1Department of Physiology and 2Division of Neuroscience, University of Alberta School of Medicine, Edmonton, Alberta T6G 2H7, Canada
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ABSTRACT |
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Nguyen, Peter V., Steven N. Duffy, and Jennie Z. Young. Differential Maintenance and Frequency-Dependent Tuning of LTP at Hippocampal Synapses of Specific Strains of Inbred Mice. J. Neurophysiol. 84: 2484-2493, 2000. Transgenic and knockout mice are used extensively to elucidate the molecular mechanisms of hippocampal synaptic plasticity. However, genetic and phenotypic variations between inbred mouse strains that are used to construct genetic models may confound the interpretation of cellular neurophysiological data derived from these models. Using in vitro slice stimulation and recording methods, we compared the membrane biophysical, cellular electrophysiological, and synaptoplastic properties of hippocampal CA1 neurons in four specific strains of inbred mice: C57BL/6J, CBA/J, DBA/2J, and 129/SvEms/J. Hippocampal long-term potentiation (LTP) induced by theta-pattern stimulation, and by repeated multi-burst 100-Hz stimulation at various interburst intervals, was better maintained in area CA1 of slices from BL/6J mice than in slices from CBA and DBA mice. At an interburst interval of 20 s, maintenance of LTP was impaired in CBA and DBA slices, as compared with BL/6J slices. When the interburst interval was reduced to 3 s, induction of LTP was significantly enhanced in129/SvEms slices, but not in DBA and CBA slices. Long-term depression (LTD) was not significantly different between slices from these four strains. For the four strains examined, CA1 pyramidal neurons showed no significant differences in spike-frequency accommodation, membrane input resistance, and number of spikes elicited by current injection. Synaptically-evoked glutamatergic postsynaptic currents did not significantly differ among CA1 pyramidal neurons in these four strains. Since the observed LTP deficits resembled those previously seen in transgenic mice with reduced hippocampal cAMP-dependent protein kinase (PKA) activity, we searched for possible strain-dependent differences in cAMP-dependent synaptic facilitation induced by forskolin (an activator of adenylate cyclase) and IBMX (a phosphodiesterase inhibitor). We found that forskolin/IBMX-induced synaptic facilitation was deficient in area CA1 of DBA/2J and CBA/J slices, but not in BL/6J and 129/SvEms/J slices. These defects in cAMP-induced synaptic facilitation may underlie the deficits in memory, observed in CBA/J and DBA/2J mice, that have been previously reported. We conclude that hippocampal LTP is influenced by genetic background and by the temporal characteristics of the stimulation protocol. The plasticity of hippocampal synapses in some inbred mouse strains may be "tuned" to particular temporal patterns of synaptic activity. From a broader perspective, our data support the notion that strain-dependent variation in genetic background is an important factor that can influence the synaptoplastic phenotypes observed in studies that use genetically modified mice to explore the molecular bases of synaptic plasticity.
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INTRODUCTION |
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Understanding the cellular and molecular
mechanisms of activity-dependent synaptic plasticity is an important
goal of neuroscience. Transgenic and gene-targeting techniques are
potent tools for defining the functions of individual genes in certain
forms of synaptic plasticity in the mammalian brain (Hogan et
al. 1994). Within particular brain regions, genes may be
expressed in elevated amounts (Mayford et al. 1996
), or
the expression of specific genes may be eliminated by targeted deletion
(Capecchi 1989
; Soriano 1995
). These
molecular strategies have been effectively applied to generate
genetically modified mice, and these animals have been used extensively
to explore the molecular mechanisms of synaptic plasticity (Chen
and Tonegawa 1997
; Mayford et al. 1995a
;
Picciotto and Wickman 1998
).
A prolific number of studies have tried to elucidate the functions of
specific genes and signaling molecules in particular forms of
activity-dependent synaptic plasticity, learning, and memory
(Micheau and Riedel 1999; Picciotto and Wickman
1998
). In these studies, a common strategy has been to use two
inbred strains of mice to generate genetically modified mice. An inbred strain is one in which matings between siblings have been performed for
at least 20 generations, resulting in a population of genetically homogeneous animals (Lyon and Searle 1989
). One inbred
strain supplies a viable genetic background for breeding and survival, while a second inbred strain provides stem cells for genetic
manipulation (Hogan et al. 1994
). An important
consideration inherent in all of these studies is that disruption or
overexpression of a single gene can lead to compensatory changes in the
expression of other genes, the presence or absence of which can vary
according to the genetic backgrounds of the mouse strains used to
generate a genetically modified line of mice (Crawley et al.
