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PubMed1993

Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia-neuron paracrine interactions.

The temporal changes in the morphological profiles of neuropeptide Y (NPY) neurons and their topographical relationship with glial cells (astrocytes) were characterized in aggregate cultures derived from fetal cortical tissue using immunocytochemical procedures. On day 6 of culture, structures labelled with NPY antibodies were small and uneven in size but many resembled neuronal cell bodies. On day 14, neuronal perikarya were well defined and several morphological types of NPY neurons could be distinguished most of which gave rise to beaded processes: unipolar or multipolar bitufted neurons whose processes branch in close proximity to the cell body; bipolar neurons; and multipolar neurons. On day 23, heavily punctate and asymmetrically labelled cell bodies were dispersed throughout the aggregate; neuronal processes were less conspicuous. At 14 and 23 days, cells expressing glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE) were abundantly distributed throughout the aggregate. Using a double immunoreaction on 14-day-old aggregates revealed that GFAP+ cells and their processes were in close apposition to and engulfing the NPY neurons. Thus, dissociated fetal NPY neurons undergo morphological differentiation in culture along with astrocytes (GFAP+) and other neuronal cell types (NSE+). Based on the topographical association of astrocytes and neurons, particularly NPY neurons, we propose that the aggregate culture system can serve as a model to study the role of paracrine interactions in the regulation of the expression of NPY.

Animals↗
PubMed1995

Neurons in the barosensory area of the caudal ventrolateral medulla project monosynaptically on to sympathoexcitatory bulbospinal neurons in the rostral ventrolateral medulla.

Neurons in the caudal ventrolateral medulla may function as interneurons in the baroreceptor reflex are by inhibiting sympathoexcitatory bulbospinal neurons in rostral ventrolateral medulla. While some caudal ventrolateral medullary neurons are excited orthodromically by baroreceptors and antidromically from the rostral ventrolateral medulla, there is no anatomical evidence to prove that these barosensory neurons of the caudal ventrolateral medulla monosynaptically innervate the bulbospinal neurons in the rostral ventrolateral medulla. To establish the presence of such a direct projection, barosensory neurons were identified in the rostral caudal ventrolateral medulla of anesthetized rats by criteria that they spontaneously discharged with a cardiac rhythm and were excited by baroreceptor stimulation. The anterograde tracer biocytin was iontophoresed onto these neurons and, in the same animal, the retrograde tracer wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles was injected by micropressure into the ipsilateral spinal (thoracic level 3) intermediolateral cell column to label bulbospinal neurons. After 18-24 h, rats were killed and sections through the rostral ventrolateral medulla were processed for both markers. By light microscopy, numerous biocytin-labeled varicose processes overlapped rostral ventrolateral medullary neurons containing wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles. By electron microscopy, biocytin was found in axons and terminals. The terminals (n = 76) were large (0.6-1.2 microns in diameter), contained numerous small, clear vesicles and formed primarily symmetric synapses on perikarya and large (1.5-4.5 microns) dendrites within the rostral ventrolateral medulla. Some of these target neurons contained wheatgerm-agglutinated apo-horseradish peroxidase conjugated to gold particles associated with lysosomes and multivesicular bodies in the cytoplasm. The results indicate that: (i) neurons in the barosensory sympathoinhibitory region of the caudal ventrolateral medulla directly synapse on bulbospinal neurons in the rostral ventrolateral medulla; and (ii) the synaptic profile (symmetric synapse) and location (perikarya and large dendrites) is consistent with the conclusion that baroreceptor neurons of the caudal ventrolateral medulla potently and monosynaptically inhibit sympathoexcitatory neurons of the rostral ventrolateral medulla. The findings support the hypothesis that the barosensory region of the rostral caudal ventrolateral medulla is an intermediate relay in the baroreceptor reflex are.

Animals↗
PubMed1996

Reduced retrograde labelling with fluorescent tracer accompanies neuronal atrophy of basal forebrain cholinergic neurons in aged rats.

