Zinc and Zolpidem Modulate mIPSCs in Rat Neocortical Pyramidal Neurons
Tony Defazio and
John J. Hablitz
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
 |
ABSTRACT |
DeFazio, Tony and John J. Hablitz. Zinc and zolpidem modulate mIPSCs in rat neocortical pyramidal neurons. J. Neurophysiol. 80: 1670-1677, 1998. Pharmacological modulation of
-aminobutyric acid-A (GABAA) receptors can provide important information on the types of subunits composing these receptors. In recombinant studies, zinc more potently inhibits 
subunits compared with the 

combination, whereas modulation by nanomolar concentrations of the benzodiazepine type 1-selective agonist zolpidem is conferred by the
1
2 subunit combination. We examined four properties of miniature inhibitory postsynaptic currents (mIPSCs) from identified necortical pyramidal cells in rat brain slices: decay time constant, peak amplitude, rate of rise, and interevent interval. Exposure to 50 µM zinc reduced the decay time constant, peak amplitude, and rate of rise with no effect on interevent interval. Zolpidem enhanced mIPSCs in a concentration-dependent manner. Both 20 and 100 nM zolpidem increased the decay time constants of mIPSCs. In some cells, both peak amplitude and rate of rise were also enhanced. All cells treated with zinc were also responsive to zolpidem. These results show that neocortical pyramidal cells have a population of GABAA receptors sensitive to both zinc and zolpidem.
 |
INTRODUCTION |
Fast, chloride-dependent inhibitory synaptic transmission in the rat neocortex is mediated by
-aminobutyric acid-A receptors (GABAARs) (Connors et al. 1988
; Howe et al. 1987
; Krnjevic and Schwartz 1967
). GABAARs are the site of action of many convulsant and anticonvulsant drugs that interact with inhibitory synaptic mechanisms in the neocortex (Weiss and Hablitz 1984
). Spontaneous inhibitory postsynaptic potentials are prominent in neocortical pyramidal cells (e.g., Luhmann and Prince 1991
). Recent electrophysiological characterization of spontaneous and miniature inhibitory postsynaptic currents (mIPSCs) indicated that there is a tonic activation of GABAARs that may regulate neuronal excitability (Salin and Prince 1996
; Xiang et al. 1998
).
Molecular cloning studies identified
14 genes encoding GABAAR subunits (Macdonald and Olsen 1994
; Smith and Olsen 1995
). Functional GABAARs of the central nervous system are presumed to be heteropentameric structures assembled from a combination of homologous subunits from several classes:
1-6,
1-4,
1-4,
, and
(Chang et al. 1996
; Nayeem et al. 1994
; Whiting et al. 1997
). The most common combination is thought to be
1
2, which makes up 43% of the GABAARs in the rat brain (McKernan and Whiting 1996
). The subunit composition(s) of native GABAARs in neocortical pyramidal neurons is presently unknown, but messenger ribonucleic acids (mRNAs) for
1,
2,
2, and
are at least moderately expressed in adult rat neocortex in addition to weak signals for the
2,
3,
4,
3, and
1 subunits (Laurie et al. 1992
; Wisden et al. 1992
). Subunit-specific antibodies show a similar pattern of expression, with diffuse but intense labeling of
1,
2/3, and
2 subunits and moderate to weak labeling of
2,
3,
5, and
subunits (Fritschy and Mohler 1995
).
Because the subunit composition of the receptor can determine sensitivity to pharmacological agents that modulate the GABAAR, it is possible that these agents could be used to identify the types of subunits composing native GABAARs. Two potentially useful agents are the imidazopyridine zolpidem and the polyvalent cation zinc. The benzodiazepine (BZ) receptor on the GABAAR is one of the many sites of action for drugs affecting these receptors. At least two classes of native BZ (BZ1 and BZ2) receptors were described by using radioligand-binding studies in native tissue (e.g., see Olsen et al. 1990
). The imidazopyridine zolpidem, the triazolopyridazine Cl 218,872, and the 1,4-benzodiazepine 2-oxo-quazepam have a greater affinity for the BZ1 receptor, whereas many of the other agonists for the BZ site lack specificity for the BZ1 and BZ2 sites (for review see Seighart 1995).
Single GABAAR subunits or specific combinations were expressed in a number of recombinant expression systems. Detailed kinetic and pharmacological analyses assessed the role of the various subunits in determining sensitivity to BZ-related compounds and zinc. Recombinant systems expressing the
1 and
2 subunits in combination with a variety of
subunits give rise to a BZ1-like receptor, whereas changing the
subunit subtype to
2,
3, or
5 gives rise to a BZ2-like pharmacology (Pritchett and Seeburg 1990
; Pritchett et al. 1989a
,b
). The
6 subunit is expressed only in cerebellar granule cells and is not sensitive to most BZ agonists when expressed with
2 and
2 subunits (Luddens et al. 1990). The
subunit is also critical because BZ-related pharmacology is absent without it (Pritchett et al. 1989b
).
