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The Journal of Neuroscience, December 15, 2000, 20(24):9071-9085
Impaired Fast-Spiking, Suppressed Cortical Inhibition, and
Increased Susceptibility to Seizures in Mice Lacking Kv3.2
K+ Channel Proteins
David
Lau1,
Eleazar
Vega-Saenz
de Miera1,
Diego
Contreras2,
Ander
Ozaita1,
Michael
Harvey1,
Alan
Chow1,
Jeffrey L.
Noebels3,
Richard
Paylor4,
James I.
Morgan5,
Christopher S.
Leonard6, and
Bernardo
Rudy1
1 Departments of Physiology and Neuroscience, and
Biochemistry, New York University School of Medicine, New York, New
York 10016, 2 Department of Neuroscience, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19106, Departments of 3 Neurology and 4 Molecular and
Human Genetics, Baylor College of Medicine, Houston, Texas, 77030, 5 Department of Developmental Neurobiology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105, and
6 Department of Physiology, New York Medical College,
Valhalla, New York 10595
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ABSTRACT |
Voltage-gated K+ channels of the Kv3 subfamily
have unusual electrophysiological properties, including activation at
very depolarized voltages (positive to  10 mV) and very fast
deactivation rates, suggesting special roles in neuronal excitability.
In the brain, Kv3 channels are prominently expressed in select neuronal
populations, which include fast-spiking (FS) GABAergic interneurons of
the neocortex, hippocampus, and caudate, as well as other
high-frequency firing neurons. Although evidence points to a key role
in high-frequency firing, a definitive understanding of the function of
these channels has been hampered by a lack of selective pharmacological
tools. We therefore generated mouse lines in which one of the Kv3
genes, Kv3.2, was disrupted by gene-targeting
methods. Whole-cell electrophysiological recording showed that the
ability to fire spikes at high frequencies was impaired in
immunocytochemically identified FS interneurons of deep cortical layers
(5-6) in which Kv3.2 proteins are normally prominent. No such
impairment was found for FS neurons of superficial layers (2-4) in
which Kv3.2 proteins are normally only weakly expressed. These data
directly support the hypothesis that Kv3 channels are necessary
for high-frequency firing. Moreover, we found that Kv3.2 / mice
showed specific alterations in their cortical EEG patterns and an
increased susceptibility to epileptic seizures consistent with an
impairment of cortical inhibitory mechanisms. This implies that, rather
than producing hyperexcitability of the inhibitory interneurons, Kv3.2
channel elimination suppresses their activity. These data suggest that
normal cortical operations depend on the ability of inhibitory
interneurons to generate high-frequency firing.
Key words:
K+ channels; neocortex; fast spiking; knock-out inhibitory interneurons; high-frequency firing; seizure
susceptibility; GABA; epilepsy
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INTRODUCTION |
Approximately 10-20% of the
neurons in the cerebral cortex are inhibitory GABAergic interneurons.
These cells play a critical role in a number of important functions,
including the gating and processing of sensory information, the
establishment and plasticity of sensory receptive fields, the
synchronization of cortical circuits, the generation of rhythms, and
the limiting of seizure activity (Fairen et al., 1984 ; Gilbert, 1993;
Jones, 1993; Amitai and Connors, 1995; Keller, 1995; Singer and Gray,
1995; Freund and Buzsaki, 1996; Jefferys et al., 1996; Steriade,
1997).
Cortical GABAergic interneurons represent a heterogenous population of
cells with subtypes differing in morphological appearance, expression
of specific markers such as calcium-binding proteins or neuropeptides,
firing patterns, synaptic properties, and axonal connectivity (Jones,
1975; Somogyi et al., 1984; Hendry et al., 1989; Freund and Buzsaki,
1996; Cauli et al., 1997; Gonchar and Burkhalter, 1997; Kawaguchi and
Kubota, 1997; Gupta et al., 2000).
The largest group of neocortical inhibitory interneurons (~50%)
consists of cells that contain the calcium-binding protein parvalbumin
(PV). These neurons are characterized by a "fast-spiking" firing
pattern, i.e., the ability to fire long trains of very brief action
potentials at high frequency with little firing frequency adaptation
(McCormick et al., 1985; Celio, 1986; Cauli et al., 1997; Kawaguchi and
Kubota, 1997). These neurons are interconnected by electrical synapses
and form a network of fast-spiking cells, suggesting a role in the
generation of synchronized cortical activity (Galarreta and Hestrin,
1999; Gibson et al., 1999).
Several lines of evidence have led to the hypothesis that specific
voltage-gated, delayed rectifier-type K+
channels composed of K+ channel
pore-forming subunits of the Kv3 subfamily (Kv3.1-Kv3.3) are critical
for the ability of neurons to fire at high frequencies in a sustained
or repetitive fashion. First, the properties of these channels, which
include activation at voltages positive to 10 mV and very fast
deactivation rates on membrane repolarization, naturally lend
themselves to a specific role in spike repolarization. Second, there is
a strong correlation between the specific expression of Kv3 RNA
transcripts and Kv3 proteins in neuronal populations that fire at high
frequencies. Third, pharmacological experiments show that blockade of
native Kv3-like currents with low concentrations of tetraethylammonium
(TEA) or 4-aminopyridine (4-AP) impairs the ability of these neurons to
fire sustained and/or repetitive-action potentials at high frequency.
Fourth, computer modeling indicates that selective blockade of Kv3
currents impairs high-frequency firing (Perney et al., 1992; Lenz et
al., 1994; Weiser et al., 1994, 1995; Du et al., 1996; Massengill et
al., 1997; Sekirnjak et al., 1997; Martina et al., 1998; Wang et al.,
1998; Chow et al., 1999; Erisir et al., 1999; Atzori et al., 2000) (for
review, see Coetzee et al., 1999; Rudy et al., 1999).
To further test the hypothesis, and given the absence of selective
channel blockers, we used gene-targeting methods to produce mice lines
that do not express Kv3.2 K+ channel
subunits (McCormack et al., 1990; Rudy et al., 1992), which are
prominently expressed in PV-containing interneurons in deep cortical
layers (Chow et al., 1999), and compared the properties of fast-spiking
neurons in the neocortex from these mice with those from normal
wild-type littermates. Results from these experiments provide direct
evidence that Kv3 channels are critical for both sustained and
repetitive high-frequency firing. Moreover, the Kv3.2 / mice show
both an enhanced susceptibility to seizures and disturbed cortical
rhythmic activity. The availability of mice in which fast-spiking is
compromised in specific neuronal populations provides a model to
investigate the consequences of this impairment on the behavior of
cortical circuits, which in turn can help in the understanding
of the function of fast-spiking, the roles of the interneurons
in cortical function, and the mechanisms by which they achieve these functions.
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MATERIALS AND METHODS |
Generation of mice lacking Kv3.2 proteins
Isolation of a mouse 129 genomic clone containing
exon I of Kv3.2. A mouse 129 genomic library
(~1 × 106 pfu) in  DashII (kind
gift from Drs. J. Rossant and A. G. Reaume, Mount Sinai Hospital,
Toronto, Canada) was screened at high stringency with a 380 bp fragment
containing the first 301 bp of the coding region of Kv3.2 and 79 bp of
the 5' untranslated region, derived from a rat Kv3.2 cDNA (McCormack et
al., 1990).
Bacteriophage DNA from positive clones was isolated with the Midi Phage DNA Prep (Qiagen, Hilden, Germany) from fresh liquid lysates.
Genomic clone inserts were excised from the bacteriophage arms by
restriction digest with NotI and subcloned into the
NotI site of the bacterial vector pBluescript (Stratagene,
La Jolla, CA). One of the isolated clones, E2, shown by hybridization
to contain sequence from the first coding exon (exon I) of Kv3.2, was
used for these studies. The restriction recognition sites of the
following enzymes were mapped on the E2 clone: BamHI,
ClaI, EcoRI, HindIII, SacI,
and XbaI. Each of the EcoRI fragments was subcloned individually into pBluescript (Stratagene) to facilitate mapping and the generation of the targeting construct. The 3' half of the clone E2, consisting of two contiguous EcoRI
fragments of 3.6 and 8.5 kb, was used for the construction of the
targeting construct and is illustrated in Figure
1A.
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Figure 1.
Generation of the Kv3.2 / mouse.
A, Targeting the Kv3.2 gene via homologous
recombination. Top, Restriction map of the mouse genome
in the area around exon I (the first coding exon) of the
Kv3.2 gene. Exon I is indicated as the solid
box and introns as lines. Arrows
under genetic elements indicate transcriptional orientation.
Middle, Kv3.2 gene-targeting vector. The neomycin
resistance gene replaced the portion of exon I downstream of the
EcoRI site and ~4 kb of intron I. PGK-Neo, Neomycin resistance gene driven by the
phosphoglycerate kinase promoter; PGK-TK, thymidine
kinase gene driven by the phosphoglycerate kinase promoter;
pBluescript, bacterial vector backbone.
Crosses indicate crossover regions in homologous
recombination. Bottom, Null Kv3.2 allele generated after
proper targeting. The 5' probe is the
XbaI-SacI fragment used as a template to
synthesize the probe for genotyping. As indicated, the 5' probe should
identify a 3.0 kb fragment in the wild-type allele and a 5.0 kb band in
the null allele when genomic DNA is digested with XbaI.
