An approach for serotonin depletion in pigs: Effects on serotonin receptor binding

Depletion of central serotonin (5‐HT) levels and dysfunction in serotonergic transmission are implicated in a variety of human CNS disorders. The mechanisms behind these serotonergic deficits have been widely studied using rodent models, but only to a limited extent in larger animal models. The pig is increasingly used as an experimental animal model especially in neuroscience research. Here, we present an approach for serotonin depletion in the pig brain. Central serotonin depletion in Danish Landrace pigs was achieved following 4 days treatment with para‐chlorophenylalanine (pCPA). On day 5, tissue concentrations of 5‐HT in seven distinct brain structures from one hemisphere: frontal and occipital cortex, striatum, hippocampus, cerebellum, rostral, and caudal brain stem, were determined. The other hemisphere was processed for receptor autoradiography. Treatments with 50 mg/kg and 100 mg/kg pCPA caused average decreases in 5‐HT concentrations of 61% ± 14% and 66% ± 16%, respectively, and a substantial loss of 5‐HT immunostaining was seen throughout the brain. The serotonin depletion significantly increased 5‐HT₄ receptor binding in nucleus accumbens, but did not alter 5‐HT_1A and 5‐HT_2A receptor or serotonin transporter binding in any brain region. In conclusion, 4 days treatment with pCPA effectively reduces 5‐HT levels in the pig brain. Further, whereas several 5‐HT markers did not change after the pCPA treatment, 5‐HT₄ receptors were consistently upregulated, indicating a greater susceptibility of this receptor to altered 5‐HT levels. This porcine model of serotonin depletion will be useful in future studies of cerebral serotonergic dysfunction. Synapse 2011. © 2010 Wiley‐Liss, Inc.     

Effect of Intravenous Captopril on c-fos Expression Induced by Sodium Depletion in Neurons of the Lamina Terminalis

Peripheral administration of the angiotensin converting enzyme (ACE) inhibitor, captopril, and the central infusion of sarile, an angiotensin II (Ang II) receptor antagonist, were used to evaluate the role of renal and brain generated Ang II in sodium depletion-induced production of Fos in cells of the subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT). Pretreatment with intravenous captopril (100 mg/kg) significantly inhibited the c-fos expression induced by sodium depletion in the SFO and OVLT. In contrast, continuous intracerebroventricular infusion of sarile (22.5 μg/4.5 h, 5 μl/h) did not affect the expected pattern of c-fos expression observed in both nuclei, 4 h after peritoneal dialysis. These results show that systemic interference with the angiotensin system of renal origen by captopril inhibited the production of Fos induced by sodium depletion in cells of the SFO and OVLT. These findings are consistent with the hypothesis that a rise in peripheral Ang II levels, triggered by sodium deficiency, could be an important mediator of the physiological and behavioral responses that lead to the restoration of sodium balance. In addition, this study suggests that increased circulating Ang II levels in response to body sodium deficit can directly stimulate neural pathways in the SFO and OVLT.     

Angiotensin II Induces Inward Currents in Subfornical Organ Neurones of Rats

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.     

Serotonergic Neurones in the Dorsal Raphe Nucleus That Project into the Medial Preoptic Area Contain Oestrogen Receptor β

Serotonin is involved in female sexual behaviour in which the medial preoptic area (MPA) has a pivotal role. The present study used immunohistochemistry, in situ hybridization and retrograde transport analysis to investigate whether serotonin neurones in the dorsal raphe nucleus (DRN) of females projecting into the MPA contained oestrogen receptor alpha or beta. The projection of serotonin neurones from the DRN to the MPA was confirmed using the microinjection of Fluoro-Gold (FG), a fluorescent retrograde tracer, into the MPA of ovariectomized (OVX-group) and OVX-rats treated with oestradiol benzoate (E2-group). A number of serotonin neurones in the DRN were labelled with FG, indicating that these serotonin neurones in DRN project their terminals into the MPA. FG-labelled serotonin neurones expressed ERbeta mRNA in the DRN, and the number of the serotonin neurones containing ERbeta mRNA between the OVX-group and the E2-treated group was not significantly different. Serotonin neurones in the DRN did not express ERalpha-immunoreactivity. Since previous findings showed that the density of serotonin-immunoreactive fibres and the concentration of serotonin within the MPA was significantly lower in the E2-group than the OVX-group, our present observations suggested that the regulatory effects of E2 on the serotonergic neurone system in the MPA may be via ERbeta within the serotonin-containing cells in the DRN of female rats.     

