John J. Hablitz

Endogenous nature of spontaneous bursting in hippocampal pyramidal neurons

The normal spontaneous bursting behavior of hippocampal pyramidal neurons was investigated. Bursting frequency was found to be membrane potential dependent, the frequency increasing with maintained depolarization and decreasing upon hyperpolarization. Short depolarizing-current pulses would trigger bursts which outlasted the stimulus, and bursting continued when synaptic transmission had been blocked. The spontaneous bursts of these neurons, in contrast to bursts induced by convulsive agents, appear to exhibit the classical behavior of endogenous bursts as observed in invertebrate neurons. The endogenous bursts in hippocampal neurons may result, also, from an interplay of intrinsic membrane currents.

Alterations in the microenvironment during spreading depression associated with epileptiform activity in the immature neocortex

Local changes in extracellular ion concentrations were measured with ion-sensitive microelectrodes in slices of mature (greater than 40 days of age) or immature (16-30 days of age) rat neocortex maintained in vitro. Repetitive stimulation resulted in increases in extracellular potassium ([K+]o) to levels of 8.85 +/- 2.1 mM in slices from adult animals and 12.77 +/- 1.8 mM in slices from immature animals. During exposure to picrotoxin, maximum levels were 11.3 +/- 2.6 and 14.8 +/- 2.5 mM in the mature and immature groups, respectively. Picrotoxin (50 microM) induced spontaneous bursts of repetitive spiking, followed by a slow, negative field potential, associated with spreading depression (SD), in slices from immature animals. [K+]o levels increased to 10.2 +/- 3.9 mM during repetitive spike discharges and reached 30.3 +/- 18.5 mM during SDs. Variations in the size of the extracellular space (ES) were examined during SD. The ES was found to reversibly decrease by 39 +/- 4.5%. Clusters of repetitive spikes were associated with 0.1-0.2 mM decreases in [Ca2+]o, whereas 1.12 +/- 0.06 mM decreases were observed during SDs. Decreases in [Na+]o and [Cl-]o of 56 +/- 10 mM and 41 +/- 9 mM, respectively, were observed during SDs suggesting that a net transmembrane movement of water occurred during SDs. These results indicate that changes in [K+]o associated with epileptiform activity in the immature nervous system are quantitatively different from those observed in the mature brain. These large increases in [K+]o may contribute to the prolonged nature of epileptiform discharges in the developing nervous system.

Hippocampal excitability and changes in extracellular potassium

Abstract Extracellular recordings were made from dendritic and somatic sites in the CA1 region of guinea pig hippocampal slices maintained in vitro . We studied the effects of increasing extracellular K + from 3.25 to 15.25 m m on potentials elicited by synaptic activation of stratum radiatum fibers. Increasing K + from 3.25 to 12.25 m m had little or no detectable effect on the excitability of presynaptic fibers, increased the amplitude of the somatic response (the population spike), and increased the size of the dendritic field EPSP in the range of 3.25 to 9.25 m m . When epileptiform activity induced by penicillin was studied as a function of [K + ] 0 , it was found that elevating K + increased the amplitude and duration of the abnormal population spike. However, significant epileptiform activity was present in 3.25 m m [K + ] 0 . All evoked activity was reversibly abolished by perfusion with 15.25 m m K + . These results indicate that [K + ] 0 has an important role in regulating neuronal excitability and may effect changes both pre- and postsynaptically.

Extracellular K+ and Ca2+ changes during epileptiform discharges in the immature rat neocortex

Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3-5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10-30 s. Measurements of extracellular potassium ([K+]o) and calcium ([Ca2+]o) were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca2+]o decreases of 0.1-0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3-0.4 mM. Measurement of [K+]o showed that individual interictal spikes were associated with increases in [K+]o up to 12 mM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K+]o were observed ca. 600 microns below the pial surface. [K+]o increases were followed by undershoots of the resting [K+]o level. The unusually high [K+]o levels associated with epileptiform discharges in the immature neocortex suggest that disturbances in [K+]o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.

Chronic exposure of developing cortical neurons to GABA down-regulates GABA/benzodiazepine receptors and GABA-gated chloride currents.

Cultures of cerebral neurons were prepared from chick embryos, 8.5 days in ovo, and maintained in vitro. Following chronic exposure of these cells to GABA, the levels of [3H]flunitrazepam binding in situ and electrophysiological responsiveness to gamma-aminobutyric acid (GABA) was examined. Treatment with 100 microM GABA for 7 days reduced [3H]flunitrazepam binding in situ by 70 +/- 8% compared to untreated controls. The binding of [3H]N-methylscopolamine was unaffected by this treatment. The reduction in [3H]flunitrazepam binding was prevented by concomitant exposure of developing neurons to the GABA antagonist R 5135, suggesting that GABAA receptor occupancy is required. The loss of bezodiazepine receptors was dependent on the GABA concentration in the culture medium and a half-saturation (IC50) value of 11.2 +/- 3.7 microM was estimated. Whole-cell patch-clamp recordings were obtained to assess the functional properties of the labile receptor pool observed in the binding studies. Neurons cultured with 100 microM GABA for 7 days showed a 60-70% reduction in the peak current amplitudes observed in response to application of 10-100 microM GABA. However, the rate of rapid desensitization, quantified by measuring changes in input conductance, was unchanged by chronic GABA exposure, yielding decay time constants of 27.1 +/- 2.1 and 34.7 +/- 4.7 s for control and treated cells, respectively. The results are consistent with a GABA modulation of the GABAA/benzodiazepine receptor complex by means of down-regulation.

