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\begin{document}

\title{Therapeutic-like activity of cannabidiolic acid methyl ester (HU-580) in
the MK-801 mouse model of schizophrenia: role for cannabinoid CB1 and
serotonin-1A receptors}



\author[1]{Brandon Richardson}%
\author[2]{Courtney Clarke}%
\author[2]{Francis Bambico}%
\affil[1]{Memorial University of Newfoundland}%
\affil[2]{Memorial University}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Schizophrenia is an incurable psychotic illness. Those diagnosed have
limited pharmacological treatment options, many of which do not provide
long term relief and come with unpleasant side effects. The
endocannabinoid and serotonergic systems are important neuromodulators
in psychotic illness. We hypothesize that cannabidiolic acid methyl
ester (HU-580) that exerts action on both these systems could have
therapeutic potential by antagonizing cannabinoid receptor-1 (CB1R) and
agonizing 5-hydroxytryptamine receptor-1A (5-HT1AR). We employed
behavioural and brain protein analyses in male and female mice exposed
to MK-801, which precipitated schizophrenia-related reactivity across a
number of behavioural dimensions. C57BL/6 mice were subjected to a
battery of behavioral tests, and we found that subchronic treatment of
MK-801 (once daily for seven days) induced positive-like, negative-like,
and cognitive-related behavioral deficits; primarily in females.
Sub-chronic treatment of MK-801 (once daily for 17 days) induced
positive- and negative-like behavioral deficits in females. Low-dose
(0.01ug/kg) but not high-dose (0.05 ug/kg) treatment rescued female mice
from schizophrenia-related behavioral deficits. Altogether, these data
suggest that HU-580 may have dose-dependent antipsychotic-like potential
that rely on mechanisms that recruit CB1R and 5-HT1AR.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Schizophrenia is a heterogenous psychotic disorder that remains
incurable. There are roughly over 21 million people living with
schizophrenia, a prevalence that has been increasing over the last two
decades (Charlson et al., 2018). Schizophrenia has multiple symptom
presentations that typically emerge in late adolescence and early
adulthood. These include positive (e.g., psychosis and delusions),
negative (e.g., anhedonia and flat affect), and cognitive (e.g., memory
deficits) symptoms (Marder \& Galderisi, 2017; Marzouk et al., 2020;
Bowie \& Harvey, 2006). Current treatment comes with unpleasant
extrapyramidal side effects and may not provide long term relief (Lally
\& MacCabe, 2015). Negative symptoms are more strongly correlated with
poorer baseline functioning (Rabinowitz et al., 2012; Leifker, Bowie, \&
Harvey, 2009), and appear to be resistant against current antipsychotic
treatment (Hanson et al., 2010; Lin, Tsai, \& Lane, 2014). Thus, new
alternative treatments are needed that are better tolerated and continue
to provide relief.

Glutamate influences the development of schizophrenia through the
N-methyl-D-aspartate receptor (NMDAR) system. NMDAR antagonists are
frequently used as models of schizophrenia in animal research, and
symptoms induced in rodents are comparable to clinical schizophrenia
(Krystal et al., 1994; Javitt \& Zukin, 1991). Dizocilpine (MK-801) is
an NMDAR antagonist often used to model schizophrenia and has been shown
to induce a number of behaviors in rodents that are indicative of
schizophrenia-like behavior such as increased locomotor activity,
changes in locomotor activity in response to stress, pre-pulse
inhibition deficits, impaired spatial memory, and social withdrawal
(Buben\selectlanguage{ngerman}íková-Vale\selectlanguage{polish}š\selectlanguage{english}ov\selectlanguage{ngerman}á et al., 2008), and similar behaviors are seen in
humans (Seillier \& Giuffrida, 2009; Bubeníková-Vale\selectlanguage{polish}š\selectlanguage{english}ov\selectlanguage{ngerman}á et al., 2008).

The neuro-modulatory serotonergic and endocannabinoid systems have been
implicated in psychotic illness as potential therapeutic pathways
(Davies \& Bhattacharyya, 2019; Yang \& Tsai, 2017). Serotonin is
implicated in schizophrenia because it is a primary regulator of
dopamine and second-generation antipsychotics antagonize
5-hydroxytryptamine receptor 2A (5-HT2AR) to provide relief for negative
symptoms more so than first-generation antipsychotics (Kim, 2021; Ohno,
2011). Indeed, drugs that are 5-hydroxytryptamine receptor 1A (5-HT1AR)
agonist provide relief for positive and negative symptoms without
extrapyramidal side effects (Lobo et al., 2022). Moreover, increased
5-HT1AR receptor density is seen in schizophrenic patients, and may be
sensitized due to overstimulating serotonin 5-HT2A receptors (Kim,
2021). Dopaminergic and serotoninergic neurons can communicate with the
endocannabinoid system to regulate excitability within the brain via
glutamate and \selectlanguage{greek}γ-\selectlanguage{english}aminobutyric acid (GABA; Peters, Cheer, \& Tonini,
2021), therefore there are complex crosstalk relationships in the
regulation of glutamate activity. Agonizing cannabinoid receptor type 1
(CB1R) results in increased glutamate and dopamine and increases
locomotive activity consistent in schizophrenic-like behavior
(Polissidis et al., 2013). Conversely, antagonizing CB1R results in
symptom relief (Ballmaier et al., 2007).

This study tested deficits induced by MK-801 in mice and investigate if
cannabidiolic acid (CBDA) methyl ester (HU-580) would provide
therapeutic relief. HU-580 is a more stable CBDA analogue that is shown
to be a strong agonist of 5-HT1AR and an antagonist of CB1R (Pertwee et
al., 2018; Navarro et al., 2020). We have ascertained that exposure to
MK-801 would induce schizophrenic-like behaviors and pathophysiology
that would be attenuated by HU-580.

\textbf{Experimental procedures}

\textbf{Animals}

This experiment utilized adult C57BL/6 mice (Charles-River
Saint-Constant, PQ, Canada) single housed weighing 25 -- 35g.
Experiments were conducted between 8:00 and 20:00. All procedures were
in accordance with the ethical guidelines by Memorial University's
institutional animal care and use committee and the Canadian Institutes
of Health Research. Mice were pseudo-randomized into two factors,
exposure (control or MK-801, n = 30 per factor, 15 males and 15 females)
and treatment (HU-580 - 0.01ug/kg, HU-580 - 0.05ug/kg, or vehicle; n =
20 per level, 10 males and 10 females), creating six groups: Ctrl+Veh,
Ctrl+LowHU, Ctrl+HighHU, MK+Veh, MK+LowHU, and MK+HighHU (n = 10 per
group, 5 males and 5 females).

\textbf{Drugs}

HU-580 was dissolved in vehicle that consisted of 5\% Tween 80, 5\%
polyethylene glycol, and 90\% saline (0.9\%). MK-801 was dissolved in
0.9\% saline. Drugs were purchased from Sigma-Aldrich. All drugs were
administered intraperitoneally (i.p.).

\textbf{MK-801 model of schizophrenia}

The effect of MK-801 was tested as a model of schizophrenia. We assessed
if MK-801 induced deficits consistent with schizophrenic-like behaviors
and receptor sensitization. Mice were given saline (sal) or MK-801
(0.3mg/kg) for 7 days. Mice were then given MK-801 or saline and
treatment (vehicle{[}veh{]}, HU-580 0.01ug/kg, or HU-580 0.05 ug/kg) for
10 days. Behaviors were tested at one week and the end of the treatment
phase.

\textbf{HU-580 Treatment}

On day 7, mice received HU-580 treatment or saline 30 minutes prior to
behavioral tests. Following, mice continued co-treatment of MK-801 or
saline and HU-580 or vehicle for ten days and were subjected to a second
battery of behavioral tests.

