Effects of GABA and baclofen on pyramidal cells in the developing rabbit hippocampus: an 'in vitro' study

Using the in vitro hippocampal slice preparation, we have investigated the effects of gamma-aminobutyric acid (GABA) and its analogue beta-(p-chlorophenyl)-GABA (baclofen) on CA1 and CA3 pyramidal cells in the developing rabbit hippocampus. Somatic applications: both GABA and baclofen, when applied to CA1 pyramidal cells from immature tissue, led to cell depolarization from resting membrane potential; this baclofen depolarization may be indirectly mediated. In contrast, CA3 pyramidal cells at the same age were primarily hyperpolarized by both drugs. In mature tissue, both GABA and baclofen applied at the soma induce cell hyperpolarizations. Dendritic applications: immature CA1 cells responded to dendritic GABA and baclofen application with depolarizations associated with increased cell excitability; here, too, the baclofen depolarization may be due to indirect 'disinhibition'. Both depolarizing and hyperpolarizing responses were recorded in immature tissue when GABA was applied to CA3 pyramidal cell dendrites: baclofen produced only hyperpolarizations. In mature CA1 cells, dendritic GABA application produced membrane depolarization, but dendritic baclofen application produced hyperpolarizations. In mature CA3 cells, dendritic GABA and baclofen application produced predominant hyperpolarizations. Mature CA1 pyramidal cells appear to retain some of the GABA-induced depolarizations characteristic of immature tissue. In contrast, mature CA3 neurons show only hyperpolarizing responses to GABA and baclofen application. In all cases, responses to GABA and baclofen are associated with a decrease in cell input resistance. We conclude that the GABAergic receptor/channel complexes mature differently in the CA1 and CA3 regions of the hippocampus.     

Postnatal maturation of Na+, K+, 2Cl- cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl- through GABAA-gated anion channels. The outward Cl- gradient that provides the driving force for Cl- efflux might be generated and maintained by the Na+, K+, 2Cl- cotransporter (NKCC) that keeps intracellular Cl- concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing.     

Shunting and hyperpolarizing GABAergic inhibition in the high-potassium model of ictogenesis in the developing rat hippocampus

Ontogenesis of GABAergic signaling may play an important role in developmental changes in seizure susceptibility in the high-potassium model of ictogenesis in vitro. The age-dependent effects of [K(+)](o) on the reversal potential of the GABA(A)-mediated responses and membrane potential in hippocampal slices in vitro were compared with the effect of GABA(A)-receptors antagonists and GABA(A) modulators on high-potassium induced seizures in the CA3 pyramidal layer of rat hippocampus in vivo. GABA(A) responses were depolarizing at P8-12 and hyperpolarizing at P17-21. In P8-12 rats, GABA(A) responses switch their polarity from depolarizing to hyperpolarizing upon elevation of extracellular potassium. At approximately 10 mM [K(+)](o), activation of GABA(A) receptors produced an isoelectric, purely shunting response characterized by no changes in the membrane potential but an increase in the membrane conductance. In P17-21 rats, the hyperpolarizing GABA(A) driving force progressively increased with elevation of [K(+)](o). In P8-12 rats in vivo, GABA(A)-receptor antagonists did not affect the occurrence of ictal discharges induced by intrahippocampal injection of 10 mM [K(+)](o), but significantly increased seizure duration. Diazepam and isoguvacine completely prevented seizures induced by 10 mM [K(+)](o). In P17-21 rats, GABA(A)-receptor antagonists strongly increased the occurrence of ictal activity induced both by 10 mM [K(+)](o). Taken together, these results suggest that anticonvulsive effects of GABA are because of the combination of shunting and hyperpolarizing actions of GABA. Although shunting GABA is already efficient in the young age group, a developmental increase in the hyperpolarizing GABA(A) driving force likely contributes to the increase in the GABAergic control of seizures upon maturation.     

