A novel cerebellar commissure and other myelinated axons in the Purkinje cell layer of a pond turtle (Trachemys scripta elegans).

Parallel fibers in the molecular layer of the vertebrate cerebellum mediate slow spike conduction in the transverse plane. In contrast, electrophysiological recordings have indicated that rapid spike conduction exists between the lateral regions of the cerebellar cortex of the red-ear pond turtle (Trachemys scripta). The anatomical basis for this commissure is now examined in that species using neuronal tracing techniques. Fluorescently tagged dextrans and lipophilic carbocyanine dyes placed in one lateral edge of this nonfoliated cortex are transported across the midline of living brains in vitro and along the axonal membranes of fixed tissues, respectively. Surprisingly, the labeled commissural axons traversed the cortex within the Purkinje cell layer, and not in the white matter of the molecular layer or the white matter below the granule cell layer. Unlike thin parallel fibers that exhibit characteristic varicosities, this commissure is composed of smooth axons of large diameter that also extend beyond the cerebellar cortex via the cerebellar peduncles. Double labeling with myelin basic protein antibody demonstrated that these commissural axons are ensheathed with myelin. In contrast to this transverse pathway, an orthogonal myelinated tract was observed along the cerebellar midline. The connections of this transverse commissure with the lateral cerebellum, the vestibular nuclear complex, and the cochlear vestibular ganglia indicate that this commissure plays a role in bilateral vestibular connectivity.

Presynaptic gating of excitation in the dorsal raphe nucleus by GABA.

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus.

[Changes of the structural organization of the nucleus raphe pallidus after the decrease of endogenous serotonin level in the prenatal period of development in rats].

The role of serotonin in the nucleus raphe pallidus (NRP) development and the dynamics of its serotonin-producing neurons were studied during various time points of the postnatal period in normal Wistar rats and in animals developing prenatally under the conditions of serotonin deficiency. It was shown that NRP contained 2 populations of serotoninergic neurons with different morphological characteristics. At the initial stages of postnatal development (Day 5) serotonin-producing neurons included only large neurons, while the synthetic activity of small neurons appeared later (by Day 10). With age, under normal conditions,the size of large neurons and their number were increased which is indicative of continuing process of differentiation and/or functional load augmentation. The size and number of small neurons were practically unchanged with age. Serotonin deficiency during prenatal development lead to the disturbance of NRP structural organization. In comparison with the control animals, the size and the number of serotonin-producing neurons of both populations was decreased, their size remained unchanged with the age. Part of the neurons underwent degeneration, resulting in the reduction of their numbers. The damage observed may change the serotoninergic innervation of the medullary nuclei, responsible for the cardiorespiratory the control, thus causing the disturbances of cardio-vascular and respiratory systems.

[Development of the habenulointerpeduncular tract in rats].

Development of the habenulointerpeduncular tract has been carried out on fixed brain preparations obtained from 21 day rat embryos and from neonatal animals on the 0 and 9 days of postnatal development by diffusion oflipophilic fluorescent carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) through neuron membranes. The marker was introduced into the nuclei of the habenula, the interpeduncular nucleus, and into the area of raphe nuclei. Neurons and fibers that contained Dil were identified on vibratome sections by fluorescent and confocal microscopy. we have found that reciprocal links between the lateral habenula nucleus and raphe nuclei are formed in the prenatal period by stage E21. Raphe nuclei innervating neurons were located in dorso- and ventrocaudal parts of the lateral habenula nucleus. Projections of the medial habenula nucleus onto interpeduncular nucleus were found only in the postnatal P2 period. Neurons that provide a source of these projections form characteristic assemblies inside the medial habenula nucleus. Therefore, the present study for the first time describes heterogenic formation of different projection systems that are involved in the habenulointerpeduncular tract of rats at perinatal ontogenesis.

Single-prolonged stress induces apoptosis in dorsal raphe nucleus in the rat model of posttraumatic stress disorder.

