Supplementary Materials for...the microdrive to enable the placement of optic fibers (200 µm core...
Transcript of Supplementary Materials for...the microdrive to enable the placement of optic fibers (200 µm core...
www.sciencemag.org/content/348/6234/560/suppl/DC1
Supplementary Materials for
Selective information routing by ventral hippocampal CA1 projection neurons
S. Ciocchi,* J. Passecker, H. Malagon-Vina, N. Mikus, T. Klausberger*
*Corresponding author. E-mail: [email protected] (S.C.); [email protected] (T.K.)
Published 1 May 2015, Science 348, 560 (2015)
DOI: 10.1126/science.aaa3245
This PDF file includes:
Materials and Methods Figs. S1 to S14 Table S1 References (31–38)
Materials and Methods
Experimental animals
Behavioural data were obtained from four Long Evans male rats (330 - 420 g, 2½ to 4 months at
the time of surgery, Charles River Laboratories); data from five additional rats were discarded
because of misplaced virus injection, tetrodes or optic fibres. Animals were housed individually
in Plexiglas cages (42 x 27 x 30 cm) and maintained under a 12 h light/12 h dark cycle with ad
libitum access to food and water. Behavioural experiments occurred in the light phase and rats
were food deprived to reach 85 % of the preoperative weight. All experimental procedures were
performed in accordance with regulations of the Austrian ministry of Science and the Medical
University of Vienna.
Surgeries: virus injection and microdrive implantation
Stereotaxic coordinates (31) used for the surgeries are found in the following table:
Procedure Targeted brain area Antero-posterior Medio-lateral Dorso-ventralOptic fiber implantation mPFC 3 0.5 -2.6Optic fiber implantation Acb 1.8 0.8 -6Optic fiber implantation Amy -2.6 4.2 -7Tetrodes implantation dCA1 -3.4 2.5 -1.5Tetrodes implantation vCA1 -4.8 4.5 -6.5
Virus injection (1st track) vCA1 -4.8 4.5 from -8.2 to -7.4Virus injection (2nd track) vCA1 -5.2 5.4 from -8.0 to -6.8
Coordinates given in millimeters and referenced to bregma
Rats were anaesthetised with isofluorane (induction 5%, maintenance 2%) in O2. Rats were fixed
in a stereotaxic frame and body temperature was stabilised with a heating pad. Local and
systemic analgesics (xylocain® 2%, metacam® 2mg/ml, 0.5 ml/kg) were applied. Iodine
solution and eye-protective cream were provided to disinfect the surgery site and protect the
corneas. Every two hours a Ringer's solution (10 ml/kg) was administered subcutaneously to
avoid dehydration. The surgery site was exposed and cleaned with saline solution. Six stainless
steel screws were anchored into the skull with two of them placed above the cerebellum to serve
as grounds and references for the electrophysiological recordings. Craniotomies were performed
in the right hemisphere above dCA1 and vCA1, mPFC, Acb and the Amy. The dura mater was
removed and a saline solution was applied to the brain surface to avoid oedemas. The
recombinant AAV2/1.syn.ChR2(H134R).EYFP (Addgene 26973P) was produced by the Penn
Vector Core at a titer of 5,9 x 1013 genome copies/ml. The virus solution was loaded into a pulled
borosilicate glass capillary and pressure-injected into the vCA1 with a Picospritzer III® (Parker
Hannifin Corporation). About 600-800 nl of virus solution was injected over 10 min along two
tracks. Rats were then implanted above dCA1 and vCA1 with a custom-made microdrive (Miba
Machine Shop, IST Austria) containing 15 independently moveable tetrodes made of four
twisted tungsten microwires (12.7 µm inner diameter, California Fine Wire Company). The
tetrode tips were gold-plated to impedances of 100 - 600 kΩ. 3 plastic tubes were integrated into
the microdrive to enable the placement of optic fibers (200 µm core diameter, 0.37 NA, Thorlabs
GmbH) in mPFC, Acb and Amy. Paraffin wax was then applied around the tetrode guide
cannulae, the remaining lower part of the microdrive was cemented (Refobacin® Bone Cement)
and the surgery site sutured. Animals were given post-operative analgesia (Dipidolor 60 mg
diluted per 500 ml drinking water) and at least 7 days of recovery time.
In vivo electrophysiology and optical stimulations
Tetrodes were progressively lowered to the vCA1 and dCA1 pyramidal layers over a period of
about 3 weeks by using SWR and theta oscillations as electrophysiological hallmarks. For each
recording day, tetrodes were moved to sample new units.
The extracellular electrical signals from the tetrodes were pre-amplified with a headstage (HS-
132A, 2 x 32 channels, Axona Ltd) to reduce cable artefacts. The output signals were amplified
1000X via a 64-channel amplifier and continuously digitised at 24 kHz at 16 bit resolution using
a 64-channel analogue-to-digital converter computer card (Axona Ltd). The signals were down-
sampled offline at 20 kHz. Single-units were extracted offline by detecting signal amplitudes 5
SD above the root mean square of the digitally filtered signal (0.8 - 5 kHz) over 0.2 ms sliding
windows. 32 data points (1.6 ms) were sampled for each single-unit. A principal component
analysis was implemented to extract the first three components of spike waveforms of each
tetrode wire (32).
Spike waveforms from individual neurons were detected using the KlustaKwik automatic
clustering software (http://klustakwik.sourceforge.net/). Individual single-units were isolated
manually by verifying the waveform shape, the modulation of waveform amplitude across
tetrode channels, the temporal autocorrelation (to assess the refractory period of a single-unit)
and crosscorrelation (to assess a common refractory period across single-units) using the
Klusters software (33). The stability of single-units was confirmed by examining spike features
over time.
To locally detect SWRs in vCA1, the local field potential signals were band-pass filtered (150 -
250 Hz) and a reference channel without SWR was subtracted from all channels to remove
common-mode noise (such as chewing artefacts). The detection threshold for SWR (the tetrode
with largest SWR power in vCA1 was selected) was set as 4 - 6 SD above the room mean square
amplitude of the filtered signal (32). SWR detection was achieved during rest/sleeping periods
immediately following behavioural experiments. The quality of SWR detection was confirmed
by visual inspection in the NeuroScope software (33).