1 subunits are thought to be localized to glia and do not support BZ-like pharmacology.
2 subunits are the most common in mammalian brain and in combination with the above-mentioned
subunits give rise to most of the high-affinity BZ receptors.
3 subunits in combination with
1 or
5 subunits give rise zolpidem-insensitive receptors with high affinity for the compound Cl 218,872 (for review see Luddens et al. 1995). Neither
nor
subunits support BZ efficacy in the absence of a
subunit (Saxena and Macdonald 1994
; Whiting et al. 1997
), and the
subunit had no significant effect on diazepam modulation when coexpressed with 

subunits (Saxena and Macdonald 1994
).
Zinc sensitivity also depends on subunit composition. Recombinant GABAARs consisting of 
subunits are very sensitive to zinc (IC50 ~1 µM) via an apparent noncompetitive mechanism (Draguhn et al. 1990
; Gingrich and Burkat 1998
; Smart et al. 1991
; Wooltorton et al. 1997
). Zinc is a mixed antagonist of recombinant receptors containing
1
2
2 subunits where it is less potent (IC50
50 µM) (Chang et al. 1995
; Gingrich and Burkat 1998
). Both the 

and 

receptors have a sensitivity to zinc intermediate to that of 
and 

receptors (Krishek et al. 1998
; Saxena and Macdonald 1994
; Whiting et al. 1997
). The
subunit subtype may also affect zinc sensitivity; maximal inhibition of the 

receptor with the
2 or
3 subunit is greater than
1-containing receptors (White and Gurley 1995
), and zinc is much more potent at
6
3
2 receptors than at
1
3
2 receptors (Fisher and Macdonald 1997
, 1998
).
In an effort to pharmacologically identify the subunits mediating GABAAR-mediated inhibitory postsynaptic currents (IPSCs) in acute brain slices of the rat neocortex, we used the whole cell patch-clamp technique to measure the effects of zinc and zolpidem on miniature IPSCs. Some of these findings were presented in abstract form (DeFazio and Hablitz 1997
).
 |
METHODS |
Slice preparation
Sprague Dawley rats (17-27 days old) were housed and handled according to approved guidelines. Rats were anesthetized with ketamine and decapitated. The brain was rapidly removed and submerged in ice-cold, oxygenated (95% O2-5% CO2) low calcium saline consisting of (in mM) 125 NaCl, 3.5 KCl, 26 NaHCO3, 3.8 MgCl2, and 10 glucose. Coronal sections (300 µm) containing somatosensory cortex were cut with a Vibratome. Slices were stored in saline consisting of (in mM) 125 NaCl, 3.5 KCl, 26 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, and 10 glucose bubbled with 95% O2-5% CO2. Recordings were begun after a stabilization period of
1 h.
Recording
Individual slices were transferred to a recording chamber mounted on the stage of a Axioskop FS microscope equipped for infrared visualization of cells. Whole cell voltage-clamp recordings were obtained from layers II/III and V neocortical pyramidal cells with patch electrodes prepared from Garner KG-33 glass. The electrodes had resistances of 2-4 M
when filled with an internal solution consisting of (in mM) 140 CsCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 CaCl2, 4 MgATP, and 0.4 NaGTP. The bathing solution consisted of the storage saline listed above with the addition of the sodium channel blocker (tetrodotoxin, 0.5 µM) and the excitatory amino acid blockers D(
)2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (0.5, 20, and 10 µM, respectively). Membrane current was amplified with an Axopatch-1B amplifier. Signals were filtered at 2 kHz and digitized at 5 kHz in 40- to 60-s duration epochs. Series resistance and input resistance were monitored regularly during recording. Cells with series resistance >20 M
or significant changes in series resistance during the experiment were not used. The mean input resistance was 215 ± 20 M
and ranged from 144-333 M
.
Zolpidem and ZnCl2 were dissolved in 100% ethanol to make concentrated stock solutions weekly and were stored at
20°C. Control experiments with ethanol alone showed no effect on any of the mIPSC parameters used in this study. All compounds were obtained from Sigma with the exception of zolpidem (RBI).
