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.     

Whole body sodium depletion modifies AT1 mRNA expression and serotonin content in the dorsal raphe nucleus

Angiotensin II (Ang II) acts on Ang II type 1 (AT1) receptors located in the organum vasculosum and subfornical organ (SFO) of the lamina terminalis as a main facilitatory mechanism of sodium appetite. The brain serotonin (5‐HT) system with soma located in the dorsal raphe nucleus (DRN) provides a main inhibitory mechanism. In the present study, we first investigated the existence of Ang II AT1 receptors in serotonergic DRN neurones. Then, we examined whether whole body sodium depletion affects the gene expression of the AT1a receptor subtype and the presumed functional significance of AT1 receptors. Using confocal microscopy, we found that tryptophan hydroxylase‐2 and serotonin neurones express AT1 receptors in the DRN. Immunofluorescence quantification showed a significant reduction in 5‐HT content but no change in AT1 receptor expression or AT1/5‐HT colocalisation in the DRN after sodium depletion. Whole body sodium depletion also significantly increased Agtr1a mRNA expression in the SFO and DRN. Oral treatment with the AT1 receptor antagonist losartan reversed the changes in Agtr1a expression in the SFO but not the DRN. Losartan injection into either the DRN or the mesencephalic aqueduct had no influence on sodium depletion‐induced 0.3 mol L^‐1 NaCl intake. The results indicate the expression of Agtr1a mRNA in the DRN and SFO as a marker of sodium depletion. They also suggest that serotonergic DRN neurones are targets for Ang II. However, the function of their AT1 receptors remains elusive.     

Regulation of the release of serotonin in the dorsal raphe nucleus by alpha1 and alpha2 adrenoceptors

To investigate the modulation of serotonin release in the dorsal raphe nucleus (DRN) by alpha(1) and alpha(2) adrenoceptors, dual-probe microdialysis was performed in conscious rats. The specific alpha(1) and alpha(2) adrenoceptor agonists and antagonists were locally infused into the DRN via retrograde microdialysis. The release of serotonin was simultaneously sampled from the DRN and prefrontal cortex (PFC). Infusion of the alpha(1) adrenoceptor agonist cirazoline into the DRN (100 microM) produced an increase in the release of serotonin in the DRN to 200% of the basal levels, but no effect was seen in the PFC. After infusion of the alpha(1) adrenoceptor antagonist prazosin into the DRN (100 microM) the release of serotonin decreased in the DRN and PFC to about 40% and 65% of the basal levels, respectively. Infusion of the alpha(2) adrenoceptor agonist clonidine into the DRN (100 microM) slightly but significantly decreased the level of serotonin in the DRN as well as in the PFC to about 70% of the basal levels. Infusion of the alpha(2A) adrenoceptor antagonist BRL 44408 into the DRN (100 microM) caused an increase of serotonin release in the DRN to 270% of the basal levels, but at the same time no changes were seen in the extracellular levels of serotonin in the PFC. The present study demonstrates that alpha(1) as well as alpha(2) adrenoceptors in the DRN modulate the release of serotonin in the DRN, and that alpha(1) adrenoceptors in the DRN are maximally stimulated during resting conditions.     

Dorsal raphe nuclei integrate allostatic information evoked by depletion-induced sodium ingestion

Structures of the lamina terminalis (LT) sense and integrate information reflecting the state of body water and sodium content. Output from the LT projects into a neural network that regulates body fluid balance. Serotonin (5-HT) and the dorsal raphe nuclei (DRN) have been implicated in the inhibitory control of salt intake (i.e., sodium appetite). Signals arriving from the LT evoked by fluid depletion-induced sodium ingestion interact with this inhibitory serotonergic system. We investigated the role of neurons along the LT that directly project to the DRN. We analyzed the pattern of immunoreactivity (ir) of LT cells double-labeled for Fos (a marker of neural activity) and Fluorogold (FG; a retrograde tracer) following sodium depletion-induced sodium intake. Seven days after injection of FG into the DRN, sodium appetite was induced by furosemide injection and overnight access to only a low sodium diet (Furo-LSD) and distilled water. Twenty-four hours later, access to 0.3 M NaCl was given to depleted or sham-depleted rats and sodium intake was measured over the following 60 min. Ninety minutes after the termination of the intake test, the animals were perfused and their brains were processed for immunohistochemical detection of Fos and FG. Compared to sham-depleted animals there was a significantly greater number of Fos-/FG-ir double-labeled cells in the subfornical organ, the organum vasculosum of the lamina terminalis and the median preoptic nucleus in rats that ingested NaCl. Projections from the LT cells may contribute to inhibitory mechanisms involving 5-HT neurons in the DRN that limit the intake of sodium and prevent excess volume expansion.     

Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens

Diverse physiological actions have been reported for 5-hydroxytryptamine (5-HT, serotonin) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Acb) suggesting that the 5-HT innervation of these forebrain areas may be derived from different populations of neurons. We examined this possibility by mapping the distribution of 5-HT-immunoreactive (ir) and non-5HT-ir neurons containing retrograde labeling following injections of different tracers into both these target regions. The analysis was focused in the dorsal raphe nucleus (DRN) of the midbrain, since 5-HT pathways to the MPFC and Acb primarily originate from this area. Volume microinjections of the fluorescent retrograde tracer, Fluoro-Gold (FG), were placed into the MPFC and microinjections of cholera toxin B subunit coupled to 15 nm gold particles (CT-Au) were placed into the Acb of the same animal. Sections through the DRN containing retrogradely labeled neurons were further processed for immunofluorescent localization of 5-HT using a rhodamine marker. Neurons retrogradely labeled from the Acb were greater in number overall than those projecting to the MPFC. In addition, Acb-projecting neurons extended into the lateral wings of the DRN, whereas MPFC-projecting neurons were more restricted to the midline. Both groups of retrogradely labeled neurons, however, were more numerous in the caudal aspect of the dorsal raphe nucleus and were scattered amongst 5-HT immunoreactive perikarya. Of783 ± 69 CT-Au labeled cells, 15% also contained the FG label and 11% contained FG and 5-HT immunoreactivity. Of613 ± 48 FG labeled cells, 24% also contained the CT-Au label and 21% were also immunoreactive to 5-HT. The results suggest a more prominent input to the Acb from both 5-HT-ir and non-5-HT-ir neurons in the caudal aspect of the DRN and further indicate that while most 5-HT-ir and non-5-HT-ir neurons project differentially to both forebrain regions, a few cells also show collateralization to the MPFC and Acb. Such collateralization of single serotonergic neurons to divergent targets mey integrate cognitive and motor activities in response to pharmacological manipulations of ascending serotonergic pathways.     

The Effects of Oestrogen and Progesterone on Serotonin and its Metabolite in the Lateral Septum, Medial Preoptic Area and Ventromedial Hypothalamic Nucleus of Female Rats

The effects of sex steroid hormones on serotonin and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) in the lateral septal nucleus (LS), the medial preoptic area (MPA) and the ventromedial nucleus of the hypothalamus (VMH) of female rats were investigated, using immunohistochemistry and high-performance liquid chromatography (HPLC). Female rats were divided into three groups: ovariectomized rats (OVX group); OVX-rats treated with estradiol benzoate alone (E2 group); and OVX-rats treated with E2 plus progesterone (E2+P group). We analysed the density of serotonin-immunoreactive fibres with a computer-assisted image analysis system, and measured the tissue concentrations of serotonin and 5-HIAA. Many serotonin-immunoreactive fibres were observed in the LS, MPA and VMH in all three groups. The density of serotonin-immunoreactive fibres in the MPA and VMH was significantly lower in the E2 and E2+P groups compared to the OVX group, whereas the LS showed no detectable differences among the three groups. In the HPLC study, the concentrations of serotonin in the MPA and VMH of the E2 and E2+P groups were significantly lower than that in the OVX group. There was no significant difference in the concentration of serotonin in the LS. The concentration of 5-HIAA and the ratio of 5-HIAA/serotonin in the LS, MPA and VMH showed no significant differences among the OVX, E2 and E2+P groups. The present results suggest that E2 priming for sexual behaviour can affect the serotonergic system by decreasing serotonin content, but not the turnover rate, in the MPA and VMH of female rats.     

