Neurons In Adult Rat Brain Research Articles

High expression of cytochrome b 5 reductase isoform 3/cytochrome b 5 system in the cerebellum and pyramidal neurons of adult rat brain.

Cytochrome b 5 reductase (Cb 5R) and cytochrome b 5 (Cb 5) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-Cb 5R isoform 3 and anti-Cb 5 antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of Cb 5R isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of Cb 5R isoform 3/Cb 5 system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration.

Bcl-2 enhances the formation of newborn striatal long-projection neurons in adult rat brain after a transient ischemic stroke

It has been reported that B-cell lymphoma 2 (Bcl-2) enhances neurogenesis as well as supporting axonal growth after injury. In the present study, we investigated whether Bcl-2 overexpression plays a role in the formation of newborn striatonigral projection neurons in the adult rat brain after transient middle cerebral artery occlusion (MCAO). We infused human Bcl-2-expressing plasmid (pBcl-2) into the lateral ventricle immediately after 30 min of MCAO, injected 5'-bromodeoxyuridine (BrdU) intraperitoneally to label proliferative cells, and microinjected fluorogold (FG) into the substantia nigra at 11 weeks of reperfusion followed by multiple immunostaining of striatonigral projection neurons at 12 weeks. We found that pBcl-2 treatment significantly increased the number of newborn neurons (BrdU(+)-NeuN(+)) in the striatum ipsilateral to the MCAO. We further detected newborn striatonigral projection neurons (BrdU(+)-FG(+)-NeuN(+)) in the ipsilateral striatum at 12 weeks. More interestingly, the number of newborn striatonigral projection neurons (BrdU(+)-FG(+)) was significantly increased by pBcl-2 treatment compared to that by pEGFP, a control plasmid. Taken together, we found that Bcl-2 overexpression in the brain enhanced the generation of newborn striatonigral projection neurons. This provides a potential strategy for promoting the reestablishment of neural networks and brain repair after ischemic injury.

Change in the radiosensitivity of developing neurons in adult rat brain

Change in the radiosensitivity of developing neurons in adult rat brain

Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion

To determine whether Bcl-2 could influence adult neurogenesis and prevent apoptosis of newborn neurons, we injected Bcl-2 expressing plasmid into the lateral ventricle of rat brain immediately following a 30-min occlusion of the middle cerebral artery (MCAO). We found that Bcl-2 increased neural progenitor cells (BrdU+-DCX+) in the ipsilateral striatum, newborn immature neurons (BrdU+-Tuj-1+) and newborn mature neurons (BrdU+-MAP-2+) in the ipsilateral striatum and frontal cortex at 1 to 4 weeks following MCAO. Bcl-2 overexpression promoted development of newborn neurons into GABAergic and cholinergic neurons in the ipsilateral striatum. Moreover, Bcl-2 significantly decreased the apoptosis of newborn neurons, determined by double staining of Tuj-1 and activated caspase-3 (Tuj-1+-Casp+). These results indicate that overexpression of Bcl-2 in adult rat brain enhances neurogenesis and survival of newborn neurons. Increasing neurogenesis and preventing the death of newborn neuron may be a strategy to aid in the repair of adult brain after stroke.

A herpesvirus vector can transduce axotomized brain neurons

If gene therapy is to be used to promote axon regeneration after spinal cord injury, a suitable vector for transgene delivery must be obtained. Replication-defective herpes simplex virus (HSV) vectors are promising candidates. We have examined whether they can express a LacZ transgene in injured neurons of adult rat brain. We transected the medial forebrain bundle, injected replication-defective HSV/ LacZ vectors close to the lesion site, and looked for transgene expression at 2–14 days after the lesion. The vectors carried the LacZ transgene controlled either by the cytomegalovirus immediate-early promoter (vector CS5) or the HSV latency-associated promoter (vector CS1). CS5 transfected many cells near the lesion at 2 days, but did not give persistent expression at 5 days. CS1, in contrast, labeled many neurons in midbrain regions remote from the injection site at 5 days, and much of this expression remained at 12–14 days. The neurons of most interest were in the substantia nigra pars compacta and parabrachial nuclei, which were axotomized by the lesion. Vector-driven β-galactosidase expression was detected in neurons in both regions. These were confirmed as axotomized by double immunofluorescence for c-Jun. By 12–14 days, many substantia nigra neurons had disappeared but some transduced neurons remained; there was no net loss of transduced neurons from the parabrachial nuclei. These results show that an HSV vector is capable of transducing axotomized cells in the central nervous system and producing transgene expression in them for at least 2 weeks after injection.

Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin ii-containing vesicles

The sec6/8 (exocyst) complex is implicated in targeting of vesicles for regulated exocytosis in various cell types and is believed to play a role in synaptogenesis and brain development. We show that the subunits sec6 and sec8 are present at significant levels in neurons of adult rat brain, and that immunoreactivity for the two subunits has a differential subcellular distribution. We show that in developing as well as mature neurons sec6 is concentrated at the inside of the presynaptic plasma membrane, while sec8 immunoreactivity shows a diffuse cytoplasmic distribution. Among established, strongly synaptophysin-positive neuronal boutons, sec6 displays highly differential concentrations, indicating a role for the complex independent of the ongoing synaptic-vesicle release activity. Sec6 is transported along neurites on secretogranin II-positive vesicles, while sec6-negative/secretogranin II-positive vesicles stay in the cell body. In PC12 cells, sec6-positive vesicles accumulate at the plasma membrane at sites of cell–cell contact. Neuronal induction of the PC12 cells with nerve growth factor shows that sec8 is not freely soluble, but may probably interact with cytoskeletal elements. The complex may facilitate the targeting of membrane material to presynaptic sites and may possibly shuttle vesicles from the cytoskeletal transport machinery to presynaptic membrane sites. Thus, we suggest that the exocyst complex serves to modulate exocytotic activity, by targeting membrane material to its presynaptic destination.

Prolonged exposure to inhalational anesthetic nitrous oxide kills neurons in adult rat brain

Short-term exposure of adult rats to nitrous oxide (N 2O), an inhalational anesthetic and NMDA ( N-methyl- d-aspartate) antagonist, causes a reversible neurotoxic vacuole reaction in neurons of the posterior cingulate/retrosplenial cortex (PC/RSC) which resembles that caused by low doses of other NMDA antagonists. Since high doses or prolonged exposure to other NMDA antagonists can cause neurons to die, we assessed whether prolonged N 2O exposure might also cause neuronal cell death. Adult female Sprague–Dawley rats were exposed to 150-vol% N 2O (approximately EC 50 for N 2O anesthesia in rats) for various durations from 1 to 16 h. The time course for onset and disappearance of the reversible vacuole reaction was studied, as was the time course and dose requirement for triggering cell death. A maximum vacuole reaction was observed in PC/RSC neurons in brains examined immediately after 3 h of 150-vol% N 2O exposure and the same magnitude of vacuole reaction was observed when brains were examined immediately after a longer period of N 2O exposure. When N 2O was terminated at 3 h and the rats were killed 1 h later, the vacuole reaction was markedly diminished and if the rats were killed 3 h later the vacuole reaction had completely disappeared. Prolonged exposure to 150-vol% N 2O (for 8 h or more) caused neuronal cell death which was detectable by silver staining 32 h later. Concurrently administered GABAergic agents, diazepam (an i.v. anesthetic), or isoflurane (an inhalational anesthetic), prevented this cell death reaction. Our findings demonstrate that short-term exposure of adult rats to N 2O causes injury to PC/RSC neurons that is rapidly reversible, and prolonged N 2O exposure causes neuronal cell death. These neurotoxic effects, including the cell death reaction, can be prevented by coadministration of GABAmimetic anesthetic agents. Duration of NMDA receptor blockade appears to be an important determinant of whether neurons are reversibly injured or are driven to cell death by an NMDA antagonist drug.

Biochemical and immunocytochemical characterization of GRIP, a putative AMPA receptor anchoring protein, in rat brain

The mechanisms by which glutamate receptors are concentrated in brain excitatory synapses are believed to involve interactions between receptor subunits and postsynaptic anchoring or scaffolding proteins. Recently GRIP, a protein containing seven PDZ domains, was identified as an AMPA receptor binding protein and implicated in the synaptic targeting of AMPA receptors. Here we show that GRIP mRNA is also expressed in some tissues outside of the brain, including testis and kidney. Specific antibodies were raised to study GRIP protein. On Western blots, GRIP protein appears as a heterogeneous band (∼130 kilodaltons) which is expressed in widespread brain regions and throughout postnatal development. Biochemical studies reveal that GRIP is largely membrane associated and enriched in the postsynaptic density (PSD), though not as highly concentrated in the PSD as is PSD-95. By immunohistochemistry, GRIP is distributed in a somatodendritic pattern in neurons of adult rat brain, with especially prominent expression in a subset of interneurons.

Expression of RESP18 in peptidergic and catecholaminergic neurons.

We examined the expression of regulated endocrine-specific protein of 18-kD (RESP18) in selected peptidergic and catecholaminergic neurons of adult rat brain. In the hypothalamic paraventricular, supraoptic, and accessory nuclei, RESP18 mRNA was highly expressed in neurons immunostained for oxytocin and vasopressin. RESP18 mRNA was also highly expressed in paraventricular nucleus neurons immunostained for corticotropin-releasing hormone, thyrotropin-releasing hormone, and somatostatin. RESP18 mRNA was expressed in POMC cells of the arcuate nucleus, in neuropeptide Y cells of the dorsal tegmental nucleus, lateral reticular nucleus, and hippocampus, and in brainstem catecholaminergic neurons. RESP18 mRNA expression was high in all paraventricular and arcuate neurons, but RESP18 protein was detectable in the perikarya of a subset of these neurons, suggesting an important post-transcriptional component to the regulation of RESP18 expression. RESP18 antisera immunostained perikarya but not axon fibers or terminals. Sub-cellular fractionation of homogenates of several hypothalamic nuclei identified RESP18 protein in fractions enriched in endoplasmic reticulum. The presence of 22- and 24-kD RESP18 isoforms in the neural lobe of the pituitary indicated that some RESP18 protein exited the endoplasmic reticulum. The post-transcriptional regulation of RESP18 expression and localization of RESP18 protein primarily to the endoplasmic reticulum suggests that RESP18 plays a regulatory role in peptidergic neurons.