Data analysis
pClamp data files were imported into CDR for event detection and subsequently exported into SCAN for analysis (CDR and SCAN programs courtesy of J. Dempster). Decay time constants, rate of rise, peak amplitudes, and interevent intervals for single IPSCs were measured with SCAN. Rate of rise was measured as the maximum rate of rise
10-90% of the peak height. Although a fraction of mIPSCs were better fit by double exponentials, for ease of analysis single exponential fits (20- to 30-ms fit window, forced to a zero baseline) were used throughout this study. Similar results were obtained by using the full width at half-maximum to measure mIPSC duration. Averaged mIPSCs were generated after aligning events on the rising phase. Cumulative probability plots (Van der Kloot 1989
) for each parameter were generated from
50 events (usually ~100) for each control and drug application. These plots were generated by sorting the values for a given parameter in ascending order and then assigning a probability between 0 and 1 based on the number of events preceding the event normalized by the total number of events in the distribution. Distributions were analyzed with the Kolmolgorov-Smirnov test (StatMost data analysis software, DataMost) and a P < 0.01 significance level for distributions with <100 events and P < 0.05 otherwise. Plots of distributions and averaged traces contained data from individual cells. Summary bar graphs reflect the average of the percent change relative to control of individual cells.

View larger version (16K):
[in this window]
[in a new window]
| FIG. 1.
Averaged miniature inhibitory postsynaptic currents (mIPSCs) illustrate modulation by 50 µM zinc. A: after 2 min to allow a complete solution change, bath-applied 50 µM zinc reduced the amplitude and decay in averaged mIPSCs from a layer II/III pyramidal cell. B: normalized traces demonstrate the changes in decay. Note the nearly complete reversal of the zinc effect after 5 min wash. The number of events averaged for the control, 50 µM zinc, and wash was 113, 117, and 128 respectively. Two 40-s epochs were analyzed for each condition.
|
|

View larger version (32K):
[in this window]
[in a new window]
| FIG. 2.
Cumulative probability plots for the events shown in Fig. 1. A: 50 µM zinc shifted the distribution of decay to the left. B and C: peak amplitude and rate of rise distributions were also reduced by zinc. D: no effect on interval was observed. The box associated with each probability plot reflects the level of significant difference between each of the distributions listed in rows and columns: , not significant; * P < 0.05; and ** P < 0.01.
|
|
 |
RESULTS |
Properties of mIPSCs
Under the recording conditions employed, pharmacologically isolated mIPSCs were recorded as inward currents at a holding potential of
60 mV. As described previously (Salin and Prince 1996
; Xiang et al. 1998
; Zhou and Hablitz 1997
), mIPSCs had rapid rise times and decayed with an exponential time course. Table 1 lists average decay time constants (
decay), rates of rise, peak amplitude, and interevent interval under control conditions. The cells common to both the zinc and zolpidem groups were only counted once for the combined group. No significant differences were observed between the two groups. The coefficient of variance (CV, the ratio of the SD to the mean) provides a measure of variability independent of the mean of a distribution. Consistent with the previous reports of the wide variation observed within individual cells, the mean CV for the peak amplitude (59.5 ± 2.5%, n = 13) was comparable with that observed in cerebellar stellate cells (Nusser et al. 1997
) and cultured retinal amacrine cells (Frerking et al. 1997
).
Zinc reduces amplitude, rate of rise, and decay time constant
Figure 1 shows averaged mIPSCs before and after bath application of 50 µM zinc. The primary effects of zinc were a reversible reduction in peak amplitude and
decay. By normalizing the peak amplitudes of the averaged traces, the reversible decrease in
decay is readily apparent (Fig. 1B). Probability distributions for
decay, peak amplitude, rate of rise, and interevent interval are shown in Fig. 2. The
decay was decreased in the presence of 50 µM zinc, as indicated by the leftward shift of the distribution (P < 0.01, Kolmogorov-Smirnov test). The effects of zinc on peak and rate of rise were more profound on larger events, possibly because of the smaller events falling below the threshold for detection. No correlation between the peak amplitude and
decay distributions was observed in any of the cells in this study (R < 0.3, n = 13), indicating that the effect on
decay was not simply due to a reduction in amplitude. No effect on interevent interval was observed (Fig. 2D).
The effects of zinc in six cells are summarized in Fig. 3. On average, zinc reduced the
decay by 14.0 ± 2.6%, whereas the rate of rise and peak amplitude were reduced by 27.5 ± 3.3% and 28.1 ± 2.6%, respectively. The slight increase in interevent interval averaged across cells was possibly due to a reduction of amplitude by zinc and subsequent loss of detection of the smallest events.

View larger version (41K):
[in this window]
[in a new window]
| FIG. 3.
