1Department of Neurology, 2Department of Physiology, and 3Department of Anatomy, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia 23298-0599; and 4Divisions of Neurology and Neuroscience, Department of Pediatrics, University of Pennsylvania School of Medicine and 5Pediatric Regional Epilepsy Program of the Children's Hospital of Philadephia, Philadelphia, Pennsylvania 19104-4318
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ABSTRACT |
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Gibbs III, John W.,
Yun-Fu Zhang,
Melissa D. Shumate, and
Douglas A. Coulter.
Regionally Selective Blockade of GABAergic Inhibition by Zinc in
the Thalamocortical System: Functional Significance.
J. Neurophysiol. 83: 1510-1521, 2000.
The
thalamocortical (TC) system is a tightly coupled synaptic circuit in
which GABAergic inhibition originating from the nucleus reticularis
thalami (NRT) serves to synchronize oscillatory TC rhythmic behavior.
Zinc is colocalized within nerve terminals throughout the TC system
with dense staining for zinc observed in NRT, neocortex, and thalamus.
Whole cell voltage-clamp recordings of GABA-evoked responses were
conducted in neurons isolated from ventrobasal thalamus, NRT, and
somatosensory cortex to investigate modulation of the GABA-mediated
chloride conductance by zinc. Zinc blocked GABA responses in a
regionally specific, noncompetitive manner within the TC system. The
regional levels of GABA blockade efficacy by zinc were: thalamus > NRT > cortex. The relationship between clonazepam and zinc
sensitivity of GABAA-mediated responses was examined to
investigate possible presence or absence of specific GABAA
receptor (GABAR) subunits. These properties of GABARs have been
hypothesized previously to be dependent on presence or absence of the
2 subunit and seem to display an inverse relationship. In
cross-correlation plots, thalamic and NRT neurons did not show a
statistically significant relationship between clonazepam and zinc
sensitivity; however, a statistically significant correlation was
observed in cortical neurons. Spontaneous epileptic TC oscillations can
be induced in vitro by perfusion of TC slices with an extracellular medium containing no added Mg2+. Multiple varieties of
oscillations are generated, including simple TC burst complexes
(sTBCs), which resemble spike-wave discharge activity. A second variant
was termed a complex TC burst complex (cTBC), which resembled
generalized tonic clonic seizure activity. sTBCs were exacerbated by
zinc, whereas cTBCs were blocked completely by zinc. This supported the
concept that zinc release may modulate TC rhythms in vivo. Zinc
interacts with a variety of ionic conductances, including GABAR
currents, N-methyl-D-aspartate (NMDA)
receptor currents, and transient potassium (A) currents.
D
2-amino-5-phosphonovaleric acid and 4-aminopyridine
blocked both s- and cTBCs in TC slices. Therefore NMDA and A
current-blocking effects of zinc are insufficient to explain
differential zinc sensitivity of these rhythms. This supports a
significant role of zinc-induced GABAR modulation in differential TC
rhythm effects. Zinc is localized in high levels within the TC system
and appears to be released during TC activity. Furthermore application
of exogenous zinc modulates TC rhythms and differentially blocks GABARs
within the TC system. These data are consistent with the hypothesis
that endogenously released zinc may have important neuromodulatory
actions impacting generation of TC rhythms, mediated at least in part
by effects on GABARs.
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INTRODUCTION |
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Zinc is present throughout the brain in nerve terminals
(Haug 1967; Wensink et al. 1988)
contained within synaptic vesicles (Ibata and Otsuka
1969
). This zinc can be released in concentrations as high as
100-300 µM during neuronal activity (Assaf and Chung 1984
). Excessive levels of zinc have been proposed to be
released during intense neuronal firing and could contribute to seizure discharge activity (Assaf and Chung 1984
) with elevated
levels associated with metabolic disorders and epileptic seizures.
Although several studies have investigated both the normal and
pathological roles of zinc release in the hippocampus
(Brooks-Kayal et al. 1998
; Buhl et al.
1996
; Gibbs et al. 1997a
; Xie and Smart
1993
), no reports to date have detailed the effects of zinc in
the TC system and the possible role of this cotransmitter in the
modulation of TC rhythms. The TC system contains zinc in the cortex and
thalamus, and particularly strong staining of zinc-containing terminals is evident in the nucleus reticularis thalami (NRT) (Haug
1973
) (see also Fig. 1). The TC
system is a tightly interconnected synaptic circuit consisting of the
reciprocally connected cortex, thalamus, and an interposed GABAergic
nucleus, NRT, which provides inhibitory innervation onto thalamus.
