Department of Cell and Molecular Biology and Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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McEchron, Matthew D., Aldis P. Weible, and John F. Disterhoft. Aging and Learning-Specific Changes in Single-Neuron Activity in CA1 Hippocampus During Rabbit Trace Eyeblink Conditioning. J. Neurophysiol. 86: 1839-1857, 2001. Rabbit trace eyeblink conditioning is a hippocampus-dependent task in which the auditory conditioned stimulus (CS) is separated from the corneal airpuff unconditioned stimulus (US) by a 500-ms empty trace interval. Young rabbits are able to associate the CS and US and acquire trace eyeblink conditioned responses (CRs); however, a subset of aged rabbits show poor learning on this task. Several studies have shown that CA1-hippocampal activity is altered by aging; however, it is unknown how aging affects the interaction of CA1 single neurons within local ensembles during learning. The present study examined the extracellular activity of CA1 pyramidal neurons within local ensembles in aged (29-34 mo) and young (3-6 mo) rabbits during 10 daily sessions (80 trials/session) of trace eyeblink conditioning. A single surgically implanted nonmovable stereotrode was used to record ensembles ranging in size from 2 to 12 separated single neurons. A total of six young and four aged rabbits acquired significant levels of CRs, whereas five aged rabbits showed very few CRs similar to a group of five young pseudoconditioned rabbits. Pyramidal cells (2,159 total) were recorded from these four groups during training. Increases in CA1 pyramidal cell firing to the CS and US were diminished in the aged nonlearners. Local ensembles from all groups contained heterogeneous types of pyramidal cell responses. Some cells showed increases while others showed decreases in firing during the trace eyeblink trial. Hierarchical clustering was used to isolate seven different classes of single-neuron responses that showed unique firing patterns during the trace conditioning trial. The proportion of cells in each group was similar for six of seven response classes. Unlike the excitatory modeling patterns reported in previous studies, three of seven response types (67% of recorded cells) exhibited some type of inhibitory decrease to the CS, US, or both. The single-neuron response classes showed different patterns of learning-related activity across training. Several of the single-neuron types from the aged nonlearners showed unique alterations in response magnitude to the CS and US. Cross-correlation analyses suggest that specific single-neuron types provide more correlated single-neuron activity to the ensemble processing of information. However, aged nonlearners showed a significantly lower level of coincident pyramidal cell firing for all cell types within local ensembles in CA1.
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INTRODUCTION |
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Aging has been shown to
produce deficits in the ability to learn hippocampus-dependent forms of
learning (Geinisman et al. 1995). Trace eyeblink
classical conditioning in the rabbit is a hippocampus-dependent task
(Kim et al. 1995
; Moyer et al. 1990
; Solomon et al. 1986
) that is often used to study the
neurophysiological mechanisms of aging (Powell et al.
1991
; Solomon et al. 1988
; Woodruff-Pak
and Thompson 1985
). In this paradigm, each trial consists of a
neutral auditory conditioned stimulus (CS) followed by a stimulus free
trace period, then a nonnoxious eyeblink-eliciting airpuff
unconditioned stimulus (US) presented to the cornea. After repeated
trials, young animals and humans learn to associate the CS and US,
whereas a subset of aged animals and humans show poor learning on this
task (Finkbiner and Woodruff-Pak 1991
; Thompson et al. 1996
; Woodruff-Pak et al. 1999
). This
ability to separate aging-impaired from -unimpaired subjects has made
trace eyeblink conditioning a very effective tool in animal and human
research on aging.
A great deal of research has been aimed at understanding the
hippocampal neurophysiology of trace eyeblink conditioning. Berger and
Thompson were the first to demonstrate that hippocampal neurons encode
learning-related information during eyeblink conditioning (Berger et al. 1976). They conducted a series of studies
examining the multiple- and single-neuron activity of the pyramidal
cell layer of the hippocampus during delay eyeblink conditioning
(Berger and Thompson 1978a
,b
; Berger et al.
1983
). Although the delay task was not hippocampus dependent,
they found that pyramidal cell activity increased during the initial
trials of training. These increases in activity paralleled the
amplitude time course of the behavioral response and preceded it
temporally. Recently, we have expanded on the work of Berger and
Thompson using variations of the stereotrode recording technique, which
allows a large number of single neurons to be isolated from a
multiple-unit record (McNaughton et al. 1983
). Our
studies have shown similar early learning-related increases in CA1
pyramidal cell activity; however, we have also found that there are a
number of different excitatory and inhibitory patterns of pyramidal
cell activity that encode learning-related information during trace
eyeblink conditioning (McEchron and Disterhoft 1997
).
These heterogeneous response profiles suggest that CA1 pyramidal
neurons may not encode trace eyeblink information in an additive
fashion but rather different patterns of single-neuron activity could
interact within a network to encode information.
There is ample evidence from several laboratories demonstrating that
CA1 neurons are affected by the process of aging (e.g., Clark et
al. 1992; Landfield and Pitler 1984
;
Potier et al. 1992
; Shen and Barnes 1996
;
West et al. 1994
). In vitro work from our laboratory
suggests that CA1 neurons of aged animals are less excitable, and this
altered excitability may play a role in the ability to learn trace
eyeblink information (Moyer et al. 2000
). A number of
studies have examined the activity of CA1 pyramidal cells in vivo and
have found that the activity of these cells is altered in aged animals
(Mizumori et al. 1996
; Shen et al. 1997
;
Tanila et al. 1997
). Although several investigations
have described how hippocampal single-neuron activity is altered as the
result of aging, there are no studies describing how the interaction of
different types of single neurons within local ensembles may play a
role in aging-related learning deficits. Therefore the present study
had two goals, to describe the different CA1 single-neuron response
types that occur during trace eyeblink conditioning and to understand
how the encoding of learning-related information within these ensembles
is affected in aged animals that are unable to learn the trace eyeblink
task. The approach of the present study was to characterize
heterogeneous single-neuron response types within ensembles. Each
ensemble was recorded from the tip of a single fixed electrode. Thus
the present study examined how heterogeneous single-neuron response
types with a close physical relationship interact to encode trace
eyeblink information. Some of the initial pilot data from this
investigation were briefly mentioned in a theoretical review
(McEchron and Disterhoft 1999
).
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METHODS |
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Subjects
Subjects were 11 young (3-6 mo) and 9 aged (30-34 mo) New
Zealand albino rabbits, Oryctolagus cuniculus. All rabbits
were females obtained from Kuiper rabbitry (Gary, IN) or from Covance Laboratories (Denver, PA). Aged rabbits were retired breeders that had
ceased all nursing for at least 3 mo. Previous work from our laboratory
(Thompson et al. 1996) has shown that the age range from
30 to 34 mo produces heterogeneous levels of impairment in trace
eyeblink conditioning that range from mild to severe. All rabbits were
housed individually and maintained on a 14/10 h light/dark cycle with
food and water provided ad libitum.
Surgery
All subjects were allowed to remain undisturbed in their cages
for 1 wk prior to any handling or surgery. Surgery was carried out
under National Institutes of Health and Northwestern University Animal
Care and Use Committee approved procedures. Animals were anesthetized with ketamine (60 mg/kg im) and xylazine (10 mg/kg im),
and the eyes were kept moist with a thin coat of antibacterial ophthalmic ointment. The skull was positioned in a stereotaxic frame
with lambda 1.5 mm below bregma. The skull was then exposed, and a
3-mm-diam hole was drilled above the left CA1 area of the hippocampus.
Five self-tapping screws (No. 2 × 1/4-in) were inserted ~2 mm into the skull to anchor the final dental cement-head assembly. In each animal, one or two nonmovable stereotrode recording bundles were stereotaxically lowered into the left CA1 area of the hippocampus (~3 mm ventral to dura) until action potentials with pyramidal cell
firing characteristics were recorded (Ranck 1973
). This
procedure ensured that the electrode tip was located within the
pyramidal cell layer of CA1. The coordinates for electrode placement
were 5.0-5.2 mm caudal to bregma and 5.2-5.4 mm lateral to midline. Dental cement was then used to secure the electrodes to the skull and
close the remaining wound area. Rabbits were given
Buprenorphine (0.3 mg/kg sc) to minimize discomfort after
recovery from anesthesia.