Summary of the effects of zinc on mIPSCs. Zinc had consistent effects on decay, rate of rise, and peak amplitude across cells and no significant effect on interevent interval in any cell. The mean reductions expressed as percent of control values for the decay, rate of rise, peak amplitude, and interevent interval were 14.0 ± 2.6%, 27.5 ± 3.3%, 28.1 ± 2.6%, and 5.4 ± 4.9%, respectively.
|
|
Zolpidem enhances decay time constant
In the cell shown in Fig. 4A, bath applications of 20 and 100 nM zolpidem significantly enhanced the peak amplitude of averaged mIPSCs in a concentration-dependent fashion. The changes in
decay became apparent when the amplitudes of the averaged mIPSCs were normalized (Fig. 4B). The effect of 100 nM zolpidem on
decay was only partially reversed after 5 min of wash. Figure 5 shows cumulative probability plots of the data from the cell shown in Fig. 4. Both concentrations of zolpidem significantly enhanced
decay. The effect of 100 nM zolpidem persisted after >5 min wash (Fig. 5A). Although in other experiments both the effects of zinc and 20 nM zolpidem could be reversed with 5 min wash, the effect of 100 nM zolpidem on the
decay was not completely reversed in any of the cells with >5 min wash (wash distribution significantly greater than control, n = 4). This lack of reversibility may be due to the lipophilic nature of zolpidem, which would make it difficult to wash out of the brain slice.

View larger version (25K):
[in this window]
[in a new window]
| FIG. 4.
Effects of zolpidem on peak amplitude and decay were apparent in averaged mIPSC traces. A: raw averages illustrate the enhancement of mIPSC peak amplitude by 20 and 100 nM zolpidem. No wash was applied between the application of 20 and 100 nM zolpidem. Note the complete reversal of the effect of 100 nM zolpidem on peak amplitude. B: normalized traces clarify the effect of zolpidem on the decay kinetics of the averaged mIPSCs; 20 nM zolpidem showed a modest enhancement of decay, and 100 nM zolpidem induced a profound increase in the decay that persisted after 5-min wash. Numbers of events averaged under each condition were 202, 272, 236, and 263. Two 60 epochs were analyzed for each condition.
|
|

View larger version (42K):
[in this window]
[in a new window]
| FIG. 5.
Effects of zolpidem on individual events are shown in cumulative probability plots. A: modest enhancement of the decay kinetics by 20 nM zolpidem is reflected in a small but significant rightward shift in the distribution of decay; 100 nM zolpidem produced a much larger rightward shift, and, consistent with the effects on averaged mIPSCs, the distribution of decay after 5-min wash was significantly greater than the control distribution. B and C: peak amplitude and rate of rise distributions also reflect enhancement by both concentrations of zolpidem, although the effect on amplitude and rate was reversible. D: no significant effects on the interevent interval distributions were observed. The number of events for each distribution are the same as in Fig. 4. As in Fig. 2, boxes associated with each plot show the level of significance of the differences between the distributions listed in rows and columns: , not significant; * P < 0.05; and ** P < 0.01.
|
|
Twenty and 100 nM zolpidem significantly and reversibly enhanced IPSC amplitude in this cell (Fig. 5B). Rate of rise was reversibly enhanced by 100 nM zolpidem (Fig. 5C). No significant effect on interevent interval was observed (Fig. 5D).
Figure 6 summarizes the effects of zolpidem on mIPSCs. In every cell exposed to either 20 nM (n = 9) or 100 nM (n = 5) zolpidem, a significant enhancement of
decay was observed. On average this resulted in a 19.5 ± 2.4 and 27.2 ± 4.8% enhancement by 20 and 100 nM zolpidem, respectively. The large error bars for rate of rise and amplitude reflect the fact that only three of nine cells exposed to 20 nM zolpidem and two of five cells exposed to 100 nM zolpidem showed a significant effect on these parameters. The slight decrease in interevent interval may be the result of an increase in the number of events detected at threshold caused by amplitude enhancement by zolpidem.

View larger version (37K):
[in this window]
[in a new window]
| FIG. 6.
Summary of the effects of zolpidem. The largest effect of zolpidem was on the enhancement of the decay distribution, 19.0 ± 2.7% and 27.4 ± 4.8% for 20 nM and 100 nM zolpidem, respectively. Lack of effect on rate of rise and peak amplitude on most cells is reflected in the SE of the average increase.
|
|
Neocortical pyramidal cells are responsive to both zinc and zolpidem
A subset of cells tested with zinc was also exposed to zolpidem and was found to be sensitive to both compounds (n = 3). An example of the response of a zinc-sensitive cell to 20 nM zolpidem is shown in Fig. 7. Zolpidem (20 nM) significantly enhanced the mIPSC
decay, as indicated by the rightward shift of the distribution of
decay in Fig. 7A (P < 0.001, Kolmogorov-Smirnov test). The minor enhancement of peak amplitude apparent in the averaged traces (Fig. 7B1) was not significant when the distributions of peak amplitudes were compared with the Kolmogorov-Smirnov test. Normalized traces shown in Fig. 7B2 illustrate the reversible increase in
decay in the presence 20 nM zolpidem.