\textbf{Behavioral assay}

Behavior was assessed using open field test (OFT), social interaction
test (SIT), novel object recognition test (NORT) and forced swim test
(FST). Behaviors were analyzed using an automated video tracking
software (EthoVision) and/or Behavioral Observation Research Interactive
Software (BORIS; Friard \& Gamba, 2016). For manual scoring, raters were
blind to mouse subject identification. Due to feasibility, testing order
was maintained. Three identical testing chambers (24 x 54.5 x 40 cm3)
were used and modified to fit each test, excluding FST. Chambers and all
objects used in each test were cleaned with 70\% ethanol between trials.

\emph{OFT:} Hyperactivity, interpreted as psychomotor agitation, was
assessed as total distance traveled in centimeters. Mice were placed in
the chamber and were recorded for 10 minutes, consisting of 5 minutes of
habituation and 5 minutes of behavioral analysis.\emph{SIT:} To test
spontaneous sociability and preference, Crawley's sociability test was
used (Kaidanovich-Beilin et al., 2011). Spontaneous social behavior was
measured in phase 1 by dividing time spent with a stranger conspecific
with time spent with a stranger mouse to produce a social behavior index
(SI). Social memory preference index (SMI) was measured in phase 2 by
dividing time spent with a stranger conspecific with time spent with a
familiar conspecific. NORT: In phase 1, two identical red plastic bulbs
(2.5 x 2.5 cm\textsuperscript{3}) were mounted at each end of the
testing chambers using Velcro. Mice were placed in the center chamber,
dividers were removed, and recording commenced for 5 minutes. In phase
2, one bulb was replaced with a gold bell or cone (2.5 x 2.5
cm\textsuperscript{3}). \emph{FST:} Mice were placed in a glass (8.5 x
20 cm) that was filled partway with water (22 -- 25 degrees Celsius). A
2-minute period of habituation occurred before recording behavior for 4
minutes.

\textbf{Enzyme-linked Immunosorbent Assay (ELISA)}

Brains were flash frozen and stored in a -80 freezer. The PFC was
removed was kept frozen throughout the entire process. The PFC was
identified using a mouse brain atlas and visual landmarks. The tissue
removed was within AP 2.93mm and 1.33mm. To prepare samples for ELISA,
1x phosphate-buffered saline (PBS) was micro-pipetted into the vials at
a concentration of 10\% tissue and 90\% PBS as per recommendations of
the kits. Samples were centrifuged at 1000 x g for 20 minutes and the
supernatant was removed. Instructions were followed for each kit
thoroughly. Each ELISA kit was purchased from MyBioSource, and this
study tested for CB1R (Catalogue: MBS2533482) and 5-HT1AR (Catalogue:
MBS287600).

\textbf{Data analysis}

Data were analyzed via SigmaPlot 15.0 using one-way or two-way ANOVA.
Dunnett's post hoc analysis was used to control for experiment-wise
error rate and to compare groups to control. Sexes were analyzed pooled
and then separated to investigate sex differences. Data are shown as
mean+/-standard error of the mean (SEM).

\textbf{Results}

Behavioral Measures

\textbf{OFT:} At 7 days, results revealed a significant interaction
between exposure and treatment in females, \emph{F} (2,24) =
4.356,\emph{p} = 0.024. Post hoc comparisons revealed that MK+Sal
(Mean{[}M{]} = 762.696, Standard Deviation{[}SD{]} = 123.944) was
trending towards traveling significantly more than Ctrl+Sal (M =
600.930, SD = 160.251), MD = 161.765, \emph{q} = 1.683, \emph{p} =
0.105, and MK+LowHU (M = 498.434, SD = 211.761) was significantly less
active than MK+Sal (M = 762.696, SD = 123.944), MD = 264.262, \emph{q} =
2.749, \emph{p} = 0.021. At 17 days, differences were significant,
\emph{F} (5,54) = 7.065,\emph{p} \textless{} 0.001. Post hoc comparisons
revealed that compared to Ctrl+Sal (M = 507.541, SD = 171.562), MK+Sal
(M = 889.621, SD = 312.669), MD = 382.080, \emph{q} = 3.755, \emph{p} =
0.002, and MK+HighHU (M = 992.224, SD = 277.479), MD = 484.683, \emph{q}
= 4.763,\emph{p} \textless{} 0.001, traveled significantly more. In
males, results were significant, \emph{F} (5,24) = 7.843, \emph{p}
\textless{} 0.001. Post hoc comparisons revealed that compared to
Ctrl+Sal (M = 479.282, SD = 198.022), MK+HighHU (M = 1157.157, SD =
300.419) traveled significantly more, MD = 677.874, \emph{q} = 5127,
\emph{p} \textless{} 0.001. In females, results were significant,
\emph{F} (5,24) = 8.487,\emph{p} \textless{} 0.001. Post hoc comparisons
revealed that MK+Sal (M = 1132.174, SD = 194.739) traveled significantly
more than Ctrl+Sal (M = 535.799, SD = 158.165), MD = 596.375, \emph{q} =
5.761, \emph{p}\textless{} 0.001, Ctrl+LowHU (M = 668.763, SD = 83.481),
MD = 463.411,\emph{q} = 4.477, \emph{p} \textless{} 0.001, Ctrl+HighHU
(M = 712.275, SD = 96.458), MD 419.899, \emph{q} = 4.057, \emph{p} =
0.002, MK+LowHU (M = 603.433, SD = 257.934), MD = 528.741, \emph{q} =
5.108, \emph{p}\textless{} 0.001, and MK+HighHU (M = 827.292, SD =
122.390), MD = 304.882, \emph{q} = 2.945, \emph{p} = 0.029 (See Figure
1).

\textbf{SIT:} At 7 days, results revealed a main effect of exposure on
SI, \emph{F} (1,53) = 6.051, \emph{p} = 0.017. Post hoc comparisons
revealed that MK+Sal mice (M = 1.845, SD = 0.22) had a significantly
lower SI than Ctrl+Sal mice (M = 2.605, SD = 0.216), MD = 0.759,\emph{q}
= 2.439, \emph{p} = 0.018. Moreover, MK+Sal (M = 1.677, SD = 0.395) had
an SI that was marginally lower than Ctrl+Sal (M = 2.764, SD = 0.375),
MD = 1.087, \emph{q} = 1.997, \emph{p} = 0.051. In males, results showed
a significant main effect of exposure on SI,\emph{F} (1,24) = 9.002,
\emph{p} = 0.006. Post hoc comparisons revealed that MK+Sal mice (M =
1.777, SD = 0.777) had a significantly lower SI than Ctrl+Sal mice (M =
3.115, SD = 1.481), MD = 1.339, \emph{q} = 3,\emph{p} = 0.006. In
females, results showed a significant interaction between exposure and
treatment, \emph{F} (2,24) = 3.777, \emph{p} = 0.037. Post hoc
comparisons revealed that MK+Sal (M = 1.596, SD = 0.244) had a
significantly lower SI than Ctrl+Sal (M = 3.046, SD = 1.834), MD = 1.45,
\emph{q} = 2.451, \emph{p} = 0.022 (See Figure 2).

