\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,polish,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Neonatal hypofunction of NMDA receptors alters perforant path synaptic
plasticity, filtering and impairs dentate gyrus-mediated spatial
discrimination}



\author[1]{Luis Alfredo  Márquez}%
\author[2]{Carolina Lopez-Rubalcava}%
\author[1]{Emilio Galvan}%
\affil[1]{CINVESTAV Departamento de Farmacobiología Sede Sur}%
\affil[2]{CINVESTAV Departamento de Farmacobiología}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose: Transient hypofunction of NMDARs represents a
convergence point for the onset and further development of psychiatric
disorders, including schizophrenia. Although the cumulative evidence
indicates dysregulation of the hippocampal formation in schizophrenia,
the integrity of the synaptic transmission and plasticity conveyed by
the somatosensorial inputs to the dentate gyrus, the perforant path
synapses, have barely been explored in this pathological condition.
Experimental Approach: We identified a series of synaptic alterations of
the lateral and medial perforant paths, in animals neonatally treated
with the NMDAR antagonist MK-801. The dysregulation here reported
suggests decreased cognitive performance, for which the dentate gyrus is
critical. Key Results: We identified alterations in the synaptic
properties of the lateral and medial perforant paths to the dentate
gyrus synapses in MK-801-treated animals. Altered glutamate release and
decreased synaptic strength precede an impairment in the induction and
expression of LTP and cannabinoid 1 receptor (CB1R)-mediated LTD.
Remarkably, by inhibiting the degradation of 2-arachidonoylglycerol, the
endogenous ligand of the CB1R, we restored the LTD in animals treated
with MK-801. Additionally, we show for the first time that spatial
discrimination, a cognitive task that requires dentate gyrus integrity,
is impaired in animals exposed to transient hypofunction of NMDARs.
Conclusion and Implications: Descriptive and mechanistic evidence
showing the dysregulation of glutamatergic transmission and synaptic
plasticity from the entorhinal cortex to the dentate gyrus is presented.
These findings may explain the cellular dysregulations underlying the
altered cognitive processing in the dentate gyrus associated with
schizophrenia.%
\end{abstract}%



\sloppy


\textbf{Neonatal hypofunction of NMDA receptors alters perforant path
synaptic plasticity, filtering and impairs dentate gyrus-mediated
spatial discrimination}

Luis A. M\selectlanguage{ngerman}árquez\textsuperscript{1}, Carolina López
Rubalcava\textsuperscript{1} and Emilio J. Galván\textsuperscript{1,2*}

\textsuperscript{1}Departamento de Farmacobiología, CINVESTAV Unidad
Sur, Ciudad de México, México.

\textsuperscript{2}Centro de Investigaciones sobre el Envejecimiento,
CIE-Cinvestav, Ciudad de México, México.

\textbf{Running title:} \emph{Transient hypofunction of NMDARs alters
dentate gyrus functionality}

*\textbf{Address} \textbf{correspondence to:}

Dr. Emilio J. Galván

\textsuperscript{1}Departmento de Farmacobiología

Centro de Investigación y Estudios Avanzados del Instituto Politécnico
Nacional

Calzada de los Tenorios No. 235, Col. Granjas Coapa C.P. 14330, México

Phone: +52(55)5483-2852

Email: \emph{ejgalvan@cinvestav.mx}

\textbf{CRediT authorship contribution statement: Márquez} :
Conceptualization, Methodology, Formal analysis, Investigation, Writing
-- original draft preparation; \textbf{Lopez-Rubalcava} : Resources and
Visualization; \textbf{Galván} : Conceptualization, Resources,
Visualization, Writing -- review and editing.\emph{}

\emph{Bullet point summary:}

\textbf{What is already known}

Hypofunction of NMDA receptors is a convergence mechanism for developing
psychiatric diseases involving hippocampal dysregulation.

Preclinical and clinical evidence suggests physiological and behavioral
dysregulation of the dentate gyrus, the hippocampus's sensorial gate.

\textbf{What this study adds}

Neonatal administration of MK-801 modifies LTP, LTD in the dentate gyrus
and reduces spatial discrimination.

Enhancing 2-AG -- CB1R signaling via MAGL inhibition restores long-term
depression in MK-801-treated hippocampal slices.

\textbf{Clinical significance}

Mnemonic discrimination performance could help identify individuals at
high risk for developing schizophrenia.

Cannabinoid system modulation may improve dentate gyrus-dependent
synaptic and behavioral capabilities in schizophrenic individuals.

\textbf{Keywords: MK-801, perforant path, dentate gyrus, cannabinoid
receptor 1, MAGL, spatial discrimination, schizophrenia}



\textbf{Abstract}

\emph{Background and Purpose} : Transient hypofunction of NMDARs
represents a convergence point for the onset and further development of
psychiatric disorders, including schizophrenia. Although the cumulative
evidence indicates dysregulation of the hippocampal formation in
schizophrenia, the integrity of the synaptic transmission and plasticity
conveyed by the somatosensorial inputs to the dentate gyrus, the
perforant path synapses, have barely been explored in this pathological
condition.

\emph{Experimental Approach} : We identified a series of synaptic
alterations of the lateral and medial perforant paths, in animals
neonatally treated with the NMDAR antagonist MK-801. The dysregulation
here reported suggests decreased cognitive performance, for which the
dentate gyrus is critical.

\emph{Key Results} : We identified alterations in the synaptic
properties of the lateral and medial perforant paths to the dentate
gyrus synapses in MK-801-treated animals. Altered glutamate release and
decreased synaptic strength precede an impairment in the induction and
expression of LTP and cannabinoid 1 receptor
(CB\textsubscript{1}R)-mediated LTD. Remarkably, by inhibiting the
degradation of 2-arachidonoylglycerol, the endogenous ligand of the
CB\textsubscript{1}R, we restored the LTD in animals treated with
MK-801. Additionally, we show for the first time that spatial
discrimination, a cognitive task that requires dentate gyrus integrity,
is impaired in animals exposed to transient hypofunction of NMDARs.

\emph{Conclusion and Implications} : Descriptive and mechanistic
evidence showing the dysregulation of glutamatergic transmission and
synaptic plasticity from the entorhinal cortex to the dentate gyrus is
presented. These findings may explain the cellular dysregulations
underlying the altered cognitive processing in the dentate gyrus
associated with schizophrenia.



\textbf{Introduction}

A growing body of evidence indicates that hypofunction of
N-methyl-D-aspartate receptors (NMDARs) represents a convergence point
for the onset and further development of psychiatric disorders,
including autism and schizophrenia (Snyder and Gao, 2013; Forsyth and
Lewis, 2017; Nakazawa and Sapkota, 2020). The genetic/chemogenetic
ablation or pharmacological blockade of NMDARs during critical periods
of brain development has successfully reproduced the negative,
cognitive, and psychotic symptoms associated with schizophrenia
(Jeevakumar et al., 2015; Kjaerby et al., 2017; Seshadri et al., 2018;
Nakao et al., 2019; Segev et al., 2020). Therefore, hypofunction of
NMDARs by neonatal blockade with antagonists such as MK-801 leads to
alterations in the release process of dopamine, glutamate, and GABA
(Nakao et al., 2019; Segev et al., 2020; Márquez et al., 2023);
alterations in the functional expression of ion channels and
postsynaptic receptors in the hippocampus (Griego et al., 2022; Márquez
et al., 2023); and behavioral deficits that resemble the negative and
cognitive symptoms of schizophrenia (Kjaerby et al., 2017; Seshadri et
al., 2018; Segev et al., 2020).

It is now accepted that the hippocampus, a brain region essential for
cognitive processing, the formation of new memories (Eichenbaum, 2004;
Nadel et al., 2012), and the development of social behaviors
(Lopez-Rojas et al., 2022), is central in the pathophysiology of
schizophrenia (Tamminga et al., 2010; Segev et al., 2020). In line with
this tenet, the dentate gyrus (DG), the somatosensorial gate of the
hippocampus, shows the greatest volumetric loss, neuroanatomic
disorganization, and altered glutamatergic/GABAergic transmission in
schizophrenic individuals and animal models (Li et al., 2015; Stan et
al., 2015; Nakahara et al., 2019; Griego et al., 2022). The DG receives
and processes somatosensorial and spatial information from the
entorhinal cortex via the lateral perforant pathway (LPP) and the medial
perforant pathway (MPP), respectively (Hunsaker et al., 2007;
Fernández-Ruiz et al., 2021), and minimizes the overlapping of new
memories with highly similar content via a theoretical mechanism known
as pattern separation (Yassa and Stark, 2011).

Additionally, multiple works have shown the relevance of the cannabinoid
1 receptor (CB\textsubscript{1}R) for the induction of long-term
depression and potentiation in the PP -- DG synapses (Wu et al., 2006;
Wang et al., 2016, 2018b; Peñasco et al., 2019; Fontaine et al., 2020).
Interestingly, dysregulation of NMDARs and CB\textsubscript{1}R is
present in animal models and schizophrenic individuals (Sz\selectlanguage{polish}ű\selectlanguage{english}cs et al.,
2016; Forsyth and Lewis, 2017; Osborne et al., 2019; M\selectlanguage{ngerman}árquez et al.,
2023). Despite the potential repercussions of the altered functionality
of NMDARs and CB\textsubscript{1}R within the LPP and MPP -- DG
synapses, little is known about the possibly altered functionality of
the DG in schizophrenia.

This study identified neurophysiological alterations endured by LPP and
MPP -- DG synapses in response to the transient hypofunction of NMDARs
during early postnatal development. The changes in the neurotransmitter
release process, altered synaptic strength, and dysregulated synaptic
filtering found in these synapses are accompanied by changes in the
induction and expression of LTP and LTD and dysfunction of the
presynaptic activity of the CB\textsubscript{1}R. We also demonstrated
that induction of LTD is restored by fostering the
2-Arachidonoylglycerol (2-AG) signaling, the endogenous ligand of the
CB1R, via negative modulation of monoacylglycerol lipase (MAGL).
Finally, we show for the first time that spatial discrimination, a
cognitive task in which the DG takes part, deteriorates in response to
neonatal hypofunction of NMDARs.\textbf{Methods}

\emph{Animals and neonatal treatment with MK-801}

All our experimental procedures were carried out in rigorous accordance
with the ''NOM-062-ZOO-1999'' local regulation for the use and care of
laboratory animals and the protocols authorized by the internal ethics
committee (CICUAL) of our institution (CINVESTAV; Protocol number
0090-14), which mandate the minimization of suffering and the number of
experimental animals used. These regulations and protocols are
consistent with the ARRIVE 2.0 guidelines, the recommendations made by
the British Journal of Pharmacology (Lilley et al., 2020), and the
National Institutes of Health (US) guidelines for animal care.

Pregnant Wistar rats were supplied by our vivarium and given continuous
veterinary care. The rats were housed in the following control
conditions: an inverted light-dark cycle (12 h; the light was turned off
at 10:00 h), a room temperature of 23 ± 1° C, and free access to food
and water. The pups' birth was designated as postnatal day 0 (P0), and
animals were kept with their mothers until P21. The experimental
procedures were performed in two cohorts of male animals ranging from 30
to 38 days old. Each experimental manipulation was carried out in at
least three different litters. From P7 to P11, the animals received
daily subcutaneous injections of either saline solution (SS, 0.9\%) or
MK-801 (0.2 mg/kg in a 2 mL/kg volume) (Griego et al., 2022; Márquez et
al., 2023). The behavioral procedures were carried out between 10:00 and
16:00 h. The experimental animals were treated with SS or MK-801,
following a single sequence of random assignments. The MK-801 was
dissolved in isotonic SS. Animal suffering was minimized during and
after the injections to reduce behavioral or neurophysiological
alterations that might affect the experimental results. In addition, the
assignment of animals to control and MK-801 groups was blind, to prevent
any potential bias.