To antidromically identify individual vCA1 projection neurons (from 4 weeks on following virus
injection), a DPSS laser (IkeCool Corporation) generating blue light (473 nm) was sequentially
connected to implanted optic fibres through a ferrule-sleeve system (Senko Ltd) with 30 - 70
mW output power delivered to the brain tissue. A screening protocol of different light
illuminations was used at the end of the behavioural experiments to optimise the identification of
individual projection neurons. This protocol included a combination of light durations of 1 or 5
ms with light intensities of 10, 20, 50 and 100% of laser output power. 50 or 100 repetitions of
each stimulation was done at 5Hz. The ability of vCA1 projection neurons to follow high
frequency stimulation was tested using 20 and 50 Hz light stimulations repeated 50x.
The pharmacological experiment was performed under urethane (1.25 g/kg body weight)
anaesthesia with additional doses of a ketamine/xylazine mixture (17 and 7 mg/ml, respectively;
0.02 - 0.1 ml). NBQX 1mM and D-AP5 2mM (Tocris Bioscience) saline solution was pressure-
injected into vCA1 of a 5 weeks virus-injected rat; in an additional experiment the drugs (NBQX
5mM and D-AP5 10mM) were injected both into the vCA1 and into the frontal cortex to block
synaptic activity also in the prefrontal cortex and nucleus accumbens. Single-unit activity was
recorded in vCA1 with simultaneous optical stimulation of the target areas using protocols
described above. Signals were recorded with 4 tetrodes and transmitted to a RA16AC headstage
(Tucker‐Davis Technologies) and to a 16-bit analogue-to-digital converter (Cambridge
Electronic Design). Single-units were isolated as described above.
Behaviour
A goal-directed navigation task, spatial exploration and an anxiety task were used in the present
study. The goal-directed navigation task and the spatial exploration required a preliminary
training procedure of about 2 - 3 weeks. One week following surgery, food deprivation started
and rats were trained in parallel on both tasks. The goal-directed navigation task was based on
consecutive learning of allocentric and egocentric rules (34). In the allocentric task, rats learnt to
find rewards at the same position at the end of one of the rewarded arms based on spatial
landmarks in the experimental room. In the egocentric rule, rats learnt to find rewards by turning
in the same direction from each of the start arms and therefore ending at opposing rewarded
arms. Rats were first habituated to the maze and to continuously run trials for sucrose pellets (3 x
20 mg, TestDiet) on a plus maze with two opposing starting and rewarded arms meeting at 90o
angles. Sucrose pellets were delivered by sensor-activated dispensers (Campden Instruments
Ltd) at the extremity of each rewarded arm. The size of the arms was 80 cm x 11 cm and the
"reward zone" corresponded to the 30 cm ahead of the dispensers. The plus maze was 55 cm
high and wooden-made. Both rewarded arms were baited during maze habituation but only one
arm during rule learning. Training to allocentric and egocentric rules started once rats
continuously performed 40 - 60 trials during the habituation phase. Rats were manually placed at
a starting arm and freely ran to one of the potentially rewarded arm. The path to the opposite
starting arm was prevented by a ceramic pot. Rats were placed in the ceramic pot between each
trial for about 5 seconds before restarting a new trial. Rats were pseudo-randomly assigned to a
starting arm before each trial: if rats reached a non-rewarded arm, the next trial restarted on the
same start arm again, otherwise, in successful trials, the consecutive trial was randomly assigned.
Rule learning was set to at least 13 correct trials out of the last 15 trials. About 4 - 8 training days
were required for rats to learn 3 - 4 rules within a single training session. The maze was cleaned
with a odourless solution every 8 trials to avoid odour-guided navigation. At least one
behavioural switch (allocentric to egocentric or egocentric to allocentric rule learning) was
presented on each experimental day. For the spatial navigational task, rats learned to forage for
chocolate flakes (Kellogg’s) randomly thrown in a 180 x 180 cm open field surrounded by 30 cm
high walls. 3 - 4 training sessions were required to reach 30 - 40 minutes of continuous open
field exploration. The light intensity in the experimental room during rule switching task and
spatial navigational task was 1 lux. Once learnt, the goal-directed navigation task and the spatial
exploration were performed in conjunction with an anxiety task, based on the exploration of a
wooden elevated-plus-maze. The elevated-plus-maze consisted of two closed and two open arms.
The dimensions of the arms were 9 x 50 cm, the walls in the closed arms were 40 cm high and
the elevated-plus-maze was elevated 70 cm above the floor. Rats were placed on the elevated-
plus-maze facing the open arm distal to the experimenter. The elevated-plus-maze sessions lasted
5 - 8 min and were done at 200 lux of room light intensity (35). To control for the specificity of
neuronal firing in the elevated-plus-maze, arms were interchanged during a consecutive elevated-
plus-maze exploration.
The three tasks were performed in the same room. The room configuration was made different
for all three tasks by changing the landmarks and the configuration of the walls surrounding the
mazes. The presentation of the three tasks was counterbalanced for order from one day to
another. The number of recording days per rat was low (3 - 6 sessions) to keep anxiety levels
high in the elevated-plus-maze task. The rats' position was monitored using an array of LEDs of
three different colours detected at 25 frames per second by an overhead video camera (Sony).
Histology
To confirm the position of the recording sites, lesions were made at the tip of the tetrodes using a
30 µA unipolar current for 10 s (Stimulus Isolator, World Precision Instruments). Rats were then
deeply anesthetised with urethane and perfused with saline followed by 20 min. fixation with 4%
paraformaldehyde, 15% (v/v) saturated picric acid and 0.05% glutaraldehyde in 0.1 M phosphate
buffer. Serial coronal sections were cut at 70 µm with a vibratome (Leica). Sections containing a
lesion were Nissl-stained. To detect the expression of ChR2, we incubated sections of interest in
serum containing mouse anti-ChR2 monoclonal antibody (1/100, mfd Diagnostics) in 0.1M Tris-
buffered saline containing 1% normal horse serum and 0.1% Triton X-100. Sections were next
incubated with a Cy5 anti-mouse fluorescent secondary antibody (1/250, Jackson
Immunoresearch Laboratories). Immunohistochemical analysis was performed on an
epifluorescence microscope (Olympus BX61).
