Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Gusev, Pavel A. and Daniel L. Alkon. Intracellular Correlates of Spatial Memory Acquisition in Hippocampal Slices: Long-Term Disinhibition of CA1 Pyramidal Cells. J. Neurophysiol. 86: 881-899, 2001. Despite many advances in our understanding of synaptic models of memory such as long-term potentiation and depression, cellular mechanisms that correlate with and may underlie behavioral learning and memory have not yet been conclusively determined. We used multiple intracellular recordings to study learning-specific modifications of intrinsic membrane and synaptic responses of the CA1 pyramidal cells (PCs) in slices of the rat dorsal hippocampus prepared at different stages of the Morris water maze (WM) task acquisition. Schaffer collateral stimulation evoked complex postsynaptic potentials (PSP) consisting of the excitatory and inhibitory postsynaptic potentials (EPSP and IPSP, respectively). After rats had learned the WM task, our major learning-specific findings included reduction of the mean peak amplitude of the IPSPs, delays in the mean peak latencies of the EPSPs and IPSPs, and correlation of the depolarizing-shifted IPSP reversal potentials and reduced IPSP-evoked membrane conductance. In addition, detailed isochronal analyses revealed that amplitudes of both early and late IPSP phases were reduced in a subset of the CA1 PCs after WM training was completed. These reduced IPSPs were significantly correlated with decreased IPSP conductance and with depolarizing-shifted IPSP reversal potentials. Input-output relations and initial rising slopes of the EPSP phase did not indicate learning-related facilitation as compared with the swim and naïve controls. Another subset of WM-trained CA1 PCs had enhanced amplitudes of action potentials but no learning-specific synaptic changes. There were no WM training-specific modifications of other intrinsic membrane properties. These data suggest that long-term disinhibition in a subset of CA1 PCs may facilitate cell discharges that represent and record the spatial location of a hidden platform in a Morris WM.
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INTRODUCTION |
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A number of specific
intracellular correlates of learning such as increased membrane
excitability (Alkon et al. 1982; Coulter et al.
1989
; Moyer et al. 1996
; Schreurs et al.
1998
; Thompson et al. 1996
), facilitated
synaptic transmission (McKernan and Shinnik-Gallagher
1997
; Power et al. 1997
), or both
(LoTurco et al. 1988
) have been found in brain slices
prepared following behavioral training of animals. Furthermore
experience-dependent plasticity has been associated with changes in
synaptic dynamics found in neocortical slices (Fennerty et al.
1999
; Rioult-Pedotti et al. 2000
). The CA1 area
of the dorsal hippocampus (HC) is critical for acquisition,
consolidation, storage, and retrieval of spatial memory (Moser
et al. 1995
; Riedel et al. 1999
; Whishaw
et al. 1994
) and contains a higher percentage of place cells
with well-defined and focused place fields (Jung et al.
1994
). Activity of these cells also has behavioral and reward
correlates (Breese et al. 1989
; Kobayashi et al.
1997
). Learning-related, long-term changes of the HC place cell
responses have been found after spatial learning in new environments
(Breese et al. 1989
; Hollup et al. 2001
;
Kentros et al. 1998
; Kobayashi et al.
1997
; Moser et al. 1999
; Nishijo et al.
1999
; Wilson and McNaughton 1993
). These
modified place cell responses are thought to encode and subsequently
store new information about spatial location of reinforcement by
increased firing rates in old and newly developed place fields
(Breese et al. 1989
; Hollup et al. 2001
;
Kobayashi et al. 1997
; Moser et al. 1999
;
Nishijo et al. 1999
). Long-term potentiation (LTP) of the excitatory synaptic inputs to the pyramidal cells (PCs) has been
considered to be a likely, but as yet not entirely confirmed, mechanism
for these place cell enhanced responses (Bliss and Collingridge 1993
; Hoh et al. 1999
; Kentros et al.
1998
; Staubli et al. 1995
,1999
; Tsien et
al. 1996
; Zamanillo et al. 1999
). Interneurons,
on the other hand, dynamically control the discharge rate (Miles
et al. 1996
) and collective activity (Cobb et al.
1995
) of the CA1 PCs during rats' exploratory behavior and
rest intervals (Csicsvari et al. 1999
; Paulsen
and Moser 1998
; Stewart 1993
; Wilson and McNaughton 1993
). To study possible membrane and/or synaptic
modifications of CA1 PCs at different stages of spatial memory
acquisition (Hoh et al. 1999
; Morris
1984
), we extended methods here that combine in vivo and in
vitro preparations (Alkon et al. 1982
; Coulter et
al. 1989
; Disterhoft et al. 1986
; LoTurco
et al. 1988
; Moyer et al. 1996
;
Sanchez-Andres and Alkon 1991
; Schreurs et al.
1998
; Thompson et al. 1996
) by recording from
slices of the dorsal HC found to be critical for water maze (WM)
spatial learning (Moser et al. 1995
; Reidel et
al. 1999
; Whishaw et al. 1994
) from animals exposed to training or control protocols. We found that CA1 PCs receive
reduced synaptic inhibition following complete acquisition of WM task.
This mechanism may contribute to the previously observed relocations of
the neuronal place fields and increases in the PCs' place-related
activity during spatial learning (Breese et al. 1989
;
Hollup et al. 2001
; Kobayashi et al.
1997
; Moser et al. 1999
; Nishijo et al.
1999
).
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METHODS |
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Training procedure
Male Wistar rats (ca 280 g) were trained on a Morris WM task in a swimming pool (1.5 m diam and 0.6 m high, filled with milky water, 23-25°C range) that was located in a well-lit room with distinct extra maze cues. A 10-cm2 transparent square platform was hidden in a constant location (quadrant center) within the pool with its top surface submerged 1.5 cm below the water level. On day 0, rats swam for 2.5 min in the pool in the absence of the platform. For the next 1 day (WMT1 group; clear water was used for the short-term WM training and swimming) or 3 days (WMT3 group), rats were trained to locate the hidden island in four trials per day. On the first day, an animal was guided to the platform if it did not find it in 2 min. Rats were then allowed to stay on the platform for 40 s before starting the next trial from another quadrant. Rats from the swim control groups swam for 2 min a day in the pool without the island for the next 1 (SW1 group) or 3 days (SW3 group), respectively. Spatial memory of rats used for in vitro recordings was assessed according to the time required to find the platform (escape latency). Transfer tests 24 h after 1 or 3 days of WM training were performed on parallel behavioral groups of the rats not used for recordings. During the transfer tests, rats were started in the quadrant opposite to the target and swam for 60 s in the pool without a platform. Spatial memory in these groups was assessed as dwell time and distance swum in four pool quadrants with video tracking system Poly-Track (San Diego Instruments). Naive rats also housed individually spent approximately the same number of days (8-10) in their home cages at the National Institutes of Health (NIH) animal facility after delivery from the Charles River Laboratory and before intracellular recordings. The animals' care was in accordance with the NIH guidelines.
