Camk2a-Cre and Tshz3 Expression in Mouse Striatal Cholinergic Interneurons: Implications for Autism Spectrum Disorder

Abstract

Camk2a-Cre mice have been widely used to study the postnatal function of several genes in forebrain projection neurons, including cortical projection neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). We linked heterozygous deletion of TSHZ3/Tshz3 gene to autism spectrum disorder (ASD) and used Camk2a-Cre mice to investigate the postnatal function of Tshz3, which is expressed by CPNs but not MSNs. Recently, single-cell transcriptomics of the adult mouse striatum revealed the expression of Camk2a in interneurons and showed Tshz3 expression in striatal cholinergic interneurons (SCINs), which are attracting increasing interest in the field of ASD. These data and the phenotypic similarity between the mice with Tshz3 haploinsufficiency and Camk2a-Cre-dependent conditional deletion of Tshz3 (Camk2a-cKO) prompted us to better characterize the expression of Tshz3 and the activity of Camk2a-Cre transgene in the striatum. Here, we show that the great majority of Tshz3-expressing cells are SCINs and that all SCINs express Tshz3. Using lineage tracing, we demonstrate that the Camk2a-Cre transgene is expressed in the SCIN lineage where it can efficiently elicit the deletion of the Tshz3-floxed allele. Moreover, transcriptomic and bioinformatic analysis in Camk2a-cKO mice showed dysregulated striatal expression of a number of genes, including genes whose human orthologues are associated with ASD and synaptic signaling. These findings identifying the expression of the Camk2a-Cre transgene in SCINs lineage lead to a reappraisal of the interpretation of experiments using Camk2a-Cre-dependent gene manipulations. They are also useful to decipher the cellular and molecular substrates of the ASD-related behavioral abnormalities observed in Tshz3 mouse models.

Targeted Tshz3 deletion in corticostriatal circuit components segregates core autistic behaviors

“…This was preferred to dual immunodetection of CHAT and TSHZ3 since the tissue fixation conditions for obtaining optimal detection of each protein are different, and also because TSHZ3 immunodetection provides weaker labeling and higher background than ß-Gal immunodetection. As reported previously [ 13 ], virtually all SCINs, both in the dorsal striatum and the nucleus accumbens, express Tshz3 (Fig. S6a, h ).…”

“…We previously reported that around 90% of the Tshz3 -expressing cells in the striatum are SCINs [ 13 ]. Using Tshz3 +/lacZ ;GAD67-GFP mice, we found here that GABAergic neurons constitute 9.7 ± 1.3% ( n = 21 sections from 3 mice) of the striatal Tshz3 population.…”

“…Here, we show that, in the cortex, the ASD-related Tshz3 gene is expressed not only in CPNs but also in about a third of GABAergic interneurons, while not in cholinergic interneurons. In contrast, in the striatum, Tshz3 -expressing cells are for their vast majority cholinergic interneurons [ 13 ] and comprise a minority of GABAergic interneurons. To disentangle the role of CPNs from that of interneurons in the ASD symptoms linked to Tshz3 deficiency, we generated and characterized Emx1-cKO mice.…”

“…In the mouse striatum, TSHZ3 is not expressed in striatal spiny projection neurons (SSPNs), which represent >90% of striatal neurons, but in a small population of cells that are likely interneurons [ 10 ]. We [ 13 ] and others [ 14 , 15 ] identified these cells as being mainly striatal cholinergic interneurons (SCINs), whose implication in ASD has been suggested by some studies [ 16 , 17 ]. We also showed that the Camk2a-Cre transgene is unexpectedly expressed in the SCIN lineage, where it efficiently elicits the deletion of Tshz3 in Camk2a-Cre mice [ 13 ].…”

“…We [ 13 ] and others [ 14 , 15 ] identified these cells as being mainly striatal cholinergic interneurons (SCINs), whose implication in ASD has been suggested by some studies [ 16 , 17 ]. We also showed that the Camk2a-Cre transgene is unexpectedly expressed in the SCIN lineage, where it efficiently elicits the deletion of Tshz3 in Camk2a-Cre mice [ 13 ]. Together, these data demonstrate that, within the corticostriatal circuitry, Tshz3 is deficient in both CPNs and SCINs, in Tshz3 +/lacZ heterozygous [ 10 ] as well as in Camk2a-cKO mice [ 12 ], which both show the full repertoire of ASD-like behavioral defects.…”