Figure legends
FIGURE 1. Neonatal treatment with MK-801 selectively reduces
synaptic strength in the MPP – DG synapse. (a) Schematic
representation of the EC – DG circuit and placement of stimulation and
recording electrodes for extracellular recordings. Stimulation of the
lateral perforant path (LPP) and the resulting LPP fEPSP were acquired
in the outer one-third section of the molecular layer of the DG (green
axons). Medial perforant path or MPP fEPSPs were acquired in the middle
one-third of the molecular layer of the DG (red axons). (b)Pharmacological sensitivity of the LPP fEPSPs and the MPP fEPSP to
different metabotropic glutamate receptors. Upper panel (green traces):
LPP fEPSP are sensitive to activation of group III mGluRs with L-AP4 (20
µM) and insensitive to stimulation of group II mGluRs with DCG-IV (5
µM). Lower panel (red traces): MPP fEPSP are insensitive to activation
of group III mGluRs. The opposite occurs in the presence of group II
mGluRs agonists. The pharmacological sensitivity of the fEPSPs to the
different mGluRs was rigorously used as an inclusion criterion for all
the experiments included in this study. (c) Input-output graphs
of the LPP fEPSPs and (d) the MPP fEPSPs in response to
increasing current pulses (100 µA steps at 0.067 Hz). Neonatal treatment
with MK-801 did not alter the magnitude of the LPP fEPSP (upper trace
and green symbols in panel c) but reduced the magnitude of the MPP fEPSP
(upper trace and red symbols in panel d), **P < 0.01.(e) Left panel: input-output curve of the LPP fiber volleys
(FV) in response to increasing current pulses; right panel: coupling
analysis of the FV vs. LPP fEPSP slope, showing no difference in
synaptic strength. (f) Left panel: input-output curve of the
MPP FV in response to increasing current pulses, showing decreased
presynaptic excitability in the MK-801 group; right panel: coupling
analysis of FV vs. MPP fEPSP slope. * P < 0.05 and
**P < 0.01. For LPP: n = 9 slices / 6 animals
for the control group and n = 10 slices / 6 animals for the
MK-801 group. For MPP: 8 slices / 6 animals for the control group
and n = 10 slices / 6 animals for the MK-801 group.
FIGURE 2. Neonatal treatment with MK-801 reduces presynaptic
neurotransmitter release at the LPP and the MPP. (a) Violin
plots contrasting the paired-pulse facilitation values (PPF; ISI 60 ms)
of the LPP (n = 28 slices / 15 animals; green symbols) and the MPP (n =
29 slices / 15 animals; red symbols) synapses in the control condition.
LPP PPF was greater than MPP PPF, ***P < 0.001.(b) Representative traces showing the pharmacological
sensitivity of the LPP and the MPP synapses. LPP PPF decreased in the
presence of L-AP4 (20 µM, upper traces), whereas the MPP PPF was
abolished by perfusion of DCG-IV (5 µM; lower traces). (c) Time
course of the LPP PPF exploring different ISI (40, 60, 100, 200, and 500
ms). Neonatal treatment with MK-801 increased the LPP PPF. *P< 0.05. For LPP PPF: n = 8 slices / 6 animals for
control and n = 9 slices / 7 animals for MK-801. (d)Representative PPF of the LPP synapses contrasting the values of control
and MK-801-treated slices. (e) Time course and (f)representative traces for MPP PPF in control (red symbols and traces)
and MK-801-treated (black symbols and traces) slices. MPP PPF: n= 9 slices / 7 animals for each condition. *P < 0.05.
FIGURE 3. Neonatal treatment with MK-801 impairs the induction
of CB1R-dependent LTD at the MPP – DG synapse. (a)Representative traces of control (upper traces) and the
CB1R antagonist, AM251 treatment (5 µM, lower traces) in
baseline, post-LFS, and the presence of DCG-IV (5 µM). (b) Time
course graph of fEPSP slope in response to LFS (900 pulses at 3 Hz) in
the MPP. In the control condition (black symbols), LFS induces LTD.