Slice preparation
Twenty-four hours after the last behavioral session, rats were
decapitated by a small animal guillotine in accordance with the
NIH-approved protocol. The whole brain was removed, and both hippocampi
were quickly dissected out in ice-cold sucrose-artificial cerebrospinal
fluid ACSF (Moyer et al. 1996; Thompson et al.
1996
) [containing (in mM) 248 sucrose, 5 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose, gassed with
95% O2-5% CO2, pH 7.4].
Transverse slices (400 µm) were cut from the dorsal parts of both HC
with a McIlwain tissue chopper and placed in an interface chamber
(31°C, Fine Science Tools) where they were incubated for 30 min under
sucrose-ACSF, and for the next 1 h under normal ACSF before
starting recordings. Slices were in contact with a solution containing
(in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, and 11 D-glucose, gassed with
95% O2-5% CO2, pH 7.4, with a perfusion speed 5-6 ml per min.
Electrophysiology
These recording conditions were chosen to minimize alterations
due to pharmacological intervention(s) and voltage-clamp manipulations that are required to record inhibitory postsynaptic currents (IPSCs). Intracellular recordings were made with microelectrodes fabricated from
thick-walled glass (2 mm OD, 1 mm ID; FHC, Bowdoinham, ME) on an
electrode puller (NE-2; Narishige, Tokyo) and filled with 3 M potassium
acetate (DC resistance, 80-120 M). An Axoclamp 2B amplifier was
employed in the bridge mode (Axon Instruments, Foster City, CA).
Electrodes were positioned with the aid of a binocular dissecting
microscope (Wild, Switzerland, magnification was up to ×50). Recording
microelectrodes were advanced by a Leitz micromanipulator (Wetzlar,
Germany). Data were acquired and analyzed with the aid of pClamp 6 software (Axon Instrument) at A/D sampling rates 5-10 kHz using a
DigiData-1200 series interface and PC Pentium (Dell, Austin, TX).
Before starting recordings, a field response in the stratum pyramidale
to a standard stimulation of Schaffer collaterals was measured.
Experiments were continued if population spikes did not show any
evidence of multiple afterpotentials. Recordings were performed from
319 neurons located in the st. pyramidale of the
CA1b area (about the middle of the length of st.
pyramidale between the fimbria and subiculum) to avoid differences in
the cell properties due to the cells' location (Masukawa et al.
1982
). Recordings were identified as somatic and from PCs based
on accepted electrophysiological criteria. CA1 neurons were included in
the study as PCs if they had an action potential amplitude
70 mV from
the spike threshold, an action potential duration 1.2 ms from rise
threshold to return to baseline membrane potential (Vm), a postburst AHP, impulse
frequency accommodation, and spontaneous activity well below 0.001 Hz
(Moyer et al. 1996
; Thompson et al. 1996
). In addition, accepted cells had a stable input
resistance
25 M
and resting Vm
more negative than
60 mV. A constant hyperpolarizing current of
0.2
nA was applied to provide stability of
Vm close to
65 mV, if necessary.
Only one cell was recorded from a given slice. We positioned a
microconcentric bipolar stimulating electrode (FHC) in the middle part
of the st. radiatum to evoke complex postsynaptic potentials (PSPs)
recorded from PCs by Schaffer collateral stimulation. We maintained the
same distance (~120-150 µ) between the stimulating and recording
electrodes throughout the experiments. Many of neuronal parameters were
measured according to criteria of previous studies performed in this as
well as other labs (Coulter et al. 1989
;
Disterhoft et al. 1986
; LoTurco et al.
1988
; Moyer et al. 1996
; Thompson et al.
1996
).
Experimental protocol to study intrinsic membrane properties
The experimental protocol to study membrane properties of PCs
was as follows: 1) 700-ms 0.5-nA hyperpolarizing current
pulses were applied to evaluate an input resistance
(Rin) and to control the balanced
bridge throughout the recording. The averages of 10 plateau voltage
deflections were used for the Rin
evaluation. 2) One-hundred-millisecond depolarizing current
pulses were applied to establish the current strength to evoke robust
one or four spikes and also to study action potential parameters. Ten
to 15 samples were collected per cell. 3) The average
maximum voltage deflection following current offsets obtained during
five injections of 100-ms current pulses was used to evaluate the slow
afterhyperpolarization (sAHP) following one and four spikes. The second
maximum was measured in the biphasic AHPs. We studied both single- and
postburst slow AHPs to avoid possible masking effects of saturation on
learning-induced changes. 4) The average numbers of action
potentials obtained during each of five 800-ms intracellular current
pulses of the same intensity that was used for the postburst sAHP study
served to evaluate spike frequency accommodation of CA1 PCs.
5) Current-voltage relations (I-V) were studied
using 700-ms current pulses with intensities ranging from 0.5 up to
+0.7 nA. Three samples of I-V traces were collected for each
cell. Average peak and plateau voltage deflections were used to
evaluate slope membrane Rin in response to hyper- and depolarizing current pulses (I < 0, I > 0).
Rin,I>0/Rin,I<0
ratios were used to evaluate membrane rectification properties.
Experimental protocol to study synaptic inputs
Stimulation of the Schaffer collateral input was employed to
assess synaptic properties of the CA1 PCs after spatial learning. The
following measurements were made: 1) input-output relations (I-O) of the excitatory component of the complex PSPs were studied by
applying 0.4-ms current pulses at 0.1 Hz to the st. radiatum. To
standardize the stimulus intensity, current intensity was used to
elicit 2-mV excitatory PSP (EPSP) increments until action potential generation. Twenty measurements of PSPs were taken for all current intensities. Average EPSP amplitudes were used to construct I-O plots
and to calculate the slopes of linear regression of the current
intensity with the EPSP amplitude. 2) The initial EPSP rising slopes were measured as EPSP amplitudes elicited in the first
milliseconds above the baseline Vm
level. Maximum subthreshold EPSPs were used in this analysis.
3) Analysis of the averaged PSPs containing maximum
subthreshold EPSPs (maximum PSPs) was performed at peaks of the EPSP
and inhibitory PSP (IPSP) phases and at 40-, 200-, and 350-ms time
points relative to the stimulus artifact. 4) Reversal
potentials (EIPSP) for the early and
late IPSP phases were evaluated at 40- and 150-ms latencies,
respectively. Averages of 5 PSPs elicited by Schaffer collateral
stimulation (approximately at half of spike threshold) at different
holding Vm were measured and plotted
against Vm. Data points for the early IPSP were fit with linear functions, and
EIPSP was determined from a
single linear fitting equation by interpolating to zero PSP amplitude
(Jensen et al. 1993). Data points for the late IPSP were
fit by one or two linear functions, and
EIPSP was determined from the second
linear fitting equation usually applied with data obtained at the
Vm held below
70 mV. And
5) changes in membrane conductance introduced during early
and late IPSPs (gIPSP) were estimated
according to the relation: gIPSP = 1/RIPSP
1/Rin where RIPSP is the total input resistance
during the IPSP, Rin is the resting
input membrane resistance (Hablitz and Thalmann 1987
). I-V relationships were plotted for the resting state, for
40- and 150-ms IPSP time points. Analyses of I-V
relationships were performed on the portion of the I-V
curves that was linear. The slopes of the resulting I-V
regression lines were used to estimate Rin and
RIPSP, respectively.
