Biomedical subjects

A Schousboe

Publications and source records attributed to A Schousboe.

At least 55 records · Page 3Linked to original sources

PubMed1998

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors.

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.

Animals↗

PubMed1998

Development of calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured neocortical neurons visualized by cobalt staining.

The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 microM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7%+/-0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 microM cyclothiazide, the developmental profile of cobalt uptake mediated by 25 microM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4%+/-0.2% at 5 DIV to 21.7%+/-1.6% at 12 DIV. The effect of 200 microM kainate was similar to that seen with 25 microM AMPA plus 50 microM cyclothiazide, resulting in 17.7%+/-0.3% stained neurons at 12 DIV. The cobalt uptake was specific to AMPA and kainate receptors because NMDA receptors and voltage-gated calcium channels were found not to mediate any cobalt staining. In addition, 10 microM 6-nitro-7-sulphamoylbenzo-[f]-quinoxaline-2,3-dione (NBQX) was able to prevent all staining at 5 and 8 DIV and most of the staining at 12 DIV, indicating that the non-NMDA ionotropic glutamate receptors are involved in cobalt uptake into the neurons. The AMPA receptor-selective antagonist GYKI 53655 was used to differentiate between cobalt influx through AMPA- or kainate-preferring receptors. After pretreatment with concanavalin A (con A), an inhibitor of kainate receptor desensitization, cobalt uptake was assessed after stimulation by 200 microM kainate in the presence of 25 microM GYKI 53655. No cobalt staining was observed under these conditions, indicating that most if not all of the cobalt influx induced by kainate was mediated through AMPA receptor channels.

Animals↗

PubMed1998

Pharmacological and functional implications of developmentally-regulated changes in GABA(A) receptor subunit expression in the cerebellum.

The cerebellum undergoes many morphological, pharmacological, and electrophysiological changes during the first 3 weeks of postnatal development. The purpose of this review is to present the most up to date synopsis of the pharmacological and functional changes in, gamma-aminobutyric acid (GABA) type A receptors during this time of cerebellar maturation. Since most of the diversity in cerebellar, GABA(A) receptor pharmacology lies within the granule cell layer, research groups have focused on this area of the cerebellum to study the developmental changes in GABA(A) receptor subunit expression and the neurodifferentiating factors involved in regulating this expression. Thus, it is important to note that developmental changes in GABA(A) receptor composition and its corresponding pharmacology will be essential for determining the type of GABA-mediated transmission that occurs between neuronal contacts in the neonatal and subsequently in the mature cerebellum.

Animals↗

PubMed1998

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABA(A) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (y-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 microM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA(A) receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 microM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA(A) antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA(A) receptor. When 100 microM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 microM lindane. The GABA(B) receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA(A) receptor.

2-Amino-5-phosphonovalerate↗

PubMed1998

Cytotoxic action of lindane in cerebellar granule neurons is mediated by interaction with inducible GABA(B) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (gamma-HCH, lindane) was studied in cultured mouse cerebellar granule neurons maintained in the presence or absence of the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). The cells were exposed for 24 hr to lindane (30-300 microM) in the culture medium. Changes in mitochondrial function were investigated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The results showed that lindane-induced cytotoxicity was concentration-dependent. In cerebellar granule cells not treated with THIP, lindane-induced cytotoxicity did not appear to be related to GABA(A) or GABA(B) receptors. However, in THIP-treated cultures, lindane-induced cytotoxicity was found to be mediated by an action of the insecticide on GABA receptors. In the latter case, GABA reduced the lindane-induced cytotoxicity, but the protective effect was not potentiated by flunitrazepam. The GABA(A) receptor agonist muscimol (50 microM) also protected the THIP-treated cultures against lindane-induced cytotoxicity. In addition, the GABA(B) receptor agonist R(+)baclofen protected the cells from lindane-induced cytotoxicity and the effect of baclofen was blocked by GABA(B) receptor antagonists. Pertussis toxin was found to reverse the protective effect of baclofen only at the highest lindane concentration (300 microM). The lindane-induced cytotoxicity could be partly explained as being secondary to excitotoxicity as a mixture of the excitatory amino acid receptor antagonists APV (D-(-)-2-amino-5-phosphonopentanoate) and CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) shifted the concentration-response curve for lindane-induced cytotoxicity to the right. It is suggested that the cytotoxic effects of lindane in THIP-treated cerebellar granule neurons are primarily related to an action of lindane on GABA(B) receptors and to a lesser extent on inducible low-affinity, benzodiazepine insensitive GABA(A) receptors.

2-Amino-5-phosphonovalerate↗

PubMed1998

Ionic mechanisms in glutamate-induced astrocyte swelling: role of K+ influx.

