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PubMed1988

Vulnerability of cultured cortical neurons to damage by excitotoxins: differential susceptibility of neurons containing NADPH-diaphorase.

Quantitative concentration-toxicity relationships were determined for the injury of cultured murine cortical neurons by several excitatory amino acid (EAA) agonists. All tested agonists produced concentration-dependent neuronal injury at concentrations between 1 and 1000 microM. With 5 min exposure, glutamate, aspartate, N-methyl-D-aspartate (NMDA), L-homocysteate (HCA), and quisqualate all had similar potencies, destroying half of the neuronal population (LD50) at concentrations of 50-200 microM, and similar efficacies, with 88-92% neuronal loss produced by exposure to high agonist concentrations. Quinolinate and kainate were substantially weaker toxins, producing only 20-30% neuronal loss after 5 min exposure to 3 mM concentrations; with prolonged (24 hr) exposure, 85-95% neuronal loss could be attained. The comparative EAA vulnerability of a specific cortical neuronal subpopulation containing high concentrations of the enzyme, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), was also examined. Glutamate had no differential toxicity on these cells, damaging them at all concentrations in proportion to the general population; however, other, more selective, agonists produced strikingly differential injuries. These NADPH-d-containing [NADPH-d(+)]neurons were selectively resistant to damage by low concentrations of the NMDA agonists quinolinate, HCA, aspartate, or NMDA itself. By contrast, NADPH-d(+)neurons were selectively destroyed by concentrations of quisqualate or kainate too low to produce much general neuronal injury. The differential susceptibility of these neurons was not absolute, as high concentrations of all tested agonists produced nonselective neuronal injury. In light of recent evidence that forebrain NADPH-d(+)neurons are selectively spared in Huntington's disease, the present study continues to support the hypothesis that neuronal loss in Huntington's disease might result from excessive NMDA-receptor stimulation by any selective NMDA agonist. Furthermore, the demonstration that the differential susceptibility of NADPH-d(+)neurons is agonist concentration-dependent, rather than absolute, could provide a basis for explaining some existing conflicting experimental data.

Animals↗
PubMed1985

[Neuronal organization of the CGLd of Cavia porcellus. A morphometric study of Nissl preparations with reference to identified neuron types].

A morphometric study has been made of the alpha-sector (Giolli and Creel 1973) of the guinea pig's dorsal lateral geniculate nucleus (dLGN) in Nissl preparations. Golgi-deimpregnation methods (FAIREN et al. 1977, Braak and Braak 1982) made it possible to identify the neuron types as geniculo-cortical relay neurons (GCR-neurons) and interneurons (I-neurons). Thus, density and somal sizes of GCR- and I-neurons and their topographical distribution patterns were determined. The density of the GCR-neurons (3 per measuring volume) is lower than in the albino rat (Wistar strain) (7 per measuring volume). The somal size varies over a wide range and is higher on average (16 hits) than for the albino rat (12 hits). As reported by Brauer and Remmler (person, comm.), the dendrites of GCR-neurons are relatively smooth. Their distal portions show only a small number of spine-like protrusions. Distinct grape-like appendages are visible in the branching zone. In one case an axon initial segment with three varicosities was observed. The number of I-neurons is considerably higher (36%) than in the albino rat (8%). The somata of I-neurons are of different size. More than 52% of the GCR-neurons are of the same size as the I-neurons. According to Brauer and Remmler (person. comm.), the two very long main dendrites were found to be little branched. The alpha-sector is apparently cytoarchitectonically homogeneous. Only after the use of statistical methods is it possible to distinguish between lateral, medial and caudal regions as in the albino rat (Werner et al. 1975), laboratory mouse (Werner et al. 1984a), and cotton rat (Werner et al., 1984b). These differ with regard to the density and somal size of the GCR-neurons and the density of the I-neurons. The densities of the GCR- and I-neurons are highest in the lateral region. In contrast with the albino rat, laboratory mouse, and cotton rat neurons of the guinea pig differ from those of the Wistar rat. The results suggest that the guinea pig's visual specialization differs at least from that of the albino rat.

Animals↗
PubMed1995

Neurons of the median neuroblast lineage of the grasshopper: a population study of the efferent DUM neurons.