During ageing, basal forebrain cholinergic neurons are prone to degeneration for unknown reasons. In this study we morphometrically evaluated the retrograde labelling of basal forebrain neurons obtained after injection of FluoroGold into multiple sites in the cerebral neocortex in aged (24-33 months) as compared with young adult (four to six months) male Sprague-Dawley rats. In addition, we looked for differences in the distribution of degenerative changes in topographic subdivisions of the basal forebrain cholinergic complex of neurons identified by immunohistochemical detection of the cholinergic markers choline acetyltransferase or low-affinity neurotrophin receptor. After injection of FluoroGold into the cerebral neocortex, the number of retrogradely labelled neurons in the horizontal diagonal band/ substantia innominata and basal nucleus was significantly lower in aged rats, by 41% and 48%, respectively. In aged rats injected with FluoroGold as well as in non-injected aged rats, the numbers of neurons immunoreactive for choline acetyltransferase and low-affinity neurotrophin receptor were significantly lower, by 23-27% in the basal forebrain system as a whole, with no significant difference in the degree of decline amongst different subdivisions (i.e. medial septum, diagonal band, substantia innominata and basal nucleus). The ratios of the number of neurons labelled with FluoroGold as compared with the number of neurons immunoreactive for either cholinergic marker were significantly lower in aged rats, by 32-37%, indicating that the decline in the number of neurons retrogradely transporting tracer was greater than the decline in the number of immunoreactive neurons in aged animals. Immunoreactive as well as retrogradely labelled neurons showed a significant shrinkage of cell surface area of 6-13% in different subdivisions of the basal forebrain cholinergic system in aged rats. These findings confirm significant loss and atrophy of basal forebrain cholinergic neurons in aged rats, and demonstrate significantly reduced retrograde labelling of these neurons with fluorescent tracer applied to their target cortex. This reduced retrograde labelling suggests an impairment of either uptake or retrograde transport mechanisms in these neurons in aged rats. Such an impairment may contribute to the degenerative changes of basal forebrain cholinergic neurons observed in ageing and age-related degenerative conditions such as Alzheimer's disease.

Acetylcholine↗
PubMed1996

Increased vulnerability of septal cholinergic neurons to partial loss of target neurons in aged rats.

To investigate whether the ageing process might affect neuron-target interactions which influence the phenotype of septal cholinergic neurons, we compared the response of these neurons to partial loss of target tissue in young adult and aged animals. Groups of young adult (four to six months) or aged (24-33 months) male Sprague-Dawley rats received unilateral infusions into the hippocampus of either the excitotoxic amino acid N-methyl-D-aspartate, or vehicle. The resulting excitotoxic lesions reduced the mean cross-sectional area of the hippocampus by 55-60%. Ipsilateral septal cholinergic neurons immunohistochemically stained for either choline acetyltransferase or low-affinity neurotrophin receptor (p75NTR) were morphometrically evaluated. In young adult rats with partial hippocampal lesions, the number and staining intensity of ipsilateral septal cholinergic neurons were not significantly different from age-matched control values, but these cholinergic neurons exhibited a significant 12% reduction in cross-sectional area. In aged rats with hippocampal lesions of equivalent size, ipsilateral cholinergic neurons showed a significant 29% reduction in cross-sectional area, a significant 19% reduction in choline acetyltransferase staining intensity as measured by densitometry, and a significant 21% reduction in the number of choline acetyltransferase- but not p75NTR-stained septal neurons, as compared with age-matched control animals. These findings show that in aged rats, septal cholinergic neurons atrophy more severely in response to the partial loss of their target neurons than in young adult rats, in the form of pronounced cell shrinkage and down-regulation of intracellular levels of the transmitter-synthesizing enzyme, choline acetyltransferase, in some cases to the point of the absence of detectable staining for this marker in some cells. The continued detection of p75NTR indicates that significant neuronal cell death did not take place. These findings suggest that basal forebrain cholinergic neurons have an increased vulnerability to disturbances of neuron-target interactions in aged animals, which may contribute to the degenerative changes exhibited by these cholinergic neurons in ageing and age-related conditions such as Alzheimer's disease.

Acetylcholine↗
PubMed2005

Co-culture of neurones with glutathione deficient astrocytes leads to increased neuronal susceptibility to nitric oxide and increased glutamate-cysteine ligase activity.