5‐HT1B autoreceptor regulation of serotonin transporter activity in synaptosomes

Serotonin‐1B (5‐HT_1B) autoreceptors are located in serotonin (5‐HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high‐affinity reuptake of 5‐HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K_m and V_max, and previous work suggests that 5‐HT_1B autoreceptors may regulate 5‐HT reuptake, in addition to modulating 5‐HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5‐HT_1B autoreceptor regulation of SERT‐mediated 5‐HT uptake into synaptosomes. The selective 5‐HT_1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild‐type but not 5‐HT_1B knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5‐HT_1B agonist CP94253. Furthermore, SERT activity varies as a function of 5‐HT_1B receptor expression—specifically, genetic deletion of 5‐HT_1B decreased SERT function, while viral‐mediated overexpression of 5‐HT_1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5‐HT_1B autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5‐HT_1B autoreceptor expression levels and is modulated by both activation and inhibition of 5‐HT_1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

Physiological regulation of brain angiotensin receptor mRNA in AT1a deficient mice

Experiments were performed to study the physiological regulation of angiotensin (Ang) AT1b receptors using Ang AT1a knockout mice (AT1aKO). Ang AT1b mRNA was analyzed in forebrain, hypothalamus, and brainstem using in situ hybridization (ISH) under baseline and water-restricted conditions. Plasma was analyzed for osmolality, vasopressin, and corticosterone. Dehydration (24 h) increased osmolality and corticosterone and decreased body weight with no difference between groups. Plasma vasopressin was not different between the groups and was not stimulated by dehydration. Under water ad libitum conditions, there were no differences in AT1b mRNA expression in medial periventricular, anterior third ventricle (AV3V), and subfornical organ (SFO) between controls and AT1aKO. In contrast, there was higher expression in the dorsal motor nucleus of the vagus (DMV) of AT1aKO vs. Controls (0.6 +/- 0.1 vs. 0.9 +/- 0.1 microCi/g, Control vs. AT1aKO in water ad libitum group). Dehydration increased AT1b expression in SFO in AT1aKO, but not in controls (0.6 +/- 0.07 vs. 0.9 +/- 0.06 microCi/g; water ad libitum vs. dehydrated). Emulsion autoradiography documents the detailed pattern of AT1b expression in brainstem of controls and AT1aKO. There was labeling in DMV, locus coeruleus, inferior olive, lateral reticular nucleus, and caudalis spinal trigemius. In conclusion, deletion of AT1a receptors produces a compensatory increase in AT1b receptor mRNA expression in brainstem, but not in hypothalamus or rostral forebrain. In addition, AT1aKO mice showed an enhanced response to dehydration in terms of AT1b mRNA expression in SFO.     

Serotonin depletion attenuates AY-9944-mediated atypical absence seizures

Summary: Purpose: To test the hypothesis that serotonin (5‐HT) plays a role in the modulation of experimental atypical absence seizures. Methods: Male Long‐Evans hooded rats were treated from postnatal day (P) 2 to P20 with the cholesterol inhibitor AY‐9944 (AY). Epidural electrodes were implanted for electrocorticography (ECoG) followed by serotonin depletion by using para‐cholorophenylalanine (PCPA). High‐performance liquid chromatography (HPLC) was used to measure the levels of serotonin and its metabolite (5‐HIAA) in various brain regions. Serotonin metabolism was computed by using the 5‐HIAA/5‐HT ratio and used to ascertain differences between groups. Results: PCPA treatment was associated with a significant decrease in the total slow spike‐and‐wave discharge (SSWD) duration in AY‐treated rats compared with controls (p < 0.01). HPLC data confirmed the PCPA depletion of 5‐HT and 5‐HIAA in cortex, thalamus, hippocampus, and brainstem compared with naïve rats. AY‐treated rats showed higher levels of 5‐HIAA and 5‐HT in the same brain regions, with a concomitant decrease in rates of serotonin turnover. Conclusions: The data indicate that serotonin depletion protects against experimental atypical absence seizures. The increased levels of 5‐HIAA and 5‐HT and altered rates of serotonin turnover suggest that the serotonergic neurotransmission may be perturbed in the AY rat.     

