The distribution of tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyltransferase immunoreactive neurons in the feline medulla oblongata

The distributions and morphological characteristics of neurons displaying immunoreactivity to the catecholamine synthetic enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) were examined in adjacent sections of the feline medulla oblongata. TH-positive neurons were found in two bilaterally symmetrical columns in the ventrolateral and dorsomedial medulla. Within the ventrolateral medulla, TH-positive neurons were found within the lateral reticular formation throughout the entire rostrocaudal extent of the medulla. In the dorsomedial medulla, TH-immunoreactive perikarya were localized to the nucleus of the tractus solitarius including the commissural subnucleus, the dorsal motor nucleus of the vagus, and the area postrema. DBH-positive neurons had distributions and morphologies similar to those of the TH-immunoreactive cells with three exceptions: TH-positive neurons far outnumbered DBH-positive neurons in the area postrema; slightly greater numbers of TH-positive neurons were seen in the commissural nucleus of the tractus solitarius; and, caudal to the obex, only TH-positive neurons were seen within the dorsal motor nucleus of the vagus. PNMT-immunoreactive neurons were found in all the nuclear regions of the medulla where both TH- and DBH-positive neurons were seen. However, the PNMT immunoreactive perikarya had a somewhat more restricted distribution along the rostrocaudal axis. In the ventrolateral medulla, PNMT-positive cells extended rostrally only as far as the retrofacial nucleus and caudally only to the obex. Within the dorsomedial medulla, PNMT immunoreactive cells were found from just rostral to the area postrema to the medullary-spinal cord junction. These findings demonstrate that the distributions of TH, DBH, and PNMT immunoreactive perikarya in the medulla of the cat are generally similar to those seen in the rat insofar as these neurons are arranged in longitudinal columns in both species. However, significant differences exist with regard to the cytoarchitectonic borders within which immunoreactive perikarya can be found and the rostrocaudal extent of the PNMT-positive cell groups in these two species.     

Enkephalinergic sympathetic and parasympathetic innervation of the rat submandibular and sublingual glands.

Enkephalinergic innervation of the rat salivary glands was investigated by immunocytochemical techniques. Based upon immunostaining for enkephalin (ENK) and tyrosine hydroxylase (TH), 4 types of neurons could be distinguished in the submandibular ganglion: cells containing both ENK and TH (9% of all ganglion cells), cells containing only ENK (17%), cells containing only TH (4%) and cells lacking both ENK and TH (70%). Almost all of the ganglion neurons were also positive for AChE and so were most of the TH-positive cells. The ENK-positive fibers outnumbered the TH-positive fibers. Although TH-positive fibers displayed concurrent ENK immunoreactivity, fibers in the blood vessel walls were only immunoreactive for TH. Excision of the superior cervical ganglion resulted in a decrease of ENK fibers and the disappearance of most of the TH fibers from the submandibular gland. Most of the remaining ENK-positive fibers were immunonegative for TH, while the remaining TH-positive fibers were also positive for ENK. The salivary gland of the postnatal 8-week-old rats had a considerable number of ENK-positive neurons and fibers in the submandibular ganglion and acini.     

Physical (in)activity‐dependent structural plasticity in bulbospinal catecholaminergic neurons of rat rostral ventrolateral medulla

Increased activity of the sympathetic nervous system is thought to play a role in the development and progression of cardiovascular disease. Recent work has shown that physical inactivity versus activity alters neuronal structure in brain regions associated with cardiovascular regulation. Our physiological studies suggest that neurons in the rostral ventrolateral medulla (RVLM) are more responsive to excitation in sedentary versus physically active animals. We hypothesized that enhanced functional responses in the RVLM may be due, in part, to changes in the structure of RVLM neurons that control sympathetic activity. We used retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) to identify bulbospinal catecholaminergic (C1) neurons in sedentary and active rats after chronic voluntary wheel‐running exercise. We then digitally reconstructed their cell bodies and dendrites at different rostrocaudal levels. The dendritic arbors of spinally projecting TH neurons from sedentary rats were more branched than those of physically active rats (P < 0.05). In sedentary rats, dendritic branching was greater in more rostral versus more caudal bulbospinal C1 neurons, whereas, in physically active rats, dendritic branching was consistent throughout the RVLM. In contrast, cell body size and the number of primary dendrites did not differ between active and inactive animals. We suggest that these structural changes provide an anatomical underpinning for the functional differences observed in our in vivo studies. These inactivity‐related structural and functional changes may enhance the overall sensitivity of RVLM neurons to excitatory stimuli and contribute to an increased risk of cardiovascular disease in sedentary individuals. J. Comp. Neurol. 522:499–513, 2014. © 2013 Wiley Periodicals, Inc.     

