Division of Neurology, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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ABSTRACT |
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Jhamandas, Jack H.,
Caroline Cho,
Balvinder Jassar,
Kim Harris,
David MacTavish, and
Jacob Easaw.
Cellular Mechanisms for Amyloid -Protein Activation of Rat
Cholinergic Basal Forebrain Neurons.
J. Neurophysiol. 86: 1312-1320, 2001.
The deposition of amyloid
-protein (A
) in the brain and the loss of cholinergic neurons in
the basal forebrain are two pathological hallmarks of Alzheimer's
disease (AD). Although the mechanism of A
neurotoxicity is unknown,
these cholinergic neurons display a selective vulnerability when
exposed to this peptide. In this study, application of
A
25-35 or A
1-40 to
acutely dissociated rat neurons from the basal forebrain nucleus
diagonal band of Broca (DBB), caused a decrease in whole cell
voltage-activated currents in a majority of cells. This reduction in
whole cell currents occurs through a modulation of a suite of potassium
conductances including calcium-activated potassium
(IC), the delayed rectifier (IK), and transient outward potassium
(IA) conductances, but not calcium or
sodium currents. Under current-clamp conditions, A
evoked an
increase in excitability and a loss of accommodation in cholinergic DBB
neurons. Using single-cell RT-PCR technique, we determined that A
actions were specific to cholinergic, but not GABAergic DBB neurons.
A
effects on whole cell currents were occluded in the presence of
membrane-permeable protein tyrosine kinase inhibitors, genistein and
tyrphostin B-44. Our data indicate that the A
actions on specific
potassium conductances are modulated through a protein tyrosine kinase
pathway and that these effects are selective to cholinergic but not
GABAergic cells. These observations provide a cellular basis for the
selectivity of A
neurotoxicity toward cholinergic basal forebrain
neurons that are at the epicenter of AD pathology.
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INTRODUCTION |
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Deposition of amyloid protein
in the form of diffuse and neuritic plaques is an important
pathological hallmark of Alzheimer's disease (AD) (Hardy
1997; Selkoe 1999
). The major component of the
neuritic amyloid plaques is amyloid
-protein (A
), a 39-43 amino
acid peptide that is generated from a larger protein, the amyloid
precursor protein (APP). There is considerable evidence to suggest that
A
and other peptide fragments derived from APP influence cellular
homeostasis and neuronal signaling through modulation of ion channel
function (for review see Fraser et al. 1997
).
The interactions of A within the membrane occur either with
preexisting ionophores or through the formation of de novo ion channels
(Arispe et al. 1996
; Fraser et al. 1997
).
A
25-35, an 11 amino acid fragment considered
to represent the neurotoxic domain of the parent A
peptide,
activates large, nonselective cation currents in bullfrog sympathetic
and rat hippocampal neurons (Furukawa et al. 1994
;
Simmons and Schneider 1993
).
A
25-35 and A
1-40
induce Ca2+ influx through voltage-gated channels
in cortical and NIE-115 neuroblastoma cells, respectively
(Davidson et al. 1994
; Weiss et al.
1994
). As yet no receptor for A
has been identified, but its
neurotoxic effects have been postulated to be mediated via plasma
membrane receptors for advanced glycation end products (RAGE), class A
scavenger receptor (SR)-related proteins, and/or the 75 kDa-neurotrophin receptor (El Khoury et al. 1996
;
Kuner et al. 1998
; Yan et al. 1996
).
Although A
modulation of ionic conductances has been studied in many
neuronal and nonneuronal systems, the linkage of these observations to
changes in neuronal excitability is less well understood.
It is now well accepted, that apart from A deposition, certain
chemically defined neurotransmitter systems, particularly the
cholinergic basal forebrain neurons, display a selective vulnerability and degeneration in AD (Price 1986
). Emerging data
support a potential link between A
peptides and the basal forebrain
cholinergic system. A
peptides can inhibit the release of endogenous
acetylcholine and high-affinity choline uptake from the hippocampus and
cortex (Kar et al. 1996
, 1998
). There is
A
-mediated inhibition of acetylcholine synthesis in cultured
cholinergic neurons (Pedersen et al. 1996
). Single
injection of A
into the basal forebrain, but not the striatum, induces damage to cholinergic neurons (Butcher et al.
1997
). These observations indicate that the chemical phenotype
of an individual cell is an important feature of A
toxicity, a
notion that is strengthened by the finding that GABA-containing neurons
of the hippocampus exhibit a relative resistance to A
in sharp
contrast to the vulnerability of cholinergic neurons exposed to this
peptide (Pike and Cotman 1993
). However, whether the
effects of A
are selective to cholinergic basal forebrain neurons,
which are at the epicenter of AD pathology, is an important but
unresolved question.
