Subchronic THC results in cerebellar conditioned learning deficits and motor coordination alteration. We followed a short schedule of systemic THC administration known to produce cannabinoid physical dependence (31) and examined whether cerebellar function would be compromised after THC exposure. Mice received THC (1, 2.5, 5, or 20 mg/kg, i.p.) twice daily over a 5-day period and once on the sixth day. We used the delayed eyeblink conditioning paradigm, since the cerebellum contributes critically to the acquisition (32) and performance (33) of this task. This learning model involves the same neuronal circuits in rodents and humans (34) and is specifically affected in current and former cannabis smokers (15, 18). The first conditioning session was performed on the last day of THC (or vehicle) treatment starting 4 hours after the last drug administration (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI67569DS1). The percentage of conditioned responses was measured every day for 9 days. Mice receiving subchronic THC at a dose of 1, 2.5, 5, or 20 mg/kg showed a dose-dependent deficit in cerebellar associative learning (Figure 1A). Specifically, this deficit was sustained from the third to the eighth day after treatment cessation in the 5 mg/kg (THC-5) or 20 mg/kg (THC-20) groups, while this was only revealed on the fifth day in the THC-2.5 group (Figure 1A). In contrast, the conditioned learning performance of the THC-1 group was similar to that of the vehicle group (Figure 1A). Considering these results, we studied the motor coordination skills on the fifth day after THC cessation in a different set of mice using the accelerating rotarod (Supplemental Figure 2) and coat-hanger tests (Figure 1B). Accelerating rotarod analysis showed learning impairment in this coordination task in THC-withdrawn mice compared with the control group at the same doses revealed in the conditioning paradigm (5 and 20 mg/kg) (Supplemental Figure 2). More robust results were observed using the coat-hanger test (Figure 1B), a more demanding test that allows for the detection of fine alterations in motor coordination function. Five days after cessation of subchronic THC treatment, the mice showed a dose-dependent alteration in several coordination parameters (fall latency, number of movements along the hanger, and extreme latency) compared with the vehicle-treated mice. We observed significant effects at the same THC doses (5 and 20 mg/kg) as in the previous paradigms (Figure 1B). However, no significant differences between groups were observed when equilibrium was assessed with the rod test (Supplemental Figure 3). Together, these data reveal the cerebellar deficits associated with THC exposure and subsequent withdrawal.

Figure 1 Cerebellar performance after subchronic THC exposure in mice. (A) Percentage of conditioned eyelid responses collected from mice that underwent subchronic THC (1, 2.5, 5, and 20 mg/kg) or subchronic VEH (n = 9–12 mice per group) treatment (see Supplemental Figure 1A for experimental chronogram). (B) Motor coordination analysis using the coat-hanger test after subchronic exposure to THC (1, 2.5, 5, and 20 mg/kg) or subchronic VEH conditions 5 days after spontaneous withdrawal (n = 11–17 mice per group). Impaired motor coordination was revealed by fall latency, number of movements to reach the end of the coat hanger, and extreme latency. *P < 0.05; **P < 0.01; ***P < 0.001 versus subchronic VEH treatment. Hab., habituation.

THC withdrawal produces molecular and cellular signs of cerebellar neuroinflammation. Five days after THC treatment cessation, cerebellar homogenates were analyzed by Western blotting and showed a THC dose–dependent increase in expression of the microglial activation marker CD11b and a decreased expression of CB1R (Figure 2A). As revealed by immunofluorescence staining and CB1R intensity analysis, the decrease in CB1R expression occurred mainly in the cerebellar molecular layer of THC-withdrawn mice (5 and 20 mg/kg) (Figure 2B) In addition, double immunostaining and colocalization analysis of IBA1 and CD11b on cerebellar slices under similar conditions demonstrated that microglial activation took place mainly in the molecular layer of the cerebellum, rather than in the granular layer (Figure 2C). In this cerebellar layer, microglia acquired a bushy morphology according to the perimeter of the soma and the length of the branches after THC treatment cessation (Figure 2D). Notably, the microglial activation phenotype was not detectable in other brain areas where CB1Rs are heavily expressed such as the hippocampus, frontal cortex, or striatum (Supplemental Figure 4).