A group of lineally related neurons in the grasshopper was studied to determine the number of efferent neurons in the group and their morphological types. The neurons arise from the median neuroblast of the third thoracic neuromere and comprise what is commonly known as the DUM or dorsal unpaired median group. Of some 92 neurons in the group, about 20 are efferent neurons, the remainder being local or intersegmental interneurons. As part of our continuing developmental studies, we wished to identify the efferent neurons within the lineage and to determine their number. Ten efferent DUM neurons had been described in earlier studies, where neurons were stained individually through microelectrodes. The remaining unidentified neurons might be novel types, multiples of known types, or both, possibilities that would not be readily distinguished through further staining of neurons individually. Rather, we used methods of retrograde staining and axon tracing that allowed us to examine the entire group of efferent DUM neurons. Nineteen efferent neurons were identified, comprising two DUM1s, five DUM3s, six DUM3,4s, three DUM3,4,5s, and three DUM5s; neurons were named according to the lateral nerves containing their axons. The efferent neurons were further divided by type according to the distribution of axonal branches in lateral nerves, the course of the primary neurite within the deep or superficial DUM tract, and the diameter of the cell body.

Animals↗
PubMed1989

Enkephalin-immunoreactive neuronal projections from the medulla oblongata to the intermediolateral cell column: relationship to substance P-immunoreactive neurons.

The present study investigated the ventral medullary distribution of enkephalin-immunoreactive neurons that project to the intermediolateral cell column and the relationship of these neurons to substance P-immunoreactive neurons. Neurons that projected to the intermediolateral cell column were identified by the presence of rhodamine-labeled microspheres within the neuronal cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Enkephalin- and substance P-immunoreactivities were identified by dual-color immunohisto-chemistry. Enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in the raphe magnus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus, and the parapyramidal region. These neurons were present throughout the rostrocaudal extent of each of these nuclei. However, in the raphe magnus the greatest number was present at more rostral levels of the nucleus. The morphology and distribution of enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were similar to those of enkephalin-immunoreactive neurons that were not observed to contain rhodamine-labeled microspheres. Substance P- and enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in similar distributions in each of the nuclei studied, except the raphe magnus. The raphe magnus contained more enkephalin- than substance P-immunoreactive neurons at rostral levels and more substance P-immunoreactive neurons than enkephalin-immunoreactive neurons at caudal levels. Coexistence of substance P- and enkephalin-immunoreactivities in ventral medullary neurons that projected to the intermediolateral cell column was rarely seen. These studies support the hypothesis that ventral medullary enkephalinergic neurons project to the intermediolateral cell column where they could act to modulate preganglionic sympathetic activity.

Animals↗
PubMed1987

Lateral tegmental field neurons of cat medulla: a source of basal activity of ventrolateral medullospinal sympathoexcitatory neurons.

We tested the hypothesis that neurons of the lateral tegmental field (LTF) of the cat medulla exert their sympathoexcitatory actions over a pathway that includes rostral ventrolateral medullospinal neurons innervating the spinal intermediolateral nucleus (IML). Thirty-one LTF neurons with sympathetic nerve-related activity [as demonstrated with spike-triggered averaging of inferior cardiac postganglionic sympathetic nerve discharge (SND)] were antidromically activated by microstimulation of the rostral ventrolateral medulla (VLM). The threshold current required to elicit the longest latency antidromic response was increased when the stimulating microelectrode was moved to more dorsal or medial sites in the rostral medulla. This observation suggests that the axons of LTF neurons projected to the rostral VLM. The firing rate of LTF neurons with sympathetic nerve-related activity was decreased during baroreceptor reflex activation. This observation is consistent with the view that these neurons subserved a sympathoexcitatory function. Twenty-five VLM neurons with sympathetic nerve-related activity were synaptically activated by microstimulation of the LTF. The modal onset latency of synaptic excitation (25.6 +/- 2.6 ms) compared favorably with the difference (31 ms on the average) between the firing times of LTF and VLM neurons relative to the peak of the cardiac-related sympathetic nerve slow wave. The firing rate of these VLM neurons decreased during baroreceptor reflex activation. Of nine VLM neurons tested, seven were antidromically activated by microstimulation of the second thoracic (T2) IML. These data are consistent with the view that LTF neurons are a source of the basal discharge of VLM-spinal sympathoexcitatory neurons. Sixteen VLM neurons with sympathetic nerve-related activity were antidromically activated by microstimulation of both the LTF and the T2 IML. In some cases, LTF stimulation activated an axonal branch rather than the main axon. This was demonstrated using time-controlled collision of the VLM neuronal action potentials initiated by LTF and T2 IML stimulation. These data raise the possibility that individual VLM neurons influence SND by actions mediated at both spinal and supraspinal levels.

