Cerebral Cortex Advance Access published March 20, 2014
Cerebral Cortex
doi:10.1093/cercor/bhu054
A Differential and Timed Contribution of Identified Hippocampal Synapses to Associative
Learning in Mice
Agnès Gruart, Raudel Sánchez-Campusano, Azahara Fernández-Guizán and José M. Delgado-García
Division of Neurosciences, Pablo de Olavide University, Seville 41013, Spain
Address correspondence to Prof. José M. Delgado-García, División de Neurociencias, Universidad Pablo de Olavide, Ctra. de Utrera, km. 1,
41013-Sevilla, Spain. Email: jmdelgar@upo.es
Although it is generally assumed that the hippocampus is involved in
associative learning, the specific contribution of the different sy-
napses present in its intrinsic circuit or comprising its afferents and
efferents is poorly defined. We studied here activity-dependent
changes in synaptic strength of 9 hippocampal synapses (corre-
sponding to the intrinsic hippocampal circuitry and to its main inputs
and outputs) during the acquisition of a trace eyeblink conditioning
in behaving mice. The timing and intensity of synaptic changes
across the acquisition process was determined. The evolution of
these timed changes in synaptic strength indicated that their func-
tional organization did not coincide with their sequential distribution
according to anatomical criteria and connectivity. Furthermore, we
explored the functional relevance of the extrinsic and intrinsic affer-
ents to CA3 and CA1 pyramidal neurons, and evaluated the distinct
input patterns to the intrinsic hippocampal circuit. Results confirm
that the acquisition of a classical eyeblink conditioning is a multisy-
naptic process in which the contribution of each synaptic contact is
different in strength, and takes place at different moments across
learning. Thus, the precise and timed activation of multiple hippo-
campal synaptic contacts during classical eyeblink conditioning
evokes a specific, dynamic map of functional synaptic states in that
circuit.
Keywords: associative learning, behaving mice, hippocampal networks,
learning-dependent synaptic plasticity, LTP
Introduction
It is commonly accepted that learning and memory processes
are able to evoke more-or-less stable changes in synaptic
weights in selected cortical and subcortical neural sites (Bliss
and Collingridge 1993; Kandel 2001; Neves et al. 2008; Wang
and Morris 2010)—that is, that newly acquired motor and/or
cognitive abilities are stored in the brain in the form of func-
tional and/or structural modifications of synaptic efficiency
(Ramón y Cajal 1909–1911; Konorski 1948; Hebb 1949). For
example, it has been shown that the hippocampal-dependent
trace eyeblink conditioning (Weiss et al. 2005; Bangasser and
Shors 2007), a form of associative learning, evokes a concomi-
tant change in strength at the hippocampal perforant pathway
(PP)–dentate gyrus (DG) (Weisz et al. 1984) and CA3–CA1
(Gruart et al. 2006) synapses of behaving mice. It has also
been shown that these changes in synaptic strength share
many properties with experimentally evoked long-term poten-
tiation (LTP) (Bliss and Collingridge 1993; Kandel 2001; Gruart
et al. 2006; Wang and Morris 2010). However, a still-open
question is whether all hippocampal synapses included in its
intrinsic circuit, and those involved in its main inputs and
outputs, present similar increases in strength and with similar
changing rates across the learning process.
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The hippocampus seems to participate in many different
functions, such as Pavlovian associative learning (Berger et al.
1983; Moyer et al. 1990; McEchron and Disterhoft 1997; Gruart
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et al. 2006), spatial orientation (Moser et al. 2008), object rec-
ognition (Clarke et al. 2010), and other forms of memory
acquisition, storage, and retrieval (Bliss and Collingridge 1993;
Neves et al. 2008; Wang and Morris 2010). In this regard, it is
unresolved whether those different functions are carried out
by localized sites within the hippocampal circuits (McHugh
et al. 2007; Nakashiba et al. 2008), or are dependent upon the
specific, timed activation of the multiple synaptic contacts
present in those circuits.
We have addressed these important questions here, studying
the evolution in strength of 9 different hippocampal synapses
during the hippocampal-dependent trace eyeblink condition-
ing in behaving mice. For this, animals were chronically im-
planted with stimulating and recording electrodes at selected
sites of the intrinsic hippocampal circuit and in the main hip-
pocampal afferent or projecting sites. First, we determined the
basic functional properties of the selected synapses—namely,
input/output curves, paired-pulse facilitation, and LTP evoked
by high-frequency stimulation (HFS) of presynaptic neurons
(see Madroñal et al. 2009). For eyeblink conditioning, animals
were presented with a tone as a conditioned stimulus (CS)
followed 500 ms from its end by an electrical shock of the
trigeminal nerve as an unconditioned stimulus (US). CRs were
determined from the electromyographic (EMG) activity of the
ipsilateral orbicularis oculi muscle. Field excitatory postsyn-
aptic potentials (fEPSPs) were evoked following CS presenta-
tions across training sessions in all of the selected synapses.
Results indicate that there is a precise and quantifiable acti-
vation sequence of the multiple hippocampal synaptic contacts
that takes place during the acquisition and extinction of a
classical eyeblink conditioning, involving a dynamic map of
synaptic-learning states.
Material and Methods
Experimental Animals
Experiments were carried out in C57Bl/6 male adult mice (3–4 months
old; 25–30 g) obtained from an official supplier (University of Granada
Animal House, Granada, Spain). Upon arrival, animals were housed in
separate cages (n = 8 per cage), but they were switched to individual
cages after surgery. Mice were kept on a 12-h light/dark cycle with con-
stant ambient temperature (21.5 ± 1 °C) and humidity (55 ± 8%). Food
and water were available ad libitum. Experiments were carried out in
accordance with the guidelines of the European Union (2003/65/CE)
and Spanish regulations (BOE 252/34367–91, 2005) for the use of lab-
oratory animals in chronic studies. All experimental protocols were
also approved by the local Ethics Committee.
 Animals were prepared for the chronic recording of the EMG
activity of the orbicularis oculi muscle and fEPSPs evoked at 9
different hippocampal synapses during classical conditioning of
eyelid responses (Figs. 1 and 2). Only animals that reached all be-
havioral criteria and with proper electrode placement and expected
field potential recordings were analyzed. We considered successful
those animals that finished the experimental protocols presenting
extracellular recordings (i.e., fEPSPs and/or LFPs) that did not
deteriorate over time. A total of 10 animals per group (i.e., per
selected synapse) were used for classical conditioning and fEPSP
recordings. Some additional animals (n = 16) were used in a prelimi-
nary study to determine the most appropriate recording and stimu-
lating sites (Fig. 3). Finally, 10 animals/selected synapse were used
for input/output curves, paired-pulse facilitation, and the LTP study
(Fig. 4). In accordance, a grand total of 196 mice were used in this
study.

