Search

Transient receptor potential melastatin 7 (TRPM7), a calcium-permeable, ubiquitously expressed ion channel, is critical for axonal development, and mediates hypoxic and ischemic neuronal cell death in vitro and in vivo. However, the downstream mechanisms underlying the TRPM7-mediated processes in physiology and pathophysiology remain unclear. In this study, we employed a mouse model of hypoxic-ischemic brain cell death which mimics the pathophysiology of hypoxic-ischemic encephalopathy (HIE). HIE is a major public health issue and an important cause of neonatal deaths worldwide; however, the available treatments for HIE remain limited. Its survivors face life-long neurological challenges including mental retardation, cerebral palsy, epilepsy and seizure disorders, motor impairments, and visual and auditory impairments. Through a proteomic analysis, we identified calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphatase calcineurin as potential mediators of cell death downstream from TRPM7 activation. Further analysis revealed that TRPM7 mediates cell death through CaMKII, calmodulin, calcineurin, p38, and cofilin cascade. In vivo, we found a significant reduction of brain injury and improvement of short- and long-term functional outcomes after HI after administration of specific TRPM7 blocker waixenicin A. Our data demonstrate a molecular mechanism of TRPM7-mediated cell death and identifies TRPM7 as a promising therapeutic and drug development target for HIE.

Minocycline is a second-generation tetracycline that has been reported to have powerful neuroprotective properties. In our previous studies, we found that d-amphetamine (AMPH) elicited action potential bursts in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. This study sought to determine the effects of minocycline on the AMPH-elicited action potential pattern changes in the central snail neuron, using the two-electrode voltage clamping method. Extracellular application of AMPH at 300 μM elicited action potential bursts in the RP4 neuron. Minocycline dose-dependently (300-900 μM) inhibited the action potential bursts elicited by AMPH. The inhibitory effects of minocycline on AMPH-elicited action potential bursts were restored by forskolin (50 μM), an adenylate cyclase activator, and by dibutyryl cAMP (N(6),2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate; 1mM), a membrane-permeable cAMP analog. Co-administration of forskolin (50 μM) plus tetraethylammonium chloride (TEA; 5mM) or co-administration of TEA (5mM) plus dibutyryl cAMP (1mM) also elicited action potential bursts, which were prevented and inhibited by minocycline. In addition, minocycline prevented and inhibited forskolin (100 μM)-elicited action potential bursts. Notably, TEA (50mM)-elicited action potential bursts in the RP4 neuron were not affected by minocycline. Minocycline did not affect steady-state outward currents of the RP4 neuron. However, minocycline did decrease the AMPH-elicited steady-state current changes. Similarly, minocycline decreased the effects of forskolin-elicited steady-state current changes. Pretreatment with H89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10 μM), a protein kinase A inhibitor, inhibited AMPH-elicited action potential bursts and decreased AMPH-elicited steady-state current changes. These results suggest that the cAMP-protein kinase A signaling pathway and the steady-state current are involved in the inhibitory effects of minocycline upon AMPH-elicited action potential bursts., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinson's disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

We have used time-lapse imaging of acute cortical slices to study the migration of neurons from their sites of origin to their positions in the developing neocortex. We found that two distinct modes of cell movement, somal translocation and glia-guided locomotion, are responsible for the radial migration of neurons generated in the cortical ventricular zone. The former is the prevalent form of radial movement of the early-born cortical neurons, while the latter is adopted by those generated later in corticogenesis. Interneurons, found to originate in the ganglionic eminence, follow tangential migratory paths to reach the developing cortex. Upon reaching the cortex, these cells seek the ventricular zone using a mode of movement that we have termed 'ventricle-directed migration', before they migrate to their positions in the cortical plate. In addition to these forms of movement, we report here a unique morphological and migratory behavior for a population of cortical neurons. These cells are multipolar in form, and are highly motile in the formation and retraction of their processes. Based on these morphological features, we refer to this type of cells as 'branching cells' and attribute the phenotype to a subset of cortical interneurons.

Layer formation in the developing cerebral cortex requires the movement of neurons from their site of origin to their final laminar position. We demonstrate, using time-lapse imaging of acute cortical slices, that two distinct forms of cell movement, locomotion and somal translocation, are responsible for the radial migration of cortical neurons. These modes are distinguished by their dynamic properties and morphological features. Locomotion and translocation are not cell-type specific; although at early ages some cells may move by translocation only, locomoting cells also translocate once their leading process reaches the marginal zone. The existence of two modes of radial migration may account for the differential effects of certain genetic mutations on cortical development.

Fluorescent Ca2+ indicator dyes can be introduced into cells through the same microelectrode used for intracellular voltage recording. Simultaneous measurement of cell membrane potential and intracellular Ca2+ concentration can be very helpful in interpreting the mechanisms of Ca2+ increases. This chapter describes fluorescence image acquisition using a CCD camera and a computer program that also records a synchronized membrane potential trace. The same program allows for preliminary data analysis. More elaborate analyses can be accomplished with commercial programs. We also describe quantitative evaluations of sources of error in the use of the statistic deltaF/F as an indicator of Ca2+ concentration. Especially important errors to minimize are changes in background fluorescence and inappropriate autofluorescence corrections. Some improvement of fluorescence images of cells deep within slices may be accomplished by masking. One method is described for making a mask based on the raw fluorescence image. With another method, highly detailed cell morphologies may be conveyed by using masks based on neurobiotin injections and camera lucida drawings.

We describe a technique for rapid labeling of a large number of cells in the nervous system with many different colors. By delivering lipophilic dye-coated particles to neuronal preparations with a "gene gun," individual neurons and glia whose membranes are contacted by the particles are quickly labeled. Using particles that are each coated with different combinations of various lipophilic dyes, many cells within a complex neuronal network can be simultaneously labeled with a wide variety of colors. This approach is most effective in living material but also labels previously fixed material. In living material, labeled neurons continue to show normal synaptic responses and undergo dendritic remodeling. This technique is thus useful for studying structural plasticity of neuronal circuits in living preparations. In addition, the Golgi-like labeling of neurons with many different colors provides a novel way to study neuronal connectivity.

