Mapping of afferent and efferent connections of phenylethanolamine N-methyltransferase-expressing neurons in the nucleus tractus solitarii.

OBJECTIVE: Phenylethanolamine N-methyltransferase (PNMT)-expressing neurons in the nucleus tractus solitarii (NTS) contribute to the regulation of autonomic functions. However, the neural circuits linking these neurons to other brain regions remain unclear. This study aims to investigate the connectivity mechanisms of the PNMT-expressing neurons in the NTS (NTSPNMT neurons). METHODS: The methodologies employed in this study included a modified rabies virus-based retrograde neural tracing technique, conventional viral anterograde tracing, and immunohistochemical staining procedures. RESULTS: A total of 43 upstream nuclei projecting to NTSPNMT neurons were identified, spanning several key brain regions including the medulla oblongata, pons, midbrain, cerebellum, diencephalon, and telencephalon. Notably, dense projections to the NTSPNMT neurons were observed from the central amygdaloid nucleus, paraventricular nucleus of the hypothalamus, area postrema, and the gigantocellular reticular nucleus. In contrast, the ventrolateral medulla, lateral parabrachial nucleus, and lateral hypothalamic area were identified as the primary destinations for axon terminals originating from NTSPNMT neurons. Additionally, reciprocal projections were evident among 21 nuclei, primarily situated within the medulla oblongata. CONCLUSION: Our research findings demonstrate that NTSPNMT neurons form extensive connections with numerous nuclei, emphasizing their essential role in the homeostatic regulation of vital autonomic functions.

Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II.

BACKGROUND: In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. RESULTS: In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. CONCLUSIONS: Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

Cyclooxygenase-1 and -2 in spinally projecting neurons are involved in CRF-induced sympathetic activation.

Corticotropin-releasing factor (CRF) in the brain has been shown to stimulate sympathetic activity, leading to elevations of blood pressure, heart rate and plasma catecholamine levels and neuronal activation of the sympathetic ganglia and adrenal medulla. We previously reported that brain cyclooxygenase (COX), the rate-limiting enzyme in the synthesis of prostanoids, is involved in centrally administered CRF-induced sympathetic activation in rats. Therefore, the present study was designed to reveal the effect of centrally administered CRF (1.5 nmol/animal) on the expression of COX isozymes, COX-1 and COX-2, in spinally projecting neurons until 6h after the administration, using rats microinjected with a monosynaptic retrograde tracer into the intermediolateral cell column of the thoracic spinal cord. Retrogradely labeled neurons were detected in the paraventricular hypothalamic nucleus (PVN), locus coeruleus (LC), raphe pallidus nucleus and rostral ventrolateral medulla. Centrally administered CRF significantly increased the number of spinally projecting PVN neurons expressing COX-1 throughout the experimental period and those expressing COX-2 during only the late phase. CRF also increased the number of spinally projecting LC neurons expressing COX-2 throughout the experimental period. In other regions, the CRF administration had no effect on COXs expression in spinally projecting neurons. These results suggest that COX-1 and COX-2 in the PVN and COX-2 in the LC play roles in the CRF-induced sympathetic regulation in rats.

Activation of ERK in the rostral ventromedial medulla is involved in hyperalgesia during peripheral inflammation.

We have previously shown that the extracellular signal-regulated kinase (ERK) is activated in the rostral ventromedial medulla (RVM) during peripheral inflammation. In the present study, the relationship between ERK signaling in the RVM and pain hypersensitivity was investigated in the rat. Microinjection of U0126, a mitogen-activated protein kinase kinase inhibitor, into the RVM decreased phosphorylated ERK at 7 h after complete Freund's adjuvant (CFA) injection into the hindpaw. The U0126 microinjection also attenuated thermal hyperalgesia in the ipsilateral hindpaw at 24 h after CFA injection. The ipsilateral paw withdrawal latency in the U0126 group (67.9%+/-5.3% vs. baseline, n=7) was significantly longer than that in the control group (52.0%+/-3.6% vs. baseline, n=8). These findings suggest that activation of ERK in the RVM contributes to thermal hyperalgesia during peripheral inflammation.

Efferent neurotransmitters in the human cochlea and vestibule.