INTRODUCTION: Post-traumatic stress disorder (PTSD) is an anxiety disorder that develops after exposure to a life-threatening traumatic experience. Meta-analyses of the brainstem showed that midsagittal area of the pons was significantly reduced in patients with PTSD, suggesting a potential apoptosis in dorsal raphe nucleus after single-prolonged stress (SPS). The aim of this study is to investigate whether SPS induces apoptosis in dorsal raphe nucleus in PTSD rats, which may be a possible mechanism of reduced volume of pons and density of gray matter. METHODS: In this study, rats were randomly divided into 1d, 7d and 14d groups after SPS along with the control group. The apoptosis rate was determined using annexin V-FITC/PI double-labeled flow cytometry (FCM). Levels of Cytochrome c (Cyt-C) was examined by Western blotting. Expression of Cyt-C on mitochondria in the dorsal raphe nucleus neuron was determined by enzymohistochemistry under transmission electron microscopy (TEM). The change of thiamine monophosphatase (TMP) levels was assessed by enzymohistochemistry under light microscope and TEM. Morphological changes of the ultrastructure of the dorsal raphe nucleus neuron were determined by TEM. RESULTS: Apoptotic morphological alterations were observed in dorsal raphe nucleus neuron for all SPS-stimulate groups of rats. The apoptosis rates were significantly increased in dorsal raphe nucleus neuron of SPS rats, along with increased release of cytochrome c from the mitochondria into the cytoplasm, increased expression of Cyt-C and TMP levels in the cytoplasm, which reached to the peak of increase 7 days of SPS. CONCLUSIONS: The results indicate that SPS induced Cyt-C released from mitochondria into cytosol and apoptosis in dorsal raphe nucleus neuron of rats. Increased TMP in cytoplasm facilitated the clearance of apoptotic cells. We propose that this presents one of the mechanisms that lead to reduced volume of pons and gray matter associated with PTSD.

Raphe serotonin neurons are not homogenous: electrophysiological, morphological and neurochemical evidence.

The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.

Ultrastructural characterization of tryptophan hydroxylase 2-specific cortical serotonergic fibers and dorsal raphe neuronal cell bodies after MDMA treatment in rat.

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to cause selective long-term serotonergic damage. OBJECTIVES: The aim of this study was to characterize the ultrastructure of serotonergic pericarya and proximal neurites in the dorsal raphe nucleus as well as the ultrastructure of serotonergic axons in the frontal cortex of adolescent Dark Agouti rats 3 days after treatment with 15 mg/kg i.p. MDMA. METHODS: Light microscopic immunohistochemistry and pre-embedding immunoelectron microscopy with a novel tryptophan hydroxylase-2 (Tph2) specific antibody, as a marker of serotonergic structures. RESULTS: Light microscopic analysis showed reduced serotonergic axon density and aberrant swollen varicosities in the frontal cortex of MDMA-treated animals. According to the electron microscopic analysis, Tph2 exhibited diffuse cytoplasmic immunolocalization in dorsal raphe neuronal cell bodies. The ultrastructural-morphometric analysis of these cell bodies did not indicate pathological changes or significant alteration in the cross-sectional areal density of any examined organelles. Proximal serotonergic neurites in the dorsal raphe exhibited no ultrastructural alteration. However, in the frontal cortex among intact fibers, numerous serotonergic axons with destructed microtubules were found. Most of their mitochondria were intact, albeit some injured axons also contained degenerating mitochondria; moreover, a few of them comprised confluent membrane whorls only. CONCLUSIONS: Our treatment protocol does not lead to ultrastructural alteration in the serotonergic dorsal raphe cell bodies and in their proximal neurites but causes impairment in cortical serotonergic axons. In these, the main ultrastructural alteration is the destruction of microtubules although a smaller portion of these axons probably undergo an irreversible damage.

Alpha4 containing nicotinic receptors are positioned to mediate postsynaptic effects on 5-HT neurons in the rat dorsal raphe nucleus.