1997
; Gerlai 1996
). Valid interpretation of the
neurophysiological phenotypes that emerge from genetically modified
mice therefore requires a knowledge of the synaptoplastic and
electrophysiological properties of relevant neurons in the parent
strains used to produce genetically modified lines of mice. Hence, the
characterization of cellular electrophysiological and synaptoplastic
properties of neurons and synapses in relevant brain structures of
inbred strains of mice is an important step toward defining the genetic
and molecular bases of synaptic plasticity.
Much is known about the behavioral characteristics and cognitive
performance capabilities of some strains of inbred mice, particularly
with reference to learning and memory (Crawley et al.
1997; Owen et al. 1997
). For example, DBA/2 and
C57BL/6 mice show markedly different performance capabilities in tests
of spatial learning and memory. DBA/2 mice are impaired in spatial
learning (Paylor et al. 1993
), and they show deficient
hippocampal synaptic plasticity that may compromise their performance
on tasks requiring integration of spatial and contextual information
(Bampton et al. 1999
; Matsuyama et al.
1997
; Nguyen et al. 2000
). In contrast, C57BL/6
mice show robust hippocampus-based learning and memory that are
correlated with intact synaptic physiology in area CA1 (Nguyen
et al. 2000
). Some of the learning deficits in DBA/2 mice may
be linked to reduced levels of hippocampal protein kinase C (PKC)
activity (Matsuyama et al. 1997
; Wehner et al.
1990
). Also, in another inbred strain, CBA/J, profound deficits
in spatial and nonspatial learning and memory are correlated with
deficiencies in the expression of some forms of hippocampal synaptic
plasticity in area CA1 (Nguyen et al. 2000
). However, a
conjoint characterization of cellular biophysical properties,
activity-dependent plasticity, and the expression of signal
transduction-mediated synaptic plasticity has not been performed on
hippocampal CA1 neurons in these strains. It is important to examine
and compare these functional properties of hippocampal neurons in these
mouse strains, because the deficits in learning and memory in DBA/2 and
CBA/J mice (Nguyen et al. 2000
; Paylor et al.
1993
) may be linked to alterations in some or all of these
cellular attributes in these strains.
Hippocampal long-term potentiation (LTP) and long-term depression (LTD)
constitute activity-dependent enhancement and reduction, respectively,
of excitatory synaptic strength (Bear and Abraham 1996;
Madison et al. 1991
). These two types of synaptic
plasticity are believed to play important roles in some forms of
learning and memory in the mammalian brain (Martin et al.
2000
). In humans and mice, area CA1 of the hippocampus is vital
for the formation of long-term memory (Tsien et al.
1996
; Zola-Morgan et al. 1986
). Genetic
modifications of key signaling molecules within area CA1 of the mouse
hippocampus can impair long-term memory and LTP (reviewed by
Micheau and Riedel 1999
). Some data on mouse
strain-specific impairments of memory and of hippocampal LTP in area
CA1 have been reported (Nguyen et al. 2000
; see also
Bampton et al. 1999
, for dentate gyrus data). However,
the cellular and molecular mechanisms underlying these strain-dependent
defects in LTP are undefined. For example, enhanced glutamatergic
postsynaptic currents have been implicated in the induction of
hippocampal LTP in area CA1 (reviewed by Kullmann and Siegelbaum
1995
and Malenka and Nicoll 1993
), and
differences in the magnitudes of these currents may exist among
particular inbred mouse strains. Also, modified membrane excitability
or altered spike frequency accommodation in hippocampal neurons may
influence the sensitivity of these neurons to excitatory synaptic input
and might thereby regulate the thresholds for induction of LTP and LTD
in these neurons. Strain-specific variations in these cellular
electrophysiological properties may impart differential expression of
cellular excitability and spike firing efficacy in hippocampal neurons,
and these electrophysiological alterations, in turn, might modify the
signal processing capabilities of hippocampal circuits.
Signal transduction pathways are also important for memory formation
and for modulating hippocampal synaptic strength (reviewed by
Elgersma and Silva 1999; Martin et al.
2000
; Micheau and Riedel 1999
; Soderling
and Derkach 2000
). Strain-dependent variations in hippocampal
protein kinase expression may influence LTP and learning and memory
(see Wehner et al. 1990
for PKC data). cAMP-dependent protein kinase (PKA) is vital for hippocampus-dependent long-term memory and for maintenance of LTP in area CA1 of C57BL/6 mice (Abel et al. 1997
; Nguyen and Kandel
1997
), but cAMP-induced forms of synaptic facilitation have not
been examined in inbred strains that display deficits in
hippocampus-dependent long-term memory (e.g., CBA/J, DBA/2J strains,
Paylor et al. 1993
; Nguyen et al. 2000
).
The primary aim of the present study was to compare the membrane
biophysical, cellular electrophysiological, and synaptoplastic properties of hippocampal neurons in particular inbred strains of mice.