Data analysis
The rats were trained, slices were prepared, and recordings were
performed by the same person. To avoid bias, data were processed only
after the final sizes of the WMT1 and SW1, WMT3 and SW3 samples of
cells were reached. Thus the records were taken without benefit of
knowledge of effects that emerged after the extensive data analyses
were performed. All data sets were first tested for the normality of
their distributions with the Kolmogorov-Smirnov test. Acquisition of
spatial memory in the Morris WM task and the effects of training on the
mean values of cellular parameters were then evaluated with a one-way
ANOVA test followed by an F test (variance equality test)
and a Fisher's post hoc test where it was applicable (Statview, SAS
Institute). Effects of behavioral training on the frequency
distributions obtained for values of cellular parameters were analyzed
with 2 test (Microsoft Excel, Microsoft). The
bins were not chosen arbitrarily. Rather the bin size for the raw data
selected to be large enough to ensure adequate sample size per bin for
statistical testing (Hays 1963
). Once selected, the same
bin size was applied, in accordance with accepted practice, to all
experimental and control groups. Physiological changes were considered
learning-specific when there was a statistically significant difference
between the WM trained rats and both control groups (comparably treated swim control and naïve animals). Comparing swim control animals to naïve control allowed us to extract nonspecific changes
related to the learning context (e.g., arousal, stress, and physical activity).
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RESULTS |
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Water-maze learning
Escape latency for the first trials of WM training was already
significantly improved in the second day of training as compared with
the first day [F(1,18) = 6.33, P < 0.05, 1-way ANOVA; Fig. 1A, trials 1 and 5]. However,
the escape latency is not a sufficient indicator of spatial learning
since it does not necessarily reflect spatial memory formation. Past
work suggests that the rats have mainly learned a strategy for the WM
task including climbing a hidden platform and searching for it
(Hoh et al. 1999). Indeed transfer tests performed
24 h after 1 day of training indicated that rats did not show
clear spatial preference for the trained quadrant as was measured by
dwell time [F(3,36) = 1.082, P > 0.3; Fig. 1B] and by distance swum in
the target and control quadrants [F(3,36) = 0.601, P > 0.6, Fig. 1C]. SW1 rats spent most of the time swimming along the walls and trying to climb them.
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Twenty-four hours after 3 days of WM training, during transfer tests,
however, rats showed strong spatial bias in their swim pathways as was
indicated by increased mean dwell time and length of swim tracks in the
trained quadrant as compared with control quadrants
[F(3,36) = 5.25, P < 0.01; P < 0.01, P < 0.02, P < 0.001, Fisher's post hoc test;
F(3,36) = 6.626, P < 0.01; P < 0.01; P < 0.001, P < 0.01 Fig. 1, B and C,
respectively]. There was no spatial bias in the dwell time and swim
track lengths in the SW1 [F(3,36) = 1.719, P > 0.2;
F(3,36) = 2.747, P > 0.05, respectively; Fig. 1, B and
C] and SW3 control groups
[F(3,36) = 1.26; P > 0.3; F(3,36) = 1.439, P > 0.2, respectively; Fig. 1, B and
C]. After 3 days, the swim controls did not change their
pattern of swimming. Swim tracks of control rats were mainly along the
walls of the pool. These behavioral results suggest that acquisition of
the WM task was complete (Morris 1984) and a spatial
representation had been formed in the HC after 3 days of WM training
but not after 1 day of WM training.
Time course of EPSP and IPSP phases and isochronal analyses
The complex PSPs induced by Schaffer collateral stimulation
include activation of several conductances underlying the EPSP and IPSP
phases initiated by glutamate release from the CA3 PC presynaptic
terminals (Andreasen and Lambert 1998; Andreasen
et al. 1989
; Collingridge et al. 1988
)
and GABA release from a number of different interneurons which target
different structural domains of the CA1 PCs (Buhl et al.
1994
; Nurse and Lacaille 1997
; Paulsen and Moser 1998
). Somatic (early) and dendritic (late) IPSPs in the HC have been implicated in the control of the output and input of
the PCs. Powerful inhibitory control by interneurons is mediated both
through PC hyperpolarization and decreases in PC membrane resistance.
Fast IPSPs arise mostly in the somata, while IPSPs with slow rise to
peak are of dendritic origin (Pearce 1993
). Early IPSPs
with fast and slow onsets are mediated by GABAA
postsynaptic receptors that control both chloride and bicarbonate
conductances (Perkins and Wong 1996
; Staley et
al. 1995
). Monosynaptic and isolated early
(GABAA) IPSPs evoked by strong stimulation of the st. radiatum mostly return to the baseline in 300-400 ms
(Billard et al. 1995
; Davies and Collingridge
1993
; Nathan and Lambert 1991
; Roepstorff
and Lambert 1994
; Xie et al. 1995
). Late
(GABAB) IPSPs last for ~600-1,000 ms, are
maximal after ~150 ms, and are mediated by diverse subclasses of
GABAB postsynaptic receptors regulating several
potassium conductances (Billard et al. 1995
; Hablitz and Thalmann 1987
; Lopantsev and
Schwartzkroin 1999
; Pham and Lacaille 1996
;
Xie et al. 1995
). Finally, IPSP diversity appears as
differences in the IPSP time courses, membrane conductance increases,
and levels of the reversal potentials.
Because of the complex and diverse nature of the PSPs in the HC, we
focused our study on those IPSPs which peaked in 70 ms. The vast
majority of the maximum IPSPs that showed this property had similar
time courses, showing large early IPSP phases and smaller amplitude
late IPSP phases (Fig. 2, B
and C). [The minor fraction of the total PSP population
with IPSP time-to-peak >70 ms had very distinct responses either with
2 almost equal size peaks or small IPSP components (Fig. 2,
B and D).]
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Given that the average half-width of isolated EPSPs is ~18-21 ms
(Andreasen and Lambert 1998; Collingridge et al.
1988
), the IPSP phase at 40 ms will not be significantly
contaminated by the EPSP, and IPSPs at 200 and 350 ms should not be
affected by EPSPs at all. In current-clamp recordings performed in
normal ASCF, brief EPSPs are mostly mediated by AMPA receptors.
Expression of the N-methyl-D-aspartate (NMDA)
component of the EPSP is prevented by concurrently activated IPSPs,
which rapidly hyperpolarize neurons into a range of
Vm at which Mg2+
blocks NMDA channels. NMDA currents have much longer half-width (~60
ms) if measured at holding Vm about
30 mV and under the presence of the GABAA
antagonist picrotoxin (Collingridge et al. 1988
).
