Intrinsic burst-firing in lamina I spinoparabrachial neurons during adolescence.

A subset of glutamatergic interneurons in the neonatal spinal superficial dorsal horn (SDH) exhibits intrinsic burst-firing (i.e. 'pacemaker' activity), which is tightly regulated by persistent, voltage-gated Na+ channels and classic inward-rectifying K+ (Kir2) channels and downregulated over the course of postnatal development. Ascending lamina I projection neurons targeting the parabrachial nucleus (PB) or periaqueductal gray (PAG) can also display pacemaker activity during early life. However, the degree to which the ionic mechanisms driving pacemaker activity are conserved across different cell types in the spinal dorsal horn, as well as whether the intrinsic bursting is restricted to newborn projection neurons, remains to be elucidated. Using in vitro patch clamp recordings from identified lamina I spinoparabrachial neurons in rat spinal cord slices, here we demonstrate that adolescent projection neurons retain their ability to generate pacemaker activity. In contrast to previous findings in lamina I interneurons, pacemaker projection neurons possessed higher membrane capacitance, lower membrane resistance, and a greater Kir-mediated conductance compared to adjacent spinoparabrachial neurons that lacked intrinsic burst-firing. Nonetheless, as previously seen in interneurons, the bath application of riluzole to block persistent Na+ channels significantly dampened pacemaker activity in projection neurons. Collectively, these results suggest that intrinsic burst-firing in the developing dorsal horn can be generated by multiple combinations of ionic conductances, and highlight the need for further investigation into the mechanisms governing pacemaker activity within the major output neurons of the SDH network.

The development of pain circuits and unique effects of neonatal injury.

Pain is a necessary sensation that prevents further tissue damage, but can be debilitating and detrimental in daily life under chronic conditions. Neuronal activity strongly regulates the maturation of the somatosensory system, and aberrant sensory input caused by injury or inflammation during critical periods of early postnatal development can have prolonged, detrimental effects on pain processing. This review will outline the maturation of neuronal circuits responsible for the transmission of nociceptive signals and the generation of pain sensation-involving peripheral sensory neurons, the spinal cord dorsal horn, and brain-in addition to the influences of the neuroimmune system on somatosensation. This summary will also highlight the unique effects of neonatal tissue injury on the maturation of these systems and subsequent consequences for adult somatosensation. Ultimately, this review emphasizes the need to account for age as an independent variable in basic and clinical pain research, and importantly, to consider the distinct qualities of the pediatric population when designing novel strategies for pain management.

Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse.

Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch. Although it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory "microcircuits" within the region. As a result, the goal of this study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique, followed by RNA-seq analysis. Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared with non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1). In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH. Differential gene expression analysis comparing pDyn nuclei across the 3 ages revealed sets of genes that were significantly upregulated (such as Cartpt, encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1, encoding the receptor for neuropeptides B/W) during postnatal development. Collectively, these results provide new insight into the potential molecular mechanisms underlying the known age-dependent changes in spinal nociceptive processing and pain sensitivity.

Somatotopic organization of central arbors from nociceptive afferents develops independently of their intact peripheral target innervation.

Functionally important regions of sensory maps are overrepresented in the sensory pathways and cortex, but the underlying developmental mechanisms are not clear. In the spinal cord dorsal horn (DH), we recently showed that paw innervating Mrgprd+ nonpeptidergic nociceptors display distinctive central arbor morphologies that well correlate with increased synapse transmission efficiency and heightened sensitivity of distal limb skin. Given that peripheral and central arbor formation of Mrgprd+ neurons co-occurs around the time of birth, we tested whether peripheral cues from different skin areas and/or postnatal reorganization mechanisms could instruct this somatotopic difference among central arbors. We found that, while terminal outgrowth/refinement occurs during early postnatal development in both the skin and the DH, postnatal refinement of central terminals precedes that of peripheral terminals. Furthermore, we used single-cell ablation of Ret to genetically disrupt epidermal innervation of Mrgprd+ neurons and revealed that the somatotopic difference among their central arbors was unaffected by this manipulation. Finally, we saw that region-specific Mrgprd+ central terminal arbors are present from the earliest postnatal stages, before skin terminals are evident. In summary, we find that region-specific organization of Mrgprd+ neuron central arbors is present shortly after initial central terminal formation, which likely develops independently of peripheral target innervation. Our data suggest that either cell-intrinsic and/or DH prepatterning mechanisms are likely to establish this somatotopic difference.

Lamina specific postnatal development of PKCγ interneurons within the rat medullary dorsal horn.