Oxytocin,but not oxytocin receptor,is rRegulated by oestrogen receptor beta in the female mouse hypothalamus

In the female rat, oestrogen receptor (ER) beta is colocalized with both oxytocin- and vasopressin-producing neurones in the paraventricular nucleus of the hypothalamus (PVN). In this study, we demonstrate that the same pattern of colocalization between ERbeta and oxytocin exists in the female mouse. Because this nucleus contains only a negligible quantity of ERalpha, it is likely that the oestrogen-dependent regulation of oxytocin and vasopressin synthesis in the PVN is mediated by ERbeta. Thus, we compared the effect of ovarian hormones on oxytocin and vasopressin mRNA expression in the PVN of wild-type (WT) and ERbeta knockout (betaERKO) mice. We also compared the effects of ovarian hormones on oxytocin receptor (OTR) expression in the medial amygdala (MeA) and ventromedial nucleus of the hypothalamus (VMN) in female WT and betaERKO mice. Ovariectomized mice underwent long-term treatment with oestradiol or oil. Progesterone was given concurrently on the final 7 days of treatment, and all mice were killed 48 h after the final progesterone injection. In the PVN, hormone treatment increased oxytocin mRNA expression in WT but not betaERKO females. These results suggest that ERbeta is necessary for the regulation of the expression of oxytocin in the PVN. Hormone treatment had no effect on vasopressin mRNA expression in the PVN, but significantly increased OTR binding in both the VMN and the MeA in both genotypes. Collectively, our data show region and peptide specific regulation by ERalpha and ERbeta in the mouse hypothalamus.     

Differential sensitivities of the two visual pathways of the chick to labelling by fluorescent retrograde tracers.

This study investigates the neurone structure-specific differences of sensitivities of fluorescent tracers. The tracers were used for retrograde labelling of contralateral projections in the two visual pathways of the chick. Rhodamine B Isothiocyanate (RITC), Fluorogold (FG) and True blue (TB) were injected into either the visual Wulst (thalamofugal pathway) or the nucleus rotundus (Rt; tectofugal pathway) and the retrogradely labelled neurones in the nucleus geniculatus lateralis pars dorsalis (GLd) or the optic tectum, respectively, were counted. Differential retrograde labelling in the two pathways was observed. In the thalamofugal pathway, both the contralateral and ipsilateral GLd cells were labelled by all three tracers (RITC, FG and TB). However, in the tectofugal pathway, whereas RITC labelled both the ipsilateral and contralateral tectal neurones, FG or TB labelled effectively only the ipsilateral tectal neurones. It was clear that FG and TB were taken up by the nerve endings and transported part-way along the axon but failed to be transported to the cell bodies of the contralateral tectal neurones. In addition, red beads and green beads were also injected into Rt and the differential labelling was also observed. Red beads labelled both ipsilateral and contralateral tectal neurones but green beads labelled only the ipsilateral tectal neurones. Since the contralateral tectal projections consist of divergent axon collaterals, the present study suggests that various retrograde tracers are not transported in these axon collaterals to label cell bodies. The contralaterally projecting neurones in the thalamofugal pathway are not axon collaterals and they were labelled by all of the tracers used.     

Oestrogen receptor alpha and beta immunoreactive cells in the suprachiasmatic nucleus of mice: distribution, sex differences and regulation by gonadal hormones

Oestrogen regulates various aspects of circadian rhythm physiology. The presence of oestrogen receptors within the suprachiasmatic nucleus (SCN), the principal circadian oscillator, indicates that some actions of oestrogen on circadian functions may be exerted at that site. The present study analysed sex differences, topographic distribution, and neurochemical phenotype of neurones expressing the alpha and beta subtypes of oestrogen receptors (ERalpha and ERbeta) in the mouse SCN. We found that relatively few neurones in the SCN are immunoreactive (IR) for ERalpha (approximately 4.5% in females and 3% in males), but five- to six-fold more SCN neurones express ERbeta. ER-IR neurones are primarily in the shell subdivision of the nucleus and show differences between the sexes, significantly greater numbers being found in females. Treatment of male or female gonadectomised mice with oestradiol benzoate for 24 h substantially reduced the number of ERbeta-IR neurones, but not ERalpha-IR neurones. Double-labelling immunocytochemical experiments to characterise the phenotype of the oestrogen-receptive neurones showed the presence of the calcium-binding proteins calretinin or calbindin D28K in approximately 12% and 10%, respectively, of ERalpha-IR neurones. A higher proportion (approximately 38%) of ERbeta-IR neurones contains calbindin D28K; a few (approximately 2%) express calretinin or vasopressin. These double-labelled cells appear primarily in the shell subdivision of the SCN. Neither vasoactive intestinal polypeptide- nor gastrin releasing peptide-immunoreactivity was observed in ER-IR neurones. These data indicate that the primary target cells for oestrogen are in the shell subdivision of the nucleus. The sexually differentiated expression and distribution of ERalpha and ERbeta in various cell populations of the SCN suggest multiple modes of oestrogen signalling within this nucleus, which may modulate circadian functions.     

