Howard Hughes Medical Institute and Division of Biology 216-76, California Institute of Technology, Pasadena, California 91125
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ABSTRACT |
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Dvorak-Carbone, Hannah and Erin M. Schuman. Patterned Activity in Stratum Lacunosum Moleculare Inhibits CA1 Pyramidal Neuron Firing. J. Neurophysiol. 82: 3213-3222, 1999. CA1 pyramidal cells are the primary output neurons of the hippocampus, carrying information about the result of hippocampal network processing to the subiculum and entorhinal cortex (EC) and thence out to the rest of the brain. The primary excitatory drive to the CA1 pyramidal cells comes via the Schaffer collateral (SC) projection from area CA3. There is also a direct projection from EC to stratum lacunosum-moleculare (SLM) of CA1, an input well positioned to modulate information flow through the hippocampus. High-frequency stimulation in SLM evokes an inhibition sufficiently strong to prevent CA1 pyramidal cells from spiking in response to SC input, a phenomenon we refer to as spike-blocking. We characterized the spike-blocking efficacy of burst stimulation (10 stimuli at 100 Hz) in SLM and found that it is greatest at ~300-600 ms after the burst, consistent with the time course of the slow GABAB signaling pathway. Spike-blocking efficacy increases in potency with the number of SLM stimuli in a burst, but also decreases with repeated presentations of SLM bursts. Spike-blocking was eliminated in the presence of GABAB antagonists. We have identified a candidate population of interneurons in SLM and distal stratum radiatum (SR) that may mediate this spike-blocking effect. We conclude that the output of CA1 pyramidal cells, and hence the hippocampus, is modulated in an input pattern-dependent manner by activation of the direct pathway from EC.
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INTRODUCTION |
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The hippocampus plays a critical role in such
high-level brain functions as learning and memory (Eichenbaum et
al. 1992; Squire and Zola-Morgan 1991
;
Wood et al. 1999
; Zola-Morgan and Squire 1990
) and spatial navigation (Muller 1996
;
Wilson and McNaughton 1993
). In order for the neural
computations performed by the hippocampus to be used by the rest of the
brain, an output from the hippocampus to neocortex is necessary. The
pyramidal cells of area CA1 are the primary population of hippocampal
principal cells projecting outside of the hippocampus, with axons
projecting to subiculum and entorhinal cortex (EC) as well as
subcortical targets (Lopes da Silva et al. 1990
;
Tamamaki and Nojyo 1995
; Van Groen and Wyss 1990
; Witter et al. 1989
). The primary
excitatory input to the CA1 pyramidal cells is the Schaffer collateral
(SC) projection from area CA3 (Amaral et al. 1990
;
Amaral and Witter 1989
; Andersen et al.
1966
; Lopes da Silva et al. 1990
). CA1 pyramidal
cells and their SC inputs are therefore crucial sites for the
regulation of hippocampal output.
Stratum lacunosum-moleculare (SLM) of area CA1 receives a number of
inputs from various other brain regions, including a direct projection
from layer III of EC (Steward and Scoville 1976), as well as projections from the nucleus reuniens of the thalamus (Wouterlood et al. 1990
) and inferotemporal cortex
(Yukie and Iwai 1988
). The function of inputs to this
distal dendritic region of CA1 pyramidal cells is not well understood
(see Soltesz and Jones 1995
). Although there is some
evidence for direct excitation via the EC projection to CA1
(Yeckel and Berger 1990
), there is also a strong
inhibitory component (Empson and Heinemann
1995a
,b
; Paré and Llinás
1995
). We investigated the effect of this inhibitory input on
the activity of CA1 pyramidal cells, focusing particularly on their
responsiveness to SC inputs. We find that the effectiveness of SC
inputs in causing pyramidal cells to fire is greatly reduced when
stratum radiatum (SR) is stimulated shortly after a burst stimulus in
SLM, a phenomenon we call spike-blocking. We investigated the
dependence of spike-blocking efficacy on the relative timing of the SR
and SLM inputs and on the number of SLM stimuli. We also investigated
whether the SLM-mediated modulation of SR inputs could itself be
modulated, by examining the effects of repeated SLM bursts on
spike-blocking. Some of these results have previously appeared in
abstract form (Dvorak and Schuman 1997
).