View larger version (16K):
[in this window]
[in a new window]
| FIG. 7.
Cells responsive to zinc were also responsive to zolpidem. A: cell from Figs. 1 and 2 was also sensitive to 20 nM zolpidem. The distribution of decay was reversibly shifted to the right. The zolpidem distribution was significantly greater than either control or wash, P < 0.01. Control and wash distributions were not significantly different. B: averaged traces (B1) and normalized (B2) averages are shown to illustrate the effects on decay. Numbers of events for each distribution and average were 128, 126, and 113 for control, 20 nM zolpidem, and wash, respectively.
|
|
 |
DISCUSSION |
The results of this study demonstrate that GABAAR-mediated mIPSCs in rat neocortical pyramidal cells can be modulated by both zinc and zolpidem. These agents had opposing effects on mIPSC
decay distributions; zinc shifted the distribution of time constants to smaller values, whereas zolpidem shifted the distribution to larger values. In contrast to the effects of ethanol on evoked hippocampal IPSCs (Weiner et al. 1997
) and the BZ receptor agonist flurazepam in cerebellar stellate cells (Nusser et al. 1997
), differential modulation of subpopulations of mIPSCs by zinc or zolpidem was not apparent in the decay time constant distributions (e.g., Figs. 2 and 5). This suggests that most of the receptors on rat neocortical pyramidal cells are responsive to both zinc and zolpidem (e.g., Fig. 7).
Properties of mIPSCs in neocortical pyramidal cells
Previous studies examined the kinetic properties of mIPSCs in neocortical interneurons and pyramidal cells (Salin and Prince 1996
; Xiang et al. 1998
; Zhou and Hablitz 1997
). Similar to previous studies in neocortex and other central neurons, mIPSC amplitudes were highly variable, ranging from 10 to 200 pA. Amplitude histograms were generally not well fit with single or sums of multiple Gaussian functions (data not shown). As measured by the coefficient of variance, amplitude variability was similar to that observed in cerebellar stellate cells and retinal amacrine cells (>50%) (Frerking et al. 1997
; Nusser et al. 1997
). The origins of this variability and non-Gaussian distribution are not well understood. Both pre- and postsynaptic mechanisms were hypothesized to underlie this phenomenon. These include variability in the number of receptors at postsynaptic sites (Nusser et al. 1997
) or variability in the amount of transmitter released from vesicles (Frerking et al. 1995
).
Zinc and zolpidem have opposite effects on mIPSCs
The current results demonstrate that GABAARs on rat neocortical pyramidal cells are sensitive to both zolpidem and zinc. Zinc (50 µM) reduced the rate of rise, amplitude, and
decay. These properties are consistent with a reduction in the affinity of the receptor for GABA, possibly via a mechanism of "mixed antagonism" similar to that observed for the recombinant
GABA receptor (Chang et al. 1995
) or
1
2 receptors (Gingrich and Burkat 1998
; Krishek et al. 1998
). Contrary to previous reports in the hippocampus (Buhl et al. 1996
), no presynaptic effect of zinc on mIPSC frequency was observed.
Zolpidem consistently enhanced the
decay and in some cells also increased the amplitude and rate of rise of mIPSCs. Zolpidem and other BZs are thought to modulate GABAARs by increasing the affinity for GABA and increasing the frequency of channel opening (Rogers et al. 1994
). Under nonsaturating conditions, this modulation would increase the amplitude of the GABA response (Frerking et al. 1995
; Lavoie and Twyman 1996
; Poncer et al. 1996
). The observation that zolpidem increased mIPSC amplitude in some cells indicates that rat neocortical neurons may have postsynaptic sites that are not saturated by quantal release of GABA (Frerking et al. 1995
; Nusser et al. 1997
). BZs may also increase single channel conductance (Eghbali et al. 1997
), although the inconsistent effect of zolpidem on amplitude suggests that this is not the case for native neocortical pyramidal cell GABAARs.
In cerebellar stellate cells, a subpopulation of mIPSCs with larger amplitudes and greater sensitivity to the BZ agonist flurazepam was observed in the probability distribution (Nusser et al. 1997
). In this study, the effects of both zinc and zolpidem on amplitude and rate of rise were greater on larger, faster events, whereas the
decay distributions tended to reflect shifts in the entire distribution. These inconsistent effects could possibly be explained by variation in the degree of saturation at different release sites. For the case of zolpidem, amplitude modulation via a change in affinity for GABA would not be observed at saturated release sites. If the smaller mIPSCs were due to release sites that were saturated because of the presence of fewer receptors, no amplitude modulation would occur. However, the decay time constant would be enhanced at both saturated and nonsaturated release sites. A similar argument can be made for zinc, assuming a zinc-mediated decrease in affinity for GABA. Another possibility is that the smallest events were lost in the baseline noise, which would force all the amplitude distributions to start at roughly the same value.
