Omega-3 Recovers Cannabinoid 1 Receptor Expression in the Adult Mouse Brain after Adolescent Binge Drinking

Adolescent binge drinking is a social problem with a long-lasting impact on cognitive functions. The cannabinoid type-1 (CB1) receptor of the endocannabinoid system (ECS) is involved in brain synaptic plasticity, cognition and behavior via receptor localization at specific subcellular compartments of the cortical, limbic and motor regions. Alcohol (EtOH) intake affects the ECS, CB1 and their functions. Evidence indicates that binge drinking during adolescence impairs memory via the abrogation of CB1-dependent synaptic plasticity in the hippocampus. However, the impact of EtOH consumption on global CB1 receptor expression in the adult brain is unknown. We studied this using optical density analysis throughout brain regions processed for light microscopy (LM) immunohistotochemistry. CB1 staining decreased significantly in the secondary motor cortex, cerebellum, cingulate cortex, amygdala and nucleus accumbens. Next, as omega-3 (n-3) polyunsaturated fatty acids (PUFAs) rescue synaptic plasticity and improve EtOH-impaired cognition, we investigated whether docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) had any effect on CB1 receptors. N-3 intake during EtOH abstinence restored CB1 immunostaining in the secondary motor cortex, cerebellum and amygdala, and ameliorated receptor density in the cingulate cortex. These results show that n-3 supplementation recovers CB1 receptor expression disrupted by EtOH in distinct brain regions involved in motor functions and cognition.


Introduction
The ECS system is mainly composed of endocannabinoids (eCBs), the enzymes involved in their synthesis and degradation, as well as cannabinoid receptors [1].The cannabinoid CB 1 , the most abundant G-protein-coupled receptor in the brain, is highly expressed in the basal ganglia, cerebral cortex, cerebellum (Cb) and hippocampus, which is consistent with its role in learning and memory, motor control, motivation, reward and emotional processing, among many others [1,2].Furthermore, CB 1 increases and changes its distribution from adolescence to adulthood, is predominantly localized in presynaptic terminals and noticeably present in glial cells and mitochondria, modulates neurotransmitter release and also participates in synaptic plasticity [1][2][3][4][5].
Binge drinking during adolescence has a long-lasting impact on cognition and behavior via interference with brain neurochemical systems, including the ECS which participates in the regulation of motivation and EtOH intake [6][7][8][9].EtOH exposure alters both CB 1 mRNA and protein [10,11], changing the availability and binding of the receptor [12,13].Conversely, gene deletion and CB 1 receptor antagonism affect drinking behavior [14].In addition, a CB 1 mRNA decrease in the adult hippocampus after adolescent binge drinking correlates with CB 1 receptor reduction in excitatory terminals of the hippocampal dentate molecular layer.These changes, together with the rise in monoacylglycerol lipase (MAGL) mRNA, the main degrading enzyme of the eCB 2-arachidonoylglycerol (2-AG), disrupt endocannabinoid-dependent long-term depression (LTD) and set off memory impairment [15].Remarkably, the 2-AG increase elicited by MAGL inhibition recovers synaptic plasticity and cognition [15].Although pharmacological manipulations are potential therapeutic strategies for treating alcohol-induced long-term cognitive deficits, nutritional supplementation might also stand up as a suitable alternative.
DHA and EPA are n-3 PUFAs that accumulate in brain cell membranes and maintain their structure and fluidity, restore glutathione levels, are anti-inflammatory, reduce oxidative stress and apoptosis, and overcome synaptic plasticity deficits, improving the cognitive impairment caused by prenatal EtOH exposure [16][17][18][19].DHA is unevenly distributed among brain regions and between neurons and glial cells, and participates in membraneassociated protein functions, cellular signaling, gene expression, lipogenesis, neurogenesis and synaptic pruning [16].EtOH decreases n-3 via different mechanisms [20,21].The negative impact of EtOH on DHA alters synaptic plasticity in the hippocampus and medial prefrontal cortex, both enriched in DHA under normal conditions [16].Noticeably, the enhancement of eCB signaling recovers emotional and cognitive functions as well as reverses the abrogated eCB-dependent synaptic plasticity caused by n-3 PUFA deficiency in brain regions processing mood and cognition [22][23][24].Moreover, DHA modulates CB 1 mRNA and protein expression [25] as well as eCBs [26,27].In fact, a new class of n-3-derived eCBs has been identified in addition to the most-known n-6-derived 2-AG and anandamide (AEA) [28].
However, despite the close relationship between EtOH, n-3 and CB 1 receptors, the direct impact of n-3 PUFAs on brain CB 1 receptor expression after EtOH intake remains unknown.In this investigation, we studied the effects of an n-3-enriched diet on CB 1 receptor expression in the adult brain after binge drinking during adolescence.In particular, we analyzed CB 1 receptor optical density in fourteen brain regions that are sensitive to EtOH damage and are known to express CB 1 receptors.