Analysis and Statistics
Optogenetic identification of vCA1 projection neurons.
To identify individual vCA1 projection neurons by antidromic spiking, we used a combination of
criteria including low spike jitter (< 0.3 ms), early latency (< 22 ms for mPFC stimulation, < 17
ms for Acb shell stimulation, < 13 ms for the Amy stimulation ) and high fidelity (> 50% of light
responsive sweeps) of light-induced spikes. Spike-collisions and the ability of vCA1 neurons to
follow 50 and/or 20 Hz photostimulation were also tested. Collision tests were classified into
three categories: neurons with a spike collision occurring in a sequence of at least 5 consecutive
antidromic spikes (binomial test, p < 0.05), neurons with a spike collision occurring without a
sequence of 5 consecutive antidromic spikes and neurons without a testable spike collision
(absence of spontaneous spikes or collisions occurring during the refractory period of a
spontaneous spike before antidromic activation (< 2 ms)). The efficacy of collisions was
dependent of the photostimulation intervals between spontaneous and antidromic spikes as
described previously (3). vCA1 neurons fulfilling at least 3 of these criteria were classified as
projection neurons to a defined target. The spike waveform similarity between antidromic spikes
and spontaneous spikes of individual vCA1 projection neurons was calculated by correlational
analysis on the averaged spike waveforms. The channel of the tetrode with the largest voltage
amplitude was used.
Analysis of place cells.
To compute firing rate maps, the arena of exploration was divided into 300 x 400 pixels. In each
pixel, the number of spikes was divided by the rat´s occupancy: the firing rate maps were
smoothed by convolving them in two dimensions with a Gaussian low-pass filter. To calculate
the stability score of place fields during the exploration of the open field, the rat´s trajectory was
divided into a first and a second half, and firing maps were computed for the 2 trajectories. A
pixel-to-pixel comparison was made by correlational analysis to assess the similarity and
therefore the stability of place fields over time. Correlation values close to 1 indicate stable place
fields whereas values closer to -1 indicate spatially anti-correlated place fields. Cells with a place
field stability score > 0.37 were considered as stable place cells. The 0.37 threshold was chosen
because it corresponded to the intersection of the bimodally distributed dCA1 place field stability
scores. The area of a place field was computed by calculating the percentage of pixels with a
firing rate value > 20% of the neuronal peak firing rate (13). The spatial tuning was calculated by
subtracting the mean firing rate from the peak firing rate of the firing rate map of each neuron.
Analysis of anxiety cells.
Elevated-plus-maze scores of firing rates of individual cells were calculated as previously
reported (4):
Elevated − plus − maze �iring rate score =A − BA + B
,where
A = 0.25 ∗ (|FL − FU| + |FL − FD| + |FR − FU| + |FR− FD|) and
B = 0.5 ∗ (|FL − FR| + |FU − FD|)
FL, FR, FU, and FD are the difference from mean firing rate (in percent) in left, right, up and
down arms, respectively. A corresponds to the mean difference in normalised firing rate between
arms of different types, while B corresponds to the mean difference in normalised firing rate for
arms of the same type. Scores with values close to 1 indicate that the firing rates are very similar
within arms of the same type (small B value) but different across arms of different types (high A
value). Negative scores indicate that the firing rates are inconsistent within arms of the same type
but very similar across arms of different types. A score of zero corresponds to a neuron with the
same firing rate in each arm of the maze. Neurons with an elevated-plus-maze score > 0.37 were
considered as anxiety responsive. Only neurons with more than 50 spikes could be classified as
anxiety cells. To quantify anxiety behaviour, we calculated offline the percentage of time spent
and the number of entries in each of the four arms based on the rat leaving or re-entering the
central part of the elevated-plus-maze. An entry was counted when the rat´s trajectory was longer
than 0.5 s in a specific arm.
Analysis of goal-directed cells.
To identify neurons excited or inhibited in the reward zone, we compared the averaged firing rate
histograms before (-2 s to 0 s, 10 bins) to after (0 s to 4 s, 20 bins) the entry into the reward zone
with the Mann–Whitney rank sum test. The trials were divided in two blocks to assess the
maintenance of reward-modulated firing. Neurons with p-values < 0.1 in each block of trials
were classified as reward modulated neurons. All reward-modulated neurons had different
activities when comparing the firing rates before and after the entry into the reward zone across
all trials (p < 0.05, Wilcoxon signed-rank test). To identify neurons that significantly changed
firing over trials, we used the change-point analysis developed by Gallistel et al., 2004 (36).
Change-point analysis enabled the identification of trials displaying a significant change in firing
relative to the preceding trials. The population z-scored firing rate histograms were computed
separately for reward zone-excited or -inhibited neurons by averaging the z-scored firing rate
histograms of individual reward zone-modulated neurons. To directly compare the time-course
of reward zone-modulated neurons in the different trial conditions of the rule switching task
(allocentric vs. egocentric; reward zone 1 vs. reward zone 2; trajectory 1 vs. trajectory 2 vs.
trajectory 3 vs. trajectory 4; rewarded vs. non-rewarded), we averaged the individual z-scored
firing rate histograms of reward zone-excited neurons with the individual and absolute z-scored
firing rate histograms of reward zone-inhibited neurons. A change-point analysis was done at the
population level to determine the earliest time point of firing rate changes during approach of the
reward zone. A state-space model developed by Smith et al., 2004 (37) was used to analyse the
behavioural learning curves of rats during the rule switching task. This model enabled the
calculation of probabilistic learning curves with confidence intervals out of binary behavioural
outcomes (correct vs. incorrect trials). The probability of correct outcome was set at 0.5.
Recruitment of cells during SWR.
To establish whether a neuron was activated during locally detected SWR, we computed z-
scored firing rate histograms for each vCA1 neuron relative to the peak voltage amplitude of
each detected SWR. Histograms ranged from -0.5 s to 0.5s and the spikes of each neuron was
binned by 0.05 s. Neurons with at least one z-score value > 2.2 in the 2 bins surrounding SWR
detection were considered as activated by SWR. Two behavioural sessions were omitted of SWR
analysis due to the absence of a rest/sleeping period.
Locomotor activity and firing rates.