Implications for the pharmacological identification of native receptors
All cells exposed to zinc and zolpidem exhibited sensitivity to both agents (e.g., Fig. 7). Enhancement of the mIPSC
decay distribution in response to 20 nM zolpidem indicates the presence of the high-affinity BZ1 receptor thought to be conferred by an
1
2 subunit combination. This is further supported by a moderate sensitivity to zinc. Previous studies with native receptors in other brain regions revealed sensitivity to both BZ agonists and moderate concentrations of zinc (Celentano et al. 1991
; Legendre and Westbrook 1991
; Smart 1992
). Alterations in zinc and BZ sensitivity were observed in several animal models of epilepsy (Buhl et al. 1996
; Gibbs et al. 1997
; Kapur and Macdonald 1997
).
However, more complicated scenarios cannot be excluded. In situ hybridization revealed mRNAs for
1,
2,
3,
4,
2,
3,
1,
2, and
subunits in layer II/III of adult rat neocortex (reviewed in Wisden et al. 1995). Similar distributions of subunits were identified with antibodies (Fritschy and Mohler 1995
). The possibility that a combination of different subunit subtypes within the heteropentameric GABAAR complex could also give rise to this dual sensitivity cannot be ignored because the pharmacology of multiple combinations of
subunits has not been thoroughly studied in recombinant systems. The
subunit also could enhance the zinc sensitivity of 

receptors (Saxena and Macdonald 1994
).
 |
ACKNOWLEDGEMENTS |
The authors thank A. Margolies for excellent technical assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-22373.
 |
FOOTNOTES |
Address reprint requests to J. J. Hablitz.
Received 27 February 1998; accepted in final form 11 June 1998.
 |
REFERENCES |
-
BUHL, E. H.,
OTIS, T. S.,
MODY, I.
Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model.
Science
271: 369-373, 1996.[Abstract]
-
CELENTANO, J. J.,
GYENES, M.,
GIBBS, T. T.,
FARB, D. H.
Negative modulation of the
-aminobutyric acid response by extracellular zinc.
Mol. Pharmacol.
40: 766-773, 1991.[Abstract] -
CHANG, Y.,
AMIN, J.,
WEISS, D. S.
Zinc is a mixed antagonist of homomeric
1
-aminobutyric acid-activated channels.
Mol. Pharmacol.
47: 595-602, 1995.[Abstract] -
CHANG, Y.,
WANG, R.,
BAROT, S.,
WEISS, D. S.
Stoichiometry of a recombinant GABAA receptor.
J. Neurosci.
16: 5415-5424, 1996.[Abstract/Free Full Text]
-
CONNORS, B. W.,
MALENKA, R. C.,
SILVA, L. R.
Two inhibitory postsynaptic potentials, and GABAA and GABAB receptor-mediated responses in neocortex of rat and cat.
J. Physiol. (Lond.)
406: 443-468, 1988.[Abstract]
-
DEFAZIO, T.,
HABLITZ, J. J.
Zinc and zolpidem modulate miniature IPSCs in rat neocortex.
Soc. Neurosci. Abstr.
23: 105, 1997.
-
DRAGUHN, A.,
VERDORN, T. A.,
EWERT, M.,
SEEBURG, P. H.,
SAKMANN, B.
Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+.
Neuron
5: 781-788, 1990.[Medline]
-
EGHBALI, M.,
CURMI, J. P.,
BIRNIR, B.,
GAGE, P. W.
Hippocampal GABAA channel conductance increased by diazepam.
Nature
388: 71-75, 1997.[Medline]
-
FISHER, J. L.,
MACDONALD, R. L.
Single channel properties of recombinant GABAA receptors containing
2 or
subtypes expressed with
1 and
3 subtypes in mouse L929 cells.
J. Physiol. (Lond.)
505: 283-297, 1997.[Abstract] -
FISHER, J. L.,
MACDONALD, R. L.
The role of an
subtype M2-M3 in regulating inhibition of GABAA receptor current by zinc and other divalent cations.
J. Neurosci.
18: 2944-2953, 1998.[Abstract/Free Full Text] -
FRERKING, M.,
BORGES, S.,
WILSON, M.
Variation in GABA mini amplitude is the consequence of variation in transmitter concentration.
Neuron
15: 885-895, 1995.[Medline]
-
FRERKING, M.,
BORGES, S.,
WILSON, M.
Are some minis multiquantal?
