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.

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.

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Direct evidence for the presence of GABAA receptors on the cytoplasmic side of the Deiters' neurone membrane.

A newly developed micromethod has been used for studying the rate of passage of 36Cl- ions across single nerve membranes from rabbit Deiters' neurones. The application of gamma-aminobutyric acid (GABA) on the cytoplasmic side of those membranes increases the rate of passage of 36Cl- ions from that side to the other one across the membrane. The maximal effect is exerted by 10(-6) M GABA and it fades at higher neurotransmitter concentrations (10(-5) M to 3.3 x 10(-3) M). The cause of this fading of the effect appears to be a receptor desensitization phenomenon. The 10(-6) M GABA effect is reversed by both 10(-4) M picrotoxin and 10(-5) M bicuculline. The overall pattern of the data indicates the presence of GABAA receptors on the cytoplasmic side of these nerve membranes.

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].

Modulation by nitric oxide of rat brain GABAA receptors.

The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L-arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells.

Holger Hydén's technique of preparation of single Deiters' neurons and study of permeability characteristics of their plasma membranes.

The protocols described here refer to Hydén's technique of isolation and microdissection of vestibular Deiters' neurons from adult mammals. The isolation of Deiters' cells from bovine is described and an example is given of the immunocytochemical visualization of their GABA(A) receptors by monoclonal antibodies against the beta(2/3) subunit. In addition, the protocol of the method for isolation of Deiters' cells from adult rabbit brain stem, the preparation of their plasma membranes and the study of their permeability characteristics is presented. Also in this case, examples of its application to the determination of chloride permeability and its modulation by GABA are given.

Preincubation of homogenates from rat brain thalamus and striatum affects the rate of [3H]GABA uptake.

The effect of preincubation of brain homogenates on the subsequent uptake of [3H]GABA was studied in rat thalamus and corpus striatum. The results show that in both brain regions preincubation causes a decrease in the initial rate of [3H]GABA influx, the phenomenon being most evident in the thalamus.

GABA and chloride permeate via the same channels across single plasma membranes microdissected from rabbit Deiters' vestibular neurones.

The permeation of labelled gamma-aminobutyric acid (GABA) across single microdissected Deiters' membranes has been studied in a microchamber system. The GABA permeation is via pores which are blocked by 4,4'-diisothiocyanato stilbene-2-2'disulphonic acid (DIDS). As this substance blocks as well chloride permeation across these membranes we tested whether GABA and chloride permeate across the same pores. Membrane pre-treatment with different doses of corticotropin releasing factor (CRF), a membrane permeant cyclic AMP analogue and phalloidin parallelly block the permeation of the two substances. Thus, it is most probable that GABA and chloride pass across the same pores. These pores may be swelling activated ones, opened by the mechanical stress on the membranes in the microchamber system. The passage of GABA across these pores may be of physiological importance in the termination of GABA inhibitory action on the vestibular Deiters' neurones.

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.

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.

Labeling of non poly(A) associated RNA in the rabbit cerebral cortex 24 hours after a single electroconvulsive shock.

The labeling pattern of non poly(A) associated (poly(A)-)RNA of rabbit cerebral cortex was studied 24 hr after a single electroconvulsive shock (ECS). The animals were injected subarachnoidally with [3H]uridine and sacrificed 1 hr later. The fractionation pattern of labeled nuclear poly(A)-RNA in the cerebral cortex of ECS treated animals was identical to that of the controls. However, microsomal poly(A)-RNA from the treated animals showed an increased labeling of 18S ribosomal RNA. Also 28S RNA displayed a higher labeling but the effect was not statistically significant. These results indicate a more efficient production of ribosomal RNA in the late post-ECS period which might be in relationship with an increased activity of brain protein synthesis machinery.

Antisecretory factor peptide derivatives specifically inhibit [3H]-gamma-amino-butyric acid/36Cl- out-->in permeation across the isolated rabbit Deiters' neuronal membrane.

AIMS: Antisecretory factor (AF) is a 41-kDa protein, its main function being the regulation of intestinal ion/water transport, but it also inhibits chloride and gamma-amino-butyric acid transport across nerve cell membranes. The present experiments were designed to evaluate whether the same AF peptide sequence mediates the permeability effects seen at the nerve cell membrane and in the rat small intestine. METHODS: Four peptides were prepared by the solid phase technique with sequences derived from positions 1-51 of the full-length antisecretory factor AF and tested on nerve cell membranes isolated from rabbit Dieter cells. RESULTS: AF peptides containing the active 36-51 peptide exerted a blocking effect of the out-->in permeation of 36Cl- as well as of [3H]-gamma-amino-butyric acid. The minimal dose causing inhibition, however, varied between 10(-11) m (AF10) and 10(-7) m (AF13). The most potent peptides have been shown previously to be active in inhibiting experimental diarrhoea in vivo in small intestinal ligated loops in rats. The non-active sequence AF23-32 did not inhibit any of the two permeation markers in vitro, a result which supports the lack of activity found also in vivo. CONCLUSION: The results suggest that AF, or AF derivatives, counteract intestinal hypersecretion by blocking anion permeation across large anionic pores. Such a blocking effect could also influence the generation of action potentials in enteric nerve cells controlling the intestinal water and ion transport system.

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.

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.

Pattern of labeling of rabbit cerebral cortex soluble proteins 24 hours after a single electroconvulsive shock.

The labeling of proteins in the particulate subcellular fractions and in the cell sap was studied in the cerebral cortex of rabbits 24 hours after a single electroconvulsive shock (ECS). To this end the animals were injected with [3H]valine subarachnoidally and sacrificed 30 minutes later. The incorporation of the labeled amino acid into proteins was slightly increased in all subcellular fractions but the increase was significant only for soluble proteins. Referring to the type of soluble polypeptides found in the cortex at this post ECS phase, no modification was found in the SDS electrophoresis pattern of steady state proteins. The fractionation pattern of labeled soluble proteins showed in one third (3 out of 9) of the experiments a remarkable stimulation of the synthesis of two polypeptides (25,000 and 54,000 Dalton MW), which were not labeled in the controls.

An electrophoretic method for the determination of the proportion of gamma-aminobutyric acid in a mixture of labeled neurotransmitter and its catabolites.

An electrophoretic method for the separation of gamma-aminobutyric acid (GABA) from its metabolites after GABA-transaminase attack is presented. The method is based on the fact that at neutral pH GABA has no net electrical charge, whereas its major metabolites, succinic acid and Krebs cycle intermediates, are negatively charged. The method appears to be especially suitable for evaluation of true-labeled neurotransmitter within the radioactivity which is found in synaptosomes after labeled GABA-uptake studies.

Components of basal and GABA activated 36Cl- influx in rat cerebral cortex microsacs.

The basal and GABA activated accumulation of labelled chloride in rat cerebral cortex microsacs has been studied as a function of incubation time. Basal accumulation has biphasic kinetics within 2 minutes of incubation with two components clearly visible. GABA activated stimulation has two phases as well, starting from (GABA) = 10(-5) M, one appearing within seconds of incubation range and the other one within tens of seconds. Both GABA-activated components are blocked by bicuculline methiodide (BMI). Practically no effect by 10(-3) M nipecotic acid was found on either the comparatively rapid or the slower phase. The two GABA activated components may correspond to two different populations of sealed vesicles with different receptor concentration per internal volume.