Animals↗
PubMed1991

Embryonic development of choline acetyltransferase in thoracic spinal motor neurons: somatic and autonomic neurons may be derived from a common cellular group.

This investigation focused on the relationship between neurotransmitter phenotype expression and rat motor neuron development, as studied with choline acetyltransferase (ChAT) immunocytochemical techniques. The development of two subclasses of motor neurons, somatic and autonomic efferents, was examined in the upper thoracic spinal cord. ChAT was first detected in a few neurons on embryonic day 12 1/2 (E12 1/2), and in numerous cells located in a single, ventrolaterally located column in the intermediate zone on E13. By E14, this group of ChAT-positive neurons was more intensely immunoreactive, and their axons could be traced to appropriate targets in developing somatic muscle and paravertebral sympathetic ganglia. During the E15-16 period, somatic and autonomic motor neurons separated into two distinct subgroups, with the latter cells being observed to translocate dorsally. By E17, these autonomic motor neurons reached their final positions in the midportion of the intermediate zone. The autonomic motor neurons were observed to extend transverse dendritic bundles across the spinal cord between E15-16, but evidence of the longitudinal bundles of sympathetic preganglionic dendrites was not observed until after birth. A recent study of cholinergic thoracic motor neurons found that both somatic and autonomic cells were generated synchronously during the E11-12 period (Barber et al., Soc Neurosci Abstr 15:588, 1989). In combination with the present results, these data indicate that no more than 1 1/2 days are necessary after motor neuron genesis before a few cells begin to express detectable levels of ChAT, and that no more than 2 days are required before large numbers express this marker of the cholinergic phenotype. Further comparisons of the present findings with those of previous investigations of the development of both somatic and autonomic motor neurons (Dennis et al., Dev Biol 81:266, 1981; Rubin, J Neurosci 5:685, 697, 1985) indicate that these cells contain ChAT at the time their axons are growing toward their respective peripheral targets 1 day before the time when physiological evidence of function is manifest. Furthermore, the present results suggest that both subclasses of motor neurons initially migrate together from the ventricular zone into a single motor column within the ventral intermediate zone, and that the autonomic neurons subsequently translocate dorsally. Thus, autonomic motor neurons appear to be an exception to the generalization that postmitotic neurons migrate directly from the germinal zone to their final positions within the central nervous system.

Animals↗
PubMed2007

The three-neuron corneal reflex circuit and modulation of second-order corneal responsive neurons.

Neurons located in the border region between the interpolaris and caudalis subdivisions of the spinal trigeminal nucleus (Vi/Vc) are second order neurons of the corneal reflex, receiving corneal afferents and projecting to the lid closing, orbicularis oculi (OO) motoneurons. Recordings of Vi/Vc neurons identified by antidromic activation from stimulation of the facial nucleus and non-identified Vi/Vc neurons reveal two neuron types, phasic and tonic. Corneal stimulation elicits Adelta latency action potentials that occur early enough to initiate OO contraction and C-fiber latency action potentials that can modulate the end of the blink in phasic Vi/Vc neurons. Tonic Vi/Vc neurons exhibit a constant irregular, low frequency discharge as well as the cornea-evoked activity exhibited by phasic neurons. For both phasic and tonic neurons, blink amplitude increases with the total number of spikes evoked by the corneal stimulus. Peak firing frequency predicts peak orbicularis oculi EMG activity. Paradigms that suppress cornea-evoked blinks differentially affect Vi/Vc neurons. Microstimulation of the border region between the spinal trigeminal caudalis subdivision and the C1 spinal cord (Vc/C1) significantly reduces the number of spikes evoked by corneal stimulation and suppresses blink amplitude. In the paired stimulus paradigm, a blink evoked by a corneal stimulus 150 ms after an identical corneal stimulus is significantly smaller than the blink elicited by the first stimulus. Vi/Vc neuron discharge, however, is slightly larger for the second blink. Our data indicate that second-order Vi/Vc neurons do not determine the specific pattern of OO muscle activity; rather Vi/Vc neurons initiate OO motoneuron discharge and program the activity of another circuit that generates the late phase of the blink. The Vc/C1 suppression of Vi/Vc neurons suggests that the Vc/C1 region provides an "internal model" of the intended blink.