Dorsal raphe serotonergic neurons promote arousal from isoflurane anesthesia

AimsGeneral anesthesia has been widely applied in surgical or nonsurgical medical procedures, but the mechanism behind remains elusive. Because of shared neural circuits of sleep and anesthesia, whether serotonergic system, which is highly implicated in modulation of sleep and wakefulness, regulates general anesthesia as well is worth investigating.MethodsImmunostaining and fiber photometry were used to assess the neuronal activities. Electroencephalography spectra and burst‐suppression ratio (BSR) were used to measure anesthetic depth and loss or recovery of righting reflex to indicate the induction or emergence time of general anesthesia. Regulation of serotonergic system was achieved through optogenetic, chemogenetic, or pharmacological methods.ResultsWe found that both Fos expression and calcium activity were significantly decreased during general anesthesia. Activation of 5‐HT neurons in the dorsal raphe nucleus (DRN) decreased the depth of anesthesia and facilitated the emergence from anesthesia, and inhibition deepened the anesthesia and prolonged the emergence time. Furthermore, agonism or antagonism of 5‐HT 1A or 2C receptors mimicked the effect of manipulating DRN serotonergic neurons.ConclusionOur results demonstrate that 5‐HT neurons in the DRN play a regulative role of general anesthesia, and activation of serotonergic neurons could facilitate emergence from general anesthesia partly through 5‐HT 1A and 2C receptors.     

Paradoxical increase in survival of newborn neurons in the dentate gyrus of mice with constitutive depletion of serotonin

Increased adult neurogenesis is a major neurobiological correlate of the beneficial effects of antidepressants. Indeed, selective serotonin (5‐HT) re‐uptake inhibitors, which increase 5‐HT transmission, enhance adult neurogenesis in the dentate gyrus (DG) of the hippocampus. However, the consequences of 5‐HT depletion are still unclear as studies using neurotoxins that target serotonergic neurons reached contradictory conclusions on the role of 5‐HT on DG cell proliferation. Here, we analysed two genetic models of 5‐HT depletion, the Pet1^?/? and the VMAT2^f/f; SERT^cre/+ mice, which have, respectively, 80 and 95% reductions in hippocampal 5‐HT. In both models, we found unchanged cell proliferation of the neural precursors in the DG subgranular zone, whereas a significant increase in the survival of newborn neurons was noted 1 and 4 weeks after BrdU injections. This pro‐survival trait was phenocopied pharmacologically with 5‐HT synthesis inhibitor PCPA treatment in adults, indicating that this effect was not developmental. Furthermore, a 1‐week administration of the 5‐HT_1A receptor agonist 8‐OH‐DPAT in Pet1^?/? and PCPA‐treated mice normalised hippocampal cell survival. Overall, our results indicate that constitutive 5‐HT depletion does not alter the proliferation of neural precursors in the DG but promotes the survival of newborn cells, an effect which involves activation of postsynaptic 5‐HT_1A receptors. The role of 5‐HT in selective neuronal elimination points to a new facet in its multiple effects in controlling neural circuit maturation.     

Molecular cloning and functional expression of a serotonin receptor fromCaenorhabditis elegans

A cDNA encoding a serotonin receptor has been isolated from aCaenorhabditis elegans mixed stage cDNA library. The nematode serotonin receptor, designated 5HT-Ce, was permanently expressed in murine Ltk-cells, where it mediates adenylate cyclase attenuation. Sequence analysis and the pharmacological profiles demonstrate its relatedness not only toDrosophila andLymnae 5HT receptors but also to mammalian 5HT1a receptors. The 5HT-Ce gene does not map close to the position of any known serotonergic mutations.     