Effects of intracellular injections of chloride and EGTA on postepileptiform-burst hyperpolarizations in hippocampal neurons

Intracellular recordings were obtained from CA1 neurons of hippocampal slices maintained in vitro. Epileptiform activity was induced by bath application of either penicillin (3.4 mM) or picrotoxin (5 X 10(-4) M). The afterhyperpolarizations (AHPs) following evoked epileptiform events induced by penicillin were inverted by diffusion of chloride ions from KCl electrodes but were insensitive to injection of EGTA. Picrotoxin produced epileptiform events qualitatively similar to those seen with penicillin, but the AHPs were reduced or abolished by injection of EGTA and were chloride insensitive. AHPs that were evoked directly by depolarizing-current pulses when penicillin or picrotoxin was present were sensitive to EGTA. These results indicate that hippocampal CA1 neurons are capable of generating two types of hyperpolarizing events and that penicillin is apparently ineffective in completely blocking the chloride-mediated event.

Long-term potentiation in frontal cortex: Role of NMDA-modulated polysynaptic excitatory pathways

The present study examined the role of N-methyl-D-aspartic acid (NMDA) receptors in synaptic plasticity in regular-spiking cells of rat frontal cortex. Intracortical stimulation, at levels subthreshold for elicitation of action potentials, evoked a late excitatory postsynaptic potential (EPSP) in layer II-III neurons that was sensitive to the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (APV). This late EPSP showed marked short-term frequency-dependent depression, suggesting that it is polysynaptic in origin. Polysynaptic late EPSPs were selectively enhanced following high-frequency stimulation. This sustained increase in synaptic efficacy, or long-term potentiation, was expressed in regular spiking cells and appeared to result from activation of NMDA receptors on excitatory interneurons. These data demonstrate the existence of an NMDA-modulated polysynaptic circuit in the neocortex which displays several types of use-dependent plasticity.

Conductance changes induced by DL-homocysteic acid and N-methyl-DL-aspartic acid in hippocampal neurons

Abstract Iontophoretic application of DL -homocysteic acid or N-methyl- DL -aspartic acid to the dendrites of hippocampal neurons maintained in vitro caused membrane depolarizations that were associated with increases as well as decreases in input resistance. Increases in resistance were most prominent when low doses of agonists were applied. Both types of conductance change persisted after bath application of tetrodotoxin and manganese.

Operant conditioning and slow potential changes from monkey cortex

Abstract The present study attempted to obtain information concerning the distribution of SP changes over monkey cortex in order to delineate the source and functional significance of contingent negative variation (CNV). Both shock-avoidance and food-reward paradigms were employed in an attempt to determine the relationship between slow potentials (SPs) and mode of reinforcement. Transcortical platinized-platinum electrodes were stereotaxically implanted in 5 rhesus monkeys. Subjects were trained to respond in order to either receive food reinforcement or avoid shock. A warning stimulus (click) was followed 1 sec later by the imperative stimulus (1500 c/sec tone of 1.5 sec duration); subjects were required to respond during the tone period by pressing a lever. Approximately 100 trials were given a day, 5 days a week, for a 2 month period. Brain electrical activity was suitably amplified and recorded on magnetic tape for subsequent computer analysis. Negative SP changes were recordable from many portions of frontal cortex. The most marked finding in the present study was the demonstration of the existence of two independent SPs related to the conditioning task. A frontal-dominant potential developed gradually with training, while the central-dominant SP was of significant magnitude from the onset of training. Each potential was of equal magnitude bilaterally and persisted for the duration of the 2 month testing period. Type of reinforcement was a significant variable in determining the distribution of cortical SPs. Shock reinforcement caused an increase in precentral regions that was not manifested in frontal areas. It was suggested that the frontal-dominant potential was analogous to CNV recorded in humans and reflected the association of the two stimuli, whereas the central-dominant SP was an indication of motor inhibition and readiness to respond. The possible relationship between these potentials was discussed in terms of triggering mechanisms for the initiation of timed movements.

Prenatal exposure to alcohol alters short-term plasticity in hippocampus.

Paired-pulse potentiation was studied in the CA1 region of rats exposed prenatally to alcohol. Pregnant rats were fed a liquid diet containing 35% ethanol-derived calories on days 3 to 21 of gestation. Control animals were fed liquid diets without ethanol. When the pups of both groups were 40 to 60 days old, hippocampal slices were prepared and maintained in vitro. Paired-pulse potentiation was examined at intervals from 5 to 400 ms. Recordings made from these slices showed that the response inhibition typically seen at short interpulse intervals was minimal or absent in the animals with prenatal exposure to alcohol. Potentiation of population spike amplitude was enhanced at intervals to 100 ms.