Age-related changes in serotonin-immunoreactive neurons in the rat nucleus raphe dorsalis and nucleus centralis superior: a light microscope study.

Comparison of serotonin-immunoreactive (SER-IR) neurons in the nucleus raphe dorsalis (NRD) and the nucleus centralis superior (NCS) of 3-month-old and 28-month-old rats was made using qualitative and quantitative immunohistochemical analysis. Significant age-related changes in size and density of the SER-IR somata as well as in the length and number of their processes were demonstrated. A different vulnerability of the SER-IR neurons in both raphe nuclei to aging was observed that may be related to their different structural and functional features.     

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。     

GABA innervation in adult rat oculomotor nucleus: A radioautographic and immunocytochemical study

Summary GABA innervation in the adult rat oculomotor nucleus (n.III) was investigated using two complementary approaches: radioautography after incubation of brain slices with tritiated GABA ([³H]GABA) along with localin vivo microinjections of the tracer, and GABA immunocytochemical procedures involving antibodies directed against a GABA-glutaraldehyde-protein conjugate. As determined by radioautography afterin vitro orin vivo labelling, the [³H]GABA uptake sites in the n.III mainly involved axon terminals. These were distributed throughout the neuropil and were often closely apposed to unlabelled motoneuron somata. A small number of glial cells also showed preferential accumulation of the tracer. The GABA-immunostaining likewise involved axon terminals throughout the nucleus, but no glial cells were immunopositive. In the dorsal region of the structure, occasional GABA-immunostained internuclear neurons were observed among unstained motoneuron cell bodies. Electron microscopic examination of [³H]GABA-labelled or GABA-immunostained profiles in n.III revealed axon terminals of around 1 m in diameter, always filled with small, round synaptic vesicles homogeneously distributed throughout the axoplasm. These boutons frequently contained mitochondria and one or more large granular vesicles. In single thin sections, 35% of [³H]GABA-labelled, and 19% of GABA-immunostained varicose profiles exhibited a synaptic differentiation, suggesting the existence of a predominantly if not entirely junctional innervation. These synapses mostly involved dendritic trunks or dendritic branches and were usually of the symmetrical type. A few, which were always symmetrical, were also observed on large somata of motoneurons. Some of the dendrites synaptically contacted by GABA-immunostained axon terminals were themselves GABA immunoreactive. These data substantiate the idea that GABA is involved in the control of motoneuron activity in n.III, and provide a structural basis for the inhibitory role of this transmitter in oculomotor function.     

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.     

Injection of capsaicin into the nucleus raphe dorsalis elicits heat loss in the rat

The acute thermoregulatory effect of microinjection of capsaicin into the nucleus raphe dorsalis (NRD) has been studied in anesthetised rats. Application of capsaicin (20 micrograms) into the NRD increased the tail skin temperature and decreased the body temperature. In animals that had previously been treated with capsaicin (50 mg/kg s.c.) the thermolytic effect of capsaicin microinjection was completely abolished. Additionally, the effects of NRD capsaicin injection on the monoamines in the preoptic region + hypothalamus (RPO-H) were studied. Levels of noradrenaline, dopamine, serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined by means of liquid chromatography. Neurochemical analysis of RPO-H showed no changes in the serotonin metabolism, but an elevated DOPAC level 30 min after the injection of capsaicin into the NRD.     

Postnatal development of noradrenergic terminals in the rat trigeminal motor nucleus: A light and electron microscopic immunocytochemical analysis

The noradrenergic (NA) innervation in the trigeminal motor nucleus (Vmot) of postnatal and adult rats was examined by light and electron microscopic immunocytochemistry using antibodies against dopamine‐β‐hydroxylase or tyrosine hydroxylase. NA fibers were identified in the Vmot as early as the day of birth (postnatal day 0; P0). A continuous increase in the density of labeled fibers was observed during development up to P20, with a slight decrease at P30 and in the adult. Electron microscopic analysis of serial ultrathin sections revealed that, at P5, nearly half (46%) of the examined NA terminals made synaptic contact with other neuronal elements with membrane specializations. The percentage of examined NA varicosities engaged in synaptic contacts increased at P15 (74%), then decreased in the adult (64%). At all developmental ages, the majority of contacts made by these boutons were symmetrical, the postsynaptic elements being mainly dendrites and occasionally somata. Interestingly, some of the NA terminals made axo‐axon contacts with other unidentified boutons. These results show that, although the density of NA fibers increases during postnatal development, functional NA boutons are present in the Vmot at early postnatal ages. Some of these fibers might exert their effects via nonsynaptic release of noradrenaline, the so‐called volume transmission, but, in the main, they form conventional synaptic contacts with dendrites, somata, and other axonal terminals in the Vmot. These results are consistent with previous electrophysiological studies that propose an important role for the NA system in modulating mastication. Anat Rec, 290:96–107, 2007. © 2006 Wiley‐Liss, Inc.     

