1Department of Environmental Science, Policy and Management, Division of Insect Biology and 2Department of Molecular and Cell Biology, Division of Neurobiology, University of California, Berkeley, California 94720
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ABSTRACT |
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Kuebler, Daniel, Haiguang Zhang, Xiaoyun Ren, and Mark A. Tanouye. Genetic Suppression of Seizure Susceptibility in Drosophila. J. Neurophysiol. 86: 1211-1225, 2001. Despite the frequency of seizure disorders in the human population, the genetic and physiological basis for these defects has been difficult to resolve. Although many genetic defects that cause seizure susceptibility have been identified, the defects involve disparate biological processes, many of which are not neural specific. The large number and heterogeneous nature of the genes involved makes it difficult to understand the complex factors underlying the etiology of seizure disorders. Examining the effect known genetic mutations have on seizure susceptibility is one approach that may prove fruitful. This approach may be helpful both in understanding how different physiological processes affect seizure susceptibility and in identifying novel therapeutic treatments. In this study, we have taken advantage of Drosophila, a genetically tractable system, to identify factors that suppress seizure susceptibility. Of particular interest has been a group of Drosophila mutants, the bang-sensitive (BS) mutants, which are much more susceptible to seizures than wild type. The BS phenotypic class includes at least eight genes, including three examined in this study, bss, eas, and sda. Through the generation of double-mutant combinations with other well-characterized Drosophila mutants, the BS mutants are particularly useful for identifying genetic factors that suppress susceptibility to seizures. We have found that mutants affecting Na+ channels, mlenapts and para, K+ channels, Sh, and electrical synapses, shak-B2, can suppress seizures in the BS mutants. This is the first demonstration that these types of mutations can suppress the development of seizures in any organism. Reduced neuronal excitability may contribute to seizure suppression. The best suppressor, mlenapts, causes an increased stimulation threshold for the giant fiber (GF) consistent with a reduction in single neuron excitability that could underlie suppression of seizures. For some other double mutants with para and ShKS133, there are no GF threshold changes, but reduced excitability may also be indicated by a reduction in GF following frequency. These results demonstrate the utility of Drosophila as a model system for studying seizure susceptibility and identify physiological processes that modify seizure susceptibility.
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INTRODUCTION |
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Seizure susceptibility is greatly
influenced by genetic factors. At present, more than a dozen genes have
been linked to various epilepsy syndromes in humans, while in mice more
than 25 genetically mutated strains have epileptic phenotypes
(McNamara 1999; Puranam and McNamara
1999
). Genetic factors can also suppress seizures and
epileptogenesis. Mice with mutations in the brain-derived neurotrophic
factor gene (BDNF) or the immediate early gene c-fos display
delayed onset of seizures following kindling (Kokaia et al.
1995
; Watanabe et al. 1996
). In addition, the
variable penetrance of human epilepsy genes (Biervert et al.
1998
; Durner et al. 1991
) indicates that other
genetic factors can, in certain cases, suppress the development of
spontaneous seizures.
The large number of disparate genes involved in seizures coupled with
the heterogeneity of seizure disorders demonstrates the complexity of
the problem and makes any study of the genetic factors that affect
susceptibility to seizures difficult. The preponderance of a wide
variety of seizure disorders (Commission on Classification and
Terminology of the International League Against Epilepsy 1989;
Hauser and Hesdorffer 1990
) indicates that finding a
simple molecular pathway responsible for the regulation of seizure
susceptibility is unlikely. In fact, the high information-processing capability of the CNS may require it to be organized in such a fashion
that it is extremely susceptible to chaotic discharges following small
and unpredictable perturbations (Bak and Chen 1991
;
Bak and Paczuski 1995
). If this is the case, then a
large number of cellular and physiological processes should be able to
affect seizure susceptibility. This is consistent with mouse knock-out
data, which has implicated genes as diverse as l-isoaspartyl methyltransferase, neuropeptide-Y, alkaline phosphatase, and a Kv1.1 potassium channel in seizure disorders
(Erickson et al. 1996
; Smart et al. 1998
;
Waymire et al. 1995
; Yamamoto et al. 1998
). Despite the formidable complexity of the problem, an
examination of the effect many well-characterized mutations have on
seizure susceptibility has several potential benefits. Such a study may facilitate the identification of both novel physiological mechanisms by
which seizure susceptibility is regulated and novel targets for the
development of anti-convulsant drugs.
One system that avails itself to such a study is Drosophila
because of the large collection of excitability and behavioral mutants
available. In addition, recent studies have demonstrated the utility of
using Drosophila as a model system for studying seizure
disorders (Kuebler and Tanouye 2000). These studies have demonstrated that seizures in Drosophila share many
characteristics with mammalian seizures and that genetic factors can
alter seizure susceptibility levels.
We have begun an initial investigation of the genetic factors that
suppress seizure susceptibility in Drosophila. This study has taken advantage of one group of mutants, the bang-sensitive (BS)
mutants (Benzer 1971; Ganetzky and Wu
1982
; Pavlidis and Tanouye 1995
) that are
particularly sensitive to seizure: they are 5-10 times more
susceptible to seizure following electrical shock than wild-type flies
(Kuebler and Tanouye 2000
). A comparison of this mutant
class to the mammalian epilepsies has been made (Benzer
1971
). The BS mutants used in this study were eas,
which encodes an ethanolamine kinase involved in one pathway of
phosphatidyl ethanolamine synthesis (Pavlidis et al.
1994
), and bss and sda, which map to the
first and third chromosomes, respectively, but whose products have not
yet been described. The BS flies provide a particularly useful
experimental tool for testing the ability of other genetic mutants to
rescue a seizure-susceptible phenotype. Our approach consists of
quantifying seizure susceptibility levels in a variety of mutant
strains. Mutants that have seizure thresholds significantly different
from wild type can then be tested in double-mutant combination with BS
strains for the ability to suppress the BS seizure-susceptible
phenotype. This approach allows us to not only identify mutations that
suppress susceptibility but also to quantify the level of that suppression.
