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The Journal of Neuroscience, August 15, 2001, 21(16):5973-5983
Experimental Localization of Kv1 Family Voltage-Gated
K+ Channel  and  Subunits in Rat Hippocampal
Formation
Michael M.
Monaghan1,
James S.
Trimmer2, and
Kenneth J.
Rhodes1
1 Neuroscience, Wyeth-Ayerst Research, Princeton, New
Jersey 08543, and 2 Department of Biochemistry and Cell
Biology and Institute for Cell and Developmental Biology, State
University of New York, Stony Brook, New York 11794
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ABSTRACT |
In the mammalian hippocampal formation,
dendrotoxin-sensitive voltage-gated K+ (Kv) channels
modulate action potential propagation and neurotransmitter release.
To explore the neuroanatomical basis for this modulation, we used in situ hybridization, coimmunoprecipitation,
and immunohistochemistry to determine the subcellular localization of
the Kv channel subunits Kv1.1, Kv1.2, Kv1.4, and Kv 2 within the
adult rat hippocampus. Although mRNAs encoding all four of these Kv
channel subunits are expressed in the cells of origin of each major
hippocampal afferent and intrinsic pathway, immunohistochemical
staining suggests that the encoded subunits are associated with the
axons and terminal fields of these cells. Using an excitotoxin lesion
strategy, we explored the subcellular localization of these subunits in
detail. We found that ibotenic acid lesions of the entorhinal cortex
eliminated Kv1.1 and Kv1.4 immunoreactivity and dramatically reduced
Kv1.2 and Kv2 immunoreactivity in the middle third of the dentate
molecular layer, indicating that these subunits are located on axons
and terminals of entorhinal afferents. Similarly, ibotenic acid lesions of the dentate gyrus eliminated Kv1.1 and Kv1.4 immunoreactivity in the
stratum lucidum of CA3, indicating that these subunits are located on
mossy fiber axons. Kainic acid lesions of CA3 dramatically reduced
Kv1.1 immunoreactivity in the stratum radiatum of CA1-CA3, indicating
that Kv1.1 immunoreactivity in these subfields is associated with the
axons and terminals of the Schaffer collaterals. Together with the
results of coimmunoprecipitation analyses, these data suggest that
action potential propagation and glutamate release at excitatory
hippocampal synapses are directly modulated by Kv1 channel complexes
predominantly localized on axons and nerve terminals.
Key words:
long-term potentiation; synaptic plasticity; A-current; dendrotoxin; ibotenic acid; perforant path; mossy fiber; Schaffer
collateral; Shaker
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INTRODUCTION |
Voltage-gated
K+ (Kv) channels play a major role in
regulating the excitability of mammalian hippocampal neurons. In these neurons, Kv channels control postsynaptic responses to excitatory input
(for review, see Johnston et al., 2000 ), modulate the amplitude of
back-propagating action potentials (Hoffman et al., 1997), control
neuronal spike properties and firing frequency (Zhang and McBain, 1995;
Golding et al., 1999), and modulate neurotransmitter release (Hu et
al., 1991; Dorandeau et al., 1997; Schechter, 1997; Southan and Owen,
1997). In mammals, Kv channels have been divided into four subfamilies,
Kv1-Kv4 (Chandy and Gutman, 1995), which differ in their primary
structure, biophysical properties, and subcellular localization. The
mammalian Shaker, or Kv1, subfamily consists of seven
members, of which at least five (Kv1.1-Kv1.4 and Kv1.6) are expressed
in the hippocampus proper (Sheng et al., 1994; Wang et al., 1994; Veh
et al., 1995; Rhodes et al., 1997). Studies using dendrotoxin
(DTX) isoforms as selective blockers of Kv1  subunits have suggested
that Kv1 channel complexes are located predominantly on the terminals
of intrinsic hippocampal circuits and subcortical afferent inputs,
where they directly modulate neurotransmitter release (Dorandeau et
al., 1997; Schechter, 1997; Southan and Owen, 1997; Geiger and Jonas,
2000). Interestingly, studies examining the effects of DTX isoforms
with distinct subunit specificity indicate that the responses of
hippocampal neurons to Kv1 channel block vary across subfields (Southan
and Owen, 1997), suggesting that the subunit composition of Kv1 channel complexes that underlie these responses also vary.
Kv1 channels are complex membrane protein oligomers consisting of four
integral membrane pore-forming subunits and four cytoplasmic subunits. It is now well established that Kv1 and subunits
differentially coassemble, giving rise to
44 channel complexes
with considerable electrophysiological and biochemical heterogeneity
(Sheng et al., 1993; Wang et al., 1993; Scott et al., 1994; Rhodes et
al., 1995, 1996, 1997). Although immunohistochemical staining for
individual Kv1 subunits in the hippocampus suggests that they are
located predominantly on axons and in terminal fields (Sheng et al.,
1993, 1994; Wang et al., 1994; Rhodes et al., 1995, 1996, 1997), there
are also reports of somatodendritic localization (Sheng et al., 1994;
Veh et al., 1995; Rhodes et al., 1996, 1997; Cooper et al.,
1998). In the few cases in which ultrastructural studies have been
performed (Wang et al., 1994; Cooper et al., 1998), there is clear
evidence for localization of Kv1 subunits along axons and at or
near axon terminals but no evidence for localization of these channels
to somatodendritic domains.
Although the exquisite laminar segregation of the major hippocampal
circuits tempts conclusions regarding protein localization in this
structure, it can be difficult to associate staining patterns with
specific pathways unless there are clear morphological criteria to
establish a the identity of a pathway (e.g., for the mossy fiber
pathway; Acsády et al., 1998). Even multiple-label anatomical techniques cannot reveal the coassociation of individual channel subunits in heteromeric channel complexes. To circumvent these issues,
we used a strategy that used circumscribed lesions,
immunohistochemistry, and coimmunoprecipitation to determine the
association of Kv1.1, Kv1.2, Kv1.4, and Kv2 with each of the major
afferent, intrinsic, and efferent pathways of the rat hippocampus. Our
data suggest that the majority of Kv1 channels present in this
structure are heteromeric and associated with the axons and terminals
of cortical afferent and intrinsic excitatory projections. Moreover, we
find that the subunit composition of Kv1 channel complexes varies
across subfields. Our results resolve some of the controversy
surrounding Kv1 subunit localization in hippocampal formation and
underscore the importance of Kv1 channels in regulating axonal
excitability and neurotransmitter release in circuits that play a key
role in memory processes and synaptic plasticity.
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MATERIALS AND METHODS |
Materials. All reagents were molecular biology grade
from Sigma (St. Louis, MO) or Roche Molecular Biochemicals
(Indianapolis, IN), except where noted otherwise.