Nicotinic acetylcholine receptors containing the alpha4 and beta2 subunits constitute the most abundant high-affinity binding site of nicotine in the brain and are critical for the addictive qualities of nicotine. 5-HT neurotransmission is thought to be an important contributor to nicotine addiction. Therefore in this study it was examined how alpha4-containing receptors are positioned to modulate the function of 5-HT neurons using ultrastructural analysis of immunolabeling for the alpha4 receptor subunit in the dorsal raphe nucleus (DR), a primary source of forebrain 5-HT in the rat. Of 150 profiles labeled for the alpha4 subunit, 140 or 93% consisted of either soma or dendrites, these were often small-caliber (distal) dendrites <1.5 microm in diameter (63/150 or 42%). The majority (107/150 or 71%) of profiles containing labeling for alpha4 were dually labeled for the synthetic enzyme for 5-HT, tryptophan hydroxylase (TPH). Within dendrites immunogold labeling for alpha4 was present on the plasma membrane or near postsynaptic densities. However, labeling for alpha4 was commonly localized to the cytoplasmic compartment often associated with smooth endoplasmic reticulum, plausibly representing receptors in transit to or from the plasma membrane. Previous studies have suggested that nicotine presynaptically regulates activity onto 5-HT neurons, however alpha4 immunolabeling was detected in only 10 axons in the DR or 7% of profiles sampled. This finding suggest that alpha4 containing receptors are minor contributors to presynaptic regulation of synaptic activity onto 5-HT neurons, but rather alpha4 containing receptors are positioned to influence 5-HT neurons directly at postsynaptic sites.

The dorsal raphe nucleus--from silver stainings to a role in depression.

Over a hundred years ago, Santiago Ramón y Cajal used a new staining method developed by Camillo Golgi to visualize, among many other structures, what we today call the dorsal raphe nucleus (DRN) of the midbrain. Over the years, the DRN has emerged as a multifunctional and multitransmitter nucleus, which modulates or influences many CNS processes. It is a phylogenetically old brain area, whose projections reach out to a large number of regions and nuclei of the CNS, particularly in the forebrain. Several DRN-related discoveries are tightly connected with important events in the history of neuroscience, for example the invention of new histological methods, the discovery of new neurotransmitter systems and the link between neurotransmitter function and mood disorders. One of the main reasons for the wide current interest in the DRN is the nucleus' involvement in depression. This involvement is particularly attributable to the main transmitter of the DRN, serotonin. Starting with a historical perspective, this essay describes the morphology, ascending projections and multitransmitter nature of the DRN, and stresses its role as a key target for depression research.

Projection patterns from the amygdaloid nuclear complex to subdivisions of the dorsal raphe nucleus in the rat.

The goal of the present study was to identify the projection from the subdivisions of the amygdaloid nuclear complex to specified subregions of the dorsal raphe (DR) nucleus and to attempt to compare the density of amygdaloid input to the DR with that of inputs from other limbic structures. Use of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), demonstrated that amygdaloid input to midline DR subdivision originates mainly from the medial portion of the medial amygdaloid nucleus, whereas that to lateral wing subdivision derives from the region extending from the lateral portion of the medial amygdaloid nucleus to the commissural stria terminalis. Use of the retrograde tracer Fluorogold (FG) produced relatively large but circumscribed injection sites comprising midline DR as well as portions of lateral wing subdivision and confirmed that the medial amygdaloid nucleus provides the major input to the DR. We also demonstrated that although amygdaloid input was not as extensive as inputs from other limbic structures such as the medial prefrontal cortex or the lateral habenular nucleus, it was comparable to input from the lateral septal nucleus. Based on these observations, we suggest that the medial amygdaloid nucleus provides substantial input to the DR and may contribute an emotional influence on sleep-wakefulness cycle or pain-stress modulation. Furthermore, it seems that the medial amygdaloid-DR projection might be anatomically and functionally distinct from the well-characterized central amygdaloid-periaqeductal gray (PAG) circuit which is essential for conditioned fear.

Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain.