Electrophysiological effects of the anticonvulsant remacemide hydrochloride and its metabolite ARL 12495AA on rat CA1 hippocampal neurons in vitro

The electrophysiological actions of the putative anticonvulsants remacemide hydrochloride and its des-glycine metabolite ARL 12495AA were examined using whole-cell recordings from CA1 hippocampal neurons in adult rat brain in vitro. Remacemide hydrochloride (4–400 μM) and ARL 12495AA (4–400 μM) limited sustained high frequency repetitive firing (SRF) induced by application of long duration depolarizing current pulses (20–400 pA, 500 msec). This SRF limitation was concentration-dependent, and equipotent IC 50 values of 66 and 60 μM were calculated for remacemide hydrochloride and ARL 12495AA, respectively. Examination of the spike configuration revealed that, over the same concentration range, each compound caused a concentration-related reduction of: (a) the action potential amplitude; and (b) the rate-of-rise. Remacemide hydrochloride or ARL 12495AA increased spike duration and decreased or eliminated the spike after-hyperpolarization. Possible mechanisms for these electrophysiological actions including modulation of sodium and/or potassium channel activity are considered. It is suggested that such multiple mechanisms, including inhibition of SRF may be relevant to the anticonvulsant properties of remacemide hydrochloride and its metabolite, ARL 12495AA. The activity of both compounds as modulators of neuronal excitability indicates that metabolic conversion of remacemide hydrochloride to ARL 12495AA could enhance the therapeutic efficacy of the former.

Immunohistochemical localization of brain-derived neurotrophic factor in adult rat brain.

To investigate the role of brain-derived neurotrophic factor in the central nervous system, we produced an anti-peptide antibody that specifically recognized brain-derived neurotrophic factor and performed immunohistochemistry for brain-derived neurotrophic factor-like immunoreactivity in normal adult rat brain. A synthetic peptide (EKVPVSKGQL), derived from mature brain-derived neurotrophic factor, was conjugated to bovine thyroglobulin at a ratio of 1:3 and used as an immunogen to produce a high-titre anti-brain-derived neurotrophic factor polyclonal antibody in Japanese white rabbits. Dot blotting demonstrated that the antiserum could detect 3.91 pmol of synthetic peptide, and Western blotting showed that the antiserum recognized one band with a molecular weight consistent with that of brain-derived neurotrophic factor. In immunohistochemistry, brain-derived neurotrophic factor-like immunoreactivity was widespread in adult rat brain, including cerebral cortex, hippocampus, basal forebrain, striatum, hypothalamus, brainstem and cerebellum. Not only neuronal somata but also nerve fibres showed positive staining. Our data suggest that brain-derived neurotrophic factor is transported through axons in a subpopulation of neurons in adult rat brain, and that brain-derived neurotrophic factor influences a great variety of neurons and acts as a neurotrophic factor in the central nervous system.

Application of brain slice cultures for investigating neuronal sprouting.

Axonal sprouting is a form of morphological plastic change of the neurons. It is proposed to underlie long-term changes in brain function and take place even in mature brain. Electriphysiological analysis revealed that chronic stress and antidepressants induce axonal degeneration and sprouting of locus coeruleus noradrenergic neurons in adult rat brain, respectively. Sprouing of hippocampal neurons was represented in the slice cultures of the hippocampus from 1-week old rat. This phenomenon was found to be specific for CA3 pyramidal neurons. Possible molecular mechanisms for neuron-specific morphological plasticity are discussed, referring to results from other laboratories.

Regulation of neuropeptide expression in the brain by neurotrophins. Potential role in vivo.

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.

Neuron-specific glucose transporter (NSGT): CNS distribution of GLUT3 rat glucose transporter (RGT3) in rat central neurons

The identity of the glucose transporters (GLUT) expressed in neurons in situ has yet to be fully established. In the present study we have isolated the GLUT3 (RGT3) cDNA and produced anti RGT3 polyclonal antibody allowing us to investigate the cellular localization and tissue distributions of RGT3 mRNA and protein in the central nervous system of the rat by the methods of in situ hybridization and immunohistochemistry. Here we demonstrate the direct evidence that RGT3 is present in neurons in adult rat brain. In situ hybridization showed the expression of RGT3 mRNA mostly in the regions of hippocampus, cerebral cortex, striatum, and the granule cell layer of the cerebellum, indicating that RGT3 mRNA is predominantly expressed within neurons. Immunohistochemistry showed that RGT3 protein is widely distributed in the rat brain, and concentrated on the plasma membrane of neurons. Double labeling studies with anti-RGT3, glial fibrillary acidic protein (GFAP), and neuron specific enolase (NSE) antibodies revealed the specific expression of RGT3 protein in neurons. Thus, RGT3 is indicated to be a neuron specific glucose transporter isoform (NSGT), and suggested to play a functionally significant role in rat central neurons.