Primary afferents transduce environmental stimuli into electrical activity that is transmitted centrally to be decoded into corresponding sensations. However, it remains unknown how afferent populations encode different somatosensory inputs. To address this, we performed two-photon Ca imaging from thousands of dorsal root ganglion (DRG) neurons in anesthetized mice while applying mechanical and thermal stimuli to hind paws. We found that approximately half of all neurons are polymodal and that heat and cold are encoded very differently. As temperature increases, more heating-sensitive neurons are activated, and most individual neurons respond more strongly, consistent with graded coding at population and single-neuron levels, respectively. In contrast, most cooling-sensitive neurons respond in an ungraded fashion, inconsistent with graded coding and suggesting combinatorial coding, based on which neurons are co-activated. Although individual neurons may respond to multiple stimuli, our results show that different stimuli activate distinct combinations of diversely tuned neurons, enabling rich population-level coding.

Whereas both GABA A receptors (GABA A Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA A R-subunits, GlyR ␣1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP ϩ and IB4 ϩ terminals were devoid of GlyR ␣1-subunit and gephyrin. The ␣1 GABA A R was also absent from these terminals. In contrast, the GABA A R ␣2/␣3/␣5 and ␤3 subunits were significantly expressed in both terminal types, as were other GABA A R-associated-proteins (␣-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA A R ␤3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynapticsites(nociceptiveafferentterminalsvsinhibitorysynapsesondorsalhornneurons).Theabsenceofgephyrinclustersfromprimary afferent terminals suggests a more diffuse mode of GABA A -mediated transmission at presynaptic than at postsynaptic sites.

BackgroundOptogenetic tools enable cell selective and temporally precise control of neuronal activity; yet, difficulties in delivering sufficient light to the spinal cord of freely behaving animals have hampered the use of spinal optogenetic approaches to produce analgesia. We describe an epidural optic fiber designed for chronic spinal optogenetics that enables the precise delivery of light at multiple wavelengths to the spinal cord dorsal horn and sensory afferents.ResultsThe epidural delivery of light enabled the optogenetic modulation of nociceptive processes at the spinal level. The acute and repeated activation of channelrhodopsin-2 expressing nociceptive afferents produced robust nocifensive behavior and mechanical sensitization in freely behaving mice, respectively. The optogenetic inhibition of GABAergic interneurons in the spinal cord dorsal horn through the activation of archaerhodopsin also produced a transient, but selective induction of mechanical hypersensitivity. Finally, we demonstrate the capacity of optogenetics to produce analgesia in freely behaving mice through the inhibition of nociceptive afferents via archaerhodopsin.ConclusionEpidural optogenetics provides a robust and powerful solution for activation of both excitatory and inhibitory opsins in sensory processing pathways. Our results demonstrate the potential of spinal optogenetics to modulate sensory behavior and produce analgesia in freely behaving animals.

GABA A /glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl −) concentration which is mainly regulated by the K +-Cl − co-transporter 2 (KCC2) in the adult central nervous system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl − extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl − load. Under high Cl − load or heightened synaptic drive, lower Cl − extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl − lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl − extrusion across laminae critically shapes plasticity for selective nociceptive modalities.

Hypoxia-inducible factor 1alpha plays a key role in the pathogenesis of AP, and the ability to down-regulate the expression of HIF-1alpha may partially explain the therapeutic effect of HBO on AP.