\textbf{FST:} At 7 days, results revealed a significant interaction
between exposure and treatment, \emph{F} (2,54) = 5.952, p = 0.005. Post
hoc comparisons revealed that MK+LowHU (M = 119.114, SD = 67.534) was
significantly less immobile than MK+Sal (M = 164.754, SD = 36.058), MD =
45.640, \emph{q} = 2.563, \emph{p} = 0.025, and Ctrl+LowHU (M = 181,
238, SD = 18.418), MD = 62.125, \emph{q} = 3.488, \emph{p} \textless{}
0.001. In females, results showed a significant interaction between
exposure and treatment, \emph{F} (2,24) = 7.180, p = 0.004. Post hoc
comparisons revealed MK+LowHU (M = 79.794, SD = 75.388) spent
significantly less time being immobile than MK+Sal (M = 161.268, SD =
25.60), MD = 81.474, \emph{q} = 3.074, \emph{p} = 0.010. At 17 days,
results were significant, \emph{F} (5,54) = 3.058 , p = 0.017. Post hoc
comparisons revealed that compared to Ctrl+Sal (M = 179.729, SD =
34.305), both MK+Sal (M = 218.972, SD = 27.616), MD = 39.243, \emph{q} =
2.984, \emph{p} = 0.018, and MK+HighHU (M = 223.827, SD = 14.747), MD =
44.098, \emph{q} = 3.353, \emph{p} = 0.007, were significantly more
immobile. In males, results trended towards a significant main effect of
exposure, \emph{F} (1,24) = 3.403, p = 0.077. Post hoc comparisons
trended towards MK+Sal mice (M = 208.368, SD = 27.631) being more
immobile than control mice (M = 186.820, SD = 35.807), MD =
21.548,\emph{q} = 1.845, \emph{p} = 0.077. In females, results trended
towards a significant main effect of exposure, \emph{F} (1,24) = 2.976,
\emph{p}= 0.097. Post hoc comparisons revealed that MK+Sal (M = 236.797,
SD = 4.435) were significantly more immobile than Ctrl+Sal (M = 196.991,
SD = 35.908), MD = 39.806, \emph{q} = 2.223, \emph{p} = 0.036, and
MK+LowHU (M = 193.755, SD = 40.899), MD = 43.042, \emph{q} = 2.404,
\emph{p} = 0.045 (See Figure 3).

\textbf{NORT:} At 7 days, results were significant, \emph{F} (5,52) =
4.148, \emph{p} = 0.003. Post hoc comparisons revealed that MK+Sal (M =
-15.35, SD = 22.633) had a significantly lower difference time with the
novel object than Ctrl+Sal (M = 15.436, SD = 22.623), MD =
30.786,\emph{q} = 2.77, \emph{p} = 0.033, Ctrl+HighHU (M = 16.712, SD =
21.955), MD = 32.062, \emph{q} = 2.885, \emph{p} = 0.024, MK+LowHU (M =
29.871, SD = 34.836), MD = 45.221, \emph{q} = 3.39, \emph{p} = 0.001,
and MK+HighHU (M = 17.847, SD = 24.296), MD = 33.197, \emph{q} =
2.907,\emph{p} = 0.023. In males, results were significant, \emph{F}
(5,24) = 3.107, \emph{p} = 0.027. Post hoc comparisons revealed that
MK+Sal (M = -24.120, SD = 24.009) had a significantly lower difference
time with the novel object than MK+LowHU (M = 40.368, SD = 43.596), MD =
64.488,\emph{q} = 3.518, \emph{p} = 0.008. In females, results were
significant, \emph{F} (5,21) = 2.942, \emph{p} = 0.036. Post hoc
comparisons revealed that MK+Sal (M = -6.75, SD = 19.592) had
significantly lower difference time with the novel object than Ctrl+Sal
(M = 32.28, SD = 10.949), MD = 38.86, \emph{q} = 2.845, \emph{p} = 0.04,
and Ctrl+HighHU (M = 31.567, SD = 20.43), MD = 38.147, \emph{q} =
2.962,\emph{p} = 0.031. At 17 days, the full analysis was not
significant. In females, results were significant, \emph{F} (5,23) =
2.781, p = 0.042. Post hoc comparisons revealed that MK+Sal (M =
-15.357, SD = 32.087) had significantly lower difference time with the
novel object than MK+LowHU (M = 66.435, SD = 42.025), MD = 81.792,
\emph{q} = 3.202, \emph{p} = 0.016 (See Figure 4).

ELISA

\textbf{CB1R} : results revealed a significant difference in CB1R
concentration, \emph{F} (3,36) = 3.405, \emph{p} = 0.028. Post hoc
comparisons revealed that MK+Sal (M = 868.171, SD = 187.744) had
significantly lower CB1R concentration than Ctrl+Sal (M = 1151.893, SD =
344.877), MD = 283.722, \emph{q} = 2.531, \emph{p} = 0.042, and MK+LowHU
(M = 1189.108, SD = 290.687), MD = 320.937, \emph{q} = 2.864, \emph{p} =
0.019. MK+Sal CB1R concentration was marginally less than MK+HighHU (M =
1127.731, SD = 111.976), MD = 259.560, \emph{q} = 2.316, \emph{p} =
0.068. In females, results revealed a significant difference in CB1R
concentration, \emph{F} (3,16) = 5.404, \emph{p} = 0.009. Post hoc
comparisons revealed that MK+Sal (M = 755.483, SD = 75.661) had
significantly lower CB1R concentration than Ctrl+Sal (M = 1188.093, SD =
299.908), MD = 432.610, \emph{q} = 3.439, \emph{p} = 0.009, and
MK+HighHU (M = 1197.826, SD = 54.206), MD = 442.343, \emph{q} =
3.516,\emph{p} = 0.008. MK+LowHU (M = 1017.288, SD = 244.288) trended
towards a greater CB1R concentration than MK+Sal, MD = 261.805, \emph{q}
= 2.081, \emph{p} = 0.13 (See Figure 5).

\textbf{5-HT1AR} : Results revealed a significant difference in 5-HT1AR
concentration, \emph{F} (3,36) = 12.893, \emph{p} \textless{} 0.001.
Post hoc comparisons revealed that MK+Sal (M = 86.386, SD = 30.55) had
significantly lower CB1R concentration than Ctrl+Sal (M = 150.418, SD =
39.378), MD = 64.032, \emph{q} = 4.963, \emph{p} \textless{} 0.001,
MK+LowHU (M = 142.419, SD = 24.399), MD = 56.033, \emph{q} =
4.343,\emph{p} \textless{} 0.001, and MK+HighHU (M = 158.752, SD =
15.809), MD = 72.366, q = 5.609, \emph{p} \textless{} 0.001. In females,
results revealed a significant difference in 5-HT1AR concentration,
F(3,16) = 158.263, \emph{p} \textless{} 0.001. Post hoc comparisons
revealed that MK+Sal (M = 58.308, SD = 2.539) had significantly lower
CB1R concentration than Ctrl+Sal (M = 114.917, SD = 10.129), MD =
56.609,\emph{q} = 13.560, \emph{p} \textless{} 0.001, MK+LowHU (M =
120.154, SD = 4.379), MD = 61.846, \emph{q} = 14.815, \emph{p}
\textless{} 0.001, and MK+HighHU (M = 146.577, SD = 6.785), MD = 88.269,
\emph{q} = 21.144,\emph{p} \textless{} 0.001. In males, results revealed
a significant difference in 5-HT1AR concentration, \emph{F} (3,16) =
32.858, \emph{p}\textless{} 0.001. Post hoc comparisons revealed that
MK+Sal (M = 114.464, SD = 11.073) had significantly lower CB1R
concentration than Ctrl+Sal (M = 185.919, SD = 15.347), MD = 71.456,
\emph{q} = 9.343,\emph{p} \textless{} 0.001, MK+LowHU (M = 164.684, SD =
9.001), MD = 50.220, \emph{q} = 6.567, \emph{p} \textless{} 0.001, and
MK+HighHU (M = 170.927, SD = 12.072), MD = 56.463, \emph{q} = 7.383,
\emph{p}\textless{} 0.001 (See Figure 5).

