This work provides the first in-depth analysis of the changes that take place in the expression of the CB₁ cannabinoid receptor in the mPCx of the obese Zucker rat. Our report demonstrates a selective increase in CB₁-expressing excitatory terminals of PL and IL of the mPCx in the obese Zucker rat. In addition, we report changes in the expression of CB₁ receptors and Gα_i2 subunits in mPCx membranes from obese Zucker rats, along with alterations in functional CB₁-G_i/o coupling. Furthermore, this study shows that the endogenous 2-AG is increased in this brain region. Importantly, the results presented here are in good agreement with previous studies characterizing the expression of CB1 receptors in the obese Zucker rat (Thanos et al., 2008b; Zarate et al., 2008a). Taking into consideration past and present results, there is robust evidence of an up-regulation of CB₁ receptors in the frontal cortical and related limbic areas of the obese Zucker rat. However, it should be noted that the changes in the expression of the CB₁ receptor observed, rather than a general phenomenon affecting all cortical areas, are limited to selective regions (e.g., mPCx). In fact, our results show that CB₁ receptors and their coupling to G_i/o proteins are increased in medial prefrontal cortical membranes of obese Zucker rats compared to lean subjects, but not in neocortical and hippocampal membranes. Moreover, no changes in CB₁-immunostaining intensity were observed in the primary motor cortex and hippocampus. Even so, because samples used for the biochemical analysis of CB₁ receptor expression encompass multiple anatomically distinct subdivisions of neocortex and hippocampus, it is conceivable that the expression of the CB₁ receptor may also be altered in neocortical areas not tested separately. In fact, our previous results (Zarate et al., 2008a) showed that CB₁ staining intensity is increased in the frontal, cingulate, and piriform cortex, but not in the parietal and temporal cortex. Nonetheless, in the study by Zarate et al. (2008a), CB₁-immunoreactivity was observed not only at axon terminals as we describe here and is widely accepted, but also at controversial somatodendritic locations. In this regard, our recent report addressing the specificity of anti-CB₁ receptor antibodies (Echeazarra et al., 2021) showed that the CB1-Go-Af450 antibody used here produces CB₁-specific immunostaining circumscribed to axonal fibers and presynaptic-like puncta, whereas the goat polyclonal K15 antibody used for immunohistochemical staining in the study by Zarate et al. (2008a) produces non-specific somatodendritic signals. In addition, the CB1-Go-Af450 antibody has proven to be adequate to describe the ultrastructural distribution of CB₁ receptors (Lafourcade et al., 2007; Peñasco et al., 2020; Bonilla-Del Río et al., 2021; Egaña-Huguet et al., 2021). In conclusion, the antibody used here has been rigorously tested and validated for specificity in a variety of applications, which is essential for a correct interpretation of the results. Indeed, cross-reactivity of antibodies with unwanted proteins is one of the main causes of the lack of reproducibility between studies (Saper, 2005; Voskuil, 2014; Taatjes and Roth, 2021).

The present study also reports findings regarding the long-term synaptic plasticity in the mPCx of the lean and obese Zucker rats. Electrophysiological recordings were taken from layers II/III of the IL area, as it was in that subregion of the mPCx that we found the strongest increase in CB₁-immunostaining in tissue sections. Although the protocol used here, consisting in prolonged stimulation at frequencies around 10 Hz, is optimal to induce eCB-eLTD in other brain regions (Lafourcade et al., 2007; Puente et al., 2011), it induced a significant CB₁-dependent LTP of evoked fEPSPs in obese rats, whereas elicited a CB₁-independent LTD in lean rats. The up-regulation of CB₁ receptors at glutamatergic synapses in the mPCx of the obese Zucker rats (as demonstrated by immunogold-silver staining and electron microscopy) could underlie these observations. However, in contrast with previous data reporting a prominent role of CB₁ receptors in LTD at the excitatory synapses of the mPCx (Lafourcade et al., 2007), here we observed that CB₁ receptors are involved in LTP elicited in obese rats by low-frequency stimulation, but not in LTD that the same stimulus produces in lean littermates. A potential source for the discrepancy between studies may arise from differences in the chosen mPCx subregion for recordings. Thus, in the study by Lafourcade et al. (2007), CB₁-LTD was observed at pyramidal synapses of layers V/VI of the PL area, whereas our recordings were made in layers II/III of the IL area. Another critical variable that may contribute to the difference could be the age and the type of the animals used; 8–12 weeks old rats here versus 4–6 weeks old mice in Lafourcade’s study.