In situ hybridization histochemistry. DNA templates for
riboprobe synthesis were prepared by PCR from the full-length cDNA clones of the corresponding subunits (Nakahira et al., 1996). All
riboprobe sequences were compared by BLAST to the GenBank database to verify that they will only recognize the appropriate targets among all deposited sequences. The riboprobe used to localize Kv2 mRNA was described previously (Rhodes et al., 1996). A riboprobe for the Kv1.1 subunit was generated using a pair of oligonucleotide primers designed to amplify a 335 bp region spanning nucleotides 1735-2070 of the rat Kv1.1 cDNA and, in addition, add the promoter sequences for T7 (forward) and T3 (reverse) polymerase. These primers
contained the following sequences:
5'-TAATACGACTCACTATAGGGAAAAAAGCACCAGGCAAGCA-3' (forward) and
5'-ATTAACCCTCACTAAAGGGAACACAGACTA CTTCATGGGC-3' (reverse). A riboprobe
for the Kv1.2 subunit was generated using a pair of primers designed to
amplify a 443 bp region spanning nucleotides 1111-1544 of the rat
Kv1.2 cDNA and, in addition, add the promoter sequence for T3
polymerase. These primers had the following sequences:
5'-GCTGATGAGCGAGATTCCCAGTTCC-3' (forward) and
5'-AATTAACCCTCACTAAAGGGATCAGTTAACATTTT-GGTAA-3' (reverse). A riboprobe
for the Kv1.4 subunit was generated using a pair of primers designed to
amplify a 496 bp region spanning nucleotides 70-566 of the rat Kv1.4
cDNA and, in addition, add the promoter sequence for T3 polymerase.
These primers had the following sequences: 5'-AAACTACCACCATGGAGGTGGCAAT-3' (forward) and
5'-AATTAACCCTCACTAAAGGGGCTCTGCCTGTGGTGGAGTT-3' (reverse). All PCR
products were gel-purified on 1.5% low-melt agarose gels. Bands
containing the PCR products were excised, phenol- and then
phenol-chloroform-extracted, and ethanol-precipitated. The pellet was
then dried and resuspended in 1× TE buffer (10 mM Tris-HCl
and 1 mM EDTA, pH 7.4). Fifty nanograms of DNA
template was used for in vitro transcription reactions using
[35S]CTP (New England Nuclear, Boston,
MA) and the Riboprobe Gemini System (Promega, Madison, WI). Each of
these riboprobes was used for in situ hybridization
histochemistry as described previously (Rhodes et al., 1996). After the
hybridization reaction, sections were apposed to Hyperfilm (Amersham
Pharmacia Biotech, Arlington Heights, IL) for 3-10 d and subsequently
dipped in nuclear track emulsion (NTB-2; Eastman Kodak, Rochester, NY)
to obtain higher-resolution autoradiograms.
To assess nonspecific labeling in the in situ hybridization
procedure, a control probe was generated from a template provided in
the Riboprobe Gemini System kit (Promega catalog #P2651). This vector
was linearized using ScaI and transcribed using T3
polymerase. The resulting transcription reaction generated two
riboprobes, one a 250 bp product and the other a 1525 bp product,
comprising only vector sequence. This control probe mixture was labeled
as described above and added to the hybridization solution at a final concentration of 50,000 cpm/µl. No specific hybridization was observed in control sections; i.e., these sections gave a weak, uniform
hybridization signal (results not shown).
Immunoprecipitation. Immunoprecipitation reactions (Rhodes
et al., 1995, 1996, 1997) were performed at 4°C using detergent lysates of a crude membrane fraction (Trimmer, 1991) isolated from
freshly dissected adult rat hippocampi. In brief, hippocampal membranes
(1 mg of membrane protein per tube) were solubilized in lysis buffer
(1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 10 mM sodium azide, 10 mM and Tris-HCl, pH 8.0)
containing a protease inhibitor mixture. Affinity-purified antibodies
specific for each subunit (Rhodes et al., 1995, 1996) were added, and
the volume was adjusted with lysis buffer to 1 ml/reaction tube.
Samples were incubated for 2 hr on a rotator, followed by addition of 50 µl of a 50% slurry of protein A-agarose and further incubation for 45 min. After incubation, protein A-Sepharose was centrifuged at
10,000 × g for 20 sec, and the resulting pellets were
washed by resuspension and centrifugation six times with lysis buffer. The final pellets were resuspended in 240 µl of reducing sample buffer.
SDS-polyacrylamide gels and immunoblotting. Samples were
size-fractionated on 9% (for analysis of subunits) or 12% (for analysis of subunits) SDS-PAGE. Crude synaptosomal membrane fractions from whole brain [rat brain membrane("RBM"); 30 µg] or from rat hippocampus [rat hippocampal membranes ("RHCM"); 20 µg] were added to SDS sample buffer, boiled, and loaded
directly. For immunoprecipitation reactions, 20 µl of sample,
representing the reaction product from 83 µg of hippocampal
membranes, was loaded. Disulfide bonds were reduced by the addition of
20 mM 2-mercaptoethanol to the sample buffer. Lauryl
sulfate (Sigma) was the SDS source used for all SDS-PAGE (Shi et al.,
1994). After electrophoretic transfer to nitrocellulose paper, the
resulting blots were blocked in Tris-buffered saline (TBS) containing
4% low-fat milk (Blotto; Johnson et al., 1984), incubated in
affinity-purified antibody diluted 1:50-1:2000 in Blotto for 1 hr or
undiluted mAb tissue culture supernatant (K17/70 anti-Kv2), and
washed three times in Blotto for 30 min total. Blots were then
incubated in HRP-conjugated secondary antibody (Cappel, West Chester,
PA; 1:2000 dilution in Blotto) for 1 hr and then washed in TBS three
times for 30 min total. The blots were then incubated in substrate for enhanced chemiluminescence (ECL) for 1 min and autoradiographed on
preflashed (to OD545 = 0.15) Fuji (Tokyo, Japan)
RX or Kodak XAR-5 film.
Experimental localization of channel subunits. All surgical
procedures were approved by the Wyeth-Ayerst Institutional Animal Care
and Use Committee and were in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals.
Before surgery, animals were deeply anesthetized with sodium
pentobarbital (50 mg/kg, i.p.) and secured in a stereotaxic carrier
(David Kopf Instruments, Tajunga, CA). For lesions of the dentate
gyrus, hippocampal subfields, or the entorhinal cortex, ibotenic acid
(0.1-0.4 µl of a 10 µg/µl solution in 0.1 M sodium phosphate buffer, pH 7.4) was injected
directly into the target structure using a 2 µl Hamilton (Reno, NV)
microsyringe mounted in a Kopf microsyringe microdrive. In some of
these animals, injections were made at two or three depths, with each
injection separated in the dorsoventral axis by 2.5 mm. To generate
lesions encompassing the entire CA3 subfield, kainic acid (1 µl of a
1 µg/µl solution in 0.1 M
NaPO4 buffer) was injected into the lateral
cerebral ventricle immediately adjacent to CA3 (procedure modified from that of Bernard and Wheal, 1995). To examine the association of individual subunits with subcortical afferents, transections of the
fornix were made unilaterally using a specially constructed Scouten
wire knife (Kopf). To produce the lesion, the guide cannula of the
knife was lowered under stereotaxic control to a point just beneath the
fornix. The wire knife was then extended 2 mm medially in the coronal
stereotaxic plane, raised 4.5 mm, and then retracted. The knife was
then again lowered, and the wire was extended 2 mm laterally in the
coronal stereotaxic plane and then raised 4.5 mm to complete the
transection. To verify that the fornix transections were complete, one
series of sections from each case was processed using
acetylcholinesterase (AChE) histochemistry, as described previously
(Tago et al., 1986).
Immunohistochemistry. After a 7 d postsurgical
survival, all animals were deeply anesthetized with sodium
pentobarbital (60 mg/kg, i.p.) and then perfused through the ascending
aorta with 0.1 M NaPO4 buffer, pH
7.4, followed by fixative containing freshly depolymerized 4%
paraformaldehyde in 0.1 M NaPO4
buffer. The remaining procedures for light microscopic
immunohistochemistry using these same subunit-specific
affinity-purified rabbit polyclonal antibodies have been described in
detail previously (Rhodes et al., 1995, 1996). Briefly,
40-µm-thick horizontal sections were incubated overnight at 4°C in
antibody vehicle containing affinity-purified rabbit polyclonal
antibodies. Detection of antibody-antigen complexes was accomplished
using the ABC Elite peroxidase reaction (Vector Laboratories,
Burlingame, CA) and visualized using a nickel-enhanced diaminobenzidine
procedure (Tago et al., 1986; Rhodes et al., 1995).
