EFFECT OF SCOPOLAMINE ON THE AUDITORY EVOKED POTENTIAL
IN THE DENTATE GYRUS OF THE AWAKE, BEHAVING RAT
by
Thomas R. Gregg
A thesis submitted to the Faculty of the
University of Delaware in partial fulfillment of the
requirements for the degree of Master of Arts in
Psychology
Spring 1996
Copyright 1996 Thomas R. Gregg
All Rights Reserved
EFFECT OF SCOPOLAMINE ON THE AUDITORY EVOKED POTENTIAL
IN THE DENTATE GYRUS OF THE AWAKE, BEHAVING RAT
by
Thomas R. Gregg
Approved:
Kenneth A. Campbell, Ph.D.
Professor in charge of thesis on behalf of the
Advisory Committee
Approved:
Evelyn Satinoff, Ph.D.
Chair of the Department of Psychology
Approved:
John Cavanaugh, Ph.D.
Interim Associate Provost for Graduate Studies
TABLE OF CONTENTS
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . iv
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . v
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . vi
Chapter
1. INTRODUCTION . . . . . . . . . . . . . . . . . . . 1
Auditory projections to the entorhinal cortex. . . 3
Primary auditory pathway . . . . . . . . . 3
Extralemniscal pathways. . . . . . . . . . 4
Muscarinic manipulations . . . . . . . . . 5
Perforant path input to the dentate gyrus. . . . . 6
Electrically-elicited field potentials . . 7
The dentate gyrus auditory evoked
potential. . . . . . . . . . . . . . . . . 8
Cholinergic input to the dentate gyrus . . . . . . 9
The current study. . . . . . . . . . . . . . . . . 10
2. METHODS. . . . . . . . . . . . . . . . . . . . . . 12
Animals and surgery. . . . . . . . . . . . . . . . 12
Testing apparatus and procedure. . . . . . . . . . 15
Data analysis. . . . . . . . . . . . . . . . . . . 17
3. RESULTS. . . . . . . . . . . . . . . . . . . . . . 19
Electrode location . . . . . . . . . . . . . . . . 19
Auditory evoked potential component definition . . 20
Effect of stimulus presentation rate on N1 . . . . 20
Effect of scopolamine on N1. . . . . . . . . . . . 21
Effects of scopolamine on motor activity . . . . . 22
4. DISCUSSION . . . . . . . . . . . . . . . . . . . . 24
TABLES . . . . . . . . . . . . . . . . . . . . . . . . 30
FIGURES. . . . . . . . . . . . . . . . . . . . . . . . 33
REFERENCES . . . . . . . . . . . . . . . . . . . . . . 40
APPENDIX: ANIMAL USE APPROVAL BY THE INSTITUTIONAL
ANIMAL CARE AND USE COMMITTEE OF THE UNIVERSITY OF
DELAWARE . . . . . . . . . . . . . . . . . . . . . . . 48
LIST OF TABLES
1. Mean and standard error of the mean (S.E.M.) of N1
amplitude and latency as a function of stimulus
presentation rate in all 8 rats. . . . . . . . . . 31
2. Effect of scopolamine on N1 amplitude and latency.
Mean (ñS.E.M.); n=8. . . . . . . . . . . . . . . . 32
LIST OF FIGURES
1. Auditory evoked potentials recorded prior to
scopolamine injection illustrate that N1 latency
is consistent (30-40 ms), but N1 amplitude is
highly variable from rat to rat (grand averages
from each of 6 rats). Calib: 100 æV, 20 ms;
positive is up.. . . . . . . . . . . . . . . . . . 34
2. Peak N1 amplitude is dependent on stimulus
presentation rate. Bars indicate +/- standard
error (n=8). . . . . . . . . . . . . . . . . . . . 35
3. Grand averages showing the effect of scopolamine
on N1. Auditory evoked potentials are shown in
the hour before (dashed line) and after injection
(solid line), averaged across all doses (n=8).
Calib: 100 æV, 20 ms. . . . . . . . . . . . . . . 36
4. Timecourse of effect of different dosage levels of
scopolamine on N1 amplitude. Mean N1 amplitude is
shown, with bars indicating ñS.E.M., at the 4
dosage levels, prior to injection and at 3
intervals following injection (n=8). Key: white=
pre-injection, light gray= 1st hr after injection,
medium gray= 3rd hr, dark gray= 24th hr. . . . . . 37
5. Auditory evoked potentials recorded in the hour
before (dashed line) and the hour after
methscopolamine injection (solid line). Grand
averages across all 8 rats are shown. Calib: 100
æV, 20 ms. . . . . . . . . . . . . . . . . . . . . 38
6. Activity levels (quadrant entries) are shown
following injections of scopolamine (scop) and
methscopolamine (mscop) at the indicated dosages
(mg/kg; n=8). Pre-injection= white, 1st hr after
injection= light gray, 24th hr=dark gray.. . . . . 39
ABSTRACT
The effect of cholinergic activity on the
modulation of auditory input to the hippocampal formation
was investigated. The auditory evoked potential (AEP)
recorded in the dentate gyrus (DG) of the hippocampal
formation in awake, freely-moving rats had a sharp
negativity (N1) at 34 ms. Subcutaneous injection of the
muscarinic antagonist scopolamine suppressed N1 peak
amplitude at doses of 0.5, 1.0, 2.0, and 5.0 mg/kg in the
first hour after injection. In the third hour after
scopolamine injection, N1 amplitude remained significantly
suppressed in an overall comparison; dosewise comparisons
showed this effect to be significant only at the highest
dose, so that the time course of N1 attenuation tended to
depend on dosage. N1 amplitude returned to control levels
by the 24th hour after injection. Control injections of
methscopolamine had no effect on N1, showing that the
scopolamine-induced effects were due to central muscarinic
blockade. Scopolamine, but not methscopolamine, also
significantly increased motor activity measured 45 min
after injection. Stimulus presentation rates of 1 Hz and
greater led to a marked decrement in N1 amplitude. The
similar sensitivity of N1 and vertex (extralemniscal) AEPs
to stimulus presentation rate and to scopolamine suggests
that N1 may reflect extralemniscal input to DG. However,
because motor activity is known to affect auditory
processing, it is possible that the scopolamine-induced N1
attenuation was due to the observed increase in motor
activity, rather than to a direct pharmacological effect
on auditory pathways.