\emph{Acute slice preparation}

Sodium pentobarbital (50 mg/kg in a 1 mL/kg volume) was used for deep
anesthesia; animals were then decapitated under its effects. Once
removed from the skulls, brains were placed into a frosty sucrose
solution containing (in mM): 210 sucrose, 2.8 KCl, 2
MgSO\textsubscript{4}, 1.25 Na\textsubscript{2}HPO\textsubscript{4}, 25
NaHCO\textsubscript{3}, 1 MgCl\textsubscript{2}, 1
CaCl\textsubscript{2}, and 10 D-glucose. The sucrose solution was
continuously bubbled with a carbogen mixture (95\%
O\textsubscript{2}/5\% CO\textsubscript{2}). Tissue blocks of cerebral
hemispheres containing the hippocampus and surrounding areas were sliced
at 385 \selectlanguage{greek}µ\selectlanguage{english}m thickness in the transversal plane using a vibrating tissue
slicer (Leica VT1000S; Nusschloc, Germany). Next, the fresh slices were
stabilized at 34\selectlanguage{ngerman}° C for 25 to 30 min in an artificial cerebrospinal
fluid solution (ACSF; pH \selectlanguage{english}[?] 7.30--7.35) containing (in mM): 125 NaCl, 2.5
KCl, 1.25 Na\textsubscript{2}HPO\textsubscript{4}, 25
NaHCO\textsubscript{3}, 4 MgCl\textsubscript{2}, 1
CaCl\textsubscript{2}, and 10 D-glucose. Next, the slices were kept at
room temperature for at least 1 hour before any experimental procedure
was performed. An individual slice was transferred to a submerged
chamber (total volume: 400 \selectlanguage{greek}µ\selectlanguage{english}L). The slice was continuously perfused with
modified ACSF at a rate of 2.2--2.5 mL/min, with the help of a
peristaltic pump (Sci Q400, Watson-Marlow, Wilmington, MA, USA). The
modified ACSF (with continuous carbogen bubbling) included the following
components (in mM): 125 NaCl, 2.5 KCl, 1.25
Na\textsubscript{2}HPO\textsubscript{4}, 25 NaHCO\textsubscript{3}, 1.5
MgCl\textsubscript{2}, 2.5 CaCl\textsubscript{2}, and 10 D-glucose. The
recordings were performed at 32.5 \selectlanguage{ngerman}±1 °C.

\emph{Extracellular recordings}

Extracellular recordings were used to determine the synaptic properties
of the MPP -- DG synapse or the LPP -- DG synapse acquired from the
supra-pyramidal blade of the DG (see schematic representation in Figure
1a). The MPP field excitatory postsynaptic potentials (MPP fEPSPs) or
the LPP fEPSPs were evoked with a bipolar nichrome electrode placed in
the middle one-third or outer one-third section of the DG's molecular
layer, respectively. The evoked fEPSPs were recorded with a borosilicate
pipette (1-2 M\selectlanguage{greek}Ω \selectlanguage{english}of tip resistance, when filled with NaCl solution, 3M)
and placed 200-300 \selectlanguage{greek}µ\selectlanguage{english}M from the stimulation electrode. The current pulses
were delivered via a high-voltage isolation unit (A365D; World Precision
Instruments, Sarasota, FL, USA), commanded with a Master-8 pulse
generator (AMPI, Jerusalem, Israel). Responses were amplified with a
Dagan BVC-700A amplifier (Minneapolis, MN, USA) connected with a 100x
gain headstage (Dagan, model 8024) and high-pass filtered at 0.3 Hz.
Electrical noise suppression was accomplished with a Humbug noise
eliminator (Quest Scientific Instruments; North Vancouver, BC, Canada).
The evoked fEPSPs were displayed on a computer-based oscilloscope and
digitalized for storage and offline analysis with LabVIEW 7.1 software
(National Instruments, Austin, TX, USA). The A/D converter device used
was the BNC-2110 (National Instrument, Austin, TX).

\emph{Determination of basal synaptic strength and paired-pulse ratio}

A series of input-output (I-O) curves of current vs. synaptic responses
was constructed by delivering increasing current pulses at 0.067 Hz from
0 to 800 \selectlanguage{greek}µ\selectlanguage{english}A (100 \selectlanguage{greek}µ\selectlanguage{english}A steeps, 100 \selectlanguage{greek}µ\selectlanguage{english}s duration) and measuring the evoked
fEPSP amplitude (in mV). From the I-O curves, we also analyzed the Fiber
volley (FV) amplitude in response to current injection to determine
presynaptic excitability. The basal synaptic strength was determined by
analyses of the FV amplitude vs. fEPSP slope fitted with a Boltzmann
function, and the differences between control and MK-801-treated slices
were compared using the resulting slope of the Boltzmann equation. For
short-term plasticity determined by paired-pulse ratio (PPR) analysis, a
paired stimulation at 0.067 Hz (15-20\% of evoked maximal fEPSP
amplitude) was obtained, with inter-stimulus intervals (ISI) of 40, 60,
100, 200, and 500 ms. The PPR was calculated as the ratio between the
amplitude of the second response (S2) and the amplitude of the first
response (S1) (PPR = S2/S1). The input-specific origins of synaptic
responses from the MPP and the LPP were corroborated by the preferential
pharmacological sensitivity to DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M) and L-AP4 (20 \selectlanguage{greek}µ\selectlanguage{english}M),
respectively (Macek et al., 1996).

\emph{Long-term plasticity: depression and synaptic potentiation}

Because homosynaptic long-term depression (LTD) has scarcely been
examined in the two anatomical divisions of the perforant path to the
DG, different patterns of low-frequency stimulation (LFS) were used to
examine the LPP's and MPP's susceptibility to express a stable LTD. A
baseline response of fEPSP slope (50-70\% of maximal fEPSP amplitude)
was acquired with paired stimulation (60 ms ISI, 100 \selectlanguage{greek}µ\selectlanguage{english}s duration of
current pulse) at 0.067 Hz for 20 min. Then, 900 unitary current pulses
at 1-3 Hz were delivered at the LPP or the MPP, and the synaptic
responses were recorded for 90 min. This was followed by pharmacological
identification with L-AP4 or DCG-IV (Macek et al., 1996). In the case of
MPP -- DG synapse, LFS at 3 Hz was examined in the presence of the
inverse agonist AM 251 (5 \selectlanguage{greek}µ\selectlanguage{english}M), to determine the dependence of
cannabinoid receptor 1 (CB1) during the induction of LTD. For long-term
potentiation (LTP) experiments, the baseline conditions and
pharmacological identification were identical to those in the LTD
experiments, except that the baseline response of the fEPSP slope was
configured at 25-35\% of its maximal amplitude. Then, a theta-burst
stimulation (TBS) protocol was delivered to the LPP or the MPP, and
synaptic responses were recorded for 90 minutes. The TBS protocol (based
on Larson and Munk\selectlanguage{ngerman}ácsy, 2015) consisted of 3 episodes repeated at 10 s,
each with 10 bursts at 5 Hz and 5 current pulses at 100 Hz (see
Supplementary Figure 2). The decay of post-tetanic potentiation (PTP)
induced by TBS was expressed as \selectlanguage{english}? value, obtained from adjusting the
best fit of individual fEPSP slope values with a nonlinear regression
function of one phase decay. Heatmaps were constructed to depict the
magnitude of LTP, and cumulative probability charts were constructed
using post-TBS fEPSP slope values from minute 11 to 90.

\emph{Pharmacological modulation of 2-AG signaling pathway}

In a subset of experiments focused on the MPP -- DG synapse from control
and MK-801-treated slices, WIN 55,212-2, a CB\textsubscript{1}R agonist
(WIN, 5 \selectlanguage{greek}µ\selectlanguage{english}M), was bath perfused for 15 minutes to examine the presynaptic
induction of LTD mediated by CB\textsubscript{1} receptor activation, as
previously reported (Fontaine et al., 2020). Additionally, physostigmine
(10 \selectlanguage{greek}µ\selectlanguage{english}M), an acetylcholinesterase inhibitor, was perfused for 15 min to
evaluate endogenous 2-AG production in the MPP -- DG synapse, as
previously demonstrated in acute hippocampal slices (Wang et al., 2018b,
2018a). Finally, in another subset of experiments, JZL 184 (1 \selectlanguage{greek}µ\selectlanguage{english}M), an
irreversible inhibitor of monoacylglycerol lipase (MAGL) that increases
the 2-AG levels (Wang et al., 2016) was perfused for 15 min on
MK-801-treated slices to examine the effects of MAGL inhibition during
the delivery of LFS at 3 Hz.

\emph{Determination of frequency-dependent filtering in the LPP -- DG
synapse}

Synaptic frequency-dependent filtering was examined in the presence of
DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M), and a baseline response of fEPSP slope configured at 50\%
of its maximal amplitude was acquired for 10-12 min. Then, a train of 10
current pulses (100 \selectlanguage{greek}µ\selectlanguage{english}s of duration) at 5, 20, and 50 Hz was delivered to
the LPP, with an interval of 10 min between trains, as previously
reported (Quintanilla et al., 2022). For each stimulation frequency, the
train's evoked synaptic responses were normalized to the slope of its
first synaptic response and plotted as the number of stimuli vs.
normalized fEPSP slope (\%).

\emph{Spatial pattern separation (OPS) task}

The spatial pattern separation (OPS) task (based on van Goethem et al.,
2018) was performed to evaluate the animals' capacity to discriminate
minimal changes in the spatial position of identical objects in a
familiar arena. To reduce the animals' stress from the experimental
manipulation, the experimenter manipulated the rats twice per day (2-3
min) for five consecutive days before starting the behavioral
evaluations. A circular arena (40 cm high and 83 cm in diameter, gray
wall) was placed on a black acrylic platform inside an experimental room
with red light. On this platform, a series of reference points'
denominated positions (P) were designated as P1 to P5, with 6 cm between
the Ps. P1 was aligned at the center of the platform, both left and
right, and then P2 to P5 were designated both upward and downward, as
depicted in Figure 9a.

The OPS task consisted of two trials (3 min duration each): the learning
trial (T1) and the discrimination trial (T2). Before T1 in each
evaluation, a habituation phase was carried out, during which the animal
was exposed to the empty arena for 5 min. In T1, the animal was
introduced to the arena with two identical objects placed in P1 from the
left and right of the platform. One hour later, in T2, the animal was
re-exposed to the same objects, but one object was displaced to a new
position; this then carried on to include all other positions (P2-P5).
Object exploration was considered for analysis when the animal explored
an object with its nose, with a minimal distance of 2 cm. From the time
of exploration for each object, we calculated the discrimination index
(DI), a quantitative measurement of the animals' preference for
exploring the displaced object over the stationary object, using the
following formula:

\begin{equation}
DI=\frac{\left(displaced\ objects\ exploration\ time-stationary\ object^{\prime}\ s\ exploration\ time\right)}{two\ objects\ total\ exploration\ time}\nonumber \\
 \end{equation}

To estimate the DI, a minimum exploration time of 7 and 10 s was
required for T1 and T2, respectively (van Goethem et al., 2018).
Likewise, the performance of each animal for all positions was
determined. Therefore, the animals were exposed 5 times to the OPS task
with an interval of 2 days and different par objects, which prevented
familiarization (van Goethem et al., 2018). The order of evaluation of
object positions was random: while one animal began with P1 (without
displacement of objects), another animal began with P5 (maximal
displacement of one object). After each trial, the platform and the
objects were cleaned with ethanol (70\%) to eliminate odor residues. The
first evaluation of the OPS task was carried out in animals of P30.