Stained sections and autoradiograms generated by in situ
hybridization were analyzed and photographed using a Zeiss (Thornwood, NY) Axiophot photomicroscope. Low-magnification photographs of these
specimens were taken using a Nikon (Melville, NY) Multiphot macrophotography system. Black-and-white 35 mm film negatives were
digitally imaged using a Nikon LS1000 35 mm film scanner. The scanned
images were arranged and labeled in Adobe (Mountain View, CA) Photoshop
with only minor adjustments of image brightness and contrast.
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RESULTS |
Localization of Kv and subunit mRNA in the
rat hippocampus
Analysis of Kv1.1, Kv1.2, Kv1.4, and Kv2 expression by in
situ hybridization revealed that mRNAs encoding these subunits are
expressed in neurons that give rise to the major cortical afferent
input and the major intrinsic projections of the rat hippocampal
formation. For example, mRNAs encoding Kv1.1, Kv1.2, Kv1.4, and Kv2
are highly expressed in the large multipolar neurons in layer II of the
entorhinal cortex that give rise to the perforant pathway, and moderate
to high levels of these four subunit mRNAs are expressed in the
granule and infragranular neurons in the dentate gyrus (Fig.
1). Similarly, there are moderate to high levels of expression of all four subunit mRNAs in the stratum pyramidale of the CA subfields, prosubiculum, and subiculum. The expression level of Kv1.1 mRNA in the CA subfields is not uniform: there is a far greater density of Kv1.1 expression in the stratum pyramidale of CA3 compared with CA1 or the subiculum (Séquier et
al., 1990; Wang et al., 1994). Analysis of emulsion autoradiograms (results not shown) indicated that in addition to their expression in
CA1-CA3 pyramidal cells, Kv1.1, Kv1.2, Kv1.4, and Kv2 mRNAs are
also expressed in some small to medium-sized interneurons in the
stratum oriens and stratum radiatum of the CA subfields.
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Figure 1.
Expression of Kv1 and subunit mRNAs in the
rat temporal lobe. These photographs of film autoradiograms show the
pattern of expression of Kv1.1, Kv1.2, Kv1.4, and Kv2 mRNA in the
rat entorhinal cortex (area 28) and hippocampal formation. Areas with
high levels of mRNA expression appear as darker regions
in these bright-field images. There is a high level of mRNA expression
for these four subunits in layer II of the entorhinal cortex
(arrowheads) and in the principal cells of all
hippocampal subfields (CA1-CA4). In contrast to the fairly uniform
expression of Kv1.2, Kv1.4, and Kv2 mRNA across hippocampal
subfields, there is a much greater density of Kv1.1 mRNA expression in
the stratum pyramidale of CA3 in comparison with the other subfields.
28, Entorhinal cortex; DG, dentate gyrus;
Sub, subiculum.
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Immunoreactivity for Kv1.1, Kv1.2, Kv1.4, and Kv2 in the
rat hippocampus
The general pattern of immunoreactivity for the Kv1.1, Kv1.2,
Kv1.4, and Kv2 polypeptides in the rat hippocampus has been described previously (Sheng et al., 1993; Wang et al., 1993, 1994; Rhodes et al., 1995, 1996, 1997; Veh et al., 1995; Cooper et al., 1998). To briefly summarize, immunoreactivity for each of these subunits appears to be concentrated in the axons and at or near the
axon terminals of cells expressing the corresponding mRNAs, with a much
lower density of staining associated with the somatodendritic membranes
of hippocampal neurons (Fig.
2B-E) (Sheng et al.,
1993; Wang et al., 1994; Rhodes et al., 1997; Cooper et al., 1998). The
exception to this pattern is Kv2. In addition to staining of axons
and terminal fields, Kv2 appears to be concentrated in the somata
and throughout the apical dendrites of dentate granule cells and
hippocampal pyramidal cells (Rhodes et al., 1997).
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Figure 2.
Effects of an ibotenic acid lesion in the
entorhinal cortex on Kv1 and subunit immunoreactivity in the
hippocampus. These photomicrographs show the pattern of
immunoreactivity for Kv1.1, Kv1.4, Kv1.2, and Kv2 in the hippocampus
of the unoperated control hemisphere (A, C, E, G) and in
the operated hemisphere (B, D, F, H) of an animal
that sustained a large unilateral ibotenic acid lesion in the
entorhinal cortex. In the control hemisphere, there is a distinct band
of immunoreactivity for all four subunits in the middle third of the
molecular layer of the dentate gyrus (arrowheads). The
location of this band corresponds to the termination zone of
projections from the medial entorhinal cortex to the dentate gyrus.
After the ibotenic acid lesion, the band of immunoreactivity for Kv1.1
and Kv1.4 in the ipsilateral dentate gyrus is eliminated (B, D,
arrowheads), and the band of immunoreactivity for Kv1.2 and
Kv2 is greatly diminished in intensity (F, H,
arrowheads). DG, Dentate gyrus;
Sub, subiculum.
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Although we can reasonably infer the subcellular distribution and
pathway association of individual subunits on the basis of a comparison
of mRNA expression and immunohistochemical staining patterns, it is not
possible to associate staining for individual subunits with specific
pathways using this type of data alone. For example, in the dentate
gyrus, there is a dense band of immunoreactivity for Kv1.1, Kv1.2,
Kv1.4, and Kv2 in the middle third of the molecular layer (Fig. 2).
The location of this band corresponds closely to the pattern of
termination of afferents from the medial entorhinal cortex (Steward,
1976; Steward and Scoville, 1976; Wyss, 1981; Rhodes et al., 1997;
Dolorfo and Amaral, 1998). Given the high levels of mRNA expression for
these four Kv1 subunits in layer II of the entorhinal cortex, one
reasonable interpretation of the immunohistochemical data is that the
band of immunoreactivity in the dentate molecular layer reflects
localization of these subunits to the axons and terminals of the medial
perforant path. Similarly, in the stratum radiatum and stratum oriens
of CA1-CA3, there is a wide zone of immunoreactivity for Kv1.1. This
zone of immunoreactivity for Kv1.1 is confined to the stratum radiatum and stratum oriens and closely matches the termination zones of the
associational and commissural components of the Schaffer collateral pathway (Swanson et al., 1978; Ishizuka et al., 1990). Given the very
high expression of Kv1.1 mRNA in CA3 pyramidal cells, one reasonable
interpretation of the immunohistochemical data is that Kv1.1 is
localized to the axons and terminals of the Schaffer collateral
projection. However, an equally reasonable interpretation of the
immunohistochemical data, in both the dentate gyrus and CA1 subfields,
is that these subunits are targeted and localized to a defined region
of granule or pyramidal cell dendrites. In the absence of other data,
it is difficult to determine which interpretation is correct. Another
level of complexity inherent in the immunohistochemical data is that
although individual subunits appear to be colocalized, the relative
abundance of individual subunits in each pathway, and the corresponding
subunit composition of the channel complexes, may be distinct. Even
where the staining patterns for individual subunits overlap, we cannot
conclude that the subunits are associated with the same pathway or even
localized to the same subcellular domain. To address these issues and
to more precisely associate the staining patterns with specific
hippocampal circuits, we destroyed the cells of origin of each circuit
with an axon-sparing neurotoxin, ibotenic acid (Kohler and Schwarcz 1983; Erselius and Wree, 1991), and then examined in sequential sections the effects on the staining pattern for all four subunits.