J. Neurophysiol.
78: 1293-1304, 1997.[Abstract/Free Full Text]
-
FRITSCHY, J. M.,
MOHLER, H.
GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits.
J. Comp. Neurol.
359: 154-194, 1995.[Medline]
-
FRITSCHY, J. M.,
PAYSAN, J.,
ENNA, A.,
MOHLER, H.
Switch in the expression of rat GABAA-receptor subtypes during postnatal development: an immunohistochemical study.
J. Neurosci.
14: 5302-5324, 1994.[Abstract]
-
GIBBS, J. W. III,
SHUMATE, M. D.,
COULTER, D. A.
Differential epilepsy-associated alterations in postsynaptic GABAA receptor function in dentate granule and CA1 neurons.
J. Neurophysiol.
77: 1924-1938, 1997.[Abstract/Free Full Text]
-
GINGRICH, K. J.,
BURKAT, P. M.
Zn2+ inhibition of recombinant GABAA receptors
an allosteric, state-dependent mechanism determined by the
-subunit.
J. Physiol. (Lond.)
506: 609-625, 1998.[Abstract/Free Full Text] -
HOWE, J. R.,
SUTOR, B.,
ZIEGLGANSBERGER, W.
Characteristics of long-duration inhibitory postsynaptic potentials in rat neocortical neurons in vitro.
Cell. Mol. Neurobiol.
7: 1-18, 1987.[Medline]
-
KAPUR, J.,
MACDONALD, R. L.
Rapid seizure-induced reduction of benzodiazepine and Zn2+ sensitivity of hippocampal dentate granule cell GABAA receptors.
J. Neurosci.
17: 7532-7540, 1997.[Abstract/Free Full Text]
-
KRISHEK, B. J.,
MOSS, S. J.,
SMART, T. G.
Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors.
J. Physiol. (Lond.)
507: 639-652, 1998.[Abstract/Free Full Text]
-
KRNJEVIC, K.,
SCHWARTZ, S.
The action of
-aminobutyric acid on cortical neurones.
Exp. Brain Res.
3: 320-336, 1967.[Medline] -
LAURIE, D. J.,
WISDEN, W.,
SEEBURG, P. H.
The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development.
J. Neurosci.
12: 4151-4172, 1992.[Abstract]
-
LAVOIE, A. M.,
TWYMAN, R. E.
Direct evidence for diazepam modulation of GABAA receptor microscopic affinity.
Neuropharmacology.
35: 1383-1392, 1996.[Medline]
-
LEGENDRE, P.,
WESTBROOK, G. L.
Noncompetitive inhibition of
-aminobutyric acid A channels by Zn.
Mol. Pharmacol.
39: 267-274, 1991.[Abstract] -
LÜDDENS, H.,
PRITCHETT, D. B.,
KOHLER, M.,
KILLISCH, I.,
KEINANEN, K.,
MONYER, H.,
SPRENGEL, R.,
SEEBURG, P. H.
Cerebellar GABAA receptor selective for a behavioural alcohol antagonist.
Nature
346: 648-651, 1990.[Medline]
-
LÜDDENS, H.,
KORPI, E. R.,
SEEBURG, P. H.
GABAA/benzodiazepine receptor heterogeneity: neurophysiological implications.
Neuropharmacology
34: 245-254, 1995.[Medline]
-
LUHMANN, H. J.,
PRINCE, D. A.
Postnatal maturation of the GABAergic system in rat neocortex.
J. Neurophysiol.
65: 247-263, 1991.[Abstract/Free Full Text]
-
MACDONALD, R. L.,
OLSEN, R. W.
GABAA receptor channels.
Annu. Rev. Neurosci.
17: 569-602, 1994.[Medline]
-
MCKERNAN, R. M.,
WHITING, P. J.
Which GABAA-receptor subtypes really occur in the brain?
Trends Neurosci.
19: 139-143, 1996.[Medline]
-
NAYEEM, N.,
GREEN, T. P.,
MARTIN, I. L.,
BARNARD, E. A.
Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.
J. Neurochem.
62: 815-818, 1994.[Medline]
-
NUSSER, Z.,
CULL-CANDY, S.,
FARRANT, M.
Differences in synaptic GABAA receptor number underlie variation in GABA mini amplitude.
Neuron
19: 697-709, 1997.[Medline]
-
OLSEN, R. W.,
MCCABE, R. T.,
WAMSLEY, J. K.
GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system.
J. Chem. Neuroanat.
3: 59-76, 1990.[Medline]
-
PONCER, J.-C.,
DÜRR, R.,
GÄHWILER, B. H.,
THOMPSON, S. M.
Modulation of synaptic GABAA receptor function by benzodiazepines in area CA3 of rat hippocampal slice cultures.
