The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat

The distribution of serotonin-immunoreactive varicosities in the paraventricular (PVH) and supraoptic (SO) nuclei was charted in normal immunohistochemical material and the probable cells of origin of these projections were identified using a combined retrograde transport-immunohistochemical method. The density of serotonergic fibers in the PVH and the SO is quite low relative to that in the immediately surrounding neuropil, in striking contrast to noradrenergic inputs to the nuclei. Immunoreactive fibers are concentrated in specific parts of the parvocellular division of the PVH, whereas in the magnocellular division of the nucleus, and in the SO, they are found mostly in regions where oxytocinergic cells predominate. These projections appear to arise from 3 distinct serotonergic cell groups (B7, B8 and B9) in the midbrain.     

Activation of the central serotonergic system in response to delayed but not omitted rewards

The forebrain serotonergic system is a crucial component in the control of impulsive behaviours. However, there is no direct evidence for natural serotonin activity during behaviours for delayed rewards as opposed to immediate rewards. Herein we show that serotonin efflux is enhanced while rats perform a task that requires waiting for a delayed reward. We simultaneously measured the levels of serotonin and dopamine in the dorsal raphe nucleus using in vivo microdialysis. Rats performed a sequential food-water navigation task under three reward conditions: immediate, delayed and intermittent. During the delayed reward condition, in which the rat had to wait for up to 4 s at the reward sites, the level of serotonin was significantly higher than that during the immediate reward condition, whereas the level of dopamine did not change significantly. By contrast, during the intermittent reward condition, in which food was given on only about one-third of the site visits, the level of dopamine was lower than that during the immediate reward condition, whereas the level of serotonin did not change significantly. Dopamine efflux, but not serotonin efflux, was positively correlated with reward consumption during the task. There was no reciprocal relationship between serotonin and dopamine. This is the first direct evidence that activation of the serotonergic system occurs specifically in relation to waiting for a delayed reward.     

Evidence that serotonergic neurons in the dorsal raphe nucleus exert a stimulatory effect on the secretion of renin but not of corticosterone

Data from previous experiments in rats indicate that release of serotonin in the central nervous system increases renin and corticosterone secretion. To determine which serotonergic neurons are involved, lesions of the dorsal or median raphe nuclei were produced by local injections of 5,7-dihydroxytryptamine (5,7-DHT) in rats, and 2 weeks later, the renin responses to parachloroamphetamine (PCA) were determined. Plasma corticosterone was also measured. PCA produced significant increases in plasma renin activity and plasma corticosterone in sham-lesioned animals and animals with median raphe lesions. The plasma corticosterone response to PCA was also normal in rats with dorsal raphe lesions but the renin response was significantly reduced. The data support the hypothesis that serotonergic neurons in the dorsal raphe nucleus are part of a neural pathway mediating increased renin secretion, and that the stimulatory effect of serotonin on corticosterone secretion is mediated by a different pathway.     

Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study

The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (> 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.     

An ascending serotonergic pain modulation pathway from the dorsal raphe nucleus to the parafascicularis nucleus of the thalamus

(1) Three types of spontaneously active neurons were found in the parafascicularis (PF) nucleus of the thalamus of the rat: slow firing units (0.5–10 spikes/s), bursting units (2–5 spikes/burst in 10–20 ms, one burst every 1–2 s) and fast firing units (15–40 spikes/s). A similar population of neurons was found in the PF of rats treated with 5,7-dihydroxytryptamine (5,7-DHT), a serotonin neurotoxin.

(2) Noxious tail pinch (TP) caused 68% of the PF neurons to increase their firing rates to 242% of their initial baseline activity, while non-noxious touch stimulation failed to induce a response. In the 5,7-DHT-treated rats, TP caused 85% of the neurons in the PF to increase their firing rates to 581% of their initial baseline activity and 22% of the neurons increased their firing in response to touching the tail. Both the number of cells responding (P < 0.05) and the percentage increase (P < 0.001) were statistically greater in serotonin-depleted rats than in controls. This indicates that serotonin (5-HT) has a tonic inhibitory influence on responses to both noxious and non-noxious sensory stimuli.