Location and distribution of tracheosyringeal motorneuron somata in the fowl

Injection of horseradish peroxidase solution (HRP) into the tracheosyringeal musculature and tongue of the domestic chick labeled their motorneuron somata in the medulla. Those of tracheosyringeal motor neurons occupied the caudal portions of the nucleus nervi hypoglossi (n. XII), whereas those for motor neurons supplying the tongue occupied the rostral part. The rostrocaudal representations of tracheolateralis, sternotrachealis, and ypsilotrachealis muscles were roughly mapped by the rostrocaudal distribution of labeled somata in n. XII. Except for sparse labeling found in the caudal part of the dorsal vagal nucleus of a few animals, no labeled cells were found outside of n. XII. Most tracheosyringeal injections labeled contralateral n. XII, but section of the contralateral tracheosyringeal nerve high on the neck prior to HRP injection abolished this labeling.     

Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray

It is well established that a group of bulbospinal neurons within the rostral ventrolateral medulla plays a crucial role in the tonic and phasic control of arterial pressure. In the cat, these neurons are confined to a discrete region which has been termed the subretrofacial (SRF) nucleus. Recent evidence suggests that this nucleus is viscerotopically organized with respect to its control over different vascular beds. These observations raise the question as to whether functionally different subgroups of SRF pressor neurons receive inputs from supramedullary cell groups that also exert a specific control over particular vascular beds. To answer this question retrogradely transported tracers (i.e. rhodamine or fluorescein-labelled microspheres, wheat germ agglutinin-horseradish peroxidase) were injected into physiologically identified sites within the rostral or caudal parts of the SRF nucleus of the cat. Separate groups of neurons in the midbrain periaqueductal gray region (PAG) were found to project specifically to subgroups of cells within the rostral and caudal parts of the SRF nucleus. These findings, together with the results of recent functional studies of the PAG suggest that these distinct projections from the PAG to the SRF nucleus are involved in the expression of different patterns of emotionally coupled cardiovascular responses.     

Quantitation of catecholamine neurons in the locus coeruleus in human brains of normal young and older adults and in depression

A quantitative study of the morphology and distribution of norepinephrinergic neurons in the human locus coeruleus (LC) is given for normal young and older adult brain. Norepinephrine (NE)-producing neurons are identified by immunocytochemistry of two NE biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), visualized by the peroxidase-antiperoxidase and immunogold-silver-staining methods. TH and DBH immunoreactions yield equivalent results. Both immunocytochemical visualization methods allow detailed analysis of neuronal morphology. The neurons of the human LC fall into four classes: large multipolar neurons with round or multiangular somata, large elliptical "bipolar" neurons, small multipolar neurons, and small ovoid "bipolar" neurons. Though most of the neurons contain neuromelanin pigment, some larger neurons lack pigmentation. Dendritic arborization of all neurons is extensive. Computer-assisted quantitative measurements of the parameters somatic size, dendritic arbor length, surface area, and volume are given. Somatic areas of LC neurons of all four classes are decreased in older adult brain, but dendritic arborization is equally extensive as in the younger. The rostrocaudal length of the LC is approximately 15 mm, and no age-dependent decrease is observed. Computer-assisted mapping of immunoreactive neurons and three-dimensional reconstruction allow division of the LC into rostral, middle, and caudal parts with characteristic distribution of neurons. Small neurons predominate in all parts, but the relative contribution of larger cells decreases in a rostrocaudal direction. A cell loss of 27-37% occurs in older adult brains and to 55% in the brain of a chronically depressed patient without dementia. Cell loss is highest in the rostral part, lower in the middle, and absent in the caudal part, and more small cells are lost than larger ones.     

