Department of Psychology and Program in Neuroscience, Harvard University, Cambridge, Massachusetts 02138
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ABSTRACT |
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Linster, Christiane, Bradley P. Wyble, and Michael E. Hasselmo. Electrical stimulation of the horizontal limb of the diagonal band of Broca modulates population EPSPs in piriform cortex. Electrical stimulation of the horizontal limb of the diagonal band of Broca (HDB) was coupled with recording of evoked potentials in the piriform cortex. Stimulation of the HDB caused an enhancement of the late, disynaptic component of the evoked potential elicited by stimulation of the lateral olfactory tract but caused a suppression of the synaptic potential elicited by stimulation of the posterior piriform cortex. The muscarinic antagonist scopolamine blocked both effects of HDB stimulation. The enhancement of disynaptic potentials could be due to cholinergic depolarization of pyramidal cells, whereas the suppression of potentials evoked by posterior piriform stimulation could be due to presynaptic inhibition of intrinsic fiber synaptic transmission by acetylcholine.
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INTRODUCTION |
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Modulatory effects of acetylcholine have been
shown to play a role in olfactory behavior. In particular, the
muscarinic antagonist scopolamine has been shown to impair short-term
memory for odors (Ravel et al. 1994), habituation to
familiar odors (Hunter and Murray 1989
), and odor-based
social recognition (Perio et al. 1989
; Soffie and
Lambert 1988
). Lesions of the cholinergic and GABAergic inputs
from the basal forebrain have also been shown to influence olfactory
habituation and investigation (Paolini and McKenzie 1993
,
1996
).
Computational models of the olfactory system have investigated how the
specific effects of cholinergic modulation on neuronal circuits could
influence processing of olfactory stimuli (Hasselmo 1993; Linster and Gervais 1996
; Linster
and Hasselmo 1997a
). The implementation of cholinergic effects
in the piriform cortex in these models has been based primarily on
experimental data from brain slice preparations. These data include
experiments showing that cholinergic agonists depolarize piriform
cortex pyramidal cells (Barkai and Hasselmo 1994
),
suppress neuronal adaptation (Barkai and Hasselmo 1994
;
Tseng and Haberly 1989
), and suppress excitatory and
inhibitory synaptic transmission in the association fiber pathway
(Hasselmo and Bower 1992
; Patil and Hasselmo
1996
; Williams and Constanti 1988a
).
Although brain slice preparations provide controlled conditions for
analyzing modulatory effects, it is important to demonstrate that these
effects appear in in vivo preparations. This ensures that the effect
appears with neuronal release of the modulator in anatomically intact
circuits. Both the olfactory bulb (OB) and piriform cortex receive
cholinergic input from the horizontal limb of the diagonal band of
Broca (HDB) (Zaborsky et al. 1986a). The neurons in the
HDB contain both ACh and GABA (Brashear et al. 1986
),
and it is known that the cholinergic and GABAergic neurons in the HDB
are at least partially spatially segregated (Brashear et al.
1986
). This spatial segregation of cholinergic and GABAergic
neurons in different regions of the HDB allows for preferential
stimulation of cholinergic neurons projecting to the OB and/or piriform
cortex (Luskin and Price 1982
; Zaborszky et al.
1986b
). Several studies of cholinergic effects in the OB have
been performed in vivo (Elaagouby et al. 1991
;
Kunze et al. 1991
, 1992
; Nickell and Shipley
1988
, 1993
; Ravel et al. 1990
), but to our
knowledge only a single study has investigated the effects of
cholinergic modulation in the piriform cortex in vivo (Zimmer et
al. 1996
).
We investigate the effect of electrical stimulation in the HDB on excitatory synaptic transmission in the piriform cortex in vivo. We investigate the effect of HDB stimulation on the population excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the lateral olfactory tract (LOT) and more posterior piriform cortex (pPC). We observe 1) no effect on the monosynaptic EPSP evoked by electrical stimulation of the LOT, 2) an increase of the disynaptic component of the population EPSP evoked by LOT stimulation, and 3) a decrease of the population EPSP evoked by electrical stimulation of intrinsic association fibers from the pPC. All the observed effects are significantly decreased under the muscarinic antagonist scopolamine, suggesting that they are at least partially mediated by cholinergic pathways.