GABAergic inhibition originating in NRT and impinging onto thalamus
synchronizes and drives both normal and oscillatory TC activity,
including sleep spindles, and the spike-wave discharges (SWDs) of
Generalized Absence (GA) epilepsy (reviewed in Steriade and
Llinás 1988
).
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Synaptically released zinc may serve as a neuromodulator, interacting
with various ligand- and voltage-gated conductances to modify cellular
responses. Zinc effects on GABARs,
N-methyl-D-aspartate (NMDA) receptors, and
voltage-gated K+ channels have all been described
(Harrison and Gibbons 1994; Westbrook and Mayer
1987
). However, the actual role of zinc in modulating function
within the CNS is poorly understood. Zinc modulatory effects on
GABA-mediated transmission have been demonstrated to be both pre- and
postsynaptic in nature. Postsynaptically, zinc blockade of GABARs has
been investigated in recombinant receptor systems and has been shown to
depend on the subunit conformation of the receptor complex
(Knoflach et al. 1996
; Saxena and Macdonald 1994
,
1996
; Smart et al. 1991
; White and Gurley
1995
). In addition, multiple putative zinc binding sites have
been characterized within GABARs (Fisher and Macdonald
1998
; Wang et al. 1995
; Wooltorton et al.
1997
). Given the distinct developmental and regional
distribution of GABAR subunits in the brain (Laurie et al.
1992
; Wisden et al. 1992
), differential
sensitivity of GABAR responses to zinc could have significant
physiological consequences in the generation and synchronization of TC rhythms.
In the present study, whole cell patch recordings were conducted in
acutely isolated neurons to investigate the modulatory effects of zinc
on GABA-evoked responses in somatosensory cortex, ventrobasal thalamus,
and NRT. Additionally, extracellular field potential recordings were
performed to examine the effects of zinc on spontaneous TC rhythms. A
preliminary report of these findings has been published in abstract
form (Gibbs et al. 1996b).
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METHODS |
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Acute isolation of neurons
Experiments were conducted on neurons acutely isolated
from adult rat somatosensory cortex, ventrobasal thalamus, and NRT using methods that have been previously described (Gibbs et al. 1996a,b
, 1998
; Oh et al. 1995
). The brain was
dissected and placed in a 4°C chilled, oxygenated
(95%O2-5%CO2) artificial
cerebrospinal fluid (ACSF) solution composed of (in mM) 201 sucrose, 3 KCl, 1.25 NaHPO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose. Coronal TC slices (400 µm) were cut and incubated for
1 h in an oxygenated medium
containing (in mM) 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 25 glucose, and 20 piperazine-N,
N'-bis [2-ethanesulfonic acid] (PIPES); with the pH adjusted to
7.0 with NaOH at 32°C. Slices were enzymed in 3 mg/ml Sigma protease
XXIII in PIPES, thoroughly rinsed, and incubated another 30-60 min in
PIPES medium before dissociation. The somatosensory cortex, thalamus,
and NRT were visualized using a dark field microscope and
microdissected out, especially making certain not to contaminate
neuronal plates containing thalamic and NRT cells, which lie in very
close proximity to one another. Chunks (1 mm2)
then were cut from each area, and cells were dispersed mechanically by
trituration of the chunks through Pasteur pipettes of decreasing bore
sizes. The resulting cell suspension then was plated onto 35-mm culture
dishes in N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (HEPES) medium composed of (in mM) 155 NaCl, 3 KCl, 1 MgCl2, 3 CaCl2, 0.0005 tetrodotoxin, and 10 HEPES-Na+, with pH adjusted
to 7.4 with NaOH.
Voltage-clamp recordings in isolated neurons
The intracellular (pipette) solution contained (in mM) 100 trizma phosphate (dibasic), 28 trizma base, 11 ethylene glycol bis-(-aminoethylether)-N, N,N',N'-tetraacetic acid, 2 MgCl2, and 0.5 CaCl2, with
pH adjusted to 7.35 with NaOH. Whole cell patch-clamp recording
experiments were conducted on a Nikon inverted microscope equipped with
Hoffman modulation contrast optics. Electrodes (4-8 M
) were pulled
on a Narishige PP-83 microelectrode two-stage puller using thin-walled
borosilicate capillary glass (WPI, Sarasota, FL). The pipette solution
also contained an intracellular ATP reconstitution consisting of 10 mM
Mg2+-ATP and 22 mM tris-phosphocreatinine 22. The
intracellular ATP maintenance solution was used to fill the shank of
the electrode but was omitted from the solution that was used to back
fill the tip of the electrode. Recordings were amplified using an
Axopatch 200A amplifier (Axon Instruments, Burlingame, CA) and filtered at 5 kHz with a 4-pole Bessel filter before digitization. All data were
displayed on a chart recorder on-line (Gould, Model 2107, Cleveland,
OH; frequency response DC-50 Hz) and stored on videotape after
digitization (at 44 kHz) with a PCM interface (Neurodata Instrument,
New York, NY). Data were played back on a chart recorder with a
frequency response of DC-25 kHz (Astro-Med DASH IV, Warwick, RI).
