Paraventricular nucleus neuronal responses following electrical stimulation of the midbrain dorsal raphe: evidence for cotransmission

Summary In order to determine the responses of paraventricular nucleus neurones following activation of central serotonergic pathways, single unit activity was recorded and responses following electrical stimulation of the midbrain dorsal raphe nucleus were examined. Excitation was recorded from approximately 50% of the cells, independent of whether they were antidromically identified as projecting to the median eminence or unidentified. Approximately 20% of cells were inhibited by the stimulation, the majority of these being unidentified. Parachlorophenylalanine-induced inhibition of serotonin synthesis reduced hypothalamic serotonin levels by 77% and caused a significant reduction in the proportion of cells excited by the stimulation, whereas the inhibitory responses were not affected. Intracerebroventricular administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine, which caused similar reductions in hypothalamic serotonin content (77%), reduced still further the proportion of excitatory responses and also reduced the proportion of cells inhibited by the stimulation. The data obtained suggest that serotonin acts as an excitatory neurotransmitter in the paraventricular nucleus; this is discussed particularly with respect to the regulation of the hypothalamo-hypophysial-adrenocortical axis. The loss of inhibitory responses in 5,7-dihydroxytryptamine treated, as opposed to the parachlorophenylalanine treated, animals suggests that the serotonergic fibers innervating the recorded cells may contain a cosecreted substance that may have important physiological actions in the control of neuronal activity in the region recorded.     

Cellular profile of the dorsal raphe lateral wing sub-region: Relationship to the lateral dorsal tegmental nucleus

As one of the main serotonergic (5HT) projections to the forebrain, the dorsal raphe nucleus (DRN) has been implicated in disorders of anxiety and depression. Although the nucleus contains the densest population of 5HT neurons in the brain, at least 50% of cells within this structure are non-serotonergic, including a large population of nitric oxide synthase (NOS) containing neurons. The DRN has a unique topographical efferent organization and can also be divided into sub-regions based on rostro-caudal and medio-lateral dimensions. NOS is co-localized with 5HT in the midline DRN but NOS-positive cells in the lateral wing (LW) of the nucleus do not express 5HT. Interestingly, the NOS LW neuronal population is immediately rostral to and in line with the cholinergic lateral dorsal tegmental nucleus (LDT). We used immunohistochemical methods to investigate the potential serotonergic regulation of NOS LW neurons and also the association of this cell grouping to the LDT. Our results indicate that >75% of NOS LW neurons express the inhibitory 5HT_1A receptor and are cholinergic (>90%). The findings suggest this assembly of cells is a rostral extension of the LDT, one that it is subject to regulation by 5HT release. As such the present study suggests a link between 5HT signaling, activation of cholinergic/NOS neurons, and the stress response including the pathophysiology underlying anxiety and depression.     

Repeated social defeat increases reactive emotional coping behavior and alters functional responses in serotonergic neurons in the rat dorsal raphe nucleus

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24 h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.     

Dye coupling between dorsal raphe neurones

Neurones in the dorsal raphe nucleus (DRN) were impaled and filled with biocytin in coronal slices of midbrain taken from young adult rats. The electrophysiological properties and gross morphology of the cells were similar to those reported previously for serotonergic neurones in the DRN. Of 27 cases in which filled neurones were recovered in histological material, almost half (48%) showed labelling of two or three cells, although only one cell had been recorded from. Coupled cells were identified as close or distantly coupled, depending on the distance from the soma of the presumed impaled cell (23.5±15 m, n=7 and 150±26.5 m, n=10 respectively). Whereas close-coupled cells may have been artefactually coupled by the penetrating electrode, coupling between distant cells is most likely to be a result of transfer of biocytin through gap junctions. Camera lucida reconstructions of pairs of labelled cells revealed extensive overlap of dendritic fields and numerous crossings between dendrites. When examined at high magnification under a light microscope, many of the crossing dendrites were found to travel in different focal planes. Nevertheless, for each pair of cells, at least one point of close apposition was observed between dendrites or between the axon and a dendrite of the presumed impaled and coupled cell. The incidence of dye coupling between neurones in the DRN may reflect a relatively high level of electrotonic coupling between the neurones. This form of coupling may be important in determining the synchronous nature of firing of neurones in the DRN.     

