Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain

We have studied the localization of serotonin- and non-serotonin-containing cell bodies in the midbrain raphe nuclei that project to the entorhinal area and the hippocampal formation in the rat brain, using the technique of combined retrograde fluorescent tracing and immunohistochemistry on the same tissue section. The branching properties of these neurons were studied by retrograde double labelling using two fluorochromes which emit fluorescence with different spectral characteristics. After injections of granular blue or propidium iodide into the medial entorhinal area, retrogradely-labelled cells were found situated bilaterally in the caudal half of the dorsal raphe nucleus, the medial part of the median raphe and throughout the rostrocaudal extension of the nucleus reticularis tegmentipontis. Injections placed successively more laterally in the entorhinal area labelled progressively less cells contralaterally in the dorsal raphe and the reticular tegmental nucleus of the pons. After fluorochrome injections into the dorsal part of the hippocampal formation, retrogradely-labelled cells were found in the caudal part of the dorsal raphe, in the peripheral part of the median raphe and to a minor extent in the medial part of this nucleus, but not in the nucleus reticularis tegmentipontis. The experiments with double retrograde fluorescent tracing showed that the raphe nuclei do not send bilateral projections to the entorhinal area in spite of the fact that many of these cells are located contralateral to the injected hemisphere in single labelling experiments. Injections of the fluorochromes into the entorhinal area and hippocampal formation showed that at least 10% of the raphe cells project to both areas simultaneously. Analysis of sections incubated with antiserum to serotonin showed that a majority of the retrogradelylabelled versus serotonin-immunoreactive cells was found to vary within different parts of the individual raphe nuclei: the ventromedial part of the dorsal, the medial part of the median and the nucleus reticularis tegmentipontis being the highest.The findings indicate that both serotonin- and non-serotonin-containing neurons in the raphe innervate the hippocampal region, that these projections may be crossed but not bilateral, and that the same neuron in the raphe may influence the neural activity in the entorhinal area and the hippocampus simultaneously.     

Coexpression of Serotonin and Nitric Oxide in the Raphe Complex: Cortical Versus Subcortical Circuit

Several lines of evidence have implicated a direct reciprocal interaction between serotonin and nitric oxide (NO). The goal of this investigation was, therefore, to examine the coexpression of tryptophan hydroxylase (TPH; the rate limiting enzyme for the synthesis of serotonin) and neuronal NO synthase (nNOS) in the ascending cortical projecting raphe nuclei (B6–B9 subgroups), when compared with the descending spinal cord projecting raphe nuclei (B1–B3 subgroups). Our data demonstrated that: (1) a significant number of raphe‐cortical projecting neurons was identified not only in the midline subgroup of dorsal raphe (B6, 7) but also in the median raphe (B8), as well as in the supralemniscal nucleus (B9); (2) serotonergic cortical projecting neurons from these three raphe nuclei exhibited a high (>80%) percentage of coexpression with nNOS immunoreactivity; (3) similarly, serotonin transporter immunoreactive fibers in the medial prefrontal cortex were also double‐labeled with nNOS immunoreactivity; (4) in contrast, the descending spinal cord projecting raphe nuclei revealed only TPH but not nNOS immunoreactivity. Our present findings suggest the existence of a direct interaction between serotonin and NO in the ascending cortical projecting raphe system. In contrast, a different strategy appears to operate the descending spinal cord projecting raphe system. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

P物质和亮-脑啡肽样免疫反应神经元在鼠中脑中缝核的分布

本实验采用PAP免疫细胞化学技术,观察P物质和亮-脑啡肽样免疫反应神经元在鼠中脑中缝核的分布并作了计数。1.含P物质样免疫反应神经元胞体仅见于中缝背核最尾侧部,含P物质阳性胞体数平均167个/只;而亮-脑啡肽样免疫反应胞体几乎遍于中缝背核全长,其阳性胞体数平均132个/只。2.亮-脑啡肽样免疫反应胞体主要定位于中缝背核中线区;而P物质样免疫反应胞体除分布于中缝背核中线区以外,也定位在其两侧的延伸。3.亮-脑啡肽样免疫反应胞体恒定地出现在中缝正中核内,平均199个/只;而P物质样免疫反应胞体仅见于2例动物。4.中缝背核内有些含P物质样免疫反应阳性胞体或突起,紧邻血管壁排列,未观察到亮-脑啡肽样免疫反应神经元有类似现象。本实验观察到鼠中缝正中核内存在含P物质样免疫反应神经元胞体。     