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METHODS |
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Surgical preparation
Adult, male Sprague Dawley rats (300-400 g) were anesthetized
with urethan (1.5 g/kg ip). Levels of anesthesia were monitored by rate
of respiration and foot withdrawal reflex and supplemented if necessary
by intraperitoneal injections. Body temperature was maintained at
37°C with a heating pad. Anesthetized animals were placed in a
stereotaxic apparatus, and the skull was exposed by scalp incision. We
used a total of 11 animals for the data presented here. In seven
animals, both stimulation of the LOT and stimulation of the pPC were
used. Holes were drilled in the skull at positions appropriate for
gaining access to the stereotaxic locations (Paxino and Watson
1986) of the LOT, the HDB, anterior piriform cortex (aPC), and pPC. Recently, it has been shown that urethan has an effect
on population EPSPs such as those observed in the experiments described
here (Cauthron and Stripling 1998
); however, the effects measured in our experiments (relative size of evoked potentials with
and without electrical stimulation of cholinergic afferents) should not
be affected by the tonic presence of urethan anesthesia.
Electrical stimulation
As summarized schematically in Fig. 2, bipolar stimulation
electrodes (100 µm stainless steel, Formvar-insulated except at the
tips, twisted together) were placed in the LOT (4.7 mm anterior; 3.4 mm
lateral; 5.2 ventral from bregma), in pPC (3.6 mm posterior, 3.0 mm
lateral (14° angled laterally); 9-10 mm ventral from bregma), and in
the HDB (0.0-0.5 mm posterior, 1.8-2.2 mm lateral, 8.6-9.0 mm
ventral from bregma). We chose the coordinates for the HDB stimulation
electrode in such a way as to ensure a high probability for stimulation
of cholinergic neurons projecting to the olfactory system, based on
anatomic data (Brashear et al. 1986; Luskin and Price 1982
; Zaborszky et al. 1986b
). Optimal
placement of the recording electrode was achieved by monitoring the
field potentials evoked in layer Ia of aPC. All stimulation currents
were delivered by a constant current stimulus isolation unit (Grass
Instruments) controlled by a Grass Instrument stimulator. Stimulation
currents for the LOT and pPC electrodes were adjusted to approximately two times threshold ([SIM]0.3 ms pulses at 100-300 µA). After placement, stimulation strength for the HDB electrode was adjusted such
as to evoke no field potential response in aPC (~100-300 µA). For
placement of the pPC electrode, we recorded the field potentials evoked
by LOT stimulation from this electrode and placed the electrode in the
deeper layers (II or III) of pPC, as determined by the polarity of the
response observed in response to LOT stimulation (Haberly
1973
). Field potentials elicited by stimulation of layer I of
the piriform cortex can easily be contaminated by antidromic LOT
activation, and the components of these field potentials are difficult
to interpret. We therefore chose to stimulate layer II-III of pPC,
evoking a population EPSP in aPC, presumably caused by direct
electrical activation of the deeper layer association fibers. This
produced a positive-going potential in layer Ia of aPC, which reversed
near layer II. The locations of stimulus electrodes and the recording
electrode are summarized schematically in Fig. 1.
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Recording
For recording of field potentials, a recording electrode (100 µm stainless steel, formvar insulated except at the tip) was placed in layer Ia of the aPC in such a way as to observe a short latency response to stimulation of the LOT. DC preamplification (Grass Instruments, ×10) was used in all recordings; the signal was then further amplified (×200) and filtered between 0.1 and 6000 Hz via a Neuralynx differential amplifier. Data acquisition, display, and control of stimulus were done by use of computer (Pentium 100 MHz and Datawave Workbench 32 software). Amplitude of field potentials was measured from the baseline (0-2 ms before the stimulus artifact) to the peak of the negative deflection (when stimulating LOT) or the positive deflection (when stimulating layer II-III of pPC).
Drug administration
Systemic injections of scopolamine (0.5 mg/kg ip in saline) were used to test whether the observed effects were due to activation of muscarinic cholinergic receptors. The difference between the baseline pulses and pulses after HDB stimulation was compared before and after the scopolamine injections.
