Spinal Lamina Research Articles

NT-3 Expression in Spared DRG and the Associated Spinal Laminae as well as Its Anterograde Transport in Sensory Neurons Following Removal of Adjacent DRG in Cats

Neurotrophin-3 plays an important role in survival and differentiation of sensory and sympathetic neurons, sprouting of neurites, synaptic reorganization, and axonal growth. The present study evaluated changes in expression of NT-3 in the spinal cord and L6 dorsal root ganglion (DRG), after ganglionectomy of adjacent dorsal roots in cats. NT-3 immunoreactivity increased at 3 days post-operation (dpo), but decreased at 10 dpo in spinal lamina II after ganglionectomy of L1-L5 and L7-S2 (leaving L6 DRG intact). Conversely, NT-3 immunoreactivity decreased on 3 dpo, but increased on 10 dpo in the nucleus dorsalis. Very little NT-3 mRNA signal was detected in the spinal cord, despite the changes in NT-3 expression. The above changes may be related to changes in NT-3 expression in the DRG. Ganglionectomy of L1-L5 and L7-S2 resulted in increase in NT-3 immunoreactivity and mRNA in small and medium-sized neurons, but decreased expression in large neurons of L6 DRG at 3 dpo. It is possible that increased NT-3 in spinal lamina II is derived from anterograde transport from small- and medium-sized neurons of L6 DRG, whereas decreased NT-3 immunoreactivity in the nucleus dorsalis is due to decreased transport of NT-3 from large neurons in the DRG at this time. This notion is supported by observations on NT-3 distribution in the dorsal root of L6 after ligation of the nerve root. The above results indicate that DRG may be a source of neurotrophic factors such as NT-3 to the spinal cord, and may contribute to plasticity in the spinal cord after injury.

Morphological characterization of spinal cord dorsal horn lamina I neurons projecting to the parabrachial nucleus in the rat

Many Rexed's lamina I neurons are nociceptive and project to the brain. Lamina I projection neurons can be classified as multipolar, fusiform, or pyramidal, based on cell body shape and characteristics of their proximal dendrites in the horizontal plane. There is also evidence that both multipolar and fusiform cells are nociceptive and pyramidal neurons nonnociceptive. In this investigation we identified which types of lamina I neurons belong to the spinoparabrachial tract in the rat and characterized them regarding the presence or absence of neurokinin-1 receptor (NK-1r) immunoreactivity. For this, cholera toxin subunit B (CTb), conjugated to a fluorescent marker was injected unilaterally into the parabrachial nucleus. Sections were additionally stained for the detection of NK-1r immunoreactivity and were examined using fluorescence and confocal microscopy. Serial confocal optical sections and 3D reconstructions were obtained for a considerable number of neurons per animal. Using immunofluorescence, we assessed the proportion of lamina I neurons belonging to the spinoparabrachial (SPB) tract and/or expressing NK-1r. The relative distribution of neurons belonging to the SPB tract was: 38.7% multipolar, 36.8% fusiform, 22.7% pyramidal, and 1.9% unclassified. Most of the SPB neurons expressing NK-1r were either multipolar or fusiform. Pyramidal SPB neurons were seldom immunoreactive for NK-1r, an observation that provides further support to the concept that most lamina I projection neurons of the pyramidal type are nonnociceptive. In addition, our study provides further evidence that these distinct morphological types of neurons differ in their phenotypic properties, but not in their projection patterns.

Contusive spinal cord injury evokes localized changes in NADPH‐d activity but extensive changes in Fos‐like immunoreactivity in the rat