Figure 2 Cerebellar responses 5 days after THC treatment cessation. (A) Immunoblot and quantification of CD11b and CB1R in cerebellar homogenates from mice processed 5 days after subchronic treatment (n = 5–6 mice per group). The optical density of CD11b and CB1R was normalized to GAPDH in the same samples. (B) Immunofluorescence and quantification of CB1R intensity in the granular and molecular layers of the cerebellum from mice processed 5 days after subchronic treatment (n = 3 mice per group, 5 images per mouse). Scale bar: 75 μm. (C) Immunolocalization and ICQ of IBA1 (red) and CD11b (green) in the molecular and granular layers of the cerebellum after treatment (n = 3 mice per group, 3 images per mouse). Scale bars: 100 μm. (D) Morphological analysis of IBA1+ microglial cells in the molecular layer of the cerebellum (n = 4 mice per group, 4 cells per mouse). See Supplemental Figure 17 for details. Scale bar: 25 μm. (E) Analysis of Cb2r mRNA expression and inflammation-related genes in cerebellar samples (n = 7–8 mice per group). (F) Flow cytometric analysis of CD11b expression and qRT-PCR analysis of acutely dissociated cerebellar cells from VEH-, THC-5–, and THC-20–treated mice (n = 3 per group). Sorted CD11b+ population (P4) and CD11b– population (P5) in THC-treated mice showed a differential expression of Cb1r, Cb2r, and Il1b. *P < 0.05; **P < 0.01; ***P < 0.001 versus subchronic VEH plus SAL (5 days).

Quantitative analysis of mRNA for Cb2r and the neuroinflammatory markers Il1b, Tnfa, Cox2, Cd11b, and Cxcl2 indicated that the expression of these genes is enhanced in the cerebellum 5 days after the cessation of subchronic THC treatment at doses of 5 and 20 mg/kg (Figure 2E).

In an additional experimental group, the cannabinoid withdrawal syndrome was precipitated after subchronic THC (20 mg/kg) treatment by rimonabant (10 mg/kg, i.p.) administered 4 hours after the last THC injection (31). Control groups that were subchronically treated with vehicle and that were receiving an acute challenge of rimonabant or its vehicle were also run in parallel. We found that under rimonabant-triggered cannabinoid withdrawal, the results were similar in terms of the reactivity of cerebellar microglia (Supplemental Figure 5, A and B) and the downregulation of cerebellar CB1R (Supplemental Figure 5C) to those reported here after spontaneous cessation of subchronic THC treatment.

We hypothesized that the neuroinflammatory phenotype observed in the cerebellar molecular layer 5 days after THC cessation would respond to a possible restricted alteration in the extracellular milieu resulting from deregulated glutamate handling in the parallel fiber terminals as a consequence of CB1R downregulation. As expected, a strong decrease in CB1R expression was also detected in the cerebellum at the end of subchronic THC treatment (Supplemental Figure 6A), in agreement with previous studies (8). In contrast, no changes in CD11b expression were detected in the cerebellar homogenates (Supplemental Figure 6A), although the microglial morphology was slightly altered (Supplemental Figure 6B). Fluoro-Jade B assay revealed no signs of cellular death in the cerebellum at the end of subchronic THC treatment, suggesting that the microglial reactivity was not associated with cytotoxicity processes (Supplemental Figure 7, A and B). Moreover, we observed no signs of microglial (Supplemental Figure 7, C and D) or astroglial (Supplemental Figure 7, E and F) proliferation in the cerebellum at the end of this treatment, as measured by cell counting and immunoblot analysis of IBA1 and glial fibrillary acidic protein (GFAP) expression. Similarly, the neuroinflammatory markers Il1b, Tnfa, Cox2, Cxcl2, and Il10 were not altered in the cerebellum at the end of THC treatment (Supplemental Figure 7G).

When the time course of these effects mediated by THC exposure was studied, we observed that CD11b expression increased in the cerebellum only 5 days after THC cessation, while the downregulation of CB1R expression reached its maximum level at the end of subchronic THC treatment, slowly recovering afterward (Supplemental Figure 8A). The time course for the effect of THC exposure on microglial morphology revealed a progressive modification of these cells to the activated phenotype (Supplemental Figure 8B). In addition, we sorted the acutely dissociated cerebellar cells, positive (CD11b+, population 4, P4) or negative (CD11b–, population 5, P5) for CD11b, from mice receiving subchronic vehicle, THC-5, or THC-20 and sacrificed 5 days after treatment cessation. These cells were analyzed by RT-PCR for mRNA expression of Cb1r, Cb2r and the proinflammatory cytokine Il1B (Figure 2F). Under these conditions, the percentage of CD11b+ (P4) cells slightly increased in the THC-20 group. In the CD11b+ (P4) population, mainly corresponding to cells positive for IBA1 staining (Supplemental Figure 9), enhanced expression of Cb2r and Il1b was observed (Figure 2F). However, CD11b– (P5) cells did not reveal modulated Cb2r or Il1b gene expression, but rather showed enhanced expression of Cb1r mRNA (Figure 2F).

