GABA-dependent Chloride Uptake Research Articles

Prenatal lorazepam exposure: 4. Persistent alterations in pentylenetetrazole-induced seizure threshhold and GABA-dependent chloride uptake after prenatal lorazepam exposure

Prenatal benzodiazepine exposure is associated with behavioral and neurochemical alterations in the early postnatal period. To determine the persistence of these effects, we evaluated pentylenetetrazole-induced seizure threshhold and GABA-dependent chloride uptake in mice at 6 and 12 months of age after prenatal lorazepam exposure. Seizure threshhold was reduced after acute lorazepam pretreatment in mice exposed to lorazepam prenatally, compared to control groups, at 6 and 12 months of age. Maximal GABA-dependent chloride uptake was also reduced in exposed mice at 6 and 12 months of age. These data indicate that behavioral and neurochemical alterations persist well into maturity after prenatal lorazepam exposure.

Interleukin-1 modulates GABAergic and glutamatergic function in brain.

Interleukin-1 is a polypeptide cytokine involved in the acute-phase response. We examined the effects of IL-1 on the major inhibitory and excitatory neurotransmitter systems in brain mediated by GABA and glutamate, respectively. IL-1 enhanced the effects of GABA as determined by GABA-dependent chloride uptake at 1 and 10 ng/ml in cortical synaptoneurosomes. This effect was attenuated by pretreatment with the IL-1 receptor antagonist. Neither IL-6 nor TNF was effective in this system. IL-1 decreased the effect of NMDA and glycine on intracellular calcium concentrations in cultured chick cortical neurons in both the presence and absence of the endogenous polyamine spermine. This effect was attenuated by pretreatment with the AMPA/kainate antagonist CNQX. These data indicate that IL-1 modulates both inhibitory and excitatory neurotransmitter function in brain.

Actions of Avermectin Analogs on γ-Aminobutyric Acid (GABA)-Sensitive and GABA-Insensitive Chloride Channels in Mouse Brain

Seven avermectin analogues that exhibited a wide range of anthelminthic and insecticidal activities were evaluated for their effects on γ-aminobutyric acid (GABA)-sensitive and GABA-insensitive components of chloride transport across mouse brain vesicle membranes. All of the compounds except the biologically inactive octahydro analogue of abamectin inhibited GABA-dependent chloride uptake into brain vesicles. The relative inhibitory potencies of the six active analogues varied over a more than 60-fold range and were generally correlated with their potencies as invertebrate toxicants. These six compounds, but not the octahydro analogue, also stimulated GABA-insensitive chloride efflux from preloaded brain vesicles. In this assay, the potencies of the six active compounds varied over a 6-fold range and were not correlated with potencies for the inhibition of GABA-dependent chloride uptake. Moreover, neither potencies nor relative efficacies for the stimulation of chloride efflux were obviously correlated with biological activity against invertebrates. These data show that avermectins are capable of modifying chloride permeability in mouse brain vesicle preparations by two distinct mechanisms, one involving GABA receptors and a second involving one or more additional chloride ionophores unrelated to GABA receptors. Both actions may contribute to the signs of intoxication observed for abamectin and other avermectins in mammals.

Modulatory action of arachidonic acid on GABAA/chloride channel receptor function in adult and aged brain cortex membranes

