ABS 094: Determining the Role of ADGRA1 in Synapse Formation and Circuit Wiring using Synaptic Tracer and Imaris Software

Swarada Kulkarni ¹ , Baris Tosun ¹ , Elizabeth Orput ¹ , Richard Sando ¹

¹ Department of Pharmacology, Vanderbilt Brain Institute,  Vanderbilt University

Van Wickle (2025) Volume 1, ABS 094

Introduction: Adhesion G Protein-Coupled Receptors (aGPCRs) are the second-largest class of GPCRs and play key roles in cell signaling, circuit formation, and synaptogenesis. Interneurons, which provide inhibitory input to excitatory neurons, are essential for maintaining excitatory inhibitory balance in neural circuits. Diseases such as epilepsy stem from an imbalance between inhibition and excitation, with too much of the latter resulting in seizures. Unpublished data from our lab shows that ADGRA1, an aGPCR, is enriched in parvalbumin and somatostatin interneurons in the mouse hippocampus. Thus, we hypothesize that ADGRA1 contributes to synaptic connectivity of local inhibitory circuits, and its absence may impair hippocampal circuit formation.
Understanding ADGRA1 may inform future therapeutic strategies for conditions linked to faulty synaptogenesis, including epilepsy, ADHD, schizophrenia, and autism (Missler et al., 2012). However, the precise localization and function of ADGRA1, and its downstream signaling partners, remain unknown.
A previous Science paper by our lab determined that latrophilin GPCRs affect synapse specificity by coincident binding of FLRTs and teneurins (Sando et al., 2019). The research involved retrograde rabies and syb2 tracing techniques to compare circuit formation with various latrophilin knockouts. For the analysis of potential circuit changes resulting from the lack of ADGRA1, similar viral synaptic tracing techniques were used alongside whole cell patch clamp electrophysiology image analysis with the IMARIS 3D reconstruction software and manual spine analysis. Preliminary results indicate that Adgra1 does not affect spine density in excitatory conditional knockout cells compared to respective controls. Additionally, the lack of Adgra1 in excitatory forebrain cells does not affect the number of primary, secondary, or tertiary dendrites in granule cells. During manual spine analysis, we observed that Cre(+) neurons may have slightly decreased spine lengths but increased dendrite lengths. Based on this, we hypothesize that ADGRA1 may regulate dendritic spine types.

Methods: We employed a combination of molecular, imaging, and electrophysiological techniques in conditional knockout (cKO) mouse models. Preliminary research specifically focused on SST and EMX1 cre conditional knockout mice.
Viral Tracing and Confocal Analysis: Bilateral stereotactic injections (AP -1.8 mm, DV -2.05 mm, ML ±1.30) were performed in the dentate gyrus of P30 mice to introduce viral constructs expressing the mClover3 and HaloTag-Syb2 fluorescent markers. Following brain sectioning and labeling, confocal imaging was used to visualize presynaptic puncta and assess synaptic connectivity in both control and ADGRA1 knockout conditions.
Electrophysiology and Imaris Morphological Reconstruction: Whole-cell patch-clamp recordings were performed in acute hippocampal slices, followed by biocytin filling of recorded neurons. Post-fixation, the filled cells were imaged and processed using Imaris 3D reconstruction software. Analyses included quantification of dendritic spine density, dendritic length, and branching complexity.

Results: Confocal images confirmed successful targeting and expression in hippocampal circuits, indicating accurate injection coordinates and developed viral components, although quantification is ongoing. In Emx1-Cre(+) ADGRA1 cKO neurons, statistically significant changes in spine density or dendritic branching patterns were not observed when compared to controls. However, visually shorter spines were noted in Cre(+) neurons, suggesting a possible influence of ADGRA1 on spine morphology, which warrants further investigation using higher-resolution imaging and automated Imaris spine classification. Dendrite lengths and branch points were slightly elevated in Cre(+) cells, suggesting possible compensatory mechanisms in response to altered synaptic input, which are being further investigated.

Discussion: These findings contribute to a growing understanding of how aGPCRs may regulate synaptic structure in the brain. Although spine density and dendritic architecture appear unchanged, subtle alterations in spine morphology suggest a more nuanced role for ADGRA1 in synaptic refinement. Given the association of synaptic dysfunction with disorders like epilepsy and autism, further exploration of ADGRA1’s signaling pathways could uncover therapeutic targets. Future work will involve higher resolution Imaris reconstructions and the addition of retrograde rabies tracing to map presynaptic inputs and determine circuit-level effects. Circuit formation will continue to be studied in both SST and PV cKO conditions.