In this study, we have focused on mutants that affect processes thought
to be involved in seizure generation. Na+ and
K+ channel mutants were chosen because they
should affect nervous system excitability (Loughney et al.
1989; Tanouye et al. 1981
). Mutants that affect
neural connections were also chosen as these may disrupt the putative
positive feedback loops thought to be involved in seizure generation
(McNamara 1994
; Traynelis and Dingledine 1988
). The data presented here quantify the ability of these
mutants to suppress seizures and provide a baseline for a more
exhaustive future study involving both forward and reverse genetic
approaches to identifying suppressor mutations. We believe
understanding how these various genetic factors suppress seizure
susceptibility may be a gateway to dissecting the tremendously complex
and heterogeneous problem of seizure disorders.
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METHODS |
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Fly stocks
WILD-TYPE STRAINS AND BS MUTANTS.
Wild-type Drosophila strains were Canton Special (CS),
Oregon-R (OR), and Berlin. Three BS mutants were used eas,
bss, and sda. The easily shocked gene
(eas) is located at map position 1-53.5 and encodes an
ethanolamine kinase (Pavlidis et al. 1994). The
bang senseless gene (bss) is located at 1-54.6
and slamdance (sda) on the third chromosome at
97D8-9 (Ganetzky and Wu 1982
; Zhang, unpublished
observations); their gene products have not been described. The mutant
behavioral phenotypes of seizure and paralysis, electrophysiological
phenotypes of seizure and synaptic failure, and the threshold for
seizure susceptibility have been described previously for the
bss1, eas1,
and sdaiso6.10 mutations (Ganetzky
and Wu 1982
; Kuebler and Tanouye 2000
;
Pavlidis and Tanouye 1995
; Pavlidis et al.
1994
).
TEMPERATURE-SENSITIVE PARALYTIC MUTANTS.
Two temperature-sensitive paralytic mutations were used,
mlenapts and para. The
maleless (mle, 2-56.2) gene encodes an RNA
helicase-like protein (Kernan et al. 1991; Lee
and Hurwitz 1993
). The no-action potential
temperature-sensitive allele (mlenapts) is
a gain-of-function mutation that causes a reduction in adult brain
voltage-gated Na+ channels as assessed by
tetrodotoxin-binding studies (Jackson et al. 1984
;
Kauvar 1982
). As described previously,
mlenapts mutants show a loss of action
potentials and behavioral paralysis at elevated (37°C) temperatures
and increased action potential refractory period at room temperature
(Wu and Ganetzky 1980
; Wu et al. 1978
).
In double-mutant combinations, mlenapts
acts as a suppressor of behavioral phenotypes for BS and Sh
mutants (Ganetzky and Wu 1982
); it causes unconditional
lethality in combination with para (Wu and Gantezky
1980
). The paralytic (para, 1-52.1) gene
encodes a voltage-gated Na+ channel alpha subunit
(Loughney et al. 1989
; Ramaswami and Tanouye 1989
). The parats1 allele causes a
loss of action potentials and behavioral paralysis at elevated (29°C)
temperatures and increased action potential refractory period at room
temperature (Siddiqi and Benzer 1976
; Suzuki et
al. 1971
; Wu and Ganetzky 1980
). The
paraST76 allele used here has been
described previously (Siddiqi and Benzer 1976
) and
appears to be less severe than parats1
based on viability in a mlenapts
background (Ganetzky 1984
). Finally, para/+
adults become paralyzed much quicker at 40°C than wild type
(Hall 1973
).
LEG-SHAKING MUTANTS.
Two behavioral leg-shaking mutants were examined Sh and
eag. The Shaker (Sh, 1-57.6) gene
encodes several types of voltage-gated K+ channel
alpha subunits (Baumann et al. 1987; Kamb et al.
1987
; Tempel et al. 1987
). For mutants under
moderate ether anesthesia, legs shake abnormally, antennae twitch, and
the abdomen pulsates. Mutations cause abnormal action potential
repolarization of the adult giant fiber, repetitive firing of action
potential in larval nerves, and prolonged transmitter release at the
larval neuromuscular junction (Ganetzky and Wu 1982
;
Jan et al. 1977
; Tanouye and Ferrus 1985
;
Tanouye et al. 1981
). Mutants are abnormal in one class of K+ current
(IA) (Gautam and Tanouye
1990
; Lichtinghagen et al. 1990
; Salkoff
and Wyman 1981
). The ShKS133
allele is an extreme mutation due to a single amino acid substitution that causes a nonfunctional channel subunit with a complete loss of
IA. The
Sh5 allele is a less extreme mutation due
to an amino acid substitution that changes
IA channel kinetics. The
ShrKO120 allele is the weakest mutation of
the three and causes a slight decrease in
IA. In double-mutant combinations,
mlenapts has been shown to suppress
Sh behavioral phenotypes (Ganetzky and Wu
1982
). The ether-a-go-go (eag, 1-50)
gene encodes a cyclic nucleotide-modulated K+
channel (Bruggemann et al. 1993
; Warmke et al.
1991
). Similar to Sh, these mutants exhibit
leg-shaking under ether and have abnormal K+
currents in larval muscles (Wu et al. 1983
). The
eag1 mutation leads to the alteration but
not the absence of four different K+ currents in
larval muscles (Zhong and Wu 1991
). In double-mutant combination with Sh, they show an enhancement of the
Sh phenotype (Ganetzky and Wu 1983
).
OTHER BEHAVIORAL MUTANTS.