Synaptic Activation of GABAA Receptors Causes a Depolarizing Potential Under Physiological Conditions in Rat Hippocampal Pyramidal Cells

Intracellular recordings with K-acetate-filled microelectrodes were performed in slices of the adult rat hippocampus maintained in vitro at 35 - 36 degrees C to analyse the potentials associated with the orthodromic inhibitory sequence generated by CA1 pyramidal cells. In 43 of 72 cells, stimuli that were delivered in the stratum radiatum induced (i) an initial excitatory postsynaptic potential (EPSP), (ii) an early, hyperpolarizing inhibitory postsynaptic potential (IPSP) (peak latency from the stimulus artefact 20 ms), (iii) an intermediate depolarizing component (peak latency=60 - 120 ms; duration=60 - 150 ms, and (iv) a late, long-lasting hyperpolarizing IPSP (peak latency=120 - 160 ms, duration >400 ms). In the remaining cells the orthodromic inhibitory response lacked the intermediate depolarization. The depolarizing component was selectively blocked by local applications of bicuculline or picrotoxin on the apical dendrites of pyramidal cells. This pharmacological procedure induced an increase in the amplitude of the EPSP that was capable of triggering 2 - 3 action potentials, but no reduction of the recurrent IPSP which is caused by GABAA receptors located close to the soma. The amplitude and duration of the depolarizing component was enhanced by lowering the temperature in the tissue chamber to 29 - 31 degrees C or by application of the GABA uptake blocker nipecotic acid, further indicating that the depolarizing component represented an active phenomenon mediated through GABA. Application of the Cl- pump blocker furosemide reduced and eventually blocked the early IPSP and the depolarizing component. These data demonstrate that under physiological conditions rat hippocampal pyramidal cells generate a depolarization that is presumably caused by an outwardly directed Cl- movement due to the activation of GABAA receptors located on the apical dendrites. This novel mechanism might modulate hippocampal excitability in both physiological and pathophysiological conditions.     

Timing of the Developmental Switch in GABAA Mediated Signaling from Excitation to Inhibition in CA3 Rat Hippocampus Using Gramicidin Perforated Patch and Extracellular Recordings

Summary:  The timing of the developmental switch in the GABA_A mediated responses from excitatory to inhibitory was studied in Wistar rat CA3 hippocampal pyramidal cells using gramicidin perforated patch-clamp and extracellular recordings. Gramicidin perforated patch recordings revealed a gradual developmental shift in the reversal potential of synaptic and isoguvacine-induced GABA_A mediated responses from –55 ± 4 mV at postnatal days P0–2 to −74 ± 3 mV at P13–15 with a midpoint of disappearance of the excitatory effects of GABA at around P8. Extracellular recordings in CA3 pyramidal cell layer revealed that the effect of isoguvacine on multiple unit activity (MUA) switched from an increase to a decrease at around P10. The effect of synaptic GABA_A mediated responses on MUA switched from an increase to a decrease at around P8. It is concluded that the developmental switch in the action of GABA via GABA_A receptors from excitatory to inhibitory occurs in Wistar rat CA3 pyramidal cells at around P8–10, an age that coincides with the transition from immature to mature hippocampal rhythms. We propose that excitatory GABA contributes to enhanced excitability and ictogenesis in the neonatal rat hippocampus.     

gamma-Aminobutyric acid (GABA) causes consistent depolarization of neurons in the guinea pig supraoptic nucleus due to an absence of GABAB recognition sites

The action of gamma-aminobutyric acid (GABA) in the supraoptic nucleus was investigated using guinea pig brain slices. GABA produced a membrane depolarization accompanied by a decrease in the input resistance. The action of GABA was concentration-dependent throughout a wide range of concentrations (10(-7)-10(-3) M). In none of the cells examined, a membrane hyperpolarization was observed. The reversal potential for the depolarization induced by GABA was about 25 mV positive to the resting membrane potential. The amplitude of the GABA-induced depolarization was increased to 1.5 X the control by reducing the external Cl- from 134.2 mM to 10.2 mM. The action of GABA was readily antagonized by relatively low concentrations of bicuculline (10(-5) M). The action of GABA in the hippocampus or in the anterior hypothalamus was markedly different from that in the supraoptic nucleus, i.e. GABA produced both depolarizing and hyperpolarizing responses in the hippocampus and consistently a hyperpolarization in the anterior hypothalamus. The depolarizing but not the hyperpolarizing response in the hippocampus was selectively blocked by picrotoxin (2 X 10(-5) M) or by bicuculline (10(-5) M). The depolarizing component was dependent on the external Cl- concentration and had a reversal potential similar to that of the depolarization induced by GABA in the supraoptic nucleus. The hyperpolarizing component was resistant to bicuculline and had a reversal potential about 30 mV negative to the resting membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depressed responses to applied and synaptically-released GABA in CA1 pyramidal cells, but not in CA1 interneurons, after transient forebrain ischemia.