Drug concentrations and method of application
GABA was prepared as a 10 mM stock solution in HEPES solution.
Zinc first was dissolved in distilled water at 100 mM and then diluted
to the final concentration in the HEPES medium, whereas clonazepam
first was dissolved in dimethyl sulfoxide (DMSO) at 100 mM and then
diluted to the final concentration in HEPES medium. The maximum
concentration of DMSO used in cellular perfusions was <0.001% and has
been shown not to alter GABA responses (Oh et al. 1995).
The applied drug concentrations were as follows: GABA (Sigma, St.
Louis, MO), 1 µM to 10 mM; clonazepam (Sigma), 100 nM; and zinc,
1-300 µM (Sigma). Solution changes were accomplished using a
13-barrel modified "sewer pipe" perfusion technique (Gibbs et al. 1997b
) in which several solutions flowed out of parallel Teflon tubes (0.2 mm ID) in a laminar fashion. Rapid (40-200 ms) and
complete solution changes at a constant flow rate then were effected by
moving the tube assembly laterally in relation to the neuron under
study. No cross-contamination was ever evident. After breaking the seal
to institute whole cell recording mode and allowing ~2-5 min to pass
to establish stable leak currents (0 to
200 pA), GABA was applied for
4-6 s and washed out with control external solution for 30-40 s. The
cell was pretreated with test drugs without GABA for 50-60 s and then
test solutions were applied together with GABA. Drug effects with
clonazepam and zinc were expressed as percentage effect on GABA-evoked
outward currents, recorded at a VHOLD
of
24 mV. Experiments were performed at 22-24°C.
Extracellular recording
TC slices were cut, and the presence of adequate TC connections
verified using a dark field microscope as described previously (Agmon and Connors 1991; Coulter and Lee
1993
; Zhang and Coulter 1996
; Zhang et
al. 1996a
,b
). Slices were transferred to an incubator containing ACSF [composed of (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 0 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose] where they were kept in
warmed (34°C) oxygenated medium. Before recording, slices were transferred to an interface recording chamber and perfused with warmed
(34°C) oxygenated (95% O2-5%
CO2) ACSF. Insulated tungsten electrodes were
positioned in TC slices with one electrode in the thalamus and one in
the cortex to confirm generalization. Signals were recorded
differentially with an AC-coupled four-channel amplifier at a gain of
1,000, with a ground electrode located nearby in the bathing medium.
Signals were bypass filtered at 10-3,000 Hz. Data were displayed
during an experiment on a four-channel chart recorder (AstroMed Dash
IV, Warwick, RI) and digitized (at 22 kHz) and stored on videotape for
later analysis using a PCM interface (Neurodata Instrument, New York, NY).
Drug application to TC slices
Zinc (100-300 µM), 4-aminopyridine (4-AP; 50 µM to 1 mM),
and D2-amino-5-phosphonovaleric acid (D-APV;
50 µM) were dissolved into the ACSF and bath applied. A 15- to 30-min
control period of stable TC activity was recorded, followed by a 10- to
30-min period of drug exposure and a 1-h wash in each experiment. Only reversible effects were analyzed.
Staining
Rat brain tissue was placed in a 1.2%
Na2S solution in a sodium phosphate buffer for
20-30 min and then transferred to a solution containing 1%
paraformaldehyde and 1.25% glutaraldehyde in a sodium phosphate buffer
and refrigerated overnight. The tissue then was placed in a 30%
sucrose solution in a sodium phosphate buffer for 2-4 h, frozen in
isopentane cooled with liquid nitrogen, and stored at 70°C until
sectioning. Tissue then was cut on a cryostat into coronal 25-µm
sections containing ventrobasal thalamus, somatosensory cortex, and
NRT, melted onto gelatin-coated slides, and stored at
70°C.
Sections then were thawed, hydrated, and developed in Timm's stain
(Haug 1973
). Slide-mounted sections then were
dehydrated, placed in xylene, and cover slipped for histological examination.
Statistical analysis on GABA response curves
All values are expressed as means ± 1 SE. Differences
between means were tested using Student's t-test.
Concentration/response curves were fitted by a Marquardt-Levenberg
nonlinear least-squares routine, using either ORIGIN (MicroCal
Software, Northhampton, MA) or ALLFIT (De Lean et al.
