Caudal Dorsal Raphe Nucleus Research Articles

Effects of Estrogen Therapy on the Serotonergic System in an Animal Model of Perimenopause Induced by 4-Vinylcyclohexen Diepoxide (VCD).

Chronic exposure to 4-vinylcycloxene diepoxide (VCD) in rodents accelerates the natural process of ovarian follicular atresia modelling perimenopause in women. We investigated why estrogen therapy is beneficial for symptomatic women despite normal or high estrogen levels during perimenopause. Female rats (28 d) were injected daily with VCD or oil for 15 d; 55-65 d after the first injection, pellets of 17β-estradiol or oil were inserted subcutaneously. Around 20 d after, the rats were euthanized (control rats on diestrus and estradiol-treated 21 d after pellets implants). Blood was collected for hormone measurement, the brains were removed and dorsal raphe nucleus (DRN), hippocampus (HPC), and amygdala (AMY) punched out for serotonin (5-HT), estrogen receptor β (ERβ), and progesterone receptor (PR) mRNA level measurements. Another set of rats was perfused for tryptophan hydroxylase (TPH) immunohistochemistry in the DRN. Periestropausal rats exhibited estradiol levels similar to controls and a lower progesterone level, which was restored by estradiol. The DRN of periestropausal rats exhibited lower expression of PR and ERβ mRNA and a lower number of TPH cells. Estradiol restored the ERβ mRNA levels and number of serotonergic cells in the DRN caudal subregion. The 5-HT levels were lower in the AMY and HPC in peristropausal rats, and estradiol treatment increased the 5-HT levels in the HPC and also increased ERβ expression in this area. In conclusion, estradiol may improve perimenopause symptoms by increasing progesterone and boosting serotonin pathway from the caudal DRN to the dorsal HPC potentially through an increment in ERβ expression in the DRN.

Afferent and efferent connections of the interpeduncular nucleus with special reference to circuits involving the habenula and raphe nuclei.

The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine.

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions

Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17β-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of behavior. These distinct associations imply that cognition, anxiety-like, and depressive-like behaviors are modulated by unique serotonergic neurocircuitry, opening the possibility of novel avenues of targeted treatment for different types of cognitive and affective disorders.

Sex-dependent programming effects of prenatal glucocorticoid treatment on the developing serotonin system and stress-related behaviors in adulthood

Prenatal stress and overexposure to glucocorticoids (GC) during development may be associated with an increased susceptibility to a number of diseases in adulthood including neuropsychiatric disorders, such as depression and anxiety. In animal models, prenatal overexposure to GC results in hyper-responsiveness to stress in adulthood, and females appear to be more susceptible than males. Here, we tested the hypothesis that overexposure to GC during fetal development has sex-specific programming effects on the brain, resulting in altered behaviors in adulthood. We examined the effects of dexamethasone (DEX; a synthetic GC) during prenatal life on stress-related behaviors in adulthood and on the tryptophan hydroxylase-2 (TpH2) gene expression in the adult dorsal raphe nucleus (DRN). TpH2 is the rate-limiting enzyme for serotonin (5-HT) synthesis and has been implicated in the etiology of human affective disorders. Timed-pregnant rats were treated with DEX from gestational days 18–22. Male and female offspring were sacrificed on the day of birth (postnatal day 0; P0), P7, and in adulthood (P80–84) and brains were examined for changes in TpH2 mRNA expression. Adult animals were also tested for anxiety- and depressive- like behaviors. In adulthood, prenatal DEX increased anxiety- and depressive- like behaviors selectively in females, as measured by decreased time spent in the center of the open field and increased time spent immobile in the forced swim test, respectively. Prenatal DEX increased TpH2 mRNA selectively in the female caudal DRN at P7, whereas it decreased TpH2 mRNA selectively in the female caudal DRN in adulthood. In animals challenged with restraint stress in adulthood, TpH2 mRNA was significantly lower in rostral DRN of prenatal DEX-treated females compared to vehicle-treated females. These data demonstrated that prenatal overexposure to GC alters the development of TpH2 gene expression and these alterations correlated with lasting behavioral changes found in adult female offspring.