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Figure 1. Experimental design. (A) Example of stimulating and recording electrodes aimed to activate the right hippocampal CA3–CA1 synapse. Superimposed recordings at the
top left illustrate fEPSPs recorded in the stratum radiatum of the CA1 area after electrical stimulation (St.) of Schaffer (Sch.) collaterals. EMG recording electrodes were implanted in
the orbicularis oculi (OO) muscle of the left upper eyelid, and stimulating electrodes were implanted on the trigeminal nerve for US presentation. For classical eyeblink conditioning,
we used a tone (20 ms; 2.4 kHz; 85 dB) as CS. Superimposed recordings at the bottom left correspond to the blink reflex evoked in the OO muscle by the electrical stimulation of
the trigeminal nerve. Note the 2 short-(R1) and long-(R2) latency components characterizing the blink reflex in mammals. DG, dentate gyrus; PP, perforant pathway. (B) A
representation of the conditioning paradigm, illustrating CS and US stimuli, and the moment at which a pair of pulses (100 μs; square; biphasic) was presented to Schaffer
collaterals (St. Hippocampus). An example of an EMG recording from the OO muscle obtained from the seventh conditioning session is illustrated, as well as an extracellular
recording of hippocampal activity from the same animal, session, and trial. Note the fEPSPs evoked in the CA1 layer following paired-pulse stimulation of Schaffer collaterals. (C)
Typical learning curve (expressed as % CRs) calculated from the EMG activity of the OO muscle. (D) At the top are illustrated fEPSPs evoked at the CA3–CA1 synapse by
paired-pulse presentations at the moments (1–3) indicated below. The graphs at the bottom show the evolution of synaptic strengths (CA3–CA1 synapse) for the 2 evoked fEPSPs
(first, left ordinate; second, right ordinate) during the successive training sessions.

2 Hippocampal Synapses and Associative Learning • Gruart et al.

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Figure 2. Location of the stimulating and recording sites. (A) Selected sites for the implantation of stimulating (red dots) and recording (blue dots) electrodes corresponding to the
9 synapses included in the study. Diagrams and coordinates relate to the Paxinos and Franklin Atlas 2001. (B,C) Representative photomicrographs illustrating the final location of
stimulating (B) and recording (C) electrodes. Electrode tracts are indicated by red (stimulating) or blue (recording) arrows. Calibration is indicated in the bottom middle
photomicrograph. Abbreviations: D, L, M, and V, represent dorsal, lateral, medial, and ventral, respectively; CPv, caudate-putamen nuclei; DG, dentate gyrus; D3V, third ventricle,
dorsal part; fmi, forceps minor of the corpus callosum; IL, infralimbic cortex; LV, lateral ventricle; M1, M2, motor cortex; PAG, periaqueductal gray; PP, perforant pathway; PrL,
prelimbic cortex; REU, thalamic reuniens nucleus; SUB, subiculum. In green are indicated the horizontal coordinates for cCA3 implanted electrodes.

Surgery
Animals were anesthetized with 0.8–1.5% isoflurane delivered via a
mouse anesthesia mask (David Kopf Instruments, Tujunga, CA, USA).
The anesthetic gas was supplied from a calibrated Fluotec 5
(Fluotec-Ohmeda, Tewksbury, MA, USA) vaporizer, at a flow rate of
1–2 L/min oxygen (AstraZeneca, Madrid, Spain). The 9 synapses
selected for this study were the following: PP–DG, DG–CA3, PP–CA3,
CA3–CA1, contralateral CA3 (cCA3)–CA1, thalamic reuniens nucleus
(REU)–CA1, PP–CA1, CA1–subiculum (SUB), and CA1–medial prefron-
tal cortex (mPFC). As shown in the diagrams included in Figure 2A,
animals were implanted with bipolar stimulating and recording elec-
trodes at the selected sites, following the corresponding stereotaxic
coordinates (Paxinos and Franklin 2001). Electrodes were made from
50 µm, Teflon-coated, tungsten wire (Advent Research, Eynsham, UK).
A bare silver wire was affixed to the occipital bone as ground. All the
implanted wires were soldered to a 6-pin socket (RS Amidata, Madrid,
Spain) and were then fixed to the skull with dental cement (Fig. 1A).
Further details of this chronic preparation are indicated elsewhere
(Gruart et al. 2006).
Recording and Stimulation Procedures
EMG recordings were carried out with the help of Grass P511 differen-
tial amplifiers within a bandwidth of 0.1 Hz–10 kHz (Grass-Telefactor,
West Warwick, RI, USA). Field EPSP recordings were carried out with a
high-impedance probe (2 × 1012 Ω, 10 pF). Electrodes were surgically
implanted in the selected hippocampal and prefrontal areas using as a
guide the field-potential depth profile evoked by paired (40 ms of in-
terval) pulses presented at the corresponding synaptic input (Fig. 3).
The recording electrode was fixed at the site where a reliable monosy-
naptic fEPSP was recorded (Fig. 2B). Synaptic field potentials were
evoked in the 9 selected synapses during habituation, conditioning,
and extinction sessions by paired pulses (100 µs in duration, 40 ms of
interpulse interval) applied to Schaffer collaterals 300 ms after CS pres-
entation. Stimulus intensities ranged from 50 to 350 µA. For each
animal, the stimulus intensity was set usually at 30–40% of the intensity
necessary to evoke a maximum fEPSP response (Gureviciene et al.
2004, Fig. 4A). An additional criterion for selecting stimulus intensity
was that the second stimulus, presented 40 ms after the conditioning
pulse, evoked a larger (>20%) synaptic field potential (Bliss and
Gardner-Medwin 1973, Fig. 1B,D).
For input/output curves (Fig. 4A), animals were stimulated with
single pulses at increasing intensities (0.02–0.4 mA, in steps of 0.02
mA). We also checked the effects of paired pulses at different (10, 20,
40, 100, 200, and 500 ms) interstimulus intervals while using intensi-
ties corresponding to 40% of the amount necessary to evoke a saturat-
ing response (Fig. 4B).

Cerebral Cortex 3
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Figure 3. Selective examples of preliminary depth-profile recordings carried out to facilitate the identification of fEPSPs evoked at the 9 synapses included in this study. (A),
Depth-profile representation of fEPSPs evoked in the hippocampal CA1 area by paired-pulse stimulation (40 ms of interpulse interval, red arrows) of ipsilateral Schaffer collaterals
(CA3 stimulation). Stimulus intensity was set at 40% of the intensity (mA) necessary to evoke maximum fEPSP responses. Recordings were carried out in steps of 100 µm. (B) A
diagrammatic representation of the location of somas and dendrites corresponding to CA1 pyramidal cells and to dentate granule neurons. The different strata are indicated. (C)
Another field-potential depth profile corresponding to fEPSPs evoked in the CA1 and dentate gyrus areas by the paired-pulse stimulation of the perforant pathway. Calibrations in (A)
are also for (C).