The antioxidant glutathione (GSH) plays an important role in protecting the mitochondrial electron transport chain (ETC) from damage by oxidative stress in astrocytes and neurones. Neurones co-cultured with astrocytes have greater GSH levels, compared to neurones cultured alone, leading to the hypothesis that astrocytes play a key role in brain GSH metabolism by supplying essential GSH precursors to neurones. A previous study has postulated that damage to the ETC following exposure to reactive nitrogen species (RNS) is less in co-cultured neurones, compared to neurones cultured alone, because of the greater GSH levels in the former cells. To investigate this further, primary culture rat neurones were co-cultured with either rat astrocytes activated with IFN-gamma and LPS to produce NO, or NO-generating astrocytes that had been depleted of intracellular GSH by 87% following incubation with the GSH synthesis inhibitor L-buthionine-S,R-sulfoximine (L-BSO). Neurones incubated with NO-generating astrocytes depleted of GSH were unable to elevate GSH levels, unlike neurones co-cultured with NO-generating astrocytes. Complexes II + III and IV of the neuronal ETC were significantly inhibited following exposure to NO-generating astrocytes depleted of GSH. No ETC damage was observed in neurones co-cultured with NO-generating astrocytes. Although neurones co-cultured with GSH depleted astrocytes did not increase cellular GSH levels, the activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, was increased by 218%, compared to neurones cultured with control astrocytes. This suggests that neuronal GCL activity could be modulated when GSH metabolism is inhibited in neighboring astrocytes.

Animals↗
PubMed2004

Co-expression pattern of neuronal nitric oxide synthase and two variants of choline acetyltransferase in myenteric neurons of porcine ileum.

Cholinergic enteric neurons were demonstrated immunohistochemically so far by using antibodies staining the common choline acetyltransferase (cChAT) in neurons of the central nervous system. The results of staining in the enteric nervous system of various species were, however, not satisfactory. We describe here findings obtained with a newly raised antibody against a peripheral variant of choline acetyltransferase (pChAT) in myenteric neurons of the pig small intestine. Triple labelling for pChAT/cChAT/neuronal nitric oxide synthase (nNOS) revealed 19.7% of 1664 neurons (within 40 ganglia) to be immunoreactive exclusively for pChAT whereas 29.6% were positive for cChAT alone and 18.8% were reactive only for nNOS. Colocalization of pChAT and cChAT was found in 22.4%, of pChAT and nNOS in 8.1% and of cChAT and nNOS in 1.4%. All three markers were simultaneously found in only 1 of 1664 neurons. To investigate the presence and possible colocalization of the above markers within morphologically defined neuron types, triple labelling of cChAT or nNOS with pChAT and a neurofilament (NF) antibody pool was applied and the coexpression patterns of pChAT and cChAT as well as of pChAT and nNOS in 120 neurons of each type were recorded. All type I, II, IV and V neurons displayed immunoreactivity either for one or both cholinergic markers. These neuron types were considered to be cholinergic. All type VI neurons, a descending neuron population, were negative for cChAT but positive for nNOS. However, 95% were immunoreactive for both pChAT and nNOS. The physiological significance of the possible co-existence of acetylcholine and nitric oxide within type VI neurons remains to be clarified. It is concluded that the pChAT and cChAT antibodies used here recognize partly different populations of enteric neurons in the pig. Thus, for total immunohistochemical characterization of cholinergic enteric neurons both forms of choline acetyltransferase have to be considered.

Animals↗
PubMed2003

Striatal neurons but not nigral dopaminergic neurons in neonatal primary cell culture express endogenous functional N-methyl-D-aspartate receptors.

Developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits were determined and compared in striatal and nigral neurons in neonatal primary cell cultures. In striatal neurons, NR1, NR2A and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal levels of NR1 mRNA and immunoreactivity expression were found at 6 day-in-vitro (DIV). NMDA receptors found at this stage in striatal neurons are likely to contain NR1 plus NR2A, NR2B and NR2D subunits. In nigral neurons, NR1 and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal level of NR1 immunoreactivity expression was found at 10 DIV. Unlike striatal neurons, NMDA receptors found in nigral neurons are likely to contain NR1 plus NR2B and NR2D subunits only. NMDA-induced toxicity assays showed that striatal neurons were most susceptible to cell death at around 10 DIV but nigral neurons were not susceptible to NMDA-induced cell death at all stages. In addition, patch clamp analysis revealed that functional NMDA receptors could only be found in striatal neurons but not in nigral dopaminergic neurons in vitro. The present results indicate that striatal and nigral neurons are programmed to express distinct NMDA receptor subunits during their endogenous development in cell cultures. Despite dopaminergic neurons in culture display NMDA receptor subunits, functional NMDA receptors are not assembled. The present findings have demonstrated that dopaminergic neurons in vitro may behave very differently to their counterparts in vivo in terms of NMDA receptor-mediated responses. Our results also have implications in transplantations using dopaminergic neurons in vitro in treatments of Parkinson's disease.

