Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.     

Ascending dopaminergic projections from the dorsal raphe nucleus in the rat

The projections of putative dopamine containing cells within the dorsal raphe nucleus (DR) were studied using a combination of tyrosine hydroxylase (TH) immunocytochemistry and fluorescent retrograde tracing. Substantial numbers of TH-immunoreactive cells in the DR were found to project to the nucleus accumbens. Progressively smaller numbers of cells were found to project to the lateral septum and medial prefrontal cortex. Very few TH-immunoreactive cells projected to the dorsal striatum, and none to the substantia nigra. TH-immunoreactive cells did not display serotonin-like immunoreactivity. These findings indicate that the projection pattern of TH-immunoreactive cells within the dorsal raphe more closely resembles that of dopaminergic cells within the ventral tegmental area (VTA) than that of serotonergic cells within the DR.     

Intraocular co-grafts of fetal dorsal raphe nucleus and suprachiasmatic nucleus

Serotonergic neurons in the fetal dorsal raphe nucleus were grafted together with fetal anterior hypothalamic tissue including the suprachiasmatic nucleus (SCN) to the anterior eye chamber of adult rats. After 6 weeks transplantation, the double grafts were immunocytochemically examined using antisera against serotonin, arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). The raphe grafts contained a large number of serotonin-immunoreactive neurons and fibers, but only a few AVP-immunoreactive fibers and VIP-immunoreactive neurons and fibers. On the other hand, numerous AVP- and VIP-immunoreactive neurons and fibers were found in the SCN of the anterior hypothalamic graft. Outgrowing serotonin-immunoreactive fibers from the raphe tissue were densely distributed in the anterior hypothalamic graft. In the SCN, however, only a few fibers were detected. The results demonstrate that the isolated anterior hypothalamic grafts can be innervated by the serotonergic neurons from the raphe grafts, but the innervation pattern of these fibers was quite different from the normal rat. The present results indicate that the isolated SCN has an inhibitory influence on the growth of serotonergic fibers.     

The locus coeruleus, norepinephrine, and memory in newborns

Use of learned odor cues by newborn rats is critical for pup survival. Rat pups acquire approach responses to maternal odors through an associative conditioning mechanism. This learned behavioral response is accompanied by a modification of olfactory bulb neural response patterns to the learned odor. Both the behavioral and neural reponse changes involved and require norepinephrine release in the olfactory bulb. The source of this norepinephrine is the locus coeruleus. It is proposed that the unique response properties of the locus coeruleus during the early postnatal period in the rat may facilitate acquisition of these critical early memories.     

Behavioral state-related changes of extracellular serotonin concentration in the dorsal raphe nucleus: a microdialysis study in the freely moving cat

For direct measurement of the extracellular concentration of serotonin (5-HT) in the dorsal raphe nucleus (DRN) over the sleep-wake cycle we used the technique of in vivo microdialysis in six freely moving, naturally sleeping cats whose behavioral state was polygraphically determined. Perfusate samples from microdialysis probes histologically localized to the DRN showed the following significantly different levels of extracellular 5-HT, with the numbers in parentheses indicating successively the mean value in fmol/5 μl perfusate sample, the % level relative to waking, and the sample n:waking (4.02, 100%, n = 38) > slow wave sleep (2.02, 50%, n = 30) > REM sleep (1.61, 38%, n = 17). These data, to our knowledge the first direct DRN 5-HT measurements during behavioral state changes, directly parallel the levels of serotonergic neuronal action potential activity and suggest that DRN extracellular 5-HT is determined by this action potential activity through synaptic release by recurrent axonal collaterals in the DRN.     

Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

Corticotropin-releasing factor administered into the locus coeruleus, but not the parabrachial nucleus, stimulates norepinephrine release in the prefrontal cortex

Previous studies have indicated that intracerebroventricular application of corticotropin-releasing factor (CRF) activates noradrenergic neurons in the brain stem locus coeruleus (LC) and norepinephrine (NE) metabolism in several brain regions. To assess whether CRF has direct effects on LC noradrenergic neurons, CRF was infused into the LC and concentrations of NE and its metabolites were measured in microdialysates collected from the medial prefrontal cortex (PFM). Infusion of 100 ng of CRF into the LC significantly increased dialysate concentrations of NE and of its catabolite MHPG in the ipsilateral PFM, whereas no significant changes were observed following infusion of artificial CSF. No response was observed when the infusions of CRF occurred outside of the LC, including those in the parabrachial nucleus. Although CRF administered into the LC slightly increased dialysate concentrations of NE in the contralateral PFM, this effect was not statistically significant. The effect of CRF injected into the LC on dialysate NE was prevented by combination with a 10-fold excess of the CRF antagonist, alpha-helical CRF_9–41 indicating some specificity in the response. These results are consistent with anatomical and electrophysiological evidence suggesting that CRF may directly activate noradrenergic neurons in or close to the LC.     

Morphological features and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus. An intracellular recording and labeling study in rat brain slices

The morphology and electrophysiological properties of serotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus (DRN) of the rat were examined in frontal brain slices. Biocytin was injected intracellularly into the intracellularly recorded neurons. Then the morphology of the recorded neurons was observed after histochemical visualization of biocytin. The recorded neurons extending their main axons outside the DRN were considered as projection neurons. Subsequently, serotonergic nature of the neurons was examined by serotonin (5-HT) immunohistochemistry. The general form of the dendritic trees is radiant and poorly branching in both 5-HT- and non-5-HT neurons. However, the dendrites of the 5-HT neurons were spiny, whereas those of the non-5-HT neurons were aspiny. The main axons of both 5-HT- and non-5-HT neurons were observed to send richly branching axon collaterals to the DRN, ventrolateral part of the periaqueductal gray and the midbrain tegmentum. In response to weak, long depolarizing current pulses, the 5-HT neurons displayed a slow and regular firing activity. The non-5-HT neurons fired at higher frequencies even when stronger current was injected. Some other differences in electrophysiological properties were also observed between the 5-HT-immunoreactive spiny projection neurons and the 5-HT-immunonegative aspiny projection neurons.     

Field potentials in primate locus coeruleus following stimulation of the dorsal noradrenergic bundle

Stimulation of the dorsal noradrenergic bundle, but not of adjacent areas, elicited field potentials in the locus coeruleus in the stumptail monkey (Macaca arctoides). This result suggests a procedure for locating the dorsal noradrenergic bundle in vivo.     

Single unit activity of noradrenergic neurons in locus coeruleus and serotonergic neurons in the nucleus raphe dorsalis of freely moving cats in relation to the cardiac cycle

Single unit activity of serotonergic (5-HT) neurons in the nucleus raphe dorsalis (NRD) and of noradrenergic (NE) neurons in the locus coeruleus (LC) was recorded in relation to the cardiac cycle in awake, freely moving cats. The discharge of NRD-5-HT neurons showed no relationship to the cardiac cycle, while LC-NE neurons displayed a cardiac periodicity such that the units were most likely to fire from 80 to 180 ms after the peak of the cardiac r-wave (diastole), and least likely to fire during the period from 40 ms before to 60 ms after the r-wave (systole). The strength of this periodicity was inversely related to the discharge rate of individual cells. Exposure to a noxious environmental stimulus (15 min of 100 dB white noise) greatly attenuated the cardiac relationship of LC-NE neurons. A blood volume expansion of approximately 15% (9.0 ml/kg b. wt.) decreased unit rate by about 25%, but did not alter either the timing or the magnitude of the LC-NE cardiac relationship. These data are discussed in terms of the participation of NRD-5-HT and LC-NE neurons in cardiovascular function, and the possible role of LC-NE neurons in short-and long-term volume homeostasis.     

Circadian activity rhythms in rats with midbrain raphe lesions

The dorsal and median mesencephalic raphe nuclei provide a robust projection to the hypothalamic suprachiasmatic nucleus, the site of a putative neuronal circadian pacemaker. Although it has been suggested that the raphe may play a role in the circadian timing system, this role has not yet been specified. In the present report, we examined the circadian activity patterns of rats with large midbrain lesions aimed at the median and dorsal raphe nuclei under conditions of light-dark entrainment, and while free-running in constant light and constant darkness. The results indicate that midbrain raphe lesions may interface with the expression of free-running circadian activity rhythms.     