1987). The significance of differences in best fit parameter
values between curves was assessed using constrained simultaneous curve
fitting testing the equality of parameters, and ALLFIT, as described in
De Lean et al. (1987)
. This method involves testing for
equality of parameters by examining the statistical consequences (via
an F test) of forcing them to be equal.
Statistical analysis on extracellular fields
After a preexposure to a medium containing no added
Mg2+, TC slices exhibited two main types of
spontaneous TC bursting activities, termed simple TC burst complexes
(sTBCs) and complex TC burst complexes (cTBCs), respectively
(Coulter and Lee 1993; Zhang and Coulter
1996
; Zhang et al. 1996a
,b
). sTBCs had
similarities to SWDs with morphology, frequency, duration, and
pharmacological sensitivity to anticonvulsant drugs resembling bursting
activity observed in GA epilepsy (5-10 Hz, 2-10 s). cTBCs had
characteristic electrophysiological similarities to generalized
tonic-clonic discharges (GTCs) with high-frequency tonic firing
followed by a period of phasic bursting lasting in total usually 30-90
s in duration. These rhythms also had a pharmacological sensitivity to
anticonvulsant drugs similar to GTCs (Zhang and Coulter
1996
). Zinc was bath applied to TC slices exhibiting
spontaneous epileptiform rhythms to examine the functional effects of
zinc in an in vitro slice preparation capable of supporting sTBCs and cTBCs.
Modulatory effects on extracellular thalamocortical rhythms were
quantified as has been described previously (Zhang and Coulter 1996; Zhang et al. 1996a
,b
). At least a 30-min
control period of activity was recorded before drug application, and in
the last 15 min of the control period, the chart record was examined to ensure that the spontaneous TC burst activity was stable. The last five
events before the solution change then were employed to measure the
average duration and amplitude of cortical bursts. The duration was
measured as the time from onset of the activity to the end of the last
burst in the complex and was measured in the cortical recording trace
because the signal-to-noise ratio was higher than in the thalamic
trace. The duration measurement also reflected the thalamic burst
complex duration because cortical and thalamic activity was coupled
tightly. The amplitude of cortical bursting was measured peak to peak
at the maximal level during the tonic burst activity in the cortical
trace. Each of these measures was taken for five sequential TC burst
complexes, and then the average of these five measurements was used for
comparison. After change to a drug-containing solution, the level of
drug effect was monitored until a maximum stable effect was achieved, and then a 15-min peak effect exposure period was monitored to ensure
activity was stable. At the end of this 15-min period, immediately
before the wash solution change, the last five TC burst complexes were
quantified as described in the preceding text. After washout of drug,
slices were monitored until a stable washout effect was achieved and
then measurements taken as described in the preceding text. For
construction of the time series bar graphs in this series of studies
(Fig. 5, B and D), the average measures of events
in each bin was continuously monitored, and the measurements averaged
per bin.
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RESULTS |
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Timm's staining in TC slices
Presence of the endogenous metal cation, zinc, can be
detected using Timm's stain for heavy metals (Haug
1973). Coronal slices of rat brain were stained using the
Timm's method to investigate the presence of zinc-containing terminals
in the cortex, ventrobasal thalamus, and the NRT (Fig. 1). Figure 1
shows a low (2×) and higher power (4×) photomicrographs of a coronal
section stained for the presence of zinc-containing synaptic terminals.
Note the prominent zinc stained terminals in the NRT (double arrows)
and thalamus (single arrow). Prominent zinc-containing terminals were also present in the cortex (triangle) as seen in the lower power view
(A).
Noncompetitive blockade of GABA-evoked responses
Acutely isolated cortical neurons were voltage-clamped at
24 mV, and GABA 1 µM-10 mM was applied alone and concurrently with zinc 30 µM (Fig. 2) in order examine
the nature of the blockade of GABA-evoked responses exerted by zinc.
Figure 2 plots the zinc 30 µM block of currents evoked by increasing
concentrations of GABA in acutely isolated cortical neurons
(n = 7). Application of increasing concentrations of
GABA or GABA concurrently with zinc resulted in sigmoidally shaped
concentration-response curves with GABA 1 mM eliciting a maximal
GABA-evoked response (Gibbs et al. 1996a
,b
; Oh et
al. 1995
). The best fit EC50s for the
GABA concentration-response relationship for GABA alone and the
concentration-response relationship for GABA and zinc 30 µM were
28.5 ± 3.9 µM and 40.3 ± 14.7 µM, respectively (not
significantly different, F test). Application of the supersaturating
concentration of GABA 10 mM concurrently with zinc 30 µM was not
sufficient to overcome the inhibitory blockade of the GABA-evoked
response exerted by zinc, resulting in a 25% reduction of the GABA
response (maximal effect 74.4 ± 7.3%). The lack of change in the
EC50 of GABA-evoked responses in the presence of
zinc and the inability of the saturating concentration GABA 10 mM to
alleviate the blockade exerted by zinc were consistent with a
noncompetitive blockade of GABAA responses by
zinc, as seen in previous studies in cultured cortical neurons
(Celentano et al. 1991
; Legendre and Westbrook
1991
). However, EC50s derived from 3 point curves are not robust and more data points could not be added in
individual cells due to limitations in the perfusion apparatus, so
possible mixed blocking effects of zinc (cf. Gingrich and Burke 1998
)
cannot be discounted.
