Functional dissociation between serotonergic pathways in dorsal and ventral hippocampus in psychotomimetic drug-induced locomotor hyperactivity and prepulse inhibition in rats

Altered hippocampal function and brain serotonin activity are implicated in the development and symptoms of schizophrenia. We have previously shown that lesions of the median raphe nucleus, but not the dorsal raphe nucleus, produced a marked enhancement of locomotor hyperactivity induced by phencyclidine and disruption of prepulse inhibition. The dorsal and ventral hippocampus receive serotonin projections predominantly from the median raphe nucleus and dorsal raphe nucleus, respectively. Therefore, we investigated the effect of local lesions of serotonin projections into the dorsal and ventral hippocampus on psychotomimetic drug-induced locomotor hyperactivity and prepulse inhibition. Male Sprague-Dawley rats were anaesthetized with pentobarbitone and stereotaxically microinjected with 5 microg of the serotonergic neurotoxin 5,7-dihydroxytryptamine into either the dorsal or the ventral hippocampus. Two weeks after surgery, dorsal hippocampus-lesioned rats showed a 100% enhancement of the locomotor hyperactivity caused by phencyclidine treatment and a slight but significant reduction of the effect of amphetamine. Prepulse inhibition was significantly disrupted in lesioned rats and serotonin levels in the dorsal hippocampus were reduced by 80%. Rats with lesions of the ventral hippocampus showed 85% depletion of serotonin and partial disruption of prepulse inhibition, but no significant changes in the effect of phencyclidine or amphetamine. These results suggest that serotonin projections from the median raphe nucleus to the dorsal hippocampus play an important role in locomotor hyperactivity and prepulse inhibition in rats, animal models of aspects of schizophrenia. This suggests that these serotonin projections may be involved in the pathophysiology of schizophrenia symptomology.     

Corticotropin-releasing factor in the dorsal raphe nucleus increases medial prefrontal cortical serotonin via type 2 receptors and median raphe nucleus activity

Interactions between central corticotropin-releasing factor (CRF) and serotonergic systems are believed to be important for mediating fear and anxiety behaviors. Recently we demonstrated that infusions of CRF into the rat dorsal raphe nucleus result in a delayed increase in serotonin release within the medial prefrontal cortex that coincided with a reduction in fear behavior. The current studies were designed to study the CRF receptor mechanisms and pathways involved in this serotonergic response. Infusions of CRF (0.5 μg/0.5 μL) were made into the dorsal raphe nucleus of urethane-anesthetized rats following either inactivation of the median raphe nucleus by muscimol (25 ng/0.25 μL) or antagonism of CRF receptor type 1 or CRF receptor type 2 in the dorsal raphe nucleus with antalarmin (25–50 ng/0.5 μL) or antisauvagine-30 (2 μg/0.5 μL), respectively. Medial prefrontal cortex serotonin levels were measured using in-vivo microdialysis and high-performance liquid chromatography with electrochemical detection. Increased medial prefrontal cortex serotonin release elicited by CRF infusion into the dorsal raphe nucleus was abolished by inactivation of the median raphe nucleus. Furthermore, antagonism of CRF receptor type 2 but not CRF receptor type 1 in the dorsal raphe nucleus abolished CRF-induced increases in medial prefrontal cortex serotonin. Follow-up studies involved electrical stimulation of the central nucleus of the amygdala, a source of CRF afferents to the dorsal raphe nucleus. Activation of the central nucleus increased medial prefrontal cortex serotonin release. This response was blocked by CRF receptor type 2 antagonism in the dorsal raphe. Overall, these results highlight complex CRF modulation of medial prefrontal cortex serotonergic activity at the level of the raphe nuclei.     

Evidence for G protein mediation of serotonin- and GABAB- induced hyperpolarization of rat dorsal raphe neurons