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METHODS |
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Tissue preparation
Slices were prepared by standard procedures from 6- to 8-wk-old male Sprague-Dawley rats. Rats were decapitated following Halothane anesthesia, and the brain rapidly removed to ice-cold, oxygenated artificial cerebrospinal fluid (ACSF; in mM: 119 NaCl, 2.5 KCl, 1.3 MgSO4, 2.4 CaCl2, 1.0 NaH2PO4, 26.2 NaHCO3, and 11.0 glucose). The posterior half of each hemisphere was glued, ventral side up, onto the stage of a cooled oscillating tissue slicer (OTS-3000-04; FHC, Brunswick, ME) and covered with chilled ACSF. Slices (500 µm) were cut. The extraneous cortical and subcortical tissue was gently dissected away with the small end of a spatula. The slices were then allowed to recover in an interface chamber at room temperature for at least 1 h before experiments were started. Further microdissection was performed in ice-cold ACSF immediately after slice preparation. All electrophysiology was done with the slices submerged and constantly perfused with oxygenated ACSF at room temperature.
To minimize the possibility of disynaptic or trisynaptic activity in
CA1, the dentate gyrus and CA3 regions were dissected away from the
slice, leaving a CA1 minislice. A cut was made through SR in distal CA1
(near the subiculum) perpendicular to the cell body layer, to prevent
antidromic activation of SC axons by the stimulating electrode in SLM
(Maccaferri and McBain 1995); SC axons do not enter SLM
to any appreciable extent (Amaral and Witter 1989
;
Tamamaki and Nojyo 1995
). Field potential recordings
were used to verify isolated activation of axons in SR or SLM
(Colbert and Levy 1992
; Dvorak-Carbone and
Schuman 1999
).
Electrophysiology
Bipolar tungsten electrodes, either concentric or paired needles, were used for stimulation. One electrode was placed in SR to stimulate the SC axons; the other was used to stimulate SLM afferents on the far side of the cut. The level of SR stimulation was set such that the resultant excitatory postsynaptic potential (EPSP) in the pyramidal cell just barely reached spike threshold; this generally required a current of 20-40 µA for 100 µs. Stimulation in SLM was generally stronger, 30-200 µA for 100 µs.
Intracellular recordings from pyramidal cells were made using sharp
electrodes whose resistance was 100-200 M when filled with 2 M
potassium acetate. Sharp electrode recordings were made blind by
lowering the electrode into stratum pyramidale until a penetration was
achieved. The voltage reading of the electrode was zeroed with the
electrode in the bath, and the bridge was balanced before penetration
and rebalanced after penetration. Capacitance compensation was applied
after penetration. Neurons included for analysis had an average resting
potential of
62.1 ± 1.0 (SE) mV, fired overshooting
action potentials, and had input resistances of 109 ± 10 M
(n = 28). Pyramidal cells were identified by the
presence of strong spike frequency accommodation in response to
positive current injection. All experiments were performed in
current-clamp mode; the cell was generally at its resting potential, although in a few (3/28) cases a small negative current was applied to
hyperpolarize the cell and prevent it from spontaneously firing action potentials.
Whole cell electrodes used for interneuron recordings had a resistance
of ~5 M when filled with intracellular solution [125 mM
KMeSO4 (City Chemical, Jersey City, NJ), 9 mM HEPES, 3.6 mM NaCl, 90 µM EGTA, 4 mM Mg-ATP, 300 µM Li-GTP, 25 mM
phosphocreatine, and 0.2-0.4% biocytin). Whole cell recordings were
made under visual guidance on an Olympus BX50WI upright microscope
equipped with a MTI VE1000 CCD camera. Positive pressure was applied to the electrode solution while advancing toward the targeted neuron, to
keep debris off the electrode as well as to clean the surface of the
neuron (Edwards 1995
). A gigaseal was obtained under
voltage-clamp conditions by applying slight negative pressure; the
patch was then clamped down to
60 mV, and whole cell configuration
was achieved by applying further negative pressure. Neurons included for analysis had resting potentials negative to
50 mV, fired overshooting action potentials, and had input resistances of 562 ± 5 M
(n = 41). All experiments were performed
in current-clamp mode, with the cell at its resting potential.
Drugs were applied by dilution of concentrated stock solutions into the perfusion medium. Stock solutions were made up in water. CGP 55845A was a kind gift from Novartis (Basel); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-OH-saclofen were obtained from RBI (Natick, MA); all other drugs were obtained from Sigma (St. Louis, MO).