Transient cerebral ischemia kills CA1 pyramidal cells of the hippocampus, whereas most CA1 interneurons survive. It has been proposed that calcium-binding proteins, neurotrophins, and/or inhibitory neuropeptides protect interneurons from ischemia. However, different synaptic responses early after reperfusion could also underlie the relative vulnerabilities to ischemia of pyramidal cells and interneurons. In this study, we used gramicidin perforated patch recording in ex vivo slices to investigate gamma-aminobutyric acid (GABA) synaptic function in CA1 pyramidal cells and interneurons 4 h after a bilateral carotid occlusion accompanied by hypovolemic hypotension. At this survival time, the amplitudes of both miniature inhibitory postsynaptic currents (mIPSCs) and GABA-evoked currents were reduced in CA1 pyramidal cells, but not in CA1 interneurons. In addition, the mean rise time of mIPSCs was reduced in pyramidal cells. The reversal potential for the GABA current (E(GABA)) did not shift toward depolarizing values in either cell type, indicating that the driving force for chloride was unchanged at this survival time. We conclude that early during reperfusion GABAergic neurotransmission is attenuated exclusively in pyramidal neurons. This is likely explained by reduced GABAA receptor sensitivity or clustering and possibly also reduced GABA release, rather than by an elevation of intracellular chloride. Impaired GABA function may contribute to ischemic neuronal death by enhancing the excitability of CA1 pyramidal cells and facilitating N-methyl-D-aspartic acid channel opening. Therefore, normalizing GABAergic function might be a useful pharmacological approach to counter excessive, and potentially excitotoxic, glutamatergic activity during the postischemic period.     

Intrinsic bursting of immature CA3 pyramidal neurons and consequent giant depolarizing potentials are driven by a persistent Na+ current and terminated by a slow Ca2+ -activated K+ current

The CA3 area of the mature hippocampus is known for its ability to generate intermittent network activity both in physiological and in pathological conditions. We have recently shown that in the early postnatal period, the intrinsic bursting of interconnected CA3 pyramidal neurons generates network events, which were originally called giant depolarizing potentials (GDPs). The voltage-dependent burst activity of individual pyramidal neurons is promoted by the well-known depolarizing action of endogenous GABA on immature neurons. In the present work, we show that a persistent Na+ current, I-Nap, accounts for the slow regenerative depolarization that triggers the intrinsic bursts in the neonatal rat CA3 pyramidal neurons (postnatal day 3-6), while a slow Ca2+ -activated K+ current, sI-K(Ca), is primarily responsible for the postburst slow afterhyperpolarization and consequent burst termination. In addition, we exploited pharmacological data obtained from intracellular recordings to study the mechanisms involved in network events recorded with field potential recordings. The data as a whole indicate that I-Nap and sI-K(Ca) are involved in the initiation and termination, respectively, of the pyramidal bursts and consequent network events underlying GDPs.     