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Regional differences in zinc blockade of GABA-evoked responses in TC neurons
Cortical, thalamic, and NRT neurons were voltage-clamped at 24
mV, and GABA 10 µM was applied alone and concurrently with varying
concentrations of zinc to examine possible regionally-selective differences in zinc blockade of GABA-evoked currents. GABA 10 µM was
on the rising phase of the concentration-response curve, allowing
augmenting effects of GABA modulators to be demonstrated, but still was
sufficient to provide robust GABA currents, facilitating study of GABA
antagonists. This allowed direct comparison of the modulatory effects
of zinc and CNZ in individual neurons. Application of zinc in
concentrations of 1-300 µM resulted in a concentration-dependent, sigmoidally increasing blockade of GABA-evoked currents in all cellular
types examined. The data could be best fitted assuming a sigmoidal
concentration/response relationship using the equation:
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Zinc was found to block GABA-evoked responses regionally in a differential fashion. The efficacy of zinc blockade order was thalamus > NRT > cortex (curves significantly different; F =34.37, P < 0.001). At 10 and 100 µM, zinc blocked GABA-evoked responses by 39.3 ± 5.7% and 68 ± 4.4% in thalamic neurons (n = 21), by 21 ± 2% and 47.4 ± 2.3% in NRT neurons (n = 45), and by 14.6 ± 1.5% and 32.3 ± 3.2% in cortical neurons (n = 39), respectively (all values significantly different, P < 0.01, t-test). Zinc 300 µM blocked of GABA-evoked responses by 84 ± 2.5%, 60 ± 1.8%, and 50 ± 1.4% in thalamic, NRT, and cortical neurons, respectively (all values significantly different, P < 0.001, t-test) (Fig. 3). In a subset of cortical neurons (<5%), zinc paradoxically weakly augmented GABA-evoked responses. These cells were not included in the analysis.
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Relationship between zinc/benzodiazepine modulation in TC neurons
Cloning expression studies of the GABAR subunit determinants
mediating differences in pharmacology have demonstrated a 2 subunit
dependence to zinc sensitivity of GABARs. Receptors containing a
2 subunit are insensitive to zinc, while receptors lacking a
2
subunit are potently blocked by zinc (Draguhn et al.
1990
; Saxena and Macdonald 1994
; Smart
and Constanti 1990
). Benzodiazepine (BZ) sensitivity of GABARs
is also conferred by the
2 subunit (Pritchett et al.
1989
). To explore whether possible regionally selective
differences in the contribution of
2 subunits to GABARs was
responsible for the above-described differences in zinc sensitivity, we
examined the correlation between zinc inhibition and CNZ augmentation of GABA-evoked responses in individual cortical, thalamic, and NRT
neurons. Neurons were voltage-clamped at
24 mV, and GABA 10 µM was
applied alone and concurrently with zinc 100 µM or the BZ, clonazepam
(CNZ) 100 nM, to examine zinc blockade-CNZ augmentation of GABA-evoked
responses in the same neuron. Figure 4
shows the relationship in TC neurons between zinc inhibition/CNZ
augmentation. The zinc/CNZ relationship showed no statistically
significant correlation in NRT and thalamic neurons (R = 0.22, P = 0.47; R =
0.44,
P = 0.39, respectively), but a significant correlation was evident in cortical neurons (R =
0.88;
P = 0.001).
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Zinc effects on sTBC and cTBC rhythms
The modulatory effects of zinc on TC rhythms were examined in 15 slices, supporting either sTBC or cTBC rhythms. Bath applied zinc (300 µM) was found to have differential modulatory effects on sTBCs and cTBCs, respectively. Figure 5 shows an example of the neuromodulatory effects of zinc 300 µM on in vitro sTBC and cTBC rhythms. Bath applied zinc 300 µM enhanced sTBC rhythms (n = 8) (Fig. 5A). The histogram in Fig. 5B shows the zinc-induced amplification of sTBC rhythms, including an enhancement of both the duration and amplitude of sTBC events. The frequency of sTBCs increased from 1.4-5.0 events/min to 2.75-9.5 events/min in the presence of zinc. In contrast, bath applied zinc 300 µM completely abolished cTBC rhythms (n = 7) (Fig. 5C). Figure 5D shows a complete reduction in both the duration and amplitude of the cTBC events.
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APV and 4-AP effects on sTBC and cTBC rhythms
Zinc has been shown to have potent neuromodulatory effects on a
variety of ionic receptors, principally including
GABAA, NMDA, and K+
channels (reviewed in Smart et al. 1994). Zinc (300 µM) will maximally block both A current and NMDA currents (Smart et al. 1994
). Therefore the selective NMDA and
K+ channel receptor blockers, D-APV
50 µM and 4-AP 50 µM-1 mM, respectively, were applied to sTBC and
cTBC rhythms in concentrations sufficient to achieve maximal block to
better characterize zinc modulatory effects on TC rhythms. Bath applied
D-APV 50 µM was found to have strong inhibitory effects
on TC rhythms. Figure 6 shows an example of the neuromodulatory effects of APV on in vitro sTBC and cTBC rhythms. Bath applied D-APV 50 µM completely abolished
both sTBC and cTBC rhythms (n = 10) (Fig. 6). The
histograms (Fig. 6, B and D) show a complete
reduction in both the duration and amplitude of the sTBCs and cTBCs.
Like D-APV, bath applied 4-AP 50 µM-1 mM was found to
have strong inhibitory effects on both sTBC and cTBC rhythms. Figure
7 shows an example of the neuromodulatory effects of 4-AP on in vitro sTBC and cTBC rhythms. Bath applied 4-AP 50 µM-1 mM attenuated both cTBC and sTBC rhythms (n = 13) (Fig. 7). The histograms (Fig. 7B) show a 50-80%
reduction in both the duration and amplitude of sTBC and cTBC events at
50 µM, and there was complete abolition of these events during
perfusion of 1 mM 4-AP (data not shown). Since s- and cTBCs were
virtually never seen in the same slices (Zhang et al.
1996a
), the reduced amplitude and duration of cTBCs shown in
Fig. 7 are probably reflecting a reduction in cTBCs, and not a
transformation of cTBCs into sTBCs. There is an inverse relationship
between the duration of an epileptiform event, and the frequency of
occurrence of these events (Zhang et al. 1996a
). In
keeping with this relationship, the smaller, shorter s- and cTBC events
in 4-AP occurred at higher frequencies (Fig. 7A). Therefore
the total time spent in epileptiform activity in control and 4-AP may
be similar.
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DISCUSSION |
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Zinc blocked GABAR-mediated responses in a regionally specific manner within the TC system, with an efficacy order of: thalamus > NRT > cortex (Fig. 3). No correlation was found between BZ augmentation and zinc sensitivity in thalamic and NRT neurons; however, a statistically significant relationship was observed in cortical neurons (Fig. 4). Zinc had differential effects on spontaneous low Mg2+-induced epileptiform TC activity in TC slices. Zinc increased the amplitude and prolonged sTBC events which resembled spike-wave discharges, while zinc blocked cTBCs, which more closely resembled generalized tonic-clonic seizures (Fig. 5). Bath application of NMDA and K+ channel blockers could not replicate the differential effects of zinc on sTBCs and cTBCs, supporting the hypothesis that regionally selective GABAergic effects of zinc (among several possible sites) may play an important role in modulating TC rhythms (Fig. 6 and 7).
Zinc modulation of GABAA-evoked responses
The GABAR is a heterooligomeric ligand-gated protein receptor
composed of differing combinations of ,
,
,
,
,
,
and
subunits (reviewed in Bonnert et al. 1999
;
Davies et al. 1997
; Hedblom and Kirkness
1997
; Macdonald and Olsen 1994
; Sieghart 1995). Differential subunit expression within the pentamer
confers unique pharmacological properties to the GABAR (cf.
Bonnert et al. 1999
; Davies et al. 1997
;
Draguhn et al. 1990
; Prichett et al.
1989
; Saxena and Macdonald 1994
; White
and Gurley 1995
) and could have important functional
consequences in generation and modulation of rhythms in the brain. In
the TC system, clonazepam differentially enhanced GABA-evoked responses
in certain areas, with an efficacy order of NRT
cortex>thalamus
(Gibbs et al. 1996a
). Clonazepam also blocked both sTBC
and cTBC TC rhythms (Zhang et al. 1996b
). Modulatory
effects on GABA-evoked responses in neurons are determined primarily by
the subunit configuration of the GABAR complex within individual
populations of cells. In cloning expression studies, it has been found
that the
2 subunit must be coexpressed with
and
subunits for
the resulting receptor to be BZ sensitive. Expression of the
subunit also impacts the zinc sensitivity of these receptors
(Draguhn et al. 1990
; Saxena and Macdonald 1994
), along with transforming the mechanism of inhibitory
block from noncompetitive to competitive (Gingrich and Burke
1998
).