双轴旋转运动刺激后大鼠中缝大核神经元活动分析

目的:分析大鼠经引发晕动病的双轴旋转运动刺激后,中缝背核(DR)内5-HT能神经元激活状况,以探讨5-HT水平与前庭刺激的关系.方法:将雄性SD大鼠随机分成两组:对照组和双轴旋转运动刺激组.所有动物装入定做的有机玻璃圆筒内,并于黑暗中进行如下处理2h:运动刺激组动物以电动马达驱动的双轴旋转运动刺激;对照组动物放置在距刺激仪器20 cm的位置.应用包含DR的冠状脑切片,以双重免疫荧光标记技术标记5-HT和Fos蛋白,并对标记神经元进行计数和统计分析.结果:对照组和旋转运动刺激组的Fos阳性神经元、5-HT阳性神经元以及Fos/5-HT双标记神经元的数目分别是:93.4 ±12.0 vs 153.1 ±21.1、528.5±36.0vs 531.1±23.4和15.1 ±11.3 vs 30.8 ±11.3.t检验分析显示两组Fos阳性神经元数目有显著性差异(P＜0.05);Fos/5-HT双标神经元数目在旋转运动刺激后有增加趋势.结论:引发晕动病的双轴旋转运动能激活中缝背核内的神经元,其中的5-HT能神经元也对刺激有反应.     

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.     

Reversible inactivation of the dorsal raphe nucleus blocked the antipanic-like effect of chronic imipramine in the elevated T-maze

Antidepressant drugs are effective in the treatment of panic in human panic disorder patients. One hypothesis is that the anti-panic effects of antidepressant drugs are mediated by an increase in the activity of serotonergic neurons within dorsal raphe nucleus (DRN) leading to an increase in serotonin-mediated inhibition of the dorsal periaqueductal gray (DPAG). In order to test this hypothesis, we investigated the effects of reversible inhibition of the DRN on behavior in the elevated T-maze (ETM) in control rats and rats chronically treated with imipramine. Rats were injected daily with imipramine (15 mg/kg i.p.) or saline for 24 days. A guide cannula was implanted in the DRN on day 14. Lidocaine (4%, 0.2 microL) or saline was injected into the DRN 10 min before the test in the ETM followed immediately by the open-field test (day 21). Three days later, the infusions were crossed-over, rats microinjected into the DRN with saline in the first trial received lidocaine and vice versa, and the behavioral tests were repeated (day 24). Chronic saline plus lidocaine in the DRN and chronic imipramine (plus saline or lidocaine in the DRN) impaired inhibitory avoidance, indicating an anxiolytic effect. In the one-way escape, lidocaine facilitated it, suggesting a panicogenic effect, while chronic imipramine impaired it, which is indicative of a panicolytic effect. Moreover, lidocaine blocked the facilitatory effect of chronic imipramine. The locomotor activity in the open field was not changed by any treatment compared to the control group. These effects were congruent with the hypothesis that the DRN has a dual effect on anxiety: increasing learned anxiety and decreasing innate anxiety. Moreover, they suggest that the DRN exerts a crucial role in the antipanic-like effect of chronic imipramine in the ETM.     

Partial co-existence of NADPH-diaphorase and acetylcholinesterase in the hypothalamic magnocellular secretory nuclei of the rat

Co-localization of NADPH-diaphorase (ND) and acetylcholinesterase (AChE) activities were explored in the magnocellular secretory nuclei of the rat hypothalamus by means of a double histochemical staining of the same sections. Partial co-existence was found in all the nuclei studied (paraventricular, supraoptic, fornicals and circular nuclei). No particular location of the neurons expressing both markers was found, although in the paraventricular nucleus all of them (ND+, AChE+ and neurons expressing both markers) were preferentially located in the magnocellular subdivisions whereas in the parvicellular ones only some neurons belonging to all three types were detected, mainly located in the periventricular and medial subdivisions. The lowest degree of co-existence was found at the level of the main magnocellular nuclei (supraoptic and paraventricular) when compared with the accessory magnocellular nuclei, especially the posterior fornical and the circular nuclei. These results extend previous data on the chemical nature of the neurons producing nitric oxide in the neurosecretory nuclei and the possible functional role of this atypical messenger in the hypothalamus.     