CONCLUSION: Current neurotransmission models based on animal studies on the mammalian inner ear not always reflect the situation in human. Rodents and primates show significant differences in characteristics of efferent innervation as well as the distribution of neuroactive substances. OBJECTIVE: Immunohistochemistry demonstrates the mammalian efferent system as neurochemically complex and diverse: several neuroactive substances may co-exist within the same efferent terminal. Using light and electron microscopic immunohistochemistry, this study presents a comparative overview of the distribution patterns of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, GABA, CGRP, and enkephalins within the peripheral nerve fiber systems of the human inner ear. MATERIALS AND METHODS: Human temporal bones were obtained post mortem and prepared according to a pre-embedding immunohistochemical technique to detect immunoreactivities to ChAT, GABA, CGRP, leu- and met-enkephalins at the electron microscopic level. RESULTS: Immunoreactivities of all the antigens were present within both the lateral and medial efferent systems of the cochlea, whereas only ChAT, GABA, and CGRP were detected in efferent pathways of the vestibular end organs.

Phylogenetic preservation of alpha3 Na+,K+-ATPase distribution in vertebrate peripheral nervous systems.

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.

Aldosterone-sensitive neurons in the nucleus of the solitary: efferent projections.

The nucleus of the solitary tract (NTS) contains a subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them uniquely sensitive to aldosterone. These neurons may drive sodium appetite, which is enhanced by aldosterone. Anterograde and retrograde neural tracing techniques were used to reveal the efferent projections of the HSD2 neurons in the rat. First, the anterograde tracer Phaseolus vulgaris leucoagglutinin was used to label axonal projections from the medial NTS. Then, NTS-innervated brain regions were injected with a retrograde tracer, cholera toxin beta subunit, to determine which sites are innervated by the HSD2 neurons. The HSD2 neurons project mainly to the ventrolateral bed nucleus of the stria terminalis (BSTvl), the pre-locus coeruleus (pre-LC), and the inner division of the external lateral parabrachial nucleus (PBel). They also send minor axonal projections to the midbrain ventral tegmental area, lateral and paraventricular hypothalamic nuclei, central nucleus of the amygdala, and periaqueductal gray matter. The HSD2 neurons do not innervate the ventrolateral medulla, a key brainstem autonomic site. Additionally, our tracing experiments confirmed that the BSTvl receives direct axonal projections from the neighboring A2 noradrenergic neurons in the NTS, and from the same pontine sites that receive major inputs from the HSD2 neurons (PBel and pre-LC). The efferent projections of the HSD2 neurons may provide new insights into the brain circuitry responsible for sodium appetite.

Cardiovascular responses and neurotransmitter changes during static muscle contraction following blockade of inducible nitric oxide synthase (iNOS) within the ventrolateral medulla.

The enzyme nitric oxide synthase (NOS) which is necessary for the production of nitric oxide from L-arginine exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our previous studies have demonstrated the roles of nNOS and eNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) in modulating cardiovascular responses during static skeletal muscle contraction via altering localized glutamate and GABA levels (Brain Res. 977 (2003) 80-89; Neuroscience Res. 52 (2005) 21-30). In this study, we investigated the role of iNOS within the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex. Bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 1.0 microM), for 60 min into the RVLM attenuated increases in mean arterial pressure (MAP), heart rate (HR), and extracellular glutamate levels during a static muscle contraction. Levels of GABA within the RVLM were increased. After 120 min of discontinuation of the drug, MAP and HR responses and glutamate/GABA concentrations recovered to baseline values during a subsequent muscle contraction. In contrast, bilateral application of AGN (1.0 microM) into CVLM potentiated cardiovascular responses and glutamate concentration while attenuating levels of GABA during a static muscle contraction. All values recovered after 120 min of discontinuation of the drug. These results demonstrate that iNOS within the ventrolateral medulla plays an important role in modulating cardiovascular responses and glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex.

Identification of neurons with acetylcholinesterase and NADPH-diaphorase activities in the centrifugal visual system of the chick.

The isthmo-optic nuclei (ION) and ectopic neurons, which constitute the centrifugal visual system (CVS), are thought to be cholinoceptive and nitrergic. However, it is not clear which neurons express these markers, namely the ones that project to the retina rather than in neurons that only participate in a local circuit. Therefore, to characterize the neurochemical patterns of the centrifugal visual system in the post-hatched chick, retinopetal cells of the isthmo-optic nuclei and the ectopic region were identified via immunolabeling for cholera toxin, a neuronal tracer, which has been injected in the ocular globe. Then, double labeled with acetylcholinesterase histochemistry to reveal cholinergic synapses, or NADPH-diaphorase histochemistry as a nitrergic marker. Briefly, acetylcholinesterase activity was present mainly in cholera toxin labeled cell bodies of the isthmo-optic nucleus and the ectopic region indicating that retinal projecting neurons of centrifugal visual system comprise a cholinoceptive pathway. On the other hand, NADPH-diaphorase histochemistry was present in the neuropile and sparse cell bodies inside of the isthmo-optic nucleus and in ectopic neurons which were not cholera toxin positive suggesting their role in an intrinsic circuit of the centrifugal visual system. These data support the idea that these two neurochemical systems are present in distinct neuronal populations in the centrifugal visual system.

