Eugene M. Barnes

Use-Dependent Regulation of GabaA Receptors

Prolonged occupancy of GABAA receptors by ligands, including GABA and benzodiazepine agonists, sets in motion a series of mechanisms that can be termed use-dependent regulation. These mechanisms can be subdivided into two distinct pathways, one for GABAA receptor downregulation and another for upregulation. Treatment of cortical neurons with GABA or benzodiazepines in cultures opens the pathway for GABAA receptor downregulation, which includes (in putative temporal order): (1) desensitization (tachyphylaxis), (2) sequestration (endocytosis) of subunit polypeptides and uncoupling of allosteric interactions between GABA and benzodiazepine binding sites, (3) subunit polypeptide degradation, and (4) repression of subunit gene expression. The end-point of GABAA receptor downregulation, a reduction in receptor number, is postulated to be established initially by degradation of the receptor protein and then maintained by a diminished level of de novo synthesis. Benzodiazepine treatment of many preparations, including cells expressing recombinant GABAA receptors, may elicit only desensitization, sequestration, or uncoupling, without a decline in receptor number. Components of the GABAA receptor downregulation pathway are also evoked by chronic administration of GABAmimetics, benzodiazepines, barbiturates, and neurosteroids in animals. This downregulation correlates with the establishment of tolerance to and physical dependence on the pharmacological effects of these drugs, suggesting a cellular model for this behavior. The upregulation of GABAA receptors is observed as one of the neurotrophic actions of GABA, primarily in cultured cerebellar granule cells. Upregulation in culture is caused by enhanced expression of genes for GABAA receptor subunits and correlates with the establishment of GABAergic circuitry in the developing cerebellum. Thus, both the upregulation and downregulation of GABAA receptors appear to represent use-dependent pathways for guiding synaptic plasticity in the vertebrate central nervous system.

Intracellular trafficking of GABAA receptors

Abstract Some of the mechanisms that control the intracellular trafficking of GABA A receptors have recently been described. Following the synthesis of α, β, and γ subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While β3, β4, and γ2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA A receptors formed from α1β or α1βγ subunits. At the neuronal surface, native GABA A receptor clustering and synaptic targeting require a γ2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA A receptor associated protein (GABARAP), may be necessary for the formation of GABA A receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA A receptor γ2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA A agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA A receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA A receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.

Chronic exposure of developing cortical neurons to GABA down-regulates GABA/benzodiazepine receptors and GABA-gated chloride currents.

Cultures of cerebral neurons were prepared from chick embryos, 8.5 days in ovo, and maintained in vitro. Following chronic exposure of these cells to GABA, the levels of [3H]flunitrazepam binding in situ and electrophysiological responsiveness to gamma-aminobutyric acid (GABA) was examined. Treatment with 100 microM GABA for 7 days reduced [3H]flunitrazepam binding in situ by 70 +/- 8% compared to untreated controls. The binding of [3H]N-methylscopolamine was unaffected by this treatment. The reduction in [3H]flunitrazepam binding was prevented by concomitant exposure of developing neurons to the GABA antagonist R 5135, suggesting that GABAA receptor occupancy is required. The loss of bezodiazepine receptors was dependent on the GABA concentration in the culture medium and a half-saturation (IC50) value of 11.2 +/- 3.7 microM was estimated. Whole-cell patch-clamp recordings were obtained to assess the functional properties of the labile receptor pool observed in the binding studies. Neurons cultured with 100 microM GABA for 7 days showed a 60-70% reduction in the peak current amplitudes observed in response to application of 10-100 microM GABA. However, the rate of rapid desensitization, quantified by measuring changes in input conductance, was unchanged by chronic GABA exposure, yielding decay time constants of 27.1 +/- 2.1 and 34.7 +/- 4.7 s for control and treated cells, respectively. The results are consistent with a GABA modulation of the GABAA/benzodiazepine receptor complex by means of down-regulation.

GABA down-regulates the GABA/benzodiazepine receptor complex in developing cerebral neurons

Neuronal cultures of the chick embryo cerebrum were used to study the chronic effects of gamma-aminobutyric acid (GABA) on the expression of the GABA/benzodiazepine receptor complex. A 7 day exposure of developing neurons to 100 microM GABA produced a 70% reduction in the level of [3H]flunitrazepam binding to intact cells, when compared to untreated controls. The reduction was due to a decrease in receptor density (Bmax) rather than the affinity. The same treatment also caused a 75% reduction in the rates of GABA-gated 36Cl- uptake by intact cells, without an effect on the basal (GABA-independent) flux. Eight days after removal of GABA from the medium of treated cultures, the neurons recovered [3H]flunitrazepam binding to levels corresponding to 74% of unexposed, age-matched controls. The results are consistent with a GABA-induced down-regulation of the GABA/benzodiazepine receptor.

Ontogeny of the GABA receptor complex in chick brain: Studies in vivo and in vitro

The ontogeny of the gamma-aminobutyric acid-A (GABAA) receptor complex in the chick brain was studied by specific binding of [3H]muscimol, [3H]flunitrazepam (Flu) and [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes. During development in ovo, the specific binding of muscimol and flunitrazepam increased from day 8 and reached 50% of adult levels of day 20, while a comparable level of TBPS binding was achieved by day 17. The increases in TBPS and Flu binding were reflected in Bmax rather than Kd changes. In embryonic brain, only a low-affinity site for muscimol (Kd = 23 nM) was observed while an additional high-affinity site (Kd = 0.4 nM), as well as the low-affinity site, was found in adult tissue. Similar studies were carried out with cultures of cerebral neurons prepared from 8-day embryos. The level of specific binding of muscimol, Flu and TBPS increased in culture, achieved one half of the maximum level by days 4-5, maximal levels by day 10 and decreased slowly thereafter. The maximal levels in culture corresponded, respectively, to 27%, 67% and 57% of these found in the 18-day embryo. The binding of Flu to membranes from neurons, embryos and adults was enhanced by addition of GABA while TBPS binding was inhibited. The EC50 and IC50 values for these effects corresponded to those for gating of chloride channels. These findings indicate a coordinated expression of receptors for GABA, benzodiazepines and convulsant/TBPS during neuronal maturation both in vivo and in vitro. The schedule for this postsynaptic ontogeny is very similar to that for presynaptic markers of GABAergic neurons (see companion paper).

Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABAA receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively. The accumulation of the beta 4 S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 microM GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their splice variants (where present) were all reduced by 47-65% compared to untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [3H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with Km = 370 microM and Vmax = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na+ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (Km = 12 microM; Vmax = 0.34 nmol/min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N6-benzyladenosine. In decreasing order of potency, N6-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.

Ontogeny of GABAergic neurons in chick brain: Studies in vivo and in vitro

The ontogeny of presynaptic elements of GABAergic neurons has been studied in the cerebrum of the chick embryo both in vivo and in vitro. The specific activity of glutamate decarboxylase (GAD) in tissue extracts followed a rising curve and approached a plateau value after 28 days in vivo. One-half of the adult levels of GAD were achieved by day 20. The specific activity of Na+-gamma-aminobutyric acid (GABA) cotransport in membrane vesicles followed a similar pattern and reached maximal levels by 28 days in vivo. One-half of the adult levels of GABA uptake were observed at day 17. The development of these markers was also studied in cultured neurons prepared from the cerebrum of 8-day-old chick embryos. The GAD activity in neuronal extracts increased linearly with time in culture up to 14 days. At this point the specific activity had reached 20% of that observed for the adult cerebrum. The specific activity of GABA uptake by intact neurons followed a pattern similar to that for GAD from days 2 to 9 in culture. Both activities increased 4-5-fold during this period, but the level of GABA transport declined thereafter. In order to compare GABA uptake values for cultured cells with those for embryonic and adult brain, membrane vesicles were prepared from cultures. At the maximal level (9-10 days in culture) the vesicular GABA uptake represented 33% of that in the 18-day embryo and 20% of adult levels. Thus the presynaptic GABAergic components developed according to similar schedules both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiration-coupled glucose transport in membrane vesicles from Azotobacter vinelandii

An inducible, carrier-mediated transport system for d-glucose is present in membrane vesicles isolated from Azotobacter vinelandii O. The active accumulation of glucose by these preparations requires the addition of oxidizable substrate: l-Malate produces a 25-fold stimulation of both the rate and steady-state levels of glucose uptake; NADH and NADPH produce a 5-fold stimulation. Phosphoenolpyruvate and ATP, in addition to a number of other metabolites, are without effect. The membrane vesicles oxidize malate to oxalacetate via l-malate dehydrogenase, and the addition of flavin adenine dinucleotide is required for maximal stimulation of glucose transport by malate. Glucose accumulation by the vesicles in the presence of l-malate requires oxygen, and is blocked by electron transport inhibitors and carbonyl cyanide m-chlorophenyl hydrazone, but not by arsenate. These findings indicate that the glucose uptake system is coupled to l-malate dehydrogenase via the respiratory chain of A. vinelandii. However, the generation of high-energy phosphate compounds or the participation of a phosphoenolpyruvate phosphotransferase system is not required for glucose accumulation by the membrane vesicles.

Reduced function of γ-aminobutyric acidA receptors in tottering mouse brain: Role of cAMP-dependent protein kinase

Abstract The single-locus mutant mouse tottering ( tg ) displays spontaneous seizures that resemble those in human petit-mal epilepsy. In order to examine alterations in GABA A receptor function which could arise as a result of this mutation, the influx of 36 C1 − was determined using microsacs (membrane vesicles) isolated from the brain of tg/tg and coisogenic C57BL/6J ( + / + ) control mice. In microsacs from both tg/tg and + / + strains, the maximum level of 36 Cl − uptake induced by 50 μM GABA was observed during five seconds of incubation at 28°C. Compared to + / +, the GABA-dependent 36 Cl − uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated 36 C1 − uptake in tg/tg microsacs were 45 ± 6.3%, 65 ± 9.9%, and 33 ± 6.1% of control ( + / + ) values for 3-, 5-, and 10-s incubations, respectively. GABA A receptor-specific agonists (30 μM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced 36 C1 − influx in the order muscimol > GABA > isoguvacine > THIP. This order was similar for both strains, but the agonist-dependent influx was always significantly lower in tg/tg compared to + / +. Treatment of the microsacs with 10 μ M H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A, PKA), was without effect on GABA-gated 36 Cl − uptake in + / +, but increased the gated uptake in tg/tg microsacs by 44 ± 16%. PKA was assayed using [ γ - 32 ]ATP and kemptide as the substrate. Triton ×-100 (0.1%) increased both the basal and 8-Br-CAMP dependent PKA activity in microsacs by 3–4 four fold, showing that most of the enzyme was intravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of + / +, while the strain difference was no longer apparent in assays containing 8-Br-cAMP. The data suggest that an abnormal elevation of protein kinase A activity in tottering mouse brain contributes to an impairment of GABA A receptor function. It is suggested that the resulting loss of inhibition could play a role in induction of the seizures which characterize the mutant phenotype.