Chapter 1
INTRODUCTION
The hippocampal formation plays a critical role in
memory (Olton, 1983); its role requires the processing of
sensory input, an activity that is modulated by the action
of acetylcholine (Foster & Deadwyler, 1992).
Acetylcholine modulates processing both in the hippocampal
formation itself and in sensory pathways that may project
to the hippocampal formation. For example, cholinergic
cells in the pedunculopontine tegmental nucleus respond to
auditory stimuli and project both to relay thalamic
nuclei, such as the medial geniculate body, and to
intralaminar and midline (nonspecific) nuclei, such as
nucleus centre median (Harrison, Woolf, & Buchwald, 1990b;
Newman & Ginsberg, 1994; Par‚, Smith, Parent, & Steriade,
1988; Steriade, Par‚, Parent, & Smith, 1988). Some of
these thalamic nuclei, including the medial geniculate
body, may project to the hippocampal formation via
multisynaptic pathways that synapse in the entorhinal
cortex (Witter, Groenewegen, Lopes DaSilva, & Lohman
1989). The goal of the current study was to characterize
further the cholinergic modulation of hippocampal sensory
input in rats by examining how the pharmacological
antagonism of cholinergic receptors affects sensory input
to the dentate gyrus (DG), which is the sensory "input
stage" for the hippocampal formation. Specifically, this
study examined the effect of systemic administration of
scopolamine, a muscarinic antagonist, on the DG averaged
auditory evoked field potential, which serves as an index
of sensory input to the hippocampal formation.
Interpretation of the results of the current study
requires consideration of previous studies of
antimuscarinic effects on both DG and the auditory
pathways that project to DG. The first part of the
background section of this paper describes the auditory
pathways projecting to the entorhinal cortex, which is the
major source of sensory input for DG, and the effects of
muscarinic blockers on these pathways. The second part
reviews the projection from the entorhinal cortex to DG
through the perforant path, focusing on the evoked field
potentials produced in DG by auditory stimulation and by
electrical stimulation of the perforant path. The last
part reviews the origins and terminations of cholinergic
fibers projecting to DG and the effects of cholinergic
manipulations on DG evoked field potentials.
Auditory projections to the entorhinal cortex
The primary, or lemniscal, auditory pathway
carries highly processed auditory information to the
primary auditory cortex; extralemniscal pathways carry
auditory information colored by arousal from the reticular
formation to several brain areas, including cortex
(Harrison et al., 1990b; Simpson & Knight, 1993). The
entorhinal cortex may receive auditory information from
either or both of these candidate pathways (Witter et al.,
1989).
Primary auditory pathway. By way of the primary
auditory pathway, auditory information is relayed from the
cochlear nuclei through the contralateral lateral
lemniscus to the nucleus of the lateral lemniscus and the
inferior colliculus (Aitkin, 1986). The cochlear nuclei
also project to the medial superior olivary nucleus, which
gives rise to the cholinergic olivocochlear bundle, a
feedback projection to the outer hair cells of the cochlea
(Guth, Norris, & Bobbin, 1976). The inferior colliculus
projects through the brachium of the inferior colliculus
to the medial geniculate body, which itself projects to
primary auditory cortex (Jones, 1985; Pickles, 1981) and
entorhinal cortex (Witter et al., 1989). The medial
geniculate body also projects to the medial and cortical
amygdaloid nuclei, which themselves provide input to the
entorhinal cortex (Haines, 1990; Witter et al., 1989).
The entorhinal cortex may receive input from auditory
cortex through pathways synapsing in association cortex,
pathways synapsing in the claustrum, or direct projections
(Witter et al., 1989).