\emph{Materials}

Except for (+)-MK-801 maleate, DCG-IV, L-AP4, and AM 251, which were
purchased from Tocris Biosciences (Minneapolis, MN, USA), the drugs and
chemicals used in this study were purchased from Sigma Aldrich (St.
Louis, MO, USA).

\emph{Statistical analysis}

Group measures are numerically expressed as mean \selectlanguage{ngerman}± SEM. The violin plots
show the median, mean, first, and third quartiles. All statistical
comparisons were performed between control values and those obtained
from MK-801-treated animals, following the guidelines on experimental
design and analysis in pharmacology (Curtis et al., 2018). The normality
distribution of data was validated with the Kolmogorov--Smirnov test
(\emph{P} ~= 0.05). No outliers were removed from the data. The
comparability among experimental conditions was assessed by two-tailed
paired (or unpaired) Student's t-test, Mann-Whitney test, Wilcoxon test,
Kruskal-Wallis test, or two-way repeated-measures (RM) analysis of
variance (ANOVA), as appropriate. The Holm--\selectlanguage{polish}Š\selectlanguage{english}id\selectlanguage{ngerman}ák post-hoc or Dunn's
post-hoc test was used for multiple comparisons among experimental
groups, only when F achieved minimal statistical significance. For all
the experiments, data were considered significant if~\emph{P}
~\textless{} 0.05.

\textbf{Results}

\emph{Criteria for isolating synaptic responses from the medial and
lateral perforant paths onto granule cells of the dentate gyrus}

The DG granule cells receive glutamatergic inputs via the axons of
stellate cells and pyramidal neurons of the lateral and medial EC. These
axons, collectively named perforant paths (PP), exhibit unique
pharmacological and synaptic properties depending on whether they
originate from the lateral or medial EC (LEC and MEC, respectively). We
first isolated and characterized their synaptic responses before
exploring whether neonatal treatment with MK-801 alters the LPP and MPP
synapses. A stimulation electrode was positioned in the outer one-third
section of the molecular layer (\selectlanguage{english}[?]200-220 \selectlanguage{greek}µ\selectlanguage{english}m above the granule cell
layer) or the middle one-third section ([?]100-120 \selectlanguage{greek}µ\selectlanguage{english}m above) of the
supra-pyramidal blade of the DG (Petersen et al., 2013), and recording
pipettes were positioned 200 -- 300 \selectlanguage{greek}µ\selectlanguage{english}M onto the stimulation electrode to
record a fEPSPs from the LPP or MPP, respectively (Figure 1a).
Presynaptic terminals of LEC-contacting granule cells selectively
express group III metabotropic glutamate receptors (mGluRs), while those
of MEC-contacting granule cells express group II mGluRs (Macek et al.,
1996; Shigemoto et al., 1997). We used these pharmacological criteria to
verify the origin of the evoked responses. Bath perfusion of DCG-IV (5
\selectlanguage{greek}µ\selectlanguage{english}M), a group II mGluRs agonist, did not depress the LPP fEPSP (92.9 \selectlanguage{ngerman}±
3.77\% of baseline response); however, the subsequent perfusion of L-AP4
(20 \selectlanguage{greek}µ\selectlanguage{english}M), a group III mGluRs agonist, abolished the synaptic response
(17.18 \selectlanguage{ngerman}± 2.8\% of baseline response; \emph{n} = 7 slices / 6 animals,
upper traces in Figure 1b). On the other hand, bath perfusion of L-AP4
did not depress the MPP fEPSP (94.8 ± 3.28\% of baseline response).
However, the synaptic response was abolished during the subsequent
application of DCG-IV (20.35 ± 3.2\% of baseline response; \emph{n} = 7
slices / 6 animals; lower traces in Figure 1b). The pharmacological
activation of mGluRs shows that responses evoked from the LPP and the
MPP converging on DG granule cells can be reliably isolated. Therefore,
we systematically used these criteria to corroborate the synaptic
origins of the evoked responses included in this study.

\emph{The transient hypofunction of NMDARs during early postnatal
development differentially affects the synaptic strength of the PP -- DG
synapses.}

It is already established that neonatal treatment with MK-801 or related
antagonists of NMDA receptors such as phencyclidine alters the
glutamatergic transmission of the hippocampus (Kjaerby et al., 2017;
Griego et al., 2022; Márquez et al., 2023). Therefore, we explored the
effects of MK-801 on the synaptic strength of the LPP and the MPP inputs
to DG granule cells. In the control condition, the averaged I-O curve of
the LPP fEPSP (0-800 \selectlanguage{greek}µ\selectlanguage{english}A; 100 \selectlanguage{greek}µ\selectlanguage{english}A steps, at 0.067 Hz) reached a maximal
amplitude of 4.16 \selectlanguage{ngerman}± 0.46 mV (black symbols in Figure 1c; \emph{n} = 9
slices / 6 animals); remarkably, neonatal treatment with MK-801 did not
alter the maximal amplitude of the LPP fEPSP (maximal amplitude: 3.54 ±
0.34 mV; green symbols in Fig 1c, \emph{n} = 10 slices / 6 animals).
Contrary to this observation, the MPP fEPSP reached a maximal amplitude
of 7.5 ± 0.68 mV (black symbols in Figure 1d; \emph{n} = 8 slices / 6
animals), and neonatal treatment with MK-801 reduced the maximal MPP
fEPSP amplitude to 3.87 ± 0.52 mV (\emph{P} \textless{} 0.001 vs.
control, two-way RM ANOVA treatment effect: F\textsubscript{(1, 16)} =
8.31; red symbols in Figure 1d; \emph{n} = 10 slices / 6 animals). Our
findings indicate that neonatal treatment with MK-801 selectively
dysregulates the MPP inputs to DG granule cells. While the MPP exhibits
a \selectlanguage{english}[?]50\% reduction in its maximal amplitude, the LPP transmission is not
affected by MK-801. Likewise, the pharmacological selectivity of MK-801
suggests a differential composition in the presynaptic and postsynaptic
components mediating glutamatergic transmission of the LPP and the MPP
onto DG granule cells. In addition, we performed an analysis of the
fEPSP kinetics from MPP and LPP synapses; these parameters corroborate
the differences in the strength of both synapses (Table 1).

Because the fEPSPs acquired for the I-O curves were preceded by
presynaptic fiber volleys (FV), we also analyzed the relationships
between stimulus intensity, FV amplitude, and fEPSP amplitude. The left
panel in Figure 1e shows the relationship between stimulus intensity (I)
and LPP FV amplitude. The right panel depicts the relationship between
FV amplitude and LPP fEPSP slope in both experimental conditions (black
and green symbols). We found that neonatal treatment with MK-801 did not
alter either relationship at the LPP -- DG synapse. However, the same
analysis at the MPP synapse uncovered a different scenario. The left
panel in Figure 1f contrasts the FV response in control vs.
MK-801-treated animals (black and red symbols). Compared to the control
slices, the MK-801-treated slices exhibited a significant decrease in FV
amplitude and faster response saturation {[}two-way RM ANOVA,
interaction effect (treatment x current intensity): F\textsubscript{(8,
88)} = 2.57, \emph{P} \textless{} 0.01; red symbols; left panel in
Figure 1f{]}. Likewise, the coupling analysis of the FV vs. MPP fEPSP
slope shows a dysregulation between FV and MPP fEPSP (slope in control:
0.87 +- 0.24 mV/ms; in MK-801: 0.16 +- 0.04 mV/ms; Mann-Whitney
test,\emph{P} \textless{} 0.01; right panel in Figure 1f). These results
suggest that neonatal treatment with MK-801 alters the propagation of
presynaptic action potentials and the concomitant synaptic response in
the MPP but not the LPP synapse.

\emph{The transient hypofunction of NMDARs during early postnatal
development modifies the paired-pulse ratio at the LPP and the MPP
synapses.}

Consistent with previous studies (Petersen et al., 2013;
Collitti-Klausnitzer et al., 2021), the PPF (60 ms inter-stimulus
interval, ISI) differs between the LPP and MPP synapses. We corroborated
that the LPP has a prominent PPF (PPF at the LPP -- DG = 1.72 +-
0.03;\emph{n} = 28 slices / 15 animals) compared to the MPP (PPF at the
MPP -- DG = 1.22 +- 0.03; \emph{n} = 29 slices / 15 animals; Student's
t-test: t\textsubscript{(55)} = 12.01, \emph{P} \textless{} 0.001).
Figure 2a shows the PPF distribution of the LPP and the MPP using a
violin graph and the responses obtained from 57 independent dorsal
hippocampal slices. Figure 2b shows representative examples of
facilitation from the LPP and the MPP and their corresponding depression
in response to selective activation of group III and II mGluRs,
respectively. Because neonatal treatment with MK-801 enhances the PPF at
the Mossy Fiber -- CA3 pyramidal cell synapse of the rat hippocampus
(Segev et al., 2020; Marquez et al., 2023), we performed a similar
exploration on the LPP and the MPP using a broader ISI range (40, 60,
100, 200, and 500 ms). The exploration of the LPP PPF revealed increased
facilitation in the MK-801 slices compared to control slices (two-way RM
ANOVA, treatment effect: F\textsubscript{(1, 16)} = 5.19,
\emph{P}\textless{} 0.05; \emph{n} = 9 slices / 7 animals, for each
condition, Figure 2c). The LPP PPF level was statistically increased at
40 and 60 ms (LPP PPF at 40 ms in control: 1.64 +- 0.07; in MK-801: 2.01+-
0.01; t\textsubscript{(16)} = 2.57, \emph{P} \textless{} 0.05; at 60 ms:
1.74 +- 0.06; in MK-801: 1.99 +- 0.06; t\textsubscript{(16)} = 2.9,
\emph{P}\textless{} 0.01; at 100 ms: 1.71 +- 0.02; in MK-801: 1.79 +-
0.04; t\textsubscript{(16)} = 1.5, \emph{P} \textgreater{} 0.05). The
temporal change in the PPF is depicted in the representative traces of
Figure 2d.

The MPP PPF also showed increased facilitation in the MK-801-treated
slices (\emph{n} = 9 slices / 7 animals) compared to control slices
(\emph{n} = 8 slices / 6 animals; two-way RM ANOVA, treatment effect:
F\textsubscript{(1, 15)} = 4.63, \emph{P} \textless{} 0.05; Figure 2e).
The facilitation was limited to the ISI of 200 ms (PPF at 60 ms in
control: 1.32 +- 0.06; in MK-801: 1.52 +- 0.08; t\textsubscript{(15)} =
1.854,\emph{P} \textgreater{} 0.05; at 100 ms: 1.3 +- 0.06; in MK-801:
1.51 +- 0.09; t\textsubscript{(15)} = 1.77, \emph{P} \textless{} 0.05; at
200 ms: 1.16 +- 0.05; in MK-801: 1.36 +- 0.04; t\textsubscript{(15)} =
2.95,\emph{P} \textless{} 0.01; representative traces in Figure 2f). The
increased PPF observed in the LPP and the MPP synapses suggests a
dysregulation in the presynaptic machinery that controls glutamate
release on the DG granule cells.

\emph{Low-frequency stimulation induces the MPP long-term depression
dependent on the cannabinoid receptor type 1.}

Thus far, our data show decreased synaptic strength and altered control
of glutamate release of the PP terminals synapsing DG granule cells.
Previous studies demonstrated that those MK-801-driven changes
negatively impact the induction of long-term synaptic plasticity in the
hippocampus (Griego et al., 2022; Marquez et al., 2023). Therefore, it
is reasonable to assume that neonatal treatment with MK-801 will
negatively impact the induction of long-term plasticity at the PP
synapses on DG granule cells of the hippocampus.