Histology and reconstruction of filled neurons
For morphological reconstructions of interneurons, the whole cell recording solution included 0.2-0.4% biocytin. To prevent nonspecific staining of damaged neurons at the slice surface, the tip of the electrode was filled with biocytin-free solution, and the electrode was then back-filled with biocytin-containing solution.
After completion of the experiments, slices were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for at least 3 days. Thin (70 µm) sections were cut on a vibratome and rinsed in PBS. The sections were incubated in an endogenous peroxidase blocker (10% MeOH, 3.5% H2O2) in PBS for 1.5 h and rinsed again in PBS. Next, sections were incubated in 2% BSA and 0.25% Triton X-100 in PBS for 45 min, followed by a wash in 2% BSA in PBS. Slices were then incubated in an avidin-HRP solution (ABC solution, Vectastain Kit PK-6100, Vector Labs, Burlingame, CA) for 2 h. After rinsing in PBS, slices were incubated in a solution of diaminobenzidine (DAB; 10 mg/20 ml PBS) with cobalt chloride (0.03%) and nickel ammonium sulfate (0.02%) for 30 min; 0.0004% H2O2 was then added, and slices were incubated till the stained neurons appeared (2-30 min). Slices were once again rinsed in PBS, mounted onto subbed slides, dehydrated in increasing alcohols, cleared in xylene, and coverslipped with Permount.
Filled neurons were observed with a ×63 oil-immersion objective on a Zeiss Axioplan upright microscope equipped with a drawing tube, which was used to reconstruct the approximate neuronal morphology. Reconstructions are strictly qualitative, and no attempt was made to measure process length or correct for tissue shrinkage.
Data acquisition and analysis
Recordings were made using an Axoclamp-2A or 2B (Axon Instruments, Foster City, CA), low-pass filtered at 3 kHz, digitized at 1 kHz, and collected directly onto an IBM-compatible Pentium-class computer using in-house software written in LabVIEW (National Instruments, Austin, TX). Intracellular responses displayed are averages of three to five individual traces.
The effectiveness of the SLM burst in blocking SR-evoked spiking was
quantified as follows. Trials of SR stimulation following an SLM burst
were interleaved with trials where SR stimuli were delivered in
isolation. For any one test, typically, 10 SR + SLM trials were
interleaved with ~25 SR-alone trials. SR-induced spike firing
probabilities in the presence or absence of the SLM burst were
calculated from these trials. Spike-blocking efficacy for each test
condition was defined as follows: spike-blocking efficacy = (probability of firing with SR stimulation alone) (probability of firing with SR + SLM stimulation). Thus spike-blocking efficacy would reach a maximum of 1 if the cell never spiked when the SR stimulus was presented following the SLM burst and always spiked when
the SR stimulus was presented in isolation, and would be 0 if the cell
was equally likely to fire in the presence or the absence of the SLM
burst. A negative value would be possible if the spike firing
probability increased in the presence of the SLM burst. In practice,
the upper bound of the measured spike-blocking efficacy was limited by
the firing probability in response to SR stimulation alone. Over all
tests, the firing probability in response to SR stimulation alone was
0.87 ± 0.01 (n = 176); therefore a spike-blocking
efficacy of ~0.9 would indicate maximal spike-blocking. In only 1 test of 176 was the SR-alone spike firing probability <0.5, and in
76% of tests, the SR-alone spike firing probability was >0.8.
All numerical values are listed as means ± SE; error bars in bar
graphs are SE. Data were analyzed and plotted using Microcal Origin.
Some statistical analyses were performed in Microsoft Excel or
STATISTICA for Windows (StatSoft, Tulsa, OK). A paired Student's
t-test was used to test statistical significance of the
spike-blocking effect, using spiking probabilities in the presence or
absence of the SLM burst as the dependent variables. For multiple
comparisons, e.g., comparing the effectiveness of spike-blocking at
different interstimulus intervals (ISIs), a repeated-measures,
one-way ANOVA was performed across that subset of the data for which
all levels (e.g., ISI) were tested on each neuron included in the
analysis; the Neuman-Keuls test was performed to assess the statistical
significance of all pair-wise post hoc comparisons. Results were
considered significant when P 0.05; P values >0.05 are reported as NS.