Extralemniscal pathways. Additionally,
extralemniscal pathways may carry auditory information to
the entorhinal cortex. These pathways diverge from the
primary auditory pathway in the brainstem, where, in cats
and probably also in rats, fibers are sent into the
reticular formation by four brainstem auditory relay
nuclei: the dorsal and ventral cochlear nuclei
(Carpenter, 1978), the nucleus of the lateral lemniscus,
and the inferior colliculus (Irvine & Jackson, 1983;
Powell & Hatton, 1969). Neurons in medullary, pontine,
and mesencephalic nuclei of the reticular formation,
including cholinergic neurons of the pedunculopontine
tegmental nucleus, project to midline and intralaminar
nuclei of the thalamus, including nucleus centralis
medialis (Newman & Ginsberg, 1994; Woolf, Harrison, &
Buchwald, 1990). The intralaminar and midline thalamic
nuclei project lightly and diffusely to most areas of the
cerebral cortex (Harrison et al., 1990b; Jones, 1985);
nucleus centralis medialis projects directly to entorhinal
cortex (Witter et al., 1989). In addition, the
norepinephrinergic locus coeruleus and the serotoninergic
raphe nuclei of the reticular formation project directly
and diffusely to cortex (Cooper, Bloom, & Roth, 1991).
The entorhinal cortex receives these reticular-formation
projections, as well as receiving indirect reticular-
formation influences via multisynaptic thalamocortical and
corticocortical connections (Harrison et al., 1990b;
Witter et al., 1989).
Muscarinic manipulations. Muscarinic blockade
appears to affect the amplitudes of signals passing
through the extralemniscal pathways but not the primary
auditory pathway, as indicated by measurements of AEP
amplitude. Sites in the primary auditory pathway from the
cochlea to neocortex receive putative cholinergic
projections or are affected by cholinergic manipulations,
including the cochlear outer hair cells (Guth et al.,
1976), inferior colliculus (Farley, Morley, Javel, &
Gorga, 1983), medial geniculate body (Woolf et al., 1990),
and auditory cortex (Metherate, Ashe, & Weinberger, 1990).
However, several studies have found that central
muscarinic antagonism due to the administration of
scopolamine or atropine has little effect on primary
auditory pathway AEPs generated in the brainstem (Church &
Gritzke, 1988; Samra, Krutak-Krol, Pohorecki, & Domino,
1985) or primary auditory cortex (Buchwald, Rubinstein,
Schwafel, & Strandburg, 1991; Dickerson & Buchwald, 1991;
Miyazato, Skinner, Reese, Boop, & Garcia-Rill, 1995).
In contrast, extralemniscal pathway AEPs are
attenuated by scopolamine. Wave A of the cat vertex AEP,
recorded above midline parietal cortex, reflects the
extralemniscal signal passing from the pedunculopontine
tegmental nucleus to nonspecific thalamus, as shown by
depth recordings in the thalamus (Hinman & Buchwald, 1983)
and by the disappearance of wave A following lesions of
the pedunculopontine tegmental nucleus (Harrison et al.,
1990b). Scopolamine attenuates wave A as recorded both at
vertex and in the thalamic nucleus centre median
(Dickerson & Buchwald, 1991; Harrison, Tung, & Buchwald,
1990a; Harrison, et al, 1990b), and attenuates a wave-A-
like component of the rat vertex AEP that is thought to
reflect extralemniscal activation (Campbell, Kalmbacher,
Specht, & Gregg, 1995; Miyazato et al., 1995; Simpson &
Knight, 1993).
Perforant path input to the dentate gyrus
The perforant path originates in the entorhinal
cortex, which receives sensory information from many areas
of the cerebral cortex. Most perforant path axons form
glutamatergic synapses with granule cell dendritic spines
in the outer two-thirds of the dendritic field in the
molecular layer of DG, although some release GABA and some
project to other hippocampal areas (Germroth,
Schwerdtferger, & Buhl, 1989; Steward, 1976; Wheal &
Miller, 1980; Witter, 1989; Witter et al., 1989). From
DG, information travels through the hippocampus by way of
the "trisynaptic circuit," the relay of projections from
the entorhinal cortex to DG granule cells, then from DG to
CA3 pyramidal cells, and then from CA3 to CA1 pyramidal
cells (Andersen, 1975); CA1 projects to the subiculum,
which projects to the basal forebrain, hypothalamus, and
cortex, including the entorhinal and retrosplenial areas
(Amaral, 1993; Swanson & Cowan, 1975; Witter et al.,
1989).
Electrically-elicited field potentials. Three
events are reflected by the components of the field
potential elicited in DG by electrical stimulation of the
medial perforant path. The first component is a compound
action potential from a population of perforant path axons
and reflects the afferent presynaptic volley arriving in
DG. This small-amplitude, triphasic (positive-negative-
positive) component has an onset latency of 0.2 ms, and
lasts 0.2 ms. The second component is similar to the N1
component of the AEP, to be described later. This
component is a population excitatory postsynaptic
potential (EPSP) with an onset latency of 2 ms and a peak
latency of 4-5 ms. It appears as a negativity in the
region of granule cell dendrites in the middle third of
the molecular layer of DG, indicating a current sink
there, but as a positivity near somas in the granule cell
layer, indicating a source there (Límo, 1971a; Rose,
1983); this distribution of sinks and sources corresponds
to the summation of EPSPs in a population of granule cells
(Deadwyler, West, & Robinson, 1981; Límo, 1971a). Intense
stimulation causes the appearance of the third component,
a population spike with 2.5 ms onset, which reflects many
simultaneous action potentials in a population of granule
cells, and is of opposite polarity to the EPSP (Límo,
1971a; Rose, 1983). The following section discusses the
similarity between the EPSP component of the electrically-
elicited field potential and the DG AEP.