Furthermore even with effective blockade of inhibition, the
I-V relation for the NMDA component does not demonstrate a conductance increase in the holding Vm
range from
80 up to
60 mV (Andreasen et al. 1989
).
To analyze learning-induced between-group differences, we chose a
sample of IPSPs with as similar time courses as possiblei.e., with
peaks
70 ms. The sampling rate (10 kHz) for the recorded PSPs was
associated with minimal error for the PSP amplitude measurement. Figure
2A illustrates the reproducibility of the maximum IPSP amplitude and waveform. Furthermore each reported value for the isochronal analyses was the average of 20 responses. Note the transition of the early IPSP phase into the late IPSP phase. The early
IPSPs were measured at 40-ms latency relative to the time of the st.
radiatum stimulation. The late IPSP phases were measured at 350-ms
latency when they were not significantly contaminated by early
responses as indicated by the difference in the
gIPSP and
EIPSP that was below
ECl
(about
85 mV) (Kaila et al. 1993
; see also Swearengen and
Chavkin 1989
) (Figs. 5 and 6, this study). Bicuculline
did not significantly affect the late IPSP conductance measured at the
late IPSP peaks (165-340 ms after strong Schaffer collateral
stimulation) (Swearengen and Chavkin 1989
). The
transitional phases between both the early and late IPSP components
were measured at a 200-ms time point.
Reduced IPSP amplitude after water-maze learning
Slices were prepared from the dorsal HC 24 h after completing one of the four behavioral procedures and from the HC of naïve control animals (n = 20, number of animals). These procedures included 1 or 3 days of WM training (WMT1 and WMT3 groups; n = 21 and 20, respectively) and 1 or 3 days of swimming without a platform in the pool (SW1 and SW3 groups; n = 20 and 20, respectively).
First, analyses of the IPSPs with time to peak 70 ms showed that the
WMT3 group had a learning-specific reduction in the mean peak IPSP
amplitude. This IPSP amplitude was smaller as compared with
naïve and SW3 controls, and WMT1 group
[F(4,219) = 3.435, P < 0.01; P < 0.01, P < 0.05, respectively, Fisher's post hoc test; Table
1]. There were no learning-specific
changes in the mean peak IPSP amplitude in the WMT1 cells as compared
with the SW1 and naïve cells (P > 0.1, P > 0.7, respectively; Table 1).
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In addition to this major learning-specific synaptic correlate of spatial memory acquisition in the WMT3 rats, we studied specifically the role of the early and late IPSPs in the observed disinhibition. The findings described in the following text are based on the results of isochronal analyses and, in general, demonstrate that animals trained for 3 days had IPSPs with reduced early and late phases. These early and late IPSP amplitude reductions were consistently manifest in significant between-group differences in IPSP distributions. These significant IPSP distribution differences occurred in the absence of learning-specific differences of passive membrane properties. Between-group differences in isochronal mean IPSP measurements, however, were not significantly different in all cases.
Analyses of the recorded PSPs from whole populations of neurons were also conducted (see following). Our main conclusions about the learning-specific synaptic changes in the dorsal HC were similar and independent of this classification of the data on the basis of IPSP dynamics.
Increased IPSP and EPSP peak latencies after water-maze learning
There was a learning-specific increase in the mean times to IPSP peaks in the WMT3 rats as compared with the naïve (P < 0.05) and SW3 rats [F(4,219) = 2.765, P < 0.05; P < 0.05; Table 1]. At the early stage of training in the WMT1 cells, this increase was not learning-specific as compared with SW1 (P > 0.9, Table 1). Representative PSP traces from the WMT3 and control (SW3, naïve) animals illustrated longer times to the IPSP peak (Fig. 4). A weaker inhibition may account for an observed learning-specific increase in the mean latency of the EPSP peak in the WMT3 group as compared with the naive [F(4,219) = 4.4, P < 0.05; P < 0.05], WMT1, SW1 groups and SW3 rats (P < 0.05, P < 0.05, P < 0.05, respectively, Table 1; see Fig. 4; Fig. 8 for representative traces).
Early IPSP phase
Because of the complexity of the membrane conductances underlying the PSPs, an isochronal measure (e.g., 40-ms time point) provided an objective means of comparing IPSP amplitudes between groups.
Analyses of IPSPs at a 40-ms latency also showed reduction of the mean
IPSP amplitude in the WMT3 group as compared with naïve, WMT1,
and SW1 rats [F(4,219) = 4.249, P < 0.01; P < 0.05, P < 0.001, P < 0.05, respectively], but not as
compared with SW3 rats. However, as was shown in previous studies,
learning-specific intracellular changes may appear only in a subset of
and not in the entire population of the recorded cells. In such cases
mean values may not sufficiently reflect learning-related modifications
(Sanchez-Andres and Alkon 1991; Schreurs et al.
1998
). In this study, therefore we analyzed between-group
differences of IPSP distributions. Based on the total number of
recordings and the range of IPSP amplitudes for each isochronal
measure, the minimum binwidth that provided a testable bin size proved
to be 20% of the overall amplitude range (Fig.
3). The distribution analyses with
2 test did reveal a learning-specific increase
in the fraction of cells with smaller early IPSPs in the WMT3 group as
compared with the SW3 group (P < 0.05; Fig.
3A). There were more IPSPs with amplitudes
7 mV in the
WMT3 slices as compared with the SW3, SW1, WMT1, and naïve
slices. Representative traces from nine PCs recorded in the dorsal
hippocampal slices obtained from the WMT3 and control (SW3,
naïve) animals are illustrated in Fig.
4. There were no learning-specific
changes in the WMT1 mean IPSP amplitude and distribution as compared
with the SW1 and naïve groups (P > 0.1, P > 0.4; P > 0.07, P > 0.5, respectively).
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Late IPSP phase
Isochronal measures (e.g., 200- and 350-ms time points) provided
an objective means of comparing late IPSP amplitudes between groups.
Late phases had reduced mean amplitudes in the WMT3 group of rats as
compared with naïve, WMT1 and SW1 rats at 200 ms
[F(4,219) = 14.947, P < 0.0001; P < 0.01; P < 0.0001, P < 0.0001, respectively; Table 1, Fig.
3B], and at 350 ms
[F(4,219) = 15.952, P < 0.0001; P < 0.05, P < 0.0001, P < 0.0001, respectively; Table 1, Fig. 3C] but not as compared with the SW3 rats. The observed
IPSP reduction was a learning-specific effect, however, as indicated by
the larger fraction of the WMT3 neurons with reduced late IPSP phases
as compared with the SW3 control neurons (P < 0.01, P < 0.01; at 200 and 350 ms, respectively; Fig. 3,
B and C; for representative traces see Fig. 4).
There were more IPSPs with amplitudes 1 mV in the WMT3 slices as
compared with the SW3, SW1, WMT1, and naïve slices. There were
no learning-specific changes in the WMT1 mean IPSP amplitude and
distribution as compared with the SW1 group at both 200- and 350-ms
time points (P > 0.3, P > 0.5;
P > 0.1, P > 0.2, respectively; also
refer to the legend of Fig. 3 for detailed discussion of the IPSP
distributions in the SW1 and SW3 groups).