Restriction enzymes are as follows: E,
EcoRI; H, HindIII;
S, SacI; X,
XbaI. B-D, Molecular characterization of
Kv3.2 knock-out mice. B, Genotyping by Southern blot
analysis. Genomic DNA was isolated from tail biopsies of juvenile mice
and digested with XbaI. Kv3.2 knock-out (/) mice
possess two copies of the engineered null allele and consequently only
show the 5.0 kb fragment after hybridization with the
32P-labeled 5' probe. Heterozygotes
(+/) show both wild-type and mutant alleles, and the
wild-type (+/+) littermates only possess wild-type
alleles. C, Northern analysis of Kv3.1 and Kv3.2 mRNA
expression in Kv3.2 null mice. Ten micrograms of total brain RNA
was loaded into each lane from a wild-type, a heterozygote, and a Kv3.2
knock-out mouse. The Northern blots were probed with Kv3.2
(right) or Kv3.1 (left)
32P-labeled cDNA probes. Notice that the Kv3.2
knock-out does not express mature Kv3.2 RNA species and the
heterozygote has lower expression levels than the wild type. In all
three Kv3.2 genotypes, Kv3.1 mRNA levels are constant. The blots were
then hybridized with  -actin cDNA probe to quantitate the amount of
RNA per lane. The sizes of the RNA standard marker for both blots are
located on the left. D, Immunoblots of Kv3.1b and Kv3.2
proteins in the Kv3.2 mutant. Solubilized brain membrane proteins from
mice of all three genotypes were electrophoresed in SDS-PAGE gels and
incubated with primary antibody against Kv3.1b (left) or
Kv3.2 (right). Kv3.2 proteins were not detectable in the
Kv3.2 null mutant, and lower levels of protein were present in the
heterozygote animal. The concentration of Kv3.1b protein was consistent
between all three genotypes. Sizes of the protein size markers are
indicated at the left of each blot.
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Generation of the targeting construct. Regions within the
3.6 and 8.5 kb EcoRI fragments of the clone E2 were selected
to be the short and long arms of homology (Fig. 1A).
The 1.9 kb short arm of homology was isolated from the 3.6 kb
EcoRI fragment by digestion with SacI and
EcoRI. The 5.0 kb long arm of homology was isolated from the
8.5 kb EcoRI fragment by restriction digestion with
XbaI and NotI (in the polylinker of pBluescript).
The neomycin resistance gene flanked by EcoRI and
XbaI sticky ends was ligated between the two arms. The
thymidine kinase gene was placed 5' of the short arm of homology, and
the entire construct was cloned into pBluescript. The final construct
was mapped by restriction digest and subsequent Southern hybridization,
as well as by sequencing of key junctions to confirm its integrity.
Homologous recombination in embryonic stem cells. W9.5
embryonic stem (ES) cells (28 × 106)
were harvested (Robertson, 1987) and resuspended in 1 ml of culture
medium in a sterile electroporation cuvette (Bio-Rad, Hercules, CA). We
mixed 40 µg of the NotI linearized targeting construct (in
sterile PBS) with the suspended cells and electroporated it with a
Gene-Pulser electroporator (Bio-Rad) at 0.23 kV, 500 µF. The pulsed
ES cells were cultured onto 60 mm feeder plates at 37°C in an
atmosphere of 5% CO2. The basic culture medium
consisted of DMEM plus 15% serum. Leukemia inhibitory
factor (106 U/ml), used to retard
ES cell differentiation, was added to all culture media except replica
plates (see below). After a day in culture, G418 (350 µg/ml
Geneticin; Life Technologies, Gaithersburg, MD) and
1-(2-deoxy-2-fluoro-1--D-arabino-furanosyl)-5-iodouracil (2 µg/ml; a gift from Eli Lilly, Indianapolis, IN) were added to the
culture medium. The medium was changed 2 d after drug introduction and then daily afterward. Five days after drug introduction,
surviving undifferentiated ES cell colonies were transferred
individually to multiwell cell culture plates (Falcon).
A total of 380 ES cell colonies were harvested. The medium was changed
2 d after harvesting and then daily. Four days later, the ES cell
cultures were trypsinized and passaged into two sterile 48-well
multiwell cell culture plates (one colony per well), a master plate
that was frozen and stored at 70°C, and a replica plate that was
further expanded. The replica plates were fed every 2 d. After 1 week in culture, the cell culture medium was discarded, and the replica
plates were washed with PBS. To each well, 250 µl of lysis buffer
(1.0 M NaCl, 10 mM EDTA, 50 mM
Tris, pH 8, 0.5% SDS, and 0.2 µg/µl proteinase K) was added, and
the plate was incubated overnight at 55°C. We added 250 µl of
isopropanol to each well and the genomic DNA pellets were
transferred individually to microcentrifuge tubes. The DNA was washed
with 70% ethanol and resuspended in 50 µl of Tris-EDTA (TE).
The DNA was used to genotype the colonies by Southern blot analysis (as
described below) to identify ES cells that had undergone homologous recombination.
Chimera generation. From the master plate, identified,
targeted ES cell colonies were expanded in culture. C57BL6
blastocysts that were 2.5 d old were harvested from the uterine
tubes of timed pregnant females, and 8-10 targeted ES cells were
introduced to the blastocoel with a beveled glass micropipette. The
injected blastocysts were implanted into pseudopregnant mothers
(Joyner, 2000). Chimeric character was estimated by coat color, and
males with >95% chimerism were selected and bred with C57BL6 females. Offspring heterozygotes were identified by Southern blot genotyping using genomic DNA obtained from tail biopsies (see below) and were bred
against C57BL6 mice for backcrossing or bred against other
heterozygotes to generate knock-out mice. All knock-out mice used in
our experiments had been backcrossed at least seven generations onto
the C57BL6 genetic background.
Genotyping by genomic Southern blot analysis
Genomic DNA from cultured ES cells or tail biopsies was digested
overnight with XbaI (Promega, Madison, WI). The digested samples were electrophoresed on 0.7% agarose-Tris-borate-EDTA gels at 7.5-9.0 V/cm until DNA fragment sizes from 2 to 6 kb were clearly separated. The gels were stained with ethidium bromide and
photographed on a UV light table with a fluorescent ruler for
orientation. The agarose gels were incubated in 5 gel volumes of
denaturing solution (1.0 M NaOH and 1.5 M NaCl) with gentle agitation for 30 min and a
change to a fresh solution after 15 min. After denaturing, the gels
were incubated in 5 gel volumes of neutralizing solution (1.0 M Tris-HCl, pH 7.5, and 1.5 M NaCl) with gentle agitation for 30 min and a
change to a fresh solution after 15 min. The DNA in the gels was
transferred onto nylon membranes (Stratagene) overnight via capillary
action. The blotted membranes were marked, and the DNA was
UV-crosslinked to the nylon membrane in a Stratalinker (Stratagene).
The membranes were stored dry at room temperature until hybridization.
The probe used for genotyping was a 1.0 kb band between the
XbaI and SacI sites in the 3.6 kb
EcoRI fragment of the E2 clone (Fig. 1A,
bottom diagram). The probe corresponds to sequences in the
intron preceding exon I of the Kv3.2 gene. Probes were labeled with [32P]dCTP with the
Redi-Prime random primer labeling kit (Amersham Pharmacia
Biotech, Arlington Heights, IL). With XbaI-digested genomic DNA, the probe hybridized to a 3.0 kb band derived from the
wild-type allele and a 5.0 kb band from the targeted null allele (Fig.
1B).
The Southern blots were prehybridized in QuikHyb (Stratagene) for 15 min at 68°C. The denatured probe was added at a final concentration
of 1.5 × 106 TCA precipitable counts
per milliliter, and the blots were hybridized in a Hybaid oven
(Labline) at 68°C for 1 hr with gentle rotation. The hybridized
Southern membranes were washed (three times) at room temperature with
2× saline-sodium phosphate-EDTA buffer (SSPE) and 0.1% SDS,
followed by a 60°C hot wash (in 0.1× SSPE and 0.1% SDS) for 30 min.
The blots were then exposed to x-ray film between two intensifying
screens at 70°C for 2 hr to 1 week, depending on the intensity of
the signal.
Isolation of genomic DNA from tail biopsies
We harvested 0.5-1.0 cm of tail from ~3-week-old mice. Tails
were digested overnight at 60°C in tail lysis buffer (100 mM NaCl, 50 mM Tris, pH 7.4, 1 mM
EDTA, 0.1% SDS, and 0.75 mg/ml proteinase K). Tail lysates were
extracted with 1 vol of chloroform, and genomic DNA was precipitated
with 2 vol of ethanol. DNA pellets were washed with 70% ethanol
and briefly air-dried to remove residual ethanol. The genomic DNA was
resuspended in TE and used for restriction enzyme digestion.
Northern blot analysis
Total RNA was obtained with the guanidine-isothiocynate method
and quantified by optical density measurements (Chomczynski and Sacchi,
1987). Total RNA (10 µg) from knock-out and wild-type mice was
electrophoresed in denaturing formaldehyde gels and transferred to
Duralon-UV membranes (Stratagene) as previously described (Rudy et
al., 1988). The Northern blots were hybridized as described for
Southern genotyping. The probe for Kv3.2 was the 380 bp probe described
previously. A full-length cDNA clone of Kv3.1b was used as a probe
template for Kv3.1 mRNA detection.
Western blot analysis
Brain membrane extracts were prepared from a P3 fraction of
tissue homogenate from adult knock-out and wild-type mice (Hartshorne and Catterall, 1984) and solubilized in Triton X-100 as previously described (Chow et al., 1999). To prepare the Western blots, membrane protein (25 µg/lane for detection with Kv3.1b-Ab and 50 µg/lane for
Kv3.2-Ab) were electrophoresed in a 9% SDS polyacrylamide gel and then
transferred onto nitrocellulose membranes (Bio-Rad) as previously
described (Chow et al., 1999). The blots were incubated with either
Kv3.1b-Ab (Weiser et al., 1995) at 1:1000-1:2000 dilution or Kv3.2-Ab
(Chow et al., 1999) at 1:50-1:100 dilution. This was followed by
incubation with horseradish peroxidase-linked anti-rabbit secondary
antibodies (Promega). Detection of the secondary antibody was performed
using chemiluminescence (Pierce, Rockford, IL). The Kv3.2-rAb was
derived from immunizing rabbits to a peptide corresponding to a
sequence present in the region of the Kv3.2 protein that is before the
first membrane-spanning domain in the N-terminal area and recognizes
all Kv3.2 isoforms (Chow et al., 1999). The Kv3.1-rAb is directed
against the C terminal of the predominant isoform of the
Kv3.1 gene, Kv3.1b (Weiser et al., 1995).