The distribution and the modulation of tyrosine hydroxylase immunoreactivity in the lateral olivocochlear system of the guinea-pig.

It was previously shown that tyrosine hydroxylase (TH) immunoreactivity in the terminals of the lateral efferents of the cochlea is decreased by acoustic trauma and that sound preconditioning counteracted this decrease [Hear Res 174 (2002) 124]. Here we identify those neurons in the lateral olivocochlear system (LOC) in the brainstem that regulates the peripheral expression of TH in the cochlea. By employing retrograde tracing techniques, dextran-labeled neurons were found predominantly in the ipsilateral LOC system including lateral superior olive (LSO), and the surrounding periolivary regions (dorsal periolivary nucleus [DPO], dorsolateral periolivary nucleus [DLPO], lateral nucleus of trapezoid body [LNTB]). Employing immunocytochemistry, it was found that a control group had 35% of the ipsilateral LOC neurons positively stained with TH. Of the total population of TH neurons, 77% were double-stained (TH and dextran) in the LOC system. Acoustic trauma decreased the number of TH positive neurons in the LSO and the surrounding DLPO, and caused a reduction of TH fiber immunolabeling in these regions. Changes were not found in the DPO or the LNTB after acoustic trauma. Sound conditioning protected against the decrease of TH immunolabeling by acoustic trauma and increased the fiber staining for TH in the LSO and DLPO, but not in the DPO or the LNTB. These results provide evidence that TH positive neurons are present in the LOC system in the guinea-pig. It is now demonstrated that protection against acoustic trauma by sound conditioning has a central component that is governed by TH in the LSO and the surrounding periolivary DLPO region.

Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat.

A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component.

Cholinergic innervation of the chick basilar papilla.

Antibodies directed against choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker of cholinergic neurons, were used to label axons and nerve terminals of efferent fibers that innervate the chick basilar papilla (BP). Two morphologically distinct populations of cholinergic fibers were labeled and classified according to the region of the BP they innervated. The inferior efferent system was composed of thick fibers that coursed radially across the basilar membrane in small fascicles, gave off small branches that innervated short hair cells with large cup-like endings, and continued past the inferior edge of the BP to ramify extensively in the hyaline cell area. The superior efferent system was made up of a group of thin fibers that remained in the superior half of the epithelium and innervated tall hair cells with bouton endings. Both inferior and superior efferent fibers richly innervated the basal two thirds of the BP. However, the apical quarter of the chick BP was virtually devoid of efferent innervation except for a few fibers that gave off bouton endings around the peripheral edges. The distribution of ChAT-positive efferent endings appeared very similar to the population of efferent endings that labeled with synapsin antisera. Double labeling with ChAT and synapsin antibodies showed that the two markers colocalized in all nerve terminals that were identified in BP whole-mounts and frozen sections. These results strongly suggest that all of the efferent fibers that innervate the chick BP are cholinergic.

Differential expression of catecholamine synthetic enzymes in the caudal ventral pons.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined.

Nitric oxide synthase in the hypothalamic paraventricular nucleus of the female rat; organization of spinal projections and coexistence with oxytocin or vasopressin.

We investigated the distributions and interrelations of neuronal nitric oxide (NO) synthase- (nNOS), oxytocin- (OT), and 8-arginine vasopressin- (AVP) immunoreactive (IR) neurons in the paraventricular nucleus (PVN), and the occurrence and distribution of nNOS spinally projecting neurons in the PVN of the female rat. Using double labelling immunohistochemistry, we mapped the distribution of nNOS-, OT- and AVP-immunoreactive (IR) neuronal cell bodies in the different parts of the PVN. About 80% of nNOS-IR cell bodies were magnocellular. About 30% of the nNOS-IR cell bodies were OT-IR, colocalization being most frequent in the rostral parts. In comparison, only approximately 3% of all nNOS-IR cell bodies were AVP-IR, evenly distributed throughout the PVN. True Blue (TB), administered unilaterally into the spinal cord, disclosed that most spinally projecting cell bodies in the PVN were localized in caudal parts. Combined TB tracing and nNOS immunohistochemistry showed that approximately 30% of spinally projecting neurons in the PVN were nNOS-IR, and that approximately 40% of these were magnocellular. Ipsilateral nNOS spinal projections were about eight times more frequent than the contralateral nNOS projections. The study describes the detailed neuroanatomical organization of nNOS neurons coexpressing OT or AVP, and of nNOS spinally projecting neurons within defined parts of the PVN. In contrast to the paraventriculo-spinal system in general, we show that the nNOS paraventriculo-spinal pathway to a large extent originates in magnocellular cell bodies. The results suggest that NO is an important messenger in the paraventriculo-spinal pathway that may in part act in concert with OT.