Calretinin and calbindin-D28k in dopaminergic neurons of the rat midbrain: a triple-labeling immunohistochemical study

We used triple-labeling immunohistochemistry in rat midbrain sections to identify dopaminergic neurons that contain either one or both of the calcium-binding proteins, calretinin (CR) and calbindin-D28k (CB). Midbrain dopaminergic neurons were immunohistochemically labeled for tyrosine hydroxylase (TH), CR, and CB. In the substantia nigra pars compacta (SNC), TH+/CR+/CB+ cells were clustered in two regions: the dorsal tier of the rostral SNC and the medial part of the intermediate SNC. The ventral tier of the rostral SNC mainly comprised both TH+/CR+/CB- and TH+/CR-/CB- cells. The lateral part of the intermediate SNC and the caudal SNC primarily consisted of TH+/CR-/CB- cells. Throughout the extent of the SNC, approximately half of the TH+ neurons were stained for neither CR nor CB, while the remaining TH+ populations were labeled for CR and/or CB. Throughout the ventral tegmental area, TH+/CR+/CB+ cells, TH+/CR+/CB- cells, TH+/CR-/CB+ cells, and TH+/CR-/CB- cells were found generally scattered, though the TH+/CR-/CB- cells were dominant in number. In the substantia nigra pars lateralis, interfascicular nucleus, and caudal linear nucleus, more than half of the TH+ cells were stained for both CR and CB. In the retrorubral field, two-thirds of the TH+ neurons contained neither protein. The present findings suggest that the SNC can be divided into subcompartments based on the distribution of dopaminergic neurons that contain calcium-binding proteins. Furthermore, because CR and CB likely contribute to calcium homeostasis by buffering intracellular calcium concentrations, midbrain dopaminergic neurons containing one or both of these calcium-binding proteins may have a higher calcium-buffering capacity than those lacking the two proteins.     

Functional organization of the suprachiasmatic nucleus of Xenopus laevis in relation to background adaptation

The process of background adaptation in the toad Xenopus laevis is controlled by neurons in the suprachiasmatic nucleus (SC) that inhibit the release of alpha-melanophore-stimulating hormone from the neuroendocrine melanotrope cells in the pituitary gland. We have identified the structural and functional organization of different neuropeptide Y (NPY)-containing cell groups in the Xenopus SC in relation to background adaptation. A ventrolateral, a dorsomedial, and a caudal group were distinguished, differing in location as well as in number, size, and shape of their cells. They also show different degrees of NPY immunoreactivity in response to different background adaptation conditions. In situ hybridization using a Xenopus mRNA probe for the exocytosis protein DOC2 revealed that melanotrope cells of black-adapted animals have a much higher expression of DOC2-mRNA than white-adapted ones. This establishes that the degree of DOC2-mRNA expression is a good parameter to measure cellular secretory activity in Xenopus. We show that in the ventrolateral SC group, more NPY-positive neurons express DOC2-mRNA in white- than in black-adapted animals. In contrast, NPY-positive neurons in the dorsomedial group have a high secretory activity under the black-adaptation condition. We propose that in black-adapted animals, NPY-positive neurons in the ventrolateral group, known to inhibit the melanotrope cells in white-adapted animals synaptically, are inhibited by NPY-containing interneurons in the dorsmedial group. NPY-positive neurons in the caudal group have similar secretory dynamics as the dorsomedial NPY neurons, indicating that they also play a role in background adaptation, distinct from that exerted by the ventrolateral and dorsomedial group.     

Modulation of sympathetic nerve activity by microinjection of the 5-HT1A receptor agonist 8-OH-DPAT into the rostroventrolateral medulla.

In the present study, renal sympathetic nerve activity was recorded simultaneously with sympathetic nerve activity to skeletal muscle vasculature to determine if the sympatho-inhibition evoked by microinjection of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)teralin (8-OH-DPAT) into the rostroventrolateral medulla (RVLM) was uniform or regional. Three patterns of sympatho-inhibition were observed in these sympathetic outflows and the type of response depended upon location of microinjection within the subretrofacial nucleus (SRF). Inhibition of renal nerve activity only was elicited by microinjections at rostral sites at the caudal pole of the facial nucleus. In contrast, inhibition of muscle sympathetic nerve activity was evoked from more caudal injections at the rostral pole of the inferior olives. Microinjection in the area between these two regions produced inhibition of both sympathetic outflows. This study demonstrates that differential inhibition of regional sympathetic outflows can be elicited by microinjection of the 5-HT1A receptor agonist 8-OH-DPAT into the RVLM. These data suggests that this modulation is due to differences in anatomical arrangement of the medullary neurons rather than differences in neuron sensitivity to the serotonergic agonist.     