GABARs show both regional and developmental alterations in subunit
conformation in the rat brain (Laurie et al. 1992;
Olsen et al. 1990
; Wisden et al. 1992
).
GABARs consisting of
and
subunits have been shown to be
potently blocked by zinc. The addition of the
subunit with
corresponding
and
subunits resulted in zinc-insensitive GABARs
(Draguhn et al. 1990
) while replacement of the
with
a
subunit enhanced zinc sensitivity (Saxena and Macdonald
1994
). However, cultured hippocampal neurons expressing "native" BZ-sensitive GABARs are sensitive to zinc (Legendre
and Westbrook 1990
; Westbrook and Mayer 1987
).
Zinc sensitivity of
2-containing GABARs has recently been shown to
depend on
subunit expression. GABARs containing
1-, in comparison to
2- and
3- subunits
coexpressed with
and
subunits were less sensitive to zinc
(White and Gurley 1995
). In addition, GABARs comprised of the
6
3
2
l subunits were more sensitive to zinc modulation than
1
3
2
l GABARs (Saxena and Macdonald 1996
).
Expression of the
subunit may also contribute to zinc and BZ
sensitivity of GABARs. GABARs containing a
subunit (replacing or
coexpressed with a
2 subunit) exhibited higher zinc sensitivity than
2-containing receptors lacking a
subunit (Saxena and
Macdonald 1994
).
In situ hybridization studies showed mRNA expression patterns in rat
ventrobasal thalamus which were strongly positive for and weakly
positive for
2 subunits. The neocortex was positive for
and
strongly positive for
2 subunits. NRT was positive for
2 and
negative for
subunit expression (Wisden et al.
1992
). Strong immunolabeling for the
2
subunit has also been demonstrated in NRT (Gutiérrez et
al. 1994
). Given the Saxena and Macdonald (1994)
findings
coupled with in situ studies, the "
/
subunit hypothesis"
of zinc sensitivity would predict the GABA blockade efficacy order to
be thalamus>cortex>NRT based on mRNA profiles and regional clonazepam
sensitivity studies in the TC system (Gibbs et al.
1996a
; Oh et al. 1995
). However, the present
study showed a zinc inhibition relationship of GABA-evoked responses to
be thalamus>NRT>cortex (Fig. 3). Given the strong CNZ effects in NRT
neurons, supporting high expression of
2 subunits within GABARs
(Gibbs et al. 1996a
) and the intermediate zinc
sensitivity effects observed in the present study, these results
would argue against the
and the
subunits as being the primary
subunits determining zinc sensitivity of native GABARs within the
TC system.
Physiological and functional consequences of zinc TC rhythmicity
Measurement of zinc levels in the hippocampus suggest that the
concentration in the extracellular space may reach as high as 300 µM
during synaptic activity (Assaf and Chung 1984).
Therefore the zinc concentrations (300 µM) employed in the present
study are comparable to what may be experienced in vivo under periods of intense neuronal firing characteristic of epileptiform behavior. In
vivo studies have shown that intracerebral injection of zinc causes
seizures (Pei and Koyama 1986
), neuronal cell death
(Lees et al. 1990
), and increased cortical neuronal
firing rates (Wright 1984
). Following zinc (50 µM)
application, cultured hippocampal neurons fired high-frequency bursts
of action potentials (Mayer and Vyklicky 1989
). In
hippocampal slices, bath applied zinc elicited giant depolarizing
potentials (GDPs) mediated by GABARs in hippocampal pyramidal neurons
(Xie and Smart 1991
). Induction of these GDPs was
blocked by agents that selectively chelate zinc (Ben-Ari and Cherubini 1991
). Like hippocampal mossy fibers, the TC system exhibits high levels of staining of zinc-containing terminals (Haug 1973
). In the present study, zinc (300 µM) was
bath applied to TC slices during spontaneous low
Mg2+-induced epileptiform activity to investigate
possible in vivo effects of endogenously released zinc on TC
rhythmicity and epileptiform behavior (Coulter and Lee
1993
; Zhang and Coulter 1996
; Zhang et
al. 1996a
,b
). Differential effects of zinc (300 µM) were
observed on sTBC and cTBC rhythms. Zinc increased the amplitude and
prolonged sTBCs, but blocked cTBCs (Fig. 5). This suggests that, if
zinc is released in the TC system during periods of high-frequency action potential firing, it could have differential modulatory effects
on various types of TC rhythms.