Histology
At the end of the experiment, current was passed through the recording and stimulation electrodes (via a 9-V battery) to deposit iron in the tissue. Transcardial perfusion was then performed with saline and potassium ferrocyanide in 10% buffered formalin. After storage in 10% formalin with 20% sucrose, brains were sectioned and stained with Neutral red for localization of the recording and stimulating electrodes.
Experimental protocol
These experiments quantified the effect of electrical
stimulation of the HDB on evoked responses in aPC (Fig. 1). As reported previously by other authors for similar experimental protocols (Nickell and Shipley 1993; Zimmer et al.
1996
), we could not observe an effect when single electrical
pulses were used for stimulation in the HDB. We therefore tested the
effect of a brief tetanus (4 pulses of 0.3-ms width separated by 10 ms)
in the HDB on the evoked responses to a subsequent single 0.3-ms pulse
in either the LOT or the pPC. A single baseline pulse was given in the
LOT or pPC electrode, and the response in the aPC was recorded
(baseline pulse). After a delay of 1 s, the tetanus in the HDB
electrode was given followed by a second single test pulse in the LOT
or pPC electrode (test pulse). The delay (
t) between the
HDB tetanus and the LOT or pPC test pulse was variable
(
t = 25, 50, 100, and 250 ms). The stimulation
current in the HDB electrode was adjusted to be just below threshold
for the observation of a population response in the aPC. In the control
experiment, the HDB stimulation was given with very low currents (<10
µA) at
t = 50 ms. For each delay
t
and for the control, a set of 10 trials was performed with a 1-min
interval between each trial. For each trial, the response to the test
pulse after HDB stimulation was compared with the response to the
baseline pulse. For the monosynaptic component of the population EPSPS,
we measured both the amplitude of the peak and the slope of the rising
phase of the potentials (measured between baseline 1-2 ms before the
stimulation artifact and the peak response). For the disynaptic phase
of the LOT-evoked response (B1), an accurate measurement of the slope
cannot be obtained; therefore only amplitude measurements were
obtained. The amplitude of the B1 response was quantified by using a
time point determined by visual inspection and measuring the distance from the prestimulus baseline to the physiological trace at that time
point (see Fig. 2). The point chosen by
visual inspection corresponded as accurately as possible to the minimum
absolute value of the first derivative of the trace (i.e., the point
where the rate change was smallest). This was done on the basis of the trace with the clearest B1 response (usually the test response) for
each experiment. In cases were a peak was clearly visible, the time
point of the local minimum was chosen. The point of measurement of B1
was usually ~8 ms after the first peak (A1). The results obtained
from amplitude and slope measurements were similar in all cases. The
results are reported as the ratio between the rising slope (or the
amplitude) of the response to the test pulse after HDB stimulation and
the response to the baseline pulse. For each delay
t,
this ratio is then compared with the ratio obtained in the control
condition (low stimulation current in HDB electrode). The experiments
(same electrode locations and stimulation currents) were repeated 30 min after injection of 0.5 mg/kg ip scopolamine. Results are reported
as average ± SE. Statistical tests (t-test, single-tailed) were performed to determine significance
(P < 0.01) of results.
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RESULTS |
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Effect of HDB stimulation on the population EPSP evoked by stimulation of the LOT
The population EPSP observed in layer Ia of the PC by stimulation
of the LOT (Fig. 2A) contains a number of different
components that have been described in detail (Haberly
1973; Ketchum and Haberly 1993
). The first
negative peak has been labeled period 1 and is composed of two phases
called A1 and B1. A1 is generated by the monosynaptic EPSP in layer Ia,
and B1 has been proposed to result from a disynaptic EPSP caused by
activation of recurrent collaterals within the piriform cortex. The
following positive component is called period 2 and is thought to
result from inhibitory input to pyramidal cells. In the experiment
described here, we observed the effect of a brief tetanus in the HDB on
period 1 of the population EPSP evoked by LOT stimulation.
No significant effect of HDB stimulation on the monosynaptic population
EPSP (A1) was observed in our experiments (n = 8; Fig.
2), as previously reported by Zimmer et al. (1996). In contrast, component B1 of the population EPSP was increased by the preceding stimulation in the HDB (Fig. 2B). As shown in Fig.