Immunohistochemistry of mouse brain
Adult mice were anesthetized with intraperitoneal injections of
sodium pentobarbital (~80-100 mg/kg) and transcardially perfused with paraformaldehyde after the loss of pain reflexes as previously described (Chow et al., 1999). The brains were removed from the animals
and processed for immunohistochemistry as described previously (Chow et
al., 1999). The Vectastain Elite ABC kit was used to immunolabel via
the horseradish peroxidase method. Kv3.1-rAb was used at 1:1000
dilution, Kv3.2-rAb was used at 1:300 dilution, and mouse monoclonal
antibodies to parvalbumin (Sigma, St. Louis, MO) were used at
1:300.
In vivo physiology
Behavioral analysis. The following behavioral tests
were all done in the laboratory of Richard Paylor (Department of
Molecular and Human Genetics, Baylor College of Medicine) with a
battery commonly used in this laboratory (Kimber et al., 1999; Peier et al., 2000). The tests were done blindly in a group of mice that included 13 mutant (four female, nine male) and nine wild-type (three
female, six male) littermates. The mice had been backcrossed seven
times to C57BL6. The tests were performed essentially as described by
Paylor et al. (1998) and included the following: (1) general
neurological screen for severe sensory and motor abnormalities, (2)
open-field test for exploratory activity and anxiety-related responses,
(3) light-dark test for anxiety-related responses, (4) rotarod test
for motor coordination and skill learning, (5) acoustic startle and
prepulse inhibition of the acoustic startle response for sensorimotor
gating, (6) habituation of the acoustic startle response for
sensorimotor adaptation, (7) contextual and auditory-cued freezing to
assess conditioned fear, and (8) the hotplate test for
analgesia-related responses. Data were analyzed using two- or three-way ANOVA.
Chronic EEG. Adult mice were anesthetized with Avertin
(1.25% tribromoethanol-amyl alcohol) by intraperitoneal injection
(0.02 ml/gm). Silver wire electrodes (0.005 inches in diameter)
soldered to a microminiature connector were implanted into the subdural space over the left and right cortical hemispheres. After several days
of recovery, EEG activity was recorded daily during random 2 hr samples
for 7-10 d using a TECA digital electroencephalograph. All
recordings were performed on mice moving freely in the test cage in the
laboratory of Jeffrey L. Noebels at Baylor College of Medicine.
EEG recording in anesthetized mice. Knock-out and wild-type
mice were anesthetized with intraperitoneal injections of a mixture of
ketamine (15 mg/kg) and xylazine (3 mg/kg). Depth of anesthesia was
ascertained by recording EEG with monopolar electrodes placed in
frontal cortex. Supplemental doses of ketamine-xylazine were given at
the slightest sign of EEG desynchronization. After the loss of tail
pinch reflexes, the mice were placed in a rodent stereotaxic apparatus
(David Kopf Instruments, Tujunga, CA) equipped with mouse head
holders. A midline sagittal incision was made along the scalp, and the
skin was reflected. Petroleum jelly was applied over the eyes to
prevent ulcers. Burr holes were drilled over the right somatosensory
cortex and the right dorsal thalamus according to the stereotaxic
coordinates (Franklin and Paxinos, 1997). Mineral oil was applied over
the exposed brain to prevent desiccation. Bipolar tungsten electrodes
were fashioned from two monopolar tungsten electrodes with resistance
of 1 M that were affixed with dental cement. The bipolar electrode
pairs were lowered into the neocortex and thalamus with fine
micromanipulators (Narishige, Tokyo, Japan) through the burr holes, and
signals were amplified with a homemade DC amplifier with head stage and
capacity compensation. In cortex, the electrodes were located in the
pial surface and in layer 6 (~0.7 mm apart); in the thalamus, the
electrodes were side by side, separated by 0.4 mm. Electrical
stimulation was also delivered through the recording electrodes with an
isolated pulse stimulator (AM Systems). Data were sampled at 1 kHz with an InstruNet (GW Systems) analog-to-digital card and analyzed in
Igor (WaveMetrics Inc., Lake Oswego, OR) with customized routines.
Seizure induction with pentylenetetrazole.
Pentylenetetrazole (PTZ) (Research Biochemicals, Natick, MA) was
dissolved in PBS and injected intraperitoneally at the indicated dose.
After injection, the animal was placed in a transparent Plexiglas cage
(30 × 20 × 25 cm) and observed for up to 30 min. Latencies
to focal (partial clonic), generalized (generalized clonic), and
maximal (tonic-clonic) behavioral seizures were recorded. The bottom of
the cage was covered with clean paper towels that were replaced for
each animal. Each cage was cleaned with water after each experiment,
before introducing a new mouse. All the animals used in this study were housed in a facility with light (12 hr light/dark cycle) and
temperature control, and all the experiments were performed in the
laboratory between 12:00 P.M. and 2 P.M. in mice of similar age (10-14 weeks).
We defined several stages in the behavioral response to PTZ injection.
Stage 1, designated as hypoactivity, was characterized by a progressive
decrease in activity until the mice stood in a crouched or prone
position with their abdomens in full contact with the bottom of the
cage. Stage 2 was isolated jerks or twitches. Stage 3 was partial or
focal clonic seizures affecting the face, head, and/or forelimbs. These
seizures were usually very brief, typically 1-2 sec. Stage 4 was
generalized clonic seizures. These usually occurred suddenly, could
last 30 sec or more, and involved generalized whole-body clonus.
Autonomic signs were frequently seen. The seizure was usually followed
by a quiescent period. Stage 5 was tonic-clonic (maximal) seizures.
Mice reaching this stage displayed wild running and jumping behavior
and then had generalized seizures characterized by tonic hindlimb
extension. Tonic-clonic maximal seizures were usually associated with death.
In vitro physiology
Slice preparation. Knock-out and wild-type mice of
ages postnatal day 15 (P15) to P21 were used for acute
brain-slice preparation (Agmon and Connors, 1991). Mice were
anesthetized with an overdose injection of sodium pentobarbital and
decapitated after the loss of pain reflexes. The brain was rapidly
removed from the skull in a bath of ice-cold artificial CSF (ACSF) (in
mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 MgSO4, 2 CaCl2, and 10 dextrose, pH 7.4, bubble-saturated
with 95% O2, 5% CO2.
Slices 250-µm-thick from the somatosensory cortex were prepared on a
vibratome (World Precision Instruments, Sarasota, FL) and placed in a
holding chamber with continuous bubbling ACSF at room temperature.
Slices were allowed to rest in the holding chamber for at least 1 hr
before transfer to the recording chamber. The submersion-type recording chamber was perfused at a rate of 2-3 ml/min with ACSF saturated with
95% O2, 5% CO2, and all
recordings were done at a temperature of 24°C controlled by an
in-line solution heater (Warner Instruments, Hamden, CT).
Whole-cell recordings. Neocortical neurons were visualized
with near-infrared light (>775 nm) at 400× magnification with a nuvicon tube camera (Dage-MTI, Michigan City, IN) and differential interference optic (DIC) on a fixed-stage microscope (Olympus Optical,
Tokyo, Japan) (Stuart et al., 1993; Erisir et al., 1999). Nonpyramidal
cells were visually selected for current-clamp experiments. Recording
microelectrodes of 6-9 M resistance were made from standard wall
borosilicate glass (Sutter Instruments, Novato, CA) with a
Flaming/Brown type micropipette puller (Sutter Instruments). The
micropipette filling solution consisted of (in
mM): 144 K-gluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 4 ATP-Mg, and 0.5 GTP-Tris. For
subsequent histochemistry, biocytin (0.2% w/v; Sigma) was added to the
internal solution just before recording. In the current-clamp mode of
an electronic bridge amplifier (Axon Instruments, Foster City, CA), repetitive firing, single spikes, and hyperpolarization responses were
recorded in the whole-cell configuration. Protocols were delivered
under the control of pClamp 7 software (Axon Instruments). Responses
were sampled at 10 kHz. Neurons were held at 70 mV with small
injections of direct current, except during protocols for spike
doublets and rebound responses when they were held at 60 mV. Action
potential shape parameters were measured from action potentials evoked
by 150 msec current steps that were just above threshold. Spike
amplitude was measured as the difference between the peak and the
threshold of the action potential. Spike threshold was determined by
finding the potential at which the second derivative of the voltage
waveform exceeded three times its SD in the period preceding spike
onset. The fast afterhyperpolarization (AHP) was measured as the
difference between the spike threshold and voltage minimum after the
action potential peak. Maximum rates of rise and decay of the action
potential were computed from the maximum and minimum of the first
derivative of the voltage waveform. Spike width was measured at half
the spike amplitude. Spike times were measured by determining the time
at which the rising phase of the action potential crossed a
fixed-threshold potential. Instantaneous frequency (one per interspike
interval) was computed from trains of action potentials evoked by 600 msec duration pulses. Steady-state firing rate was the average of
instantaneous frequency for the last five intervals of a train. Current
strength was increased until spike failure occurred within the 600 msec
duration pulse. The maximum steady-state firing rate was the
steady-state firing rate from the train evoked by the current strengths
(at increments of 100 pA) before that which produced spike failure.
Firing frequency adaptation was calculated by dividing the steady-state
firing rate by the first instantaneous frequency of the train. All
analysis was performed in customized routines in Igor and Sigma Plot.