Experimental analysis of Kv subunit expression using ibotenic
acid lesions
Ibotenic acid lesions of the entorhinal cortex
Large unilateral ibotenic acid lesions of the entorhinal cortex
were made in eight animals. In each animal, four separate injections of
ibotenic acid were made into the right entorhinal cortex, with each
injection spaced 2.5 mm apart in the dorsoventral axis. Although these
lesions destroyed almost the entire entorhinal cortex, analysis of
sections stained for Nissl substance revealed that the most ventral and
lateral entorhinal areas and the entire dentate gyrus were spared in
all cases.
An example of the effects of one of these large entorhinal ibotenic
acid lesions is shown in Figure 2. This lesion resulted in virtually
complete destruction of cells in the entorhinal cortex and, in concert
with the degeneration of the axons and terminals of entorhinal
afferents, led to profound gliosis within the middle third of the
molecular layer of the dentate gyrus and in the stratum moleculare of
CA1-CA3 (results not shown). When compared with the nonoperated
control hemisphere (Fig. 2A), it is clear that this
lesion produced a dramatic effect on Kv1.1, Kv1.2, Kv1.4, and Kv2
immunoreactivity in the dentate gyrus (Fig.
2A-H). For example, the dense band of Kv1.1
and Kv1.4 immunoreactivity in the middle third of the dentate molecular
layer was eliminated (Figs. 2A-D,
3A,B), and there appeared to
be a shift in the staining pattern for Kv1.2 and Kv2 such that the
bands of Kv1.2 and Kv2 immunoreactivity became more diffuse and
occupied the outer half of the molecular layer instead of being
concentrated in the middle third (Figs. 2E-H,
3C,D). Interestingly, in the ipsilateral dentate gyrus there
was an increase in the overall intensity of Kv1.1, Kv1.2, and Kv1.4
immunoreactivity (see below). This entorhinal cortex lesion also
reduced the density of Kv1.1, Kv1.2, Kv1.4, and Kv2 immunoreactivity
in the stratum moleculare of CA1-CA3. This loss of immunoreactivity
was caused by the loss of axons and terminals of entorhinal layer II
and III neurons that form the ammonic component of the perforant path
(Steward and Scoville, 1976; Witter et al., 1988).
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Figure 3.
Effects of an ibotenic acid lesion
in the entorhinal cortex on Kv1 and subunit immunoreactivity
in the dentate gyrus. These high-magnification
photomicrographs were taken from the same animal shown in Figure
2 and demonstrate the pattern of immunoreactivity for Kv1.1,
Kv1.4, Kv2, and Kv1.2 in the dentate gyrus of the unoperated control
hemisphere (A-D) and in the hemisphere that
sustained a large ibotenic acid lesion in the entorhinal cortex
(A'-D'). Each pair of photomicrographs is aligned on
the dentate granule cell layer (gc) to facilitate
comparison of the staining patterns in the two hemispheres. After the
entorhinal cortex lesion, the distinct band of immunoreactivity for
Kv1.1 (A') and Kv1.4 (B') in the middle
third (mt) of the molecular layer is virtually
eliminated in the operated hemisphere. In the inner third
(it) and outer third (ot) of the
molecular layer, however, the density of Kv1.1 and Kv1.4
immunoreactivity appears to increase. The effects of the entorhinal
cortex lesion on Kv2 (C) and Kv1.2
(D) immunoreactivity were somewhat different from
for Kv1.1 and Kv1.4. The density of Kv2 and Kv1.2 in the middle
third of the molecular layer is clearly diminished, but there
remains a band of Kv2 and Kv1.2 immunoreactivity in the outer half
of the molecular layer.
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The changes observed after ibotenic acid lesions of the entorhinal
cortex suggest that in the dentate gyrus, all four subunits are located
on the axons and at or near the terminals of entorhinal afferents.
These results are consistent with the electron microscopic observations
of Wang et al. (1994) and Cooper et al. (1998), who demonstrated that
in the dentate gyrus, Kv1.1 Kv1.2, and Kv1.4 immunoreactivity is
contained within axons and axon terminals in the middle third of the
molecular layer. Our results also indicate that, on the side of the
lesion, there is a compensatory increase in the density of Kv1.1,
Kv1.2, Kv1.4, and Kv2 immunoreactivity in the inner and outer thirds
of the dentate molecular layer. However, it is not clear from these
data whether these changes in immunoreactivity are attributable to the
degeneration of presynaptic axons and terminals from the entorhinal
cortex or to postsynaptic changes in granule cell dendrites (i.e., loss
or reorganization of synapses) evoked by loss of entorhinal input, or
both. To distinguish between these possibilities, we made ibotenic acid
lesions in the dentate gyrus and examined the effects of this
complementary lesion on the distribution and density of Kv1 subunit immunoreactivity.
Ibotenic acid lesions of the dentate gyrus
Seven animals sustained a unilateral lesion of the dentate gyrus.
In each animal, a single injection of ibotenic acid was made at
approximately the midpoint of the dorsoventral axis of the hippocampus.
In most animals the lesion destroyed neurons within a small sphere of
tissue that included the dentate gyrus and spread across the
hippocampal fissure into the distal CA1 subfield and subiculum (Fig.
4). For these dentate gyrus lesions, we
restricted our analyses to animals from which analysis of sections stained for Nissl substance revealed that there was little or no spread
of the toxin into the CA3 subfield. This lesion destroyed dentate
granule cells and hilar neurons and, in addition, killed neurons in the
distal CA1 subfield and the subiculum. There was also some shrinkage of
the dentate gyrus itself (Fig. 4, compare A, B), presumably
because of loss of dentate granule cells and their dendritic
volume.
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Figure 4.
Effects of an ibotenic acid lesion in the dentate
gyrus on Kv1.1 and Kv1.4 immunoreactivity in the dentate gyrus and CA3
subfield. Photomicrographs show sections taken from an animal that
sustained a unilateral injection of ibotenic acid into the dentate
gyrus. As shown in the sections stained for Nissl substance (A,
B), this ibotenic acid lesion killed virtually all dentate
granule cells as well as neurons in CA4, the distal end of CA1, and the
subiculum. Neurons in the CA3 subfield were not affected by this
lesion. This lesion did not appear to reduce the density of Kv1.1
(compare C, D) or Kv1.4 (compare E,
F) immunoreactivity in the middle third of the dentate
molecular layer (asterisks). Moreover, this lesion did
not alter the density of Kv1.2 (G, H) or Kv2
(I, J) immunoreactivity in the molecular layer of
the dentate gyrus. These results are consistent with those shown in
Figures 2 and 3 and indicate that the dense band of Kv1.1 and
Kv1.4 immunoreactivity in the dentate molecular layer is associated
with entorhinal afferents and not the dendrites of dentate granule
cells. Interestingly, this dentate gyrus lesion virtually eliminated
the band of Kv1.1 and Kv1.4 immunoreactivity in the stratum lucidum of
CA3 (arrowheads). Because the axons and terminals of
dentate granule cells (the mossy fibers) were destroyed by this
ibotenic acid lesion, this result indicates that Kv1.1 and Kv1.4 are
associated with mossy fiber axons. DG, Dentate gyrus;
Sub, subiculum.