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RESULTS |
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Burst stimulation in SLM results in a large IPSP and blocks SC-induced spiking in a GABAB-dependent manner
A single stimulus in SLM usually resulted in a biphasic response
in the postsynaptic pyramidal cell, with a small EPSP (0.9 ± 0.1 mV, peaking 31.3 ± 1.7 ms after the stimulus artifact,
n = 23), presumably mediated by excitatory axons from
layer III of EC (Empson and Heinemann 1995a,b
), nucleus
reuniens thalami (Dolleman-Van Der Weel and Witter 1996
;
Wouterlood et al. 1990
), or inferotemporal cortex
(Yukie and Iwai 1988
), followed by a slow, small
inhibitory postsynaptic potential (IPSP) (
1.1 ± 0.1 mV, peaking
281 ± 10 ms after the stimulus artifact, n = 28;
Fig. 1A). With the
addition of the GABAA antagonist bicuculline (20 µM), the
time-to-peak and amplitude of the EPSP and late IPSP were increased,
suggesting the presence of a GABAA receptor-mediated IPSP
(Empson and Heinemann 1995a
,b
). Burst stimulation in
SLM, i.e., 10 stimuli at 100 Hz, resulted in a significantly larger IPSP (
4.4 ± 0.4 mV, peaking 391 ± 13 ms after the
stimulus artifact, P < 0.0001, n = 27; Fig. 1B). This
burst-elicited IPSP was mediated by GABAB receptors,
because it was significantly reduced in the presence of the
GABAB antagonist 2-OH-saclofen (100 µM; peak IPSP amplitude,
2.0 ± 0.3 mV, significantly different from the IPSP in the same cell under control conditions, P < 0.05, n = 4) and virtually eliminated in the
presence of the more potent GABAB antagonist CGP 55845A (2 µM; peak IPSP amplitude
0.5 ± 0.3 mV, significantly different
from the IPSP in the same cell under control conditions,
P < 0.05, n = 3; Fig.
1C).
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We wished to examine whether the inhibition evoked by high-frequency stimulation in SLM could have a functional effect on the output of the hippocampus, namely action potentials in CA1 pyramidal cells. SC stimulation strength in SR was set to such a level that the response was just suprathreshold for action potential generation (Fig. 1D). Bursts of stimuli in SLM were tested for their ability to block spiking evoked by SR stimulation. Spike-blocking efficacy can range from 0 (no block: spiking equally likely in presence or absence of SLM stimulation) to 1 (maximal block: spikes never evoked following SLM stimulation), or be negative if spiking probability increased following SLM stimulation (see METHODS). When the SR stimulus was delivered in the middle of the SLM burst-evoked IPSP, spike generation was blocked (Fig. 1E); spike-blocking efficacy 400 ms after an SLM burst of 10 stimuli at 100 Hz was 0.73 ± 0.05 (n = 19). This spike-blocking effect was mediated by GABAB receptors, because spike-blocking efficacy was reduced from 0.69 ± 0.01 to 0.15 ± 0.003 in the presence of 100 µM 2-OH-saclofen (P < 0.01, n = 3) and was reduced from 0.82 ± 0.02 to 0.17 ± 0.03 in the presence of 2 µM CGP 55845A (P < 0.001, n = 3; Fig. 1F).
Spike-blocking efficacy is dependent on relative timing of the SLM and SR stimuli
The dependence of spike-blocking efficacy on the relative timing
of SR and SLM stimulation may suggest under what circumstances spike-blocking may occur in vivo. Dependence on ISI (measured from the
1st stimulus in the SLM burst to the SR stimulus) was tested in 23 neurons using an SLM burst pattern of 10 stimuli at 100 Hz; ISIs
measured ranged from 25 ms (with the SR stimulus thus arriving during
the SLM burst) to 1,500 ms. Spike-blocking efficacy reached a maximum
of 0.77 ± 0.04 (n = 19; significantly different
from 0, P < 0.00001) at an ISI of 400 ms, a time
interval consistent with the slow time course of the G
protein-mediated GABAB signaling pathway
(Misgeld et al. 1995; Mott and Lewis
1994
), and dropped off at shorter or longer ISIs (Fig.
2A). At 1,500 ms, the
spike-blocking effect was not significant (spike-blocking efficacy,
0.15 ± 0.15, NS, n = 4).
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To compare spike-blocking efficacy at different ISIs, an ANOVA was performed on data from nine neurons on which ISIs of 100, 200, 400, 600, and 800 ms had been tested. There was significant variation in spike-blocking efficacy between ISIs (F = 6.78, DF = 4, P < 0.001). Spike-blocking efficacy at 100, 200, and 800 ms was significantly lower than that at 400 ms (Newman-Keuls test, P < 0.01, n = 9), and spike-blocking efficacy was also significantly lower at 800 than at 600 ms ISI (Newman-Keuls test, P < 0.01, n = 9). This confirms the observation that spike-blocking efficacy was greatest at intermediate ISIs.
