GABAA-Dependent chloride influx modulates GABAB-mediated IPSPs in hippocampal pyramidal cells.

The relationship between postsynaptic inhibitory responses [the fast GABA(A)-mediated inhibitory postsynaptic potential (IPSP) and the slow GABA(B)-mediated IPSP] were investigated in hippocampal CA3 pyramidal cells. Mossy fiber-evoked GABA(B)-mediated IPSPs were, paradoxically, of greater amplitude in cells with resting membrane potential of -62 mV (13.6 +/- 0.5 mV; mean +/- SE) as compared with cells with resting membrane potential of -54 mV (7.0 +/- 0.8 mV). In addition, when a cell's membrane potential was artificially manipulated, GABA(B)-mediated IPSPs were reduced at relatively depolarized levels (-55 mV) and enhanced at relatively hyperpolarized potentials (at least -60 mV). In contrast, the preceding GABA(A)-mediated IPSPs were larger at the more positive membrane potentials and smaller as the cell was hyperpolarized. Similar voltage dependency was obtained when monosynaptic GABA(A)- and GABA(B)-mediated IPSPs were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABA(B)-mediated IPSPs isolated in the presence of glutamatergic and GABA(A) receptor antagonists were not reduced at the more positive membrane potentials, and were significantly larger in amplitude than GABA(B)-mediated IPSPs preceded by a monosynaptic GABA(A)-mediated IPSP. The amplitude of the isolated monosynaptic GABA(B)-mediated IPSPs recorded with potassium chloride-containing microelectrodes was significantly smaller than the comparable potential recorded with potassium acetate microelectrodes without chloride. We conclude that voltage-dependent chloride influx, via GABA(A) receptor-gated channels, modulates postsynaptic GABA(B)-mediated inhibition in hippocampal CA3 pyramidal cells.     

Use-dependent depression of IPSPs in rat hippocampal pyramidal cells in vitro

We have used intracellular recording techniques to study the use-dependence of evoked inhibitory postsynaptic potentials (IPSPs) in rat CA1 hippocampal pyramidal cells. We determined reversal potentials and conductance changes associated with IPSPs and responses to directly applied gamma-aminobutyric acid (GABA). The IPSP depression could be seen after a single conditioning stimulus. This depression appeared to be due primarily to a 50% decrease in IPSP conductance (gIPSP). Trains of stimulating pulses (50 pulses at 5 or 10 Hz) produced more pronounced effects than a single conditioning pulse. Suprathreshold repetitive stimulation of stratum radiatum (SR) produced epileptiform burst firing and greater depression of IPSPs than did alvear (ALV) or subthreshold SR stimulation. During suprathreshold SR stimulation the IPSP was nearly abolished and the membrane potential could become less negative than the resting potential. A masking effect of facilitated depolarizing potentials on IPSPs was unlikely since IPSPs accompanied by little or no depolarizing potential were also depressed by SR trains. The 75% reduction in IPSP conductance found after repetitive stimulation confirmed that an overlapping conductance was not responsible for the depression of the IPSP. The GABA-induced conductance increase was not depressed by identical trains. Trains of stimulation induced depolarizing shifts in equilibrium potentials for the IPSP (EIPSP) and GABA (EGABA) of approximately 10 mV. These shifts were always greater after SR trains than after ALV trains. Simultaneous recordings of membrane potential and extracellular potassium concentration ([K+]o) with K+-sensitive microelectrodes revealed a direct correlation between the two during a stimulus train. Membrane potential depolarized as much as 18 mV from the peak of the IPSP and [K+]o could increase to a maximum of 10 mM during some trains. A depressant effect (of approximately 50%) of K+ on IPSPs was demonstrated by brief pressure ejection of K+ near the soma. We conclude that repetitive stimulation depresses gIPSP and shifts EIPSP in the depolarizing direction. Whereas gIPSP began to decline after a single conditioning pulse, the additional depression of IPSPs produced by stimulus trains was due in large part to shifts in EIPSP. Depression of gIPSP was not due to desensitization or block of ionic conductances, since gGABA was not reduced. The EIPSP may change as a result of increases in [K+]o.     