Electrophysiological analysis of midbrain periaqueductal gray influence on cardiovascular neurons in the ventrolateral medulla oblongata

Stimulation of sites in the rostral or caudoventral periaqueductal gray (PAG) results in substantial increases in mean blood pressure (MBP) and heart rate (HR). The efferent pathways from these PAG subregions possibly include a relay in the ventrolateral medulla oblongata (VLM), where neurons involved in maintaining vasomotor tone are located. Extracellular recordings were made from 21 cardiovascular neurons in the rostral VLM (RVLM) and from 6 cardiovascular neurons in the caudal VLM (CVLM) of the rat. These neurons showed barosensitivity and cardiac rhythmicity. In addition, the activity of 54 noncardiovascular and nonrespiratory units was recorded. Responses to electrical stimulation of sites in the (rostral or caudal) PAG were studied in 16 of the 21 cardiovascular RVLM neurons, the 6 CVLM neurons, and 46 of the 54 noncardiovascular neurons. Eight of the RVLM neurons were excited by rostral PAG stimulation; the poststimulus time histograms showed a constant latency in five units (32 ± 3 ms). This suggests the presence of relatively direct (although not monosynaptic) excitatory pathways from the rostral PAG to cardiovascular neurons in the RVLM, consisting of slowly conducting fibers (0.2-0.3 m/s). Five RVLM neurons did not respond to rostral PAG stimulation. Three units were tested with caudal PAG stimulation: one was excited, one inhibited, and one was unresponsive. The six cardiovascular CVLM neurons did not respond to PAG stimulation. Of the 46 noncardiovascular neurons, 14 cells were excited, 7 inhibited, and 2 cells antidromically activated. These results confirm earlier findings, extending them to the rostral PAG. They supply further evidence for the influence of the PAG on the cardiovascular function-related neuronal circuitry in the VLM.     

Evidence for a rostrocaudal organization in dorsal root ganglia during development as demonstrated by intra-uterine WGA-HRP injections into the hindlimb of rat fetuses.

The development of the sensory innervation of the rat hindlimb was studied with special attention to the dorsal root ganglia and the lumbar plexus. Injections of wheat germ agglutinin-horseradish peroxidase were made into the hindlimb of 30 rat fetuses of gestational ages ranging from embryonic day 15-18. Additionally wheat germ agglutinin-horseradish peroxidase was applied to the sciatic nerves of 8 neonatal rats and 3 adults. The saphenous nerves of 2 neonatal rats were labeled. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the hindlimb of the fetuses result in completely and partially labeled dorsal root ganglia. Partial labeling always concerns the rostral or caudal part of a dorsal root ganglion. The associated dorsal roots of partially labeled dorsal root ganglia are also partially labeled in a corresponding rostrocaudal fashion. Reconstructions of the labeled nerves following injections into the hindlimb suggested that the somata of the sensory neurons of a particular nerve can be restricted to the rostral or caudal half of a dorsal root ganglion. For example: the rostral half of the fourth lumbar dorsal root ganglion belongs to the femoral nerve and its caudal half to the sciatic nerve. The results of application of wheat germ agglutinin-horseradish peroxidase to the central ends of the cut sciatic and saphenous nerves in neonatal rats confirmed these observations. So the rostrocaudal organization in the dorsal root ganglia stems from the distribution pattern of the peripheral nerves.     

Immunohistochemical study of catecholamine enzymes and neuropeptide Y (NPY) in the rostral ventrolateral medulla and bulbospinal projection

The purpose of this study was to determine whether neuropeptide Y (NPY) terminals in the intermediolateral spinal cord originate from the rostral ventrolateral medulla (RVLM). Immunohistochemical staining of tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and NPY in the rat brainstem and spinal cord were performed in this study in order to examine consequences of lesions of the RVLM and of intracisternal injections of 6-hydroxydopamine (6-OHDA) on catecholamine and NPY immunoreactivity in the intermediolateral column (IML) of rats. In addition, ricin, a retrograde neurotoxin, was applied in the superior cervical ganglion (SCG) to determine its effect on catecholamine and NPY immunoreactivity in the IML. Computer-aided image analysis was used to quantify the immunohistochemical changes in the RVLM and spinal cord. The results demosntrated that many catecholamine- and NPY-containing neurons and/or fibers existed in the RVLM and their teminals were found in the IML. After administration of 6-OHDA intracisternally, the catecholamine and NPY immunoreactivities were decreased both in the brainstem and IML of the spinal cord. Following unilateral microinjection of 6-OHDA into the RVLM, the number of NPY- and catecholamine-containing neurons decreased and there was a reduction in neuron terminals on the ipsilateral side. After injection of ricin into the SCG, the catecholamine and NPY neurons of the medulla were not affected, whereas their terminals in the IML decreased ipsilaterally. These results indicate that most of the catecholamine- and NPY-immunoreactive terminals found in the IML originated in the RVLM. These terminals appear to project towards the superior cervical ganglia. © 1993 Wiley-Liss, Inc.     