Behavioral training
Rabbits were allowed 5-7 days of recovery before any handling
or testing. For all training sessions, animals were placed in a cloth
restraining jacket and Plexiglas restraining box and then placed in a
sound-attenuating chamber. The right eye was held open in a comfortable
position with eyelid hooks attached to a velcro strap. The ends of
rubber tubes (1-cm diam) were placed comfortably in each ear and served
to deliver the auditory tone CS (100 ms; 90 dB; 6 kHz; 5-ms rise/fall
time) from headphones. An airpuff tube was placed 1 cm from the
animal's right eyeball and served to deliver the airpuff US (150 ms; 3 psi). The airpuff was supplied by compressed air and controlled by a
regulator and solenoid valve. The airpuff intensity was adequate to
reliably evoke an eyeblink that extended the nictitating membrane
across the globe of the eyeball. A lightweight infrared sensor was used to transduce extensions of the nictitating membrane (Thompson et
al. 1994). The sensor was fastened to the dental acrylic on the
animal's head. This enabled the animal's head to move comfortably within the restrainer. Changes in voltage from the infrared sensor were
sampled by a computer which also controlled the delivery of CSs and USs
(Akase et al. 1994
).
Animals were first given one 30-min acclimation session during which no
stimuli were presented. One day following acclimation, trace-conditioned rabbits received daily sessions (10 session total)
consisting of 80 CS-US trials presented at an intertrial interval of
30-60 s (mean, 45 s). Each trial consisted of the CS, followed by
a 500-ms stimulus free trace period, then the US. For the first 4 days
of training, these animals also received a US-alone test trial on
trials 11, 32, 53, and 74. These test trials were
used to determine if the CS information on CS-US trials augmented the
unconditioned behavioral response, similar to reflex facilitation
described by Gormezano et al. (1983). The reflex facilitation analyses used only the CS-US trials and US-alone trials
where the onset of blinks occurred following the US onset.
Pseudoconditioned control rabbits also received acclimation followed by
daily sessions consisting of 80 CS-alone trials and 80 US-alone trials
(intertrial interval of 15-30 s). Trace and pseudoconditioning
sessions lasted ~1 h and consisted of the same number of CSs and USs.
A significant blink response produced a change in voltage 10 ms in
duration and
4 SD of the mean baseline voltage. For trace-conditioned
animals, conditioned responses (CRs) were defined as significant
eyeblinks that were anticipatory of the US, that is, occurring within
the 300-ms period prior to US onset. This ensured that the conditioned
responses in the trace-conditioned group were not due to sensitization.
However, conditioned responses for the pseudoconditioned group were
defined as significant blinks occurring within the 600-ms period
following CS onset. Trace-conditioned animals were considered learners
if the percentage of CRs exceeded 50% on any day, and animals were
considered learning impaired (nonlearners) if the daily CR percentage
never exceeded 25%. Animals that failed to show 25% CRs by the 10th
day of training were administered an additional 5 days of training. All
of the nonlearners failed to exceed 25% CRs during these 15 days of training.
Single-neuron recording
Single neurons were recorded from rabbits during trace eyeblink
conditioning using surgically implanted nonmoveable electrodes that
were cemented in place. This minimized the amount of drift or movement
of the electrode tip during a single training session. Each implanted
recording electrode consisted of six channels with a total diameter of
~80 µm. Each channel was a Teflon-coated tungsten microwire (18 µm diam bare; 25-µm diam coated). The channels were bonded tightly
together in parallel with epoxylite to form a 25-µm center-to-center
spacing. The tip of the electrode was cut at a 45° angle with sharp
scissors to maximize the number of single neurons recorded from the
electrode. During recording, two-wire stereotrode combinations were
selected from the implanted probe that provided the largest and most
heterogeneous ensembles of single neurons (2-12 neurons). This is an
enhanced version of the stereotrode technique, which has been shown to
allow large numbers of single neurons to be recorded and separated with
much greater accuracy than single electrodes (McNaughton et al.
1983). Similar ensemble techniques have been used for recording
tightly clustered groups of single neurons from a single probe (e.g., Apkarian et al. 2000
).
Single-neuron analog signals were amplified (10,000 times), filtered
(band-pass, 300 Hz to 10 kHz), and collected with a DT 2821 Data
Translation board (Marlboro, MA) attached to a 200-MHz Pentium
computer, which sampled each channel at 30 kHz. Single-neuron data were
collected 1 s prior and 2.75 s following CS onset using software from DataWave Technologies (Longmont, CO). The software recorded 1.5-ms epochs of data whenever a single neuron discharged a
definable action potential. The action potentials of each of the
different single neurons recorded on an electrode were separated off-line using a template-matching program developed in our laboratory. This software allowed template windows to be defined for each single
neuron's characteristic waveform. All action potential waveforms that
fell within the boundaries of a single template-window belonged to an
individual single neuron. The template window could account for any
unique segment along the single-neuron waveform, and the template
window could be minimized anywhere along the waveform to exclude other
electrophysiological data that did not fit the exact shape of an
individual single neuron. The template could be widened to account for
drifting and changes in the single-neuron waveform across a recording
session. The software compared the waveform of every single-neuron
discharge to all other electrophysiological activity on the probe. All
action-potential waveforms on a probe were also compared visually to
ensure that the characteristic waveform of each individually defined
single neuron was different from the waveforms of all other defined
single neurons on the probe. This conservative approach ensured that
the ensembles recorded from each probe were made up of unique single
neurons that could be accurately followed throughout a single training
session. Individual hippocampal pyramidal cells have been reported to
exhibit complex spikes within a burst of activity where the action
potentials of a single neuron decrease in height (Ranck
1973). Based on parameters described by Quirk and Wilson
(1999)
, the software was able to track patterns of activity
that might represent complex spike activity. This prevented the complex
spike activity of a single neuron from overlapping with more than one
individually defined single neuron.
Single-neuron activity was analyzed from a single day of training only if the ensemble of single neurons on a stereotrode remained consistent throughout the entire 1-h training session. This ensured that the electrode did not drift during the recording session, which might produce an overlap of activity from more than one single neuron. However, it is important to note that the configuration of single neurons on a stereotrode changed from 1 day of recording to the next in almost all cases. A conservative approach was used, and neurons were treated as the same neuron on consecutive days only if the same template window yielded the same configuration of single neurons on one probe. This does not rule out the possibility that small drifts in the electrode between recording sessions may have allowed new configurations of single neurons to form which included one or two of the neurons from the previous recording day.
Following spike separation, an average waveform was computed for each
single neuron to determine if the neuron was a pyramidal or theta cell.
Action-potential widths were calculated from each average waveform as
the peak time minus the valley time. Pyramidal cells were separated
from theta cells using measurements of action-potential width and
background firing rate. Using criteria similar to those described by
Fox and Ranck (1981), cells with a spike duration
0.3
ms and background firing rate <6 Hz were classified as pyramidal cells, and cells with a spike duration <0.3 ms and a background firing
rate
6 Hz were classified as interneurons.
Analyses
All statistical analyses were performed with the aid of Microsoft Visual Basic routines developed in our laboratory and Minitab statistical software v10.0 (State College, PA). Analyses of background firing rate were performed by calculating the mean single-neuron discharge rate prior to the delivery of each trial used in training. Changes in single-neuron action potential firing were measured using standard t-test scores. For each neuron, standard test scores were computed for time periods from 200 to 600 ms in duration following either the CS or US to capture discrete short-latency increases or decreases in activity. The standard test scores were computed by subtracting the number of action potentials in the period preceding CS onset from the number of action potentials in the period following CS or US onset. The difference calculated for each period was divided by the sample standard deviation during baseline. The test score measures could then be averaged across trials or across neurons or to compare changes in activity across days of training.
A hierarchical clustering analysis was used to classify the response
profile of each single neuron recorded during trace eyeblink conditioning. This is a multivariate statistical technique where each
observation, or single neuron in this case, is assumed to be a unique
cluster for the first step of the analysis. In the first step, the two
most similar observations are joined together to form a cluster. In the
second step, either a third observation joins the two previous
observations or two of these observations join together into a
different cluster. The analysis proceeds by joining together
observations that are most similar, each step results in one less
cluster than the preceding step. The result of the analysis is a
limited number of unique clusters of observations, where the
observations within each cluster share the most similarities. Theoretical and mathematical descriptions of this analysis are supplied
by Everitt and Dunn (1991), and Lance and William
(1967)
. Briefly, the clustering analysis in this study used a
Pearson distance matrix where the distance between observations
i and k are as follows with
vj the variance of variable j
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The hierarchical clustering analysis sought to classify cells based on their activity during trace conditioning. Single-neuron responses recorded during pseudoconditioning were considered a unique class of responses because they were not recorded during any CS-US trials; therefore responses from these cells were not included in the initial clustering analyses. Therefore, a final categorization of all trace and pseudoconditioned cells was conducted by matching the average trial activity of each cell to a template of each category produced by the original hierarchical clustering analysis. The template for each category was created by summing the 80 trials of each trace conditioned single neuron's daily activity into 100-ms bins and averaging each summed bin across all neurons within a category. An average profile of single-neuron activity was then created for each cell recorded during trace conditioning using 100-ms bins. For pseudoconditioning this was done by summing across the pseudoconditioned trials of each single neuron's daily activity into 100-ms bins for the following periods: the baseline period prior to CS onset on CS-alone trials, the 600-ms period following CS onset on CS-alone trials, and the period following the onset of the US on US-alone trials. This created a daily average of pseudoconditioned activity to the CSs and USs that could then be matched to one of the trace conditioned templates from the clustering analysis. A dependent t-test was used to measure the difference between the 100-ms bins of the template and the daily average of a single neuron's pseudoconditioned activity. Both trace and pseudoconditioned cells were then matched into the trace conditioned category with the least difference in activity.
