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Temporary Inactivation of the Retrosplenial Cortex Causes a
Transient Reorganization of Spatial Coding in the Hippocampus
Brenton G. Cooper and Sheri J. Y. Mizumori
Department of Psychology, University of Utah, Salt Lake City, Utah 84112
The ability to navigate accurately is dependent on the integra-
tion of visual and movement-related cues. Navigation based on
metrics derived from movement is referred to as path integra-
tion. Recent theories of navigation have suggested that poste-
rior cortical areas, the retrosplenial and posterior parietal cor-
tex, are involved in path integration during navigation. In
support of this hypothesis, we have found previously that tem-
porary inactivation of retrosplenial cortex results in dark-
selective impairments on the radial maze (Cooper and Mizu-
mori, 1999). To understand further the role of the retrosplenial
cortex in navigation, we combined temporary inactivation of
retrosplenial cortex with recording of complex spike cells in the
hippocampus. Thus, behavioral performance during spatial
memory testing could be compared with place-field responses
before, and during, inactivation of retrosplenial cortex. In the
first experiment, behavioral results confirmed that inactivation
of retrosplenial cortex only impairs radial maze performance in
darkness when animals are at asymptote levels of performance.
A second experiment revealed that retrosplenial cortex inacti-
vation impaired spatial learning during initial light training. In
both experiments, the normal location of hippocampal “place
fields” was changed by temporary inactivation of retrosplenial
cortex, whereas other electrophysiological properties of the
cells were not affected. The changes in place coding occurred
in the presence, and absence, of behavioral impairments. We
suggest that the retrosplenial cortex provides mnemonic spatial
information for updating location codes in the hippocampus,
thereby facilitating accurate path integration. In this way, the
retrosplenial cortex and hippocampus may be part of an inter-
active neural system that mediates navigation.
Key words: navigation; place cells; path integration; spatial
memory; head direction; posterior cingulate cortex
Navigation requires the continual updating of one’s current loca-
tion based on stable visual features of the environment and
movement through space (Gallistel, 1990; Etienne, 1992). Under-
standing the neural basis of navigation requires, at the very least,
identification of the different structures involved in integrating
visual cues with movement during navigation.
Hippocampal pyramidal cells fire robustly when animals tra-
verse particular locations in space; these locations are called the
“place field” of the cell (O’Keefe and Dostrovsky, 1971). The
contribution of visual cues to place fields has been illustrated by
rotating the salient cue(s) in a testing arena (O’Keefe and Con-
way, 1978; Muller et al., 1991). Most place fields are bound to the
distal cues, and simultaneously recorded neurons rotate synchro-
nously suggesting that neuronal ensembles follow distal cues
(O’Keefe and Speakman, 1987; Knierim et al., 1995). However,
place fields can develop in blind rats (Hill and Best, 1981; Save et
al., 1998), in the absence of visual cues (Quirk et al., 1990), and
maintain spatial firing when visual cues are removed (Muller et
al., 1991; Markus et al., 1994; Mizumori et al., 1999). Accordingly,
there has been an increased interest in the role of movement-
related cues in hippocampal spatial processing. Manipulations
designed to disrupt or manipulate self-motion cues can disrupt or
systematically alter place fields of hippocampal neurons (Sharp et
al., 1990, 1995; Knierim et al., 1995; Gothard et al., 1996; Jeffery
et al., 1997; Jeffery and O’Keefe, 1999).
McNaughton et al. (1996) suggest that posterior parietal and
retrosplenial cortex may send self-motion information to the
hippocampus. On the basis initially of anatomical observations,
we further hypothesized that retrosplenial cortex facilitates the
experience-dependent integration of visual and self-motion cues.
Visual projections from both the geniculostriate and tectocortical
pathways converge in retrosplenial cortex (van Groen and Wyss,
1990a; Zilles and Wree, 1995). Information related to one’s
movement may arrive in retrosplenial cortex via direct projec-
tions between posterior parietal cortex and anterior thalamic
nuclei (ATN) (van Groen and Wyss, 1995; Zilles and Wree,
1995). A subset of cells in retrosplenial cortex is sensitive to the
direction an animal is facing in an environment; these cells are
commonly referred to as “head-direction cells.” Retrosplenial
cortex head-direction cells are modulated by visual and move-
ment cues (Chen et al., 1994a,b). In agreement with our initial
hypothesis, temporary inactivation of retrosplenial cortex causes
behavioral impairments when animals perform a well learned
spatial memory task in darkness but does not disrupt perfor-
mance when visual cues are available (Cooper and Mizumori,
1999).
To understand better the contribution of retrosplenial cortex to
navigation and hippocampal spatial processing, temporary inac-
tivation of retrosplenial cortex was combined with recording of
hippocampal cells in well trained rats during light and dark maze
performance. We predicted that place cells would change their
Received May 10, 2000; revised Jan. 19, 2001; accepted March 12, 2001.
This work was supported by National Institutes of Health Predoctoral Fellowship
MH 11998 to B.G.C. and Grant M H 58755 to S.J.Y.M. We thank Gena Ettinger and
Theodore F. Manka for help in behav ioral training, Wayne Pratt and Alex Guazzelli
for helpful comments on thi s manuscript, James Canfield for extraordi nary technical
assistance on a variety of aspects of this project, and Stefan Leutgeb and Alex
Guazzelli for outstanding programming assistance.
Correspondence should be addressed to Dr. Sheri J. Y. Mizumori, Department of
Psychology, Box 351525, Universit y of Washington, Seattle, WA 98195-1525. E-mail:
mizumori@u.washington.edu.
B. G. Cooper’s present address: Department of Biology, University of Utah, Salt
Lake City, UT 84112.
Copyright © 2001 Society for Neuroscience 0270-6474/01/213986-16$15.00/0
The Journal of Neuroscience, June 1, 2001, 21(11):3986–4001
spatial coding during inactivation, especially when behavioral
impairments were evident. In a second experiment, retrosplenial
cortex was inactivated before the animals initially learned the
spatial memory task in light conditions. If retrosplenial cortex
contributes to mnemonic integration of visual and self-motion
information, we predicted that inactivation would cause spatial-
learning impairments and place-field instability during initial
visual spatial learning.
MATERIALS AND METHODS
Subjects. Eleven male Long–Evans rats obtained from Simonson’s Lab-
oratories (Gilroy, CA) were used in the experiments. Animals were kept
in a temperature- and humidity-controlled room with a 12 hr light cycle
(lights on at 7:00 A.M.). One week was given to allow the rats to
acclimate to the laboratory before onset of experimental procedures.
During this time animals were handled and weighed daily. During
behavioral testing and training, animals were food restricted and main-
tained at 80% of their free-feeding weights. Animal testing was con-
ducted in an Association for Accreditation of Laboratory Animal C are-
approved facility, within the guidelines of National Institutes of Health
animal care and use.
Behavioral method. Animals were trained to perform a spatial memory
task on an eight-arm radial maze using procedures similar to those
described elsewhere (Mizumori et al., 1989; Cooper et al., 1998; Cooper
and Mizumori, 1999). Briefly, the maze is elevated (77 cm) above the
floor and consists of eight arms (59.5 ⫻5 cm) with 0.5 cm railings
radiating from a center platform (19.5 cm in diameter). Arm access is
afforded or restricted via remote control by raising or lowering, respec-
tively, the proximal portion of the maze arm. A camera positioned above
the maze allowed the experimenter to monitor the animals from an
adjacent room. In darkness, movement of the animal was monitored by
observing a pair of infrared diode arrays attached to the head of the rat.
Two mazes with identical physical dimensions were located in two
different testing rooms. One room was used solely for behavioral training
(room 1). Room 1 was a large open room containing numerous distal
cues surrounding the maze. The maze for electrophysiological recording
was enclosed in a controlled cue environment consisting of black curtains
forming a square around the maze (room 2). A canopy-style ceiling
started at the camera and draped down to the four walls of the enclosure.
The room contained numerous distal cues for use by the animals to
determine their location and directional heading within the environment
[for further description of room 2, see Cooper et al. (1998)].
In room 1, animals were habituated to the maze and then trained to
retrieve chocolate milk from the end of randomly selected maze arms.
On each trial, the experimenter randomly selected eight maze arms,
which were individually presented until the animal had visited all maze
arms. After this “forced-choice” nonspatial memory training, animals
were then trained to perform a “win-shift” spatial memory task in room
2. The spatial memory task consisted of two phases. In the first phase
animals were presented with four randomly selected arms individually
and sequentially. While animals were on the fourth arm, the second
phase began by raising all eight maze arms. Animals were allowed to
chose freely among the eight arms; arm reentries were considered errors.
This partial forced-choice procedure minimizes the possibility that ani-
mals will develop a response-based strategy for solving the task.
Electrode construction and surg ical procedure. Hippocampal single-unit
activity was recorded using the stereotrode recording technique (Mc-
Naughton et al., 1983b). Two lacquer-coated tungsten wires (25
m;
California FineWire) were twisted together, dipped in Epoxylite, and
baked. The stereotrode was then threaded through a 30 ga stainless steel
tube, and two to four cannulas were mounted on a moveable microdrive;
two microdrives were implanted per animal.
Similar to procedures used previously, guide cannulas were cut from 25
ga stainless steel tubing at a length of 1.2 cm (Mizumori et al., 1989, 1990,
1994; Cooper and Mizumori, 1999). Removable stylets were constructed
from 33 ga stainless steel tubing and were placed inside of the guide
cannulas to prevent occlusion of the tubing. By the use of a stereotaxic
drill assembly, holes were drilled in 0.15 mm nylon sheeting, and guide
cannulas were glued in the holes 2 mm apart from each other. Injection
needles were made of 33 ga stainless steel tubing glued inside of 25 ga
stainless steel tubing. The injection needles protruded ⬃0.5 mm beyond
the guide cannulas. Stylets remained in the guide cannulas after surgery
and between injections.