In the present study, a novel GluR2 receptor-specific immunotoxin was produced. The immunotoxin was produced by conjugation of molecules of trichosanthin, a ribosome inactivating protein, with goat anti-mouse immunoglobulin molecules. The secondary antibody was then combined with a commercially available GluR2 specific primary antibody to form an immunotoxin. The immunotoxins were unilaterally injected either into the neostriatum or into the lateral ventricle of rats. After one week, ipsilateral turning movements were observed after apomorphine treatments in those animals injected by the striatal route. In perfuse-fixed sections of the neostriatum, immunoreactivity for GluR2 was found to decrease in the striatal-lesioned animals. Most of the GluR2-immunoreactive perikarya in the neostriatum, the presumed medium spiny neurons, were depleted. In addition, immunoreactivity for GluR2/3, GluR5/6/7 and NMDAR1 was found to decrease to a different extent in the lesioned neostriatum. The number of GluR1-immunoreactive perikarya in the neostriatum, a group of striatal interneurons, was not affected by the GluR2 lesion. Ventricular administration of the GluR2 immunotoxin however, was found to be less potent. These results demonstrate for the first time that an indirect immunotoxin is useful for immunolesioning. A difference in potency was also observed in different routes of administration. The depletion of GluR2-containing medium spiny neurons in the neostriatum may upset the balance of the output systems of the basal ganglia and has a profound effect in movement control of the animals.

The growth inhibitory and differentiation inducing effects of euxanthone (1,7-dihydroxyxanthone) from the medicinal plant Polygala caudata on the neuroblastoma (Neural 2A, subclone BU-1) were investigated. At the concentration range of 0-100 microM, euxanthone inhibits the growth of BU-1 cells in a dose dependent manner. The 50% growth inhibitory concentration (IC50) was 41 microM. Significant induction of morphological differentiation and neurite growth was observed at the concentration of 100 microM. Frequency of proliferative neuroblastoma cells was determined after induction of differentiation. The frequency of proliferating BU-1 cells was markedly reduced from 1/1.1 to <1/99. Confocal microscopy also confirmed that the morphological differentiation of BU-1 was associated with the expression of neurite specific marker MAP-2 protein in neurites. These data suggest that euxanthone may be one of the neuropharmacological active compounds in the medicinal plant Polygala caudata.

In the present study, a comparison of potency between a commercially available immunotoxin, 192-immunoglobulin-SAP (192-IgG), and a novel immunotoxin produced in our laboratory, anti-p75-anti-mouse IgG-trichosanthin conjugates (p75-TCS), was conducted. Both of the immunotoxins were specific for nerve growth factor p75 receptor. Cholinergic neurons in the rat basal forebrain and in the neostriatum were depleted after the injection of either 192-IgG or p75-TCS. These indicate that both types of immunotoxins are potent and useful in performing immunolesioning experiments. In addition, there were variations in potency among the two immunotoxins in different routes of administration. The 192-IgG was more potent than the p75-TCS in the case of ventricular injections. In case of striatal injections, 192-IgG caused serious tissue necrosis and considerable tissue damage in the brain region. In contrast, p75-TCS was potent and caused a selective and specific depletion of cholinergic neurons in the neostriatum. These results indicate that indirect immunotoxins may be more useful for performing immunolesioning experiments in case of brain parenchyma administration.

Complex but coordinated interactions involving ensembles of neuronal cells result in the accurate processing of information in the adult central nervous system. However, recent studies monitoring the global patterns of activity of neuronal populations have demonstrated that immature neurons also interact to produce coordinated patterns of activity during the early stages of development. In particular, these patterns of coordinated activity occur during the period when neuronal connections are established, thus leading us to believe that such activity patterns might underlie the precision to which many neural pathways are wired up. Multielectrode recording and calcium imaging are two of the techniques that have been instrumental in revealing the spatial and temporal properties of the coordinated activity of developing neural networks in vitro. While multielectrode arrays measure the action potential activity of the cells, calcium imaging permits changes in intracellular calcium levels to be monitored over time. Both techniques have been used successfully to monitor the activity of cellular networks in culture, but they have also been applied in assessing the patterns of activity in intact or semi-intact pieces of neural tissues, such as the developing retina, neocortex and spinal cord. More recently, it has also been possible to correlate the structure and function of the cellular components of the networks by combining intracellular dye filling with the multineuronal recordings. In this review, brief descriptions and the applications of the two techniques will be presented, and the advantages and limitations of multielectrode array will be compared with that of calcium imaging using recordings of the developing mammalian retina as the primary example.

Glutamate excitocytotoxicity is implied in the cause of neuronal degeneration in the neostriatum, in which the toxicity may be mediated by different families of glutamate receptors. The precise cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunits (GluR1-4), one of the major family that involves in the mechanisms of glutamate excitocytotoxicity, in different populations of striatal neurons is therefore of special interest. Immunoreactivity for GluR2/3 subunits was detected in the medium-sized spiny neurons. By double labelling experiments, immunoreactivity for GluR1 and GluR4 was detected only in aspiny striatal neurons that display parvalbumin immunoreactivity, but not in the other neuron populations that display choline acetyltransferase or muscarinic m2 receptor immunoreactivity, nor neurons that display nitric oxide synthase immunoreactivity or nicotinamide adenine dinucleotide phosphate-diaphorase activity. These results indicate that GluR1 and GluR4 immunoreactivity is displayed only in the GABAergic interneurons in the neostriatum. In addition, almost all of the GluR1-immunoreactive neurons were found to display GluR4 immunoreactivity. This finding indicates for the first time that the striatal GABAergic interneurons co-express GluR1 and GluR4 subunits. The results of the present study indicate that there is a differential localization of AMPA-type glutamate receptor subunits in different populations of striatal neurons and they may have a different susceptibility to glutamate excitocytotoxicity.