The dentate gyrus auditory evoked potential.
Auditory stimulation, tooth-pulp stimulation, and
electrical stimulation of the medial perforant path all
evoke time-locked responses in DG (Brankack & Buzs ki,
1986; Foster & Deadwyler, 1992). The AEP recorded in DG
of the naive rat has a single component (N1), a sharp
waveform of 30 ms peak latency and 100 to 500 æV peak
amplitude (Deadwyler et al., 1981, Foster & Deadwyler,
1992).
Three findings show that N1 reflects EPSPs
generated by a population of granule cells in response to
medial perforant path activation. First, N1 and the
electrically-elicited EPSP both reach a well-defined
maximal negativity in the termination zone of medial
perforant path axons, which is located in the middle third
of the molecular layer (Deadwyler et al., 1981; Witter,
1989). Second, the amplitudes of N1 and the electrically-
elicited EPSP show parallel augmentations and reductions
in response to behavioral and cholinergic manipulations,
suggesting that both potentials arise from a single set of
neural elements (Foster, Hampson, & Deadwyler, 1984;
Foster & Deadwyler, 1992). Third, N1 is eliminated by
lesions of the entorhinal cortex, suggesting that the N1-
producing auditory signal reaches DG via the entorhinal
cortex and perforant path (Deadwyler et al., 1981).
Cholinergic input to the dentate gyrus
The molecular layer of rostral DG receives most of
its cholinergic innervation via the fornix from the
vertical limb of the diagonal band of Broca, as shown by
immunostaining for choline acetyltransferase (Amaral &
Kurz, 1985; Frotscher, Nitsch, & L‚r nth 1989).
Cholinergic neurons intrinsic to DG and a cholinergic
projection from the supramammillary nucleus of the
hypothalamus also contribute cholinergic terminals
(Harley, LaCaille, & Galway, 1983; Frotscher et al., 1989;
Mizumori, McNaughton, & Barnes, 1989). The molecular
layer and the hilus, which are the DG areas receiving the
densest cholinergic innervation, contain mostly muscarinic
receptors (Kuhar, 1975; Spencer, Horv th, & Traber, 1986).
The application of acetylcholine or cholinergic
agonists to DG reduces the amplitude of the EPSP evoked in
DG by electrical stimulation of the medial perforant path
in the awake, behaving rat, the anesthetized rat, or DG
slices in vitro (Foster & Deadwyler, 1992; Kahle & Cotman,
1989; Konopacki, MacIver, Bland, & Roth, 1987; Valentino &
Dingledine, 1981). N1 also exhibits this cholinergically-
elicited depression (Foster & Deadwyler, 1992).
Specifically, both N1 and the EPSP component of the
electrically-elicited field potential in DG are attenuated
by application of acetylcholine to the middle third of the
outer molecular layer in the freely moving rat (Foster &
Deadwyler, 1992). This decrement may result from an
activation of muscarinic receptors that increases the
permeability of granule cell membranes to potassium ions
(Nabekura, Ebihara, & Akaike, 1993).
The current study
The current study examined the effects of
muscarinic antagonism on the N1 component of the DG AEP,
using subcutaneous injection of scopolamine.
Methscopolamine, which does not cross the blood-brain
barrier, was injected instead of scopolamine on some
occasions in order to test whether peripheral
anticholinergic action affected the AEP. If muscarinic
antagonism affects N1 amplitude through a central, not
peripheral, mechanism, then scopolamine but not
methscopolamine should have affected N1.
This study will show how the blockade of
cholinergic receptors affects sensory input to the
hippocampal formation; however, it will not show whether
the effect is due to the receptor blockade in ascending
auditory pathways, in DG, or in other brain areas. The
scopolamine-treated brain provides a model for the
cholinergic deficits present in early Alzheimer's Disease
(Buchwald et al. 1991); thus, in addition to clarifying
the effect of cholinergic systems on input to the
hippocampal formation in the normal brain, the current
study may suggest how this input is changed in early
Alzheimer's Disease.
Chapter 2
METHODS
Animals and surgery
Eight male albino Wistar rats (Hilltop Laboratory
Animals, Scottsdale, PA), were 180-360 days of age and
weighed 400-700 g at the time of surgery. They were
singly housed on a 12:12 light/dark cycle (lights on at
7:30 a.m.) with ad libitum access to food and water. Some
subjects had been deprived of food previously for operant
conditioning experiments.
Each subject was prepared for stereotaxic surgery
by administering atropine sulfate (1.08-2.16 mg/kg i.p.)
prior to induction of surgical anesthesia with sodium
pentobarbital (50 mg/kg i.p.). Lidocaine (4-6 mg s.c.)
was administered for local anesthesia of the scalp.
Chloral hydrate in 0.9% saline solution (160 mg/ml) was
administered as needed in intraperitoneal injections of 8-
100 mg/kg.