Biochemical experiments intended at characterizing the levels and G_i/o coupling of CB₁ receptors in the mPCx of the obese Zucker rats provided very consistent results. Thus, immunoblot signal intensity for CB₁ receptor increased by 1.57-fold in membranes of obese Zucker rats compared to lean subjects, whereas WIN 55,212-2-stimulated specific binding of [³⁵S]GTPγS increased by 1.62-fold. This is consistent with the ability of the WIN 55,212-2-bound CB₁ receptor to recruit more G_i/o proteins in the mPCx of the obese Zucker rat and suggests that the availability of Gα_i/o proteins is not rate limiting. The absence of changes in the basal levels of [³⁵S]GTPγS binding in membranes of the mPCx of the obese Zucker rats as compared with their lean littermates suggests that the CB₁ receptor may be not constitutively active and producing agonist-independent receptor activation. Although results of the biochemical experiments performed here cannot discriminate signals originating from CB₁ receptors located specifically at glutamatergic or GABAergic terminals, we presume that the increase of CB₁-immunoreactivity and maximal efficacy of WIN 55,212-2 to stimulate [³⁵S]GTPγS binding in mPCx membranes of obese rats is due to the up-regulation of CB₁ receptors at glutamatergic terminals (as demonstrated here by electron microscopy). In the same way, we infer that the increase in the WIN 55,212-2 maximal efficacy observed in mPCx membranes is a consequence of an increased coupling of glutamatergic CB₁ receptors to G_i/o proteins. Indeed, as noted above, the strengthening of CB₁ receptor-mediated G_i/o signaling as a result of its up-regulation at glutamatergic synapses can explain the emergence of CB₁-dependent LTP of evoked fEPSPs in the mPCx of the obese Zucker rats. The observed up-regulation of CB₁ receptor expression concomitantly with an increase in the efficacy of agonist-stimulated CB₁ receptor coupling to G_i/o proteins reinforces the classical idea that CB₁ receptor density is the main element determining the magnitude of responses to eCBs. In fact, we have recently demonstrated that the extent of the coupling of CB₁ receptors to G_i/o proteins in frontal cortical and hippocampal synaptosomes of specific (glutamatergic and GABAergic) neurotransmitter phenotypes correlates with the abundance of CB₁ receptors (Saumell-Esnaola et al., 2021). However, despite the traditional receptor theory would not predict a decrease in potency for CB₁ agonists under conditions of increased CB₁ receptor levels, WIN 55,212-2 was 4.6-fold less potent in stimulating [³⁵S]GTPγS binding in the mPCx membranes of the obese Zucker rat than in those from lean littermates. Also, the possibility that changes in the transduction machinery downstream of the CB₁ receptor could account for the observed phenomenon has to be considered. In this sense, our results show for the first time that the Gα_i2 subunit is down-regulated in the mPCx of obese rats. However, the situation bears comparison with previously published results in membranes of hepatocytes and adipocytes of obese Zucker rats (Bushfield et al., 1990; Strassheim et al., 1991), hepatocytes from obese (ob/ob) mice (McFarlane-Anderson et al., 1992) and fat cells obtained from obese human subjects (Ohisalo and Milligan, 1989). The results indicate a lower expression and function of Gα_i/o subunits in obese individuals leading to a reduction of the potency of p[NH]ppG, a non-hydrolyzable GTP analog, to inhibit forskolin-stimulated adenylate cyclase activity. Moreover, Bushfield et al. (1990) concluded that their results may be explained by an increased protein kinase C activity in hepatocytes of obese Zucker rats leading to phosphorylation-induced inactivation of Gα_i2 proteins. The possibility that this phenomenon could be present in the brains of obese animals only has been evaluated in whole brain homogenates of obese (ob/ob) mice with negative results (McFarlane-Anderson et al., 1992). However, as in our present study, it is possible that a separate analysis of discrete areas of the brain could find differences between the obese and lean phenotypes. In summary, one may wonder whether a decreased availability of Gα_i2 proteins could plausibly explain the potency loss of WIN 55,212-2 observed in obese rats. This possibility claims that WIN 55,212-2-bound CB₁ receptor activates multiple G_i/o proteins simultaneously, and both the efficacy and potency of the agonist to activate individual Gα_i/o subunits may vary considerably. In this context, the CB₁ receptor provides a key example of ligand-directed GPCR functional selectivity that extends even to its actions on the classic pertussis toxin-sensitive G_i/o signaling (Hudson et al., 2010). Thus, several laboratories have found agonist-specific differences in potency and/or intrinsic activity of the CB₁ receptor toward different Gα_i/o subtypes (Glass and Northup, 1999; Prather et al., 2000; Mukhopadhyay and Howlett, 2005). For instance, stimulation of CB₁ receptors by WIN 55,212-2 results in the activation of a distinct pattern of at least five different Gα_i/o subunits in several brain regions and, most importantly, the concentration of WIN 55,212-2 required to half maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different Gα_i/o subunits (Prather et al., 2000). Therefore, we are tempted to speculate that the rightward shift in the concentration-response curve for WIN55,212-2-stimulated [³⁵S]GTPγS binding in mPCx membranes of obese Zucker rats is related to the recruitment of a population of Gα_i/o proteins in which the contribution of Gα_i2 has been reduced and has been compensated by other Gα_i/o proteins with lower affinity for the WIN55,212-2-bound CB₁ receptor. A detailed pharmacological analysis comparing the potencies and efficacies of various cannabinoid agonists, and especially the endogenous agonist 2-AG, in activating individual Gα_i/o proteins should help address this question and is currently an experimental objective in our laboratory.