Animals were food and water deprived for 24 hr before surgery and
then anesthetized with sodium pentobarbital (33 mg/kg). After animals
were deeply anesthetized, they were given 0.2 ml of Atropine to prevent
respiratory distress. Ten burr holes were drilled, and self-tapping anchor
screws were inserted into the holes. Hippocampal electrodes were placed
in the dorsal hippocampus at two recording sites. Recordings from the
right hippocampus were at 1.8–2.2 mm posterior to bregma and 1.8 mm
lateral. In the left hemisphere, electrodes were placed 2.5– 4.0 mm
posterior and 2.0–2.5 mm lateral. Dura was cut, and electrodes were
lowered 1.5 mm ventral to the surface of the brain, just dorsal to the
hippocampus. The right and left hemisphere placements were selected to
maximize the amount of room available around the guide cannulas,
providing easy access for insertion of the injection needles. Retrosplenial
cortex craniotomies were drilled at 6.0 mm posterior to bregma and 1.0
mm lateral to the midline. Dura was cut, and guide cannulas were
implanted 1.0 mm ventral to the surface of the brain. A reference
electrode (114
m Teflon-coated stainless steel wire) was placed near the
corpus callosum, and a ground lead (125
m Teflon-coated stainless steel
wire) was soldered to a stainless steel jeweler’s screw that was fastened to
the skull. Vacuum grease was packed around the electrodes and guide
cannulas to protect the surface of the brain from exposure to dental
cement. The microdrives and guide cannulas were permanently attached
to the head of the rat by application of dental cement. The electrode
wires were connected to an 18-pin plug that was cemented behind the
microdrives and guide cannulas. After surgery, 0.1 ml of Bicillin (300,000
U/ml) was administered intramuscularly in each hindlimb to guard
against infection. Animals were given 1 week of free feeding before the
onset of food restriction and experimental procedures.
Unit recording and behavioral monitoring. The rat was connected to a
head stage for all recording sessions. The head stage contained 13–16
field effect transistors and a pair of infrared-emitting diode arrays. The
location of the animal on the maze was monitored via an automatic
tracking system (Dragon Tracker, Boulder, CO) that sampled position
data at a frequency of 20 Hz. The tracking system identified two diode
arrays simultaneously, distinguished them on the basis of size of the
array, and gave x–ycoordinates in a 256 ⫻256 grid for each array. The
front array was placed directly over the nose of the animal. It was
comprised of five to seven infrared diodes that identified the location of
the animal. A smaller rear diode array (made of one or two diodes) was
placed 12 cm behind the front array. The pair of diode arrays was used
to calculate the heading direction of the animal.
Single-unit activity was recorded simultaneously and independently on
each wire of the stereotrode pair. Each signal was amplified (3,000 –
10,000⫻), filtered at half amplitude between 600 and 6 kHz, and then
passed through a window discriminator such that a 1 msec sampling
period began when either input surpassed a predetermined threshold.
The DataWave “Discovery” data acquisition system recorded each ana-
log trace at a frequency of 20 –32 kHz depending on the number of
simultaneously recorded electrodes. The system software allowed the
experimenter to isolate individual units from the otherwise multiunit
record by comparing the spike characteristics recorded simultaneously
on two closely spaced electrodes (xand y). Scatterplots of waveform
features recorded on xand yelectrodes were displayed. Multiple wave-
form parameters were used to separate individual cells from each other
and from background noise. Particularly useful features included spike
amplitude, spike width (time differences between the peak and subse-
quent trough of an action potential signal), and the latency differences
between the spike peak and valley on the xand the yelectrodes. In
addition, a template-matching algorithm was used off-line to facilitate
spike separation further. For each cell, the experimenter stepped
through a series of two-dimensional cluster plots identifying the combi-
nation of spike characteristics that were most likely associated with a
single-spike generator. After being identified, the specific cluster bound-
aries that characterized each cell were saved for use in subsequent
recording sessions. This provided reasonable certainty that the same cell
was being recorded across multiple test days.
Experiment 1
The present experiment was intended to replicate and extend our pre-
vious behavioral findings of a dark-selective spatial memory impairment
after retrosplenial cortex inactivation (Cooper and Mizumori, 1999). We
sought to extend those results by addressing potential neural mechanisms
underlying dark spatial memory impairments. Therefore, we examined
changes in hippocampal cellular activity during retrosplenial cortex in-
activation in light and dark testing conditions.
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3987
Behavioral and injection methods. After 2 weeks of spatial memory
training (in room 2) and error rates had decreased to an average of less
than one error per trial, animals were considered to be at asymptote
performance. At this point animals were run every third day unless
hippocampal unit activity was encountered. When well isolated units
were located, animals performed the spatial memory task in light and
dark testing conditions with or without the retrosplenial cortex tempo-
rarily inactivated.
Previous work has demonstrated that tetracaine (2%) is active for ⬃20
min, which is the equivalent of approximately five maze trials (Mizumori
et al., 1989, 1990, 1994; Cooper and Mizumori, 1999). Therefore, most of
the behavioral and electrophysiological analyses are based on five control
and five inactivation trials. All injections were accomplished by hand via
a Hamilton syringe. Tetracaine was slowly inf used over the course of 2–3
min, and 1 min was allowed for diffusion.
Figure 1 displays the experimental design used for inactivation during
light and dark testing. For the light inactivation testing, animals first ran
five control spatial memory trials. They were then removed from the
maze and taken into an adjacent room, and 1
l of tetracaine was inf used
bilaterally into the retrosplenial cortex. Animals were then returned to
the maze room and performed trials 6 –10 under normal lighting condi-
tions. Trials 1–5 were considered control trials and were compared with
trials 6–10 (Fig. 1 A). The final set of five trials was used to monitor the
extent to which behavioral and electrophysiological changes returned
toward control levels.
Consistent with our previous work, two versions of the dark injection
procedure were used to control for the time of injection during dark trials
(Cooper and Mizumori, 1999). For the dark I testing procedure, animals
ran five maze trials in the light, were removed from the maze, and were
taken into an adjacent room, and 1
l of tetracaine was infused bilater-
ally into the retrosplenial cortex. One minute was allowed for diffusion,
and animals were returned to the maze in the light. Room lights were
turned off immediately before the onset of testing, and animals per-
formed 10 maze trials in darkness. For the dark I inactivation procedure,
trials 6–10 were dark inactivation trials, and trials 11–15 were control
dark trials (Fig. 1B). In the dark II inactivation procedure, animals ran
five light trials, were removed from the maze, and were taken into an
adjacent maze room; injection cannulas were inserted, but tetracaine was
not infused at this time. Animals were returned to the maze in the light,
room lights were extinguished, and then animals performed trials 6 –10 in
darkness. After trial 10 (during dark trials), room lights were turned on,
and animals were removed from the maze. Tetracaine (1
l) was infused
bilaterally into the retrosplenial cortex in an adjacent room, and animals
were then returned to the maze to perform trials 11–15 in darkness
(inactivation trials). For the dark II procedure, trials 6 –10 were control
dark trials, and trials 11–15 were dark inactivation trials (Fig. 1C).
Regardless of which dark testing procedure was used, animals always ran
five control and five inactivation dark trials. The control trials in dark ness
occurred either after tetracaine had worn off (i.e., Dark I) or before it was
injected (i.e., Dark II). The two types of injection procedures were used
to control for the potential confounds of strategy switching that may
occur with initial, or prolonged, dark testing.
Behavioral and electrophysiological data analyses. The experimenter
recorded the number of errors (i.e., repeat arm entries) and the time
taken to complete each trial during the course of the behavioral testing.
An average number of errors per trial during control and inactivation
phases of testing was computed for each animal during light and dark
testing. Time per choice was calculated by dividing the time to complete
each trial by the number of arms visited within the trial. Changes in error
rates and time per choice were evaluated by computing a two-way repeated
measures ANOVA comparing light and dark trials with
␣
⫽0.05.
Hippocampal cells can be divided into pyramidal complex spike (CS)
and interneuron single-spike (
) cell populations (Fox and Ranck, 1975,
1981). These separate populations are readily identifiable on the basis of
their unique spike characteristics. CS cells have broader spikes (⬎300
sec from peak to valley) than do
cells and fire at a relatively low rate.
In addition, CS cells show a characteristic bursting pattern of three to
four action potentials occurring within 2– 4 msec of each other.
Cell
spikes occur ⬃8 msec apart, with a much higher overall firing rate than
CS cells have, and they have a narrower spike width (⬍300
sec from
peak to valley). These spike characteristics are derived from stored
analog traces of the waveforms, autocorrelations, and interspike interval
histograms of the individual cells (Markus et al., 1994).
All spatial analyses of the electrophysiological data are based on
reducing the 256 ⫻256gridtoa64⫻64 grid, resulting in quadratic
pixels ⬵2.4 ⫻2.4 cm. Spatial specificity was quantified by first calculating
the mean firing rate of the cell as the rat moved toward the center
platform (inbound) or away from the center platform (outbound) on each
of the eight maze arms yielding 16 average rates. The spatial specificity
score was determined by dividing the highest of the 16 rates by the
average of the remaining 15 (McNaughton et al., 1983a; Mizumori et al.,
1989, 1992, 1994). Reliability was the proportion of trials that the cell
fired maximally on the same arm and direction (inbound or outbound)
across trials. In agreement with previous studies, cells with a spatial
specificity score ⱖ2.5 and a reliability of 0.40 were considered to have a
place field. This indicates that a cell discharged at least 2.5 times its
average rate as the rat moved on one arm in one direction for at least two
of five trials. In addition to the spatial specificity score, information
content and sparsity were calculated. Theoretically, information content
is a measure of how well the firing rate of the cell predicts the location
of an animal within an environment (Skaggs et al., 1993). Larger infor-
mation content scores reflect smaller place fields. Information content is
defined as:
information content ⫽⌺P
j
(R
j
/R)log2(R
j
/R).