Effect of arachidonic acid on binding parameters of two binding sites on the GABAA receptor and on GABA activated Cl- uptake was investigated in synaptic plasma membrane and in synaptoneurosomes from brain cortex of adult (4-months old) and aged (27-months old) rats. The ligands used were [3H]muscimol, a GABA agonist and [35S]-t-butylbicyclophosphorothionate ([35S]TBPS), a convulsant that binds to the site near the chloride channel. Arachidonic acid increases significantly GABAA agonist binding and concomitantly decreases [35S]TBPS binding in a concentration dependent manner. The analysis of binding parameters in adult brain showed a significant decrease by AA of KD value for low but not for high affinity of [3H]muscimol binding. Concomitantly, AA enhances Bmax values for high affinity binding and has no effect on Bmax value for low affinity binding in synaptic plasma membrane (SPM) from adult brain. In synaptic plasma membrane from aged brain AA increases low and high affinity binding of agonist to GABAA receptor, modified significantly KD value by about 30 and 66%, respectively. On the other hand, AA significantly decreases of [35S]TBPS binding to chloride channel recognition site. Scatchard's analysis indicates that this inhibition results from a decrease of total number of binding sites. Moreover, the affinity of [35S]TBPS binding was increased (KD = 37.0 nM for AA-treated membrane, as compared to 69.3 nM KD in control membrane). GABA-dependent chloride uptake into synaptoneurosomes is also inhibited by AA in a concentration dependent manner in adult brain. In aged brain synaptoneurosomes AA has similar inhibitory effect on GABA-activated chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of Phenylpyrazole Insecticides at the GABA-Gated Chloride Channel

The GABA-gated chloride channel is the proposed target for phenylpyrazole insecticides based on studies comparing four potent insecticidal compounds with two herbicidal but noninsecticidal analogs. The four insecticides used are 1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazoles with the following heterocyclic substituents: 3-cyano-4-[(trifluoromethyl)sulfinyl]-5-amino (fipronil, 1), 4-[(trifluoromethyl)thiol (2), 4-[(trifluoromethyl)sulfinyl] (3), 4-[(trifluoromethyl)sulfonyl] (4). Pyrazole substituents of the two herbicidal analogs examined are 5-amino-4-nitro (5), 5-(2-chloropropanamide)-4-nitro (6). Piperonyl butoxide strongly antagonizes the toxicity of thioether 2 to houseflies, does not alter the potency of sulfoxide 3 and sulfone 4, and strongly synergizes sulfoxide 1, indicating the importance of oxidative activation and detoxification to insecticidal activity. Insecticides 1-4, but not herbicides 5 and 6, yield poisoning signs in house flies and mice similar or identical to those produced by trioxabicyclooctanes such as 1-[(4-ethynyl)phenyl]-4-n-propy-2,6,7-trioxabicyclo[2.2.2]octane (EBOB), which is known to block the γ-aminobutyric acid (GABA)-gated chloride channel. A dieldrin-resistant house fly strain with a low-affinity EBOB binding site is also tolerant to 1 and 2. The first four phenylpyrazoles are more potent than the latter two as inhibitors of [3H]EBOB binding with 50% inhibition levels of 2-20 nM in house fly head membranes and 1700-10,100 nM in mouse brain membranes. Compounds 1 and 2 are much more potent than 5 and 6 in inhibiting ivermectin-induced neurotransmitter release from cricket central nervous system synaptosomes. Compound 1 is also more effective than 2 and 5 at 20 μM in blocking GABA-dependent chloride uptake in mouse brain microvesicles; in this assay 6 is as effective as 1 although they act by different mechanisms in vivo in mice based on the symptomology. Neither 1 nor 2 inhibits [3H]avermectin binding in house fly head membranes. Phenylpyrazoles 1 and 2 block [3H]EBOB binding in house fly head membranes by reducing the Bmax but not the Kd, indicating that they are irreversible or poorly reversible inhibitors or act at a distinct but coupled binding site. The insecticidal phenylpyrazoles appear to block the GABA-gated chloride channel with higher potency for a site in house fly brain than in mouse brain, thereby providing useful selective toxicity. These findings are consistent with an earlier pharmacological investigation on the action of a structurally related phenyltriazole as a potent antagonist of GABA-activated chloride currents in insects.

Increased GABAA-dependent chloride uptake in mice selectively bred for low aggressive behavior.