A secondary objective of our study was to compare the expression of
cAMP-induced synaptic facilitation in these same strains, since PKA and
cAMP are known to play important roles in the expression of
hippocampus-based long-term memory and hippocampal LTP (Abel et
al. 1997; Huang and Kandel 1994
; Slack
and Pockett 1991
; Weisskopf et al. 1994
;
Wong et al. 1999
). The strains examined here were
selected because their performance capabilities on
hippocampus-dependent learning and memory tasks have been previously
documented (Crawley et al. 1997
; Nguyen et al.
2000
; Owen et al. 1997
; Paylor et al. 1993
), and also because these strains have been used
extensively to generate genetically modified mice for studies of
synaptic plasticity and cognitive functions, including learning and
memory (Piciotto and Wickman 1998
). We addressed the
following fundamental questions: Is hippocampal LTP in some inbred
strains optimally "tuned" to, or induced by, specific temporal
patterns of imposed synaptic activity? Are there strain-dependent
differences in spike firing efficacy, postsynaptic glutamatergic
currents, and membrane biophysical properties in hippocampal neurons?
Do hippocampal neurons show significant strain-dependent variations in
cAMP-induced synaptic facilitation? Some of these data have been
published in abstract form (Duffy et al. 2000
).
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METHODS |
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Animals
All mice were male, aged 4-8 wk (Jackson Labs, Maine). We
examined four inbred strains: C57BL/6J, CBA/J, DBA/2J, and
129/SvEms+Ter?/J. For LTD experiments, we used
hippocampal slices from mice aged 4-5 wk, while all other experiments
were performed on slices cut from mice aged 7-8 wk. Animals were
housed on a 12-h light/dark cycle at the University of Alberta Health
Sciences Animal Care Facility, using guidelines approved by CCAC
(Canadian Council on Animal Care).
Electrophysiology: extracellular field recordings
Transverse hippocampal slices (400-µm thickness) were cut and
maintained in an interface chamber at 28°C (for more details, see
Nguyen and Kandel 1997). The artificial cerebrospinal
fluid (ACSF) used for dissection and superfusion contained (in mM) 125 NaCl, 4.4 KCl, 1.5 MgSO4, 1 NaH2PO4, 26 NaHCO3, 10 glucose, 2.5 CaCl2. Extracellular excitatory postsynaptic
field potentials (fEPSP) were recorded in stratum radiatum of area CA1
with glass microelectrodes (2-4 M
resistances, filled with ACSF).
The Schaffer collateral pathway was stimulated with bipolar nickel
chromium electrodes positioned in stratum radiatum, and evoked fEPSPs
were recorded, digitized, and analyzed using pClamp 7 software (Axon Instruments). Baseline fEPSPs were elicited once per minute by applying
a stimulus intensity (0.08-ms pulse width) sufficient to evoke fEPSP
amplitudes that were 40% of maximal sizes. For some experiments,
fEPSPs were measured during stimulation (once per minute at test
intensity) of a second, independent pathway in stratum radiatum
(Nguyen et al. 1994
). This control pathway did
not receive high-frequency stimulation and it was monitored before and
after induction of LTP in a neighboring pathway in stratum radiatum of
the same slice. Various stimulation protocols were used to induce LTP
and LTD; these are described at appropriate parts of the
RESULTS section. Rp-cAMPS, an inhibitor of PKA, was dissolved in ACSF. Forskolin and IBMX (from RBI) were prepared as 50 mM
stock solutions in DMSO and were diluted in ACSF to obtain a final DMSO
concentration of 0.1%. This DMSO concentration did not affect synaptic
responses under our experimental conditions.
Whole-cell patch-clamp recordings
Hippocampal CA1 neurons were patch-clamped in the "blind"
whole-cell mode (Blanton et al. 1989) using an
Axopatch-1D amplifier and pClamp-7 acquisition software (Axon
Instruments). Whole-cell patch-clamp electrodes were produced on a
multistage puller (Sutter Instruments) to yield resistances of 6-9
M
. All cells were in the CA1 pyramidal cell layer, as judged by
visual placement of the patch-clamp electrode. Spike-frequency
accommodation was used as the physiological criterion for identifying
these neurons as pyramidal cells (Kandel and Spencer
1961
; Schwartzkroin 1977
; Schwartzkroin
and Mathers 1978
). Cells with resting potentials more
depolarized than
60 mV, or spike amplitudes <80-90 mV, were rejected.
-aminobutyric acid-A (GABAA)
currents were blocked with bath-applied 10 µM picrotoxin (RBI).