For the correlational analysis of firing rates with locomotor activity, the open field exploration of
each rat was divided in 1 s time segments and firing rates, speeds (first derivative of the position
with respect to time) and accelerations (2nd derivative of the position with respect to time)
values were calculated and correlated for each vCA1 neuron over each time segments.
Bootstrap analysis.
We used bootstrap analysis to determine whether a vCA1 projection type was significantly
enriched or impoverished in task-responsive neurons. For each projection type and its
corresponding number of projections, we randomly assigned a number of task-responsive
neurons from the grand total. This operation was repeated 10000 times which led to a bootstrap
distribution of resampled data and the original dataset was compared to the bootstrapped
distribution. Data points beyond 95% of the confidence interval of the bootstrapped distribution
were considered as significantly enriched or impoverished in task-responsive neurons.
All calculations were made in MATLAB (version 7.9) and statistical analysis was done in the
commercially available software SPSS (version 22.0) and SigmaPlot (version 12.5). All
significant correlational analysis was significant at alpha = 0.05 with both Spearman’s and
Pearson’s methods.
Statistics were performed for each experiment as follows:
Fig. 1 (F). Box plots display the median, the first and third quartiles and the ranges of the
latencies distribution of individual cells.
Fig. 2 (C). (Left) Percentage of dCA1 and vCA1 anxiety neurons were unequally represented.
The null hypothesis, that the percentage of dCA1 and vCA1 anxiety neurons was similar, was
rejected by the Pearson Chi-Square test: χ2 (1, 499) = 14.61, p < 0.001. Thus, there were
significantly more anxiety neurons in vCA1 (n = 43 out of 233, 18.45 %) compared to dCA1 (n
= 19 out of 266, 7.14 %).
Fig. 2 (C). (Right) Percentage of anxiety neurons among vCA1 projection types. Bootstrap
analysis revealed that the percentage of anxiety neurons in the vCA1 → mPFC projection (n = 6
out of 14, 42.86 %, p < 0.05) lay above confidence intervals. Thus, there were significantly more
anxiety neurons in the vCA1 → mPFC projection than chance level. All of the other projection
types lay within confidence intervals (p > 0.05).
Fig. 2 (D). Difference of elevated-plus-maze scores between vCA1 projection neurons with an
axon collateral in mPFC or the Amy (without counting axon collaterals in both targets). Two-
tailed, unpaired t-test revealed a significant difference (p < 0.05) between elevated-plus-maze
scores of vCA1 projection neurons with an axon collateral in mPFC (n = 43, elevated-plus-maze
score = 0.12 ± 0.04 ) and vCA1 projection neurons with an axon collateral in the Amy (n = 12,
elevated-plus-maze score = -0.08 ± 0.06). Values are expressed as mean ± s.e.m..
Fig. 2 (G). (Left) Percentage of dCA1 and vCA1 place cells was different. The null hypothesis
that the percentage of dCA1 and vCA1 place cells was similar was rejected by the Pearson Chi-
Square test: χ2 (1, 499) = 71.85, p < 0.001. Thus, there were significantly less place cells in
vCA1 (n = 33 out of 233, 14.16 %) compared to dCA1 (n = 133 out of 266, 50 %).
Fig. 2 (G). (Right) Percentage of place cells among vCA1 projection types revealed that none of
the projection types lay beyond confidence intervals (Bootstrap analysis, p > 0.05 for all
projection types).
Fig. 3 (C). (Top) Percentage of reward zone-excited neurons among vCA1 projection types.
Bootstrap analysis revealed that the percentage of reward zone-excited neurons in the vCA1 →
mPFC/Acb projection (n = 6 out of 29, 20.67 %, p < 0.05) lay above confidence intervals Thus,
there were significantly more reward zone-excited neurons in the vCA1 → mPFC/Acb projection
than chance level. All of the other projection types lay within confidence intervals (p > 0.05).
Fig. 3 (C). (Bottom) Percentage of reward zone-inhibited neurons among vCA1 projection types.
Bootstrap analysis revealed that the percentage of reward zone-inhibited neurons in the vCA1 →
Acb projection (n = 10 out of 35, 28.57 %, p < 0.05) lay above confidence intervals. Thus, there
were significantly more reward zone-inhibited neurons in the vCA1 → Acb projection compared
to chance level. All of the other projection types lay within confidence intervals (p > 0.05).
Fig. 3 (D). Percentage of reward zone-excited, reward zone-inhibited and non responsive
neurons in vCA1 projections to mPFC/Acb and Acb were differently represented. The null
hypothesis that reward zone responses had similar proportions across or within vCA1 projections
to mPFC/Acb and Acb was rejected by the Pearson Chi-Square test: χ2 (2, 64) = 10.55, p < 0.01.
Thus, there were significantly more reward zone-excited neurons in vCA1 → mPFC/Acb
projections (n = 6 out of 29, 20.67 %) than reward zone-excited neurons in vCA1 → Acb
projections (n = 1 out of 35, 2.86 %), and than reward zone-inhibited neurons within vCA1 →
mPFC/Acb projections (n = 1 out of 29, 3.45 %, post-hoc Bonferroni correction, p < 0.05 for
both comparisons). Also, there were significantly more reward zone-inhibited neurons in
vCA1→ Acb projections (n = 10 out of 35, 28.57 %) than reward zone-inhibited neurons in
vCA1→ mPFC/Acb projections (n = 1 out of 29, 3.45 %), and than reward zone-excited neurons
within vCA1 → Acb projections (n = 1 out of 35, 2.86 %, post -hoc Bonferroni correction, p <
0.05 for both comparisons). The proportions of non-responsive neurons were not significantly
different from the other groups.
Fig. 3 (E). Comparison of absolute difference in firing rate between reward zone and maze for
vCA1 projections with an axon collateral in Acb (n = 79, 1.12 ± 0.23 Hz) compared to vCA1
neurons without Acb projections (n = 20, 0.47 ± 0.19 Hz). Two-tailed, unpaired t-test revealed a
significant difference (p < 0.05). Values are expressed as mean ± s.e.m..
Fig. 4 (A). (Left) Percentage of overall task responsiveness was higher than chance levels in
vCA1 → mPFC/Amy/Acb projections (Bootstrap analysis, p < 0.05). None of the othe r
projection types lay beyond confidence intervals (Bootstrap analysis, p > 0.05 for all the
projection types).
