Protein synthesis in the hippocampal pyramidal cells of rats during a behavioral test.

Protein synthesis was studied in the pyramidal nerve cells of the CA3 region in the hippocampus of rats uring a behavioral test involving transfer of handedness. A new electrophoretic technique was used for separation of 10(-7) to 10(-9) gram of protein and radiometric determination of the various protein fractions. After interventricular administration of (3)H-leucine, protein synthesis of two fast-moving fractions was significantly higher bilaterally in the hippocampus of the trained rats. There was also a trend to lateralization of the highest protein synthesis to the learning side.

Enzyme changes in neurons and glia during barbiturate sleep.

During barbiturate sleep of rabbits, the succinoxidase activity in isolated neurons and glia from the caudal part of the reticular formation was lower than that during physiological sleep. No rhythmical, inverse enzyme changes were detected in barbiturate sleep in the neuron-glia unit, such as were found in physiological sleep.

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.

Fractionation on the microscale of brain polyribosomes by electrophoresis on acrylamide gels.

A method is described for brain polyribosome fractionation by acrylamide gel electrophoresis. Brain polyribosomes were run in 2.0% gels in quartz capillaries of 800 mum inner diameter where the gels were supported by capillary force. The gels could then be ultraviolet-scanned in situ. Amounts of brain polyribosomes as small as 10-10(-3) A260nm unit could be analysed by this method. The method was checked by running a macroscale-prepared brain polyribosome sample. The various electrophoretic bands obtained showed a favourable A260nm: A280 ratio. A short RNase treatment caused the disappearance of the slowly migrating bands and the emergence of a predominant band migrating faster than the dimer. The various polyribosomal bands were then identified by comparison with the mobility of polyribosome fractions taken from a sucrose gradient fractionation. Finally, the electrophoretic pattern of brain polyribosomes compared favourably with the pattern obtained by the classic method of sucrose gradient sedimentation. The electrophoretic fractionation of polyribosomes prepared from one rat hippocampus (80 mg) is presented.

Inhibition by sodium valproate of the transport of GABA through the Deiters' neurone plasma membrane.

The transport of GABA through the microdissected plasma membrane Deiters' neurone reflects the physiological event of postsynaptic uptake of GABA by its uptake carrier. Sodium valproate at concentrations greater than or equal to 2.4 mM was able to decrease markedly (57%) such a transport. This effect, which reduces the efficiency of the GABA postsynaptic inactivation process, might be a mechanism for the potentiation by valproate of the synaptic action of GABA.

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.

Evaluation of the electrophysiological consequences of GABA removal from the synaptic cleft by Na+ ion transport-coupled neuronal uptake.

The pre- and postsynaptic electrophysiological consequences of a carrier-mediated, Na+ ion transport-coupled removal of gamma-aminobutyric acid (GABA) from the relevant synaptic clefts are discussed. Assuming for the GABA internalization process a stoichiometry like GABAo + 3NA+o + K+i in equilibrium GABAi + 3Na+i + K+o and a synaptic cleft GABA maximal concentration of 100 microM we calculated the presynaptic depolarization associated with GABA removal between 11.5 and 38.2 mV. At the postsynaptic level the effect appears to be less marked.

A calculation method for evaluating the time course of GABA removal from a synaptic cleft by presynaptic uptake systems.

A calculation method for evaluating the time course of gamma-aminobutyric acid (GABA) removal from a synaptic cleft by presynaptic uptake is suggested. The evaluation of the actual time required to remove GABA requires the knowledge of: (a) KM's and Vmax's (mol/min/mg protein) of the synaptosomal uptake systems in a certain brain area; (b) the synaptosomal volume per mg of protein in the synaptosomal preparation used; (c) the mean sphere diameter for synaptic boutons in the brain area considered and the proportion of GABAergic nerve terminals.

Membrane-bound S-100 protein on nerve cells and its distribution.

In this study we present data supporting a heterogeneous distribution of the brain-specific S-100 protein on nerve cell plasma membranes from isolated neurons. This has been shown in a previous study by immunoelectron microscopy using peroxidase-conjugated anti-S-100 antiserum and in this study by immunofluorescence using fluorescein-conjugated anti-S-100 antiserum, the specificity of which is discussed. Incubation of the cells at +23 degrees C or at +4 degrees C did not change the distribution pattern of S-100 over the cell membranes, nor did incubation in antimetabolites, or incubation with univalent Fab fragments. We conclude that the membrane-bound S-100 protein with its heterogeneous and polar distribution on the nerve cells is a sign of a protein differentiation of the neuron.

Alteration of the pattern of hippocampal nerve cell RNA labelling during training in rats.

