Optogenetic Interrogation of Electrophysiological Dendritic Properties and Their Effect on Pacemaking Neurons from Acute Rodent Brain Slices.

Understanding dendritic excitability is essential for a complete and precise characterization of neurons' input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology. Key features • A method for studying the effects of electrotonic and nonlinear dendritic properties on the sub- and suprathreshold responses of pacemaking neurons. • Combines somatic patch clamp or perforated patch recordings with optogenetic activation in acute brain slices to investigate dendritic linear transformation without patching the dendrite. • Oscillatory illumination at various frequencies estimates spectral properties of the dendrite using subthreshold voltage-clamp recordings and studies entrainment of pacemakers in current clamp recordings. • This protocol uses Poisson white noise illumination to estimate dendritic phase response curves and peri-stimulus time histograms.

Striatal Neurons Are Recruited Dynamically into Collective Representations of Self-Initiated and Learned Actions in Freely Moving Mice.

Striatal spiny projection neurons are hyperpolarized-at-rest (HaR) and driven to action potential threshold by a small number of powerful inputs-an input-output configuration that is detrimental to response reliability. Because the striatum is important for habitual behaviors and goal-directed learning, we conducted a microendoscopic imaging in freely moving mice that express a genetically encoded Ca2+ indicator sparsely in striatal HaR neurons to evaluate their response reliability during self-initiated movements and operant conditioning. The sparse expression was critical for longitudinal studies of response reliability, and for studying correlations among HaR neurons while minimizing spurious correlations arising from contamination by the background signal. We found that HaR neurons are recruited dynamically into action representation, with distinct neuronal subsets being engaged in a moment-by-moment fashion. While individual neurons respond with little reliability, the population response remained stable across days. Moreover, we found evidence for the temporal coupling between neuronal subsets during conditioned (but not innate) behaviors.

Acetylcholine waves and dopamine release in the striatum.

Striatal dopamine encodes reward, with recent work showing that dopamine release occurs in spatiotemporal waves. However, the mechanism of dopamine waves is unknown. Here we report that acetylcholine release in mouse striatum also exhibits wave activity, and that the spatial scale of striatal dopamine release is extended by nicotinic acetylcholine receptors. Based on these findings, and on our demonstration that single cholinergic interneurons can induce dopamine release, we hypothesized that the local reciprocal interaction between cholinergic interneurons and dopamine axons suffices to drive endogenous traveling waves. We show that the morphological and physiological properties of cholinergic interneuron - dopamine axon interactions can be modeled as a reaction-diffusion system that gives rise to traveling waves. Analytically-tractable versions of the model show that the structure and the nature of propagation of acetylcholine and dopamine traveling waves depend on their coupling, and that traveling waves can give rise to empirically observed correlations between these signals. Thus, our study provides evidence for striatal acetylcholine waves in vivo, and proposes a testable theoretical framework that predicts that the observed dopamine and acetylcholine waves are strongly coupled phenomena.