Activation within dorsal medullary nuclei following stimulation in the hypothalamic paraventricular nucleus in rats

The influence of the hypothalamic paraventricular nucleus (PVN) on neurones in the dorsal medulla has been examined in 71 urethane/sagatal-anaesthetised rats. Of 536 neurones localised and tested for responses to electrical stimulation of both the vagus and/or the PVN, 378 were synaptically or antidromically activated following vagal stimulation 72 of which were synaptically activated by stimulation within PVN. The majority of those were located at the border between NTS and dorsal motor nucleus of the vagus in caudal NTS. None showed cardiac or ventilatory rhythm. Neurones showing such rhythms were not affected from PVN. Of 89 neurones in dorsal motor nucleus of the vagus, ten were synaptically activated and two synaptically depressed from PVN. PVN activated neurones in NTS tested for responses to stimulation of arterial baroreceptors and carotid body chemoreceptors were either unaffected or inhibited, but gastric inflation excited them. The results suggest a powerful PVN influence on the dorsal medulla, which is largely confined to the ventral and caudal NTS. There is little evidence for an effect on neurones with a cardiovascular function, but the abdominal vagal influence suggests a link with feeding.     

Projections from the ventral tegmental area and mesencephalic raphe to the dorsal raphe nucleus in the rat

Summary The origins of the dopaminergic innervation of the rat dorsal raphe nucleus (NRD) have been investigated using a combination of fluorescent retrograde tracing and fluorescence histochemistry. Stereotaxic microinjections of True Blue were placed in the central, caudal and lateral portions of the NRD, and after 6–12 days survival the brains were processed for fluorescence histochemical detection of catecholamines. Retrogradely labeled neurons were searched for in the diencephalic A11 and A13 dopaminergic cell groups, substantia nigra, ventral tegmental area (VTA) and the linear, central superior and dorsal raphe nuclei. The various NRD injections consistently resulted in retrograde labeling of a small number of catecholamine-containing, presumed dopaminergic cell bodies, confined mainly to three regions: the VTA, the linear and central superior raphe nuclei and the NRD itself. The present findings indicate that not only dopaminergic neurons in the VTA but also the system of catecholamine-containing cells, extending dorsally and caudally from the VTA within the midline raphe area, project to the NRD. Although often similar in size, shape and distribution to the catecholaminergic neurons the majority of retrogradely labeled cells in these regions were, however, found to be non-catecholaminergic.Abbreviations 3 Principal oculomotor nucleus - 4 Trochlear nucleus - Aq Cerebral aqueduct - cp cerebral peduncle - cst cortico-spinal tract - dscp decussation of the superior cerebellar peduncle - DTg Dorsal tegmental nucleus - fr fasciculus retroflexus - IF Interfascicular nucleus - IP Interpeduncular nucleus - LL nucleus of the lateral lemniscus - ml medial lemniscus - mlf medial longitudinal fasciculus - mNV mesencephalic trigeminal nucleus - NLC Nucleus linearis caudalis - NLR Nucleus linearis rostralis - NRD Dorsal raphe nucleus - PAG Periaqueductal grey - PN Pontine nucleus - PRN Pontine raphe nucleus - R Red nucleus - RCS Nucleus raphe centralis superior - SN Substantia nigra - VTA Ventral tegmental area - VTg Ventral tegmental nucleus     

高频电刺激大鼠丘脑底核抑制背侧中缝核5-羟色胺表达

本研究探讨高频电刺激丘脑底核对大鼠背侧中缝核5-羟色胺(5-HT)表达的影响。实验动物分两组,刺激组给予高频电流(130Hz,100μA,60μs)刺激大鼠右侧丘脑底核,对照组大鼠右侧丘脑底核植入电极,但无电流输出。刺激结束后,用免疫组织化学方法染色背侧中缝核5-HT能神经元,检测背侧中缝核5-HT能神经元的数量和平均灰度值。结果显示电刺激组背侧中缝核5-HT阳性神经元数目与对照组比明显减少(t(13)=3.786,P=0.002),并且神经递质5-HT表达量减少,平均灰度值显著增高(t(13)=7.917,P<0.001)。本实验结果表明高频电刺激丘脑底核对背侧中缝核5-HT能神经元有抑制作用,在应用高频电刺激丘脑底核治疗Parkison病运动障碍时出现的情绪障碍可能与其有关。     