Spike-blocking efficacy is dependent on the number of stimuli in the SLM burst
We sought to determine how spike-blocking efficacy varied with the number of stimuli in the SLM burst. IPSP amplitude and duration increased when the number of stimuli in the burst was increased (e.g., see Fig. 2B, inset). We tested spike-blocking with single SLM stimuli as well as bursts consisting of 2-15 stimuli delivered at 100 Hz in 9 neurons. With only a single SLM stimulus, there was no significant spike-blocking effect (spike-blocking efficacy, 0.12 ± 0.07, NS different from 0, n = 9); spike-blocking efficacy was greatly increased by repetitive stimulation (Fig. 2B). There was a significant effect of number of stimuli/burst on spike-blocking efficacy (F = 6.04, DF = 8, P < 0.01). One stimulus was significantly less effective in spike-blocking than three or more stimuli (Newman-Keuls test, P < 0.05 for all comparisons). Two stimuli were significantly less effective than 8 or 10 stimuli (Newman-Keuls test, P < 0.05). No other significant differences were observed between different numbers of stimuli.
Repeated presentation of the SLM burst results in a reduction of the IPSP and of spike-blocking efficacy
Having characterized the effect of SLM-evoked inhibition on
excitatory SC transmission, we wished to examine whether this inhibitory effect could itself be modulated. GABA-mediated responses are known to undergo frequency-dependent depression by means of presynaptic GABAB autoreceptors (e.g.,
Davies et al. 1990). We found that repeated presentation
of the SLM burst (10 stimuli at 100 Hz) at 1 Hz resulted in an
exponential decay of IPSP amplitude, with a time constant of 3.7 ± 0.2 s (n = 8; Fig.
3).
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Is spike-blocking efficacy modulated along with IPSP amplitude? To determine how spike-blocking efficacy varied with position in the train of SLM bursts, we used a modified stimulation paradigm. We presented 10 SC stimuli at 1 Hz alone, followed by 10 SC stimuli at 1 Hz offset by 400 ms from 10 SLM bursts at 1 Hz (Fig. 4A), followed by 10 SC stimuli again. This set of stimuli was repeated 5-10 times at ~5-min intervals to allow for recovery of the GABAB response. Spike-firing probability for SC stimulation alone and in the presence of the SLM bursts was determined for each position in the train of bursts (Fig. 4B; n = 8). Spike-blocking efficacy decayed in an exponential manner with repeated SLM burst stimulation, from 0.73 ± 0.07 during the first burst, to 0.08 ± 0.04 during the last burst (Fig. 4C; n = 8). The time constant of this decay was 2.8 ± 0.3 s, similar to the time course of IPSP depression. Spike-blocking efficacy varied significantly with position in the train (repeated-measures ANOVA, F = 11.10, DF = 9, P < 0.0001); post hoc analysis showed that spike-blocking efficacy was significantly greater during the first burst in the train than at any other position (Neuman-Keuls test, P < 0.05) and significantly greater during the second burst than in any of the 4th through 10th bursts (Neuman-Keuls test, P < 0.05 for all comparisons).
|
Because of our observation that SC spike firing probability was not constant during a train of 10 stimuli at 1 Hz (Fig. 4B), we wished to verify that the apparent decrease in spike-blocking efficacy was not due, rather, to a facilitation in the SC response owing to repeated stimulation. To test this, we compared spike-blocking efficacy during the first and last SLM bursts by presenting only two SC stimuli nine seconds apart, thus occurring during the first and last IPSPs of a train of bursts. Spike-blocking efficacy was 0.87 ± 0.13 during the first burst and 0.17 ± 0.12 during the last burst, a significant difference (P < 0.05, n = 3), indicating that spike-blocking efficacy did in fact decrease over the course of the burst train.
Recovery from the decay of spike-blocking was measured by performing single probe tests 2 min following a train of SLM bursts. After 2 min, spike-blocking efficacy had returned to 0.46 ± 0.12 (n = 5; Fig. 5); spike-blocking efficacy at 5-10 min following the previous burst train was 0.72 ± 0.08 (n = 5), very similar to the 0.74 ± 0.05 spike-blocking efficacy observed when single bursts were tested at 400 ms ISI (compare with Fig. 2A).