Ultrastructural evidence of kisspeptin-gonadotrophin-releasing hormone (GnRH) interaction in the median eminence of female rats: implication of axo-axonal regulation of GnRH release

The present study was conducted to determine the morphological and functional interaction between kisspeptin and gonadotrophin-releasing hormone (GnRH) neuronal elements at the median eminence in female rats to clarify a possibility that kisspeptin directly stimulates GnRH release at the nerve end. A dual immunoelectron microscopic study of kisspeptin and GnRH showed that the kisspeptin-immunoreactive nerve element directly abutted the GnRH-immunoreactive nerve element, although no obvious synaptic structure was found between kisspeptin and GnRH neurones in the median eminence. The current retrograde tracing study with FluoroGold (FG) indicates that kisspeptin neurones are not in contact with fenestrated capillaries because no FG signal was found in kisspeptin neurones when the FG was injected peripherally. This peripheral FG injection revealed the neuroendocrine neurones projecting to the median eminence because FG-positive GnRH neuronal cell bodies were found in the preoptic area. Synthetic rat kisspeptin (1-52)-amide stimulated GnRH release from the median eminence tissues in a dose-dependent manner. Thus, the present results suggest that kisspeptin at least partly exerts stimulatory effects on GnRH release from the neuronal terminals of GnRH neurones by axo-axonal nonsynaptic interaction in the median eminence.     

Correlation of estrogen beta-receptor messenger RNA with endogenous levels of plasma estradiol and progesterone in the female rat hypothalamus,the bed nucleus of stria terminalis and the medial amygdala

Estrogen receptor beta (ERbeta) has been previously mapped in the rat central nervous system. This study aims to explore the regulation of ERbeta mRNA as it is expressed in the intact and cycling female rat brain. Young adult female rats (90+ day, N=20) were screened for estrous phases via vaginal cytology and sacrificed. Brains and blood were collected and processed for in situ hybridization and estradiol (E2) and progesterone (P4) hormone assays, respectively. ERbeta mRNA levels exhibited significant correlations with ovarian steroid ratios (E2/P4) in various brain regions, including the bed nucleus of stria terminalis, the medial nucleus of amygdala, and the anteroventral periventricular nuclei but not the paraventricular and the supraoptic nuclei or the preoptic area of the hypothalamus. No regulatory changes were detected in the cortex. Specifically, in the affected regions, higher P4 levels were significantly correlated with higher ERbeta mRNA expression. In contrast, there was a tendency for higher E2 levels to be correlated with lower ERbeta mRNA expression, but this tendency reached significance only in the bed nucleus of stria terminalis. These results suggest that ERbeta mRNA is regulated in the intact and cycling female rat hypothalamic as well as extrahypothalamic brain regions, and the circulating ovarian hormones play a critical role.     

Lateral parabrachial afferent areas and serotonin mechanisms activated by volume expansion