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Despite the loss of dentate granule cells, the distribution of
immunoreactivity for Kv1.1, Kv1.2, Kv1.4, and Kv2 in the dentate molecular layer was not affected by this lesion (Fig.
4C-J). The normally dense band of Kv1.1, Kv1.2,
Kv1.4, and Kv2 immunoreactivity in the middle third of the molecular
layer remained. Moreover, in the middle third of the molecular layer,
there actually appeared to be an increase in the density of reaction
product for all four subunits, suggesting that there may have been a
compensatory increase in Kv1 subunit expression in response to this
lesion. Together with the data described above, these results strongly
suggest that in the middle third of the molecular layer of the dentate gyrus, the changes in immunoreactivity observed after entorhinal ibotenic acid lesions were attributable to loss of entorhinal afferents
and not loss of postsynaptic targets.
Another pronounced effect of ibotenic acid lesions in the dentate gyrus
was that the high density of Kv1.1 and Kv1.4 immunoreactivity in the
stratum lucidum of CA3 was completely eliminated (Fig. 4D,F). Because dentate granule cells were
destroyed by this lesion but CA3 pyramidal cells appeared unaffected,
this result indicates that these two subunits are located on the axons
of the mossy fiber pathway. This finding is consistent with our
previous interpretations of this staining pattern (Rhodes et al., 1995,
1997) and with the results of recent electron microscopic analyses
(Cooper et al., 1998). However, it is conceivable that these changes in
Kv1.1 and Kv1.4 immunoreactivity in the CA3 subfield resulted from
postsynaptic changes in CA3 pyramidal cells resulting from loss of
mossy fiber input. To address this issue, we made ibotenic acid lesions
of the CA3 subfield and examined the effects on the distribution and
density of Kv1 channel subunits.
Ibotenic acid lesions of the CA3 subfield
Nine animals sustained a large unilateral ibotenic acid lesion of
the CA3 subfield. In each animal, a single injection of ibotenic acid
was made at a middorsoventral level of the hippocampus. In most animals
the lesion destroyed a 1-2 mm sphere of tissue that included the CA3
subfield, a small amount of CA2, and the proximal third of CA1. In
other animals, the CA3 lesion was smaller and entirely confined to CA3
(Fig. 5B).
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Figure 5.
Effects of an ibotenic acid lesion in the CA3
subfield on Kv1.1 and Kv1.4 immunoreactivity in the CA3 and CA1
subfields. Photomicrographs show sections taken from an animal that
sustained a unilateral injection of ibotenic acid in CA3. As shown in
the sections stained for Nissl substance (A, B), this
ibotenic acid lesion killed neurons in the CA3 subfield only. This
lesion did not appear to reduce the density of Kv1.1 (compare C,
D) or Kv1.4 (compare E, F)
immunoreactivity in the stratum lucidum of CA3. This result is
consistent with the data in Figure 4, which indicated that in
stratum lucidum of CA3, Kv1.1 and Kv1.4 immunoreactivity is associated
with axons of the mossy fiber pathway and not with the dendrites of CA3
pyramidal cells. As described in Results, this discrete lesion in CA3
did not alter the density of Kv1.1 or Kv1.4 (or Kv1.2 or Kv2;
results not shown) immunoreactivity in other hippocampal subfields.
DG, Dentate gyrus; Sub, subiculum.
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Ibotenic acid lesions within the CA3 subfield had little effect on the
distribution or density of Kv1.1 and Kv1.4 immunoreactivity in the
stratum lucidum (Fig. 5D,F), although there was a
dramatic loss of CA3 pyramidal cells within the boundaries of the
lesion (Fig. 5B). This result confirms that the
immunoreactivity for Kv1.1 and Kv1.4 in the stratum lucidum is
associated with mossy fiber axons, and that the loss of
immunoreactivity for Kv1.1 and Kv1.4 observed after ibotenic acid
lesions of the dentate gyrus was attributable to destruction of the
cells of origin of the mossy fiber pathway (the dentate granule cells)
and not to a loss of postsynaptic targets in CA3.
Surprisingly, ibotenic acid lesions in the CA3 subfield produced an
increase in the density of Kv1.1 immunoreactivity within the boundaries
of the lesion (Fig. 5D). Although the reason for this
increased staining is unclear, it may be attributable to a compensatory
increase in Kv1.1 protein expression in the axons of the Schaffer
collaterals originating from cells outside the boundaries of this lesion.
As described above and in our earlier publications (Rhodes et al.,
1997), one interpretation of the Kv1.1 staining pattern in the stratum
radiatum of CA3 and CA1 is that this subunit is located on the axons
and at or near the axon terminals of the Schaffer collateral pathway.
This interpretation is consistent with the very high level of Kv1.1
mRNA expression in the CA3 subfield and with electron microscopic
analyses demonstrating Kv1.1 immunoreactivity in axons and presynaptic
terminals in the stratum radiatum of CA1 but not in the dendrites of
CA1 pyramidal cells (Wang et al., 1994). Because of this, we were
somewhat surprised to see that ibotenic acid lesions in CA3 did not
produce a detectable decrease in Kv1.1 immunoreactivity in the stratum
radiatum of CA3 or CA1. One explanation for the lack of an effect is
that the axons of CA3 pyramidal cells forming the Schaffer collateral
pathway are highly branched and form extensive connections along the
septotemporal axis of the hippocampus (Tamamaki and Nojyo, 1988;
Ishizuka et al., 1990). As a result, our circumscribed ibotenic acid
lesions may not have destroyed enough CA3 pyramidal cells to
significantly reduce the number of Schaffer collateral axons in CA1
even at levels immediately adjacent to the lesion.
Ibotenic acid lesions in the CA1 subfield
Unilateral ibotenic acid lesions of the CA1 subfield were made in
three animals. In each animal, a single injection of ibotenic acid was
made at a middorsoventral level of the hippocampus. In most animals,
the ibotenic acid lesion destroyed neurons within a 1-3 mm sphere of
tissue and involved virtually all of the CA1 subfield plus some of the
adjacent CA2 subfield, prosubiculum, and subiculum. In some cases,
these lesions also destroyed a portion of the dentate gyrus. However,
analysis of sections stained for Nissl substance revealed that in all
of these cases the CA3 subfield was almost entirely spared.
An example of the effects of a CA1 ibotenic acid lesion is shown in
Figure 6. In this animal, the lesion
destroyed the entire CA2 and the proximal three-fourths of the CA1
subfield but spared all but the most distal tip of CA3 and completely
spared the subiculum. This lesion eliminated immunoreactivity for
Kv2 in the somata and dendrites of CA1 pyramidal cells (Fig.
6H). As described above for the ibotenic acid lesion
in CA3, the density of Kv1.1 immunoreactivity in the stratum radiatum
and stratum oriens increased within the boundaries of the lesion, but
the density of Kv1.2 (results not shown) and Kv1.4 (Fig.
6F) immunoreactivity was unchanged. Similarly, within
the boundaries of the lesion, the density of Kv1.2 and Kv1.4
immunoreactivity in the stratum moleculare of CA1 was unchanged. Together with the results of the other lesions, these data suggest that
in the stratum oriens, stratum radiatum, and stratum moleculare of CA1,
Kv1.1, Kv1.2, and Kv1.4 are located presynaptically on afferent inputs.