The subfornical organ: A novel site for prolactin action

Prolactin (PRL ) is a peptide hormone that performs over 300 biological functions, including those that require binding to prolactin receptor (PRL ‐R) in neurones within the central nervous system (CNS ). To enter the CNS , circulating PRL must overcome the blood‐brain barrier. Accordingly, areas of the brain that do not possess a blood‐brain barrier, such as the subfornical organ (SFO ), are optimally positioned to interact with systemic PRL . The SFO has been classically implicated in energy and fluid homeostasis but has the potential to influence oestrous cyclicity and gonadotrophin release, which are also functions of PRL . We aimed to confirm and characterise the expression of PRL ‐R in the SFO , as well as identify the effects of PRL application on membrane excitability of dissociated SFO neurones. Using a quantitative real‐time polymerase chain reaction, we found that PRL ‐R mRNA in the SFO of male and female Sprague Dawley rats did not significantly differ between juvenile and sexually mature rats (P  = .34), male and female rats (P  = .97) or across the oestrous cycle (P  = .54). Patch‐clamp recordings were obtained in juvenile male rats to further investigate the actions of PRL at the SFO . Dissociated SFO neurones perfused with 1 μmol L^‐1 PRL resulted in 2 responsive subpopulations of neurones; 40% depolarised (n = 15/43, 11.3 ± 1.7 mV ) and 14% hyperpolarised (n = 6/43, ?6.7 ± 1.4 mV ) to PRL application. Within the range of 10 pmol L^‐1 to 1 μmol L^‐1, the concentrations of PRL were not significantly different in either the magnitude (P  = .53) or proportion (P  = .19) of response. Furthermore, PRL application significantly reduced the transient K⁺ current in 67% of SFO neurones in voltage‐clamp configuration (n = 6/9, P  = .02). The stability in response to PRL and expression of PRL ‐R in the SFO suggests that PRL function is conserved across physiological states and circulating PRL concentrations, prompting further investigations aiming to clarify the nature of PRL function in the SFO.     

Sodium appetite elicited by low‐sodium diet is dependent on p44/42 mitogen‐activated protein kinase (extracellular signal‐regulated kinase 1/2) activation in the brain

Sodium appetite is regulated by several signalling molecules, among which angiotensin II (Ang II) serves as a key driver of robust salt intake by binding to Ang II type 1 receptors (AT1R) in several regions in the brain. The activation of these receptors recruits the mitogen‐activated protein kinase (MAPK) pathway, which has previously been linked to Ang II‐induced increases in sodium appetite. Thus, we addressed the involvement of MAPK signalling in the induction of sodium appetite after 4 days of low‐sodium diet consumption. An increase in extracellular signal‐regulated kinase (ERK) phosphorylation in the laminae terminalis and mediobasal hypothalamus was observed after low‐sodium diet consumption. This response was reduced by i.c.v. microinjection of an AT1R antagonist into the laminae terminalis but not the hypothalamus. This result indicates that low‐sodium diet consumption activates the MAPK pathway via Ang II/AT1R signalling on the laminae terminalis. On the other hand, activation of the MAPK pathway in the mediobasal hypothalamus after low‐sodium diet consumption appears to involve another extracellular mediator. We also evaluated whether a low‐sodium diet could increase the sensitivity for Ang II in the brain and activate the MAPK pathway. However, i.c.v. injection of Ang II increased ERK phosphorylation on the laminae terminalis and mediobasal hypothalamus; this increase achieved a response magnitude similar to those observed in both the normal and low‐sodium diet groups. These data indicate that low‐sodium diet consumption for 4 days is insufficient to change the ERK phosphorylation response to Ang II in the brain. To investigate whether the MAPK pathway is involved in sodium appetite after low‐sodium diet consumption, we performed i.c.v. microinjections of a MAPK pathway inhibitor (PD98059). PD98059 inhibited both saline and water intake after low‐sodium diet consumption. Thus, the MAPK pathway is involved in promoting the sodium appetite after low‐sodium diet consumption.     