Perfusion of AM 251 (5 µM, white symbols) LFS prevented the induction of
LTD. (c) Representative traces of the control (upper black
traces) and MK-801-treated group (lower red traces) in baseline,
post-LFS, and the presence of DCG-IV (5 µM). (d) Time course
graph of LTD. In the MK-801-treated group, LFS failed to induce LTD; an
abnormal potentiation (red symbols) was observed that was sensitive to
DCG-IV (5 µM). (e) Bar graph summarizing the magnitude of
normalized fEPSP slopes at 90 min after LFS and in the presence of
DCG-IV in the control (symbols and black bars), AM-251 (symbols and
white bars), and MK-801 (symbols and red bars) groups. *P< 0.05. Each symbol within the bars represents one independent
experiment; bars represent the media ± SEM. (f) Bar graph
showing the changes in PPR before and after LFS. The control group
exhibited increased PPR after LFS, suggesting a reduction in presynaptic
release at the MPP. *P < 0.05; n = 6 slices / 6 animals
for control and MK-801; n = 4 slices / 4 animals for AM 251.(g) Cumulative probability distribution chart of normalized
fEPSPs post-LFS in the control (black line), AM-251 (gray line), and
MK-801 (red line) groups. The individual values for this analysis (bins
configured at 0.8 value) correspond to 10 to 90 minutes of the time
course in panels b and c .
FIGURE 4. Neonatal treatment with MK-801 reduces the functional
expression of CB1R but not the synthesis of 2-AG at the
MPP – DG synapse. (a) Representative traces of control and MK-801
fEPSPs during baseline (1), 90 min post-WIN 55,212-2 (2), and DCG-IV
(3). (b) Time course graph of normalized fEPSP slope in
response to the perfusion of the CB1R agonist, WIN
55,212-2 (5 µM for 15 min), in control and MK-801-treated groups (black
and red symbols, respectively). (c) Representative traces and(d) time course graph of normalized fEPSPs in the presence of
the cholinesterase inhibitor physostigmine (10 µM for 15 min) in control
and MK-801-treated groups (black and red symbols, respectively). Slices
of the MK-801-treated group exhibited synaptic depression in response to
physostigmine perfusion. (e) Bar graph contrasting the synaptic
depression elicited with WIN 55,212-2 in control vs. MK-801-treated
groups. *P < 0.05, n = 6 slices / 5 animals for control and n
= 6 slices / 6 animals for MK-801. (f) Bar graph contrasting
the magnitude of synaptic depression in response to physostigmine. The
MK-801-treated group (red bar) exhibited reduced synaptic depression;
however, this difference lacked statistical significance compared to the
control group (black bar). n = 6 slices / 6 animals for control and n =
5 slices/ 5 animals for MK-801. (g) Bar graph contrasting the
magnitude of synaptic depression in the MK-801-treated groups in
response to WIN 55,212-2 or physostigmine. The synaptic depression
induced by physostigmine is similar to the magnitude of depression
induced by activation of CB1R with WIN, suggesting that
2-AG synthesis is not affected by neonatal treatment with MK-801.
FIGURE 5. Pharmacological inhibition of monoacylglycerol lipase
(MAGL) enzyme restores the impaired CB1R-dependent LTD
at the MPP – DG synapse. (a) Representative fEPSP traces from
MK-801-treated slices (red traces) or those preincubated with JZL 184
(blue traces) in the conditions indicated by the numbers. (b)Time course graph of normalized fEPSPs from MK-801-treated slices in the
presence of the irreversible inhibitor of monoacylglycerol lipase (MAGL
JZL 184, 1 µM; blue symbols). LFS induced stable LTD in the presence of
JZL 184 MK-801-treated slices. (c) Bar graph. Perfusion of JZL
184 rescues LTD at the MPP – DG synapse. *P < 0.05; n = 6
slices / 6 animals for MK-801-treated group and n = 5 slices / 5 animals
for MK-801 + JZL 184 group. (d) Bar graphs showing restored LTD
is accompanied by increased PPR. * P < 0.05. (e)Cumulative probability distribution plot of the fEPSP slope values after
LFS (red line) or LFS + JZL (blue line) in MK-801-treated slices. The
individual values for this analysis (bins configured at 0.8 value)
correspond to 10 to 90 minutes of the time course in panel b.
FIGURE 6. Neonatal treatment with MK-801 impairs the TBS-induced
LTP at the LPP – DG synapse. (a) Representative traces of fEPSP from
control (black traces) and MK-801 (green traces) in the conditions
indicated by the numbers. (b) Time course of normalized fEPSP
slope from control and MK-801 in response to TBS. Arrowhead indicates
the delivery of the TBS train (10 bursts at 5 Hz; each burst consisted
of 5 pulses at 100 Hz). Inset bar graph comparing the post-tetanic
potentiation decay calculated by adjusting a best-fit single exponential
decay function. The PTP dropped faster in the MK-801-treated group.