Increased anxiety in corticotropin-releasing factor type 2 receptor-null mice requires recent acute stress exposure and is associated with dysregulated serotonergic activity in limbic brain areas

BackgroundCorticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate successful recovery from stress to maintain mental health. They are abundant in the midbrain raphe nuclei, where they regulate serotonergic neuronal activity and have been demonstrated to mediate behavioural consequences of stress. Here, we describe behavioural and serotonergic responses consistent with maladaptive recovery from stressful challenge in CRFR2-null mice.ResultsCRFR2-null mice showed similar anxiety levels to control mice before and immediately after acute restraint stress, and also after cessation of chronic stress. However, they showed increased anxiety by 24 hours after restraint, whether or not they had been chronically stressed.Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were quantified and the level of 5-HIAA in the caudal dorsal raphe nucleus (DRN) was increased under basal conditions in CRFR2-null mice, indicating increased 5-HT turnover. Twenty-four hours following restraint, 5-HIAA was decreased only in CRFR2-null mice, suggesting that they had not fully recovered from the challenge. In efferent limbic structures, CRFR2-null mice showed lower levels of basal 5-HT in the lateral septum and subiculum, and again showed a differential response to restraint stress from controls.Local cerebral glucose utilization (LCMRglu) revealed decreased neuronal activity in the DRN of CRFR2-null mice under basal conditions. Following 5-HT receptor agonist challenge, LCMRglu responses indicated that 5-HT1A receptor responses in the DRN were attenuated in CRFR2-null mice. However, postsynaptic 5-HT receptor responses in forebrain regions were intact.ConclusionsThese results suggest that CRFR2 are required for proper functionality of 5-HT1A receptors in the raphe nuclei, and are key to successful recovery from stress. This disrupted serotonergic function in CRFR2-null mice likely contributes to their stress-sensitive phenotype. The 5-HT content in lateral septum and subiculum was notably altered. These areas are important for anxiety, and are also implicated in reward and the pathophysiology of addiction. The role of CRFR2 in stress-related psychopathologies deserves further consideration.

Effects of continuously enhanced corticotropin releasing factor expression within the bed nucleus of the stria terminalis on conditioned and unconditioned anxiety

The lateral division of the bed nucleus of the stria terminalis (BNST), which forms part of the circuitry regulating fear and anxiety, contains a large number of neurons expressing corticotropin releasing factor (CRF), a neuropeptide that plays a prominent role in the etiology of fear- and anxiety-related psychopathologies. Stress increases CRF expression within BNST neurons, implicating these cells in stress- and anxiety-related behaviors. These experiments examined the effect of chronically enhanced CRF expression within BNST neurons on conditioned and unconditioned anxiety-related behavior by using a lentiviral vector containing a promoter that targets CRF gene over-expression (OE) to CRFergic cells. We found that BNST CRF over-expression did not affect unconditioned anxiety-like responses in the elevated plus maze or basal acoustic startle amplitude. CRF OE induced prior to training weakened sustained fear (conditioned anxiety); when induced after conditioning, CRF OE increased expression of the conditioned emotional memory. Increased BNST CRF expression did not affect plasma corticosterone concentration but did decrease CRFR1 receptor density within the BNST and CRFR2 receptor density within the dorsal portion of the caudal dorsal raphe nucleus. These data raise the possibility that the observed behavioral effects may be mediated by enhanced CRF receptor signaling or compensatory changes in CRF receptor density within these structures. Together, these studies demonstrate that CRF neurons within the lateral BNST modulate conditioned anxiety-like behaviors and also suggest that enhanced CRF expression within these neurons may contribute to inappropriate regulation of emotional memories.

Serotonin 1B Autoreceptors Originating in the Caudal Dorsal Raphe Nucleus Reduce Expression of Fear and Depression-Like Behavior