Therefore, the inflammatory phenotype in the cerebellum was progressively enhanced after subchronic THC cessation and correlated with the poor cerebellar functioning.

Blockade of microglial activation ameliorates cerebellar deficits. We used the tetracycline antibiotic minocycline, an immunosuppressant with inhibitory effects on microglial activation (35), to evaluate the role of this process in the motor coordination deficit described above after cessation of the subchronic THC treatment. Minocycline (40 mg/kg, i.p.) was administered 1 hour after the last THC injection (5 or 20 mg/kg, i.p.) on the sixth day of THC treatment, and once a day for 5 days. Subchronic minocycline treatment reversed the enhancement of CD11b levels in the cerebellum that had been promoted by subchronic THC administration (Figure 3A). Moreover, minocycline treatment normalized microglial morphology to control levels with regard to both the perimeter of the soma and the length of the branches (Figure 3B). Cerebellar CB1R expression was not affected after minocycline administration, as detected by immunoblotting of cerebellar samples (Figure 3C) and by immunofluorescence detection in the molecular layer of the cerebellum (Figure 3D).

Figure 3 MIN administration after subchronic THC exposure prevents the activation of microglia in the cerebellum. (A) Immunoblot detection and quantification of cerebellar CD11b (n = 5 mice per group) at the end of subchronic exposure to MIN or SAL under subchronic THC (5 and 20 mg/kg) and subchronic VEH treatment conditions. (B) Morphological analysis of IBA1+ cells in the cerebellar cortex (n = 3–4 mice per group, 4–5 cells per mouse). Scale bar: 25 μm. (C) Immunoblot detection and quantification of cerebellar CB1R (n = 6 mice per group) at the end of MIN or SAL exposure under subchronic THC (5 and 20 mg/kg) and subchronic VEH treatment conditions. (D) Immunolocalization and quantification of CB1R intensity in the cerebellar molecular layer at the end of subchronic exposure to MIN or SAL under subchronic THC (5 and 20 mg/kg) and subchronic VEH treatment conditions (n = 4 mice per group; 3–4 images per mouse). Note the downregulation of CB1R in the molecular layer of the cerebellum 5 days after the end of THC-5 and THC-20 subchronic treatments. Scale bar: 75 μm. *P < 0.05; **P < 0.01; ***P < 0.001 versus subchronic VEH plus SAL (5 days); #P < 0.05; ##P < 0.01; ###P < 0.001 versus subchronic THC (5 or 20 mg/kg) plus SAL (5 days) treatment.

The compromised cerebellar conditioned learning of the THC-withdrawn mice was normalized after minocycline blockade of microglial activation. As described above, the first conditioning session (C1) was performed on the last day of subchronic THC (5 or 20 mg/kg) or vehicle treatment, which coincided with the first day of minocycline (or saline) administration (C1) (Figure 4, A and C, and Supplemental Figure 1B). As shown previously, the groups that had received subchronic THC showed a dose-dependent deficit in associative learning compared with the control groups during the conditioning phase (Figure 4, A and C). The administration of minocycline increased the percentage of conditioned responses starting on the third day of treatment (C3) (Figure 4, A and C). In another experimental set, THC-withdrawn mice were tested for motor coordination on the fifth and last day of minocycline administration using the coat-hanger test. Minocycline also reversed the motor coordination impairment induced by THC, improving coat-hanger test performance compared with that of the subchronically THC–treated mice receiving saline (Figure 4, B and D). Therefore, microglial activation blockade correlates with a reduction in the motor coordination deficits observed in spontaneous THC-withdrawn mice. In an additional experiment, we verified that acute minocycline administration did not modify the somatic manifestations of rimonabant-mediated (10 mg/kg, i.p.) withdrawal syndrome after subchronic THC (20 mg/kg, i.p.) treatment, which discards a possible acute effect of minocycline on the manifestation of withdrawal symptoms (Supplemental Figure 10).

