(D-143) AAV-mDLX5/6 Vectors Transduce Cortical Neurons Expressing Parvalbumin-Expressing but not Calretinin-Expressing Interneurons

Introduction:: Inhibitory interneurons play essential roles in shaping cortical circuits. These roles are critically important for brain function but are poorly understood because inhibitory interneurons come in a variety of distinct types. Inhibitory interneurons can be classified on the basis of non-overlapping molecular markers. For example, the calcium-binding proteins parvalbumin (PV) and calretinin (CR) are expressed in two distinct populations of cortical GABAergic interneurons in both rodents and primates[1].
Dysfunction of GABAergic neurons can result in an imbalance between excitatory and inhibitory brain activity, which can contribute to various central nervous system (CNS) disorders. Gene therapy uses viruses to modify gene expression and treat CNS illnesses. Recombinant adeno-associated viruses (rAAVs) are commonly used as vectors for gene therapy. These can be modified to express opsins, which enable optogenetic manipulation of neural activity. Cell-type-specific enhancers can also be introduced to enable targeted expression of transgenes in specific cells. For example, the mDLX5/6 enhancer is active selectively in GABAergic neurons and rAAVs containing this enhancer have been used to transduce GABAergic neurons selectively. Whether this enhancer drives expression equally strongly ins all types of GABAergic interneurons is unclear[2].
To answer that question, we injected two monkeys with rAAV1 and rAAV9 vectors expressing Channelrhodopsin-mCherry under the control of the mDLX5/6 enhancer. To compare the results between rodents and primates, a rat was injected with a rAAV1 containing the mDLX5/6 enhancer driving the expression of ChrimsonR-tdTomato.

Materials and Methods:: The rat was injected with 1000 nl of AAV vector using a Hamilton syringe attached to an automated pump. Injections were made at a rate of 200 nl/min at three locations spaced 0.5 cm apart along a single track. Each injection was followed by a 2-minute waiting period. After a six-week period, the brain was extracted, and 30 mm-thick sections were subjected to immunohistochemical staining using PV/CR antibodies. Labelled neurons were imaged with a LEICA SP8X confocal microscope and counted using Imaris software. The two monkeys underwent similar procedures.


Results, Conclusions, and Discussions:: We counted the.number of PV+ and CR+ neurons that were transduced by the rAAVs. We found that a large fraction of of PV neurons were transduced with the rAAV vector. In contrast,hardly any CR neurons were transduced. These results were consistent in both rats and primates.

The difference in efficiency between PV and CR neurons may be related to their distinct developmental pathways, as their subtype identity is determined by their origin time and location. PV interneurons are derived from the ventral medial ganglionic eminence (MGE) region, while some CR interneurons are derived from the caudal ganglionic eminence (CGE)[3]. Previous studies have shown that the DLX5/6 enhancer plays a significant role in promoting the growth and differentiation of PV neurons in the CNS[4]. Perhaps the mDLX5/6 enhancer has greater activity in MGE-derived neurons that in CGE-derived neurons.

Another possible explanation for our result is that PV and CR-expressing neurons are differently susceptible to rAAV. This could be due to differences in specific receptors and cellular machinery required for rAAV uptake and transduction. The ability of rAAV vectors to penetrate different types of cells can also depend on several factors such as the viral capsid used, the route of administration, and the target cell type[2]. Further research is required to better comprehend the factors that influence rAAV transduction efficiency in different cell types.


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1. Medalla, M., Mo, B., Nasar, R., Zhou, Y., Park, J., & Luebke, J. I. (2023). Comparative Features of Calretinin, Calbindin and Parvalbumin Expressing Interneurons in Mouse and Monkey Primary Visual and Frontal Cortices. bioRxiv, 2023-02.


2. Duba-Kiss, R., Niibori, Y., & Hampson, D. R. (2021). GABAergic Gene Regulatory Elements Used in Adeno-Associated Viral Vectors. Frontiers in Neurology, 12, 745159.


3. Rudy, B., Fishell, G., Lee, S., & Hjerling‐Leffler, J. (2011). Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons. Developmental neurobiology, 71(1), 45-61.


4. Wang Y, Dye CA, Sohal V, Long JE, Estrada RC, Roztocil T, et al. Dlx5 and Dlx6 regulate the development of parvalbumin-expressing cortical interneurons. J Neurosci. (2010) 30:5334–45. doi: 10.1523/JNEUROSCI.5963-09.2010