One of the key questions concerning glutamate toxicity is how a transient NMDA exposure can lead to a delayed death of neurons. To address this issue, we performed whole-cell recording on acutely isolated hippocampal CA1 neurons to monitor the membrane response after NMDA exposure. Transient NMDA exposure (100 microM, 10 min) induced an inward current (postexposure current; Ipe) which was associated with a Ca2+- and Na+-permeable cation conductance. Ipe continuously increased (in the absence of NMDA) until death of the neuron occurred. Application of NMDA in the absence of extracellular calcium failed to trigger Ipe and neuronal death. Postexposure suppression of Ipe protected against NMDA toxicity. These results indicate that a cation current, which is induced by an increase in intracellular calcium concentration ([Ca2+]i) and is itself partly carried by Ca2+, links the initial NMDA exposure to neuronal death.

The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3 dicarboxylic acid (ACPD) at concentrations above 60 microM produced stereotypic oscillatory activity in CA3 pyramidal cells of rat hippocampal slices. This oscillatory activity consisted of trains of depolarizations with overriding action potentials. On average, individual trains lasted 7 sec and recurred at intervals of 24 sec. During each train, the constituent depolarizations achieved a maximum frequency of 27 Hz, then slowed to 8 Hz toward the end of the train. Extracellular and dual intracellular recordings suggested that this ACPD-induced oscillatory activity occurred synchronously in the CA3 population. The oscillations persisted in the presence of GABAA, GABAB, and NMDA receptor antagonists. In contrast, the oscillations were blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10-30 microM). Likewise, the oscillations were blocked by the metabotropic glutamate receptor antagonists (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG; 1 mM], (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG; 1mM] and (S)-4-carboxyphenylglycine [(S)-4CPG; 1 mM]. The results suggest that activation of metabotropic glutamate receptors can result in a permissive state that allows AMPA/kainate receptor-mediated conductances to mediate synchronized activity among hippocampal CA3 neurons.

The grease-gap chamber makes it possible to perfuse two parts of a brain slice separately. Hence, it lies between bath application and iontophoresis or pressure ejection in terms of the size of the region that receives controlled drug dosing. Modifications of a commercially available chamber are described which have permitted the first extracellular and intracellular single-unit recordings to be taken from brain slices in a grease-gap preparation. In addition, we describe the electrical resistance of the gap as a measure of the integrity of the barrier and a simple method for monitoring this resistance continuously. The resistance monitor is particularly useful during low flow rate conditions that improve mechanical stability. These techniques extend the grease-gap method to electrophysiological studies of single cells in slices.

It is well known that across species, the relative size of the cortical area representing a particular sensory surface is proportional to how important that sense is for the animal. Furthermore, we are commonly aware of the observation that the loss of one sense, such as sight, appears to lead to an increase in sensitivity of the remaining senses, although the physiological basis for this is not entirely clear. Now, several studies, including that of Zheng and Purves (11), have suggested that the cortical area devoted to a particular sensory system can be modulated by neuronal activity during development. The fact that use, or disuse, of a sensory organ can lead to significant changes in its area of representation in the developing cortex is intriguing and calls for further investigations aimed at understanding the functional significance and the mechanisms underlying these changes. What remains to be determined is whether enhanced "growth" also means enhanced performance by that sensory system and, if so, whether this is the result of selective changes in neuronal connectivity and/or synaptic efficacy. It is too early to tell, but, whatever the outcome, it is refreshing to consider neuronal growth in the light of enhanced neural activity, in parallel to the results of activity deprivation, to which we are more accustomed.

1. Ca2+ current was examined in acutely isolated hippocampal cells with the use of whole cell voltage-clamp recording and under continuous intracellular perfusion. A persistent Ca2+ current was activated by depolarization to -10 mV from a holding potential of -50 mV. 2. The persistent Ca2+ current was suppressed upon a wash out of the intracellular Mg(2+)-ATP. Adenosine 3',5'-cyclic monophosphate (cAMP) introduced intracellularly potentiated the Ca2+ current, and kinase A inhibitor blocked the current. 3. Reversible suppression of the persistent Ca2+ current was also observed by elevating intracellular Ca2+. This Ca(2+)-dependent suppression was retarded by the addition of a phosphatase inhibitor, okadaic acid, to the intracellular solution. 4. N-methyl-D-aspartate (NMDA) elicited inward current (NMDA response) in the isolated cells. The persistent Ca2+ current was transiently suppressed after the NMDA response. Suppression of the Ca2+ current by NMDA was reduced when intracellular Ca2+ buffering capacity was increased by increasing the concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) from a concentration of 1-10 mM. 5. Substitution of ATP in the intracellular solution with ATP-gamma-S or the addition of okadaic acid to the intracellular solution reduced the suppressive effect of NMDA on the Ca2+ current. 6. The results suggest that the persistent Ca2+ current in the hippocampal cells is maintained by a kinase A-mediated phosphorylation. Increases in the intracellular Ca2+ concentration suppressed the Ca2+ current via a mechanism involving a phosphatase. Ca2+ entry through the NMDA receptor channel suppressed the Ca2+ channel by activating the phosphatase.