Fig. 4 (A). (Right) Percentage of overall task responsive neurons was higher in the vCA1 →
mPFC/Amy/Acb projections compared to all the remainder of the vCA1 projections pooled
together (Pearson Chi-Square test: χ2 (1, 297) = 4.72, p < 0.05).
Fig. 4 (B). (Left) Comparison of the averaged firing rates of the vCA1 projection types across the
three behavioural tasks. One-way ANOVA test (F(7,16) = 8.53, P < 0.001) revealed a significant
difference among groups (Amy: 2.15 ± 0.34 Hz, Acb: 2.41 ± 0.25 Hz, mPFC: 1.00 ± 0.31 Hz,
mPFC/Acb: 1.62 ± 0.26 Hz, mPFC/Amy: 1.32 ± 0.58 Hz, Acb/Amy: 1.68 ± 0.62 Hz,
mPFC/Amy/Acb: 5.30 ± 1.29 Hz, non identified projection: 5.24 ± 0.17 Hz). Pair wise multiple
comparisons with the Bonferroni t-test revealed significant difference between the vCA1 triple
projections and all the other projection types (P < 0.05), with the exception of the non-identified
projection group (p > 0.05).
Fig. 4 (C). Percentage of SWR-active neurons was higher than chance levels in the vCA1 →
mPFC/Amy/Acb projections (Bootstrap analysis, p < 0.05). None of the other projection types
lay beyond confidence intervals (Bootstrap analysis, p > 0.05 for all the projection types).
Fig. S5. There were no differences in the jitter of antidromic spikes before (0.24 ± 0.03 ms; mean
± s.e.m.) and after drug application (0.20 ± 0.02 ms, n = 4 cells, p > 0.05, two-tailed paired t-
test).
Fig. S8 (A). Comparison of percentage of time and entries in open and closed arms of the
elevated-plus-maze. Two-tailed, paired t-test revealed a significant difference for the percentage
of time spent in open arms vs. closed arms (open arms: 41.42 ± 4.96 %, closed arms: 58.58 ±
4.96 %, n = 16 elevated-plus-maze sessions, p < 0.05) and of entries in open vs. closed arms
(open arms: 44.57 ± 3 .48 %, closed arms: 55.43 ± 3.48 %, n = 16 elevated-plus-maze sessions, p
< 0.05). Values are expressed as mean ± s.e.m..
Fig. S8 (B). Comparison of the firing rate differences between each closed arm and each open
arm of the elevated-plus-maze for all vCA1 neurons. Difference of firing rate from mean firing
on the elevated-plus-maze was calculated for each closed arm (closed arm 1: -0.26 ± 0.09 Hz,
closed arm 2: -0.27 ± 0.08 Hz) and each open arm (open arm 1: 0.31 ± 0.09 Hz, open arm 2: 0.22
± 0.07 Hz) for all 233 vCA1 neurons. Due to non-normally distributed data, the Kruskal–Wallis
one-way ANOVA ranks test was used and revealed significant differences between the different
arm groups (H = 56.84, 3 degrees of freedom, P < 0.001). Pair wise multiple comparisons with
the Tukey test revealed significant differences between closed arm 1 and each of the open arms
(p < 0.05) and between closed arm 2 and each of the open arms (p < 0.05). There were no
significant differences between closed arm 1 and closed arm 2, and between open arm 1 and
open arm 2 (p > 0.05 for both). Values are expressed as mean ± s.e.m..
Fig. S9. (Left) Comparison of place field size between vCA1 (n = 233, 59.23 ± 1.99 %) and
dCA1 (n = 266, 48.83 ± 2.02 %) revealed significantly larger place fields in vCA1 vs. dCA1
(Mann-Whitney Rank Sum Test, p < 0.001). Values are expressed as mean ± s.e.m..
Fig. S9. (Middle) Comparison of the spatial tuning of place fields between vCA1 (n = 233, 4.26
± 0.40 Hz) and dCA1 (n = 266, 7.94 ± 0.42 Hz) revealed significantly lower spatial tuning in
vCA1 vs. dCA1 (Mann-Whitney Rank Sum Test, p < 0.001). Values are expressed as mean ±
s.e.m..
Fig. S9. (Right) Comparison of the spatial stability of place fields between vCA1 (n = 233, 0.11
± 0.02 %) and dCA1 (n = 266, 0.38 ± 0.02 %) revealed significantly less stable place fields in
vCA1 vs. dCA1 (Mann-Whitney Rank Sum Test, p < 0.001). Values are expressed as mean ±
s.e.m..
Fig. S10 (B). (Top left) A two-way repeated measures ANOVA test revealed no significant
difference within the strategy factor (allocentric and egocentric, F(1,754) = 0.88, p > 0.05).
Fig. S10 (B). (Top right) A two-way repeated measures ANOVA test revealed a significant
difference within the reward zone factor (reward zone 1, reward zone 2, (F(1,754) = 4.93, p <
0.05). Yet, none of interaction between reward zone and time led to significant results (post-hoc
Bonferroni test, p > 0.05).
Fig. S10 (B). (Bottom left) A two-way repeated measures ANOVA test revealed no significant
difference within the trajectory factor (trajectory 1, 2, 3 4, F(3,2262) = 0.54, p > 0.05).
Fig. S10 (B). (Bottom right) A two-way repeated measures ANOVA test revealed no significant
difference within the reward outcome factor (rewarded and non rewarded, F(1,754) = 3.08, p >
0.05).
Fig. S13. (Left) Percentage of task responsive neurons was higher than chance levels in vCA1 →
mPFC/Amy/Acb and vCA1 → mPFC/Acb projections (Bootstrap analysis, p < 0.05). (Right)
Percentage of task responsive neurons was higher than chance levels in double and triple vCA1
projections (Bootstrap analysis, p < 0.05). None of the other projection types lay beyond
confidence intervals (Bootstrap analysis, p > 0.05 for all the projection types).
Fig. S1. Location of the recording sites in vCA1 and dCA1 and endpoints of the optic fibres during photostimulation in mPFC, Acb and Amy.