To examine the relationship of single-cell firing patterns within local
ensembles, cross-correlation histograms (adapted from Perkel et
al. 1967), also called cross-correlograms (CCRs), were constructed from the activity of pairs of cells recorded from each
single stereotrode. These CCRs were constructed by measuring the time
interval (resolution of 1 ms) between each action potential-discharge of one cell of the pair with each discharge of the other cell. Intervals were measured for all spike discharges between the two cells,
and the interval durations were plotted in a CCR using binwidths
ranging from 10 to 100 ms. The height of each bin within the CCR was
adjusted by dividing it by the total number of action potential
discharges recorded from both of the cells within the pair. This
correction of CCR height normalized for larger measures of coincident
activity that might be due to faster firing rates of one cell or both
cells within the pair and thus allowed for the level of coincidence to
be compared between cell pairs. The magnitude of correlated activity or
coincidence was compared between cell pairs by examining the height of
the two tallest adjacent bins of the adjusted CCR. This method for
calculating a cross-correlation coefficient was in part based on
methods used previously by Eggermont (1992)
and
Roy and Alloway (1999)
. The goal of this study was to
examine differences in coincident firing between groups. Therefore this
normalized cross-correlation measure was not used for judging the
significance of a CCR but rather for comparing the magnitude of
coincident activity between groups.
Although the number of cells recorded from an individual stereotrode
ranged from 2 to 12 cells, analyses were limited to the stereotrode-ensembles that contained 4 cells. The magnitude of coincident activity was calculated for each possible cell pair within a
stereotrode ensemble. This approach allowed us to examine how one
specific neuron within the stereotrode ensemble coordinated single-neuron discharges with the other cells within the ensemble. An
average magnitude of coincident activity was also calculated for each
stereotrode ensemble, and because each cross-correlation coefficient
was normalized for firing rate, the magnitude of ensemble-correlated activity could be compared between groups.
Statistical tests used ANOVAs with a general linear model. Repeated-measures ANOVAs were used to examine behavioral data across days of training. However, factorial rather than repeated-measures ANOVAs were used to measure changes in single-neuron activity across days of training because only 26 individual neurons could be tracked from 1 day of training to the next. Significant interactions were subjected to follow-up one-way ANOVAs using the MSerror term from the main ANOVA. Significant main effects were followed up with Newman-Keuls post hoc tests. An alpha of 0.05 was required for all significant analyses.
Histology
Marking lesions were placed at the tips of all electrodes by passing DC current (25 µA) for 20 s. Animals were overdosed with pentobarbital sodium and perfused transcardially with saline (0.9% NaCl) followed by 10% formaldehyde. Brains were then frozen, sectioned coronally (50 µm thick), mounted on albumin/gelatin-coated slides, and stained with neutral red. A light microscope (×25 and ×50) was then used to locate electrode tips.
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RESULTS |
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Histology
All of the animals used in this study had electrode tips placed directly in the pyramidal cell body layer of the CA1 area of the dorsal hippocampus. Data from two animals were not included in analyses because the electrode tips were placed 100-200 µm below the pyramidal cell layer.
Trace eyeblink conditioning
Greater than half of the aged animals showed (n = 5) few if any CRs, similar to the young pseudoconditioned group
(n = 5). These aged nonlearners failed to show daily CR
percentages 25% even though behavioral training was administered for
15 days. The remaining aged animals (n = 4) showed
normal acquisition very similar to that of the young animals
(n = 6). Figure
1A shows the mean percentage
of trace eyeblink CRs across the 10 days of training for these four
groups: young, aged learners, aged nonlearners, and young
pseudoconditioned. A repeated measures analysis of these data revealed
a significant group × days interaction, F(27, 144) = 4.8, P = 0.0001. Follow-up tests showed that the young
group exhibited a greater percentage of CRs than the aged nonlearners and the pseudoconditioned group during the last 3 days of training. These tests also revealed that the aged learners showed a greater percentage of CRs than the aged nonlearners and the pseudoconditioned group during the last 4 days of training. The level of
pseudoconditioning shown in this study was similar to the level of
pseudoconditioned eyeblink responses shown in other rabbit studies (for
review, see Gormezano et al. 1983
).
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Each animal received four US-alone test trials during each of the first
4 days of training. Figure 1B shows that the average unconditioned behavioral responses (non-CR responses) on the CS-US trials and the US-alone trials during the first 4 days of training were
identical for the three groups that received trace eyeblink conditioning. This figure shows that the average unconditioned responses of the three trace groups were augmented similarly on CS-US
trials. This effect is similar to reflex facilitation described by
Gormezano et al. (1983) and Weisz and McInerney
(1990)
. Thus the CS and US information was able to enter the
CNS of all trace conditioned groups; however, the aged nonlearners were
still unable to make the necessary associations between these stimuli
for learning to occur. The three trace-conditioned groups also showed
behavioral responses to the US-alone trials that were similar to the
pseudoconditioned group. One-way ANOVAs compared the unconditioned
behavioral responses on CS-US trials during the first 4 days of
training for the three trace-conditioned groups on the following
measures: maximum amplitude, area under the curve, response onset
latency, and peak voltage latency. None of these ANOVAs revealed
significant effects. One-way ANOVAs also compared all four groups
on the unconditioned behavioral responses on US-alone trials during the
first 4 days of training. The only significant ANOVA was on the measure
of peak voltage latency, F(3, 17) = 3.57, P = 0.04. The follow-up test showed that the
pseudoconditioned animals showed a shorter peak latency compared with
the trace-conditioned animals. The result of this analysis was most
likely due to the pseudoconditioned animals having received numerous
additional US-alone trials prior to the US-alone test trials.
Average single-neuron response profiles
A total of 2,159 pyramidal cells were recorded from the four
groups of animals. Each cell was recorded throughout one entire session
from at least 1 of the 10 days of training shown in Fig. 1. A total of
16 other cells were excluded from the analyses because they exhibited
firing characteristics similar to interneurons (see Fox and
Ranck 1981). The young, aged learners, aged nonlearners, and
pseudoconditioned groups each contained 693, 563, 361, and 542 pyramidal cells, respectively. Almost all of these single neurons were
tracked for only a single day of training. Thus on average, 54 cells
were recorded for each of the 10 days of training for each group. The
channel configuration of the electrodes was optimized prior to each day
of recording to sample the largest ensemble of CA1 neurons.
Furthermore, the number and firing pattern of the pyramidal cells on
each stereotrode changed significantly from one day of training to the
next. As a result in most cases it was difficult to track isolated
pyramidal cells from one day of training to the next. However, 26 of
the 2,159 neurons were tracked for two successive days of training. It
is possible that many additional neurons were not tracked as the same
single neuron because of significant changes in the waveform and firing
pattern from one day of training to the next. Regardless, this does not detract from the goal of this study, to describe the different CA1
single-neuron response types that occur during trace eyeblink conditioning and to understand how the encoding of learning-related information within these ensembles is affected in aged animals that are
unable to learn the trace eyeblink task.
The single-neuron activity of each of the four groups was characterized initially by examining the average single-neuron response pattern during the early and late stages of training. This allowed for an examination of single-neuron activity before and after the acquisition of the trace eyeblink conditioned response. Figure 2 shows perievent histograms of spike discharge activity averaged across all of the single neurons recorded from the first 3 and last 3 days of training. Each of the groups exhibited an average increase in single-neuron activity following the CS and US both early and late in training. However, early in training the single-neuron increase following the CS and US was much smaller in the aged nonlearners. A factorial ANOVA with a group and days factor (early vs. late) was applied to the change score measures of single-neuron activity during the 400-ms period following the onset of the CS. The interaction was significant, F(3, 1269) = 4.23, P = 0.006, and follow-up ANOVA and post hoc tests revealed that the single-neuron increase in activity following the CS was significantly smaller for the aged nonlearners compared with the other groups early in training, F(3, 640) = 4.20, P = 0.007. A factorial ANOVA for the 400-ms period following the US revealed a similar interaction, F(3, 1258) = 3.55, P = 0.014. Follow-up ANOVA and post hoc tests revealed that the single-neuron increase in activity following the US was significantly smaller for the aged nonlearners compared with the other groups early in training, F(3, 640) = 5.50, P = 0.001. These analyses show that the average increase in activity of CA1 pyramidal cells is reduced early in training in aged animals that are not able to acquire trace eyeblink conditioning.