In vitro intracellular recording techniques in the rat brain slice preparation demonstrate that both serotonin (5-HT) and baclofen (a GABA_B-receptor agonist) inhibit 5-HT neurons in the dorsal raphe nucleus by inducing a hyperpolarization of membrane potential and a decrease in apparent input resistance (R_in). Similar to previous results with 5-HT, baclofen-mediated inhibition of 5-HT neurons also shows an apparent reversal potential (E_rev) of approximately-90 mV, consistent with mediation by K channels. In slices from rats that had previously received a local injection of pertussis toxin (0.5 μg) immediately rostral to the dorsal raphe nucleus, there was a virtually complete blockade of inhibition induced by both the serotonin autoreceptor and the GABA_B-receptor. Intracellular injection of the stable GTP analog (guanosine-5−O-(3-thiotriphosphate); GTPγS) mimicked the actions of both 5-HT and baclofen. The inhibitory actions of GTPγS were not additive with those of either 5-HT or baclofen, suggesting they share some common effector system. The stable cAMP analog (8-bromo-adenosine-3′, 5′-cyclic monophosphate (8-Br-cAMP) had no effect on membrane potential or apparent input resistance and did not block the inhibitory actions mediated by 5-HT or baclofen. The local injection of pertussis toxin (0.5 γg) caused a far greater blockade of 5-HT and baclofen-mediated inhibition than the intracerebroventricular (i.c.v.) injection of pertussis toxin (1.0 γg). In parallel sets of animals with i.c.v. and local injections, we measured the pertussis toxin-mediated ADP-ribosylation of G proteins in membranes prepared from dorsal raphe nucleus. These biochemical studies showed that sensitivities to 5-HT and baclofen correlated with the concentration of remaining non-ADP-ribosylated G proteins following in vivo pertussis toxin injection. In summary, these results provide evidence for the role of a G protein(s) in the mediation of the cAMP-independent increase in potassium conductance in 5-HT neurons of dorsal raphe nucleus induced by both 5-HT_1A and GABA_B-receptors.     

Role of serotonin in the apomorphine-induced increase of adrenomedullary ornithine decar☐ylase activity

The role of serotonin in the regulation of adrenomedullary ornithine decar☐ylase (ODC) activity has been explored in rats after systemic administration of p-chlorophenylalanine (PCPA) and intraventricular 5,6-dihydroxytryptamine (DHT) or in animals with electrolytic lesions of the medial and dorsal raphe nuclei. None of the treatments produced any alteration in endogenous ODC activity. However, all except lesion of the dorsal raphe nucleus significantly potentiated the induction of adrenomedullary ODC produced by apomorphine (APM) administration. It is suggested that serotonergic fibers originating partly in the medial raphe nucleus exert a tonic inhibitory action over the APM-induced increase in adrenomedullary ODC activity.     

Neuronal projections from the mesencephalic raphe nuclear complex to the suprachiasmatic nucleus and the deep pineal gland of the golden hamster (Mesocricetus auratus)

Neuronal projections from the mesencephalic raphe system to the suprachiasmatic nucleus and the pineal complex were mapped in this study of the golden hamster, by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and the retrograde tracer cholera toxin subunit B. From the median raphe nucleus, a rostral projection ascended in the ventral part of the mesencephalon to continue in the medial forebrain bundle of the forebrain. Nerve fibres from this bundle innervated the ventral and medial parts of the suprachiasmatic nucleus. At the level of the interpeduncular nucleus of the mesencephalon, fibres of the ventral bundle bent dorsally to reach the epithalamic area and to continue in the forebrain in a periventricular position. Some of these fibres innervated the dorsal tip of the suprachiasmatic nucleus. The dorsal raphe nucleus was the origin of a nerve fibre bundle, located in the periaqueductal gray of the mesencephalon, innervating the deep pineal gland and pineal stalk. Injection of cholera toxin B into the suprachiasmatic nucleus labelled cells in the median raphe. Combination of the retrograde tracing from the suprachiasmatic nucleus and serotonin transmitter immunohistochemistry showed that some of the cholera toxin B–immunoreactive nerve cells also contained serotonin. Thus, this study of the golden hamster shows a serotonergic projection from the median raphe nucleus to the suprachiasmatic nucleus and a projection from the dorsal raphe nucleus to the deep pineal gland supporting physiological indications of an influence of serotonin on the photoreceptive circadian system of the brain. J. Comp. Neurol. 399:73–93, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for G protein mediation of serotonin- and GABAB-induced hyperpolarization of rat dorsal raphe neurons