- 4.6 mm - 4.8 mm - 5.2 mm
- 4.4 mm- 3.8 mm- 3.1 mm
- 2.6 mm1.8 mm3 mm
Recording site Optical stimulation site
1 mm
mPFC Acb Amy
dCA1 dCA1 dCA1
vCA1 and dCA1 vCA1 vCA1
Fig. S2. For each projection type three neurons with light-induced spikes from multisynaptic pathways are shown (green rectangles), which have a longer latency and larger jitter compared to light-induced antidromic spikes (red rectangles).
-50 -40-30-20-10 0 10 20 30 40 50
Tria
ls
0
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10
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ls
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ls
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50
Optic fibre targeting the Amy:three different vCA1 cells
Optic fibre targeting the Acb :three different vCA1 cells
Optic fibre targeting the mPFC:three different vCA1 cells
Latency (ms) Latency (ms) Latency (ms)
= light-induced antidromic spikes= light-induced spikes from mutli-synaptic pathways
Fig. S3. For each projection type five neurons with increasing antidromic activation latencies are shown. Note that the jitter of the antidromically evoked spikes does not increase with longer latencies. Activation latencies reflect axonal geometries, paths and conduction time, and might be different for individual vCA1 pyramidal cells (22).
-50 -40-30-20-10 0 10 20 30 40 50
Tria
ls
0
10
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Tria
ls
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-50-40-30-20-10 0 10 20 30 40 500
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ls
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-50-40-30-20-10 0 10 20 30 40 500
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Tria
ls
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10
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50
-50-40-30-20-10 0 10 20 30 40 500
10
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50
-50-40-30-20-10 0 10 20 30 40 50
Tria
ls
0
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50
Latency (ms)
-50 -40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
Latency (ms)
-50-40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
Latency (ms)
Latency: 4 ms
Latency: 9 ms
Latency: 12 ms
Latency: 7 ms
Latency: 11 ms
Latency: 7 ms
Latency: 9 ms Latency: 11 ms
Latency: 13 ms
Latency: 15 ms Latency: 17 ms
Latency: 15 ms
Latency: 8 ms
Latency: 13 ms
Latency: 11 ms
Five different vCA1 Amyproj. neurons
Five different vCA1 Acbproj. neurons
Five different vCA1 mPFCproj. neurons
Fig. S4. (A) For each projection type the spikes of a neuron induced with different laser powers are shown. Note that the latencies increased with lower laser power. (B) For the majority of projecting neurons, such a shift in spike latencies with different laser powers were observed which is consistent with a previous report on optogenetically induced spikes (38).
-50 -40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
-50-40-30-20-10 0 10 20 30 40 50
Tria
ls
0
10
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50
-50-40-30-20-10 0 10 20 30 40 500
10
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-50-40-30-20-10 0 10 20 30 40 50
Tria
ls
0
10
20
30
40
50
-50-40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
Latency (ms) Latency (ms)
Estimated power: 15 mWLatency: 8.0 ms
Late
ncy
chan
gew
ithpo
wer
chan
ge(%
neur
ons)
0
50
100
vCA1 Amy projections vCA1 Acb projections vCA1 mPFC projections
-50-40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
-50-40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
-50-40-30-20-10 0 10 20 30 40 500
10
20
30
40
50
Latency (ms)
Estimated power: 3 mWLatency: 10.8 ms
vCA1 mPFCproj. neuron
vCA1 Acbproj. neuron
vCA1 Amyproj. neuron
-50 -40-30-20-10 0 10 20 30 40 50
Tria
ls
0
10
20
30
40
50Estimated power: 30 mWLatency: 4.3 ms
Estimated power: 20 mWLatency: 8.2 ms
Estimated power: 7 mWLatency: 8.8 ms
Estimated power: 30 mWLatency: 7.4 ms
Estimated power: 20 mWLatency: 11.6 ms
Estimated power: 7 mWLatency: 12.9 ms
Estimated power: 30 mWLatency: 10.5 ms
0
50
100
0
50
100
17/21 64/79 39/52
A
B
= light-induced antidromic spikes
Fig. S5. Spontaneous and light-induced spiking of four vCA1 neurons before and after infusion of synaptic transmission blockers in vCA1 and the frontal cortex of an anesthetised rat. In the presence of NBQX and APV, spontaneous spiking and light-induced orthodromic spikes (B, green box) are abolished, while light-induced antidromic spiking (A; red box) remains stable. There are no differences in the jitter of antidromic spikes before (0.24 ± 0.03 ms; mean ± s.e.m.) and after drug application (0.20 ± 0.02 ms, n = 4 cells, p > 0.05, two-tailed paired t-test).
light period, same laser power before and after NBQX+APV
NBQX+ APV
-100 -50 0 50 1000
25
50
-100 -50 0 50 1000
25
50
NBQX+ APV
-100 -50 0 50 1000
25
50
Latency (ms)
-100 -50 0 50 1000
25
50
Latency (ms)
A
B
= light-induced antidromic spikes
= light-induced orthodromic spikes
NBQX+ APV
-100 -50 0 50 1000
25
50
-100 -50 0 50 1000
25
50
Latency (ms)Latency (ms)
NBQX+ APV
-100 -50 0 50 1000
25
50
-100 -50 0 50 1000
25
50
Tria
lsTr
ials
Tria
lsTr
ials
Fig. S6. (A) Spike waveform comparison between spontaneous and light-induced activity in an optogenetically identified vCA1 projection neuron. Scale bars: 400 µs, 50 µV. (B) Correlation values of spontaneous and light-induced spike waveforms of all vCA1 projection neurons.
Waveform similarity (r)0.90 0.95 1.00
Cou
nt
0
10
20
30
r = 0.994
LightSpont.
A B
Fig. S7. Measures of antidromic spike responses across individual vCA1 projection neurons targeting Amy, Acb or mPFC. (A) Percentage of vCA1 → Amy projection neurons with spike collision, low spike jitter, early spike latency, high spike fidelity and with the ability to follow high frequency stimulations. (B) Same quantification as in (A) for vCA1 projection neurons targeting Acb. (C) Same quantification as in (A) for vCA1 projection neurons targeting mPFC. Projection neurons without spontaneous activity during stimulation or with collisions occurring during the refractory period of a spontaneous spike before antidromic activation (< 2 ms) are defined as not testable collisions. Absolute numbers per category are indicated.