Fos expression in serotonergic neurons in the rat brainstem following noxious stimuli: an immunohistochemical double-labelling study

In order to detect whether there were different expression patterns of Fos protein induced by somatic or visceral noxious stimulation in the serotonergic neurons in the rat brainstem, an immunohistochemical double-labelling technique for serotonin (5-HT) and Fos was employed after subcutaneous or stomach injection of formalin. The two stimuli were matched in pilot experiments to produce maximum Fos expression. The expression of Fos protein in 5-HT-containing neurons (5-HT/Fos co-localized neurons) could be observed in the ventrolateral subdivision of the midbrain periaqueductal grey, interpeduncular nucleus, paramedian raphe nucleus, all of the brainstem raphe nuclei, the alpha part of the gigantocellular reticular nucleus and the lateral paragigantocellular reticular nucleus. The locations of the 5-HT/Fos co-localized neurons in the brainstem of animals subjected to somatic noxious stimulation were similar to those subjected to visceral noxious stimulation. However, the number and proportion of the 5-HT/Fos co-localized neurons in the median raphe nucleus and nucleus raphe obscurus of the rat subjected to visceral noxious stimulation were statistically greater than those in rats subjected to somatic noxious stimulation. These results suggest that serotonergic neurons in median raphe nucleus and nucleus raphe obscurus have a tendency to higher neuronal activity after visceral noxious stimulation.     

Serotonergic and nonserotonergic neurons in the dorsal raphe nucleus send collateralized projections to both the vestibular nuclei and the central amygdaloid nucleus

Using a combination of double retrograde tracing and serotonin immunofluorescence staining, we examined whether individual serotonergic and nonserotonergic neurons in the dorsal raphe nucleus are sources of collateralized axonal projections to vestibular nuclei and the central amygdaloid nucleus in the rat. Following unilateral injections of Diamidino Yellow into the vestibular nuclei and Fast Blue into the central amygdaloid nucleus, it was observed that approximately one-fourth of the dorsal raphe nucleus neurons projecting to the vestibular nuclei send axon collaterals to the central amygdaloid nucleus. Immunofluorescence staining for serotonin revealed that more than half of the dorsal raphe nucleus neurons from which these collateralized projections arise contain serotonin-like immunoreactivity. These findings indicate that a subpopulation of serotonergic and nonserotonergic dorsal raphe nucleus cells may act to co-modulate processing in the vestibular nuclei and the central amygdaloid nucleus, regions implicated in the generation of emotional and affective responses to real and perceived motion.     

Topographic principles in the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat

The spinal projections from the raphe-associated brainstem areas containing serotonergic neurons were studied with aldehyde-induced fluorescence in combination with the retrograde fluorescent tracer True Blue in the rat. This technique makes it possible to determine simultaneously the projections of individual neurons and to detect whether serotonin is present in the same neurons. After tracer injections into the spinal cord retrogradely labeled serotonergic and non-serotonergic neurons were found in the medullary raphe nuclei and adjacent regions and to a lesser extent in association with the dorsal and median raphe nuclei in the mesencephalon. Large True Blue injections that covered one side of the spinal cord at mid-cervical level labeled about 60% of the ipsilaterally situated serotonergic neurons in the medullary raphe regions while the corresponding figure contralaterally was about 25%. On both sides a larger number of labeled non-serotonergic neurons were found; these were sometimes located dorsal to, but often intermingled with, the serotonergic cells. While the serotonergic projection from the mesencephalon could not be labeled from injections below cervical levels, the labeling in more caudal brainstem regions exhibited only minor variations depending on the rostrocaudal level of the spinal segment injected. Furthermore, quantitative data from injections at different levels indicate that the majority of the spinal-projecting neurons traverse most of the length of the cord. Summarizing the results obtained from small injections restricted to subregions of the cord we feel that it is possible to distinguish three fairly distinct pathways for spinal projections from the medullary raphe and adjacent regions: The dorsal pathway originates mainly from cells in the caudal pons and rostral medulla oblongata (rostral part of nucleus raphe magnus, nucleus raphe magnus proper, nucleus reticularis gigantocellularis pars alpha and nucleus paragigantocellularis). This pathway, which contains a large non-serotonergic component, descends through the dorsal part of the lateral funiculus and terminates mainly in the dorsal horn at all spinal cord levels. The intermediate pathway is largely serotonergic with its cell bodies located within the arcuate cell group (situated just ventral and lateral to the pyramids very close to the ventral surface of the brainstem) and in the nucleus raphe obscurus and pallidus and terminates in the intermediate grey at thoracolumbar and upper sacral levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