However, since long-term depression has been scarcely explored in the PP
-- DG synapses, we first determined a reliable stimulation protocol to
induce LTD. Our exploratory experiments show that low-frequency
stimulation (LFS, 900 pulses, 1 Hz), a protocol commonly used to induce
glutamatergic LTD in the hippocampus (Dudek and Bear, 1992), fails to
induce stable synaptic depression in both the LPP -- DG and the MPP --
DG synapse. In the LPP -- DG synapse, LFS did not alter the baseline
response (fEPSP at 50 min post-LFS: 108 +- 7.73\% of baseline, \emph{n} =
4 slices / 4 animals; white symbols and upper traces in Figures 1a and
1b). In the MPP -- DG synapse, LFS caused transitory synaptic
depression, and the fEPSP returned to baseline values within 50 min
(fEPSP at 50 min post-LFS: 93.15 +- 5, \emph{n} = 5 slices / 4 animals;
white symbols and upper traces in supplementary Figure 1d and 1e).

Stellar cells and pyramidal neurons of EC layer II discharge volleys in
the delta and theta range (0.4 -- 10 Hz) to DG granule cells (Gloveli et
al., 1997; Deshmukh et al., 2010). Therefore, we explored whether a
stimulation paradigm within this physiological range induces LTD.
Contrary to our prediction, we found that 900 pulses delivered at 3 Hz
triggered synaptic potentiation in the LPP -- DG synapse (fEPSP at 50
min post-LFS: 121.2 +- 9.32\% of baseline, \emph{n} = 3 slices / 3
animals; black circles and lower traces in supplementary figures 1a and
1b). This form of synaptic potentiation requires additional
investigation beyond the scope of the present study.

In sharp contrast, the delivery of 900 pulses at 3 Hz in the MPP -- DG
synapse induced stable glutamatergic LTD that lasted up to 50 min (fEPSP
at 50 min post-LFS: 69.64 +- 6.65\% of baseline, \emph{n} = 4 slices / 4
animals; Mann-Whitney test, \emph{P} \textless{} 0.05 vs. LFS at 1 Hz;
black circles and lower traces in Figures 1d and 1e). Because the
delivery of 900 pulses at 3 Hz induced a reliable LTD in the MPP but not
in the LPP synapse, subsequent experiments were restricted to the MPP --
DG synapse.

Figure 3a-b summarizes the induction of LTD in the MPP -- DG synapse and
its sensitivity to perfusion DCG-IV (fEPSP at 90 min post-LFS: 69 +-
6.25\% of baseline response,\emph{n} = 6 slices / 6 animals; fEPSP in
the presence of DGC-IV: 15.66 +- 2.55\% of baseline response, Wilcoxon
test, \emph{P} \textless{} 0.05 vs. baseline response). Moreover, the
induction of MPP LTD was accompanied by an increment in the PPF (MPP PPF
in control condition: 1.18 +- 0.08; at 90 min post-LFS: 1.54 +- 0.17;
Wilcoxon test, \emph{P}\textless{} 0.05; black bars in Figure 3f),
indicating presynaptic locus for expression of LTD.

According to a previous study, induction of LTD in the MPP synapse
requires the delivery of 6,000 pulses at 10 Hz while simultaneously
blocking GABA\textsubscript{A} receptors (Penasco et al., 2019). Under
these experimental conditions, the authors reported that this form of
LTD requires activation of the cannabinoid receptor type 1
(CB\textsubscript{1}R). Therefore, our next experiment aimed to
determine if CB\textsubscript{1}R is required for LTD induced with 3 Hz
while maintaining active GABAergic transmission. Consistent with Penasco
et al., we found that perfusion of the CB\textsubscript{1}R antagonist,
AM 251 (5 \selectlanguage{greek}µ\selectlanguage{english}M), blocks the induction of LTD (MPP fEPSP at 90 min
post-LFS: 119.7 \selectlanguage{ngerman}± 10.94\% of baseline response, \emph{n} = 4 slices / 4
animals; lower traces and white symbols in Figure 3a-b and white bars in
Figure 3e). The subsequent perfusion of the DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M) at the end of
experiments corroborated the MPP origin of the synaptic responses (fEPSP
in the presence of DCG-IV: 15.7 \selectlanguage{ngerman}± 5.22\% of baseline; white bar in
Figure 3e). Likewise, perfusion of AM 251 prevented the changes in the
MPP PPF observed after the induction of LTD (MPP PPF in AM 251 treated
slices in control condition: 1.22 ± 0.1; at 90 min post-LFS: 1.2 ± 0.08;
Wilcoxon test, \emph{P} \textgreater{} 0.05; white bars in Figure 3f).
Together, these results confirm that the MPP -- DG synapse exhibits a
stable LTD with a presynaptic locus of expression and requires
CB\textsubscript{1}R activation.

\emph{The transient hypofunction of NMDARs during early postnatal
development impairs the CB\textsubscript{1}R-dependent LTD at the MPP --
DG synapse.}

If CB\textsubscript{1}R activation is a necessary step for the induction
of MPP LTD, and previous studies have documented altered
CB\textsubscript{1}R functionality in several schizophrenia models
(Kaminitz et al., 2014; Sz\selectlanguage{polish}ű\selectlanguage{english}cs et al., 2016; Osborne et al., 2019), it is
plausible that MK-801-treated slices could exhibit altered LTD
induction. We tested this prediction by applying LFS (900 pulses at 3
Hz) to the MK-801-treated slices. As shown in Figure 3d, LFS failed to
induce LTD at the MPP -- DG synapse. Moreover, the stimulation protocol
induced potentiation of the MPP fEPSP (fEPSP at 90 min post-LFS in
MK-801-treated slices: 139.7 \selectlanguage{ngerman}± 23.12\% of baseline, \emph{n} = 6 slices
/ 6 animals, Kruskal-Wallis test, Dunn's post-hoc test, \emph{P}
\textless{} 0.05 vs. control; traces and red bars in Figure 3c-d). The
synaptic response was sensitive to perfusion of DCG-IV, confirming its
presynaptic MPP nature (fEPSP in the presence of DCG-IV: 42.13 ± 8.5\%
of baseline, Kruskal-Wallis test, Dunn's post-hoc test, \emph{P}
\textless{} 0.05 vs. control; red bars in Figure 3e). Interestingly, the
anomalous synaptic potentiation observed in the MK-801-treated slices
did not alter the MPP PPF (PPR in control condition: 1.43 ± 0.07; at 90
min post-LFS: 1.5 ± 0.09; Wilcoxon test, \emph{P}\textgreater{} 0.05;
red bars in Figure 3f), suggesting altered functionality of postsynaptic
glutamate receptors. The cumulative probability chart in Figure 3g
summarizes the magnitude of the MPP LTD observed in control slices
(black line), the blockade of LTD in the presence of AM 251 (gray line),
and the synaptic response of MK-801-treated slices (red line). These
results demonstrate that transient hypofunction of NMDARs during early
postnatal development impairs the presynaptic-mediated,
CB\textsubscript{1}R-dependent LTD and triggers aberrant forms of
synaptic plasticity in the MPP -- DG synapse.

\emph{The transient hypofunction of NMDARs during early postnatal
development alters the functional expression of CB\textsubscript{1}R in
the MPP -- DG synapse.}

Given CB\textsubscript{1}R's critical role in the induction of LTD, we
next examined the functional expression of CB\textsubscript{1}R in the
MPP -- DG synapse. We hypothesized that pharmacological activation of
CB\textsubscript{1}R would fail to induce synaptic depression in the
MK-801-treated slices. To test this prediction, we acquired a stable
baseline of MPP fEPSPs for 20 min prior to perfusing WIN 55,212-2, a
CB\textsubscript{1}R agonist (WIN, 5 \selectlanguage{greek}µ\selectlanguage{english}M; 15 min). Recording continued up
to 90 min, followed by perfusion of DCG-IV. In the control condition, we
found that WIN triggers a stable synaptic depression in the MPP -- DG
synapse, sensitive to DCG-IV (fEPSP 90 min post-WIN: 43.01 \selectlanguage{ngerman}± 6.53\% of
baseline, \emph{n} = 6 slices / 5 animals; fEPSP in the presence of
DCG-IV: 11.23 ± 4.89\% of baseline; traces and black bars in Figure
4a-b, e). By contrast, perfusion of WIN in the MK-801-treated slices
decreased the magnitude of the synaptic depression of the MPP fEPSP
(fEPSP 90 min post-WIN: 74.11 ± 12.16\% of baseline, \emph{n} = 6 slices
/ 6 animals, Student's t-test: t\textsubscript{(10)} = 2.25, \emph{P}
\textless{} 0.05 vs. control; fEPSP in the presence of DCG-IV: 28.89 ±
4.61\% of baseline; traces and red bars in Figure 4a-b, e), suggesting
functional dysregulation of presynaptically expressed
CB\textsubscript{1}R.

Because presynaptic activation of CB\textsubscript{1}R requires
postsynaptic synthesis and release of 2-AG, we next investigated if
neonatal treatment with MK-801 interferes with the postsynaptic
synthesis and release of 2-AG. We stimulated the local production of
2-AG by perfusing the cholinesterase inhibitor physostigmine (10 \selectlanguage{greek}µ\selectlanguage{english}M) and
recording the MPP fEPSPs. After a stable baseline was acquired for 20
min, physostigmine was perfused (10 \selectlanguage{greek}µ\selectlanguage{english}M for 15 min). Recording continued
up to 90 min and was followed by the perfusion of DCG-IV. In the control
condition, physostigmine perfusion induced synaptic depression,
sensitive to DCG-IV (fEPSP 90 min post-physostigmine: 52.45 \selectlanguage{ngerman}± 7.06\% of
baseline, \emph{n} = 6 slices / 6 animals; fEPSP in the presence of
DCG-IV: 9.38 ± 1.2\% of baseline; traces and black bars in Figure 4c-d,
f). Strikingly, in the MK-801-treated slices, physostigmine induced a
seemingly diminished synaptic depression, sensitive to DCG-IV (fEPSP 90
min post-physostigmine: 77.65 ± 9.65\% of baseline, \emph{n} = 5 slices
/ 5 animals, Student's t-test: t\textsubscript{(9)} = 2.15; \emph{P}
\textgreater{} 0.05 vs. control; fEPSP in the presence of DCG-IV: 30.27
± 1.97\% of baseline; traces and red bars in Figure 4c-d, f). The
magnitude of the synaptic depression observed in both WIN and
physostigmine in the MK-801-treated slices was similar (Figure 4g),
suggesting that 2-AG production in granule cells is not affected by
transient hypofunction of NMDARs during early postnatal development.
Likewise, the reduced synaptic depression observed in the MK-801 in the
presence of physostigmine may be ascribed to dysregulated
CB\textsubscript{1}R functionality. This possibility requires further
exploration.