In this study, we investigated the actions of A on acutely
dissociated rat cholinergic basal forebrain neurons from the nucleus of
the diagonal band of Broca (DBB) using a combination of whole cell
patch-clamp and single-cell reverse transcription polymerase chain
reaction (RT-PCR) analysis. Our data show that the blockade of specific
potassium conductances is a potential underlying mechanism for the
action of A
on DBB neurons and may explain its effects in regulating
their excitability. Finally, we identify tyrosine phosphorylation as an
intracellular signal transduction pathway for these actions.
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METHODS |
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Dissociation procedures
Details of the procedure for acute dissociation of neurons from
the DBB are described in Jassar et al. (1999). Briefly,
brains were quickly removed from decapitated male Sprague-Dawley rats (15-25 day postnatal) and placed in cold artificial cerebrospinal fluid (ACSF) that contained (in mM) 140 NaCl, 2.5 KCl, 1.5 CaCl2, 5 MgCl2, 10 HEPES,
and 33 D-glucose 33 (pH 7.4). Brain slices (350 µm thick)
were cut on a vibratome, and the area containing the DBB was dissected
out. Although most of the tissue contained the horizontal limb of the
DBB, some slices may have included a component of the vertical limb of
the DBB. Acutely dissociated neurons were prepared by enzymatic
treatment of slice with trypsin (0.65 mg/ml) at 30°C, followed by
mechanical trituration for dispersion of individual cells. Cells were
then plated on poly-L-lysine (0.005% wt/vol)-coated cover
slips and viewed under an inverted microscope (Zeiss Axiovert 35). All
solutions were kept oxygenated by continuous bubbling with pure oxygen.
Electrophysiological recordings
Whole cell patch-clamp recordings were performed at room
temperature (20-22°C) using an Axopatch-1D amplifier. Series
resistance compensation was continuously adjusted to >80% and
monitored and readjusted as necessary during the course of each
experiment. Junction potential was nulled with the pipette tip immersed
in the bath. Internal patch pipette solution contained (in mM) 140 K-methylsulfate, 10 EGTA, 5 MgCl2, 1 CaCl2, 10 HEPES, 2.2 Na2-ATP, and 0.3 Na-GTP (pH 7.2). Putative
acutely dissociated DBB neurons were initially identified for recording
by visual inspection. Current-voltage relationships and excitability
characteristics were used to distinguish neurons from glial or other
cell types. Whole cell recordings were also done in the bridge
current-clamp mode using an Axoclamp-2B amplifier to examine the
effects of A on current-evoked changes in excitability of the
acutely dissociated DBB neurons. Action potentials were evoked by brief
current injection (0.6-1.5nA, 600 ms duration) through the patch
pipette. The resting membrane potential (RMP), number of spikes
elicited, and interspike intervals were recorded for comparison under
different experimental conditions. The membrane currents (voltage-clamp
experiments) or the membrane voltages (current-clamp experiments) were
recorded and analyzed on computer using pCLAMP software (version
6.0.3).
After whole cell configuration was established, we waited at least 5 min for steady-state currents to stabilize. The filter was set at 20 kHz during data acquisition. Cells were held in voltage clamp at 80
mV, which was close to the RMP observed in earlier studies on neurons
from basal forebrain slices (Alonso et al. 1994
;
Easaw et al. 1997
). A 1-s long hyperpolarizing command to
110 mV was applied to remove inactivation of
K+ channels so that the maximum current could be
activated during the subsequent slow voltage ramp to +30 mV (20 mV/s)
that followed it. No obvious tail currents were observed at the end of
the ramp when the command potential was returned to
80 mV, suggesting that the ramp elicited mainly steady-state currents.
Cell size was estimated electronically using the whole cell capacitance
compensation circuit on the Axopatch-1D amplifier. Maximum
voltage-clamp error in recording a current of 10 nA using a patch
electrode with an electrode resistance of 5 M was 10 mV. This
reflects the average maximum error since the currents recorded were
usually smaller than 10 nA. In figures displaying difference currents,
capacitance transients have been truncated.
To examine the effects of A on the contribution of
Ca2+ to voltage-dependent ionic currents, we
utilized an external solution that was nominally
Ca2+-free and contained 50 µM
Cd2+. In this solution
CaCl2 was replaced with an equimolar
concentration of MgCl2. To record currents
through calcium channels, we used Ba2+ as a
charge carrier as previously described (Easaw et al.
