Probes for INS

In response to cortical stroke and unilateral corticospinal tract degeneration, compensatory sprouting of spared corticospinal fibers is associated with recovery of skilled movement in rodents. To date, little is known about the molecular mechanisms orchestrating this spontaneous rewiring. In this study, we provide insights into the molecular changes in the spinal cord tissue after large ischemic cortical injury in adult female mice, with a focus on factors that might influence the re-innervation process by contralesional corticospinal neurons. We mapped the area of cervical grey matter re-innervation by sprouting contralesional corticospinal axons after unilateral photothrombotic stroke of the motor cortex in mice using anterograde tracing. The mRNA profile of this re-innervation area was analyzed using whole-genome sequencing to identify differentially expressed genes at selected time points during the recovery process. Bioinformatic analysis revealed two phases of processes: Early after stroke (4-7 days post injury), the spinal transcriptome is characterized by inflammatory processes, including phagocytic processes as well as complement cascade activation. Microglia are specifically activated in the denervated corticospinal projection fields in this early phase. In a later phase (28-42 days post injury), biological processes include tissue repair pathways with up-regulated genes related to neurite outgrowth. Thus, the stroke-denervated spinal grey matter, in particular its intermediate laminae, represents a growth-promoting environment for sprouting corticospinal fibers originating from the contralesional motor cortex. This data set provides a solid starting point for future studies addressing key elements of the post-stroke recovery process, with the goal to improve neuroregenerative treatment options for stroke patients.SIGNIFICANCE STATEMENTWe show that the molecular changes in the spinal cord target tissue of the stroke-affected corticospinal tract are mainly defined by two phases: an early inflammatory phase during which microglia are specifically activated in the target area of re-innervating corticospinal motor neurons; and a late phase during which growth-promoting factors are upregulated which can influence the sprouting response, arborization and synapse formation. By defining for the first time the endogenous molecular machinery in the stroke-denervated cervical spinal grey matter with a focus on promotors of axon growth through the growth-inhibitory adult CNS, this study will serve as a basis to address novel neuroregenerative treatment options for chronic stroke patients.

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system.

The use of human tissue to validate putative analgesic targets identified in rodents is a promising strategy for improving the historically poor translational record of preclinical pain research. We recently demonstrated that in mouse and human sensory neurons, agonists for metabotropic glutamate receptors 2 and 3 (mGluR2/3) reduce membrane hyperexcitability produced by the inflammatory mediator prostaglandin E2 (PGE2). Previous rodent studies indicate that mGluR2/3 can also reduce peripheral sensitization by suppressing inflammation-induced sensitization of TRPV1. Whether this observation similarly translates to human sensory neurons has not yet been tested. We found that activation of mGluR2/3 with the agonist APDC suppressed PGE2-induced sensitization of TRPV1 in mouse, but not human sensory neurons. We also evaluated sensory neuron expression of the gene transcripts for mGluR2 (Grm2), mGluR3 (Grm3), and TRPV1 (Trpv1). The majority of Trpv1+ mouse and human sensory neurons expressed Grm2 and/or Grm3, and in both mouse and human, Grm2 was expressed in a greater percentage of sensory neurons than Grm3. Although we demonstrated a functional difference in the modulation of TRPV1 sensitization by mGluR2/3 activation between mouse and human, there were no species differences in the gene transcript colocalization of mGluR2 or mGluR3 with TRPV1 that might explain this functional difference. Taken together with our previous work, these results suggest that mGluR2/3 activation suppresses only some aspects of human sensory neuron sensitization caused by PGE2. These differences have implications for potential healthy human voluntary studies or clinical trials evaluating the analgesic efficacy of mGluR2/3 agonists or positive allosteric modulators.