Results are reported as mean ± SEM. TEA (Research Biochemicals)
was bath-applied. Only one fast-spiking neuron was recorded per brain slice.
Histochemistry and immunolabeling of recorded neurons
After electrophysiological characterization, brain slices were
fixed for 1-2 hr at room temperature in 4% paraformaldehyde in PBS.
The slices were transferred into 30% sucrose with 0.02% sodium azide
and stored at 4°C. Slices were washed three times in PBS to remove
sucrose and incubated in a blocking-permeablization solution [1%
(w/v) BSA, 0.4% (v/v) Triton X-100, and 10% (v/v) normal goat serum]
for 1 hr. For primary labeling, streptavidin conjugated to Cy2 (1:250
dilution; Jackson ImmunoResearch, West Grove, PA) and mouse monoclonal
parvalbumin IgG (1:400) (Sigma) were incubated with the brain slices in
10% blocking solution in PBS for 7 d at 4°C. The slices were
washed twice in PBS and incubated with the secondary antibody,
Cy3-conjugated anti-mouse IgG, for 5 d at 4°C. After three
washes in PBS, the slices were mounted onto glass slides in 0.001 M phosphate buffer and allowed to air dry. The
sections were coverslipped with a polyvinyl alcohol-glycerol medium
with 2% 1,4-diazabicyclo[2,2,2]octane (Goslin and Banker, 1991). The
sections were examined and scored on a Zeiss (Oberkochen, Germany)
Axiophot epifluorescence microscope. Sections containing biocytin-labeled neurons were examined for PV immunoreactivity without
knowledge of the physiological characteristics of the recorded neuron.
Only sections with distinct PV immunoreactivity present at the depth of
the biocytin-labeled somata, as determined using a 40× objective, were
considered for scoring. This precaution was taken to reduce the
possibility of falsely scoring double-labeled cells as PV-negative
because of incomplete antibody or chromophore penetration. Digital
images were acquired on an Zeiss Axiovert 35 M confocal microscope with
a 40× objective lens, a scanning laser attachment, and a
krypton-argon mixed-gas laser, and transferred into a graphics program
(Photoshop 5.0).
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RESULTS |
Generation of mice lacking Kv3.2 proteins
Gene targeting by homologous recombination in ES cells (Thomas and
Capecchi, 1987) was used to generate mouse lines in which the
Kv3.2 gene has been disrupted. The targeting construct used to modify the mouse Kv3.2 gene was derived from a mouse 129 genomic clone and is shown diagrammatically in Figure
1A, which also illustrates the structure of the gene
in the mutated area before and after the targeting. The 3' end portion
of the first coding exon (exon I) of Kv3.2 was deleted and
replaced by a neomycin gene. The portion of exon I that was deleted
encodes the subunit (or tetramerization) domain (T domain) that is
critical for the oligomerization of Kv channel subunits (Li et al.,
1992; Shen and Pfaffinger, 1995; Xu et al., 1995). Therefore, if a
truncated protein were to be made at the normal starting methionine of
Kv3.2, it would lack the T domain and would not oligomerize with
products of other Kv3 genes and produce dominant negative effects
(McCormack et al., 1991; Babila et al., 1994; Ribera et al., 1996). ES
cells with a targeted allele were selected and implanted in foster
mothers. Several chimeras were obtained, from which two independent
lines of Kv3.2 / mice were established. Both have been backcrossed (7 and 10 times so far) to C57BL6 mice and are being maintained in this
genetic background. Kv3.2 / mice lack Kv3.2 mRNA and protein
products (Figs. 1C,D,
2), whereas heterozygous mice have reduced mRNA and protein levels (Fig. 1C,D). In
contrast, the levels of products of the closely related
Kv3.1 gene were not affected (Fig.
1C,D). We also determined whether the
distribution of Kv3.1 protein was altered in the Kv3.2 / mice by
immunohistochemistry (Fig. 2). Kv3.1 proteins have a wider expression
pattern than Kv3.2 proteins (Weiser et al., 1995; Rudy et al., 1999)
and overlap in several neuronal populations, including PV-containing
neurons in the neocortex, globus pallidus, and hippocampus, in which
Kv3.1 and Kv3.2 proteins may form heteromeric Kv3 channels (Chow et al., 1999; Hernandez-Pineda et al., 1999). In the Kv3.2 / mice, Kv3.1 proteins were detected in both these regions of overlap and the
other structures in which they are normally found (Fig. 2). The overall
brain histology (Fig. 2; see also Fig. 4) and the barrel structure
(data not shown) of somatosensory cortex also appeared normal in these
mice.
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Figure 2.
Normal distribution of Kv3.1 protein and lack of
Kv3.2 protein in the Kv3.2 / mouse. Immunoperoxidase detection of
Kv3.2 and Kv3.1 proteins in brain sections from: Top row
Kv3.2 wild-type (+/+); bottom row, Kv3.2
knock-out (/) littermates. Sections were overexposed
to emphasize the lack of Kv3.2 staining in knock-out mice. Kv3.2
products have a highly specific pattern of expression in brain and have
not been detected outside the CNS (Rudy et al., 1999). In the brain,
Kv3.2 proteins are prominently expressed in thalamocortical
projections, the axons of the thalamic relay neurons in the dorsal
thalamus (Moreno et al., 1995). The immunostaining of the collaterals
of these axons in the reticular thalamic (RT)
nucleus produces the labeling seen in this structure, and the staining
of the thalamocortical terminals produces the labeled barrel structure
seen in layer IV of the neocortex (Ctx).
The staining of the hippocampus (Hip) and deep
neocortical layers is produced by the prominent immunolabeling of the
somas and axons of all PV-containing and a subset of
somatostatin-containing GABAergic interneurons (Chow et al., 1999;
Atzori et al., 2000). Kv3.2 proteins are also present in GABAergic
neurons in other forebrain structures, including the caudate, basal
forebrain, and globus pallidus (GP). Kv3.2 proteins are
found as well in yet to be identified neurons in the inferior
colliculus, the nucleus of the lateral lemniscus, and dorsal cochlear
(DCh), trigeminal, deep-cerebellar (Den),
and vestibular nuclei (Weiser et al., 1994; Moreno et al., 1995; Chow
et al., 1999; Hernandez-Pineda et al., 1999; Atzori et al., 2000). Note
the absence of Kv3.2 proteins and the normal distribution of Kv3.1
proteins in the knock-out mice. Cer, Cerebellum;
Gr, granule cell layer of the cerebellar cortex;
Mol, molecular layer of the cerebellar cortex;
VB, ventrobasal nucleus of the thalamus;
VCh, ventral cochlear nucleus.
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Phenotypic characterization of Kv3.2 / mice: increased
susceptibility to epileptic seizures
Kv3.2 / mice in the mixed 129-C57BL6 or in the C57BL6
background have a healthy appearance and grow normally. Both male and
female, homozygote (/) and heterozygote (+/) mice are fertile. All the behavioral and functional analysis of the mice has been done in
the nearly pure C57BL6 background. The mice show no evidence of severe
sensory or motor abnormalities during neurological screens. Moreover,
there were no significant differences (p > 0.05) detected in overall total distance traveled in the open-field
test, light-dark test, rotarod test, prepulse inhibition, startle
habituation, conditioned fear, spatial learning, or hotplate test (see
Materials and Methods). There was one significant difference in the
open-field test. Knock-out mice had a significantly lower
(p < 0.04) center-to-total distance ratio,
which is an indicator of anxiety in the open field. However, there were
no statistically significant differences in the light-dark exploration
box (an independent test of anxiety), so one must be cautious about
making too much of the anxiety phenotype in the open field. Future
experiments will be needed to determine whether there is a possible
anxiety phenotype by evaluating the mice in other assays of anxiety
such as the elevated plus-maze.
Spontaneous, epileptic episodes lasting 5-40 sec and characterized by
tonic-clonic convulsions have been observed in behaving Kv3.2 /
mice (n = 14 out of several hundred Kv3.2-deficient mice under similar manipulations) but never in wild-type littermates. These always occurred while the animals were being manipulated but
could not be reliably provoked by routine handling or auditory or
photic stimuli. The electrographic record during one of these spontaneous episodes is shown in Figure
3. Spontaneous epileptic episodes in the
Kv3.2 / mice are rare, and usually there are none during typical
studies with the mice. The waking background EEG activity in these
mutants is unremarkable, and no abnormal patterns of spike-wave
discharge were observed in particular. However, the seizures suggest
cortical excitability increases in the Kv3.2 / mouse, a hypothesis
that was supported by a series of experiments described later in this
paper.
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Figure 3.
Electrographic pattern of a spontaneous seizure in
a Kv3.2 / mouse. Continuous EEG recording of a generalized
tonic-clonic behavioral convulsive episode shows bilateral seizure
activity arising shortly after a single interictal discharge. Abnormal
synchronous activity increases in frequency for ~40 sec and ends
abruptly with no postictal depression of the EEG.
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Impaired fast spiking in cortical interneurons from Kv3.2
/ mice
We used the Kv3.2 / mice to directly test the hypothesis that
K+ channels containing Kv3 proteins are
required for sustained high-frequency firing in fast-spiking cortical
interneurons. Because PV-containing cortical interneurons in the deep
layers prominently express both Kv3.2 and Kv3.1 proteins (probably in
heteromeric channels), whereas PV-containing neurons in superficial
layers express mainly Kv3.1 subunits (Chow et al., 1999), we predicted
that PV-containing neurons in the deep layers would be more affected in
Kv3.2 / mice than neurons in superficial layers. We confirmed that
the levels (data not shown) and distribution (Fig.
4) of PV immunoreactivity in the cortex
were not affected in the Kv3.2 / mouse.
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Figure 4.