Animals↗
PubMed2005

Neuronal regeneration partially compensates the delayed neuronal cell death observed in the hippocampal CA1 field of soman-poisoned mice.

Soman poisoning induces long-term neuropathology characterized by the presence of damaged neurons up to 2 months after exposure in various central brain areas, especially the hippocampal CA1 layer. Rapid depletion of this layer could therefore be expected. Surprisingly, the CA1 layer remained consistently visible, suggesting delayed death of these damaged neurons, potentially accompanied by neuronal regeneration. To address this issue, mice were exposed to a convulsive dose of soman (110 microg/kg followed by 5.0mg/kg of atropine methyl nitrate (MNA) 1 min later) and brains were collected from day 1 to day 90 post-exposure. Damaged and residual healthy neurons were quantified on brain sections using hemalun-phloxin and fluorojade staining or neuronal nuclei antigen (NeuN) immunohistochemistry. On post-soman day 1, a moderate neuronal cell death was noticed in the hippocampal CA1 layer. In this area, an important and steady quantity of damaged neurons (about 48% of the whole pyramidal neurons) was detected from post-soman day 1 to day 30. Thus, throughout this period, damaged neurons seemed to survive, as confirmed by the unmodified depth of the hippocampal CA1 layer. The dramatic disappearance of the damaged neurons occurred only later during the experiment and was almost complete at day 90 after soman exposure. Interestingly, between day 30 and day 90 following poisoning, an increase in the number of residual healthy pyramidal neurons was observed. These different kinetic patterns related to the density of total, damaged and residual healthy neurons after soman poisoning demonstrate that neuronal regeneration is delayed in the hippocampal CA1 layer and is concomitant to the death of damaged neurons.

Animals↗
PubMed2000

Pharmacological inactivation of the vesicular monoamine transporter can enhance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration of midbrain dopaminergic neurons, but not locus coeruleus noradrenergic neurons.

The vesicular monoamine transporter in the brain can sequester the neurotoxin 1-methyl-4-phenylpyridinium into synaptic vesicles and protect catecholamine-containing neurons from degeneration. Mouse nigrostriatal dopaminergic neurons, and to a lesser extent locus coeruleus noradrenergic neurons, are vulnerable to toxicity produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The present study sought to determine whether pharmacological inactivation of the vesicular monoamine transporter in the brain would enhance the degeneration of substantia nigra dopaminergic neurons and locus coeruleus noradrenergic neurons in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated animals. Mice were treated subacutely with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine alone, or in combination with vesicular monoamine transporter inhibitors (tetrabenazine or Ro4-1284), and 10-24 days later striatal dopamine and cortical norepinephrine levels were measured using chromatographic methods. In the same animals, substantia nigra and locus coeruleus catecholaminergic neurons were counted using tyrosine hydroxylase immunohistochemical staining with computer imaging techniques. Mice in which pharmacological blockage of the vesicular monoamine transporter enhanced the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in the depletion of striatal dopamine concentrations also exhibited enhanced degeneration of substantia nigra dopaminergic neurons. In the same animals, however, vesicular monoamine transporter blockade did not enhance the effects of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine in the locus coeruleus noradrenergic system. These data are consistent with the hypothesis that the vesicular monoamine transporter can protect catecholamine-containing neurons from 1-methyl-4-phenylpyridinium-induced degeneration by sequestration of the toxin within brain vesicular monoamine transporter-containing synaptic vesicles. Since the amount of vesicular monoamine transporter in locus coeruleus neurons is more than in substantia nigra neurons, and because 1-methyl-4-phenylpyridinium is sequestered within locus coeruleus neurons to a far greater extent than within substantia nigra neurons, it may be that a greater amount of vesicular monoamine transporter inhibition is required for 1-methyl-4-phenylpyridinium to be toxic to locus coeruleus neurons than to substantia nigra dopaminergic neurons.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

The development of postganglionic sympathetic neurons: coordinating neuronal differentiation and diversification.