P
j
is the probability that a rat will occupy bin j,R
j
is the mean firing rate
for bin j, and Ris the mean firing rate across the entire maze. Sparsity is
a measure of the size of the place field and is defined as:
sparsity ⫽共⌺P
j
R
j
)
2
/⌺P
j
R
j
2
.
P
j
is the probability that the rat occupied bin j, and R
j
is the mean firing
rate for bin j.
The animal moving down the first arm identified the beginning of each
trial, and the end was determined when the animal had visited each of the
eight maze arms. The measures of spatial selectivity (spatial specificity,
Figure 1. The injection procedure used for light inactivation and dark I
and dark II inactivation. A dashed ver tical line and Inject denote the time
of injection; Inact indicates the trials used for the inactivation condition.
A, In light inactivation, the first five trials serve as control trials for
comparison with trials 6 –10 that are inactivation trials. The final set of
five trials was used to ensure that behavioral and electrophysiological
changes returned toward control levels. B, In dark I inactivation, the
injection occurred before the onset of dark trials. C, In dark II inactiva-
tion, the injection took place after the first five dark trials.
3988 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
information content, etc.) were calculated for each trial, and then means
were determined for the five control, five inactivation, and five recovery
trials.
If the place field of a cell changes location relative to a baseline
sampling period and remains stable for several trials, the measures of
spatially localized firing described above might not show significant
differences across phases of testing. Accordingly, a spatial correlation was
computed that examined the extent to which firing-rate maps were
correlated within and across control and inactivation trials. Figure 2
displays the trials from the light inactivation (Fig. 2 A), dark I inactivation
(Fig. 2B), and dark II inactivation (Fig. 2C) that were used for the spatial
correlation analysis. The analysis was based on a pixel-by-pixel correla-
tion of average firing rates during the first two trials of the control
condition compared with the average of the last two trials of the control
condition (Fig. 2, “Con to Con”). To examine the effects of retrosplenial
cortex inactivation on the place fields, the spatial correlation was com-
puted comparing the average rates of the first two trials of the control
condition and the first two inactivation trials (Fig. 2, “Con to Inact”). If
the spatial distribution of a place field is similar during control and
inactivation trials, the correlation between con to con and con to inact
should be comparable. In this case, the place field would be considered
stable across the conditions. If the place field changes location, enlarges
in size, or reduces in size or the firing rate of the cell changes, then the
con-to-con correlation would be higher than the con-to-inact correlation.
In this case, the field would be considered unstable across conditions. A
within-subjects ttest was used to determine whether the CS place-field
correlation changed significantly from the control to inactivation
conditions.
Two phases of testing were analyzed with the spatial correlation, a
control phase was compared with another control phase, or a control
phase was compared with an inactivation phase. The spatial correlation
only included bins that were visited in both phases for at least 200 msec.
The joint occupancy requirement ensured that only areas of the maze
that were sampled consistently across phases of testing were used in the
correlation analysis. The use of two trial segments from each testing
phase was chosen for two reasons. First, previous research has shown that
the most reliable and pronounced electrophysiological effects of inacti-
vation in freely behaving animals occur during the first two trials after
injection (Mizumori et al., 1989, 1994). Last, the two trial segments
provided the largest number of trials that allowed each phase (control
and inactivation) to be divided into an equal number of trials for
assessing place-field stability within and across phases of testing. The
spatial correlation used in the present study is similar to analyses used in
other reports (Knierim et al., 1995, 1998; Barnes et al., 1997). However,
the absolute numbers may vary substantially across experiments depend-
ing on the amount of time sampled for the correlation and the size of the
pixels (binning resolution). Therefore, relative changes in the spatial
correlation are appropriate for comparison with other studies.
Experiment 2
Behavior and injection procedure. Six animals were first trained to retrieve
chocolate milk from the end of randomly presented maze arms in room
1 using a forced-choice nonspatial task (see Behavioral method in Ma-
terials and Methods). During this initial training, animals were checked
daily for hippocampal single-unit activity. After multiple complex spike
cells were identified and animals were running consistently on the maze
in room 1, rats were randomly assigned to either tetracaine (n⫽3) or
control (n⫽3) groups. The tetracaine group received daily injections (1
l/hemisphere) before the onset of spatial memory training in room 2.
Similarly, the control group received vehicle control injections (1
l/
hemisphere) before the onset of spatial memory training in room 2. After
the injection of tetracaine or the vehicle control, 1 min was allowed for
diffusion, and animals were immediately carried into the testing room.
Spatial memory trials commenced as quickly as possible after the injec-
tion. Animals performed five trials or ran maze trials for a maximum of
20 min, whichever occurred first. Training continued for 5 consecutive
days, while hippocampal unit activity was recorded. Efforts were made to
identify carefully the same cells across days during the acquisition, but
not all cells could be recorded for 5 continuous days.
Behavioral and electrophysiological data analyses. Behavioral data were
analyzed by computing the mean number of errors committed on each
trial during each test day. A mixed-design ANOVA comparing average
errors between the tetracaine and saline groups across days (
␣
⫽0.05)
was used to analyze the acquisition data. Electrophysiological measures
were also analyzed using a mixed-design ANOVA. The analysis requires
that each “subject” contributes a score for each day. Some cells were
recorded across days, whereas new cells were also encountered on dif-
ferent days. Thus, a single cell could not always contribute a score for
each day in the repeated measures component of the ANOVA. To
resolve this issue, a mean score for each animal based on the cells
recorded from that day was used in the analysis. Mean spatial specificity,
reliability, information content, sparsity, mean rate, and the spatial
correlation measure were calculated for each day during acquisition. Two
criteria were necessary to calculate the spatial correlation score. First,
each animal had to run at least four trials on each day of acquisition of
the spatial memory task. In this case, the first two trials were correlated
with the last two trials. When animals ran five trials during acquisition
training, trials 1 and 2 were compared with trials 4 and 5; the majority of
the analyses are based on this comparison. Second, at least one CS cell
had to be recorded from each animal during each day of the spatial
memory task. These criteria were met on days 2– 4 of spatial memory
acquisition. Therefore, the statistical analyses for all measures of spatial
coding by hippocampal neurons were restricted to these days. However,
the behavioral analysis compares all5dofacquisition.
Figure 2. The trials used for the spatial correlation analysis during
control and inactivation trials are displayed in the figure. The last five
trials during light inactivation and the first five light trials for dark I and
dark II inactivation are omitted from the spatial analysis; accordingly they
are not included in the figure. A, For light inactivation, the firing rates on
visited pixels during the first t wo trials of the control trials were correlated
with the rates on visited pixels during the last two trials of the control
condition (Con to Con). This provided the control stability assessment of
place fields. To assess the effects of inactivation, the first two trials of the
control trials were correlated with the first two inactivation trials (Con to
Inact). B, The first two dark trials after inactivation were compared with
the last two dark trials performed (Con to Inact) for the spatial correla-
tion. Trials 11 and 12 were compared with trials 14 and 15 for the Con to
Con spatial correlation. C, For dark II inactivation, the first two dark trials
(6, 7) were compared with trials 9 and 10 for the Con to Con spatial
correlation. The Con to Inact spatial correlation was derived from compar-
ing trials 6 and 7 with the first two trials after inactivation (trials 11, 12).
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3989
RESULTS
Experiment 1
Histology
Figure 3Adisplays the locations of the tips of the cannulas in the
retrosplenial cortex from six animals (Paxinos and Watson, 1986).
Three other animals were excluded from the behavioral and most
of the electrophysiological analyses because of the placements of
the cannulas. In one animal the tip of the cannula was placed in
the superior colliculus. This animal did not contribute control
injection data and was excluded from all behavioral and electro-
physiological analyses. In two other animals, only one of the two
cannulas penetrated the cortex. The animals with unilateral guide
cannulas were excluded from behavioral and electrophysiological
analyses of retrosplenial cortex inactivation, but their data were
kept for all control injections. In several cases, permanent ink was
injected before killing the rat. In these animals, ink spread into
the retrosplenial granular and agranular areas, cingulum bundle,
and medial areas of posterior parietal cortex (Oc2MM), consis-
tent with our previous work (Cooper and Mizumori, 1999). The
ink did not spread into any subregions of the hippocampus.
The recording sites in the hippocampus from the six animals
are displayed in Figure 3B. Each filled circle corresponds to
approximate recording sites of two to eight cells. Electrode tracks
that passed through similar areas across animals were grouped
together for the graphical display of recording sites. Most of the
recordings were from the left hemisphere in hilar/CA3 and to a
lesser extent CA1. The recording sites were evenly distributed
across animals, with the exception that one animal contributed
the cells recorded in the right hemisphere. A total of 10 CS cells
and 1
cell were recorded in the right hemisphere. In the left
hemisphere, 12 CS cells and 3
cells were recorded in CA1, and
36 CS cells and 7
cells were recorded in CA3 during inactivation
of retrosplenial cortex. The larger number of cells in CA3 was an
inadvertent consequence of the fact that some of the animals took
part in experiment 1 after completing experiment 2 (described
below). Therefore, some of the recording electrodes passed
through CA1 before commencing the current experiment. Retro-
splenial cortex inactivation did not result in obvious differences in
electrophysiological changes between hippocampal subregions or
across animals. Therefore, the data were combined for the elec-
trophysiological analyses.
Behavioral data
Experiment 1 replicated our previous behavioral data (Cooper
and Mizumori, 1999). A two-way repeated measures ANOVA
demonstrated that inactivation of retrosplenial cortex resulted in
a significant increase in errors when retrosplenial cortex was
inactivated and animals were tested during dark spatial memory
performance. There were significant main effects of injection
condition [F
(1,8)
⫽14.74; p⬍0.01] and lighting condition [F
(1,1)
⫽12.04; p⬍0.02]. The dark-specific changes in behavioral
performance caused by inactivation of retrosplenial cortex are
illustrated by the significant interaction between lighting condi-
tion and trials during the injection condition [F
(1,4)
⫽4.30; p⬍
0.05]. Figure 4Adisplays the absence of a change in error rates
when retrosplenial cortex was inactivated during light testing.