The Role of Angiotensin II on Sodium Appetite After a Low‐Sodium Diet

The present study aimed to investigate the role of angiotensin II (Ang II) on sodium appetite in rats subjected to a normal or a low‐sodium diet (1% or > 0.1% NaCl) for 4 days. During sodium restriction, a reduction in water intake, urinary volume and sodium excretion was observed. After a low‐sodium diet, we observed decreased plasma protein concentrations and haematocrit associated with a slight reduction in arterial pressure, without any significant changes in heart rate, natraemia, corticotrophin‐releasing hormone mRNA expression in the paraventricular nucleus and corticosterone levels. After providing hypertonic saline, there was an increase in saline intake followed by a small increase in water intake, resulting in an enhanced saline intake ratio and the recovery of arterial pressure. Sodium deprivation increased plasma but not brain Ang I and II concentrations. A low‐sodium diet increased kidney renin and liver angiotensinogen mRNA levels but not lung angiotensin‐converting enzyme mRNA expression. Moreover, Ang II type 1a receptor mRNA expression was increased in the subfornical organ and the dorsal raphe nucleus and decreased in the medial preoptic nuclei, without changes in the paraventricular nucleus and the nucleus of solitary tract after a low‐sodium diet. Blockade of AT₁ receptors or brain Ang II synthesis led to a reduction in sodium intake after a low‐sodium diet. Intracerebroventricular injection of Ang II led to a similar increase in sodium and water intake in the control and low‐sodium diet groups. In conclusion, the results of the present study suggest that Ang II is involved in the increased sodium appetite after a low‐sodium diet.     

Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb

Olfactory processing is well known to be regulated by centrifugal afferents from other brain regions, such as noradrenergic, acetylcholinergic, and serotonergic neurons. Serotonergic neurons widely innervate and regulate the functions of various brain regions. In the present study, we focused on serotonergic regulation of the olfactory bulb (OB), one of the most structurally and functionally well‐defined brain regions. Visualization of a single neuron among abundant and dense fibers is essential to characterize and understand neuronal circuits. We accomplished this visualization by successfully labeling and reconstructing serotonin (5‐hydroxytryptamine: 5‐HT) neurons by infection with sindbis and adeno‐associated virus into dorsal raphe nuclei (DRN) of mice. 5‐HT synapses were analyzed by correlative confocal laser microscopy and serial‐electron microscopy (EM) study. To further characterize 5‐HT neuronal and network function, we analyzed whether glutamate was released from 5‐HT synaptic terminals using immuno‐EM. Our results are the first visualizations of complete 5‐HT neurons and fibers projecting from DRN to the OB with bifurcations. We found that a single 5‐HT axon can form synaptic contacts to both type 1 and 2 periglomerular cells within a single glomerulus. Through immunolabeling, we also identified vesicular glutamate transporter 3 in 5‐HT neurons terminals, indicating possible glutamatergic transmission. Our present study strongly implicates the involvement of brain regions such as the DRN in regulation of the elaborate mechanisms of olfactory processing. We further provide a structure basis of the network for coordinating or linking olfactory encoding with other neural systems, with special attention to serotonergic regulation. J. Comp. Neurol. 523:262–280, 2015. © 2014 Wiley Periodicals, Inc.     

Serotonin2C receptors in the nucleus accumbens are involved in enhanced alcohol-drinking behavior

Dopamine and serotonin (5‐HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol‐drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol‐exposed mice, the expression of 5‐HT_2C receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5‐HT_2C receptor significantly increased in the ACC. The expression of 5‐HT₇ receptor mRNA increased in the ACC and DRN. Contents of 5‐HT decreased in the ACC shell (ACC_S) and DRN of the alcohol‐exposed mice. The basal extracellular releases of dopamine (DA) and 5‐HT in the ACC_S increased more in the alcohol‐exposed mice than in alcohol‐naïve mice. The magnitude of the alcohol‐induced ACC_S DA and 5‐HT release in the alcohol‐exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC_S of the 5‐HT_2C receptor antagonist, SB‐242084, suppressed voluntary alcohol‐drinking behavior in the alcohol‐exposed mice. But the i.p. administration of the 5‐HT₇ receptor antagonist, SB‐258719, did not have significant effects on alcohol‐drinking behavior in the alcohol‐exposed mice. The effects of the 5‐HT_2C receptor antagonist were not observed in the air‐exposed control mice. These results suggest that adaptations of the 5‐HT system, especially the upregulation of 5‐HT_2C receptors in the ACC_S, are involved in the development of enhanced voluntary alcohol‐drinking behavior.     