(3) In control rats, electrical stimulation of the dorsal raphe nucleus (DR) decreased the firing rates of PF neurons. In contrast, the same DR stimulation induced an increase in PF firing rates during stimulation in serotonin-depleted rats and this increase in firing rates remained several seconds after cessation of stimulation. This indicates that the DR may use at least two different neurotransmitters in its projections to forebrain structures.

(4) In control rats, the TP stimulation induced an increase in firing rates of PF neurons while DR stimulation attenuated the excitation induced by TP stimulation. In serotonin-depleted rats, DR stimulation and TP both caused an increase in firing rates. This effect was not additive indicating that there may be a serotonergic projection from the DR to the PF which modifies responses to somatosensory stimuli.

(5) The inhibitory effects elicited by electrical stimulation were limited to the immediate area of the DR. Stimulation of the adjacent reticular formation 1 mm lateral to the DR produced the opposite effect, an increase in firing rate often accompanied by driven spike activity in the PF.

Decreased GABAergic innervation of the pituitary intermediate lobe after rostral hypothalamic cuts

The effects of hypothalamic cuts at various rostro-caudal levels on the GABAergic innervation of the neurointermediate lobe of the pituitary gland have been studied. The GABAergic innervation was visualized through glutamate-decarboxylase (GAD) immunocytochemistry. Caudal hypothalamic cuts which transected the pituitary stalk completely abolished the GAD immunoreactive plexus. Rostral cuts which separated about one-third of the median eminence and arcuate nucleus from the pituitary gland decreased the GAD-immunoreactive network in the intermediate lobe but did not affect the neural lobe significantly. Although the precise location of the cell bodies giving rise to the GABAergic innervation of the neurointermediate lobe remains unknown, our findings indicate that their projections are descending ones. They are severed by rostral hypothalamic cuts and show a rostrocaudal arrangement. It is likely that the GABAergic endings of the intermediate lobe originate in the rostral hypothalamus, probably in the rostral part of the arcuate nucleus and/or in the anterior periventricular area. The GABAergic fibers in the neural lobe have a more caudal origin than those innervating the intermediate lobe.     

Aminergic and cholinergic afferents to REM sleep induction regions of the pontine reticular formation in the rat

Microinjection of cholinergic agonists in a dorsolateral part of the mesopontine tegmentum has been shown to induce a rapid eye movement (REM) sleep-like state. Physiological evidence indicates that not only acetylcholine but also various amine transmitters, including those implicated in behavioral state regulation, affect neuronal activity in this region of the pontine reticular formation. In the present study, sources of select aminergic and cholinergic inputs to this REM sleep induction zone were identified and quantitatively analyzed by using fluorescence retrograde tracing combined with immunofluorescence in the rat. In addition to previously demonstrated cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei, the REM sleep induction zone received various aminergic inputs that originated in widely distributed regions of the brainstem and hypothalamus. Serotoninergic afferents represented a mean of 44% of all aminergic/cholinergic source neurons projecting to the REM sleep induction zone, which was comparable to the mean percentage of 39% represented by cholinergic afferent neurons. The serotoninergic afferents originated from the raphe nuclei at all brainstem levels, with heavier projections from the pontine than from the medullary raphe nuclei. Unexpectedly, an additional major serotoninergic input was provided by serotoninergic neurons in the nucleus prosupralemniscus (B9). Noradrenergic afferent neurons represented a mean of 14% of all aminergic/cholinergic source neurons, which was only about one-third of the mean percentage of either cholinergic or serotoninergic source neurons. These noradrenergic projection neurons were located not only in the locus ceruleus (8%) but also in the lateral tegmentum, including the A5 (4%) and A7 (2%) cell groups. Histaminergic neurons in the tuberomammillary hypothalamic nucleus represented a minor group of afferent neurons (3%), and a still smaller input came from adrenegic C1 neurons. The pattern of these transmitter-specific afferent connections appeared to be similar regardless of the longitudinal level within the REM sleep induction zone. The present results are consistent with previous behavioral and physiological evidence for a role of the pontine REM sleep induction zone in triggering REM sleep. The regulation of REM sleep induction would be best understood in terms of a state-dependent interplay of cholinergic, serotoninergic, and other inputs all acting convergently upon neurons in the REM sleep-inducing region of the pontine reticular formation.     