Efferent innervation in the goldfish saccule examined by acetylcholinesterase histochemistry.

In contrast to the abundance of information available regarding the anatomy and physiology of afferents within the goldfish saccule, the efferent system of this auditory endorgan has been scarcely studied morphologically. In this study, acetylcholinesterase histochemistry with diaminobenzidine enhancement was used to describe the morphology of efferents. Under light microscopy, labeled fibers appeared in the distal portion of the saccular nerve, penetrated the basement membrane and formed a horizontal mesh-like plexus near the base of hair cells. Many vertical branchlets with terminal swellings protruded upward toward hair cells from the plexus. Under electron microscopy, dense extracellular labeling was present around efferent terminals, which often formed clusters on hair cells. Labeling was also present around unmyelinated fibers of passage within the sensory epithelium and the distal saccular nerve. These fibers contained coarse microtubules and small vesicles, and often ran in a bundle with other similar fibers. Based on their position within the epithelium, histochemistry and ultrastructural characteristics, these fibers were concluded to be efferents. These fibers became myelinated and unlabeled in the proximal saccular nerve. These results suggest that acetylcholinesterase can be a marker of entire distal unmyelinated portions of efferent fibers and demonstrated abundant efferent innervation in the goldfish saccule.

Developmental regulation of tryptophan hydroxylase messenger RNA expression and enzyme activity in the raphe and its target fields.

Tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin and during development, brain serotonin levels and tryptophan hydroxylase activities increase. Increased tryptophan hydroxylase activity could result from alterations in tryptophan hydroxylase messenger RNA levels, translation, and/or post-translational regulation. Tryptophan hydroxylase messenger RNA levels in the dorsal raphe nucleus increased 35-fold between embryonic day 18 and postnatal day 22, measured by quantitative in situ hybridization, then decreased by 40% between postnatal days 22 and 61. These changes correlated with tryptophan hydroxylase enzyme activities in the raphe nuclei as expected, but not in cortical or hippocampal targets. Tryptophan hydroxylase messenger RNA expression in the nucleus raphe obscuris increased 2.5-fold between postnatal days 8 and 22 but did not correlate with enzyme activity in the spinal cord. Using an in vitro model of serotonergic raphe neuron differentiation, serotonergic differentiation was associated with an increase in both tryptophan hydroxylase promoter activity and protein expression. In vivo, tryptophan hydroxylase messenger RNA levels per single cell and per brain section were correlated during development up to postnatal day 22, but not beyond for both the dorsal raphe nucleus and nucleus raphe obscuris. Between postnatal days 22 and 61 single cell levels of tryptophan hydroxylase messenger RNA in the dorsal raphe nucleus did not change yet the levels per brain section significantly decreased by 40%. During the same period in the nucleus raphe obscuris, tryptophan hydroxylase messenger RNA levels per single cell signifcantly increased by 30% yet levels per brain section did not change. Comparison of tryptophan hydroxylase messenger RNA levels per cell and per brain section indicated a serotonergic loss between postnatal days 22 and 61 in both the dorsal raphe nucleus and nucleus raphe obscuris and may reflect either a loss of neurotransmitter phenotype or cell death. This study is the first to characterize the expression of brain tryptophan hydroxylase messenger RNA during rat development. In addition, this study is the first to report the activity of tryptophan hydroxylase in the spinal cord and hippocampus in the embryonic and neonatal rat. Together, the data provide a better understanding of the intricate relationship between patterns of tryptophan hydroxylase messenger RNA expression and enzyme activity.

Co-localization of glutamate, choline acetyltransferase and glycine in the mammalian vestibular ganglion and periphery.