Action Potentials↗
PubMed1993

Simultaneous intracellular recordings from enteric neurons reveal that myenteric AH neurons transmit via slow excitatory postsynaptic potentials.

Simultaneous intracellular electrical recordings were made from pairs of neurons separated circumferentially by 100-200 microns of the myenteric plexus of the guinea-pig ileum in vitro. The recording electrodes were filled with the dye neurobiotin which was injected into impaled nerve cells, and later revealed histochemically. Intracellular current pulses were used to evoke action potentials via the recording electrode in one type of myenteric neuron, in most cases an AH neuron, while a second electrode was used to record from a simultaneously impaled S neuron or AH neuron. AH neurons are thought to be primary sensory neurons, whereas S neurons are interneurons and motor neurons. Ninety pairs of neurons were adequately tested for interaction. From these, 17 S neurons and three AH neurons that responded to AH neuron stimulation were detected. In each case, the response was a slow depolarization that was seen only in response to a train of stimuli at 10 Hz. The slow depolarizations were enhanced by passing depolarizing current and diminished by hyperpolarization. Responses were also diminished by lowering external Ca.2+ and elevating Mg2+. In all cases in which intracellular recording indicated communication between neurons, morphological evidence of connection was seen. In no case was there communication without connection, but in four instances, morphological connections appeared to exist, although no physiological evidence of communication was obtained.

Action Potentials↗
PubMed1999

Transcranial magnetic stimulation identifies upper motor neuron involvement in motor neuron disease.

OBJECTIVE: To evaluate the sensitivity of transcranial magnetic stimulation (TMS) to identify upper motor neuron involvement in patients with motor neuron disease. BACKGROUND: Diagnosis of ALS depends on upper and lower motor neuron involvement. Lower motor neuron involvement may be documented with electromyography, whereas definite evidence of upper motor neuron involvement may be elusive. A sensitive, noninvasive test of upper motor neuron function would be useful. METHODS: TMS and clinical assessment in 121 patients with motor neuron disease. RESULTS: TMS revealed evidence of upper motor neuron dysfunction in 84 of 121 (69%) patients, including 30 of 40 (75%) patients with only probable upper motor neuron signs and unsuspected upper motor neuron involvement in 6 of 22 (27%) patients who had purely lower motor neuron syndromes clinically. In selected cases, upper motor neuron involvement identified with TMS was verified in postmortem examination. Increased motor evoked potential threshold was the abnormality observed most frequently and was only weakly related to peripheral compound muscle action potential amplitude. In a subset of 12 patients reexamined after 11+/-6 months, TMS showed progression of abnormalities, including progressive inexcitability of central motor pathways and loss of the normal inhibitory cortical stimulation silent period. CONCLUSIONS: TMS provides a sensitive means for the assessment and monitoring of excitatory and inhibitory upper motor neuron function in motor neuron disease.

Adult↗
PubMed1996

Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei.