Location and electrophysiological characterization of rostral medullary adrenergic neurons that contain neuropeptide Y mRNA in rat medulla

The objective of this study was to characterize the projection pattern and electrophysiological properties of the rostral medullary adrenergic neurons (C(1)) that express neuropeptide Y (NPY) mRNA in rat. NPY mRNA was found in a variable fraction of tyrosine hydroxylase immunoreactive (TH-IR) neurons depending on the medullary level. By retrograde labeling (Fast Blue, FluoroGold), NPY mRNA was detected in virtually all C(1) cells (96%) and C(3) cells (100%) with hypothalamic projections but in only 9% of C(1) cells and 58% of C(3) cells projecting to thoracic segment 3 (T(3)) or T(6) of the spinal cord. To identify the electrophysiological properties of the C(1) cells that express NPY mRNA, we recorded from baroinhibited neurons within the C(1) region of the ventrolateral medulla (RVLM) and tested for projections to segment T(3), the hypothalamus, or both. By using the juxtacellular method, we labeled these cells with biotinamide and determined whether the recorded neurons were TH-IR and contained NPY mRNA. At rostral levels (Bregma -11.8 mm), barosensitive neurons had a wide range of conduction velocities (0.4-6.0 m/second) and discharge rates (2-28 spikes/second). Most projected to T(3) only (27 of 31 cells), and 4 projected to both the hypothalamus and the spinal cord. Most of the baroinhibited cells with spinal projections but with no hypothalamic projections had TH-IR but no NPY mRNA (11 of 17 cells). Only 1 cell had both (1 of 17 cells), and 5 cells had neither (5 of 17 cells). Both TH-IR and NPY mRNA were found in neurons with dual projections (2 of 2 cells). At level Bregma -12.5 mm, baroinhibited neurons had projections to the hypothalamus only (13 of 13 cells) and had unmyelinated axons and a low discharge rate. Four of five neurons contained both TH-IR and NPY mRNA, and 1 neuron contained neither. In short, NPY is expressed mostly by C(1) cells with projection to the hypothalamus. NPY-positive C(1) neurons are barosensitive, have unmyelinated axons, and have a very low rate of discharge. Most bulbospinal C(1) cells with a putative sympathoexcitatory role do not make NPY.     

Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study

Microinjection of the excitatory amino acid D,L-homocysteic acid (40 nmol, in 200 nl) made into the ventrolateral part of the caudal half (A2.5-P1.5) of the midbrain periaqueductal gray (PAG) of the decerebrate cat evoked a hypotensive reaction associated with a slowing of the heart and a decrease in either external iliac or renal vascular resistance. The decrease in iliac vascular resistance was elicited from the pretentorial portion (A2.5-A0.6) of the PAG hypotensive area, whereas the decrease in renal vascular resistance was elicited from the subtentorial portion (A0.6-P1.5). Anatomical experiments using the method of retrograde transport of rhodamine-labelled microspheres or wheat germ agglutinin-horseradish peroxidase demonstrated topographically organized projections from the ventrolateral PAG to the subretrofacial (SRF) pressor nucleus in the rostral ventrolateral medulla. The pretentorial part of the ventrolateral PAG projected mainly to the caudal part of the SRF nucleus, which preferentially controls iliac vascular resistance. The subtentorial part of the ventrolateral PAG projected mainly to the rostral part of the SRF nucleus, which preferentially controls renal vascular resistance. Taken together, these findings suggest: (i) that neurons within the ventrolateral PAG are viscerotopically organized; and (ii) that their hypotensive function may be mediated by an inhibition of SRF pressor neurons. The results are discussed in relation to the recently described PAG hypertensive area which also is viscerotopically organized and projects to the SRF nucleus.     