Whole population of the recorded IPSPs
The vast majority of the maximum IPSPs peaked in 70 ms and had a
large early phase followed by a late smaller phase (see Fig. 2 for
methods). While we focused our study on this major class of IPSPs, we
did check the whole population of the recorded IPSPs regardless of
their classification based on IPSP kinetics and peak latency. While
there were no learning-specific differences of mean IPSP amplitudes
(using the reference parameters discussed in the preceding text), there
was a larger fraction of cells with small IPSP in the WMT3 group as
compared with the SW3 group (P < 0.01 and
P < 0.01 at 200- and 350-ms latencies respectively; means and distributions are not shown). This difference was present despite the somewhat more hyperpolarized
Vm in the SW3 group (not shown). The
same tendency was observed in distributions of PSP amplitudes measured
at 40-ms latency, although difference in this case was not
statistically significant (P = 0.07).
Vm
The data analysis revealed a small but statistically significant difference in the mean values of the baseline Vm among the five groups of cells during PSP study [F(4,219) = 8.043, P < 0.0001]. However, there were no learning-specific changes in Vm (Table 1). Conductance and reversal potential studies were not dependent on the Vm level.
Membrane conductance evoked during IPSP phase (gIPSP)
Analysis of the membrane conductance changes evoked during the IPSP phases provided compelling evidence of the reduced inhibition after WM learning. IPSPs have a powerful control over neuron excitability not only by determining the level of the Vm but mainly due to their shunting effect on membrane resistance. This shunting significantly reduces the EPSPs' ability to bring Vm close to an action potential threshold. Given that learning-induced changes in the isochronal IPSP amplitudes were revealed by the distribution analyses, we concentrated on relationships between the isochronal IPSP amplitude and conductance to assess the effect(s) of the IPSP decrease on neuronal properties.
Although there were no learning-specific changes in mean
gIPSP measured both at 40- and 150-ms
time points [F(4,156) = 0.551, P > 0.6; F(4,151) = 3.965, P < 0.01, respectively; Table 1], analyses
performed on scatter plots of the
gIPSP versus maximum IPSP amplitudes
showed a significant linear correlation between these parameters. IPSPs
with smaller amplitudes were associated with smaller increases in
membrane conductance (Fig. 5).
Furthermore complete acquisition of the WM task was associated with an
increase in the fraction of neurons with reduced IPSP amplitude and
reduced gIPSP measured for the early
and late IPSP phases (Fig. 5, A and B). The mean
value for the WMT3 group was chosen as a basis for between-group
comparisons because this mean was the most reduced of all the group
means. The proportion of the cells with early IPSP amplitude 7.5 mV
(mean IPSP amplitude at 40 ms in the WMT3 group) and
gIPSP
22 nS (mean
gIPSP at 40 ms in the WMT3 group) was
larger in neurons from the WMT3 rats as compared with the naïve
and SW3 controls (P < 0.05; P < 0.05;
Fig. 5A). The proportion of the cells with late IPSP
amplitude
2.8 mV (mean IPSP amplitude at 200 ms in the WMT3 group)
and gIPSP
3.1 nS (mean
gIPSP at 150 ms in the WMT3 group) was
also larger in neurons from the WMT3 rats as compared with the
naïve and SW3 controls (P < 0.05; P < 0.05; Fig. 5B). At the same time,
gIPSP was not different among SW3,
naïve, WMT1, and SW1 animals at both latencies (Fig. 5,
A and B). Therefore long-term learning-specific
disinhibition in a subset of the CA1 PCs was accompanied by reduced
gIPSP evoked during the early and late IPSP
phases, and, consequently by reduced shunting effect on neuron
Rin.
|
IPSP reversal potential (EIPSP)
To examine the ionic basis of the disinhibition of PCs as a result
of spatial learning, we measured IPSP
EIPSP in all groups. The effects of
the WM training could not be seen in the mean
EIPSP measured at 40-ms latency
[F(4,156) = 2.03, P > 0.05; Table 1] and at 150-ms latency
[F(4,151) = 2.11, P > 0.05; Table 1]. There was, however, a significant linear
correlation between IPSP amplitude and the level of the early
EIPSPi.e., smaller IPSPs had more depolarized EIPSP (Fig.
6). To identify learning-specific changes within the isochronal IPSP distributions, we focused our analysis on
those cells with early IPSP amplitudes that were
7.5 mV (mean IPSP
amplitude at 40 ms in the WMT3 group) and
EIPSP
78 mV (mean
EIPSP at 40 ms in the WMT3 group) in
all five groups. This subset was larger in neurons from the WMT3 rats
as compared with the naïve and SW3 controls (P < 0.05; P < 0.05; Fig. 6A). At the same
time, both SW1 and SW3 controls were not different from naïve
subjects (P > 0.6, P > 0.6; Fig.
6A) and WMT1 group was not different from SW1 control
(P > 0.06).
|
Similar analysis of the relationships between the late IPSP amplitude
and late EIPSP revealed that only the
group showing complete acquisition of the spatial memory task also
showed an increased fraction of cells with both reduced late IPSPs and
late depolarized EIPSPs. The larger
fraction of CA1 PCs that had late IPSP amplitudes 2.8 mV (mean IPSP
amplitude at 200 ms in the WMT3 group) was associated with depolarized
EIPSP at 150 ms (
mean EIPSP
98 mV in the WMT3 group) only
in the WMT3 rats as compared with the SW3 and naïve groups
(P < 0.05; P < 0.01, respectively; Fig. 6B). Thus complete learning of the water maze (WMT3
group) was associated with an increase in the fraction of CA1 PCs with reduced early and late IPSPs as well as depolarized early and late
EIPSP.
Learning-specific relation between EIPSP and gIPSP
To further characterize the possible mechanisms of the
learning-induced depolarizing shifts in the
EIPSP, we analyzed for a correlation
between the EIPSP and
gIPSP for the above-described early
and late-phase IPSP components. For both early and late IPSPs, there
was a statistically significant linear correlation between the
EIPSP and
gIPSP in the WMT3 group
(r = 0.55, slope of the linear regression =
1.09; r =
0.43, slope =
0.1). This is
another major finding that emerged without any segregation into
subpopulations for exactly the same early IPSP and late IPSP components
that showed learning-specific reduction of amplitudes. This correlation
was not characteristic of the SW3 [r =
0.07, slope =
0.18, NS; r = 0.11, slope = 0.03, nonsignificant (NS)] and naïve group (r =
0.33, slope =
0.86; r = 0.19, slope = 0.05, NS; plots not shown).