Two other behavioral mutants were examined slo and
shak-B. The slowpoke (slo, 3-86) gene
encodes a Ca2+-activated K+
channel (Atkinson et al. 1991). The mutants exhibit
temperature-sensitive paralysis and are sluggish at room temperature
(Elkins et al. 1986
). The defect leads to a large
reduction in the fast Ca2+-activated
K+ current (ICF)
in adults (Elkins et al. 1986
) and larvae (Singh and Wu 1989
). The shaking-B (shak-B,
1-64) gene encodes gap-junction proteins (Crompton et al.
1995
; Krishnan et al. 1993
; Phelan et al.
1998
). The shak-B2 allele prevents
the formation of electrical synapses in the giant fiber (GF) system, in
the flight circuit, and likely in many other parts of the nervous
system (Phelan et al. 1996
; Trimarchi and Murphey
1997
). Mutations also display aberrant neuronal branching patterns and neuroconnectivity defects in the GF system
("Passover" defects) (Thomas and Wyman
1984
). These lead to deficits in signaling (weak or no
response) between the GF axon and the TTM motoneuron and in the GF to
dorsal longitudinal muscle (DLM) pathway. Behaviorally, mutants show no
escape response: flies are unable to jump into the air and fly away at
a light off stimulus.
DOUBLE MUTANTS. The double mutants used in this study were tested to verify the presence of both the BS mutation as well as the putative suppressor mutation. The presence of either para or mlenapts in the homozygous double-mutant stocks was verified by paralysis following 1-min exposure to 37°C, which is characteristic of these mutations. The BS mutants do not paralyze under such conditions. The presence of ShKS133 in the homozygous double-mutant stock was verified by rapid leg shaking under ether, the behavioral characteristic of these mutants. The BS mutants do not exhibit rapid leg shaking under ether. The presence of shak-B2 in the homozygous double-mutant stocks was verified by the absence of the normal giant fiber pathway response that is characteristic of shak-B2 mutants.
The presence of the homozygous BS mutation in the double-mutant stocks was verified by back-crossing each double-mutant stock to males of the appropriate BS genotype. Female flies from these crosses, which should be homozygous for the BS mutation and heterozygous for the suppressor mutation, were then tested for the BS seizure phenotype. All of the following genotypes arising from backcrosses resembled BS homozygous flies in seizure threshold phenotype: bss/bss;mlenapts/+ (9 of 10 exhibited seizures at 4.5 V), bss/bss shak-B2/+ (10 of 10 exhibited seizures at 4.5 V), eas/eas shak-B2/+ (10 of 10 exhibited seizures at 6 V), shak-B2/+;sda/sda (8 of 10 exhibited seizures at 10 V), and mlenapts/+;sda/sda (10 of 10 exhibited seizures at 10 V). The lack of any obvious effects among the different genetic backgrounds tested also indicated that alterations in the seizure thresholds reported here were due to the homozygous presence of suppressors in the double-mutant combinations and not likely due to nonspecific genetic background differences. Flies of the genotype ShKS133/+;sda/sda arising from the ShKS133;sda double-mutant backcross displayed a seizure threshold that was above the BS range. These flies have a seizure threshold of 13.8 ± 2.7 V, indicating a single copy of the semi-dominant ShKS133 allele can suppress seizures to a limited extent. The males from this backcross were examined to ensure the homozygous presence of sda in the original double mutant. All of the males from this cross were identical to sda homozygotes. Finally, flies of the genotype para/+;sda/sda arising from the para;sda double-mutant backcross displayed a seizure threshold, 14.7 ± 3.9 V, that was above the sda homozygote threshold. This value is much lower than the double-mutant threshold of 38.9 ± 8.0 V, but it does indicate that a single copy of para can suppress seizures to a limited extent. The males from this backcross were examined to ensure the homozygous presence of sda in the original double mutant.Electrophysiology
The GF circuit was used to assess nervous system function. All
experiments were performed at room temperature 22-24°C. Methods for
handling and mounting flies, stimulating the GF with single pulses
(0.2-ms duration, 0.5 Hz), and recording of evoked DLM potentials have
been described previously (Kuebler and Tanouye 2000;
Pavlidis and Tanouye 1995
). During the course of these
experiments, the GF was stimulated continuously to assess GF circuit
function. Care was taken not to use flies from overcrowded vials as the reduced size of these flies could artificially lower the seizure threshold.
To determine following frequency of the GF circuit, 20 consecutive suprathreshold stimulus pulses (1.2-1.4 times the GF threshold) were delivered to the GF at a particular frequency. The following frequency was determined as the highest frequency at which the DLM responded to at least 19 of the 20 pulses. Between the different trials, flies were allowed to rest for at least 1 min. GF following frequencies were not determined for shak-B2 (also called Passover) because the GF pathway is disrupted in this mutant.
Seizures were elicited by short wavetrains of high-frequency (HF)
electrical stimuli (0.4-ms pulses at 200 Hz) delivered to the brain for
300 ms. Seizures consist of HF activity in at least seven different
muscle groups and over 30 muscle fibers in the thorax (Kuebler
and Tanouye 2000). Seizures are followed by a period of
synaptic failure in the GF pathway. The figures used for this paper
display recordings from DLM indirect flight muscles. The aberrant HF
activity seen in the DLMs corresponds both with DLM motoneuron activity
(Pavlidis and Tanouye 1995
) and with similar activity in
motoneurons innervating other muscle groups throughout the fly
(Kuebler and Tanouye 2000
). Methods for determining
seizure thresholds and latency to seizure were as described
(Kuebler and Tanouye 2000
). Some of the less susceptible
genotypes examined here did not seize using 300-ms HF stimuli of our
standard procedure. For these strains, a threshold could be determined
following 400-ms HF stimuli as noted.