Our results also show that 2-AG levels in cortical areas of obese Zucker rats are increased by 2- to 4-fold as compared to their lean littermates. Previous results also showed and increased accumulation (2-fold) of 2-AG in the hypothalamus of obese Zucker rats with respect to their lean controls (di Marzo et al., 2001). Moreover, according to our present and previous data (di Marzo et al., 2001), both the pattern of the regional differences in the levels of 2-AG (i.e., hypothalamus versus frontal cortex) and the absolute hypothalamic and cortical 2-AG levels (nmoles/g brain tissue) in lean Zucker rats are similar to those reported in Sprague–Dawley and Wistar rats (Bisogno et al., 1999; di Marzo et al., 2001; Richardson et al., 2007). The biosynthesis of 2-AG involves only one family of lipids, the sn-1-acyl-2-arachidonoylglycerols (DAGs), which are mostly produced by the hydrolysis of phosphoinositides (PI) via PI-specific PLC activity. DAGs are used as biosynthetic precursors of 2-AG through the action of either of the two sn-2-selective DAG lipases DAGLα and DAGLβ (di Marzo and de Petrocellis, 2012). With respect to the inactivation of 2-AG, the prominent role of monoacylglycerol lipase (MAGL) has been unequivocally established (Dinh et al., 2002, 2004). Although the mechanism of increased 2-AG levels in the prefrontal cortex of the obese Zucker rats reported here is still unclear increased levels of both DAGL and MAGL activities were reported in the spinal cord of obese Zucker rats (Farooqui et al., 1987). Additionally, recently it has been defined a novel mechanism for DAGLα regulation by calcium/calmodulin-dependent protein kinase II (CaMKII) (Shonesy et al., 2013). The authors show that CaMKIIα binds to and phosphorylates DAGLα, and inhibits 2-AG synthesis in vitro. Moreover, behavioral studies indicate that CaMKIIα is a negative regulator of 2-AG signaling in vivo (Shonesy et al., 2013). Interestingly, down-regulation of phosphorylated and total CaMKII has been reported in the hippocampal CA1 region of obese Zucker rats (Alzoubi et al., 2005) and in animal models of chronic psychosocial stress (Gerges et al., 2004).