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Heterogeneous stereotrode ensembles
To further understand the altered CA1 pyramidal cell activity in the aged nonlearners, the ensemble profile of single neurons recorded on each of the stereotrodes was examined. One or two stereotrodes were implanted in each animal, and the ensemble profile on each stereotrode almost always changed from one day to the next. Therefore each of the 10 days of recording was treated as a new stereotrode ensemble. This approach allowed 354 different stereotrode ensembles to be analyzed in this study. The number of neurons isolated from each stereotrode ensemble recorded in CA1 ranged from 2 to 12, with an average of 6.5 neurons/stereotrode. The group averages of neurons/stereotrode were nearly identical, as confirmed by a nonsignificant one-way ANOVA. The smallest average was obtained from the aged learners [6.2 ± 2.2 (SD) neurons/stereotrode], and the largest average was from the young group (6.6 ± 2.6 neurons/stereotrode). This suggests that group differences in single-neuron activity were not due to differences in the sampling or the morphological distribution of cells.
Mean change score measures of single-neuron firing were used to examine the profile of single-neuron responses on the stereotrodes implanted in CA1. These measures provided a descriptive analysis of the increases (excitatory) and decreases (inhibitory) in single-neuron activity that make up the profile of each stereotrode ensemble. Examination of the daily mean change in single-neuron activity following the onset of the CS revealed that 79% of the 354 stereotrode recordings used in this study had at least one excitatory and one inhibitory cell within their ensemble. A similar examination of the activity following the CS revealed that 48% of the stereotrodes had at least two excitatory and two inhibitory cells within the ensemble, and 25% had at least three excitatory and three inhibitory cells within the ensemble. Examination of the daily mean change in single-neuron activity following the onset of the US revealed similar heterogeneous stereotrode profiles. The measures of activity following the US revealed that 75% of the stereotrodes had at least one excitatory and one inhibitory cell, 42% had at least two excitatory cells and two inhibitory cells, and 22% had at least three excitatory and three inhibitory cells within an ensemble. Thus the stereotrode ensembles consisted of heterogeneous profiles of single-neuron activity. There was no group difference in the amount of heterogeneity on the stereotrodes. These analyses of inhibition and excitation suggest that the overall group analyses from Fig. 2 may be due in large part to a specific class or classes of cells. Therefore the aim of our subsequent analyses was to characterize the different profiles of single-neuron activity that occurred during trace eyeblink conditioning.
Figure 3 shows an example of a
heterogeneous ensemble of 10 individual single neurons recorded
simultaneously from a single stereotrode from a young animal on a
single day of training (day 5 in this example). A total of 12 neurons
were recorded from this stereotrode ensemble; however, only 10 of the
neurons are displayed because 2 of the neurons never fired during the
3.75-s window of analysis shown in the figure. These neurons were
recorded simultaneously from the tip of an implanted stereotrode that
never moved during training. The perievent time histograms show that at
least three of these neurons (neurons 4, 6, and
9) show on average excitatory increases in activity to the
US; however, three other neurons in this ensemble show inhibitory
decreases in activity (neurons 2, 5, and 7)
immediately after US onset. Figure 4
demonstrates how multiple-neuron average analyses overlook a
significant part of the information about the ensemble. The summed
activity of the individual single neurons from Fig. 3 (shown in Fig.
4A), similar to a multiple-unit record, is largely dictated
by the activity of the number 4 single neuron in Fig. 3. This ensemble effect could account for the early findings of Berger and
Thompson (1978), who used multiple-unit recordings and found
that CA1 cells show large bimodal increases in activity to the stimuli
used in eyeblink conditioning. However, our previous work
(McEchron and Disterhoft 1997
) and especially the
present investigation showed that very few single neurons exhibit this
pattern of activity. Figure 4, A and B, also
suggests that a small number of neurons with faster firing rates can
have a significant influence on multineuron or ensemble analyses.
Figure 4C shows the number of action potentials summed
across all of the neurons shown in Fig. 3 excluding neurons 4 and 6. Figure 4C demonstrates that neurons
with very low firing rates (e.g., neuron 6) can also
influence the overall shape of multineuron or ensemble analyses. Thus
single-neuron response patterns of activity in CA1 are very
heterogeneous and ensemble activity can be influenced by the excitatory
or inhibitory pattern of activity of an individual single-neuron
response pattern (e.g., Fig. 4C) or the overall firing rate
of a single neuron (e.g., Fig. 4, A and B).
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Heterogeneous single-neuron response profiles
To further understand these heterogeneous ensembles, it was necessary to go beyond the simple excitatory/inhibitory dimension of activity and describe single neurons based on the change in activity across the entire trace eyeblink conditioning trial. Thus a multidimensional approach was used to understand the learning-related activity of specific classes of cells within the ensemble. An hierarchical clustering analysis categorized each single neuron based on the average firing pattern of activity during the trace eyeblink conditioning trial. Specifically, this analysis compared the daily single-neuron firing-pattern of each cell (summed across the 80 CS-US trials of a single day of training) to the daily activity of all of the other cells recorded during trace eyeblink conditioning. The analysis then determined the similarity of each of the single-neuron firing patterns to classify these single-neuron response profiles into a limited number of unique categories.
These clustering analyses sought to classify cells based on their activity during trace conditioning, therefore cells from pseudoconditioned animals were not included in the initial analysis. The hierarchical clustering analysis was performed on the daily average of activity of 1,367 single neurons recorded from the young and aged animals that received trace eyeblink conditioning. The original hierarchical analysis produced nine clusters; however, analysis of the daily average of activity of each of these clusters revealed that several clusters had identical average patterns of activity for the trace eyeblink trial. Therefore several identical clusters were combined into the next highest cluster within the hierarchical tree. This process revealed six unique patterns of activity. Each trace conditioned cell was then matched to a template that corresponded to one of the six patterns of activity. The average daily responses of these cell categories are shown in Fig. 5, A-F. An additional 250 cells, shown in Fig. 5G, were not included in the clustering analysis because they fired three spikes or less during all 80 trace eyeblink conditioning trials of a single day of training. These cells were considered a unique category of cells because they tended not to fire after the onset of the CS and US. Furthermore, when these cells were included in the original clustering analysis, they were categorized along with cell types in C and D, which had distinctively higher background firing rates compared with the cell type in G. The firing pattern of the cell type in G, shown in Fig. 5 appears to be inhibitory; however, the possibility cannot be ruled out that this effect could be a random placement of a very small number of spike discharges.
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The dendogram in Fig. 5 shows the resulting distance of the single-cell
response categories A-F that resulted from the clustering analysis. These distances can also be viewed as the amount of dissimilarity between the single-cell categories (see Everitt and Dunn 1991). Categories A and
B are similar to each other and C and
D are similar to each other, but A-D
are more similar to each other compared with E. Finally,
category F appears to have the least similarity with the
other categories in the analysis. Below the dendogram is the percentage
of cells from each of the trace conditioned groups belonging to each
single-cell response category A-F. For each single-cell
response category, a
2 test compared the
percentage of cells falling into each of the trace conditioned groups.
There was no group difference in the percentage of cells falling into
cell categories A-F; however, the aged nonlearners had a
significantly higher percentage (22%) of cells in the single-cell
response category G compared with the other
trace-conditioned groups, the young (9.2%) and aged learners (17.2%),
2 = 6.125, P < 0.05. Analyses also
revealed that there was no significant difference in the expression of
any one of the cell types between the early and late stages of training
(first and last 5 days of training).
Several interesting observations can be made from the cellular
response categories shown in Fig. 5. Type F single neurons made up a very small percentage of the neurons encountered in this
study. An example histogram of an individual type F neuron is shown as neuron 4 in Fig. 3. Although there were a small
percentage of these neurons encountered, 28% of the stereotrode
ensembles contained at least one type F single neuron. This
suggests that a small percentage of these faster firing neurons could
have a significant impact on the pattern of the multiunit ensemble
activity in CA1, as shown by neuron 4 in Fig. 3. Although
type F neurons fired much faster than the other
single-neuron response types, their average firing rate (i.e.,
0.75 ± 0.65 Hz) was still well below the firing rate of theta
cells (i.e., 6-8 Hz) as described by Ranck (1973).