The fine-tuned operation of the nervous system is accomplished by a diverse set of neurons which differ in their morphology, biochemistry and, consequently, their functional properties. The accurate interconnection between different neuron populations and their target tissues is the prerequisite for physiologically appropriate information processing. This is exemplified by the regulatory action of the autonomic nervous system in vertebrates to sustain homeostasis under changing physiological demands. For this purpose, the coordination of divergent regulatory responses is required in a multitude of tissues spread over the entire body. To meet this task, diverse neuronal populations interact at different levels. In the sympathetic system. chemical relations between preganglionic and postganglionic neurons appear to differ along the rostrocaudal axis. In addition, postganglionic neurons innervating different target tissues at a segmental level have distinct properties. Differences in their preganglionic innervation and their integrative membrane properties result in diverse activation patterns upon reflex stimulation. Moreover, postganglionic neurons differ in the transmitter molecules they employ to convey information to the target tissues. The segregation of noradrenaline and acetylcholine to different populations of postganglionic sympathetic neurons is well established. A combination of cellular and molecular approaches has begun to uncover how such a complex system may be generated during development. Growth and transcription factors involved in noradrenergic and cholinergic differentiation are characterised. Interestingly, they can also promote the expression of proteins involved in transmitter secretion. As the proteins participating in the vesicle cycle are expressed in many neuron populations, whereas the enzymes of transmitter biosynthesis are restricted to subpopulations of neurons, the findings suggest that early in neuronal development subpopulation-specific and more widely expressed neuronal properties can be commonly induced. Still, many details concerning the signals involved in the induction of the neurotransmitter synthesis and release machinery remain to be worked out. Likewise, the regulatory processes resulting in differences of electrophysiological membrane properties and the specific recognition between pre- and postganglionic neurons have to be determined. Ultimately, this will lead to an understanding at the molecular level of the development of a nervous system with diverse neuronal populations that are specifically interconnected to distinct input neurons and target tissues as required for the performance of a complex regulatory function.

Animals↗
PubMed1999

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective.

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.

Animals↗
PubMed2002

Suppression of death receptor signaling in cerebellar Purkinje neurons protects neighboring granule neurons from apoptosis via an insulin-like growth factor I-dependent mechanism.

Neuronal apoptosis contributes to the progression of neurodegenerative disease. Primary cerebellar granule neurons are an established in vitro model for investigating neuronal death. After removal of serum and depolarizing potassium, granule neurons undergo apoptosis via a mechanism that requires intrinsic (mitochondrial) death signals; however, the role of extrinsic (death receptor-mediated) signals is presently unclear. Here, we investigate involvement of death receptor signaling in granule neuron apoptosis by expressing adenoviral, AU1-tagged, dominant-negative Fas-associated death domain (Ad-AU1-deltaFADD). Ad-AU1-deltaFADD decreased apoptosis of granule neurons from 65 +/- 5 to 27 +/- 2% (n = 7, p < 0.01). Unexpectedly, immunocytochemical staining for AU1 revealed that <5% of granule neurons expressed deltaFADD. In contrast, deltaFADD was expressed in >95% of calbindin-positive Purkinje neurons ( approximately 2% of the cerebellar culture). Granule neurons in proximity to deltaFADD-expressing Purkinje cells demonstrated markedly increased survival. Both granule and Purkinje neurons expressed insulin-like growth factor-I (IGF-I) receptors, and deltaFADD-mediated survival of granule neurons was inhibited by an IGF-I receptor blocking antibody. These results demonstrate that the selective suppression of death receptor signaling in Purkinje neurons is sufficient to rescue neighboring granule neurons that depend on Purkinje cell-derived IGF-I. Thus, the extrinsic death pathway has a profound but indirect effect on the survival of cerebellar granule neurons.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival.

Astrocytes are intimately involved in both glutamate and gamma-aminobutyric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2- 13C]acetate combined with ex vivo 13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.

Acetate-CoA Ligase↗
PubMed1987

Functional associations among simultaneously monitored lateral medullary respiratory neurons in the cat. I. Evidence for excitatory and inhibitory actions of inspiratory neurons.