Centrifugal visual system of Crocodylus niloticus: a hodological, histochemical, and immunocytochemical study

The retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approximately 6,000 in number, these neurons are distributed in seven regions extending from the mesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being located contralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. The retinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticularis lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleus subcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commissura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleus subcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopic centrifugal visual neurons of birds, although a small number of monopolar neurons resembling those of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in the griseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in the nuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally, approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide synthase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactive for choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Fibers immunoreactive to TH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) were frequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. These findings are discussed in relation to previous results obtained in other reptilian species and in birds.     

Rat medulla oblongata. II. Dopaminergic, noradrenergic (A1 and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes

The aim of this study was to determine the anatomical relationships between catecholaminergic neurons and cytoarchitectonically defined nuclei in the caudal medulla oblongata. Previous studies have demonstrated the existence of noradrenergic cell bodies (designated as the A1 and A2 cell groups) in the caudal medulla oblongata of the rat (Dahlstr?m and Fuxe, '64), including the nTS. There is no information currently available with regard to details of the distribution of these noradrenergic neurons in the functionally distinct subnuclei of the medulla oblongata. In this study the location of catecholamine-synthesizing enzymes was examined in the serial sections of the caudal medulla oblongata of the rat: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT). The immunoperoxidase method of Sternberger ('79) was used to demonstrate the location of immunoreactive neurons, nerve fibers, and presumptive terminal processes. This was followed by Nissl staining of the same sections to localize accurately the immunoreactivity. Noradrenergic neurons (TH- and DBH-positive and PNMT-negative) were localized in a number of subnuclei of the nucleus of the tractus solitarius (nTS), the area postrema (ap), and in the dorsal motor nucleus of the vagus (dmnX). The distribution of these noradrenergic cells was different at different rostrocaudal levels. In addition, adrenergic neurons (TH-, DBH-, and PMNT-positive) were identified dorsal to the tractus solitarius (TS), in the dorsal strip region (ds), the periventricular region (PVR), the dorsal parasolitarius region (dPSR), and the dmnX (rostral to obex). In addition, dopaminergic neurons (TH-positive and DBH- and PNMT-negative) were found in the ap and dmnX. The A1 cell group in the ventrolateral medulla consisted almost exclusively of noradrenergic neurons (TH- and DBH-positive and PNMT-negative). These results indicate that in the rat the A2 cell group is a mixed population of catecholaminergic neurons that are localized in well-defined regions of the dorsal medulla oblongata. The distribution of these neurons is very specific both in terms of rostrocaudal levels and cytoarchitectonic subdivisions of regions of the medulla known to be involved in central autonomic control. This supports the hypothesis that monoaminergic neurons in the dorsal medulla play important roles in the central regulation of visceral function.     

Characterization of the central cell groups regulating the kidney in the rat

Retrograde, transneuronal viral tracing technique combined with neurotransmitter immunohistochemistry was used to identify the type of neurons in spinal cord and brain that project to the rat's kidney. Pseudorabies virus (PRV) injections were made into the left kidney. After an incubation of 4 days postinjection, PRV-infected neurons were located immunocytochemically in the ipsilateral intermediolateral (IML) cell column of the spinal cord and several brainstem cell groups: medullary raphe nuclei, ventromedial medulla (VMM), rostral ventrolateral medulla (RVLM), A5 cell group and the hypothalamic paraventricular nucleus (PVH). In the medulla, serotonin (5-HT)-immunoreactive neurons of the caudal raphe nuclei, substance P (SP)-immunoreactive neurons of the raphe obscurus (ROb) nuclei and tyrosine hydroxylase (TH)-immunoreactive neurons of A5 cells were infected. In the VMM and RVLM, immunoreactive phenylethanolamine-N-methyltransferase (PNMT) neurons were infected. Some PRV-infected neurons in VMM contain 5-HT immunoreactivity. In the hypothalamus, immunoreactive vasopressin (VP) and oxytocin (OT) neurons were infected with PRV. This work indicates that sympathetic outflow to kidney is regulated by different types of neurons and the bulbospinal pathways regulating sympathetic outflow to the kidney are not obviously different from those regulating the other visceral, e.g., adrenal, heart, etc.     