In vitro intracellular recording techniques in the rat brain slice preparation demonstrate that both serotonin (5-HT) and baclofen (a GABAB-receptor agonist) inhibit 5-HT neurons in the dorsal raphe nucleus by inducing a hyperpolarization of membrane potential and a decrease in apparent input resistance (Rin). Similar to previous results with 5-HT, baclofen-mediated inhibition of 5-HT neurons also shows an apparent reversal potential (Erev) of approximately -90 mV, consistent with mediation by K channels. In slices from rats that had previously received a local injection of pertussis toxin (0.5 microgram) immediately rostral to the dorsal raphe nucleus, there was a virtually complete blockade of inhibition induced by both the serotonin autoreceptor and the GABAB-receptor. Intracellular injection of the stable GTP analog (guanosine-5'-O-(3-thiotriphosphate); GTP gamma S) mimicked the actions of both 5-HT and baclofen. The inhibitory actions of GTP gamma S were not additive with those of either 5-HT or baclofen, suggesting they share some common effector system. The stable cAMP analog (8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP] had no effect on membrane potential or apparent input resistance and did not block the inhibitory actions mediated by 5-HT or baclofen. The local injection of pertussis toxin (0.5 microgram) caused a far greater blockade of 5-HT and baclofen-mediated inhibition than the intracerebroventricular (i.c.v.) injection of pertussis toxin (1.0 micrograms). In parallel sets of animals with i.c.v. and local injections, we measured the pertussis toxin-mediated ADP-ribosylation of G proteins in membranes prepared from dorsal raphe nucleus. These biochemical studies showed that sensitivities to 5-HT and baclofen correlated with the concentration of remaining non-ADP-ribosylated G proteins following in vivo pertussis toxin injection. In summary, these results provide evidence for the role of a G protein(s) in the mediation of the cAMP-independent increase in potassium conductance in 5-HT neurons of dorsal raphe nucleus induced by both 5-HT1A- and GABAB-receptors.     

Infusion of flesinoxan into the amygdala blocks the fear-potentiated startle

In a previous study it was demonstrated that flesinoxan, a selective serotonin (5-HT)1A receptor agonist, had anxiolytic properties in the fear-potentiated startle paradigm. The present study investigated the putative site of action of flesinoxan in this paradigm. Flesinoxan infused either into the dorsal raphe nucleus or the median raphe nucleus did not affect startle potentiation. Bilateral infusion of flesinoxan into the central nucleus of the amygdala on the other hand, dose-dependently blocked the fear-potentiated startle response. These data indicate that flesinoxan exerts it anxiolytic effects in the fear-potentiated startle paradigm via the central nucleus of the amygdala, whereas the dorsal and median raphe nuclei are not directly involved in this process.     

An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat

The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after ³H-proline micro-injections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudate-putamen and the parieto-temporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body. Further studies were done to test whether the fiber tracts travelling in the MFB contained 5-HT. Unilateral (left) injections of 5,7-dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of ³H-proline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5-HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5-hydroxytryptophan administration. The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5-HT. The results agree with the neuroanatomical distribution of the 5-HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.     

Serotonergic and nonserotonergic projections from the rat interpeduncular nucleus to the septum, hippocampal formation and raphe: a combined immunocytochemical and fluorescent retrograde labelling study of neurons in the apical subnucleus

This study examined the subnuclear distribution and transmitter content of neurons in the interpeduncular nucleus (IPN) that projected to the septum, dorsal hippocampal formation, and/or raphe. Following the injection of fast blue into the medial septum/diagonal band nucleus and rhodamine-conjugated microspheres into the dorsal hippocampal formation (or vice versa), retrogradely-labelled cells were found throughout the apical subnucleus of the IPN. Incubation of these sections with 5-hydroxytryptamine antiserum indicated that a small number of fast blue- or rhodamine-positive cells also contained serotonin. Occasional apical cells contained both fast blue and rhodamine, indicating a dual projection via collaterals to both the septum and hippocampus. Injection of either dye into the raphe also retrogradely labelled cells in the apical subnucleus, none of which contained serotonin. These results suggest that the IPN may function to integrate the activity within subcortical limbic nuclei via widespread serotonergic and non-serotonergic projections.     

Role of serotonin in the apomorphine-induced increase of adrenomedullary ornithine decarboxylase activity

The role of serotonin in the regulation of adrenomedullary ornithine decar?ylase (ODC) activity has been explored in rats after systemic administration of p-chlorophenylalanine (PCPA) and intraventricular 5,6-dihydroxytryptamine (DHT) or in animals with electrolytic lesions of the medial and dorsal raphe nuclei. None of the treatments produced any alteration in endogenous ODC activity. However, all except lesion of the dorsal raphe nucleus significantly potentiated the induction of adrenomedullary ODC produced by apomorphine (APM) administration. It is suggested that serotonergic fibers originating partly in the medial raphe nucleus exert a tonic inhibitory action over the APM-induced increase in adrenomedullary ODC activity.     