Selective breeding for aggressive behavior alters GABA-dependent chloride uptake and behavioral response to benzodiazepine treatment. Pharmacological and biochemical studies examined subjects from three lines of adult male ICR mice selectively bred for either high levels or low levels of aggressive behavior, as well as unselected controls. Selective breeding produced two lines of behaviorally distinct males. During 5-min dyadic confrontations with an outbred stimulus animal, untreated low-aggressive mice spent more time in walking, rearing, and social interaction than untreated high-aggressive mice. The three lines also showed different responsiveness to the aggression increasing and decreasing effects as well as the sedative effects of benzodiazepine treatment. High doses of chlordiazepoxide (17, 30 mg/kg) reduced motor behaviors (walk, rear and groom) in the low-aggressive line without altering these behaviors in the high aggressive line. In the high-aggressive line, the same doses of chlordiazepoxide (17, 30 mg/kg) produced a behavioral shift; aggressive behaviors were reduced while social behaviors increased to levels similar to the untreated low-aggressive line. In contrast, only the unselected line pursued and threatened more after a low dose of chlordiazepoxide (3 mg/kg). The three lines also showed alterations at the GABAA-benzodiazepine receptor complex. Specific uptake of [3H]Ro-15-1788 was increased in cerebral cortex, hypothalamus and hippocampus in the low-aggressive line, and was reduced in these areas in high-aggressive line when compared with unselected controls. Similarly, GABA-dependent chloride uptake in cortical synaptoneurosomes was augmented in low-aggressive mice and decreased in high-aggressive mice when compared to unselected controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic lethality of chloride-channel-directed insecticides and convulsants in mammals

The intrinsic lethality of a series of chloride-channel-directed convulsants and insecticides was determined by intracerebral injection into mice. The toxicities of the cyclodiene insecticides and picrotoxinin were potentiated by intracerebral injection, when compared to their reported intraperitoneal LD 50 s. In contrast, the toxicities of lindane, abamectin, and the bicyclic convulsants TBPS and a TBOB analog were approximately the same by either route of administration. These results suggest that the brain is the primary target site for the cyclodienes and picrotoxinin, while the peripheral nervous system may be relatively more important in the toxic action of lindane, abamectin, and bicyclic convulsants such as TBPS. With the exception of picrotoxinin, these compounds showed, overall, a good correlation between acute intracerebral toxicity and potency for inhibiting GABA-dependent chloride uptake. The evidence that multiple chloride-channel subtypes serve as targets for these compounds and the potential impact this had on the results of these studies are discussed.

Chronic benzodiazepine administration: IX. Attenuation of alprazolam discontinuation effects by carbamazepine

Clinical studies suggest that carbamazepine may attenuate effects of alprazolam discontinuation. Since discontinuation of chronic alprazolam in a mouse model is associated with behavioral alterations and upregulation at the y-aminobutyric acid A (GABA A) receptor, we studied the effects of carbamazepine administration after alprazolam (2 mg/kg/day) discontinuation. Open-field activity was increased in mice 4 days after alprazolam discontinuation, but this effect was reduced significantly by continuous infusion of carbamazepine, 25 or 100 mg/kg/day. Benzodiazepine receptor binding in vivo was increased in cortex at 2 and 4 days after alprazolam discontinuation, and in hypothalamus at 4 days; with carbamazepine, 100 mg/kg/day, binding in both regions at these time points was similar to control values. Similar results were observed in cortex with benzodiazepine receptor binding in vitro. GABA-dependent chloride uptake was also increased at 4 days after alprazolam administration. Treatment with carbamazepine attenuated (P < 0.10) this increase. Carbamazepine alone after vehicle did not alter benzodiazepine binding or GABA-dependent chloride uptake. These results indicate that carbamazepine administration after alprazolam discontinuation attenuates behavioral and neurochemical alterations associated with discontinuation.

Activation of γ‐aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a

The interaction of avermectin B1a (AVMB1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with 36Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB1a-stimulated chloride release required the presence of intact vesicles. The efflux of 36Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB1a produced half-maximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 +/- 0.3 microM and a 35.4 +/- 1.4% maximal loss of intravesicular chloride at saturating concentrations. gamma-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB1a-stimulated chloride efflux. These data demonstrate that AVMB1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.