N-3 and Brain CB 1 Receptor Expression
The brain and cerebellar CB 1 immunostaining patterns in H 2 O, EtOH, n-3-EtOH and n-3-H 2 O mice corresponded well with previous observations of the CB 1 receptor distribution in the mouse brain and cerebellum.CB 1 immunoreactive punctate appeared concentratedin certain brain regions as well as in some cerebral and cerebellar cortical layers.Thus, strong CB 1 receptor immunoreactivity was observed in the granule cell layer of the olfactory bulb (OB) [29] and remarkable staining was also revealed in the striatum, cerebral cortex (layers II-III and V-VI), olfactory tubercle, substantia nigra pars reticulata (SN), amygdala (Amg) and hippocampus [30], as well as the cerebellar molecular and Purkinje cell layers.More moderate immunoreactivity was detected in the nucleus accumbens (Acb) [31].

Long-Lasting Effect of Adolescent Binge Drinking on CB1 Receptor Expression
We have shown that binge drinking during adolescence reduces CB1 receptor immunostaining in certain regions of the mature mouse brain, in particular, the M2, Cb, cg, Amg and Acb.Interestingly, n-3 supplementation during abstinence restores CB1 receptor expression measured using optical density in the M2, Cb and Amg and ameliorates density levels in the cg.