Normal distribution of cortical PV
immunoreactivity in Kv3.2-deficient mice. Immunoperoxidase detection of
PV in Kv3.2 wild-type (A-C) and knock-out
(D-F) littermates. PV is localized in a
subpopulation of neurons in the neocortex and hippocampus
(A, D). In the neocortex
(B, E), PV-positive neurons are scattered
throughout all cortical layers. In neocortical interneurons in
wild-type and knock-out animals (C,
F), PV is present in multipolar neurons (also
known as basket cells) and is expressed throughout the cell, including
dendrites and axons. Pyramidal cells (some indicated by
arrows) are not stained for PV but are surrounded by
immunopositive puncta (the baskets), the terminal boutons from
the GABAergic interneurons. Scale bar: A,
D, 1 mm; B, E, 250 µm;
C, F, 50 µm.
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The shape of action potentials and the repetitive firing properties of
cells from knock-out and wild-type littermate mice in both superficial
and deep cortical layers were compared using whole-cell recording
methods from nonpyramidal neurons visualized by IR-DIC optics in slices
of somatosensory cortex. Nonpyramidal neurons in both knock-out and
wild-type mice had different firing patterns that could be classified
into three types, similar to those previously reported in rat and mouse
neocortex: regular spiking (RS), low-threshold spiking (LTS), and fast
spiking (FS) (Kawaguchi and Kubota, 1993, 1997; Cauli et al., 1997;
Erisir et al., 1999; Gibson et al., 1999). Under our recording
conditions, FS neurons were characterized by having short-duration
action potentials and large, brief AHPs. In response to sustained
current injection, they fired high-frequency spike trains with abrupt onset and little spike frequency adaptation. These neurons were easily
distinguished from regular spiking neurons that sustained much lower
maximum frequencies ( 50 vs >100 spikes/sec) and adapted much more
(mean rates at the end of a 600 msec pulse were 40% of initial rates
in RS neurons compared with  70% in FS cells). FS neurons could also
be distinguished from LTS cells, which showed pronounced spike
frequency adaptation (steady-state rates were, on average, <40% of
initial rates) and generated low-threshold spikes or spike bursts in
response to depolarization from hyperpolarized potentials [as
previously described in rat by Kawaguchi and Kubota (1993) and Gibson
et al. (1999)]. FS neurons had firing thresholds 10-15 mV more
positive than RS and LTS cells and had significantly lower input
resistance than the other two types of cells (133 ± 6.2 M,
n = 22 for FS, compared with 222.1 ± 22 M,
n = 52 for RS, and 349.7 ± 30.2 M,
n = 21 for LTS cells) [similar to observations in rat
by Kawaguchi and Kubota (1993)].
The action potential and repetitive firing characteristics of a typical
multipolar, PV-positive, layer 5 neuron from a wild-type mouse are
compared with those of a multipolar, PV-positive, layer 5 neuron from a
knock-out littermate in Figures 5 and
6. The action potential from the
knock-out mouse was broader (width at half maximum of 1.1 vs 0.72 msec)
(Fig. 5A1) and had a slower maximum rate
of repolarization (66 vs 110 mV/msec) (Fig.
5A2, dashed line) than the
neuron from the wild-type littermate. In addition, the deceleration
of the membrane potential as it entered into the AHP was smaller in the
Kv3.2 / neuron, suggesting that the repolarization current decays
more slowly (Fig. 5A3, dashed
line). These and other differences in action potential shape are
summarized in Table 1 and indicate that
spike repolarization was impaired in the deep-layer FS neurons from
knock-out animals. Despite these differences, the maximum rate of rise
of the spike (for single spikes or the initial spike in a train) was
similar in the neurons from the two genotypes (Table 1), indicating
that the mechanisms responsible for initiating the action potential
were unimpaired. To verify that recordings were made from PV-containing
neurons, slices were fixed and processed for PV immunohistochemistry
after the recording and filling of neurons with biocytin (Du et al., 1996; Erisir et al., 1999). An example from a Kv3.2 / mouse is
shown in Figure 5B. All of the FS cells that were scored for PV immunoreactivity (see details in Materials and Methods) were PV-positive (n = 14 from wild-type mice and 16 from
knock-outs), and the inverse was also true; all of the neurons that
were scored positive for PV had been classified as fast-spiking
electrophysiologically.
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Figure 5.
FS neurons from Kv3.2-deficient mice have broader
action potentials with slower rates of repolarization.
A, Representative action potentials from a
PV-immunoreactive deep-layer neuron from a Kv3.2 wild-type
(WT) mouse are compared with the action
potentials from a representative PV-immunoreactive deep-layer neuron
from a Kv3.2 / (KO) mouse. Shown for each neuron are
two action potentials (A1) and their
first (A2) and second
(A3) derivatives. The action
potentials were wider (1.1 vs 0.72 msec at half maximum), and their
maximum rates of repolarization were smaller in the knock-outs
(second peak in first derivative; dashed
line in A2). In addition, the
peak deceleration of the membrane potential as it enters into the AHP
(third peak in the second derivative; dashed
line in A3) was much smaller
in the knock-out. B, The knock-out neuron whose data are
shown in A was biocytin-labeled
(B2) and was immunoreactive for
parvalbumin (B1; see also
B3, in which the superimposition of
the images with the two chromophores is shown), indicating that it was
an FS neuron. Data from this cell are also shown in Figure 6.
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Figure 6.
Impaired high-frequency firing in Kv3.2 /
mice. A, Repetitive firing of an FS PV-positive
deep-layer neuron from a wild-type mouse in response to two current
steps (375 and 975 pA). Firing frequency increases with increased
depolarizing current, and there is very little firing frequency
adaptation throughout the pulse. B, Repetitive firing of
an FS PV-positive deep-layer neuron from a Kv3.2 / mouse (same cell
as in Fig. 5) in response to same current steps as in A.
Firing frequency is much less than in the neuron from the wild-type
mouse, and there is more firing frequency adaptation. There is spike
failure during the largest current step. Also notice that the AHPs are
faster in the neuron from the wild-type mouse. C,
Instantaneous firing frequency plotted as a function of time from onset
of the current pulse of 875 pA for the knock-out and 975 for the
wild-type mice. Notice that there is much more adaptation of the firing
frequency in the knock-out than in the wild type. D,
Steady-state firing frequency versus injected current. Firing frequency
increases with current injection much more in the neuron from the
wild-type than the neuron from the knock-out mouse, and spike failure
occurs with lower current strengths (indicated by the last point
shown). In both cases the steady-state firing frequency reaches a
saturating (steady-state) value before failure.
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Fast-spiking neurons from wild-type and knock-out mice also differed in
their repetitive firing characteristics. Records from a typical
PV-positive neuron in deep cortical layers of each genotype are shown
in Figure 6. Both cells fired repetitively during steady depolarizations, and in both cases the steady-state firing rate increased as a function of injected current reaching near-saturation values before spike failure took place (Fig. 6D).
However, the wild-type neuron was able to sustain higher steady-state
firing frequencies (133 vs 66 spikes/sec) and showed significantly less firing frequency adaptation (mean firing rate at the end of a 600 msec
pulse was 75% of initial rates for the wild-type cell and 46% for the
cell from the Kv3.2 / mouse) than the neuron from the knock-out
(Fig. 6A-C). Spike failure also occurred
at much lower current strengths in the knock-out neuron than in the wild type (Fig. 6B,D).
These differences between wild-type and knock-out deep-layer neurons
were reproducible and statistically different when neurons from a large
number of mice of each genotype were compared (Fig. 7, Table 1). In scatter plots comparing
steady-state firing frequency and firing frequency adaptation (Fig.
7A) or steady-state firing frequency and action potential
width at half maximum (Fig. 7B), the cells from each
genotype showed a different, although overlapping, distribution
(p < 0.01 for the firing rate;
p < 0.02 for the degree of adaptation;
p < 0.001 for the spike width; one-way ANOVA). Most of
the neurons from the knock-out were in a cluster of cells with lower
steady-state frequencies, higher spike frequency adaptation, and wider
spikes. Yet, even in the knock-out mice, fast-spiking cells fired
faster and adapted less than regular spiking (Fig. 7A) or LTS (data not shown) neurons. However, some of the
fastest firing neurons from the Kv3.2 / animals fired nearly as
fast as the fastest firing neurons from the wild-type animals. This may
be related to the different relative levels of Kv3.1 and Kv3.2 proteins
in individual neurons, given that the expression of Kv3.1 remained
unaffected in the Kv3.2 / animals. Support for this idea was
obtained from experiments with low TEA concentrations described
below.
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Figure 7.
Disruption of the Kv3.2 gene affects the action
potential duration and fast-spiking properties (but not the input
resistance) of FS interneurons from deep cortical layers but not from
superficial layers. The fraction of the initial firing frequency
remaining at the end of a 600 msec depolarization
(A), the width at half amplitude
(B), and the input resistance
(C) of FS layer V-VI neurons from wild-type
(open symbols) and knock-out (filled
symbols) mice are plotted against the maximum steady-state
firing rate of each cell. Parameters from deep-layer regular spiking
nonpyramidal (RSNP) neurons of both genotypes are included in
A for comparison. Mean values for each parameter are
indicated by the symbol with the error bars that
indicate the SEM. The values from knock-out neurons clustered at lower
maximum firing rates (A, B), increased
firing frequency adaptation (A), and longer
action potential duration (B). However, there was
no difference in the input resistance of FS neurons from wild-type and
knock-out animals (C). Also notice the
quasi-linear relationship between steady-state firing rate and
firing frequency adaptation (A) or spike width
(B) in both wild-type and knock-out mice,
indicating that these parameters depend on common underlying factors.
D, Same as A for layer II-III neurons
from knock-out and wild-type mice, illustrating the lack of effect of
the mutation on superficial layer FS neurons. Similarly, no differences
were detected in action potential duration (Table 1).