Figure 4 MIN treatment prevents the cerebellar deficits produced by THC exposure. (A) Percentage of conditioned eyelid responses collected from mice receiving MIN or SAL after subchronic THC-5 or VEH treatment (n = 9–11 mice per group) (see Supplemental Figure 1B for experimental chronogram). (B) Motor coordination analysis at the end of subchronic exposure to MIN or SAL in mice that had previously received THC-5 or VEH (n = 15 mice per group). Alterations in motor coordination evaluated in the coat-hanger test were ameliorated by subchronic MIN administration. (C) Percentage of conditioned eyelid responses collected from mice receiving MIN or SAL after subchronic THC-20 or VEH treatment (n = 9–11 mice per group). See Supplemental Figure 1B for experimental chronogram. (D) Motor coordination analysis at the end of subchronic exposure to MIN or SAL in mice that had previously received THC-20 or VEH (n = 15 mice per group). Impairment in motor coordination skills measured by the coat-hanger test was prevented with subchronic MIN treatment. *P < 0.05; **P < 0.01; ***P < 0.001 versus subchronic VEH plus SAL (5 days) treatment. #P < 0.05; ##P < 0.01; ###P < 0.001 versus subchronic THC (5 or 20 mg/kg) plus SAL (5 days) treatment.

Together, these results suggest the involvement of microglial activation on cerebellar conditioned learning and motor coordination impairments promoted by subchronic THC administration, given that optimal performance on both functional tests requires intact cerebellar-mediated functioning.

CB1R downregulation is critical for cerebellar neuroinflammation and function. To evaluate the relevance of CB1R downregulation in the neuroinflammatory and behavioral alterations reported here, we investigated these responses in CB1R KO mice (Cb1–/–). Mild motor coordination deficits have been previously reported in Cb1–/– adult mice (3–5 months of age), as demonstrated by rotarod (36) and eyeblink conditioning tests (17). We observed increased CD11b expression in the cerebellum, but not in the hippocampus of naive Cb1–/– mice (Figure 5A). Moreover, the percentage of acutely dissociated cerebellar cells identified as CD11b+ (P4) were more abundant in the Cb1–/– mice than in the controls and expressed enhanced mRNA levels of Cb2r and Il1b (Figure 5B).

Figure 5 Genetically downregulated CB1R promotes cerebellar neuroinflammation and reversible cerebellar deficits. (A) CD11b detection in WT and Cb1r KO (Cb1–/–) mice (n = 5 per group). CD11b quantification was normalized to GAPDH. *P < 0.05 versus WT. (B) Flow cytometric analysis of CD11b expression and quantitative RT-PCR (qRT-PCR) analysis of acutely dissociated cerebellar cells from WT and Cb1–/– mice (n = 3–4 per group). (C) CD11b in cerebellar homogenates in WT and Cb1–/– mice after treatment (n = 6 mice per group). CD11b detection was normalized to GAPDH. *P < 0.05 versus WT plus SAL; #P < 0.05 versus Cb1–/–plus SAL. (D) Morphological analysis of IBA1+ cells in the cerebellar cortex (n = 3–5 mice per group, 5 cells per mouse) of WT and Cb1–/– mice after treatment. Scale bar: 25 μm. ***P < 0.001 versus WT plus SAL; ###P < 0.001 versus Cb1–/– plus SAL. (E) Analysis of Cb2r mRNA expression by qRT-PCR and inflammation-related genes in the cerebellum (n = 5–6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. (F) Percentage of conditioned eyelid responses collected from WT and Cb1–/– mice treated with MIN or SAL (n = 7–10 mice per group). See Supplemental Figure 12A for experimental chronogram. **P < 0.01; ***P < 0.001 versus WT plus SAL; #P < 0.05, ##P < 0.01, ####P < 0.001 versus Cb1–/– plus SAL. (G) Motor coordination analysis in WT and Cb1–/– mice after treatment (n = 13–22 mice per group). **P < 0.01, ***P < 0.001 versus WT plus SAL; #P < 0.05, ###P < 0.001 versus Cb1–/– plus SAL.

The neuroinflammatory phenotype observed in the Cb1–/– mice was also sensitive to minocycline treatment, since CD11b expression (Figure 5C) and the morphological changes highlighted by IBA1 staining of microglia (Figure 5D) were normalized in the cerebellum after subchronic minocycline administration. Moreover, enhanced mRNA expression of Cb2r and inflammatory mediators, such as Il1b, Il10, Tnfa, Cox2, and Cxcl2, was detected in the cerebellum of Cb1–/– mice, and these changes were sensitive to minocycline treatment (Figure 5E). Additionally, no changes in GFAP expression were observed in the cerebellum of Cb1–/– mice (Supplemental Figure 11).

