Chemogenetic activation of excitatory neurons alters hippocampal neurotransmission in a dose-dependent manner

Abstract

Equal contributing corresponding authors Author contributions: SP, JCL, and VV designed the experiments. SP carried out slice physiology experiments. SS performed immunofluorescence staining to detect HA-tag. SS, and AB performed primary cortical neuron cultures. SP, MK and SM performed and analyzed ratiometric two-photon calcium imaging. SP and VV wrote the paper. Abstract Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic tools are extensively used to manipulate neuronal activity in a cell-type specific manner. Whole-cell patch-clamp recordings indicate membrane depolarization, coupled with increased neuronal firing rate, following administration of the DREADD ligand, Clozapine-N-Oxide (CNO) to activate the Gq-coupled DREADD, hM3Dq. Although hM3Dq has been used to enhance neuronal firing in order to manipulate diverse behaviors, often within thirty minutes to an hour post-CNO administration, the physiological effects on excitatory neurotransmission remain poorly understood. We investigated the influence of CNO-mediated hM3Dq DREADD activation on distinct aspects of hippocampal excitatory neurotransmission at the Schaffer collateral-CA1 synapse in hippocampal slices derived from mice expressing hM3Dq in Ca 2+/ calmodulin dependent protein kinase α (CamKIIα)-positive excitatory neurons. Our results indicate a clear dose-dependent effect on fEPSP slope, with no change noted at the lower dose of CNO (1 µM) and a significant, long-term decline in fEPSP slope observed at higher doses (5-20 µM). Further, we noted a robust theta burst stimulus (TBS) induced long-term potentiation (LTP) in the presence of the lower CNO (1 µM) dose, which was significantly attenuated at the higher CNO (20 µM) dose. Whole-cell patch clamp recording revealed both complex dose-dependent regulation of excitability, and spontaneous and evoked activity of CA1 pyramidal neurons in response to hM3Dq activation across CNO concentrations. Our data indicate that CNO-mediated activation of the hM3Dq DREADD results in dose-dependent regulation of excitatory hippocampal neurotransmission, and highlight the importance of careful interpretation of behavioral experiments involving chemogenetic manipulation. 5 administration. Taken together, our results suggest that hM3Dq activation in hippocampal CamKIIα-positive excitatory neurons produces clear dose-dependent effects on neurotransmission, highlighting the importance for careful interpretation of behavioral studies using chemogenetic activation. 9 Materials and Methods Animals CamKIIα-tTA transgenic mice were received as a gift from Dr. Christopher Pittenger, Department of Psychiatry (Mayford et al., 1996), Yale School of Medicine. TetO-hM3Dq mice (Cat. No. 014093; Tg(tetO-CHRM3*)1Blr/J) were purchased from Jackson Laboratories, USA. CamKIIα-tTA and TetO-hM3Dq bigenic mice were bred, and genotypes of double positive animals were confirmed using PCR based analysis. The background strain C57Bl6/J was used for control experiments. Male mice were used for all experiments....

A neural substrate of compulsive alcohol use

“…CNO (4936, TOCRIS) was dissolved in artificial cerebrospinal fluid (3525, TOCRIS) as previously described (69,70) at a concentration of 1 mM. Rats received 0.3 l per side through the 33-gauge injectors (2 mm below the cannula placement) at a rate of 0.15 l/min, 15 min before the punishment session.…”

Stress undermines reward-guided cognitive performance through synaptic depression in the lateral habenula

“…Is the stress-driven weakening of habenular excitatory transmission required for the increased error incidence in the T-maze task? Initially, we aimed to emulate the stress-driven synaptic adaptation by capitalizing on the concept of homeostatic downscaling, whereby prolonged neuronal hyperactivity reduces the strength of synaptic transmission ( Pan-Vazquez et al., 2020 ; Pati et al., 2019 ; Turrigiano, 2008 ). We injected an hM3Dq-encoding AAV (rAAV 8.2 -hSyn1-hM3Dq-mCherry) into the LHb, allowing neuronal depolarization after exposure to the specific ligand clozapine N-oxide (CNO) ( Figures S4 A and S4B).…”

“…Reportadaptation by capitalizing on the concept of homeostatic downscaling, whereby prolonged neuronal hyperactivity reduces the strength of synaptic transmission(Pan-Vazquez et al, 2020;Pati et al, 2019;Turrigiano, 2008).…”