Spontaneous recurrent network activity in organotypic rat hippocampal slices

Organotypic hippocampal slices were prepared from postnatal day 4 rats and maintained in culture for >6 weeks. Cultured slices exhibited from 12 days in vitro spontaneous events which closely resembled giant depolarizing potentials (GDPs) recorded in neonatal hippocampal slices. GDP-like events occurred over the entire hippocampus with a delay of 30-60 ms between two adjacent regions as demonstrated by pair recordings from CA3-CA3, CA3-CA1 and interneurone-CA3 pyramidal cells. As in acute slices, spontaneous recurrent events were generated by the interplay of GABA and glutamate acting on AMPA receptors as they were reversibly blocked by bicuculline and 6,7-dinitroquinoxaline-2,3-dione but not by dl-2-amino-5-phosphonopentaoic acid. The equilibrium potentials for GABA measured in whole cell and gramicidin-perforated patch from interconnected interneurones-CA3 pyramidal cells were -70 and -56 mV, respectively. The resting membrane potential estimated from the reversal of N-methyl-D-aspartate-induced single-channel currents in cell-attach experiments was -75 mV. In spite of its depolarizing action, in the majority of cases GABA was still inhibitory as it blocked the firing of principal cells. The increased level of glutamatergic connectivity certainly contributed to network synchronization and to the development of interictal discharges after prolonged exposure to bicuculline. In spite of its inhibitory action, in a minority of cells GABA was still depolarizing and excitatory as it was able to bring principal cells to fire, suggesting that a certain degree of immaturity is still present in cultured slices. This was in line with the transient bicuculline-induced block of GDPs and with the isoguvacine-induced increase of GDP frequency.     

Differential bicuculline-induced epileptogenesis in rat neonatal, juvenile and adult CA3 pyramidal neurons in vitro.

The GABA(A) receptor antagonist bicuculline methiodide (BMI, 10 microM) transformed the evoked synaptic responses, recorded intracellularly from the CA3 area of neonatal (postnatal days 3-7, P3-P7), juvenile (P8-P20) and adult hippocampal slices, into long-lasting paroxysmal depolarizations (PDs), with repetitive action potentials (APs). In the same preparation, GABA(A)-mediated fast-IPSPs were depolarizing at resting membrane potential (RMP), with a reversal potential shifting to a hyperpolarizing direction with age (n=15, P6-P17). BMI provoked also spontaneous PDs in juvenile (20/30) and adult (7/10) but not in neonatal (0/12) neurons. PDs were depressed by either the NMDA receptor antagonist CPP (10 microM) or the non-NMDA antagonist CNQX (10 microM), but were blocked only by the combination of the two (n=6), indicating that activation of either NMDA or non-NMDA receptors can independently sustain PDs in immature hippocampus. In conclusion, these findings show that endogenous GABA tonically inhibits CA3 synaptic responses in neonatal life despite the depolarizing nature of GABA(A)-mediated potentials. Moreover, they suggest that during the 1st postnatal week, disinhibition alone is not sufficient to provoke spontaneous epileptiform discharges in CA3 hippocampal area.     

Dissociation of the IPSP and response to GABA during spreading depression-like depolarizations in hippocampal slices

We have compared the sensitivity of CA1 and CA3 hippocampal pyramidal cells, in mature and immature tissue, to spreading depression-like depolarization episodes. Using hippocampal slices from rabbit, we have found that mature and immature tissue, and CA1 and CA3 neurons, were differentially prone to depolarization episodes, depending on the method used to produce the depolarization. CA1 region was generally more sensitive than CA3. Spontaneous and stimulus-evoked depolarizations were seen more frequently in immature tissue than in mature slices, but anoxia-induced depolarizations were much more likely to occur in mature tissue. Synaptic transmission and responses to somatic gamma-aminobutyric acid (GABA) ejection were compared during anoxia-induced depolarizations in mature slices. The early component of the inhibitory postsynaptic potential (IPSP) normally had the same reversal potential as the GABA response. During anoxia-induced depolarization, both the drug response and the PSPs were lost. Synaptic transmission generally disappeared before the response to exogenous GABA application; the GABA response reappeared before synaptic function was restored. During the recovery of resting potential (RMP) following depolarization, the reversal potential of the early IPSP differed significantly from that of the GABA response; when the cell had recovered to RMP, the IPSP was depolarizing, whereas GABA application produced a 'normal' cell hyperpolarization. IPSPs and GABA-mediated responses attained their pre-depolarization form within a few minutes of RMP recovery. These observations suggest that, at least under special circumstances, the early component of the IPSP and GABA-mediated hyperpolarizations can be dissociated. Therefore, the early IPSP may be mediated by more complex mechanisms than a simple alteration in chloride conductance due to GABA-receptor interactions.     