Zinc has been shown to modulate a variety of ligand-gated and voltage-
sensitive neuronal channels (reviewed in Smart et al. 1994) including
GABARs (Celentano et al. 1991
; Legendre and
Westbrook 1991
), NMDA receptors (Westbrook and Mayer
1987
), and an inactivating K+ current
(IA) (Harrison et al. 1993
).
Differential modulatory actions of zinc on multiple ionic channels
could have elicited the opposite effects of zinc 300 µM on sTBC and
cTBC rhythms. To more conclusively determine which of the cellular
effects of zinc were important in modulation of TC rhythms, the
selective NMDA antagonist, D-APV, and selective
IA blocker, 4-AP, were bath applied to TC slices
generating sTBC and cTBC rhythms to compare effects of these agents to
zinc effects. While zinc (300 µM) increased and inhibited sTBC and
cTBC rhythms, respectively, bath application of APV (50 µM) or 4-AP
(50 µM-1 mM) blocked both sTBCs and cTBCs (Fig. 6 and 7).
These NMDA and A current blockers did not mirror the effects of zinc on
TC rhythms. Therefore zinc effects on sTBCs and cTBCs were not
exclusively due to modulation of NMDA or IA channels (Fig. 6 and 7).
While D-APV and 4-AP both blocked all variants of TC
rhythms, the differential effects of zinc on TC rhythms were more
difficult to interpret. Zinc, D-APV, and 4-AP suppressed
cTBC rhythms in a similar fashion. In vitro cTBC rhythms induced by a
media containing no added Mg2+ have been shown
previously to be predominantly a cortical rhythm, triggered by a
paroxysmal depolarizing shift, with no thalamic component necessary for
generation of cTBC rhythmicity (Coulter and Lee 1993).
Zinc blockade of cTBC rhythms by could be due to zinc effects on NMDA
receptors in cortical neurons, as has been previously reported in
cultured hippocampal neurons (Westbrook and Mayer 1987
)
(Fig. 5).
Zinc effects on sTBC rhythms did not resemble those of
D-APV or 4-AP. sTBCs require an intact TC circuit for
expression (Coulter and Lee 1993). They appear to be
driven by an underlying thalamic oscillation synchronized by GABAergic
inhibitory IPSPs from NRT (Coulter and Zhang 1996
).
These IPSPs then trigger the activation of a low threshold calcium
current (IT) in thalamus (reviewed in Steriade
and Llinás 1988
; McCormik and Bal 1997
). Thalamic relay nuclei rely on at least two transient currents,
IA and IT, which regulate
the cellular excitability of the thalamic neuronal membrane potential
at rest, with both the IA and
IT conductances deinactivated by
hyperpolarization and activated by depolarization (Huguenard et
al. 1991). Zinc enhancement of sTBC rhythms may be due to a
combination of effects: blockade of IA in cortex
and thalamus, and "disinhibition" of GABAergic synaptic circuitry in NRT, increasing NRT output, which would in turn increase the amplitude of GABAB-mediated IPSPs from NRT onto
thalamus, increasing deinactivation of the thalamic T current, and
augmenting excitability (Fig. 3). This hypothetical effect of zinc on
sTBCs is analogueous to bicuculline's effects in enhancing spontaneous
oscillations within the ferret lateral geniculate slices (von Krosigk
et al. 1993
). Firstly, blockade of IA by zinc
would tend to disrupt the delicate balance between the
IA and IT currents in
thalamic relay neurons. Removal of the hyperpolarizing influence of
IA would induce more robust and faster activation
of IT, promoting activation of low-threshold
spikes (LTS) in thalamus. Additionally, the regionally selective zinc
blockade of GABA-evoked responses could impact the genesis of sTBC
rhythms (Fig. 3). A "disinhibition" of the GABAA-mediated intra-NRT inhibitory synaptic
connections (Ulrich and Huguenard 1996
; von
Krosigk et al. 1993
) by zinc could also contribute to the
amplification of sTBC rhythms. This would increase the inhibitory
GABAergic output from NRT onto thalamic relay neurons, thereby
promoting the activation of IT (cf.
Destexhe 1998
; Kim et al. 1997
), which
serves as a critical amplifier underlying sTBC and SWD TC rhythms
(Coulter et al. 1989
).
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ACKNOWLEDGMENTS |
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We thank G. B. Schroder for technical assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-31000 to D. A. Coulter, the MCV MD/PhD program to J. W. Gibbs and M. D. Shumate, and the Sophie and Nathan Gumenick Neuroscience and Alzheimer's Research Fund.
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FOOTNOTES |
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Present address and address for reprint requests: D. A. Coulter, 3516 Civic Center Blvd., Abramson Research Center, Room 410B, Philadelphia, PA 19104-4318.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 November 1998; accepted in final form 15 September 1999.
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REFERENCES |
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