Chronic exposure to a benzodiazepine partial agonist does not alter GABA A receptor function in cultured neurons

The benzodiazepine partial agonist Rol6-6028 does not lead to GABA A receptor downregulation in vivo. To assess effects of this compound in vitro, cultured neurons were exposed to Rol6-6028, 1 and 10 μM. GABA-dependent chloride uptake was unaffected at either dose of Rol6-6028 from 2 to 10 days, in contrast to decreased function observed with clonazepam, 1 μM, at 10 days. Ro16-6028 exposure did not alter GABA-independent chloride uptake, total neuronal protein, or cellular protein synthesis.

Prolonged exposure to gaba activates GABA-gated chloride channels in the presence of channel-blocking convulsants

1. In assays of 36Cl − uptake into mouse brain vesicles, 100 μM GABA markedly increased both the initial rate of 36Cl − uptake and the total amount of chloride taken up over a 120-sec incubation period. Specific GABA-dependent 36Cl − uptake (the difference between total and background uptake) was essentially complete within 15 sec of incubation. 2. Incubation with GABA following preincubation with 10 μM endrin, a polychlorocycloalkane insecticide and established blocker of GABA-gated chloride channels, showed a stimulation of uptake over background levels that was much slower in onset than that observed with GABA alone but nevertheless achieved virtually the same level of stimulation above background levels after 90 sec of incubation with GABA. 3. In electrophysiological assays of GABA receptors expressed in Xenopus oocytes following injection with rat brain mRNA, endrin (20 μM) effectively blocked the transient currents elicited by brief exposure of oocytes to GABA (200 μM). However, prolonged exposure to GABA in the absence of perfusion produced a large, slowly-developing inward current. 4. The actions of several known GABA antagonists were also compared as inhibitors of GABA-dependent 36Cl − uptake into mouse brain vesicles at short (4 sec) and long (120 sec) incubation times using concentrations of inhibitors known to produce approximately 70–90% inhibition of GABA-dependent chloride uptake in 4-sec incubations. Picrotoxinin and TBPS, like endrin, were completely ineffective as inhibitors in 120-sec incubations. In contrast, bicuculline was almost as effective at 120 sec as at 4 sec, and avermectin B 1a produced approximately 50% inhibition of the GABA response after 120 sec 5. These data suggest that the potent inhibitory action of picrotoxinin, bicyclic phosphates and orthocarboxylates, and polychlorocycloalkane insecticides on GABA-gated chloride channels may be restricted to a subpopulation of receptors that mediates transient, rapidly-densitizing responses to GABA.

Differential effects of chronic lorazepam and alprazolam on benzodiazepine binding and GABAA‐receptor function

1. Chronic benzodiazepine administration has been associated with tolerance and with downregulation of gamma-aminobutyric acidA (GABAA)-receptor binding and function. However, effects of individual benzodiazepines on brain regions have varied. 2. To compare the effects of chronic lorazepam and alprazolam, we have administered these drugs to mice for 1 and 7 days (2 mg kg-1 day-1) and determined benzodiazepine receptor binding in vivo with and without administration of CL 218,872, 25 mg kg-1 i.p., and GABA-dependent chloride uptake in 3 brain regions at these time points. 3. Benzodiazepine binding was decreased in the cortex and hippocampus at day 7 compared to day 1 of lorazepam, with an increase in CL 218,872-resistant (Type 2) sites in both regions. Maximal GABA-dependent chloride uptake was also decreased in the cortex and hippocampus at day 7. 4. Binding was decreased only in the cortex after 7 days of alprazolam, with no significant change in Type 2 binding. Maximal GABA-dependent chloride uptake was also decreased only in the cortex. 5. These data suggest that the effects of chronic benzodiazepine administration on the GABAA-receptor may be both region-specific and receptor subtype-specific.