As described above and previously (Rhodes et al., 1997), it remains a
possibility that a small proportion of the immunoreactivity for Kv1.1,
Kv1.2, and Kv1.4 is located postsynaptically on the cell bodies and
dendrites of CA1 pyramidal cells and interneurons.
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Figure 6.
Effects of an ibotenic acid lesion in the CA1
subfield on Kv1 and subunit immunoreactivity in CA1.
Photomicrographs show sections taken from an animal that sustained a
unilateral injection of ibotenic acid in CA1. As shown in the sections
stained for Nissl substance (A, B), this ibotenic acid
lesion killed neurons in CA1, CA2, and the distal tip of CA3. This
lesion produced a slight increase in the density of Kv1.1
immunoreactivity within the boundaries of the lesion (D,
asterisk) but did not affect the density of Kv1.4 (E,
F). This result is consistent with our interpretation
that the majority of Kv1.1 (and Kv1.4) immunoreactivity in CA1 is
associated with the axons and terminals of the Schaffer collateral
pathway and not with the apical dendrites of CA pyramidal cells. This
CA1 lesion also dramatically reduced the density of Kv2
immunoreactivity within the boundaries of the lesion (H,
asterisk). This result is consistent with our previous
interpretations (Rhodes et al., 1996, 1997) that Kv2 is concentrated
in the somata and apical dendrites of hippocampal pyramidal cells.
DG, Dentate gyrus; Sub, subiculum.
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Kainic acid lesions of the CA3 subfield
Given the very high levels of Kv1.1 mRNA expression in CA3
pyramidal cells, and because CA3 and CA1 ibotenic acid lesions failed
to alter the density of Kv1.1 immunoreactivity in CA1, we decided to
produce a more complete lesion of CA3. To accomplish this, we took
advantage of the observation that CA3 pyramidal cells are highly
sensitive to the neurotoxin kainic acid (Bernard and Wheal, 1995).
Unilateral CA3 lesions using kainic acid, injected into the lateral
cerebral ventricle immediately adjacent to the CA3 subfield, were made
in six animals. An example of the effects of such a lesion is shown in
Figure 7. This lesion destroyed the entire CA3 subfield, as evidenced by the loss neurons in Nissl-stained sections and by the necrotic tissue in the stratum radiatum of CA3
(Fig. 7B). This lesion also destroyed the large polymorphic neurons in the hilus of the dentate gyrus as well as a subpopulation of
pyramidal cells in the subiculum. Importantly, pyramidal cells in CA1
appeared to be spared by this lesion, and there did not appear to be
any neuronal loss in the contralateral hippocampus.
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Figure 7.
Effects of a large kainic acid lesion in the CA3
subfield on Kv1 and subunit immunoreactivity in CA3 and CA1. As
shown in the sections stained for Nissl substance (A,
B), this kainic acid lesion killed neurons along the entire
mediolateral and dorsoventral extent of CA3 but spared neurons within
the CA1 subfield. The major effect of this lesion was to dramatically
reduce the density of Kv1.1 immunoreactivity within the stratum
radiatum of CA3 and CA1 (compare C, D, asterisks).
Interestingly, this lesion also appeared to reduce slightly the density
of Kv1.4 and Kv1.2 immunoreactivity in the stratum radiatum of CA3 and
CA1. Together, these results suggest that in the stratum radiatum of
CA1, Kv1.1, Kv1.4, and Kv1.2 are all associated with the axons and
terminals of the Schaffer collateral pathway. Consistent with the
results presented in Figures 5 and 6, this large CA3 lesion did not
appear to reduce the density of Kv1.1 (D) or
Kv1.4 (F) immunoreactivity in the stratum lucidum
of CA3 (arrowheads). Because this lesion destroyed CA3
pyramidal cells, this result also confirms that Kv1.1 and Kv1.4
immunoreactivity in CA3 is associated with mossy fiber axons.
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Unlike the circumscribed ibotenic acid lesion (Fig. 5), the complete
CA3 kainic acid lesion eliminated the vast majority of Kv1.1
immunoreactivity in the stratum radiatum of CA1, suggesting that Kv1.1
is located on the axons and terminals of the Schaffer collateral
pathway (Fig. 7, compare C, D). Some Kv1.1 immunoreactivity remained after these large CA3 lesions; this weak Kv1.1 staining appeared to be diffusely distributed in the dendrites of hippocampal pyramidal cells. Surprisingly, the density of Kv1.2 and Kv1.4 immunoreactivity in the stratum radiatum of CA3 and CA1 was also reduced after this lesion (Fig. 7E-H), suggesting
that these two subunits may also be located on axons and terminals of
the Schaffer collateral pathway. Together, these results suggest that
in the stratum radiatum of CA1, a large proportion of Kv1.1
immunoreactivity is located on the axons and terminals of the Schaffer
collateral pathway and that these terminals also contain, albeit at a
far lower density, Kv1.2 and Kv1.4.
Effects of fornix transections on Kv1 and subunit immunoreactivity
As described previously (Rhodes et al., 1996), Kv2 mRNA is
expressed in large neurons in the medial septal and diagonal band nuclei. This pattern corresponds well to the patterns of Kv1.1, Kv1.2,
and Kv1.4 expression in these structures (K. J. Rhodes, M. M. Monaghan, N. X. Barrezueta, and J. S. Trimmer, unpublished observations), suggesting that all four of these subunits are expressed
in neurons known to provide subcortical cholinergic input to the
hippocampal formation (Mesulam et al., 1983). To explore the
association of Kv1 family and subunits with septal and other
subcortical afferents to the hippocampus, seven animals sustained a
unilateral transection of the fornix. Although the fornix transections
completely eliminated histochemical staining for AChE within the
ipsilateral dentate gyrus and CA subfields, these lesions did not have
any detectable effect on the distribution or density Kv1.1, Kv1.2,
Kv1.4, or Kv2 immunoreactivity within the hippocampal formation
(results not shown). This result indicates that these subunits are not
located presynaptically on cholinergic afferents or on other
subcortical inputs that reach the hippocampus via the fornix.
Association of Kv and subunits in hippocampal
K+ channel complexes
The pattern of immunoreactivity for Kv1.1, Kv1.2, Kv1.4, and
Kv2 in hippocampal formation and the results of our lesion analyses predict that these subunits are associated with one another in heteromultimeric K+ channel complexes
located on excitatory projections. To determine the extent of
association of Kv1.1, Kv1.2, Kv1.4, and Kv2 in rat hippocampal
K+ channel complexes, we performed
reciprocal coimmunoprecipitation experiments using subunit-specific
antibodies. Detergent lysates were prepared from rat hippocampal
membranes under conditions previously shown to preserve -
subunit interactions (Rhodes et al., 1995, 1996, 1997; Nakahira et al.,
1996; Shi et al., 1996). For each subunit, immunoprecipitation
reactions were performed under conditions of antibody excess, which was
verified by subsequent removal and analysis of the depleted supernatant
after pelleting the immunoprecipitation reaction product. In each case
all recoverable antigen was removed by the initial immunoprecipitation
reaction. Immunoprecipitation products, representing immunopurified
channel complexes, were then subjected to immunoblot analyses to assay for the presence of the specific and subunit polypeptides (Fig.
8).
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Figure 8.