Recent evidence has shown that the serotonergic mechanism of the lateral parabrachial nucleus (LPBN) participates in the regulation of renal and hormonal responses to isotonic blood volume expansion (BVE). We investigated the BVE-induced Fos activation along forebrain and hindbrain nuclei and particularly within the serotonergic clusters of the raphé system that directly project to the LPBN. We also examined whether there are changes in the concentration of serotonin (5HT) within the raphé nucleus in response to the same stimulus. With this purpose, we analyzed the cells doubly labeled for Fos and Fluorogold (FG) following BVE (NaCl 0.15 M, 2 ml/100 g b.w., 1 min) 7 days after FG injection into the LPBN. Compared with the control group, blood volume-expanded rats showed a significant greater number of Fos-FG double-labeled cells along the nucleus of the solitary tract, locus coeruleus, hypothalamic paraventricular nucleus, central extended amygdala complex, and dorsal raphé nucleus (DRN) cells. Our study also showed an increase in the number of serotonergic DRN neurons activated in response to isotonic BVE. We also observed decreased levels of 5HT and its metabolite 5-hydroxyindoleacetic acid (measured by high-pressure liquid chromatography) within the raphé nucleus 15 min after BVE. Given our previous evidence on the role of the serotonergic system in the LPBN after BVE, the present morphofunctional findings suggest the existence of a key pathway (DRN-LPBN) that may control BVE response through the modulation of 5HT release.     

Modulation of oestrogen receptor-beta mRNA expression in rat paraventricular and supraoptic nucleus neurones following adrenal steroid manipulation and hyperosmotic stimulation

Magnocellular neurosecretory neurones in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei express oestrogen receptor beta (ERbeta) but not ERalpha. In the PVN, ERbeta is strongly expressed in the ventromedial parvocellular neurones projecting to the brainstem. We used quantitative in situ hybridization, with (35)S-labelled riboprobes, to study heterologous regulation by manipulating adrenal steroid hormones (72 h after adrenalectomy +/- corticosterone replacement; repeated stress: halothane inhalation, environmental cold, immobilization, each daily for 3 days) in male rats. Adrenalectomy increased ERbeta mRNA expression in the magnocellular PVN and SON, by 2.2 and 2.5-fold, respectively, with no effect in the ventromedial parvocellular PVN neurones. Corticosterone replacement partially prevented the increases in ERbeta mRNA expression in magnocellular PVN and SON neurones. Repeated stress over 72 h had no effect on ERbeta mRNA expression in the magnocellular PVN or SON, but increased expression 1.4-fold in the ventromedial parvocellular PVN neurones. Although consequences of hydromineral balance derangement after adrenalectomy may stimulate magnocellular neurones, strongly stimulating the neurones by giving intact male rats 2% saline to drink for 72 h decreased ERbeta mRNA expression in the magnocellular PVN and SON neurones by approximately 60%, and in the ventromedial parvocellular PVN neurones by 13%. Thus, ERbeta mRNA expression is negatively regulated by basal glucocorticoid secretion in magnocellular PVN and SON neurones, and positively regulated by stress in ventromedial parvocellular PVN neurones. However, ERbeta mRNA expression in magnocellular neurones is negatively linked to hyperosmotic stimulation of the neurones. The 6.25-fold variation in ERbeta mRNA expression in magnocellular neurones from salt-loading to adrenalectomy could alter their sensitivity to oestrogens. Consequently, regulation of oxytocin and vasopressin neurone activity via ERbeta is expected to vary according to their functional state and, in particular, on basal glucocorticoid actions.     

Chemical neuroanatomy of the dorsal raphe nucleus and adjacent structures of the mouse brain

Serotonin neurons play a major role in many normal and pathological brain functions. In the rat these neurons have a varying number of cotransmitters, including neuropeptides. Here we studied, with histochemical techniques, the relation between serotonin, some other small‐molecule transmitters, and a number of neuropeptides in the dorsal raphe nucleus (DRN) and the adjacent ventral periaqueductal gray (vPAG) of mouse, an important question being to establish possible differences from rat. Even if similarly distributed, the serotonin neurons in mouse lacked the extensive coexpression of nitric oxide synthase and galanin seen in rat. Although partly overlapping in the vPAG, no evidence was obtained for the coexistence of serotonin with dopamine, substance P, cholecystokinin, enkephalin, somatostatin, neurotensin, dynorphin, thyrotropin‐releasing hormone, or corticotropin‐releasing hormone. However, some serotonin neurons expressed the γ‐aminobutyric acid (GABA)‐synthesizing enzyme glutamic acid decarboxylase (GAD). Work in other laboratories suggests that, as in rat, serotonin neurons in the mouse midline DRN express the vesicular glutamate transporter 3, presumably releasing glutamate. Our study also shows that many of the neuropeptides studied (substance P, galanin, neurotensin, dynorphin, and corticotropin‐releasing factor) are present in nerve terminal networks of varying densities close to the serotonin neurons, and therefore may directly or indirectly influence these cells. The apparently low numbers of coexisting messengers in mouse serotonin neurons, compared to rat, indicate considerable species differences with regard to the chemical neuronatomy of the DRN. Thus, extrapolation of DRN physiology, and possibly pathology, from rat to mouse, and even human, should be made with caution. J. Comp. Neurol. 518:3464–3494, 2010. © 2010 Wiley‐Liss, Inc.     