The comparative effects of environmental enrichment with exercise and serotonin transporter blockade on serotonergic neurons in the dorsal raphe nucleus

We have previously reported that inhibition of the serotonin transporter (SERT) by selective serotonin reuptake inhibitor (SSRI) fluoxetine significantly reduces the number of tryptophan hydroxylase (TPH)-positive cells in the dorsal raphe nucleus (DRN). We have been interested in exploring whether this SSRI-induced change in TPH might be modified by housing in an enriched environment. Like SSRI antidepressants, environmental enrichment (EE) and physical exercise have been found to have efficacy in the prevention and alleviation of depression. We postulated that EE with exercise and SERT inhibition would similarly affect TPH regulation and that EE with exercise might modify the effect of fluoxetine on TPH. Three week old male Sprague-Dawley rats were housed in either a standard cage (SE) or an enriched environment (EE). SE animals were singly housed with no access to enrichment objects. EE animals were group housed and were provided with various enrichment objects (e.g. running wheel) that were changed and rearranged regularly. Nine weeks after the experiment began, the rats were randomly assigned to one of four treatment groups: (1) SE control; (2) SE fluoxetine; (3) EE control; or (4) EE fluoxetine. Fluoxetine (5 mg/kg/day) was placed in the drinking water. Sections of DRN were processed for TPH immunohistochemistry. The number of TPH-positive cells was determined by blinded, manual counting. Results were analyzed by analysis of variance (ANOVA) followed by post-hoc Tukey tests. Significance was set at P < 0.05. For animals housed in a standard environment, fluoxetine induced a significant 29% reduction in the number of TPH-immunoreactive cells in the DRN. A similar reduction in TPH immunoreactivity was observed in animals that were housed in an enriched environment but not exposed to fluoxetine (39%). The number of TPH-positive cells in the DRN for animals housed in an enriched environment and exposed to fluoxetine was not significantly different than animals housed in an enriched environment and not exposed to fluoxetine. The reduction of TPH immunoreactivity in the DRN by EE with exercise suggests that a modified housing environment and voluntary exercise affects regulation of TPH, possibly via a mechanism similar to that of SERT inhibitors. This downregulation of serotonin biosynthesis by fluoxetine and EE with exercise may ultimately play a role in the therapeutic action of both interventions.     

Evidence that the medial and dorsal raphe nuclei mediate serotonergically-induced increases in prolactin release from the pituitary

Electrolytic lesions of the medial (MR) or dorsal (DR) raphe nucleus significantly antagonized serum prolactin elevations produced by 5-hydroxytryptophan (5-HTP) in rats pretreated with fluoxetine or citalopram, (serotonin (5-HT) uptake blockers). Lesioned animals in which total blockade of serum prolactin elevations was observed also had total blockade of 5-HT accumulation in the median eminence. However, the increase in serum prolactin levels produced by 5-HTP plus 5-HT reuptake blockade in lesioned rats was not significantly different from sham-operated rats if as little as 15–20% of control median eminence accumulation was present. Serum prolactin elevations produced by quipazine, a direct acting 5-HT agonist, were not significantly affected by MR lesions. On the basis of these results, we suggest that: (1) serum prolactin elevations following 5-HT reuptake blockade plus 5-HTP are correlated with 5-HT concentration in the median eminence; (2) lesion-induced antagonism of 5-HTP-induced prolactin elevation is critically dependent upon complete blockade of median eminence 5-HT accumulation; and (3) 5-HT neurons arising from cell bodies located in the MR and DR are necessary for endogenous serotonergically mediated effects on prolactin secretion in the rat.     