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SLM interneurons may contribute to spike-blocking
We made recordings from 41 SLM interneurons, potential mediators of this spike-blocking effect, to determine their responses to high-frequency SLM stimulation. Some (14/41) interneurons were not very responsive at all to stimulation in SLM, despite having dendrites in that layer. Of this subset (14/41), some interneurons could be driven directly (i.e., spiking due to direct depolarization or antidromic activation of the axon) by SLM stimulation. Another set of interneurons (3/41) responded solely with an IPSP to temporoammonic (TA) stimulation. The last set (24/41) of SLM interneurons responded with a large EPSP to stimulation in SLM and could be made to spike by repeated SLM stimulation at high frequencies; an example of such an interneuron is shown in Fig. 6. The spike-blocking effect we characterized is likely to be mediated by interneurons that were driven synaptically, as well as those that were driven directly by the stimulating electrode.
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DISCUSSION |
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We have shown that high-frequency stimulation in SLM can regulate
the output activity of CA1 pyramidal cells in response to excitatory SR
inputs. This result suggests a role for the direct projection from EC
to SLM of CA1, the TA pathway (Fredens et al. 1984;
Maccaferri and McBain 1995
; Reeves et al.
1997
), in regulation of the output of the hippocampus.
Individual EC layer III neurons, whose axons are activated by SLM
stimulation, can naturally fire at the high frequencies used in this
study (Finch et al. 1986
; but see Mizumori et al.
1992
; but see Stewart et al. 1992
; but see
Dickson et al. 1997
; Gloveli et al.
1997
). EC layer II/III neurons also fire in high-frequency
population bursts (Chrobak and Buzsáki 1998
). A
similar inhibitory phenomenon has been observed in area CA3 of the
hippocampus, where a single stimulus to the perforant pathway in SLM
can block spontaneous firing of CA3 pyramidal cells (Kehl and
McLennan 1985a
,b
) and reduce the amplitude of a
population spike evoked by subsequent stimulation of the fimbria (Kehl and McLennan 1985a
,b
).
Previous reports have suggested that the TA pathway may inhibit SC
responses in area CA1 (Empson and Heinemann 1995a,b
;
Levy et al. 1998
). However, the relative timing of SR
and SLM stimulation in these studies was based on the difference in
synaptic delays between the mono- and trisynaptic inputs from EC to
CA1. This approach assumes that the cells of origin of the two
pathways, which consist of discrete populations within EC
(Steward and Scoville 1976
), are active simultaneously.
Because of the difficulty of identifying the layer of origin of single
units recorded in vivo (e.g., see Chrobak and Buzsáki
1998
; Quirk et al. 1992
; Stewart et al.
1992
), it is not known whether this is the case. Because afferent inputs are segregated to different layers of the EC (e.g., see
Witter 1993
), it is likely that the cells of layers II
and III of EC have different activity patterns. The slow and long time
course of the SLM-mediated inhibitory effect reported in our study
suggests that spike-blocking may act as an overall damping of the
output of the hippocampus, rather than a more synapse-specific or
temporally restricted effect.
The dependence of the spike-blocking effect on GABAB
receptors was confirmed by its elimination in the presence of the
GABAB receptor antagonist CGP 55845A (Fig. 1). The
spike-blocking effect may be mediated by presynaptic GABAB
receptors located on the SC axon terminals, as seen elsewhere (e.g.,
Isaacson et al. 1993), by the hyperpolarization evoked
by activation of postsynaptic GABAB receptors on the
pyramidal cells (e.g.,Connors et al. 1988
), or a
combination of both factors; we did not address this issue in our study.
The variation of spike-blocking efficacy with ISI is consistent with
the time course of GABAB-mediated phenomena (reviewed in
Mott and Lewis 1994). The IPSP evoked by SLM burst
stimulation peaked at 397 ± 14 ms, and spike-blocking efficacy
was greatest at an ISI of 400 ms. Some spike-blocking was observed as
early as 100 ms, at which point the somatic membrane potential was near its resting potential, suggesting that postsynaptic hyperpolarization may not be essential to the spike-blocking effect. At short ISIs, shunting of the SC input by the GABAA or EPSP components of
the SLM response may have contributed to the spike-blocking effect. We
found that the spike-blocking effect also had a longer time course than
the IPSP, because there was no significant difference between
spike-blocking efficacy at 400 and 600 ms ISI, and spike-blocking could
still be observed even 800-1,000 ms after the SLM burst. Spike-blocking at long ISIs may have been mediated by the activation of
presynaptic GABAB receptors on SC axon terminals
(Isaacson et al. 1993
).