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Several parameters that help distinguish fast-spiking neurons from
other interneurons in wild-type animals remain unchanged in the mutant
mice (Fig. 7C, Table 1) and were therefore also useful to
distinguish the neurons electrophysiologically. As in the case of
neurons from wild-type mice (see above), FS neurons in knock-out
animals had lower input resistance than RS and LTS cells (142 ± 8.6 M, n = 29; 231.8 ± 16.4 M,
n = 52; and 329.5 ± 19.7 M, n = 21, respectively), as well as higher firing thresholds (10-15 mV).
There usually was more spontaneous synaptic activity observed in
records from FS neurons than from the other cell types. LTS cells could
also be distinguished from RS and FS neurons in normal and knock-out
animals by the presence of low-threshold spikes when depolarized from
hyperpolarized potentials, as in normal animals (Kawaguchi and Kubota,
1993, 1997; Gibson et al., 1999).
There were no differences between knock-out and wild-type littermates
in the firing properties of regular spiking neurons (Fig.
7A). Furthermore, in contrast to the large differences in action potential shape and repetitive firing properties of deep-layer FS neurons from wild-type and knock-out mice, no significant
differences were observed when FS neurons in superficial layers were
compared (Fig. 7D, Table 1).
Low TEA concentrations eliminate the differences between wild-type
and Kv3.2 / fast-spiking neurons
The differences in the action potential and repetitive firing
properties of fast-spiking neurons from wild-type and knock-out mice
resemble the effects produced by application of low concentrations of
TEA (<1 mM) to neurons from normal mice (Erisir et al.,
1999). However, although low TEA concentrations also nearly completely blocked the AHP (Erisir et al., 1999), the AHPs in knock-out mice were
on the average only ~25% smaller than in wild-type mice (Table 1).
Moreover, although submillimolar concentrations of TEA affect the
magnitude but not significantly the kinetics of the AHP
(Erisir et al., 1999), we found that the kinetics of the AHP was
different in Kv3.2-deficient and wild-type animals (Fig.
5A1,A3). The
fast AHP characteristic of fast-spiking neurons from wild-type mice was
replaced by a slower AHP in fast-spiking neurons from Kv3.2 / mice
(Fig. 5A) (Fig. 6, compare
A,B). We hypothesize that the slow
AHP in FS neurons from Kv3.2 / mice is generated by the increased
activation of an unidentified K+
conductance (perhaps mediated by
Ca2+-activated
K+ channels), which deactivates at rates
slower than those of Kv3 channels. There is increased activation of
this conductance in Kv3.2-deficient mice because of the increase in the
duration of the action potential. We would further like to suggest that
this is not seen when TEA is used to block Kv3 channels because the drug also blocks this unidentified
K+ conductance. Consistent with this
idea, low TEA concentrations blocked the slow AHP in neurons from
knock-out mice (Fig. 8).
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Figure 8.
Low TEA concentrations block the AHP in FS neurons
from wild-type (WT) and knock-out
(KO) mice. The AHP is slower in the FS neuron from the
Kv3.2 / mouse. However, low TEA concentrations block the AHP nearly
completely in both cases.
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Other than these differential effects on the AHP, all of the effects of
the Kv3.2 / mutation on the spike and repetitive firing properties
of deep-layer fast-spiking neurons resembled the effects of a partial
block of Kv3 channels with TEA (~0.2 mM) on wild-type FS
neurons (Erisir et al., 1999). We expected the mutation to be
equivalent to a partial block of Kv3 channels because FS neurons in the
Kv3.2 / mice still express normal levels of Kv3.1 proteins (Figs.
1, 2), and Kv3.1 and Kv3.2 proteins express similar currents in
heterologous expression systems (Hernandez-Pineda et al., 1999; Rudy et
al., 1999). To test this hypothesis, we compared the effects of TEA on
the spike width, steady-state firing rate, and degree of adaptation of
fast-spiking neurons from wild-type and knock-out mice. The values of
these parameters in FS neurons from knock-out animals in the absence of
the channel blocker were close to those in wild-type animals in the
presence of submillimolar concentrations of TEA. For example, the spike
width of deep-layer FS neurons from Kv3.2-deficient mice (0.94 ± 0.04 msec, n = 20) was comparable with the spike width
of wild-type mice in 0.2 mM TEA (0.95 ± 0.03 msec, n = 17). Similarly, the steady-state firing frequency and firing frequency adaptation of deep-layer FS neurons from
Kv3.2 / mice (116.6 ± 10.2 spikes per second,
n = 20; and 0.68 ± 0.03, n = 20, respectively) were close to those for wild-type neurons in 0.2
mM TEA (92.9 ± 2.9, n = 16;
and 0.62 ± 0.04, n = 15, in 0.2 mM TEA) (Fig. 9).
Furthermore, although TEA still had effects on spike width and
repetitive firing properties of FS neurons from knock-out animals, the
effects were smaller than those observed in neurons from wild-type
animals (Fig. 9). In 1.0 mM TEA, a drug
concentration expected to block 80% of the total Kv3 channels,
knock-out and wild-type neurons had similar properties (e.g., 63 ± 6 spikes/sec, n = 15; and 55 ± 3 spikes/sec, n = 18, for the steady-state firing rate of wild-type
and knock-out FS neurons, respectively, in 1 mM
TEA) (Fig. 9C). This would be expected if the mutation and
the drug (at concentrations of 1 mM) are
acting, for the most part, on the same conductance. These data are also
consistent with the notion that we are recording from similar neurons
in the two types of animals and that, other than the lack of the
mutated channel proteins, the cells have not changed significantly in
active conductances or other membrane properties. Moreover, these
results also suggest that the dispersion in steady-state firing
frequency values between different FS neurons might be the result of
differences in the number of active Kv3 channels. Because Kv3
channel activity can be modulated by neurotransmitters and second
messengers (Moreno et al., 1995; Atzori et al., 2000), different
cortical neurons could have different proportions of active channels
depending on having been recent targets of these modulators.
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Figure 9.
Low TEA concentrations affect the firing
properties of FS neurons from wild-type mice more than those from
knock-out mice. The steady-state firing frequency and the preservation
of the initial firing frequency during a 600 msec pulse are plotted for
a number of deep-layer FS neurons in Kv3.2 / mice
(filled symbols) and wild-type littermates
(open symbols) in the absence of TEA
(A), and in the presence of 0.2 (B) and 1 mM
(C) TEA. An amplified view of the data in
C is shown in the insert at the
right. These neurons are a subset of the population of
neurons shown in Figure 7A-C. Different
symbols (as indicated in A) have been
used for the FS neurons for which we were able to establish the PV
immunoreactivity. Note that 0.2 mM TEA shifts more the
values of the neurons from wild-type than knock-out mice and that, in
the presence of 1 mM TEA, FS neurons from wild-type and
Kv3.2-deficient mice have similar properties. Similar results were
obtained in plots of action potential duration (half width) and
steady-state firing frequency (data not shown).
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FS neurons can repetitively fire spike doublets at 40 Hz, an
ability that requires Kv3 channels
Under certain physiological conditions, fast-spiking neurons may
not undergo long, steady depolarizations such as those used in the
previous experiments. For example, it has been shown in both the
neocortex and the hippocampus (Traub et al., 1996, 1999; Steriade et
al., 1998) that, when stimulated, fast-spiking neurons generate fast
rhythmic (~40 Hz) spike bursts. The bursts consist of two (spike
doublets) to three spikes with intraburst frequencies that are similar
to the steady-state frequencies observed during long, steady
depolarizations (100-200 Hz at room temperature; 300-400 Hz at
37°C). We asked whether the ability to generate such rhythmic bursts
repetitively is impaired in FS neurons from the Kv3.2 / mice.
Cortical neurons were stimulated repetitively with brief
depolarizations that generated one or more spikes per stimulus,
repeated at various frequencies. We focus on the results at 40 Hz, the characteristic frequency observed in the EEG during periods of
brain activation (Bouyer et al., 1981; Llinas and Ribary, 1993; Murthy
and Fetz, 1996a,b; Steriade et al., 1996). In wild-type mice, both
regular-spiking and fast-spiking GABAergic interneurons in both
superficial and deep cortical layers could follow 40 Hz stimuli that
generated a single spike per stimulus for periods up to 1 min (the
longest tested; data not shown). However, only fast-spiking neurons
could follow larger stimuli that generated a spike doublet without
failing (Fig. 10). In contrast,
deep-layer, fast-spiking neurons from knock-out mice failed in their
ability to fire spike doublets repetitively much more rapidly than did neurons from wild-type animals (Fig.
10A,B). In fact, in Kv3.2 /
mice, during the first stimulus, the second spike was already smaller
than the first spike, and it got smaller, slower, and more delayed with
subsequent depolarizations (Fig. 10A), suggesting a
reduction in the available Na+ current.
This is consistent with the hypothesis that Kv3 channels facilitate
fast spiking by increasing Na+ channel
recovery from inactivation (Erisir et al., 1999). This behavior was
similar to that observed after application of low concentrations of TEA
on deep-layer FS neurons from wild-type mice (data not
shown).
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Figure 10.
Deep-layer FS neurons from wild-type, but not
from Kv3.2-deficient, mice can repetitively fire action potential
doublets for long periods. A, FS neurons from wild-type
(WT) and Kv3.2 / (KO) mice
were stimulated repetitively at 40 Hz with brief depolarizing current
pulses that generated a spike doublet (for a total duration of 1 min).