Data were obtained from 45 anesthetized (Dial), paralyzed, artificially ventilated, bilaterally vagotomized cats. Arrays of extracellular electrodes were used to monitor simultaneously the activities of lateral medullary respiratory neurons located in the rostral and caudal regions of the ventral respiratory group. The average discharge rate as a function of time in the respiratory cycle was determined for each neuron and concurrent phrenic nerve activity. Most cells were tested for axonal projections to the spinal cord or the ipsilateral vagus nerve using antidromic stimulation techniques. Seven hundred and sixty-one pairs of ipsilateral respiratory neurons that contained at least one neuron whose maximum discharge rate occurred during the inspiratory phase were analyzed by cross-correlation of the simultaneously recorded spike trains. Twenty-three percent of the 410 pairs of inspiratory (I) neurons showed short time scale correlations indicative of functional association due to paucisynaptic connections or shared inputs. Eight per cent of the 351 pairs composed of an I cell and and expiratory (E) neuron were correlated. We found evidence for excitation of both bulbospinal I neurons and I cells that were not antidromically activated by stimulation of the spinal cord and vagus nerve (NAA neurons) by NAA I cells. We also obtained data suggesting inhibitory actions of cells whose maximum discharge rate occurred in the first half of the I phase (I-DEC neurons). These actions included inhibition of other I-DEC neurons, inhibition of cells whose greatest firing rate occurred in the last half of the I phase (I-AUG neurons), inhibition of E-DEC neurons, and inhibition of E-AUG cells. Sixty-two percent (31/50) of the correlations that could be interpreted as evidence for an excitatory or inhibitory paucisynaptic connection were detected in pairs composed of a caudal and a rostral ventral respiratory group neuron. Eighty-eight percent (14/16) of proposed intergroup excitatory connections involved a projection from the rostral neuron of the pair to the caudal cell, whereas 73% (11/15) of proposed inhibitory connections involved a caudal-to-rostral projection. These results support and suggest several hypotheses for mechanisms that may help to control the development of augmenting activity in and the timing of each phase of the respiratory cycle.

Animals↗
PubMed1997

Metabotropic glutamate receptor agonists alter neuronal excitability and Ca2+ levels via the phospholipase C transduction pathway in cultured Purkinje neurons.

Selective agonists for metabotropic glutamate receptor (mGluR) subtypes were tested on mature, cultured rat cerebellar Purkinje neurons (> or = 21 days in vitro) to identify functionally relevant mGluRs expressed by these neurons and to investigate the transduction pathways associated with mGluR-mediated changes in membrane excitability. Current-clamp recordings (nystatin/perforated-patch method) were used to measure the membrane response of Purkinje neurons to brief microperfusion pulses (1.5 s) of the group I (mGluR1/mGluR5) agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (300 microM), quisqualate (5 microM), and (R,S)-3,5-dihydroxyphenylglycine (50-500 microM). All group I mGluR agonists elicited biphasic membrane responses and burst activity in the Purkinje neurons. In addition, the group I mGluR agonists produced alterations in the active membrane properties of the Purkinje neurons and depressed the OFF response after hyperpolarizing current injection. In parallel microscopic Ca2+ imaging experiments, application of the group I mGluR agonists to fura-2-loaded cells elicited increases in intracellular Ca2+ in both the somatic and dendritic regions. The group II (mGluR2/mGluR3) agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (10 microM) and the group III (mGluR4/mGluR6/mGluR7/mGluR8) agonists L(+)-2-amino-4-phosphonobutyric acid (1 mM) and O-phospho-L-serine (200 microM) had no effect on the membrane potential or intracellular Ca2+ levels of the Purkinje neurons. The cultured Purkinje neurons, but not granule neurons or interneurons, showed immunostaining for mGluR1alpha in both the somatic and dendritic regions. All effects of the group I mGluR agonists were blocked by (+)-alpha-methyl-4-carboxyphenylglycine (1 mM), an mGluR antagonist. Furthermore, the phospholipase C inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (2 microM) blocked the group I mGluR agonist-mediated electrophysiological response and greatly attenuated the Ca2+ signal elicited by group I mGluR agonists, particularly in the dendrites. The inactive analogue 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidine-dione (2 microM) was relatively ineffective against the electrophysiological response and Ca2+ signal. These results indicate that functional group I mGluRs (but not group II or III mGluRs) can be activated on mature Purkinje neurons in culture and result in changes in neuronal excitability and intracellular Ca2+ mediated through phospholipase C. These data obtained from a defined neuronal type, the Purkinje neuron, confirm biochemical and molecular studies on the transduction mechanisms of group I mGluRs and show that this transduction pathway is linked to neuronal excitability and intracellular Ca2+ release in the Purkinje neurons.

