Marc A. Dichter

Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture.

(1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride. (2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter. (3) The neurons are much less sensitive to glycine and insensitive to taurine. (4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects. (5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.

Electrical excitability of cultured adrenal chromaffin cells.

1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro‐electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded ‐50mV. 3. Chromaffin cells were capable of generating all‐or‐nothing over‐shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(‐6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae.

Biochemical correlates of GABA function in rat cortical neurons in culture

Serial biochemical studies of a rat cortical tissue culture system in which synapses regularly form showed that gamma-aminobutyric acid (GABA) is present in the cultures and increases with their maturation. The tissue GABA concentration in mature cultures is similar to that of adult rat cortex in vivo. The synthetic enzyme, glutamate decarboxylase, also increases with age as does high affinity GABA uptake. GABA uptake was blocked by L-2,4-diaminobutyrate (DABA) and had the properties of neuronal GABA uptake. Specific release by depolarizing media of both exogenous [3H]GABA and GABA synthesized from D-[U-14C]glucose was demonstrated. The GABA released by high potassium media had higher specific activity and a greater contribution from glucose (as compared to acetate) than GABA found in the medium in the absence of depolarization. Calcium dependency of evoked GABA release could be shown only after pretreatment of cultures with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N-tetraacetic acid or EGTA. Synaptosomes may exhibit greater calcium dependence of evoked transmitter release than intact cells in culture because their intracellular calcium stores are depleted during preparation. Glycine uptake by the cultures was much less in amount than was GABA uptake, and specific release of glycine could not be demonstrated. Specific binding of both a GABA agonist ([3H]muscimol) and an antagonist ([3H]bicuculline) was shown by membranes prepared from the cultures. By contrast, when [3H]muscimol binding to intact cells was studied, essentially all binding was sodium dependent and had the properties of GABA uptake binding. We conclude that the use of [3H]muscimol for receptor studies is valid only after the elimination of GABA uptake systems. Biochemical data from these studies support the concept that GABA is the transmitter for many cortical synapses. Glycine and taurine are not likely to be transmitters in these cortical cultures. When considered together with physiological data from the preceding paper, we have satisfied Wermans criteria (see ref. 36) for accepting GABA as the major inhibitory transmitter in the cortical culture system.

Anticonvulsant Sensitivity of Absence Seizures in the Tottering Mutant Mouse

Summary: Homozygous tottering mice (tg, autosomal recessive) exhibit frequent spontaneous “absence” seizures accompanied by bilaterally synchronous spike‐and‐wave or polyspike electrocorticographic discharges. In adult tottering mice, the antiepileptic effects of a single dose of ethosuximide, diazepam, phenobarbital, or phenytoin were assessed using continuous electrocorticographic recording to monitor seizure incidence. The dose chosen for each drug was selected to correspond to an effective antiepileptic dose in standard murine models. Ethosuximide, 150 mg/kg, diazepam, 1.4 mg/kg, and phenobarbital, 25 mg/kg were effective against absence seizures. In contrast, phenytoin at 5, 10, 30, or 60 mg/kg produced no significant reduction in the incidence of absence seizures. These results suggest that absence seizures in the tottering mutant may represent a relatively specific pharmacological model for the identification of drugs effective for clinical absence epilepsy, and emphasize the potential value of this new model for the study of fundamental mechanisms of absence of epilepsy and the actions of antiepileptic drugs.

Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography

Autoradiographic studies of rat cortical cultures were conducted with tritiated transmitters and related drugs. Autoradiographs prepared from cultures incubated in [3H]GABA showed selective labeling: dense accumulations of silver grains over the somas and all processes of approximately 30-50% of the neuronal population, few grains over the non-neuronal cells. This labeling was blocked by diaminobutyric acid (DABA) and sodium-free media but not by beta-alanine and thus has the characteristics of GABA uptake in other neuronal systems. There were no obvious differences in the size, shape, number of processes or distribution in the culture between neurons which accumulated GABA and those which did not. Similar cultures incubated in either [3H]glycine or [3H]glutamate and processed by autoradiography resulted in a much different distribution of silver grains than that seen for [3H]GABA. Following incubation in [3H]glycine, silver grains were distributed uniformly over all cells in the culture, both neuronal and non-neuronal. This distribution suggests a metabolic and not a neurotransmitter role for glycine in the cultures, as would be expected of neuronal cells derived from cerebral cortex. Glutamate incubations resulted in the appearance of silver grains over only the non-neuronal cells with very few over the neuronal population. Autoradiograms were also prepared following incubation in the potent GABA receptor agonist [3H]muscimol. These autoradiograms were indistinguishable from those obtained following [3H]GABA incubation. Thus, a finite population of neurons was densely labeled, the labeling was blocked by the GABA uptake inhibitors DABA, nipecotic acid, guvacine and Na+-free media, while substances which interact with the GABA receptor, bicuculline methiodide, THIP, isoguvacine and the noncompetitive antagonist, picrotoxin, were without effect. These results demonstrate that the affinity of muscimol for the GABA uptake site far outweighs its affinity for the GABA receptor site in autoradiographic experiments where intact cells are employed, presumably because its binding to receptors is fleeting. Therefore, muscimol autoradiography may not be informative about GABA receptor localization. These autoradiographic studies suggest that nearly half the neurons in our culture system are GABA neurons but disclosed no morphological handle for GABA neurons.

Benzodiazepine binding and interactions with the GABA receptor complex in living cultures of rat cerebral cortex

Benzodiazepines bind to living cultures of dissociated rat cerebral cortex. This binding is saturable, and kinetic analyses indicate that the binding is to a single class on sites with kinetic constants very close to those obtained using neuronal membrane preparations. The efficacy of a number of benzodiazepines, xanthine derivatives and other drugs in competition experiments is similar to that seen in neuronal membrane preparations, and suggests that the benzodiazepine binding site studied in these investigations is the same as that found in neuronal membrane preparations and believed to be the pharmacologically active benzodiazepine binding site. GABA agonists increase the binding of benzodiazepines, and this increase has the same order of efficacy as their ability to hyperpolarize the neurons when applied at known concentrations with muscimol greater than GABA greater than THIP. At high concentrations THIP potentials benzodiazepine binding to the same level as GABA. Diazepam increases the ability of both GABA and THIP to hyperpolarize the neurons as well as the amplitude of spontaneous IPSPS which, in this system, are GABA-mediated. The competitive GABA antagonist bicuculline methiodide slightly decreased benzodiazepine binding and also antagonized the increase due to GABA. The non-competitive GABA antagonist picrotoxinin had no effect on benzodiazepine binding but did antagonize the GABA-induced increase in benzodiazepine binding. Replacement of Cl- in the incubation medium by acetate, which does not permeate the GABA-mediated Cl-- ionophore, increases benzodiazepine binding, and GABA no longer increases the binding. Picrotoxinin decreases the increase in benzodiazepine binding is Cl--free media, and this decrease is blocked by GABA. These results are discussed in terms of interactions at the GABA receptor complex consisting of a GABA recognition site, a benzodiazepine recognition site, a picrotoxinin recognition site, and a Cl- ionophore.

Chick sympathetic neurons develop receptors for α-bungarotoxin in vitro, but the toxin does not block nicotinic receptors

