Fractalkine and minocycline alter neuronal activity in the spinal cord dorsal horn

Fractalkine (FKN) evokes nociceptive behavior in nai ve rats, whereas minocycline attenuates pain acutely after neuronal injury. We show that, in nai ve rats, FKN causes hyperresponsiveness of lumbar wide dynamic range neurons to brush, pressure and pinch applied to the hindpaw. One day after spinal nerve ligation (SNL), minocycline attenuates after-discharge and responses to brush and pressure. In contrast, minocycline does not alter evoked neuronal responses 10 days after SNL or sciatic constriction, but increases spontaneous discharge. We speculate that microglia rapidly alter sensory neuronal activity in nai ve and neuropathic rats acutely, but not chronically, after injury.     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.     

Oxytocin prevents neuronal network pain-related changes on spinal cord dorsal horn in vitro

Recently, oxytocin (OT) has been studied as a potential modulator of endogenous analgesia by acting upon pain circuits at the spinal cord and supraspinal levels. Yet the detailed action mechanisms of OT are still undetermined. The present study aimed to evaluate the action of OT in the spinal cord dorsal horn network under nociceptive-like conditions induced by the activation of the N-methyl-d-aspartate (NMDA) receptor and formalin injection, using calcium imaging techniques. Results demonstrate that the spontaneous Ca²⁺-dependent activity of the dorsal horn cells was scarce, and the coactivity of cells was mainly absent. When NMDA was applied, high rates of activity and coactivity occurred in the dorsal horn cells; these rates of high activity mimicked the activity dynamics evoked by a neuropathic pain condition. In addition, although OT treatment increased activity rates, it was also capable of disrupting the conformation of coordinated activity previously consolidated by NMDA treatment, without showing any effect by itself. Altogether, our results suggest that OT globally prevents the formation of coordinated patterns previously generated by nociceptive-like conditions on dorsal horn cells by NMDA application, which supports previous evidence showing that OT represents a potential therapeutic alternative for the treatment of chronic neuropathic pain.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum

The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.     

Modulation of dorsal horn neuronal activity by spinal cord stimulation in a rat model of neuropathy: The role of the dorsal funicles

We recorded the spike activity from spinal neurons In rats with a model of neuropathy after ligation of then. ishiadicus. A significantly increased frequency of background discharges and responsiveness to nonnoxious stimuli were observed in dorsal horn wide-dynamic range (convergent) neurons in a group of allodynic rats, as compared with non-allodynic and intact rats. Spinal cord stimulation (SCS) Induced a significant depression of both the principal responses and afterdischarges in allodynic rats. The frequency of background discharges was markedly decreased in approximately one third of the neurons. These effects outlasted SCS by about 10 rain. The moderating effect of SCS is considered a result of activation of distinctly different and complementary mechanisms: segmental and transsupraspinal. The former appears to be the most important in allodynic animals.     

Normal anatomy and physiology of the spinal cord dorsal horn

The dorsal horn of the spinal cord receives afferent input from innocuous primary afferent neurons via collaterals from the dorsal columns. This input is integrated and relayed primarily by neurons in laminae III-VI. Dorsal horn neurons which encode innocuous inputs project to the medulla and the cervical spinal cord via the dorsal columns and the dorsolateral funiculus. Nociceptive primary afferent neurons enter the spinal dorsal horn via collaterals from Lissauer's tract. Nociceptive input is integrated and relayed by neurons in laminae I, II and V which project to the reticular formation and thalamus via the anterolateral tract.     

Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord

The roof plate resident BMPs have sequential functions in the developing spinal cord, establishing cell fate and orienting axonal trajectories. These activities are, however, restricted to the dI1–dI3 neurons in the most dorsal region of the spinal cord. What limits the extent of the action of the BMPs to these neurons? To address this question, we have examined both the distribution of the inhibitory Smads (I-Smads), Smad6 and Smad7 in the spinal cord and the consequence of ectopically expressing the I-Smads in chicken embryos. Our studies suggest that the I-Smads function in vivo to restrict the action of BMP signaling in the dorsal spinal cord. Moreover, the I-Smads have distinct roles in regulating the diverse activities of the BMPs. Thus, the ectopic expression of Smad7 suppresses the dI1 and dI3 neural fates and concomitantly increases the number of dI4–dI6 spinal neurons. In contrast, Smad6 most potently functions to block dI1 axon outgrowth. Taken together, these experiments suggest that the I-Smads have distinct roles in spatially limiting the response of cells to BMP signaling.     

Role of NO in modulating neuronal activity in superficial dorsal horn of spinal cord during exercise pressor reflex

Static contraction of hindlimb skeletal muscle in cats induces a reflex pressor response. The superficial dorsal horn of the spinal cord is the major site of the first synapse of this reflex. In this study, static contraction of the triceps surae muscle was evoked by electrical stimulation of the tibial nerve for 2 min in anesthetized cats (stimulus parameters: two times motor threshold at 30 Hz, 0.025-ms duration). Ten stimulations were performed and 1-min rest was allowed between stimulations. Muscle contraction caused a maximal increase of 32 +/- 5 mmHg in mean arterial pressure (MAP), which was obtained from the first three contractions. Activated neurons in the superficial dorsal horn were identified by c-Fos protein. Distinct c-Fos expression was present in the L6-S1 level of the superficial dorsal horn ipsilateral to the contracting leg (88 +/- 14 labeled cells per section at L7), whereas only scattered c-Fos expression was observed in the contralateral superficial dorsal horn (9 +/- 2 labeled cells per section, P < 0.05 compared with ipsilateral section). A few c-Fos-labeled cells were found in control animals (12 +/- 5 labeled cells per section, P < 0.05 compared with stimulated cats). Furthermore, double-labeling methods demonstrated that c-Fos protein coexisted with nitric oxide (NO) synthase (NOS) positive staining in the superficial dorsal horn. Finally, an intrathecal injection of an inhibitor of NOS, N-nitro-L-arginine methyl ester (5 mM), resulted in fewer c-Fos-labeled cells (58 +/- 12 labeled cells per section) and a reduced maximal MAP response (20 +/- 3 mmHg, P < 0.05). These results suggest that the exercise pressor reflex induced by static contraction is mediated by activation of neurons in the superficial dorsal horn and that formation of NO in this region is involved in modulating the activated neurons and the pressor response to contraction.     

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.     

Immunohistochemical studies of peptidergic neurons in the dorsal horn of the spinal cord

The indirect immunofluorescence technique was used to localize substance P, somatostatin, methionine--enkephalin, neurotensin, and oxytocin in the dorsal horn of the rat spinal cord. The unique distribution of each peptide is described and the relative amount of each peptide in laminae I--III of the dorsal horn and the dorsal part of the lateral funniculus qualitatively assessed. Colchicine treatment and dorsal rhizotomy were used to determine, in part, the origin of immunoreactive fibers and terminals observed in the dorsal horn.     

P2X receptor-mediated purinergic sensory pathways to the spinal cord dorsal horn

P2X receptors are expressed on different functional groups of primary afferent fibers. P2X receptor-mediated sensory inputs can be either innocuous or nociceptive, depending on which dorsal horn regions receive these inputs. We provide a brief review of P2X receptor-mediated purinergic sensory pathways to different regions in the dorsal horn. These P2X purinergic pathways are identified in normal animals, which provides insights into their physiological functions. Future studies on P2X purinergic pathways in animal models of pathological conditions may provide insights on how P2X receptors play a role in pathological pain states.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.