Vesicle-containing dendrites in the nucleus raphe dorsalis of the cat. A serial section electron microscopic analysis

Summary The nucleus raphe dorsalis of the cat was examined by serial section electron microscopy and the presence of vesicle-containing dendrites is reported. Such dendrites were divided into two classes according to their synaptic contact. Dendrites containing round and/or pleomorphic vesicles associated with a clear synaptic specialization which was generally intermediate between a Gray's type I or II were classified as presynaptic dendrites. These presynaptic dendrites were presynaptic to conventional dendrites and dendritic spines. In addition, some profiles containing a sparse population of vesicles which may be dendritic in nature were observed involved in serial synaptic arrangements. A second class of dendrites were characterized by the presence of vesicles which were never found associated with any synaptic membrane specialization. Commonly, the vesicles were densely packed and associated with unusual densities. Serial section analysis of these densities showed that they were not presynaptic dense projections. We suggest that the existence of vesicle-containing dendrites in the nucleus raphe dorsalis of the cat constitute the morphological support for the dendritic release of neurotransmitters.     

Cooling of the nucleus raphe dorsalis induces sleep in the cat

Localized moderate cooling (+10°C) of the anterior and ventral part of the raphe dorsalis induced cortical synchronization and ponto-geniculo-occipital (PGO) waves followed sometimes by paradoxical sleep. Cooling the region of the raphe magnus produced cortical arousal. After depletion of brain 5-hydroxytryptamine (5-HT) by para-chlorophenylalanine, cooling the raphe dorsalis resulted in muscle atonia and bursts of high frequency PGO waves but not in cortical synchronization. Following restoration of brain 5-HT by intravenous administration of 5-hydroxytryptophan, cooling again produced cortical synchronization and PGO waves as in the control experiments, but with a diminishing efficacy during repetitive cooling episodes.     

K+ channel and 5-hydroxytryptamine1A autoreceptor interactions in the rat dorsal raphe nucleus: an in vitro electrophysiological study.

Extracellular recordings were made from serotonergic neurons of the rat dorsal raphe nucleus in a slice preparation. In the presence of phenylephrine (3 microM) to restore the pacemaker activity of otherwise silent serotonergic neurons, superfusion with the 5-hydroxytryptamine1A agonist ipsapirone depressed the firing of these neurons with an IC50 of approximately 50 nM. Complete inhibition was achieved with 100-300 nM of the drug. Concomitant superfusion with the 5-hydroxytryptamine1A antagonists spiperone (100 nM) or propranolol (10 microM) markedly reduced the inhibitory effect of ipsapirone (100 nM). Superfusion with K+ channel blockers such as apamin (50-100 nM), charybdotoxin (100 nM) or Ba2+ (1 mM) did not induce any changes in the electrical activity of serotonergic neurons. However, 4-aminopyridine (0.1-1 mM) disrupted the regularity of their discharge without affecting the mean firing rate. The ipsapirone-induced inhibition was unchanged by apamin and charybdotoxin, but was markedly reduced by Ba2+ and 4-aminopyridine. Thus the IC50 of ipsapirone was shifted to approximately 150 nM in the presence of 1 mM of 4-aminopyridine. These results indicate that, in serotonergic neurons within the dorsal raphe nucleus, the K+ channel opened through the stimulation of 5-hydroxytryptamine1A autoreceptors is 4-aminopyridine-sensitive.     

Enkephalin fibers synapse on cholinergic neurons in the rat sacral intermediolateral nucleus: a double-immunostaining at the light and electron microscopic levels

Relationships between leucine-enkephalin fibers and cholinergic neurons in the rat sacral intermediolateral nucleus were examined by light and electron microscopy using double-immunostaining method. Cholinergic neurons in the sacral intermediolateral nucleus were labeled by a rat-mouse monoclonal antibody to choline acetyltransferase and stained bluish green with 5-bromo-4-chloro-3-indolyl-beta-D- galactoside reaction products using beta-galactosidase as a marker. On the same sections, leucine-enkephalin fibers were labeled by a rabbit polyclonal antiserum to leucine-enkephalin and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the sacral intermediolateral nucleus. In the same region, leucine-enkephalin-like immunoreactive cells. In the electron microscope, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products were in the form of coarse electron dense deposits in the choline acetyltransferase-like immunoreactive structures and could be distinguished from the much finer grained diaminobenzidine reaction products. Choline acetyltransferase-like immunoreactive neurons received synaptic inputs from leucine-enkephalin fibers-like immunoreactive terminals. These findings suggest that leucine-enkephalin fibers may affect the activity of cholinergic parasympathetic preganglionic neurons.     

gamma-Aminobutyric acid decreases levels of messenger ribonucleic acid encoding prolactin in the rat pituitary

The effect of gamma-aminobutyric acid (GABA) on levels of messenger ribonucleic acid (mRNA) encoding prolactin (PRL) was studied in cultured anterior pituitary cells, in vitro and in intact rats, in vivo. As quantitated by hybridization to a 32P-labeled rat PRL complementary deoxyribonucleic acid (cDNA) probe, levels of PRL mRNA in cultured pituitary cells were decreased by about 50% following 3 days exposure to 10(-5) M GABA. This effect was mimicked by muscimol (10(-6) M) and antagonized by bicuculline (10(-5) M). An increase of endogenous GABA levels in vivo effected by injection of GABA transaminase blockers (aminooxyacetic acid, 20 mg/kg, twice daily; vinyl GABA, 800 mg/kg) into rats resulted in a similar decrease in rat PRL mRNA levels in the adenohypophysis 3-4 days following commencement of the drug treatment. These findings suggest that GABA might inhibit PRL gene expression by a direct action on lactotrophs of the adenohypophysis.     