Distinct types of ionic modulation of GABA actions in pyramidal cells and interneurons during electrical induction of hippocampal seizure-like network activity

It has recently been shown that electrical stimulation in normal extracellular fluid induces seizure-like afterdischarge activity that is always preceded by GABA-dependent slow depolarization. These afterdischarge responses are synchronous among mature hippocampal neurons and driven by excitatory GABAergic input. However, the differences in the mechanisms whereby the GABAergic signals in pyramidal cells and interneurons are transiently converted from hyperpolarizing to depolarizing (and even excitatory) have remained unclear. To clarify the network mechanisms underlying this rapid GABA conversion that induces afterdischarges, we examined the temporal changes in GABAergic responses in pyramidal cells and/or interneurons of the rat hippocampal CA1 area in vitro. The extents of slow depolarization and GABA conversion were much larger in the pyramidal cell group than in any group of interneurons. Besides GABA(A) receptor activation, neuronal excitation by ionotropic glutamate receptors enhanced GABA conversion in the pyramidal cells and consequent induction of afterdischarge. The slow depolarization was confirmed to consist of two distinct phases; an early phase that depended primarily on GABA(A)-mediated postsynaptic Cl- accumulation, and a late phase that depended on extracellular K+ accumulation, both of which were enhanced by glutamatergic neuron excitation. Moreover, extracellular K+ accumulation augmented each oscillatory response of the afterdischarge, probably by further Cl- accumulation through K+-coupled Cl- transporters. Our findings suggest that the GABA reversal potential may be elevated above their spike threshold predominantly in the pyramidal cells by biphasic Cl- intrusion during the slow depolarization in GABA- and glutamate-dependent fashion, leading to the initiation of seizure-like epileptiform activity.     

Differences in developmental changes in GABAergic response between bushy and stellate cells in the rat anteroventral cochlear nucleus

Many mammalian central nervous system neuron responses mediated by GABA(A) receptors undergo a developmental transition from excitation to inhibition, but little is known about the time of this switch in specific cell types in the developing anteroventral cochlear nucleus (AVCN). In the present study, bushy and stellate cells, two major cell types in the AVCN, were identified according to their morphology and electrophysiology. The equilibrium potential of GABA-evoked currents (E(GABA)) was examined using the gramicidin-perforated patch-clamp technique. We found that the action of GABA in bushy and stellate cells switched from predominantly depolarizing to predominantly hyperpolarizing with respect to their resting membrane potential (V(rest)) at different postnatal ages. Such a switch in the GABA response of bushy cells occurred before the first postnatal week, whereas that in stellate cells happened at the end of the second postnatal week. Furthermore, we discovered that bushy cells had a more depolarized V(rest) than did stellate cells before the second postnatal week; however, the E(GABA) of bushy and stellate cells was not significantly different. Thus, the discrepancy in the timing of the developmental shift from depolarizing to hyperpolarizing GABA responses between bushy and stellate cells may be due to the difference in their V(rest), but not due to E(GABA) itself. These results suggest that GABAergic inhibition functions earlier in bushy than in stellate cells. In contrast, the longer excitatory action of GABA on stellate cells possibly renders them more vulnerable than bushy cells to excitotoxic substances during early development.     

The dendritic response to GABA in CA1 of the hippocampal slice

Application of GABA in the dendritic region of pyramidal cells elicits a depolarization which, in fact, is the sum of a hyperpolarizing and a depolarizing process. At the reversal potential of the depolarizing response (-42 mV) the GABA-induced current fluctuations do not have a minimum. Consequently, a conductance change to more than one ion is involved. Cl- is in part responsible, Ca2+ is not because Mn2+ and Mg2+ do not change the response. Whether Na+ is involved is uncertain. Substitution with choline had no effect but choline may permeate through the membrane during the depolarizing response. Nipecotic acid inhibits a Na+-GABA uptake mechanism but does not change the dendritic response.     