Phencyclidine-induced locomotor hyperactivity is enhanced in mice after stereotaxic brain serotonin depletion

The aim of this study was to investigate the role of forebrain serotonin projections in behavioural models with relevance to schizophrenia. Mice received stereotaxic micro-injections of the serotonin neurotoxin 5,7-dihydroxytryptamine into the median raphe nucleus (MRN). Two weeks later, MRN-lesioned mice were hyperactive at baseline and showed enhanced locomotor hyperactivity induced by phencyclidine. In contrast, no lesion effect was observed on the locomotor hyperactivity induced by amphetamine treatment or on prepulse inhibition. Lesioned mice showed a 68% depletion of serotonin in the hippocampus and 31% depletion in the striatum. These data confirm previous studies in rats that selective serotonin depletion in the brain enhances the effect of phencyclidine, but not amphetamine, on locomotor activity. This enhanced action of phencyclidine is likely to be mediated by the absence of serotonin-mediated behavioural inhibition in the hippocampus, leaving the psychostimulant effects of phencyclidine unopposed. Taken together with previous studies in rats, these studies in mice suggest that serotonin release in the dorsal hippocampus constitutes a behavioural inhibitory pathway normally involved in dampening excessive behavioural stimulation. Dysfunction of this pathway could be involved in psychosis and its stimulation could be a potential mechanism of action of antipsychotic drugs.     

Effects of electrical stimulation of the lateral habenula on single-unit activity of raphe neurons.

Recent anatomical studies with horseradish peroxidase injections into the anterior raphe have demonstrated that the nucleus raphe dorsalis in the rat receives a major afferent input from the lateral habenula (LHb). The present study examined electrophysiologically the effects of electrical stimulation of the LHb on the spontaneous activity of midbrain and anterior pontine raphe units in anesthetized rats. The results showed that: (a) LHb stimulation (1 or 10 Hz, 0.5 to 1.0 mA) suppressed the activity of most raphe units, with the effects outlasting the duration of the stimulation in some instances; the raphe cells which showed periods of suppression during LHb stimulation were both those of the classical serotonin type (N = 26), characterized by slow regular baseline firing rates, and other raphe cells (N=52) with faster baseline rates (to 60/s); (b) inhibition of unit activity was much less pronounced for non-raphe cells lateral to the midline; (c) anatomical control stimulation points dorsal to the LHb did not alter raphe unit activity; and (d) the pathway from the habenula to the raphe may involve a dorsal route. After a knife cut through the superior colliculus-central gray at the level of the interpeduncular nucleus, the effects of habenular stimulation were substantially reduced. Conversely, stimulation of the superior colliculus just posterior to the habenula (presumably containing descending fibers from the habenula) markedly suppressed raphe unit activity. In summary, the present electrophysiologic findings were consistent with the view that activation of habenular afferent fibers to the raphe exerted a major inhibitory influence on the spontaneous activity of midbrain and pontine raphe neurons. Considerably smaller effects were exerted on lateral reticular cells. A dorsal pathway may be involved in mediating the habenular effects on raphe activity.     

Further studies on the activation of rat median raphe serotonergic neurons by inescapable sound stress

Previous studies, using a biochemical measure of serotonergic neuronal function, show that inescapable, randomly presented sound pulses activate serotonergic neurons in the rat median raphe but not dorsal raphe nucleus. The present study reveals that this activation also occurs in serotonin projection areas, in hippocampus, nucleus accumbens and cortex but not in caudate nucleus. The selectivity of this response is examined by comparing the response to sound stress with that produced by morphine, a treatment known to selectively activate dorsal raphe but not median raphe serotonergic neurons. Two approaches are used in Sprague-Dawley rat to measure the activation of serotonergic neurons: (1) determination ex vivo of accumulation of 5-hydroxytryptophan (5-HTP) in tissue from the dorsal and median raphe nuclei, hippocampus, cortex, caudate nucleus, and nucleus accumbens following in vivo inhibition of aromatic amino acid decarboxylase; and (2) measurement of extracellular serotonin levels in hippocampus, caudate nucleus, and nucleus accumbens. Sound stress increases 5-HTP accumulation in median raphe nucleus, hippocampus, cortex, and nucleus accumbens, but not dorsal raphe nucleus or caudate nucleus. Sound stress also enhances extracellular serotonin levels in hippocampus and nucleus accumbens, but not caudate nucleus. In contrast, the morphine treatment enhances 5-HTP accumulation in dorsal raphe nucleus, cortex and caudate nucleus, but not in median raphe nucleus, hippocampus or nucleus accumbens. Furthermore, it increases extracellular serotonin levels in only the caudate nucleus. The combined effects of sound stress and morphine on 5-HTP accumulation are identical to those obtained by each treatment individually. These findings provide further support for the presence of serotonergic neurons within the median raphe nucleus that have a unique response profile. These neurons may have an important role in responses or adaptations to stress.     

