Date Published: May 24, 2019
Publisher: Public Library of Science
Author(s): Aarti Nagayach, Anshuman Singh, Angel L. De Blas, Alfred I. Geller, Jian Jing.
Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses.
Neocortical GABAergic inhibitory interneurons play critical roles in synaptic plasticity, circuit physiology, and learning. Moreover, a number of neurological disorders are associated with defects in GABAergic transmission in the neocortex, including schizophrenia, autism, and other intellectual disabilities . Of note, advanced cognitive tasks are encoded in distributed forebrain circuits that span multiple neocortical areas. Within a neocortical area, complex local circuits support information processing, and neurons are interconnected into functional columns [2, 3]. These local circuits contain tens to hundreds or thousands of different neuron types, and each type forms precise synaptic connections with other neuron types . GABAergic neurons comprise ten to thirty percent of the neurons in a specific neocortical area, and GABAergic synapses represent only five to fifteen percent of neocortical synapses [5–7]. Thus, understanding local circuit information processing is challenging due to the size and complexity of these circuits. Genetic strategies for analyzing circuit physiology have tremendous potential for studying this problem, however, these strategies usually affect an entire circuit, or a large part of a circuit [8–11]. Thus, understanding local circuit physiology and information processing would benefit from a gene transfer technology that can selectively deliver different genes into the pre- and post-synaptic neurons that are located in specific neocortical layers, and connected by GABAergic synapses.
We have developed a technology to selectively deliver different genes into pre- or post-synaptic local interneurons that are connected by GABAergic synapses. This synapse type selectivity is conferred by a synthetic peptide neurotransmitter that is expressed in the presynaptic neurons, and has three domains: Processing and release of the recombinant protein as a peptide neurotransmitter is supported by the N-terminal, DCV sorting domain; the middle domain binds to GABAA receptors on the postsynaptic neurons; and a His tag is the C-terminal domain. Upon release, this synthetic peptide neurotransmitter binds to postsynaptic neurons that contain GABAA receptors. The second gene transfer is selective for these neurons, and uses antibody-mediated, targeted gene transfer and anti-His tag antibodies.