GABA uptake by rabbit restiform body homogenates.

Restiform body (inferior cerebellar peduncle) preparations were obtained from rabbit brain stem slices and homogenized. When challenged with labelled GABA, these homogenates took it up briskly. We have characterized pharmacologically this uptake which resulted almost equally neuronal and glial. The neuronal component of the GABA uptake might be due to the adjacent cochlear nuclei coming along in the preparations, whereas the glial component probably belongs to the restiform body proper. Another possibility is that actually both components are due to the myelinated fibers and glia which make up the restiform body.

Modulation by acute stress of chloride permeation across microdissected vestibular neurons membranes: different results in two rabbit strains and CRF involvement.

Free hand isolation of adult rabbit vestibular Deiters' neurons and dissection of their single membranes allows the study of their ionic permeability characteristics in a microchambers device. In the case of hare-like rabbits, the dissection of such membranes presents evidence of a high basal permeation of labelled chloride, possibly related to mechanical disturbance of the plasma membrane-related cytoskeleton and activation of chloride channels. This did not apply to the laboratory strain of white New Zealand rabbits. However, membranes from hare-like rabbits which were stressed by being rotated on a platform before the experiment, behaved like those from the New Zealand strain. Vice versa, habituation to handling day after day of New Zealand rabbits resulted in a chloride permeation equal to that of unstressed hare-like rabbits. We propose that the stressful conditions result in the release of neurochemical messages to the vestibular Deiters' cells which influence their electrophysiological behavior. The corticotropin releasing factor (CRF), a stress-related peptide present in the climbing fibers, actually blocks the basal chloride permeation across the Deiters' membranes and this effect is partially reversed by its receptor antagonist, alpha-helical CRF [9-41].

Antiserum against S-100 protein prevents long term potentiation through a cAMP-related mechanism.

Long term potentiation (LTP) was induced in the CA1 region of rat hippocampal slices by tetanization of the Schaffer collaterals. Local pretreatment of CA1 with serum of rabbits immunized against S-100 prevented the potentiation. However, treatment of the slices with a membrane permeant cAMP analogue, such as 8-Br-cAMP, could protect against the blocking effect of anti S-100 serum. We suggest that in the rat endogenous S-100b is involved in transduction mechanisms during LTP induction, via its ability to stimulate adenylate cyclase. Possible mechanisms of this action are discussed.

Unraveling of important neurobiological mechanisms by the use of pure, fully differentiated neurons obtained from adult animals.

An important, and often overlooked, problem in the neurochemical approach to neurobiological problems is that analysis of tissue involves almost always a heterogeneous population of cells (neurons, glia and other types of tissue cells). The use of cell cultures has obvious limitations such as that they derive from embryonic or immediately postnatal animals; in addition, the cell culture conditions most certainly are quite different from the real tissue environment for the nerve cells. We underline here an alternative strategy, which is not new, but which, in our view, has already given formidable contributions to neurobiological studies and still is giving results of great importance. This is the technique proposed and used since the late fifties and early sixties by the senior author (H. Hydén). The method involves the isolation of the big vestibular neurons from the adult rabbit vestibular nucleus. The neurons, fully differentiated and performing a precisely defined function, are obtained rapidly and completely free from surrounding glial cells. The separate microbiochemical study of these cells and their surrounding glia has yielded already in 1962, the information that modifications in gene expression are associated with plastic modifications of the function of the relevant neurons, which take place in the behavioral event of learning. Another important concept was formulated in the same time period following determination of the activities of energy metabolism related enzymes separately in vestibular neurons and their glia under vestibular stimulation. This is the concept that, under increased functional activity glia increases its anaerobic metabolism and passes then on the resulting metabolites to the neurons for aerobic metabolism. Both these concepts (RNA and memory; metabolic cooperation between glia and neurons) are nowadays widely accepted. In addition, this approach with pure big nerve cells has allowed in recent years the discovery of a novel mechanism for chloride extrusion in these cells. This mechanism utilizes structures similar to GABA activated chloride channels in cyclic modifications resulting in the final extrusion of chloride ions. The energy for the process is provided by a protein phosphorylation step. Future approaches are warranted such as the possibility of recognizing by RT-PCR specific neuronal mRNAs and their modification in expression in relation to function and plastic modifications (learning). Another possible interesting application appears to be the recognition of the mRNAs for GABA(A) receptor subunits expressed here in these neurons in relation to the physiological and pharmacological characteristics of these native neuronal GABA(A) receptors.