Almost half of the single neurons showed an excitatory increase in
activity to the US (48.3%; types A, C, E, and
F), while only a quarter of the single neurons show some
type of excitation to the CS (25.4%; types A, B, E, and
F). This suggests that many of the cells in CA1 encode
information about the US, whereas far fewer cells play a role in
encoding CS information. In contrast to these increases, more than
two-thirds of the cells show some type of inhibitory decrease in
activity during trace eyeblink conditioning (67.2%; types C,
D, and G). This shows that the majority of the cells in
CA1 do not transmit bimodal increases in unit activity, rather the
cells in CA1 appear to be composed of heterogeneous ensembles or
networks of individual cells with unique roles in transmitting
information. It is also interesting to note that 26 neurons were
tracked for two consecutive days of training. The response categories
(A-G) of 21 of these 26 neurons remained the same for two
successive days, suggesting that pyramidal cells have relatively
specific and inflexible response patterns with respect to trace
conditioning. Although the response type remained consistent for these
21 cells, the magnitude of the excitatory or inhibitory response did
change from one day to the next.
Single neurons from pseudoconditioned animals were not included in the clustering analysis to ensure that the single-neuron response profile of each category was defined by only trace-conditioned activity. However, it was necessary to compare trace-conditionted and pseudoconditioned activity for specific categories of single neurons. Therefore, both trace and pseudoconditioned cells were matched to templates corresponding to each of the trace-conditioning categories produced by the original clustering analysis. This allowed cell types from all groups to be analyzed in subsequent analyses.
Baseline levels of single-neuron activity
Analyses of background firing rate were performed by calculating the mean single-neuron discharge rate during the 1-s period prior to the delivery of each trial used in training. Overall, the mean background firing rate of the pyramidal cells was very low, 0.27 ± 0.4 Hz, with a range from 0 to 2.9 Hz. A factorial ANOVA was used to compare baseline firing of the seven single-cell categories shown in Fig. 5 and the four groups of animals in this study. The analysis revealed a significant interaction of group × cell type, F(18, 2086) = 15.91, P = 0.0001. Follow-up one-way ANOVAs and post hoc tests revealed that the difference in baseline activity between the groups was similar for the cell categories A-D. Figure 6 shows that for these specific cell types, the baseline single-neuron firing of the aged nonlearners was significantly less than the other groups. There was no difference in baseline activity between the young and aged learners. However, the pseudoconditioned baseline activity was greater than all of the other groups. The enhanced baseline single-neuron firing in the pseudoconditioned group was most likely the result of the large number of discrete trials and the shortened intertrial interval used during daily training. Although the trace and pseudoconditioned animals received the same number of CSs and USs during a daily session, the pseudoconditioned animals received twice as many discrete trials (CS- and US-alone trials) and had half the amount of time between these trials. Similar group differences in baseline firing can also be seen prior to the CS onset in the histograms in Fig. 2.
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Changes in single-neuron activity
Standardized change scores of single-neuron firing were used to examine daily increases and decreases in single-neuron activity for each of the single-cell response categories depicted in Fig. 5. The change scores revealed that the aged nonlearners showed unique increases and decreases in single-neuron activity for specific cell types. A factorial ANOVA was conducted using the change scores computed for the 600-ms period following the onset of the CS. This analysis revealed a significant interaction of group × cell type, F(18, 2131) = 12.14, P = 0.0001. Follow-up analyses for the activity following the CS revealed that group effects were specific to the single-cell response categories B-D, F, and G. A similar factorial ANOVA was conducted using the change scores computed for the 600-ms period following the onset of the US. This analysis revealed a significant interaction of group × cell type, F(18, 2131) = 18.45, P = 0.0001. Follow-up analyses for the activity following the US revealed that group effects were specific to the single-cell response categories B, C, F, and G. Figures 7 and 8 show the changes in activity following the CS and US across days of training for the cell types that showed significant group differences in activity.
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Figure 7 shows the change in single-neuron activity for cell types B and F, the excitatory cell types that showed significant group differences in single-neuron firing. This figure shows the change in activity for the 600-ms periods following both the CS and US combined. Changes in activity were analyzed for both of these periods together because the pattern of activity across the groups and days was nearly identical for the periods following the CS and US. The top panel of this figure shows that early in training type B neurons from the young and aged learners showed greater increases in activity following the CS and US compared with the same type of neurons from the aged nonlearners and pseudoconditioned animals. A [2 (early, late) × 4 (group)]-factorial ANOVA conducted on the change in type B single-neuron responses in the top panel of Fig. 7 revealed a significant interaction, F(3, 132) = 4.10, P = 0.008. Follow-up tests revealed that during the first half of training the type B cells from the young and aged learners showed a greater increase in activity following the CS and US compared with the cells from the aged nonlearners and pseudoconditioned animals. The bottom panel of Fig. 7 shows that type F neurons from all of the groups that received trace eyeblink conditioning exhibited greater increases in activity following both the CS and US compared with the same type of neurons from the pseudoconditioned animals. A two [early, late ×4 (Group)]-factorial ANOVA conducted on the type F single-neuron responses in the lower panel of Fig. 7 revealed a significant group main effect, F(3, 93) = 13.18, P = 0.0001. Follow-up tests revealed that the type F neurons from all of the groups that received trace eyeblink conditioning showed greater increases in activity following the CS and US compared with the same type of neurons from the pseudoconditioned animals.
Figure 8 shows the change in single-neuron activity for cell types C, D, and G, the inhibitory cell types that showed significant group differences in single-neuron firing. This figure shows that the inhibitory cell types from the aged nonlearners exhibited smaller decreases in activity following the CS (top) and US (bottom) compared with the other three groups. A factorial ANOVA conducted on the single-neuron responses of type C, D, and G cells in the top panel of this figure revealed a significant group main effect, F(3, 1461) = 4.04, P = 0.007. Post hoc tests revealed that type C, D, and G cells from the aged nonlearners showed a smaller decrease in single-neuron activity following the CS compared with the other groups. A factorial ANOVA conducted on the type D and G single-neuron responses in the bottom panel of Fig. 8 revealed a significant group main effect, F(3, 941) = 9.66, P = 0.0001. Post hoc tests revealed that type D and G cells from aged nonlearners showed a smaller decrease in single-neuron activity following the US compared with the other groups.
Ensemble single-neuron activity
Cross-correlation analyses were used to examine the coordinated firing of the different cell types (A-G) and to compare the coordinated firing of the single-neuron ensembles among the four groups in this study. Coordinated activity of cell pairs within a stereotrode ensemble was measured using CCRs. Only pairs recorded from the same stereotrode were analyzed with this method. The bins of each CCR were normalized according to the number of action potentials recorded from both cells in the pair. Figure 9, A and B, shows normalized CCRs for two example cell pairs from a single five-neuron steretrode ensemble. This ensemble was obtained from a young animal on the 6th day of training. The CCRs from this stereotrode ensemble are similar to many others recorded in this study, where the activity of one neuron was correlated with the activity of specific neurons within the stereotrode ensemble. For example, Fig. 9A shows a cell type F single neuron, which exhibited a large amount of coincident activity at a very short time delay (near 0) with a cell type D single neuron simultaneously recorded within the ensemble. Figure 9B shows that the same cell type F single neuron exhibited a much smaller amount of coincident activity with a cell type A single neuron simultaneously recorded from the same ensemble.
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The magnitude of correlated activity, or coincidence, was compared between cell pairs on each stereotrode by examining the number of events in the two tallest adjacent bins of the adjusted CCR. This allowed a mean cross-correlation coefficient to be calculated for each cell type (A-G) within a stereotrode ensemble. For example, a stereotrode ensemble that contained the cell types A-C would allow one mean for cell type A to be computed for this stereotrode ensemble, which included the cell pairs A-B and A-C. Means would also be computed for cell types B and C on this stereotrode. Cross-correlation coefficients were computed using 10 different CCR binwidths ranging from 10 to 100 ms. The means were examined using a factorial ANOVA (group × cell type), which was applied individually to the means computed for each binwidth (i.e., 10, 20 ··· 100 ms). These ANOVAs all revealed significant effects of group and cell type but no interaction. The significant group effects for the 10- and 100-ms bin ANOVAs were F(3, 982) = 5.56, P = 0.001 and F(3, 982) = 6.85, P = 0.0001, respectively. The significant cell type effects for the 10- and 100-ms bin ANOVAs were F(6, 982) = 6.70, P = 0.0001 and F(6, 982) = 15.77, P = 0.0001, respectively. The ANOVAs for the other binwidths revealed similar levels of significance but were not reported for purposes of simplicity. Figure 9C shows the mean adjusted correlation coefficient for each of the groups at each of the binwidths. Follow-up tests revealed that the young, pseudoconditioned, and aged learners all showed significantly greater levels of coordinated activity within the stereotrode ensembles compared with the aged nonlearners. The effect was similar for all binwidths. The differences between the young group and the aged learners and the young group and the pseudoconditioned group were not significant but did approach significance at all binwidths.
