Abstract
Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.
Keywords: Channelrhodopsin, Dopamine, Cotransmission, Patch-clamp, Immunofluorescence, Adeno associated virus
Background
To establish the function of specific neural circuits it is necessary to determine the anatomical connectome, the mapping of anatomical connections, and its functional connectome, the mapping of the strength, incidence and neurotransmitter nature of connections. The use of viral transsynaptic tracing techniques that are monosynaptically restricted, allows for the description of complex anatomical connections of neural circuits, including the dopamine system (Callaway and Luo, 2015; Faget et al., 2016). The functional connectivity of these circuits has been harder to determine due to the intermingling of axons that make selective electrical stimulation impossible. With the advent of optogenetics it became possible to stimulate genetically defined populations of cells selectively. This allowed for the identification of new connections made by striatal medium spiny neurons (Chuhma et al., 2011), ventral midbrain glutamate neurons (Hnasko et al., 2012; Root et al., 2014) and dopamine/glutamate neurons (Mingote et al., 2015). Moreover, optogenetics used in a systematic and comprehensive manner to map the incidence and strength of connections of specific neuronal populations to defined postsynaptic target regions, determines the functional connectome of defined neuronal populations (Chuhma et al., 2011; Mingote et al., 2015). In this protocol, we describe how to determine the functional connectome of any genetically defined neuronal population. As an example, we focus on the excitatory functional connectome of dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015).
Materials and Reagents
Equipment
Procedure
Viral transfection delivers multiple copies of ChR2 and induces strong expression. However, ChR2 expression varies among animals so the specificity and efficacy of the transfection needs to be determined and taken into account when determining a functional connectome. Visualization of immunostaining of brains sections for the EYFP tag enables tracing dopaminergic projections, assessing their dopaminergic status and by guiding recordings provides a comprehensive basis for systematic recordings.
Software
Data analysis
Notes
Recipes
Acknowledgments
This protocol was used to obtain the data published in the Journal of Neuroscience (Mingote, S., Chuhma, N., Kusnoor, S. V., Field, B., Deutch, A. Y. and Rayport, S. [2015]). Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J Neurosci 35[49]: 16259-16271). Procedures involving mice were conducted in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the Institutional Animal Care and Use Committees of Columbia University and New York State Psychiatric Institute. The work was supported by a NARSAD Young Investigator award (SM), DA017978 and MH087758 (SR).
References
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