1. Intracellular recordings were obtained from pyramidal, granule and hilar cells in transverse slices of guinea-pig hippocampus to examine synaptic interactions between GABAergic neurones. 2. In the presence of the convulsant compound 4-aminopyridine (4-AP), after fast excitatory amino acid (EAA) neurotransmission was blocked pharmacologically, large amplitude inhibitory postsynaptic potentials (IPSPs) occurred rhythmically (every 4-8 s) and synchronously in all principal cell populations (triphasic synchronized IPSPs). In the presence of the GABAA receptor blocker picrotoxin (PTX), a large amplitude IPSP continued to occur spontaneously in all principal cells simultaneously (monophasic synchronized IPSP). 3. Burst firing occurred simultaneously in a group of hilar neurones (synchronized bursting neurones) coincident with triphasic synchronized IPSPs in principal cells. After PTX was added, the bursts and the underlying depolarizing synaptic potentials were completely suppressed in some of the synchronized bursting neurones (type I hilar neurones), while others (type II hilar neurones) continued to fire in bursts coincident with monophasic synchronized IPSPs in principal cells. Intense hyperpolarization blocked burst firing and revealed underlying attenuated spikes of less than 10 mV, but did not uncover any underlying depolarizing synaptic potentials. 4. In type II hilar neurones, during sufficient hyperpolarization, spontaneous activity consisted of attenuated spikes. With depolarization, the small spikes began to trigger full size action potentials. These data suggest the presence of electrotonically remote spike initiation sites. 5. The morphology of synchronized bursting neurones was revealed by intracellular injection of the fluorescent dye Lucifer Yellow. Attempts to inject dye into one type II hilar neurone often resulted in the labelling of two to four cells (dye coupling). Dye coupling was not observed in type I hilar neurones. 6. These findings indicate that excitatory interactions synchronizing the firing of GABAergic neurones can occur in the absence of fast EAA neurotransmission. GABAergic neurones can become synchronized via their recurrent collaterals through the depolarizing action of synaptically activated GABAA receptors. In addition, a subpopulation of GABAergic neurones can become synchronized by a mechanism probably involving electrotonic coupling.

The emergence of precise and orderly sets of neuronal connections often depends upon coordinated electrical activity during the early stages of development. In recent years, an increasing number of reports have shown that neurons of immature sensory systems can spontaneously generate electrical activity that occurs synchronously amongst adjacent cells. These patterns of correlated activity seem to be well suited to the role of providing the cues that are necessary for the activity-dependent refinement of the neural connections in the developing visual, auditory and somatosensory pathways.

1. Intracellular recordings, taken from CA1 pyramidal cells in guinea-pig hippocampal slices, were used to examine the origins of repetitive and burst firing in these cells. Single action potentials were elicited by depolarizing current injection at somatic recording sites. In contrast, current injection during intradendritic recordings initiated burst firing in the dendrites. Burst firing could be elicited in the soma by direct depolarization of distal apical dendrites (> 150 microns from the cell body layer) with large extracellular polarizing electrodes. 2. Intracellular recordings were taken simultaneously from the apical dendrites and pyramidal cell somata with the intention of impaling the same neurone with both electrodes. Paired dendrite-soma recordings confirmed that rhythmic single action potentials were generated at the cell soma, whereas bursts of action potentials were initiated in the distal apical dendrites (> 150 microns from the cell body layer). Fast spikes in the dendrite often triggered fast spikes in the soma, but not all fast spikes in the dendritic burst were 'relayed' to the soma. 3. In paired recordings, when a dendritic action potential failed to elicit a full somatic action potential, a 'd-spike' was commonly recorded in the soma. Somatic d-spikes were uniform all-or-none responses that could be shown, in some cases, to trigger the full somatic action potentials. 4. Attenuated spikes could be recorded in the dendrites, triggered by action potentials initiated at the cell soma. Dendritic responses to somatic stimulation sometimes varied in amplitude, but always showed a direct correspondence with somatic action potentials. 5. Dendritic recordings taken closer to the pyramidal cell bodies (< 150 microns from the cell body layer) showed a 'transitional' region where single action potentials rather than burst discharges could be evoked. After-potentials of these single spikes differed from those associated with somatic spikes in that proximal dendritic spikes had depolarizing after-potentials. The observed shift from after-hyperpolarization to depolarizing after-potentials in intradendritic recordings taken progressively further from the cell body corresponds to the change from repetitive to burst firing. 6. The results indicate that activity of the CA1 pyramidal cell soma, presumably a reflection of its output, can be either burst or repetitive firing. Somatic 'bursts,' unlike the burst discharges seen in the apical dendrites or the burst discharges reported in CA3 cells, are not initiated locally. Rather, they appear to be simply a rapid spike-for-spike response by the soma to the fast spikes that form part of the apical dendritic burst.(ABSTRACT TRUNCATED AT 400 WORDS)

Tetraethylammonium (TEA) effects on K currents were examined on either side of the membrane of hippocampal CA1 neurons by means of whole-cell voltage-clamp recording and intracellular perfusion. Recording media contained ion channel blockers to allow the selective activation of voltage-dependent K currents which consisted of a rapidly decaying component (A-current) and a delayed component. Voltage protocols were applied to separate the A-current from the delayed component. Results show that 10 mM extracellular TEA suppressed 50 +/- 11% (S.D., n = 4) of the delayed current at different levels of depolarization but had little effect on the A-current. In contrast 10 mM TEA applied by intracellular perfusion suppressed the A-current by 42 +/- 10% (S.D., n = 4) in addition to inhibiting the delayed currently 55 +/- 15% (S.D., n = 4). Both the intracellular and extracellular actions of TEA on K currents showed no voltage- nor time-dependency. The results suggest that voltage-dependent transient current (A-current) is mediated through a separate group of ionic channels distinct from those that sustained the delayed current. Furthermore, the asymmetrical effects of intracellular and extracellular TEA on the transient current are similar to those described for the A-current in molluscan neurons. This observation supports the notion that the structure of the ion channel mediating the A-current is closely conserved across different species.

