The role of cannabinoid 1 receptor expressing interneurons in behavior

Abstract

Schizophrenia is a devastating neurodevelopmental disorder that affects approximately 1% of the population. Reduced expression of the 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of the disease and is encoded by the GAD1 gene. In schizophrenia, GAD67 downregulation occurs in multiple interneuronal subpopulations, including the cannabinoid receptor type 1 positive (CNR1+) cells, but the functional consequences of these disturbances are not well understood. To investigate the role of the CNR1-positive GABA-ergic interneurons in behavioral and molecular processes, we employed a novel, miRNA-mediated transgenic mouse approach. We silenced the Gad1 transcript using a miRNA engineered to specifically target Gad1 mRNA under the control of Cnr1 bacterial artificial chromosome. Behavioral characterization of our transgenic mice showed elevated and persistent conditioned fear associated with an auditory cue and a significantly altered response to an amphetamine challenge. These deficits could not be attributed to sensory deficits or changes in baseline learning and memory. Furthermore, HPLC analyses revealed that Cnr1/Gad1 mice have enhanced serotonin levels, but not dopamine levels in response to amphetamine. Our findings demonstrate that dysfunction of a small subset of interneurons can have a profound effect on behavior and that the GABA-ergic, monoamine, and cannabinoid systems are functionally interconnected. The results also suggest that understanding the function of various interneuronal subclasses might be essential to develop knowledge-based treatment strategies for various mental disorders including schizophrenia and substance abuse.

Keywords: Amphetamine; Behavior; Cnr1; Dopamine; GAD67; Gad1; Interneuron; Schizophrenia; Serotonin; Transgenic; miRNA.

Figure 1. Generation and validation of Cnr1/Gad1 transgenic mouse line

(A) Schematic representation of the construct used to generate the Cnr1/Gad1 transgenic mouse line. A centrally located minigene, containing a truncated part of the first exon, intervening intron, and second exon of the β-globin gene were the non-coding, but transcribed backbone which served as a ‘carrier’ for a synthetic miRNA directed against the Gad1 mRNA. The transcribed-translated part of the construct was the Gfp sequence inserted in frame with the start codon of the BAC-encoded Cnr1 gene, marking the cells in which the construct was activated. (B–C–D) Immunohistochemical assessment of Tg mice. (B) Triple immunohistochemistry showing the near-perfect co-localization of GFP with CNR1 in the hippocampus, stratum, substantia nigra, amygdala and cerebellum. Larger images on the left denote merged CNR1-GFP-DAPI triple-labeled images, small image stripes on the right denote enlarged part of the image on the left with signge-channel label (CNR1, GFP, DAPI and combined). DAPI co-labeling was performed to reveal overall tissue structure. Note that the GFP (produced from our transgene) and the co-expressed CNR1 (from the endogenous source) showed a staining pattern that is consistent with the previously described CNR1 expression across the rodent brain(Pettit et al., 1998; Tsou et al., 1998), suggesting that our transgene achieved the right spatial targeting. (C–D) To examine the cell-type specificity of the construct effects, we performed GFP-GAD67-PV triple-labeling of neocortical (C) and hippocampal (D) tissue sections. Larger pictures represent merged triple-labeled images, smaller monochrome images denote single channel fluorescence, higher magnification images (PV, GAD67 and GFP) originating from the larger picture (denoted by the white rectangle). As expected, the GFP expressing neurons (thus CNR1-expressing neurons) lacked GAD67 expression (yellow circles), suggesting that we achieved our goal of eliminating GAD67 production in the CNR1+ neuronal population. In contrast, GAD67 expression in the parvalbumin+ interneurons (blue circles) was not affected (retained both GAD67-PV immunostaining), suggesting that our construct specifically affected the CNR1-GAD67 subpopulation of interneurons. Abbreviations. Neocortex: Roman numerals denote neocortical laminae; WM - white matter; Hippocampus: so – stratum oriens, sp - stratum pyramidale, sr - stratum radiatum, slm - stratum lacunosum moleculare; Striatum: LGP - lateral globus pallidus ; CPu - caudate putamen; Substantia nigra: SNR - pars reticulata, SNC - pars compacta ; Amygdala: La – lateral amygdaloid nucleus, BLA - basolateral amygdaloid nucleus, ec - external capsule, En – endopiriform nucleus; Cerebellum: ml – molecular layer, plc – Purkinje cell layer, igl - internal granule layer. Calibration bar=100μm.