Although spike-blocking efficacy was not directly dependent on the IPSP
amplitude, the size of the IPSP still appears to be a good indicator of
the amount of GABAB activation. Consistent with this, an
increase in the number of stimuli in the SLM burst resulted in
increases both in IPSP amplitude (and duration) and in spike-blocking
efficacy. Increasing the number of SLM stimuli/burst could result both
in the recruitment of more interneurons, because of EPSP summation, and
in more action potentials per interneuron. Previous studies have shown
that activation of individual SLM interneurons does not result in a
GABAB response visible at the soma of CA1 pyramidal cells,
even when a train of action potentials is elicited in the interneuron
(Ouardouz and Lacaille 1997; Vida et al.
1998
). In general, it is believed that several interneurons must be activated to evoke a GABAB-mediated response
(Fortunato et al. 1996
; Isaacson et al.
1993
; Lambert and Wilson 1994
), possibly because
of a requirement for GABA "spill-over" to extrasynaptically located
GABAB receptors (Isaacson et al. 1993
).
Patterns of activity in the nervous system do not occur in a vacuum;
responses to synaptic activity are conditioned by the prior history of
the synapse. The balance of excitation and inhibition in the
hippocampus varies constantly due to activity-dependent regulation of
synaptic transmission. Here, we have shown that short-term depression
of an SLM-activated spike-blocking effect can shift the balance between
excitation and inhibition in the inputs onto CA1 pyramidal cells.
Spike-blocking evoked by burst SLM stimulation was depressed when
bursts were repeated at 1 Hz (Figs. 4 and 5). Down-regulation of the
inhibitory TA input may contribute to frequency-dependent facilitation
of the trisynaptic pathway (Herreras et al. 1987). In
the presence of natural patterns of activity, such as theta rhythms,
the efficacy of spike-blocking could be continuously up- and
downregulated. The long-term depression of excitatory TA responses
following low-frequency (1 Hz) stimulation (Dvorak-Carbone and
Schuman 1999
) may also contribute to a rebalancing of
inhibitory and excitatory transmission in this pathway.
Activity-dependent depression of inhibitory responses has been well
characterized (Ben-Ari et al. 1979; Davies et al.
1990
; Deisz and Prince 1989
; McCarren and
Alger 1985
; Thompson and Gähwiler 1989
)
and is mediated by GABAB autoreceptors (Davies et
al. 1990
; Roepstorff and Lambert 1994
) as well
as by a GABAB-independent process, possibly synaptic
vesicle depletion (Fortunato et al. 1996
; Lambert
and Wilson 1994
). Although most studies of modulation of
inhibition have focused on GABAA responses,
GABAB responses are also reduced with repeated stimulation
(Ling and Benardo 1994
; Williams and Lacaille
1992
). We found that repeated presentation of the SLM burst
stimulus at 1 Hz resulted in a decrease in the IPSP amplitude along
with a decrease in spike-blocking efficacy. In addition to processes
intrinsic to interneuron axon terminals, a decrease in recruitment of
SLM interneurons may also have contributed to the decreased IPSP size
(Congar et al. 1995
). Also, the repeated high-frequency
stimulation used may have resulted in depletion of the available pool
of synaptic vesicles (Liu and Tsien 1995
; Stevens
and Tsujimoto 1995
). The slow time course of recovery from
activity-dependent depression is consistent with synaptic vesicle
depletion (Lass et al. 1973
; Liu and Tsien
1995
; Wiley et al. 1987
).