5-ht5B receptor mRNA in the raphe nuclei: coexpression with serotonin transporter

We used double-label in situ hybridization to examine the cellular localization of 5-ht(5B) receptor mRNA in relation to serotonin transporter mRNA in the rat dorsal raphe (DR) and central superior nucleus (CS, median raphe nucleus). 5-ht(5B) receptor mRNA hybridization signal was often found on serotonin transporter mRNA-positive neuron profiles. The degree of cellular colocalization of these mRNAs notably varied among the different regions of the raphe nuclei. In the DR, cell bodies showing 5-ht(5B) receptor mRNA expression were abundant in the medial portions of the nucleus, all of them being also labeled for serotonin transporter mRNA. In contrast, in the ventrolateral regions (lateral wings) of the DR, we observed serotonin transporter mRNA-positive cells, but they were devoid of 5-ht(5B) receptor mRNA signal. In the CS, the level of coexpression of 5-ht(5B) receptor mRNA with serotonin transporter mRNA was high in the intermediate portions of the nucleus; however, we were unable to detect specific 5-ht(5B) receptor mRNA hybridization signal in its caudal extent. Our results support the presence of 5-ht(5B) receptor in serotonergic neurons in the DR and CS, suggesting an autoreceptor role for this receptor subtype.     

Effects of ovarian steroids on serotonin metabolism within grossly dissected and microdissected brain regions of the ovariectomized rat

Brain serotonin (5-HT) levels and accumulation rate within the dorsal raphe nucleus (DR) were elevated in ovariectomized (OVX) rats after ovarian steroid treatment, which consisted of estradiol benzoate (EB) followed by progesterone (P) 48 hr later. Some animals were also given the monoamine oxidase (MAO) inhibitor pargyline at designated times 10–40 min prior to sacrifice. Decapitation occurred 5 hr following the second steroid injection and 4–6 hr into the dark phase of a 14:10 light/dark cycle. Basal 5-HT levels and the rate of 5-HT accumulation (5-HT_r) following MAO inhibition were estimated by radioenzymatic assay of brain tissue punched from frozen sections or dissected freehand. Within the DR, 5-HT levels and 5-HT_r rates were respectively 13.8% (p < 0.001) and 23.2% (p < 0.025) higher in EB + P treated females compared with oil controls. Differences in basal 5-HT were found within the median raphe nucleus, but these were inconsistent from experiment to experiment. No differences were found in tissue immediately adjacent to these nuclear regions or within other brain regions including the ventral medial nucleus and suprachiasmatic nucleus of the hypothalamus. Increased levels of 5-HT were also found in grossly dissected tissue containing the mesencephalic raphe nuclei.     