Long-Lasting Effect of Adolescent Binge Drinking on CB 1 Receptor Expression
We have shown that binge drinking during adolescence reduces CB 1 receptor immunostaining in certain regions of the mature mouse brain, in particular, the M2, Cb, cg, Amg and Acb.Interestingly, n-3 supplementation during abstinence restores CB 1 receptor expression measured using optical density in the M2, Cb and Amg and ameliorates density levels in the cg.
The CB 1 receptor expression pattern matched the brain receptor distribution in the cortical, limbic and motor regions [2,30].However, long-term changes in CB 1 immunostaining after adolescent alcohol intake were restricted to some brain regions and the Cb that seem to correlate with the impact of EtOH intake on brain structure and function [6,32,33].EtOH alters grey matter throughout the cortex, including the olfactory areas, Amg and Cb [33][34][35].Also, the mesocorticolimbic system is affected.We observed that the longlasting decrease in CB 1 receptors normally expressed in the Acb [36] did not recover over time after adolescent binge drinking, remaining low even when the animals were under n-3 supplementation.As CB 1 receptors intervene in brain maturation, it is plausible that the CB 1 receptor expression deficits revealed in the Acb negatively contribute to the shape of the mesocorticolimbic system during the adolescent period, ultimately promoting brain vulnerability and alcohol addiction [35].
This study was conducted using male mice.Though males might be more vulnerable to withdrawal [37], females are more sensitive to EtOH [38,39].Also, the effectiveness of treatments differs between males and females [40].It is plausible that the EtOH impact on CB 1 receptor expression and the effects of the n-3-enriched diet may vary between males and females, a possibility that will be explored in our future investigations.
The LM immunohistochemistry applied in the present study has some obvious limitations that deserve attention.Immunohistochemical techniques for LM were used in the 1990s to describe the pattern of CB 1 receptor-like immunoreactivity in the brain [41,42].Consequently, the strong immunostaining observed in certain brain regions (motor, limbic, reward, cortical) endorsed the advance in the knowledge of CB 1 receptor functions in brain circuits.However, low CB 1 receptor expression in cell types cannot be visualized using LM immunohistochemistry [30].In addition, the tendency to diffusion of the 3-3 diaminobenzidine (DAB) reaction product used as chromogen in LM immunohistochemistry could lead to potential unspecific staining or false positives due to endogenous biotinylated proteins.These pitfalls can only be ruled out by using appropriate controls.In this study, we have used CB 1 -knockout brain tissue to discard bias, confirming the specificity of the CB 1 staining observed throughout the brain in our experimental conditions.Ultimately, high-resolution immunoelectron microscopy that has been shown to be an excellent tool for unveiling the precise subcellular localization of CB 1 receptors in the brain, would definitely identify the subcellular compartments and the CB 1 receptor pools that were conspicuously reduced by adolescent EtOH intake, and recovered by n-3 in the specific brain regions identified in this study.
Endocannabinoid levels, membrane fluidity and EtOH-degrading enzymatic machinery could contribute to the altered CB 1 receptor pattern observed in the adult brain after adolescent binge drinking.In addition, EtOH intake during adolescence causes memory impairment that can last into adulthood [43,44].Our model of binge drinking during adolescence used in this study has previously been shown to be associated with hippocampal memory deficits in adulthood [15,45].Although CB 1 receptor optical density was not significantly affected by binge drinking, subtle subcellular changes in receptor expression were detected in the dentate molecular layer that should contribute to the abrogation of cannabinoid-dependent synaptic plasticity at the excitatory medial perforant path synapses and related memory loss [15].Remarkably, the deleterious cognitive binge drinking effects were recovered by increasing the endocannabinoid 2-AG or by environmental conditions [15,45].Furthermore, the M2 and Cb, both affected by EtOH, are brain regions involved in motor coordination [46,47] and EtOH intake leads to motor incoor-dination and ataxia [43].Our present results show a significant decrease in CB 1 staining in the cerebellar molecular layer, where the receptor is mostly localized to the excitatory granule cell parallel fiber terminals [48].However, the lack of CB 1 receptors does not seem to cause evident cerebellar motor deficits [49], despite their role in motor learning [50].Nevertheless, cannabinoid-dependent motor control is also exerted from the cortex [51], where we detected deficits in CB 1 receptors upon adolescent binge drinking.Interestingly, young adult mice under enriched environment recovered motor coordination and balance after adolescent binge drinking [45].It is likely that the ECS participates in this motor improvement, as it is the case in the memory recovery elicited by environmental enrichment via the restoration of endocannabinoid-dependent excitatory synaptic plasticity, in which CB 1 receptors, group I metabotropic glutamate receptors and 2-AG were involved [9].
EtOH modifies synaptic membrane fluidity [52] and stimulates arachidonic acid (AA) production from membrane phospholipids by increasing phospholipase A 2 (PLA 2 ) [53].The availability of more AA for AEA synthesis may be responsible for the decrease in CB 1 agonist binding and gene expression elicited by chronic EtOH in certain brain regions [54,55]).In fact, the drop in CB 1 receptor immunostaining in the cerebellar molecular layer correlates with AEA transport inhibition and a 2-AG increase in granule cells after chronic EtOH [56,57].Cannabinoids internalize CB 1 receptors and reduce their mobility, having an impact on receptor availability at the synapse [58].We have demonstrated previously that ∆-9-tetrahydrocannabinol (THC) causes a selective CB 1 receptor labeling decrease in certain subcellular compartments (excitatory and inhibitory terminals, mitochondria, astrocytes) of several brain regions [59].This distinct impact of THC could be related to the different THC levels and metabolites detected among brain regions after acute THC administration [60].A similar phenomenon could befall our model of binge drinking.In fact, brain EtOH metabolism by class III alcohol dehydrogenase (ADH) generates acetaldehyde that accumulates in the hippocampus, cortex and Cb, where the enzyme is more expressed [61].Interestingly, the enzyme distribution coincides in brain regions with high CB 1 receptor expression, such as the cortex and Cb, both strikingly affected by adolescent binge drinking.EtOH decreases glutathione in these same regions, thus increasing oxidative processes and brain damage in a model of prenatal EtOH exposure [17].As class III ADH is a glutathione-dependent formaldehyde dehydrogenase, it is likely that the glutathione reduction elicited by EtOH and the consequent oxidative state leads to enzyme malfunction, jeopardizing EtOH elimination in regions where the enzyme is more abundant.

N-3 Recovers CB 1 Receptor Expression in the Brain
DHA and AA are major phospholipid components of brain cell membranes [16,19].EtOH reduces DHA in the brain [20,21] and its deficit impacts on both cell membranes, altering their biophysical properties, and related membrane proteins, such as enzymes and receptors [19].N-3 deficiency lowers CB 1 receptors in different brain regions [62] and impairs endocannabinoid-mediated synaptic plasticity [22].The Fr3, OB, Cb, hippocampus, midbrain and striatum rank in high-to-low order among the brain regions with more DHA [63].However, the negative effect on CB 1 receptor expression in the adult brain after adolescent binge drinking was particularly outstanding in the M2, Cb and Acb (ventral striatum), with no effect in the hippocampus, dorsal striatum, some cortical areas, OB and SN.A DHA-enriched diet counteracts the low brain n-3 PUFA levels due to EtOH intake [21] and reverses EtOH-induced impairment of synaptic plasticity [18].It also restores aquaporin-4, PLA-2 and glutathione affected by EtOH [17,21,64,65].The recovery of CB 1 receptor immunostaining by n-3 supplementation during the abstinence period points to the normalization of cell membrane homeostasis.
In conclusion, abusive EtOH consumption during adolescence alters CB 1 receptor immunostaining optical density in some brain regions of the adult mouse, and an n-3enriched diet recovers the reduced CB 1 expression in limbic and motor structures following binge drinking.Uncovering the PUFA effects and mechanisms by which the n-3-enriched diet can impact on brain cannabinoid receptor expression (as shown in this paper) and function after adolescent binge drinking, could be an appropriate non-pharmacological approach to counteract the EtOH impact on cannabinoid-dependent synaptic plasticity, cognition and behavior.