1999
). The external solution contained (in mM) 150 tetraethylammonium chloride, 2 BaCl2, 10 HEPES,
and 30 glucose (pH to 7.4 with TEA-OH). The internal patch pipette
solution consisted of (in mM) 130 Cs-methanesulfonate, 2 MgCl2, 10 HEPES, 10 BAPTA, 4 Mg-ATP, 0.3 Na-GTP,
and 0.1 Leupeptin (pH to 7.2 with CsOH). Depolarizing voltage steps
from
80 to +70 mV (increment 10 mV/step; 20 ms duration) were applied
to voltage-clamped DBB neurons under control conditions and in the presence of A
. Leak currents were minimal under our recording conditions. They did not change during the recordings and were not
affected by application of A
. Therefore we did not subtract these in
subsequent measurements of steady-state barium currents.
We also studied the effects of A on
INa. To isolate
INa, the external solution contained
(in mM) 125 NaCl, 20 TEA-Br (to block K+
currents), 2 MgCl2, 5 MnCl2
(to block Ca2+ currents), 10 HEPES, and 20 glucose (pH 7.4 with Tris-OH), and the internal solution consisted of
(in mM) 130 cesium-methanesulfonate, 10 HEPES, 10 BAPTA, 5 Mg-ATP, and
0.3 Na2-GTP (pH 7.2 with Cs-OH). The currents
were evoked by 10-ms voltage steps from
80 mV to a maximum of +60 mV
(increment 10 mV/step; 10 ms duration) in the presence and absence of
A
.
Drugs and solutions
All chemicals were purchased from Sigma (St. Louis, MO) except
the following: A1-40,
A
25-35, A
35-25 (QCB Biosource International, Camarillo, CA); genistein and daidzein (ICN, Costa Mesa, CA); and tyrphostin B-44 (Calbiochem, San Diego, CA).
Stock solutions of A
peptides were prepared by dissolving the
peptides at 1 mM in deionized water and stored in aliquots at
70°C
(Ueda et al. 1997
). On the day of the experiment, A
peptides were diluted in external perfusing medium just before the time
of application. All drugs and chemicals were applied via bath perfusion
at the rate of 3-5 ml per minute, which allowed complete exchange in
less then half a minute. Data are presented as means ± SE.
Student's two-tailed t-test (paired when appropriate) was
utilized for determining significance of effect.
Single-cell RT-PCR for chemical phenotyping
Where possible, neurons were harvested after
electrophysiological recordings were completed and readied for RT-PCR
according to a previously described protocol (Surmeier et al.
1996). In brief, contents of the electrode containing the cell
and 5 µl of internal solution were expelled into a 0.2-ml PCR tube
containing 5 µl sterile water (Sigma water W-4502), 0.5 µl
dithiothreitol 0.1 M (DTT), 0.5 µl RNasin (10 U/µl), and 1 µl
oligo-dT (0.5 µg/µl). The tube was then placed on ice.
Single-stranded cDNA was then synthesized from mRNA by adding a
solution containing 1 µl SuperScript II RT (200 U/µl), 2 µl
10 × PCR buffer, 2 µl 25 mM MgCl2, 1.5 µl 0.1 M DTT, 1 µl 10 mM dNTPs, and 0.5 µl RNasin (10 U/µl).
The PCR tube was gently mixed and incubated in a Techne Progene thermal cycler at 42°C for 50 min. The process was then terminated by heating
to 72°C for 15 min, and the tube cooled to 4°C. Subsequently 2 µl
of the RT product was taken and combined with 5 µl 10 × PCR buffer, 5 µl 25 mM MgCl2, 0.5 µl
Taq polymerase (5 U/µl), 31.5 µl sterile water (sigma
water W-4502), 1 µl 25 mM dNTP mixture, and 1.5 µl of a specific
set of primers (15 µM). All reagents were purchased from GIBCO BRL.
Primer sequences for choline acetyltransferase (ChAT) and for glutamate
decarboxylase (GAD) have been previously described (Surmeier et
al. 1996
; Tkatch et al. 1998
), and that for
-actin was obtained from GenBank (the lower primer 5'-GAT AGA GCC
ACC AAT CCA C, the upper primer 5'-CCA TGT ACG TAG CCA TCC A). All
primers were synthesized at the University of Alberta Department of
Biochemistry. The contents were mixed together and placed in the
thermal cycler. The PCR amplification protocol was as follows: step 1:
94°C 4 min; step 2: 94°C 1 min, 53°C 1 min, 72°C 45 s
(step 2 was repeated 35 times); step 3: 72°C 15 min; step 4: held at
4°C. A portion of the product was then run on a 2% TEA agarose gel,
and the gel was then placed in a bath containing 2 µg/ml of ethidium
bromide, after 10-min DNA bands were visualized with ultraviolet light
box and photographed with a Polaroid camera.