Serotonin 1B (5-HT(1B)) autoreceptors regulate release of serotonin from terminals of dorsal raphe nucleus (DRN) projections. Expression of 5-HT(1B) in the DRN inversely correlates with behavioral measures of emotion, and viral-mediated overexpression of 5-HT(1B) receptors in the middle DRN inversely reduces measures of fear and anxiety in unstressed rats. Because the caudal subregion of the DRN is important in translating stress into emotional dysregulation, we explored behavioral functions of 5-HT(1B) autoreceptors in the caudal DRN. We manipulated 5-HT(1B) autoreceptor function in rats using either viral-mediated gene transfer into the caudal DRN or systemic injections of the 5-HT(1B) agonist 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP-94253). Rats were tested in forced swim test, open field test, and contextual fear conditioning. Overexpression of 5-HT(1B) in the caudal DRN increased swimming in the forced swim test. It did not alter locomotion or thigmotaxis in the open field test but did reduce conditioned freezing. Freezing was reduced when 5-HT(1B) overexpression was present only during testing but not training. The CP-94253 exerted an inverted U-shaped dose response curve on conditioned freezing, with most pronounced effects seen at 1 mg/kg. At this dose, CP-94253 administered before a fear retention test reduced freezing both during that session and in subsequent drug-free testing, but only when drug was paired with re-exposure to the fear context. The 5-HT(1B) autoreceptors originating in the caudal DRN regulate behavioral expression of helplessness and fear. Because systemic pharmacologic treatment with a 5-HT(1B) agonist facilitates reductions in fear, 5-HT(1B) receptors may be a target for the treatment of certain anxiety disorders.

Overexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner

Previous studies showed that chronic estrogen treatment increases tryptophan hydroxylase-2 (TpH2) mRNA in the caudal dorsal raphe nucleus (DRN), and this increase was associated with decreased anxiety. The present study explored the interaction of estrogen and targeted, bidirectional manipulation of TpH2 expression in the caudal DRN by knockdown or viral overexpression, to decrease or increase tryptophan hydroxylase expression respectively, on anxiety behavior. Rats were ovariectomized and replaced with empty or estradiol capsules (OVX, OVX/E, respectively). Animals received microinfusions of either antisense TpH2 or control morpholino oligonucleotides into caudal DRN and were later tested in the open field test. A separate group of animals were microinfused with TpH2-GFP or GFP-only herpes simplex viral vectors into caudal DRN and tested in the open field. The bidirectional impact of manipulations on TpH2 expression was confirmed using a combination of quantitative protein and mRNA measurements; TpH2 expression changes were limited to discrete subregions of DRN that were targeted by the manipulations. Estradiol decreased anxiety in all behavioral measures. In the OVX/E group, TpH2 knockdown significantly decreased time spent in the center of the open field, but not in the OVX group, suggesting that TpH2 knockdown reduced the anxiolytic effects of estrogen. Conversely, TpH2 overexpression in the OVX group mimicked the effects of estrogen, as measured by increased time spent in the center of the open field. These results suggest that estrogen and TpH2 in the caudal DRN have a critical interaction in regulating anxiety-like behavior.

Brief pup exposure induces Fos expression in the lateral habenula and serotonergic caudal dorsal raphe nucleus of paternally experienced male California mice (Peromyscus californicus)

Fathers play a substantial role in infant care in a small but significant number of mammalian species, including humans. However, the neural circuitry controlling paternal behavior is much less understood than its female counterpart. In order to characterize brain areas activated by paternal care, male California mice were separated from their female mate and litter for 3 h and then exposed to a pup or a control object (a glass pebble with the approximate size and oblong shape of a newborn pup) for 10 min. All males receiving a pup showed a strong paternal response towards it, whereas males receiving a pebble interacted with it only occasionally. Despite the clear behavioral differences, exposure to a pup did not increase Fos-like immunoreactivity (Fos-LIR) compared to a pebble in brain areas previously found to be associated with parental care, including the medial preoptic nucleus and medial bed nucleus of the stria terminalis. Pup exposure did, however, significantly increase Fos-LIR in the lateral habenula (LHb) and in predominantly serotonergic neurons in the caudal dorsal raphe nucleus (DRC), as compared to pebble exposure. Both the LHb and DRC are known to be involved in the behavioral responses to strong emotional stimuli; therefore, these areas might play a role in controlling parental behavior in male California mice.