Glutamate (Glu) is considered to be the main transmitter at the central synapses of primary vestibular afferents (PVA) and glycine (Gly) is assumed to play a modulatory role. In the vestibular periphery a transmitter role for acetylcholine (ACh) has been attributed chiefly to vestibular efferents (VE), however only a subset of VE neurons displays immunoreactivity (ir) for choline acetyltransferase (ChAT) and acetylcholine esterase (AChE). Controversial results exist on the presence of these two enzymes in PVA. In this study the presence of Glu, ChAT, Gly and their co-localization in the vestibular ganglia (VG) and end organs of mouse, rat, guinea pig and squirrel monkey were investigated. In the VG all bipolar neurons display strong Glu-ir and the majority of cells show a graded ChAT-ir and Gly-ir in all species examined. ChAT and Gly are present in highly overlapping neuronal populations and with a similar gradation. In the end organs ChAT and Gly are again co-localized in the same sets of fibers and endings. In conclusion, in the vestibular ganglion and end organs ChAT appears also to be present in primary afferents rather than being restricted to efferent processes. ChAT in primary afferents might indicate a modulatory or co-transmitter function of acetylcholine.

[Immunohistochemical study of structural basis of inhibition in central cerebellar nuclei]. / Immunogistokhimicheskoe izuchenie strukturnykh osnov tormozheniia v tsenral'nykh iadrakh mozzhechka u myshei.

Glutamatic acid decarboxylase (GAD): a synthetic enzyme for inhibitory neurotransmitter GABA, was studied in the central cerebellar nuclei. Synapses were found: morphological structures providing of inhibition in the central cerebellar nuclei.

Input from central nucleus of the amygdala efferents to pericoerulear dendrites, some of which contain tyrosine hydroxylase immunoreactivity.

Light microscopic anterograde tracing studies indicate that neurons in the central nucleus of the amygdala (CNA) project to a region of the dorsal pontine tegmentum ventral to the superior cerebellar peduncle which contains noradrenergic dendrites of the nucleus locus coeruleus (LC). However, it has not been established whether the efferent terminals from the CNA target catecholamine-containing dendrites of the LC or dendrites of neurons from neighboring nuclei which may extend into this region. To examine this question, we combined immunoperoxidase labeling of the anterograde tracer biotinylated dextran amine (BDA) from the CNA with immunogold-silver labeling of the catecholamine-synthesizing enzyme tryrosine hydroxylase (TH) in the rostrolateral LC region of adult rats. By light microscopy, BDA-labeled processes were dense in the dorsal pons within the parabrachial nuclei as well as in the pericoerulear region immediately ventral to the superior cerebellar peduncle. Higher magnification revealed that BDA-labeled varicose fibers overlapped TH-labeled processes in this pericoerulear region. By electron microscopy, anterogradely labeled axon terminals contained small, clear as well as some large dense core vesicles and were commonly apposed to astrocytic processes along some portion of their plasmalemma. BDA-labeled terminals mainly formed symmetric type synaptic contacts characteristic of inhibitory transmitters. Of 250 BDA-labeled axon terminals examined where TH immunoreactivity was present in the neuropil, 81% contacted unlabeled and 19% contacted TH-labeled dendrites. Additionally, amygdala efferents were often apposed to unlabeled axon terminals forming asymmetric (excitatory type) synapses. These results demonstrate that amygdaloid efferents may directly alter the activity of catecholaminergic and non-catecholaminergic neurons in this pericoerulear region of the rat brain. Furthermore, our study suggests that CNA efferents may indirectly affect the activity of pericoerulear neurons through modulation of excitatory afferents. Amygdaloid projections to noradrenergic neurons may help integrate behavioral and visceral responses to threatening stimuli by influencing the widespread noradrenergic projections from the LC.

Distribution of choline acetyltransferase mRNA in the efferent vestibular neurons of the chinchilla.

The distribution of choline acetyltransferase messenger RNA (mRNA) among efferent vestibular neurons in the chinchilla was investigated. mRNA coding for choline acetyltransferase, the enzyme that synthesizes acetylcholine, was used as a marker for the cholinergic system. In order to retrogradely label the efferent vestibular neurons, Fluoro-gold was injected through the oval window into the inner ear of anesthetized young male chinchillas (6 to 12 months old). The animals were anesthetized and perfused through the heart 2 days post injection with 4% paraformaldehyde in phosphate buffer. Retrogradely labeled efferent vestibular neurons were mapped in brainstem sections prior to processing for in situ hybridization histochemistry using radiolabeled ribonucleic acid probes complementary to the 3' end of the choline acetyltransferase mRNA. At the levels of the ascending facial nerve and the genu of the facial nerve, we found that approximately 90% of the Fluoro-gold labeled cells in group E1 contained choline acetyltransferase mRNA. All of the group E2 cells that were labeled with Fluoro-gold were found to be cholinergic (contain choline acetyltransferase mRNA). Finally, 60% of the Fluoro-gold-labeled cells in the caudal pontine reticular nucleus contained choline acetyltransferase mRNA.