Effect of thalamic parafascicularis nucleus stimulation in regulation of serotoninergic transmission in the cat caudate nucleus: involvement of autoreceptors in the dorsalis raphe nucleus

The mechanisms involved in parafascicularis nucleus control on serotoninergic neurons projecting into the caudate nucleus were investigated in "encéphale-isole" cats. The effects of unilateral stimulation of the parafascicularis nucleus on the release of newly synthesized [3H]serotonin were simultaneously determined in the ipsilateral caudate nucleus and the dorsalis raphe nucleus using push-pull cannulae. The actions of various pharmacological treatments performed either in the caudate nucleus or in the dorsalis raphe nucleus were also examined. The electrical or chemical stimulation of the parafascicularis nucleus induced a decrease in striatal [3H]serotonin release and an increase in [3H]serotonin release in the dorsalis raphe nucleus. The blockade of cholinergic (mecamylamine) and glutamatergic (PK 26124) transmissions at the striatal level did not modify the thalamic stimulation-induced effect on serotonin release in the caudate nucleus or in the dorsalis raphe nucleus. However, a decrease induced by parafascicularis nucleus stimulation in serotonin release in the caudate nucleus could not be observed when the autoreceptors present on serotoninergic nerve cell bodies localized in the dorsalis raphe nucleus were blocked by a methiothepin perfusion within the nucleus. These results indicate that the parafascicularis nucleus controls striatal serotonin transmission by inducing changes in the nerve activity of serotoninergic neurons in the dorsalis raphe nucleus via somatodendritic serotonin release and autoreceptors.     

Changing distribution of monoaminergic markers in the developing human cerebral cortex with special emphasis on the serotonin transporter

This article reviews the current knowledge of the early onset of the monoaminergic innervation in the developing cerebral cortex in humans and of changes in the distribution of tyrosine hydroxylase (TH) immunoreactivity in different neuronal populations of the developing telencephalon. The early genesis of the central monoaminergic neurons in mammals has led to postulations of a trophic role of monoamines in brain morphogenesis--especially in the cerebral cortex. The developmental effects of amines can be linked to the transient expression of different molecules linked to dopamine or serotonin neurotransmission. We present novel data on the immunocytochemistry of the vesicular monoamine transporter (VMAT2) and of the high-affinity serotonin transporter (SERT) in human fetuses. SERT is a marker of the serotoninergic axons and allows visualization of the serotonin afferents of the raphe in the human telencephalon. In addition, during a restricted time period corresponding to 12-14 postovulatory weeks, we found SERT-immunolabeled fibers in the rostral and caudal limbs of the internal capsule that do not correspond to serotoninergic fibers, but do coincide with the calbindin D28k-labeled thalamocortical fiber tracts. The present observations are correlated with findings in rodents, in which a transient expression of SERT is visible in the thalamocortical axons during early postnatal life. The function of this transporter has been shown to be important for the fine-tuning of cortical sensory maps during the critical period of development of these maps. Although the present observation does not allow ascertainment of which neurons transiently express SERT, it lends support to the notion that serotonin and serotonin uptake could have important developmental roles, during the formation of brain connections in humans, as they have in rodents.     

Origin and influence of the serotoninergic innervation of the subcommissural organ in the rat

The origin of the serotoninergic innervation of the rat subcommissural organ was studied using radioautography of tritiated serotonin and biochemical determination of endogenous serotonin content after electrolytic lesions of raphe nuclei. The results suggest that this innervation is mainly derived from nuclei raphe centralis superior and raphe dorsalis, each nucleus contributing about one-third of the input. A possible contribution from nucleus raphe pontis is also suggested. Given the different patterns of innervation revealed by silver staining of nerve fibers and the different patterns of secretory activity observed with histochemical methods after the electrolytic lesions, the following working hypothesis is formulated. Nucleus raphe dorsalis would inhibit the synthesis of secretory material in the rat subcommissural organ via medium-sized serotoninergic fibers restricted to the hypendymal region, whereas nucleus raphe centralis superior might inhibit the release of secretory material via rather thin serotoninergic fibers reaching the nuclear level of the ependyma. This hypothesis is in line with the inhibitory effect postulated for the serotoninergic innervation in the rat subcommissural organ in early investigations using serotonin neurotoxins.     