Cellular resting potential (Em), input
resistance (Rin), spike-frequency
accommodation (SFA), and membrane depolarization (following 100-Hz, 1-s
stimulation) were measured in current-clamp mode with an electrode
solution containing (in mM) 130-140 potassium gluconate, 10 NaCl, 5 MgCl2, 10 HEPES, 2 NaATP, 0.3 NaGTP, 0.5 EGTA,
0.05 CaCl2, pH 7.3. Synaptically evoked
glutamatergic currents were measured in voltage-clamp mode using an
electrode solution containing (in mM) 130-140 KF, 10 HEPES, 10 NaCl,
10 EGTA, pH 7.3.
Data analysis
We assessed induction and maintenance of LTP or LTD in slices
from the four strains. "Induction" was assessed as the fEPSP slope
measured 5 min after the end of high-frequency stimulation (for LTP
experiments) or immediately after 1-Hz stimulation (for LTD
experiments). Interstrain comparisons of induction efficacy were done
by pooling and averaging the fEPSP slopes measured at these time points
within each strain and by comparing the resulting mean slopes across
strains. "Maintenance" was assessed by calculating the ratio of the
fEPSP slope measured 50 min (for theta-burst LTP experiments), 100 min
(for all other LTP protocols), or 45 min (for LTD experiments) after
high- or low-frequency stimulation, to the fEPSP slope measured 5 min
after initiation of potentiation or immediately after initiation of
depression. The 50- and 100-min time points were chosen because the
inhibitory effects of PKA antagonists, which block maintenance (but not
induction) of LTP, are clearly evident at these time points
(Frey et al. 1993; Huang and Kandel 1994
;
Nguyen and Kandel 1997
). The ratio ("maintenance index") of the fEPSP slopes measured at these early and late time points was calculated for each slice and expressed as a percentage, and
the mean ratio was obtained for each strain by pooling and averaging
the ratios produced by a given stimulation protocol. Hence, a
maintenance index of zero would signify complete decay of fEPSP slopes
to control baseline values, and an index of 100 would signify no decay
from initial fEPSP slope values measured soon after high- or
low-frequency stimulation. Averaged data for induction and maintenance
of LTP and LTD are presented in Tables 1
and 2. Biophysical data are summarized in
Table 3.
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A single-factor analysis of variance (ANOVA) was used to assess whether at least one group (strain) was significantly different from the others, within a given stimulation regimen. To identify which particular pairs of strains showed significant differences in our measured electrophysiological parameters, we applied Student's t-test to those cases where the ANOVA revealed a significant difference between groups.
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RESULTS |
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CBA, DBA, and 129/SvEms mice are deficient in theta-burst LTP
Theta patterns of electrical activity in the hippocampus can occur
during environmental exploration and behavioral conditioning (Grastyan et al. 1959; Green et al. 1960
;
Vanderwolf 1969
), and LTP induced by theta-burst
patterns of stimulation (TBS) may be linked to the exploratory behavior
of rodents in spatially novel environments (Bland 1986
;
Larson et al. 1986
; Otto et al. 1991
; Staubli and Lynch 1987
).
In CBA, DBA, and 129/SvEms slices, TBS (15 bursts of four pulses at 100-Hz, delivered at an interburst interval of 200 ms) elicited initial increases in fEPSP slopes (measured 5 min after theta-burst stimulation) that were significantly less than those measured in BL6 slices (Fig. 1, Table 1). In slices from CBA and DBA mice, maintenance of theta-burst LTP was also significantly less robust than in BL/6J slices (Fig. 1, Table 2). These results indicate that genetic background may significantly influence the induction and persistence of theta-burst LTP in area CA1.
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Strain-specific rescue of LTP by a temporally compressed pattern of stimulation
Synaptoplastic processes in the hippocampus are believed to be
critically modulated by the temporal pattern of synaptic stimulation (Bear and Abraham 1996; Bito et al. 1996
;
Hawkins et al. 1993
; Madison et al.
1991
). Multiple bursts of high-frequency stimulation can elicit
long-lasting potentiation in the rodent hippocampus (see review by
Huang et al. 1996
). Previous studies have shown strain-dependent deficits in the expression of LTP following temporally spaced patterns of stimulation (e.g., "tetra-burst" stimulation: four 1-s bursts of 100-Hz, with bursts spaced 5 min apart;
Nguyen et al. 2000
). However, temporally compressed
patterns of stimulation can also induce robust and long-lasting LTP in
mouse hippocampal slices (Woo et al. 2000
), and these
compressed patterns more closely resemble the firing patterns of
hippocampal neurons in vivo (Kandel and Spencer 1961
).
