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.