Forebrain and brainstem afferents to the arcuate nucleus in the rat: potential pathways for the modulation of hypophyseal secretions

Doxorubicin, an anti-oncogenic agent, was used as a retrograde marker to identify arcuate nucleus afferent projections. Injections of this tracer into the arcuate nucleus indicated that the subfornical organ, the organum vasculosum of the lamina terminalis, the nucleus raphe dorsalis and median raphe send projections to the arcuate nucleus. Immunocytochemical procedures were used to demonstrate that the raphe projections to the arcuate nucleus are serotoninergic. This anatomical investigation provides evidence that neural pathways exist between forebrain body fluid and mineral nuclei, mesencephalic serotonin nuclei and the arcuate nuclei.     

Collateral projections of nucleus raphe dorsalis neurones to the caudate-putamen and region around the nucleus raphe magnus and nucleus reticularis gigantocellularis pars alpha in the rat

It was examined whether or not the nucleus raphe dorsalis (RD) neurons projecting to the caudate-putamen (CPu) might also project to the motor-controlling region around the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (Gia) in the rat. Single RD neurons projecting to the CPu and NRM/Gia by way of axon collaterals were identified by the retrograde double-labeling method with fluorescent dyes, Fast Blue and Diamidino Yellow, which were injected respectively into the CPu and NRM/Gia. Then, serotonin (5-HT)-like immunoreactivity of the double-labeled RD neurons was examined immunohistochemically; approximately 60% of the double-labeled RD neurons showed 5-HT-like immunoreactivity. The results indicated that some of serotonergic and non-serotonergic RD neurons might control motor functions simultaneously at the levels of the CPu and NRM/Gia by way of axon collaterals.     

脑干内5-HT能神经元至小脑皮质的纤维投射

本文用单纯免疫细胞化学和免疫电镜方法研究了大白鼠小脑皮质内5-HT纤维的分布及存在形式,并用HRP逆行追踪和免疫细胞化学结合法研究了脑干内5-HT能神经元向小脑皮质的纤维投射。实验证实:5-HT纤维广泛存在于小脑皮质各部、各层,其中Ⅴ～Ⅷ叶较集中,蚓部纤维密度较半球高。皮质各层中,颗粒细胞外层及Purkinje细胞层纤维密度最高,呈树枝状分布;分子层次之,呈“T”型分布;颗粒细胞内层纤维密度最低,向垂直皮质表面方向行走。电镜观察到:小脑皮质分子层内有多个5-HT免疫反应阳性末梢,其中多数为无突触末梢,部分分布在毛细血管壁附近,少数为轴-轴型突触末梢。实验还证实:小脑内5 HT纤维有多个来源,它们可来自延髓外侧网状核、中缝隐核、巨细胞网状核、楔外核、脑桥被盖网状核。     

The serotonin innervation of the cerebral cortex in the rat—an immunohistochemical analysis

The serotonergic innervation of the cerebral cortex in the rat has been studied by immunohistochemistry employing an antibody directed against the neurotransmitter, serotonin. The dorsal raphe, median raphe and B9 cell groups contain intensely labelled neuronal perikarya. Bundles of large diameter axons suggestive of fibers of passage are observed in successive sections as they ascend through the midbrain tegmentum, medial forebrain bundle, diagonal band and supracallosal stria en route to the cortex. In addition, a lateral pathway to the cerebral cortex traversing the ansa peduncularis is visualized. All regions of the cerebral cortex appear to be innervated by serotonergic axons which have a distinctive morphology: they are fine (0.1–0.5 μm), varicose, and extremely convoluted. Serotonergic axons of passage are thicker and comparatively straight. Throughout the lateral neocortex, as well as in the anterior cingulate cortex, serotonergic axons form a densely arborizing plexus through all cortical layers. Contrary to earlier reports, based on histofluorescence, describing a sparse innervation of the cortex with most of the fibers found in the molecular layer, the present study reveals that the innervation is relatively uniform across all cortical layers. In most of the cortex the density of serotonin-containing axons exceeds that of noradrenergic fibers. A distinctive and different pattern of serotonin innervation is found in the posterior cingulate cortex (cytoarchitectonic field RSg): the serotonergic axons are restricted largely to lamina I and III. A restricted laminar pattern also characterizes the innervation of the hippocampus; dense axonal plexuses occur in the outer rim of the dentate hilus and in the stratum lacunosum-moleculare. The serotonergic afferents to the cortex appear to have at least two different modes of distribution, a relatively uniform pattern in the anterior cingulate and the lateral neocortex and a restricted, laminar pattern in the posterior cingulate and the hippocampus.The density and extent of the serotonin innervation is such that the raphe neurons may contact every cell in the cortex. The widespread arborization of serotonin axons contrasts with the spatially restricted termination of thalamic afferents. The distribution of serotonin-containing fibers also differs substantially from the terminal patterns of noradrenergic and dopaminergic fibers. The differences in axonal morphology and distribution amongst the monoamine afferents reflect differences in their contributions to cortical circuitry. The present findings indicate that the serotonin-containing neurons may exert a profound and global, but not necessarily uniform, influence upon cortical function.     