大鼠中缝背核及中缝正中核内的VIP、GABA样触液神经元

本文将ＣＢ－ＨＲＰ注入侧脑室，用ＣＢ－ＨＲＰ逆行迫踪与免疫细胞化学相结合的方法，对大鼠脑干内的中缝背核及中缝正中核的远位触液神经元进行了定性研究。结果表明：中缝背核内存在ＶＩＰ样、ＧＡＢＡ样免疫反应阳性的触液神经元；中缝正中核内亦存在少量ＶＩＰ样、ＧＡＢＡ样免疫反应阳性触液神经元。它们的形态和数量各异。本文首次报道中缝背核和中缝正中核内远位触液神经元的化学性质，为探索其机能意义提供了形态学依据。     

Projections from the medial septum and diagonal band of Broca to the dorsal and central superior raphe nuclei: a non-cholinergic pathway

Summary The anatomical organization of projections from the medial septal nucleus (MS), and the vertical (VDB) and horizontal limb (HDB) of the diagonal band of Broca to the dorsal raphe nucleus (NRD) and the central superior raphe nucleus (RCS) of the rat were studied by anterograde [³H]-leucine, and True Blue and Fluoro Gold fluorescent retrograde tracing. Projections from the MS were found to enter the basal mesencephalon at the rostro-medial aspect of the pontine nuclei, curve dorsally and terminate throughout the RCS and in the caudal portion of the NRD. Fibers from the VDB were found to enter these raphe nuclei by two separate routes; some fibers reached the basal mesencephalon, curved dorsally and terminated in the RCS and NRD. Other fibers entered the pedunculopontine nucleus, curved medially and reached the NRD. Presumed terminal labelling was found overlaying the RCS and NRD throughout their rostro-caudal extensions. The brain stem projections from HDB entered the mesencephalon by the same routes as those from VDB, but the labelling over RCS was sparse, and the NRD labelling was preferentially distributed to the rostral portion of the nucleus. The present data indicate a crude topographic organization of the projections from the septal region to the NRD and RCS. In general, the distribution of presumed terminal labelling appeared to be more closely associated with the distribution of NRD and RCS 5-HT immunoreactive cell bodies, than with the cytoarchitectonically defined extensions of these raphe nuclei. By sequential evaluation of the distribution of retrogradely labelled and acetylcholine esterase-stained cells on the same section, and by selective tracing with radiolabelled choline, it appears that the vast majority, if not all, of the neurons in MS and diagonal band which project to the rostral raphe are non-cholinergic.Abbreviations CLi caudal linear raphe nucleus - DTg dorsal tegmental nucleus - flm medial longitudinal fasciculus - HDB horizontal limb of the diagonal band of Broca - IP interpeduncular nucleus - MS medial septal nucleus - NRD dorsal raphe nucleus - PAG periaqueductal gray - Pn pontine nucleus - R red nucleus - RCS central superior raphe nucleus - RMg raphe magnus nucleus - RPn pontine raphe nucleus - SN substantia nigra - VDB vertical limb of the diagonal band of Broca - VTg ventral tegmental nucleus - 5 trigeminal motor nucleus     

豚鼠中脑中缝背核和中缝正中核5-羟色胺能神经元向视交叉上核的直接投射

实验借助HRP逆行定位和免疫细胞化学定性相结合的双重标记技术,探讨豚鼠中脑中缝背核和中缝正中核向下丘脑视交叉上核的直接投射.结果首次确定了:(1)中缝背核/中缝正中核内5-羟色胺和非5-羟色胺能神经元对视交叉上核的直接投射和分布特点;(2)投射神经元主要集中在中缝背核;(3)中缝背核对视交叉上核的投射神经元几乎均为5-羟色胺能,而中缝正中核中5-羟色胺和非5-羟色胺能投射神经元约各占半数.     