\textbf{Discussions}

MK-801 was effective in inducing schizophrenia-like behaviors consistent
with previous literature (Mabunga et al., 2019; Zou et al., 2008; Langen
et al., 2012; Nilsson et al., 2007). Indeed, hyperlocomotion (OFT),
social deficits (SIT), immobility (FST), and cognitive deficits (NORT)
that were shown in this study are consistent effects of NMDAR
antagonists as a pharmacological model of schizophrenia (Lee \& Zhou,
2019). These behaviors are hypothesized to be related to MK-801 inducing
positive-like (i.e., hyperlocomotion), negative-like (i.e., social
withdrawal, immobility), and cognitive-related (i.e., memory impairment)
symptoms of schizophrenia (Powell \& Miyakawa, 2006). These behavioral
deficits occur likely because hypofunction of the NMDAR on parvalbumin
(PV)-type gamma-amino-butyric acid (GABA)-producing interneurons impairs
cortical activity (Balu, 2016). In turn, several regions and pathways in
the brain are either hypo- or hyper-activated and result in altered
dopamine, serotonin, and glutamate signaling (Balu, 2016). This results
in behavioral changes seen in this study.

The effects of MK-801 were dependent on treatment length and sex. Some
studies have shown that MK-801 effects may be dose- and approach-
dependent in that a high dose (0.5mg/kg), but not a chronic low dose
(0.1mg/kg) increases locomotor activity (Eyjolfsson et al., 2006).
Indeed, previous literature does show sex-dependent effects of MK-801
(D'Souza, Harlan, and Garcia, 2002). One example of this is the
motor-induced differences of MK-801. In females, it is typical of MK-801
to cause increased activity measured as hyperlocomotion (Feinstein \&
Kritzer, 2013). However, males do not exhibit the same effects. Instead,
males show deficits in other aspects of motor-coordination that may need
to be measured in other tests (Feinstein \& Kritzer, 2013). Therefore,
it is without confidence that we could conclude that MK-801 didn't
induce the motor-related effects in males as females; only that the OFT
was more sensitive in detecting deficits in females. However,
unexpectedly our results showed a synergistic-like effect of MK-801 and
HU-580 in the OFT, but only in males. Previous literature shows that
cannabinoids can affect locomotive activity in the OFT (Kasten, Zhang,
\& Boehm, 2019), and effects of cannabis in C57BL/6 can be sex-dependent
(Peterson et al., 2023). In addition, stress has been shown to have
sex-dependent effects in rodents that can increase locomotive activity
in males (Jakovcevski, Schachner, \& Morellini, 2008; Franceschelli et
al., 2014). It is possible that the synergistic effect observed here is
a complex set of sex-dependent effects building upon each other. For
example, stress has been shown to alter eCB signalling, increasing the
presence of endogenous cannabinoids (eCBs) (Rademacher et al., 2008).
However, in the presence of endogenous cannabinoids, compounds such as
CBD and CBDA tend to convert into a CB1R inverse agonist (Navarro et
al., 2020; An et al., 2020), likely producing an anxiogenic effect (Sink
et al., 2010).

As expected, HU-580 attenuated hyperlocomotion, immobility, and
cognitive-deficits. Due to limited literature, the effects of HU-580
must be compared to what is known of its successor compounds CBD and
CBDA. HU-580 is comparable with similar mechanisms of action with
greater potency suggesting possible greater efficacy (Pertwee et al.,
2018; Navarro et al., 2020). These results are consistent with previous
studies that showed CBD can block the effects of MK-801 (Kruk-Slomka \&
Biala, 2021). While CBD and CBDA alone do not seem to affect locomotor
activity (Calapai et al., 2022), its regulation of NDMAR via CB1R and
5-HT1AR is likely to be one of the mechanisms that reverses
hyperlocomotion (Rodr\selectlanguage{ngerman}íguez-Muñoz et al., 2016; Yuen et al., 2005).
Previous research was able to show that NMDAR antagonism can induce
immobility in the FST (Langen et al., 2012), and our study corroborates
the findings that compounds such as CBD and HU-580 can significantly
reduce immobility behavior (Sales et al., 2019; Sales et al., 2020;
Dlugosz et al., 2023). Moreover, CBD (Felipe et al., 2013; Osborne et
al., 2017; Leweke et al., 2021) and CBDA (Kim et al., 2023) have been
shown to improve or rescue cognitive performance in psychiatric and
neurological disorders. Regarding behavioral amelioration, conclusions
about receptor correlates seem to be complex. CB1R and 5HT1AR are both
implicated in positive, negative, and cognitive symptom functioning of
schizophrenia as potential therapeutic targets or pathologies (Dickens
et al., 2020; Ceccarini et al., 2013; Borgan et al., 2019; Kim, 2021;
Kishi, Meltzer, \& Iwata, 2013; Ohno, 2011; \selectlanguage{polish}Š\selectlanguage{english}vob \selectlanguage{polish}Š\selectlanguage{english}trac, Pivac, \&
M\selectlanguage{ngerman}ück-\selectlanguage{polish}Š\selectlanguage{english}eler, 2016). Therefore, until follow-up studies isolate the
therapeutic mechanisms for each behavior in HU-580, further definitive
conclusions cannot be made. In our lab, these investigations are under
way.

MK-801 down-regulated both the CB1R and 5-HT1AR in the PFC. One such
explanation for this is that MK-801 causes an imbalance in excitation
and inhibition activity by hypofunction of NMDAR and recruits CB1R
regulation to restabilize excitatory-inhibitor (E/I) balance
(S\selectlanguage{ngerman}ánchez-Blázquez, Rodríguez-Muñoz, \& Garzón, 2014). Such dysregulation
causes both stress-related responses (Liang, Chen, \& Cheng, 2022) and
the employment of regulatory receptors (Sánchez-Blázquez,
Rodríguez-Muñoz, \& Garzón, 2014). Stress induced by MK-801 could be
inducing an increase in eCB production and/or release (e.g., 2-AG;
Hillard, 2014; Morena et al., 2016). Such increase would agonize CB1Rs,
and if done so chronically, should induce receptor sensitization
(Hillard, 2014). Additionally, it is possible that MK-801 is
down-regulating CB1R via oxidative stress pathways as chronic stress is
known to downregulate CB1R (Liang, Chen, \& Cheng, 2022; Morena et al.,
2016).

HU-580 blocked the downregulation of CB1R by MK-801 exposure. NMDAR and
CB1Rs have a complex relationship, and CB1Rs are known to regulate
glutamatergic activity (Sánchez-Blázquez, Rodríguez-Muñoz, \& Garzón,
2014). Thus, it is likely that CB1R antagonism mitigated some of the
increased regulatory mechanisms of NMDAR hypofunction enough so to
prevent internalization and down-regulation (Rodríguez-Muñoz et al.,
2016). By introducing an antagonist to mitigate downregulation, it is
possible that receptor sensitization is kept normal, and effective
regulation of dysfunctional NMDAR activity is maintained. Alternatively,
it is possible that by antagonizing CB1R, the oxidative stress response
of MK-801 and CB1R are being mitigated and preventing down-regulation
(Liang, Chen, \& Cheng, 2022; Mukhopadhyay et al., 2010). However, there
are limited studies on this topic and further research is needed to
elucidate the relationship between NMDAR dysfunction, and eCB synthesis,
production, and release.