A temporal analysis of the origin and distribution of serotoninergic afferents in the cerebellum of pouch young opossums

Summary In the present study, a temporal analysis of the pattern of distribution of serotoninergic fibers and varicosities within the cerebellum of pouch young opossums was carried out. Particular attention was focused on animals ranging in age from postnatal day (PD) 21-PD 72, because there is a transient expression of serotonin immunoreactivity in the cerebellar cortex during that interval. Between PD 1–33, there is a progressive increase in serotoninergic immunoreactivity throughout the cerebellar cortex. After PD 33, there is a decrease in the relative number of immunostained fibers followed by a reorganization into the adult pattern of distribution.A double labeling paradigm, in which horseradish peroxidase, used as a retrograde marker, combined with serotonin immunohistochemistry was employed to localize serotoninergic neurons that project to the developing cerebellum. Initially (PD 9), serotoninergic cells in the medullary reticular formation and dorsolateral pontine tegmentum are double labeled. After PD 77, only neurons in the medullary reticular formation were double labeled.The course taken by serotoninergic axons from the brainstem to the cerebellum also was analyzed. Between PD 1 and PD 42, serotoninergic axons enter the cerebellum via four different routes: 1) the inferior cerebellar peduncle; 2) a pathway located lateral and rostral to the inferior cerebellar peduncle; this bundle of serotonin axons contains immunoreactive fibers that also enter the tectum (this tract is referred to as the tecto-cerebellar bundle in this report); 3) the medial aspect of the superior cerebellar peduncle; and 4) the tela choroidea. After PD 40, the latter two pathways are the primary routes by which serotoninergic fibers enter the cerebellum. The loss of serotoninergic fibers in the first two pathways coincides with the decrease in serotoninergic immunoreactivity seen in the cerebellar cortex described above.In summary, the results suggest that the serotoninergic projection to the opossum's cerebellum is remodelled during development. It is proposed that the serotonin fibers present at early stages of development may play a role in regulating specific events in cerebellar maturation. In contrast, the serotoninergic axons which have a more restricted pattern of distribution later in development, and in the adult, likely modulate neuronal activity within the cerebellum.Abbreviations BP basilar pons - CB cerebellum - CF cephalic flexure - CN cerebellar nuclei - CRI crus I - CRII crus II - DAO dorsal accessory olive - DN dentate nucleus - EGL external granule cell layer - F flocculus - FP primary fissue - ICP interior cerebellar reduncle - IO inferior olive - IOC inferior olivary complex - IV fourth ventricle - LS lobus simplex - MCP middle cerebellar peduncle - MED medulla - MID midbrain - PF pontine flexure - PFL paraflocculus - PML paramedian lobule - PN pontine nuclei - PT pontine tegmentum - RA raphe - RGc _v nucleus reticularis gigantocellularis pars ventralis - SO superior olive - SV superior medullary velum - TC tela choroidea - TE tectum - VII facial nucleus - Roman numerals I–X cerebellar lobules     

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.     

Serotonin hyperinnervation in the adult rat ventral mesencephalon following unilateral transection of the medial forebrain bundle. Correlation with reactive microglial and astroglial populations.

