The high-affinity sulphonylurea receptor regulates KATP channels in nerve terminals of the rat motor cortex

The coexpression of sulphonylurea binding sites and ATP-sensitive K+(KATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive KATP channels and charybdotoxin-sensitive, large-conductance calcium-activated K+ BKCa channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium-independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and KATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the KATP channel may serve to limit glutamate release under conditions of metabolic stress.     

Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K+] before glutaraldehyde fixation. At normal [K+], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K+ depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca2+-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.     

The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter

The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies. However, major components of this system remain to be identified. We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters. HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots. HvHAK1 also mediated low-affinity Na+ uptake. Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated. Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family. Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters.     

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.     

Localization of preprogalanin messenger RNA in rat brain: identification of transcripts in a subpopulation of cerebellar Purkinje cells

Galanin, a 29 amino acid peptide, is widely distributed throughout both the peripheral and central nervous systems and is thought to be involved in multiple physiological functions including smooth muscle relaxation, stimulation of feeding, blood pressure regulation, control of hormone secretion and modulation of nociception. Galanin has been shown to co-exist with several neurotransmitters throughout the neuroaxis and in some cases to modify their presynaptic and postsynaptic actions. In the present study, the anatomical distribution of preprogalanin messenger RNA in rat brain was examined by in situ hybridization histochemistry using specific 35S-labelled oligonucleotide probes. Neurons expressing preprogalanin messenger RNA were found throughout the brain and were particularly abundant in the hypothalamus. High densities of preprogalanin messenger RNA-positive neurons were found in the anteroventral preoptic, supraoptic, paraventricular and dorsomedial nuclei of the hypothalamus, in the locus coeruleus and in the nucleus of the solitary tract. Moderate densities of preprogalanin messenger RNA-positive cells were apparent in the periventricular and arcuate nuclei of the hypothalamus, in the dorsal raphe and dorsal cochlear nuclei. Low densities of preprogalanin messenger RNA-expressing neurons were observed in the piriform cortex, medial septum and the retrochiasmatic area. These findings are consistent with results of previous in situ localization studies of preprogalanin messenger RNA and also with studies reporting the distribution of galanin-like immunoreactivity in rat brain. A novel finding, however, was the detection of preprogalanin messenger RNA in Purkinje cells in the caudal cerebellar vermis (lobules 6 to 10) and the flocculus and paraflocculus of the lateral hemispheres of the cerebellum. Galanin is presumably co-localized in these cells with GABA, which is normally present in Purkinje cells and possibly with tyrosine hydroxylase, which has recently been detected in a similar subpopulation of cerebellar Purkinje cells in both rat and mouse. Thus, the present study reveals a previously unreported site of galanin gene expression in the cerebellum which represents a novel, putative site of action for galanin to add to its already varied physiological roles.     

The stimulus-evoked release of glutamate and GABA from brain subregions following transient forebrain ischemia in the rat

The release of glutamate and GABA in response to K+ depolarization was determined for tissue prisms prepared from brain subregions removed from rats following 30 min of forebrain ischemia or recirculation periods up to 24 h. There were statistically significant effects of this treatment on release of both amino acids from samples of the dorsolateral striatum, an area developing selective neuronal degeneration. However, for at least the first 3 h of recirculation the calcium-dependent and calcium-independent release of both amino acids in this region were similar to pre-ischemic values. Differences were observed under some conditions at longer recirculation times. In particular there was a decrease in calcium-dependent GABA release at 24 h of recirculation and a trend towards increased release of glutamate at 6 h of recirculation and beyond. No statistically significant differences were seen in samples from the paramedian neocortex, a region resistant to post-ischemic damage. These results suggest that changes in the ability to release glutamate and GABA in response to stimulation are not necessary for the development of neurodegeneration in the striatum but rather that release of these amino acids may be modified as a result of the degenerative process.     

Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain

An inhibitory neurotransmitter in mature brain, gamma-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl- accumulation in immature neurons and oligodendrocytes.     