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     

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     

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。     

5-羟色胺转运体在酗酒人脑干头侧中缝核群的表达

目的:观察酗酒人脑标本脑干头侧中缝核群5-羟色胺转运体(SERT)的表达变化。方法:应用免疫放射自显影的方法,显示SERT免疫反应强度在酗酒人脑标本脑干头侧中缝核群的变化,并与健康人脑标本比较。结果:健康组SERT免疫反应强标记信号在脑桥头侧和中脑主要集中分布在与中缝核群相同的区域。在酗酒人脑标本,正常分布于脑干头侧中缝核群的SERT免疫反应标记信号减弱;中缝正中核、中缝背核尾侧部、中缝背核束间部、中缝背核腹侧部、中缝背核背侧部的SERT免疫反应含量强度与健康组相应区域比较显著降低。结论:酗酒者头侧中缝核群SERT蛋白表达降低。     

大鼠中缝背核接触脑脊液神经元化学性质的研究

用CB-HRP追踪与免疫细胞化学结合的方法,对大鼠中缝背核接触脑脊液神经元(简称触液神经元)的化学性质进行了研究.将CB-HRP注入第三脑室后,中缝背核内观察到CB-HRP标记细胞,标记细胞分布于中缝背核的背侧部和腹侧部.在CB-HRP与P物质或5-羟色胺免疫细胞化学结合的切片上,中缝背核内出现三种标记细胞:HRP单标细胞,P物质或5-羟色胺免疫反应阳性单标细胞,HRP/SP或HRP/5-HT双标细胞,双标细胞为中、小型梭形或圆形细胞.上述结果提示:中缝背核存在SP能和5-HT能触液神经元.     

大鼠中缝背核一氧化氮合酶神经元投射纤维在大脑皮质微血管的分布

目的：研究通过损毁脑干中缝背核(DR)，探讨中缝背核NOS阳性神经元是否投射分布于大脑皮质微血管。方法：将16只SD雄性成年大鼠分为实验组与对照组。对实验组大鼠中缝背核微量注射喹啉酸，饲养1w，灌注固定，然后将大脑及脑干作冠状冰冻切片，NADPH—d组化染色。结果：实验组大鼠的中缝背核被有效损毁，其NOS阳性神经元的数量减少了59．1％(P＜0．001)。额、顶叶皮质NOS阳性纤维终末减少了32．1％(P＜0．05)，其中附着于皮质微血管的NOS阳性纤维终未了减少了37．8％(P＜0．01)。而枕额叶皮质NOS阳性纤维终末也减少了32．8％(P＜0．05)，其中附着于皮质微血管的阳性纤维终末减少了39．4％(P＜0．01)。结论：位于中缝背核的NOS阳性神经元投射分布于大脑皮质微血管，可能参与大脑皮质血流量的调节。     

Distribution of dopaminergic cell bodies in the median raphe nucleus of the rat brain

An increasing amount of data suggests that a dysfunction in dopamine (DA) neurotransmission is involved in the pathophysiology of various neurological and psychiatric disorders. With this in mind, the distribution and connectivity of the dopaminergic system in the rat brain has been studied extensively. So far, little is known about the distribution of DA containing neurons in the median raphe nucleus (MnR). This nucleus is mainly defined by a large population of serotonin containing neurons. Using quantitative immunohistochemistry, we observed the presence of a small number of DA containing neurons in the rat MnR, which was in contrast to a previous report.     

Effects of electrical stimulation of the raphe area on the micturition reflex in cats

The raphe nucleus has a variety of physiological functions, including emotion, regulation of skeletal muscle motoneurons, spinal transmission of nociceptive signals, sleep, respiration, gastric motility, and cardiovascular function. Recent evidence has shown that centrally administered serotonin has modulatory effects on micturition function, and that decreased brain serotonin might underlie depression and an overactive bladder. We applied high-frequency stimulation (HFS; 0.2-ms duration, 100 Hz) in the raphe nucleus and the adjacent midline area in 20 supracollicular decerebrate cats, which mostly elicited inhibition of the micturition reflex. The effective amplitude of the electrical stimulation for evoking inhibitory responses was less than 50 muA. We also examined single neuronal activities in the raphe nucleus in response to isovolumetric spontaneous micturition reflexes. In total, 79 neurons were recorded in the raphe nucleus that were related to urinary storage/micturition cycles. Of the neurons recorded, the most common were tonic storage neurons (48%), followed by tonic micturition neurons (28%), phasic storage neurons (18%), and phasic micturition neurons (6%). In addition to the tonic/phasic as well as storage/micturition classification, the neurons showed diverse discharge patterns: augmenting, constant and decrementing, with the constant discharge pattern being most common. Among neurons in the raphe nucleus, the neurons with a decrementing discharge pattern were concentrated in the rostral portion, whereas the augmenting and constant neurons existed diffusely. The storage and micturition neurons were intermingled in the rostral portion, whereas they were separate in the caudal portion. In conclusion, the results of the present study indicate that HFS of the raphe area inhibits the micturition reflex and that there are micturition-related neuronal firings in the raphe area in cats, suggesting that the raphe nucleus is involved in neural control of micturition.     