Involvement of S-100 protein in anoxic long-term potentiation.

In in vitro rat hippocampal slices a short period (2 min) of hypoxia resulted in lasting potentiation of the population spike transynaptically evoked in CA1 by stimulation of Schaffer collaterals ("anoxic LTP"). Pretreatment of slices with antiserum against S-100 protein fully prevented this anoxic LTP. Since also "classical" (i.e., induced by high-frequency electrical stimulation) long-term potentiation is prevented by anti S-100 serum, this represents one more important similarity between these events.

Intracellular GABA-activated in-->out permeation of chloride across the Deiters' neuron membrane: modulation by phosphorylating activities.

The modulation of intracellular GABA activated 36Cl- in-->out permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg2+/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg2+ to the intracellular side buffer: a complete block of the stimulation by GABA of 36Cl- in-->out permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg2+ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg2+/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on 36Cl- in-->out permeation.

An electrogenic ionic pump derived from an ionotropic receptor: assessment of a candidate.

1. Data obtained studying permeability characteristics of single Deiters' membranes in a microchamber system show that intracellular GABA can activate chloride in-->out passage with a GABAA pharmacology. 2. The overall data suggest the presence of a chloride extrusion pump in these neurons based on intracellular GABA activated chloride channels. 3. This conclusion takes up a previous theoretical suggestion that ionic channels could work as ionic pumps provided an energy input modifies the energy profile along the permeation path. 4. According to our quantitative evaluation, this pumping mechanism works with a low yield and along a cycle with a strongly asymmetric behavior, being far from equilibrium due to powerful "leakage" pathways for chloride in these neurons.

Chloride permeation across the Deiters' neuron plasma membrane: activation by GABA on the membrane cytoplasmic side.

Single plasma membranes were microdissected from Deiters' neurons freshly obtained from the lateral vestibular nucleus of the rabbit and their chloride permeability was studied in a microchamber system. The basal in-->out 36Cl- permeation initially found was brought to zero by Zn2+, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and iodide. GABA on the membrane cytoplasmic side resulted in a measurable in-->out 36Cl- passage, which was blocked by the GABA(A) antagonists bicuculline and picrotoxin. This effect peaked at 1 microM GABA on the inner side of the membrane. At higher GABA concentrations, a strong desensitization of the effect was found. Stimulation of Cl- permeability by GABA on the extracellular side of the membrane peaked at much higher GABA concentrations, 10-100 microM. This excludes an effect due to passage of the neurotransmitter from the inner to the outer compartment in our microchamber device. Moreover, this possibility is also dismissed by the fact that 1 microM GABA on the membrane outside did not evoke any 36Cl- in-->out permeation. In addition, pentobarbitone by itself could also stimulate 36Cl- in-->out permeation when added on the cytoplasmic side of Deiters' membrane. On these bases and in agreement with our previous reports, we propose that structures behaving pharmacologically as GABA(A) receptors respond to low levels of GABA on the cytoplasmic side of these neurons' membranes. We suggest that these structures are devices that, at the expense of ATP consumed in their phosphorylation, extrude Cl- after postsynaptic GABA uptake into the Deiters' neuron.

Stimulation of chloride in-->out permeation across the Deiters' neuron membrane by pentobarbital on the cytoplasmic side: additional evidence of GABA(A) receptors acting as chloride extrusion pumps.

