Robert H. Thalmann

Taurine in hippocampus: Localization and postsynaptic action

Both immunocytochemical and electrophysiological methods have been employed to determine whether the localization of the taurine synthetic enzyme, cysteine sulfinic acid decarboxylase, (CSAD) and the postsynaptic action of taurine in the CA1 region of rat hippocampus are consistent with the hypothesis that taurine may be used as a neurotransmitter by some hippocampal neurons. At the light microscopic level, CSAD-immunoreactivity (CSAD-IR) was found in the pyramidal basket cells, and around pyramidal cells in stratum pyramidale and stratum radiatum. At the electron microscopic level, CSAD-IR was seen most often in the soma and the dendrites and was rather infrequent in the axon or the nerve terminals. Electrophysiological observations on the in vitro hippocampal slice demonstrated that pyramidal neurons respond to artificially applied taurine with inhibition that depended in large part upon an increased chloride conductance. Although electrophysiological observations are consistent with a neurotransmitter role for taurine, results from immunocytochemical studies suggest a minor role for taurine as a neurotransmitter. In fact, immunocytochemical observations suggested that taurine may be used as a neurotransmitter only by a small number of pyramidal basket interneurons, the vast majority of CSAD-positive neurons may use taurine for other functions.

Biphasic response of hippocampal pyramidal neurons to GABA

GABA released either iontophoretically or synaptically near pyramidal neurons in the CA1 region of the rat hippocampal slice could produce a biphasic response: a hyperpolarization followed by a depolarization. The depolarizing component elicited by either method was accompanied by an increased membrane conductance, and a reduction in neuronal discharge. The depolarization was reversed at a potential which was less negative than the resting membrane potential; it was blocked by antagonists of GABA action such as picrotoxin; it was sensitive to manipulation of extracellular chloride concentration; and it persisted in the presence of concentrations of cobalt or manganese which were sufficient to block evoked synaptic activity. Iontophoresis of GABA near the apical dendrites elicited an initial depolarization rather than an initial hyperpolarization, suggesting a dendritic origin for the depolarizing component. Together, these results suggest that GABA can produce, in the same neuron, both hyperpolarizing and depolarizing responses which depend at least in part upon changes in chloride conductances.

Some factors that influence the decrement in the response to GABA during its continuous iontophoretic application to hippocampal neurons.

The response decrement that occurs during continuous iontophoretic application of GABA to hippocampal neurons was characterized by intracellular methods in the rat hippocampal slice. Using several paradigms that compared the responses to GABA with those to poorly transported analogues, we then identified a large component of this decrement that appeared to be independent of GABA uptake and metabolism, and that is probably independent of intracellular chloride accumulation as well. This decrement, which both developed and recovered with half times that average between 3 and 5s, is too brief to directly account for long-term plasticity of the GABA synapse. However, its time course is appropriate to participation in the development of cellular responses to brief flurries of GABA-mediated inhibitory postsynaptic potentials that may occur normally, or that may occur abnormally during a seizure or artificial tetany.

Reversal properties of an EGTA-resistant late hyperpolarization that follows synaptic stimulation of hippocampal neurons

A late hyperpolarization (LH) which follows low level to moderate synaptic stimulation of neurons in the hippocampal formation neurons was previously shown to consist primarily of a potassium conductance. The present experiments showed that when extracellular cesium was present in order to reduce anamolous rectification, the relation between LH amplitude and membrane potential was linear over a range of negative membrane potentials between approximately -60 and -120 millivolts. In addition, the LH was resistant to blockade by the calcium chelator EGTA. Each of these results differ with those expected if the underlying conductance of the LH were similar to several known potassium conductances of vertebrate neurons, including presently known potassium conductances that are activated by calcium.

Regulation of γ-aminobutyric acidB (GABAB) receptors in cerebral cortex during the estrous cycle

We examined binding of the GABAB receptor agonist baclofen to brain synaptic membranes as a function of the natural variations in gonadal steroids that occur during the estrous cycle of the adult rat. We found that the binding of baclofen to neocortical membranes varied systematically as a function of the estrous cycle, with the lowest binding occuring during the estrus stage. Binding to archicortical (hippocampal) and hypothalamic preparations also varied with the estrous cycle, except that the lowest level of binding in these latter cases occurred during the diestrus stage. The variation of [3H]baclofen binding during the estrous cycle was different with respect to the binding of [3H]muscimol, an agonist for GABAA receptors, and [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist for serotonin 5-HT1A receptors that shares similar G proteins and effectors with GABAB receptors. Saturation binding studies of cortical GABAB receptors showed that apparent receptor density (Bmax) rather than affinity (Kd)best accountd for the change in binding during the estrous cycle in that Bmax), like total specific binding, was at a minimum during the estrus stage. The robust regulation of GABAB receptors in neocortex was unexpected and its functional significance is at present unknown. However, the correlation of the menstrual cycle with mood and other behavioral changes, and the correlations of the estrous and menstrual cycles with seizure susceptibility, may somehow depend upon hormonal regulation of transmitter systems such as the one we have observed here.