Previous experiments that examined seizure susceptibility were
performed on female flies because they are larger in size
(Kuebler and Tanouye 2000; Pavlidis and Tanouye
1995
; Pavlidis et al. 1994
). However, here we
find that males have consistently lower seizure thresholds and have
focused on them exclusively to facilitate our analysis of
high-threshold genotypes. As example, CS females have a seizure
threshold of 44.5 ± 4.4 (SD) V (Kuebler and
Tanouye 2000
) while the male CS threshold is 30.1 ± 3.8 V
(Table 1). Likewise, OR males were found
to have a lower threshold (39.3 ± 6.6 V; Table 1) than females
(48.4 ± 3.6 V) (Kuebler and Tanouye 2000
). Similar
differences were observed in several of the mutant strains that we
examined. For clarity here, only male thresholds are reported. In
addition to sex differences in seizure threshold, we also found males
had lower GF thresholds than females. The threshold of this neuron,
which is located in the brain, can be monitored with relative ease due
to its synaptic connections to the large indirect flight muscles in the
thorax. In all cases examined, the GF threshold in males (Table 1) was
lower than that found previously in females (Kuebler and Tanouye
2000
).
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RESULTS |
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Seizure susceptibility in the BS mutants
Seizures in Drosophila following HF stimuli consist of
aberrant HF firing in all muscle fibers and motoneurons that have been examined in the fly (Kuebler and Tanouye 2000) followed
by a period of quiescence typified by synaptic failure. Previous
studies have indicated that the ability of HF stimuli to elicit these
seizures can be modified by genetic mutations (Kuebler and
Tanouye 2000
). BS mutants, including the three examined here
bss, eas, and sda, have been shown to
be more susceptible to seizures than wild type (Kuebler and
Tanouye 2000
). These mutants have seizure thresholds ranging
from 3 to 7 V as opposed to the wild-type strains, Berlin, CS, and OR,
which have seizure thresholds ranging from 26 to 39 V (Table 1, Figs.
1 and
2). Because of
their phenotype, the BS mutants are a useful tool for investigating the
types of genetic factors that can suppress seizure susceptibility.
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Seizure thresholds in Na+ channel mutants
The first class of mutants we investigated consisted of
two mutants that affect voltage-gated Na+
channels, mlenapts and para.
These mutants seemed likely candidates for having alterations in
seizure susceptibility as many anti-convulsants such as phenytoin, carbamazepine, and lamotrigine suppress seizures through interactions with voltage-gated Na+ channels (Kuo
1998; Kuo et al. 1997
). Both of these mutants
displayed decreased levels of seizure susceptibility compared with
wild-type strains (Table 1). Examples of
mlenapts and para
seizurescan be seen in Fig. 2. The seizure threshold found
for para was 65 ± 7.2 V, well above the wild-type
range, while mlenapts had no appreciable
seizures following the standard 300-ms HF stimuli. With 400-ms stimuli,
the seizure threshold for mlenapts was
72 ± 7.3 V.
Both mlenapts and para suppress the BS seizure-susceptible phenotype
Because both mlenapts and para were much less susceptible to seizures than wild type, both mutants were tested for the ability to suppress BS seizures (Table 2). The para mutation was able to suppress the BS seizure phenotype as the addition of sda into a para background raised the seizure threshold to wild-type levels (38.9 ± 8.0 V), well above the 6.2 ± 0.8 V sda seizure threshold. The mlenapts mutation was able to rescue the seizure susceptibility defect in two BS mutants tested, bss and sda. The double-mutant combination bss;mlenapts has a seizure threshold of 29 ± 4.7 V, which is in the range of wild type, as opposed to a 3.2 ± 0.6 V threshold for bss (Fig. 2). The mlenapts;sda double mutant had no appreciable seizures following 300- and 400-ms HF stimuli had to be used. The value obtained, 89 ± 10.2 V, is much greater than wild-type values and is even slightly higher than that found for mlenapts alone (Table 2). In both mlenapts double mutants, bss;mlenapts and mlenapts;sda, there was an absence of spontaneous seizure activity on recovery from the initial seizure and failure that may be related to the reduction in seizure susceptibility seen in these mutants. The BS flies always display intense spontaneous seizure activity during this period; however, the activity was absent in both mlenapts double mutants bss;mlenapts (Fig. 2) and mlenapts;sda (not shown).
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Seizure thresholds in K+ channel mutants
The second group of mutants examined consisted of
Drosophila K+ channel mutants. We
postulated mutants may have increased seizure susceptibility levels as
K+ channel defects can lead to seizure
disorders in both mice and humans (Charlier et al. 1998;
Singh et al. 1998
; Smart et al. 1998
). We
tested a variety of K+ channel mutants including
eag, a mutant that affects a member of a family of
voltage-gated K+ channels (Wu and Ganetzky
1992
) and slo, a mutant that affects the
calcium-activated K+ current,
ICF (Atkinson et al.
1991
). Surprisingly, we found that both eag and
slo have seizure thresholds above the wild-type range, 62 ± 10.3 V for eag and 52.9 ± 7.4 V for
slo (Table 1).
We also tested three different mutant alleles of the Sh K+ channel gene and found that all three mutants were resistant to seizures in comparison to wild-type strains (Table 1). The most defective Sh mutant, ShKS133, was resistant to seizures following 300-ms HF stimuli, and its threshold following 400-ms HF stimuli, 84 ± 12.8 V, was one of the highest seen in this study. The ShrKO120 mutant was also resistant to seizures compared with wild type as it was found to have a seizure threshold of 87 ± 8.5 V following 300-ms HF stimuli. The Sh5 mutant did not display much resistance to seizures; it has a seizure threshold of 51 ± 6.1 V, a value just above the wild-type range.