Pre- and post-natal ontogeny of serotonergic projections to the rat spinal cord

The development of 5-hydroxytryptamine (5-HT) innervation in the spinal cord was studied from embryonic day 14 (E14) to adulthood. Sprague-Dawley rats were fixed by perfusion with 5% glutaraldehyde in cacodylate-sodium metabisulfite buffer, and vibratome sections were processed for immunocytochemistry with a 5-HT antiserum. For electron microscopy, the sections were flat-embedded in araldite, and thin sectioning was performed. 5-HT neurons caudally directed from raphe nuclei invade the spinal cord at E14 and reach the caudalmost levels by E16-E17. In longitudinal sections, axons are seen by E15, at cervical and upper thoracic levels, to invade the presumptive gray matter from the anterior and lateral funiculi. The invasion process occurred either by sharp angulation of the axon or by branching of a collateral. By E16, at thoracic level the anterior horn and the intermediolateral columns are profusely innervated by very thin, varicose fibers; synapses are seen at E17 and E18 using EM. 5-HT immunoreactive boutons are involved here. After birth, 5-HT innervation of these two areas evolves progressively from a diffuse network to a more restricted pattern, especially at the thoracic level for the intermediolateral column and at cervical and lumbar levels for the anterior horn. The adult pattern is reached by postnatal day 21 (P21). The growth of axons toward the dorsal horn becomes noticeable by E19 at all spinal levels, when fibers invade the neck of the horn from the lateral funiculus, and innervation proceeds diffusely until P5. At P7, thin fibers course dorsally and laterally along the border of the gray matter and ramify profusely in layers I and II. The adult pattern is also reached in the dorsal horn by P21. These results are discussed in relation to the postnatal maturation of motor and sensory circuits and to the development of transplanted raphe neurons in the rat spinal cord.     

Immunocytochemical localization of serotonin in the rat dentate gyrus following raphe transplants

The ultrastructural features of the serotoninergic innervation of the rat dentate gyrus in normal adults and in animals receiving raphe nuclear area transplants was investigated using an antibody to serotonin (5-HT). Neonatal rats received a lesion of the fimbria-fornix and entorhinal cortex. Three days later, a portion of embryonic (E-16-18) raphe nuclear area was transplanted to the entorhinal cavity and the animals were allowed to survive for 60 days. Animals were processed for the immunocytochemical localization of 5-HT using the peroxidase-antiperoxidase method. Light microscopic observation showed that 5-HT-containing fibers from transplanted raphe neurons densely innervated the hilar and molecular zones of the dentate gyrus. Electron microscopic analysis showed that 5-HT immunoreactivity was contained only in axons and axon varicosities. There were no differences in the ultrastructural characteristics of axons and axon terminals between normal animals and those which had received raphe transplants. A mixture of both conventional synaptic junctions and non-synaptic axonal swellings were found in both groups.     

Estrogen enhances light-induced activation of dorsal raphe serotonergic neurons

The serotonergic system has been implicated in the modulation of physiological processes including circadian rhythms, learning, memory, mood and food intake. In females, cessation of ovarian function produces deleterious changes in all of these processes and estrogen treatment often ameliorates these conditions. Estrogen may produce these effects by acting on the midbrain raphe, an estrogen-sensitive region that receives direct projections from sensory systems. Here we examined the ability of estradiol to modulate neuronal responses of neurons within raphe nuclei to photic stimulation. Ovariectomized rats treated with estradiol or cholesterol were killed 1 h after the normal onset of light (Zeitgeber time 0) or after a 2-h phase advance (Zeitgeber time 22). In a second study, estradiol-treated ovariectomized rats under constant dark conditions were exposed to light 2 h before the subjective onset of circadian time [(CT)22] and killed 1 h later (CT23). The brains from all animals were processed for Fos and/or serotonin (5-HT) immunocytochemistry. Comparisons showed that the phase shift increased Fos immunoreactivity in all dorsal raphe nucleus (DRN) regions. Although estradiol did not alter the overall number of Fos-positive nuclei, it significantly increased the number of Fos/5-HT double-labelled cells in the medial and lateral DRN. In contrast, neither a phase shift nor estradiol altered the number of Fos-immunoreactive cells or the proportion of Fos-positive 5-HT cells in the median raphe nucleus. Results reveal that the DRN 5-HT system responds to changes in the light : dark cycle and that these responses are modulated by estrogen.     