During surgery, skull holes were drilled to
accommodate electrodes and 6 support screws. The
recording microelectrode (0.5-1.5 Mê impedance) had been
made by etching 125 æm tungsten wire to a sharp tip,
approximately 9 æm in diameter, as measured at a point 12
æm from the tip, and insulating with Epoxylite; it was
bent into a right-angled "z"-shape, the crossbar of which
would be located above the skull and encased in dental
cement to ensure stability of location in the brain. This
electrode was positioned in the left DG (2.2 mm posterior
and 1.0 mm lateral from bregma, and 2.7 mm ventral from
the dura mater). A skull screw reference electrode, made
of a stainless steel screw (0-80) soldered to 0.25 mm
stainless steel wire, was threaded through the skull above
the right olfactory bulb, 7.0 mm anterior and 1.5 mm
lateral from bregma. A twisted-pair bipolar stimulating
electrode was made of 127 æm insulated stainless steel
wire cut to form blunt tips; the tips were vertically
separated by 1 mm. The stimulating electrode was placed
in the left perforant path, 3.5 mm lateral from the
midline, 1.5 mm anterior to the transverse lambdoid
suture, 3.0 mm ventral from dura mater, and angled
laterally at 10 degrees from vertical in the coronal
plane. Two grounding screws were also threaded into the
skull. Accurate placement of the recording microelectrode
in the middle third of the DG molecular layer was achieved
by maximizing the negative-going population EPSP produced
by stimulating the perforant path with a 200 æA, 0.1 ms DC
pulse (Deadwyler, West, Cotman, & Lynch, 1975; Límo,
1971a). After electrode placements, connector pins were
snapped into a plastic housing, and the assembly was
cemented to the skull with dental acrylic.
To reduce the risk of infection, the incision was
treated with iodine and antibiotic ointment, gentamicin
was administered (5 mg/kg i.m.), and each subject was
monitored for signs of infection for 3 days following
surgery. Butorphanol tartrate (0.2 mg/kg) was used as a
post-surgical analgesic. Each subject was allowed 1 week
to recover before recording began.
After recovery, subjects were habituated to the
testing procedure, then assessed for stability of the
electrically-elicited DG field potential over a period of
3 days. Recording always began during the light portion
of the subjects' light/dark cycles. Subjects were tested
in a Plexiglas enclosure (25 cm x 25 cm x 48 cm) situated
inside a sound-attenuating chamber. An audio speaker with
a 10 cm diaphragm was mounted behind a perforated portion
of one wall of the enclosure, 30 cm from the floor.
The recording electrode was connected to a field-
effect transistor operational amplifier in follower mode
with unity gain. From there, the signal was routed
through shielded wires and a commutator to an AC-coupled
differential amplifier, then to a second stage of
amplification, for a total gain of 1000 with bandpass 2-
125 Hz. The signal beginning 400 ms prior to tone onset
and lasting 2000 ms was digitized by a 12-bit analog-to-
digital converter (Data Translation, Inc.), recorded using
a sampling rate of 500 samples per second, and stored on a
personal computer.
Testing apparatus and procedure
The first phase of the experiment examined the
effects of presentation rate on N1. AEPs were recorded in
response to abrupt-onset tones of 10 kHz frequency and 50
ms duration. The tones ranged between 70 and 85 dB SPL in
the space accessible to the rat, as measured with a sound
level meter (Radio Shack, 33-2050), and rarely produced a
startle response. The tones were presented at each of 6
presentation rates (0.1, 0.2, 0.5, 1, 2, and 4 Hz) in
ascending order. Each AEP consisted of the averaged
records of DG electrical activity recorded during a block
of 100 trials.
For the second, drug-testing phase of the
experiment, (-)scopolamine hydrobromide or scopolamine
methyl bromide (Sigma Chemical Co.) was dissolved in
sterile vehicle (0.9% saline) to make concentrations
allowing a constant injection volume of 1 ml/kg, for
subcutaneous intrascapular injection. For AEP collection,
interstimulus interval varied pseudorandomly from 21 to 51
s (mean = 35 s), tone duration was 1 s, and blocks
consisted of 120 trials. Each subject received two 2-day
sequences of recording sessions for each of 4 doses of
scopolamine administered in ascending order (0.5, 1.0,
2.0, and 5.0 mg/kg), and one dose of methscopolamine,
administered last (5.0 mg/kg). On the first day, a block
of 120 individual evoked potential trials was recorded
prior to scopolamine injection, and after post-injection
intervals of 10-15 min (first hour after injection), 2 hr
(third hour after injection), and 4 hr. On the second day
a block of trials was recorded during the 24th hour after
injection; then saline vehicle was injected, and blocks of
trials were again recorded in the first and third hours
after injection. After an interval of at least 2 days,
another 2-day sequence began. Motor activity was
monitored in each subject for three 2-min intervals--5 min
before injection, 30 min after injection, and 24 hr after
injection--by counting the number of times the subject's
head crossed floor quadrant boundaries (quadrant entries).
At the end of the experiment, cathodal DC lesions
were made with the recording and stimulating electrodes
(50 uA, 10 s). After waiting 2 days to allow gliosis to
occur, the rat was euthanized and perfused, and the brain
was removed and stored in a solution of 10% formalin and
30% sucrose for histology.