ShKS133 can partially suppress the BS seizure-susceptible phenotype
We tested the ability of the ShKS133 to suppress the BS seizure-susceptible phenotype even though K+ channels had not previously been thought of as targets for anti-convulsants. We found that the introduction of sda into a ShKS133 background led to partial suppression of the sda seizure-susceptible phenotype. The ShKS133;sda double mutant has a seizure threshold (18.8 ± 5.7 V) that is above the values for the BS mutants yet below the wild-type range (Table 2, Fig. 3). ShKS133;sda flies also exhibited a decrease in the GF failure time following HF stimuli. The double mutant failed for 25 ± 7.4 s (Fig. 3), while sda failed for 64 ± 10.5 s (Fig. 1). The presence of ShKS133 appeared to suppress both failure and seizures although the ShKS133;sda flies were still more susceptible than wild type.
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Mutant that affects neural connections is resistant to seizures
The mutant we investigated is shak-B2
that affects neural connections. Because neural connections and
pathways are thought to be critical in both the generation and spread
of seizures throughout the nervous system, mutations that disrupt these
connections may affect seizure susceptibility. In addition, the
shak-B2 mutant, which prevents the
formation of electrical synapses throughout the nervous system
(Phelan et al. 1996; Trimarchi and Murphey 1997
), may inhibit the synchronization of neuronal firing, a
trademark of seizure activity (Carlen et al. 1996
;
Perez-Velazquez et al. 1994
). Here we found that the
shak-B2 mutant has decreased
susceptibility to seizures as the seizure threshold in this mutant,
95 ± 10.2 V, is well above wild-type levels (Fig.
4, Table 1).
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Shak-B2 mutation suppresses different BS mutants to varying degrees
The shak-B2 mutant was tested for its ability to suppress seizures in all three of the BS mutants studied here, bss, eas, and sda (Table 2, Fig. 4). In the case of bss, the presence of the shak-B2 mutation did not significantly alter the seizure susceptibility. There was little difference in the seizure thresholds between bss and bss shak-B2 double mutants: bss shak-B2 mutants have a threshold of 3.6 ± 0.7 V, while bss mutants have a threshold of 3.2 ± 0.6 V. The eas shak-B2 double mutant displayed partial suppression as it has a seizure threshold (15.3 ± 3.2 V) that falls between eas and the wild-type range (Tables 1 and 2). Finally, the addition of sda to a shak-B2 background raised the seizure threshold to wild-type levels (31.4 ± 5.2 V), well above the 6.2 ± 0.8 V seizure threshold seen in the sda mutant (Table 2, Fig. 4). The reduction in excitability in shak-B2;sda mutants was also evident by the absence of spontaneous seizure activity during the recovery described previously for bss;mlenapts and mlenapts;sda.
Increased latency to seizures and reduction of spontaneous seizures in bss shak-B2
Despite the lack of ability to alter the seizure threshold in
bss flies, the presence of the
shak-B2 mutation did lead to a significant
increase in the latency to seizure onset in bss
shak-B2 flies following 4-V HF stimuli. In the
case of the double mutant, the latency to seizure onset was 768 ± 371 ms, while the latency for bss was 295 ± 81 ms
(n = 20). Previous studies (Kuebler and Tanouye
2000) have shown that in bss, low-voltage HF stimuli
give rise to seizures with longer latencies possibly because it takes some time for the activity in the few neurons activated by the 4-V HF
stimulus to be amplified through positive feedback loops to generate a
seizure. When higher voltage HF stimuli are used, we proposed that more
neurons are recruited directly by the stimulus and therefore less time
is taken for this activity to be amplified into a seizure. This also
appears to occur in the double mutant as the latency to seizure
decreases to 288 ± 25 ms (n = 5) following 12-V
HF stimuli. It is possible that the absence of functional electrical
connections in bss shak-B2 flies increases
the amount of time necessary to generate a seizure at 4 V by disrupting
the amplification of the neural activity induced by the HF stimuli.
A reduction in excitability in the bss shak-B2 mutant could also be seen on examining the activity seen on recovery from synaptic failure. Following electrically induced seizure and synaptic failure, bss mutants often undergo additional bouts of seizure and GF failure. These additional bouts of seizure and GF failure occurred in 87% of bss flies during recovery from a 4-V HF stimulus, while this activity only occurred in 12% of the bss shak-B2 mutants. The absence of the electrical connections may disrupt the amplification or synchronization of the activity that occurs following synaptic failure such that it is much more difficult to generate further bouts of seizure and GF failure in these double mutants.
Double mutants: general features
Our general findings are that although all of the BS mutations examined could be suppressed in double-mutant combinations, they did not all appear to be suppressed equally well. The bss mutant was the most difficult to suppress, sda appeared to be the easiest to suppress, while eas was somewhere in between. Likewise, the mutations that we used to set the genetic background had varying abilities to act as suppressors. For the mutants tested, mlenapts acted as the best suppressor, ShKS133 appeared to be the weakest of the suppressors, while the para and shak-B2 mutations fell somewhere in between. In addition, the ability of a strain to suppress the BS phenotype could not be predicted purely on the basis of the seizure threshold. For example, while mlenapts and ShKS133 had very similar seizure thresholds, they are at opposite ends of the spectrum in terms of their ability to suppress the BS seizure phenotype.
The double-mutant combinations displayed seizure thresholds that spanned an extremely large range. Despite this large range, the double mutants can be classified into four categories. The first consisted of those with a partially suppressed BS seizure-susceptibility phenotype and included eas shak-B2 and ShKS133;sda. These flies had seizure thresholds that were above the BS level but below wild type. The second category consisted of those with a completely suppressed BS phenotype and included bss;mlenapts, shak-B2;sda, and para;sda. These flies had seizure thresholds that were comparable to wild type. The final two categories each had only one representative and consisted of those double mutants that had seizure phenotypes that were consistent with one of the parental types. The mlenapts;sda strain was the only double mutant that had a seizure threshold nearly identical to the parental suppressor strain, in this case mlenapts. On the other hand, the bss shak-B2 strain was the only double mutant that had a seizure threshold comparable to the parental BS mutant, although the seizure phenotype of bss shak-B2 does differ slightly from bss.