Animals↗
PubMed2001

Influence of non-neuronal cells on establishment of neuropeptide Y expression by sympathetic neurons in vitro.

Sympathetic ganglion precursors were used to test the role of non-neuronal cell-derived factors in the establishment of mature neuronal phenotypes. In control conditions, characteristic neuronal populations with and without neuropeptide Y (NPY) expression developed after 1 week, but following removal of non-neuronal cells, virtually all neurons expressed NPY. Thus, ganglionic non-neuronal cells downregulated NPY expression in some but not all neurons in vitro. Conditioned medium from non-neuronal cells restored heterogeneous NPY expression, a finding that suggests the factors are soluble. A non-neuronal cell-derived cytokine, leukemia inhibitory factor, dramatically reduced NPY expression in all neurons, but no concentration tested could recapitulate heterogeneous neuronal NPY expression. Sympathetic precursors that incorporated BrdU early in the culture were more likely to develop NPY expression than neurons that became postmitotic later in vitro. Together, these findings support the notion that the environment in which neurons become postmitotic contributes to neuronal phenotype.

Animals↗
PubMed1998

[Involvement of proteinases produced by both neurons and microglia in neuronal lesion and death pathways].

Much attention has been paid to proteinases derived from not only neurons but also microglia in relation to neuronal death. There is accumulating evidence that intra- and extracellular proteinases in these cells are part of the basic machinery of neuronal death pathways. Some members of the ced-3/interleukin-1 beta converting enzyme (ICE) (caspase) family of cysteine proteinases have been thought to play a major role in apoptosis of not only non-neuronal cells but also neurons. Calpain has also been demonstrated to be a mediator of the neurodegenerative response. Recent studies have shown that excitotoxic and ischemic neuronal injury could be attenuated by inhibitors of caspases and calpain. Several recent studies have suggested the involvement of endosomal/lysosomal proteinases, including cathepsins B, D and E, in neuronal death induced by excitotoxins and ischemia. Furthermore, it has been reported that the extracellular tissue-type plasminogen activator/plasmin proteolytic cascade is involved in excitotoxic injury of the hippocampal neurons. In addition to such neuronal proteinases, microglial proteinases are believed to be important for the modification of neuronal functions positively or negatively. Cathepsins E and S derived from microglia have been suggested to contribute to neuronal survival through degradation and removal of beta-amyloid, damaged neurons and cellular debris. On the other hand, 6-hydroxydopamine-induced microglial cell death was inhibited by inhibitors of aspartic proteinases and caspases, suggesting the involvement of cathepsins E and D and caspases in microglial cell death. Therefore, identification of which proteinases play a causative role in neuronal death execution and clarification of the regulators and substrates for such proteinases is very important for understanding the molecular basis of the neuronal death pathways and to develop novel neuroprotective agents.

Aging↗
PubMed1999

Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation.

Caspases play a pivotal role in neuronal cell death during development and after trophic factor withdrawal. However, the mechanisms regulating caspase activity and the role played by caspase activation in response to neuronal injury is poorly understood. The tumor suppressor gene p53 has been implicated in the loss of neuronal viability caused by excitotoxic and DNA damaging agents. In the present study we determined if p53-mediated neuronal cell death required caspase activation. DNA damage increased caspase activity in both cultured embryonic telencephalic and postnatal cortical neurons in a p53-dependent manner. Caspase inhibitors protected embryonic telencephalic neurons, but not postnatal cortical neurons, from DNA damage-induced cell death as measured by direct cell counting and annexin V staining. In marked contrast to the caspase inhibitors, an inhibitor of the DNA repair enzyme, poly(ADP-ribose) polymerase, conferred significant protection from genotoxic and excitotoxic cell death on postnatal cortical neurons but had no effect on embryonic neurons. Glutamate-mediated excitotoxicity in postnatal neurons was not associated with measurable changes in caspase activity, consistent with the failure of caspase inhibitors to prevent cell death under these conditions. Moreover, adenovirus-mediated overexpression of p53 killed embryonic and postnatal neurons without activating caspases. Thus, p53-mediated neuronal cell death may occur via both caspase-dependent and caspase-independent pathways. These results demonstrate that p53 is required for caspase activation in response to some forms of neuronal injury. However, the relative importance of caspase activation in neurons depends on the developmental status of the cell and the specific nature of the death stimulus.

Adenoviridae↗