Figure 4Cshows the significant increase in error rates during dark
testing when retrosplenial cortex was inactivated. The dark-
specific behavioral deficit did not depend on the time of injection
during dark trials (data not shown).
Figure 4, Band D, displays the average time per choice across
trials in light and dark testing conditions. A two-way repeated
measures ANOVA did not show main effects for lighting condi-
tion [F
(1,8)
⫽0.16; NS] or injection condition [F
(1,1)
⫽0.75; NS],
which demonstrates that maze run times did not change with
either dark testing or inactivation of retrosplenial cortex. One
animal did not undergo inactivation while recording hippocampal
cells but contributed to control injection data; therefore the
statistical analyses are based on five of the six animals with
bilateral guide cannulas placed in the retrosplenial cortex.
Figure 3. Location of the tips and recording sites of the guide cannulas.
A, Each filled circle corresponds to a single guide cannula. Previous work
and ink injections have shown that the spread of tetracaine is just slightly
more thana1mmcircumference around the injection site. Therefore,
retrosplenial granular and agranular areas, cingulum bundle, and Oc2MM
of posterior parietal cortex were likely affected by injections of tetracaine.
B, Each filled circle corresponds to two to eight cells recorded in that
location. For electrode tracks that passed through the same area in
different animals, a single filled circle is used to signify the recording site
of multiple cells. The majority of cells (n⫽43) were recorded from CA3
in the left hemisphere; a smaller number of cells (n⫽15) were also
recorded in CA1.
3990 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
Inactivation effects on
cells in light and dark
Seven
cells were recorded during inactivation of retrosplenial
cortex in light testing conditions, and there were no significant
changes in reliability, information content, or selectivity (Table
1). The average firing rate of
cells increased from 13.70 (⫾1.98)
Hz during control light trials to 15.02 (⫾2.17) Hz during light
inactivation trials [t(6) ⫽⫺3.04; p⬍0.05; Table 1]. Eight
cells
were tested with inactivation of retrosplenial cortex in darkness.
There were no significant differences between injections in dark I
or dark II; therefore the data were combined for the statistical
analyses. There were no significant changes in any of the mea-
sures of spatial coding or mean rate when retrosplenial cortex was
inactivated during dark trials (Table 1).
Inactivation effects on CS cells in light
Thirty-six CS cells were recorded from five animals during ret-
rosplenial cortex inactivation in light testing conditions. Table 2
displays the absence of significant changes in reliability, informa-
tion content, sparsity, and mean rate between control and inac-
tivation trials of spatial memory performance. Spatial specificity
showed a marginally significant decrease during inactivation
[t(35) ⫽2.04; p⫽0.05]. Spatial specificity, information content,
and sparsity are all measures of place-field size, but only spatial
specificity is sensitive to firing on single versus multiple arms
across trials. Therefore, the significant decrease in spatial speci-
ficity may reflect place fields firing on multiple arms after inacti-
vation of retrosplenial cortex (see further discussion of this issue
below).
Twenty-four of the 36 C S cells had place fields in either the
control light or inactivation light trials (see description of place-
field criteria in Materials and Methods of Experiment 1). For
these cells, we examined changes in place coding during control
and inactivation trials using the spatial correlation measure (see
Fig. 2 and Behavioral and electrophysiological data analyses in
Materials and Methods of Experiment 1). Despite the sustained
behavioral accuracy during light inactivation, Figure 5Adisplays
the significant decrease in mean spatial correlation from con-to-
con to con-to-inact testing conditions. The mean con-to-con cor-
relation was 0.20 (⫾0.04), and it dropped to 0.10 (⫾0.02) in the
con-to-inact correlation [t(23) ⫽2.61; p⬍0.02]. To relate the
change in spatial correlation of place cells to the behavior during
inactivation, a Pearson correlation was computed between the
con-to-inact spatial correlation and average errors per trial during
the inactivation phase of testing. Figure 5Bdisplays the nonsig-
nificant correlation between the spatial correlation scores and
performance on the maze during light testing [r(24) ⫽0.28; NS].
To establish whether the place fields began to return to their
original location during the inactivation condition, the spatial
correlation that was computed between trials 1 and 2 of the
control condition were compared with that of trials 9 and 10 of the
inactivation condition. The spatial correlation was 0.15 (⫾0.04),
which was not significantly different from the control spatial
correlation score [t(23) ⫽0.44; NS]. In the light trials, animals
continued to run maze trials (trials 11–15); therefore we com-
puted an additional spatial correlation between the first two
control trials and the last two trials of the testing condition. The
spatial correlation between these trials was 0.15 (⫾0.04), which is
comparable with the spatial correlation comparing the control
light trials with the end of the inactivation trials. Thus, the
recovery that occurs during the inactivation trials does not sub-
stantially change with additional maze trials. This suggests that
the most pronounced changes in spatial coding after inactivation
occur during the initial trials and that by the end of the inactiva-
tion condition the effects are less prevalent and do not change
substantially with repeated maze trials. This pattern of data is
consistent with previous work using injections of tetracaine and
this testing procedure (Mizumori et al., 1989, 1994).
To examine further when the place fields are stable and unsta-
ble during inactivation of retrosplenial cortex, we computed the
spatial correlation comparing individual trials with each other
before and after the injection. For establishing the stability of
control light trials, the spatial correlation between trials 1 and 2 of
the control trials was calculated. To examine stability of spatial
coding during and after inactivation of retrosplenial cortex, we
computed the spatial correlation between the first two inactiva-
tion trials (trials 6 and 7) and the last two trials of the inactivation
condition (trials 9 and 10). Between the first two baseline trials
the spatial correlation was 0.29 (⫾0.06), during the first two
inactivation trials the correlation was 0.13 (⫾0.04), and during
the last two trials of the inactivation condition the correlation was
0.19 (⫾0.05). A repeated measures ANOVA revealed that there
was a significant change in the spatial correlation values observed
across trials [F
(1,2)
⫽4.09; p⬍0.02]. A Newman–Keuls post hoc
analysis demonstrated that only the first two trials during inacti-
vation were significantly different from the control trials or the
last pair of inactivation trials ( p⬍0.05). These data suggest that
inactivation of retrosplenial cortex caused initial instability of the
location-coding properties of place fields, but they tend to regain
spatially coherent firing after repeated maze trials.
The spatial correlation examined two inactivation trials com-
pared with two control trials. However, the spatial firing measures
presented in Table 2 were based on analyses between five maze
trials. To ensure that the data presented in Table 2 have sufficient
Figure 4. Temporary inactivation of retrosplenial cortex only impairs
dark spatial memory performance. A, C, The average number of errors
(⫾SEM) for control and inactivation trials is displayed. Inactivation of
retrosplenial cortex did not change performance during light testing ( A)
but caused a significant impairment when animals were tested in darkness
with retrosplenial cortex inactivated (C). B, D, The average time per
choice on the maze was not affected by retrosplenial cortex inactivation
during light ( B) or dark (D) inactivation. Because the data obtained from
dark I and dark II were similar, they were combined for the present
analysis.
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3991
resolution to detect changes in location coding during retrosple-
nial cortex inactivation, we performed the identical analysis com-
paring the first two control trials with the first two inactivation
trials. There were no significant changes in spatial specificity,
information content, sparsity, or mean rate (data not shown). It
should be noted, however, that the two trial analyses might not
provide sufficient resolution to detect changes in place fields.
The light inactivation effects could be caused by nonspecific
factors, such as maze removal or stress from the injections. To
confirm that this was not the case, 14 cells with place fields were
tested with vehicle control injections. In these cases, the average
correlation did not change as a function of injection. During the
con-to-con phases of testing the spatial correlation was 0.18 (⫾
0.02), and it was 0.22 (⫾0.04) during con-to-vehicle-control
phases [t(13) ⫽⫺0.35; NS]. This provides additional evidence
that the change in the spatial correlation is likely caused by
temporary inactivation of retrosplenial cortex and not caused by
nonspecific effects of the injection procedure.
These data suggest that inactivation of retrosplenial cortex
changes the preferred firing location of the place cells. Figure 6
displays two simultaneously recorded place cells during couplets
of control trials, inactivation trials, and recovery trials (next day).
The cell in Figure 6 Ashowed a preference for the west end of the
maze arm during the control trials 1 and 2 and trials 4 and 5.
These trials were used for the con-to-con spatial correlation.
During inactivation trials 6 and 7, the original location of the
place field changed to firing on the west and north arms of the
maze. These trials were compared with the control trials 1 and 2
for the con-to-inact spatial correlation. The original location of
the place field did not return during trials 9 and 10; however, the
correlate did return on the following test day. In Figure 6 B,a
simultaneously recorded cell shows a response similar to that of
the cell displayed in Figure 6 A. The graphical display of the fields
may appear somewhat “noisy” because the data displayed repre-
sent only two trials. During this time, the animal only passed
through each maze arm a total of four times. This compares with
displays in other reports that represent 20 –30 min of sampling,
and more visits, to particular locations (Mizumori et al., 1990,
1994).
Inactivation effects on CS cells in dark
Thirty-two CS cells from four animals were recorded during
retrosplenial cortex inactivation in darkness. Eighteen C S cells
were recorded during dark I inactivation, and fourteen CS cells
were recorded in dark II inactivation. There were no significant
differences in errors between the dark testing conditions, so the
data were combined for the electrophysiological analyses. Similar
to the light testing, spatial specificity, reliability, information
content, sparsity, and mean rate did not change significantly
between control and inactivation trials (Table 2). However, the
preferred firing location of place cells did not remain stable
during inactivation in darkness.