Development of the rat sublingual gland: A light and electron microscopic immunocytochemical study

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP‐1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous‐like apical granules that labeled with anti‐mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti‐SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP‐1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti‐SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti‐mucin, and serous cells had numerous dense granules containing CSP‐1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti‐SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well‐formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells. Anat Rec 266:30–42, 2002. © 2002 Wiley‐Liss, Inc.     

VIP neurons as prime synaptic targets for serotonin afferents in rat suprachiasmatic nucleus: a combined radioautographic and immunocytochemical study

Summary Cellular relationships between serotonin (5-HT) axon terminals and neurons containing vasoactive intestinal peptide (VIP) were characterized by combined radioautography and immunocytochemistry in rat suprachiasmatic nucleus (SCN). Light microscopic immunoradioautographs showed significant overlap between (³H)5-HT uptake sites and VIP-immunoreactive elements in the ventral half of the SCN. Of the 255 (³H)5-HT-labelled axonal profiles detected in a systematic electron microscopic survey of single thin sections from this area, 75 (30%) were directly apposed to VIP-immunoreactive nerve cell bodies and/or dendrites. Radioautographically labelled 5-HT varicosities often showed well-differentiated, symmetrical or asymmetrical synaptic junctions, 60% of which were established on VIP-immunoreactive nerve cell bodies or dendrites. In a separate sampling of 198 (³H)5-HT-labelled terminals seen in apposition with VIP-immunoreactive elements, 50 showed a junctional complex at the site of contact. Postsynaptic immunoreactive elements were mostly dendrites but also included nerve cell bodies. Despite the methodological limitations inherent to the present double labelling approach, these data strongly support the view that VIP neurons are prime synaptic targets for 5-HT afferents in the SCN. VIP/5-HT interactions are thus likely to play an important functional role in this nucleus and may in particular subserve the 5-HT mediated regulation of certain circadian rhythms, including that of pituitary hormone secretion.     

Quantitative electron microscopy on the injured hypoglossal nucleus in the rat

Summary The hypoglossal nuclei of adult male rats which had received left hypoglossal axotomies, were studied quantitatively by making area measurements of structures on twenty electron micrographs per hypoglossal nucleus. Boutons and dendrites were analysed in further detail by counting and measuring profiles, counting synaptic thickenings per profile, and counting degenerating profiles. Results of light microscopy were confirmed and extended. In injured nuclei, microglia increased from 2 days to 2 weeks after axotomy, wrapping around neuron perikarya and parts of the dendrites between boutons and their post-synaptic sites. As microglia diminished after 2 weeks, astrocyte processes increased and replaced them, reaching peak growth at 5 weeks after axotomy. From 7 days to 5 weeks after axotomy there were fewer synaptic junctions. Neurons became stripped of boutons, but dendrites retained some. Dendrites shrank and some degenerated. Boutons shrank and decreased in numbers. At 10 and 12 weeks, all parameters returned to normal, by which time the hypoglossal nerve had reconnected with the tongue.Possibly the chronological succession from boutons to microglial processes to astrocyte processes to boutons covering the neuron perikarya reflects changes in the neuronal glycocalyx during repair.     

gamma-Aminobutyric acid as an inhibitory neurotransmitter in the rat supraoptic nucleus: intracellular recordings in the hypothalamic slice

Intracellular recordings have been made from rat supraoptic neurones in the hypothalamic slice preparation. Application of gamma-aminobutyric acid (GABA) caused all neurones to hyperpolarise and this was accompanied by an increase in membrane conductance. GABA application examined on a variety of cells was found to have a potent influence on patterning of electrical activity, always consistent with an inhibitory action.     

Development of the rat sublingual gland: a light and electron microscopic immunocytochemical study.

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP-1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous-like apical granules that labeled with anti-mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti-SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP-1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti-SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti-mucin, and serous cells had numerous dense granules containing CSP-1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti-SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well-formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells.     

The immunocytochemical demonstration of renin in a juxtaglomerular cell tumour by light and electron microscopy

This is a study of a juxtaglomerular cell tumour using an antibody to pure human renin and the peroxidase-antiperoxidase technique. We report the distribution of immunoreactive renin in the tumour visualized by light microscopy of both paraffin and resin embedded tissue sections. We found that the cells which contained most renin granules were endocrine-like cells in the parenchyma of the tumour. Ultrastructural immunocytochemistry showed that immunoreactive renin was present not only in crystalline protogranules and in round membrane bound granules, but also in intermediate forms.