For evoking LTP, each animal was presented with five 200 Hz,
100 ms trains of pulses at a rate of 1/s. This HFS protocol was pre-
sented 6 times in total, at intervals of 1 min. The 100 µs, square, bipha-
sic pulses used to evoke LTP were applied at the same intensity used
for the single pulse presented during baseline and post-HFS record-
ings. Baseline records were collected at a rate of 3/min for the 15 min
preceding HFS presentation. After the HFS protocol, recordings at the
same stimulus intensity and frequency were collected for 2 h, and then
for 15 min on each of the 2 following days (Fig. 4C).
Classical Eyeblink Conditioning
For recordings, 2 animals at a time were placed in separate small (5 ×
5 × 10 cm) plastic chambers located inside a Faraday box (30 × 30 ×
20 cm). Classical conditioning was achieved using a trace paradigm
(Fig. 1B). For this, a tone (20 ms, 2.4 kHz, 85 dB) was presented to
the animals as a CS. The US consisted of a 500 µs, 3× threshold,
square, cathodal pulse. The US started 500 ms after the end of the CS.
A total of 2 habituation, 10 conditioning, and 5 extinction sessions
were carried out for each animal (Fig. 1C). A conditioning session
consisted of 66 CS–US presentations, and lasted ≈30 min. In 10% of
cases, the CS was presented alone. CS–US presentations were separ-
ated at random by 30 ± 5 s. For habituation and extinction sessions,
only the CS was presented, also for 60 times per session at intervals of
30 ± 5 s. As criterion, we considered a “CR” the presence, during the
CS–US period, of EMG activity lasting >10 ms and initiated >50 ms
after the CS onset. In addition, the integrated EMG activity recorded
during the CS–US interval had to be at least 2.5 times greater than the
averaged activity recorded immediately before CS presentation
(Gruart et al. 2006).
Field EPSPs were evoked at the 9 selected synapses 300 ms after CS
presentations across every habituation, conditioning, and extinction
sessions (Figs 1B,D and 5A,B).
Histology
At the end of the experiments, mice were deeply re-anesthetized
(sodium pentobarbital, 50 mg/kg) and perfused transcardially with
saline and 4% phosphate-buffered paraformaldehyde. Selected sec-
tions (50 µm) including the implanted wires were mounted on gelati-
nized glass slides and stained using the Nissl technique with 0.1%
Toluidine Blue, to determine the proper location of stimulating and
recording electrodes (Fig. 2B).
Data Collection and Analysis
EMG and hippocampal activity, and 1 V rectangular pulses correspond-
ing to CS and US presentations, were stored digitally on a computer
through an analog/digital converter (CED 1401 Plus; Cambridge Elec-
tronic Design, Cambridge, UK), at a sampling frequency of 11–22 kHz
and an amplitude resolution of 12 bits. Commercial computer pro-
grams (Spike 2 and SIGAVG, from Cambridge Electronic Design) were
modified to represent EMG and extracellular synaptic field potential re-
cordings. Data were analyzed off-line for quantification of CRs and the
fEPSP slopes (Gruart et al. 2006). The slope of evoked fEPSPs was

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Figure 4. Functional properties of the 9 synapses included in this study. Illustrated data were collected from implanted animals (n = 10 per group) that passed the 3 successive
experimental tests: input/output curves, paired-pulse facilitation, and LTP. (A), Input/output curves evoked at the 9 selected synapses. Each circle represents the mean value (with its
corresponding SEM) computed from 50 stimulus presentations (5 per experimental animal). (B), Field EPSP facilitation evoked in the 9 synapses. Data shown are mean ± SEM
slopes of the second fEPSP expressed as a percentage of the first for the 6 interpulse intervals. Significant fEPSP facilitation was evoked at short (10–40 ms) interstimulus intervals
in most of the synapses, but some of them presented facilitation at large intervals (*, P < 0.05). (C) An attempt was made to evoke LTP in the 9 synapses by HFS of the
corresponding presynaptic sites. The HFS consisted of 5 trains (200 Hz, 100 ms) of pulses at a rate of 1/s. This protocol was presented 6 times in total, at intervals of 1 min. In
order to obtain a baseline, animals were stimulated every 20 s for 15 min in the afferent pathways. After HFS, the same single stimulus was presented at the initial rate (3/min) for
another 120 min. Recording sessions were repeated on 2 additional days (15 min each). Note that while most synapses presented significant increases in fEPSP slopes after HFS,
some of them presented a low (PP–CA1) or delayed (CA1–mPFC) potentiation, or a significant depression (REU–CA1). *, P < 0.05.