The time of birth of subpopulations of dorsal root ganglion (DRG) neurons was studied with immunohistochemistry for 5-bromodeoxyuridine (BrdU). Pregnant rats were injected with BrdU i.p. to label the neurons on one of the embryonic days (E) E11-E16. When they were adults, the rats were given injections of Fluoro-Gold (FG) into the gracile nucleus to identify DRG neurons projecting to this structure. Following a 5 day survival period, the animals were perfused with aldehyde fixative. Sections from the L3-L5 DRGs were processed for BrdU immunohistochemistry followed by either immunostaining for the antineurofilament antibody RT97, as marker of the light neuronal subpopulation, or histochemical staining for the B4 isolectin from Griffonia simplicifolia I, as marker of the small dark subpopulation. The results indicated that the DRG neurons were generated between E12 and E16. The RT97+ neurons were generated on E12-E15, with a peak at E13. FG+ neurons, the majority of which were RT97+, were also generated on E12-E15. The B4+ neurons were generated on E13-E16, with a peak around E14. The overall pattern of neurogenesis of the DRG neurons showed that the RT97+ neurons were produced prior to the B4+ neurons. These findings are in agreement with earlier observations that the large DRG neurons are generated earlier than the small dark neurons. Our findings also suggest the existence of a third neuronal subpopulation that might be produced at the latest period of DRG neurogenesis at E15-E16.

Animals↗
PubMed1999

Toxicity of tunicamycin to cultured brain neurons: ultrastructure of the degenerating neurons.

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 microg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.

Animals↗
PubMed2001

Neuronal body size correlates with the number of nucleoli and Cajal bodies, and with the organization of the splicing machinery in rat trigeminal ganglion neurons.

Trigeminal ganglion neurons comprise three main cell body-size types. This cell size heterogeneity provides an excellent neuronal model to study the cell size-dependent organization and dynamics of the nucleoli, Cajal (coiled) bodies (CBs), and nuclear speckles of pre-mRNA splicing factors, nuclear structures that play a key role in the normal neuronal physiology. We have analyzed the number of nucleoli and CBs and the structural and molecular organization of CBs and nuclear speckles in the three neuronal types by using immunofluorescence with antibodies that recognize nucleoli (fibrillarin), CBs (coilin), and nuclear speckles (snRNPs), confocal microscopy, and electron microscopy. Whereas the mean number of nucleoli per neuron decreases as a function of cell size, the number of CBs per cell significantly increases in large neurons in comparison with the small ones. In addition, large neurons have a higher proportion of CBs associated with the nucleolus. In all neuronal types, CBs concentrate coilin, fibrillarin, snRNPs, and the survival motor neuron protein (SMN). Immunostaining for snRNPs shows small speckle domains and extensive areas of diffuse nucleoplasmic signal in large neurons, in contrast with the large nuclear speckles found in small neurons. Furthermore, flow cytometric analysis shows that all neurons are in the range of diploid cells. These findings indicate that the fusion behavior of nucleoli, the formation of CBs and their relationships with the nucleolus, as well as the compartmentalization of the pre-mRNA splicing machinery, is related to cell body size in the trigeminal ganglion neurons. Because transcriptional activity is a basic determinant mechanism of cell size in diploid cells, we suggest that our findings reflect a distinct transcription-dependent organization of the nucleolus and splicing machinery in the three cell types of trigeminal ganglion neurons.

Animals↗
PubMed2002

Patterns of expression of calcium binding proteins and neuronal nitric oxide synthase in different populations of hippocampal GABAergic neurons in mice.

We examined the expression of calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin D28K (CB), and neuronal nitric oxide synthase (nNOS) in gamma-aminobutyric acid (GABA)ergic neurons of the mouse hippocampus, with particular reference to areal and dorsoventral differences. First, we estimated the colocalization of the calcium binding proteins and nNOS. GABAergic neurons containing both PV and nNOS, i.e., PV-immunoreactive (-IR)/nNOS-IR neurons, were rare in Ammon's horn but frequent in the dentate gyrus (DG). CR-IR/nNOS-IR neurons and CB-IR/nNOS-IR neurons were frequent in Ammon's horn but rare in the DG. In the entire hippocampus, the percentage of CR-IR neurons containing nNOS was significantly higher at the ventral level (44.3%) than at the dorsal level (17.0%). The percentage of CB-IR neurons containing nNOS was also significantly higher at the ventral level (42.3%) than at the dorsal level (29.3%). Next, we estimated the numerical densities (NDs) of calcium binding protein-containing GABAergic neurons. The ND of PV-IR neurons was comparable at the dorsal (1.16 x 10(3)/mm(3)) and ventral levels (1.23 x 10(3)/mm(3)), respectively. The ND of CR-IR neurons was less at the dorsal level (0.52 x 10(3)/mm(3)) than at the ventral level (0.64 x 10(3)/mm(3)). The ND of CB-IR neurons was also less at the dorsal level (0.91 x 10(3)/mm(3)) than at the ventral level (1.57 x 10(3)/mm(3)). Overall, approximately half of the GABAergic neurons contained one of the three calcium binding proteins (45% at the dorsal level and 47% at the ventral level). These data establish a baseline for examining potential roles of GABAergic neurons in hippocampal network activity in mice.