Estrogen receptor beta regulates the expression of tryptophan-hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei

Dysfunctions of the brain 5-HT system are often associated with affective disorders, such as depression. The raphe nuclei target the limbic system and most forebrain areas and constitute the main source of 5-HT in the brain. All 5-HT neurons express tryptophan hydroxylase-2 (TPH2), the brain specific, rate-limiting enzyme for 5-HT synthesis. Estrogen receptor (ER) beta agonists have been shown to attenuate anxiety- and despair-like behaviors in rodent models. Therefore, we tested the hypothesis that ERbeta may contribute to the regulation of gene expression in 5-HT neurons of the dorsal raphe nuclei (DRN) by examining the effects of systemic and local application of the selective ERbeta agonist diarylpropionitrile (DPN) on tph2 mRNA expression. Ovariectomized (OVX) female rats were injected s.c. with DPN or vehicle once daily for 8 days. In situ hybridization revealed that systemic DPN-treatment elevated basal tph2 mRNA expression in the caudal and mid-dorsal DRN. Behavioral testing of all animals in the open field (OF) and on the elevated plus maze (EPM) on days 6 and 7 of treatment confirmed the anxiolytic nature of ERbeta activation. Another cohort of female OVX rats was stereotaxically implanted bilaterally with hormone-containing wax pellets flanking the DRN. Pellets contained 17-beta-estradiol (E), DPN, or no hormone. Both DPN and E significantly enhanced tph2 mRNA expression in the mid-dorsal DRN. DPN also increased tph2 mRNA in the caudal DRN. DPN- and E-treated rats displayed a more active stress-coping behavior in the forced-swim test (FST). No behavioral differences were found in the OF or on the EPM. These data indicate that ERbeta acts at the level of the rat DRN to modulate tph2 mRNA expression and thereby influence 5-HT synthesis in DRN subregions. Our results also suggest that local activation of ERbeta neurons in the DRN may be sufficient to decrease despair-like behavior, but not anxiolytic behaviors.

Trafficking of neurokinin‐1 receptors in serotonin neurons is controlled by substance P within the rat dorsal raphe nucleus

Substance P (SP) modulates serotonin neurotransmission via neurokinin-1 receptors (NK1rs), and exerts regulatory effects on mood through habenular afferents to the dorsal raphe nucleus (DRN). We have previously demonstrated that, in the caudal DRN of rat, some serotonin neurons are endowed with NK1rs that are mostly cytoplasmic, whereas these receptors are mostly membrane bound in non-serotonin neurons. Here, we first examined by double-labeling immunocytochemistry the relationships between SP axon terminals and these two categories of DRN neurons. Almost half of the SP terminals were synaptic and many were in close contact with serotonin dendrites, but never with non-serotonin dendrites. In additional double-immunolabeling experiments, most if not all dendrites bearing membranous NK1rs appeared to be GABAergic. Treatment with the selective neurokinin-1 antagonist RP67580 modified the subcellular distribution of NK1rs in serotonin neurons. At 1 h after administration of a single dose, the receptor distribution was unchanged in both dendritic types but, after daily administration for 7 or 21 days, the plasma membrane and cytoplasmic density of NK1rs were increased in serotonin dendrites, without any change in non-serotonin dendrites. These treatments also increased NK1r gene expression in the caudal DRN. Lastly, a marked increase in the membrane (but not cytoplasmic) density of NK1rs was measured in serotonin dendrites after bilateral habenular lesion. These results suggest that the trafficking of NK1rs represents a cellular mechanism in control of the modulation of serotonin neuron activity by SP in DRN.

From here to paternity: Neural correlates of the onset of paternal behavior in California mice ( Peromyscus californicus)

In a minority of mammalian species, including humans, fathers play a significant role in infant care. Compared to maternal behavior, the neural and hormonal bases of paternal care are poorly understood. We analyzed behavioral, neuronal and neuropeptide responses towards unfamiliar pups in biparental California mice, comparing males housed with another male (“virgin males”) or with a female before (“paired males”) or after (“new fathers”) the birth of their first litter. New fathers approached pups more rapidly and spent more time engaging in paternal behavior than virgin males. In each cage housing two virgin males, one was spontaneously paternal and one was not. New fathers and paired males spent more time sniffing and touching a wire mesh ball containing a newborn pup than virgin males. Only new fathers showed significantly increased Fos-like immunoreactivity in the medial preoptic nucleus (MPO) following exposure to a pup-containing ball, as compared to an empty ball. Moreover, Fos-LIR in the bed nucleus of the stria terminalis (STMV and STMPM) and caudal dorsal raphe nucleus (DRC) was increased in new fathers, independent of test condition. No differences were found among the groups in Fos-LIR in oxytocinergic or vasopressinergic neurons. These results suggest that sexual and paternal experiences facilitate paternal behavior, but other cues play a role as well. Paternal experience increases Fos-LIR induced by distal pup cues in the MPO, but not in oxytocin and vasopressin neurons. Fatherhood also appears to alter neurotransmission in the BNST and DRC, regions implicated in emotionality and stress-responsiveness.