Animal Treatment
Four-week-old C57BL/6J male mice (Janvier Labs, Le Genest-Saint-Isle, France) were housed in pairs and habituated to a dark cycle (8 a.m.-8 p.m.).Then, they were exposed to water or drinking-in-the-dark (DID) during adolescence, as previously described [45].Briefly, the mice were individualized and exposed to a bottle of 10 mL tap water or EtOH 20% (Boter S.L., Barcelona, Spain) four days a week during four weeks (postnatal day (PND) 32 to 56).They had free access to the bottle for 2 h the first three days, and 4 h the fourth day.Mice were resting and kept in pairs with food and water ad libitum for the last three days of the week (Figure 4A-C).On PND 56, a blood sample was collected from the lateral tail vein using a capillary tube 30 min after 4 h of EtOH exposure (Sarstedt, Nümbrecht, Germany).Blood samples were analyzed for EtOH concentration using a commercial OH assay kit (Abcam ab65646, Madrid, Spain), following manufacturer instructions.Half of the mice were fed 2% EPA and DHA (2.2% EPA and 1.5% DHA of total fats; SAFE, Augy, France) during withdrawal (PND 56-73).Twice a week, mice and food were weighed to measure EPA and DHA intake (mg/kg/day) (Figure 4D).Three mice/group were culled on PND 73.They were deeply anesthetized using 4% chloral hydrate (10 mL/kg body weight, i.p.) and perfused through the left ventricle with 30 mL phosphate-buffered saline (PBS, 0.1 M, pH 7.4), followed by 80 mL of the fixative (4% formaldehyde depolymerized from paraformaldehyde, 0.2% picric acid, 0.1% glutaraldehyde) prepared in PBS at room temperature (RT).Brains were removed and post-fixed as described elsewhere in detail [59].

Immunohistochemistry for Light Microscopy
This was performed following the protocol previously published [30].Briefly, coronal and sagittal sections cut at 50 µm on a vibratome (Leica VT 1000s, Wetzlar, Germany) were taken rostro-caudally from the whole brain and Cb, respectively, and collected in Int. J. Mol.Sci.2022, 23, x FOR PEER REVIEW 4 of 15

Figure 2 .
Figure 2. CB1 receptor-like immunoreactivity in the M2, Cb, cg, Amg and Acb of adult male mice exposed to H2O, EtOH, n-3-EtOH or n-3-H2O.Pre-embedding immunoperoxidase method for light microscopy.The typical CB1 staining pattern is observed: abundant dotty elements distributed in the superficial (II-III) and deep (V) layers of the M2 cortex as well as in the cingulum (cg) and amygdala (Amg); uniform immunostaining in the cerebellar molecular (M) layer, strong basket cell terminal labeling around Purkinje cell (PC) bodies in the PC layer, and lack of staining in the granule (G) cell layer; very faint staining in the Acb with only some positive varicose fibers in control (H2O).Scale bar: 200 µm.

Figure 2 .
Figure 2. CB 1 receptor-like immunoreactivity in the M2, Cb, cg, Amg and Acb of adult male mice exposed to H 2 O, EtOH, n-3-EtOH or n-3-H 2 O. Pre-embedding immunoperoxidase method for light microscopy.The typical CB 1 staining pattern is observed: abundant dotty elements distributed in the superficial (II-III) and deep (V) layers of the M2 cortex as well as in the cingulum (cg) and amygdala (Amg); uniform immunostaining in the cerebellar molecular (M) layer, strong basket cell terminal labeling around Purkinje cell (PC) bodies in the PC layer, and lack of staining in the granule (G) cell layer; very faint staining in the Acb with only some positive varicose fibers in control (H 2 O).Scale bar: 200 µm.

Table 3 .
Primary antibody used for immunohistochemistry.