2C, a significant effect (169 ± 7.3% of the baseline
response; n = 8; P < 0.01) was
observed, only for
t = 50 ms. This stimulation
induced increase in B1 was blocked by scopolamine; 30 min after
injection of 0.5 mg/kg scopolamine, no significant effect was observed
after the tetanus in the HDB electrode with a delay of
t = 50 ms (110 ± 6% of test response;
n = 6; P > 0.2). Scopolamine had no
effect on the response to the test pulse in the LOT. Thus the baseline used for comparison did not change. Figure 2 summarizes the effects on
the population EPSP evoked by stimulation of the LOT.
Modulation of the population EPSP evoked by stimulation of the pPC
For these measurements, the stimulation electrode was placed into
layers II-III of pPC. Electrical stimulation of the deeper layers of
pPC evokes a complex population response recorded in layer Ia of aPC,
of which we considered only the first peak, which is presumably the
monosynaptic response evoked by stimulation of intrinsic association
fibers and which could be reliably evoked in our experiments. When
recorded in layer Ia of aPC, the first deflection of the response
evoked by stimulation of the deeper layers is positive. Consistent with
anatomic data, this potential reverses from positive to negative around
the same location as the response evoked by stimulation of the LOT
reverses from negative to positive. The first peak is negative if
recorded in the deeper layers (layers II-III) of aPC, suggesting that
we are stimulating association fibers that terminate in layers II-III
or in the most proximal portion of layer Ib (Haberly
1985). In most animals tested (8/10), a significant decrease in
the population EPSP evoked by stimulation of the deeper layers of pPC
was observed after a tetanus in the HDB (Fig.
3A). The maximal decrease was
observed when the pPC pulse occurred 50 ms after the HDB stimulation
(78% ± 0.6% of the test response; P < 0.0001, n = 10) (Fig. 3, A and C). In contrast to the effect observed for LOT stimulation, the effect on the
pPC-evoked EPSP was still significant at a delay of 100 ms (84% ± 0.8% of test response) and at a delay of 250 ms (92% ± 1.2% of test
response). Injection of 0.5 mg/kg scopolamine completely abolished the
observed decrease in size induced by HDB stimulation (n = 8). Interestingly, scopolamine by itself had an effect on the
amplitude of the test pulse. When the deeper layers were stimulated, 30 min after the administration of 0.5 mg/kg scopolamine, the average
rising slope of the population EPSP increased to 146 ± 5.2% of
the response before scopolamine injection, suggesting that these
potentials are under tonic suppression by muscarinic receptor
activation.
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Stimulation sites
After each experiment, all recording and stimulation sites were
verified histologically. Only animals in which the stimulation sites
were located in the area designated as HDB (Paxino and Watson 1986) were considered for the analysis of the results
described here. Figure 4 shows a summary
of the lesion sites produced by the HDB electrode (Fig. 4A)
and the pPC electrode (Fig. 4B). The locus of stimulation
was in the area of the HDB shown to contain a high density of
cholinergic neurons projecting to the olfactory system (Brashear
et al. 1986
; Luskin and Price 1982
;
Zaborszky et al. 1986b
).
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DISCUSSION |
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The results presented here demonstrate that electrical activation
of cholinergic input from the horizontal limb of the diagonal band
modulates evoked synaptic potentials in the piriform cortex, causing
1) suppression of monosynaptic potentials evoked by
stimulation of pPC, 2) enhancement of the disynaptic
activity induced by LOT stimulation, and 3) no change in the
monosynaptic activity induced by LOT stimulation. These results are
consistent with brain slice physiological experiments showing
cholinergic suppression of intrinsic synaptic potentials in layer Ib
with little or no effect on synaptic potentials elicited by afferent
fiber stimulation in layer Ia (Hasselmo and Bower 1992).
The synaptic potentials elicited by stimulation of the pPC probably
arise from associative connections from pPC to aPC, in contrast to the
potentials elicited by LOT stimulation, which involve induction of both
afferent synaptic currents in layer Ia and associative synaptic
currents in layers Ib and in deeper layers (Biella and DeCurtis
1993; Ketchum and Haberly 1993
). Our results
suggest that the observed suppression of the population EPSP is
mediated by muscarinic receptors, as systemic injections of scopolamine
reduce the suppression here in the same manner that scopolamine blocks
suppression of potentials induced by carbachol in slice preparations of
the piriform cortex (Hasselmo and Bower 1992
;
Williams and Constanti 1988a
,b
).