We have previously studied changes in the serotoninergic and dopaminergic nigrostriatal systems following transection of the medial forebrain bundle and found a long-term axotomy-induced increase in the levels of serotonin and its main metabolite, 5-hydroxyindolacetic acid in substantia nigra [Venero et al. (1997) J. Neurochem. 68, 2458-2468]. In an attempt to find a rationale for this effect, we have performed an immunohistochemical study. Transection of the medial forebrain bundle of the rat interrupted most of the ascending serotoninergic pathways from the raphe nuclei as revealed by serotonin immunoreactivity. While serotonin immunostaining was almost absent in striatum, it doubled in the ventral mesencephalon at 21 days postlesion. This axotomy-induced increase was accompanied by an increased density of the serotonin nerve terminal network in the ipsilateral substantia nigra and ventral tegmental area. The increase in serotonin immunoreactivity was in line with the measured levels of serotonin and 5-hydroxyindolacetic acid in substantia nigra. In addition, the distribution pattern of glial fibrillary acidic protein-immunoreactive astrocytes and OX42-immunoreactive microglia correlated highly with the location of increased serotonin fibre density in the ventral mesencephalon, especially in ventral tegmental area and in the most medial part of substantia nigra. We suggest that a pruning effect may underly the axotomy-induced increase in serotonin immunoreactivity in the ventral mesencephalon, and further, that activated astroglia and microglia may play a role in directing serotoninergic axonal regeneration following axotomy.     

The role of serotonin release and autoreceptors in the dorsalis raphe nucleus in the control of serotonin release in the cat caudate nucleus

Using a push-pull cannula technique and an isotopic method for estimating [3H]serotonin continuously synthesized from [3H]tryptophan, the effects of changes in the release of serotonin in the dorsalis raphe nucleus on in vivo release of [3H]serotonin in the cat caudate nucleus were investigated. The increase in the release of serotonin in the dorsalis raphe nucleus caused by local application of parachlorophenylethylamine (10(-6) M) reduced striatal [3H]serotonin release. This inhibition in serotonin release in the striatum was blocked by the prior and continuous local superfusion of the dorsal raphe with methiothepin (10(-6) M), a serotonin autoreceptor antagonist. GABA (5 x 10(-5) M) applied to the dorsalis raphe reduced both local and striatal release of [3H]serotonin. However, picrotoxin (10(-5) M), a GABA A receptor antagonist applied locally in the dorsalis raphe nucleus increased [3H]serotonin release while decreasing striatal [3H]serotonin release. This decrease in serotonin release in the striatum was again blocked by continuous superfusion of the raphe with methiothepin. Furthermore, superfusion of serotonergic cell bodies of the dorsalis raphe nucleus with methiothepin alone never altered local release or striatal release of [3H]serotonin. These data strongly suggest that the release of serotonin from the cell body in the dorsalis raphe nucleus phasically controls release of the amine at the axonal nerve ending through serotonergic autoreceptors located on serotonergic nerve cell bodies in the dorsalis raphe nucleus. The origin of the serotonin released in the dorsalis raphe nucleus and the possibility that this type of regulation could be related to changes in nerve impulse conduction of the serotonergic raphe-striatal system are discussed.     

多重脑震荡大鼠脑干中缝核团内5-羟色胺免疫阳性表达变化的研究

为了探讨多重脑震荡(multiple cerebral concussion,MCC)后大鼠中缝核团内5-羟色胺(5-HT)能神经元的变化规律,本实验采用自制单摆式机械打击装置复制MCC大鼠模型,研究伤后大鼠脑干中缝核团内5-HT及5-HT合成过程中的限速酶-色胺酸羟化酶(TPH)的表达。将56只大鼠随机分为7组:对照组、伤后1、2、4、8、16和24d组(n=8)。用免疫组织化学染色技术及图像分析法定量分析伤后大鼠脑干中缝核团内5-HT和TPH的表达变化。结果显示:(1)TPH免疫反应阳性产物在中缝背核、正中中缝核的表达在伤后2d时达到高峰,与正常对照组相比有显著性差异(P<0.05);中缝大核和中缝苍白核分别以伤后1d组和4d组阳性反应最强;(2)5-HT免疫反应阳性产物在中缝背核、正中中缝核的表达也在伤后2d时达到高峰,16、24d组基本恢复至正常水平;而中缝大核和中缝苍白核内5-HT的免疫反应性在各损伤组与正常对照组之间均无显著性差异(P>0.05)。以上结果表明,多重脑震荡后中缝核团内TPH和5-HT的表达增高,这为研究5-HT对MCC后认知障碍的影响提供了形态学依据。     

The organization of serotonergic projections to cerebral cortex in primates: retrograde transport studies.

Retrograde axonal transport and immunocytochemical methods were utilized to determine the origin of serotonergic afferents to selected primary projection and association areas of cerebral cortex in macaque monkeys. After injections of Fast Blue or Diamidino Yellow in primary motor, somatosensory, or visual cortex, retrogradely labeled neurons are found in both the dorsal and median raphe nuclei. The sets of dorsal raphe neurons which innervate these cortical areas differ in their spatial distributions along the rostrocaudal axis of the brainstem; a coarse rostrocaudal topographic relationship is found between these groups of dorsal raphe neurons and their cortical targets. In contrast, neurons in the median raphe which innervate these primary projection areas are not differentially distributed along the rostrocaudal axis. However, in both the median and dorsal raphe nuclei, most neurons projecting to primary visual cortex are situated lateral to the cells which project to motor and somatosensory areas; many of these visually projecting neurons lie among the fascicles of the medial longitudinal fasciculus. For comparison with the serotonergic innervation of primary projection areas, the locations of raphe cells projecting to three areas of association cortex were examined: dorsolateral prefrontal cortex, area 5 and area 7b. Neurons projecting to each of these association areas are found throughout the dorsal and median raphe nuclei. Their distributions are similar to one another; however, more cells projecting to dorsolateral prefrontal cortex are in the rostral part of the dorsal raphe. The dorsal and median raphe neurons projecting to these association areas are intermingled with neurons projecting to motor and somatosensory cortex, but are medial to most of those projecting to visual cortex. Thus, separate cortical areas are innervated by different sets of raphe neurons; these sets partially overlap, yet differ in their rostrocaudal and mediolateral distributions. Ascending serotonergic projections to cerebral cortex form a widely distributed system which exhibits a highly intricate anatomic organization. The present observations support the hypothesis that the dorsal raphe nucleus is comprised of distinct sets of neurons whose output is distributed to multiple, interconnected cortical areas; these serotonergic projections may play a role in the coordination of excitability in functionally related areas of cortex. In contrast, the serotonergic projections arising from the median raphe appear to be more divergent and are likely to have a global influence on cortical activity. Since these individual raphe nuclei have different projection patterns, they are likely to have distinct functional roles.     

大鼠孤束核内5－羟色胺能纤维的来源──HRP逆行追踪与5－羟色胺免疫组织化学双标记洁研究

用ＨＲＰ注入孤束核逆行追踪和５－羟色胺（５－ＨＴ）免疫组织化学相结合的双标记法，观察大鼠孤束核内的５－ＨＴ能纤维和终末的来源。结果在脑干内见到许多ＨＲＰ单标、５－ＨＴ单标和ＨＲＰ／５－ＨＴ双标细胞，其中ＨＲＰ／５－ＨＴ双标细胞主要分布于脑桥被盖核（２６．５７％）、脑桥网状核（２４．１９％）、中缝大核（２１．１７％），其次为中缝隐核（１２．７４％）、中缝苍白核（７．７８％），少数见于中缝背核、中脑导水管周围灰质和中缝脑桥核等处。而在结状神经节内仅见到ＨＲＰ单标细胞，未见到５－ＨＴ单标和ＨＲＰ／５－ＨＴ双标细胞。以上结果表明，大鼠孤束核的５－ＨＴ能纤维和终末主要来源于脑桥与延髓的５－ＨＴ神经元的投射。本研究提示脑桥与延髓内上述核团的５－ＨＴ神经元通过向孤束核的直接投射参与孤束核内脏功能的调控。     

Effects of beta-amyloid protein on serotoninergic, noradrenergic, and cholinergic markers in neurons of the pontomesencephalic tegmentum in the rat

The effects on serotoninergic, noradrenergic and cholinergic markers on neurons of the pontomesencephalic tegmentum nuclei were studied in rats following local administration of fibrillar beta-amyloid peptide (Abeta1-40) into the left retrosplenial cortex. Focal deposition of Abeta in the retrosplenial cortex resulted in a loss of serotoninergic neurons in the dorsal and median raphe nuclei. The dorsal raphe nucleus showed a statistically significant reduction of 31.7% in the number of serotoninergic neurons and a decrease (up to 17.38%) in neuronal density in comparison with the same parameters in uninjected controls. A statistically significant reduction of 50.3%, together with a significant decrease of 53.94% in the density of serotoninergic neurons, was also observed in the median raphe nucleus as compared with control animals. Furthermore, a significant reduction of 35.07% in the number of noradrenergic neurons as well as a statistically significant decrease of 56.55% in the density of dopamine-beta-hydroxylase-immunoreactive neurons were also found in the locus coeruleus as compared with the corresponding hemisphere in uninjected controls. By contrast, a reduction of 24.37% in the number of choline acetyltransferase-positive neurons and a slight decrease (up to 22.28%) in the density of cholinergic neurons, which were not statistically significant, was observed in the laterodorsal tegmental nucleus in comparison with the same parameters in control animals. These results show that three different neurochemically defined populations of neurons in the pontomesencephalic tegmentum are affected by the neurotoxicity of Abeta in vivo and that Abeta might indirectly affect serotoninergic, noradrenergic and cholinergic innervation in the retrosplenial cortex.     