Data analysis
In order to reduce electrical artifacts in data
collected in the drug-testing phase, individual AEPs were
screened to automatically reject the 10-15% of trials in
which the voltage level exceeded pre-set boundaries of a
window in the 400 ms of the pre-tone interval. Average
AEPs were then computed for each of 4 blocks of time per
recording session: the hour before injection and the
first, 3rd, and 24th hours after injection. Average AEPs
were adjusted to a zero-volt baseline by subtracting the
mean voltage level in the 20 ms preceding tone onset from
the entire waveform; then N1 amplitude and latency values
were extracted. N1 peak amplitude was defined as the most
negative value in the period 28-46 ms after tone onset.
The overall within-subjects effects of scopolamine
injection on the 3 dependent variables (N1 amplitude, N1
latency, and activity) were assessed using separate
repeated-measures analyses of variance. The data were fit
to a mixed linear model using the SAS MIXED procedure (SAS
Institute; Cary, NC). Mixed linear models utilize linear
algebra to expand the usual general linear regression
model to allow random effects to be counted as predictor
variables, instead of residuals, in the regression
equation, thus increasing statistical power. These models
allow the expected covariance structure of the error
matrix of the data to be specified, and allow for good
management of missing observations (SAS Institute Inc.,
1992). The compound symmetry model was found to fit the
covariance structure of the error matrix of the AEP data;
the autoregressive model fit the matrix of the activity
data; thus these models were used for further statistical
tests. The overall effect of scopolamine on N1 recorded
in the first, third, and 24th hours after injection was
analyzed at a significance criterion of p < 0.05.
Corresponding tests for the effect of scopolamine at each
dose required the criterion to be adjusted for the number
of comparisons (Dunn's procedure; p < .00417). Tests for
linear and quadratic trends across doses were also
performed (p < 0.05).
Chapter 3
RESULTS
Electrode location
The recording electrode had been positioned in the
DG outer molecular layer during stereotaxic surgery by
optimization of the negative-going extracellular EPSP
elicited by perforant path stimulation. At the time of
AEP recording, auditory stimulation produced the expected
AEP waveform in all 8 subjects. Although the
electrically-elicited EPSP waveforms indicated accurate DG
electrode placement in 3 subjects, they were degraded and
uninterpretable in 5 subjects. In one rat, the electrode
shifted greatly during the middle of the experiment, as
shown by AEPs; data collected after the change in AEP
waveform were not used.
Histology confirmed that lesions made by the
recording electrodes were in or near DG in all 8 rats.
The lesion was located in the DG molecular layer in 1 rat,
the granule cell layer in 1 rat, the hilus in 4 rats, area
CA1 in 1 rat, and CA3 in 1 rat. Of the 5 stimulating
electrode lesions found, 4 were located in the forceps
major of the corpus callosum, at about 7.5 mm posterior to
bregma, and 1 was in the adjacent neocortex.
Auditory evoked potential component definition
AEPs recorded prior to scopolamine administration
in DG of untrained rats contained a prominent negative-
going N1 component with 30-40 ms latency to peak (Fig. 1).
The parameters of N1 were generally comparable to those
previously reported in trained and untrained rats
(Deadwyler et al., 1981): an onset latency of
approximately 20 ms, mean latency to peak of 33.6 ms (ñ1.1
ms S.E.M.), peak amplitude of -267.6 æV (ñ51.2 æV), and
duration of approximately 20 ms. N1 latency was
relatively consistent across animals, but N1 peak
amplitude varied from -126 æV to -499 æV among subjects
(Fig. 1). In addition to N1, two other components
occurred in some rats: a small initial positivity, with
20 ms mean peak latency and 37 æV mean amplitude; and/or a
late-onset, long-duration positivity (P2; Fig. 1).
Effect of stimulus presentation rate on N1
Peak N1 amplitude was dependent on stimulus
presentation rate: as presentation rate increased from
0.1 Hz to 0.2 Hz, N1 amplitude remained high, but as
presentation rate increased from 0.2 Hz to 4 Hz, N1
diminished, reflecting a negatively accelerated function
with an inflection point at approximately 0.3 Hz (Fig. 2).
N1 latency appeared not to vary as a function of
presentation rate (Table 1).
Effect of scopolamine on N1
Overall, scopolamine produced a significant
reduction in N1 amplitude in the first hour after
injection (Figs. 3 and 4). The N1 amplitude attenuation
was significant at each dose level. However, the
different scopolamine doses did not differ in magnitude of
effect on N1 amplitude, that is, no dosewise linear or
quadratic trend was evident.
Two hours after injection, N1 amplitude remained
significantly suppressed overall. However, in dosewise
tests, this suppression achieved significance only at the
highest dose of scopolamine (5 mg/kg; Fig. 4, Table 2).
The different scopolamine doses appeared to differ in
time-course of effect, with higher doses resulting in
greater attenuations in N1 amplitude (Fig. 4), but this
effect did not reach significance. N1 latency was not
affected by scopolamine (Table 2).
Methscopolamine administration (5.0 mg/kg) did not
affect N1 amplitude or latency (Fig. 5). Peak N1
amplitude prior to methscopolamine injection was -259 æV
(ñ 54 æV) at 32 ms; post-injection peak amplitude was -247
æV (ñ 58 æV) at 30 ms. This implies that the N1
attenuation produced by scopolamine was not due to
peripheral antimuscarinic effects, the injection
procedure, the mere passage of time, or repeated exposure
to the tone (habituation). Saline vehicle injections also
had no effect on N1.