Distribution of GABA sensitive areas on hippocampal pyramidal cells

Summary In isolated transverse hippocampal slices GABA (gamma-amino-butyric acid) was applied iontophoretically to various parts of the dendritic tree of CA1 pyramidal cells. Indices of GABA effects were reduced amplitude of orthodromically driven CA1 population spikes and inhibition of single CA1 units driven by orthodromic stimulation or by application of L-glutamate.Weak iontophoretic currents of GABA (3–6 nA, backing current -3 nA) effectively reduced the amplitude of the population spike and arrested unit activity when applied in a position close to the soma. The effect was halved when GABA was applied 25–30 m away at right angles to the main dendritic axis. In the direction of the main dendritic axis, GABA was effective as far as 250 and 150 m from the soma in the apical and basal dendritic directions, respectively, corresponding to about 50% of the total dendritic length. The best effect was usually found at a depth corresponding to that of the recording electrode, probably because the main dendritic axis was parallel to the slice surface.The sharp localization of GABA sensitivity when applied in the pyramidal layer supports earlier evidence that GABA mediates the basket cell inhibition on the soma of the pyramidal hippocampal cells. In the dendritic tree, GABA may also have an inhibitory function with an effectiveness matching that of the soma application.     

GABA(B) receptor activation modulates GABA(A) receptor-mediated inhibition in chicken nucleus magnocellularis neurons

Neurons of nucleus magnocellularis (NM), a division of avian cochlear nucleus that performs precise temporal encoding, receive glutamatergic excitatory input solely from the eighth nerve and GABAergic inhibitory input primarily from the ipsilateral superior olivary nucleus. GABA activates both ligand-gated Cl^– channels [GABA_A receptors (GABA_ARs)] and G protein-coupled receptors (GABA_B receptors). The net effect of GABA_AR-mediated input to NM is inhibitory, although depolarizing. Several studies have shown that this shunting, inhibitory GABAergic input can evoke action potentials in postsynaptic NM neurons, which could interfere with their temporal encoding. While this GABA-mediated firing is limited by a low-voltage-activated K⁺ conductance, we have found evidence for a second mechanism. We investigated modulation of GABA_AR-mediated responses by GABA_BRs using whole cell recording techniques. Bath-applied baclofen, a GABA_BR agonist, produced dose-dependent suppression of evoked inhibitory postsynaptic currents (eIPSCs). This suppression was blocked by CGP52432 a potent and selective GABA_BR antagonist. Baclofen reduced the frequency but not the amplitude of miniature IPSCs (mIPSCs) and did not affect postsynaptic currents elicited by puff application of a specific GABA_AR agonist muscimol, suggesting a presynaptic mechanism for the GABA_BR-mediated modulation. Firing of NM neurons by synaptic stimulation of GABAergic inputs to NM was eliminated by baclofen. However, endogenous GABA_BR activity in the presynaptic inhibitory terminals was not observed. We propose that presynaptic GABA_BRs function as autoreceptors, regulating synaptic strength of GABA_AR-mediated inhibition, and prevent NM neurons from generating firing during activation of the inhibitory inputs.     

Direct depolarization and antidromic action potentials transiently suppress dendritic IPSPs in hippocampal CA1 pyramidal cells

Whole-cell current-clamp recordings were made from distal dendrites of rat hippocampal CA1 pyramidal cells. Following depolarization of the dendritic membrane by direct injection of current pulses or by back-propagating action potentials elicited by antidromic stimulation, evoked gamma-aminobutyric acid-A (GABA(A)) receptor-mediated inhibitory postsynaptic potentials (IPSPs) were transiently suppressed. This suppression had properties similar to depolarization-induced suppression of inhibition (DSI): it was enhanced by carbachol, blocked by dendritic hyperpolarization sufficient to prevent action potential invasion, and reduced by 4-aminopyridine (4-AP) application. Thus DSI or a DSI-like process can be recorded in CA1 distal dendrites. Moreover, localized application of TTX to stratum pyramidale blocked somatic action potentials and somatic IPSPs, but not dendritic IPSPs or DSI induced by direct dendritic depolarization, suggesting DSI is expressed in part in the dendrites. These data extend the potential physiological roles of DSI.     

Reversal potential for glutamate responses in hippocampal pyramidal cells

L-Glutamic acid was iontophoresed onto hippocampal CA1 pyramidal neurons. Intracellular recordings revealed that glutamate reliably depolarized all neurons examined. The depolarization evoked by glutamate was voltage sensitivity and had an apparent reversal potential close to 0 mV.     

Distribution of functional glutamate and GABA receptors on hippocampal pyramidal cells and interneurons

The distribution of functional neurotransmitter receptors is an important determinant of neuronal information processing. To map the location of functional glutamate and GABA receptors on individual hippocampal neurons, we photolyzed "caged" glutamate and GABA while measuring the electrical currents resulting from activation of these receptors. Responses to uncaged neurotransmitters were spatially nonuniform and varied according to the type of receptor and type of neuron. Every region of CA1 pyramidal cells responded to glutamate and GABA, but glutamate and GABA receptors increased in density along the length of their distal dendrites. Similar gradients of glutamate receptors were found in stratum radiatum interneurons, while GABA responses were detectable only in the perisomatic region of these interneurons. These regional variations in receptor distribution indicate the selective targeting of receptors on central neurons and may reflect a mechanism for local regulation of synaptic efficacy.     