MK-801 induced down regulation of 5-HT1AR that was blocked by HU-580.
Findings of increased 5-HT1AR in the PFC of mice corroborates past
literature that showed MK-801 increases 5-HT1AR in the rat brain
(Wedzony et al., 1997). The pathology of 5-HT1AR in schizophrenia is
extremely complex with regionally specific sensitization of 5-HT1AR
(Nikolaus, Müller, \& Hautzel, 2016; Razakarivony, Newman-Tancredi, \&
Zimmer, 2021). Our results corroborate with some literature showing
decreased 5-HT1AR in the PFC (Nikolaus, Müller, \& Hautzel, 2016).
However, literature is not consistent, and some studies report increased
5-HT1AR in the PFC (Bantick et al., 2001). Although these results are at
odds with what have been reported (Bantick et al., 2001), 5-HT1AR
pathological changes may also be subject to compensatory processes that
are contingent upon the animal model used, the duration of MK-801
exposure, the model species and strain used, specific experimental
procedures and conditions, and the brain structures of interest in
question. Moreover, Inconsistent findings of 5-HT1AR sensitization may
be a cause for heterogeneity and/or differential manifestation of
schizophrenia symptomatology and there may be a difference in 5-HT1AR
sub-types. 5-HT1AR heteroreceptors and autoreceptors are sub-types of
5-HT1AR that mediate serotoninergic signalling and could be
differentially sensitized in schizophrenia (Albert, 2012). Therefore, a
procedural approach that will be able to distinguish these subtypes is
recommended and is being investigated in our lab. Another possible
explanation for 5-HT1AR downregulation by MK-801 is that mice in this
study began as healthy adults with an already established serotonin
system. Serotonin receptors, especially 5-HT1AR, are involved in the
regulation of both NMDARs and the stress response (Biswal et al., 2015;
Flügge et al., 1998; Yuen et al., 2005). Similar to the explanation of
CB1R downregulation, 5-HT1Rs are likely over activated in an attempt to
regulate NMDAR dysfunction and this may subsequently cause
internalization (Yuen et al., 2005).

Overall, our results point towards the occurrence of extremely complex
crosstalks among the serotonin, eCB, and glutamatergic systems
(Haj-Dahmane \& Shen, 2011). Although it may be straightforward to
discuss each receptor independently, its also possible that there are
on-going interactions among these neurotransmitter and receptor systems
that MK-801 or HU-580 may be perturbing in a complex manner (Haj-Dahmane
\& Shen, 2011). However, the dual CB1R-5-HT1AR mechanism of HU-580 posed
an enormous challenge to draw conclusions regarding very specific
effects on individual mechanisms occurring in an intact brain in vivo.
Thus, follow-up studies are needed with pharmacological challenges to
the receptor systems (i.e., opposing agonism/antagonism) to investigate
precise mechanisms involved in sensitization.

Our study shows preliminary support for both MK-801 as a model of
schizophrenia in mice, and that HU-580 would reverse MK-801-induced
deficits. These effects appear to be sex- or dose- dependent and further
investigation is required to fully elucidate the therapeutic potential
of HU-580. Further, we provide support for the role of the CB1R and
5-HT1AR in regulating NMDAR activity in schizophrenia.



\textbf{References}

Albert P. R. (2012). Transcriptional regulation of the 5-HT1A receptor:
implications for mental illness. \emph{Philosophical transactions of the
Royal Society of London. Series B, Biological sciences, 367} (1601),
2402--2415. https://doi.org/10.1098/rstb.2011.0376

An, D., Peigneur, S., Hendrickx, L. A., \& Tytgat, J. (2020). Targeting
Cannabinoid Receptors: Current Status and Prospects of Natural
Products.\emph{International journal of molecular sciences, 21} (14),
5064. https://doi.org/10.3390/ijms21145064

Ballmaier, M., Bortolato, M., Rizzetti, C., Zoli, M., Gessa, G., Heinz,
A., \& Spano, P. (2007). Cannabinoid Receptor Antagonists Counteract
Sensorimotor Gating Deficits in the Phencyclidine Model of
Psychosis.\emph{Neuropsychopharmacology} , \emph{32} (10), 2098--2107.
https://doi.org/10.1038/sj.npp.1301344

Balu D. T. (2016). The NMDA Receptor and Schizophrenia: From
Pathophysiology to Treatment. \emph{Advances in pharmacology (San Diego,
Calif.), 76} , 351--382. https://doi.org/10.1016/bs.apha.2016.01.006

Biswal, M. R., Ahmed, C. M., Ildefonso, C. J., Han, P., Li, H., Jivanji,
H., Mao, H., \& Lewin, A. S. (2015). Systemic treatment with a 5HT1a
agonist induces anti-oxidant protection and preserves the retina from
mitochondrial oxidative stress. \emph{Experimental eye research, 140} ,
94--105. https://doi.org/10.1016/j.exer.2015.07.022

Borgan, F., Laurikainen, H., Veronese, M., Marques, T. R.,
Haaparanta-Solin, M., Solin, O., Dahoun, T., Rogdaki, M., Salokangas, R.
K., Karukivi, M., Di Forti, M., Turkheimer, F., Hietala, J., Howes, O.,
\& METSY Group (2019). In Vivo Availability of Cannabinoid 1 Receptor
Levels in Patients with First-Episode Psychosis. \emph{JAMA psychiatry,
76} (10), 1074--1084. https://doi.org/10.1001/jamapsychiatry.2019.1427

Bowie, C. R., \& Harvey, P. D. (2006). Cognitive deficits and functional
outcome in schizophrenia. \emph{Neuropsychiatric Disease and Treatment,
2} (4), 531-536. https://doi.org/10.2147/nedt.2006.2.4.531

Bubeníková-Vale\selectlanguage{polish}š\selectlanguage{english}ov\selectlanguage{ngerman}á, V., Horá\selectlanguage{polish}č\selectlanguage{english}ek, J., Vrajov\selectlanguage{ngerman}á, M., \& Höschl, C. (2008).
Models of schizophrenia in humans and animals based on inhibition of
NMDA receptors. \emph{Neuroscience \& Biobehavioral Reviews, 32} (5),
1014-1023. https://doi.org/10.1016/j.neubiorev.2008.03.012

Calapai, F., Cardia, L., Calapai, G., Di Mauro, D., Trimarchi, F.,
Ammendolia, I., \& Mannucci, C. (2022). Effects of Cannabidiol on
Locomotor Activity. \emph{Life (Basel, Switzerland), 12} (5), 652.
https://doi.org/10.3390/life12050652

Ceccarini, J., De Hert, M., Van Winkel, R., Peuskens, J., Bormans, G.,
Kranaster, L., Enning, F., Koethe, D., Leweke, F. M., \& Van Laere, K.
(2013). Increased ventral striatal CB1 receptor binding is related to
negative symptoms in drug-free patients with
schizophrenia.\emph{NeuroImage, 79} , 304--312.
https://doi.org/10.1016/j.neuroimage.2013.04.052

Charlson, F. J., Ferrari, A. J., Santomauro, D. F., Diminic, S.,
Stockings, E., Scott, J. G., McGrath, J. J., \& Whiteford, H. A. (2018).
Global Epidemiology and Burden of Schizophrenia: Findings From the
Global Burden of Disease Study 2016. \emph{Schizophrenia bulletin, 44}
(6), 1195--1203. https://doi.org/10.1093/schbul/sby058

Davies, C., \& Bhattacharyya, S. (2019). Cannabidiol as a potential
treatment for psychosis. \emph{Therapeutic Advances in
Psychopharmacology} , \emph{9.} https://doi.org/10.1177/2045125319881916

Dickens, A. M., Borgan, F., Laurikainen, H., Lamichhane, S., Marques,
T., Rönkkö, T., Veronese, M., Lindeman, T., Hyötyläinen, T., Howes, O.,
Hietala, J., \& Ore\selectlanguage{polish}š\selectlanguage{english}i\selectlanguage{polish}č, \selectlanguage{english}M. (2020). Links between central CB1-receptor
availability and peripheral endocannabinoids in patients with first
episode psychosis. \emph{NPJ schizophrenia, 6} (1), 21.
https://doi.org/10.1038/s41537-020-00110-7

Dlugosz, L., Zhou, H. Z., Scott, B. W., \& Burnham, M. (2023). The
effects of cannabidiol and \selectlanguage{greek}Δ\selectlanguage{english}9-tetrahydrocannabinol, alone and in
combination, in the maximal electroshock seizure model. \emph{Epilepsy
research, 190} , 107087.
https://doi.org/10.1016/j.eplepsyres.2023.107087