Perc
enta
ge
0
25
50
75
100
Not testable collision
Spike collision (w/o a sequenceof 5 consecutive antidromic spikes)
Spike collision (in a sequenceof at least 5 consecutiveantidromic spikes)
11
4
6
Spikes follow 50 or 20 Hzphotostimulations
Spikes do not follow 50 or 20 hzphotostimulations
Jitter < 0.3 ms Spike fidelity > 50%
Spike fidelity < 50%
Photostimulationnot tested
12
9
21
8
8
5
Amy < 13 ms, Acb < 17 ms,mPFC < 22 ms
21
A vCA1 to Amy projections B vCA1 to Acb projections C vCA1 to mPFC projections
Collis
ion
Jitte
rLa
tenc
yFi
delity HFS
Perc
enta
ge
0
25
50
75
100
38
27
14
44
35
79
47
15
17
79
Collis
ion
Jitte
rLa
tenc
yFi
delity HFS
Perc
enta
ge
0
25
50
75
10032
11
9
31
21
5231
12
9
52
Collis
ion
Jitte
rLa
tenc
yFi
delity HFS
Spike collision: Spike jitter: Spike latency: Spike fidelity: High frequency stimulation(HFS):
Fig. S8. Specificity of anxiety-related firing in the vCA1. (A) (Left) Trajectory of a rat during elevated-plus-maze exploration and (right) percentage of time spent and entries in the open and closed arms of the elevated-plus-maze for all anxiety sessions (n = 16). Error bars indicate mean ± s.e.m. (B) (Left) Firing rate map of an individual vCA1 neuron with higher discharge in each of the open arms compared to the closed arms. (Right) Averaged firing rates
Perc
enta
ge
0
20
40
60
80**
Open armsClosed arms
Time Entries
Closed arm
Open arm
Rat's trajectory ΔFi
ring
rate
(di ff
.fro
mm
ean,
Hz)
-0.50
-0.25
0.00
0.25
0.50
Closedarms
n = 233
**
Openarms
(open arm 1)
-8 -4 0 4 8
(ope
nar
m2)
-5
0
5
(closed arms)
-15 -10 -5 0 5 10
(ope
nar
ms)
-10
-5
0
5r2 = 0.33p < 0.001
r2 = 0.27p < 0.001
Δ Firing rate(closed arm 1)
-15 -10 -5 0 5 10
ΔFi
ring
rate
(clo
sed
arm
2)
-10
-5
0
5 r2 = 0.21p < 0.001
Min
Firingrate
Max25 cm
25 cm
BA
C Correlational analysis within standard EPM
D Correlational analysis across standard and altered EPM
Δ Firing rate(standard maze)
-12 -8 -4 0 4
Firin
gra
te(a
ltere
dm
aze)
-15-10
-505 r2 = 0.24
p < 0.001
(standard maze)
-8 -4 0 4 8
Firin
gra
te(a
ltere
dm
aze)
-15
0
15
30 r2 = 0.27p < 0.001
open - openclosed - closed
EPM score(standard maze)
-0.5 0.0 0.5 1.0
EPM
scor
e(a
ltere
dm
aze)
-0.5
0.0
0.5
1.0 r2 = 0.19p < 0.001
ΔFi
ring
rate
ΔFi
ring
rate
Δ Firing rate Δ Firing rateΔ Δ
Δ Firing rate
Cel
ls(%
)
0
25
50
**
33/23315/266
*
17/1343/70/80/24/295/144/350/4
Cel
ls(%
)
0
25
50
dCA1 vCA110/2334/266
Amy Acb mPFC mPFCAcb
mPFCAmy
AcbAmy
mPFCAmyAcb
non ident.projection
7/1340/70/80/21/291/141/350/4
Cells firing in open arms
Cells firing in closed arms
F
E
Mean EPM score ofEPM-responsive neurons
0.3 0.6 0.9
0
50
100
r2 = 0.28p < 0.05
Ope
nar
ms
-clo
sed
arm
s(%
time)
(difference from mean) of the entire vCA1 neuronal population in each arm of the elevated-plus-maze. A higher firing is observed in open arms. Error bars indicate mean ± s.e.m. (C) Correlational analysis of firing rates for all vCA1 units (n = 233) between (left) open arms, (middle) closed arms and (right) across open and closed arms. Firing rates are positively correlated within the closed and open arms, but negatively correlated across arms of different types. (D) Correlational analysis of (left) elevated-plus-maze scores, and of firing rates across (middle) closed arms and (right) open arms in the standard and altered elevated-plus-maze (n = 58 units). Elevated-plus-maze scores and firing rates are positively correlated across mazes. (E) Most of the neurons with significant elevated-plus-maze-score increase their firing when the animals are on the open arms. The histogram from Fig. 2C is here split into neurons firing more on the open or closed arms. (F) Elevated-plus-maze-scores (averaged for each behavioural session) correlate with behavioural anxiety levels measured as time spent in open or closed arms (n = 16 elevated-plus-maze sessions). Note that no differences in linear speed or acceleration (data not shown) are observed between open and closed arms (speed open arms: 22.1 ± 1.0 m/s, speed closed arms: 21.9 ± 1.3 m/s, acceleration open arms: 1.2 ± 1.4 m/s2 acceleration closed arms: -1.2 ± 1.4 m/s2, n = 16 sessions, p > 0.05, data are mean ± s.e.m.. * P < 0.05, ** P < 0.01 significance levels.
Fig. S9. Measures of place cells across the longitudinal axis of the hippocampus. (Left) Quantification of place field size, (middle) spatial tuning and (right) spatial stability in dCA1 (n = 266) and vCA1 (n = 233) neurons. vCA1 neurons have larger place fields, lower spatial tuning and stability compared to dCA1 neurons. Box plots indicate median, quartiles (boxes) and range (whiskers). *** P < 0.001 significance levels.