Blockade of a late inhibitory postsynaptic potential in hippocampal CA3 neurons in vitro reveals a late depolarizing potential that is augmented by pentobarbital

These experiments show that blockade of a late inhibitory postsynaptic potential (IPSP) in rat hippocampus by injection of GTP gamma s into a single monitored neuron, or by injection of pertussis toxin into the hippocampus, exposed a synaptic potential that was depolarizing relative to the early, GABAA mediated IPSP. The reversal potential of this late depolarizing potential (LDP) was 10-12 mV positive to that of the early IPSP. The response was augmented by 40-60 microM pentobarbital, and the augmented response appeared to be sensitive to picrotoxin, an antagonist of GABAA action. The LDP is comparable to a depolarizing GABAA synaptic response that had been previously observed only when synaptic behavior of slices was grossly altered by exposure to pentobarbital or 4-aminopyridine.

Effect of 2-hydroxy-saclofen, an antagonist of GABAB action, upon the binding of baclofen and other receptor ligands in rat cerebrum

2-Hydroxysaclofen (2-OH-saclofen), a newly available compound which blocks certain physiological actions of the gamma-aminobutyric acidB (GABAB) agonist, baclofen, was found to displace [3H]baclofen at least 10-fold more potently than did phaclofen, a previously available antagonist of GABAB action. 2-OH-Saclofen reduced both the affinity and apparent density of baclofen binding sites and displaced baclofen binding at least 60-fold more potently than it displaced the binding of ligands for 3 other transmitters present in the rat cerebral cortex.

Effect of Guanosine 5′-O-(3-Thiotriphosphate) and Calcium on γ-Aminobutyric AcidB Binding as a Function of Postnatal Development

Abstract: We have examined the development of γ‐aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of ∼50% with the GABAB ligand (–)‐[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium‐ and guanosine triphosphate‐binding protein (G protein)‐regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTPγS was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptor affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTPγS may affect the GABAB receptor by different mechanisms.

Effects of Dihydropyridine Calcium Channel Ligands on Rat Brain γ-Aminobutyric AcidB Receptors

This study shows that low nanomolar concentrations of the calcium channel antagonist nifedipine displaced [3H]baclofen labeling of γ‐aminobutyric acidB (GABAB) receptors, whereas similar concentrations of two calcium channel agonists stimulated this GA‐BAB receptor labeling. Neither effect was likely to be due to dihydropyridine (DHP) binding to baclofen recognition sites, because the inhibitory ligand nifedipine primarily affected apparent receptor density rather than affinity. Although these results could reflect the coupling of GABAB receptors with calcium channels, they do not rule out other, possibly more direct interactions between GABAB receptors and DHP binding sites. These DHP effects occur at much lower concentrations and display other significant differences from previously reported effects of DHPs on other transmitter receptors.

Regulation of cyclic AMP level by progesterone in ovariectomized rat neocortex

Exposure of neocortical slices to progesterone, without prior treatment with estrogen, augmented forskolin-induced cyclic AMP within 15 min. 30 nM progesterone produced approximately 1/2 the maximal effect but as little as 10 nM progesterone produced a detectable increase in cyclic AMP. When forskolin was replaced by dideoxyforskolin, an analog that does not directly stimulate adenylyl cyclase but shares many of its other actions, progesterone did not augment cyclic AMP. Progesterone also failed to affect increased cyclic AMP that followed exposure to norepinephrine or isoproterenol. The effect of progesterone upon cyclic AMP was also evident when tetrodotoxin was added to block voltage-dependent sodium channels, suggesting that intercellular communication that is dependent upon action potentials was not necessary. The effect of progesterone was at least partially blocked by antagonists of GABAA receptor action, suggesting the involvement of GABAA or GABAA-like receptors. The effect of progesterone was also not homogeneous over the neo cortex. While forskolin-stimulated cyclic AMP was augmented by progesterone in the parietal and occipital regions, it was suppressed in the frontal region. These results are envisioned as a progesterone action upon a small and perhaps compartmentalized component of the cellular cyclic AMP system, an effect that is made detectable in our whole-tissue assay by the well known ability of forskolin to potentiate many hormonal effects upon cyclic AMP.