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Localization of cannabinoid CB1 receptor mRNA in neuronal subpopulations of rat striatum: A double‐label in situ hybridization study

Double‐label in situ hybridization was used to identify the phenotypes of striatal neurons that express mRNA for cannabinoid CB1 receptors. Simultaneous detection of multiple mRNAs was performed by combining a 35S‐labeled ribonucleotide probe for CB1 mRNA with digoxigenin‐labeled riboprobes for striatal projection neurons (preprotachykinin A, prodynorphin, and preproenkephalin mRNAs) and interneurons (vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), somatostatin, and glutamic acid decarboxylase (Mr 67,000; GAD67) mRNAs). To ascertain whether CB1 mRNA was a marker for striatal efferents, digoxigenin‐labeled probes for mRNA markers of both striatonigral (prodynorphin or preprotachykinin A mRNAs), and striatopallidal (proenkephalin mRNAs) projection neurons were combined with the 35S‐labeled probe for CB1. A mediolateral gradient in CB1 mRNA expression was observed at rostral and mid‐striatal levels; in the same coronal sections the number of silver grains per cell ranged from below the threshold of detectability at the medial and ventral poles to saturation at the dorsolateral boundary bordered by the corpus callosum. At the caudal level examined, CB1 mRNA was denser in the ventral sector relative to the dorsal sector. Virtually all neurons expressing mRNA markers for striatal projection neurons colocalized CB1 mRNA. Combining a 35S‐labeled riboprobe for CB1 with digoxigenin‐labeled riboprobes for both preproenkephalin and prodynorphin confirmed localization of CB1 mRNA to striatonigral and striatopallidal neurons expressing prodynorphin and preproenkephalin mRNAs, respectively. However, CB1 mRNA‐positive cells that failed to coexpress the other markers were also apparent. CB1 mRNA was localized to putative GABAergic interneurons that express high levels of GAD67 mRNA. These interneurons enable functional interactions between the direct and indirect striatal output pathways. By contrast, aspiny interneurons that express preprosomatostatin mRNA and cholinergic interneurons that coexpress ChAT and VAChT mRNAs were CB1 mRNA‐negative. The present data provide direct evidence that cannabinoid receptors are synthesized in striatonigral neurons that contain dynorphin and substance P and striatopallidal neurons that contain enkephalin. By contrast, local circuit neurons in striatum that contain somatostatin or acetylcholine do not synthesize cannabinoid receptors. Synapse 37:71–80, 2000. Published 2000 Wiley‐Liss, Inc.