Pentobarbital stimulates 36Cl- permeation across single Deiters' membranes in a microchamber system, acting on classical, extracellularly facing, GABA(A) receptors. However, when applied on the membrane cytoplasmic side it activates per se labeled chloride in-->out permeation. No effect was found on chloride out-->in permeation. Similarly, at lower concentrations it facilitates the increase of 36Cl- in-->out permeation by application of GABA on the membrane inside, again via asymmetric chloride channels allowing in-->out but not out-->in passage. These data confirm that on the Deiters' membrane cytoplasmic side there are structures behaving pharmacologically as GABA(A) receptors whose function is that of a Cl- extrusion pump. This mechanism involves a cycle of activation-phosphorylation/desensitization-reactivation of the receptor complexes.

Modulation by calcium of Deiters' neuron GABAA receptors in the rabbit.

The function of classical GABAA receptors of the rabbit Deiters' neurons has been studied at the single membrane level by a biochemical micromethod involving the study of labelled chloride permeation. In particular, labelled chloride permeation across microdissected fresh single membranes was studied in a microchamber system. The stimulation of 36Cl- out-->in permeation by "extracellular" GABA was determined under different conditions in the respect of Ca++. When the conditions were such that "intracellular" Ca++ was 0.02 microM there appeared to be an optimal effect by GABA on chloride passage. Conditions presumably resulting in an increase of [Ca++]i beyond the level reported above led to a decreased GABA effect, especially at the highest GABA concentrations used (> or = 10(-4)M). However, complete removal of Ca++ by a high (12 mM) intracellular EGTA concentration erased completely the GABA effect. These results indicate that in these neurons an optimal GABAA receptor function requires [Ca++]i levels well below micromolar. The high [EGTA]i effect seems to imply that too low a [Ca++]i is also harmful to the proper function of these GABAA receptors. However, an alternative explanation is possible.

S-100 beta has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method.

The localisation of S-100 in mammalian CNS neurons has been under debate for more than two decades. We address the question with two polyclonal and two new monoclonal antibodies. The specificity and the distribution in rat brain is based on an antigen retrieval method. We present evidence that aldehyde fixatives mask S-100 beta in neurons, and that the immunoreactivity is retrieved after trypsinisation. Neuronal S-100 beta is also detected in unfixed and ethanol fixed sections. The neuronal immunoreactivity is partly solubilised from unfixed tissue sections with 2.5 mM EDTA and is completely extracted with 2.5 mM EDTA and 1% Triton X-100. Most of the glial S-100 beta is washed out from unfixed tissue sections with saline. S-100 beta has distinct distribution in neurons of the hindbrain, i.e., the brainstem and cerebellum, but is not observed in the forebrain. One of the monoclonal antibodies immunostained neither neurons nor glia when it had been absorbed with S-100 crosslinked to nitrocellulose membranes. The distribution of neuronal S-100 beta differed from that of other neuronal calcium binding proteins, such as calbindin and parvalbumin. It was confined mainly to cholinergic neurons of the hindbrain. The presence of S-100 beta in distinct neuronal populations may indicate neurotrophic effects of S-100 beta. The notion is supported by the capability of S-100 to cause neurite outgrowth in vitro.

The increase in Cl- permeation across the Deiters' neuron membrane by GABA on its cytoplasmic side is abolished by protein kinase C (PKC) activators.

1. Cl- ion outward permeation across microdissected Deiters' neuron plasma membranes is augmented by GABA on the membrane cytoplasmic side. When these neurons are preincubated with a PKC activator, phorbol-12,13-dibutyrate (PdBu), there is a complex pattern of effects on basal and GABA-activated 36Cl- in-->out permeation. A distinct fact is an increase in basal Cl- passage and a disappearance of the 10(-6) M GABA effect at [PdBu] = 0.1 microM. 2. Likewise, 0.1 microM oleylacetylglycerol (OAG) treatment erases the effect completely, further supporting a role for PKC in modulating GABA-stimulated Cl- in-->out permeation. 3. The inactive ester, phorbol-12,13-didecanoate (Pdd), at 0.1 microM, does not affect GABA stimulation of Cl- passage. 4. High concentration (15-20 microM) of OAG and PdBu block the "intracellular" GABA efefct. However, the 20 microM PdBu effect is reversed by 30 microM H7. 5. These results indicate a role of endogenous PKC in Cl- extrusion by GABAA receptors on the cytoplasmic side of the Deiters' neuron membrane.