Figure 9D shows the same data as in Fig. 9C
excluding all cell pairs with a background firing rate 0.67 Hz. This
criterion represented the mean pyramidal cell background firing rate
(0.27 Hz) plus 1 SD (0.4 Hz). Excluding these faster firing cells
eliminated 27% of the total 21,200 cell pairs. Nearly identical group
effects are shown in Fig. 9, C and D. This
provides evidence that the group effect in Fig. 9C was not
due to a subset of cells with faster firing rates. The factorial ANOVAs
for the data in Fig. 9D were similar to those in Fig.
9C with significant effects of group and cell type but no
interaction. The significant group effects for the 10- and 100-ms bin
ANOVAs were F(3, 900) = 5.90, P = 0.001 and F(3, 900) = 8.11, P = 0.0001, respectively. The significant cell type effects for the 10- and 100-ms
bin ANOVAs were F(6, 900) = 3.83, P = 0.0001 and F(6, 900) = 12.41, P = 0.0001, respectively. Similar to the data in Fig. 9C,
follow-up tests for the data in Fig. 9D revealed that the
young, pseudoconditioned, and aged learners all show significantly
greater levels of coordinated activity within the stereotrode ensembles
compared with the aged nonlearners. The effect was the same for all
binwidths. The differences between the young group and the aged
learners, and the young group and the pseudoconditioned group were not
significant but did approach significance at all binwidths.
Figure 9E shows the same data as in Fig. 9C excluding all cell pairs where one cell of the pair fired >25% more action potentials than the other cell. Excluding the pairs with a discordant number of spikes eliminated 81% of the total number of cell pairs. Nearly identical group effects are shown in Fig. 9, C and E. Not shown are analyses that also excluded all cell pairs with one cell of the pair firing >50% more action potentials than the other cell. This excluded only 62% of the total cell pairs and produced group effects identical to those in Fig. 9E. The factorial ANOVAs for the data in Fig. 9E were similar to those in Fig. 9C with significant effects of group and cell type but no interaction. The significant group effects for the 10- and 100-ms bin ANOVAs from Fig. 9C were F(3, 554) = 4.43, P = 0.004 and F(3, 554) = 5.28, P = 0.001, respectively. The significant cell type effects for the 10- and 100-ms bin ANOVAs were F(6, 554) = 3.83, P = 0.037 and F(6, 554) = 10.73, P = 0.0001, respectively. Similar to the data in Fig. 9C, follow-up tests for the data in Fig. 9E revealed that the young, pseudoconditioned, and aged learners all showed significantly greater levels of coordinated activity within the stereotrode ensembles compared with the aged nonlearners. The effect was the same for all binwidths. Because the same effects were revealed with the exclusion of the pairs with discordant firing rates, this provides evidence that the group effect in Fig. 9C was not due to a small number of cells within the ensemble with a much faster firing rate. In summary, Fig. 9, C-E, shows that aged animals that are unable to learn trace eyeblink conditioning show a significantly lower level of coincident pyramidal cell firing within local ensembles in CA1. This effect was not due to differences in background firing between the groups or heterogeneous firing rates within the ensembles.
Analyses of the coincident activity from Fig. 9, C-E,
revealed a significant effect of cell type (types A-G in
Fig. 5) but no interaction of group and cell type. Means for each of
the cell types are shown in Fig. 9F. The pattern of
correlation coefficients between each of the cell types shown in Fig.
9F was similar for all of the analyses in Fig. 9,
C-E. The means in Fig. 9F were taken from the
CCR measures computed using 50-ms bins from the data in Fig.
9C. The ANOVA for the 50-ms bin in Fig. 9C
revealed significant effects of group, F(3, 982) = 6.60, P = 0.0001, and cell type, F(6,
982) = 13.99, P = 0.0001. Follow-up tests on the cell type effect revealed that coordinated activity of cell type F with the other cells in the ensemble (pairs that contained
1 cell type F) was greater than the coordinated activity
of all other cell type combinations. The follow-up tests also showed that pairs that contained cell type G showed less
coordinated activity than all other cell type combinations.
Furthermore, pairs that contained cell type C or
D showed more coordinated activity than pairs that contained
cell type E. These analyses suggest that specific cell types
will provide more coincident activity to the ensemble processing of
information. For example, cell type F will contribute more
coincident events to an ensemble compared with cell type E.
This may be due, in part, to the density pattern of action potentials.
However, it is very important to note that the deficient coincident
activity in the aged nonlearners was not due to a lack of one or
several cell types, as analyses from Fig. 5 demonstrated that the cell
types (except for G) are expressed evenly among the groups.
The analyses of the correlation coefficients in Fig. 9,
C-E, all revealed a similar group pattern for each of the
cell types (A-G). Finally, the group effects for coincident activity in Fig. 9, C-E, were not due to group differences
in firing rate because measures of coincidence were corrected by the
number of action potentials discharged by a cell pair, and the Pearson
correlation between total number of action potential discharges and the
corrected correlation coefficient measure was negligible,
r = 0.119.
It is important to note that outlier mean correlation coefficients from
two stereotrode ensembles from the young group were not included in the
analyses in Fig. 9, C-E, because these means were 4 SD of the mean. Analyses for the data in Fig. 9,
C-E, were performed at 10 different binwidths ranging from
10 to 100 ms, and identical effects were revealed at all binwidths.
These multiple analyses were performed to ensure that group effects were not due to binwidths that might select for cell pairs with a
specific firing rate or pattern. Many ANOVAs were required for this
approach, which could inflate the probability of a type I error;
however, all significant group effects were below the alpha level of
0.005. The measures of correlated single-neuron firing used in this
study were not aimed at examining the overall magnitude of the
correlations, nor were they used to examine the significance of the
correlated firing of an individual pair of single neurons. On the other
hand, the measures of coordinated firing used in this study were
designed specifically to correct for faster firing rates of single
neurons and to be able to compare the coordinated firing of the
different groups.
These cross-correlation analyses suggest that specific cell types provide more coincident activity to the ensemble processing of information. However, regardless of the cell types within the ensemble, aged animals that were unable to learn trace eyeblink conditioning showed a significantly lower level of coincident pyramidal cell firing within local ensembles in CA1.
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DISCUSSION |
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The results of the present study showed that more than half of the aged rabbits (30-34 mo of age) were unable to acquire trace eyeblink CRs while the remaining aged animals learned this task as well as young animals. Similar to all the other groups of animals, aged nonlearners showed larger unconditioned eyeblink responses on paired trials compared with US-alone trials, suggesting that the animals were able to sense the auditory-CS information. Increases in CA1 pyramidal cell firing to the CS and US were diminished in the aged animals that were learning impaired. The local ensembles of pyramidal cells from all groups exhibited heterogeneous types of increases and decreases in single-neuron firing during the trace eyeblink trial. Hierarchical clustering was used to isolate seven different classes of single-neuron responses which showed unique firing patterns during the trace eyeblink conditioning trial. Unlike the excitatory patterns of modeling reported in previous studies, three of seven single-neuron response types in this study (67% of the total number of cells) exhibited some type of inhibitory decrease to the CS, US, or both stimuli during training. The proportion of cells in the young and aged groups was similar for six of seven of these single-neuron response classes. Several of the single-neuron response types in the aged learning-impaired animals showed an altered response magnitude to the CS and US compared with the same cell types in the other groups of animals. Measurements of correlated firing within local ensembles demonstrated that the aged nonlearners showed a significantly lower level of coincident pyramidal cell firing for all of the cell types within local ensembles in CA1.