Of the many different kinds of GABAergic interneurons in the
hippocampus (see Freund and Buzsáki 1996 for
review), the interneurons of SLM are likely candidates for mediators of
the spike-blocking effect. SLM interneurons can be driven either
synaptically (Fig. 6) (Lacaille and Schwartzkroin
1988a
,b
; Williams et al. 1994
) or by
direct depolarization (unpublished observations) in response to
stimulation in SLM. Focal stimulation in SLM has been used to evoke
GABAB-mediated synaptic responses in pyramidal cells, presumably by the activation of SLM interneurons (Benardo
1995
; Miles et al. 1996
; Williams and
Lacaille 1992
). Trains of action potentials in SLM interneurons
can block action potentials from being evoked by depolarizing current
injection in pyramidal cells (Lacaille and Schwartzkroin
1988a
,b
). Other types of interneurons may also contribute to
SLM-activated spike-blocking, including vertically oriented
oriens/alveus interneurons (McBain et al. 1994
),
stratum pyramidale basket cells (Han 1996
;
Sík et al. 1995
), and chandelier cells
(Buhl et al. 1994
; Li et al. 1992
), all
of which have dendritic arborizations in SLM. Basket and chandelier cells have been identified as postsynaptic targets of TA axons (Kiss et al. 1996
).
Under what physiological circumstances is the spike-blocking effect
likely to be evoked? If SLM interneurons are indeed responsible for
spike-blocking, then it needs to be determined under what circumstances
they are active. SLM receives projections from layer III of EC
(Steward and Scoville 1976), nucleus reuniens thalami (Dolleman-Van Der Weel and Witter 1996
), inferotemporal cortex (Yukie and Iwai 1988
), and amygdala (Petrovich et
al. 1997
; Pikkarainen et al. 1999
), all of which
might activate SLM interneurons. Disynaptic inhibition of CA1 pyramidal
cells via the TA input has been demonstrated in vitro (Empson
and Heinemann 1995a
,b
), and the projection from nucleus
reuniens thalami has been shown to activate SLM interneurons in vivo
(Dolleman-Van der Weel et al. 1997
).
Burst firing of SLM interneurons may be required for spike-blocking;
SLM interneurons may fire in bursts when recovering from hyperpolarization (Lacaille and Schwartzkroin 1988a,b
),
possibly due to low-threshold, transient Ca2+ currents
(Fraser and MacVicar 1991
; but see Williams et
al. 1994
). SLM interneurons are also depolarized and fire
action potentials in the presence of the muscarinic acetylcholine
receptor agonist carbachol (Chapman and Lacaille 1998
);
the SR/SLM border receives substantial cholinergic innervation
(Matthews et al. 1987
), suggesting that SLM interneurons
in vivo may be activated by cholinergic inputs.
SLM-evoked blockade of SC excitation of pyramidal cells may be
important for selective regulation of excitatory inputs to CA1.
Although SC inputs are a primary source of excitatory input to area CA1
(Amaral et al. 1990; Amaral and Witter
1989
; Andersen et al. 1966
; Lopes da
Silva et al. 1990
), under some circumstances, the TA pathway
can also have a strong excitatory effect (Buzsáki et al.
1995
; Yeckel and Berger 1990
). Responses in CA1
to SC or TA inputs are differentially sensitive to the
GABAB agonist baclofen, with SC field responses greatly
reduced while TA responses are unaffected (Colbert and Levy
1992
). A similar differential suppression of SC versus TA
inputs to CA1 in the presence of carbachol has been demonstrated
(Hasselmo and Schnell 1994
); such regulation is proposed
to be important in switching between encoding and retrieval modes of
associative memory systems (Hasselmo and Schnell 1994
).
GABAB-mediated selective suppression of inputs to CA1, such
as that shown here during SLM activity, could also mediate such a
switch (Hasselmo et al. 1996
). Other models of memory
decoding in area CA1 require a strong excitatory input from the EC to
CA1 (McClelland and Goddard 1996
), further suggesting
the importance of selective suppression of SC inputs. A test of the
possible role of GABAB-mediated spike-blocking in selecting
between SC and TA inputs to CA1 pyramidal cells will require a better
understanding of the circumstances under which TA input to CA1 is
sufficiently strong to cause pyramidal cells to fire.
In conclusion, we have shown that afferent inputs to SLM, including the direct projection from EC, are ideally positioned to gate information flow through the trisynaptic pathway by means of appropriately timed inputs. In vivo studies, where fiber tracts are intact and can be stimulated independently, would be helpful to determine which of the many afferents to SLM mediate the inhibitory spike-blocking effect, and under what circumstances the gating is physiologically effective.
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ACKNOWLEDGMENTS |
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The authors thank Dr. A. M. Suter at Novartis for a kind gift of CGP 55845A, and the Howard Hughes Medical Institute for funding, including a predoctoral fellowship to H. Dvorak-Carbone.
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FOOTNOTES |
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Address reprint requests to E. M. Schuman.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 June 1999; accepted in final form 27 August 1999.
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REFERENCES |
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