The analysis of cerebellar function after minocycline treatment in Cb1–/– mice revealed a remarkable recovery of the disrupted response in cerebellar conditioned learning (Figure 5F and Supplemental Figure 12A). Similarly, the motor coordination impairment revealed in Cb1–/– mice by the coat-hanger test was also significantly improved with minocycline treatment (Figure 5G), demonstrating the relevance of microglial activation in the phenotype promoted by CB1R deletion.

IL-1 receptor signaling blockade resolves cannabinoid-mediated cerebellar deficits. As described above, cerebellar Il1b expression was enhanced 5 days after subchronic THC treatment cessation in CD11b+ (P4) cells, and was constantly altered in Cb1–/– mice. Local microinjection of IL-1β into the cerebellum is known to produce ataxia (37) and to enhance the firing rates of cerebellar Purkinje neurons (38) acting on the IL-1 receptors (IL-1Rs) that are strongly expressed in Purkinje neurons (39). We therefore tested whether an inhibitor of IL-1R signaling, an end-protected L-valyl derivative with antagonistic effects on IL-1R (IL-1RA), would improve cerebellar performance under our experimental conditions. For delayed eyeblink conditioning analysis, mice received IL-1RA (100 mg/kg, i.p.) 4 days after subchronic THC treatment cessation for 3 consecutive days 4 hours before the test sessions (C5–C7) (Figure 6A and Supplemental Figure 1C). Mice receiving IL-1RA showed an increase in the number of conditioned responses that reached statistical significance from the second day of IL-1RA administration for the THC (5 mg/kg) group and from the first day of administration for the THC (20 mg/kg) group (Figure 6A). Similarly, Cb1–/– mice showed transiently improved performance in the delayed eyeblink conditioning paradigm after IL-1RA administration (Figure 6B and Supplemental Figure 12B). Correspondingly, acute IL-1RA administration 4 hours before the coat-hanger test normalized the performance of the mice that received subchronic THC (5 or 20 mg/kg) treatment to that of the control mice (Figure 6C). A similar treatment with IL-1RA in Cb1–/– mice significantly improved their performance in the coat-hanger test (Figure 6D). Interestingly, no effect of IL-1RA was observed in the control groups (subchronic vehicle [5 days] and WT mice), either with the conditioned cerebellar learning test or the coat-hanger test (Figure 6). Moreover, no alteration of cerebellar microglia morphology was detected after acute IL-1RA administration (Supplemental Figure 13), pointing to a specific role of this blockade strategy under neuroinflammatory conditions.

Figure 6 Acute IL-1RA administration resolves the cerebellar deficit resulting from cannabinoid receptor deregulation and neuroinflammation. (A) Percentage of conditioned eyelid responses collected from mice subchronically treated with THC (5 and 20 mg/kg) and VEH (n = 5–10 mice per group). See Supplemental Figure 1C for experimental chronogram. *P < 0.05, **P < 0.01, ***P < 0.001 versus subchronic VEH treatment plus DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001 versus subchronic THC (5 or 20 mg/kg) treatment plus DMSO. (B) Percentage of conditioned eyelid responses collected from WT and Cb1–/– mice (n = 5–10 per group). See Supplemental Figure 12B for experimental chronogram. *P < 0.05, **P < 0.01 versus WT plus DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Cb1–/– plus DMSO. (C) Motor coordination analysis with the coat-hanger test in subchronic THC (5 and 20 mg/kg) and subchronic VEH conditions 5 days after spontaneous withdrawal (n = 9–15 mice per group). Four hours before the test, mice received an acute injection of IL-1RA (100 mg/kg, i.p.) or its VEH (DMSO). *P < 0.05, **P < 0.01 versus subchronic VEH. (D) Motor coordination analysis with the coat-hanger test in WT and Cb1–/– mice (n = 8–13 per group). Four hours before the test, mice received an injection of IL-1RA or its VEH. **P < 0.001 versus WT plus DMSO; ##P < 0.01 versus Cb1–/– plus DMSO.