\emph{The inhibition of monoacylglycerol lipase responsible for 2-AG
breakdown restores the impaired CB\textsubscript{1}R-dependent LTD in
MK-801-treated slices.}

We reasoned that the impaired LTD may be partially explained by
insufficient production or accelerated breakdown of 2-AG during synaptic
stimulation. Previous studies have documented increased activity of
monoacylglycerol lipase (MAGL), the enzyme responsible for 2-AG
degradation, in schizophrenic individuals and experimental models of
schizophrenia (Du et al., 2013; Kaya et al., 2019). Therefore, we
examined whether increasing 2-AG signaling by inhibiting MAGL activity
might restore the electric induction of LTD in the MK-801-treated
slices. For these experiments, MK-801-treated slices were perfused with
the irreversible MAGL inhibitor, JZL 184 (1 \selectlanguage{greek}µ\selectlanguage{english}M, perfused for the last 10
minutes of the baseline response and the first 5 minutes during LFS).
Before LFS, the application of JZL 184 did not modify the baseline
response. More importantly, JZL 184 reverted the loss of LTD at the MPP
-- DG synapse of the MK-801-treated slices for up to 90 min (fEPSP 90
min post-LFS: 58.46 \selectlanguage{ngerman}± 11.68 \% of baseline, \emph{n} = 5 slices / 5
animals, Student's t-test: t\textsubscript{(9)} = 2.94,
\emph{P}\textless{} 0.01 vs. MK-801; traces and blue bars in Figure
5a-c). Perfusion of DCG-IV at the end of the recordings corroborated the
MPP origin of the synaptic response (fEPSP in the presence of DCG-IV: 27
± 7.43\% of baseline). Likewise, induction of MPP LTD in the
MK-801-treated slices was accompanied by increased MP PPF (PPR in
control condition: 1.38 ± 1.11; at 90 min post-LFS: 1.77 ± 0.09; paired
Student's t-test: t\textsubscript{(4)} = 2.97, \emph{P} \textless{}
0.05; blue bars in Figure 5d), which exhibited similar facilitation
magnitude to the control slices (see black bars in Figure 3f). The
cumulative probability chart in Figure 5e summarizes the magnitude of
the MPP LTD observed in the JZL 184-treated slices (blue line) and the
lack of LTD in the MK-801-treated slices (red line). These findings
demonstrate that LTD induction in the MPP -- DG synapse of animals
treated with MK-801 is restored by using MAGL blockade to reduce 2-AG
breakdown and thus enhance its signaling. This supports the notion that
MAGL blockade has potentially neuroprotective effects (Ren et al.,
2020).

\emph{Neonatal hypofunction of NMDARs during early postnatal development
impairs the induction of long-term potentiation in the LPP -- DG
synapse.}

Granule cells undergo LTP through multiple stimulation patterns that
mimic PP activity impinging on the DG. These stimulation protocols are
physiologically and behaviorally relevant (Lopez-Rojas et al., 2016);
among them, theta-burst stimulation (TBS) has been successfully used to
induce stable LTP at the MPP and LPP synapses (Lopez-Rojas et al., 2016;
Vyleta and Snyder, 2021). Therefore, we applied TBS to the LPP to test
the effects of neonatal MK-801 on LTP induction. A stable baseline of
LPP fEPSP slope was recorded for 20 min prior to TBS {[}3 episodes
repeated at 10 s; each episode included 5 pulses delivered at 100 Hz and
repeated 10 times at 5 Hz (Larson and Munkácsy, 2015){]}. Recording
continued for 90 min and was followed by perfusion of L-AP4 (20 \selectlanguage{greek}µ\selectlanguage{english}M). In
the control condition, TBS triggered a marked post-tetanic potentiation
(LPP PTP), followed by long-lasting LPP fEPSP slope potentiation (fEPSP
at PTP: 213.4 \selectlanguage{ngerman}± 23.13\% of baseline; fEPSP 90 min post-TBS: 144.6
±13.28\% of baseline; fEPSP in presence of L-AP4: 23.18 ± 3.82\% of
baseline; \emph{n} = 7 slices / 6 animals, traces and black bars in
Figure 6a-b). On the other hand, on the MK-801-treated slices, TBS
caused a strong PTP but did not induce a sustained increase in the slope
of the fEPSP, as illustrated in the green traces and green symbols in
Figure 6a-b (fEPSP at PTP: 239.9 ± 18.28\% of baseline; fEPSP 90 min
post-TBS: 105.5 ± 10.9\% of baseline, Mann-Whitney test,
\emph{P}\textless{} 0.05 vs. control; fEPSP in presence of L-AP4: 28.01
± 4.33\% of baseline; \emph{n} = 7 slices / 7 animals). Interestingly,
by adjusting a best-fit single exponential decay function, we found that
decay of PTP (tau, \selectlanguage{english}?) is faster in MK-801-treated slices than control
slices, suggesting short-term plasticity dysregulation (\selectlanguage{english}? in control:
114 +- 23.38 s; \selectlanguage{english}? in MK-801: 47.03 +- 5.88 s, Mann-Whitney test,
\emph{P}\textless{} 0.05: inset in Figure 6b).

The bar graphs in Figure 6c summarize the mean PTP, potentiation, and
sensitivity to L-AP4, while the symbols represent the individual
experiments for the control condition and MK-801-treated slices. The
heatmaps in Figure 6d contrast the magnitude of the potentiation
obtained in each slice, and the cumulative probability graph in Figure
6e shows the overall potentiation for control slices (black symbols) vs.
MK-801-treated slices (green symbols). Together, these experiments show
that the potentiation of the LPP fEPSP is sensitive to the transient
hypofunction of NMDARs during early postnatal development.

\emph{Neonatal hypofunction of NMDARs during early postnatal development
does not interfere with the expression of LTP in the MPP -- DG synapse.}

We also explored the synaptic potentiation of the MPP from control and
MK-801-treated slices with the same TBS protocol we delivered at the LPP
synapse. A stable baseline response of MPP fEPSPs was recorded for 20
min. This was followed by TBS and 90 min of continuous recording and
then, finally, perfusion of DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M). In the control slices, MPP
TBS triggered a PTP and a robust, long-lasting enhancement of the MPP
fEPSP slope, sensitive to DCG-IV (fEPSP at PTP: 199.8 \selectlanguage{ngerman}± 11.65\% of
baseline; fEPSP 90 min post-TBS: 171.4 ± 16\% of baseline; fEPSP in
presence of DCG-IV: 18.27 ± 2.24\% of baseline; \emph{n} = 8 slices / 7
animals; traces and black symbols in Figure 7a-b). Interestingly, MPP
TBS delivered on the MK-801-treated slices also triggered PTP, followed
by a sustained increase in the slope of the fEPSP not statistically
different from the synaptic potentiation observed in the control slices
(fEPSP at PTP: 198.9 ± 29.47\% of baseline; fEPSP 90 min post-TBS: 135 ±
9.43\% of baseline, Mann-Whitney test, \emph{P} \textgreater{} 0.05 vs.
control; fEPSP in presence of DCG-IV: 32.46 ± 5.16\% of
baseline;\emph{n} = 7 slices / 7 animals; traces and red symbols in
Figure 7a-b). Likewise, we did not find differences in \selectlanguage{english}? values between
the control and MK-801-treated slices (\selectlanguage{english}? in control: 65 +- 3 s; \selectlanguage{english}? in
MK-801: 83.94 +- 16.34 s, Mann-Whitney test, \emph{P} \textgreater{}
0.05: inset in Figure 7b).

The average post-TBS responses at 90 min for the slices analyzed in the
control condition and MK-801-treated slices are depicted in the bar
graphs in Figure 7c. The heatmaps in Figure 7d contrast the magnitude of
the synaptic potentiation obtained in each slice, and the cumulative
probability graph in Figure 7e shows the potentiation for control slices
(black symbols) vs. MK-801-treated slices (red symbols). These results
suggest that transient hypofunction of NMDA receptors during early
postnatal development does not suppress the MPP -- DG synapse's ability
to undergo LTP in young rats.

\emph{Transient hypofunction of NMDARs during early postnatal
development impairs the frequency-dependent synaptic filtering in the
LPP -- DG synapse.}

A specialized feature of the LPP -- DG synapse is that it operates as a
low-pass filter (Quintanilla et al., 2022). That is, while low
frequencies, such as those in the theta range, are efficiently
transferred to the DG, the high frequency patterns, i.e., the gamma
range, are greatly attenuated upon arrival to the DG. Therefore, in the
next experiment, we examined the frequency-dependent filtering property
of the LPP -- DG synapse by delivering stimulation frequencies relevant
to hippocampal cognitive processing, such as the theta (5 Hz), beta (20
Hz), and low gamma (50 Hz) stimulation ranges (Engel and Fries, 2010;
Colgin, 2016).

Consistent with Quintanilla et al. (2022), in control slices, a theta
train (10 pulses at 5 Hz) induced sustained facilitation all over the
evoked synaptic responses (\emph{n} = 9 slices / 6 animals; traces and
black symbols in Figures 8a1 and 8b1). The traces in Figure 8c1 also
depict the facilitated response, which contrasts the amplitude of the
first response vs. the last response (S1/S10). In contrast, the
MK-801-treated slices exhibited greater facilitation in response to 5 Hz
stimulation (\emph{n} = 7 slices / 7 animals). The green symbols of the
scatter plot in Figure 8a1 show the increased facilitation of the LPP
fEPSPs in MK-801-treated slices compared to control slices (RM two-way
ANOVA, treatment effect: F\textsubscript{(1, 14)} = 7.553,
\emph{P}\textless{} 0.05; green traces in Figure 8b1), and the increased
facilitation in the amplitude of last response vs. first response is
depicted in the green traces in Figure 8c1.

On the other hand, in response to 20 Hz stimulation, control slices
exhibited increased facilitation on the first part of the train (second
to fifth response), which reverted in the last five responses,
generating a Gaussian-type facilitation distribution (\emph{n} = 8
slices / 6 animals; symbols and black traces in Figure 8a2-c2). Contrary
to the ability to attenuate the synaptic responses, the MK-801-treated
slices exhibited greater facilitation. i.e., decreased filtering
ability. However, this phenomenon, illustrated in Figure 8a2, did not
show significant differences compared to control slices (RM two-way
ANOVA, treatment effect: F\textsubscript{(1, 15)} = 3.386, P
\textgreater{} 0.05; \emph{n} = 8 slices / 7 animals for MK-801). The
representative traces are depicted in Figures 8b2 and 8c2.

The diminished filtering ability of the LPP -- DG synapse in
MK-801-treated slices was further revealed by stimulation in the gamma
frequency range (50 Hz). Under this experimental paradigm, the control
slices exhibited enhanced facilitation in the initial portion of the
stimulation train (second to fourth response). The facilitation reverted
in the middle section and was strongly attenuated in the final section
of the train (\emph{n} = 8 slices / 6 animals; symbols and black traces
in Figure 8a3-c3). Although filtering ability was present in the
MK-801-treated slices, there was still an easily identifiable decrease
under this condition compared to control slices (RM two-way ANOVA,
treatment effect: F\textsubscript{(1, 14)} = 5.285, \emph{P} \textless{}
0.05, \emph{n} = 8 slices / 7 animals for MK-801; green symbols in
Figure 8a3 and green traces in Figures 8b3 and 8c3). The dysregulation
in frequency-dependent filtering of LPP in MK-801-treated slices can
also be observed in the scatter plot graphs, which show the change in
amplitude between the first response and tenth response for each slice
at 5, 20, and 50 Hz train (Figure 8d-f), demonstrating reduced synaptic
filtering in MK-801-treated slices. These results show that the synaptic
property of low-pass filtering of the LPP -- DG synapse is dramatically
altered in response to the transient hypofunction of NMDARs during early
postnatal development.

\emph{Transient hypofunction of NMDA receptors impairs spatial
discrimination in rats.}

It is hypothesized that after the arrival of cortical information
conveying two similar experiences or two events close in time, the DG
uses a computational process called pattern separation to orthogonalize
(or maximize) the differences in the incoming information onto area CA3
(Yassa and Stark, 2011; Santoro, 2013). This neural mechanism is
believed to facilitate the proper storage of similar neuronal
information as independent events and prevent the overlapping of new
memories. We hypothesized that, if the DG's integrity is compromised due
to transient hypofunction of NMDARs, the behavioral activity in which
the DG participates will also be compromised. To explore this
possibility, we evaluated MK-801-treated animals' ability to
discriminate small changes in the spatial configuration of identical
objects maintained in a familiar environment (Santoro, 2013). The
discrimination index (DI) of this behavioral test was evaluated by
alternating the spatial configuration of identical objects in a familiar
environment (see Figure 9a for a schematic representation). A series of
spatial positions (P) was used to determine the minimal displacement
position of one object vs. another at which the animal perceived the
change in spatial position. This cognitive ability increases the demand
for spatial pattern separation activity (van Goethem et al., 2018).