Modulation by intracellular Ca++ of GABA activated Cl- extrusion from Deiters' neuron.

The increase in 36Cl- in --> out permeation across plasma membranes from the Deiters' neurons by GABA on the cytoplasmic side is modulated by Ca++. The GABA stimulation was maximal with Ca++ on the intracellular side at the physiologically likely concentration of 3 x 10(-8) M. A lower effect was found in the 10(-7) - 10(-5) M [Ca++]i range with a total disappearance of it at [Ca++] of .2 mM. This Cl- extrusion mechanism is likely to be involved in the establishment of a Cl- out --> in electrochemical gradient. Modulation of it by intracellular Ca++ transients may be of physiological importance.

Further evidence for the presence of gamma-aminobutyric acidA (GABAA) receptors on the cytoplasmic side of Deiters' membrane.

1. The permeation of labeled Cl- across single nerve membranes microdissected from rabbit Deiters' neurons was studied in a microchamber system. The in----out permeation of the ions was evaluated under control conditions and in the presence of either 10(-6) M GABA or 10(-6) M GABA plus 10(-5) M bicuculline methiodide (BMI) on the membrane cytoplasmic side. 2. In 32 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied with the addition of GABA. Within each experiment all the membranes were obtained from the same animal. 3. In an additional 10 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied in the presence of GABA plus bicuculline methiodide on the membrane cytoplasmic side. 4. The data show that 10(-6) M GABA on the Deiters' membrane cytoplasmic side stimulates Cl- permeation in----out by 42% (P = 0.0000001). When 10(-5) M BMI was present together with GABA, no stimulation of Cl- in----out permeation occurred.

Can Cl- ions be extruded from a gamma-aminobutyric (GABA)-acceptive nerve cell via GABAA receptors on the plasma membrane cytoplasmic side?

1. In this commentary we discuss results obtained by a micromethod for the study of Cl- permeability across single nerve membranes from rabbit Deiters' neurons. 2. These results showed the presence of GABAA receptors on the nerve cell membrane cytoplasmic side. 3. We could show that these receptor complexes have a higher affinity for GABA than their extracellularly facing counterparts. Moreover, they present a phenomenon of desensitization. Another distinct property is that upon activation by GABA, they expose positive charges at their cytoplasmic mouths. 4. We propose that these receptor complexes could function in situ as a device for extruding Cl- anions from the nerve cell interior. This phenomenon would create an electrochemical gradient for Cl- penetration into the cell upon the action of extracellular GABA, after its presynaptic release.

Stimulation of 36Cl- influx into rabbit cerebral cortex microsacs by the endogenous antigen S-100.

The rate of accumulation of 36Cl- ions into rabbit cerebral cortex microsacs has been studied under various conditions. Such a parameter is increased by 164% by 10(-4) M GABA, the effect being reversed in the presence of 10(-4) M bicuculline and 10(-4) M picrotoxin. Incorporation of the endogenous antigen S-100, by a preincubation of the fraction with the protein in the presence of 2.5 mM Ca++, brings about a 56% stimulation of 36Cl- influx rate. This effect does not seem to be additive with that of GABA. The S-100 effect is reversed by picrotoxin but not by bicuculline. The overall pattern of data suggests an action of the antigen directly at the GABAA receptor complex and in particular at the GABA activated Cl- channel.

Stimulation of 36Cl- permeation in the in----out direction across the Dieters' neuron membrane by GABA on its cytoplasmic side: effect of different ionic conditions.