Acoustic stimulation in vivo and corticotropin-releasing factor in vitro increase tryptophan hydroxylase activity in the rat caudal dorsal raphe nucleus

Exposure of rats to unpredictable loud sound pulses increases activity of the rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase (TPH), in the median raphe nucleus (MnR) and a mesolimbocortical serotonergic system. Corticotropin-releasing factor (CRF)-induced activation of a subset of serotonergic neurons in the caudal dorsal raphe nucleus (DR) may underlie stress-related increases in TPH activity in the MnR and a mesolimbocortical serotonergic system. An in vivo acoustic stimulation paradigm and an in vitro brain slice preparation were designed to test the hypothesis that stress-related stimuli and CRF receptor activation have convergent actions on TPH activity in the caudal DR (DRC). We measured 5-hydroxytryptophan (5-HTP) accumulation as an index of TPH activity following inhibition of aromatic amino acid decarboxylase (using NSD-1015). To examine effects of acoustic stimulation on TPH activity, male Wistar rats, pretreated with NSD-1015, were exposed to a 30 min sham, predictable or unpredictable acoustic stimulation paradigm; brains were frozen and microdissected for analyses of tissue 5-HTP concentrations in subregions of the DR. To examine the effect of CRF receptor activation on TPH activity, freshly prepared brain slices were exposed to CRF (0–2000 nM) for 10 min in the presence of NSD-1015, then frozen and microdissected for analysis of tissue 5-HTP concentrations. Increases in TPH activity in the DRC, but not other subregions, were observed in both paradigms. These findings are consistent with the hypothesis that stress-related increases in TPH activity are mediated via effects of CRF or CRF-related neuropeptides on a mesolimbocortical serotonergic system originating in the DRC.

Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: Relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus

Serotonergic systems arising from the mid-rostrocaudal and caudal dorsal raphe nucleus (DR) have been implicated in the facilitation of anxiety-related behavioral responses to anxiogenic drugs or aversive stimuli. In this study we attempted to determine a threshold to engage serotonergic neurons in the DR following exposure to aversive conditions in an anxiety-related behavioral test. We manipulated the intensity of anxiogenic stimuli in studies of male Wistar rats by leaving them undisturbed (CO), briefly handling them (HA), or exposing them to an open-field arena for 15-min under low-light (LL: 8–13lx) or high-light (HL: 400–500lx) conditions. Rats exposed to HL conditions responded with reduced locomotor activity, reduced time spent exploring the center of the arena, a lower frequency of rearing and grooming, and an increased frequency of facing the corner of the arena compared to LL rats. Rats exposed to HL conditions had small but significant increases in c-Fos expression within serotonergic neurons in subdivisions of the rostral DR. Exposure to HL conditions did not alter c-Fos responses in serotonergic neurons in any other DR subdivision. In contrast, rats exposed to the open-field arena had increased c-Fos expression in non-serotonergic cells throughout the DR compared to CO rats, and this effect was particularly apparent in the dorsolateral part of the DR. We conclude that exposure to HL conditions, compared to LL conditions, increased anxiety-related behavioral responses in an open-field arena but this stimulus was at or below the threshold required to increase c-Fos expression in serotonergic neurons.

Activity-dependent heterogeneous populations of nitric oxide synthase neurons in the rat dorsal raphe nucleus