GF threshold is not altered in most strains
One possible explanation for the altered seizure thresholds seen
in many of the double mutants is that the threshold of each individual
neuron in these strains could be altered. If this was the case,
different HF stimulus voltages could be required to recruit the same
number of neurons in different genotypes. This would account for the
different seizure thresholds seen here. To investigate this
possibility, we examined the threshold of the GF neuron to determine if
individual neuron thresholds were significantly altered in these
strains. We found that the GF threshold differed in only three cases,
mlenapts,
mlenapts;sda, and
bss;mlenapts (Table
3). In all three cases, the GF threshold
is elevated compared with wild type, a factor that may contribute to
the fact that mlenapts is the best
suppressor we have studied. Despite this, the GF threshold did not
necessarily correlate with the seizure threshold in these three cases:
the double-mutant bss;mlenapts
has a higher GF threshold than mlenapts
alone despite the fact that the double mutant has a much lower seizure
threshold (Tables 1 and 2). In addition, another neuron in
mlenapts flies, the DLM motoneuron, showed
no changes in stimulation threshold compared with wild type
(Kuebler and Tanouye 2000).
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Based on the preceding data, it is difficult to see a consistent pattern concerning individual neuron thresholds that could explain the wide range of seizure thresholds found here. It does remain a formal possibility though that other neurons not examined here may display altered thresholds that correspond more closely to the seizure threshold.
Following frequency alterations
During seizures, abnormal HF neuronal spiking spreads throughout the nervous system. If the nervous system is not capable of supporting HF spiking activity, then seizure suppression might occur. To examine this possibility, we chose the measurement of the GF following frequency, the maximum stimulation frequency the GF pathway can reliably follow, as an additional method for dissecting the excitability of the nervous system in the various strains. The first indication that GF following frequency may affect the seizure threshold is seen on examining the mutants with the highest thresholds, ShKS133 and mlenapts. These mutants do not display seizures following 300-ms HF stimuli and both have following frequencies that are well below the wild-type range (Table 4, Fig. 5). Likewise, the ShrKO120 and para mutants, which have seizure thresholds that fall between wild-type and the high-threshold mutants, have GF following frequencies that were intermediate between these two groups.
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Further evidence is seen in the fact that all the double-mutant combinations that show a decrease in seizure susceptibility also display a decrease in the GF following frequency. The bss; mlenapts, and mlenapts;sda double mutants have GF following frequencies that are greatly reduced compared with the following frequencies seen in bss and sda respectively (Table 4, Fig. 6). In both cases the following frequency was reduced to levels similar to mlenapts. In the case of para;sda, the double mutant has a following frequency that is lower than either sda or para alone. Finally, the addition of sda to a ShKS133 background reduced the following frequency well below the value found for sda. In all of these cases, the reduction in GF following frequency corresponded to a complete or partial suppression of the seizure susceptible phenotype normally seen in the BS mutants.
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Despite these correlations, GF following frequency was not an absolute predictor of seizure susceptibility. For example, para;sda has a lower following frequency than mlenapts;sda despite having a higher level of seizure susceptibility (Table 4, Fig. 6). In fact, a low following frequency did not always correspond with a high seizure threshold indicating that other factors are involved. The ShKS133;sda double mutant is a case in point as it has one of the lowest following frequencies yet has a seizure threshold that is below wild type (Table 4). If the reduction in following frequency does indeed have an anti-convulsant effect, this effect could be partially compensated for in ShKS133;sda mutants by other hyperexcitable defects associated with the ShKS133 or sda mutations.
We also tested the GF following frequency of the BS mutants and found that bss, eas, sda had following frequencies similar to wild type (Table 4). It is clear that the seizure susceptibility seen in these mutants is not due to increased following frequencies, indicating other mechanisms must account for the phenotype seen in the BS mutants. In addition, the following frequencies of two BS mutants, bss and eas, are slightly lower than CS wild-type flies, despite the lower seizure threshold in these mutants. It is clear that the following frequency does not always predict the seizure susceptibility level; however, it appears that modifications in following frequency could be one factor that contributes to the overall seizure susceptibility of the nervous system.
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DISCUSSION |
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We have demonstrated that certain genetic mutations can both elevate the seizure threshold in Drosophila and suppress the seizure-susceptible phenotype seen in BS mutants. All of these mutants, in double-mutant combination with BS mutants, provided a genetic background that elevated the seizure threshold to values above those seen in the BS mutants alone. This is the first demonstration that mutations in Na+ channels, K+ channels, and a connexin protein can suppress seizures. Understanding how these mutations contribute to the seizure phenotype will be instrumental in furthering our knowledge of the complex array of factors that contribute to seizure susceptibility.
Na+ channel mutants suppress seizures
Seizures were found to be suppressed by altering
voltage-gated-Na+ channels through mutations such
as mlenapts and para that lead
to a reduction of functional Na+ channels
(Kuroda et al. 1991; Loughney et al.
1989
). The data presented here and in previous studies
(Nelson and Wyman 1990
) indicate that the reduction in
Na+ channels leads to a decreased ability of the
GF pathway to support HF firing. The loss of HF firing may contribute
to the ability of these mutations to suppress BS seizures following HF
stimuli. In every case, the suppression of seizures in these double
mutants corresponds to a decrease in the GF following frequency
compared with the following frequency seen in BS mutants alone.