Shown are the pulses and voltage responses for the first two stimuli
and the first two after 1, 5, and 20 sec of continuous stimulation. The
wild-type neuron never failed to respond with a spike doublet. In
contrast, the knock-out neuron failed to generate a spike doublet
rapidly after the onset of the stimulation. Note that, in the
knock-out, the second spike is smaller than the first
spike from the first current pulse, and it becomes smaller, slower, and
more delayed with subsequent depolarizations until the cell fails to
generate a second spike. B, Summary of the results of
this test applied to 11 knock-out neurons and 10 wild-type neurons. The
plot indicates the maximum firing rate versus the time at which the
cell failed to produce a second spike for FS neurons from
wild-type (open symbols) and Kv3.2-deficient mice
(filled symbols). In approximately half of the
wild-type neurons, the cell continued to fire spike doublets for the
duration of the experiment (1 min). The other half failed at times
considerably longer (40 ± 6.9 sec) than those that produced
failure in neurons from the Kv3.2-deficient mice (10 ± 2.4 sec).
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Increased cortical excitability in the Kv3.2 / mouse
The cellular analysis showed that the ability of fast-spiking
interneurons to fire at high frequencies for long durations or
repetitively is impaired in Kv3.2 / mice. The knock-out mice are
thus useful to test hypotheses of the function of these neurons and the
significance of fast spiking in their performance. The presence of
sporadic epileptic seizures in Kv3.2-deficient mice suggests that the
mice have an increased susceptibility to seizures. Here we present a
series of observations that provide further evidence of increased
cortical excitability and susceptibility to seizures in the Kv3.2 / mouse.
The first experiment consisted of recording spontaneous EEG from cortex
and thalamus in ketamine-xylazine anesthetized Kv3.2 / and
wild-type mice. Results were obtained with multisite extracellular and
field potential recordings using arrays of high-impedance tungsten
electrodes. As shown in Figure
11A, wild-type mice
(n = 21) showed the characteristic slow rhythm observed
during natural slow-wave sleep and during ketamine-xylazine anesthesia
in other species (Steriade et al., 1993a; Contreras and Steriade,
1995). In wild-type mice, the slow rhythm was characterized by
recurring sequences at <1 Hz of depth-negative followed by
depth-positive waves in the EEG, which occurred in synchrony with the
corresponding thalamic territory. Such sequences correspond
intracellularly to neuronal depolarization and hyperpolarization,
respectively (Steriade et al., 1993a; Contreras and Steriade, 1995).
The EEG of the Kv3.2 / mice (n = 18) showed,
superimposed on the underlying slow rhythm, spontaneous
high-amplitude sharp potentials, lasting from 30 to 100 msec and with
the same polarity as the slow oscillation (depth-negative and
surface-positive). Such sharp potentials pervaded throughout all phases
of the slow oscillation and produced a very irregular slow rhythm (Fig.
11B). Nevertheless, the slow rhythm is clearly
present, suggesting that the cortical synchronization responsible for
this rhythm still occurs in the knock-out. The "spikiness" of the
slow rhythm in Kv3.2-deficient mice, illustrated with the example in
Figure 11B, is the most characteristic effect of the
mutation that we have observed until now. All Kv3.2 / mice tested
showed similar irregularities on the EEG, which we have never seen in
wild-type littermates or in mice from commercial sources. The high
degree of spatiotemporal synchrony of the slow rhythm is achieved by
massive reentrant corticocortical circuits, as shown by the absence of
effects of massive ipsilateral thalamectomy followed by sectioning of
the callosum (Steriade et al., 1993b). We propose that the sharp
potentials in the Kv3.2 / mouse reflect a poor local control of the
strong synchronized corticocortical inputs that reach the cortex during
the slow oscillations.
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Figure 11.
Distortions in the cortical EEG in the Kv3.2
/ mouse. Bipolar EEG from the S1 cortex and the ventrobasal nucleus
of the thalamus (VB) in a wild-type (A)
and a knock-out (B) mouse. Both EEGs show a
predominant slow rhythm (<1 Hz) typical of ketamine-xylazine
anesthesia. Whereas the EEG of the normal mouse shows a regular pattern
in synchrony with the thalamus, the EEG of the knock-out mouse is
pervaded by high-amplitude, short-lasting waves in all phases of the
slow oscillation. Such sharp waves occur in synchrony with burst firing
in the thalamic recording. Because we observed no significant
electrophysiological differences in the thalamus of wild-type and Kv3.2
/ mice (data not shown), we ascribed the generation of burst firing
in thalamus to an increased corticothalamic drive probably caused by
increased firing of cortical cells during the sharp potentials.
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In the course of the previous experiments, we found that seizures were
prevalent in Kv3.2 / mice under ketamine-xylazine anesthesia.
Under these conditions, most (12 of 18) Kv3.2 / mice, but not their
wild-type littermates (0 of 21), showed electrographic seizures that
occurred spontaneously (typically every 10-20 min) (Fig.
12A). Similar
seizures were easily induced in Kv3.2 /mice (12 of 12 mice tested)
by electrical stimulation of the thalamus (Fig. 12B).
In contrast, we were never able to evoke seizures in wild-type animals
(n = 19), even with stimuli that were 10 times stronger
or longer in duration (and in a variety of patterns) than those
required to evoke seizures in knock-out mice. Both spontaneous and
evoked seizures were often associated with motor manifestations that
varied from small jerks of the limbs and movements of the whiskers to
full convulsions. In principle, these seizures could result from
increased thalamic input into the cortex or from reduced cortical
control in Kv3.2-deficient mice. This is an important consideration,
given the prominent expression of Kv3.2 in thalamic relay neurons and
thalamocortical projections (Fig. 2) (Rudy et al., 1992; Weiser et al.,
1994; Moreno et al., 1995). However, the failure to induce seizures
when large doses of bicuculline are injected into the thalamus
(Steriade and Contreras, 1998) shows that the feedforward and feedback
inhibitory mechanisms of the cortex are capable of handling the
entrance into the cortex of thalamic stimuli of increased magnitude.
Therefore, the presence of thalamic-induced seizures in the Kv3.2 /
mouse is likely to result from increased cortical excitability.
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Figure 12.
Seizures in the ketamine-xylazine anesthetized
Kv3.2 / mouse. A and B represent data
from two different animals. EEG was recorded from the depth of the
primary somatosensory cortex (S1). The seizure in
A occurred spontaneously. The seizure in
B was triggered by high-frequency stimulation (7 short
trains of 100 Hz repeated at ~10 Hz) delivered to the ventrobasal
nucleus of the thalamus (VB stim). Both spontaneous and
evoked seizures were initiated abruptly by a paroxysmal depolarizing
shift (PDS) and consisted of runs of 5-7 Hz
low-amplitude waves followed by higher amplitude waves usually at <1
Hz. Seizures also terminated abruptly and were followed by a variable
period (10-30 sec) of postictal depression with flat EEG. The
background activity preceding seizures was like that seen in Figure
11B. Note the change in gain during the record
shown in B. The gain in A had been
changed earlier and was 10 times smaller before the seizure.
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The last set of experiments consisted of testing seizure susceptibility
to chemical convulsants in vivo. Kv3.2 / mice were more
sensitive to the convulsant PTZ, a GABAA
receptor antagonist. When challenged with a single dose of PTZ (50 mg/kg), Kv3.2 / mice showed more severe responses than wild-type
littermates (Fig. 13A). For
example, at this dose, 52% of Kv3.2 / mice (n = 23) compared with 16% of wild types (n = 19)
developed severe stage 4 tonic-clonic convulsions associated with
death. Similarly, the delay between drug application and the first
evidence of epileptic activity was significantly shorter in the
knock-out mice (Fig. 13B).
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Figure 13.
Susceptibility of Kv3.2-deficient mice to
PTZ-induced seizures. A, The maximum response of
wild-type (WT; n = 19) and knock-out
(KO; n = 23) mice after a single
injection of 50 mg/kg PTZ intraperitoneally was scored as follows:
1, only isolated twitches; 2, only
partial or focal seizures (stage 3, see Materials and Methods);
3, generalized clonic seizures; and 4,
large tonic-clonic epileptic seizures. Knock-out mice tend to progress
to more severe stages than wild-type littermates. p < 0.01 (Mann-Whitney rank sum test). B, Latency to
first seizure. Plotted is the time from the PTZ injection to the first
sign of seizure. The latency was shorter for the knock-out mice.
p < 0.02, Student's t
test.
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Together, the data provide strong evidence that mice deficient in Kv3.2
subunits have susceptibility to seizures probably reflecting an
increase in cortical excitability. Because Kv3.2 proteins are expressed
in inhibitory but not in excitatory neurons in the cortex (Chow et al.,
1999), and assuming that compensatory changes in other cortical
elements have not taken place, the inferred increase in cortical
excitability most likely arises from impaired cortical inhibition.
Deficits in cortical inhibition may occur in the Kv3.2 / mouse if
the fast-spiking ability of GABAergic interneurons is necessary to
achieve proper levels of inhibition (see Discussion). This increased
cortical excitability is likely to contribute to the spontaneous
seizures observed in awake animals.
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DISCUSSION |
Kv3 channels and high frequency firing
Three (Kv3.1-Kv3.3) of the four Kv3 genes are prominently
expressed in brain tissue and show different but overlapping patterns of expression (Perney et al., 1992; Rudy et al., 1992; Vega-Saenz de
Miera et al., 1994; Weiser et al., 1994). Many, if not most, of the
neuronal populations expressing these genes fire spikes at high
frequency. This correlation led to the suggestion that Kv3 channels may
play an important role in high-frequency firing (see introductory
remarks). Experiments using the K+ channel
blockers TEA and 4-AP, at concentrations that block Kv3 channels in
heterologous expression systems (Grissmer et al., 1994; Coetzee et al.,
1999; Rudy et al., 1999), and Kv3-like currents in native neurons (Du
et al., 1996; Wang et al., 1998; Erisir et al., 1999; Hernandez-Pineda
et al., 1999) support the idea that Kv3 channels play a dominant role
in repolarizing the action potential of expressing neurons and are
necessary to maintain high-frequency firing (Du et al., 1996;
Massengill et al., 1997; Martina et al., 1998; Wang et al., 1998;
Erisir et al., 1999). However, the possibility that these drugs are
also blocking other types of K+ channels
has been difficult to eliminate.