Augmentation of GABA A receptor function by chronic exposure to GABA-neutral and GABA-negative benzodiazepine ligands in cultured cortical neurons

Chronic benzodiazepine agonist administration may lead to decreases in γ-aminobutyric acid A (GABA A) receptor binding and function, but little information is available concerning chronic GABA-neutral or GABA-negative benzodiazepine exposure. We evaluated effects of chronic exposure to flumazenil (Ro15-1788) and FG 7142 ( N-methyl- β-carboline-3-carboxamide) on GABA-dependent chloride uptake in chick cerebral cortical neurons in primary culture. Acute flumazenil treatment (1 μM) had no effect on chloride uptake, but uptake was increased after 2 days of exposure. Similar increases were observed after 4 and 10 days. Flumazenil, 0.1 μM, had no effect after 10 days, and a 10 μM concentration had a similar effect as the 1 μM concentration. Acute FG 7142 (1 μM) decreased chloride uptake, but uptake was increased markedly after 2, 4, and 10 days of treatment. No effect was observed after treatment for 10 days with 0.1 μM, but a 10 μM concentration showed similar enhancement to the 1 μM concentration. Concurrent treatment with 0.3 μM flumazenil which did not affect chloride uptake and 1 μM FG 7142 for 10 days substantially attenuated the effects of FG 7142, suggesting that FG 7142 effects are mediated at the benzodiazepine site. Benzodiazepine receptor binding was increased in cultures treated for 10 days with 1 μM flumazenil or FG 7142, with an increase in receptor number in both cases but no change in apparent affinity. Neither flumazenil nor FG 7142 (1 μM for 10 days) altered GABA-independent chloride uptake, total cellular protein, protein synthesis or degradation, or neuronal survival. These results indicate that both chronic GABA-neutral and GABA-negative benzodiazepine exposures in cultured cortical neurons lead to increases in GABA-dependent chloride uptake and benzodiazepine binding. Effects of GABA-negative benzodiazepine exposure appear to be greater than those observed with GABA-neutral benzodiazepine exposure.

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 μM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2–10 μM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 μM) but not APV (50 μM) blocked the increase in chloride uptake due to kainate or quisqualate (10 μM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KCl) or ouabain pretreatment (5 μM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABA A receptor complex.

Chronic morphine administration augments benzodiazepine binding and GABAA receptor function

Behavioral and neurochemical evidence indicates links between the opioid and GABA neurotransmitter systems. To assess effects of chronic opiates on the major site of postsynaptic GABAergic activity, the GABAA receptor, we administered chronic morphine and naltrexone to mice and evaluated binding at the benzodiazepine and t-butylbicyclophosphorothionate (TBPS) sites and GABA-dependent chloride uptake. After morphine (3 days), benzodiazepine receptor binding in vivo but not in vitro was increased in cortex compared to placebo-treated mice. TBPS binding was unchanged in cortex, but muscimol-stimulated chloride uptake was increased at low doses of muscimol. Benzodiazepine and TBPS binding and muscimol-stimulated chloride uptake were unchanged in naltrexone-(8 days) compared to placebo-treated mice. When naltrexone was administered previously to block opiate sites, the increases in benzodiazepine binding and chloride uptake observed with chronic morphine were reversed. These results indicate that chronic morphine but not naltrexone enhances benzodiazepine binding and GABAA receptor function, perhaps by an action at opioid receptors.

Neurotoxic insecticides inhibit GABA-dependent chloride uptake by mouse brain vesicles

The neurotoxic insecticides endrin, dieldrin, aldrin, lindane (γ-1,2,3,4,5,6,-hexachlorocyclohexane) and deltamethrin inhibited γ-aminobutyric acid-dependent 36Cl − uptake by mouse brain vesicles. Of the insecticides examined, the chlorinated cyclodienes endrin and dieldrin were the most potent, producing 50% inhibition at 2.8 and 13.9 μM, respectively. Lindane and deltamethrin were less effective, and with deltamethrin the effect was incompletely stereospecific. These results demonstrate the disruption of γ-aminobutyric acid receptor-chloride ionophore function in mammalian brain by neurotoxic insecticides and provide evidence that this complex is the principal site of cyclodiene action.

Chemical Nature of Synaptic Transmission in Vertebrates

Chemical Nature of Synaptic Transmission in Vertebrates