In control animals, we corroborated that the DI depends on the magnitude
of displacement of one object (Figure 9b), a phenomenon previously
reported in adult animals (van Goethem et al., 2018). On the other hand,
MK-801-treated animals efficiently differentiated the change in the
spatial position of objects when displacement was maximal (DI in P5 from
control vs. MK-801: 0.41 +- 0.035 vs. 0.43 +- 0.07; \emph{n} = 10 for the
control group and \emph{n} = 12 for MK-801 group), and exhibited
comparable DI values in the absence of object displacement (DI in P1
from control vs. MK-801: 0.001 +- 0.021 vs. -0.01 +- 0.011). However, when
the magnitude of one object's displacement was gradually reduced from P4
to P2, the MK-801-treated animals' discrimination ability was reduced
(DI in P4 from control vs. MK-801: 0.38 +- 0.04 vs. 0.17 +- 0.05; DI in P3
from control vs. MK-801: 0.25 +- 0.03 vs. 0.1 +- 0.04; DI in P2 from
control vs. MK-801: 0.07 +- 0.03 vs. 0.001 +- 0.01). These differences in
DI values were significant at P3 (two-way RM ANOVA, treatment effect:
F\textsubscript{(1, 20)} = 8.225, \emph{P} \textless{} 0.01; Holm--\selectlanguage{polish}Š\selectlanguage{english}id\selectlanguage{ngerman}ák
post-hoc test, \emph{P} \textless{} 0.05). The heatmaps in Figure 9c
show the spatial discrimination performance for both experimental
conditions. Although MK-801-treated animals efficiently discriminate new
object positions when the magnitude of the displacement is maximal (P5),
their mnemonic ability for spatial discrimination is reduced when the
magnitude of displacement of one object is gradually narrowed, a
condition that increases the demand of spatial pattern separation
(Figure 9d). Together, these results suggest that impaired spatial
discrimination in response to transient blockade of NMDARs may reflect
impaired pattern separation associated with psychiatric disorders such
as schizophrenia (Faghihi and Moustafa, 2015).

\textbf{Discussion}

This study provides experimental evidence of a series of synaptic
alterations of the lateral and the medial perforant paths in response to
the transient hypofunction of NMDARs. We documented persistent
dysregulation in the glutamate release process from the LPP synapse,
blunted induction of LPP LTP, and decreased synaptic filtering
capability. In the MPP-DG synapse, the altered glutamate release was
accompanied by impaired CB\textsubscript{1}R-dependent LTD and weakened
LTP. Mechanistically, the impairment of MPP LTD was partly due to
decreased functional expression of the CB\textsubscript{1}R. More
importantly, enhancing the 2-AG signaling pathway via pharmacological
inhibition of the MAGL enzyme restored the strength of the MPP LTD. At
the behavioral level, we show for the first time that transient
hypofunction of NMDARs impairs spatial discrimination, a cognitive task
in which DG plays a critical role.

\textbf{Changes in synaptic strength and presynaptic release at the PP
synapses}

Functionally, the glutamatergic inputs to the DG convey distinct types
of somatosensorial information. The MPP conveys spatial information from
the MEC, and the LPP transfers multisensorial information from the LEC
(Hunsaker et al., 2007; Fernández-Ruiz et al., 2021). Moreover, the
synaptic transfer is sustained by axons with unique electrophysiological
properties that determine plasticity capabilities and the pace and
strength of neurotransmitter release(Petersen et al., 2013;
Collitti-Klausnitzer et al., 2021). Those synaptic features have been
robustly demonstrated in this study. Consistent with previous works
(Segev et al., 2020; Márquez et al., 2023), our FV and PPF analyses
revealed that~MK-801 alters the FV amplitude (or presynaptic action
potentials) and glutamate release. Dysregulation in the propagation of
the FV and the subsequent activation of the molecular machinery
underlying glutamate release may explain the reduced strength of the
glutamatergic transmission found in the MPP synapse. In line with this
possibility, transient hypofunction of NMDARs interferes with the
functional expression of presynaptic proteins that control
neurotransmitter release in animal models of schizophrenia (Maher and
LoTurco, 2012; Saggu et al., 2013) and schizophrenic individuals (Egbujo
et al., 2016). Finally, the altered neurotransmitter release process in
the MPP compared to the LPP synapse of MK-801-treated animals may
suggest a marked dysregulation in transferring spatial information but
not non-spatial information from the PP to the DG, a phenomenon that
requires additional investigation.

\textbf{Changes in the induction and expression of synaptic plasticity}

Because glutamatergic LTD has been scarcely explored in the PP -- DG
synapses, a relevant finding of this study was the establishment of a
protocol to induce reliable LTD. The exploratory experiments designed to
this end showed that electrical stimulation of MPP, but not LPP, induces
a stable LTD in response to 900 pulses delivered at 3Hz, a stimulation
frequency that mimics the delta--theta range of volley activity of
stellar cells and pyramidal neurons of EC layer II synapsing DG granule
cells (Gloveli et al., 1997; Deshmukh et al., 2010). In a previous
study, Peñasco et al. demonstrated that MPP LTD can be induced with 6000
pulses at 10 Hz, but this required simultaneous blockade of
GABA\textsubscript{A} receptors and involved activation of the
CB\textsubscript{1}R (Peñasco et al., 2019). In the present study, LTD
was induced with less electrical stimulation, and GABAergic transmission
remained active; the resulting LTD was stable for up to 90 minutes
without visible signs of a return to the baseline fEPSP value. The LTD
was accompanied by increased PPR, indicating a presynaptic locus for its
expression. As in Peñasco et al., the MPP LTD reported in our study
requires postsynaptic production of 2-AG.

In slices from MK-801-treated animals, LFS induced synaptic potentiation
instead of LTD. The absence of LTD may result from presynaptic
dysregulation in CB\textsubscript{1}R activity, accelerated breakdown of
2-AG, or altered synthesis of 2-AG at the postsynaptic level. The latter
is unlikely, since physostigmine induced a synaptic depression like that
observed with activation of CB\textsubscript{1}R in the MK-801
condition. These findings indicate that MK-801-treated animals maintain
functional postsynaptic production of 2-AG, suggesting a potential
dysregulation locus at the presynaptic MPP terminals. In this regard, a
critical finding of this study was that the blockade of the MAGL enzyme
reverted LTD loss in MK-801-treated animals. MAGL is a presynaptically
expressed enzyme that metabolizes 2-AG and is essential in multiple
physiological processes, including neuroplasticity, cognitive
performance, and behavior (Wang et al., 2018a; Zanfirescu et al., 2021).
Our results suggest that transient hypofunction of NMDARs interferes
with the expression of the MAGL enzyme, affecting the MPP's ability to
express long-term depression of glutamatergic transmission.

We also documented that transient hypofunction of NMDARs hinders the
induction and expression of TBS-induced LTP in the LPP synapse and
attenuates the magnitude but not the expression of LTP in the MPP
synapse. While MPP LTP may be induced in MK-801-treated slices through a
series of redundant signaling pathways, including CaMKII and PKA-ERK1/2
(Wu et al., 2006; Welsby et al., 2009), that assure its postsynaptic
expression, in the LPP -- DG synapse, LTP requires postsynaptic
synthesis of 2-AG (Wang et al., 2016), presynaptic activation of
CB\textsubscript{1}R, and increased neurotransmitter release mediated by
the non-canonic CB\textsubscript{1}R /FAK/ROCK pathway (Wang et al.,
2018b). Although we did not investigate the mechanisms underlying the
blunted induction of LPP LTP, decreased functional expression of
CB\textsubscript{1}R receptors or accelerated degradation of 2-AG may
explain the absence of LTP observed in this study.

\textbf{Changes in the synaptic filtering at the lateral PP}

It has been hypothesized that low-pass filtering of synaptic inputs to
the DG underlies the encoding information of auditory, olfactory, and
visual cues (Scullin and Partridge, 2012; Madar et al., 2019). In this
regard, frequency-dependent synaptic filtering of the LPP has been
thoroughly characterized in adult mice (Quintanilla et al., 2022). We
first confirmed that juvenile rats possess this physiological property;
then, we demonstrated that synaptic filtering in the LPP is drastically
altered in slices from MK-801-treated animals. Our experimental findings
and multiple theoretical models (Chance et al., 1998; Quintanilla et
al., 2022) imply that the diminished filtering capacity of the LPP
synapse may result from a decreased presynaptic release. The impaired
low-pass filtering capability of the LPP could represent the cellular
substrate that explains the abnormal processing of sensory information
conveyed by the LPP, which is excessively amplified in the DG of
schizophrenic individuals (Arnold, 1999; Prasad et al., 2004; Behrendt,
2016) instead of being properly filtered. Although appealing, this idea
requires experimental examination.

\textbf{Behavioral dysregulation}

Our spatial discrimination tests suggest that transient hypofunction of
NMDARs alters the functional integrity of the DG and thus pattern
separation activity, a model of neuronal activity that enables the
recognition of independent events despite highly similar information
content (Yassa and Stark, 2011). Previous studies have documented
reduced volume and alterations in the cellular architecture of the
hippocampus and surrounding areas of schizophrenic individuals, with the
greatest volume loss in the DG (Nakahara et al., 2019). Volumetric
dysregulation also implies changes in the information processing in
which DG participates, including pattern separation activity (Faghihi
and Moustafa, 2015). In line with this, a recent study has shown
deficits in mnemonic discrimination in schizotypal and first-episode
psychotic individuals (Kraguljac et al., 2021). We demonstrate for the
first time that transient hypofunction of NMDARs impairs spatial
discrimination in juvenile male rats. This finding substantiates the
idea that impaired mnemonic discrimination is present during the early
or prodromal phase of schizophrenia. Consistent with this, recent work
showed that children and adolescents at high risk of developing
schizophrenia exhibited impaired mnemonic discrimination compared to a
control group (\selectlanguage{polish}İ\selectlanguage{english}mamo\selectlanguage{polish}ğ\selectlanguage{english}lu et al., 2023). Given the similar impairments in
the mnemonic discrimination task found in our animal model and
individuals at high risk for developing schizophrenia, we propose that
mnemonic discrimination can be used as a clinical hallmark of the
cognitive status of individuals with a high risk of developing
schizophrenia. Early screening could allow early therapeutic
interventions and modify the course of the illness, as previously
suggested (Insel, 2010). At the preclinical level, the animal models
that presumably mimic the phenotype schizophrenia should reproduce this
impaired mnemonic discrimination in the juvenile stage of animals.

\textbf{Conclusion}

Transient hypofunction of NMDARs with MK-801 alters the synaptic
transfer of information from the entorhinal cortex to the dentate gyrus.
These changes impact the lateral and medial synapses, dysregulating
presynaptic glutamate release, induction of LTD and LTP, and synaptic
filtering. Consistent with these alterations, spatial discrimination
that depends on pattern separation activity is hindered in the
experimental group. These physiological and behavioral dysregulations
might account for the reduced cognitive performance observed in
schizophrenia. Given its therapeutic potential, future research should
consider the modulation of the endocannabinoid system (i.e.,
CB\textsubscript{1}R, 2-AG, or MAGL activity) to restore the synaptic
strength and cognitive abilities of individuals at higher risk of
developing schizophrenia.

\textbf{CONFLICT OF INTEREST}

The authors declare no competing financial interests.