The data discussed so far support the hypothesis that the obese Zucker rats may represent a preclinical model of genetic vulnerability to obesity, due to hyperactivity of endocannabinoid signaling in the mPCx that would shift the E/I balance, thus increasing the inhibitory tone in this brain area. Previous studies using conditional mutant mice lacking CB₁ receptors either in cortical glutamatergic neurons (Glu-CB₁-KO) or GABAergic neurons (GABA-CB₁-KO) (Monory et al., 2006, 2007) revealed some clues to explain how CB₁ receptors located in excitatory and inhibitory terminals of mPCx could contribute to the behavioral phenotype of the obese Zucker rat. When the conditional mutant mice Glu-CB₁-KO and GABA-CB₁-KO were subjected to experimental protocols that allowed for assessing food consumption, the opposite phenotypes appeared clearly (Lafenêtre et al., 2009; Bellocchio et al., 2010). Glu-CB₁-KO mice were hypophagic, and the phenotype was pharmacologically related to the glutamate actions on NMDA receptors. Conversely, GABA-CB₁-KO mice were more hyperphagic than their wild-type littermates, and the phenotype was a consequence of an increased GABA-A receptor activation (Bellocchio et al., 2010). Moreover, when the experimental protocol was designed to assess the behavioral inhibition component of impulsivity (Pattij et al., 2007), the results suggested that the deletion of CB₁ receptors in glutamatergic neurons leads to an increased behavioral inhibition in the approach to novel palatable food, whereas GABA-CB₁-KO mice did not show any safety behavior toward novel food, and immediately consumed the maximal amount (Lafenêtre et al., 2009). As discussed by the authors (Lafenêtre et al., 2009; Bellocchio et al., 2010), a reduction of glutamatergic transmission through activation of the endocannabinoid system would decrease behavioral inhibition, and thereby increase impulsivity in the approach to food. In support of these findings, most recent data demonstrated that the lack of CB₁ receptors in dorsal telencephalic glutamatergic neurons abrogated the overconsumption of palatable food and the development of obesity (Ruiz de Azua et al., 2021) by promoting a resilient phenotype to food addiction (Domingo-Rodriguez et al., 2020). Consistently, in standard rodent animals, the systemic blockade of glutamatergic transmission leads to an increase in impulsive behavior (Mirjana et al., 2004; Floresco et al., 2008).

One interesting question arising from the results presented here is related to the potential impact that the hyperactivity of CB₁ receptor signaling at the glutamatergic terminals of the PL and IL subregions of mPCx could have on the firing activity of subcortical monoaminergic neurons (i.e., ventral tegmental area-VTA). It is in this frame of reasoning, it must be kept in mind that both the cell body of the mesolimbic dopaminergic neurons and their terminal projection areas (i.e., nucleus accumbens, NAcc) are innervated by glutamatergic afferents from the prefrontal cortex (Sesack et al., 1989; Takagishi and Chiba, 1991; Berendse et al., 1992). Although the mechanisms and pathways by which mPCx modulates VTA dopamine neurons projecting to the NAcc need to be clarified, previous results have demonstrated that direct glutamate injected into the mPCx selectively increased burst firing of single dopamine cells in the VTA area and enhanced the release of dopamine from nerve terminals in the NAcc (Murase et al., 1993). Moreover, the specific pharmacological blockade of NMDA receptors in the mPCx is able to produce the neurochemical and motor changes associated with the dysfunction of the corticostriatal circuit, including an increase in dopamine and acetylcholine release in the NAcc and also an increase in motor activity (del Arco et al., 2008). Importantly, the chemogenetic inhibition of the glutamatergic neuronal activity of a specific prelimbic-nucleus accumbens pathway, which is indeed also under the control of CB₁ receptors, induces compulsive food seeking (Domingo-Rodriguez et al., 2020). Although we focus on the role of the prefrontal cortex on inhibitory control and impulsivity (Volkow et al., 2008a,b; Boeka and Lokken, 2011), we are aware that the functions of the prefrontal cortex that contribute to obesity could be those that are sensitive to modulation by the neurotransmitter dopamine via striatal prefrontal pathways. Clinical neuroimaging studies in obese subjects have provided evidence that the association of striatal D₂ dopamine receptors with inhibitory control and with impulsivity is mediated in part by their modulation of prefrontal regions (Epstein et al., 2007; Volkow et al., 2008b). Similarly, preclinical studies have shown that animals with low striatal D₂ dopamine receptor levels, including the obese Zucker rat, are more impulsive than their littermates with higher D₂ dopamine receptor levels (Dalley et al., 2007; Thanos et al., 2008a; Boomhower et al., 2013). Finally, additional studies are needed to clarify whether the observed up-regulation of CB₁ receptors at glutamatergic terminals of the PL and IL subregions of the medial prefrontal cortex reported here serves an adaptive purpose, as it has been demonstrated in a murine model of obesity with leptin deficiency (Cristino et al., 2013). In that study, authors reported that orexigenic neurons of the lateral hypothalamic area (LH) of the ob/ob mice undergo a shift from predominant control by CB₁-expressing excitatory to CB₁-expressing inhibitory inputs, a phenomenon that was partly reversed by leptin administration via the mTOR branch of the leptin receptor signaling cascade (Cota et al., 2006).

In conclusion, the results of the present study show that the Zucker lines (lean and obese) represent two homogeneous populations that can be used to test the hypothesis on the involvement of hyperactivity of the medial prefrontal endocannabinoid system at glutamatergic terminals in the individual vulnerability to obesity.