Adenosine release during seizures attenuates GABAA receptor-mediated depolarization

Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA(A) receptor (GABA(A)R) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA(A)R signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA(A)R responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA(A)R responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA(A)Rs, or by a change in the conductance of GABA(A)Rs. Rather, A(1)R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA(A)R currents. As depolarizing GABA(A)R signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA(A)R signaling may represent an important mechanism by which adenosine can limit seizure activity.     

Cholinergic synaptic potentials in the supragranular layers of auditory cortex

Receptive-field plasticity within the auditory neocortex is associated with learning, memory, and acetylcholine (ACh). However, the interplay of elements involved in changing receptive-fields remains unclear. Herein, we describe a depolarizing and a hyperpolarizing potential elicited by repetitive stimulation (20-100 Hz, 0.5-2 sec) and dependent on ACh, which may be involved in modifying receptive-fields. These potentials were recorded, using whole cell techniques, in layer II/III pyramidal cells in the rat auditory cortex in vitro. Stimulation at low stimulus intensities can give rise to a hyperpolarizing response and stimulation at higher stimulus intensities can elicit a depolarizing response. The depolarizing response had a reversal potential of -35 mV, and was reduced by the combination of AMPA/kainate and NMDA glutamate receptor antagonists (AMPA/kainate: CNQX, DNQX, and GYKI 52466; NMDA: APV, MK-801) and by the muscarinic ACh receptor antagonist atropine. The hyperpolarizing response had a reversal potential of -73 mV and could be reduced by atropine, GABA(A) receptor antagonists (bicuculline and a Cl(-) channel blocker picrotoxin), and to a small extent a GABA(B) receptor antagonist (saclofen). This suggests that the hyperpolarizing response is likely to be mediated by ACh acting on GABAergic interneurons. Extracellular recordings, also made from layer II/III of cortical slices, yielded a negative-going potential which was reduced by ionotropic glutamate receptor antagonists (same as above) and by the ACh receptor antagonists atropine and scopolamine, suggesting that this potential was the extracellular representation of the depolarizing response.     

Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus

The immature brain is prone to seizures but the underlying mechanisms are poorly understood. We explored the hypothesis that increased seizure susceptibility during early development is due to the excitatory action of GABA. Using noninvasive extracellular field potential and cell-attached recordings in CA3 of Sprague-Dawley rat hippocampal slices, we compared the developmental alterations in three parameters: excitatory actions of GABA, presence of the immature pattern of giant depolarizing potentials (GDPs) and severity of epileptiform activity generated by high potassium. The GABA(A) receptor agonist isoguvacine increased firing of CA3 pyramidal cells in neonatal slices while inhibiting activity in adults. A switch in the GABA(A) signalling from excitation to inhibition occurred at postnatal day (P) 13.5 +/- 0.4. Field GDPs were present in the form of spontaneous population bursts until P12.7 +/- 0.3. High potassium (8.5 mm) induced seizure-like events (SLEs) in 35% of slices at P7-16 (peak at P11.3 +/- 0.4), but only interictal activity before and after that age. The GABA(A) receptor antagonist bicuculline reduced the frequency or completely blocked SLEs and induced interictal clonic-like activity accompanied by a reduction in the frequency but an increase in the amplitude of the population spikes. In slices with interictal activity, bicuculline typically caused a large amplitude interictal clonic-like activity at all ages; in slices from P5-16 rats it was often preceded by one SLE at the beginning of bicuculline application. These results suggest that, in the immature hippocampus, GABA exerts dual (both excitatory and inhibitory) actions and that the excitatory component in the action of GABA may contribute to increased excitability during early development.     