Topographic organization of serotonergic dorsal raphe neurons projecting to the superior colliculus in the Mongolian gerbil (Meriones unguiculatus).

Recent evidence suggests that the dorsal raphe nucleus (DRN) of the brainstem is a collection of neuronal clusters having different neurochemical characteristics and efferent projection patterns. To gain further insight into the neuroanatomic organization of the DRN, neuronal populations projecting to the superior colliculus (SC) were mapped in a highly visual rodent, the Mongolian gerbil (Meriones unguiculatus). Retrograde tracers Fluoro-Gold (FG) or cholera toxin subunit-B (CTB) were injected into the superficial layers of the SC, and serotonin (5-hydroxytryptamine, 5-HT) -positive cells were identified by using immunocytochemistry in the FG-injected animals. Based on its projections to the SC, the DRN was divided into five rostrocaudal levels. In the rostral and middle levels of the DRN, virtually all FG-filled cells occurred in the lateral DRN, and 36-55% of 5-HT-immunoreactive (5-HT-ir) cells were also double-labeled with FG. Caudally, FG-filled cells occurred in the lateral, ventromedial, and interfascicular DRN; and 44, 12, and 31% of 5-HT-ir cells, respectively, were also FG-filled. The dorsomedial DRN contained only a small proportion of FG-filled cells at its most caudal level and was completely devoid of FG-filled cells more rostrally. The CTB-injected animals showed a similar distribution of retrogradely labeled cells in the DRN. Topographically, the dorsal tegmental nucleus and the laterodorsal tegmental nucleus appeared to be closely associated with 5-HT-ir cells in the caudal DRN. These results suggest that the lateral DRN and the ventromedial/interfascicular DRN may be anatomically, morphologically, and neurochemically unique subdivisions of the gerbil DRN.     

Destruction of a sub-population of cortical neurones by suicide transport of volkensin, a lectin from Adenia volkensii.

A method for the destruction of a sub-population of neocortical pyramidal neurones is described. The technique uses unilateral striatal injections of volkensin, a toxic lectin from Adenia volkensii, which undergoes retrograde axonal transport from the site of injection to destroy subcortically projecting pyramidal cells within the neocortex. Striatal volkensin injections produce a significant reduction in the number of large pyramidal neurones of the infragranular layer. The selectivity of the lesion was demonstrated by the preservation of glutamic acid decarboxylase mRNA positive cells, considered to be cortical interneurones within ipsilateral cortex. The binding of the serotonin 1A receptor agonist [3H]-8-hydroxy-2-(n-dipropylamino)tetralin, visualised by autoradiography, was reduced in areas showing loss of large cells, indicating that these receptors may be present on subcortically projecting pyramidal cells. Ricin, another toxin lectin, but effective as a suicide transport agent in only the peripheral nervous system, produced local striatal damage but no cortical cell loss. The selective destruction by volkensin of neocortical pyramidal neurones with subcortical projection targets should aid the neurobiological investigation of such cells. Additionally, data obtained using this technique may provide insights into the interpretation of biochemical findings in neurodegenerative disease in which pyramidal cell loss is a significant feature.     

Differential in vivo clearance of serotonin in rat dorsal raphe nucleus and CA3 region