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABAA receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABAA receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABAA receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABAA receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

1. We have developed a 19-compartment cable model of a guinea pig CA3 pyramidal neuron. Each compartment is allowed to contain six active ionic conductances: gNa, gCa, gK(DR) (where DR stands for delayed rectifier), gK(A), gK(AHP), and gK(C). THe conductance gCa is of the high-voltage activated type. The model kinetics for the first five of these conductances incorporate voltage-clamp data obtained from isolated hippocampal pyramidal neurons. The kinetics of gK(C) are based on data from bullfrog sympathetic neurons. The time constant for decay of submembrane calcium derives from optical imaging of Ca signals in Purkinje cell dendrites. 2. To construct the model from available voltage-clamp data, we first reproduced current-clamp records from a model isolated neuron (soma plus proximal dendrites). We next assumed that ionic channel kinetics in the dendrites were the same as in the soma. In accord with dendritic recordings and calcium-imaging data, we also assumed that significant gCa occurs in dendrites. We then attached sections of basilar and apical dendritic cable. By trial and error, we found a distribution (not necessarily unique) of ionic conductance densities that was consistent with current-clamp records from the soma and dendrites of whole neurons and from isolated apical dendrites. 3. The resulting model reproduces the Ca(2+)-dependent spike depolarizing afterpotential (DAP) recorded after a stimulus subthreshold for burst elicitation. 4. The model also reproduces the behavior of CA3 pyramidal neurons injected with increasing somatic depolarizing currents: low-frequency (0.3-1.0 Hz) rhythmic bursting for small currents, with burst frequency increasing with current magnitude; then more irregular bursts followed by afterhyperpolarizations (AHPs) interspersed with brief bursts without AHPs; and finally, rhythmic action potentials without bursts. 5. The model predicts the existence of still another firing pattern during tonic depolarizing dendritic stimulation: brief bursts at less than 1 to approximately 12 Hz, a pattern not observed during somatic stimulation. These bursts correspond to rhythmic dendritic calcium spikes. 6. The model CA3 pyramidal neuron can be made to resemble functionally a CA1 pyramidal neuron by increasing gK(DR) and decreasing dendritic gCa and gK(C). Specifically, after these alterations, tonic depolarization of the soma leads to adapting repetitive firing, whereas stimulation of the distal dendrites leads to bursting. 7. A critical set of parameters concerns the regulation of the pool of intracellular [Ca2+] that interacts with membrane channels (gK(C) and gK(AHP)), particularly in the dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of intracellular Ca2+ (Ca2+i) on K+ currents in hippocampal cells were examined using acutely isolated cells obtained from adult guinea pigs. Whole-cell voltage-clamp recordings were carried out in a configuration that allowed a continuous perfusion of the intracellular medium. Recording media were made to block inward currents and allowed selective activation of K(+)-dependent outward currents. Voltage-dependent outward currents consisted of an initial rapidly decaying component followed by a sustained component. The time constant of decay of the transient current was about 25 msec, and previous studies (Numann et al., 1987) showed that the kinetic and pharmacological properties of this current closely resembled the A current recorded in invertebrate neurons (Connor and Stevens, 1971; Thompson, 1982). Intracellular perfusion of hippocampal cells with a solution containing elevated Ca2+ (about 4.5 x 10(-4) M) elicited outward currents at the holding potential (-45 to -55 mV) and produced changes in voltage-dependent K+ currents. The transient outward current (IA) activated by depolarization was suppressed with increases in Ca2+i. Delayed, sustained K+ currents were greatly potentiated. Data also showed that, among the 3 effects elicited by Ca2+i, suppression of IA was most sensitive to Ca2+i elevation. Previous results (Numann et al., 1987) showed that IA had a lower threshold (about -45 mV) than sustained currents (about -40 mV). By using low levels of depolarization (-40 mV), IA can be selectively activated, and the suppressive effect of Ca2+i on IA was confirmed on the kinetically isolated IA.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Neurones were isolated from the CA1 region of the guinea-pig hippocampus and subjected to the whole-cell mode of voltage clamping, to determine the kinetics of voltage-gated Ca2+ channel activation. 2. Isolated neurones had an abbreviated morphology, having lost most of the distal dendritic tree during the isolation procedure. The electrical compactness of the cells facilitates voltage clamp analysis. 3. Block of sodium and potassium currents revealed a persistent current activated on depolarization above -40 mV, which inactivated slowly when the intracellular medium contained EGTA. The current was blocked by Co2+ and Cd2+, augmented by increases in Ca2+ and could be carried by Ba2+, suggesting that the current is borne by Ca2+. 4. Steady-state activation of the Ca2+ current was found to be well described by the Boltzman equation raised to the second power. 5. The open channel's current-voltage (I-V) relationship rectified in the inward direction and was consistent with the constant-field equation. 6. The kinetics of Ca2+ current onset followed m2 kinetics throughout the range of its activation. Tail current kinetics were in accord with this model. A detailed Hodgkin-Huxley model was derived, defining the activation of this current. 7. The kinetics of the currents observed in this regionally and morphologically defined class of neurones were consistent with the existence of a single kinetic class of channels.

Excitatory synaptic interactions between CA3 neurones in slices from guinea-pig hippocampus were examined. Recurrent excitatory post-synaptic potentials (e.p.s.p.s) were evoked by action potentials in a single presynaptic neurone or by the antidromic activation of part of the CA3 pyramidal cell population. The peak amplitude of unitary e.p.s.p.s was 1-2 mV at potentials between -64 and -70 mV. Their time to peak was 7-12 ms and the initial phase of their decay was slower than that of a somatically injected voltage pulse. Recurrent e.p.s.p.s were often followed by a small (0.3 mV) hyperpolarization, or undershoot. Recurrent e.p.s.p.s were compared with e.p.s.p.s evoked by stimulating mossy fibres, which terminate proximally on apical dendrites of CA3 pyramidal cells. They were of slower time course and reversed at a more positive potential than mossy fibre e.p.s.p.s. Some synaptic terminals made by recurrent axon collaterals apparently terminate at distant locations on apical dendrites. The decay of both recurrent e.p.s.p.s and dendritic voltage pulses was prolonged by membrane depolarization within a 10-15 mV subthreshold potential range. Voltage-dependent inward currents activated by the synaptic depolarization may contribute to the slow initial decay of these synaptic events. The undershoot did not occur when transmission of a unitary e.p.s.p. failed and was of slower time course than the hyperpolarization due to an inhibitory post-synaptic potential (i.p.s.p.). It was suppressed by intracellular application of K+ channel blockers and probably reflects an intrinsic outward current activated as a consequence of the synaptic depolarization. Considerable temporal summation of synaptic potentials occurred when recurrent synapses were activated twice at an interval of 5-10 ms, typical of the spontaneous burst firing pattern of CA3 neurones. The mean facilitation of a second e.p.s.p. at this interval was about 0.6. The efficacy of a third and subsequent e.p.s.p.s at similar interval was reduced. Presynaptic bursts of three to five action potentials evoked summed e.p.s.p.s of amplitude 2-4 mV, with time to peak 20-40 ms and decaying phase of similar duration. Their rising phase was relatively smooth and summed events were succeeded by an undershoot. Presynaptic bursts could cause a post-synaptic neurone to discharge.

