Recombinant Human GAD65 Protein [His, biotin]

The dorsal periacqueductal gray (dPAG) is a midbrain structure having an essential role in coordinating defensive behaviors in response to aversive stimulation. However, the question of whether dPAG neurons can respond to aversive conditioning and retrieval, properties involved in emergence of negative emotional state, is still under debate. Here we used calcium imaging by fiber photometry to record the activity of dPAG(VGluT2+) and dPAG(GAD2+) neuronal populations during unconditioned and conditioned aversive stimulation. Then, following an unconditioned stimulation we performed a retrieval experiment to quantify memory-like responses of dPAG neurons. This shown that whilst both dPAG(VGluT2+) and dPAG(GAD2+) neuronal populations respond to direct US stimulation, and to CS stimulation during conditioning, only the dPAG(VGluT2+) population persisted in responding to the CS stimulation during retrieval. Finally to better understand these divergences in dPAG(VGluT2+) and dPAG(GAD2+) responses, we investigated their respective connectivity patterns by performing a cell specific monosynaptic retrograde rabies virus tracing experiment. This revealed that different patterns of fibers projects to dPAG(VGluT2+) and dPAG(GAD2+), which could explain part of their response specificities. This may indicate that glutamatergic subpopulation is a main contributor of aversive memories in dPAG.

Gamma-aminobutyric acid (GABA) neurotransmission in the lateral septum (LS) is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD), the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2) and GAD67 (GAD1), and used fluorescence in Situ hybridization (FISH) with tyramide signal amplification (TSA) to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg) and medial preoptic area (MPOA) in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%), while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%). Using the neuronal marker NeuN, almost every neuron in LS (> 90%) was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB) or calretinin (CR), but not parvalbumin (PV); almost all CB-or CR-immunoreactive neurons (98-100%) were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI) (approximately 58% for CB and 35% for CR). These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of the LS.