Previously we defined neuronal subclasses within the mouse peripheral nervous system using an experimental strategy called "constellation pharmacology." Here we demonstrate the broad applicability of constellation pharmacology by extending it to the CNS and specifically to the ventral respiratory column (VRC) of mouse brainstem, a region containing the neuronal network controlling respiratory rhythm. Analysis of dissociated cells from this locus revealed three major cell classes, each encompassing multiple subclasses. We broadly analyzed the combinations (constellations) of receptors and ion channels expressed within VRC cell classes and subclasses. These were strikingly different from the constellations of receptors and ion channels found in subclasses of peripheral neurons from mouse dorsal root ganglia. Within the VRC cell population, a subset of dissociated neurons responded to substance P, putatively corresponding to inspiratory pre-Bötzinger complex (preBötC) neurons. Using constellation pharmacology, we found that these substance P-responsive neurons also responded to histamine, and about half responded to bradykinin. Electrophysiological studies conducted in brainstem slices confirmed that preBötC neurons responsive to substance P exhibited similar responsiveness to bradykinin and histamine. The results demonstrate the predictive utility of constellation pharmacology for defining modulatory inputs into specific neuronal subclasses within central neuronal networks.calcium imaging | NK1 receptor | glutamate | acetylcholine | conotoxin P rogress in understanding the mammalian brain has been impeded by the extraordinary complexity of cell types comprising the circuitry and the difficulty in bridging different levels of biological organization from the molecular to the cellular and systems level (1-4). Systems neuroscientists study the circuitry and high-level functions of the brain, whereas molecular neuroscientists study the molecular components. The large divide between these two branches of neuroscience clearly needs to be bridged to understand fully neuronal and behavioral functions in health and disease. To this end, we recently demonstrated an experimental approach we call "constellation pharmacology" to identify different neuronal subclasses by the combinations (constellations) of receptors and ion channels functionally expressed in each subclass (5-8). This experimental approach initially was applied to somatosensory neurons of the peripheral nervous system (PNS). In the present study, we use constellation pharmacology to identify neuronal subclasses of the CNS and to characterize these subclasses at the network level. Specifically, we have used constellation pharmacology to define the diverse cell types found in the mouse ventral respiratory column (VRC) and surrounding brainstem tissue.The VRC contains a variety of neurons that are active during either inspiratory or expiratory phases of breathing. One key network within the VRC is the pre-Bötzinger complex (preBötC), which contains the circuitry e...

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.

In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17–34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.

The ventral respiratory column (VRC) is a region in the brainstem shown to control breathing patterns in mammals. Using a mouse model, cells from this region are dissociated, plated and incubated with a dye that indicates changes in cytoplasmic calcium levels. Hundreds of cells are monitored as a diverse set of pharmacological agents are applied. The spectrum of cellular responses provides a profile of the receptors found on each of these neurons. Previously, varieties of cell classes were shown to contain NMDA receptors. However, specific compositions of subunits within these receptors are not known. These receptors are ligand gated ion channels composed of four non‐covalently bound protein subunits. Each subunit has a different response profile determined by interactions of agonist and antagonists. Conantokins (peptides isolated from snail venom) and other compounds afford understanding which NMDA receptor subtypes are present in different cell‐classes. This project has continued to classify the subunit compositions of NMDA receptors with the ultimate goal of defining which NMDA receptor subunits are present in each class. The results indicate that different neuronal subclasses express different NMDA response subtypes. This will provide valuable information on the individual neurons in the networks of the VRC, and will allow for pharmacological interventions to change behavior in this region. Grant Funding Source: Supported by GM48677

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