Although individual neuron (i.e., GF) excitability following
single-pulse stimulation is altered in some of these double mutants it
does not closely correspond to the changes in seizure susceptibility.
The reduction in the availability of active Na+
channels in mlenapts and para
suggests that they may inhibit seizures in much the same way as the
anti-convulsants phenytoin and carbamazepine (Kuo 1998;
McNamara 1999
). During repetitive firing, these drugs
are thought to stabilize the Na+ channel in the
inactive state thereby reducing the number of channels that can be
activated. This leads to an inhibition of HF firing (McLean and
Macdonald 1983
, 1986
) similar to the GF following frequency
defect in mlenapts and para.
The level of Na+ channel inactivation also
accounts for seizures in patients who suffer from generalized epilepsy
with febrile seizures plus (GEFS+), a disease resulting from a mutation
in the Na+ channel beta1 subunit that causes the
channel to inactivate much slower (Wallace et al. 1998
).
K+ channel mutants suppress seizures
The finding that Sh and eag mutants
displayed increased seizure thresholds compared with wild type was
surprising given the hyperexcitability defects seen in these strains
(Jan et al. 1977; Kaplan and Trout 1969
;
Tanouye et al. 1981
). In fact, the Sh seizure phenotype in Drosophila is in stark contrast to the
spontaneous seizure phenotype seen in a mouse knockout of the Shaker
family channel Kv1.1 (Smart et al.
1998
). The difference in Sh mutant phenotypes could
be due to the differences between the Sh loci in flies and
mice. In mice, there are several Sh loci that are expressed
differentially (Drewe et al. 1992
; Lock et al.
1994
; Wang et al. 1994
) such that a mutation in
one would presumably affect only a subset of neurons and could be
partially compensated for by other loci. In flies, there is only one
identified Sh locus that gives rise to a variety of
Sh transcripts (Kamb et al. 1988
) such that
mutations affect most if not all the Sh channels in the fly.
It is clear from mouse knockout studies that the specific Sh
channel affected by mutation is critical to the seizure phenotype. For
example, the Sh family Kv4.1 knockout
does not have a seizure susceptible phenotype (Ho et al.
1997
), in contrast to the Sh Kv1.1 knockout described in the preceding text.
It is possible that mutations that affect one or a small subset of
Sh transcripts could lead to a seizure susceptible defect in
flies; however, mutations of this type have not been identified.
It is not obvious why the K+ channel mutants
studied here have higher seizure thresholds than wild type nor why
ShKS133 can suppress sda
seizures. One possible explanation centers on the ability of
neural pathways in these mutants to support the type of HF activity
seen during seizures. In ShKS133 mutants,
the ability of the GF to follow HF stimulation is severely impaired: it
is reduced three to four times that of wild type. This is likely the
result of delayed repolarization in
ShKS133 neurons (Tanouye et al.
1981). Among several possibilities, delayed repolarization
could prolong Na+ channel inactivation, thereby
inhibiting HF activity in the GF pathway and possibly other pathways as well.
Despite this correlation, it is apparent that other mechanisms must
contribute to the seizure phenotype seen in these mutants for two
reasons: Sh5 and
ShrKO120 have similar GF following
frequencies despite having different seizure thresholds and
eag has no reduction in GF following frequency despite
having an increased seizure threshold. Another possible contributing mechanism is that K+-channel
mutants may disrupt the synchronization of synaptic components in
positive feedback loops that are thought to be involved in the
generation of the oscillations seen during seizures (Huguenard and Prince 1994; Traynelis and Dingledine 1988
;
Warren et al. 1994
). The altered repolarization kinetics
seen in these strains (for a review, see Wu and Ganetzky
1992
) could affect firing patterns of certain neurons critical
to the generation of seizures by causing components of the positive
feedback loop to fire out of synch.
Mutations that affect neuronal connections
The involvement of positive feedback loops in seizure generation
suggests that mutations altering neuronal connections within these
loops might be expected to affect seizure susceptibility. One way to
disrupt neuronal connections is to use
shak-B2 flies. These flies are defective
in a neural-specific connexin gene that leads to the absence of
electrical synapses or neuronal gap junctions in the fly
(Crompton et al. 1995; Krishnan et al. 1993
; Trimarchi and Murphey 1997
). The absence
of these leads to an increase in the seizure threshold in
shak-B2 mutants and an ability to suppress
seizures in double-mutant combinations.
The effect shak-B2 mutations have on
seizure susceptibility may be related to the role neuronal gap
junctions play in the synchronous firing of populations of neurons. It
is known that gap junctions are involved in the generation of
synchronous bursting in CA1 pyramidal cells in the in vitro
calcium-free epilepsy model. The synchronous seizure-like discharges
normally seen in this model are inhibited by methods that block gap
junctions (Perez-Velaquez et al. 1994). In fact,
electrical coupling of neurons mediated by gap junctions has long been
proposed as a factor involved in generating the synchronous bursts seen
during seizures (Carlen et al. 1996
; Dudek et al.
1986
).
The latency to seizure in bss shak-B2
mutants as compared with bss is sometimes up to 2 s
longer even though the voltage necessary to trigger seizure in the two
strains is similar. This could be due to a lack of
gap-junction-mediated synchronization in the positive feedback loops.
In this case, the longer latency in bss shak-B2 flies would occur because it takes
longer for the activity triggered by the HF stimulus to be amplified
within the feedback loop in the absence of the proper synchronizing
connections. A similar mechanism could also explain the requirement of
higher intensity HF stimuli to activate enough neurons synchronously to
trigger a seizure in the shak-B2,
eas shak-B2, and
shak-B2;sda mutants as
described previously (Kuebler and Tanouye 2000).