L-Glutamate (L-GLU) induced astrocyte swelling in a time- and concentration-dependent, as well as Na+- and Ca2+-dependent, and Cl(-)-independent manner. Swelling was prevented by MK-801, cystine, and ouabain. Since L-GLU swelling is ionically dependent, we determined the role of various ions in such swelling. Our results indicate that K+ uptake plays a major role in the mechanism of L-GLU-induced astrocyte swelling. Like swelling, K+ uptake is dependent on Ca2+ and Na+, but not on Cl-. Likewise, K+ uptake was inhibited by MK-801, cystine, and ouabain. The K+ channel blockers, Ba2+ and tetraethylammonium, partially prevented L-GLU-induced swelling. In addition to K+ channels, K+ influx may also be mediated through Na+/K+-ATPase, as its activity is increased by L-GLU uptake along with the required Na+. Taken together, the data suggest that K+ influx plays a key role in the mechanism of L-GLU-mediated astrocyte swelling.

Animals↗

PubMed1998

Spectral components of cytosolic [Ca2+] spiking in neurons.

We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application. Delayed complex responses of large [Ca2+]c spiking observed in cells from a different set of cultures were synthesized by a set of frequencies within the range 0.018-0.117 Hz. Differential frequency patterns are suggested as characteristics of the [Ca2+]c spiking responses of neurons under different conditions.

Animals↗

PubMed1998

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex.

Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2',2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl- gradient by removal of extracellular Cl- significantly increased the rate of pHi recovery. In HCO3(-)-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl-, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H+ influx in the recovery of pHi after alkalinization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

Alternative Splicing↗

PubMed1998

Hippocampal stimulation produces neuronal death in the immature brain.

We re-examined the proposed resistance of the immature brain to seizure-induced damage. In awake, freely moving rat pups, intermittent perforant path stimulation produced selective hippocampal cell loss and reduction in paired-pulse inhibition. During 16 h of stimulation, animals showed frequent wet dog shakes and hind-limb scratching movements but no convulsive motor activity. In situ end-labelling performed 2 h after the end of stimulation showed an intense band of positively-labelled eosinophilic cells with condensed profiles bilaterally in the dentate granule cell layer of stimulated animals. Control animals showed no in situ end-labelling positivity in the dentate gyrus. These cells were not observed 24 h later, suggestive of rapidly scavenged apoptotic cells. One day after the end of stimulation, many necrotic interneurons with eosinophilic cytoplasm and pyknotic nuclei were observed in the hilus of the stimulated dentate gyrus in all rats tested. Hippocampal pyramidal cells in CA1, CA3 and subiculum showed bilateral damage greater on the side of stimulation, and prepiriform cortex sustained bilateral symmetrical lesions. One month after perforant path stimulation, Cresyl Violet staining showed the number of large hilar interneurons (>15 microm) was reduced on the stimulated side (54.1 +/- 12.2) compared to the non-stimulated side (100.5 +/- 10.2 cells, P<0.01). Immunohistochemical analysis showed significant losses in somatostatin (8.5 +/- 1.6 stimulated side, 22.8 +/- 3.8 unstimulated side, P<0.05) and neuropeptide Y (12.8 +/- 3.2 stimulated side, 17.0 +/- 4.1 unstimulated side, P<0.05) immunoreactive cells in the stimulated hilus but no loss of parvalbumin-immunoreactive cells. Significant reductions in paired-pulse inhibition were found after stimulation but there was some return of inhibition by one month. These combined data demonstrate that the immature brain can incur damage as a result of prolonged seizure-like hippocampal activity mimicking status epilepticus in immature rats. The hippocampal damage produced by perforant path stimulation is associated with the immediate loss of physiological inhibition suggesting important modification of excitatory control in an extremely epileptogenic region of the brain.

Animals↗

PubMed1998

Functional properties of glycine receptors expressed in primary cultures of mouse cerebellar granule cells.

Expression of the glycine receptor was investigated in membranes prepared from primary cultures of mouse cerebellar granule cells and postnatal mouse cerebellum using the antagonist [3H]strychnine for ligand binding. Scatchard analysis of the binding data obtained from P17 cerebellum showed a single population of binding sites (K(D) approximately 6 nM) and [3H]strychnine binding to membranes prepared from cultured neurons and P17 cerebellum was found to have the same sensitivity to the glycinergic agonists glycine, beta-alanine and taurine. The development of [3H]strychnine binding sites in cultured cerebellar granule cells and cerebellum showed opposing profiles. [3H]strychnine binding to primary cultures increased significantly during the culture period whereas during development in vivo the number of binding sites decreased over time and was hardly detectable in the adult cerebellum. Release of preloaded D-[3H]aspartate evoked by 40 mM K+ from granule cells cultured for seven days was inhibited by glycine by about 50%. Beginning after seven days in culture the ability of glycine to inhibit transmitter release declined to no inhibition after 17 days in culture. Experiments with the non-competitive antagonist, picrotoxinin, showed no blocking effect of 150 microM picrotoxinin on the glycine-induced inhibition of transmitter release. This contrasted with the inhibitory effect of 100 microM picrotoxinin in whole-cell patch-clamp recordings on responses to 500 microM glycine (56% block). Furthermore, it was demonstrated that the amplitude of the glycine activated peak current had the same size after six to seven days and after 16-17 days in culture. Northern blot analysis, and co-injection of messenger RNA plus antisense oligonucleotides into Xenopus oocytes revealed glycine receptor alpha2 and beta messenger RNAs in the cultured granule cells. These findings suggest that granule cells in culture express glycine receptor isoforms containing alpha2 picrotoxinin-sensitive and alpha2/beta picrotoxinin-insensitive receptors.