Protein kinase C gamma (PKCγ) interneurons, located in the superficial spinal (SDH) and medullary dorsal horns (MDH), have been shown to play a critical role in cutaneous mechanical hypersensitivity. However, a thorough characterization of their development in the MDH is lacking. Here, it is shown that the number of PKCγ-ir interneurons changes from postnatal day 3 (P3) to P60 (adult) and such developmental changes differ according to laminae. PKCγ-ir interneurons are already present at P3-5 in laminae I, IIo, and III. In lamina III, they then decrease from P11-P15 to P60. Interestingly, PKCγ-ir interneurons appear only at P6 in lamina IIi, and they conversely increase to reach adult levels at P11-15. Analysis of neurogenesis using bromodeoxyuridine (BrdU) does not detect any PKCγ-BrdU double-labeling in lamina IIi. Quantification of the neuronal marker, NeuN, reveals a sharp neuronal decline (∼50%) within all superficial MDH laminae during early development (P3-15), suggesting that developmental changes in PKCγ-ir interneurons are independent from those of other neurons. Finally, neonatal capsaicin treatment, which produces a permanent loss of most unmyelinated afferent fibers, has no effect on the development of PKCγ-ir interneurons. Together, the results show that: (i) the expression of PKCγ-ir interneurons in MDH is developmentally regulated with a critical period at P11-P15, (ii) PKCγ-ir interneurons are developmentally heterogeneous, (iii) lamina IIi PKCγ-ir interneurons appear less vulnerable to cell death, and (iv) postnatal maturation of PKCγ-ir interneurons is due to neither neurogenesis, nor neuronal migration, and is independent of C-fiber development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 102-119, 2017.

Development of putative inhibitory neurons in the embryonic and postnatal mouse superficial spinal dorsal horn.

The superficial spinal dorsal horn is the first relay station of pain processing. It is also widely accepted that spinal synaptic processing to control the modality and intensity of pain signals transmitted to higher brain centers is primarily defined by inhibitory neurons in the superficial spinal dorsal horn. Earlier studies suggest that the construction of pain processing spinal neural circuits including the GABAergic components should be completed by birth, although major chemical refinements may occur postnatally. Because of their utmost importance in pain processing, we intended to provide a detailed knowledge concerning the development of GABAergic neurons in the superficial spinal dorsal horn, which is now missing from the literature. Thus, we studied the developmental changes in the distribution of neurons expressing GABAergic markers like Pax2, GAD65 and GAD67 in the superficial spinal dorsal horn of wild type as well as GAD65-GFP and GAD67-GFP transgenic mice from embryonic day 11.5 (E11.5) till postnatal day 14 (P14). We found that GABAergic neurons populate the superficial spinal dorsal horn from the beginning of its delineation at E14.5. We also showed that the numbers of GABAergic neurons in the superficial spinal dorsal horn continuously increase till E17.5, but there is a prominent decline in their numbers during the first two postnatal weeks. Our results indicate that the developmental process leading to the delineation of the inhibitory and excitatory cellular assemblies of pain processing neural circuits in the superficial spinal dorsal horn of mice is not completed by birth, but it continues postnatally.

Inward-rectifying K+ (Kir2) leak conductance dampens the excitability of lamina I projection neurons in the neonatal rat.

Spinal lamina I projection neurons serve as a major conduit by which noxious stimuli detected in the periphery are transmitted to nociceptive circuits in the brain, including the parabrachial nucleus (PB) and the periaqueductal gray (PAG). While neonatal spino-PB neurons are more than twice as likely to exhibit spontaneous activity compared to spino-PAG neurons, the underlying mechanisms remain unclear since nothing is known about the voltage-independent (i.e. 'leak') ion channels expressed by these distinct populations during early life. To begin identifying these key leak conductances, the present study investigated the role of classical inward-rectifying K+ (Kir2) channels in the regulation of intrinsic excitability in neonatal rat spino-PB and spino-PAG neurons. The data demonstrate that a reduction in Kir2-mediated conductance by external BaCl2 significantly enhanced intrinsic membrane excitability in both groups. Similar results were observed in spino-PB neurons following Kir2 channel block with the selective antagonist ML133. In addition, voltage-clamp experiments showed that spino-PB and spino-PAG neurons express similar amounts of Kir2 current during the early postnatal period, suggesting that the differences in the prevalence of spontaneous activity between the two populations are not explained by differential expression of Kir2 channels. Overall, the results indicate that Kir2-mediated conductance tonically dampens the firing of multiple subpopulations of lamina I projection neurons during early life. Therefore, Kir2 channels are positioned to tightly shape the output of the immature spinal nociceptive circuit and thus regulate the ascending flow of nociceptive information to the developing brain, which has important functional implications for pediatric pain.

Maintenance of glia in the optic lamina is mediated by EGFR signaling by photoreceptors in adult Drosophila.

The late onset of neurodegeneration in humans indicates that the survival and function of cells in the nervous system must be maintained throughout adulthood. In the optic lamina of the adult Drosophila, the photoreceptor axons are surrounded by multiple types of glia. We demonstrated that the adult photoreceptors actively contribute to glia maintenance in their target field within the optic lamina. This effect is dependent on the epidermal growth factor receptor (EGFR) ligands produced by the R1-6 photoreceptors and transported to the optic lamina to act on EGFR in the lamina glia. EGFR signaling is necessary and sufficient to act in a cell-autonomous manner in the lamina glia. Our results suggest that EGFR signaling is required for the trafficking of the autophagosome/endosome to the lysosome. The loss of EGFR signaling results in cell degeneration most likely because of the accumulation of autophagosomes. Our findings provide in vivo evidence for the role of adult neurons in the maintenance of glia and a novel role for EGFR signaling in the autophagic flux.