The histological detection of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a marker for nitric oxide-producing cells, was used to evaluate ongoing changes in the neural biochemistry of the rat spinal cord 1 week following contusive spinal cord injury (SCI). In addition, the immunohistochemical detection of the immediate-early gene c-fos was used to identify basal patterns of neural activity at this time. The numbers and laminar locations of NADPH-d- and c-fos-positive cells were examined in spinal segments adjacent to the site of injury (T12-S3) as well as those distant from the injury (C3-C5) in both SCI and un-injured rats. Our data show that contusive SCI results in a significant reduction in NADPH-d labelling in the superficial dorsal horn, and a significant increase in NADPH-d expression in small bipolar neurons and large motoneurons in the ventral horn at the site of the injury. In spinal segments distant to the injury site (C3-C5), NADPH-d activity did not differ from that of uninjured controls. Furthermore, significant reductions in the levels of c-fos expression were observed in SCI rats, in spinal segments both at and distant to the site of injury for all spinal laminae. The only exception was a dramatic increase observed in the sacral parasympathetic nucleus. These data suggest that increased NADPH-d expression is related to conditions specific to the site of injury, whereas the changes in c-fos expression probably indicate more global changes in neuronal activity following SCI.

Differential wiring of local excitatory and inhibitory synaptic inputs to islet cells in rat spinal lamina II demonstrated by laser scanning photostimulation

The substantia gelatinosa (lamina II) of the spinal dorsal horn contains inhibitory and excitatory interneurons that are thought to play a critical role in the modulation of nociception. However, the organization of the intrinsic circuitry within lamina II remains poorly understood. We used glutamate uncaging by laser scanning photostimulation to map the location of neurons that give rise to local synaptic inputs to islet cells, a major class of inhibitory interneuron in lamina II. We also mapped the distribution of sites on the islet cells that exhibited direct (non-synaptic) responses to uncaging of excitatory and inhibitory transmitters. Local synaptic inputs to islet cells arose almost entirely from within lamina II, and these local inputs included both excitatory and inhibitory components. Furthermore, there was a striking segregation in the location of sites that evoked excitatory versus inhibitory synaptic inputs, such that inhibitory presynaptic neurons were distributed more proximal to the islet cell soma. This was paralleled in part by a differential distribution of transmitter receptor sites on the islet cell, in that inhibitory sites were confined to the peri-somatic region while excitatory sites were more widespread. This differential organization of excitatory and inhibitory inputs suggests a principle for the wiring of local circuitry within the substantia gelatinosa.

Vanilloid receptor-1 (TRPV1)-dependent activation of inhibitory neurotransmission in spinal substantia gelatinosa neurons of mouse

Inhibitory neurotransmission in spinal cord dorsal horn is mainly mediated by γ-amino butyric acid (GABA) and glycine. By patch clamp recordings and correlative immunocytochemistry, we studied here the effect of 2 μM capsaicin-induced vanilloid receptor-1 (TRPV1) activation on IPSCs in spinal lamina II neurons from post-natal mice. Specificity was confirmed after pre-incubation with the competitive antagonist SB366791 (10 μM). After a single capsaicin pulse, an intense increase of spontaneous IPSC (sIPSC) frequency was observed in the presence of NBQX 10 μM (62/81 neurons; ∼76%) or NBQX 10 μM + AP-5 20–100 μM (27/42 neurons; ∼64%). Only a subpopulation (∼40%) of responsive neurons showed a significant amplitude increase. Seventy-two percent of the neurons displayed pure GABA A receptor-mediated sIPSCs, whereas the remaining ones showed mixed GABAergic/glycinergic events. After two consecutive capsaicin pulses, frequency rises were very similar, and both significantly higher than controls. When the second pulse was given in the presence of 4 μM L732,138, a selective antagonist of the substance P (SP) preferred receptor NK1, we observed a significant loss in frequency increase (63.90% with NBQX and 52.35% with NBQX + AP-5). TTX (1 μM) largely (∼81.5%) blocked the effect of capsaicin. These results show that TRPV1 activation on primary afferent fibers releases SP. The peptide then excites inhibitory neurons in laminae I, III and IV, leading to an increased release of GABA/glycine in lamina II via a parallel alternative pathway to glutamate.