Animals↗

PubMed1998

Cellular distribution and kinetic properties of high-affinity glutamate transporters.

L-glutamic acid is a key chemical transmitter of excitatory signals in the nervous system. The termination of glutamatergic transmission occurs via uptake of glutamate by a family of high-affinity glutamate transporters that utilize the Na+/K+ electrochemical gradient as a driving force. The stoichiometry of a single translocation cycle is still debatable, although all proposed models stipulate an inward movement of a net positive charge. This electrogenic mechanism is capable of translocating the neurotransmitter against its several thousand-fold concentration gradient, therefore, keeping the resting glutamate concentration below the treshold levels. The five cloned transporters (GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5) exhibit distinct distribution patterns and kinetic properties in different brain regions, cell types, and reconstitution systems. Moreover, distinct pharmacological profiles were revealed among the species homologues. GLAST and GLT1, the predominant glutamate transporters in the brain, are coexpressed in astroglial processes, whereas neuronal carriers are mainly located in the dendrosomatic compartment. Some of these carrier proteins may possess signal transducing properties, distinct from their transporter activity. Some experimental conditions and several naturally occurring and synthetic compounds are capable of regulating the expression of glutamate transporters. However, selective pharmacological tools interfering with the individual glutamate carriers have yet to be developed.

ATP-Binding Cassette Transporters↗

PubMed1998

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species.

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.

Animals↗

PubMed1998

Metabolism of lactate in cultured GABAergic neurons studied by 13C nuclear magnetic resonance spectroscopy.

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), gamma-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).

Animals↗

PubMed1998

Mitochondrial heterogeneity in the brain at the cellular level.

Classically, compartmentation of glutamate metabolism in the brain is associated with the fact that neurons and glia exhibit distinct differences with regard to metabolism of this amino acid. The recent use of 13C-labeled compounds to study this metabolism in conjunction with the availability of cell type-specific tissue culture modes has led to the notion that such compartmentation may even be present in individual cell types, neurons as well as glia. To better understand and explain this, it is proposed that mitochondrial heterogeneity may exist resulting in tricarboxylic acid cycles with different properties regarding cycling rates and ratio as well as coupling to amino acid biosynthesis, primarily involving glutamate and aspartate. These hypotheses are evaluated in the light of current knowledge about mitochondrial structure and function.

Animals↗

PubMed1998

Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13C-NMR spectroscopy.

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, gamma-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.

Animals↗

PubMed1998

Effects of gamma-aminobutyric acid (GABA) on synaptogenesis and synaptic function.

The correct establishment and function of synapses depend on a variety of factors, such as guidance of pre- and postsynaptic neurons as well as receptor development and localization. gamma-Aminobutyric acid (GABA) has a pronounced effect on these events and elicits differentiation of neurons; that is, GABA acts as a trophic signal. Accordingly, activating preexisting GABA receptors, a trophic GABA signal enhances the growth rate of neuronal processes, facilitates synapse formation, and promotes synthesis of specific proteins. Transcription and de novo synthesis are initiated by the GABA signal, but the intracellular link between GABA receptor activation and DNA transcription is largely unknown. GABA also controls the induction and development of functionally and pharmacologically different GABAA receptor subtypes. The induced receptors are likely to be inserted only into the synaptic membrane domain. However, this ability to target the induced GABAA receptors is probably coupled to the maturation of neurons and not to the action of GABA per se. The induced GABAA receptors apparently mediate a pronounced inhibition of neurotransmitter release, whereas other subtypes of GABAA receptors may be modulatory rather than inhibitory.

Animals↗

PubMed1997

Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors.

Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the alpha6 GABA(A) receptor subunit. Membranes prepared from these cultures were photolabeled with the imidazobenzodiazepine [3H]Ro15-4513. In THIP-treated cultures at 4 days in vitro (DIV), photolabeled [3H]Ro15-4513 binding in membranes was significantly increased for both the 51 kilodalton, kDa, (alpha1 subunit) and 56-kDa (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however, no significant change in [3H]Ro15-4513 binding was observed for the 56-kDa polypeptide. Immunolabeling of the alpha6 subunit using silver-enhanced, immuno-gold staining of granule cells showed a significant effect with THIP treatment only at 4 DIV and not at 8 DIV. Examination by light microscopy demonstrated that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest that THIP has a trophic effect on alpha6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABA(A) agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABA(A) receptor subunits in the developing cerebellum.

Animals↗