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 (Rodríguez-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; Švob Štrac, Pivac, &
Mück-Šeler, 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
(Sánchez-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.