The relationship between the degree of neurodegeneration of rat brain 5-HT nerve terminals and the dose and frequency of administration of MDMA ('ecstasy')

The effect of varying the dose and frequency of administration of 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') on both the acute hyperthermic response and the long term neurodegeneration of 5-hydroxytryptamine (5-HT) nerve terminals in the brain has been studied in Dark Agouti rats. A single injection (4-15 mg/kg i.p.) of MDMA produced immediate dose-related hyperthermia and a dose-related decrease in 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and [3H]paroxetine binding in regions of the brain 7 days later, with a dose of 4 mg/kg having no degenerative effect. This dose was also without effect when given once daily for 4 days, but produced a marked loss of [3H]paroxetine binding and indole concentration ( approximately 55%) when given twice daily for 4 days. When a dose of 4 mg/kg was given twice weekly for 8 weeks it had no effect on these serotoninergic markers, despite a clear anorectic effect of the drug being seen. These data demonstrate that MDMA-induced neurodegeneration is related to both the dose and frequency of administration and indicate that damage to 5-HT neurones can occur in the absence of a hyperthermic response to the drug. We suggest that damage occurs when endogenous free radical scavenging mechanisms become overwhelmed or exhausted.     

Selective excitatory amino acid uptake in glutamatergic nerve terminals and in glia in the rat striatum: quantitative electron microscopic immunocytochemistry of exogenous (D)-aspartate and endogenous glutamate and GABA

To characterize glutamate/aspartate uptake activity in various cellular and subcellular elements in the striatum, rat striatal slices were exposed to 10 and 50 mu M exogenous (D)-aspartate. After fixation with glutaraldehyde/formaldehyde the distribution of (D)-aspartate was analysed by postembedding immunocytochemistry and the ultrastructural distribution was compared with the distributions of endogenous glutamate and GABA. Light microscopically, (D)-aspartate-like immunoreactivity was localized in conspicuous dots along very weakly labelled dendritic profiles and neuron cell bodies. At the electron microscope level gold particles signalling (D)-aspartate occurred at highest density in nerve terminals making asymmetrical contacts with postsynaptic spines (i.e. resembling synapses of cortical afferents). Astrocytic processes also contained gold particles, but at a lower density than nerve endings. In contrast, dendritic spines were only weakly (D)-aspartate-positive. The difference in labelling at 10 and 50 mu M (D)-aspartate was consistent with 'high-affinity' uptake. Neighbouring sections processed with other antibodies showed that the D-aspartate labelling. Occurred in nerve terminals strongly immunoreactive for glutamate, rather than in terminals very weakly glutamate-immunopositive or in nerve endings immunoreactive for GABA. Glutamate labelling of perfusion-fixed striatum confirmed that terminals forming asymmetrical synaptic contacts with spines were enriched with gold particles, suggesting that these terminals use glutamate as a transmitter. This study demonstrates that high-affinity uptake sites for excitatory amino acids in the striatum are most strongly expressed on presumed glutamatergic nerve terminals and on astrocytes.     

Indicators of oxidative stress in aging rat brain. The effect of nerve growth factor

INTRODUCTION AND OBJECTIVE: Increased oxidative stress during ageing and the neurodegenerative disorders associated with this has been described. The central nervous system is particularly vulnerable to oxidative damage because of its high energy requirements, high oxygen consumption, high tissue concentration of iron and relatively low levels of some antioxidant systems. Treatment with neurotrophic factors may reverse neurone deterioration and stimulate cholinergic activity in aged rats. It may have a similar neuroprotector effect against damage due to ischaemic reperfusion, hypoglycaemia, inflammation and other pathological conditions involving oxidative stress. In this study we determined some indicators of oxidative stress in rat brains during ageing and evaluated this in response to a plan of treatment with murine nerve growth factor (FCN) for 38 days. MATERIAL AND METHODS: Biochemical techniques were used for determination of oxidative stress indicators. RESULTS AND CONCLUSIONS: We found that with age there was a significant increase in phospholipase A2 and superoxide dysmutase activity and concentration of hipoperoxidases, whilst the concentration of reduced glutathion fell. Catalase activity increased in the hippocampal and striate regions and decreased in the cortex and septal area. There was less oxidative stress in rats treated with FCN. In view of our results, we conclude that the level of oxidative stress increases with ageing, with significant differences between areas of the brain. The region most vulnerable to damage from species reactive to oxygen was the hippocampus, and the protective effect of FCN may be related to potentiation of antioxidant defenses.     