Animals↗
PubMed2005

Synaptic connections between pigment-dispersing factor-immunoreactive neurons and neurons in the pars lateralis of the blow fly Protophormia terraenovae.

In females of the blow fly Protophormia terraenovae, neurons with cell bodies in the pars lateralis (PL) projecting to the retrocerebral complex (designated as PL neurons) are necessary for the induction of reproductive diapause under short-day and low-temperature conditions. In the present study, neural connections between PL neurons and pigment-dispersing factor (PDF)-immunoreactive neurons were examined via immunolight microscopy and immunoelectron microscopy combined with backfills through the cardiac-recurrent nerve. Immunolight microscopy showed that fibers of PL neurons overlapped with PDF-immunoreactive fibers in the dorsolateral region of the superior protocerebral neuropil. Immunoelectron microscopy showed that PDF-immunoreactive fibers formed output synapses with fibers of PL neurons and unlabeled neurons in a region dorsoanteriorly located with respect to the calyx of the mushroom body. The distribution of synaptic connections between PDF-immunoreactive fibers and the fibers of PL neurons was sparse. According to the projection patterns, PDF-immunoreactive fibers with synaptic connections with PL neurons appeared to originate from PDF-immunoreactive neurons with cell bodies at the base of the medulla of the optic lobe (medulla PDF neurons), which are putative circadian clock neurons in P. terraenovae. PDF immunoreactivity was restrictively detected in dense-core vesicles but not in clear synaptic vesicles. The present results suggest that medulla PDF neurons convey time or photoperiodic information to PL neurons for diapause induction through direct synaptic connections.

Animals↗
PubMed1991

GABA-synthesizing neurons in the medulla: their relationship to serotonin-containing and spinally projecting neurons in the rat.

GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase-antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems.

Animals↗
PubMed2002

Generation and characterization of human hybrid neurons produced between embryonic CNS neurons and neuroblastoma cells.

A human hybrid neuronal cell line A1 has been generated by somatic fusion between a human fetal cerebral neuron and a human neuroblastoma cell, and RT-PCR, immunochemical, and electrophysiological studies of the hybrid cells indicated that the cells express faithfully of morphological, immunochemical, physiological, and genetic features of human cerebral neurons. A1 hybrid neurons express neuron-specific markers such as neurofilament-L (NF-L), NF-M, NF-H, MAP-2, and beta tubulin III. A1 human hybrid neurons express messages for various cytokines and cytokine receptors which are similar to parental human CNS neurons and different from the other parental cell line, SK-SH-SY5Y neuroblastoma. A1 hybrid neurons also express messages for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD), indicating that they could differentiate into various subsets of neuronal types. Whole-cell patch clamp experiments showed that A1 hybrid neurons expressed Na+ currents, which were completely blocked by tetrodotoxin. In addition, depolarizing and hyperpolarizing voltage clamp steps evoked respective outward and inward K+ currents in these cells. When A1 hybrid neurons were exposed to beta amyloid for 72 hr, there was three-fold increase in TUNEL positive cells over controls, indicating that beta amyloid is neurotoxic to A1 hybrid neurons. The present study indicates that the A1 human hybrid neuronal cell line should serve as a valuable in vitro model for studies of biology, physiology, and pathology of human neurons in health and disease.

Amyloid beta-Peptides↗
PubMed1988

Excitatory input to burst neurons from the labyrinth and its mediating pathway in the cat: location and functional characteristics of burster-driving neurons.