Distribution of GABA, glycine, and glutamate in neurons of the medulla oblongata and their projections to the midbrain tectum in plethodontid salamanders

In the medulla oblongata of plethodontid salamanders, GABA-, glycine-, and glutamate-like immunoreactivity (ir) of neurons was studied. Combined tracing and immunohistochemical experiments were performed to analyze the transmitter content of medullary nuclei with reciprocal connections with the tectum mesencephali. The distribution of transmitters differed significantly between rostral and caudal medulla; dual or triple localization of transmitters was present in somata throughout the rostrocaudal extent of the medulla. Regarding the rostral medulla, the largest number of GABA- and gly-ir neurons was found in the medial zone. Neurons of the nucleus reticularis medius (NRM) retrogradely labeled by tracer application into the tectum revealed predominantly gly-ir, often colocalized with glu-ir. The NRM appears to be homologous to the mammalian gigantocellular reticular nucleus, and its glycinergic projection is most likely part of a negative feedback loop between medulla and tectum. Neurons of the dorsal and vestibular nucleus projecting to the tectum were glu-ir and often revealed additional GABA- and/or gly-ir in the vestibular nucleus. Regarding the caudal medulla, the highest density of GABA- and gly-ir cells was found in the lateral zone. Differences in the neurochemistry of the rostral versus caudal medulla appear to result from the transmitter content of projection nuclei in the rostral medulla and support the idea that the rostral medulla is involved in tecto-reticular interaction. Our results likewise underline the role of the NRM in visual object selection and orientation as suggested by behavioral studies and recordings from tectal neurons.     

Evidence for tonic disinhibition of RVLM sympathoexcitatory neurons from the caudal pressor area

Previous studies in the rat have shown that a significant proportion of the tonic activity of presympathetic neurons in the rostral ventrolateral medulla (RVLM) is dependent on the tonic activity of neurons within the caudal pressor area (CPA), located in the most caudal part of the caudal ventrolateral medulla (CVLM). In this study, we determined the extent to which tonically active neurons in the CPA contribute to sympathetic vasomotor tone, and we also investigated the pharmacological mechanisms by which these neurons affect the tonic activity of RVLM presympathetic neurons. In anaesthetised rabbits, bilateral injections of the neuroinhibitory compound muscimol into the CVLM at the level of the most caudal part of the lateral reticular nucleus, which corresponds to the anatomical location of the CPA as mapped in the rat, resulted in an immediate profound hypotension and almost complete abolition of renal sympathetic nerve activity (rSNA). In contrast, microinjections into surrounding regions had little or no effect or else evoked a delayed hypotensive response. The hypotensive and sympathoinhibitory response evoked by inhibition of the CPA was greatly delayed by prior injections of the GABA receptor antagonist bicuculline into the RVLM. In contrast, injections of the glutamate receptor antagonist kynurenic acid into the RVLM did not alter the hypotensive and sympathoinhibitory response. The results indicate that neurons within the CPA tonically inhibit other neurons, which, in turn, inhibit RVLM sympathoexcitatory neurons, via a GABAergic synapse. This disinhibition of RVLM neurons by CPA neurons is essential for maintaining resting sympathetic vasomotor tone.     

Neural precursor cells derived from human embryonic brain retain regional specificity

Recent studies have revealed that neural precursor cells can be expanded not only from the subventricular zone and hippocampus but also from other regions of the human embryonic brain. To determine the regional differences of these precursor cells, we divided the brain of a 9-week-old human embryo into four parts, i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon. All cultures of the tissues yielded neurospheres, and these spheres gave rise to neurons, astrocytes, and oligodendrocytes. An analysis of clonal populations revealed that these precursor cells were multipotent, and two region-specific differences in neural precursor cells were revealed: 1) The precursor cells from the rostral part of the brain tended to proliferate faster than those from the caudal part, and 2) the precursor cells from the diencephalon and mesencephalon gave rise to more tyrosine hydoxylase (TH)-positive neurons than those from the telencephalon and rhombencephalon. When 50-day-cultured spheres were caused to differentiate, the percentage of TH-positive cells per total cell population was 1.2% for diencephalic and mesencephalic precursors, whereas it was 0.4% for telencephalic and rhombencephalic ones. Furthermore, the TH-positive cells from diencephalic and mesencephalic precursors were large, multipolar, and gamma-aminobutyric acid (GABA)-negative, which suggested that these cells were midbrain dopaminergic neurons. In contrast, TH-positive cells from telencephalic and rhombencephalic precursors were small, bipolar, and GABA-positive. These results suggest that human neural precursor cells might have the potential to differentiate into a variety of cells but retain regional specificity.