*P < 0.05. (c) Bar graph contrasting the
magnitude of PTP, LTP, and the effect of L-AP4 (20 µM) on the LPP
synapse. *P < 0.05; n = 7 slices / 6 animals for control and n
= 7 slices / 7 animals for MK-801. (d) Heatmaps showing the
magnitude of LTP in each slice from both experimental groups.(e) Cumulative probability distribution plot of the fEPSP slope
values. The individual values for this analysis (bins configured at 0.8
value) correspond to 10 to 90 minutes of the time course in panel b.
FIGURE 7. Neonatal treatment with MK-801 does not interfere with
the induction of TBS-induced LTP at the MPP – DG synapse. (a)Representative traces of fEPSP from control (black traces) and MK-801
(red traces) in the conditions indicated by the numbers. (b)Time course of normalized fEPSP slope in response to TBS. Inset bar
graph comparing the post-tetanic potentiation decay calculated by
adjusting a best-fit single exponential decay function. No difference
was found in either group. (c) Bar graph contrasting the
magnitude of PTP, LTP, and the effect of DCG-IV (5 µM). n = 8 slices / 7
animals for control and n = 7 slices / 7 animals for MK-801.(d) Heatmap showing the magnitude of LTP in each slice from
both experimental groups. (e) Cumulative probability
distribution plot of the fEPSP slope values.
FIGURE 8. Neonatal treatment with MK-801 impairs
frequency-dependent synaptic filtering at the LPP. (a1-a3, left panels)Scatter plot showing the evoked fEPSPs slope from control (black
symbols) and MK-801-treated (green symbols) groups in response to 5, 20,
and 50 Hz trains. In control slices, 5 Hz induced synaptic facilitation
throughout the stimulation train, 20 Hz caused a mild synaptic
depression at the end of the train, and 50 Hz induced massive
attenuation of the synaptic response (horizontal dashed lines represent
facilitation value vs. S1). In the MK-801-treated slices,
frequency-dependent filtering was impaired at 5 Hz and 50 Hz since
synaptic facilitation was increased. *P < 0.05. Train at 5 Hz:
n = 9 slices/animals for control and n = 7 slices / 7 animals for
MK-801. Train at 20 Hz and 50 Hz: n = 8 slices / 6 animals for control
and n = 8 slices / 7 animals for MK-801. (b1-b3) Representative
traces in response to trains at 5 Hz, 20 Hz, and 50 Hz. (c1-c3)Over-imposed traces showing the first synaptic response (S1) vs. the
tenth response (S10) in the three frequencies examined. (d-f)Bar graphs contrasting the magnitude of the fEPSP amplitude (measured in
mV) from S1 and S10 in both experimental conditions at the three
frequencies tested.
FIGURE 9. Neonatal treatment with MK-801 impairs spatial
discrimination in male rats. (a) Schematic representation of the object
spatial pattern separation (OPS) task. The task comprised a learning
trial (T1) and a discrimination trial (T2). It was performed in a
circular arena with two equidistant identical objects (40 cm) placed in
five positions (P), with 6 cm between positions. These positions were
designated both left and right from the center of the arena. In T1, the
animals were exposed to two objects placed in P1. In T2, both objects
were placed in P1 (null displacement), or one was placed in P2, P3, P4,
or P5 (maximal displacement). The discrimination index (DI) was
indicative of performance in this task. All animals were evaluated in
all P values with intervals of two days. Objects in green or gray prism
form represent identical par objects. (b) Bar graph summarizing
the DI values obtained from control (black bars) and MK-801-treated
(blue bars) animals during the OPS task. MK-801-treated animals could
not distinguish the displacement of one object to P3, indicating
impaired spatial discrimination. *P < 0.05, n = 10 for control
and n = 12 for MK-801. (c) Heatmap showing all DI values for
each animal from both experimental conditions. While the color scale in
the P5 columns is similar in control and MK-801-treated animals, the
color pattern in the P3 columns differs between experimental conditions,
indicating reduced DI values in MK-801-treated animals. (d)Line chart summarizing DI performance from control (black line) and
MK-801 (blue line) in function of the magnitude of displacement of one
object in T2. In control animals, when the magnitude of displacement of
one object is decreased, the cognitive demand of pattern separation is
theoretically increased, which supports the spatial discrimination of
the position of objects. However, when the magnitude of displacement of
one object is maximal (24 cm), MK-801-treated animals exhibit similar
behavioral performance to control animals. When the cognitive demand of
pattern separation increases, the MK-801-treated animals exhibit
impaired performance in spatial discrimination.