In vivo chronoamperometric recordings were used to determine if the majority of serotonin transporters (SERTs) in the dorsal raphe nucleus (DRN) are functionally active. This was achieved by comparing the clearance of exogenously applied serotonin (5-HT) from the extracellular fluid (ECF) of the DRN to that in the CA3 region of the hippocampus, an area with lower SERT density. Serotonin was pressure ejected into these regions in anesthetized rats and reproducible electrochemical signals measured by carbon fiber microelectrodes were recorded. Consistent with SERT density as measured by [3H]cyanoimipramine binding in these brain regions (DRN>CA3), clearance of 5-HT was significantly faster in DRN compared to that in the CA3 region. The selective serotonin reuptake inhibitor, fluvoxamine, prolonged 5-HT clearance in both CA3 and DRN. It is known that the norepinephrine transporter (NET) contributes to clearance of 5-HT in the dentate gyrus (DG) but not in CA3. Given that the DRN receives noradrenergic innervation, it was also determined if the NET contributes to 5-HT clearance in the DRN. Destruction of the NET with the neurotoxin 6-hydroxydopamine failed to alter 5-HT clearance parameters in the DRN. These data support the hypothesis that serotonin transporters are functionally active in the DRN, that serotonin clearance is mediated primarily by the SERT in the DRN and that the faster clearance of 5-HT from this region is related to its greater density of functional SERTs.     

Altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter-deficient mice.

BACKGROUND: As a key regulator of serotonergic activity and target of many antidepressant treatments, the serotonin transporter (SERT) represents a potential mediator of anxiety- and depression-related behaviors. Using mice lacking the SERT (SERT KO), we examined the role of SERT function in anxiety- and depression-related behaviors and serotonergic neuron function. METHODS: Serotonin transporter knockout mice were evaluated in paradigms designed to assess anxiety-, depression-, and stress-related behaviors. Dorsal raphe nucleus (DRN) function was assessed by quantitative serotonergic cell counting and extracellular electrical recording of neuronal firing properties. RESULTS: Serotonin transporter knockout mice showed an increase in latency to feed in a novel situation, more immobility in a forced swim, increased escape latency in a shock escape paradigm, and decreased immobility in tail suspension. No differences in anxiety-related behaviors were seen in the open field and the elevated plus maze. Serotonin transporter knockout mice exhibit a 50% reduction in serotonergic cell number and a fourfold decrease in firing rate in the DRN. CONCLUSIONS: Developmental loss of SERT produces altered behaviors in models of depression that are generally opposite to those produced by antidepressant treatment. The reduced serotonergic cell number and firing rate in the DRN of adult SERT KO mice suggest a mechanism for these altered behaviors.     

Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus

Estradiol and progesterone modulate central serotonergic activity; however, the mechanism(s) of action remain unclear. Recently, estradiol-induced progestin receptors (PRs) have been localized within the majority of serotonin (5-HT) neurons in the female macaque dorsal raphe nucleus (DRN; Bethea [1994] Neuroendocrinology 60:50–61). In the present study, we investigated whether estrogen receptors (ERs) and/or PRs exist within 5-HT and/or non-5-HT cells in the female and male rat DRN and whether estradiol treatment alters the expression of these receptors. Young adult female and male Sprague-Dawley rats were gonadectomized, and 1 week later, half of the animals received a subcutaneous Silastic implant of estradiol-17β. Animals were transcardially perfused 2 days later with acrolein and paraformaldehyde, and sequential dual-label immunocytochemistry was performed on adjacent sections by using either a PR antibody or an ERα antibody. This was followed by an antibody to either the 5-HT-synthesizing enzyme, tryptophan hydroxylase (TPH), or to the astrocytic marker, glial fibrillary acidic protein (GFAP). Cells containing immunoreactivity (ir) for nuclear ERs or PRs were identified within the rat DRN in a region-specific distribution in both sexes. No colocalization of nuclear ER-ir or PR-ir with cytoplasmic TPH-ir or GFAP-ir was observed in either sex or treatment, indicating that the steroid target cells are neither 5-HT neurons nor astrocytes. Females were found to have approximately 30% more PR-labeled cells compared with males throughout the DRN (P < 0.05), but no sex difference was detected in the number of neurons demonstrating ER-ir. In both sexes, 2 days of estradiol exposure decreased the number of cells with ER-ir, whereas it greatly increased the number of cells containing PR-ir in several DRN regions (P < 0.005). Collectively, these findings demonstrate the existence of nonserotonergic cells that contain nuclear ERs or PRs within the female and male rat DRN, including estradiol-inducible PRs. These findings point to a species difference in ovarian steroid regulation of 5-HT activity between the macaque and the rat, perhaps transsynaptically via local neurons in the rat brain. J. Comp. Neurol. 391:322–334, 1998. © 1998 Wiley-Liss, Inc.