We tested the hypothesis that there may be strain-dependent differences in the responsiveness of hippocampal synapses to distinct temporal patterns of synaptic activity. Slices from the four strains were stimulated with four 100-Hz bursts (1-s burst duration) at 20-s interburst intervals, and fEPSPs were tracked for 100 min after high-frequency stimulation. As shown in Fig. 2 and Table 1, induction of LTP was significantly less robust in slices from 129/SvEms mice than in BL6 slices. In CBA and DBA slices, the level of potentiation seen 5 min following tetra-burst stimulation with a 20-s interburst interval was not significantly different from the levels observed in BL6 slices (Table 1). In contrast, CBA and DBA slices showed significantly less robust maintenance of LTP than BL6 slices following tetra-burst stimulation with a 20-s interburst interval (Table 2). Furthermore, CBA slices showed significantly less robust maintenance of LTP than slices from 129/SvEms mice (Table 2).
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Extremely compressed tetra-burst stimulation (with just a 3-s
interburst interval) can induce long-lasting and robust LTP in
hippocampal slices prepared from PKA mutant mice (Woo et al. 2000). These mice show deficient LTP following temporally
spaced patterns of stimulation (Abel et al. 1997
). This
suggests that this compressed pattern of stimulation may be optimal for
recruiting subcellular processes that are important for LTP expression
at CA1 synapses. To test this hypothesis on these four inbred strains, we applied the same tetra-burst protocol as before, but we further reduced the interburst interval to just 3 s. With this protocol, we observed significantly greater induction and maintenance of LTP in
129/SvEms slices than with the 20-s interburst interval in this same
strain (Tables 1 and 2, see also Fig.
3A; compare with Fig.
2A). Furthermore, the initial level of potentiation seen in
129/SvEms slices with the 3-s protocol was no longer significantly different from that seen in BL6 slices (Table 1). Thus, the defect in
LTP induction seen in 129/SvEms slices with an interburst interval of
20 s can be rescued by reducing the interburst interval to just
3 s, without changing the total amount of imposed activity. In
contrast, this 3-s protocol decreased (though not significantly) the
average level of initial potentiation in CBA and DBA slices, as
compared with values measured following the 20-s protocol (Table 1).
Also, like the 20-s protocol, the 3-s regimen elicited significantly less robust maintenance of LTP in CBA and DBA slices as compared with
BL6 and 129/SvEms slices (Table 2). These results show that the
plasticity of hippocampal synapses in some inbred mouse strains is
sensitive to the temporal pattern of imposed activity, and they support
the notion that some temporally compressed patterns of stimulation may
be optimal for recruiting subcellular processes important for the
induction of robust and persistent LTP.
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Strain-independent induction of LTD
In contrast to LTP, induction and maintenance of LTD, elicited by 1-Hz stimulation for 15 min, were not significantly different between slices from BL/6J and each of the other strains (Fig. 4, Tables 1 and 2). Thus, LTP induction and maintenance were impaired in some of the strains examined here, while LTD was not significantly different between these same strains.
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Biophysical properties of hippocampal CA1 pyramidal cells are similar between strains
The observed differences in LTP expression among these four mouse strains may result from strain-dependent variations in the biophysical and electrophysiological properties of hippocampal neurons. Strain-selective alterations in cellular biophysical and electrophysiological properties could modify LTP expression by altering Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptor channels. For example, lower membrane input resistances would produce smaller postsynaptic membrane depolarizations for a given synaptic input, leading to weaker LTP induction and attenuated LTP expression. Similarly, a smaller contribution of NMDA currents to the total postsynaptic glutamatergic current, or a fewer number of action potentials evoked during sustained postsynaptic membrane depolarization (i.e., greater spike-frequency accommodation), may reduce postsynaptic Ca2+ influx and attenuate LTP expression.
We measured membrane resting potential (Em), membrane input resistance (Rin), spike-frequency accommodation, membrane depolarization in response to 100-Hz stimulation, and synaptically evoked glutamatergic currents in CA1 pyramidal neurons (in slices) using blind whole-cell patch-clamp techniques. Measurements of these biophysical properties are illustrated in Fig. 5, and the data are summarized in Fig. 6 and Table 3.
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Membrane excitability can be assessed by determining the spike firing
efficacy of a cell over a range of injected current amplitudes. To
measure membrane excitability in CA1 pyramidal neurons, we injected
current pulses (50-300 pA) (Fig. 5A) and counted the number
of spikes evoked at each current magnitude. Spike frequency
accommodation is a reduction in spike firing rate during a sustained
stimulus (Schwartzkroin 1977) and accommodation was
assessed by determining the delay of each spike in a spike train evoked
by 300-pA current injection.
Non-NMDA and NMDA receptor-mediated glutamatergic currents were
measured by stimulating the presynaptic Schaffer collateral pathway
(with an extracellular electrode) while varying the holding membrane
potential between 100 and +40 mV (Fig. 5B,
left). The non-NMDA receptor-mediated current was measured
as the early peak current, while the NMDA receptor-mediated current was
identified by measuring the evoked current magnitude 35 ms after the
peak. The I-V graph (Fig. 5B, right)
displays the rectifying current (NMDA component) measured 35 ms after
the peak, and the early linear current (non-NMDA component).