D'Souza, D. D., Harlan R. E., and Garcia, M. M. (2002). Sexually
dimorphic effects of morphine and MK-801: sex steroid-dependent and
-independent mechanisms. \emph{Journal of Applied Physiology, 92} (2),
493-503. https://doi.org/10.1152/japplphysiol.00565.2001

Eyjolfsson, E. M., Brenner, E., Kondziella, D., \& Sonnewald, U. (2006).
Repeated injection of MK-801: An animal model of
schizophrenia?\emph{Neurochemistry International, 48} , 541-546.
https://doi.org/10.1016/j.neuint.2005.11.019

Feinstein, I., \& Kritzer, M. F. (2013). Acute N-methyl-d-aspartate
receptor hypofunction induced by MK801 evokes sex-specific changes in
behaviors observed in open-field testing in adult male and proestrus
female rats. \emph{Neuroscience} , \emph{228} , 200--214.
https://doi.org/10.1016/j.neuroscience.2012.10.026

Felipe, V. G., Llorente, R., Del-Bel, E., Lopez-Gallardo, M., Viveros,
M. P., \& Francisco, S. G. (2013). Chronic Cannabidiol Attenuates
behavioral and Glial Changes Induced by Repeated Treatment with the NMDA
Receptor Antagonist MK-801 in Mice. \emph{Behavioural Pharmacology 24} ,
e25, https://doi.org/10.1097/01.fbp.0000434770.13614.41

Fitzgerald, M. L., Chan, J., Mackie, K., Lupica, C. R., \& Pickel, V. M.
(2012). Altered dendritic distribution of dopamine D2 receptors and
reduction in mitochondrial number in parvalbumin-containing interneurons
in the medial prefrontal cortex of cannabinoid-1 (CB1) receptor knockout
mice. \emph{Journal of Comparative Neurology} , \emph{520} (17),
4013--4031. https://doi.org/10.1002/cne.23141

Fl\selectlanguage{ngerman}ügge, G., Kramer, M., Rensing, S., \& Fuchs, E. (1998).
5HT1A-receptors and behaviour under chronic stress: selective
counteraction by testosterone. \emph{The European journal of
neuroscience, 10} (8), 2685--2693.

Franceschelli, A., Herchick, S., Thelen, C., Papadopoulou-Daifoti, Z.,
\& Pitychoutis, P. M. (2014). Sex differences in the chronic mild stress
model of depression. \emph{Behavioural Pharmacology, 25} (5-6),
372--383. https://doi.org/10.1097/FBP.0000000000000062

Friard, O., \& Gamba, M. (2016). BORIS: a free, versatile open-source
event-logging software for video/audio coding and live
observations.\emph{Methods in Ecology and Evolution} , 7(11), 1325-1330,
https://doi.org/10.1111/2041-210X.12584

Haj-Dahmane, S., \& Shen, R. Y. (2011). Modulation of the serotonin
system by endocannabinoid signaling. \emph{Neuropharmacology, 61} (3),
414--420. https://doi.org/10.1016/j.neuropharm.2011.02.016

Hanson, E., Healey, K., Wolf, D., Kohler, C. (2010). Assessment of
pharmacotherapy for negative symptoms of schizophrenia, \emph{Current
Psychiatry Reports} , \emph{12} (6), 563--571.
https://doi.org/10.1007/s11920-010-0148-0

Hen-Shoval, D., Amar, S., Shbiro, L., Smoum, R., Haj, C. G., Mechoulam,
R., Zalsman, G., Weller, A., \& Shoval, G. (2018). Acute oral
cannabidiolic acid methyl ester reduces depression-like behavior in two
genetic animal models of depression. \emph{Behavioural Brain Research}
,\emph{351} , 1--3. https://doi.org/10.1016/j.bbr.2018.05.027

Hillard C. J. (2014). Stress regulates endocannabinoid-CB1 receptor
signaling. \emph{Seminars in immunology, 26} (5), 380--388.
https://doi.org/10.1016/j.smim.2014.04.001

Jakovcevski, M., Schachner, M., \& Morellini, F. (2008). Individual
variability in the stress response of C57BL/6J male mice correlates with
trait anxiety. \emph{Genes, Brain, and Behavior, 7} (2), 235--243.
https://doi.org/10.1111/j.1601-183X.2007.00345.x

Javitt, D. C., Zukin, S. R. (1991). Recent advances in the phencyclidine
model of schizophrenia. \emph{American Journal of Psychiatry, 148} (10),
1301-1308. https://doi.org/10.1176/ajp.148.10.1301. PMID: 1654746.

Kaar, S. J., Angelescu, I., Marques, T. R., \& Howes, O. D. (2019).
Pre-frontal parvalbumin interneurons in schizophrenia: A meta-analysis
of post-mortem studies. \emph{Journal of Neural Transmission}
,\emph{126} (12), 1637--1651. https://doi.org/10.1007/s00702-019-02080-2

Kaidanovich-Beilin, O., Lipina, T., Vukobradovic, I., Roder, J., \&
Woodgett, J. R. (2011). Assessment of social interaction
behaviors.\emph{Journal of visualized experiments: JoVE} , (48), 2473.
https://doi.org/10.3791/2473

Kasten, C. R., Zhang, Y., \& Boehm, S. L., 2nd (2019). Acute
Cannabinoids Produce Robust Anxiety-Like and Locomotor Effects in Mice,
but Long-Term Consequences Are Age- and Sex-Dependent. \emph{Frontiers
in Behavioral Neuroscience, 13} , 32.
https://doi.org/10.3389/fnbeh.2019.00032

Kim, J., Choi, P., Park, Y. T., Kim, T., Ham, J., \& Kim, J. C. (2023).
The Cannabinoids, CBDA and THCA, Rescue Memory Deficits and Reduce
Amyloid-Beta and Tau Pathology in an Alzheimer's Disease-like Mouse
Model. \emph{International Journal of Molecular Sciences, 24} (7), 6827.
https://doi.org/10.3390/ijms24076827

Kim S. A. (2021). 5-HT1A and 5-HT2A Signaling, Desensitization, and
Downregulation: Serotonergic Dysfunction and Abnormal Receptor Density
in Schizophrenia and the Prodrome. \emph{Cureus, 13} (6), e15811.
https://doi.org/10.7759/cureus.15811

Kishi, T., Meltzer, H. Y., \& Iwata, N. (2013). Augmentation of
antipsychotic drug action by azapirone 5-HT1A receptor partial agonists:
a meta-analysis. \emph{The International Journal of
Neuropsychopharmacology, 16} (6), 1259--1266.
https://doi.org/10.1017/S1461145713000151

Kruk-Slomka, M., \& Biala, G. (2021). Cannabidiol Attenuates
MK-801-Induced Cognitive Symptoms of Schizophrenia in the Passive
Avoidance Test in Mice. \emph{Molecules (Basel, Switzerland), 26} (19),
5977. https://doi.org/10.3390/molecules26195977

Krystal, J. H., Karper, L. P., Seibyl, J. P., Freeman, G. K., Delaney,
R., Bremner, J. D., Heninger, G. R., Bowers, M. B. Jr., \& Charney, D.
S. (1994). Subanesthetic effects of the noncompetitive NMDA antagonist,
ketamine, in humans. Psychotomimetic, perceptual, cognitive, and
neuroendocrine responses. \emph{Archives of General Psychiatry, 51} (3),
199-214. https://doi.org/10.1001/archpsyc.1994.03950030035004

Lally, J., \& MacCabe, J. H. (2015). Antipsychotic medication in
schizophrenia: A review. \emph{British Medical Bulletin} , \emph{114}
(1), 169--179. https://doi.org/10.1093/bmb/ldv017