Leptin uptake by serotonergic neurones of the dorsal raphe

The effects of leptin on food intake, metabolism, sleep patterns and reproduction may be mediated, in part, by the midbrain serotonergic systems. Here, we report on the distribution of neurones that accumulate leptin in the raphe nuclei of male and female rats after intracerebroventricular administration of mouse recombinant leptin labelled with digoxigenin. Direct leptin-targeted cells were present in the periventricular grey, pontine and raphe nuclei. Confocal microscopy revealed that raphe neurones which accumulated leptin were predominantly serotonergic. The temporal pattern of leptin accumulation by raphe neurones showed a marked gender difference: 6 h after leptin administration, all male and female rats showed massive leptin binding in the dorsal raphe, while 30 min after leptin treatment, only 10% of male rats exhibited leptin-labelled cells in contrast to 50% of females. The present observations reveal that leptin can be selectively accumulated by serotonergic neurones in the raphe nuclei and that this mechanism is gender specific. These findings support the idea that the midbrain serotonergic system is an important mediator of the effects of leptin on brain function and may provide an explanation for gender differences in metabolism regulation and its coordination with higher functions of the brain.     

Somatodendritic autoreceptor regulation of serotonergic neurons: dependence on L-tryptophan and tryptophan hydroxylase-activating kinases

The somatodendritic 5-HT(1A) autoreceptor has been considered a major determinant of the output of the serotonin (5-HT) neuronal system. However, recent studies in brain slices from the dorsal raphe nucleus have questioned the relevance of 5-HT autoinhibition under physiological conditions. In the present study, we found that the difficulty in demonstrating 5-HT tonic autoinhibition in slice results from in vitro conditions that are unfavorable for sustaining 5-HT synthesis. Robust, tonic 5-HT(1A) autoinhibition can be restored by reinstating in vivo 5-HT synthesizing conditions with the initial 5-HT precursor l-tryptophan and the tryptophan hydroxylase co-factor tetrahydrobiopterin (BH(4)). The presence of tonic autoinhibition under these conditions was revealed by the disinhibitory effect of a low concentration of the 5-HT(1A) antagonist WAY 100635. Neurons showing an autoinhibitory response to L-tryptophan were confirmed immunohistochemically to be serotonergic. Once conditions for tonic autoinhibition had been established in raphe slice, we were able to show that 5-HT autoinhibition is critically regulated by the tryptophan hydroxylase-activating kinases calcium/calmodulin protein kinase II (CaMKII) and protein kinase A (PKA). In addition, at physiological concentrations of L-tryptophan, there was an augmentation of 5-HT(1A) receptor-mediated autoinhibition when the firing of 5-HT cells activated with increasing concentrations of the alpha(1) adrenoceptor agonist phenylephrine. Increased calcium influx at higher firing rates, by activating tryptophan hydroxylase via CaMKII and PKA, can work together with tryptophan to enhance negative feedback control of the output of the serotonergic system.     