The enhancement of disynaptic activity evoked by LOT stimulation
probably results from the direct cholinergic depolarization of piriform
cortex neurons described in vitro (Barkai and Hasselmo 1994; Tseng and Haberly 1989
), which seems to
cause a sufficient increase in spiking of pyramidal cells and
disynaptic transmission to overcome the simultaneous suppression of
intrinsic synaptic transmission.
Relation to previous in vivo results
The data presented here complements results from previous work in
the OB showing that stimulation of the HDB suppresses synaptic potentials elicited in the OB by stimulation of the anterior commissure (Nickell and Shipley 1993). The commissural connections
from one OB to the granule cells in the contralateral OB could be
considered analogous to intrinsic connections between the pPC and the
aPC, although suppression of transmission at these synapses has not been investigated in slice preparations of the OB. The enhancement of
disynaptic potentials elicited by LOT stimulation here could result
from an increase in excitability of pyramidal cells; indeed previous
results reported in the piriform cortex (Zimmer et al. 1996
) show an enhancement of spontaneous activity in piriform cortex pyramidal cells after electrical stimulation of the HDB.
A similar cholinergic modulation of synaptic transmission has been
shown in in vivo experiments in the hippocampal formation. Iontophoretic injections of acetylcholine or muscarine into stratum radiatum of region CA1 have been shown to suppress population EPSPs
elicited by stimulation of the fimbria (Rovira et al. 1982, 1983
). Repetitive stimulation of the medial septum also causes suppression of evoked synaptic potentials in stratum radiatum of region
CA1 (Rovira et al. 1983
) in an experimental paradigm similar to the experiments presented here. Systemic injections of
scopolamine blocked the observed effect in that study as well, suggesting that it is at least partially mediated by muscarinic receptors. Other studies have demonstrated the stimulation of the
medial septum causes enhancement of population spikes in the pyramidal
cell layer of region CA1 (Ben Ari et al. 1981
;
Krnjevic and Ropert 1981
, 1982
) and the granule cell
layer of the dentate gyrus (Bilkey and Goddard 1985
;
Fantie and Goddard 1982
). This effect shows a rapid time
course similar to the effect described here and could result from
cholinergic depolarization of pyramidal cells, as suggested by our
results for piriform cortex pyramidal cells.
Functional significance
Our results support the notion that cholinergic modulation of
population EPSPs occurs within the intact animal. We observe an
enhancement of the disynaptic potential elicited by LOT stimulation, which may result from depolarization of pyramidal cells. We also observe a suppression of the population EPSP evoked by stimulation of
intrinsic association fibers. These two effects are complementary; a
depolarization of pyramidal cells accompanied by a suppression of
intrinsic association fiber transmission can increase the
signal-to-noise ratio in response to afferent input from the LOT
(Hasselmo et al. 1997). Previous computational modeling
work suggests that this cholinergic modulation might allow afferent
input to have a dominant influence on physiological activity in the
piriform cortex (Barkai et al. 1994
; Hasselmo and
Linster 1997
; Hasselmo et al. 1992
). The results
presented here show that during activation of the HDB the influence of
intrinsic connections arising from pPC is considerably weakened
relative to the unchanged magnitude of monosynaptic afferent input. A
dominant influence of afferent input, enhanced by the depolarization of
the pyramidal cells, would be appropriate for learning of new
information with minimal interference from previously stored
representations (Hasselmo and Bower 1993
;
Hasselmo and Linster 1997b
). This would be particularly appropriate for the proposed function of the piriform cortex as an
associative memory network (Haberly 1985
; Haberly
and Bower 1989
) because neural network models of associative
memory require greater influence of afferent input during storage of
information than during retrieval.
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ACKNOWLEDGMENTS |
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The authors are thankful to J. Goodman, L. Zimmer, and Dr. Celeste Wiersig-Wiechman for advice on histology and in vivo electrophysiology.
C. Linster was supported by National Science Foundation (NSF) Grant IBN-9723947. B. P. Wyble was supported by a predoctoral award from the NSF.
Present address and addres for reprint requests: C. Linster, Dept. of Psychology, Boston University, 64 Cumminton St., Boston, MA 02215.
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FOOTNOTES |
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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 27 July 1998; accepted in final form 10 February 1999.
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REFERENCES |
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