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RESULTS |
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Most of the acutely dissociated neurons from the DBB had
neuronlike morphology (i.e., large cells with a conspicuous nucleus, nucleolus, and a few blunt processes that were truncated
axon/dendrites). Under our recording conditions, the average input
conductance measured from the slope of the current-voltage
(I-V) relationships between 60 and
110 mV was 1.20 ± 0.16 nS (mean ± SE, n = 51).
Based on the previous observations (Jassar et al. 1999),
we utilized a voltage-ramp protocol where the cells were held at
80
mV and subjected to voltage ramps from
110 to +30 mV at the rate of
20 mV/s after conditioning at
110 mV for 1 s.
Effects of A on potassium currents
Whole cell potassium currents were recorded under control
conditions and in the presence of A (range of concentration 0.1-2.0 µM). The concentration of A
(1 µM) that we used in the present experiments is among the lowest used in previous electrophysiological studies of this peptide in acutely dissociated neurons (range 2-100
µM) (reviewed in Fraser et al. 1997
) or in brain
slices (range 200 nM to 2 µM) (Pettit et al. 2001
;
Wu et al. 1995
). In 51 DBB neurons, the outward currents
in the voltage range from
30 to +30 mV were decreased by both the
longer isoform of the peptide (A
1-40) and the
truncated active peptide fragment (A
25-35).
Maximal effects of A
on whole cell currents were observed within
90 s application, and the response did not desensitize with
repeated applications of the peptide. Figure 1, A and B, shows
the reversible decrease in outward currents caused by application of
A
25-35 (1 µM) and
A
1-40 (1 µM), respectively. The amplitude
of the currents at +30 mV was decreased significantly in the presence
of A
25-35 (control = 6.67 ± 0.24 nA, A
25-35 = 5.72 ± 0.23 nA,
n = 51, P < 0.001) or
A
1-40 (control = 7.52 ± 0.72 nA,
A
1-40 = 6.43 ± 0.70 nA,
n = 12, P < 0.001). The percent
reduction in amplitude of currents at +30 mV was also similar with both
peptides (A
25-35 = 14.21 ± 1.34%,
A
1-40 = 14.88 ± 4.16%, P = 0.8). The scrambled peptide fragment (reverse
fragment, A
35-25) had no effect on the whole
cell current-voltage relationships (not illustrated). Since the effects
of A
1-40 and A
25-35 on whole cell currents were essentially identical, we therefore utilized active peptide fragment A
25-35 (1 µM) in all subsequent experiments. In 11 DBB neurons,
A
25-35 did not evoke any change in whole cell
current or caused an increase or decrease in whole cell current that
was <5% from control values at +30 mV. An example of a cell that was
nonresponsive to A
is shown in Fig. 1C.
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The outward potassium currents are a mixture of calcium-activated and
noncalcium-activated components. Calcium-activated currents include the
voltage-sensitive conductances called maxi
gK(Ca) (IC) and the voltage-insensitive ones
that underlie action potential afterhyperpolarization
(IAHP). Noncalcium-activated
conductances consist of the delayed rectifier, M- and A-conductances
(IK,
IM, and
IA, respectively) and sodium currents.
In DBB neurons, there was little evidence for
IM relaxation in the currents evoked
by hyperpolarizing commands (10 mV/step) to 110 mV from a holding potential of
30 mV. Therefore IM was
not investigated in detail in this study. We studied the effects of
A
on IC,
IK, and
IA as well as on calcium
(ICa) and sodium
(INa) currents.
EFFECTS OF A ON CALCIUM-ACTIVATED POTASSIUM CURRENTS.
Of the two main Ca2+-activated potassium
currents, under whole cell recording conditions,
IAHP makes little contribution and majority of the currents flow through the voltage-sensitive
Ca2+-activated potassium channels,
IC (Jassar et al.
1999
). To elucidate the contributions of these conductances to
A
effects, we examined A
actions under conditions where the
external perfusion solution was replaced with the one that was
Ca2+-free (0 mM Ca2+) and
contained 50 µM Cd2+. Figure
2A shows the average of
current-voltage relationships obtained from 17 neurons under control
conditions, with 0 mM Ca2+ external medium, and
with A
in the presence of 0 mM Ca2+ external.