\textbf{DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design and Analysis, and Animal
Experimentation, and as recommended by funding agencies, publishers and
other organisations engaged with supporting research.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{References}

Arnold, O.H. (1999). Schizophrenia -- A Disturbance of Signal
Interaction between the Entorhinal Cortex and the Dentate Gyrus? The
Contribution of Experimental Dibenamine Psychosis to the Pathogenesis of
Schizophrenia: A Hypothesis. Neuropsychobiology 40: 21--32.

Behrendt, R.P. (2016). Hallucinatory experience as aberrant event memory
formation: Implications for the pathophysiology of schizophrenia. Prog.
Neuro-Psychopharmacology Biol. Psychiatry 71: 203--209.

Chance, F.S., Nelson, S.B., and Abbott, L.F. (1998). Synaptic Depression
and the Temporal Response Characteristics of V1 Cells. J. Neurosci. 18:
4785--4799.

Colgin, L.L. (2016). Rhythms of the hippocampal network. Nat. Rev.
Neurosci. 17: 239--249.

Collitti-Klausnitzer, J., Hagena, H., Dubovyk, V., and Manahan-Vaughan,
D. (2021). Preferential frequency-dependent induction of synaptic
depression by the lateral perforant path and of synaptic potentiation by
the medial perforant path inputs to the dentate gyrus. Hippocampus 31:
957--981.

Curtis, M.J., Alexander, S., Cirino, G., Docherty, J.R., George, C.H.,
Giembycz, M.A., et al. (2018). Experimental design and analysis and
their reporting II: updated and simplified guidance for authors and peer
reviewers. Br. J. Pharmacol. 175: 987--993.

Deshmukh, S.S., Yoganarasimha, D., Voicu, H., and Knierim, J.J. (2010).
Theta Modulation in the Medial and the Lateral Entorhinal Cortices. J.
Neurophysiol. 104: 994--1006.

Du, H., Kwon, I.K., and Kim, J. (2013). Neuregulin-1 Impairs the
Long-term Depression of Hippocampal Inhibitory Synapses by Facilitating
the Degradation of Endocannabinoid 2-AG. J. Neurosci. 33: 15022--15031.

Dudek, S.M., and Bear, M.F. (1992). Homosynaptic long-term depression in
area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor
blockade. Proc. Natl. Acad. Sci. 89: 4363--4367.

Egbujo, C.N., Sinclair, D., and Hahn, C.G. (2016). Dysregulations of
Synaptic Vesicle Trafficking in Schizophrenia. Curr. Psychiatry Rep. 18:
1--10.

Eichenbaum, H. (2004). Hippocampus: Cognitive Processes and Neural
Representations that Underlie Declarative Memory. Neuron 44: 109--120.

Engel, A.K., and Fries, P. (2010). Beta-band oscillations --- signalling
the status quo? Curr. Opin. Neurobiol. 20: 156--165.

Faghihi, F., and Moustafa, A.A. (2015). A computational model of pattern
separation efficiency in the dentate gyrus with implications in
schizophrenia. Front. Syst. Neurosci. 9: 132405.

Fern\selectlanguage{ngerman}ández-Ruiz, A., Oliva, A., Soula, M., Rocha-Almeida, F., Nagy, G.A.,
Martin-Vazquez, G., et al. (2021). Gamma rhythm communication between
entorhinal cortex and dentate gyrus neuronal assemblies. Science (80).
372.

Fontaine, C.J., Gräfe, E.L., Pinar, C., Bonilla-Del Río, I., Grandes,
P., and Christie, B.R. (2020). Endocannabinoid receptors contribute
significantly to multiple forms of long-term depression in the rat
dentate gyrus. Learn. Mem. 27: 380--389.

Forsyth, J.K., and Lewis, D.A. (2017). Mapping the Consequences of
Impaired Synaptic Plasticity in Schizophrenia through Development: An
Integrative Model for Diverse Clinical Features. Trends Cogn. Sci. 21:
760--778.

Gloveli, T., Schmitz, D., Empson, R.M., and Heinemann, U. (1997).
Frequency-dependent information flow from the entorhinal cortex to the
hippocampus. J. Neurophysiol. 78: 3444--9.

Goethem, N.P. van, Hagen, B.T.J. van, and Prickaerts, J. (2018).
Assessing spatial pattern separation in rodents using the object pattern
separation task. Nat. Protoc. 13: 1763--1792.

Griego, E., Hernández-Frausto, M., Márquez, L.A., Lara-Valderrabano, L.,
López Rubalcava, C., and Galván, E.J. (2022). Activation of D1/D5
receptors ameliorates decreased intrinsic excitability of hippocampal
neurons induced by neonatal blockade of N-methyl-d-aspartate receptors.
Br. J. Pharmacol. 179: 1695--1715.

Hunsaker, M.R., Mooy, G.G., Swift, J.S., and Kesner, R.P. (2007).
Dissociations of the Medial and Lateral Perforant Path Projections Into
Dorsal DG, CA3, and CA1 for Spatial and Nonspatial (Visual Object)
Information Processing. Behav. Neurosci. 121: 742--750.

\selectlanguage{polish}İ\selectlanguage{english}mamo\selectlanguage{polish}ğ\selectlanguage{english}lu, A., Wahlheim, C.N., Belger, A., and S. Giovanello, K. (2023).
Impaired mnemonic discrimination in children and adolescents at risk for
schizophrenia. Schizophrenia 9: 1--8.

Insel, T.R. (2010). Rethinking schizophrenia. Nature 468: 187--193.

Jeevakumar, V., Driskill, C., Paine, A., Sobhanian, M., Vakil, H.,
Morris, B., et al. (2015). Ketamine administration during the second
postnatal week induces enduring schizophrenia-like behavioral symptoms
and reduces parvalbumin expression in the medial prefrontal cortex of
adult mice. Behav. Brain Res. 282: 165--175.

Kaminitz, A., Barzilay, R., Segal, H., Taler, M., Offen, D., Gil-Ad, I.,
et al. (2014). Dominant negative DISC1 mutant mice display specific
social behaviour deficits and aberration in BDNF and cannabinoid
receptor expression. World J. Biol. Psychiatry 15: 76--82.

Kaya, M.C., Bulut, M., Kaplan, \selectlanguage{polish}İ., \selectlanguage{english}and Gunes, M. (2019). Levels of
endocannabinoid metabolizing enzymes are not related with BDNF levels in
patients with schizophrenia: a case-controlled study. Psychiatry Clin.
Psychopharmacol. 29: 441--445.

Kjaerby, C., Hovels\selectlanguage{ngerman}ø, N., Dalby, N.O., and Sotty, F. (2017).
Phencyclidine administration during neurodevelopment alters network
activity in prefrontal cortex and hippocampus in adult rats. J.
Neurophysiol. 118: 1002--1011.

Kraguljac, N.V., Carle, M., Frölich, M.A., Tran, S., Yassa, M.A., White,
D.M., et al. (2021). Mnemonic Discrimination Deficits in First-Episode
Psychosis and a Ketamine Model Suggest Dentate Gyrus Pathology Linked to
NMDA Receptor Hypofunction. Biol. Psychiatry Cogn. Neurosci.
Neuroimaging 6: 1185--1192.

Larson, J., and Munkácsy, E. (2015). Theta-burst LTP. Brain Res. 1621:
38--50.

Li, J.T., Zhao, Y.Y., Wang, H.L., Wang, X.D., Su, Y.A., and Si, T.M.
(2015). Long-term effects of neonatal exposure to MK-801 on recognition
memory and excitatory-inhibitory balance in rat hippocampus.
Neuroscience 308: 134--143.

Lilley, E., Stanford, S.C., Kendall, D.E., Alexander, S.P.H., Cirino,
G., Docherty, J.R., et al. (2020). ARRIVE 2.0 and the British Journal of
Pharmacology: Updated guidance for 2020. Br. J. Pharmacol. 177:
3611--3616.

Lopez-Rojas, J., Heine, M., and Kreutz, M.R. (2016). Plasticity of
intrinsic excitability in mature granule cells of the dentate gyrus.
Sci. Rep. 6: 21615.

Lopez-Rojas, J., Solis, C.A. de, Leroy, F., Kandel, E.R., and
Siegelbaum, S.A. (2022). A direct lateral entorhinal cortex to
hippocampal CA2 circuit conveys social information required for social
memory. Neuron 110: 1559-1572.e4.

Macek, T.A., Winder, D.G., Gereau IV, R.W., Ladd, C.O., and Conn, P.J.
(1996). Differential involvement of group II and group III mGluRs as
autoreceptors at lateral and medial perforant path synapses. J.
Neurophysiol. 76: 3798--3806.

Madar, A.D., Ewell, L.A., and Jones, M. V. (2019). Temporal pattern
separation in hippocampal neurons through multiplexed neural codes. PLOS
Comput. Biol. 15: e1006932.

Maher, B.J., and LoTurco, J.J. (2012). Disrupted-in-Schizophrenia
(DISC1) Functions Presynaptically at Glutamatergic Synapses. PLoS One 7:
e34053.

Márquez, L.A., Griego, E., López Rubalcava, C., and Galván, E.J. (2023).
NMDA receptor activity during postnatal development determines intrinsic
excitability and mossy fiber long-term potentiation of CA3 pyramidal
cells. Hippocampus 33: 906--921.

Nadel, L., Hupbach, A., Gomez, R., and Newman-Smith, K. (2012). Memory
formation, consolidation and transformation. Neurosci. Biobehav. Rev.
36: 1640--1645.

Nakahara, S., Turner, J.A., Calhoun, V.D., Lim, K.O., Mueller, B.,
Bustillo, J.R., et al. (2019). Dentate gyrus volume deficit in
schizophrenia. Psychol. Med. 1--11.

Nakao, K., Jeevakumar, V., Jiang, S.Z., Fujita, Y., Diaz, N.B., Annan,
C.A.P., et al. (2019). Schizophrenia-Like Dopamine Release Abnormalities
in a Mouse Model of NMDA Receptor Hypofunction. Schizophr. Bull. 45:
138--147.

Nakazawa, K., and Sapkota, K. (2020). The origin of NMDA receptor
hypofunction in schizophrenia. Pharmacol. Ther. 205: 107426.

Osborne, A.L., Solowij, N., Babic, I., Lum, J.S., Newell, K.A., Huang,
X.F., et al. (2019). Effect of cannabidiol on endocannabinoid,
glutamatergic and GABAergic signalling markers in male offspring of a
maternal immune activation (poly I:C) model relevant to schizophrenia.
Prog. Neuro-Psychopharmacology Biol. Psychiatry 95: 109666.

Peñasco, S., Rico-Barrio, I., Puente, N., Gómez-Urquijo, S.M., Fontaine,
C.J., Egaña-Huguet, J., et al. (2019). Endocannabinoid long-term
depression revealed at medial perforant path excitatory synapses in the
dentate gyrus. Neuropharmacology 153: 32--40.

Petersen, R.P., Moradpour, F., Eadie, B.D., Shin, J.D., Kannangara,
T.S., Delaney, K.R., et al. (2013). Electrophysiological identification
of medial and lateral perforant path inputs to the dentate gyrus.
Neuroscience 252: 154--68.

Prasad, K.M.R., Patel, A.R., Muddasani, S., Sweeney, J., and Keshavan,
M.S. (2004). The entorhinal cortex in first-episode psychotic disorders:
A structural magnetic resonance imaging study. Am. J. Psychiatry 161:
1612--1619.

Quintanilla, J., Jia, Y., Lauterborn, J.C., Pruess, B.S., Le, A.A., Cox,
C.D., et al. (2022). Novel types of frequency filtering in the lateral
perforant path projections to dentate gyrus. J. Physiol. 600:
3865--3896.

Ren, S. yu, Wang, Z. zhen, Zhang, Y., and Chen, N. hong (2020).
Potential application of endocannabinoid system agents in
neuropsychiatric and neurodegenerative diseases---focusing on FAAH/MAGL
inhibitors. Acta Pharmacol. Sin. 41: 1263--1271.