The brainstem dorsal raphe nucleus (DRN) contains an abundant distribution of nitric oxide (NO) synthase (NOS)-containing neuronal profiles in two distinct populations: faint- and intense-immunoreactive cells in midline (ventromedial and dorsomedial) and lateral wing subregions, respectively. This study tested the hypothesis that different functional dynamics underlie the topography of NOS-containing cells in the DRN rostrocaudal and mediolateral neuraxis by using a capsaicin challenge paradigm (50 mg/kg, subcutaneous). Compared with vehicle, capsaicin significantly and preferentially increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, an index of constitutive NOS) reactivity in the rostral midline and caudal lateral wing subregions. Furthermore, capsaicin activated more Fos-positive cells than vehicle within all subregions of the DRN but with a caudal versus rostral predominance in activation pattern. In addition, a high proportion of capsaicin-induced Fos cells in the midline but almost none in lateral wing stained for NADPH-d. These observations suggest the existence of two functionally distinct populations of NOS neurons in the DRN. Furthermore, capsaicin increased galanin immunoreactivity with predominant staining in cell soma and fiber processes in midline and lateral wing subregions of the nucleus, respectively. The total capsaicin-induced galanin immunoreactivity was higher in rostral versus caudal DRN, and a high proportion of galanin-positive cells in the midline also contained NADPH-d and neuronal NOS, thus suggesting a potential NO–galanin interaction in these neurons. The differential pattern of Fos/NADPH-d colocalization across the nucleus suggests that midline and lateral wing NOS neurons of the DRN express their neuromodulatory actions on discrete efferent targets via different intracellular mechanisms.

More tryptophan hydroxylase in the brainstem dorsal raphe nucleus in depressed suicides

Deficient serotonin neurotransmission in suicide is indicated by reduced brainstem serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), fewer 5-HT 1A autoreceptors and reduced cortical serotonin transporter binding in suicide victims. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of 5-HT, and alterations in TPH could explain some of these findings. We sought to determine the amount of TPH immunoreactivity (TPH-IR) in the dorsal (DRN) and median (MRN) raphe nuclei in suicides and controls. Brainstems of suicide victims and controls ( n = 11 pairs) were collected at autopsy, matched for age, sex and postmortem interval, frozen and sectioned (20 μm). Immunoautoradiography, using an antibody to label TPH, was performed, slides exposed to film and autoradiograms quantified by a computer-based image analysis system. We examined sections every 1000 μm throughout the whole length of the nucleus, performing statistical analysis only on those subjects for whom the raphe was complete ( n = 8 pairs). TPH-IR (μCi/g) was higher in suicides than controls (S: 300.8 ± 70.8 vs. C: 259.6 ± 40.7, t = 2.57, df = 7, P = 0.04) in the dorsal raphe nucleus (DRN), and not different between suicides and controls (S: 251.3 ± 44.2 vs. C: 235.9 ± 27.4, t = 1.49, df = 7, P = 0.18) in the MRN. DRN TPH-IR was higher in male suicide victims (MS) compared to male controls (MC; MS: 318.4 ± 54.4 vs. MC: 271.9 ± 22.5, t = 2.66, df = 6, P = 0.03). The analysis of TPH-IR area and density at each DRN rostrocaudal levels showed higher area and density in suicides compared to controls in the rostral DRN and lower area and density in the caudal DRN. TPH-IR, an index of the amount of TPH enzyme, in the DRN is higher in depressed suicides. More TPH may be an upregulatory homeostatic response to impaired serotonin release or less autoreceptor activation. Alternatively, the serotonin impairment in suicide may be due to hypofunctional serotonin-synthesizing enzyme.

Serotonin-related gene expression in female monkeys with individual sensitivity to stress

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. In this study, the expression of four genes pivotal to serotonin neural function was assessed in monkeys previously categorized as highly stress resistant ( n=3; normal menstrual cyclicity through two stress cycles), medium stress resistant ( n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e. stress-sensitive; n=4; anovulatory as soon as stress is initiated). In situ hybridization and quantitative image analysis was used to measure mRNAs coding for SERT (serotonin transporter), 5HT1A autoreceptor, MAO-A and MAO-B (monoamine oxidases) at six levels of the dorsal raphe nucleus (DRN). Optical density (OD) and positive pixel area were measured with NIH Image software. In addition, serotonin neurons were immunostained and counted at three levels of the DRN. Finally, each animal was genotyped for the serotonin transporter long polymorphic region (5HTTLPR). Stress sensitive animals had lower expression of SERT mRNA in the caudal region of the DRN ( P<0.04). SERT mRNA OD in the caudal DRN was positively correlated with serum progesterone during a pre-stress control cycle ( P<0.0007). 5HT1A mRNA OD signal tended to decline in the stress-sensitive group, but statistical difference between averages was lacking in analysis of variance. However, 5HT1A mRNA signal was positively correlated with control cycle progesterone ( P<0.009). There was significantly less MAO-A mRNA signal in the stress-sensitive group ( P<0.007) and MAO-A OD was positively correlated with progesterone from a pre-stress control cycle ( P<0.007). MAO-B mRNA exhibited a similar downward trend in the stress-sensitive group. MAO-B OD also correlated with control cycle progesterone ( P<0.003). There were significantly fewer serotonin neurons in the stress-sensitive group. All animals contained only the long form of the 5HTTLPR. Thus, all serotonin-related mRNAs examined in the dorsal raphe to date were lower (SERT, MAO-A) or exhibited a lower trend (5HT1A, MAO-B) in the stress sensitive animals, which probably reflects the lower number of serotonin neurons present.