Heteromeric Kv1 and subunit channel
complexes in the rat hippocampus. Detergent lysates of adult rat brain
membranes (RBM, 30 µg) and rat hippocampal membranes
(RHCM, 20 µg), and aliquots of products of
immunoprecipitation reactions performed on detergent extracts of 83 µg of RHCM with the indicated rabbit polyclonal antibodies were
size-fractionated by 9% ( subunit blots) or 12% (Kv2 blot)
SDS-PAGE. Samples were transferred to nitrocellulose and probed with
affinity-purified rabbit anti-Kv1 subunit-specific polyclonal
antibodies or mouse anti-Kv2 mAb K17/70. Bound antibody was detected
by ECL and autoradiography. Arrows point to the band
resulting from specific detection of the antigen listed to the
right. For clarity, only the section of the immunoblot
containing the cognate antigen is shown.
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Reciprocal immunoblots were performed using antibodies
specific for each of the and subunit polypeptides. Each of the affinity-purified rabbit polyclonal anti- subunit antibodies could
directly immunoprecipitate the respective subunit from the brain
membrane extracts (Fig. 8). Overall, clear evidence for association of
Kv1.1, Kv1.2, Kv1.4, and Kv2 in hippocampal K+ channel complexes was obtained.
Although quantitative analyses were not performed, comparison of the
relative intensities of bands visible on our immunoblots, which were
performed under conditions of antibody excess, indicted that most of
the Kv1.1, Kv1.2, and Kv1.4 pools in the hippocampus are associated
with the cytoplasmic Kv2 subunit. The pool of Kv1.1 seemed split
between that associated with Kv1.2 and Kv1.4, whereas Kv1.2 was
predominantly associated with Kv1.1 and to a lesser extent with Kv1.4.
Consistent with this, complexes immunopurified with anti-Kv1.1
antibodies contained the greater part of hippocampal Kv1.4, with less
of the Kv1.4 present in complexes containing Kv1.2. Kv2.1, which has a
somatodendritic localization on both principal cells and
interneurons in the hippocampus (Rhodes et al., 1995, 1997), could not
be detected in hippocampal K+ channel
complexes containing Kv1.1, Kv1.2, Kv1.4, or Kv2. Overall, these
results suggest that Kv1.1, Kv1.2, and Kv1.4 associate with one another
in hippocampal K+ channel complexes and
that many of these complexes contain at least one Kv2 subunit.
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DISCUSSION |
In summary, the results described above indicate that Kv1 channels
containing the Kv1.1, Kv1.2, Kv1.4, and Kv2 subunits are associated
with the axons and terminal fields of the major cortical afferent input
and the major intrinsic projections within the rat hippocampal
formation (Fig. 9). Although the mRNA for
each subunit is expressed in the cells of origin of these projections, the comparative lack of immunoreactivity in the somata of principal cells indicates that these subunits are efficiently transported out of
the cell body and targeted to axonal and nerve terminal domains. This
relationship between mRNA expression and protein localization is
similar to that reported for the cerebellum (McNamara et al., 1993,
1996; Rhodes et al., 1996, 1997) and spinal cord (Rasband and Trimmer,
2001). Ibotenic or kainic acid lesions altered the density of Kv1 and subunit immunoreactivity in the termination fields of cells
destroyed by the lesion. Lesions placed within those termination
fields, however, were generally without effect on the distribution of
immunoreactivity within the boundaries of the lesion. Two clear
exceptions to this general pattern are the loss of Kv2
immunoreactivity in the apical dendrites of CA1-CA3 pyramidal cells
caused by lesions within these subfields and the increased density of
Kv1.1 immunoreactivity within the boundaries of ibotenic acid lesions
in CA3 or CA1. The reason for the selective increase in Kv1.1
immunoreactivity within the boundaries of these two lesions is unclear
but is unlikely to represent a staining artifact, because it did not
occur with the other rabbit polyclonal antibodies or in control
sections in which the primary antibody was omitted. Therefore, the
increase in Kv1.1 density may represent an attempt by these circuits to
alter the subunit composition of Kv1 channels in response to
perturbations of the Schaffer collateral pathway. Nonetheless, the use
of complementary lesions allows us to distinguish between changes in
immunoreactivity resulting from axonal degeneration and changes in
immunoreactivity caused by altered morphology in the postsynaptic
target. This is a powerful approach for associating immunohistochemical
staining patterns with specific anatomical pathways (Rouse and Levey,
1997).
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Figure 9.
Summary of the localization of Kv1.1, Kv1.2,
Kv1.4, and Kv2 across hippocampal subfields. This summary figure is
derived from the results of our analysis of systematic lesions and the
effects of these lesions on the staining patterns for the indicated
subunit. In each lamina, the subunits are ordered according to their
relative staining intensity, from high to low. Although the
distribution of Kv1 was not examined in the present study, we have
reported the distribution of Kv1 previously (Rhodes et al., 1997)
and included our interpretation of its localization in this summary
figure. C/A, Commissural/associational pathway;
lPP, lateral perforant path; mPP, medial
perforant path; PP, perforant path; SC,
Schaffer collaterals; MF, mossy fiber zone;
ot, outer-third of the molecular layer;
mt, middle third of the molecular layer;
it, inner third of the molecular layer;
gc, granule cell layer; ig, infragranular
layer; l-m, stratum lacunosum moleculare;
rd, stratum radiatum; py, stratum
pyramidale; or, stratum oriens.
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Although the results from the present study are consistent with
previous interpretations of Kv1 and subunit staining patterns in the hippocampus (Wang et al., 1993; Rhodes et al., 1995, 1996, 1997;
Cooper et al., 1998), they are at odds with the conclusions of Veh et
al. (1995) and Sheng et al. (1994). In particular, Veh et al. (1995)
concluded that the majority of Kv1.1 immunoreactivity in the dentate
gyrus and CA subfields is associated with the dendrites of granule and
pyramidal cells, respectively, and Sheng et al. (1994) reported intense
Kv1.2 immunoreactivity throughout the apical dendritic arbors of
hippocampal pyramidal cells and in mossy fiber terminals in CA3.
Although the reasons for the differences in the data are unclear, one
reasonable explanation is that Veh et al. (1995) interpreted their
staining as dendritic when in fact it was concentrated in axons and
terminals. As mentioned above, interpretation of staining patterns in
the absence of direct experimental support can be difficult,
particularly in regions such as CA3 and CA1, where there is both a
dense ramification of dendritic arbors and dense synaptic innervation.
A second reasonable explanation of the differences between these data
are that the antibodies and immunohistochemical procedures (e.g.,
fixation and tissue sectioning) were different. Regardless of which
explanation is correct, our results, which demonstrate a clear
relationship among mRNA expression patterns, immunohistochemical
staining, coimmunoprecipitation, and the results of systematic and
complementary lesions, suggest that the majority of Kv1 subunit
immunoreactivity is associated with axons and terminals of excitatory
pathways and not with somata and dendrites of hippocampal neurons.