Whole-cell patch study of GABAergic inhibition in CA1 neurons of immature rat hippocampal slices

Inhibitory processes mediated by gamma-aminobutyric acid (GABA) were studied in immature rat hippocampal slices using the whole-cell patch clamp technique. Orthodromically evoked hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were observed in CA1 neurons of postnatal 2-5 (P2-5) and 7-13 (P7-13) day old rats under conditions of low internal [Cl-]. In the whole-cell voltage-clamp mode, applications of GABA evoked outwards currents which reversed at -55 mV and -62 mV in P2-5 and P7-13 CA1 neurons, respectively, with comparable reversal potential for the IPSPs for each age. An increase in internal [Cl-] caused a depolarizing shift of the GABA reversal potential which followed the Nernst equation. In both groups of neurons, the IPSPs and GABA currents were blocked with the bath applications of bicuculline (10 microM) and picrotoxin (100 microM). We conclude that the GABAA-mediated inhibitory synaptic process exists in P2-5 CA1 neurons and hypothesize that the absence of such IPSPs noted in previous studies of immature CA1 neurons was likely due to higher internal [Cl-] in the more immature neurons.     

Theta oscillations in somata and dendrites of hippocampal pyramidal cells in vivo: Activity-dependent phase-precession of action potentials

Theta frequency field oscillation reflects synchronized synaptic potentials that entrain the discharge of neuronal populations within the ≈100–200 ms range. The cellular-synaptic generation of theta activity in the hippocampus was investigated by intracellular recordings from the somata and dendrites of CA1 pyramidal cells in urethane-anesthetized rats. The recorded neurons were verified by intracellular injection of biocytin. Transition from non-theta to theta state was characterized by a large decrease in the input resistance of the neuron (39% in the soma), tonic somatic hyperpolarization and dendritic depolarization. The probability of pyramidal cell discharge, as measured in single cells and from a population of extracellularly recorded units, was highest at or slightly after the negative peak of the field theta recorded from the pyramidal layer. In contrast, cyclic depolarizations in dendrites corresponded to the positive phase of the pyramidal layer field theta (i.e. the hyperpolarizing phase of somatic theta). Current-induced depolarization of the dendrite triggered large amplitude slow spikes (putative Ca²⁺ spikes) which were phase-locked to the positive phase of field theta. In the absence of background theta, strong dendritic depolarization by current injection led to large amplitude, self-sustained oscillation in the theta frequency range. Depolarization of the neuron resulted in a voltage-dependent phase precession of the action potentials. The voltage-dependent phase-precession was replicated by a two-compartment conductance model. Using an active (bursting) dendritic compartment spike phase advancement of action potentials, relative to the somatic theta rhythm, occurred up to 360 degrees. These data indicate that distal dendritic depolarization of the pyramidal cell by the entorhinal input during theta overlaps in time with somatic hyperpolarization. As a result, most pyramidal cells are either silent or discharge with single spikes on the negative portion of local field theta (i.e., when the somatic region is least polarized). However, strong dendritic excitation may overcome perisomatic inhibition and the large depolarizing theta rhythm in the dendrites may induce spike bursts at an earlier phase of the extracellular theta cycle. The magnitude of dendritic depolarization is reflected by the timing of action potentials within the theta cycle. We hypothesize that the competition between the out-of-phase theta oscillation in the soma and dendrite is responsible for the advancement of spike discharges observed in the behaving animal. Hippocampus 1998;8:244–261. © 1998 Wiley-Liss, Inc.     

Postnatal differentiation of cortical interneuron signalling

Most GABAergic interneurons in the cortex are born at embryonic stages in the ganglionic eminences and migrate tangentially to their final destination. They continue, however, to differentiate and functionally integrate in the circuitry until later postnatal stages of the rodent brain. Recent investigations show that interneurons undergo marked changes in their morphological, intrinsic and synaptic properties as they mature. Action potential shape and its propagation, the period of transmitter release and the time course of the postsynaptic GABA(A) receptor-mediated conductance become faster during the first three to four postnatal weeks, resulting in a developmental switch of interneurons from slow to fast signalling units. At the same time, the nature of GABAergic signalling is classically considered to shift from depolarizing to hyperpolarizing. However, recent studies oppose this view as interneuron synapses can be shunting, excitatory or hyperpolarizing in the mature cortex, demonstrating the coexistence of diverse developmental rules for the emerging effects of GABAergic synapses. Thus, mature interneuron signalling comes in many forms and is apparently optimized to the network in which the neurons are embedded.