Cerebral Cortex 5
collected as the ratio between the difference of amplitudes (in mV) and
the corresponding difference of times (in ms) for the selected points
from fEPSP recordings (i.e., mV/ms). The relatively different evolution
of fEPSP slopes evoked by the 2 pulses presented during the CS–US in-
tervals was calculated as a second/first × 100 ratio (see Madroñal et al.
2009).
Only data from successful animals (i.e., those that allowed a com-
plete study with a proper functioning of both recording and stimulat-
ing systems) were computed and analyzed. All the analyses were
developed with the help of homemade quantification and represen-
tation programs. The computational tools, the analytical approach of
synaptic-learning-state functions, and the timing–strength evolution
indices proposed here (equations 1–3) were designed and developed
with the help of modified versions of the MATLAB (The MathWorks,
Natick, MA, USA) functions, as well as, by means of new applications
(see Sánchez-Campusano et al. 2009; 2011; 2012) and customized
scripts (Figs 5–8).
Computed results were processed for statistical analysis using the
Sigma Plot 11.0 package (Sigma Plot, San Jose, CA, USA) and the
MATLAB Toolbox (“Multivariate Statistics” and “Circular Statistics”
[Berens 2009] tools) for Windows. For multivariate statistics assess-
ments, both parametric (analysis of variance [ANOVA] F-tests, with or
without repeated-measures [RM]) and nonparametric (ANOVA tests on
Ranks, with RM [Friedman RM ANOVA] or without RM [Kruskal–Wallis
ANOVA]) methods were used to assess the statistical significance of differ-
ences among groups, followed by the appropriate test (Holm-Sidak, or
Tukey, or Student–Newman–Keuls, in this order of priority) for all the
pairwise multiple-comparison analyses.
When the normality (Shapiro–Wilk, or Kolmogorov–Smirnov test)
and equal variance of the errors (Levene Median test) assumptions
were satisfied, the statistic F½ðm1Þ;ðm1Þðn1Þ;ðlmÞ ; with its correspond-
ing orders m (number of groups), n (number of animals), and l
(number of multivariate observations), was reported (Sánchez-
Campusano et al. 2009). For ANOVA F-test with/without RM, the first
number (m−1) represents the variability between groups (i.e., the
variability due to the differences among the column means) and deter-
mines the numerator degree of freedom in the F-distribution table. The
second and the third numbers in the bracket following the F statistic
represent the variability within groups (i.e., the variability due to the
differences between the data in each column and the column mean).
For ANOVA F-test with RM the second number (m − 1) × (n − 1) deter-
mines the denominator degree of freedom in the F-distribution table,
while the third number informs about the number of multivariate
observations. For ANOVA F-test without RM, the second number
informs about the number of experimental subjects, while the third
number (l − m) determines the denominator degree of freedom in the
F-distribution table (Hogg and Ledolter 1987). The factors related to
the statistical model proposed here were the training phase and days,
corresponding to 1-way or 2-way ANOVA F-tests.
When the normality assumption was not verified, the significance
(P value) of χ 2 statistic was calculated using the ranks of the data rather
than their numeric values. Ranks are found by ordering the data from
smallest to largest across all groups, and taking the numeric index of
this ordering. The rank for a tied observation is equal to the average
rank of all observations tied with it. Note that ANOVA test on Ranks is a
nonparametric version of the classical ANOVA F-test, and an extension
of the Wilcoxon rank sum test to >2 groups. Thus, the usual F statistic
is replaced by a χ 2 statistic (Hollander and Wolfe 1999). Finally, the
parametric and nonparametric methods were also applied to the data
taking in to account the sessions/days as repeated measures.
For the circular statistics inferences (Batschelet 1981; Mardia and
Jupp 1999; Berens 2009; Sánchez-Campusano et al. 2011), we used
both the Rayleigh and the Watson hypothesis tests, in addition to the
von Mises distribution (the circular analog of the normal distribution),
for the probability density of the circular mean of the time data and the
corresponding circular dispersion indices: s  s for the simultaneous
timing–strength characterization of each synapse (see eq. 1, Figs 6A
and 7D, and Supplementary Table 1 and Supplementary Material,
Appendix A) and relative dispersion index (RDI) for the relative
dispersion patterns between different synapses (see eq. 2, Figs 6A
and 7D, and Supplementary Table 2 and Supplementary Material,
6 Hippocampal Synapses and Associative Learning • Gruart et al.
Appendix A). Regression analysis was used to study the evolutions of
the percentage of CRs (Figs 1C, 5B, and 7C) and the fEPSP slopes
(Figs 1D, 5A, and 7A,B) across conditioning, and for the trend of the sy-
naptic evolution index (SEI) (see eq. 3, Figs 6B,C, and 8, and Sup-
plementary Table 3 and Supplementary Material, Appendix B).
In general, for all the statistical tests (multivariate, regression, and
circular statistics), the significance level (P) was indicated. It is
common to declare a result significant if the P value is <0.05 (*), 0.01
(**), or 0.001 (***). In particular, for each regression test, the correlation
coefficient (r), and the parameters and equation of the best poly-
nomial/sigmoidal fit, were reported. Unless otherwise indicated, data
are represented by the mean ± standard error of mean (SEM). The
different timing–strength evolution indices proposed in this work are
defined below:
Timing–strength dispersion index s  sðijrÞ for the ith-synapse or for
the rth-circular pattern of the % CRs:
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1  r s (ijr)
s
 s (ijr) ¼ 2 : ð1Þ
2½C s (ijr)
Relative dispersion index RDIi1,i2|r between the circular timing–
strength patterns of 2 different (i1 and i2) synapses or between the
i1th-synapse and the rth-circular pattern for the level of expression of
the CRs:
  2
s
 s(i1) 1  rs(i1) C s(i2jr)
RDI i 1;i2jr ¼ ¼ : ð2Þ
s
 s (i2jr) 1  rs(i2jr) Cs(i1)
SEI (in the same unit of measurement as the fEPSP slope —i.e., mV/
ms, or the % of baseline) was designed to estimate the changes
between different synaptic-learning states of the same synapse or to
measure the dynamic changes between different synaptic-learning
states of different synapses:
1;T 2
T2
Si2 - SiT1
SEI Ti 1;i2 ¼ 1
: ð3Þ
T 2- T 1
In equations 1 and 2, rs is the circular kurtosis and C s is the radius of
the circumference that describes the centroid with respect to the
origin. In equation 3, Si1 T1
and STi22 represent the synaptic strengths
(fEPSP slopes, as % of baseline) at the synaptic-learning states
[Si1 ð j1Þ; T ð j1Þ and ½Si2 ð j2Þ; T ð j2Þ; respectively. Notice that the evol-
ution index SEI measures the simultaneous changes in the 2 physio-
logical variables of each synapse—that is, the timing T( j) and the
synaptic strength Si ( j), as well as the relative dynamic evolution
between different synapses at different learning states. For further
details about these analytical approaches, the readers may refer to Sup-
plementary Material (see Supplementary Tables 1–3, and Appendices
A and B).
Results
Basic Functional Properties of Hippocampal Synapses
Included in This Study
Mice were prepared for the chronic recording of fEPSPs evoked
at the 9 selected synapses belonging to the hippocampal intrin-
sic circuit or involved in its main inputs and outputs (Fig 1A)
during classical conditioning of eyelid responses. The final
location of recording and stimulating sites was checked with his-
tological procedures at the end of the recording sessions
(Fig. 2). Nevertheless, during surgery, the electrode location in
the selected recording or stimulating sites was decided with the
help of field-potential depth profiles collected in a preliminary
set of experiments (Fig. 3).
In a first series of experiments (n = 10 mice/group) we
checked the general functional properties of the 9 selected sy-
napses. Input/output curves presented increased fEPSP slopes
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Figure 5. Synaptic-learning states (colored rectangles) and multiple-comparison analyses of their synaptic strengths. (A,B) Evolution of fEPSP slopes (as % of baseline, see A) collected
at 9 hippocampal synapses, and CRs (% CRs, see B) across the successive training sessions. Selected synapses were: 1, perforant pathway (PP)–dentate gyrus (DG); 2, DG–CA3; 3,
PP–CA3; 4, CA3–CA1; 5, contralateral CA3 (cCA3)–CA1; 6, thalamic reuniens nucleus (REU)–CA1; 7, PP–CA1; 8, CA1–subiculum (SUB); and 9, CA1–medial prefrontal cortex (mPFC).
Note that some synapses increased in learning-dependent strength across training (PP–DG, CA3–CA1, cCA3–CA1, REU–CA1, and CA1–SUB), whilst for others their strengths increased
at the beginning of the conditioning sessions and decreased at the end (DG–CA3, and PP–CA3), did not present any significant change (PP–CA1), or decreased across training (CA1–
mPFC). See code color bars to the right of (A) and (B). (C) A diagram of hippocampal synapses included in this study, indicating their main input and output connections. (D) Mean
values of maximum synaptic strength during learning for the 9 synapses. According to the statistical analyses, 4 synapses (1, 2, 4, and 8) presented maximum synaptic strengths
significantly different [F4,36,45 = 11.70; P < 0.001] from those evoked by synapse 9 (left histogram). Note the potentiation [F1,19,18 = 30.37; P < 0.001] of synapse 8 in contrast to the
inhibition of synapse 9 during the acquisition process. A similar analysis (right histogram) showed that synapses 3, 4, 6, and 5 presented significantly different [F4,36,45 = 6.22;
P < 0.001] maximum synaptic strengths from those shown by synapse 7. However, synapses 1, 4, 5, and 8 presented similar synaptic strengths [F4,36,45 = 1.86; P > 0.05] at the
asymptotic level (session C10, see magenta arrows) of the acquisition process. For the multiple-comparison analyses (Tukey’s test) with respect to the synapses 9 (left histogram) and 7
(right histogram), the significance level is indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars represent SEM.