Parkinson’s disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre- and postganglionic neurons

Clinical signs frequently recognized in early phases of sporadic Parkinson's disease (PD) may include autonomic dysfunctions and the experience of pain. Early disease-related lesions that may account for these symptoms are presently unknown or incompletely known. In this study, immunocytochemistry for alpha-synuclein was used to investigate the first relay stations of the pain system as well as parasympathetic and sympathetic pre- and postganglionic nerve cells in the lower brainstem, spinal cord, and coeliac ganglion in 100 microm polyethylene glycol embedded sections from six autopsy individuals, whose brains were staged for PD-associated synucleinopathy. Immunoreactive inclusions were found for the first time in spinal cord lamina I neurons. Lower portions of the spinal cord downwards of the fourth thoracic segment appeared to be predominantly affected, whereas the spinal trigeminal nucleus was virtually intact. Additional involvement was seen in parasympathetic preganglionic projection neurons of the vagal nerve, in sympathetic preganglionic neurons of the spinal cord, and in postganglionic neurons of the coeliac ganglion. The known interconnectivities between all of these components offer a possible explanation for their particular vulnerability. Lamina I neurons (pain system) directly project upon sympathetic relay centers, and these, in turn, exert influence on the parasympathetic regulation of the enteric nervous system. This constellation indicates that physical contacts between vulnerable regions play a key role in the pathogenesis of PD.

Transformation of the Output of Spinal Lamina I Neurons After Nerve Injury and Microglia Stimulation Underlying Neuropathic Pain

BackgroundDisinhibition of neurons in the superficial spinal dorsal horn, via microglia – neuron signaling leading to disruption of chloride homeostasis, is a potential cellular substrate for neuropathic pain. But, a central unresolved question is whether this disinhibition can transform the activity and responses of spinal nociceptive output neurons to account for the symptoms of neuropathic pain.ResultsHere we show that peripheral nerve injury, local spinal administration of ATP-stimulated microglia or pharmacological disruption of chloride transport change the phenotype of spinal lamina I output neurons, causing them to 1) increase the gain of nociceptive responsiveness, 2) relay innocuous mechanical input and 3) generate spontaneous bursts of activity. The changes in the electrophysiological phenotype of lamina I neurons may account for three principal components of neuropathic pain: hyperalgesia, mechanical allodynia and spontaneous pain, respectively.ConclusionThe transformation of discharge activity and sensory specificity provides an aberrant signal in a primarily nociceptive ascending pathway that may serve as a basis for the symptoms of neuropathic pain.

Physiological properties of spinal lamina II GABAergic neurons in mice following peripheral nerve injury

Aberrant GABAergic inhibition in spinal dorsal horn may underlie some forms of neuropathic pain. Potential, but yet unexplored, mechanisms include reduced excitability, abnormal discharge patterns or altered synaptic input of spinal GABAergic neurons. To test these hypotheses, we quantitatively compared active and passive membrane properties, firing patterns in response to depolarizing current steps and synaptic input of GABAergic neurons in spinal dorsal horn lamina II of neuropathic and of control animals. Transgenic mice were used which expressed enhanced green fluorescent protein (EGFP) controlled by the GAD67 promoter, thereby labelling one-third of all spinal GABAergic neurons. In all neuropathic mice included in this study, chronic constriction injury of one sciatic nerve led to tactile allodynia and thermal hyperalgesia. Control mice were sham-operated. Membrane excitability of GABAergic neurons from neuropathic or sham-treated animals was indistinguishable. The most frequent firing patterns observed in neuropathic and sham-operated animals were the initial burst (neuropathic: 46%, sham-treated: 42%), the gap (neuropathic: 31%, sham-treated: 29%) and the tonic firing pattern (neuropathic: 16%, sham-treated: 24%). The synaptic input from dorsal root afferents was similar in neuropathic and in control animals. Thus, a reduced membrane excitability, altered firing patterns or changes in synaptic input of this group of GABAergic neurons in lamina II of the spinal cord dorsal horn are unlikely causes for neuropathic pain.