Substance P afferents from the habenula innervate the dorsal raphe nucleus

Summary The lateral habenula nuclei of the diencephalon innervate the median and dorsal raphe nuclei of the brainstem. Habenula lesions lead to decreased substance P levels in the dorsal but not median raphe within 24 hours. From this data, we propose a peptidergic innervation of the dorsal raphe nucleus by the habenula nuclei.     

Effect on memory processes of anti-vasopressin serum microinjected into the dorsal raphe nucleus: The role of catecholaminergic neurotransmission

Summary Anti-arginine⁸-vasopressin serum was microinjected into the mesencephalic dorsal raphe nucleus immediately after the learning trial, in a one-trial learning passive avoidance reaction. The treatment attenuated passive avoidance behavior 24 h after treatment, suggesting a role of the endogenous vasopressin of this area in memory processes. On the other hand, the antiserum did not influence passive avoidance behavior if 6-hydroxydopamine was microinjected into the raphe region. The data suggest that the antiserum may have primarily interacted with catecholaminergic terminals, which enter the dorsal raphe nucleus.This work was supported by the Scientific Research Council, Ministry of Health, Hungary [Grant No. 4-08-0302-03-0(T)]     

Involvement of local orphanin FQ in the development of analgesic tolerance induced by morphine microinjections into the dorsal raphe nucleus of rats

It is well known that dorsal raphe nucleus (DRN) is one of the key structures for the development of opioid analgesia and tolerance. An increased activity of ‘antiopioids’ like orphanin-FQ (OFQ) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of DRN-located OFQ in the opioid analgesic tolerance induced by repeated microinjections of morphine (MOR) into DRN. Male rats were implanted with chronic guide cannulae aimed at the DRN. Microinjection of MOR (0.5 μg in 0.5 μl) into DRN caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the OFQ receptor antagonist nocistatin (NST) (1 ng in 0.5 μl) into the same DRN site, the microinjections of MOR always produced antinociception and did not induce tolerance. If NST microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single NST microinjection into the same DRN site was enough to restore the antinociceptive effect of MOR. On the other hand, if OFQ (1 ng in 0.5 μl) was microinjected into DRN, then MOR microinjection administered 15 min later into the same DRN site did not elicit antinociception. Finally, opioid tolerance induced by repeated systemic MOR injections (5 mg/kg, ip) was reversed by a single microinjection of NST into DRN. This emphasizes the central importance of DRN-located OFQ in the MOR analgesic tolerance.     

Behavioral inhibition induced by electrical stimulation of the median raphe nucleus of the rat.

Earlier studies using brain lesions or drugs that impair 5-hydroxytryptamine (5-HT) neurotransmission suggest that the ascending serotonergic system is involved in behavioral inhibition. Confirming this hypothesis, the present study demonstrates that electrical stimulation of the median raphe nucleus (NMR) of the rat induces behavioral inhibition. Rats implanted with brain electrodes in the NMR were trained to lever press under a variable-interval 1 min schedule of water presentation. At variable time intervals averaging 7 min, periods of 90 sec of brain stimulation were superimposed on VI responding without any exteroceptive stimulus change. Electrical stimulation of the NMR caused suppression of ongoing lever-pressing behavior, proportional to the stimulus intensity. In addition, NMR stimulation elicited defecation, also directly related to the stimulus intensity, and a cluster of somatic and autonomic changes, such as crouching, micturition, piloerection and teeth chattering, that are characteristic of the rat's emotional behavior in stressful situations. In four out of six rats, the response-suppressant effect of NMR stimulation was counteracted by the 5-HT synthesis inhibitor, para-chlorophenylalanine (PCPA), three to five days following the administration of a single dose of 500 mg/kg of the drug. The effect of PCPA tended to disappear after fifteen days and in one rat, was reversed by 5-hydroxytryptophan, given after benserazide, on the third day from PCPA injection. These results support the suggestion that the mesolimbic serotonergic pathway originating in the NMR and projecting to the septal area and the hippocampus is a substrate of behavioral inhibition, in the rat.     