Cerebral Cortex 7
Figure 6. Evolution in timing and strength and functional organization of the 9 selected synapses across learning. (A) Timing–strength dispersion patterns between the mean Downloaded from http://cercor.oxfordjournals.org/ at Serials Section Norris Medical Library on March 31, 2014
(green arrows) and maximum (red arrows) values of synaptic strengths across conditioning. The normalized fEPSP slope determined the strength/magnitude of the vector, while the
training session (H2, C1–C10, or E1) determined the timing/orientation of the vector. Three synapses showed a dispersion pattern similar [one way ANOVA F-test, F3,27,36 = 3.68;
P > 0.01] to that of eyelid activity (% CRs, 100%; PP–DG, 96.20%; CA3–CA1, 121.22%; CA1–SUB, 112.54%), whilst other synapses (cCA3–CA1, 31.16%; REU–CA1, 28.81%; PP–
CA1, 9.89%) presented smaller [one way ANOVA F-test, F3,27,36 = 341.91; P < 0.001] timing–strength dispersion patterns, and the synapses DG–CA3 (11.55%), PP–CA3
(10.37%), and CA1–mPFC (12.68%) showed smaller values [one way ANOVA F-test, F3,27,36 = 415.68; P < 0.001] of the dispersion indices but with angular displacements in
opposite directions (see blue bent arrow inside each circumference). (B, C) Color map representations of synaptic strengths (fEPSP slopes, as % of baseline) between mean and
maximum (max) values (white dashed arrows) for all the synapses. (B) Preliminary distribution of the 9 synapses according to anatomical criteria and connectivity (Fig. 5C). (C)
Proposed functional distribution of synapses according to the timing–strength dispersion index for each synapse ( ss) (see eq. 1 and Supplementary Table 1), the RDI between 2
circular patterns (see eq. 2 and Supplementary Table 2), and the trend (see black dashed arrow) of the SEI (see eq. 3, Supplementary Table 3 and Fig. 8) across synapses.

with increasing intensities (in mA), although some synapses
(REU–CA1, CA1–SUB, and CA1–mPFC) reached lower asymp-
totic values than the others (Fig. 4A), indicating some impor-
tant functional differences between them. In addition,
perforant pathway inputs evoked differential effects on their
hippocampal targets. Specifically, input/output relationships
were steeper for the PP–DG synapse than those evoked at the
PP–CA3 and PP–CA1 synapses, a fact already reported in an-
esthetized guinea pigs (Bartesaghi et al. 2006).
We also checked the paired-pulse facilitation present in the
selected synapses. Paired-pulse stimulation is a form of short-
term synaptic modulation used as an indirect measurement of

8 Hippocampal Synapses and Associative Learning • Gruart et al.

 Downloaded from http://cercor.oxfordjournals.org/ at Serials Section Norris Medical Library on March 31, 2014

Figure 7. Synaptic-learning states (see the color rectangles) and their timing–strength synaptic evolutions. (A, B) Evolution of the synaptic strengths (fEPSP slopes, as % of
baseline) collected in the hippocampal circuit following a paired-pulse stimulation paradigm (first and second St.) during a trace eyeblink conditioning. Selected synapses were: perforant
pathway (PP)–dentate gyrus (DG); DG–CA3; PP–CA3; CA3–CA1; contralateral CA3 (cCA3)–CA1; thalamic reuniens nucleus (REU)–CA1; PP–CA1; CA1–subiculum (SUB); and CA1–
medial prefrontal cortices (mPFC). (C) Evolution of the percentage of conditioned responses (% CRs) across the successive habituation (n = 2), conditioning (n = 10), and extinction
(n = 5) sessions. Colors correspond to the quantitative color bars to the right of the panels (A,B). (D) The timing–strength dispersion patterns between the mean (green arrow) and
maximum (red arrow) values of synaptic strength (for the normalized fEPSPs 2) across 12 training sessions (H2, C1–C10, and E1). Three synapses showed a dispersion pattern similar
[one-way ANOVA F-test, F3,27,36 = 1.92; P > 0.05] to that of the eyelid activity (% CRs, 100%; PP–DG, 98.19%; CA3–CA1, 121.09%; CA1–SUB, 108.01%), other synapses (cCA3–CA1,
9.67%; REU–CA1, 29.64%; PP–CA1, 3.11%) presented smaller [one-way ANOVA F-test, F3,27,36 = 436.49; P < 0.001] timing–strength dispersion patterns; and the synapses DG–CA3
(11.31%), PP–CA3 (10.23%), CA1–mPFC (11.52%) showed smaller values [one-way ANOVA F-test, F3,27,36 = 433.80; P < 0.001] of the dispersion indices ( ss, see eq. 1 and
Supplementary Table 1; RDI, see eq. 2 and Supplementary Table 2) but with angular displacements in opposite directions (see blue bent arrows inside each circumference).

Cerebral Cortex 9
 Downloaded from http://cercor.oxfordjournals.org/ at Serials Section Norris Medical Library on March 31, 2014
Figure 8. A 3D learning-dependent array constructed in accordance with the SEI. (A) The evolution of the mean timing between the mean and maximum values of the synaptic
strength (see black dashed arrow in Fig. 6C). (B) Trends of SEI in relation to the proposed functional distribution of the synapses (Fig. 6C). The color-dashed curves represent the
contours of the different regions (R1–R4 and R1.2–R3.4) where the SEI values are distributed with respect to the zero reference line (in red) and the regression line (in black). (C)
The regression analysis showed a strong dependence (r = 0.95; P < 0.01) between the mean timing and the SEI values. For all the regression analyses (in A–C) the linear
equations, the correlation coefficients (r), and the significance levels (P) are indicated. (D) At the left is illustrated a 3D-array representing the synaptic-learning states. The proposed
distribution of the synapses according to the SEI (in this order: 2, 3, 4, 8, 1, 5, and 6) allowed establishing an alternative model of functional connections (see the right diagram of
connections) in the hippocampal circuit. The colors of the arrows in the diagram of connections correspond with those of the contours of the regions (in B).