Sustained depolarizing shift of the GABA reversal potential by glutamate receptor activation in hippocampal neurons

The inhibitory action of GABA is a consequence of a relatively hyperpolarized Cl⁻ reversal potential (E_Cl), which results from the activity of K⁺–Cl⁻ cotransporter (KCC2). In this study we investigated the effects of glutamate and glutamatergic synaptic activity on E_Cl. In dissociated culture of mature hippocampal neurons, the application of glutamate caused positive E_Cl shifts with two distinct temporal components. Following a large transient depolarizing state, the sustained depolarizing state (E_Cl-sustained) lasted more than 30 min. The E_Cl-sustained disappeared in the absence of external Ca²⁺ during glutamate application and was blocked by both AP5 and MK801, but not by nifedipine. The E_Cl-sustained was also induced by NMDA. The E_Cl-sustained was blocked by furosemide, a blocker of both KCC2 and NKCC1, but not bumetanide, a blocker of NKCC1. On the other hand, in immature neurons having less expression of KCC2, NMDA failed to induce the sustained depolarizing E_Cl shift. In organotypic slice cultured neurons, repetitive activation of glutamatergic afferents also generated a sustained depolarizing E_Cl shift. These results suggest that Ca²⁺ influx through NMDA receptors causes the down-regulation of KCC2 and gives rise to long lasting positive E_Cl shifts, which might contribute to hyperexcitability, LTP, and epileptiform discharges.     

Sustained depolarizing shift of the GABA reversal potential by glutamate receptor activation in hippocampal neurons

The inhibitory action of GABA is a consequence of a relatively hyperpolarized Cl(-) reversal potential (E(Cl)), which results from the activity of K(+)-Cl(-) cotransporter (KCC2). In this study we investigated the effects of glutamate and glutamatergic synaptic activity on E(Cl). In dissociated culture of mature hippocampal neurons, the application of glutamate caused positive E(Cl) shifts with two distinct temporal components. Following a large transient depolarizing state, the sustained depolarizing state (E(Cl)-sustained) lasted more than 30 min. The E(Cl)-sustained disappeared in the absence of external Ca(2+) during glutamate application and was blocked by both AP5 and MK801, but not by nifedipine. The E(Cl)-sustained was also induced by NMDA. The E(Cl)-sustained was blocked by furosemide, a blocker of both KCC2 and NKCC1, but not bumetanide, a blocker of NKCC1. On the other hand, in immature neurons having less expression of KCC2, NMDA failed to induce the sustained depolarizing E(Cl) shift. In organotypic slice cultured neurons, repetitive activation of glutamatergic afferents also generated a sustained depolarizing E(Cl) shift. These results suggest that Ca(2+) influx through NMDA receptors causes the down-regulation of KCC2 and gives rise to long lasting positive E(Cl) shifts, which might contribute to hyperexcitability, LTP, and epileptiform discharges.     

Chemical hypoxia in hippocampal pyramidal cells affects membrane potential differentially depending on resting potential

The aim of the present study was to analyze the effect of chemical hypoxia (cyanide) on the membrane potential of hippocampal CA1 neurons and to elucidate the reason for previously found differences in the reaction to hypoxia in these cells. Recordings were performed in brain slices from 8-19-day-old rats with whole-cell patch clamp on cells identified with near-infrared video microscopy. Cyanide (0.1-2.0 mM) caused different responses depending on the resting potential of the cells: hyperpolarization (or an initial depolarization followed by hyperpolarization) was generally seen in cells with less negative resting potential (-56+/-6.1 mV), and depolarization in cells with more negative resting potential (-62+/-3.4 mV). After 10 min in cyanide the membrane potential in all cells had reached approximately the same level (-62+/-5.8 mV), the direction and size of the voltage response having an inverse linear relation to the resting potential (k=-0.98, r=0.71). The direction of the cyanide response was not reversed by current injection (depolarization by 12 mV) in cells with more negative resting potential (-60+/-2.8 mV). Wash out of cyanide caused hyperpolarization in 70% of the cells. Presence of ouabain (2 microM) resulted in pronounced depolarization during cyanide perfusion, and potentiated the hyperpolarization during wash out indicating that this part of the effect is not dependent on a reactivation of the Na/K pump. In conclusion, chemical hypoxia with cyanide changes the membrane potential in CA1 cells in size and direction depending on the original resting potential of the cells. The present findings suggested that cyanide activated not only K+ channels but in addition increased a Na+ current which has a more positive equilibrium potential.     