Replacing the external solution with 0 mM Ca2+
decreased the currents by 18.58 ± 2.46% (control = 6.94 ± 0.47 nA, 0 mM Ca2+ = 5.62 ± 0.40 nA, n = 17). Application of A
under these
conditions further reduced the currents by 8.77 ± 1.72% (0 mM
Ca2+ A
= 5.16 ± 0.41 nA,
n = 17, P < 0.001). We also studied
A
effects in the presence of charybdotoxin (CTX) and iberiotoxin,
specific blockers of IC channels.
Figure 2B shows the average of I-V relationships obtained from 19 neurons under control conditions, in the presence of
CTX (25 nM) and A
application in the presence of CTX. CTX reduced the outward currents at +30 mV by 12.89 ± 2.56%
(control = 6.62 ± 0.37 nA, CTX = 5.76 ± 0.37 nA,
n = 19). Application of A
in the presence of CTX
further reduced the currents by 7.81 ± 1.83% (CTX + A
= 5.31 ± 0.33 nA, n = 19, P < 0.001). The percent reduction of currents by A
in the presence of
CTX was not significantly different from that obtained by omitting
Ca2+ from the external perfusate
(P = 0.09). In seven cells, we also examined the
effects of A
in the presence of iberiotoxin (50 nM). A
reduction
of whole cell currents with iberiotoxin was similar (10.2 ± 2.5%, not illustrated) to that observed with CTX.
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EFFECTS OF A ON CALCIUM CURRENTS.
Since A
reduction of whole cell currents is attenuated by
approximately 50% in the presence of CTX or by the removal of external Ca2+, Ca2+-activated
currents appear to play an important role in the response of DBB
neurons to A
application. This can result from either an effect on
Ca2+-dependent conductances, i.e.,
IC or, a more upstream effect on Ca2+ channels that in turn may activate potassium
conductances. To examine this issue, we recorded barium currents
(IBa) flowing through
Ca2+ channels. Figure 2C shows the
average I-V relationships of
IBa recorded from nine neurons. A
did not significantly affect the IBa
(control = 3.32 ± 0.23 nA, A
= 3.19 ± 0.25 nA,
n = 9, P = 0.12, at
10 mV).
EFFECTS OF A ON TRANSIENT OUTWARD
(IA) AND THE DELAYED RECTIFIER
(IK) POTASSIUM CURRENTS.
IA and
IK are voltage-sensitive currents, and
their activation and inactivation are strongly voltage dependent.
IA requires the holding potential to
be relatively hyperpolarized (approximately
110 mV) for removal of
its inactivation, whereas it is inactivated at
40 mV. On the other
hand, IK is not inactivated at
40
mV. These biophysical properties of IA
and IK can thus be utilized to isolate
these currents. Therefore a conditioning pulse to
40 mV will activate
IK without any significant
contamination by IA (Connor and
Stevens 1971
; Easaw et al. 1999
). A conditioning
pulse to
120 mV will activate both
IA and
IK. The difference currents obtained
by subtracting the currents evoked by depolarizing pulses following a
conditioning pulse to
40 mV from those evoked following a
conditioning pulse to
120 mV provide an accurate estimate of IA (Connor and Stevens
1971
; Easaw et al. 1999
). Figure 2D
shows the currents recorded from a neuron with a conditioning pulse to
40 mV for 150 ms, representing mainly
IK, under control conditions, in the
presence of A
and recovery on wash out of A
. A
reduced IK by 10.93 ± 2.4%
(control = 7.14 ± 0.42 nA, A
= 6.41 ± 0.45 nA,
n = 34, P < 0.001 at +30 mV). Figure
2E shows the difference currents recorded from the same
neuron representing mainly IA, under
control conditions, in the presence of A
, and on wash out. IA was reduced significantly by A
by 16.96 ± 3.89% (control = 5.42 ± 0.58 nA, A
= 4.58 ± 0.55 nA, n = 34, P < 0.001). We have previously shown that the residual sustained current
remaining at the end of the 100-ms test pulse (shown in Fig.
3B) consists mainly of
IK and
IC (Easaw et al. 1999
),
both of which are also reduced by A
.
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EFFECTS OF A ON SODIUM CURRENTS.
