The development of BAC transgenic mice expressing promoter-specific fluorescent reporter proteins has been a great asset for neuroscience by enabling detection of neuronal subsets in live tissue. For the study of basal ganglia physiology, reporters driven by type 1 and 2 dopamine receptors have been particularly useful for distinguishing the two classes of striatal projection neurons – striatonigral and striatopallidal. However, emerging evidence suggests that some of the transgenic reporter lines may have suboptimal features. The ideal transgenic reporter line should (1) express a reporter with high sensitivity and specificity for detecting the cellular subset of interest and that does not otherwise alter the biology of the cells in which it is expressed, and (2) involve a genetic manipulation that does not cause any additional genetic effects other than expression of the reporter. Here we introduce a new BAC transgenic reporter line, Drd1a-tdTomato line 6, with features that approximate these ideals, offering substantial benefits over existing lines. In this study, we investigate the integrity of dopamine-sensitive behaviors and test the sensitivity and specificity of tdTomato fluorescence for identifying striatonigral projection neurons in mice. Behaviorally, hemizygous Drd1a-tdTomato line 6 mice are similar to littermate controls; while hemizygous Drd2-EGFP mice are not. In characterizing the sensitivity and specificity of line 6 mice, we find that both are high. The results of this characterization indicate that line 6 Drd1a-tdTomato+/− mice offer a useful alternative approach to identify both striatonigral and striatopallidal neurons in a single transgenic line with a high degree of accuracy.
Background SAP90/PSD-95-associated protein 3 (SAPAP3, also DLGAP3 or GKAP3) is an excitatory postsynaptic protein implicated in the pathogenesis of obsessive-compulsive behaviors. In mice, genetic deletion of Sapap3 causes obsessive-compulsive disorder (OCD)-like behaviors that are rescued by striatal expression of Sapap3, demonstrating the importance of striatal neurotransmission for the OCD-like behaviors. In the striatum, there are two main excitatory synaptic circuits, corticostriatal and thalamostriatal. Neurotransmission defects in either or both of these circuits could potentially contribute to the OCD-like behaviors of Sapap3 knockout (KO) mice. Previously we reported that Sapap3 deletion reduces corticostriatal AMPA-type glutamate receptor (AMPAR)-mediated synaptic transmission. Methods Whole-cell electrophysiological recording techniques in acute brain slices were used to measure synaptic transmission in the corticostriatal and thalamostriatal circuits of Sapap3 KO mice and littermate controls. Transgenic fluorescent reporters identified striatopallidal and striatonigral projection neurons. SAPAP isoforms at corticostriatal and thalamostriatal synapses were detected using immunostaining techniques. Results In contrast to corticostriatal synapses, thalamostriatal synaptic activity is unaffected by Sapap3 deletion. At the molecular level, we find that another SAPAP family member, SAPAP4, is present at thalamostriatal, but not corticostriatal synapses. This finding provides a molecular rationale for the functional divergence we observe between thalamic and cortical striatal circuits in Sapap3 KO mice. Conclusion These findings define the circuit-level neurotransmission defects in a genetic mouse model for OCD-related behaviors, focusing attention on the corticostriatal circuit for mediating the behavioral abnormalities. Our results also provide the first evidence that SAPAP isoforms may be localized to synapses according to circuit-selective principles.
Synaptic transmission mediated by AMPA-type glutamate receptors (AMPARs) is regulated by scaffold proteins in the postsynaptic density. SAP90/PSD-95-associated protein 3 (SAPAP3) is a scaffold protein that is highly expressed in striatal excitatory synapses. While loss of SAPAP3 is known to cause obsessive compulsive disorder-like behaviors in mice and reduce extracellular field potentials in the striatum, the mechanism by which SAPAP3 regulates excitatory neurotransmission is largely unknown. This study demonstrates that Sapap3 deletion reduces AMPAR-mediated synaptic transmission in striatal medium spiny neurons (MSNs) through postsynaptic endocytosis of AMPARs. Striatal MSNs in Sapap3 KO mice have fewer synapses with AMPAR activity and a higher proportion of silent synapses. We further find that increased metabotropic glutamate receptor 5 (mGluR5) activity in Sapap3 KO mice underlies the decrease in AMPAR synaptic transmission and excessive synapse silencing. These findings suggest a model whereby the normal role of SAPAP3 is to inhibit mGluR5-driven endocytosis of AMPARs. The results of this study provide the first evidence for the mechanism by which the SAPAP family of scaffold proteins regulates AMPAR synaptic activity.
Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.
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