The mechanism of action of commonly used antidepressants remains an issue of debate. In the experiments reported here we studied the effects of three representative compounds, the selective serotonin reuptake inhibitor fluoxetine, the selective serotonin reuptake enhancer tianeptine and the selective norepinephrine reuptake inhibitor desipramine on the structure of central serotonin pathways after a 4-week administration. We found that the serotonin modulators fluoxetine and tianeptine, but not desipramine, increase the density of 5-HT and serotonin transporter (SERT)-immunoreactive axons in the neocortical layer IV and certain forebrain limbic areas, such as piriform cortex and the shell region of nucleus accumbens. These changes were noted in the absence of a significant effect of serotonin antidepressants on the expression of tryptophan hydroxylase (TPH-2), i.e. the rate-limiting enzyme for 5-HT biosynthesis and of SERT at the mRNA level. In addition, we found that anterogradely filled terminal axons from injections of biotinylated dextran amine into the dorsal raphe showed significantly more branching in animals treated with fluoxetine compared with animals treated with liposyn vehicle. Our findings suggest that antidepressants may exert very selective structural effects on their cognate monoamine systems in normal animals and raise the possibility that neurotrophic mechanisms may play a role in their clinical efficacy.

Two populations of glutamatergic axons in the rat dorsal raphe nucleus defined by the vesicular glutamate transporters 1 and 2.

Most glutamatergic neurons in the brain express one of two vesicular glutamate transporters, vGlut1 or vGlut2. Cortical glutamatergic neurons highly express vGlut1, whereas vGlut2 predominates in subcortical areas. In this study immunohistochemical detection of vGlut1 or vGlut2 was used in combination with tryptophan hydroxylase (TPH) to characterize glutamatergic innervation of the dorsal raphe nucleus (DRN) of the rat. Immunofluorescence labeling of both vGlut1 and vGlut2 was punctate and homogenously distributed throughout the DRN. Puncta labeled for vGlut2 appeared more numerous then those labeled for vGlut1. Ultrastructural analysis revealed axon terminals containing vGlut1 and vGlut2 formed asymmetric-type synapses 80% and 95% of the time, respectively. Postsynaptic targets of vGlut1- and vGlut2-containing axons differed in morphology. vGlut1-labeled axon terminals synapsed predominantly on small-caliber (distal) dendrites (42%, 46/110) or dendritic spines (46%, 50/110). In contrast, vGlut2-containing axons synapsed on larger caliber (proximal) dendritic shafts (> 0.5 microm diameter; 48%, 78/161). A fraction of both vGlut1- or vGlut2-labeled axons synapsed onto TPH-containing dendrites (14% and 34%, respectively). These observations reveal that different populations of glutamate-containing axons innervate selective dendritic domains of serotonergic and non-serotonergic neurons, suggesting they play different functional roles in modulating excitation within the DRN.

Regional, cellular, and subcellular localization of RGS10 in rodent brain.

The regulator of G protein signaling type 10 (RGS10) modulates Galphai/o signaling by means of its GTPase accelerating activity and is abundantly expressed in brain and in immune tissues. To elucidate RGS10 function in the nervous system, we mapped RGS10 protein in rat and mouse brain using light microscopic (LM) and electron microscopic (EM) immunohistochemical techniques. The LM showed that RGS10-like immunoreactivity (LIR) labels all cellular subcompartments of neurons and microglia, including their nuclei. There were several differences between RGS10-LIR distributions in rat and mouse, the most striking of which were the far denser immunoreactivity in rat dentate gyrus and dorsal raphe. The EM analysis corroborated and extended our findings from LM. Thus, EM confirmed the presence of dense RGS10-LIR in the euchromatin compartment of nuclei. The EM analysis also resolved dense staining on terminals at symmetric synapses onto pyramidal cell somata. Dual immunofluorescence showed that forebrain interneurons densely labeled with RGS10-LIR partially colocalized with parvalbumin-LIR. Dual-labeling histochemistry in caudoputamen demonstrated that densely labeled striatal cells were biased to the indirect-projecting output pathway. Dual-labeling immunofluorescence also showed that densely labeled RGS10-LIR cells in the dentate gyrus subgranular zone were not proliferating but that newly born cells could differentiate to express RGS10-LIR. Taken together, these data support a role for RGS10 in diverse processes that include modulation of pre- and postsynaptic G-protein signaling. Moreover, enrichment of RGS10 in transcriptionally active regions of the nucleus suggests an unforeseen role of RGS10 in modulating gene expression.

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus.

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.