1. Spikes of single neurons were recorded extracellularly in the cat prepositus hypoglossi nucleus and the underlying reticular formation and were identified as type II neurons by horizontal rotation. Among these neurons, those activated by contralateral vestibular nerve stimulation with short latencies (1.5-3.0 ms) were selected for further study. 2. A class of these identified neurons was antidromically activated from the contralateral excitatory burst neuron (EBN) area immediately rostral to the abducens nucleus. Systematic tracking for antidromic stimulation revealed a wide distribution of effective spots in and near the EBN area, with varied latencies and thresholds, suggesting terminal branching in that area. The same neurons were also antidromically activated from the contralateral inhibitory burst neuron (IBN) area, the region near the midline, and the nucleus reticularis tegmenti pontis. 3. These neurons exhibited a characteristic firing pattern related to nystagmus: with contralateral rotation the firing rate gradually increased during the slow phase (type II response) and further steeply increased in a burst fashion before and during the contraversive quick phase. Since the time of occurrence of burst activity in these neurons was similar to that of contralateral ENBs and IBNs that received their axonal projection, it is suggested that they send excitatory input to burst neurons, and can thus be called burster-driving neurons (BDNs). 4. Intracellular study revealed that stimulation of the BDN area produced monosynaptic EPSPs in contralateral EBNs. The monosynaptic connection of BDNs with EBNs was confirmed by detecting unitary extracellular synaptic currents of EBNs with the spike-triggered averaging technique. 5. In contrast to BDNs, another class of nystagmus-related type II neurons in the prepositus hypoglossi and medullary reticular formation showed a discharge pattern similar to that of abducens motoneurons on the same side. None of them was antidromically activated from the contralateral pontine reticular formation including the EBN area. Some neurons responded antidromically to stimulation of the ipsilateral dorsomedial pontine reticular formation. 6. In conclusion, the input from the horizontal canal during rotation reaches the contralateral prepositus hypoglossi nucleus and the underlying reticular formation through the vestibular nuclei, and a class of neurons in these structures (BDNs) responds to the canal input in a burst fashion following a tonic type II activity. The axons of BDNs cross the midline and monosynaptically excite EBNs on the side of the canal stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Quantitative cytology of ganglion neurons and satellite glial cells in the superior cervical ganglion of the sheep. Relationship with ganglion neuron size.

Neurons and glial cells of the superior cervical ganglion of sheep were investigated with morphometric methods in the light and electron microscope. The nerve cell sectional area (measured on nucleated cell profiles) ranged from 165 to 2500 microns2, which corresponds to range in cell diameter from 14 to 56 microns and a range in cell volume from 1600 to 93,000 microns3, i.e. a 60-fold volume difference between smallest and largest neurons. The distribution of cell sizes appeared unimodal, with a predominance of small neurons; there were no variations in different parts of the ganglion. This wide range in nerve cell sizes is discussed in the light of the suggestion that large neurons innervate a greater amount of target tissue (e.g. smooth muscle) and are less readily excitable than smaller neurons: it is thus possible that there is differential recruitment of ganglion neurons in autonomic reflexes. The ultrastructural features of ganglion neurons in the sheep were similar to those observed in small laboratory animals. The relative volumes of perikaryal cytoplasm occupied by mitochondria and Golgi apparatus were 8.5% and 4.8%, respectively, but the average values were the same in small and large neurons. Subsurface cisternae of endoplasmic reticulum were common in the perikaryon, while in the dendrites clusters of synaptic vesicles were found beneath the plasma membrane; the absence of a glial wrapping at the latter sites suggests that they are points of (non-synaptic) release of transmitters. The extent of the capsule that satellite cells form around each neurons was compared in size-based classes of neurons. There was no difference in the size of glial nuclei, and this suggests that glial cells are probably of uniform size. However, glial cells were more densely packed over the surface of large neurons than over the surface of small neurons--in fact the packing density was proportional to the ganglion neuron volume, rather than to its surface. The average thickness of the glial capsule was significantly greater around large than around small neurons. It is suggested that the matching of glial cell number and nerve cell volume is achieved during development by glial cell mitosis taking place long after the nerve cells have ceased dividing.

Animals↗