Serotonin projection patterns to the cochlear nucleus

The cochlear nucleus is well known as an obligatory relay center for primary auditory nerve fibers. Perhaps not so well known is the neural input to the cochlear nucleus from cells containing serotonin that reside near the midline in the midbrain raphe region. Although the specific locations of the main, if not sole, sources of serotonin within the dorsal cochlear nucleus subdivision are known to be the dorsal and median raphe nuclei, sources of serotonin located within other cochlear nucleus subdivisions are not currently known. Anterograde tract tracing was used to label fibers originating from the dorsal and median raphe nuclei while fluorescence immunohistochemistry was used to simultaneously label specific serotonin fibers in cat. Biotinylated dextran amine was injected into the dorsal and median raphe nuclei and was visualized with Texas Red, while serotonin was visualized with fluorescein. Thus, double-labeled fibers were unequivocally identified as serotoninergic and originating from one of the labeled neurons within the dorsal and median raphe nuclei. Double-labeled fiber segments, typically of fine caliber with oval varicosities, were observed in many areas of the cochlear nucleus. They were found in the molecular layer of the dorsal cochlear nucleus, in the small cell cap region, and in the granule cell and external regions of the cochlear nuclei, bilaterally, of all cats. However, the density of these double-labeled fiber segments varied considerably depending upon the exact region in which they were found. Fiber segments were most dense in the dorsal cochlear nucleus (especially in the molecular layer) and the large spherical cell area of the anteroventral cochlear nucleus; they were moderately dense in the small cell cap region; and fiber segments were least dense in the octopus and multipolar cell regions of the posteroventral cochlear nucleus. Because of the presence of labeled fiber segments in subdivisions of the cochlear nucleus other than the dorsal cochlear nucleus, we concluded that the serotoninergic projection pattern to the cochlear nucleus is divergent and non-specific. Double-labeled fiber segments were also present, but sparse, in the superior olive, localized mainly in periolivary regions; this indicated that the divergence of dorsal and median raphe neurons that extends throughout regions of the cochlear nucleus also extended well beyond the cochlear nucleus to include at least the superior olivary complex as well.     

Altered Cav1.2 function in the Timothy syndrome mouse model produces ascending serotonergic abnormalities

Polymorphism in the gene CACNA1C, encoding the pore‐forming subunit of Cav1.2 L‐type calcium channels, has one of the strongest genetic linkages to schizophrenia, bipolar disorder and major depressive disorder: psychopathologies in which serotonin signaling has been implicated. Additionally, a gain‐of‐function mutation in CACNA1C is responsible for the neurodevelopmental disorder Timothy syndrome that presents with prominent behavioral features on the autism spectrum. Given an emerging role for serotonin in the etiology of autism spectrum disorders (ASD), we investigate the relationship between Cav1.2 and the ascending serotonin system in the Timothy syndrome type 2 (TS2‐neo) mouse, which displays behavioral features consistent with the core triad of ASD. We find that TS2‐neo mice exhibit enhanced serotonin tissue content and axon innervation of the dorsal striatum, as well as decreased serotonin turnover in the amygdala. These regionally specific alterations are accompanied by an enhanced active coping response during acute stress (forced swim), serotonin neuron Fos activity in the caudal dorsal raphe, and serotonin type 1A receptor‐dependent feedback inhibition of the rostral dorsal raphe nuclei. Collectively, these results suggest that the global gain‐of‐function Cav1.2 mutation associated with Timothy syndrome has pleiotropic effects on the ascending serotonin system including neuroanatomical changes, regional differences in forebrain serotonin metabolism and feedback regulatory control mechanisms within the dorsal raphe. Altered activity of the ascending serotonin system continues to emerge as a common neural signature across several ASD mouse models, and the capacity for Cav1.2 L‐type calcium channels to impact both serotonin structure and function has important implications for several neuropsychiatric conditions.     

Serotonergic retinopetal projections from the dorsal raphe nucleus in the mouse demonstrated by combined [H] 5-HT retrograde tracing and immunolabeling of endogenous 5-HT

The present study demonstrated a direct serotonergic retinopetal projection in the mouse stemming from the lateral portion of the dorsal raphe nucleus bilaterally. A double-labeling technique was employed combining: (1) radioautography and retrograde axonal tracing following intraocular injection of [³H] 5-HT and (2) immunocytochemical identification of endogenous 5-HT. Radiolabeled neurons were only observed within the dorsal raphe nucleus and were always double-labeled with the 5-HT antibody. The radiolabeling appeared to be specific resulting from the retrograde transport of a radioactive 5-HT derivative product following uptake of the neurotransmitter by intraretinal terminals.     

Evidence for a tonic GABAergic control of serotonin neurons in the median raphe nucleus

We examined whether serotonin (5-HT)-containing neurons of the midbrain raphe nuclei are subject to an inhibitory control by GABA. We found that injection into the median raphe nucleus of the GABA antagonists picrotoxin and bicuculline and the GABA agonist muscimol increase and decrease, respectively, the 5-HT turnover and the steady-state content of 5-hydroxyindoleacetic acid. The results provide biochemical evidence of a tonic inhibition by GABA of 5-HT neuronal activity in the median raphe nucleus; this inhibitory effect is potentiated by benzodiazepines.