痛抑郁二联征模型大鼠中缝背核不同水平5羟色胺表达差异

目的 探讨痛抑郁二联征模型大鼠中缝背核（DRN）5羟色胺（5-HT）阳性细胞的分布特点。 方法 20只雄性SD大鼠随机分为对照组和模型组。模型组大鼠连续3d皮下注射利血平,每日1次,以诱发痛抑郁二联征模型。模型制作后第1天和第2天检测左后足机械缩足阈观察大鼠痛阈变化,模型制作后第2天旷场实验和高架O迷宫实验检测大鼠情绪变化,免疫荧光技术观察DRN前囟后6.8mm、7.3mm和7.8mm水平的5-HT表达。 结果 模型组大鼠经利血平注射后机械痛阈显著下降,并产生抑郁样行为。对照组的DRN前囟后6.8mm、7.3mm和7.8mm水平的5-HT阳性细胞表达量分别为106.00±10.21、96.67±24.50和195.67±2.33。模型组相应的表达量分别为61.67±14.53,72.33±34.35和53.67±26.77。除前囟后7.8mm水平模型组与对照组相比有显著性差异（P<0.05）之外,其他两个水平5-HT阳性细胞表达量模型组和对照组之间差异无显著性（P>0.05）。 结论 利血平诱导的痛抑郁二联征大鼠模型以及中缝背核5-HT表达的降低,可能主要与DRN前囟后7.8mm水平的5-HT阳性细胞数变化有关,而与前囟后6.8mm和后7.3mm 水平无关。     

Potential noradrenergic regulation of serotonergic neurons in the median raphe nucleus

Summary Pharmacological and morphological evidence suggests that the functional activity of serotonergic neurons may be regulated by catecholamines. We have attempted to reveal a potential pathway by which this interaction might occur. Rats received bilateral knife cut lesions of the ventral noradrenergic bundle which severed the A-1 and A-2 cell body contributions to this projection. Controls received a sham lesion into the cerebellum. Two weeks later all animals were sacrificed, and norepinephrine and serotonin levels were measured in discrete nuclei of the brain. Lesion placement was confirmed histofluorometrically. Serotonin levels in the median raphe nucleus were significantly reduced by 40%, but levels of serotonin were unaffected in the dorsal raphe nucleus and 8 serotonergic terminal regions. The lesions did not affect levels of norepinephrine in the locus coeruleus, cingulate cortex, or habenula. This study suggests that a noradrenergic projection to the median raphe nucleus from the A-1 and A-2 cell body groups may modulate serotonergic neuronal function.This work was presented, in part, at the Society for Neuroscience Annual Meeting, Anaheim, California, 1977     

Anterograde tracing of projections from the dorsal raphe nucleus to the vestibular nuclei

This study used the anterograde transport of biotinylated dextran amine (BDA) to identify the course and terminal distribution of projections from the dorsal raphe nucleus (DRN) to the vestibular nuclei in rats. After iontophoretic injection of BDA into the medial and lateral regions of DRN, anterogradely labeled fibers descend within the medial longitudinal fasciculus and the ventricular fiber plexus to terminate within two discrete regions of the vestibular nuclear complex. One terminal field was located primarily ipsilateral to the injection site and involved rostrodorsal aspects of the vestibular nuclei, including superior vestibular nucleus and rostral portions of the medial vestibular nucleus (MVN) and lateral vestibular nucleus (LVN). The other terminal field involved caudoventral aspects of both ipsilateral and contralateral MVN and LVN and was less heavily innervated. These findings confirm that the vestibular nuclei are targeted by a regionally-selective projection from the DRN. The segregation of DRN terminals into anatomically distinct fields indicates that the DRN-vestibular nucleus projections are organized to selectively modulate processing within specific functional domains of the vestibular nuclear complex. In particular, these terminal fields may be organized to modulate vestibular regions involved in eye movement-related velocity storage, coordination of vestibular and affective responses, and the bilateral coordination of horizontal eye movement reflexes.     

Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey

Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18-25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus.