1Department of Neurology, Medical College of
Virginia, Richmond, Virginia 23298; 2Department
of Medicine,
Caddick, Sarah J.,
Chunsheng Wang,
Colin F. Fletcher,
Nancy A. Jenkins,
Neal G. Copeland, and
David A. Hosford.
Excitatory but not inhibitory synaptic transmission is reduced in
lethargic (Cacnb4lh) and tottering
(Cacna1atg) mouse thalami. Recent
studies of the homozygous tottering (Cacna1atg)
and lethargic mouse (Cacnb4lh) models of absence
seizures have identified mutations in the genes encoding the A variety of genetic models of absence epilepsy
exhibit spontaneous spike-wave discharges (SWDs) that share
electrophysiological, behavioral and pharmacological properties with
absence seizures in humans (Berkovic 1997a Positional cloning techniques were used recently to identify the genes
causing absence seizures in two models: the gene encoding the Because the mutated Mouse colonies
Colonies of Cacnb4lh homozygotes and
their nonepileptic background control strain [designated +/+:
descended from (C57BL/6JEi × C3H/HeSnJ) F1s] were
maintained in the Duke University vivarium. Male lethargic
heterozygotes (Cacnb4lh/+) were bred with female
lethargic heterozygotes to produce 25% homozygous
Cacnb4lh in the progeny. By 14 days of age,
these Cacnb4lh homozygotes were distinguished
from their phenotypically normal heterozygous lethargic and +/+
littermates by the presence of an ataxic gait.
Colonies of Cacna1atg homozygotes and their
background control strain (C57BL/6J) were maintained at the Advanced
Biosciences Labs-National Cancer Institute vivarium. Through
crosses in which the tg allele was maintained in repulsion
to the semidominant allele Os, which causes
oligosyndactylism, Cacna1atg homozygotes could
be recognized at birth by verifying the lack of oligosyndactylism.
Matched Cacna1atg homozygotes and C57BL/6J mice
were shipped to D. A. Hosford's lab for study.
All mice received food and water ad libitum, and they were maintained
on a 12 h/12 h light/dark cycle. Animal care and use adhered to
guidelines proposed in Guide for the Care and Use of Laboratory Animals
(National Institutes of Health 1985 In vitro slice preparation
The experiments in this study were performed on male,
age-matched (P14-28) pairs of either Cacnb4lh
homozygotes and +/+ mice or Cacna1atg
homozygotes and C57BL/6J mice. Briefly, mice were anesthetized under
halothane before removing the brains in oxygenated (95% O2-5% CO2) cold (4°C) artificial
cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 25 NaHCO3, 10 glucose, 1 MgCl2, 2 CaCl2, 3.3 KCl, and 1.25 NaH2PO4.
Brains were glued, ventral surface facing down, to the stage of a
vibratome, and horizontal brain slices (450 µm) were prepared and
subsequently incubated in an ACSF-filled chamber held at room
temperature for 1 h before use. Slices were transferred as needed
to a submersion chamber constantly perfused with oxygenated ACSF.
Chamber bath temperature was maintained at 32 ± 1°C.
Intracellular recordings: all mice
Whole cell voltage-clamp recordings of VB cells were made using
the blind patch technique (Blanton et al. 1989 EPSCs and IPSCs
CACNB4lh HOMOZYGOTES.
In initial experiments EPSCs were isolated by adding bicuculline
methiodide (BMI, 5 µM) and CGP 55845A (1 µM) to the bathing medium;
along with QX-314 (1 mM) already present in the internal solution,
these agents blocked all GABA-mediated responses. Cells were held at
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1A and
4 subunits, respectively, of voltage-gated Ca2+ channels
(VGCCs).
subunits normally regulate Ca2+ currents via a
direct interaction with
1 (pore-forming) subunits of VGCCs, and
VGCCs are known to play a significant role in controlling the release
of transmitter from presynaptic nerve terminals in the CNS. Because the
gene mutation in Cacnb4lh homozygotes results in
loss of the
4 subunit's binding site for
1 subunits, we
hypothesized that synaptic transmission would be altered in the CNS of
Cacnb4lh homozygotes. We tested this hypothesis
by using whole cell recordings of single cells in an in vitro slice
preparation to investigate synaptic transmission in one of the critical
neuronal populations that generate seizure activity in this strain, the
somatosensory thalamus. The primary finding reported here is the
observation of a significant decrease in glutamatergic synaptic
transmission mediated by both
N-methyl-D-aspartate (NMDA) and non-NMDA
receptors in somatosensory thalamic neurons of
Cacnb4lh homozygotes compared with matched,
nonepileptic mice. In contrast, there was no significant decrease in
GABAergic transmission in Cacnb4lh homozygotes
nor was there any difference in effects mediated by presynaptic
GABAB receptors. We found a similar decrease in glutamatergic but not GABAergic responses in
Cacna1atg homozygotes, suggesting that the
independent mutations in the two strains each affected P/Q channel
function by causing defective neurotransmitter release specific to
glutamatergic synapses in the somatosensory thalamus. This may be an
important factor underlying the generation of seizures in these models.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,b
;
Williams 1953
). Before the genes causing seizures in
these models were identified, phenomenologic approaches were used to
delineate three neuronal populations comprising the cellular network
that generates SWDs in animal models: neocortical pyramidal neurons,
thalamic relay neurons, and GABAergic neurons of the nucleus
reticularis thalami (NRT) (Caddick and Hosford 1996a
;
Crunelli and Leresche 1991
; Huguenard and Prince
1994
; Snead 1995
; Steriade et al.
1993
; von Krosigk et al. 1993
). SWDs are
generated when a physiological (tonic) mode of neuronal firing shifts
to a burst-firing mode (see Crunelli and Leresche 1991
; Steriade and Llinás 1988
; Steriade
et al. 1993
). An intrinsic conductance critical in the shift
from tonic to burst-firing mode is IT, the
low-threshold calcium current, (Coulter et al. 1989a
,b
; Crunelli and Leresche 1991
; Roy et al.
1984
; Suzuki and Rogawski 1989
; White et
al. 1989
).
1A
subunit of voltage-gated calcium channels (VGCCs) in the homozygous
tottering (Cacna1atg) mouse (Fletcher et
al. 1996
) and the gene encoding the
4 subunit of VGCCs in
the homozygous lethargic (Cacnb4lh) mouse
(Burgess et al. 1997
). These findings permit
investigations of the molecular mechanisms through which these gene
mutations cause the SWDs of absence seizures. The
1A subunit of
VGCCs forms the pore of P/Q-type calcium channels (Mori et al.
1991
; Randall and Tsien 1995
; Sather et
al. 1993
; Starr et al. 1991
; Stea et al.
1994
; Zhang et al. 1993
), which subserve a
variety of functions including neurotransmitter release at presynaptic
sites (reviewed by McCleskey 1994
). The
4 subunit is
one of at least four subtypes of
subunits, each of which enhances
calcium flux by binding to regulatory domains on
1 subunits
(Castellano et al. 1993
; Josephson and Varadi
1996
; Lacerda et al. 1994
; Massa et al.
1995
; Pérez-García et al.
1995
; for review, see Castellano and Perez-Reyes 1994
; Catterall 1991
; McCleskey
1994
; Miller 1992
).
4 subunit in Cacnb4lh
homozygotes lacks the binding domain necessary for regulation of
1
subunits (Pragnell et al. 1994
), we hypothesized that
4 subunits in these mice would be rendered nonfunctional, resulting
in decreased calcium influx after activation of VGCCs during synaptic
transmission. In principle, decreased availability of calcium in the
nerve terminal would result in decreased transmitter release with a
consequent reduction in postsynaptic responses (for review, see
Dolphin 1995
, 1996
; Tareilus and Breer
1995
). In this study, we addressed this hypothesis by examining
excitatory and inhibitory synaptic transmission at a synapse that is
critical to the generation of absence seizures in
Cacnb4lh homozygotes, the ventrobasal (VB)
thalamic nucleus (Caddick and Hosford 1996a
;
Hosford et al. 1995a
). We also tested the related hypothesis that postsynaptic responses would be affected in a similar
manner in Cacna1atg homozygotes, by virtue of
aberrant P/Q-channel function. The primary finding reported here is a
significant decrease in glutamatergic synaptic transmission in both
mutant strains in contrast to a lack of effect on GABAergic
transmission at presynaptic or postsynaptic sites. Because the decrease
in excitatory responses affects both N-methyl-D-aspartate (NMDA) and non-NMDA
responses in Cacnb4lh homozygotes, these
findings suggest a selective decrease in calcium flux at excitatory
synapses in VB neurons. Preliminary reports of these findings have been
presented elsewhere (Caddick and Hosford 1997
;
Caddick et al. 1997
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
) and to guidelines
of the American Physiological Society. Animal use was monitored by the
local Institute Animal Care and Use Committees (IACUCs) of Duke
University and the Durham Veterans Affairs Medical Center.
). Glass
microelectrodes (WPI) were pulled on a Narashige PB-7 with resistance
of 2-5 M
. For initial experiments measuring excitatory and
inhibitory postsynaptic currents (EPSCs and IPSCs), microelectrodes
were filled with solution containing (in mM) 100 N-methyl-D-glucamine, 100 methanesulfonic acid,
40 CsFl, 2 MgCl2, 10 HEPES, and 1 lidocaine
N-ethyl-bromide (QX-314; pH 7.2 with CsOH). In later
experiments examining NMDA and non-NMDA responses, microelectrodes were
filled with solution containing (in mM) 120 cesium gluconate, 10 HEPES,
10 cesium EGTA, 4 QX-314, and 2 MgATP (pH 7.25; osmolality 280-290
mOsm). The QX-314 served not only to occlude postsynaptic
GABAB responses but also to prevent the generation of
action potentials that could contaminate measurements of EPSCs or
IPSCs. Recordings were made using a whole cell amplifier (Warner PC
501-A) and stored on-line using the Strathclyde Software acquisition
and analysis package (J. Dempster, Strathclyde University). Cells
initially were held at
60 mV and input resistance determined by a
series of voltage steps (I-V) from
100 to
40 mV. An
adjacent stimulating electrode (monopolar tungsten; WPI) delivered
current ranging from 5 to 40 µA (80 µS) every 15 s. The
current at which the maximal response was obtained was noted and used
for subsequent stimulations (see following text). Series resistance was
monitored, and cells were rejected if this increased >15 M
.
Additional pharmacological blockers then were added to the external
bathing medium as described in the next section, depending on the
responses to be studied.
60 mV. In later experiments that were designed to measure the
respective contributions of NMDA and non-NMDA responses to the EPSCs,
cells were held at +30 mV to remove magnesium block and to minimize
rundown, thereby maximizing NMDA responses; 6,7-dinitroquinoxaline (DNQX; 20 µM) also was added to the bathing medium to eliminate non-NMDA responses. Stimulus-response (I/O) curves were obtained for
all cells, and subsequent stimulation was set to achieve
maximal-amplitude events.
CACNA1Atg HOMOZYGOTES.
EPSCs and IPSCs were measured at the same time; GABAB
responses were occluded by including QX-314 (1 mM) in the internal
solution. Cells were held at 60 mV. Stimulus-response (I/O) curves
were obtained for all cells and subsequent stimulation was set to
achieve maximal-amplitude events.
Analysis/statistics
Data were averaged and compared using an unpaired Student's t-test. All numerical means and all error bars are expressed as means ± SE.
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RESULTS |
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Recordings were obtained from 58 +/+ and 60 homozygous
Cacnb4lh VB cells (Fig.