Finally, we measured the membrane depolarizations of CA1 pyramidal
neurons in response to a single 1-s burst of 100-Hz stimulation of
Schaffer collateral fibers (Fig. 5C). This measurement
assesses the degree of membrane depolarization experienced by the soma following activation of dendritic excitatory synapses. The
amount of somal depolarization seen after a single brief burst of
stimulation is dependent on the amount of cable filtering of EPSPs
imposed by the dendritic conductance load of a cell. The degree of
dendritic filtering of EPSPs, and thereby the net depolarization
experienced by the soma following brief stimulation, may vary according
to genetic background. Repeated bursts of 100-Hz stimulation were not
used because such repeated patterns of synaptic stimulation can lead to
rapid postsynaptic receptor modifications (e.g., AMPA receptor
mobilization/phosphorylation, Banke et al. 2000;
Shi et al. 1999
), which may alter the degree of somal
depolarization in a manner that may be independent of genetic background.
The number of action potentials counted at any given magnitude of injected current was not statistically different between strains (Fig. 6A, P > 0.2, single-factor ANOVA). To determine the average spike frequency accommodation, we fitted individual spike delay plots (Fig. 6B, left) with a fifth-order polynomial (R2 was always >0.98 after using a fifth-order polynomial) and averaged the plots for each strain (Fig. 6B, left). The ratio of initial slope (t = 0-50 ms) to late slope (t = 600-1000 ms) of these plots is a measure of the mean reduction in average spike frequency (Fig. 6B, right) during current injection, and the ratios obtained for these strains were not statistically different from each other (P > 0.1, single-factor ANOVA).
The ratio of the peak size of the synaptically evoked glutamatergic
current (measured at Em = 100 mV) to the
current amplitude 35 ms after this peak was used as an estimate of the
relative contribution of NMDA current to the total evoked glutamatergic current (Fig. 6C, Table 3). Again, no strain-dependent
differences were observed (P > 0.2, single-factor
ANOVA), suggesting that altered non-NMDA/NMDA receptor currents do not
account for the LTP deficiencies seen in some of the tested strains.
Membrane depolarizations during a single 1-s burst of 100-Hz stimulation (Figs. 5C and 6D) were variable, but they were not significantly different among the tested strains (P > 0.2, single-factor ANOVA; Table 3). This result suggests that strain-specific reduction of postsynaptic depolarization during high-frequency presynaptic stimulation cannot explain the observed strain-dependent differences in LTP induction.
cAMP-induced synaptic facilitation is deficient in selected strains
Transgenic mice with reduced hippocampal PKA activity have
defective maintenance of LTP (Abel et al. 1997). Are
there strain-specific differences in cAMP-dependent synaptic
facilitation? We explored this question by measuring the magnitudes of
synaptic facilitation induced by chemical activation of the cAMP-PKA
pathway (Chavez-Noriega and Stevens 1992
). Transient
co-application of the adenylate cyclase activator, forskolin, and the
phosphodiesterase inhibitor, IBMX (50 µM each), elicited variable
increases in fEPSP slope in some strains (Fig.
7). While 129/SvEms slices showed an
increase in fEPSP slope that was not significantly different from BL/6J
slices (BL/6J, 264 ± 37%; 129/SvEms, 212 ± 34%; P > 0.1), DBA and CBA slices showed significantly smaller synaptic
facilitation than BL/6J slices (DBA: 123 ± 15%, P < 0.01; CBA: 152 ± 30%, P < 0.05). The
forskolin/IBMX-induced facilitation in BL/6J slices was blocked by
Rp-cAMPS (100 µM, an inhibitor of PKA) (Fig. 7A), thereby
confirming that this form of synaptic facilitation is mediated by
activation of PKA. Hence, these data point to a strain-selective
deficit in PKA-dependent synaptic facilitation.
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DISCUSSION |
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Our study has revealed significant variations in the induction and maintenance of LTP in area CA1 of four selected strains of inbred mice. Hippocampal slices from CBA/J and DBA/2J mice showed less robust induction and maintenance of LTP than BL/6J slices following either tetra-burst high-frequency stimulation or theta-burst stimulation. Hence, defective induction and maintenance of LTP observed in mutant mice generated from CBA/J or DBA/2J strains may result from the genetic background of the parent strain rather than from the genetic manipulation per se.