Langen, B., Dost, R., Egerland, U., Stange, H., \& Hoefgen, N. (2012).
Effect of PDE10A inhibitors on MK-801-induced immobility in the forced
swim test. \emph{Psychopharmacology, 221} (2), 249--259.
https://doi.org/10.1007/s00213-011-2567-y

Lee, G., \& Zhou, Y. (2019). NMDAR Hypofunction Animal Models of
Schizophrenia. \emph{Frontiers in Molecular Neuroscience, 12} , 185.
https://doi.org/10.3389/fnmol.2019.00185

Leifker, F. R., Bowie, C. R., \& Harvey, P. D. (2009). Determinants of
everyday outcomes in schizophrenia: The influences of cognitive
impairment, functional capacity, and symptoms. \emph{Schizophrenia
Research, 115} (1), 82--87. https://doi.org/10.1016/j.schres.2009.09.004

Levine, A., Liktor-Busa, E., Lipinski, A. A., Couture, S.,
Balasubramanian, S., Aicher, S. A., Langlais, P. R., Vanderah, T. W., \&
Largent-Milnes, T. M. (2021). Sex differences in the expression of the
endocannabinoid system within V1M cortex and PAG of Sprague Dawley
rats.\emph{Biology of Sex Differences, 12} (1), 60.
https://doi.org/10.1186/s13293-021-00402-2

Leweke, F. M., Rohleder, C., Gerth, C. W., Hellmich, M., Pukrop, R., \&
Koethe, D. (2021). Cannabidiol and Amisulpride Improve Cognition in
Acute Schizophrenia in an Explorative, Double-Blind, Active-Controlled,
Randomized Clinical Trial. \emph{Frontiers in Pharmacology, 12} ,
614811. https://doi.org/10.3389/fphar.2021.614811

Liang, J. Q., Chen, X., \& Cheng, Y. (2022). Paeoniflorin Rescued
MK-801-Induced Schizophrenia-Like Behaviors in Mice via Oxidative Stress
Pathway. \emph{Frontiers in Nutrition, 9} , 870032.
https://doi.org/10.3389/fnut.2022.870032

Lin, C.Y., Tsai, G. E., \& Lane, H.Y. (2014). Assessing and treating
cognitive impairment in schizophrenia: current and future. \emph{Current
Pharmaceutical Design} , \emph{20} (32), 5127--5138.
https://doi.org/10.2174/1381612819666140110120015

Litvin, Y., Phan, A., Hill, M. N., Pfaff, D. W., \& McEwen, B. S.
(2013). CB1 receptor signaling regulates social anxiety and
memory.\emph{Genes, Brain, and Behavior, 12} (5), 479-489.
https://doi.org/10.1111/gbb.12045

Liu, X., Zhao, G., Wang, F., \& Wang, L. (2020). Sexual dimorphic
distribution of cannabinoid 1 receptor mRNA in adult C57BL/6
mice.\emph{Journal of Comparative Neurology, 528} (12), 1986-1999.
https://doi.org/10.1002/cne.24868

Lobo, M. C., Whitehurst, T. S., Kaar, S. J., \& Howes, O. D. (2022). New
and emerging treatments for schizophrenia: a narrative review of their
pharmacology, efficacy and side effect profile relative to established
antipsychotics. \emph{Neuroscience and Biobehavioral Reviews, 132} ,
324--361. https://doi.org/10.1016/j.neubiorev.2021.11.032

Mabunga, D. F. N., Park, D., Ryu, O., Valencia, S. T., Adil, K. J. L.,
Kim, S., Kwon, K. J., Shin, C. Y., \& Jeon, S. J. (2019). Recapitulation
of Neuropsychiatric Behavioral Features in Mice Using Acute Low-dose
MK-801 Administration. \emph{Experimental Neurobiology, 28} (6),
697--708. https://doi.org/10.5607/en.2019.28.6.697

Marder, S. R., \& Galderisi, S. (2017). The current conceptualization of
negative symptoms in schizophrenia. \emph{World Psychiatry, 16} (1),
14-24. https://doi.org/10.1002/wps.20385

Marzouk, T., Winkelbeiner, S., Azizi, H., Malhotra, A., \& Homan, P.
(2020). Transcranial magnetic stimulation for positive symptoms in
schizophrenia: A systematic review. \emph{Neuropsychobiology, 79} (6),
384-396. https://doi.org/10.1159/000502148

Morena, M., Patel, S., Bains, J. S., \& Hill, M. N. (2016).
Neurobiological Interactions Between Stress and the Endocannabinoid
System. \emph{Neuropsychopharmacology: official publication of the
American College of Neuropsychopharmacology, 41} (1), 80--102.
https://doi.org/10.1038/npp.2015.166

Mukhopadhyay, P., Rajesh, M., Bátkai, S., Patel, V., Kashiwaya, Y.,
Liaudet, L., Evgenov, O. V., Mackie, K., Haskó, G., \& Pacher, P.
(2010). CB1 cannabinoid receptors promote oxidative stress and cell
death in murine models of doxorubicin-induced cardiomyopathy and in
human cardiomyocytes. \emph{Cardiovascular Research, 85} (4), 773--784.
https://doi.org/10.1093/cvr/cvp369

Nahar, L., Delacroix, B. M., \& Nam, H. W. (2021). The Role of
Parvalbumin Interneurons in Neurotransmitter Balance and Neurological
Disease. \emph{Frontiers in Psychiatry} , \emph{12} , 679960.
https://doi.org/10.3389/fpsyt.2021.679960

Navarro, G., Varani, K., Lillo, A., Vincenzi, F., Rivas-Santisteban, R.,
Raïch, I., Reyes-Resina, I., Ferreiro-Vera, C., Borea, P. A., Sánchez de
Medina, V., Nadal, X., \& Franco, R. (2020). Pharmacological data of
cannabidiol- and cannabigerol-type phytocannabinoids acting on
cannabinoid CB1, CB2 and CB1/CB2 heteromer
receptors.\emph{Pharmacological Research} , \emph{159} , 104940.
https://doi.org/10.1016/j.phrs.2020.104940

Nawata, Y., Hiranita, T., \& Yamamoto, T. (2010). A cannabinoid CB1
Receptor Antagonist Ameliorates Impairment of Recognition Memory on
Withdrawal from MDMA (Ecstasy). \emph{Neuropsychopharmacology, 35} ,
515-520. https://doi.org/10.1038/npp.2009.158

Nikolaus, S., Müller, H. W., \& Hautzel, H. (2016). Different patterns
of 5-HT receptor and transporter dysfunction in neuropsychiatric
disorders--a comparative analysis of in vivo imaging
findings.\emph{Reviews in the Neurosciences, 27} (1), 27--59.
https://doi.org/10.1515/revneuro-2015-0014

Nilsson, M., Hansson, S., Carlsson, A., \& Carlsson, M. L. (2007).
Differential effects of the N-methyl-d-aspartate receptor antagonist
MK-801 on different stages of object recognition memory in
mice.\emph{Neuroscience, 149} (1), 123--130.
https://doi.org/10.1016/j.neuroscience.2007.07.019

Ohno, Y. (2011). Therapeutic Role of 5-HT1A Receptors in The Treatment
of Schizophrenia and Parkinson's Disease: Therapeutic Role of 5-HT1A
Receptors. \emph{CNS Neuroscience \& Therapeutics} , \emph{17} (1),
58--65. https://doi.org/10.1111/j.1755-5949.2010.00211.x

Osborne, A. L., Solowij, N., \& Weston-Green, K. (2017). A systematic
review of the effect of cannabidiol on cognitive function: Relevance to
schizophrenia. \emph{Neuroscience \& Biobehavioral Reviews, 72} ,
310--324. https://doi.org/10.1016/j.neubiorev.2016.11.012