Effects of scopolamine on motor activity
At the beginning of each recording session, the
rat explored the enclosure for several minutes, rearing,
sniffing, and locomoting. In later parts of the session,
activity levels were low; the rat often remained
motionless in a single location, with its head in a normal
upright position approximately 2 cm from the box floor.
Ten minutes prior to scopolamine injection, movement
averaged 3.4 (ñ 0.7) quadrant entries per 2-min period.
Injection of scopolamine produced a large, dose-
dependent increase in mean activity level, to 40.3 (ñ 4.3)
quadrant entries per 2-min period. Activity returned to
control levels by 23 h after injection (Fig. 6).
Methscopolamine injections did not affect locomotion,
although eyeblinking activity appeared to increase,
probably due to peripheral anticholinergic effects (Fig.
6).
The scopolamine-induced increase in activity
consisted mostly of two behaviors, one being rapid
unidirectional turning in the enclosure, during which the
rat oriented its head toward each corner consecutively,
often punctuating this movement with a nosepoke into the
corner. The other consisted of the rat standing in one
place and quickly moving its head between two locations a
few cm apart, as if orienting to each location repeatedly.
Scopolamine also appeared to increase the number of
grooming, rearing, and stretching behaviors. Due to the
increase in rearing, the average position of the rat's
head in the cage was slightly higher than normal after
administration of scopolamine, but not methscopolamine.
Chapter 4
DISCUSSION
The AEP evoked in DG by 10 kHz tones had a
prominent negative-going N1 component, with a 34 ms peak
latency; N1 parameters were in general accordance with
previous reports (Deadwyler et al., 1981). Systemic
scopolamine injection at all doses attenuated N1 in the
first hour after injection. Methscopolamine, which does
not cross the blood-brain barrier (Brown, 1990), did not
affect N1, indicating that the scopolamine-induced
attenuation was due to central muscarinic antagonism. In
addition, scopolamine, but not methscopolamine, greatly
increased motor activity. Consequently, scopolamine may
have reduced N1 amplitude due to either a direct action on
auditory pathways or indirectly, via the augmentation of
motor activity.
If scopolamine acted directly on the auditory
pathways projecting to DG, it may have acted on any of
three candidate sites. First, muscarinic antagonism in DG
may have contributed to the decrease in N1 amplitude.
However, other evidence suggests that muscarinic
antagonism in DG should increase N1 amplitude.
Cholinergic agonists in DG reduce both the amplitude of N1
and of the field potential elicited in DG by stimulation
of the perforant path (Foster and Deadwyler, 1992; Kahle
and Cotman, 1989; Konopacki et al., 1987). Because
antimuscarinics and cholinergic agonists would be expected
to have opposite effects, these results suggest that the
scopolamine-induced decrease in N1 amplitude in the
present study may not be due to the antagonism of
muscarinic receptors in DG. On the other hand, atropine
also attenuates the electrically-elicited field potential
in DG slices (Konopacki et al., 1987), leaving open the
possibility that muscarinic blockade in DG may have
contributed to the scopolamine-induced N1 attenuation.
Second, scopolamine may have acted on the primary
auditory pathway. However, because scopolamine and
atropine have little effect on AEPs generated in the
brainstem (Church & Gritzke, 1988; Samra et al., 1985) or
primary auditory cortex (Buchwald, et al., 1991; Dickerson
& Buchwald, 1991; Miyazato et al., 1995), it is
questionable whether an effect of scopolamine on the
primary auditory pathway could have been responsible for
the N1 attenuation observed in the current study.
A third set of candidate sites includes the
extralemniscal pathways that ascend from brainstem sites
such as the pedunculopontine tegmental nucleus to
nonspecific thalamus and cortex, and may ultimately
project to entorhinal cortex and DG. Muscarinic blockade
is known to attenuate extralemniscal AEPs recorded in
nonspecific thalamus in the cat (Harrison et al., 1990a)
and at vertex in both the cat (Dickerson & Buchwald, 1991;
Harrison et al., 1990a,b) and rat (Campbell et al., 1995;
Miyazato et al., 1995). An extralemniscal modulation of
input to DG could reflect this attenuation, providing a
possible explanation for the N1 attenuation observed after
scopolamine in the present study. Other evidence that
extralemniscal pathways may contribute a significant
proportion of input to DG is provided by examining the
effect of stimulus presentation rate on evoked potential
amplitude at different recording sites. Primary auditory
pathway AEPs and the electrically-elicited EPSP in DG
follow relatively fast rates of repeated stimulation
(e.g., 10 Hz) before decreasing below half-maximal
amplitude (Erwin & Buchwald, 1986; Límo, 1971b; Miyazato
et al., 1995). In contrast, N1 recorded from DG in the
present study and extralemniscal (vertex) AEPs both
exhibit slow recovery, with amplitude falling off rapidly
at stimulation rates above 1 Hz (Buchwald, Hinman, Norman
& Huang, 1981; Campbell et al., 1995; Miyazato et al.,
1995). These results, plus anatomical evidence showing
that the entorhinal cortex may receive extralemniscal
projections (Witter et al., 1989), provide converging
evidence that extralemniscal pathways may contribute
significantly to N1. If so, then the length of the N1
recovery cycle may have been due to extralemniscal input
to DG, and the scopolamine-induced attenuation of N1 may
have been due to the action of scopolamine on
extralemniscal pathways.