The deficient induction of theta-burst LTP seen in DBA/2J, CBA/J, and
129/SvEms/J slices correlates with these strains' poor performance on
the Barnes circular platform maze, a test of spatial learning and
memory (Nguyen et al. 2000). LTP induced by theta-burst stimulation may be linked to the exploratory behavior of rodents in
spatially novel environments, and theta patterns of electrical activity
in the hippocampus can occur during such exploratory behavior
(Bland 1986
; Larson et al. 1986
;
Otto et al. 1991
). Although we have demonstrated that
maintenance of theta-burst LTP is strain-dependent, the exact
mechanisms responsible for these strain-specific deficits are
undefined. One possible cellular basis for the strain-specific variation in theta-burst LTP seen here is that strain- and
activity-dependent differences in the strength or duration of
feed-forward synaptic inhibition (Alger and Nicoll 1982
;
McCarren and Alger 1985
) within the in vitro slice
preparation may affect theta-burst LTP induction. Further research is
needed to explore the role of synaptic inhibition in regulating LTP in
these inbred strains.
Previous studies have shown differential expression of LTP in the
dentate gyrus of specific inbred strains of anesthetized mice (129 Ola,
C3H, C57 albino, DBA/2, and FVB/N strains: see Bampton et al.
1999). These studies used temporally compressed theta-pattern
stimulation for longer periods of time than the regimen used in our
present study. The study by Bampton et al. (1999)
, and
our present findings, suggest that a strain-specific deficit in
theta-burst LTP induction may exist in multiple distinct regions of the
hippocampus both in in vitro and in vivo preparations. The collective
results suggest that the learning and memory deficits reported for
DBA/2 mice may be linked to defective theta-burst LTP in more than one
sub-region of the hippocampus.
Although we observed no significant differences in LTD among slices of
these four strains, it should be noted that we have only used one
stimulation protocol to induce LTD (1 Hz, 15 min). This protocol was
examined because of its reliability in producing measurable LTD at
rodent hippocampal synapses (Dudek and Bear 1992). We
did not examine the effects of different temporal patterns of
low-frequency stimulation on LTD in these strains, since delivering the
same number of stimulus pulses at higher frequencies may engage processes that elicit LTP (Dudek and Bear 1992
) in a
manner that may be dependent on other factors, such as kinase
activation dynamics (see Mayford et al. 1995b
).
Hippocampal synapses may be "tuned" to particular temporal patterns of electrical activity, and the subcellular processes responsible for activity-dependent plasticity at these synapses may be optimally engaged by select patterns of electrical activity. An important finding presented here is the observation that the temporal pattern of synaptic stimulation can critically modulate the induction and maintenance of hippocampal LTP in a strain-specific manner. Induction and maintenance of LTP were enhanced in 129/SvEms/J slices following repeated stimulation using 3-s interburst intervals, while the same total amount of imposed activity produced less robust induction and maintenance of LTP when a 20-s interburst interval was applied. In the other three strains tested, changing the interburst interval from 20- to 3-s, while keeping the total imposed activity constant, did not significantly affect the induction or maintenance of LTP. Extremely compressed stimulation patterns, such as the 3-s interval protocol used here, might be more effective (than spaced stimulation) at recruiting subcellular processes important for LTP induction. Our results indicate that such activity-sensitive processes may be expressed in a manner that is dependent on genetic background. In other words, the hippocampal neurons of some mouse strains may be more optimally tuned to particular temporal patterns of synaptic activity because of strain-related variations in genetic background. Our data also underscore the importance of using various temporal patterns of synaptic stimulation when searching for altered synaptic plasticity in genetically modified mice.
The biophysical membrane properties and cellular electrophysiological
attributes of hippocampal CA1 pyramidal neurons did not significantly
vary between the four strains examined here. Our data complement and
further extend previous research that showed no marked strain-dependent
variations in synaptic input-output coupling in area CA1 (Nguyen
et al. 2000; see also Bampton et al. 1999
for
dentate gyrus data). We observed no significant strain-specific differences in membrane input resistance, spike frequency
accommodation, and membrane depolarization (during 100-Hz stimulation)
in CA1 pyramidal cells. Thus, variation in genetic background among
these four inbred strains does not appear to cause significant
variations in membrane biophysical properties or spike firing efficacy
of CA1 neurons in these strains.
Glutamatergic receptors, such as NMDA and non-NMDA receptors, are
important for the induction of hippocampal LTP (Malenka and
Nicoll 1993), but no significant strain-dependent variation in
the sizes of synaptically evoked NMDA and non-NMDA currents was
observed in the present study. These data are consistent with those
reported by Jia et al. (1998)
, which showed that, for
some inbred and hybrid strains (129 Sv, 129 Sv × C57BL/6, and 129 Sv × CD1), LTP of AMPA- and NMDA-type currents was independent of genetic background. We conclude that the genetic backgrounds of the
strains examined here do not significantly alter the amplitudes of
synaptically evoked glutamatergic currents as measured under the
experimental conditions used here. However, there may be more subtle
strain-dependent differences in the expression or modulation of these
receptors which we have not examined in the present study.