Pertwee, R. G., Rock, E. M., Guenther, K., Limebeer, C. L., Stevenson,
L. A., Haj, C., Smoum, R., Parker, L. A., \& Mechoulam, R. (2018).
Cannabidiolic acid methyl ester, a stable synthetic analogue of
cannabidiolic acid, can produce 5-HT \textsubscript{1A}receptor-mediated
suppression of nausea and anxiety in rats: HU-580, CBDA, 5-HT
\textsubscript{1A} receptor, nausea and anxiety.\emph{British Journal of
Pharmacology} , \emph{175} (1), 100--112.
https://doi.org/10.1111/bph.14073

Peters, K. Z., Cheer, J. F., \& Tonini, R. (2021). Modulating the
Neuromodulators: Dopamine, Serotonin, and the Endocannabinoid
System.\emph{Trends in Neurosciences, 44} (6), 464-477.
https://doi.org/10.1016/j.tins.2021.02.001

Peterson, C., Sallam, N., Baglot, S., Kohro, Y., Trang, T., Hill, M. N.,
\& Borgland, S. L. (2023). Sex differences in pharmacokinetics, central
accumulation, and behavioural effects of oral cannabis consumption in
male and female C57BL/6 mice. \emph{BioRxiv} ,
2023.05.10.540248(preprint). https://doi.org/10.1101/2023.05.10.540248

Polissidis, A., Galanopoulos, A., Naxakis, G., Papahatjis, D.,
Papadopoulou-Daifoti, Z., \& Antoniou, K. (2013). The cannabinoid CB1
receptor biphasically modulates motor activity and regulates dopamine
and glutamate release region dependently. \emph{International Journal of
Neuropsychopharmacology} , \emph{16} (2), 393--403.
https://doi.org/10.1017/S1461145712000156

Powell, C. M., \& Miyakawa, T. (2006). Schizophrenia-relevant behavioral
testing in rodent models: a uniquely human disorder?. \emph{Biological
Psychiatry, 59} (12), 1198--1207.
https://doi.org/10.1016/j.biopsych.2006.05.008

Rabinowitz, J., Levine, S. Z., Garibaldi, G., Bugarski-Kirola, D.,
Berardo, C. G., \& Kapur, S. (2012). Negative symptoms have greater
impact on functioning than positive symptoms in schizophrenia: Analysis
of CATIE data. \emph{Schizophrenia Research, 137} (1), 147-150.
https://doi.org/10.1016/j.schres.2012.01.015

Rademacher, D. J., Meier, S. E., Shi, L., Ho, W. S., Jarrahian, A., \&
Hillard, C. J. (2008). Effects of acute and repeated restraint stress on
endocannabinoid content in the amygdala, ventral striatum, and medial
prefrontal cortex in mice. \emph{Neuropharmacology, 54} (1), 108--116.
https://doi.org/10.1016/j.neuropharm.2007.06.012

Razakarivony, O., Newman-Tancredi, A., \& Zimmer, L. (2021). Towards in
vivo imaging of functionally active 5-HT1A receptors in schizophrenia:
concepts and challenges. \emph{Translational Psychiatry, 11} (1), 22.
https://doi.org/10.1038/s41398-020-01119-3

Rodrigues da Silva, N., Gomes, F. V., Sonego, A. B., Silva, N. R. da, \&
Guimarães, F. S. (2020). Cannabidiol attenuates behavioral changes in a
rodent model of schizophrenia through 5-HT1A, but not CB1 and CB2
receptors. \emph{Pharmacological Research} , \emph{156} , 104749.
https://doi.org/10.1016/j.phrs.2020.104749

Rodríguez-Muñoz, M., Sánchez-Blázquez, P., Merlos, M., \& Garzón-Niño,
J. (2016). Endocannabinoid control of glutamate NMDA receptors: the
therapeutic potential and consequences of dysfunction. \emph{Oncotarget,
7} (34), 55840--55862. https://doi.org/10.18632/oncotarget.10095

Sales, A. J., Fogaça, M. V., Sartim, A. G., Pereira, V. S., Wegener, G.,
Guimarães, F. S., \& Joca, S. R. L. (2019). Cannabidiol Induces Rapid
and Sustained Antidepressant-Like Effects Through Increased BDNF
Signaling and Synaptogenesis in the Prefrontal Cortex. \emph{Molecular
Neurobiology, 56} (2), 1070--1081.
https://doi.org/10.1007/s12035-018-1143-4

Sales, A. J., Guimarães, F. S., \& Joca, S. R. L. (2020). CBD modulates
DNA methylation in the prefrontal cortex and hippocampus of mice exposed
to forced swim. \emph{Behavioural Brain Research, 388} , 112627.
https://doi.org/10.1016/j.bbr.2020.112627

Sánchez-Blázquez, P., Rodríguez-Muñoz, M., \& Garzón, J. (2014). The
cannabinoid receptor 1 associates with NMDA receptors to produce
glutamatergic hypofunction: implications in psychosis and
schizophrenia.\emph{Frontiers in Pharmacology, 4} , 169.
https://doi.org/10.3389/fphar.2013.00169

Seillier, A., \& Giuffrida, A. (2009). Evaluation of NMDA receptor
models of schizophrenia: divergences in the behavioral effects of
sub-chronic PCP and MK-801. \emph{Behavioural Brain Research, 204} (2),
410--415. https://doi.org/10.1016/j.bbr.2009.02.007

Sink, K. S., Segovia, K. N., Sink, J., Randall, P. A., Collins, L. E.,
Correa, M., Markus, E. J., Vemuri, V. K., Makriyannis, A., \& Salamone,
J. D. (2010). Potential anxiogenic effects of cannabinoid CB1 receptor
antagonists/inverse agonists in rats: comparisons between AM4113, AM251,
and the benzodiazepine inverse agonist FG-7142. \emph{European
Neuropsychopharmacology: the journal of the European College of
Neuropsychopharmacology, 20} (2), 112--122.
https://doi.org/10.1016/j.euroneuro.2009.11.002

\selectlanguage{polish}Š\selectlanguage{english}vob \selectlanguage{polish}Š\selectlanguage{english}trac, D., Pivac, N., \& M\selectlanguage{ngerman}ück-\selectlanguage{polish}Š\selectlanguage{english}eler, D. (2016). The serotonergic
system and cognitive function. \emph{Translational neuroscience, 7} (1),
35--49. https://doi.org/10.1515/tnsci-2016-0007

Wedzony, K., Ma\selectlanguage{polish}ć\selectlanguage{english}kowiak, M., Czyrak, A., Fija\selectlanguage{polish}ł, \selectlanguage{english}K., \& Michalska, B.
(1997). Single doses of MK-801, a non-competitive antagonist of NMDA
receptors, increase the number of 5-HT1A serotonin receptors in the rat
brain. \emph{Brain Research, 756} (1-2), 84--91.
https://doi.org/10.1016/s0006-8993(97)00159-5

Yang, A., \& Tsai, S.-J. (2017). New Targets for Schizophrenia Treatment
beyond the Dopamine Hypothesis. \emph{International Journal of Molecular
Sciences} , \emph{18} (8), 1689. https://doi.org/10.3390/ijms18081689

Yuen, E. Y., Jiang, Q., Chen, P., Gu, Z., Feng, J., \& Yan, Z. (2005).
Serotonin 5-HT1A receptors regulate NMDA receptor channels through a
microtubule-dependent mechanism. \emph{The Journal of Neuroscience: the
official journal of the Society for Neuroscience, 25} (23), 5488--5501.
https://doi.org/10.1523/JNEUROSCI.1187-05.2005

Zou, H., Zhang, C., Xie, Q., Zhang, M., Shi, J., Jin, M., \& Yu, L.
(2008). Low dose MK-801 reduces social investigation in
mice.\emph{Pharmacology, Biochemistry, and Behavior, 90} (4), 753--757.
https://doi.org/10.1016/j.pbb.2008.06.002\selectlanguage{english}
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