Isolated neurons from adult central ganglia of the snail Helisoma were cultured in vitro in modified Liebowitz L-15 medium. Such neurons displayed electrical excitability comparable to that in acutely dissected ganglia. Isolated neurons remained spherical in defined medium throughout culture durations up to 2 weeks. This static morphology was contrasted by the significant neuritic outgrowth which occurred from neurons maintained in medium with co-cultured intact Helisoma brains or in brain conditioned medium. A morphological sequence of growth cone formation and neurite extension occurred only in the presence of a conditioning factor(s) with a mode of action which included tight binding of the conditioning factor to the substratum. Under these conditions, the two primary neuronal phenotypes, electrical excitability and complex neuronal architecture, could be affected independently in adult molluscan neurons cultured in vitro.

In the mammalian cortex, glutamate and gamma-aminobutyric acid (GABA) are the principal transmitters mediating excitatory and inhibitory synaptic events. Glutamate activates cation conductances that lead to membrane depolarization whereas GABA controls chloride conductances that produce hyperpolarization. Here we report that the GABAA-activated conductance in hippocampal pyramidal cells is enhanced by glutamate at concentrations below that required for its excitatory action. The GABA-potentiating effect can be induced, with comparable potency, by several glutamate analogues such as quisqualate, N-methyl-D-aspartate (NMDA), kainate and, surprisingly, by D-2-amino-5-phosphonovalerate (APV), an antagonist for NMDA receptors. Data from dose-response curves show that glutamate enhances the GABAA conductance without significantly changing GABA binding affinity. The low concentration of glutamate needed to enhance GABAA responses raises the possibility that glutamate modulates the strength of GABA-mediated transmission in the cortex.

Stimulation of the amygdala in coronal slices of the amygdala-pyriform region elicited burst responses in the pyriform cortex cells. The burst responses, recorded intracellularly, consisted of a train of action potentials riding on a depolarizing envelope that lasted for an average of 0.4 s. When a similar stimulus was applied to slices prepared from rats previously subjected to amygdala kindling, burst responses were significantly prolonged (average of 6.6 s). Our results indicate that the increased excitability of the chronic epileptic site developed in vivo was retained in the excised amygdala-pyriform slice.

The neurons of the developing and adult ganglion cell layer of the cat retina may be morphologically divided into two major populations. One population, the classic neurons, is mainly composed of ganglion cells, and of a small percentage of displaced amacrines, the bar cells. The remaining neurons are microneurons, which make up the majority of the displaced amacrine population. The loss of ganglion cells during the development has been attributed to cell death. It has alternatively been suggested that some ganglion cells may lose their axon and be transformed into displaced amacrine cells, without degeneration of the cell soma. Reexamination of foetal and postnatal cat retinas confirms the presence of degenerating cells in the ganglion cell layer. Their number appears to be at a maximum on embryonic day (E) 57 but declines rapidly until birth. The peak of cell death thus coincides with the decline in optic nerve fibre counts and classical neuron or ganglion cell numbers. Some cells in early stages of degeneration resemble classical neurons, but the original morphology of those advanced stages of degeneration could not be identified, nor was it possible to identify pyknotic microneurons at any stage. Substantial degeneration of the microneurons is not suggested but if it occurs, it is masked by an overall increase in the population of these cells before birth. Cell death in the microneuron population thus cannot yet be ruled out. It has been argued in the literature that fragments of degenerating cells in developing neural tissue are cleared by microglia within 10-14 hours. In order to test the hypothesis that operation of cell death can alone account for the observed loss of classical neurons in the foetal cat retina, we have modelled the effect of various presumed clearance times on corresponding neuronal population magnitudes. It is found that a constant clearance time of 10-24 hours would be consistent with the observed loss of classical neurons before birth. If this is true, then no ganglion cells would remain for transformation into amacrine cells. The absolute density of degenerating or pyknotic cells is found to be relatively constant across the retina. However their density expressed as a percentage of the local population of classical neurons is markedly higher in peripheral than central retina. In the former region, they compose more than 10% of classical neurons at stage E57. On the same day, the percentage distribution maps define an elongated central area containing only 3-5% pyknotic profiles. This region corresponds to the location of the future visual streak.(ABSTRACT TRUNCATED AT 400 WORDS)

The in vitro neocortical brain slice technique was used to study electrophysiological properties of neurons from brain biopsies in 10 patients undergoing neurosurgical treatment for a variety of conditions, including focal epilepsy. The principal finding was the occurrence of orthodromically evoked depolarization shifts (DSs) and burst discharges in a proportion of neurons in slices from epileptogenic cortex. These evoked depolarizations and bursts had a number of properties in common with those from experimental epileptogenic foci in neocortex, including large amplitude and prolonged duration; long and variable latencies; and all or none, threshold type behavior, dependent on the parameters of orthodromic stimulation. Also DSs could not be evoked by intracellular stimulation, or blocked by hyperpolarizing current pulses once they had been orthodromically evoked. Responses of DSs to current thus differed markedly from those of neurons in epileptogenic guinea pig hippocampal slices. The results of these experiments suggest that intracellular events in human neurons involved in epileptogenesis are similar in appearance to those in various animal models. Neurons in chronic epileptogenesis are similar in appearance to those in various animal models. Neurons in chronic epileptogenic foci retain some of their abnormal properties within brain slices maintained in vitro.