Ultrastructural evidence for a role of gamma-aminobutyric acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system.

The dorsal raphe nucleus (DRN) serotonin (5-HT) system has been implicated in acute responses to stress and in stress-related psychiatric disorders such as anxiety and depression. Substantial findings suggest that the neuropeptide corticotropin-releasing factor (CRF) is instrumental in modulating the activity of this system during stress. Because the DRN is neurochemically heterogeneous, dual immunoelectron microscopy was used to examine cellular substrates for interactions between CRF and either 5-HT or gamma-aminobutyric acid (GABA) in the dorsolateral and ventromedial DRN. CRF immunoreactivity was identified primarily within axon terminals, where immunolabeling was particularly enriched in dense-core vesicles. Although CRF terminals targeted 5-HT-containing dendrites in the dorsolateral DRN (16%; n = 251 terminals), synaptic contacts with dendrites that lacked detectable 5-HT immunolabeling were more numerous (48%). In contrast, dual labeling for CRF and GABA (n = 240 terminals) in the dorsolateral DRN revealed that substantially more CRF terminals contacted GABA dendrites (42%) as opposed to unlabeled dendrites (29%). In the ventromedial DRN, contacts between CRF axon terminals and either 5-HT-labeled dendrites or GABA-containing dendrites were fewer than in the dorsolateral DRN. As in the dorsolateral DRN, CRF terminals more frequently contacted GABA dendrites than 5-HT dendrites (30% vs. 8%, respectively). The findings support physiological studies suggesting that CRF has both direct and indirect effects on DRN-5-HT neurons and further implicate GABA as a primary mediator by which CRF and stressors alter the activity of the DRN-5-HT system.

Median raphe mediates estrogenic effects to the hippocampus in female rats.

Subcortical regions such as the medial septum-diagonal band of Broca and supramammillary area have been shown to mediate indirect oestrogenic effects on hippocampal morphology and function. Here, the role of the median raphe (MR), a serotonergic subcortical structure, is studied. To this end, 17beta-estradiol-filled 30-gauge cannulae were implanted into the MR of female ovariectomized rats; cholesterol-filled cannulae served as controls. After seven days, using unbiased electron microscopic stereological calculations and semiquantitative analysis, the spine synapse density and surface density of glial fibrillary acidic protein-positive astrocyte processes, respectively, were determined in the stratum radiatum of the CA1 region of the hippocampus. Changes in the serotonergic innervation of the hippocampal CA1 region were determined by immunohistochemistry and subsequent morphometric analysis. In the stratum radiatum of the CA1 region, local estradiol application into the MR resulted in a 47% increase in spine synapse density. Simultaneously, the density of glial fibrillary acidic protein-positive fibers decreased by 16%. The density of serotonin (5-HT) innervation of the strata lacunosum moleculare and radiatum of the CA1 region of the hippocampus was reduced in response to estradiol, as shown by a decrease in the length of fibers (27.6 and 48.3% decrease, respectively) and the number of large varicosities (32.5 and 38.8% decrease, respectively). These observations suggest a major role of the MR in mediating oestrogenic effects on the hippocampus and an involvement of the serotonergic system.

Prefrontal cortical projections to the rat dorsal raphe nucleus: ultrastructural features and associations with serotonin and gamma-aminobutyric acid neurons.

Studies of human brain indicate that both the ventromedial prefrontal cortex (PFC) and the dorsal raphe nucleus (DRN) may be dysfunctional in major depressive illness, making it important to understand the functional interactions between these brain regions. Anatomical studies have shown that the PFC projects to the DRN, although the synaptic targets of this excitatory pathway have not yet been identified. Electrophysiological investigations in the rat DRN report that most serotonin neurons are inhibited by electrical stimulation of the PFC, suggesting that this pathway is more likely to synapse onto neighboring gamma-aminobutyric acid (GABA) neurons than onto serotonin cells. We tested this hypothesis by electron microscopic examination of DRN sections dually labeled for biotin dextran amine anterogradely transported from the PFC and immunogold-silver labeling for tryptophan hydroxylase (TrH) or for GABA. In the DRN, the majority of PFC axons either synapsed onto unlabeled dendrites or failed to form detectable synapses in single sections. Other PFC axons synapsed onto either TrH- or GABA-immunolabeled processes. Considerably more tissue sampling was necessary to detect PFC synapses onto TrH- than onto GABA-labeled dendrites, suggesting that the latter connections are more common. In other cases, PFC terminals and TrH- or GABA-immunoreactive dendrites either were closely apposed, without forming detectable synapses, or were separated by glial processes. These results provide potential anatomical substrates whereby the PFC can both directly and indirectly regulate the activity of serotonin neurons in the DRN and possibly contribute to the pathophysiology of depression.