Of the 32 CS cells tested during inactivation in darkness, 20
showed a place field in either the control or inactivation dark
trials. For these 20 cells, we computed the spatial correlation
between the con-to-con dark trials and the con-to-inact dark trials
(Fig. 5C). The mean spatial correlation was 0.27 (⫾0.06) in the
con-to-con condition and dropped significantly to 0.03 (⫾0.02) in
the con-to-inact phase of testing [t(19) ⫽3.387; p⬍0.01]. As with
the light trials, we also computed a Pearson correlation between
Table 1.
Cell responses to inactivation during light and dark testing
Cells (n⫽11
a
)
Light (n⫽7) Dark (n⫽8)
Control Inactivation Control Inactivation
Spatial Spec. 1.65 ⫾0.10 1.77 ⫾0.18 1.34 ⫾0.04 2.68 ⫾0.93
Rel 0.31 ⫾0.07 0.36 ⫾0.06 0.41 ⫾0.07 0.50 ⫾0.14
Info Cont. 0.67 ⫾0.13 0.61 ⫾0.14 0.68 ⫾0.18 0.70 ⫾0.21
Spars. 0.53 ⫾0.06 0.55 ⫾0.06 0.54 ⫾0.06 0.56 ⫾0.07
Mean Rate 13.70* ⫾1.98 15.02 ⫾2.17 15.81 ⫾4.50 16.46 ⫾4.69
The average spatial specificity, reliability, information content, sparsity, and mean rate for the
cells recorded during light
and dark inactivation are shown.
a
The total number of cells does not match the sum of the numbers in the light and dark conditions because four
cells were
recorded in both conditions.
*p⬍0.05.
Info Cont., Information content; Rel., reliability; Spars., sparsity; Spatial Spec., spatial specificity.
Table 2. CS cell responses to inactivation during light and dark testing
CS cells (n⫽58
a
)
Light (n⫽36) Dark (n⫽32)
Control Inactivation Control Inactivation
Spatial Spec. 7.32 ⫾1.12 5.23* ⫾0.86 6.74 ⫾1.50 6.53 ⫾1.92
Rel 0.47 ⫾0.04 0.42 ⫾0.04 0.36 ⫾0.04 0.38 ⫾0.05
Info Cont. 3.85 ⫾0.40 3.63 ⫾0.39 4.61 ⫾0.35 4.68 ⫾0.34
Spars. 0.05 ⫾0.02 0.06 ⫾0.01 0.06 ⫾0.01 0.05 ⫾0.01
Mean Rate 0.71 ⫾0.14 0.72 ⫾0.15 0.65 ⫾0.15 0.64 ⫾0.15
The average spatial specificity, reliabilit y, information content, sparsity, and mean rate for the CS cells recorded during light
and dark inactivation are shown.
a
The total number of cells does not match the sum of the totals from the light and dark conditions because 10 C S cells were
recorded in both conditions.
*p⬍0.05.
3992 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
the con-to-inact spatial correlation and errors committed during
inactivation. Figure 5Ddisplays a negative relationship between
errors and the spatial correlation [r(20) ⫽⫺0.46; p⬍0.05]. The
negative correlation suggests that animals made more errors
when there was a larger change in hippocampal spatial coding
(i.e., a lower spatial correlation). We analyzed the C S cells (n⫽
5) recorded in the first dark testing session of each animal. These
cells showed a pattern of data in the spatial correlation measure
comparable with that shown by the remaining cells in subsequent
dark trials.
As we did in the light trials, we compared the first two control
trials with the first two inactivation trials for the measures of
spatial firing by hippocampal place cells. Similar to the data
collapsing across five trials, there were no significant changes
between control and inactivation phases of testing for spatial
specificity, information content, sparsity, or mean rate (data not
shown). The absence of significant changes in place-field mea-
sures, but a significant change in the spatial correlation, suggests
that the place fields changed their preferred firing location after
inactivation of retrosplenial cortex.
As with the light trials, the duration of place-field changes
during dark trials was evaluated by computing the spatial corre-
lation between individual trials. The last two dark trials (dark I
inactivation) or the first two dark trials (dark II inactivation) were
used to establish control stability in darkness. The control stabil-
ity was compared with the spatial correlation obtained from the
first two trials after inactivation and with the spatial correlation
from the last two trials of the dark inactivation condition. This
trial-by-trial correlation provides information about the stability
of the place fields between individual trials of the treatment
conditions. The correlation for the baseline dark trials was 0.52
(⫾0.82), during the first two trials after inactivation the corre-
lation was 0.09 (⫾0.05), and during the last two trials of the dark
inactivation condition the correlation was 0.33 (⫾0.09). A re-
peated measures ANOVA revealed that there was a significant
change in the spatial correlation values across trials [F
(1,2)
⫽6.22;
p⬍0.05]. A Newman–Keuls post hoc analysis showed that the
spatial correlation during the first two inactivation trials was
significantly different from that of both the control dark trials and
the last trials of the inactivation condition ( p⬍0.05). These data
suggest that, similar to light trials, there is a significant decrease
in intertrial stability in spatial coding by place cells immediately
after inactivation but that this instability is eventually replaced
with a period of stability after several maze trials.
Two different cells that showed dark field reorganization are
displayed in Figure 7, Aand B(dark I and dark II injection
procedures, respectively). In Figure 7A, the two light trials are
shown for a cell with a field localized to the end of the north maze
arm. The location of the preferred field was disrupted during the
pairs of dark inactivation trials, trials 6 and 7 and trials 9 and 10.
The preferred location of the field did not return to the original
location until the last pair of dark control trials (trials 14 and 15).
Another cell recorded during the dark II inactivation procedure
is displayed in Figure 7B. This place cell showed a preferred
firing location on the northern edge of the center platform during
light trials and was generally maintained during the pairs of dark
trials used for the con-to-con spatial correlation analysis. In
contrast to the center platform field location during control light
and dark trials, after inactivation the field shifted to the west and
southwest maze arms. The field slowly, although incompletely,
began to return to the center platform during the final pair of
dark inactivation trials. In all of the cases with dark testing on the
subsequent day, the place field returned to the original control
location.
Comparison between light and dark
A total of 10 CS cells were tested in both light and darkness. Of
these 10 cells, five CS cells showed place fields in both lighting
conditions. To determine whether inactivation caused a greater
change in place fields in darkness than in light, we compared the
data from these five cells. The average control light spatial cor-
relation was 0.22 (⫾0.11), and the control dark spatial correlation
was 0.23 (⫾0.17). A within-subjects ttest showed that these
values were not significantly different [t(4) ⫽⫺0.05; NS]. The
average light inactivation spatial correlation was 0.10 (⫾0.05),
and the average dark inactivation spatial correlation was ⫺0.01
(⫾0.005). A within-subjects ttest showed that this difference
between light and dark approached statistical significance [t(4) ⫽
2.65; p⫽0.06].
An example of a cell with a place field that showed a similar
pattern of reorganization in light and dark, but greater reorgani-
zation in darkness, is displayed in Figure 8. This cell was re-
corded across several days and maintained a primary field in the
light and dark on the southeast maze arm. Figure 8Adisplays the
effects of inactivation during light testing. The top spatial plot
shows five control light trials, and the spatial plots below are the
individual trials after inactivation of retrosplenial cortex (with the
Figure 5. Temporary inactivation of retrosplenial cortex decreased after
inactivation of retrosplenial cortex in light and dark but was only related
to behavior during dark testing. A, The spatial correlation during con to
con (Control ) is significantly higher than that during con to inact (Inact;
p⬍0.01). This suggests that hippocampal place cells changed their
preferred firing location during light inactivation. B, In light inactivation,
there was no relationship between errors and the spatial correlation,
suggesting that changes in place coding by hippocampal cells do not
predict behavioral performance in the light. C, The spatial correlation in
dark inactivation decreased significantly when retrosplenial cortex was
inactivated ( p⬍0.01). D, There was a significant correlation between
errors in darkness and changes in place coding by hippocampal place
cells. *p⬍0.05; **p⬍0.01. Corr, Correlation.
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3993
number next to the plot indicating the trial number after inacti-
vation). During the first trial after light inactivation, the field
shifted to the north arm of the maze. This location is maintained
until the fourth light trial. During the fourth light trial, the field
returned to the southeast arm of the maze and maintained that
location for the subsequent trials (only one more trial is displayed
in the figure).
In Figure 8B, the five light trials preceding the injection of
tetracaine are displayed in the top spatial plot. As with the
previous day, the field was located primarily on the southeast
maze arm. During dark I inactivation, the field changed locations
across the first two dark trials. On dark trial three, the place field
began to stabilize on the north maze arm and maintained that
preference for the majority of the trials in darkness (data not
shown). This is consistent with the location of the field during
light inactivation (shown in Fig. 8 A). The original location of the
field did not return to the southeast maze arm until the last trial
in darkness (trial 10). For this cell, when visual information was
available during retrosplenial cortex inactivation, the reorganiza-
tion of spatial coding by hippocampal neurons was less persistent
compared with testing without visual information.