Induced homotypic sprouting of serotonergic fibers in hippocampus. II. An immunocytochemistry study

The dorsal hippocampus of the rat normally receives its 5-HT innervation from two homologous groups of cells in the median raphe nucleus via the cingulum bundle-induseum griseum (CB-IG) and the fornix-fimbria (FF) (J. Comp. Neurol., 179 (1978) 641-667 and Brain Res. Bull., 10 (1983) 445-451). 5-HT immunoreactive (IR) fibers are distributed in a laminar pattern in the hippocampus. These fibers have large varicosities and are densely distributed in the infragranular layer of dentate gyrus, in the stratum lacunosum-moleculare of the cornu Ammonis and in the area fasciola cinerea (FC). The present study provides evidence that the density of the 5-HT-IR fibers in the dorsal hippocampus is greatly decreased but maintained a similar laminar pattern 3 days after lesioning by microinjection of 4 micrograms of 5,7-dihydroxytryptamine (5,7-DHT) into the CB-IG. An apparently normal density and distribution pattern of the 5-HT-IR fiber is seen by 42 days postlesion. The FC in the hippocampus is among the first regions reinnervated by 5-HT-IR fibers with very dense and large varicosities. The restitution of 5-HT-IR fibers in the dorsal hippocampus after the 5,7-DHT lesion in the CB-IG is accompanied by a marked increase in the number and intensity of 5-HT-IR fibers in the FF. No evidence of a regeneration of 5-HT-IR fibers is seen distal to the injection site in the CB-IG. These observations provide direct evidence for homotypic collateral sprouting in the CNS induced by removal of a single fiber type.     

The cells of origin of a sexually dimorphic serotonergic input to the medial preoptic nucleus of the rat

The medial preoptic nucleus (MPN) is a sexually dimorphic complex composed of 3 distinct cytoarchitectonic subdivisions, and a sexually dimorphic distribution of presumably serotonergic fibers is associated with the lateral part of the nucleus (MPNl). In this study the probable cells of origin for these serotonergic fibers were identified by using a combined fluorescent retrograde tracer and immunofluorescence method. Serotonergic afferents to the MPN appear to arise exclusively from the dorsal raphe nucleus (B7), the median raphe nucleus (B8), and the region adjacent to the medial lemniscus (B9).     

A new, possibly serotonergic, neuron in the lamina of the blowfly optic lobe: an immunocytochemical and Golgi-EM study

With a modified Golgi impregnation technique a new neuron type, with extensive tangential processes, was discovered in the optic lamina of the blowfly, Calliphora erythrocephala. This cell is unusual since the majority of its varicose processes reside in a layer distal to the synaptic layer of the lamina. Using antibodies to 5-HT it was found that this new cell type was 5-HT immunoreactive, and varicosities of Golgi impregnated neurons analyzed electron-microscopically contain dense core vesicles.     

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.     

Chloral hydrate anesthesia alters the responsiveness of central serotonergic neurons in the cat

The influence of chloral hydrate anesthesia on the spontaneous activity and responsiveness of serotonergic neurons was examined by administering chloral hydrate (300 mg/kg, i.p.) to freely moving cats from which serotonergic unit activity in the dorsal raphe nucleus (DRN) was being recorded. Although chloral hydrate administration produced a surgical level of anesthesia within 15 min following injection, it produced only a small decrease ( 20%) in the spontaneous activity of DRN serotonergic neurons. In contrast, the responsiveness of these same neurons was greatly altered by chloral hydrate administration. By examining the same neuron before and after chloral hydrate injection, it was found that chloral hydrate anesthesia completely abolished the excitatory responses of DRN serotonergic neurons to auditory and visual stimuli, as well as their excitatory response to electrical stimulation of the gigantocellular tegmental field (FTG) in the pontine reticular formation. On the other hand, the inhibition of serotonergic neuron firing resulting from systemic administration of WB 4101 (1.0 mg/kg, i.p.), a selective α₁ adrenergic receptor antagonist, was greatly potentiated by chloral hydrate anesthesia. Therefore, these data indicate that chloral hydrate anesthesia produces profound changes in the physiological and pharmacological responses of central serotonergic neurons which are not predictable by examination of spontaneous activity alone. Furthermore, as discussed, if it not clear to what extent these confounding influences might generalize to other anesthetized or immobilized preparations. Thus, beyond the obvious advantage which allows for the study of relationships between neuronal activity and behavior, single unit studies conducted in awake, freely moving animals also may be of greater value for basic physiological and pharmacological studies.