The permeation of labelled Cl- ions across single plasma membranes microdissected from rabbit Deiters' neuron was studied in a microchamber system. In particular, we studied 36Cl- in----out permeation and its stimulation by 10(-6) M GABA on the cytoplasmic side under different ionic conditions on both sides of the membrane. Three main results were found: a) The GABA effect turns up at a cytoplasmic side [Cl-], 21 mM, in the range of the normal steady state intracellular Cl- concentration. It disappears both at tracer level of Cl- on the cytoplasmic side and when [Cl-] is high there (140 mM). b) The increase in ionic strength due to the equinormal substitution of monovalent anions (Cl- and acetate) with the trivalent impermeant anion citrate on both sides of the membrane erases the GABA effect even if Cl- is at the optimal cytoplasmic side concentration, 21 mM. c) Citrate ions reduce to the limit of significance the GABA effect even when they are only on the membrane extracellular side and [Cl-] is at the optimal level on the other side. These results confirm that GABA stimulated Cl- permeation in the in----out direction is via Cl- channels exposing positive charges at their cytoplasmic mouth. In addition, they point out that such GABA activated channels are endowed with a partial electrical positivity at their extracellular exit.

Further studies on the effect of ASF factor on Cl- permeability across the Deiters' neurone plasma membrane.

Experiments have been performed in order to assess whether the antisecretory factor (ASF) can influence the permeation of Cl- ions across the Deiters' neurone plasma membrane in the outward direction. ASF is a naturally occurring, acidic protein with a molecular weight of about 60,000, which specifically inhibits enterotoxin-induced intestinal secretion. Both basal and "intracellular" GABA activated Cl- permeability was studied, and the result confirmed the existence of GABAA receptors on the Deiters' membrane cytoplasmic side. ASF completely abolished the effect of "intracellular" GABA on Cl- permeability in the outward direction. However, ASF did not influence the basal Cl- permeability in the outward direction.

The mechanism by which intracellular GABA increases Cl- outward permeability across Deiters' neurone plasma membranes.

The present experiments aimed to clarify how the interaction of gamma-amino-butyric acid (GABA) with its receptors on the cytoplasmic side of the Deiters' neurone plasma membrane causes Cl- permeability to be higher in the in----out than in the opposite direction. To this end, the rate of 36Cl- in----out passage was studied in basal conditions, with GABA on the cytoplasmic side and in the presence of both GABA and an increased ionic strength buffer on that side. The results show that an increase in ionic strength reverses the GABA effect of 36Cl- permeability. The reversion being caused by changes in the buffer on the intracellular but not on the extracellular side. Our interpretation of this result is that the interaction of GABA with its cytoplasmic side receptors induces an exposure of positive charges only at the intracellular mouth of the intraneuronal GABA gated Cl- channels. This asymmetry would be the basis of the reported higher Cl- permeability in the in----out direction.

GABA A receptors on the cytoplasmic side of the Deiters' neurone plasma membrane: mechanism and functional implications.

A micromethod has been used for studying the passage of 36Cl- ions between two microchambers across the GABA acceptive plasma membrane of the rabbit Deiters' neurone, under various different conditions. The presence of 3.3 mM GABA, steady state intracellular concentration in situ (Okada & Shimada, 1976), on the cytoplasmic side of those membranes prolongs remarkably the time for the achievement of 36Cl- equilibrium across the membranes (from 4 minutes to 30 minutes). The effect appears to be mediated by "intracellularly oriented" GABAA receptor complexes as studied with GABAA antagonists such as bicuculline and picrotoxin. Intracellular GABA is still active in this effect at 10(-6) M. This phenomenon is discussed as, when coupled with the intracellular ATP producing machinery, being of importance for synaptically released GABA hyperpolarizing action on these neurones. The action of intracellular GABA on "internally oriented" receptors appears to involve the exposure of positively charged loci at the cytoplasmic side of the membrane. The consequent accumulation of intracellular Cl- at the membrane inner side would account for the higher overall permeability of those ions in the in----out direction. This circumstance was assessed by blocking the intracellular GABA effect either working at a relatively basic pH (8.4) inside or increasing the ionic strength in the intracellular compartment.