Stability across days
The five cells that contributed to the light–dark comparison were
examined for stability across days. Baseline spatial stability was
assessed by calculating the spatial correlation for trials 1 and 2
Figure 6. Responses of two simulta-
neously recorded place cells during light
inactivation. Pairs of trials are shown to
illustrate the spatially selective activity
that occurred during the con-to-con and
con-to-inact spatial correlation. A,In
this case the cell showed a preferred
field on the western maze arm during
trials 1 and 2 and during trials 4 and 5
(con-to-con trials). During retrosplenial
cortex inactivation, the location of the
field changed to firing on two arms and
then began to fire on the northwestern
maze arm in the subsequent trials. The
preferred location for the cell did not
return until the subsequent test day. B,
This cell showed a similar consistent
field during control light trials; during
inactivation the field changed locations
and then began to fire on the southeast-
ern maze arm. Interestingly, the simul-
taneously recorded cells both rotated
their preferred fields during inactiva-
tion but by different amounts. In both
cases, the preferred field did not return
until the next test day. All of the spatial plots omit cellular activity that is ⬍20% of the maximum rate of the cell during the trials. The maximum rate
is shown as dark areas, and shaded areas correspond to intermediate rates. This form of presentation is the same for Figures 7, 8, and 10. It should be
noted that the small sample size for the spatial plots reduces the variability in the firing of the place cell (the animal only passes through the place field
a total of four times, and if the field is directional the cell only has two opportunities to be active for a given plot). Accordingly, the firing-rate distribution
is reduced substantially because of the small sample size.
Figure 7. Responses of two different
place cells recorded in dark I and dark
II inactivation conditions. A, This cell
showed a preferred field on the north
maze arm during the initial light trials,
and the preferred location changed
across trials during the inactivation con-
dition. The field did not return to the
original location until the last pair of
control dark trials. B, The preferred
field of this cell was on the northern
portion of the center platform during
light trials and control dark trials. Dur-
ing inactivation, the field shifted to fir-
ing on two maze arms and began to
return during the last inactivation trials.
3994 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
and trials 4 and 5 for day 1. To determine whether the spatial
firing changed across days, the spatial correlation was computed
between trials 1 and 2 of day 1 and trials 1 and 2 of day 2. The
baseline correlation for day 1 was 0.35 (⫾0.12), and the across-
days correlation value was 0.36 (⫾0.20) [t(4) ⫽⫺0.09; NS]. Thus,
the spatial-coding properties of place cells in hippocampal neu-
rons are stable across days, suggesting that the changes in spatial
coding after inactivation of retrosplenial cortex are transient.
Partial versus complete reorganization
It is presently unclear under what environmental conditions place
fields develop entirely new location-selective codes (i.e., complete
reorganization) or maintain similar, but not identical, spatial
firing patterns (i.e., partial reorganization). To address this issue
within the context of the contribution of retrosplenial cortex, we
compared the spatial correlation measures in light and dark
during inactivation for multiple simultaneously recorded place
cells. We set an a priori criteria for “reorganization” as a spatial
correlation score for a given cell that was less than the mean for
all of the cells minus one SEM [e.g., reorganization ⫽spatial
correlation for a cell ⬍(mean of all cells ⫺SEM)].
During light testing with inactivation of retrosplenial cortex,
there were five data sets with multiple simultaneously recorded
place cells (four to six cells per data set). In four of the five data
sets, at least one cell did not show spatial reorganization, despite
the fact that the remaining simultaneously recorded cells showed
reorganization. Thus, not all cells responded in the same way
during inactivation in light testing, which suggests that there was
partial reorganization of spatial coding during light inactivation
trials (for individual data during inactivation in light, see Fig. 5B).
During dark inactivation, there were four data sets with multiple
place cells recorded during inactivation of retrosplenial cortex
(three to four cells per data set). In three of the four data sets, all
of the cells recorded showed reorganization. However, in one
data set, two of the four cells simultaneously recorded showed
reorganization, and the remaining two did not. Interestingly, this
one case occurred when inactivation of retrosplenial cortex did
not result in behavioral impairments in darkness (see Fig. 5Dfor
individual cell responses during inactivation in dark). In sum, the
data are consistent with the interpretation that there is partial,
and not complete, reorganization during inactivation of retrosple-
nial cortex in light and dark testing.
Experiment 2
Histology
Six animals were used in this experiment. One animal assigned to
the control injection group received a unilateral guide cannula
placement. The data from this animal were virtually identical to
those of the other control animals and were included in the
control group. The injection sites for the remaining five animals
were in the same areas displayed in Figure 3A(four of the six
animals were included in both experiments). Recording sites were
in CA1 and CA3 of the hippocampus in regions similar to those
displayed in Figure 3B. Approximately half of the cells in the
tetracaine and control groups were in CA3 and CA1. Because of
the small sample size, the data were combined for the analyses.
Figure 8. Recovery after inactivation requires more time in darkness
than in light. The top spatial plots in Aand Bdisplay five light trials
preceding tetracaine injection. Individual trials after inactivation of ret-
rosplenial cortex are displayed below the injection line; the number to the
left of the spatial plot corresponds to the trial number after injection. A,
After inactivation of retrosplenial cortex, the field shifted from the south-
east maze arm to firing on the north arm of the maze and maintained that
firing pattern until the fourth light trial. The field then maintained this
location for the majority of the remaining light trials (only one more trial
is displayed in the figure). B, For the dark I inactivation procedure, the
cell did not show a consistent preferred firing location until the third dark
trial after inactivation. For this trial and the majority of the remaining
trials, the cell continued to fire on the north arm of the maze. This is the
same location found to be the preferred firing pattern during light inac-
tivation trials. The original preferred location for this cell did not return
4
until the last dark trial (10 trials later). Thus, without visual information
to update the place-coding system, the cellular correlate requires more
trials to return to the original location compared with when visual infor-
mation is available.
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3995
Behavioral data
Two of the animals assigned to the control group ran two and
three trials, respectively, during the first day of spatial memory
training. During the next 4 d, all of the control animals ran five
spatial memory trials. All of the animals receiving tetracaine
injections ran at least four trials on the first day of acquisition and
five trials on the remaining 4 d. The mean number of errors for
each day is based on the number of trials that each animal
performed during each test day. Figure 9Adisplays the significant
behavioral impairment during acquisition of the spatial memory
task in light testing conditions. A mixed-design repeated mea-
sures ANOVA revealed a significant main effect of treatment
group [F
(1,4)
⫽34.42; p⬍0.02], a significant within effect of
training day [F
(1,4)
⫽5.30; p⬍0.02], but no significant interaction
between group and training [F
(1,4)
⫽2.40; p⬎0.05]. A Newman–
Keuls post hoc analysis of the within effect showed that days 1 and
2 were significantly different from days 4 and 5 ( p⬍0.05).
Therefore, tetracaine injections into retrosplenial cortex caused
an initial performance deficit during spatial memory acquisition,
but animals in both treatment groups were able eventually to
acquire the task.
Electrophysiological data
In the inactivation group (n⫽3), nine CS cells were recorded on
day 2 of acquisition, seven were recorded on day 3, and six were
recorded on day 4. In the control group (n⫽3), 13 cells were
recorded on day 2 of acquisition, 12 were recorded on day 3, and
10 were recorded on day 4. In agreement with the data obtained
from experiment 1, inactivation of retrosplenial cortex did not
result in significant changes between groups in spatial specificity,
reliability, information content, sparsity, or mean rate during the
3 d of acquisition (see Table 3). In contrast to the absence of
changes in these measures, Figure 9Bdisplays the significant
decrease across 3 d of acquisition for the spatial correlation of C S
cells in the inactivation group compared with the control group.
A mixed-design ANOVA revealed a significant main effect of
treatment group [F
(1,4)
⫽21.37; p⫽0.01] and interaction of
group by training [F
(1,4)
⫽5.14; p⬍0.05] but not a significant
within effect of training [F
(1,4)
⫽0.84; NS].
To relate the spatial correlation scores with behavioral perfor-
mance for each animal, we computed a Pearson correlation be-
tween the average spatial correlation score for each day and the
average number of errors committed on each test day. Thus, there
were nine correlation scores for the control and inactivation
groups. A negative correlation between errors and the spatial
correlation score would be indicative of improved performance
with increasing stability of hippocampal representations of space.
Figure 9Cdisplays the absence of a significant relationship be-
tween errors and the spatial correlation [r(9) ⫽⫺0.22; NS]. For
the inactivation group, there was a significant correlation between
the spatial correlation and errors [r(9) ⫽0.85; p⬍0.01] (Fig. 9D).
The positive correlation suggests that field stability in the inacti-
vation group is indicative of poorer performance.
In the tetracaine group, only seven C S cells were recorded
across 2 d, and of those three were recorded for 3 consecutive
days. Five CS cells in the control group were recorded for 3
continuous days. An interesting pattern was observed in cells
recorded from animals in the inactivation group; all of the cells
showed different preferred firing locations across days (as mea-
sured by the maximal rate on one arm of the maze across the five
trials). In contrast, only two of the place fields recorded from
control animals changed their preferred spatial firing across days.
Figure 10Adisplays a cell from the inactivation group that
showed a different preferred location across days. Figure 10 B
displays a cell from the control group across days 2–4 of acqui-
sition that remained in the same location across days.
DISCUSSION
Experiment 1
The present results confirmed our previous findings that inacti-
vation of retrosplenial cortex impairs spatial memory perfor-
mance in darkness. Retrosplenial cortex inactivation also caused
hippocampal place cells to change their spatial firing patterns in
light and dark testing conditions. The change in spatial coding
occurred in the presence, and absence, of behavioral impairments
(this issue will be explored further in General Discussion).
Nonspecific effects of the injection are not likely explanations
for the behavioral and electrophysiological changes. First, vehicle
control injections did not change hippocampal spatial coding in
the light. In addition, instability in single-cell recording is not a
potential cause of the observed changes in spatial coding, because
the original place field of the cell returned during control trials or
the subsequent day. Spread of tetracaine into the hippocampus is
Figure 9. Inactivation of retrosplenial cortex impairs spatial learning and
place-field stability. A, Spatial memory acquisition is impaired when
tetracaine is infused into the retrosplenial cortex immediately before
testing. The mean number of errors is significantly ( p⬍0.01) higher in
the tetracaine group (Inact) compared with the control group (Con).
There is a significant improvement across test days in both groups ( p⬍
0.05). B, The spatial correlation also shows a significant difference be-
tween tetracaine and control groups ( p⬍0.05). Only test days 2– 4 are
displayed because those are the only days during which animals ran five
trials and multiple hippocampal CS cells were recorded from the animals.