Role of the dorsal raphe nucleus in morphine-induced immediate early gene expression in the rat striatum

Serotonin (5-HT) is thought to be involved in morphine action in the brain. To determine if the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN) are involved in morphine-induced c-Fos and JunB expression in the caudate-putamen (CPu), the mu receptor antagonist, beta-funaltrexamine (beta-FNA), was unilaterally infused into the PAG adjacent to DRN prior to morphine. Behaviorally, beta-FNA prevented morphine-induced loss of righting and Straub tail. In the CPu of beta-FNA treated rats, morphine-induced c-Fos and JunB were attenuated compared to vehicle-infused rats. These results suggest that morphine acts within the PAG-DRN to exert rapid behavioral effects and to induce c-Fos and JunB in the striatum.     

The role of the habenular complex in the elevation of dorsal raphe nucleus serotonin and the changes in the behavioral responses produced by uncontrollable stress

Previous research indicates that the serotonergic neurons of the caudal dorsal raphe nucleus (DRN) are activated to a greater degree by inescapable shock (IS) as compared to escapable shock (ES), causing a greater release of serotonin (5-HT) in the DRN and in target regions. This differential activation is necessary for the behavioral changes that occur after exposure to IS, but not to ES (i.e. learned helplessness/behavioral depression). Although the critical role of the DRN in learned helplessness is clear, the neural inputs to the caudal DRN which result in this selective activation are unknown. One structure that may be involved in the activation of the DRN and the induction of learned helplessness/behavioral depression is the habenular complex. In experiment 1, habenula lesions eliminated the differential rise in DRN extracellular 5-HT levels in response to IS and ES exposure by severely attenuating the rise in 5-HT for both groups. In experiment 2, sham operated and habenula lesioned rats were exposed to either ES, IS or no stress (home cage control; HCC). Twenty-four hours later, sham rats previously exposed to IS exhibited longer escape latencies as compared to both ES and HCC rats (i.e. learned helplessness). The habenular lesion eliminated the differences in escape latency between groups, thus eliminating the induction of learned helplessness/behavioral depression. These results suggest that the habenula is necessary for the differential activation of the DRN and the escape deficits produced by IS.     

The nucleus locus coeruleus modulates local cerebral glucose utilization during noise stress in rats

Local cerebral glucose utilization (LCGU), estimated by the quantitative autoradiographic 2-deoxyglucose technique, was studied in rats with bilateral 6-hydroxydopamine lesions of the locus coeruleus (LC) and in vehicle-injected controls. Unanesthetized animals were studied during exposure to stressful levels of white noise (95 dB) or in relative silence (50 dB). Results indicated that noise caused greater and more widespread increases in LCGU in animals with LC lesions than in vehicle-injected controls. Lesions alone had little or no effect in animals not subjected to noise. Analyses of variance revealed significant treatment interaction effects (intact/lesion x silence/noise) for 37 of 109 regions measured. The pattern of results suggests that the LC acts during stress to limit unnecessary cerebral activity that might interfere with efficient sensory processing and/or the organization of appropriate behavioral responses. In this respect LC function may be similar to those actions of the peripheral sympathetic nervous system that suppress vegetative functions during stress to allow for the performance of coping responses.     

Burst-firing activity of presumed 5-HT neurones of the rat dorsal raphe nucleus: electrophysiological analysis by antidromic stimulation

We recently reported raphe neurones which frequently fired spikes in short bursts. However, the action potentials were broad and the neurones fired in a slow and regular pattern, suggesting they were an unusual type of 5-hydroxytryptamine (5-HT) neurone. In the present study, we investigated whether these putative burst-firing 5-HT neurones project to the forebrain and whether all spikes fired in bursts propagate along the axon. In anaesthetised rats, electrical stimulation of the medial forebrain bundle evoked antidromic spikes in both burst-firing neurones and in single-spiking, classical 5-HT neurones recorded in the dorsal raphe nucleus. Although the antidromic spike latency of the single-spiking and burst-firing neurones showed a clear overlap, burst-firing neurones had a significantly shorter latency than single-spiking neurones. For both burst-firing neurones and classical 5-HT neurones, antidromic spikes made collisions with spontaneously occurring spikes. Furthermore, in all burst-firing neurones tested, first, second and third order spikes in a burst could be made to collide with an antidromic spike. Interestingly, in a small number of burst-firing neurones, antidromic stimulation evoked spike doublets, similar to those recorded spontaneously. From these data we conclude that burst-tiring neurones in the dorsal raphe nucleus project to the forebrain, and each spike generated by the burst propagates along the axon and could thereby release transmitter (5-HT).     