Although the complementary lesion analysis described above can account
for and explain the origin of most Kv1 and subunit immunoreactivity in the hippocampus, one area of uncertainty is the
inner third of the molecular layer of the dentate gyrus. We observed
that the density of Kv1.1 and Kv1.4 immunoreactivity increases after
ibotenic acid lesions of the entorhinal cortex and remains unchanged
after circumscribed ibotenic acid lesions in the dentate gyrus. The
most parsimonious explanation for these results is that in the inner
third, Kv1.1 and Kv1.4 are associated with subcortical afferents to the
dentate gyrus or with axons of the commissural/associational (C/A)
pathway. The latter pathway is formed by the mossy cells and other
interneurons in the dentate hilus (Laatsch and Cowan, 1967; Amaral and
Witter, 1989). The lack of an effect of fornix lesions on Kv1.1 and
Kv1.4 immunoreactivity makes it unlikely that subcortical afferents are
the source of the staining in the inner third. Although we did not
attempt to disrupt the C/A pathway directly, there is some evidence
from our data to indicate that Kv1.1, Kv1.4, and Kv1.2 are associated with axons and terminals of this projection. As described in Results, our kainic acid lesions of the CA3 subfield also destroyed neurons in
the hilus of the dentate gyrus that give rise to the C/A pathway. Careful examination of these cases (Fig. 7) indicates that in the
ipsilateral dentate gyrus there is a reduction in the density of Kv1.1,
Kv1.2, and Kv1.4 immunoreactivity in the inner third of the molecular
layer, suggesting that these subunits are located on the terminals of
the dentate C/A pathway. This observation should be followed up with
direct surgical lesions (Rouse and Levey, 1997).
Together with the immunohistochemical staining, the results of our
coimmunoprecipitation analyses indicate that there is a complex and
heterogeneous association of Kv1 subunits across hippocampal
subfields. Interestingly, our data indicated that the total pool of
Kv1.1 is split between complexes containing Kv1.4 or Kv1.2 and that the
majority of Kv1.4-containing complexes also contained Kv1.1, with less
Kv1.4 coassociated with Kv1.2. These results correspond well with the
immunohistochemical findings, in that Kv1.4 is colocalized with Kv1.1
in the perforant path, mossy fiber pathway, and Schaffer collaterals,
and Kv1.2 is colocalized with Kv1.1 in the perforant path and Schaffer
collaterals. In areas such as the dentate gyrus, Kv1.4 and Kv1.2 may be
coassociated on some, but not all, perforant path terminals, although
it appears that these two subunits are colocalized. In the middle third
of the dentate molecular layer, for example, it is clear that the staining for Kv1.2 and Kv1.4 overlaps, but entorhinal lesions have
distinct effects on their distribution, indicating that these two
subunits may be associated with separate or only partly overlapping sets of entorhinal afferents. Although the coimmunoprecipitation results indicate that most of the Kv1 subunit pool in the
hippocampus is coassociated with the Kv2 subunit, the
immunohistochemical data clearly indicate that some Kv2
immunoreactivity is concentrated in domains that do not have a
corresponding high density of Kv1.1, Kv1.2, or Kv1.4. Although the
strong staining for Kv2 in the apical dendrites of CA pyramidal
cells indicates that there is likely to be a Kv1 family subunit
located postsynaptically in hippocampal pyramidal cells, it is not
clear which Kv1 subunit this may be. Alternative explanations for the
"excess" Kv2 are that in these locations Kv2 interacts with
subunits from other Kv channel families (Yang et al., 2001), or
that it functions autonomously, perhaps as an oxidoreductase enzyme
(McCormack and McCormack, 1994; Gulbis et al., 1999). Thus it is
possible that in pyramidal cell dendrites, Kv2 serves functions
unrelated to its role as an integral component of Kv1 channels.
Although the data reported here are consistent with the demonstration
that DTX-sensitive Kv channels modulate neurotransmitter release by
direct action at nerve terminals (Dorandeau et al., 1997; Schechter,
1997; Southan and Owen, 1997; Geiger and Jonas, 2000), they do not
indicate that Kv1.1 and Kv1.2 contribute to the DTX-sensitive transient
K+ conductance that has been observed in
the dendrites of rat and mouse hippocampal pyramidal cells (Halliwell
et al., 1986; Storm 1990; Wu and Barish, 1992; Golding et al., 1999).
There are several likely explanations for this discrepancy. One is that
there may be other DTX-sensitive subunits not studied in this
report, such as Kv1.6, located on the dendrites of hippocampal
pyramidal cells. A second explanation is that the minor component of
the Kv1.1 or Kv1.2 immunoreactivity that remains after our lesions is
located on the dendrites of hippocampal pyramidal cells in sufficient
density to form measurable DTX-sensitive currents.
The coimmunoprecipitation and immunohistochemical data described here
and in previously published work suggest that the biophysical properties of Kv1 and subunits vary across hippocampal
pathways. In the mossy fiber pathway, the colocalization and likely
coassociation of Kv1.1 and Kv1.4, in a complex that also contains
Kv1 (Rhodes et al., 1997), would be expected to give rise to a
rapidly inactivating DTX-sensitive K+
conductance located on mossy fiber axons and terminals. In fact, a
DTX-sensitive A-type current has been directly observed in patch-clamp analyses of mossy-fiber terminals in rat hippocampal slices (Geiger and
Jonas, 2000). A DTX-sensitive A-type current having a similar subunit
composition would be expected on some perforant path terminals in the
dentate gyrus, whereas a separate population of perforant path
terminals may contain a Kv1.2 in association with Kv 2, forming a
slowly inactivating DTX-sensitive K+
current. In the CA1 subfield, the coassociation and colocalization of
Kv1.1, Kv1.2, and Kv1.4, together with Kv1 (Rhodes et al., 1997),
would be expected to form another DTX-sensitive A-type current
modulating glutamate release from terminals of the Schaffer collateral
pathway. Clearly, the heteromultimerization of Kv1 subunits provides a
molecular substrate for presynaptic modulation of glutamate release in
a pathway-specific manner. Moreover, the biophysical properties of
channels containing these subunits can be altered by phosphorylation
(Levitan, 1999), providing a potent mechanism for dynamic regulation of
neurotransmitter release.
The importance of DTX-sensitive Kv1-family
K+ channels to hippocampal function has
long been recognized. The pioneering studies of Halliwell and Dolly
(1982) and Halliwell et al. (1986) showed that the epileptogenic
effects of DTX were attributable to increased neurotransmitter release,
and that this toxin binds to components present at terminal regions to
mediate its preferential effect in the hippocampus. These studies also
identified a transient K+ conductance as a
major target for the effects of DTX in CA1 pyramidal cells (Halliwell
et al., 1986). Subsequent studies revealed an important role for
DTX-sensitive transient or A-type conductance in the hippocampus
(Storm, 1990). The importance of axonal A-type currents in hippocampal
mossy fiber terminals has also been shown in more recent studies, which
have implicated rapidly inactivating, DTX-sensitive axonal Kv channels
as the main molecular determinants of activity-dependent spike
broadening (Geiger and Jonas, 2000). Such dynamic modification of
action potential shape impacts both Ca2+
influx and glutamate release at mossy fiber terminals and as such would
be expected to augment synaptic transmission at mossy fiber synapses.
Our results showing the association of Kv1.1 and Kv1.4 in hippocampal
membranes, and the colocalization of these and other Kv1-family subunits in hippocampal circuits, point toward a significant
contribution of heteromeric Kv1 channels to activity-dependent spike
broadening and regulation of excitatory transmitter release within
mammalian hippocampal formation.
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FOOTNOTES |
Received April 10, 2001; revised June 1, 2001; accepted June 4, 2001.
This work was supported by Wyeth-Ayerst Research and the Center for
Biotechnology at Stony Brook, funded by the New York State Science and
Technology Foundation and by National Institutes of Health Grant
NS34383 (J.S.T.). We thank Nestor Barrezueta for technical support with
the in situ hybridization studies.
Correspondence should be addressed to Dr. Kenneth J. Rhodes,
Neuroscience, Wyeth-Ayerst Research, CN 8000, Princeton, NJ
08543. E-mail: rhodesk2{at}war.wyeth.com.
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TOP
ABSTRACT
INTRODUCTION