Sodium currents are involved in the fast depolarizing phase of the
action potential. Enhancement of these currents can increase the
excitability and vice versa. We recorded sodium currents in isolation
to assess if A
has any effects on these currents. The sodium
currents were TTX sensitive. Figure 3B shows the average I-V relationships obtained from 11 cells under control
conditions, in the presence of A
, and recovery on wash out of
A
. A
did not influence sodium currents in DBB neurons
(control =
6.34 ± 0.59 nA, A
=
6.08 ± 0.57 nA, P = 0.12 at +20 mV).
Effects of A on excitability of DBB neurons
Application of A results in depolarization of the RMP, increase
in excitability, and loss of accommodation. Figure
4A depicts a DBB neuron
showing accommodation under control conditions. On application of A
,
the number of spikes evoked by injecting the same amount of current as
under control conditions in the same neuron was increased, indicating
an increase in excitability (Fig. 4B). Under control
conditions, the average RMP was
65.5 ± 2.2 mV, which
depolarized to
56.4 ± 1.8 mV (n = 21, P < 0.001) on A
application and recovered to
72.0 ± 3.3 mV on wash out. The average number of spikes elicited
by current injection was 8.9 ± 1.5 under control
conditions, 13.9 ± 1.4 in the presence of A
, and 6.4 ± 1.8 on wash out (n = 21, P < 0.001).
In addition to the increase in excitability, A
also caused a loss of
accommodation. The interspike interval between the first two and the
last two action potentials provides a measure of accommodation. Under
control conditions the first interspike interval was 46.5 ± 2.3 ms, and the last interspike interval was 90.1 ± 16.3 ms
(n = 6, Fig. 4C). In the presence of A
,
the first interspike interval was 33.3 ± 1.8 ms, and the last
interspike interval was 42.1 ± 2.9 ms. On recovery the first
interspike interval was 48.1 ± 1.5 ms, and the last interspike
interval was 93.6 ± 18.1 ms.
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A larger difference between the first and last interspike intervals
indicates a greater degree of accommodation. A significantly reduced
the difference between the first and the last interspike intervals
consistent with a loss of accommodation (P < 0.005).
Chemical phenotype of the A responsive neurons
There are two main chemical neurotransmitter phenotypes
represented in the DBB neurons: GABAergic and cholinergic. Whole cell recordings were made from a heterogeneous population of DBB neurons. Definitive determination of the chemical phenotype was done by single-cell RT-PCR analysis. ChAT was used as a specific marker for
cholinergic neurons, and GAD was used as a specific marker for
GABAergic neurons. Figure 5 shows the
photograph of a gel indicating RT-PCR products from an A-responsive
cell shown in Fig. 1A and also an A
-nonresponsive neuron
(Fig. 1C). The A
responsive cell on the left reveals a
band corresponding to the molecular weight of the ChAT primer, and the
A
nonresponsive cell in the middle of the gel shows a band
corresponding to the molecular weight of GAD primer. Results from 81 DBB neurons that were recorded in either voltage- or current-clamp
modes and in which PT-PCR reaction was unequivocal are summarized in
Fig. 5, bottom. All cells that responded A
with a
reduction in whole cell currents or an increase in excitability were
ChAT positive (n = 63) and GAD negative. On the other
hand, all the A
-nonresponsive neurons were GAD positive
(n = 18) and ChAT negative.
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Previous studies have suggested that larger dissociated cells from the
basal forebrain are more likely to be cholinergic (Griffith et
al. 1994; Jassar et al. 1999
). In the present
study the average membrane capacitance estimated electronically was
16.7 ± 0.5 pF (n = 171; range 12-29 pF).
ChAT-positive cells had an average membrane capacitance of 16.4 ± 0.7 pF (range, 13-21 pF), whereas GAD-positive cells were found to
have a capacitance of 18.2 ± 1.4 pF (range, 14-22 pF).
Therefore, on the basis of our results, it would seem that cell size
correlates poorly with the chemical identity of a particular cell as
determined by single cell RT-PCR.
Involvement of protein tyrosine phosphorylation in A response
Protein tyrosine phosphorylation modulates voltage- and
ligand-gated channels to influence neuronal function (Raymond et
al. 1993; Wang and Salter 1994
). In PC 12 cells
and olfactory neuroblasts, application of
A
1-40 and A
25-35
induces a rapid and dose-dependent tyrosine phosphorylation that is
accompanied by a rise in cytosolic Ca2+
(Luo et al. 1995
). The DBB cells are enriched with
protein tyrosine kinase (PTK) activity, and we have previously
shown that GABA responses in these cells are modulated by PTK
phosphorylation (Jassar et al. 1997
). We investigated
whether PTK may play a role in A
-evoked responses in DBB neurons.