RNA synthesis in the CA3 region of brain hippocampus was studied in rats trained to a handedness reversal task. The newly synthesized RNA, labelled by intraventricular injection of radioactive orotic acid, was extracted and analyzed by micromethods. The result was following. In the trained animals the incorporation of the labelled precursor into RNA, corrected by the pool radioactivity, was almost double in comparison with the controls. In the trained animals there was a stimulation of the incorporation in the high molecular weight (greater than 18S) RNA region and in two specific low molecular weight regions: 8-9 and 16-17S. Taking in account previous results of other authors, these data appear to imply a stimulation of brain mRNA synthesis as a result of training.

Increased binding of GABA to its post-synaptic carrier sites on the plasma membrane of Deiters' neurons after a learning experiment in rats.

A new micromethod for studying the interaction of gamma-aminobutyric acid (GABA) with its post-synaptic uptake in a defined type of nerve cell is described. The method involves the isolation by free hand microdissection of Deiters' nerve cells from the rabbit lateral vestibular nucleus and their incubation with tritiated GABA in the presence of 100 mM Na+ at 4 degrees C. From the binding data a Kd was calculated for this interaction of 104 nM and a BMax of 8.4 X 10(5) sites/neuron. The method was applied to the study of the modifications of the binding of GABA to Deiters' neurons from control rats and rats trained to balance on a steel wire in order to reach food. This performance is a powerful stimulation to the vestibular system. The results show that, in the binding experiment with 100 nM [3H]GABA, the amount of GABA which specifically binds to the Deiters' neurons is increased by 38% in the trained rats. Analogously, when the incubation with GABA was performed at 37 degrees C, involving an intake of GABA into the neurons, the amount of GABA taken up increases by 50% in the trained group. No GABA-binding or uptake increase was found when animals were subjected to intense vestibular stimulation for a short period without learning. These results indicate that when rats learn a behavioral test which involves an improved vestibular control, there is a specific neurochemical modification in the neurons of the lateral vestibular nucleus. This modification seems to be of importance for the physiology of the neuronal circuits controlling the vestibular function in the rat.

gamma-Aminobutyric acid stimulates chloride permeability across microdissected Deiters' neuronal membrane.

Fluxes of 36Cl- across freshly prepared Deiters' neuronal membranes have been studied in a two-compartment microchamber simulating the extra- and the intracellular space. The rate of 36Cl- influx was enhanced by gamma-aminobutyric acid (GABA) (10(-4) M), the effect being reversed by picrotoxin (10(-4) M) and by bicuculline (10(-5) M). Diazepam (10(-8)-10(-7) M) did not potentiate the response to GABA and rather depressed it. However, a barbiturate site is most probably present in the GABA receptor complexes since pentobarbitone (10(-4) M) was able to stimulate 36Cl- permeability to the same extent as GABA itself.

Increase in chloride ion permeability across the nerve cell membrane after the endogenous antigen S-100 incorporation.

36Cl fluxes through microdissected Deiters' neuronal membranes have been studied in a microchamber device simulating the extra- and intracellular compartments. GABA stimulates Cl- permeability through the membranes by 24%. Also, S-100/Ca2+ incorporation into the Deiters' membrane increases 36Cl- permeation to a similar extent. The two effects do not appear to be additive. This circumstance is interpreted as indicating that S-100/Ca2+ exerts its effects via postsynaptic GABAA receptor complexes.

Protein pattern alterations in hippocampal and cortical cells as a function of training in rats.

The study reports changes in the protein pattern and incorporation of L-[U-14C]-leucine in brain cells of the hippocampus and sensorimotor cortex of rats. The following subcellular fractions were analyzed by SDS-acrylamide gel electrophoresis: plasma membranes, synaptosomal membranes and synaptic mitochondria. Recurring reversal training gave an increased synthesis of synaptosomal membrane proteins with mol. wt. 35,000-45,000 and 60,000 and 100,000 in trained animals compared to active controls. Lesser changes were observed in plasma membrane and synaptic mitochondria fractions. Of the brain areas studied, the hippocampal synaptosomal fraction showed an initial, temporary response, and the cortical cell fractions responded subsequently. Judged from the time sequence of the protein response, it seems that recurrent reversal training induces a change in synaptic protein towards higher molecular weights, suggesting that these changes reflect a modification of the distribution of synaptic protein.

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].

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.

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.

Incorporation of amino acids into protein in different brain areas of rat, subjected to enriched and restricted environment.

The incorporation of [14C]valine into acid-insoluble brain material from rats living in enriched and restricted environment, respectively, was studied. Quantitative estimations showed higher incorporation into brain protein of frontal, entorhinal, cerebellar and visual cortex and the hippocampus in enriched environment animals compared to restricted environment animals. This finding was confirmed by autoradiography.