Studies were carried out on the development and physiological role of receptors for alpha-bungarotoxin (alphaBT) on chick embryo sympathetic neurons maintained in dissociated cell culture. Neurons from embryos of 13 days incubation (E13) developed alphaBT receptors in vitro with a time course and to a maximum level per cell similar to that previously observed for such neurons in vivo. In vitro receptor development by E11 and E8 neurons was also present, but (in comparison with E13 neurons) reached somewhat lower maximal levels. Receptor development in vitro was not affected by exclusion of non-neuronal cells from the cultures. In the present and in previous studies, binding of alphaBT to chick sympathetic neurons was blocked by a variety of ligands of nicotinic acetylcholine receptors. However, saturating concentrations of toxin were found here to be ineffective in blocking either (a) release of [3H]norepinephrine from the cultured neurons elicited via nicotinic stimulation of acetylcholine receptors or (b) depolarizing responses of the cultured neurons elicited by iontophoretically applied acetylcholine and nicotine. Kinetic studies further revealed that, while the idssociation of alphaBT from the cultured neurons is considerably enhanced in the presence of a cholinergic ligand (100 micrometer nicotine), the rate of this dissociation (t1/2 congruent to 30 min) appears to be too slow to account for the inability of the toxin to block nicotinic responses. Such findings show that chick embryo sympathetic neurons can develop receptors for alphaBT both in vivo and in vitro, but that the toxin does not block activation of their nicotinic acetylcholine receptors. The physiologic nature of specific binding sites for alphaBT on such neurons is thus presently unclear.

Effects of norepinephrine on rat neocortical neurons in dissociated cell culture

Intracellular recordings were made from neurons in dissociated cell culture of neocortex during application of norepinephrine (NE) or other adrenergic agonists. In the population of neurons generally studied, greater than 18 micron in diameter, adrenergic agonists from 1 nM to 50 microM produced no change in membrane potential or input resistance 120 cells). Adrenergic agonists increased synaptic activity impinging on the impaled cell in 25/120 neurons (21%). In neurons in cocultures of locus coeruleus and cerebral cortex, again the same synaptic response to perfusion with NE was noted in 13/93 neurons (14%). In addition, direct effects of NE were noted on 6/93 neurons recorded from in cocultures, all close to the explant. In these cells, NE hyperpolarized the membrane in association with a small decrease in input resistance (11%). These responsive cells may have originated within the explant. A paradigm was used for testing the possibility of a responsive element in the cultures distinct from the impaled soma. Hot spots were found using concentrations of isoproterenol as low as 10 nM.

Glycogen accumulation in rat cerebral cortex in dissociated cell culture

Incorporation of [3H]glucose into [3H]glycogen was demonstrated in individual coverslip cultures of dissociated rat cerebral cortex. The time course of incorporation of [3H] glucose into [3H]glycogen was investigated, and it was found that [3H]glycogen accumulation monotonically increased during at least the first hour of incubation with [3H]glucose. In order to identify which cells accumulate glycogen in these cultures we attempted to demonstrate cytochemically the localization of glycogen. We found, however, that conventional aqueous methods of glycogen cytochemistry did not reliably or consistently stain the cultures. By labelling glycogen using [3H]glucose, we were able to show that the entire [3H]glycogen compartment was extractable by water after ethanol fixation. Therefore we developed a non-aqueous technique which preserves tissue glycogen by exploiting its solubility properties. Using this technique we were able to cytochemically demonstrate glycogen in at least two different cell types in the cultures.

Physiology and pharmacology of olfactory bulb neurons in dissociated cell culture

Cells from olfactory bulbs of embryonic rats were grown in dissociated cell culture for up to 5 weeks. Both neurons and non-neuronal cells grew in these cultures, with a variety of neuronal populations appearing. A population of 20-25% of the neurons were GABAergic by the criterion of [3H]GABA uptake. Electrophysiologic measurements were made of the baseline activity of the cultured neurons. Cells showed a mean resting potential of 60.1 +/- 1.2 mV and a mean input resistance of 87.6 +/- 9.5 M omega. All cells were sensitive to microperfusion of GABA with half-maximal effect occurring at about 20 microM. Glutamate was universally excitatory but with variations in degree. Carnosine (beta-Ala-L-His), tested over the concentration range of 10 nM to 100 microM, had no effect on input resistance, resting potential, action potential shape, on-going synaptic activity or the responsiveness to either GABA or glutamate. These results are further evidence against a role for carnosine as the excitatory transmitter of the primary olfactory afferents.