In this study, we used the genetic elimination of the Kv3.2
gene, which is prominently expressed in deep-layer FS neurons in the
neocortex, to test directly the role of Kv3 genes in fast spiking. The
results show that fast spiking is impaired in neocortical deep-layer FS
neurons but not in superficial layer FS neurons in which Kv3.2 is
weakly expressed. These results provide strong, independent evidence
that Kv3 channels play a critical role in enabling high-frequency
firing in FS neurons. Together with data showing that the PKA-dependent
modulation of the firing frequency of FS interneurons in the
hippocampus is absent in Kv3.2 / mice (Atzori et al., 2000), the
experiments described here provide the most direct evidence for a
critical role of Kv3 channels in sustained or repetitive high-frequency firing.
The ability of Kv3 channels to facilitate sustained or repetitive
high-frequency firing is a direct consequence of the special properties
of these channels that distinguish them from other voltage-gated
K+ channels. (1) By activating at very
depolarized potentials, the cell can use large numbers of Kv3 channels
to produce fast-spike repolarization and a large AHP with minimum
effects on input resistance, threshold, or rise time, thus keeping
action potentials brief without compromising action potential
generation. (2) By keeping action potentials brief, Kv3 channels
minimize the inactivation of the Na+
conductance during the spike. (3) By generating a large AHP, Kv3
channels accelerate recovery from the Na+
channel inactivation that did take place during the spike. (4) The
brief duration of the AHP (produced by the fast deactivation of Kv3
channels) restores high-input resistance quickly. The increase in the
number of Na+ channels that have recovered
from inactivation and the fast termination of the AHP minimize the
duration of the refractory period, allowing the cell to reach firing
threshold sooner than in the absence of Kv3 channels (Sekirnjak et al.,
1997; Erisir et al., 1999).
The effects of blocking Kv3 channels become stronger during long trains
or repetitive activity because there is accumulation of
Na+ channel inactivation resulting from
the repeated activation of the channels. The larger increase in spike
rise time during repetitive activity in Kv3.2-deficient mice (Figs.
5A1,A2, 10)
supports this view. The results with Kv3.2-deficient mice also suggest
that the increased activation of slower K+
conductances active during the AHP, produced by the increase in action
potential duration, may also contribute to reducing firing frequency
and increasing firing frequency adaptation after Kv3 channel removal.
Evidence that Kv3 channels are necessary for sustained or repetitive
high-frequency firing has now been obtained for FS neurons in the
neocortex and hippocampus (Martina et al., 1998; Erisir et al., 1999;
Atzori et al., 2000; this study), for neurons in the medial vestibular
nucleus (MVN) (S. du Lac and C. Sekirnjak, unpublished
observations) and the medial nucleus of the trapezoid body (MNTB) in
the auditory brainstem (Wang et al., 1998). Kv3 gene products are found
in many other neurons capable of high-frequency firing (Weiser et al.,
1994), suggesting a similar role in these neurons. Although it appears
that Kv3 channels are necessary for sustained or repetitive
high-frequency firing in many (if not all) high-frequency firing
neurons, their presence is clearly not sufficient. The effects on
excitability of the Kv3 channels will depend on the other conductances
active on the cell and their precise localization in the neuron. In
fact, the three cell types (FS interneurons in the neocortex and
hippocampus, neurons in the MNTB, and neurons in the MVN) for which a
role of Kv3 channels in high-frequency firing has been suggested have
different firing properties. Both FS interneurons and MVN neurons (du
Lac and Lisberger, 1995; du Lac, 1996) fire long, high-frequency spike
trains in response to steady depolarizations. However, FS interneurons
produce long, high-frequency spike trains abruptly, and the
steady-state firing frequency saturates as a function of injected
current (as in the examples shown here). In contrast, MVN neurons are
spontaneously active, and the relationship between injected current and
firing frequency is nearly linear (du Lac and Lisberger, 1995; du Lac, 1996). The presence of low voltage-activating or persistent
Na+ channels and differences in other
conductances probably account for these differences in firing properties.
On the other hand, the MNTB neurons produce one or, at most, two action
potentials independently of the duration or strength of the
depolarization, probably because depolarization activates large, low
voltage-activating "D"-type K+
currents that limit repetitive activity (Brew and Forsythe, 1995). However, these neurons can fire action potentials entrained to very
high-frequency inputs (>600 Hz), the property which appears to depend
on Kv3 (probably Kv3.1 and Kv3.3) channels (Wang et al., 1998).
Kv3 channels are an excellent example of how the specialized properties
of certain channels contribute to functional specificity. This helps to
explain the biological significance of K+
channel diversity as a key factor contributing to the diversity of the
electrophysiological properties of neurons and to the specificity of
neuromodulator actions (Adams and Galvan, 1986; Llinas, 1988; Rudy,
1988; Baxter and Byrne, 1991; Hille, 1992).
Increased cortical excitability in the Kv3.2
/ mouse
Several observations indicate that Kv3.2-deficient mice have
increased cortical excitability. Increased excitability, resulting in
increased susceptibility to seizures, if not a full epileptic behavior,
has also been observed in two other K+
channel knock-out mice lines: Kv1.1 (Smart et al., 1998) and one of the
G-protein-gated inward rectifier K+
channel genes (GIRK2) (Signorini et al., 1997).
Moreover, mutations in the K+ channel
genes KCNQ2 and KCNQ3, subunits of M-type
K+ channels, have been found in humans
with benign familial neonatal convulsions (Charlier et al., 1998; Singh
et al., 1998). A strong association between increased excitability and
K+ channel dysfunction may not seem
surprising. K+ channels typically suppress
and limit cell excitability by competing with depolarizing currents,
and thus, reduced K+ channel expression is
expected to generate hyperexcitability. Indeed, 4-AP and other
K+ channel blockers are often used as
convulsants in experimental animals (Rutecki et al., 1987; Velluti et
al., 1987).
However, it is very likely that the underlying mechanism responsible
for the increased cortical excitability of Kv3.2-deficient mice is
different from that producing the hyperexcitability phenotypes in the
other examples. K+ channels containing
Kv1.1, GIRK2, and KCNQ2 and KCNQ3 subunits operate close to the resting
potential and act as breaks dampening the effects of depolarizing
inputs. Moreover, these channels are prominently expressed in
excitatory neurons in neocortex, hippocampus, and many other brain
areas. Hyperexcitability of these cells, resulting from suppressing
these channels, is likely to underlie the hyperexcitability behavior
(Signorini et al., 1997; Smart et al., 1998). However, in the neocortex
and hippocampus, Kv3.2 is only expressed by inhibitory neurons (Chow et
al., 1999; Atzori et al., 2000). The defects in the function of these
cells are likely to be key contributors to the observed
increases in cortical excitability, although this remains to be
conclusively demonstrated.
Hyperexcitability of these interneurons should produce increased
cortical inhibition and therefore suppression of hyperexcitable phenotypes, contrary to what we see in the Kv3.2 / mouse. The solution to this apparent paradox lies in the unique role of Kv3 channels in neuronal excitability as illustrated by the results obtained with the Kv3.2-deficient mouse, which suggest that suppression of this K+ channel results in impaired
cellular firing instead of hyperexcitability. Kv3 channels are not
active anywhere near threshold potentials, and therefore their removal
is not expected to affect much the responsiveness to synaptic inputs.
As shown here and in previous pharmacological studies (Du et al., 1996;
Massengill et al., 1997; Erisir et al., 1999), suppression of Kv3
channels produces broadening of the action potential. This could
increase GABA release from these cells, which should result in
increased cortical inhibition. On the other hand, cortical inhibition
might be reduced if the ability to sustain repetitive firing at high
frequencies is even more important in determining inhibitory levels.
Our data suggest that this is the case and that impaired fast spiking
decreases the performance of the inhibitory circuits in the cortex.
This view is consistent with observations that FS neurons fire long, high-frequency trains or repetitive high-frequency bursts of action potentials in response to physiological stimuli (Kawaguchi and Kubota,
1993; Benardo, 1994; Zhu and Connors, 1999). Future studies on the
performance of inhibitory synaptic transmission in knock-out and
wild-type animals should provide further tests of this view.
The fact that a single type of K+ channel
plays a dominant role in enabling high-frequency firing has allowed us
to generate mice lines in which this property is impaired in selective
neuronal populations. Preliminary experiments show cellular changes
similar to those described here on FS neurons in superficial cortical layers in Kv3.1-deficient mice (Ho et al., 1997), and larger effects resembling those produced by complete Kv3 channel block (and throughout all layers of the cortex) are expected on mice deficient in both genes.
GABAergic interneurons are thought to play important roles in many
cortical functions. These mice lines will be interesting models to test
hypotheses on the role of GABAergic interneurons in these functions.
For example, preliminary observations in the hippocampus revealed a
decrease in high-frequency oscillations in Kv3.2 / mice, suggesting
impaired synchronization (Atzori et al., 2000), one of the key roles
attributed to cortical GABAergic interneurons.
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FOOTNOTES |
Received Aug. 18, 2000; revised Sept. 7, 2000; accepted Sept. 18, 2000.
This work was supported by grants from the National Institutes of
Health, American Lebanese Syrian Associated Charities, and the National
Science Foundation. D.L. was supported by the Medical Scientists
Training Program grant at New York University School of Medicine.
Correspondence should be addressed to Dr. Bernardo Rudy, Department of
Physiology and Neuroscience, New York University School of Medicine,
550 First Avenue, New York, NY 10016. E-mail:
Rudyb01{at}med.nyu.edu.
| |
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