Alternatively, the effect of scopolamine on N1 may
have been due to an intervening behavioral variable such
as arousal or motor activity, particularly because motor
activity increased considerably after antimuscarinic
injection in the current study. Other studies also report
that antimuscarinics increase motor activity (Aquilonius,
Lundholm & Winbladh 1972; Mueller & Peel, 1990; Toide,
1989) and this effect has been localized to muscarinic
receptors in the pedunculopontine or laterodorsal
tegmental nuclei (LaViolette & Yeomans, 1995; Mathur &
Yeomans, 1993).
Motor activity attenuates AEP amplitude in cats
through two separate mechanisms: increased activity of
the middle ear muscles and an unknown effect at higher
levels (Starr, 1964). In rats, frontal cortex AEPs
decline during exploration and grooming (Bringmann and
Klingberg, 1990), and vertex AEPs are smaller during
movement than during still, alert states (Adler, Rose, &
Freedman, 1986). Thus, the literature is consistent with
the hypothesis that a scopolamine-induced increase in
motor activity, such as the one observed in the current
study, may attenuate auditory signal strength, leading to
a decrease in N1 amplitude.
Further studies could evaluate this possibility.
If scopolamine-induced motor activity attenuates lemniscal
AEPs, then it could be concluded that the scopolamine-
induced N1 attenuation found in the current study was
partially or completely due to the increased motor
activity. If, on the other hand, scopolamine-induced
motor activity does not attenuate lemniscal AEPs, then it
could be concluded that the scopolamine-induced
attenuation of DG AEPs was specific to the extralemniscal
system and/or DG, due to either motor activity or
cholinergic modulation of auditory processing. In that
case, one methodological strategy to eliminate the
confound due to movement would involve the use of physical
restraint or neuromuscular blockade, to reduce or prevent
scopolamine-induced movement during N1 recording. Another
strategy would involve injecting scopolamine into the
intralaminar thalamic nuclei to block muscarinic receptors
that are activated by pedunculopontine tegmental
projections; this procedure would be expected to produce
the extralemniscal effects of scopolamine without
increasing motor activity (Dickerson & Buchwald, 1991;
Mathur & Yeomans, 1993).
In the current study, subcutaneous scopolamine
injection was found to augment motor activity and to
attenuate the DG N1 potential in the first hour after
injection in the freely-moving rat. Because motor
activity may decrease the strength of auditory signals in
the brain, the scopolamine-induced movement increase may
have contributed to the N1 attenuation. Alternatively, if
the relevant effect of scopolamine was on auditory
pathways, then the results would indicate a role for
acetylcholine in the transmission of auditory information
to the hippocampus. In this latter case, cholinergic
synapses, perhaps those located in the extralemniscal
pathways, may modulate auditory input to the hippocampus;
muscarinic blockade may alter this modulation so as to
reduce the amplitude of auditory signals reaching the
hippocampus. TABLES Table 1. Mean and standard error of the mean (S.E.M.) of
N1 amplitude and latency as a function of stimulus
presentation rate in all 8 rats. Table 2. Effect of scopolamine on N1 amplitude and
latency. Mean (ñS.E.M.); n=8. FIGURES
Figure 1. Auditory evoked potentials recorded prior to
scopolamine injection illustrate that N1 latency is
consistent (30-40 ms), but N1 amplitude is highly variable
from rat to rat (grand averages from each of 6 rats).
Calib: 100 æV, 20 ms; positive is up.
Figure 2. Peak N1 amplitude is dependent on stimulus
presentation rate. Bars indicate +/- standard error
(n=8).
Figure 3. Grand averages showing the effect of
scopolamine on N1. Auditory evoked potentials are shown
in the hour before (dashed line) and after injection
(solid line), averaged across all doses (n=8). Calib:
100 æV, 20 ms.
Figure 4. Timecourse of effect of different dosage levels
of scopolamine on N1 amplitude. Mean N1 amplitude is
shown, with bars indicating ñS.E.M., at the 4 dosage
levels, prior to injection and at 3 intervals following
injection (n=8). Key: white= pre-injection, light gray=
1st hr after injection, medium gray= 3rd hr, dark gray=
24th hr.
Figure 5. Auditory evoked potentials recorded in the hour
before (dashed line) and the hour after methscopolamine
injection (solid line). Grand averages across all 8 rats
are shown. Calib: 100 æV, 20 ms.
Figure 6. Activity levels (quadrant entries) are shown
following injections of scopolamine (scop) and
methscopolamine (mscop) at the indicated dosages (mg/kg;
n=8). Pre-injection= white, 1st hr after injection= light
gray, 24th hr=dark gray.
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APPENDIX: ANIMAL USE APPROVAL BY THE INSTITUTIONAL ANIMAL
CARE AND USE COMMITTEE OF THE UNIVERSITY OF DELAWARE
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