Neuropharmacology
35: 1169-1179, 1996.[Medline]
-
PRITCHETT, D. B.,
LUDDENS, H.,
SEEBURG, P. H.
Type I and type II GABAA-benzodiazepine receptors produced in transfected cells.
Science
245: 1389-1392, 1989a.[Medline]
-
PRITCHETT, D. B.,
SONTHEIMER, H.,
SHIVERS, B. D.,
YMER, S.,
KETTENMANN, H.,
SCHOFIELD, P. R.,
SEEBURG, P. H.
Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.
Nature
338: 582-585, 1989b.[Medline]
-
PRITCHETT, D. B.,
SEEBURG, P. H.
-Aminobutyric acidA receptor
5-subunit creates novel type II benzodiazepine receptor pharmacology.
J. Neurochem.
54: 1802-1804, 1990.[Medline] -
RABOW, L. E.,
RUSSEK, S. J.,
FARB, D. H.
From ion currents to genomic analysis: recent advances in GABAA receptor research.
Synapse
21: 189-274, 1995.[Medline]
-
ROGERS, C. J.,
TWYMAN, R. E.,
MACDONALD, R. L.
Benzodiazepine and
-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.
J. Physiol. (Lond.)
475: 69-82, 1994.[Abstract] -
SALIN, P. A.,
PRINCE, D. A.
Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex.
J. Neurophysiol.
75: 1573-1588, 1996.[Abstract/Free Full Text]
-
SAXENA, N. C.,
MACDONALD, R. L.
Assembly of GABAA receptor subunits: role of the
subunit.
J. Neurosci.
14: 7077-7086, 1994.[Abstract] -
SIEGHART, W.
Structure and pharmacology of
-aminobutyric acidA receptor subtypes.
Pharmacol. Rev.
47: 181-234, 1995.[Medline] -
SMART, T. G. A
novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.
J. Physiol. (Lond.)
447: 587-625, 1992.[Abstract]
-
SMART, T. G.,
MOSS, S. J.,
XIE, X.,
HUGANIR, R. L.
GABAA receptors are differentially sensitive to zinc: dependence on subunit composition.
Br. J. Pharmacol.
103: 1837-1839, 1991.[Abstract]
-
SMITH, G. B.,
OLSEN, R. W.
Functional domains of GABAA receptors.
Trends Pharmacol. Sci.
16: 162-168, 1995.[Medline]
-
VAN DER KLOOT, W.
Statistical and graphical methods for testing the hypothesis that quanta are made up of subunits.
J. Neurosci. Methods
27: 81-89, 1989.[Medline]
-
WEINER, J. L.,
GU, C.,
DUNWIDDIE, T. V.
Differential ethanol sensitivity of subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal neurons.
J. Neurophysiol.
77: 1306-1312, 1997.[Abstract/Free Full Text]
-
WEISS, D. S.,
HABLITZ, J. J.
Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex.
Cell. Mol. Neurobiol.
4: 301-317, 1984.[Medline]
-
WHITE, G.,
GURLEY, D. A.
subunits influence Zn2+ block of
2 containing GABAA receptor currents.
Neuroreport
6: 461-464, 1995.[Medline] -
WHITING, P. J.,
MCALLISTER, G.,
VASSILATIS, D.,
BONNERT, T. P.,
HEAVENS, R. P.,
SMITH, D. W.,
HEWSON, L.,
O'DONNELL, R.,
RIGBY, M. R.,
SIRINATHSINGHJI, D. J.,
MARSHALL, G.,
THOMPSON, S. A.,
WAFFORD, K. A.
Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties.
J. Neurosci.
17: 5027-5037, 1997.[Abstract/Free Full Text]
-
WISDEN, W.,
LAURIE, D. J.,
MONYER, H.,
SEEBURG, P. H.
The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon.
J. Neurosci.
12: 1040-1062, 1992.[Abstract]
-
WOOLTORTON, J.R.A.,
MCDONALD, B. J.,
MOSS, S. J.,
SMART, T. G.
Identification of a Zn2+ binding site on the murine GABAA receptor complex-dependence on the second transmembrane domain of
subunits.
J. Physiol. (Lond.)
505: 633-640, 1997.[Abstract] -
XIANG, Z. X.,
HUGUENARD, J. R.,
PRINCE, D. A.
GABAA receptor-mediated currents in interneurons and pyramidal cells of rat visual cortex.
J. Physiol. (Lond.)
506: 715-730, 1998.[Abstract/Free Full Text]
-
ZHOU, F. M.,
HABLITZ, J. J.
Metabotropic glutamate receptor enhancement of spontaneous IPSCs in neocortical interneurons.
J. Neurophysiol.
78: 2287-2295, 1997.[Abstract/Free Full Text]