Ensembles and response types
The present study demonstrated that there were a number of
different excitatory and inhibitory patterns of CA1 single neuron responding during the trace eyeblink trial. Our previous single-neuron investigations of CA1 used single electrodes along with single-neuron classification software to describe several excitatory and inhibitory single-neuron response profiles during trace eyeblink conditioning (e.g., McEchron and Disterhoft 1997). However, the
initial delay eyeblink conditioning studies conducted by Berger and
Thompson (e.g., Berger and Thompson 1978a
,b
;
Berger et al. 1983
) reported that most, if not all,
pyramidal cells showed increases in firing during the delay eyeblink
conditioning trial that paralleled the amplitude time course of the
behavioral eyeblink response. They used multiple-unit recording
techniques in one study and single-unit recording techniques in the
other. It is possible that the single-electrode/multiple-unit recording
techniques used in their studies did not allow for the sampling of
heterogeneous types of single-neuron responses, whereas the microwire
stereotrode recording method used in this study allowed single neurons
to be separated within the multiple unit record. It is possible that
the trace procedures used in the present study contributed to the
heterogeneous firing in CA1 that was not present in Berger and
Thompson's earlier delay eyeblink conditioning studies. It is also
important to mention that their work does show a few examples of other
types of responses besides modeling, and their single-neuron work
clearly states that many single neurons were not studied because they
did not meet a number of their electrophysiological criteria. Thus
Berger and Thompson's work may have focused more on cell types
B or F, especially cell type F, which
has more frequent background firing and a larger evoked firing rate.
Our example10-neuron ensemble in Fig. 3 demonstrated how one single
neuron with a faster background and evoked firing rate could influence
the multiple-unit record. This dominant single neuron was classified as
an F-type cell. The F-type cells showed a pattern
of responses that resembled the modeling responses described by
Berger and Thompson (1978)
. Also congruent with their
work, both the B and the F type cells in
trace-conditioned animals showed greater increases in activity following both the CS and US compared with the cells recorded from
pseudoconditioned animals.
The heterogeneous single-neuron response types described in the present
study were recorded simultaneously within ensembles located at the tips
of single nonmovable stereotrodes. The majority of the ensembles showed
heterogeneity, and there was no evidence that placement of the
electrodes was related to the occurrence of any one single-neuron
response type. This suggests that the heterogeneous single-neuron
response types had a close physical relationship. Several mechanisms
could account for the different single-neuron response patterns of
individual pyramidal cells encountered in CA1. There is some
speculation that unique physiological attributes of individual CA1
pyramidal cells could contribute to a heterogeneous population of
pyramidal cells (Lisman and Harris 1993), which in turn
could contribute to a network of different response types. There are a
number of reports of heterogeneous types of synaptic responses measured
on individual CA1 pyramidal cells in vitro (Turner et al.
1997
; Yuste et al. 1999
). Other in vitro work
has shown heterogeneous levels of synaptic plasticity for different
pyramidal cells (Debanne et al. 1999
). There is also
evidence for a heterogeneous distribution of receptors on pyramidal
cell dendrites (Lujan et al. 1997
). On the other hand, anatomical studies suggest that CA1 pyramidal cells are remarkably homogeneous with respect to dendritic branching and efferent and afferent projections (Ishizuka et al. 1995
;
Swanson et al. 1981
; Tamamaki and Nojyo
1990
). This may suggest that heterogeneous responses in CA1
emanate, at least in part, from the activity of interneurons. It is
clear that there are heterogeneous types of interneurons in the CA1 of
the hippocampus (Halasy et al. 1996
; Oliva et al.
2000
; Pearce 1993
; Tietz et al.
1999
; van Hooft et al. 2000
). Furthermore,
studies have shown that there is a large amount of heterogeneity in the
magnitude of synaptic plasticity in these distinct types of
interneurons in CA1 (Maccaferri and McBain 1996
). Other
studies have speculated that interneurons may play a key role in
network processing of learning related information (Banks et al.
2000
; Fukuda and Kosaka 2000
). Together this
evidence suggests that future electrophysiological recording studies
should record simultaneously from interneurons and pyramidal cells to
understand the how this learning network operates.
We cannot rule out the possibility that action potentials from several
interneurons were recorded in our large sample of isolated cells.
However, Ranck's pyramidal cell-criteria were carefully adhered to in
this study, and the firing rate of all cells was <3 Hz, averaging <1
Hz (Ranck 1973). Furthermore, all electrode tips were
placed directly in the pyramidal cell layer. This suggests that we
recorded very few, if any interneurons. Nevertheless, if a small number
of interneurons mistakenly meet our pyramidal cell criteria, it is
unlikely that this small number would influence the categorization of
pyramidal cell types, which used a very large sample of 1,367 neurons.
It was difficult to resolve from the present experimental design
whether the response characteristics of individual pyramidal cells are
hardwired and inflexible during trace eyeblink conditioning or if
individual pyramidal cells can flexibly perform multiple response
patterns. The channel-configuration of the electrodes in this study was
optimized on each day of training so that the largest ensemble of CA1
neurons was recorded, and as a result in most cases, it was difficult
to track isolated pyramidal cells from 1 day of training to the next.
However, in the few cases (26 neurons) where neurons were tracked for
more than 1 day of training, similar response types (i.e.,
A-G) were almost always measured from one day to the next.
Although this is a very small number of cells, it may suggest that
pyramidal cells have specific and relatively inflexible response
patterns with respect to trace conditioning. Clearly, future work
should directly address whether the heterogeneous response types of
individual pyramidal cells are hardwired and consistent. Consistent
responses for individual cells might imply that a functional
topographical map exists in CA1 for encoding trace eyeblink
information. Unfortunately, this study was not designed to directly
address functional mapping because all of the electrodes in this study
were placed consistently in one region of the dorsal hippocampus.
Within this arrow region there did not appear to be any relationship
between the characteristics of the CA1 ensembles and the placement of
these electrodes. A consistent and inflexible response pattern of
activity during trace conditioning would be analogous to a premapped
role of CA1 pyramidal cells, as suggested in numerous spatial recording
studies (e.g., O'Keefe and Speakman 1987). Spatial
studies have shown that pyramidal cells in the hippocampus fire bursts
of action potentials when an animal is in a specific place in the
environment. These pyramidal cell place fields are fairly stable across
long periods of time (Thompson and Best 1990
),
consistent with the notion that the hippocampus consists of a
relatively hardwired cognitive map (O'Keefe and Nadel
1978
). In addition to these spatial studies, there is a large
body of literature that has shown that hippocampal neurons also encode
nonspatial information during learning (Hampson et al.
1999
; McEchron and Disterhoft 1999
; Wood et al. 1999
; Young et al. 1994
). In one of these
studies, Hampson and colleagues (1999)
have demonstrated
that populations of neurons that respond to spatial and/or nonspatial
learning-related information show an orderly topographical arrangement
in the hippocampus. This may be consistent with the notion that the
heterogeneous cell types in this study are part of a larger hardwired
network responsible for encoding learning-related information.
Spatial studies are well suited for in vivo electrophysiological
investigations of the hippocampus because pyramidal cell responses are
fairly homogeneous during spatial behavior. Specifically, an individual
pyramidal cell increases its firing rate when an animal is in one
location of the environment and shows a lower rate of firing when the
animal is not in that location. It may appear that the activity of
pyramidal cells in the present nonspatial trace eyeblink conditioning
task is entirely different and more complex than the firing patterns
found in spatial studies, such that pyramidal cells show a number of
different excitatory and inhibitory responses during the trace eyeblink
trial. Alternatively, there may be an interesting parallel between the
CA1 encoding of trace eyeblink conditioning and the encoding of spatial
information. Specifically, each individual pyramidal cell encodes a
different location in space, and it is believed that all of the
individual place cells together form a map of space (O'Keefe
and Nadel 1978). There may be a parallel function with trace
eyeblink conditioning such that all of the combined excitatory and
inhibitory single-neuron response profiles form a temporal map to
encode learning related information about the trace eyeblink
conditioning trial. Others have proposed similar notions of temporal
mapping where discharges of different single neurons occur close
together in time to bridge the gap between temporally discontiguous
events (e.g., Levy 1996
; Wallenstein et al.
1998
). However, these temporal mapping theories tend to suggest
that single-neuron events are evenly distributed between the stimuli
being associated. The cell types described in this investigation do not
appear to show an even distribution of activity between the CS and US
but rather seem to encode the CS and especially the US in unique
patterns of activity. Regardless of this issue, the heterogeneous
response types found in trace eyeblink conditioning might provide a
unique advantage for trying to understand how hippocampal networks
process learning-related information. Moreover, in trace eyeblink
conditioning animals are held in a fixed position and a limited number
of discrete stimuli are presented. This nonspatial learning paradigm
may be optimal for examining how different response elements interact to encode learning-related information.
The present study revealed that only cell type B within the local ensembles showed learning-related changes in activity. This suggests that cell type B is one of the critical components of the hippocampal network involved in the transmission of learning-related information. However, cell type F showed significant increases in activity that were specific only to the paired trace conditioning procedure, that is, only the pseudoconditioned animals did not show increases in cell type F activity. Thus it appears that the activity of these cells is the product of the associative presentation of the CS and US. The associative activity of the F cells may be an important precursor to the events of learning. The F cell type also provides the most coordinated activity within the CA1 ensembles. Thus the activity of these cells may serve to provide the critical associative overlapping activity with other cells in the ensemble, possibly allowing heterosynaptic changes in plasticity. Other cell types in the ensemble, such as C and D, may have an opposing role by down-regulating the amount of overlapping activity within the network. Thus it is possible that all of the cellular response types described in this study (A-G), and perhaps others not encountered, are each critical components of a network that interact to encode learning-related trace eyeblink information.
