Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays.

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from naïve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

Transgenic Cross-Referencing of Inhibitory and Excitatory Interneuron Populations to Dissect Neuronal Heterogeneity in the Dorsal Horn.

The superficial dorsal horn (SDH, LI-II) of the spinal cord receives and processes multimodal sensory information from skin, muscle, joints, and viscera then relay it to the brain. Neurons within the SDH fall into two broad categories, projection neurons and interneurons. The later can be further subdivided into excitatory and inhibitory types. Traditionally, interneurons within the SDH have been divided into overlapping groups according to their neurochemical, morphological and electrophysiological properties. Recent clustering analyses, based on molecular transcript profiles of cells and nuclei, have predicted many more functional groups of interneurons than expected using traditional approaches. In this study, we used electrophysiological and morphological data obtained from genetically-identified excitatory (vGLUT2) and inhibitory (vGAT) interneurons in transgenic mice to cluster cells into groups sharing common characteristics and subsequently determined how many clusters can be assigned by combinations of these properties. Consistent with previous reports, we show differences exist between excitatory and inhibitory interneurons in terms of their excitability, nature of the ongoing excitatory drive, action potential (AP) properties, sub-threshold current kinetics, and morphology. The resulting clusters based on statistical and unbiased assortment of these data fell well short of the numbers of molecularly predicted clusters. There was no clear characteristic that in isolation defined a population, rather multiple variables were needed to predict cluster membership. Importantly though, our analysis highlighted the appropriateness of using transgenic lines as tools to functionally subdivide both excitatory and inhibitory interneuron populations.

Functional and Molecular Analysis of Proprioceptive Sensory Neuron Excitability in Mice.

Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP-) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (Ih) was observed in all GFP+ neurons but was infrequently noted in the GFP- population (100% vs. 11%). For GFP+ neurons, Ih activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP- neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP- cells differ markedly, providing evidence for modality-specific signaling between the two groups. However, the GFP+ DRG population demonstrates considerable internal heterogeneity when hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) properties and subunit expression are considered. We propose this heterogeneity reflects the existence of different peripheral receptors such as tendon organs, muscle spindles or mechanoreceptors in the putative proprioceptive neuron population.

The search for novel analgesics: re-examining spinal cord circuits with new tools.

In this perspective, we propose the absence of detailed information regarding spinal cord circuits that process sensory information remains a major barrier to advancing analgesia. We highlight recent advances showing that functionally discrete populations of neurons in the spinal cord dorsal horn (DH) play distinct roles in processing sensory information. We then discuss new molecular, electrophysiological, and optogenetic techniques that can be employed to understand how DH circuits process tactile and nociceptive information. We believe this information can drive the development of entirely new classes of pharmacotherapies that target key elements in spinal circuits to selectively modify sensory function and blunt pain.