An in vitro organ culture system for buccal ganglia of the adult snail, Helisoma, is described. The system supports: (1) maintenance of characteristic electrophysiological parameters of identified neurons over seven days of culture; (2) choline metabolism including uptake and synthesis over the same duration; (3) sprouting and growth of neurons in response to axotomy; (4) the formation of novel central electrotonic connections between identified neurons as a result of sprouting and growth. These observations on neuronal growth and the formation of connections are similar to those made with in vivo culture. The use of in vitro culture allows precise manipulations not previously possible. When buccal ganglia are cultured in vitro with the cut distal ends of peripheral nerve trunks held closely apposed, axons of neurons 5R and 5L in the nerve trunks are capable of forming electrotonic connections similar to central connections. The capability of these neurons to form electrotonic connections via their peripheral axons implies that special structures (i.e., central neurites) are not required for the formation of connections; and neither are special environments (i.e., the central neuropile) required for these connections.

One goal of mammalian neurobiology is to understand the generation of neuronal activity in large networks. Conceptual schemes have been based on either the properties of single cells or of individual synapses. For instance, the intrinsic oscillatory properties of individual thalamic neurons are thought to underlie thalamic spindle rhythms. This issue has been pursued with a computer model of the CA3 region of the hippocampus that is based on known cellular and synaptic properties. Over a wide range of parameters, this model generates a rhythmic activity at a frequency faster than the firing of individual cells. During each rhythmic event, a few cells fire while most other cells receive synchronous synaptic inputs. This activity resembles the hippocampal theta rhythm as well as synchronized synaptic events observed in vitro. The amplitude and frequency of this emergent rhythmic activity depend on intrinsic cellular properties and the connectivity and strength of both excitatory and inhibitory synapses.

Synchronized discharge of populations of cortical neurons are often observed to underly both the interictal spikes and tonic seizures generated in experimental epilepsy studies. Recently it has been shown that similar synchronized discharges occur in cortical brain slices treated with convulsants such as penicillin, picrotoxin, or bicuculline. The favorable experimental conditions offered by the in vitro preparation have facilitated a detailed examination on the cellular basis for the generation of the epileptic neuronal synchrony. In this chapter we shall review some experimental observations on the neuronal synchronization and describe a mechanism for its generation based on the computer simulation approach. Three factors are considered to be essential for epileptic synchronization observation in vitro. First, cortical neurons may intrinsically generate bursts of action potentials. Second, recurrent excitatory connections exist that are sufficiently powerful that bursting activity may spread between synaptically connected neurons. Third, inhibition within the local neuronal circuit must be adequately attenuated to allow excitation to spread through the recurrent excitatory connections. Computer simulation studies have been based on these assumptions, using neuronal networks where each cell is connected to more than one postsynaptic neuron. Bursting initiated in one cell excites all its follower cells, and the sequential recruitment of an increasing number of cells eventually leads to a simultaneous discharge of the population. A number of recent experimental observations lend credence to the proposed scheme for neuronal synchrony. Simultaneous paired intracellular recordings provided direct evidence that a burst of action potentials in a presynaptic cell can activate action potentials postsynaptically. Furthermore, it is shown that the rhythm of spontaneous discharge in a neuronal population can be influenced by the activity of one neuron within the population.

1. Exposure to the gamma-aminobutyric acid antagonist, picrotoxin, causes the discharge of hippocampal pyramidal cells to become synchronized. Synaptic mechanisms underlying the development of synchrony were investigated by recording from pairs of cells in the CA3 region of guinea-pig hippocampal slices. 2. Picrotoxin suppressed unitary inhibitory synaptic events. It appeared not to affect monosynaptic excitatory connections. Picrotoxin revealed latent excitatory connections in seven out of twenty-one dual recordings from burst-firing cells. 3. Post-synaptic events revealed by picrotoxin were elicited rarely by single action potentials. They were evoked with mean latencies of at least 8 ms and with more than 30% failures of transmission by bursts of three or more action potentials. They were suppressed by increasing extracellular Ca2+. They were considered to be mediated by polysynaptic excitatory pathways. 4. Polysynaptic excitatory post-synaptic potentials (e.p.s.p.s) had a smooth rising phase with time-to-peak of 15-40 ms and a falling phase of similar duration. Their amplitude was 2-3 mV at membrane potentials close to -70 mV. This shape was similar to that of summed e.p.s.p.s evoked by a burst of three to six action potentials at monosynaptic connections between CA3 cells. 5. One cell could evoke excitatory synaptic events in more than one follower cell, suggesting that axon collaterals mediating recurrent excitation were divergent. More than one polysynaptic excitatory pathway could exist between two cells. 6. We examined the role of recurrent excitatory synapses in the development of synchrony. As inhibition was suppressed by picrotoxin, simultaneous excitatory synaptic events appeared in recordings from pairs of cells. They occurred rhythmically at intervals of 0.5-3 s and grew in amplitude with time. Synchronous neuronal discharges were observed when the threshold for action potential generation was exceeded. 7. Firing induced in one cell could sometimes evoke a sequence of post-synaptic events in another cell as inhibition was suppressed. Initially, no connection was detected. On adding picrotoxin, a polysynaptic e.p.s.p. was revealed and with time longer-latency components were recruited to the synaptic event. The amplitude of later components grew until firing threshold was reached. 8. We suggest that synchronous firing develops due to the loss of inhibitory control over the spread of firing between CA3 pyramidal cells via divergent, polysynaptic, recurrent excitatory pathways.

