Classifying neuronal subclasses of the cerebellum through constellation pharmacology

Abstract

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain.

An integrative approach to the facile functional classification of dorsal root ganglion neuronal subclasses

“…Constellation Pharmacology. Experiments were performed as described previously (1)(2)(3)(4)(5)(6)22) and detailed in SI Appendix, Materials and Methods. Briefly, lumbar DRG neurons from CGRP-GFP mice (in a CD1 genetic background) from ages postnatal (P)41 to P89 were dissociated by treating DRGs with trypsin followed by mechanical trituration and plated on polylysine-coated coverslips.…”

“…As key mediators of inter-and intracellular signaling events in the nervous system, ion channels and G protein-coupled receptor (GPCR) are useful for classifying neuronal cell types. We previously developed constellation pharmacology as a calcium imaging-based method using highly selective pharmacological agents to identify combinations of ion-channel and GPCR subtypes in specific neuronal classes (1)(2)(3)(4)(5)(6). Small-diameter DRG neurons often differ in their functional expression of a variety of transient receptor potential (TRP) channels (e.g., TRPV1, TRPA1, and TRPM8).…”

RIP2 Gates TRAF6 Interaction with Death Receptor p75NTR to Regulate Cerebellar Granule Neuron Survival

“…CGNs outnumber all other brain neurons combined and are thought to encompass heterogeneous subpopulations (Curtice et al, 2016). Recent findings have revealed their capacity of encoding complex aspects of reward and predictive information, in addition to classical sensorimotor encoding (Giovannucci et al, 2017;Wagner et al, 2017), beginning to provide an explanation for their large numbers.…”

Chronicling changes in the somatosensory neurons after peripheral nerve injury

“…The baseline calcium level for each cell trace was observed, and a response was assigned as positive calcium influx (value = 1), if the magnitude of calcium influx was higher than a threshold of 0.05 units (of 340/380 ratio) above the baseline. In addition, the time course of the 340:380 ratio for each regions of interest (ROI [trace]) was analyzed using a set of R functions (R Core Team 2013) for quantitative analysis of peak heights, clustering, and determination of cell-phenotype percentages as described previously (29).…”