Pharmacological characterisation of amylin-related peptides activating subfornical organ neurones

Amylin, calcitonin gene-related peptide (CGRP) and calcitonin are structurally related peptides with overlapping peripheral and central actions. Amylin and calcitonin excite the majority of neurones in the subfornical organ (SFO), where high densities of so-called C-type G-protein-coupled receptors have been detected. Subcutaneous injection of these hormones stimulates drinking similar to angiotensin II (ANGII), a dipsogen acting via the SFO. We now show that in addition to amylin and rat calcitonin (rCT), CGRP and salmon calcitonin (sCT) also excite SFO neurones. In extracellular recordings of an in vitro slice preparation of the SFO, 78% of all neurones (n=31) superfused with CGRP (10⁻⁶ M) were excited. The excitatory effect was dose-dependent and reversible with an average threshold concentration of 5×10⁻⁷ M, which is approximately 15-fold higher than reported for amylin-induced excitations. sCT (10⁻⁷ M), which behaves as a non-competitive agonist at amylin as well as calcitonin receptors, caused irreversible excitatory responses in 96% of all recordings (n=26). Amylin-, CRGP- and rCT-induced excitations could be blocked by the selective amylin receptor antagonist AC187 (10⁻⁵ to 10⁻⁶ M), whereas sCT-induced excitations were not inhibited. The receptor antagonist human CGRP(8-37) (10⁻⁶ M) partly caused agonistic responses, but did not block CGRP-induced excitations. The pharmacological profile observed in the present work, and in a recent publication using the same preparation, indicating (1) that CGRP is a weaker agonist in the SFO than amylin, (2) that sCT excites SFO neurones, and (3) that responses are blocked by AC187 but not by CGRP(8-37), is inconsistent with activation via CGRP receptors, but is instead consistent with involvement of amylin (C3) and calcitonin (C1) receptors, which are co-localized to a high degree on the same subset of SFO-neurones. We propose that it is unlikely that blood-borne CGRP has a significant effect on neurones in the SFO.     

Serotonergic and Catecholaminergic Interactions with Co-Localised Dopamine-Melatonin Neurones in the Hypothalamus of the Female Turkey

Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co‐localised dopamine‐melatonin (DA‐MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA‐MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1′dioctadecyl‐3,3,3′3′‐tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DBH) and phenyl N‐methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA‐MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA‐MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.     

A role for nitric oxide in serotonin neurons of the midbrain raphe nuclei

Neuronal nitric oxide synthase (nNOS) catalyses the production of the neurotransmitter nitric oxide. nNOS is expressed in the dorsal raphe nucleus (DRN), a source of ascending serotonergic projections. In this study, we examined the distribution nNOS and the function of nitric oxide in the DRN and adjacent median raphe nucleus (MRN) of the rat. We hypothesized that nNOS is differentially expressed across the raphe nuclei and that nitric oxide influences the firing activity of a subgroup of 5‐HT neurons. Immunohistochemistry revealed that, nNOS is present in around 40% of 5‐HT neurons, throughout the DRN and MRN, as well as in some non‐5‐HT neurons immediately adjacent to the DRN and MRN. The nitric oxide receptor, soluble guanylyl cyclase, was present in all 5‐HT neurons examined in the DRN and MRN. In vitro extracellular electrophysiology revealed that application of the nitric oxide donor, diethylamine NONOate (30–300 µM) inhibited 60%–70% of putative 5‐HT neurons, excited approximately 10% of putative 5‐HT neurons and had no effect on the rest. The inhibitory response to nitric oxide was blocked by [1H‐[1,2,4]oxadiazolo‐[4, 3‐a]quinoxalin‐1‐one (ODQ, 30 or 100 µM), indicating mediation by soluble guanylyl cyclase. Juxtacellular labelling revealed that nitric oxide inhibits firing in both putative 5‐HT neurons which express nNOS and those which do not express nNOS. Our data are consistent with the notion that nitric oxide acts as both a trans‐synaptic and autocrine signaller in 5‐HT neurons in the DRN and MRN and that its effects are widespread and primarily inhibitory.