABAA receptor in the hippocampal CA1 region, which is in line with the hypothesis that the attenuation of GABAergic activity at this level could be responsible for the improvement of cognitive performance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe neuroprotective effects of PRL have already been studied \u003cem\u003ein silico\u003c/em\u003e and associated with an increased expression of various transcription factors and genes involved in learning and memory [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Besides this later finding, other studies trying to explain the role of PRL on cognition include the PRL-releasing peptide, which seems to reduce the expression of inflammatory markers in the microglia by decreasing the activity of the NADPH oxidase-regulated NLRP3 inflammasome in rats [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], the reduction of Ca2\u0026thinsp;+\u0026thinsp;input under conditions of Glu-induced excitotoxicity, the overexpression of anti-apoptotic proteins, etc. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, all this evidence suggests that PRL possesses beneficial effects on learning and cognition, but its underlaying mechanisms remain to be elucidated. The cellular mechanisms involved in the pro-cognitive effects of PRL and its association with changes in the structural plasticity of regions related to learning such as the prefrontal cortex and hippocampus are still poorly understood. Nevertheless, some evidence has emerged suggesting a close relationship between these variables. For instance, Leuner and Gould [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] reported in mother rats an increase of dendritic spines density in pyramidal cells of the medial prefrontal cortex and hippocampal neurons from CA1 and linked this finding to an improvement of cognitive flexibility. More recently, Wang et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] studying voles during the parental care stage found similar morphological changes in the same brain regions, which was close related to the PRL levels reached during this parental care stage in these animals. Such an increase in dendritic spines had already been observed during the lactation period, in tuberoinfundibular dopaminergic cells; a phenomenon associated with an intensification in excitatory inputs into this neuronal group [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the piriform cortex and posterolateral cortical amygdala, an increased density of mushroom and stubby dendritic spines has also been found during early and late gestation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. At the same stage, PRL has been reported to promote increased progenitor cell expression in the subventricular region in mice [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The findings of the present research and data reported in the literature suggest that PRL not only promotes the appearance of plastic changes in different neuronal groups but also facilitates the process of neurogenesis.\u003c/p\u003e \u003cp\u003eIt seems that the molecular events associated with changes in neuronal morphology after PRL exposure are related to its ability to induce, in addition to cell differentiation, neuronal and dendritic axonal growth through the expression of proteins associated with cytoskeleton modeling such as nestin and MAP2 at CA1 hippocampal level [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thus, it is likely that the high arborization together with the high density of dendritic spines, especially of the mushroom type, found in the dorsal hippocampal CA1 of HPRL rats may be due to PRL\u0026rsquo;s ability to induce the expression of cytoskeletal proteins together with neurotrophic factors such as GDNF and BDNF, as demonstrated by Arnold et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], who used the same method of the current study (implanting two pituitary homografts), to induce HPRL rodents. In general, these morphological changes were blocked by BRC, which implies the important role of PRL in promoting plastic changes at the hippocampus and their close relationship with the improvement of both STM and LTM.\u003c/p\u003e \u003cp\u003eOur finding that the procognitive actions of PRL were not completely blocked by BRC may be due to the fact that the dose of BRC tested was not sufficient to fully impair the production of the hormone or only did it for a short period. Given that the dose used here had no impact on the rat\u0026acute;s motor skills (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), further experiments increasing the dose of BRC and blocking other neurotransmitter systems traditionally involved in the regulation of recognition memory are being carried out to clarify this point.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no conflicts of interest to declare\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was partially supported by COFAA and SIP-IPN (Mexico). We thank Histotechnologist Carmen Baltazar for her invaluable support with Golgi techniques.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMarano RJ, Ben-Jonathan N (2014) Minireview: Extrapituitary prolactin: An update on the distribution, regulation, and functions. 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J Neurosci 34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.2452-13.2014\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.2452-13.2014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CA1, learning, prolactin, dendritic morphology, ovariectomy","lastPublishedDoi":"10.21203/rs.3.rs-4774359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4774359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pituitary hormone prolactin (PRL) has been traditionally associated to reproductive functions, specifically to lactogenesis. Currently, this hormone has been linked to a great diversity of brain processes such as neurogenesis, neuroprotection, learning and memory formation. Regarding this later, literature is controversial since some studies indicate that PRL, like estrogens, improve some types of memory, while other report the opposite.\u003c/p\u003e \u003cp\u003eImplanting pituitary homografts under the renal capsule induces a 10-fold increase of plasmatic PRL in male rats, but its effect on females is unknown. On the other hand, long-term (15 weeks) removal of ovaries produces a clear deficit in memory function by avoiding the action of estrogens and progesterone on this process. The present study was aimed at exploring the putative pro-cognitive actions of endogenous PRL and its role on the morphology of pyramidal neurons from hippocampus. The novel object recognition test, a hippocampal and cortical-dependent memory task was used for evaluating short- and long-term memory, while bromocriptine, a dopaminergic agonist, was assayed to block the actions of PRL on behavioral and morphological parameters.\u003c/p\u003e \u003cp\u003eMain results indicate that PRL prevents the cognitive deficit observed in ovariectomized rats and increases the mushroom spines in CA1 pyramidal neurons; an effect that was partially impeded by bromocriptine.\u003c/p\u003e","manuscriptTitle":"Hyperprolactinemia prevents short- and long-term memory deficits in ovariectomized rats and modifies the neuronal morphology of hippocampal CA1 neurons.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 14:33:23","doi":"10.21203/rs.3.rs-4774359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9eacc8f3-3079-4bb8-9682-84058c47d04a","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-11T13:15:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-02 14:33:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4774359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4774359","identity":"rs-4774359","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}