抑制一氧化氮合成酶对大鼠睡眠及5－羟色胺神经元免疫反应的影响

采用多导描记及免疫组化方法，观察了侧脑室和腹腔给予硝基左旋精氨酸（Ｌ－ＮＮＡ）对慢性植入电极大鼠睡眠量及中缝背核５－羟色胺（５－ＨＴ）神经元免疫阳性反应的影响。结果表明：Ｌ－ＮＮＡ显著抑制慢波睡眠和快眼动睡眠，与对照相比较，觉醒时间延长。脑室给予Ｌ－ＮＮＡ使中缝背核５－ＨＴ神经元阳性细胞增多。提示一氧化氮合成抑制所致的睡眠抑制可能与中缝背核５－ＨＴ神经元免疫阳性反应增强有关。     

Non-serotonergic dorsal and median raphe projection onto parvalbumin- and calbindin-containing neurons in hippocampus and septum

The median raphe nucleus is involved in controlling and maintaining hippocampal activity through its projection to inhibitory neurons in medial septum and hippocampus. It has been shown that anterogradely axonal-traced fibers originating in the median raphe nucleus project onto calbindin-containing neurons in hippocampus and parvalbumin-containing neurons in medial septum. Parallel immunohistochemistry studies showing serotonin fibers contacting calbindin- and parvalbumin-positive neurons have led to the assumption that raphe fibers projecting on these types of neurons are mainly serotonergic. However, in both dorsal and median raphe nucleus there is a large amount of non-serotonergic neurons which also are projecting neurons, indicating that a part of the raphe fibers projecting to hippocampus and septum may be non-serotonergic. Our aim was to determine whether there is a non-serotonergic projection from the raphe nucleus onto calbindin- and parvalbumin-containing neurons in hippocampus and septum. Biotin dextran amine was used as the anterograde neuronal tracer and injected into either dorsal or median raphe nucleus. By use of triple immunofluorescence-labeling we analyzed the serotonergic content of the biotin dextran amine-labeled fibers contacting parvalbumin- and calbindin-positive neurons. Surprisingly, we found a significant non-serotonergic projection from both dorsal and median raphe nuclei onto calbindin- and parvalbumin-containing interneurons in septum and hippocampus, with a preference in hippocampus for projecting onto calbindin-positive neurons. These results indicate that the raphe nuclei may exert their control on hippocampal and septal activity not only through a serotonergic projection, but also through a significant non-serotonergic pathway.     

Role of serotonin transporter inhibition in the regulation of tryptophan hydroxylase in brainstem raphe nuclei: time course and regional specificity

Drugs that selectively inhibit the serotonin transporter (SERT) are widely prescribed for treatment of depression and a range of anxiety disorders. We studied the time course of changes in tryptophan hydroxylase (TPH) in four raphe nuclei after initiation of two different SERT inhibitors, citalopram and fluoxetine. In the first experiment, groups of Sprague–Dawley rats received daily meals of rice pudding either alone (n=9) or mixed with citalopram 5 mg/kg/day (n=27). Rats were sacrificed after 24 h, 7 days or 28 days of treatment. Sections of dorsal raphe nucleus (DRN), median raphe nucleus (MRN), raphe magnus nucleus (RMN) and caudal linear nucleus (CLN) were processed for TPH immunohistochemistry. Citalopram induced a significant reduction in DRN TPH-positive cell counts at 24 h (41%), 7 days (38%) and 28 days (52%). Similar reductions in TPH-positive cell counts were also observed at each timepoint in the MRN and in the RMN. In the MRN, citalopram resulted in significant reductions at 24 h (26%), 7 days (16%) and 28 days (23%). In the RMN, citalopram induced significant reductions of TPH-positive cell counts at 24 h (45%), 7 days (34%) and 28 days (43%). By contrast, no significant differences between control and treatment groups were observed in the CLN at any of the time points that we studied. To investigate whether these changes would occur with other SERT inhibitors, we conducted a second experiment, this time with a 28-day course of fluoxetine. As was observed with citalopram, fluoxetine induced significant reductions of TPH cell counts in the DRN (39%), MRN (38%) and RMN (41%), with no significant differences in the CLN. These results indicate that SERT inhibition can alter the regulation of TPH, the rate limiting enzyme for serotonin biosynthesis. This persistent and regionally specific downregulation of serotonin biosynthesis may account for some of the clinical withdrawal symptoms associated with drugs that inhibit SERT.