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus.

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.

Chronic excess of corticosterone increases serotonergic fibre degeneration in aged rats.

Evidence is presented for the potentiating role of corticosterone on axonal degeneration of serotonergic neurones during ageing. Aged rats, 24 months old, were implanted subcutaneously with 2 x 100 mg pellets of corticosterone. Serotonergic and cholinergic (ChAT- and NADPHd-positive) fibre degenerations in the anteroventral thalamic nucleus (AVT) were measured 2 months after corticosterone implantation. Numbers of immunoreactive serotonergic raphe and mesolimbic cholinergic neurones were also quantified. Basal plasma corticosterone and adrenocorticotropin (ACTH) concentrations were assayed at 2, 4, 6, and 8 weeks after implantation in the plasma and at 1, 2, 4 and 6 weeks in urine. The degree of serotonergic fibre aberrations in the AVT increased significantly after corticosterone exposure, while that of ChAT-positive and NADPHd-stained axon aberrations showed a modest but nonsignificant increase. A positive correlation between the magnitudes of serotonergic and cholinergic fibre aberrations appeared in the AVT, but only in the corticosterone-treated rats. The number of serotonin immunopositive neurones in the raphe nuclei after corticosterone decreased marginally, while that of mesopontine ChAT-positive neurones was not influenced. Measurements of basal plasma corticosterone and ACTH, as well as urine corticosterone, revealed that the steroid implantation increased the plasma corticosterone level for at least 4 weeks and decreased ACTH level for at least 6 weeks. By the week 8, the pituitary-adrenal function was apparently restored. However, at sacrifice, both the weight of adrenal glands and that of thymus remained reduced, indicating the long-lasting effects of corticosterone on target tissues. It is concluded that the raphe serotonergic neurones and their projecting fibres are sensitive to corticosterone excess in aged rats and become more vulnerable to degeneration processes than under normal ageing conditions. Cholinergic neurones of brainstem origin, which also express massive NADPHd activity, are more resistant against corticosterone, but their axon degeneration correlates to serotonergic fibre degeneration.

Serotonin immunoreactivity in the retinal projecting isthmo-optic nucleus and evidence of brainstem raphe connections in the pigeon.

Serotonin (5-HT) immunoreactive (-ir) profiles within the isthmo-optic nucleus (ION) of the centrifugal visual system (CVS) were studied in the pigeon using light microscopic immunohistofluorescent and electron microscopic immunocytochemical pre-embedding techniques. The brainstem origin of the 5-HT input upon the ION was determined by combining 5-HT immunohistofluorescence (FITC) and retrograde transneuronal tracing after intraocular injection of Rhodamine beta-isothiocyanate. The light microscopic results showed that 5-HT endings were mainly localised within the neuropillar zones of the ventral ION. The 5-HT-ir cell bodies, belonging to a lateral extension of the dorsal raphe system, were observed within the same region as the centrifugal ectopic neurons (EN) underlying the ION and some displayed dendritic processes which penetrated the nucleus. Double-labeled neurons, representing 5-HT-ir afferents to the ION, were identified only within the n. linearis caudalis region of the ventral raphe. The electron microscopic results confirmed the presence of 5-HT-ir dendritic processes within the ventral part of the nucleus and showed that they were contacted by axon terminals belonging to intrinsic interneurons. The functional organisation of the ION and the possible contribution of serotonergic raphe afferents and efferents are discussed in relation to present hypotheses linking the avian CVS to mechanisms of visual attention.