changes in the probability of release of neurotransmitter at the
presynaptic terminal (Thomson 2000; Zucker and Regehr
2002). According to the multiple-comparison analyses, most of
the selected synapses presented a significant (P < 0.05) paired-
pulse facilitation at short intervals (10–40 ms), but some of
them extended the facilitation to 100 ms (DG–CA3, PP–CA3,
cCA3–CA1, and CA1–mPFC), or even 200 ms (REU–CA1) of
interpulse interval (Fig. 4B).
Finally, an LTP was evoked in all of the synapses by HFS of
the corresponding presynaptic terminal (see Materials and
Methods). When evoked in behaving mice, most LTPs pre-
sented profiles similar to those already described for the CA3–
CA1 synapse in the same species (Gruart et al. 2006; Madroñal
et al. 2007). However, and as reported recently (Jurado-Parras
et al. 2012), the CA1–mPFC synapse presented a significant
(P < 0.05) delayed building up of the expected synaptic poten-
tiation, a fact also reported recently in behaving mice (Eleore
et al. 2011). Finally, the REU–CA1 synapse showed an evident
and significant (P < 0.05) long-term depression in response to
the presentation of the HFS protocol (Fig. 4C). On the whole,
these results suggested the presence of different functional
properties of the selected synapses that could make them act in
diverse, but complementary, ways during the acquisition of
actual associative learning tasks.
Differential Changes in Strength Evoked in the Selected
Hippocampal Synapses by Classical Eyeblink
Conditioning of Behaving Mice
In a second series of experiments, animals were prepared for
the classical conditioning of eyelid responses using a
hippocampal-dependent trace conditioning paradigm (Fig. 1A,
B; Weiss et al. 2005; Bangasser and Shors 2007). Mice (n = 10
per group) acquired this type of associative learning progress-
ively, reaching 50% of CRs by the fourth conditioning session