Plac
efie
ldsi
ze(%
area
)
0
50
100
vCA1dCA1 Spat
ialt
unin
g(Δ
Hz)
0
10
20
vCA1 dCA1
Spat
ials
tabi
lity
(r)
-0.5
0.0
0.5
1.0
vCA1dCA1
*** *** ***
Fig. S10. Goal-directed navigation task, behavioural performance and general goal-directed activity in vCA1 projection neurons. (A) (Left) Experimental design of the goal-directed navigation with changing rules. (Right) Behavioural learning curve of a rat during strategy switching predicted by a state–space model (37). Rule change was not signalled to the rat. (B) Normalised population firing rate histograms of reward zone-modulated neurons subdivided into (top left) allocentric and egocentric trials, (top right) reward zone 1 and reward zone 2 trials, (bottom left) trajectory 1, 2, 3 or 4 trials and (bottom right) rewarded and non-rewarded trials. No change is observed in any of the conditions suggesting that goal-directed firing is a general phenomenon for a subset of vCA1 projection neurons. Data are mean ± s.e.m.
Start
Start
RewardReward
Start
Start
Allocentric("go to west")
Egocentric("turn right")
Reward >< <Trials
0 20 40 60Prob
abili
tyof
aco
rrec
ttria
l
0.0
0.5
1.0egocentricallocentricA
Switch
mean performance95th confidenceinterval
incorrect trial
correct trial
-2 -1 0 1 2 3 4
-1012
-2 -1 0 1 2 3 4
-1012
-2 -1 0 1 2 3 4
-1012 allocentric
egocentric
Firin
gra
te(Z
-sco
reno
rm.)
Time (s)
-2 -1 0 1 2 3 4
-1012
traj. 1
-2 -1 0 1 2 3 4
-1012
-2 -1 0 1 2 3 4
-1012
-2 -1 0 1 2 3 4
-1012
traj. 3traj. 2traj. 4
Time (s)
-2 -1 0 1 2 3 4
-1012 rewarded
non-rewarded
reward zone 1reward zone 2
-2 -1 0 1 2 3 4
-1012
-2 -1 0 1 2 3 4
-1012
B
Firin
gra
te(Z
-sco
reno
rm.)
Firin
gra
te(Z
-sco
reno
rm.)
Firin
gra
te(Z
-sco
reno
rm.)
<
Fig. S11. Correlational analysis of firing rates versus locomotor activity in vCA1. (Left) Low correlation values of firing rates (n = 233 units) with the speed or (right) the acceleration of the rats.
Firing rate vs.acceleration correlation (r)
-1.0 -0.5 0.0 0.5 1.0
Cou
nt0
50100150200250
Firing rate vs.speed correlation (r)
-1.0 -0.5 0.0 0.5 1.0
Cou
nt
050
100150200250
Fig. S12. The number of vCA1 projection neurons and the percentages of anxiety-related, place and reward zone-responsive cells are shown for each of the four recorded rats individually. Projections significantly enriched in tasks responsive neurons are marked with an arrow. Note that the observed differences in task responsiveness are not due to a strong sampling bias originating from a single animal. Note that the percentages of anxiety-related, place and reward zone-responsive cells were not different (data not shown) when comparing the cells with faster antidromic activation latencies (0-50th percentile) to all cells (chi-square tests, p > 0.05).
Anxi
ety
cells
(%)
0
10
20
30
40
50
Plac
ece
lls(%
)
0
20
40
60
Rew
ard-
zone
mod
ulat
edce
lls(%
)
0
20
40
60
Amy Acb mPFC mPFCAcb
mPFCAmy
mPFCAmyAcb
AcbAmy
Num
ber o
f pro
ject
ions
0
10
20
30
40
rat 1 rat 2 rat 3 rat 4
Fig. S13. Higher overall behavioural responsiveness of vCA1 triple projections compared to chance level and (right) to the other vCA1 projection types pooled together. Numbers indicate the ratio of task-responsive neurons in each group. This is the same plot as in Fig. 4A but here each neuron counts only as 1 irrespective if it responds to one, two or all three tasks. Note the stepwise increase in task-responsiveness along with increase in target numbers. * P < 0.05 significance level.
Task
resp
.(%
)
0
20
40
60
80
100
Amy Acb mPFC mPFCAcb
mPFCAmy
mPFCAmyAcb
non ident.projection
AcbAmy
*
58/1346/75/81/218/296/1416/351/4
*
58/1346/724/3923/53
**
Tripleproj.
non ident.projection
Singleproj.
Doubleproj.
Fig. S14. Percentages of anxiety-related, place, reward zone-excited or reward zone-inhibited neurons for each projection type. Bootstrap analysis indicates that reward zone-inhibited cells are significantly enriched in neurons projecting to Acb, while anxiety-related cells are significantly enriched in neurons projecting to mPFC (p < 0.05). This analysis for single projections across different tasks reaches similar conclusion as the analysis of different projections for a single task shown in Figs. 2 and 3.
Task
resp
.cel
ls(%
)
0
20
40
60
Amy(n = 4)
Acb(n = 35)
mPFC(n = 14)
mPFCAcb
(n = 29)
mPFCAmy(n = 2)
mPFCAmyAcb
(n = 7)
non ident.projection
(n = 134)
AcbAmy(n = 8)
**
0 1 0 1 5 4 1 10 6 1 1 1 5 3 6 1 0 1 0 0 0 0 1 3 3 3 1 1 24 20 11 22
Anxiety cells Place cells Reward zone-excited cells
Reward zone-inhibited cells
Table S1. The overlap of task-responsive cells is listed for each projection type. Note the low number of cells responding to more than one task. In particular, out of the 11 vCA1-> Acb reward zone-modulated neurons, only 3/11 are also responsive in the other two tasks. Similarly, out of the 6 vCA1-> mPFC anxiety neurons, only 1/6 is also responsive in the other two tasks. Out of the 7 vCA1-> mPFC/Acb reward zone-modulated neurons, only 1/7 is also responsive in the other two tasks.
Amy(n = 4)
Acb(n = 35)
mPFC(n = 14)
mPFCAcb
(n = 29)
mPFCAmy(n = 2)
mPFCAmyAcb(n = 7)
non ident.projection
(n = 134)
AcbAmy(n = 8)
Anxiety/place
Anxiety/goal
Place/goal
Anxiety/place/goal
Ove
rlap
of ta
skre
spon
sive
cells
0 1 1 1 0 0 0 3
1 2 1 1 0 0 1 2
0 2 2 1 0 0 1 10
0 1 0 1 0 0 0 0
Projection type
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