Anti-GNB4 (internal region) polyclonal antibody

We used proximity-dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT-1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in mixed primary rat brain cultures affected both the clustering of GLT-1 at the astrocytic processes and the transport kinetics, increasing its uptake activity at low micromolar glutamate concentrations in a manner that was dependent on the effector kinase PAK1 and the actin cytoskeleton. Interestingly, the same manipulations had a different effect on another glial glutamate transporter, GLAST, inhibiting its activity. Importantly, glutamate acts through metabotropic receptors to stimulate the activity of Rac1 in astrocytes, supporting the existence of cross-talk between extracellular glutamate and the astrocytic form of the GLT-1 regulated by Rac1. CDC42EP4/BORG4 (a CDC42 effector) was also identified in the BioID screen, and it is a protein that regulates the assembly of septins and actin fibers, influencing the organization of the cytoskeleton. We found that GLT-1 interacts with septins, which reduces its lateral mobility at the cell surface. Finally, the G-protein subunit GNB4 dampens the activity of GLT-1, as revealed by its response to the activator peptide mSIRK, both in heterologous systems and in primary brain cultures. This effect occurs rapidly and thus, it is unlikely to depend on cytoskeletal dynamics. These novel interactions shed new light on the events controlling GLT-1 activity, thereby helping us to better understand how glutamate homeostasis is maintained in the brain.

BACKGROUND: To achieve goals, organisms are often faced with complex tasks that require enhanced control of cognitive faculties for optimal performance. However, the neural circuit mechanisms underlying this ability are unclear. The claustrum is proposed to mediate a variety of functions ranging from sensory binding to cognitive control of action, but direct functional assessments of this telencephalic nucleus are lacking. METHODS: Here, we employed the Gnb4 (guanine nucleotide-binding subunit beta-4) cre driver line in mice to selectively monitor and manipulate claustrum projection neurons during 1-choice versus 5-choice serial reaction time task performance. RESULTS: Using fiber photometry, we found elevated claustrum activity prior to an expected cue during correct performance on the cognitively demanding 5-choice response assay relative to the less demanding 1-choice version of the task. Claustrum activity during reward acquisition was also enhanced when task demand was higher. Furthermore, optogenetically inhibiting the claustrum prior to the onset of the cue reduced choice accuracy on the 5-choice task but not on the 1-choice task. CONCLUSIONS: These results suggest that the claustrum supports a cognitive control function necessary for optimal behavioral performance under cognitively demanding conditions.