10 Hippocampal Synapses and Associative Learning • Gruart et al.

and reaching top values (75% of CRs) by the 10th (Fig. 1C). In
parallel with the increase in the percentage of CRs, and as
already reported for this synapse (Gruart et al. 2006), we were
able to record a learning-dependent change in fEPSP slopes
evoked by a pair of pulses presented to the CA3–CA1 synapse
during the CS–US interval (Fig. 1B,D). It has already been
shown that this synaptic potentiation is significantly related
with the increase in the percentage of CRs (Gruart et al. 2006).
We used the same conditioning paradigm to check the evol-
ution of activity-dependent changes in strength for the 9 sy-
napses included in this study. As illustrated in Figure 5A,B,
changes in synaptic strength reached maximum (or minimum)
values at different times and rates. Although most synapses
presented a progressive, sustained increase in fEPSP slopes,
others presented an early potentiation that also disappeared
very soon (DG–CA3), were progressively depressed (CA1–
mPFC), or were not affected by the training (PP–CA1).
The appropriate statistical analysis (combination of para-
metric multivariate methods [ANOVA F-test] and multiple-
comparison analyses [Tukey’s test]) carried out with the col-
lected data indicated that some synapses (PP–DG, DG–CA3,
CA3–CA1) belonging to the intrinsic hippocampal circuit
(Amaral 1993) presented maximum synaptic changes signifi-
cantly [F3,27,36 = 16.51; P < 0.001] different (in both strength
and session presentation) from those evoked by the CA1–
mPFC synapse (Fig. 5D, left histogram).
We also observed important differences in the changes
evoked at the 2 hippocampal output pathways (CA1–SUB and
CA1–mPFC) included in this study (Fig. 5A,D). Thus, one-way
ANOVA F-test with factors (days) revealed that fEPSPs evoked
at the subiculum (CA1–SUB) were potentiated [F1,9,18 = 30.37;
P < 0.001] during the conditioning sessions relative to baseline,
whilst effects on the prefrontal cortex (CA1–mPFC) were
depressed [F4,36,45 = 11.70; P < 0.001] in comparison with the
maximum synaptic strengths in other synapses (intrinsic hip-
pocampal circuit and CA1–SUB).
A similar statistical approach (Fig. 5D, right histogram)
showed that the PP–CA1 synapse did not follow the changes in
strength [F4,36,45 = 6.22; P < 0.001] taking place in the other in-
trinsic synapses (PP–CA3, CA3–CA1, cCA3–CA1, and REU–
CA1) of the hippocampal circuit. Finally, 4 synapses (PP–DG,
CA3–CA1, cCA3–CA1, and CA1–SUB) presented similar synap-
tic strengths [F4,36,45 = 1.86; P > 0.05] at the asymptotic level
(10th conditioning session) of the acquisition process.
Different Timing–Strength Evolution Patterns Evoked
at the Selected Synapses During Classical Eyeblink
Conditioning
As illustrated in Figure 5A, the classical conditioning of eyelid
responses carried out in the experimental animals not only
evoked specific changes in strength in the 9 selected synapses,
but also those changes took place at precise moments across
the successive training sessions. In accordance, we decided to
analyze both processes simultaneously. Thus, we also analyzed
the timing–strength dispersion pattern (Batschelet 1981;
Mardia and Jupp 1999; Sánchez-Campusano et al 2011) ob-
served between the mean and maximum synaptic strength
values across conditioning (Fig. 6A). In this analytical ap-
proach, the normalized fEPSP slope determined the strength/
magnitude of the vector (red or green arrow in Fig. 6A), while
the training session determined the timing/orientation of the
vector.
According to the data illustrated in Figure 6A, 3 synapses
showed a dispersion pattern similar [F3,27,36 = 3.68; P > 0.01] to
that of eyelid activity during conditioning sessions (% CRs,
100%; PP–DG, 96%; CA3–CA1, 121%; and CA1–SUB, 112%),
whilst other synapses (cCA3–CA1; REU–CA1; PP–CA1) pre-
sented smaller [<32%; F3,27,36 = 341.91; P < 0.001] timing–
strength dispersion patterns, suggesting a lower degree of cor-
relation with the acquisition process (see Supplementary
Tables 1 and 2 and Supplementary Material, Appendix A).
Finally, 3 synapses (DG–CA3, PP–CA3, and CA1–mPFC) pre-
sented the smallest values [<13%; F3,27,36 = 415.68; P < 0.001] of
the dispersion indices, with angular displacements in opposite
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directions (see blue bent arrows in Fig. 6A).
The SEI was designed to estimate the changes between differ-
ent synaptic-learning states of the same synapse or to measure
the dynamic changes between different synaptic-learning states
of different synapses. In particular, for each synapse, the analy-
sis of the SEI between the mean and maximum values of the sy-
naptic strength and their corresponding times of occurrence
(see Supplementary Table 3 and Supplementary Material,
Appendix B) enabled us to propose an alternative distribution
of the synapses (in this order: 2, DG–CA3; 3, PP–CA3; 4, CA3–
CA1; 8, CA1–SUB; 1, PP–DG; 5, cCA3–CA1; 6, REU–CA1) (see
Fig. 6B,C), and a hypothetical model of input–output physio-
logical connections at the hippocampal circuit (see the diagram
of connections in Fig. 6C) while the animal learns the associative
learning task.
For comparative purposes, in Figure 7 are illustrated the
different changes in synaptic strength evoked by the second of
the pair of pulses presented to each of the selected synapses
during the training sessions, in comparison with the effects
evoked by the first (fEPSPs 1 illustrated in Fig. 7A; and fEPSPs
2 illustrated in Fig. 7B). Changes evoked by the second pulse
evolved across training in a way similar to those evoked by the
first one. Thus, some synapses increased in learning-
dependent strength across training (PP–DG, CA3–CA1, cCA3–
CA1, REU–CA1, and CA1–SUB); other synapses increased their
synaptic strengths at the beginning of the conditioning ses-
sions and decreased at the end (DG–CA3 and PP–CA3); others
did not present any significant change in this type of associat-
ive learning (PP–CA1); and, finally, others decreased in their
synaptic strength (CA1–mPFC). Although the general evolution
of changes in synaptic strength was similar for the fEPSPs
evoked by the 2 pulses, some differences (≥25%; P ≤ 0.05) in
intensity (PP–DG, PP–CA3, CA3–CA1, and CA1–SUB) were
also evident (Fig. 7A,B). In addition, some minor differences
in the timing–strength dispersion pattern were observed in 2
of the synapses (PP–CA3 and PP–CA1) for the evolution of
fEPSPs evoked by the first (Fig. 6A) and the second (Fig. 7D)
pulses across learning.
In accordance with an early report (Madroñal et al. 2009)
paired-pulse facilitation (i.e., the relative changes in strength of
the second vs. the first pulse) was significantly modified (P ≤
0.05) in all of the studied synapses (apart the PP–CA1 one)
across learning and extinction (not illustrated). As a whole, and
as already proposed for many other synaptic circuits (see
Thomson 2000; Zucker and Regehr 2002 for details), the differ-
ent synaptic effects evoked by the pair of pulses suggest that
some presynaptic mechanisms are involved in classical eye-
blink conditioning. In this regard, it has been already proposed
Cerebral Cortex 11
the involvement of presynaptic components of the CA3–CA1
synapse in the same type of associative learning in mice (Madro-
ñal et al. 2009).
The analysis of the timing–strength synaptic evolutions
across conditioning (Figs 5–8), including the calculation of the
dispersion patterns (ss and RDI) (see equations 1 and 2, and
Supplementary Tables 1 and 2) and the SEI (see eq. 3 and Sup-
plementary Table 3), allowed us to propose the putative func-
tional organization of the 9 synapses included in the present
study, which did not coincide with their sequential organization
according to anatomical criteria and connectivity (Figs 5C and
6B,C; Amaral 1993). The picture emerging (Figs 5D, 6C, and 8)
is that following an early, short-lasting activation of the DG–CA3
synapse, there is an increasing process of activation of the
intrinsic hippocampal circuit (CA3–CA1), including one of the
hippocampal main outputs (CA1–SUB) and its contralateral
counterpart (cCA3–CA1). Perforant pathway inputs (PP–CA3
and PP–DG synapses) are activated in a delayed form, a fact that
has also been reported during a tone-food association test
carried out in behaving rats (Segal and Olds 1972; Segal et al.
1972), whilst the PP–CA1 synapse seems not to participate ac-
tively in this type of associative learning (Figs 5A,D, 6C, and
8C). Interestingly, whereas synaptic projections to the subicu-
lum (CA1–SUB) are potentiated, those to the medial prefrontal
cortex (CA1–mPFC) are disfacilitated (Figs 5A,D and 6C).
Discussion
The functional and dynamic approach used here to better un-
derstand the plastic changes taking place in 9 selected hippo-
campal synapses during the actual acquisition and extinction
of a classical eyeblink conditioning task allows us to propose
the hippocampus as a neuronal structure enabling the specific,
timed combinations in synaptic changes in strength character-
izing this type of associative learning (Berger et al. 1983;
Moyer et al. 1990; McEchron and Disterhoft 1997; Weiss et al.
2005; Gruart et al. 2006; Bangasser and Shors 2007; Madroñal
et al. 2007). It can be assumed that other cognitive functions
carried out by hippocampal networks, such as spatial orien-
tation, object recognition, and memory storage and recall
(Moser et al. 2008; Neves et al. 2008; Clarke et al. 2010; Wang
and Morris 2010), are carried out by spatial-temporal patterns
of synaptic activities different to the ones described here for
classical eyeblink conditioning.
The collected results have enabled us to evaluate the trends
of the proposed SEI (see eq. 3, Fig. 8A–C, and Supplementary
Table 3) and to define a 3D learning-dependent array (Fig. 8D)
characterizing the contribution of the hippocampal synapses
(included in its intrinsic circuit and those involved in its main
inputs and outputs) to trace eyeblink conditioning. In this