GABA(B)-receptor modulation of short-term synaptic depression at an excitatory input to murine hippocampal CA3 pyramidal neurons

GABA(B) agonists inhibit excitatory transmission to hippocampal CA3 neurons during low frequency stimulation. We examined whether GABA(B) receptor activation can also enhance synaptic efficacy, when investigated at an input with high initial release probability. Short-term depression of field excitatory postsynaptic potential (EPSP) amplitude was observed during trains of stimuli applied to associational/commissural inputs (10-50 Hz; 22 degrees C). Baclofen (10 microM) reduced the amplitude of initial EPSPs in a train, and also reduced the degree of short-term depression. EPSPs recorded late in a train were significantly larger in baclofen than those recorded in control solution. These dual effects were mimicked by another selective GABA(B) agonist (SKF 97541, 10 microM), and abolished by a GABA(B)-selective antagonist (SCH 50911, 20 microM). The effects of baclofen were similar at a higher recording temperature (32 degrees C), where short-term depression was observed at higher stimulation frequencies. These results are consistent with the idea that a reduction of transmitter release probability could increase the fidelity of high-frequency transmission at this input, an effect that could help account for excitatory effects of GABA(B) agonists in some seizure models.     

Membrane potential of CA3 hippocampal pyramidal cells during postnatal development

A depolarized resting membrane potential has long been considered to be a universal feature of immature neurons. Despite the physiological importance, the underlying mechanisms of this developmental phenomenon are poorly understood. Using perforated-patch, whole cell, and cell-attached recordings, we measured the membrane potential in CA3 pyramidal cells in hippocampal slices from postnatal rats. With gramicidin perforated-patch recordings, membrane potential was -44 +/- 4 (SE) mV at postnatal days P0-P2, and it progressively shifted to -67 +/- 2 mV at P13-15. A similar developmental change of the membrane potential has been also observed with conventional whole cell recordings. However, the value of the membrane potential deduced from the reversal potential of N-methyl-d-aspartate channels in cell-attached recordings did not change with age and was -77 +/- 2 mV at P2 and -77 +/- 2 mV at P13-14. The membrane potential measured using whole cell recordings correlated with seal and input resistance, being most depolarized in neurons with high, several gigaohms, input resistance and low seal resistance. Simulations revealed that depolarized values of the membrane potential in whole cell and perforated-patch recordings could be explained by a shunt through the seal contact between the pipette and membrane. Thus the membrane potential of CA3 pyramidal cells appears to be strongly negative at birth and does not change during postnatal development.     

Membrane potential and impedance changes in hippocampal pyramidal cells during theta rhythm

Summary Intracellular recordings were made from hippocampal pyramidal cells identified by their depths and their responses to commissural stimulation. Recordings were made during spontaneous bouts of hippocampal theta rhythm in urethane anesthetized rats. Membrane potentials (V _m) of pyramidal cells varied with the phase of the theta rhythm, that is, there was an intracellular theta rhythm. The changes in V _m averaged about 2 mV peak to peak. Averaged intracellular theta waves showed that CA1 pyramids were most depolarized at the time of the positive peak of the extracellular theta rhythm recorded in (and superficial to) the CA1 pyramidal cell layer (CA1 theta). Peak depolarizations for CA3/4 pyramids were more broadly distributed, but occurred mainly in the interval just before the positive peak to just before the negative peak of the CA1 theta. Input impedance minima that were measurable at frequencies as high as 100 Hz occurred at about the same phases of the extracellular theta rhythm as the peak depolarizations (positive-going zero crossing to negative-going zero crossing of the CA1 theta). Such impedance changes imply conductance changes on the soma. The magnitude and localization of the conductance changes suggests that somatic IPSPs make major contributions to the intracellular theta rhythm. The phase relation between the intracellular and extracellular theta rhythms could be reversed by long duration current pulses that depolarized the cells slightly. This implies that either the intracellular theta-related IPSPs are depolarizing potential changes, or that they occur simultaneously with EPSPs. The phase of the intracellular theta rhythm was generally unaffected by long duration hyperpolarizing current pulses. Chloride leakage that reversed the evoked IPSPs usually had no effect on the phase of the intracellular theta rhythm, although in one case it appeared to cause its amplitude to increase.     

Activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells

Amacrine cells in the vertebrate retina receive glutamatergic input from bipolar cells and make synapses onto bipolar cells, ganglion cells, and other amacrine cells. Recent studies indicate that amacrine cells express metabotropic glutamate receptors (mGluRs) and that signaling within the inner plexiform layer (IPL) of the retina might be modulated by mGluR activity. This study tests the hypothesis that activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells. Whole cell voltage-clamp recordings were combined with pharmacology to establish the identity of the ionotropic GABA receptors expressed in primary cultures of chick amacrine cells and to determine how mGluR5 activity affected the behavior of those receptors. Application of GABA (20 microM) produced currents that reversed at -58.2 +/- 0.9 mV, near the predicted Cl(-) reversal potential of -59 mV. The GABA(A) receptor antagonist, bicuculline (50 microM), completely blocked the GABA-gated currents. cis-4-Aminocrotonic acid (CACA; 100 microM), a GABA(C) receptor agonist, produced small currents that were not blocked by the GABA(C) antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA; 20 microM), but were completely blocked by bicuculline. These results indicate that cultured amacrine cells express GABA(A) receptors exclusively. Activating mGluR5 with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 300 microM) enhanced GABA-gated currents by 10.0 +/- 1.5%. Buffering internal Ca(2+) with BAPTA (10 mM) blocked the CHPG-dependent enhancement. Activation of PKC with the cell-permeable PKC activators (-)-7-octylindolactam V, phorbol 12-myristate 13 acetate (PMA), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GABA-gated currents in a dose-dependent manner. Preactivation of PKC occluded the mGluR5-dependent enhancement, and inhibition of Ca-dependent PKC isotypes with G?6976 (35 nM) suppressed the effects of mGluR5 activation, suggesting that mGluR5 and PKC are part of the same pathway. To determine if mGluR5-dependent enhancement occurred at synaptic GABA(A) receptors, postsynaptic currents were recorded in the presence of CHPG. On average, the mean amplitudes of the quantal events were increased by about 18% when mGluR5 was activated. These results indicate that activation of mGluR5 enhances GABA-gated current in cultured amacrine cells in a manner that is both Ca(2+)- and PKC-dependent. These results support the possibility that glutamate released from bipolar cells can modulate the function of GABAergic amacrine cells and alter signaling in the inner plexiform layer.     

Excitatory GABA input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.     

D-Serine differently modulates NMDA receptor function in rat CA1 hippocampal pyramidal cells and interneurons

The organization of the neuronal hippocampal network depends on the tightly regulated interaction between pyramidal cells (PCs) and interneurons (Ints). NMDA receptor (NMDAR) activation requires the binding of glutamate and co-activation of the 'glycine site'. It has been reported that d -serine is a more potent endogenous agonist than glycine for that site. While many studies have focused on NMDAR function in PCs, little is known regarding the modulation of NMDARs in Ints. We studied the modulatory effect of d -serine on NMDAR EPSCs in PCs and in stratum radiatum Ints using whole-cell patch-clamp recording in rat acute hippocampal slices. We found that d -serine enhances NMDAR function and differently modulates NMDAR currents in both cell types. The augmentation of NMDAR currents by d -serine was significantly larger in PCs compared with Ints. Moreover, we found differences in the kinetics of NMDAR currents in PCs and Ints. Our findings indicate that regulation of NMDAR through the 'glycine site' depends on the cell types. We speculate that the observed differences arise from assemblies of diverse NMDAR subunits. Overall, our data suggest that d -serine may be involved in regulation of the excitation-inhibition balance in the CA1 hippocampal region.     

Depolarizing IPSPs and depolarization by GABA of rat neostriatum cells in vitro

Summary In neostriatal slices pretreated with sodium pentobarbital (100 M) and 4-aminopyridine (50 M), intrastriatal stimulation elicited EPSPs followed by a slowly decaying depolarization which lasted about 200 ms and was associated with a membrane conductance increase and a suppression of spike potentials. This depolarizing inhibitory synaptic action could be blocked by picrotoxin (50 M) or bicuculline (50 M). The reversal potential for the slowly decaying depolarization was –57 to –62 mV, i.e. it was positive with respect to the resting membrane potential (¯x = –67 mV). GABA, injected into the tissue in the vicinity of the recording electrode by pressure application, or added to the perfusate (10 M–1 mM), depolarized the cells and reduced both the membrane resistance and the amplitude of EPSPs. The reversal potential of GABA depolarization was found in a potential range approximating that of the slowly decaying depolarization. These results are compatible with the assumption that GABA is the transmitter of an intrinsic inhibition in rat neostriatum, but indicate that GABA-mediated IPSPs of neostriatal cells in vitro are depolarizing at the resting membrane potential. The possible reasons for this are discussed.