1) and from 6 C57BL/6J and 5 homozygous
Cacna1atg VB cells. On breakthrough, cells were
held in voltage clamp at 60 mV and subjected to a series of voltage
steps from
100 to
40 mV. Input resistance of each cell then was
calculated. Input resistance was not significantly different between
Cacnb4lh homozygotes and +/+ nor between
Cacna1atg homozygotes and C57Bl/6J (Table
1), similar to observations in a previous
study (Caddick and Hosford 1996a
).
|
|
Evaluation of glutamatergic synaptic transmission: Cacnb4lh homozygotes
In the initial set of experiments using slices from
Cacnb4lh homozygotes, VB cells were clamped at
60 mV in the presence of BMI (5 µM). Synaptically evoked EPSCs were
recorded by applying current locally through an adjacent stimulating
electrode. Increasing intensities of stimulus were given to assess the
stimulus necessary to produce a maximal response amplitude. This
stimulus then was applied every 15 s, and the responses were
collected. Under these conditions, the peak amplitude and latency to
peak were measured and compared between +/+ and
Cacnb4lh homozygotes. There was no significant
difference in the latency to peak (+/+: 5 ± 0.2 ms,
n = 10; Cacnb4lh homozygotes:
6 ± 0.5 ms, n = 11). However, there was a
significant increase in the peak amplitude of the EPSC in +/+ versus
Cacnb4lh homozygotes, (+/+: 1.1 ± 0.1 nA;
Cacnb4lh homozygotes: 0.4 ± 0.08 nA;
P < 0.001; Fig. 1). The reduction in EPSC amplitude in
Cacnb4lh homozygotes was evident at all stimulus
intensities tested (Fig. 2) and can be
seen as a significant shift in the input/output curve. This result
indicates that the difference in amplitude can be found regardless of
the strength of synaptic stimulation that may be present under
physiological or pathological conditions and regardless of the number
of synaptic inputs stimulated.
|
The inclusion of QX-314 in the recording electrode assured the lack of action potential generation, which if present could contaminate EPSC measurements. To verify that the EPSCs were not contaminated by other voltage-gated currents, we examined the reversal potential of the EPSCs and found that it was ~0 mV. Because a considerably more positive reversal potential would be expected if the EPSCs were contaminated by other voltage-gated currents such as INa, these data indicate that the EPSCs were uncontaminated.
Evaluation of NMDA and non-NMDA synaptic transmission: Cacnb4lh homozygotes
Further experiments were carried out to determine the glutamate subtype(s) responsible for reduced EPSC amplitudes in VB neurons from Cacnb4lh homozygotes. The rationale for these experiments was to provide information that would help determine whether the reduction in EPSC amplitudes stemmed from presynaptic or postsynaptic mechanisms. Conditions were optimized for NMDA responses by holding cells at a sufficiently depolarized potential (+30 mV) to remove the magnesium block (see METHODS). Under these conditions, we reproduced the finding that EPSCs in VB neurons in Cacnb4lh homozygotes were significantly smaller than in +/+ mice (+/+: 353 ± 75 pA, n = 11; Cacnb4lh homozygotes: 152 ± 37 pA, n = 11; P <0.001; Fig. 3). Cells then were examined under conditions that would occlude non-NMDA responses (addition of 20 µM DNQX to the bathing medium). The amplitudes of non-NMDA EPSCs were calculated as the difference between the amplitudes of total and NMDA EPSCs. The amplitudes of NMDA EPSCs (+/+: 73 ± 12 pA, n = 8; Cacnb4lh homozygotes: 22 ± 14 pA, n = 9; P <0.05; Fig. 4) and non-NMDA EPSCs (+/+: 280 ± 58 pA; Cacnb4lh homozygotes: 130 ± 28 pA; P <0.05) were significantly reduced in Cacnb4lh homozygotes compared with +/+ mice.
|
|
Evaluation of GABAergic synaptic transmission: Cacnb4lh homozygotes
VB cells in slices from Cacnb4lh homozygotes were clamped at 0 mV in the presence of DL-APV and DNQX (20 µM). Synaptically evoked IPSCAs were recorded by applying current locally through an adjacent stimulating electrode, and stimuli of increasing intensities were given to assess maximal response amplitude. The stimulus then was applied every 15 s and the responses collected (Fig. 5). Under these conditions, the peak amplitude and latency to peak were measured and compared between +/+ and Cacnb4lh homozygotes. There was no significant difference in the latency to peak (+/+: 6 ± 0.2 ms; Cacnb4lh homozygotes: 6 ± 0.1 ms) nor was there a significant difference in peak amplitude of the IPSCA in +/+ versus Cacnb4lh homozygotes (+/+: 2.7 ± 0.4 nA; Cacnb4lh homozygotes: 1.8 ± 0.3 nA).
|
The contribution of presynaptic GABAB receptors to GABAergic transmission was assessed by comparing paired-pulse depression (PPD) of IPSC amplitudes using a 200-ms interpulse interval. There was no significant difference in the percent of PPD between strains (+/+: 29 ± 3%; Cacnb4lh homozygotes: 35 ± 15%; n = 9 pairs; Fig. 6). Likewise, there was no significant difference in the portion of PPD that was blocked by CGP 55845A and hence GABAB receptor dependent (+/+: 14 ± 5%; Cacnb4lh homozygotes: 18 ± 15%; n = 9 pairs).
|
Comparison of EPSC/IPSCA and EPSP/IPSPA pairs in Cacna1atg and C57BL/6J mice
There was no difference in resting membrane potentials, input resistances, or membrane time constants in Cacna1atg homozygotes (n = 5) compared with C57BL/6J (n = 6) cells (Table 2). This paralleled the lack of differences in these measures in VB cells from Cacnb4lh homozygotes and +/+ mice (Table 1). Maximal stimulus-evoked EPSCs were significantly smaller in Cacna1atg homozygotes than C57BL/6J VB cells (Cacna1atg homozygotes: 0.36 ± 0.08 nA; C57BL/6J: 0.61 ± 0.26 nA; P < 0.05; Fig. 7). In contrast, there was no significant difference in maximal evoked IPSCAs (Cacna1atg homozygotes: 0.32 ± 0.12 nA; C57BL/6J: 0.33 ± 0.09 nA; Fig. 7).
|
|
To further compare excitatory and inhibitory responses in VB neurons from Cacna1atg homozygotes and C57BL/6J, locally evoked synaptic potentials also were recorded in these neurons under current clamp. The maximal amplitude of the EPSP recorded in VB neurons in Cacna1atg homozygotes (6.3 ± 1.3 mV) was significantly reduced (P < 0.05) compared with C57BL/6J (11.7 ± 1.3 mV; Fig. 8). In contrast, there was no significant difference in maximal amplitude of the IPSPA between the strains (Cacna1atg: 6.7 ± 1.1 mV; C57BL/6J: 4.2 ± 2.3 mV; Fig. 8).
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DISCUSSION |
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The principal finding emerging from this study was a significant decrease in the amplitude of EPSCs but not IPSCs in VB neurons of both Cacnb4lh and Cacna1atg homozygotes compared with their respective nonepileptic controls. The observed decrease in EPSC amplitudes was apparent in both NMDA and non-NMDA components of Cacnb4lh homozygotes, strongly suggesting that the decrease stemmed primarily from a presynaptic defect in transmitter release from glutamatergic terminals. A second finding was a lack of any difference in effects mediated by presynaptic GABAB receptors on inhibitory afferents to VB neurons in Cacnb4lh homozygotes, underscoring the selectivity of altered excitatory transmission at this synapse.
Possible links between mutated 4 subunit and decreased glutamate
release from nerve terminals in Cacnb4lh homozygotes
There are a number of plausible mechanisms that could link a
mutated 4 subunit with a presynaptic defect in glutamate release. For example, VGCCs play a critical role in the cascade of events that
occur between the arrival of an action potential at a nerve terminal
and neurotransmitter release (see Kelly 1993
). In
principle, decreased calcium influx through a VGCC composed of
defective subunits could result in decreased neurotransmitter release
(see Dolphin 1995
, 1996
; Tareilus and Breer
1995
). Thus it is possible that defective
4 subunits,
lacking the binding domain required for modulation of calcium flux,
could contribute to decreased neurotransmitter release in synapses in
Cacnb4lh homozygotes.
Supporting the idea that the mutation of VGCC subunits in
Cacnb4lh homozygotes causes decreased calcium
influx, earlier findings from our lab showed a significant reduction in
KCl-induced 45Ca+2 uptake in thalamic and
neocortical synaptosomes in Cacnb4lh homozygotes
compared with matched, nonepileptic mice (Lin et al.
1995). This finding supports the premise that mutated
4
subunits can lead to reduced calcium entry into depolarized nerve
terminals in Cacnb4lh homozygotes, resulting in
decreased neurotransmitter release.
However, if presynaptic VGCCs lead to reduced neurotransmitter release
in synapses of Cacnb4lh homozygotes, then it is
surprising that glutamate but not GABA release is reduced significantly
at synapses of VB thalamic neurons. There are two possible explanations
for this result: glutamate but not GABA release from terminals in VB
synapses is modulated by VGCCs that are affected by the mutated 4
subunit or glutamate release (significant, 54% decrease in EPSCs) is
affected to a much greater extent than GABA release (nonsignificant but
detectable 33% decrease in IPSCs).
The first of these explanations would imply that VGCC subtypes with
differing requirements for a functional 4 subunit mediate glutamate
and GABA release at terminals in VB synapses. A precedent for this idea
is provided by findings that suggest that selected VGCC subtypes
mediate glutamate as opposed to GABA release. In hippocampal CA1
synapses, glutamate release was more dependent on P/Q-type than N-type
VGCCs (Burke et al. 1993
; Potier et al. 1993
), whereas GABA release was more dependent on N-type than P/Q-type VGCCs (Potier et al. 1993
). In cultured,
synaptically connected thalamic neurons, glutamate release was mediated
by non-N- and non-L-type high-threshold calcium currents
(Pfrieger et al. 1992
), suggesting the possibility that
P/Q-type VGCCs were among the population of high-threshold VGCCs that
mediated glutamate release. Likewise, in fibers synapsing on Purkinje
cells, P- and Q-type VGCCs mediated excitatory transmission, whereas
non-P/Q -type VGCCs mediated GABAergic transmission (Doroshenko
et al. 1997
).
Alternatively, a second explanation for our findings is that both
glutamate and GABA release are affected by mutated 4 subunits in
Cacnb4lh homozygotes but that the effect is more
pronounced on glutamate release. If so, then increasing the sample size
of neurons studied might enable a smaller reduction of the
GABAA IPSC to be measured at the 0.05 level of
significance. However, this scenario is actually a subset of the first
possibility discussed earlier because in either case EPSC amplitudes
are affected to a greater extent than IPSCA amplitudes.
Therefore a plausible molecular explanation for the substantially
greater reduction in glutamate than GABA release is that VGCC subtypes
with differing requirements for a functional
4 subunit underlie
release of glutamate and of GABA at VB synapses.
Evidence that 4 mutations in Cacnb4lh homozygotes
selectively affect P/Q channels: evidence from Cacna1atg
homozygotes
Given the idea that glutamate neurotransmission is subserved
primarily by P/Q channels, is it possible that the P/Q channel subtype
is affected selectively by the defective 4 subunit in Cacnb4lh homozygotes? Indirect evidence
supporting this possibility emerges from the similar phenotypic
features of seizures in Cacna1atg and
Cacnb4lh homozygotes (Hosford et al.
1992
; Noebels 1986
; Sidman et al. 1965
). The gene mutation that causes seizures in homozygous
Cacna1atg and its variant, leaner
(Cacna1atg-la), affects the
1A subunit of
VGCCs (Fletcher et al. 1996
), which comprises the pore
of the P/Q-type VGCC (see Catterall 1991
; Dolphin 1995
; McCleskey 1994
; Miller
1992
). Moreover, whole cell recordings of dissociated Purkinje
cells in homozygous Cacna1atg-la mice showed a
reduction in P-type currents (Lorenzon et al. 1998
). Hence it is possible that mutations affecting P/Q-type VGCCs in Cacna1atg and
Cacna1atg-la homozygotes produce absence
seizures that are phenotypically similar to those in
Cacnb4lh homozygotes because of independent
mutations that affect the function of P/Q-type VGCCs.
More direct evidence is provided by our findings from Cacna1atg homozygotes in this study, showing that excitatory but not GABAergic responses are reduced in VB neurons compared with C57BL/6J controls. Together with the phenotypic similarity in seizures in Cacnb4lh and Cacna1atg homozygotes, the similar reduction in excitatory responses in the two strains strongly suggests that P/Q channels are selectively affected by these independent gene mutations.
Our observation of reduced excitatory responses in VB thalamic
neurons in Cacna1atg homozygotes provides the
first evidence supporting reduced glutamatergic transmission in
neuronal populations critical to generation of absence seizures in this
model. However, our findings accord with observations by Helekar
and Noebels (1994), who found reduced non-NMDA conductances
during paroxysmal depolarizing shifts in CA3 pyramidal neurons in
Cacna1atg homozygotes compared with C57BL/6J
controls. It is likely that the mutation of
1A subunits of VGCCs in
Cacna1atg homozygotes affects P/Q-channel
function in multiple neuronal populations, irrespective of their
theoretical role in the generation of absence SWDs.
Possible links between VGCC function and generation of absence seizures
It has been thought for some time that an imbalance between
excitation and inhibition within the thalamocortical loop might be
critical for the genesis of absence seizures (Gloor et al. 1990). Indeed the preservation of GABAergic inhibition within this network distinguishes absence seizures from generalized convulsive seizures (Gloor and Fariello 1988
). It is possible to
speculate then that the pronounced decrease in EPSCs and smaller
reduction in IPSCAs may tip the balance in favor of
GABAergic inhibition of VB neurons in Cacnb4lh
and Cacna1atg homozygotes. As a number of
research groups have demonstrated, enhanced GABAergic input in
thalamocortical neurons can synchronize these neurons into a
burst-firing mode (see Steriade and Lance 1988
; also
Crunelli and Leresche 1991
; Huguenard and Prince
1994
; Steriade et al. 1993
; von Krosigk
et al. 1993
). A plausible underlying mechanism is that enhanced
GABAergic transmission at thalamocortical neurons can deinactivate
T-type VGCCs, enabling them to generate an IT
during the decay from the GABA-induced IPSC. Synchronized generation of
IT by a population of thalamocortical neurons
then may generate the burst activity of the SWD, producing an absence seizure (Crunelli and Leresche 1991
).
Thus in principle, the generation of absence seizures in
Cacnb4lh or Cacna1atg
homozygotes may stem from synchronization provided by a net enhanced GABAergic input to thalamocortical neurons. This has been suggested as
a general mechanism for absence seizures in both genetic and pharmacological models (Banerjee and Snead 1995;
Caddick and Hosford 1996b
; Crunelli and Leresche
1991
; Gloor 1990
; Snead 1995
).
Alternative mechanisms may underlie the connection between reduced
excitatory drive onto VB neurons and the generation of thalamocortical
oscillations. For example, it is possible that the reduced excitatory
drive is caused in some manner by the mutated 4 subunit of VGCCs,
and yet the reduced excitatory drive may be unrelated entirely to
generation of oscillations. Likewise other mechanisms besides those
given in the preceding text may link thalamic responses to absence
seizures. To help distinguish between the many speculative
possibilities, further experiments are underway to characterize the
extent to which net enhanced GABAergic transmission predominates in
synapses critical to the generation of absence seizures, both in these
mutant mice and other models, and the mechanisms linking decreased
function of P/Q-type VGCCs to the generation of absence seizures in
these models. It is hoped that the results forthcoming may facilitate the development of new approaches in the treatment of patients with
absence epilepsy.
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ACKNOWLEDGMENTS |
---|
The authors thank Dr. Douglas Coulter for valuable suggestions, Drs. Coulter, Cathleen M. Lutz, and Wilkie A. Wilson, Jr. for critical reviews of the manuscript, and S. Sneed for administrative assistance. CGP 55845A was a gift from Novartis.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-30977, a Veterans Affairs Merit Review, and a Klingenstein Epilepsy Fellowship Award to D. A. Hosford. This study was also supported by the National Cancer Institute, Department of Health and Human Services, under contract to Advanced Biosciences Labs (C. F. Fletcher, N. A. Jenkins, and N. G. Copeland).
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FOOTNOTES |
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Address for reprint requests: D. A. Hosford, Dept. of Medicine (Neurology), Bldg. 16, Rm. 38, Duke University and Durham V. A. Medical Center, 508 Fulton St., Durham, NC 27705.
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 28 July 1998; accepted in final form 15 January 1999.
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REFERENCES |
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