respect, it is very important to consider that the evolution
index SEI measures the simultaneous changes in the 2 physio-
logical variables of each synapse—that is, the timing and the
synaptic strength, as well as the relative dynamic evolution
between different synapses at different learning states. In
general, the 3 indices defined here ( ss, eq. 1; RDI, eq. 2; and
SEI, eq. 3) enable a better determination of the relationships
between different functional states—that is, between different
synaptic-learning states involved in the acquisition of an
associative learning task. The present results strongly suggest
that this type of associative learning is a multisynaptic process
in which the contribution of each synaptic contact is different
12 Hippocampal Synapses and Associative Learning • Gruart et al.
in strength and takes place at a different acquisition moment
across the conditioning process.
The significance of the dynamic patterns set up by the intrin-
sic and/or extrinsic afferents to both CA3 and CA1 networks
was also established. Synaptic terminals impinging upon CA3
postsynaptic pyramidal neurons (DG–CA3 and PP–CA3) pre-
sented similar activation profiles (Fig. 5A,D) across condition-
ing; but quite different circular means of time distributions
(Fig. 6A) and different mean values of the SEI (Fig. 8 and Sup-
plementary Table 3). These differing data suggest the need for
2 distinct input systems to the hippocampal CA3 network
while the animal learns this classical conditioning task. The
computational relevance of these 2 afferents to CA3 pyramidal
neurons has been suggested by other authors (Treves and Rolls
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1992; Treves 2004; Rolls and Kesner 2006) and supported by
the present results. We found that the synapses from the
dentate granular cells onto CA3 neurons (DG–CA3 synapse) via
mossy fibers (first input system) presented an early and signifi-
cant potentiation (Fig. 5A,D) and very high absolute values of
the SEI (Fig. 8 and Supplementary Table 3) in order to force, at
the beginning of the learning process, the CA3 cells into a
pattern of activity relatively independent of any input being re-
ceived from CA3 recurrent collaterals.
Interestingly, at the precise moment that the synaptic
strength of the DG–CA3 synapse began to decrease, the PP–
DG synapse presented a progressive, sustained increase in
fEPSP slopes (Fig. 5A,D). In fact, the effects of these dynamic
changes were reflected as different dispersion patterns ( ss;
Figure 6A and Supplementary Tables 1 and 2) and very differ-
ent values of the proposed evolution index (SEI) (Fig. 8 and
Supplementary Table 3). In this respect, we can propose that
following an early, short-lasting activation of the DG–CA3
synapse, there is an increasing process of activation of the PP–
DG synapse in order to regulate the intensity of the DG–CA3
synapse and to relay enough specific signals to initiate the re-
trieval process, via perforant path input (second input system)
to CA3 pyramidal cells (PP–CA3 synapse, see Fig. 8D). In this
regard, it has already been shown that single neuron changes
in CA3 are not directly dependent upon those in DG, and that
the sequence of changes taking place in those neurons does
not directly confirm the classical intrinsic hippocampal circuit
(Segal and Olds 1972; Segal et al. 1972). In addition, it was
shown years ago that the PP–DG synaptic activity is poten-
tiated during nictitating membrane response conditioning in
rabbits (Weisz et al. 1984).
According to this dynamic approach, synapses impinging
upon the same set of CA1 postsynaptic pyramidal cells (PP–
CA1, CA3–CA1, cCA3–CA1, and REU–CA1) seemed to present
different activation profiles (Fig. 5A,D) at the asymptotic level
(10th conditioning session) of the acquisition process.
Additionally, these synapses have different dispersion patterns
ss) (Fig. 6A and Supplementary Table 1) and different values
(
of the SEI (Fig. 8 and Supplementary Table 3). This evidence
allows us to suggest the need for at least 3 different operant
input systems (from CA3, cCA3, and REU) to the hippocampal
CA1 network (see diagram in Fig. 8D) during the acquisition of
the trace eyeblink conditioning. The information relayed by
such different afferent connections determines the timing and
intensity of the fEPSPs evoked in CA1 neurons. Thus, we can
propose that whereas CA3 cells project both to CA1 via the
Schaeffer collaterals (first input system, CA3–CA1 synapse) and
to other CA3 cells via recurrent collaterals, those from the
contralateral counterpart (second input system) project to CA1
pyramidal cells (cCA3–CA1 synapse) with maximum poten-
tiation (Fig. 5A,D), as well as with an extensive dispersion
pattern ( ss) (Fig. 6A, and Supplementary Tables 1 and 2) and
the highest values of the SEI (Fig. 8 and Supplementary
Table 3). In contrast, the synapse formed by the extrinsic affer-
ents from the thalamic reuniens nucleus (third input system) to
CA1 neurons (REU–CA1) presented a lower activation profile
than the cCA3–CA1 synapse (Figs 5A,D and 6C).
Furthermore, and following the dynamic approach devel-
oped here, the dispersion patterns of both REU–CA1 and CA3–
CA1 synapses with respect to the level of CR expression were
significantly different ( ss; Supplementary Tables 1 and 2); and
the calculation of the synaptic evolution index between REU–
CA1 and cCA3–CA1 synapses indicates that there are important
differences (SEI; Figs 5D and 8) in the progressive potentiation
of these synapses. The absence of a definite LTP (Eleore et al.
2011), or even the presence of a long-term depression, instead
of the expected LTP, in the REU–CA1 synapse when activated
by the HFS protocol, is coherent with its late activation during
the extinction sessions (see Fig. 5A). In this regard, the exist-
ence has been proposed of a closed REU–CA1 circuit that
allows the reuniens nucleus to modulate the activity in CA1, de-
pending on hippocampal output (Dolleman-Van der Weel
et al. 1997).
Whereas the CA1–SUB synapse is potentiated, the synaptic
projections to the medial prefrontal cortex (CA1–mPFC) are
disfacilitated, and the differences between them were highly
significant (Fig. 5A,D). These differences were reflected di-
rectly in the dispersion patterns (ss) (Fig. 6A, and Supplemen-
tary Tables 1 and 2) and in the SEI values (Fig. 8 and
Supplementary Table 3). The facilitation of learning-related
commands sent to the subiculum could be related to the diffu-
sion across sensory-motor circuits of the increased CS–US
associative strength in order to generate the corresponding
overt motor responses (i.e. the CRs), whilst the disfacilitation
of medial prefrontal circuits could contribute to the proper
release of the newly formed CR. As recently shown (Alexander
and Brown 2011; Leal-Campanario et al. 2013), the mPFC
needs to be inhibited in order for the CR to occur. As already
reported Jurado-Parras et al. (2012), and confirmed here, the
small, delayed LTP observed in the CA1–mPFC pathway is
indicative of the peculiar role played by this synapse in the
release of newly acquired motor responses (Alexander and
Brown 2011; Leal-Campanario et al. 2013).
Finally, the PP–CA1 synapse seems to play a specific role
(Izumi and Zorumski 2008) erasing unwanted memories, and
is thus not involved in this type of associative learning (see SEI
values in Fig. 8 and Supplementary Table 3). In addition, and
as already suggested for the hippocampal CA3–CA1 synapse
(Madroñal et al. 2009), collected data suggest the presence of
some presynaptic mechanisms, at selected synapses of the hip-
pocampal circuitry, in the acquisition of this type of associative
learning.
As already shown for the CA3–CA1 synapse during classical
eyeblink conditioning using a trace paradigm (Gruart et al.
2006), significant changes in synaptic strength also took place
in some of the selected hippocampal synapses during the ex-
tinction process (Figs 5 and 7). These results further confirm
that extinction is not a mere passive process during which ac-
quired memories are erased. To the contrary, extinction seems
to be an active process involving the activation of selective
synaptic patterns and underlying molecular events (Inda et al.
2005).
In summary, we can propose that the results obtained with
the dynamic approach presented here for determining the
functional organization of hippocampal circuits during actual
learning are incompatible with the excessive localization of
hippocampal functions proposed by others (McHugh et al.
2007; Nakashiba et al. 2008). Thus, depending on the specific,
timed activation of its multiple synaptic contacts, the hippo-
campus can be involved in many different functions, such as
spatial orientation (Moser et al. 2008), object recognition
(Clarke et al. 2010), and various other forms of memory acqui-
sition and retrieval (Bliss and Collingridge 1993; Neves et al.
2008; Wang and Morris 2010). It can therefore be proposed
Downloaded from http://cercor.oxfordjournals.org/ at Serials Section Norris Medical Library on March 31, 2014
that for each associative and/or cognitive function carried out
by the hippocampal circuit there is a corresponding functional
map of different synaptic weights characterizing it. In future
studies, it will be important to systematically manipulate each
of individual components of the hippocampal circuits,
piece-by-piece, using powerful genetic approaches, to reveal
their respective roles in learning and memory processes.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxford
journals.org/
Notes
The authors thank J.M. González-Martín for his technical assistance
and Roger Churchill for his editorial help. Conflict of Interest: None de-
clared.
Funding
This study was supported by Spanish MINECO (BFU2011-
29089 and BFU2011-29286) and Junta de Andalucía (BIO122
and CVI7222) grants to A.G. and J.M.D.-G.
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