A more detailed analysis of the relationship between
EIPSP and
gIPSP further showed that the fraction of
cells with early gIPSP 22 nS (mean
gIPSP in the WMT3 cells) and
EIPSP
78 mV (mean
EIPSP in the WMT3 cells) was larger in
the WMT3 group as compared with SW3 and naïve cells
(P < 0.05, P < 0.01). The fraction of
cells with late gIPSP
3.1 nS (mean
gIPSP in the WMT3 cells) and
EIPSP
98 mV (mean
EIPSP in the WMT3 group) was larger in the WMT3 group as compared with SW3 (P < 0.05),
although, it was not statistically significant larger as compared with
naïve cells (P = 0.11).
Representative cells in Fig. 7 demonstrate depolarized EIPSP and smaller estimated changes in the membrane conductance gIPSP during the early and late IPSP phases after acquisition of the water maze task as compared with the SW3 and naïve control groups.
|
Thus correlation analyses suggest that a learning-induced reduction in
the total gIPSP is also associated
with an increased role of the conductance(s) that have reversal
potentials more depolarized than ECl and
EK+. At this stage, we cannot distinguish
between such candidates as HCO
Relations between EIPSP and EPSP rising slope
We observed an ~28-mV range of the early
EIPSP (from 88 mV up to
60 mV;
Fig. 6A) and a 45-mV range for the late
EIPSP (from
120 mV up to
75 mV;
Fig. 6B) in all experimental and control groups. One of the
possible explanations for such variability could be difference in
summation of the EPSP- and IPSP-related Na+,
Ca2+, Cl
, and
K+ conductances (Andreasen and Lambert
1998
; Collingridge et al. 1988
; Hablitz
and Thalmann 1987
; Jensen et al. 1993
;
Otis et al. 1993
). As in previous studies
(Lopantsev and Schwartzkroin 1999
; Otis et al.
1993
), we found here that
EIPSPs for some late IPSPs were more
negative than the theoretical equilibrium potential for
K+ current (
98 mV at 2.5 mM
K+ in ACSF) (Otis et al. 1993
). It
has been suggested that a clear late
EIPSP is not demonstrable under
physiological levels of K+ because of the
rectification in membrane properties at hyperpolarized Vm and activation of a parallel
shunting conductance (Hablitz and Thalmann 1987
).
Bilinear late IPSP versus Vm plots in
the present study (not shown) as well in another studies may reflect this possible effect of rectification (Lopantsev and
Schwartzkroin 1999
; Otis et al. 1993
).
Potentiation of both non-NMDA and NMDA EPSP components is expressed as
an increase of the EPSP rising slopes (Blitzer et al. 1995; Buonomano 1999
; Huang and Hsu
1999
; Manabe et al. 2000
). There was, however,
no correlation between the IPSP peak amplitude and the EPSP initial
slope (r =
0.16, P > 0.2) nor was there a
correlation between the EIPSP at 40 ms
and the EPSP initial rising slope (r =
0.24,
P > 0.1) in the WMT3 group. These data do not support
the hypothesis therefore that learning-specific depolarizing shifts in
the EIPSP and IPSP amplitude reduction were caused by EPSP facilitation. We cannot entirely rule out, however,
the possibility that potentiated EPSPs may be masked by a potentiated
IPSP conductance i.e., one offsetting the other.
EPSP input-output relationships and initial rising slope
The following findings from the five groups of neurons provide additional evidence that the Schaffer collateral-induced excitatory inputs of the CA1 PCs were not facilitated as a result of the spatial learning. EPSP amplitudes were measured from the baseline Vm.
1) Slopes from linear regression equations for stimulating current-EPSP amplitude (I-O) relations were determined individually for each cell in all groups and comparisons were performed separately for the same ranges of stimulating currents. There were no learning-specific differences in the intensity of stimulating current applied to Schaffer collateral input. The intensities of both average and maximum stimulating currents to elicit EPSPs in the WMT3 and SW3 groups were not different from naïve control and from each other (P > 0.9, P > 0.9, P > 0.9, P > 0.9, respectively; Table 2). However, ANOVA and post hoc analyses showed higher intensities of the stimulating current to elicit EPSPs in slices in the WMT1 and SW1 groups in comparison to naïve, WMT3, and SW3 slices [F(4,214) = 7.81, P < 0.0001; P < 0.05, P > 0.05; P < 0.05, P < 0.05, P < 0.01, P < 0.01 for average current, respectively, F(4,238) = 8.3, P < 0.0001; P < 0.01, P > 0.05, P < 0.01, P < 0.01, P < 0.05; P < 0.01 for maximum current, respectively; Table 2]. Considering all stimulating current ranges, however, one-way ANOVA did not reveal any specific effect of WM training on the linear regression slopes of stimulating current versus EPSP amplitude after 1 day of training [F(2,70) = 0.1946, P > 0.8; F(1,58) = 0.0622, P > 0.8; Table 2, WMT1, SW1, and naïve groups] or after 3 days of WM training [F(2,29) = 0.6133, P > 0.5; F(2,56) = 0.5666, P > 0.5; F(2,26) = 0.3393, P > 0.7; Table 2, WMT3, SW3, and naïve groups]. Synaptic responses and stimulus-response relationships for the EPSP phase from the WMT3 and SW3 representative cells are illustrated in Fig. 8.
|
|
2) The initial rising slopes of the maximum subthreshold
EPSPs frequently used as an indicator of EPSP potentiation
(Blitzer et al. 1995; Buonomano 1999
;
Huang and Hsu 1999
; Manabe et al. 2000
)
were also not affected by WM training
[F(4,219) = 1.179, P > 0.3 for the subgroup of cells; Table 1,
F(4,256) = 0.5666, P > 0.5 for the whole population; not shown]. Representative traces from all five groups of cells and EPSP rising slope frequency distributions demonstrate no effect of the behavioral procedures (Fig.
9, A and B).
|
3) While EPSPs had larger mean amplitudes in slices from the
WMT3 and SW3 rats as compared with EPSPs recorded in slices from the
WMT1 and SW1 rats [F(4,255) = 6.227, P < 0.0001; P < 0.0001, P < 0.01, P < 0.0001, P < 0.01 for total PSP population, not shown; F(4,219) = 7.11, P < 0.0001; P < 0.01, P < 0.001, P < 0.05 for the PSP with IPSP peaks at 70 ms; Table
1], they were not different from naïve neurons
(P > 0.1; P > 0.3 for the whole
population, not shown; P > 0.07; P > 0.8 for PSP with early IPSP peaks, Table 1). Mean EPSP amplitude and
EPSP distribution in the WMT3 group were also not different from the
SW3 EPSPs [P > 0.1, P > 0.7; P > 0.4, P > 0.1, respectively for
the whole population (not shown) and subset of cells, Table 1]. There
may have also been a nonspecific enhancement of EPSP amplitude due to
the slightly hyperpolarized Vm in the
WMT3 and SW3 groups as compared with the WMT1 and SW1 rats (Table 1).
Based on the preceding studies of the complex PSPs, we conclude, therefore that learning-specific disinhibition in a subset of the dorsal CA1 PCs is due to reduced synaptic inhibition with smaller membrane conductances evoked during IPSPs and depolarizing-shifted reversal potentials. At the same time, it is unlikely that IPSP reduction can mask EPSP facilitation.