Another explanation of the cell types in this study is that a limited
number of these cell types may encode specific critical properties of
learned information in the hippocampus, while the other cell types are
performing less critical auxiliary functions. The notion of a small
number of cells encoding learning-related information is similar to the
idea of sparse coding where a neural network represents
multidimensional data but only a small number of neurons are
significantly activated at one time (Olshausen and Field
1996). The present study did not allow us to determine if
sparse coding is operating in CA1 because nearly all of the cells
recorded, at least types A-F, showed some type of activity related to the stimuli in the trace eyeblink trial. However, this does
not necessarily mean that all of these patterns are playing a critical
role in trace eyeblink learning. For example, only one of the cell
types (B), representing 7.4% of the neurons recorded showed
conditioning-specific increases in activity, lending support for a
sparse coding hypothesis. Furthermore, a larger percentage of cells
(66%) showed some type of decrease in activity during the trace
eyeblink trial, which may allow for an increase in the signal-to-noise
ratio controlled by the small number of cells (e.g., cell type
B) regulating learning-related information. Alternatively, the different single-neuron response types encountered showed very
robust yet different patterns of activity within a local ensemble
during the trace eyeblink trial. This may imply that no one cell type
controls more information than the others, and these closely spaced
cells with very different firing patterns are somehow working together
to encode learning-related information during trace eyeblink conditioning.
Aging and pyramidal cell responses
The expression of most of the classes of single-neuron responses
(A-F) was similar for the young and aged groups in this
study; however, the response magnitude of a specific subset of these single-neuron response types was affected in the aged animals that were
unable to learn trace eyeblink conditioning. Specifically, the
excitatory response of cell type B to the CS and US and the inhibitory responses of cell types C, D, and G
were smaller in the aged nonlearners compared with the other trace
conditioning groups. Alternatively, measures of coordinated ensemble
firing demonstrated that the aged nonlearners showed less coordinated ensemble firing for all of the cell types. This suggests that the aged
nonlearners may have possessed an inherent physiological deficit in the
hippocampal cellular networks that produced deficient levels of
coordinated activity. Moreover, it would appear that a sufficient level
of coordinated activity in CA1 is a critical feature of healthy
hippocampal networks that allows ensembles of cells to process
learning-related information. It is impossible to determine from this
study if these cellular response deficits are specific and local to CA1
or if they are the result of altered transmission from other areas in
the hippocampus, entorhinal cortex, or medial septum. Evidence from
many studies suggests that the deficits are probably distributed in all
of these areas of the circuit. For example, Smith and colleagues
(2000) used synaptophysin immunoreactivity to show that in aged
nonlearning animals, there is considerable synapse loss in the dentate
gyrus and CA3 regions, which provide inputs to the CA1. In addition to
these areas, other cortical structures such as the entorhinal cortex,
perirhinal cortex, and subiculum of aged nonlearning animals have been
shown to have an increased level of binding sites for galanin, a
cholinergic inhibitor (Krzywkowski et al.
1993
). Armstrong and colleagues (1993)
have also described cholinergic alterations in the medial septum which
are aging and learning specific. Findings from our laboratory have
shown that the membrane properties of CA1 cells show decreases in
excitability in aged animals (Moyer et al. 1992
). We
have also shown that a calcium channel blocker, cholinesterase inhibitor, and muscarinic agonist enhance learning in aging rabbits and
produce a significant increase in neuronal excitability in aging CA1
neurons (Deyo et al. 1989
; Kronforst-Collins et
al. 1997
; Moyer et al. 1992
; Oh
et al. 1999
; Weiss et al. 2000
). Together this
evidence suggests that the deficits in single-neuron and ensemble
firing in the aged nonlearners in this study were the result of
deficits that are distributed throughout the hippocampal learning
network and most likely involve a number of neurotransmitter systems.
Excitatory and inhibitory cellular response types were uniquely
affected in the aged-nonlearners, suggesting that several different
aging-related mechanisms may be operating on hippocampal networks to
produce learning deficits. The inhibitory cell types in the aged
nonlearners showed less inhibition of firing to the CS and US during
the trace eyeblink trial. This could be explained by several lines of
evidence that suggest that aging produces a reduction in the inhibitory
synaptic transmission mediated by GABA mechanisms (Billard et
al. 1995; Jouvenceau et al. 1998
; Potier
et al. 1992
). The firing response of the excitatory cell type B
to the CS and US was significantly less in the pseudoconditioned and
aged nonlearners compared with the young and aged learners. This
decrease in responsivity for cell type B may be a reflection of the
widely reported finding of aging-related decreases in synaptic responsiveness in CA1 (Barnes et al. 1997
;
Jouvenceau et al. 1998
; Landfield et al.
1978
). Jouvenceau and coworkers (1998)
have
found that glutamate receptor subtypes in CA1 are differentially
affected by aging. Their study examined CA1 field potentials in vitro
and found that N-methyl-D-aspartate (NMDA)
receptor-mediated transmission was not altered in aged animals, but
non-NMDA receptor mediated transmission was depressed. Other
experiments from this group have shown that cholinergic denervation in
young rats potentiates glutamatergic synaptic transmission in CA1
(Jouvenceau et al. 1997
). Together, their work suggests
that aging produces a deficit in cholinergic synaptic transmission in
the hippocampus, which others have shown as well (i.e., Shen and
Barnes 1996
), which in turn produces a compensatory maintenance
of NMDA-receptor-mediated synaptic transmission in CA1. This could
explain why the B-type excitatory cellular responses were affected in
aged nonlearners while the A, E, and F excitatory
responses were not affected. Our laboratory has provided numerous
examples showing that pharmacological compensation for cholinergic
changes reverses aging-related deficits in trace eyeblink conditioning
(Disterhoft et al. 1999
; Kronforst-Collins et al.
1997
; Oh et al. 1999
; Weiss et al.
2000
).
The baseline firing rate of several cell types was affected in an aging
and learning specific manner. The baseline firing rate of the pyramidal
cell types A-D was significantly decreased in the aged
nonlearners compared with the other groups. These aged
learning-impaired animals also showed a higher expression of cell type
G, which exhibited a very low rate of firing (<3 spikes
during 80 trials). It is important to note that these group differences
in baseline firing were not responsible for the group differences in
coordinated ensemble activity. It is conceivable that firing rate could
influence correlation measures; however, this does not appear to be the
case in this study since measures of correlated unit activity were
adjusted for firing rate using several different methods, and there was
no correlation between the number of spikes fired by a cell pair and
the magnitude of the adjusted correlation coefficient. Regardless of
this issue, the group differences in baseline single-neuron firing rate
suggest that specific subsets of pyramidal cells in the CA1 of aged
learning-impaired animals are less excitable than the pyramidal cells
of young and aged learners. These observations are consistent with the
in vitro electrophysiological evidence which has shown that CA1
pyramidal cells from aged nonlearning animals are less excitable.
Specifically, both the afterhyperpolarization and accommodation
measured in the in vitro slice are increased in CA1 neurons from aging
rabbits (Moyer et al. 1992) and rats (Landfield
and Pitler 1984
; Potier et al. 1992
) compared
with young animals. A series of studies from our laboratory has shown
that a calcium channel blocker, cholinesterase inhibitor, and
muscarinic agonist enhance learning in aging rabbits and produce a
significant increase in neuronal excitability in aging CA1 neurons
(Deyo et al. 1989
; Kronforst-Collins et al.
1997
; Moyer et al. 1992
; Oh et al.
1999
; Weiss et al. 2000
).
In summary, we have described a number of different excitatory and inhibitory pyramidal cell response patterns in CA1 which encode learning-related information during trace eyeblink conditioning. The interaction of these different pyramidal cell response types within local ensembles may be important for encoding learning-related information during trace eyeblink conditioning. Single-neuron response types were differentially affected in the aged animals that were unable to learn trace eyeblink conditioning. Aged learning-impaired animals also showed alterations in the coordinated firing of all of these cell types within these CA1 ensembles.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants RO1 AG-08796, RO1 MH-47340, and F32 AG-05711.
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FOOTNOTES |
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Present address and address for reprint requests: M. D. McEchron, Dept. of Behavioral Science, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033.
Received 14 July 2000; accepted in final form 11 June 2001.
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REFERENCES |
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