Although there is a significant difference between groups in the spatial
correlation, there was not a significant within-group effect of training.
These data suggest that although behavior improves across trials, the
spatial correlation does not. C, The spatial correlation did not relate to
behavioral performance during acquisition in the animals receiving vehi-
cle control injections [r(9) ⫽⫺0.22; NS]. D, In contrast to control animals,
tetracaine injections into retrosplenial cortex resulted in a highly signifi-
cant correlation between errors and place-field stability [r(9) ⫽0.85; p⬍
0.01]. This suggests that when place fields remain in the same location
across trials after inactivation of retrosplenial cortex there is a greater
likelihood for behavioral impairments (**p⬍0.01).
3996 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
not a likely cause of the observed effects because the mean rate of
the cells did not change significantly and dye was not observed in
the hippocampus after ink injections. Motor disturbances are an
unlikely explanation for the current results because maze run
time did not change as a function of injections and retrosplenial
cortex inactivation had no effect on performance during light
trials.
A disruption in processing local maze cues is not a probable
source of the dark performance deficit. Permanent removal of
retrosplenial cortex results in behavioral impairments on the
water maze, a task that substantially reduces the prevalence of
local maze cues (Sutherland et al., 1988; Sutherland and Hoesing,
1993). In addition, head-direction cells are observed in retrosple-
nial cortex, and they are controlled by visual and movement-
related information rather than local cues (Chen et al., 1994b).
The cingulum bundle and Oc2MM were probably affected by
the injections. However, inactivation of the adjacent cingulum
bundle may not be responsible for the behavioral and electrophys-
iological changes, because permanent removal of the cingulum
bundle, but not the retrosplenial cortex, impairs spatial memory
performance on the radial maze in light conditions (Neave et al.,
1997). Posterior parietal cortex integrates information from the
external environment with motor movements (Colby and Gold-
berg, 1999). It has been suggested that movement-based informa-
tion from posterior parietal cortex is transformed to an extended
spatial coordinate reference frame in retrosplenial cortex (Vogt
and Miller, 1983; Chen et al., 1994b). Retrosplenial cortex re-
ceives information from Oc2MM and has anatomical connections
with the hippocampus via the entorhinal cortex and postsubicu-
lum (Vogt and Miller, 1983; Wyss and van Groen, 1992). Accord-
ingly, we suggest that the most parsimonious explanation of the
current data is that retrosplenial cortex provides experience-
dependent spatial information relevant for spatial memory per-
formance in darkness and hippocampal spatial processing.
Behavioral studies suggest that dark performance on the radial
maze may require the integration of mnemonic information,
self-motion cues, and static background cues to solve the task
(Brown and Bing, 1997). Save (1997) has demonstrated that water
maze performance in darkness is enhanced with longer preexpo-
sure to the visual cues. This suggests that memory of the visual
environment improves spatial localization in darkness. We sug-
gest that when there is a strong requirement for spatial memory
to guide processing of self-motion information (as is the case
during dark performance), retrosplenial cortex plays a particu-
larly important role in navigational accuracy. When visual cues
are available, animals may rely on currently viewed spatial fea-
tures of the environment to navigate accurately.
Navigation relying on nonvisual information is thought to be
dependent primarily on processing movement-related cues. This
type of navigation is referred to as path integration and is subject
Table 3. Electrophysiological characteristics of CS cells during acquisition in tetracaine and control
groups
Measure Day 2 Day 3 Day 4
Spatial Spec. Tet 2.55 ( ⫾0.27) 3.27 ( ⫾1.11) 2.51 ( ⫾0.42)
Con 3.89 ( ⫾1.03) 3.90 ( ⫾1.03) 5.45 ( ⫾2.44)
Rel Tet 0.40 ( ⫾0.13) 0.45 ( ⫾0.09) 0.44 ( ⫾0.28)
Con 0.46 ( ⫾0.127) 0.50 ( ⫾0.09) 0.41 ( ⫾0.12)
Info Cont. Tet 2.20 ( ⫾1.27) 2.60 ( ⫾0.43) 2.37 ( ⫾0.70)
Con 3.86 ( ⫾0.48) 3.09 ( ⫾0.52) 3.93 ( ⫾0.40)
Spars. Tet 0.20 ( ⫾0.08) 0.15 ( ⫾0.03) 0.16 ( ⫾0.06)
Con 0.13 ( ⫾0.05) 0.19 ( ⫾0.07) 0.08 ( ⫾0.005)
Mean Rate Tet 3.35 ( ⫾2.34) 1.56 ( ⫾0.54) 1.09 ( ⫾0.31)
Con 1.62 ( ⫾0.59) 4.22 ( ⫾1.91) 2.76 ( ⫾1.49)
The measures of spatially selective firing and mean firing rate during days 2– 4 of spatial memory acquisition for the
tetracaine and vehicle control groups are shown.
Con, Vehicle control group; Tet, tetracaine group.
Figure 10. Inactivation of retrosplenial cortex causes place fields to be
less stable across days. A, The location of a place field recorded across 3
consecutive days of acquisition recorded from an animal undergoing
retrosplenial cortex inactivation is shown. The field changes the preferred
firing location across days from the north arm to the eastern edge of the
center platform and to the east maze arm on days 2–4 of acquisition. B,
Most of the cells recorded from an animal receiving vehicle control
injections remained in the same location across days. The place field
remains on the northern arm for 3 consecutive days of acquisition. Place
fields appeared less stable after tetracaine injections than what was
observed for animals receiving control injections. To illustrate recording
stability, a set of 50 waveforms from each day is displayed above each
spatial plot.
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3997
to cumulative error without an external source of information to
update the navigational system (Gallistel, 1990; Etienne, 1992).
One manipulation for disrupting self-motion cues (a component
of the path integration process) is to carry an animal in an opaque
box and slowly rotate the box while transporting the rat to the
testing room. This manipulation causes spatial-learning impair-
ments and place fields to be unstable across testing sessions
(Knierim et al., 1995; Dudchenko et al., 1997; Martin et al., 1997).
These data suggest that self-motion cues may be the metric for
establishing stable representations of the visual environment (cf.
McNaughton et al., 1996; Samsonovich and McNaughton, 1997;
but see Redish and Touretzky, 1997). These results illustrate the
importance of the normal integration of visual and self-motion
cues for ensuring stable place coding and normal spatial learning.
During initial learning, animals may navigate by comparing
their current position, direction, and velocity relative to remem-
bered locations in space. This may be similar to the proposed role
of memory in updating movement information when testing oc-
curs in darkness (Brown and Bing, 1997; Save, 1997). If retro-
splenial cortex contributes to the integration of mnemonic spatial
information with self-motion cues, then inactivation of retrosple-
nial cortex would be expected to disrupt initial learning about
visual environments. In addition, place fields would be expected
to be less stable during acquisition in animals undergoing inacti-
vation of retrosplenial cortex. We sought to explore our hypoth-
esis further by examining behavioral and electrophysiological
changes after inactivation of retrosplenial cortex during acquisi-
tion of the spatial memory task with room lights available.
Experiment 2
Experiment 2 revealed that retrosplenial cortex is necessary for
normal performance of the spatial memory task with visual cues
available. Furthermore, place-field stability within and across days
of testing is dependent, in part, on the normal activity of retro-
splenial cortex. If place fields in tetracaine-treated animals re-
mained in the same location across trials within each day of
acquisition, then performance was impaired (Fig. 9D). This pat-
tern of data paralleled the behavioral data that demonstrated that
animals in the tetracaine group were eventually able to acquire
the spatial memory task. Unreliable spatial coding within a day is
a unique phenomenon because it is commonly assumed that
stable coding of space leads to more accurate performance (Mi-
zumori et al., 1994, 1996; Barnes et al., 1997). However, the
current testing conditions are not directly analogous to previous
work. First, animals are performing a task while a brain area is
temporarily inactivated, and second, the effectiveness of tetra-
caine likely decreases with time (see Experiment 1). The small
sample size limits broad conclusions, but the finding that perfor-
mance on the maze improved across days suggests that there may
be behavioral and neural compensation. This compensation, in
turn, may be reflected by this unique pattern of hippocampal
spatial coding. If animals use the same location code established
in the first trials after the inactivation condition during all of the
maze trials, then they are less likely to navigate accurately.
However, if there is a different spatial firing pattern in the later
trials compared with that of the first two trials, animals are able
to perform as well as controls. These data provide interesting
evidence of f uture research evaluating how hippocampal process-
ing of space is modified by behavioral or neural compensation
during learning. Furthermore, they provide evidence of flexible
hippocampal processing enabling spatial learning.
Previous lesion studies have demonstrated that permanent re-
moval of retrosplenial cortex impairs spatial learning and memory
in a water maze (Sutherland et al., 1988; Sutherland and Hoesing,
1993; but see Warburton et al., 1998) and disrupts orientation
responses (Ellard et al., 1990; Kwon et al., 1990; Ellard and
Chapman, 1991). However, retrosplenial cortex lesions do not
result in spatial memory deficits on the eight-arm radial maze task
when room lights are available (Neave et al., 1997). This provides
an apparent paradox in the contribution of retrosplenial cortex to
spatial learning and memory. Perhaps the paradox can be re-
solved by comparing differences in the requirement for visual
memory and self-motion integration across tasks. The water
maze is relatively devoid of local cues for use during spatial
localization. Therefore, identif ying locations based on movement
relative to previously experienced visual cues is critical for solv-
ing this task. Orientation responses are typically elicited with
visual stimuli and examine movement toward (approach) or away
from (avoidance) the stimulus. Avoidance responses, in particu-
lar, may use spatial memory for recalling locations of potential
refuge within the environment, and this mnemonic information
may be integrated with the visual threat stimulus to mediate rapid
flight responses. The radial maze in darkness is solved by a
combination of mnemonic spatial cues, self-motion information,
and local cues (Brown and Bing, 1997). The pattern of deficits
across the tasks suggests that the integration of visual memory
and movement-related cues is a common element underlying
impairments after temporary or permanent damage to retrosple-
nial cortex.