Hoxb8 intersection defines a role for Lmx1b in excitatory dorsal horn neuron development, spinofugal connectivity, and nociception.

Spinal cord neurons respond to peripheral noxious stimuli and relay this information to higher brain centers, but the molecules controlling the assembly of such pathways are poorly known. In this study, we use the intersection of Lmx1b and Hoxb8::Cre expression in the spinal cord to genetically define nociceptive circuits. Specifically, we show that Lmx1b, previously shown to be expressed in glutamatergic dorsal horn neurons and critical for dorsal horn development, is expressed in nociceptive dorsal horn neurons and that its deletion results in the specific loss of excitatory dorsal horn neurons by apoptosis, without any effect on inhibitory neuron numbers. To assess the behavioral consequences of Lmx1b deletion in the spinal cord, we used the brain-sparing driver Hoxb8::Cre. We show that such a deletion of Lmxb1 leads to a robust reduction in sensitivity to mechanical and thermal noxious stimulation. Furthermore, such conditional mutant mice show a loss of a subpopulation of glutamatergic dorsal horn neurons, abnormal sensory afferent innervations, and reduced spinofugal innervation of the parabrachial nucleus and the periaqueductal gray, important nociceptive structures. Together, our results demonstrate an important role for the intersection of Lmx1b and Hoxb8::cre expression in the development of nociceptive dorsal horn circuits critical for mechanical and thermal pain processing.

Neuropathic pain is constitutively suppressed in early life by anti-inflammatory neuroimmune regulation.

Peripheral nerve injury can trigger neuropathic pain in adults but not in infants; indeed, for unknown reasons, neuropathic pain is rare before adolescence. We show here that the absence of neuropathic pain response in infant male rats and mice following nerve injury is due to an active, constitutive immune suppression of dorsal horn pain activity. In contrast to adult nerve injury, which triggers a proinflammatory immune response in the spinal dorsal horn, infant nerve injury triggers an anti-inflammatory immune response, characterized by significant increases in IL-4 and IL-10. This immediate anti-inflammatory response can also be evoked by direct C-fiber nerve stimulation in infant, but not adult, mice. Blockade of the anti-inflammatory activity with intrathecal anti-IL10 unmasks neuropathic pain behavior in infant nerve injured mice, showing that pain hypersensitivity in young mice is actively suppressed by a dominant anti-inflammatory neuroimmune response. As infant nerve injured mice reach adolescence (postnatal day 25-30), the dorsal horn immune profile switches from an anti-inflammatory to a proinflammatory response characterized by significant increases in TNF and BDNF, and this is accompanied by a late onset neuropathic pain behavior and increased dorsal horn cell sensitivity to cutaneous mechanical and cold stimuli. These findings show that neuropathic pain following early life nerve injury is not absent but suppressed by neuroimmune activity and that "latent" pain can still emerge at adolescence, when the neuroimmune profile changes. The data may explain why neuropathic pain is rare in young children and also why it can emerge, for no observable reason, in adolescent patients.

Plasma glucocorticoids differentially modulate phasic and tonic GABA inhibition during early postnatal development in rat spinal lamina II.

Nociceptive processing is tuned by GABAA receptor-mediated inhibition in the spinal cord dorsal horn that undergoes postnatal maturation in rodents. These GABAergic inhibitory postsynaptic currents (IPSCs) are modulated by 3α5α-reduced steroids during early postnatal development in spinal cord lamina II. Thus an enhanced phasic inhibition is present in neonates and decreases over time. GABA can also activate extrasynaptic receptors, giving rise to tonic inhibition. In this study, we characterized the contribution of plasma corticosterone (CORT) to postnatal maturation of spinal phasic and, for the first time, tonic GABAergic inhibitions. We used Fisher and Lewis rat strains displaying respectively high and low hypothalamic-pituitary-adrenal axis reactivity, compared to control Sprague-Dawley rats. Measured plasma CORT levels were significantly higher in Fisher rats, which also displayed significantly higher mechanical nociceptive thresholds, supporting the hypothesis of an antinociceptive action of CORT. Recorded GABAA IPSCs shortened during maturation in all strains while remaining larger in Fisher rats. Blocking the 5α-reduction of steroids in Fisher rats produced a further decrease of IPSC deactivation time constant. In contrast, GABAA tonic inhibition progressively increased during maturation, without any difference among strains. In conclusion, we show that both phasic and tonic GABAergic inhibitions undergo postnatal maturation in lamina II. Moreover spinal production of 3α5α-reduced steroids that presumably derive from plasma CORT is correlated to spinal GABAA phasic (but not tonic) inhibition and to mechanical nociceptive thresholds.