The effects of temperature and synaptic blockade in vitro on neurons of the horizontal limb of the diagonal band of Broca in the rat basal forebrain

We studied the thermosensitivity of neurons in the rat horizontal limb of the diagonal band of Broca (HDB) in vitro under normal conditions and under conditions of a low calcium/high magnesium synaptic blockade (SB). Of 52 HDB neurons tested, 34 neurons (65%) were warm-sensitive (WS), three neurons (6%) were cold-sensitive (CS), 11 neurons (21%) were temperature-insensitive (TI) and four additional neurons (8%) were both warm- and cold-sensitive (WS/CS). Of 34 neurons tested for thermosensitivity under SB, 11 were WS, 4 were CS and 19 were TI. Nearly half (48%) of the WS neurons maintained warm sensitivity under SB, 43% became TI and 9% became CS. Baseline firing rates of neurons significantly decreased during SB and then increased during recovery from SB. In addition, a distinct anatomical distribution of thermosensitive neurons was found in the HDB. The most ventral aspect of the HDB (interaural +0.9-1.3 mm) had proportionally fewer temperature sensitive neurons (65% vs. 88%) than areas more dorsal (interaural +1.3-1.7 mm), and only one of seven ventral HDB neurons (14%) remained thermosensitive during SB. In the dorsal HDB, 65% of the neurons maintained thermosensitivity during SB. These results demonstrate that the HDB contains inherently thermosensitive neurons, and that a difference in thermal characteristics exists between the ventral and dorsal HDB neurons.     

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.     

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies.     

Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of alpha7-containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic alpha7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC50 of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at alpha7 nAChRs. In contrast, choline did not activate alpha4beta2 agonist-bearing nAChRs on hippocampal neurons, and acted as a partial agonist at alpha3beta4-containing nAChRs on PC12 cells. The ethyl alcohol moiety of choline is required for the selective action on alpha7 nAChR. Exposure of cultured hippocampal neurons for 10 min to choline (10-100 microM) resulted in desensitization of the native alpha7 nAChRs. Moreover, chronic exposure (10 days) of the cultured hippocampal neurons to a desensitizing concentration of choline (approximately 30 microM) decreased their responsiveness to ACh. The selective action of choline on native alpha7 nAChRs suggests that this naturally occurring compound may act in vivo as an endogenous ligand for these receptors. Putative physiological actions of choline include retrograde messenger activity during the development of the mammalian central nervous system and during periods of elevated synaptic activity that leads to long-term potentiation.     

The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals

Clathrin-mediated endocytosis was shown to be arrested in mitosis due to a block in the invagination of clathrin-coated pits. A Xenopus mitotic phosphoprotein, MP90, is very similar to an abundant mammalian nerve terminal protein, epsin, which binds the Eps15 homology (EH) domain of Eps15 and the alpha-adaptin subunit of the clathrin adaptor AP-2. We show here that both rat epsin and Eps15 are mitotic phosphoproteins and that their mitotic phosphorylation inhibits binding to the appendage domain of alpha-adaptin. Both epsin and Eps15, like other cytosolic components of the synaptic vesicle endocytic machinery, undergo constitutive phosphorylation and depolarization-dependent dephosphorylation in nerve terminals. Furthermore, their binding to AP-2 in brain extracts is enhanced by dephosphorylation. Epsin together with Eps15 was proposed to assist the clathrin coat in its dynamic rearrangements during the invagination/fission reactions. Their mitotic phosphorylation may be one of the mechanisms by which the invagination of clathrin-coated pits is blocked in mitosis and their stimulation-dependent dephosphorylation at synapses may contribute to the compensatory burst of endocytosis after a secretory stimulus.     

Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.     

The CRF1 receptor mediates the excitatory actions of corticotropin releasing factor (CRF) in the developing rat brain: in vivo evidence using a novel, selective, non-peptide CRF receptor antagonist

One of two groups of sheep was immunosuppressed with the glucocorticoid, dexamethasone, at the time of the first but not of the second of two booster vaccinations with tetanus toxoid given at an interval of 28 days. Treatment with dexamethasone decisively reduced the anti-tetanus antibody response to the first booster vaccination and affected both IgM and IgG1 antibody. However, antibody titres increased after the second booster vaccination in the treated sheep and were similar in size to those in the untreated sheep which rose in stepwise fashion after each booster vaccination. The differences in response imply that processes involved in displaying an anamnestic response and recalling previously established memory are sensitive to glucocorticoids. Accordingly, they can be separated from the glucocorticoid-resistant processes that lead to the expansion of immunological memory following multiple exposures to an antigen.     

The superoxide production mediated by the redox cycling of xenobiotics in rat brain microsomes is dependent on their reduction potential

Responding effectively to trauma survivors who engage in self-injury can be challenging, even for experienced therapists. This paper outlines therapeutic goals and appropriate clinical responses, including remaining present at and open to communication about disclosures of self-injury, helping clients to intervene in their own process of self-injury, and working with clients to resolve underlying issues. Alternatives to self-injury are discussed and cautions are offered about common therapeutic responses likely to be particularly unhelpful.