Genistein and tyrphostin B-44 are relatively specific
membrane-permeable blockers of PTK (Valenzuela et al.
1995
; Wang and Salter 1994
). Figure
6A shows the average
I-V relationships from 7 neurons under control conditions, in the presence of genistein (100 µM), and A
in the presence of
genistein. Figure 6B shows the average I-V
relationships from six neurons under control conditions, in the
presence of tyrphostin B-44 (50 µM), and A
in the presence of
tyrphostin. These compounds decreased the whole cell outward currents
by 60.3 ± 4.97% (control = 6.76 ± 0.55 nA,
genistein = 2.66 ± 0.42 nA, n = 7) and
55.01 ± 3.04% (control = 5.87 ± 0.39 nA,
tyrphostin = 2.63 ± 0.21 nA, n = 6),
respectively. However, A
failed to affect these outward potassium
currents in the presence of genistein or tyrphostin (genistein + A
= 2.44 ± 0.34 nA, 63.48 ± 4.06%,
P > 0.05; tyrphostin + A
= 2.48 ± 0.18 nA, 57.54 ± 2.30%, P > 0.07). The response to
A
, a reduction of 11.45 ± 0.82% of the outward currents at +30 mV (n = 6), was not significantly affected in the
presence of daidzein (100 µM), an inactive analogue of genistein
(Fig. 6C, control = 6.85 ± 0.32 nA, daidzein = 6.44 ± 0.36 nA, daidzein + A
= 5.71 ± 0.34 nA,
n = 6, P < 0.001).
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DISCUSSION |
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These experiments demonstrate four major findings. First, A
reduces a suite of potassium currents in basal forebrain neurons, including calcium-activated potassium
(IC), the delayed rectifier (IK), and transient outward potassium
(IA) conductances, but not calcium or
sodium currents. Second, under current-clamp conditions, application of
A
evoked an increase in excitability and a loss of accommodation in
cholinergic DBB neurons. Third, using single-cell RT-PCR analysis, we
show that A
actions are specific to cholinergic, but not GABAergic
DBB neurons. Finally, the A
effects on cholinergic DBB neurons
appear to be mediated via activation of protein tyrosine kinase
signaling pathway.
A modulation of ionic conductances and neuronal excitability
A and related amyloidogenic metabolic fragments have been shown
to alter cellular ionic conductances with existing channels or by de
novo channel formation (Fraser et al. 1997
). Such
alteration in ionic homeostasis has been linked to the ability of A
to induce cell death and may provide a molecular mechanism for
neurodegeneration seen in AD (Mattson et al. 1992
;
Yu et al. 1998
). In hippocampal neurons, sAPP, a larger
fragment of the parent APP, activates K+ channels
and reduces intracellular Ca2+ through cGMP
production and protein dephosphorylation (Furukawa et al.
1996
). Suppression of excitability and membrane
hyperpolarization by such mechanisms have been advanced to support, in
part, a "neuroprotective" role for sAPP. However, although A
has
been shown to activate a wide variety of K+ and
Ca2+ conductances, there is limited information
on its role in influencing neuronal excitability. Our data show that
A
decreases IC currents without
influencing calcium currents and leads us to conclude that A
has a
direct effect on IC channels. Since
IC is responsible, in part, for the
repolarization phase of the action potential and plays an important
role in the process of spike frequency adaptation (accommodation)
(Vergara et al. 1998
), A
-induced blockade of
IC that we have observed could explain
the increase in excitability and loss of accommodation seen with A
.
We have previously shown that in DBB neurons at RMP, inhibition of
IC with either charybdotoxin or
iberiotoxin results in an increase in excitability and loss of
accommodation similar to that observed for A
in the present study
(Easaw et al. 1999
; Jassar et al. 1999
).
Fast-inactivating potassium channels
(IA) are also important in modulating
neuronal excitability. A-induced reduction of IA observed in our study is consistent
with a similar effect observed in rat hippocampal neurons (Good
et al. 1996
). Blockade of IA by A
could lead to increased duration of depolarization during an
action potential, which in turn could increase
Ca2+ influx. However, unlike several previous
reports, we did not observe an effect of A
on
Ca2+ currents (Brorson et al.