Membrane properties after water-maze training
In addition to the receptor-regulated (e.g., GABA) postsynaptic excitability, intrinsic neuronal excitability parameters were also examined.
Action potential
There was a learning-specific increase in the variance of spike
amplitude in the WMT3 group as compared with the SW3 (43.4 vs. 25.8, ratio = 1.68; P < 0.05, F test) and
naïve (43.4 vs. 21.7, ratio = 2; P < 0.01) controls due to an apparent increase in the fraction of neurons
with enhanced spike amplitudes after 3 days of WM training (Table
3; Fig.
10). There were no learning-specific differences in the spike width (Table 3). There was no observed learning-specific change of the input resistance that could account for
the observed increase of action potential amplitude (see Table 4). The observed learning-specific
increase of spike amplitude might be explainable by a decreased input
conductance following long-term disinhibition. However, such an
explanation can be excluded by the absence of a significant correlation
between action potential amplitude and the amplitude of early
(r = 0.07, P > 0.6) and late IPSPs
(r = 0.06, P > 0.6) in the WMT3
neurons.
|
|
|
These results suggest that one subset of CA1 PCs showed learning-specific reduction of IPSPs (see preceding text), but no changes of intrinsic membrane properties. Another subset of the PCs in the WMT3 slices showed increased spike amplitude but no synaptic changes.
Intrinsic membrane excitability
There were no learning-specific changes in spike threshold, resting Vm level, or input resistance values (Table 3). Nor were there learning-specific changes in the sAHP following a single spike or a four-spike train (Fig. 11A, Table 3; see Fig. 12A for representative traces). There were also no learning-specific changes in the spike frequency accommodation during the 800-ms depolarizing current pulses (Fig. 11B, Table 3; see Fig. 12B for representative traces). Enhancement of the sAHP and spike frequency accommodation was not learning-specific. The mean values of the peak latencies for the one- and four-spike sAHPs, mean values of the decay times of the postburst sAHP, interspike time intervals between first, second, third, and fourth action potentials (not shown), and mean values of the total duration of four-spike bursts were also not affected by behavioral treatment (Table 3).
|
|
Reduced sAHPs and spike frequency accommodation were observed, however,
in the CA1 and CA3 PCs with a different learning paradigm, namely
acquisition of the classical delayed and trace eyeblink conditionings
in rabbits (Coulter et al. 1989; Disterhoft et
al. 1986
; Moyer et al. 1996
;
Sanchez-Andres and Alkon 1991
; Thompson et al.
1996
).
Although membrane properties of neurons can be affected by synaptic
activity, it has been shown previously (Alger and Nicoll 1980; Gusev and Alkon 1998
), that sAHP is not
contaminated by the evoked IPSP. Averaging large numbers of samples for
each individual neuron's measurement should minimize effects of
spontaneous synaptic activity on measurements.
Membrane slope input resistance and rectification
To exclude the possibility that synaptic modifications could simply reflect a difference in passive membrane properties, we performed a detailed analysis of current-voltage relations for the pyramidal cells. Slope membrane input resistance measured with depolarizing current injections and rectification in passive membrane properties, both measured at peaks and plateau of voltage responses, were not affected by behavioral procedures in the WMT3 and SW3 cells [F(2,154) = 1.4989; P > 0.2; F(2,157) = 1.4509; P > 0.2; F(2,154) = 0.188, P > 0.8; F(2,157) = 0.1295, P > 0.8; Table 4]. Both WMT3 and SW3 groups, however, showed somewhat smaller slope Rin in response to hyperpolarizing current pulses measured at the peak [F(4,275) = 3.637, P < 0.01] as well as at the plateau [F(4,275) = 3.78; P < 0.01] of voltage responses as compared with naïve rats (P < 0.01, P < 0.001, P < 0.01, P < 0.001, respectively; Table 4, Fig. 13). In addition, Rin in the SW3 PCs was smaller as compared with the SW1 (P < 0.05, P < 0.05) and WMT1 (P <0.05, P < 0.05) PCs. There were no differences in the times to peak in voltage responses to hyperpolarizing 0.5-nA current pulses [F(4,275) = 0.333; P > 0.8], suggesting that membrane capacitance of the PCs was not modified by WM training. Therefore learning-specific reduction in the IPSP conductance could not be a consequence of changes in Rin and membrane rectification in the WM-trained rats.
|
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DISCUSSION |
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Our principal learning-specific findings are reduction of the mean
peak IPSP amplitude as well as increased mean latencies to the IPSP and
EPSP peaks and correlation of the depolarizing-shifted IPSP reversal
potentials and reduced IPSP-evoked membrane conductance. Furthermore
this long-term disinhibition in a subset of the dorsal CA1 PCs was a
clear and significant intracellular correlate of spatial memory
acquisition. Disinhibition became apparent as the rats progressed from
strategy learning (Hoh et al. 1999) to the complete
representation of the spatial water maze (Morris 1984
). At this advanced stage of the spatial training (WMT3 group),
learning-specific synaptic correlates of the WM task acquisition
included reduced early and late IPSP phases in the complex PSPs and
delayed IPSP and EPSP peaks. Reduced early and late IPSPs were
significantly correlated with decreased IPSP-evoked membrane
conductance and depolarized IPSP reversal potential. At the early stage
of WM learning (WMT1 group), we did not observe any learning-specific changes, although there was a statistically nonsignificant tendency to
an increased proportion of the cells with a depolarized reversal potential for the early IPSP.
We defined the maximum IPSP as that elicited with a stimulus intensity that also elicits a preceding EPSP just subthreshold for an action potential. The changes in the maximum IPSP amplitude were not influenced by the level of pyramidal cell excitability since we found no learning-specific differences in intrinsic membrane excitability. Namely, there were no learning-specific differences in the current intensity required to evoke a single action potential or four-spike burst, single-spike, or postburst sAHP amplitudes, spike frequency accommodation, membrane resistance, capacity or rectification, resting Vm, and spike threshold.
These reductions in the WMT3 IPSPs were not correlated with EPSP facilitation. Analyses of the EPSP facilitation indices such as I-O relations, initial rising slopes and amplitudes of the EPSP did not indicate learning-specific changes. Therefore we suggest that disinhibition occurred primarily due to the reduction in the IPSPs' amplitudes and conductances and shifts in the balance of ionic conductances responsible for the GABA-mediated responses.