It is also possible that the removal of gap junctions in the nervous system of shak-B2 flies controls seizures in much the same manner as the surgical removal of neural connections controls seizures in some patients that suffer from intractable epilepsy. This implies that seizures may occur in shak-B2 mutants at the site of the stimulating electrodes, but due to lack of electrical connections in the nervous system, the seizure does not spread throughout the fly. This possibility is unlikely because seizures that spread to the thorax can be generated in shak-B2 flies, albeit at high-voltages, and in bss shak-B2 flies at very low voltages.
Model of Drosophila seizure susceptibility
A simple model that accounts for several major features of Drosophila seizure susceptibility and its modification by genetic mutations is depicted in Fig. 7. A neuron (labeled Sei) is capable of delivering seizure throughout the Drosophila CNS. Seizures are triggered in Sei by three presynaptic input neurons (labeled Wt, Bs, and Su). Although Sei, Wt, Bs, and Su are shown as single neurons, each may represent a population of neurons with similar properties or an extensive neural circuit. For convenience, the three input neurons are given separate names; however, they may actually be similar or identical to each other; we have not found features that distinguish them. Synaptic inputs from the presynaptic neurons show temporal and spatial summation in Sei.
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In a normal wild-type fly, stimulation of two neurons (say: Bs and Wt) with a HF electrical stimulus wavetrain triggers a seizure. Synaptic potentials from Bs and Wt summate temporally and spatially in Sei to trigger the seizure. In a BS mutant, it is much easier to trigger a seizure and stimulation of only a single neuron (say: Bs) is sufficient. A low-voltage HF stimulus is sufficient to drive Bs, and its synaptic potentials summate temporally in Sei to trigger the seizure. In a suppressor mutant, it is much more difficult to trigger a seizure and necessary to stimulate all three neurons (Bs, Wt, and Su), thereby requiring a higher voltage HF stimulus. In a double mutant, susceptibility has been restored to the wild-type level and a seizure is triggered by stimulating only two neurons (say: Bs and Wt). These observations suggest that different synaptic inputs interact spatially and temporally to trigger seizures.
Suppressor mutations compromise nervous system signaling capabilities.
Several mutations, mlenapts,
para, and Sh, may suppress seizures because of an
effect on firing frequencies. Kuebler and Tanouye (2000)
showed that the effectiveness of the triggering stimulus is dependent
on the frequency of stimulus pulses in the HF wavetrain. A stimulus
frequency of 200 Hz appears to be ideal, while a stimulus frequency of
150 Hz is substantially less effective. Mutations that compromise following frequency suppress seizures because 200-Hz HF frequencies cannot be maintained in input neurons and there is decreased temporal summation at the triggering synapses. To elicit seizures, additional inputs must be stimulated to increase spatial summation. For
shak-B2, nervous system signaling
capabilities are compromised because of neuroconnectivity defects. If
the number of synapses or synaptic strength of Sei inputs are
compromised, then decreased spatial summation is expected. To elicit
seizures, additional inputs must be stimulated by higher voltage HF
stimuli to compensate.
This simple model accounts for several major features of Drosophila seizure susceptibility and its modification by genetic mutations. However, it also appears to be an over-simplification because of some observations that are not well explained. The most fundamental of these is how BS mutations act to reduce the triggering requirements for Sei neuron activation from two neurons (Bs and Wt) to just one (Bs alone). For the most part, the nervous systems of BS mutants function completely normally generating predominantly normal behaviors. In all parts of the nervous system that we can record from, BS mutants do not show a higher capacity for following frequency; there are no obvious alterations in chemical synaptic transmission and there are no obvious supernumerary synaptic connections that would enhance synaptic strength. Despite our failure to discover an obvious cellular physiological defect, BS mutations have a profound effect on overall seizure sensitivity.
Other complexities that are not well explained by this simple model
include high-voltage seizure suppression and changes in the latency
between HF stimulus and the start of the seizure (Kuebler and
Tanouye 2000). There is no indication of the potential
importance of synchronous firing of neurons that could be important in
seizure genesis and that could be disrupted in
shak-B2 mutants leading to suppression.
There is no indication whether or not synaptic failure (the quiescent
period) might also play a role in seizure genesis (Pavlidis and
Tanouye 1995
).
Utility of using Drosophila to identify seizure suppressor genes
It is clear from this study that genetic factors can decrease
seizure susceptibility in flies, while previous studies have demonstrated that genetic factors can increase seizure susceptibility (Kuebler and Tanouye 2000). The ability to identify
these factors in mammals, especially in humans, is quite limited
compared with the genetically tractable system of
Drosophila. The generation of Drosophila double
mutants described here allows one to test the ability of a variety of
mutations to alter the seizure-susceptibility levels seen in BS
mutants. In each case in which the BS seizure susceptible phenotype was
altered, a single mutation, which becomes a possible target for the
development of novel anti-convulsants, is responsible. Because the
genetic defects as well as the phenotypic consequences of these
mutations are known, these studies also determine how various
physiological processes contribute to the overall seizure
susceptibility of the fly. Further experiments of this type as well as
the isolation of new suppressors may bring us closer to unraveling the
complexity of seizure disorders.
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ACKNOWLEDGMENTS |
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The authors thank S. Faulhaber for assistance in maintenance of Drosophila stocks. We thank Drs. Jeremy Lee and Charlie Oh for discussion throughout this project and Drs. David Bentley and Geoff Owen for insightful comments on the manuscript.
Part of this work was supported by National Institute of Neurological Disorders and Stroke Grant NS-31231 to M. A. Tanouye.
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FOOTNOTES |
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Address for reprint requests: M. A. Tanouye, Dept. of Environmental Science, Policy and Management, 201 Wellman Hall, University of California, Berkeley, CA 94720 (E-mail: tanouye{at}uclink4.berkeley.edu).
Received 20 September 2000; accepted in final form 30 May 2001.
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REFERENCES |
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