Serotonergic inhibition of the dorsal lateral geniculate nucleus

Electrophysiological studies were conducted on chloral hydrate-anesthetized rats to determine if the dorsal raphe nucleus (DR) exerts an inhibitory influence upon the dorsal lateral geniculate nucleus (dLGN), and if this inhibition is mediated by the release of serotonin (5-HT). Conditioning stimuli presented to the DR 100-400 ms before an optic tract (OT) shock significantly lowered the amplitude of OT shock-elicited, postsynaptic, field potentials of less than 3 ms latency. Rare, long-latency, field potentials (greater than 5 ms) were diminished in amplitude when preconditioning intervals were less than 15 ms. Six days after intracerebral injection of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (8 micrograms), into the dLGN, significant reductions were observed in 5-HT and 5-hydroxyindole acetic acid in the dLGN. Field potentials recorded on the sixth day in indoleamine-depleted dLGN were significantly less inhibited by DR preconditioning. Intracerebral injections of a control solution neither altered monoamine levels nor the degree of inhibition by DR preconditioning. These data provide further evidence that inhibition of dLGN by DR is mediated by release of 5-HT.     

Dorsal raphe nucleus stimulation modulates the response of layers IV and V barrel cortical neurons in rat

The effect of dorsal raphe nucleus (DRN) electrical stimulation on response properties of layers IV and V barrel cortical neurons was studied. To assess the receptive field characteristics of cortical neurons, responses of neurons were recorded following the displacement of principal and adjacent whiskers individually or in a condition test paradigm. Then neuronal responses to the displacement of whiskers were analyzed following DRN stimulation at 0, 50, 100, 200 and 400 ms inter-stimulation intervals. Considering On responses, DRN stimulation suppressed the response magnitude of layer V neurons to principal whisker deflection, while it slightly increased that of layer IV neurons (not statistically significant). The response latency of layer IV neurons increased when DRN was stimulated 200 or 400 ms before principal whisker deflection, while the response latency of layer V was not changed. DRN stimulation had no effect on either magnitude or latency of neuronal response to the adjacent whisker deflections. We observed a decrease in the inhibitory effect of the adjacent whisker deflection on the magnitude of neuronal response to the principal whisker deflection in layer IV when DRN was stimulated 200 ms before the principal whisker deflection. Off responses did not show any significant effect of DRN stimulation. Our results suggest a modulating role for DRN in processing of the incoming information into barrel cortex. This effect might be location dependent.     

Glutamatergic afferent projections to the dorsal raphe nucleus of the rat

Based on WGA-apo-HRP-gold (WG) retrograde tracing, the present study revealed that different subdivisions of the dorsal raphe (DR) such as dorsomedial, ventromedial, lateral wing, and caudal regions receive unique, topographically organized afferent inputs, that are more restricted than previously reported. Phaseolus vulgaris leucoagglutinin anterograde tracing studies confirmed that the medial prefrontal cortex provides the major afferent input to each subdivision of the DR. Double-labeling studies combining WG tracing and glutamate immunostaining indicated that the medial prefrontal cortex, various hypothalamic nuclei including perifornical, lateral, and arcuate nuclei, and several medullary regions such as lateral and medial parabrachial nuclei, and laterodorsal tegmental nucleus provide the major glutamatergic input to each subregion of the DR. It should be noted that the degree of glutamatergic input from these afferent sites was specific for each DR subdivision. The present findings indicated that dorsomedial, ventromedial, lateral wing, and caudal subdivisions of the DR receive excitatory inputs from both cortical and subcortical sites which might be involved in regulation or modulation of a broad range of systems, including sensory and motor functions, arousal and sleep-wake cycle, biorhythmic, cognitive, and affective behaviors.