Developmental organization of raphe serotonin neuron groups in the rat

Summary The pre- and early postnatal development of serotonin neurons in the rat brainstem was studied using the fluorescence histochemical method. The technique utilized does not require drug pretreatment to visualize an intense serotonin fluorophore localized in neuronal perikarya, dendrites, and axons. All the serotonin neuron groups develop as bilateral nuclei which extend from the midbrain through the medulla. Six of the nine groups undergo a midline fusion from embryonic day 18 (E 18) through postnatal day 6 (P 6) in a rostrocaudal gradient. Cells of the nucleus raphe dorsalis fuse first (by P1), whereas the serotonin neurons located in nucleus raphe pallidus do not fuse until P 6. This gradient is comparable to the one described for the first observable fluorescence in the serotonin neurons groups. After final cell division, the serotonin neurons undergo a primary migration from the ventricular zone along the midline, where they are situated during embryogenesis, and a secondary migration extending into postnatal life which concludes with fusion in the midline. The bilateral origins of the serotonin cell groups are maintained in the adult. This is expressed by the apparent ipsilateral projections of some of the raphe neurons determined recently in our laboratory utilizing autoradiographic and horseradish peroxidase techniques.     

Distribution of the pro-opiomelanocortin-immunoreactive axons in relation to the serotoninergic neurons in the dorsal raphe nucleus of the rat.

The anatomical relationships between pro-opiomelanocortin-containing axons and serotonin neurons in the nucleus raphe dorsalis (NRD) of the rat were examined at the light microscope level with antibodies against CLIP (corticotropin-like intermediate lobe peptide), alpha-MSH (alpha-melanocyte-stimulating hormone) and serotonin. Sequential double labeling was performed with either immunofluorescence or peroxidase-antiperoxidase techniques. It was observed that the network of POMC-immunoreactive axons displayed a gradient of decreasing density from rostral to caudal levels and from dorsal to ventral parts or the NRD. The examples of close proximity between immunoreactive axons and serotonin cell bodies or dendrites were rather scarce. On the whole, the immunoreactive fibers seemed to run quasi-independently of the serotonin neurons.     

GAP-43 Immunoreactivity in the brain of the developing and adult wallaby ( Macropus eugenii)

We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions.In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.     

Atropine, a muscarinic cholinergic receptor antagonist increases serotonin, but not dopamine levels in discrete brain regions of mice

We investigated the effects of atropine, a muscarinic acetylcholine (ACh) receptor antagonist, on the level of serotonin in discrete brain regions, the nucleus raphe dorsalis (NRD), nucleus caudatus putamen (NCP), cerebral cortex and the cerebellum. Biogenic amines were assayed employing HPLC electrochemistry in these regions 30 min following different doses of atropine (5, 10, 25mg/kg; i.p.), and at various time points (15, 30, 60, 120 min) after 25mg/kg of the drug. The cholinergic receptor antagonist caused a dose-dependent alteration in the level of serotonin in NRD, but the increase was not dose-dependent for other regions studied. The metabolite of serotonin, 5-hydroxyindoleacetic acid was unaffected. Atropine did not affect the levels of dopamine or its metabolites dihydroxyphenyl acetic acid and homovanillic acid. The present study suggests significant effect of this antimuscarinic agent on the synthesis of serotonin in the central serotoninergic pathways, which may have clinical relevance.     

Double and triple labeling of neurons with fluorescent substances; The study of collateral pathways in the ascending raphe system

A retrograde labeling procedure utilizing fluorescent substances (Granular Blue, Nuclear Yellow and propidium iodide) was used to establish the presence of branching axons in the ascending raphe system of young rats. After injections in septum, medial thalamus and olfactory cortex, the number of double-labeled cells in various combinations was found to be relatively large in the dorsal raphe nucleus, whereas triple-labeled cells occurred more rarely. Each class of neurons, i.e. single-, double- and triple-labeled, were shown to have a predominant distribution within specific parts of the nucleus.