Saggu, S., Cannon, T.D., Jentsch, J.D., and Lavin, A. (2013). Potential
molecular mechanisms for decreased synaptic glutamate release in
dysbindin-1 mutant mice. Schizophr. Res. 146: 254--263.

Santoro, A. (2013). Reassessing pattern separation in the dentate gyrus.
Front. Behav. Neurosci. 7: 59930.

Scullin, C.S., and Partridge, L.D. (2012). Modulation by pregnenolone
sulfate of filtering properties in the hippocampal trisynaptic circuit.
Hippocampus 22: 2184--2198.

Segev, A., Yanagi, M., Scott, D., Southcott, S.A., Lister, J.M., Tan,
C., et al. (2020). Reduced GluN1 in mouse dentate gyrus is associated
with CA3 hyperactivity and psychosis-like behaviors. Mol. Psychiatry 25:
2832--2843.

Seshadri, S., Klaus, A., Winkowski, D.E., Kanold, P.O., and Plenz, D.
(2018). Altered avalanche dynamics in a developmental NMDAR hypofunction
model of cognitive impairment. Transl. Psychiatry 2017 81 8: 1--12.

Shigemoto, R., Kinoshita, A., Wada, E., Nomura, S., Ohishi, H., Takada,
M., et al. (1997). Differential Presynaptic Localization of Metabotropic
Glutamate Receptor Subtypes in the Rat Hippocampus. J. Neurosci. 17:
7503--7522.

Snyder, M.A., and Gao, W.J. (2013). NMDA hypofunction as a convergence
point for progression and symptoms of schizophrenia. Front. Cell.
Neurosci. 7: 1--12.

Stan, A.D., Ghose, S., Zhao, C., Hulsey, K., Mihalakos, P., Yanagi, M.,
et al. (2015). Magnetic resonance spectroscopy and tissue protein
concentrations together suggest lower glutamate signaling in dentate
gyrus in schizophrenia. Mol. Psychiatry 20: 433--439.

Sz\selectlanguage{polish}ű\selectlanguage{english}cs, E., Dvor\selectlanguage{ngerman}ácskó, S., Tömböly, C., Büki, A., Kékesi, G., Horváth,
G., et al. (2016). Decreased CB receptor binding and cannabinoid
signaling in three brain regions of a rat model of schizophrenia.
Neurosci. Lett. 633: 87--93.

Tamminga, C.A., Stan, A.D., and Wagner, A.D. (2010). The hippocampal
formation in schizophrenia. Am. J. Psychiatry 167: 1178--93.

Vyleta, N.P., and Snyder, J.S. (2021). Prolonged development of
long-term potentiation at lateral entorhinal cortex synapses onto
adult-born neurons. PLoS One 16: e0253642.

Wang, W., Cox, B.M., Jia, Y., Le, A.A., Cox, C.D., Jung, K.M., et al.
(2018a). Treating a novel plasticity defect rescues episodic memory in
Fragile X model mice. Mol. Psychiatry 23: 1798--1806.

Wang, W., Jia, Y., Pham, D.T., Palmer, L.C., Jung, K.M., Cox, C.D., et
al. (2018b). Atypical Endocannabinoid Signaling Initiates a New Form of
Memory-Related Plasticity at a Cortical Input to Hippocampus. Cereb.
Cortex 28: 2253--2266.

Wang, W., Trieu, B.H., Palmer, L.C., Jia, Y., Pham, D.T., Jung, K.M., et
al. (2016). A Primary Cortical Input to Hippocampus Expresses a
Pathway-Specific and Endocannabinoid-Dependent Form of Long-Term
Potentiation. ENeuro 3: 10049--10053.

Welsby, P.J., Rowan, M.J., and Anwyl, R. (2009). Intracellular
mechanisms underlying the nicotinic enhancement of LTP in the rat
dentate gyrus. Eur. J. Neurosci. 29: 65--75.

Wu, J., Rowan, M.J., and Anwyl, R. (2006). Long-term potentiation is
mediated by multiple kinase cascades involving CaMKII or either PKA or
p42/44 MAPK in the adult rat dentate gyrus in vitro. J. Neurophysiol.
95: 3519--3527.

Yassa, M.A., and Stark, C.E.L. (2011). Pattern separation in the
hippocampus. Trends Neurosci. 34: 515--525.

Zanfirescu, A., Ungurianu, A., Mihai, D.P., Radulescu, D., and
Nitulescu, G.M. (2021). Targeting monoacylglycerol lipase in pursuit of
therapies for neurological and neurodegenerative diseases. Molecules 26:
5668.



\textbf{Figure legends}

\textbf{FIGURE 1. ~Neonatal treatment with MK-801 selectively reduces
synaptic strength in the MPP -- DG synapse.} \textbf{(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).
\textbf{(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 \selectlanguage{greek}µ\selectlanguage{english}M) and insensitive to stimulation of group II mGluRs with
DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M). 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. \textbf{(c)}
Input-output graphs of the LPP fEPSPs and \textbf{(d)} the MPP fEPSPs in
response to increasing current pulses (100 \selectlanguage{greek}µ\selectlanguage{english}A 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),
**\emph{P} ~\textless{} 0.01.\textbf{(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. \textbf{(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. *~\emph{P} ~\textless{}
0.05 and **\emph{P} ~\textless{} 0.01. For LPP:~~\emph{n} ~= 9 slices /
6 animals for the control group and~\emph{n} ~= 10 slices / 6 animals
for the MK-801 group. For MPP: 8 slices / 6 animals for the control
group and~\emph{n} ~= 10 slices / 6 animals for the MK-801 group.

\textbf{FIGURE 2. Neonatal treatment with MK-801 reduces presynaptic
neurotransmitter release at the LPP and the MPP.} \textbf{(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, ***\emph{P} \textless{}
0.001.\textbf{(b)} Representative traces showing the pharmacological
sensitivity of the LPP and the MPP synapses. LPP PPF decreased in the
presence of L-AP4 (20 \selectlanguage{greek}µ\selectlanguage{english}M, upper traces), whereas the MPP PPF was
abolished by perfusion of DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M; lower traces). \textbf{(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.
*\emph{P}\textless{} 0.05. For LPP PPF: \emph{n} = 8 slices / 6 animals
for control and \emph{n} = 9 slices / 7 animals for MK-801.
\textbf{(d)}Representative PPF of the LPP synapses contrasting the
values of control and MK-801-treated slices. \textbf{(e)} Time course
and \textbf{(f)}representative traces for MPP PPF in control (red
symbols and traces) and MK-801-treated (black symbols and traces)
slices. MPP PPF: \emph{n}= 9 slices / 7 animals for each condition.
*\emph{P} \textless{} 0.05.

\textbf{FIGURE 3. Neonatal treatment with MK-801 impairs the induction
of CB\textsubscript{1}R-dependent LTD at the MPP -- DG synapse.
(a)}Representative traces of control (upper traces) and the
CB\textsubscript{1}R antagonist, AM251 treatment (5 \selectlanguage{greek}µ\selectlanguage{english}M, lower traces) in
baseline, post-LFS, and the presence of DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M). \textbf{(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 \selectlanguage{greek}µ\selectlanguage{english}M, white symbols) LFS prevented the induction of
LTD. \textbf{(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 \selectlanguage{greek}µ\selectlanguage{english}M). \textbf{(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 \selectlanguage{greek}µ\selectlanguage{english}M). \textbf{(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.
*\emph{P}\textless{} 0.05. Each symbol within the bars represents one
independent experiment; bars represent the media \selectlanguage{ngerman}± SEM. \textbf{(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. *\emph{P} \textless{} 0.05; n = 6 slices / 6 animals
for control and MK-801; n = 4 slices / 4 animals for AM 251.\textbf{(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
\textbf{b} and \textbf{c} .

\textbf{FIGURE 4. Neonatal treatment with MK-801 reduces the functional
expression of CB\textsubscript{1}R 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). \textbf{(b)} Time course graph of normalized fEPSP slope in
response to the perfusion of the CB\textsubscript{1}R agonist, WIN
55,212-2 (5 \selectlanguage{greek}µ\selectlanguage{english}M for 15 min), in control and MK-801-treated groups (black
and red symbols, respectively). \textbf{(c)} Representative traces
and\textbf{(d)} time course graph of normalized fEPSPs in the presence
of the cholinesterase inhibitor physostigmine (10 \selectlanguage{greek}µ\selectlanguage{english}M 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. \textbf{(e)} Bar graph contrasting
the synaptic depression elicited with WIN 55,212-2 in control vs.
MK-801-treated groups. *P \textless{} 0.05, n = 6 slices / 5 animals for
control and n = 6 slices / 6 animals for MK-801. \textbf{(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. \textbf{(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 CB\textsubscript{1}R with WIN,
suggesting that 2-AG synthesis is not affected by neonatal treatment
with MK-801.

\textbf{FIGURE 5. Pharmacological inhibition of monoacylglycerol lipase
(MAGL) enzyme restores the impaired CB\textsubscript{1}R-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.
\textbf{(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 \selectlanguage{greek}µ\selectlanguage{english}M; blue symbols). LFS induced stable LTD in the
presence of JZL 184 MK-801-treated slices. \textbf{(c)} Bar graph.
Perfusion of JZL 184 rescues LTD at the MPP -- DG synapse. *P
\textless{} 0.05; n = 6 slices / 6 animals for MK-801-treated group and
n = 5 slices / 5 animals for MK-801 + JZL 184 group. \textbf{(d)} Bar
graphs showing restored LTD is accompanied by increased PPR. * P
\textless{} 0.05. \textbf{(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.

\textbf{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. \textbf{(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.
*\emph{P} \textless{} 0.05. \textbf{(c)} Bar graph contrasting the
magnitude of PTP, LTP, and the effect of L-AP4 (20 \selectlanguage{greek}µ\selectlanguage{english}M) on the LPP
synapse. *P \textless{} 0.05; n = 7 slices / 6 animals for control and n
= 7 slices / 7 animals for MK-801. \textbf{(d)} Heatmaps showing the
magnitude of LTP in each slice from both experimental
groups.\textbf{(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.

\textbf{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.
\textbf{(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. \textbf{(c)} Bar graph contrasting the
magnitude of PTP, LTP, and the effect of DCG-IV (5 \selectlanguage{greek}µ\selectlanguage{english}M). n = 8 slices / 7
animals for control and n = 7 slices / 7 animals for MK-801.\textbf{(d)}
Heatmap showing the magnitude of LTP in each slice from both
experimental groups. \textbf{(e)} Cumulative probability distribution
plot of the fEPSP slope values.

\textbf{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 \textless{} 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. \textbf{(b1-b3)} Representative
traces in response to trains at 5 Hz, 20 Hz, and 50 Hz.
\textbf{(c1-c3)}Over-imposed traces showing the first synaptic response
(S1) vs. the tenth response (S10) in the three frequencies examined.
\textbf{(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.

\textbf{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. \textbf{(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 \textless{} 0.05, n = 10 for control
and n = 12 for MK-801. \textbf{(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. \textbf{(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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-01/Figure-01}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-02/Figure-02}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-03/Figure-03}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-04/Figure-04}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-05/Figure-05}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-06/Figure-06}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-07/Figure-07}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-08/Figure-08}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-09/Figure-09}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`Sept 04 Table 1.docx`  available at \url{https://authorea.com/users/659540/articles/663448-neonatal-hypofunction-of-nmda-receptors-alters-perforant-path-synaptic-plasticity-filtering-and-impairs-dentate-gyrus-mediated-spatial-discrimination}

\selectlanguage{english}
\FloatBarrier
\end{document}