Figure 2. Altered fear attenuation in Cnr1/Gad1 transgenic mice

(A) Experimental design. The experiment was performed over 3 days (Smith et al., 2007). Day one: habituation phase for 12 min. Day two: training phase with 6 tone- shock pairs. Day three: context testing phase in the familiar environment with no tones over a 15 min period, followed by novel environment for 3 min, and concluded with 10 cue trials separated by 80 second intervals. (B) After a day of environment habituation on day two mice were conditioned to associate a tone followed by a foot shock. Over the course of 6 shock pairings (X-axis) both the Wt and the Tg learned the task equally well judged by the % of freezing response (Y-axis). Arrows denote the presentation of the conditioning stimulus. (C) Percent of time spent freezing in familiar environment was comparable between Wt and Tg mice. X axis denotes 1-minute intervals following the presentation of the condition stimulus (tone), Y-axis denotes % of freezing for each of the 15 intervals. None of the data points showed a significant difference between the Wt and Tg animals. (D) To assess hippocampus-dependent associative memory, we measured freezing % in the same familiar environment (FE) where training occurred, compared to a new novel environment (NE). This type of memory appears to be unaffected in our Tg line compared to Wt littermates. (E) In the novel environment, where a tone (but no shock) is presented, Wt mice display relatively little fear/freezing (Y-axis). In contrast, Tg are still freezing almost as much as at the end of the training day even after 10 trials with no shock. Asterisk denotes statistically significant differences for the cycles (p<0.05). (F) Total time spent freezing in response to tone alone on day 3, showing a significant increase in the time spent freezing in Tg animals in comparison to Wt littermates (p =0.001) (F-value =21.8). n= 13 Wt; n=11 Tg in all panels.

Figure 3. Baseline learning, memory, and anxiety are normal in Cnr1/Gad1 transgenic mice

(A) Comparison of time spent in the center or periphery of an open field box (Y-axis) as a measure of exploration. Tg mice show normal preference for the periphery that is similar to that seen in Wt mice. (B) Zero-maze evacuation of anxiety also shows that Tg and Wt mice both preferred to spend time in closed arms over open arm. (C) Y-maze evaluation of hippocampus-dependent memory shows that Tg and Wt mice were indistinguishable. (D) Assessment of order recognition revealed that both Wt and Tg spent less time investigating non-socially relevant odor (orange extract) then socially relevant odor (bedding from unfamiliar male cage) that decreased over time. (E) Social interaction assessment was measured by time spent investigating a novel mouse. Both Tg and Wt mice spent significantly greater time investigating a novel mouse than a novel object (an empty pencil cup). (F) When a novel object was replaced with a novel mouse while simultaneously presenting the now familiar mouse, Tg mice showed a significant preference (p= 0.01) for increased interaction with the novel mouse over the Wt littermates. n= 13 Wt; n=11 Tg in all panels.

Figure 4. Effect of a CNR1 agonist, CP55490 on prepulse inhibition (PPI) and acoustic startle response (ASR)

(A) Percent prepulse inhibition (Y-axis) was positively correlated to acoustic sound intensity (X-axis) in both Wt and Tg animals. (B) This response was not significantly affected by IP administration of 25mg/kg CP55940 for any of the individual sound intensities, however, when the average group responses were compared across the 4 tone intensities (70, 76, 82, 88 dB), Tg animals showed a significantly increased startle (p=0.005).(C) Acoustic startle response was also significantly elevated after IP injection of CP55940 in Tg animals (p=0.01) (F-value =16.4). n= 13 Wt; n=11 Tg in all panels.

Figure 5. Cnr1/Gad1 transgenic mice have decreased response to amphetamine (AMPH) challenge

(A) AMPH challenge with 3mg/kg IP injection and evaluation of motor activity in beam break cage in 5 min bins (Y-axis) show significant and long lasting suppression of AMPH -induced motor activity (p= 0.01). (B–C) Cumulative ambulations as well as total ambulation is significantly reduced in AMPH treated Tg mice compared to WT littermate controls (p= 0.001) (F-value =14). n= 13 Wt; n=11 Tg in all panels.

Figure 6. Serotonin levels in the striatum are increased after CNR1 receptor activation and AMPH challenge

(A) Activation of CNR1+ neurons with CP55940 did not significantly alter dopamine levels in the striatum in the Wt or Tg animals. However, activation of CNR1+ neurons with CP55940 significantly increased serotonin levels in both Tg and Wt animals (p=0.001) (B). Furthermore, this elevation of 5-HT levels was significantly higher in the Tg mice than in the Wt littermates (p=0.01). A similar experiment was performed on the AMPH-treated Tg and Wt mice (C–D), with a comparable outcome: striatal response to AMPH was similar in both Wt and Tg mice, but Tg animals responded with increased levels of 5-HT in the stratum (p= 0.001). n=10 Wt; n=10 Tg in all panels.