Distinct Populations of Spinal Cord Lamina II Interneurons Expressing G-Protein-Gated Potassium Channels

Noxious stimuli are sensed and carried to the spinal cord dorsal horn by A delta and C primary afferent fibers. Some of this input is relayed directly to supraspinal sites by projection neurons, whereas much of the input impinges on a heterogeneous population of interneurons in lamina II. Previously, we demonstrated that G-protein-gated inwardly rectifying potassium (GIRK) channels are expressed in lamina II of the mouse spinal cord and that pharmacologic ablation of spinal GIRK channels selectively blunts the analgesic effect of high but not lower doses of intrathecal mu-opioid receptor (MOR) agonists. Here, we report that GIRK channels formed by GIRK1 and GIRK2 subunits are found in two large populations of lamina II excitatory interneurons. One population displays relatively large apparent whole-cell capacitances and prominent GIRK-dependent current responses to the MOR agonist [D-Ala2,N-MePhe4,Gly-ol5] -enkephalin (DAMGO). A second population shows smaller apparent capacitance values and a GIRK-dependent response to the GABA(B) receptor agonist baclofen, but not DAMGO. Ultrastructural analysis revealed that GIRK subunits preferentially label type I synaptic glomeruli, suggesting that GIRK-containing lamina II interneurons receive prominent input from C fibers, while receiving little input from A delta fibers. Thus, excitatory interneurons in lamina II of the mouse spinal cord can be subdivided into different populations based on the neurotransmitter system coupled to GIRK channels. This important distinction will afford a unique opportunity to characterize spinal nociceptive circuitry with defined physiological significance.

Differential contribution of TRPV1 to thermal responses and tissue injury-induced sensitization of dorsal horn neurons in laminae I and V in the mouse

Our previous recordings from dorsal root ganglion and spinal lamina V neurons from TRPV1-mutant mice showed dramatic decreases in responses to temperatures near the activation threshold of this channel (43–49 °C). Somewhat unexpectedly, we only observed behavioral deficits in these mice at higher temperatures (50–58 °C). In the present study, we tested the hypothesis that the noxious heat-evoked pain behavior that persists in TRPV1-mutant mice reflects residual responsiveness of neurons in the superficial, but not deep, dorsal horn. To this end, we performed in vivo extracellular recordings of spinal nociresponsive neurons in laminae I and V in wild type (WT) and TRPV1 mutant mice. Neurons in WT and mutant mice from both laminae did not differ in their spontaneous activity or evoked responses to mechanical or cold stimuli. By contrast, most lamina I neurons from mutant mice responded to noxious heat with significantly higher thresholds than in WT mice. In contrast, lamina V neurons from mutant mice were virtually unresponsive to noxious heat before and after topical mustard oil-induced tissue injury. Interestingly, lamina I neurons in mutant mice displayed thermal sensitization following tissue injury, comparable in magnitude, but of shorter duration, than in WT mice. We conclude that TRPV1 is necessary for noxious heat-evoked responses of lamina V neurons, both before and after tissue injury. It is also an essential contributor to the normal activation threshold of lamina I neurons to noxious heat and for the full duration of thermal sensitization of lamina I neurons following injury. Finally, our results suggest that the processing of noxious thermal messages by neurons in lamina I involves convergent inputs from a heterogeneous population of primary afferent thermal nociceptors.

Neurotrophin‐induced preprotachykinin‐A gene promoter modulation in organotypic rat spinal cord culture

To study regulation of the preprotachykinin-A gene promoter, we utilised a biolistic gene transfer protocol to deliver a DNA construct that incorporates a portion of the preprotachykinin-A gene promoter and an enhanced green fluorescent protein reporter gene into neonatal rat spinal cord organotypic slices. The ability of the neurokinin-1 receptor agonist [Sar9,Met(O2)11]-substance P, nerve growth factor and brain derived neurotrophic factor to modulate positively preprotachykinin-A gene promoter construct activity, as indicated by de novo enhanced green fluorescent protein expression, was determined. Treatment of organotypic slices with [Sar9, Met(O2)11]-substance P (10 microm, P < 0.05), nerve growth factor (200 ng/mL, P < 0.001) or brain derived neurotrophic factor (200 ng/mL, P < 0.02) significantly increased the proportion of cytomegaloviral promoter-DsRed transfected cells (used to visualise total transfected cells) that co-expressed enhanced green fluorescent protein. The distribution of enhanced green fluorescent protein/DsRed-positive neurones across spinal laminae was broadly in line with the known distribution of spinal Trk and neurokinin-1 receptors. These data suggest a modulated activity of the preprotachykinin-A gene promoter in spinal neurones in vitro by substance P and/or neurotrophins. The functional consequences of such transcriptional changes within central peptidergic circuitry and their relevance to chronic pain are considered.