An improved flat-mount procedure demonstrates that the developing ganglion cell layer of the cat retina contains two morphologically distinct populations of presumed neurons at all ages between embryonic day 36 (E36) and adulthood. One population resembles the adult "classical neurons" composing the ganglion cells and bar-cells of Hughes, while the remaining cells, which are smaller and possess much less Nissl substance, presumably correspond to precursors of the adult microneurons. Although the total neuron population of the retinal ganglion cell layer remains quite constant at all studied ages, its component subpopulations alter significantly during prenatal development; some 50% of classical neurons disappear before birth and the microneuron population doubles during the same period. An obvious centroperipheral gradient exists for classical neurons by stage E47, but the microneuron density gradient only becomes apparent at birth. A 2:1 centroperipheral ratio for the total neuron population is also apparent at E47. Centroperipheral neuronal density gradients continue to increase during postnatal growth. Loss of classical neurons during prenatal life as a result of cell death or transformation into microneurons, has been postulated as a mechanism for determining neuron density gradients. Cell death does occur in the ganglion cell population but it is not yet established whether microneurons of the ganglion cell layer originate from ganglion cell transformation or migrate as a differentiated class from the ventricular layer. However, it can be concluded that not all microneurons originate from ganglion cell transformation, because the total loss of classical neurons is less than the increase in microneuron numbers during development. The population magnitudes of both neuronal classes in the ganglion cell layer stabilise after birth. However, it is during the postnatal period that the adult cruciate density topography is achieved by both populations. It is concluded that differential areal growth is the prime mechanism for postnatal cell redistribution.

Spatially resolved measurements of intracellular free calcium and of the changes produced by excitatory amino acids were made in neurons isolated from adult mammalian brain. Extremely long-lasting (minutes) Ca2+ gradients were induced in the apical dendrites of hippocampal CA1 neurons after brief (1 to 3 seconds), local application of either glutamate or N-methyl-D-aspartate (NMDA). These gradients reflect the continuous flux of Ca2+ into the dendrite. The sustained gradients, but not the immediate transient response to the agonists, were prevented by prior treatment with the protein kinase C inhibitor sphingosine. Expression of the long-lasting Ca2+ gradients generally required a priming or conditioning stimulus with the excitatory agonist. The findings demonstrate a coupling between NMDA receptor activation and long-lasting intracellular Ca2+ elevation that could contribute to certain use-dependent modifications of synaptic responses in hippocampal CA1 neurons.

We have attempted to suppress voltage-dependent conductances in hippocampal neurons by introducing various intracellular agents. Voltage-clamp studies were carried out using acutely dissociated hippocampal neurons from adult guinea pigs. Synaptic events were examined using intracellular recordings in the slice preparation. Sodium conductance was suppressed when the quaternary lidocaine derivative QX 314 was introduced intracellularly. Potassium conductances were blocked by intracellular cesium or Tris. We also found that the anion fluoride could affect calcium conductance by an intracellular action. When anions other than fluoride were used for intracellular recordings, the voltage-dependent calcium current inactivated slowly and showed persistent activation at membrane potentials between -40 and -10 mV. In contrast, when fluoride was present intracellularly, the inactivation kinetics of the calcium current were accelerated and the persistent component of the current was largely suppressed. Intracellular recordings in the hippocampal slice showed that when electrodes contained cesium, QX 314, and fluoride, the spiking and nonlinear responses of the neuronal membrane to depolarization were blocked. In these conditions the time course and voltage-dependence of EPSPs could be examined in detail without complications due to voltage-dependent currents of the postsynaptic cell.

A method is described for the isolation of neurons from defined regions of the mammalian central nervous system, by a combination of mechanical and enzymatic means. The procedure liberates neurons free of cellular debris and glial investments, allowing the formation of giga-ohm seals with patch clamp electrodes. The characteristic morphology of neurons is maintained, together with the diversity of active channels evident in the intact nervous system.

1. Neurones were isolated from the hippocampus of adult guinea-pigs by enzymatic and mechanical treatment. The electrophysiological properties of these cells were examined immediately after dissociation by intracellular recordings using low-resistance electrodes (2-5 M omega). 2. Pyramidal-shaped cells were identified visually. Intracellular recordings showed that these cells have input resistances ranging from 200 to 1300 M omega. Passive voltage responses to hyperpolarizing current injection were fitted by single exponentials decaying with time constants ranging from 15 to 60 ms. This suggests that the electrotonic structure of these cells is compact such that injected current elicited isopotential intracellular responses. 3. Outward currents activated by depolarization were examined in these cells using voltage-clamp techniques. The amplitude and the time course of the outward currents were profoundly affected by the holding potential. For cells held at -50 mV or more positive, depolarizing steps produced a slowly rising outward current. At holding potentials negative to -55 mV depolarizing pulses produced an additional early transient outward current followed by a slowly rising component which decayed gradually during sustained depolarizations. 4. The outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), tetraethylammonium (TEA) and 4-aminopyridine (4-AP). 5. The transient current peaked within 6 ms of the onset of depolarizing pulses. It decayed exponentially with a time constant of 20-40 ms. The amplitude of the current activated by a fixed depolarization increased gradually as the duration or the amplitude of the hyperpolarizing pre-pulse increased. The current activated by a fixed depolarization reached its half-maximal level when the hyperpolarizing pre-pulse was at -83 mV. 6. 4-AP exerted two actions on the transient current. Firstly, the time constant of the falling phase decreased by about a factor of two. Secondly, the current was blocked in a time- and voltage-dependent manner: the block increased when the hyperpolarizing pre-pulse lengthened. TEA, up to 10 mM, did not affect the amplitude of the transient current. Co2+ suppressed this current. The effects of Co2+ consisted of a shift to the positive direction of the voltage dependence of the current. 7. The delayed currents can be divided into Ca2+-dependent and Ca2+-independent components. The component persistent in the Co2+ solution (K-current) decayed slowly with maintained depolarization (time constant greater than 3 s).(ABSTRACT TRUNCATED AT 400 WORDS)