Subdivisions of the dorsal raphe nucleus projecting to the lateral geniculate nucleus and primary visual cortex in the Mongolian gerbil

The mammalian dorsal raphe nucleus (DRN) is composed of sub-divisions with different anatomical and functional properties. Using cholera toxin subunit B as a retrograde tracer, DRN subdivisions projecting to the lateral geniculate nucleus and to the primary visual cortex were examined in the Mongolian gerbil. DRN neurons projecting to the lateral geniculate nucleus were observed in the lateral DRN (rostrally) and in the ventromedial DRN (caudally), while DRN cells projecting to the primary visual cortex were observed at all rostral-caudal levels in the ventromedial DRN. These results demonstrate a significant overlap between the DRN projections to the lateral geniculate and superior colliculus, and show that only the caudal ventromedial DRN projects to all three major visual targets: the lateral geniculate nucleus, primary visual cortex, and superior colliculus. Since the DRN is involved in depression and other neuropsychiatric disorders, as well as is affected by many psychotropic substances, these data may help to develop new treatments and therapies targeting specific DRN subdivisions.

Corticotropin Releasing Hormone Type 2 Receptors in the Dorsal Raphe Nucleus Mediate the Behavioral Consequences of Uncontrollable Stress

Uncontrollable shock produces a constellation of behavioral changes that are not observed after equivalent escapable shock. These include interference with escape and potentiation of fear conditioning. The activation of corticotropin-releasing hormone (CRH) receptors within the caudal dorsal raphe nucleus (DRN) during inescapable tailshock (IS) has been shown to be critical for the development of these behavioral changes. CRH binds to two receptor subtypes, both of which are found in the DRN. The present set of studies examined which CRH receptor subtype mediates the effects of IS. Intra-DRN administration of the CRH(2) receptor antagonist anti-sauvagine-30 before IS dose-dependently blocked IS-induced behavioral changes; the CRH(1) receptor antagonist 2-methyl-4-(N-propyl-N-cycloproanemethylamino)-5-chloro-6-(2,4,6-trichloranilino)pyrimidine (NBI27914), administered in the same manner, did not. Moreover, the highly selective CRH(2) receptor agonist urocortin II (Ucn II) dose-dependently caused behavioral changes associated with IS in the absence of shock. Ucn II was effective at doses 100-fold lower than those previously required for CRH. The relationship between CRH(2) receptors and DRN 5-HT is discussed.

The Role of Corticotropin-Releasing Hormone in the Dorsal Raphe Nucleus in Mediating the Behavioral Consequences of Uncontrollable Stress

Inescapable shock (IS) produces subsequent interference with escape behavior and increased fear conditioning that has been linked to increased activity and release of serotonin (5-HT) from neurons within the caudal dorsal raphe nucleus (DRN) both at the time of IS and later behavioral testing. Extrahypothalamic corticotropin-releasing hormone (CRH) has been implicated in many stress-related phenomena and has recently been shown to increase DRN 5-HT activity in the same caudal DRN area at which IS increases 5-HT activity. The current set of studies therefore examined the role of CRH in mediating the behavioral sequelae of IS. Intra-DRN microinjection of the nonselective CRH receptor antagonist d-Phe CRH (12-41) blocked the IS-induced behavioral changes when administered before IS but not when administered before later behavioral testing. Furthermore, intra-DRN administration of CRH in the absence of IS dose-dependently mimicked the effects of IS and interfered with escape behavior and increased fear conditioning 24 hr later. This effect was specific to injection of CRH into the caudal DRN and was not produced by microinjection into the rostral DRN. Intracerebroventricular CRH produced escape deficits and potentiated fear conditioning 24 hr later at only much higher doses, further confirming the site specificity of the effects. The potential role of the caudal DRN in states of anxiety is discussed.