The most intriguing finding of the present study is the lack of robust
cAMP/PKA-dependent synaptic facilitation in CBA/J and DBA/2J slices.
Forskolin enhances synaptic transmission in hippocampal area CA1 in a
manner that requires adenylate cyclase activity (Chavez-Noriega
and Stevens 1992). Co-application of forskolin with IBMX was
used in the present study because such treatment increases the
reliability of induction of robust synaptic facilitation in area CA1
(Chavez-Noriega and Stevens 1992
). IBMX blocks adenosine receptors and inhibits phosphodiesterases in the hippocampus
(Smellie et al. 1979
). Both actions can increase cAMP
levels, since tonic activation of adenosine receptors in area CA1
(Dunwiddie and Hoffer 1980
) has been correlated with
inhibition of adenylate cyclase (Chavez-Noriega and Stevens
1992
; Dunwiddie and Fredholm 1989
; Fredholm et al. 1983
). Our block of
forskolin/IBMX-induced synaptic facilitation by a specific inhibitor of
PKA (Rp-cAMPS) confirms that this enhancement of synaptic transmission
was mediated through PKA.
It is noteworthy that CBA/J and DBA/2J mice are defective in some forms
of hippocampus-dependent spatial and nonspatial memory (Nguyen
et al. 2000). PKA is critical for the maintenance of
hippocampal LTP in area CA1 and for hippocampus-based memory
consolidation (Abel et al. 1997
). Our data suggest that
the deficits in spatial and nonspatial memory seen in CBA/J and DBA/2J
mice (Nguyen et al. 2000
) may be correlated with
deficient cAMP-induced (and PKA-dependent) synaptic facilitation in
hippocampal area CA1. Ours is the first study to demonstrate natural,
genotype-related variations in PKA-dependent forms of synaptic
facilitation in the hippocampus. Further research is needed to
determine whether this strain-specific deficit in PKA-dependent
synaptic facilitation is correlated with altered hippocampal expression
of particular PKA subunits (for preliminary data, see Duffy et
al. 2000
) or modified levels of hippocampal PKA activity.
In summary, our findings underscore the importance of careful
selection of strains for use in generating genetically modified mice.
These inbred strains should be screened for physiological phenotypes
that may confound the interpretation of results derived from the
electrophysiological analysis of genetically modified mice produced
through the cross-breeding of these strains. Our study has shown that
activity-dependent synaptic plasticity is significantly influenced by
genetic background and by the particular temporal pattern of
stimulation used for examining synaptoplastic mechanisms in hippocampal
slices. Hippocampal synapses in particular strains, such as
129/SvEms/J, appear to be tuned to specific temporal patterns of
synaptic activity. In contrast, hippocampal synapses in other strains,
such as C57BL/6J, CBA/J, and DBA/2J, appear to be less susceptible to
subtle changes in the temporal pattern of imposed synaptic activity.
Pyramidal neurons in area CA1 of the four strains tested here showed no
significant differences in membrane biophysical properties, spike
firing efficacy, or glutamatergic synaptic transmission (as measured
with whole-cell patch-clamp techniques). However, in some strains,
cAMP-dependent synaptic facilitation was attenuated, suggesting that
some of the observed strain-specific deficits in LTP may (directly or indirectly) result from altered signaling through the cAMP-PKA pathway.
We suggest that genetic background can critically influence the
efficacy or expression of intracellular synaptoplastic mechanisms involving signal transduction processes such as phosphorylation and
dephosphorylation. These mechanisms may include components of specific
kinase/phosphatase cascades, or substrate proteins regulated by, but
located downstream from, these modulatory cascades. In contrast, the
electrophysiological responses of synaptic glutamatergic receptors and
membrane excitability appear to be less susceptible to significant
alterations caused by variations in genetic background. Hence,
strain-dependent variations in genetic background may selectively affect intracellular processes in hippocampal neurons, and
alterations of some of these processes (e.g., cAMP-mediated signal
transduction) may underlie some of the documented strain-dependent
deficits in hippocampus-based learning and memory (Crawley et
al. 1997; Nguyen et al. 2000
; Paylor et
al. 1993
; Wehner et al. 1990
).
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ACKNOWLEDGMENTS |
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P. V. Nguyen held Scholarship awards from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Foundation for Medical Research (AHFMR). S. N. Duffy is a Postdoctoral Fellow of the AHFMR.
This work was supported by grants and funds (to P. V. Nguyen) from the AHFMR, the CIHR, and the University of Alberta Faculty of Medicine.
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FOOTNOTES |
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Address for reprint requests: P. V. Nguyen, Dept. of Physiology, University of Alberta School of Medicine, Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
Received 26 April 2000; accepted in final form 4 August 2000.
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REFERENCES |
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