Cerebellar neuroinflammation modulates ARC/ARG3.1 expression. The activity of cerebellar Purkinje neurons can be evaluated by measuring the expression of the activity-regulated cytoskeleton-associated protein, also known as ARG3.1 (ARC/ARG3.1) (40). ARC/ARG3.1 is expressed in Purkinje cell neurons, where it colocalizes with calbindin immunoreactivity (Figure 7A). We measured ARC/ARG3.1 expression in cerebellar homogenates to assess the Purkinje cell activity under the experimental conditions studied above. Mice after subchronic THC treatment cessation (Figure 7B) and Cb1–/– mice (Figure 7C) showed enhanced expression of ARC/ARG3.1 compared with control mice. Interestingly, a clear reduction of ARC/ARG3.1 expression was observed after subchronic minocycline treatment in both experimental conditions (Figure 7, B and C).

Figure 7 Cerebellar neuroinflammation secondary to cannabinoid downregulation modulates ARC/ARG3.1 expression. (A) Immunofluorescence detection of ARC/ARG3.1 and calbindin in Purkinje neurons in the cerebellar cortex. Scale bar: 50 μm. (B) Immunoblot detection and quantification of ARC/ARG3.1 in cerebellar homogenates obtained at the end of subchronic exposure to MIN or SAL under subchronic THC (5 and 20 mg/kg) and subchronic VEH treatment conditions (n = 5–6 mice per group). *P < 0.05, **P < 0.01 versus subchronic VEH plus SAL (5 days); #P < 0.05, ##P < 0.01 versus subchronic THC-5 plus SAL (5 days) or subchronic THC-20 plus SAL (5 days). (C) Immunoblot detection and quantification of ARC/ARG3.1 in cerebellar homogenates from Cb1–/– and WT mice at the end of subchronic exposure to MIN or SAL (n = 5–6 mice per group). *P < 0.05 versus subchronic WT plus SAL; #P < 0.05 versus WT plus SAL. (D) ARC/ARG3.1 immunostained images from mice that were subchronically treated with THC-5, THC-20, or VEH and that received MIN or SAL for 5 days. ARC/ARG3.1 intensity was measured along a 300-μm line stretched along the 3 cerebellar layers. Plot represents ARC/ARG3.1 intensity alongside the layers quantified with ImageJ software. Scale bar: 100 μm. (E) ARC/ARG3.1 immunostained images from Cb1–/– and WT mice at the end of subchronic exposure to MIN or SAL. ARC/ARG3.1 intensity was measured along a 300-μm line stretched along the 3 cerebellar layers. Plot represents ARC/ARG3.1 intensity alongside the layers quantified with ImageJ software. Scale bar: 100 μm.

Immunofluorescence analysis for ARC/ARG3.1 revealed a localized increase in ARC/ARG3.1 expression in the Purkinje cells of the cerebellum both after subchronic THC treatment cessation (Figure 7D) and in Cb1–/– mice (Figure 7E). This result was corroborated by the intensity analysis performed across the different layers of the cerebellar cortex (Figure 7, D and E). These results point to a relevant involvement of the neuroinflammatory process, which is produced by cerebellar CB1R downregulation, in the activity-related changes (expression of ARC/ARG3.1) promoted in Purkinje cells.

CB1R deregulation in parallel fibers recreates the cerebellar deficit. To better evaluate whether the downregulation of CB1R in cerebellar parallel fibers would affect microglial responses and cerebellar performance, we used a conditional Cb1a6– mouse model lacking CB1R in cerebellar granular cells (12). In this mouse line, CB1Rs were absent from the parallel fibers, but were preserved in other cerebellar locations where CB1Rs are less abundantly expressed, such as the climbing fibers (Supplemental Figure 14). In this mouse line, immunofluorescence detection of CB1Rs on microglial cells located in the molecular layer of the cerebellum was negligible and indistinguishable from the background staining (Supplemental Figure 15). Microglial morphology revealed by staining with IBA1 showed an activated phenotype (Figure 8A) of these cells in the cerebellar molecular layer of the Cb1a6– mice. These mice displayed an enhanced cerebellar mRNA expression of Cb2r, Il1b, and Tnfa (Figure 8B). The increased Cb2r and Il1b expression was restricted to CD11b+ (P4) cells, but Cb2r and Il1b were not detected in CD11b– (P5) cells acutely isolated from the cerebellum (Figure 8C). Similar to previous data involving Cb1–/– mice, Cb1a6– mice showed enhanced expression of cerebellar ARC/ARG3.1 (Figure 8, D and E) and, compared with their control littermates, exhibited a significant motor coordination deficit in the coat-hanger test (Figure 8F) that was prevented by acute IL-1RA administration (Figure 8G). The use of this novel genetic tool provides direct evidence for the role of CB1R in cerebellar parallel fibers in modulating these morphological and behavioral responses.