1995
; Mattson et al. 1992
; Ueda et al. 1997
) or Na+ currents. Although A
did
not influence Ca2+ currents, prolonged
depolarization resulting from a blockade of K+
currents (both IA and
IK) could still lead to increased
total Ca2+ influx. In the present study we noted
that A
decreases the TEA-sensitive IK conductance, which is different
from the observation in septal cell line (SN56) and murine cortical
cultures where A
exposure resulted in an enhancement of
IK (Colom et al. 1998
;
Yu et al. 1998
). However, in the latter studies, no
acute effects of A
on IK were
noted, but 7-11 h after A
exposure, the
IK was enhanced, and this effect could
be blocked by TEA. These delayed effects of A
on potentiation of
IK have been postulated to explain the ability of A
to induce apoptotic cell death in cultures (Yu
et al. 1998
).
Selective effects of A on cholinergic neurons
Our data indicate that cell size is not a reliable index for
distinguishing cholinergic from GABAergic neurons in the DBB. However,
single-cell RT PCR offers an effective and reliable means to make a
distinction between these two major chemical phenotype of cells in the
basal forebrain. At present, the cause of preferential degeneration of
forebrain cholinergic neurons remains unclear. The neurotoxic potential
of A peptides could possibly mediate the degeneration of cholinergic
neurons in AD brains. This notion is supported by data that a single
injection of A
peptide into the septal nucleus induces damage to
cholinergic but not parvalbumin-containing (presumably GABAergic)
neurons (Harkany et al. 1995
). Cholinergic neurons in
hippocampal cultures seem particularly susceptible to injury following
exposure to A
, whereas GABA-containing neurons are relatively
resistant to A
-induced neurotoxicity (Pike and Cotman
1993
). Our experiments show that the effects of A
in
modulating K+ channel conductances and neuronal
excitability are specific to cholinergic and not GABAergic neurons.
This finding apart from demonstrating, at a cellular level, a link
between A
peptides and cholinergic function, may help explain the
selective vulnerability of cholinergic neurons to A
that is observed
in vivo and in cell cultures. A
's ability to render cholinergic
neurons hyperexcitable through loss of accommodation could result in
prolonged depolarization and eventual cell death resulting from
excessive Ca2+ influx. Indeed A
has previously
been shown to markedly potentiate glutamate-induced cell death in human
cortical neurons through an increase in Ca2+
influx (Mattson et al. 1992
).
A and protein tyrosine phosphorylation
In the present study we have observed that A-mediated reduction
in whole cell K+-currents in DBB neurons is
occluded by bath application of PTK inhibitors genistein and tryphostin
B-44(
), but unaffected by daidzein, which is structurally similar to
genistein but has no effect on PTK activity. It is possible that the
effects of genistein, apart from its properties as a PTK inhibitor,
could be also be attributed to its ability to directly block
voltage-gated K+ conductances (Ogata et
al. 1997
; Smirnov and Aaronson 1995
). However,
A
effects were also blocked by tyrphostin B-44(
), another PTK
inhibitor, which has not been shown to block K+
conductances. These results suggest that endogenous PTKs may play an
important role in coupling the A
effects on K+
conductances in cholinergic forebrain neurons. There is considerable evidence that protein tyrosine kinases and phosphatases, acting on
potassium channels, can regulate neuronal excitability (Jonas and Kaczmarek 1996
).
Conclusions
The fact that A is constitutively produced in the brain
(Shoji et al. 1992
) and is capable of influencing ion
channel function of cholinergic neurons as demonstrated in the present
study suggests that this peptide may have a neuromodulatory role within
the DBB in vivo apart from its neurotoxic effects in the context of AD. The underlying mechanism for the specificity of A
response toward cholinergic, and not GABAergic, basal forebrain cells is an important question that remains unresolved. It is possible that the difference in
responsiveness of cholinergic versus GABAergic cells to A
that we
have observed in the present study may be due to the presence of a
receptor for A
, which has yet to be identified, on cholinergic but
not GABAergic neurons. Further studies of the receptor and molecular
mechanisms underlying the coupling of A
effects to potassium
channels through PTK signaling pathways in cholinergic basal forebrain
neurons may shed additional important insights into cholinergic
hypofunction seen in AD.
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ACKNOWLEDGMENTS |
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We thank Dr. C. W. Bourque for helpful comments and suggestions and C. Krys for assistance with preparation of the manuscript.
This research was supported by grants from the Canadian Institutes of Health Research (MT-10473) and the University Hospital Foundation. C. Cho, J. Easaw, and B. Jassar were supported by training awards from the Alberta Heritage Foundation for Medical Research.
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FOOTNOTES |
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Address for reprint requests: J. H. Jhamandas, Div. of Neurology, 530 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail: jack.jhamandas{at}ualberta.ca).
Received 7 February 2001; accepted in final form 24 May 2001.
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REFERENCES |
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