The learning-specific synaptic changes observed here during concurrent
stimulation of the excitatory and inhibitory synapses suggest that a
subset of the CA1 PCs will be less inhibited and thus show more
effective temporal summation of excitatory inputs. The smaller membrane
conductance during the late IPSPs would be expected to increase the
weight of the excitatory inputs on the CA1 PC dendrites mediated by
Schaffer collaterals. Thus conditions may be provided for opening of
the NMDA channels and induction of the NMDA-dependent changes in
excitatory synapses (Andreasen and Lambert 1998;
Andreasen et al. 1989
; Bliss and Collingridge 1993
; Collingridge et al. 1988
). The smaller
membrane conductance during early somatic IPSPs would be expected to
facilitate spike generation (Buhl et al. 1994
;
Miles et al. 1996
; Nurse and Lacaille 1997
; Paulsen and Moser 1998
). Backpropagating
action potentials would, in turn, enhance Ca2+
entry into dendrites and possibly would trigger further modifications such as LTP of excitatory synapses (Markram et al. 1997
;
Tsubokawa and Ross 1996
). Our previous intracellular
study of rabbit eyeblink conditioning demonstrated learning-specific
changes of EPSP summation but not of individual EPSPs (LoTurco
et al. 1988
). The lack of a learning-specific change in EPSPs
or intrinsic neuronal excitability is most likely not due to the
pyramidal cell selection, since only 7 of 326 neurons were rejected
based on predetermined criteria (see METHODS). Although our
data do not indicate a net facilitation of the EPSPs recorded from
PCs' soma, to make a conclusion about the real distribution of
learning-related EPSP modifications, multiple dendritic intracellular
recordings would be necessary.
Nevertheless long-term disinhibition could contribute to the previously
observed place-related increases in the activity of place cells after
learning the position of a platform to escape from the water
(Hollup et al. 2001; Moser et al. 1999
).
This is consistent with in vivo observations of suppressed
interneuronal activity during exploration of an unfamiliar environment
(Paulsen and Moser 1998
) and learning of a novel spatial
representation (Wilson and McNaughton 1993
).
Approximately 20% of place cells recorded in vivo during spatial
learning gradually increased their firing rates in already established
or newly acquired place fields (Kobayashi et al. 1997; Nishijo et al. 1999
). Although we cannot claim that a
subset of PCs with reduced IPSPs are themselves the place cells or that disinhibition itself creates the place cells, we suggest that long-term
disinhibition may promote the place cells' ability to transiently
increase their firing rates and to discharge at early phases of theta
cycles when rats cross these place fields (Hollup et al.
2001
; Kamondi et al. 1998
; Kentros et al.
1998
; Kobayashi et al. 1997
; Magee
1999
; Moser et al. 1999
; O'Keefe and
Recce 1993
; Wilson and McNaughton 1993
) and
anticipate a reward (Nishijo et al. 1999
). Finally,
long-term disinhibition could also facilitate involvement of the
particular PCs in neuronal ensemble reactivation and depolarization of
specific downstream CA1 targets during the animal's rest cycle and
period of sharp waves in the HC electroencephalogram (Csicsvari
et al. 1999
; Kudrimoti et al. 1999
;
Nadasdy et al. 1999
).
An increase in bicarbonate flow through the GABAA
receptor-mediated channels or increase in proportional conductance of
GABAD (depolarizing) channels (Perkins and
Wong 1996) may account for the observed depolarizing shifts of
EIPSP and reduced amplitudes of the
early IPSPs. Consistent with these possibilities, positive shifts of
EIPSPs were correlated with reduction
of the early IPSPs. Long-term postsynaptic transformation of the fast
IPSPs into the EPSPs may also contribute to the observed
learning-related long-term disinhibition (Collin et al.
1995
). It has been shown that pairings of GABA applications or
basket cell stimulation with the CA1 PC depolarization caused IPSP
transformation associated with depolarizing shifts in
EIPSP. A sole reduction in chloride
conductance did not cause a depolarizing shift in the early
EIPSP in the CA3 area (Thompson
and Gahwiler 1989b
) due, for instance, to an increase in the
EPSP conductance. Pharmacological isolation of the IPSPs in future
studies may reveal or exclude more subtle interactions of the EPSP- and
IPSP-related conductances.
Decreases in the IPSP conductance could result from either presynaptic
processes through the decrease in the evoked GABA release, or
postsynaptic processes, such as desensitization (Thompson and Gahwiler 1989a,b
). Disinhibition due to decreased GABA
release from the terminals targeting spiking CA1 PCs or due to both
decreased GABA release and driving force for Cl
ions in CA3 PCs (following repetitive mossy fiber stimulation) lasted
only for minutes (Alger et al. 1996
; Thompson and
Gahwiler 1989a
). It is unlikely therefore that these pre- and
postsynaptic changes may underlie the persistent disinhibition observed
in slices after WM learning. GABAA receptor
phosphorylation is a possible postsynaptic mechanism for reduced IPSP
conductance (Moss et al. 1992
).
On the other hand, modified relations between hyperpolarizing potassium
currents and depolarizing current(s) all regulated by
GABAB receptors (Pham and Lacaille
1996) may cause the observed positive shifts of the
late EIPSP and reduced amplitudes of
the late IPSPs. Because the fast EPSPs are not present at the 150-ms time point and the NMDA component is suppressed with recording conditions used here (Andreasen and Lambert 1998
;
Andreasen et al. 1989
; Collingridge et al.
1988
), we suggest that EPSP-evoked conductances do not
contribute to the observed depolarizing shifts in the late
EIPSP. Therefore our present data
indicate a learning-induced depolarizing shift in the balance of
postsynaptic ionic conductances underlying the late GABA-mediated
synaptic inhibition in a subset of cells with reduced IPSPs. The
relative importance of the post- versus presynaptic mechanisms of
learning-specific disinhibition will be a subject of future study.
After complete learning of the water maze following 3 days of training,
we also found learning-specific increased action potential amplitudes
in a different subset of neurons. Reduction in the IA-like potassium current similar to
that observed after classical conditioning in rabbit Purkinje cell
dendrites (Schreurs et al. 1998) and the mollusk
Hermissenda (Alkon et al. 1982
) may account for the increased action potential amplitude that we observed in the
CA1 PCs.
The nonspecific reduction of PCs' membrane excitability and somewhat
higher current intensities applied to elicit EPSPs may be related to
stress-induced increase in the slow
Ca2+-dependent K+
conductance (IAHP) and depression of
amino acid-mediated transmission (Joels and de Kloet 1992,
1993
; Teschemacher et al. 1996
).
In conclusion, our data provide the first intracellular synaptic correlate of spatial memory acquisition in the dorsal CA1 area of the HC. These results suggest that neuronal ensembles may be formed during associative learning by prolonged reduction of somatic and dendritic inhibition in a subset of the PCs. This prolonged disinhibition represents, therefore a novel long-term synaptic change that can contribute importantly to hippocampal function during learning and memory.
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ACKNOWLEDGMENTS |
---|
We thank B. Schreurs for help with experimental setup and critical review of the manuscript, D. Buck and N. Meiri for help with behavioral experiments, and A. Lyckman for comments on earlier drafts of the manuscript.
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FOOTNOTES |
---|
Present address and address for reprint requests: P. A. Gusev, Blanchette Rockefeller Neurosciences Institute, 9601 Medical Center Dr., Academic and Research Building, 3d Floor, Rockville, MD 20850 (E-mail: gusevpa{at}brni-jhu.org).
Received 12 January 2001; accepted in final form 11 April 2001.
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REFERENCES |
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