Taken together, experiments 1 and 2 suggest that the retrosple-
nial cortex and hippocampus are interactive partners mediating
spatial memory and behavior. Retrosplenial cortex importantly
contributes to behavior when recalled information about the
spatial environment needs to be integrated with self-motion cues
to learn a spatial task. The reorganization of hippocampal spatial
coding observed in the present experiments is consistent with
previous work showing changes in spatial coding after manipula-
tions of visual and self-motion cues (Sharp et al., 1995; Knierim
et al., 1998) and with computational models that place these cues
in conflict with each other (Guazzelli et al., 1999; Redish and
Touretzky, 1999). These issues are developed below.
General discussion
Experiment 1 confirmed that retrosplenial cortex inactivation
selectively impairs dark spatial memory performance, and exper-
iment 2 showed that retrosplenial cortex is necessary for initial
visual spatial learning. In contrast to the selective pattern of
behavioral effects, hippocampal spatial coding was altered when
performance was normal (E xperiment 1, light testing) and im-
paired (Experiment 1, dark testing; E xperiment 2, acquisition in
the light). Hippocampal reorganization, as reflected by individual
neurons, does not necessarily predict behavioral impairments.
Instead, changes in hippocampal spatial coding may be related to
modifications in the reliance on sensory modalities relevant for
different cognitive strategies used to solve the task (cf. Markus et
al., 1995). We suggest that the pattern of data from both exper-
iments is consistent with the hypothesis that retrosplenial cortex
contributes to the integration of visual and self-motion cues for
use in navigation and for hippocampal spatial coding. When place
cells change their reliance on sensory information for spatial
coding, hippocampal reorganization likely occurs. Without the
normal integration of visual and movement-related cues, animals
may use different sources of sensory information for navigation
and hippocampal spatial coding.
3998 J. Neurosci., June 1, 2001, 21(11):3986–4001 Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization
It is possible that the hippocampal reorganization observed in
the present experiments was caused by the entire visual environ-
ment being perceived as novel after inactivation of retrosplenial
cortex. The novelty explanation appears unlikely. Animals trans-
ferred to a novel environment tend to make more errors (in the
light) than they do in a familiar environment at asymptote levels
of performance (Mizumori et al., 1995), which was not observed
in the current study. Nonspecific sensory and motor deficits are
not probable explanations for the observed effects. Changes in
spatial coding did not occur during vehicle control injections,
performance was preserved during light testing at asymptote, and
maze run time did not change as a function of injections.
Visual and self-motion integration, place-field reorganization,
and behavior
Knierim et al. (1998) placed visual and self-motion cues in
conflict with each other by rotating the animal relative to a
previously stable visual cue and recorded place cells before and
after the rotation. When the rotation was ⬍45°, place cells fol-
lowed the new location visual cue; when the rotation was ⬎45°,
many place cells unpredictably changed their normal spatial firing
patterns relative to the distal cue. Thus, when the perceived
landmarks are mildly incongruent with the expected orientation
of the animal, hippocampal place cells and perhaps the naviga-
tional system in general correct and update in agreement with the
cues. If the landmarks are substantially deviated from the remem-
bered configuration, then hippocampal cells show spatial reorga-
nization. Similar results have been demonstrated in behavioral
studies of the golden hamster. Rotation of a single visual land-
mark can control homing behavior when it is rotated ⬍45° from
the standard location. However, rotations by ⬎45° caused animals
to rely on internal sources of information to compute the return
trajectory (Etienne et al., 1990). These experiments taken to-
gether with the current results suggest that (1) the hippocampus
may reorganize spatial representations when there is substantial
deviation between visual and self-motion cues and (2) despite the
presence of hippocampal reorganization, behavior may be intact
because animals can rely on either visual or self-motion cues to
mediate navigation.
In addition to changes in place fields during light testing, there
was an even more pronounced reorganization when retrosplenial
cortex was inactivated before dark trials. This suggests that with
visual cues available, the navigational system may correct and
update more quickly than when they were not visible (see Fig. 8).
It remains a possibility that inactivation of retrosplenial cortex
may cause a deficit based solely on self-motion processing. This
appears unlikely because vestibular and visual information are
integrated in brainstem levels of the CNS (for review, see Smith,
1997), and the integration is preserved in all levels of the limbic
system (for review, see Taube, 1998). Therefore, it appears more
likely that retrosplenial cortex contributes to the integrative pro-
cess rather than a single domain in isolation.
Spatial memory and place-field reorganization
Recent theories of navigation have suggested that during initial
learning animals establish visuospatial representations relative to
their movement through space (McNaughton et al., 1996; Sam-
sonovich and McNaughton, 1997). Accordingly, we examined the
role of retrosplenial cortex during visuospatial learning. Perfor-
mance deficits were observed during initial learning after retro-
splenial cortex inactivation (E xperiment 2). In addition, place
fields showed reorganization across trials and across days during
acquisition. Previous work has demonstrated that normal integra-
tion of visual and self-motion cues is critical for appetitively me-
diated spatial learning and for establishing the preferred firing
fields of hippocampal place cells (Knierim et al., 1995; Dudchenko
et al., 1997; Martin et al., 1997). The results from experiment 2 are
consistent with the interpretation that retrosplenial cortex provides
spatial memory for use in guiding and directing movements during
spatial learning and hippocampal coding of space.
In agreement with our interpretation that memory deficits may
account for the hippocampal reorganization observed in the
current experiments, Barnes et al. (1997) suggested that impaired
spatial memory in aged animals might be related to changes in
hippocampal reorganization. Hippocampal place coding in old
animals periodically reorganized when they were reintroduced to
a familiar environment (Barnes et al., 1997). The bimodal distri-
bution of place-field responses may explain the behavioral per-
formance of old animals on the water maze task, in which they
displayed either accurate or inaccurate paths to the hidden plat-
form (Barnes et al., 1997). Redish and Touretzky (1999) modeled
this phenomenon by weakening the long-term potentiation-
dependent integration of visual cues with the resetting of path
integration when simulated animals were introduced to the envi-
ronment. In their computational model, a similar pattern of
periodic spatial recoding of a familiar environment was observed.
The experiment and the model are both consistent with our
interpretation that spatial memory impairments may disrupt the
normal integration of visual and self-motion cues and lead to
hippocampal reorganization.
Unique contribution of retrosplenial corte x to navigation
Anatomical findings and experimental results suggest that there is
a thalamic differentiation of visual and self-motion processing that
can be relayed to retrosplenial cortex (van Groen and Wyss, 1992,
1995; van Groen et al., 1993). The lateral dorsal nucleus of the
thalamus (LDN) and ATN may be related to visual and vestibular
processing, respectively, of the head-direction signal. The LDN
head-direction cells are visually sensitive, because they do not
maintain their preferred firing direction for extended periods of
time in darkness (Mizumori and Williams, 1993). In contrast, ATN
head-direction cells are dependent on vestibular input (Blair and
Sharp, 1996; Stackman and Taube, 1997) and persist for a substan-
tial amount of time in darkness (Knierim et al., 1998). On-line
updating of visual directional information may be processed
through LDN input to retrosplenial cortex, whereas changes in
directional heading derived from vestibular activation are relayed
to retrosplenial cortex from ATN. Posterior parietal cortex could
provide proprioceptive feedback to retrosplenial cortex.
Memory-guided navigation may be accomplished via a com-
parison between the currently experienced visual, vestibular, and
proprioceptive inputs relative to past experiences within the
environment. This comparison may allow for updating of the
directional heading relative to the currently experienced sensory
information (Mizumori et al., 2001). The mnemonic spatial in-
formation from retrosplenial cortex may then be relayed to the
hippocampus directly via entorhinal input or indirectly through
the postsubiculum (van Groen and Wyss, 1990b; Wyss and van
Groen, 1992). Retrosplenial–hippocampal interactions may be
critical for behavior when spatial localization requires mnemonic
information about the environment.
It has been suggested that the hippocampus functions as a
“path integrator” and allows animals to navigate successfully on
the basis of movement through space (Maaswinkel et al., 1999;
Cooper and Mizumori •Retrosplenial Cortex and Hippocampal Reorganization J. Neurosci., June 1, 2001, 21(11):3986–4001 3999
Whishaw and Gorny, 1999; but see Alyan and McNaughton,
1999) and that path integration information may be received from
posterior cortical areas (Chen et al., 1994b; McNaughton et al.,
1996; Guazzelli et al., 1999). In this study, changes in hippocam-
pal coding of space were correlated with behavioral performance
when path integration strategies may have been used (e.g., dark-
ness and acquisition). However, there are examples when the
recoding of space occurred despite high levels of performance
(e.g., light testing). Therefore, the present results neither confirm
nor reject the possibility that the hippocampus contributes to path
integration. We suggest that although the hippocampus may be
involved in navigation based on nonvisual cues, it likely does so in
concert with retrosplenial cortex.
Summary and conclusions
The goals of the present study were to replicate and extend our
previous findings of dark-selective behavioral impairments after
inactivation of retrosplenial cortex and to examine putative neu-
ral mechanisms of the behavioral impairments. Retrosplenial
cortex may contribute to navigation when animals are required to
update spatial information relative to remembered features of the
environment. This process likely requires interactions between
the retrosplenial cortex and hippocampus as evidenced by
changes in spatial coding when retrosplenial cortex was not active.
Furthermore, the duration of hippocampal reorganization ap-
pears to be more persistent when visual cues are not immediately
available to update the navigational system. Therefore, retrosple-
nial cortex interactions with the hippocampus may allow past
experience to update and correct cumulative errors that occur
during path integration.
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