Possible sources and sites of action of the nitric oxide involved in synaptic plasticity at spinal lamina i projection neurons

The synaptic long-term potentiation between primary afferent C-fibers and spinal lamina I projection neurons is a cellular model for hyperalgesia [Ikeda H, Heinke B, Ruscheweyh R, Sandkühler J (2003) Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 299:1237–1240]. In lamina I neurons with a projection to the periaqueductal gray, this long-term potentiation is dependent on nitric oxide. In the present study, we used immunohistochemistry to detect possible sources and sites of action of the nitric oxide necessary for the long-term potentiation at lamina I spino-periaqueductal gray neurons in rats. None of the three isoforms of the nitric oxide synthase was expressed in a significant number of lamina I spino-periaqueductal gray neurons or primary afferent C-fibers (as evaluated by staining of their cell bodies in the dorsal root ganglia). However, endothelial and inducible nitric oxide synthase were found throughout the spinal cord vasculature and neuronal nitric oxide synthase was present in a number of neurons in laminae II and III. The nitric oxide target soluble guanylyl cyclase was detected in most lamina I spino-periaqueductal gray neurons and in approximately 12% of the dorsal root ganglion neurons, all of them nociceptive as evaluated by coexpression of substance P. Synthesis of cyclic 3′,5′-guanosine monophosphate upon stimulation by a nitric oxide donor confirmed the presence of active guanylyl cyclase in at least a portion of the spino-periaqueductal gray neuronal cell bodies. We therefore propose that nitric oxide generated in neighboring neurons or blood vessels acts on the spino-periaqueductal gray neuron and/or the primary afferent C-fiber to enable long-term potentiation. Lamina I spino-parabrachial neurons were stained for comparison and yielded similar results.

Reduction of Anion Reversal Potential Subverts the Inhibitory Control of Firing Rate in Spinal Lamina I Neurons: Towards a Biophysical Basis for Neuropathic pain

BackgroundReduction of the transmembrane chloride gradient in spinal lamina I neurons contributes to the cellular hyperexcitability producing allodynia and hyperalgesia after peripheral nerve injury. The resultant decrease in anion reversal potential (i.e. shift in Eanion to less negative potentials) reduces glycine/GABAA receptor-mediated hyperpolarization, but the large increase in membrane conductance caused by inhibitory input can nonetheless shunt concurrent excitatory input. Without knowing the relative contribution of hyperpolarization and shunting to inhibition's modulation of firing rate, it is difficult to predict how much net disinhibition results from reduction of Eanion. We therefore used a biophysically accurate lamina I neuron model to investigate quantitatively how changes in Eanion affect firing rate modulation.ResultsSimulations reveal that even a small reduction of Eanion compromises inhibitory control of firing rate because reduction of Eanion not only decreases glycine/GABAA receptor-mediated hyperpolarization, but can also indirectly compromise the capacity of shunting to reduce spiking. The latter effect occurs because shunting-mediated modulation of firing rate depends on a competition between two biophysical phenomena: shunting reduces depolarization, which translates into reduced spiking, but shunting also shortens the membrane time constant, which translates into faster membrane charging and increased spiking; the latter effect predominates when average depolarization is suprathreshold. Disinhibition therefore occurs as both hyperpolarization- and shunting-mediated modulation of firing rate are subverted by reduction of Eanion. Small reductions may be compensated for by increased glycine/GABAA receptor-mediated input, but the system decompensates (i.e. compensation fails) as reduction of Eanion exceeds a critical value. Hyperexcitability necessarily develops once disinhibition becomes incompensable. Furthermore, compensation by increased glycine/GABAA receptor-mediated input introduces instability into the system, rendering it increasingly prone to abrupt decompensation and even paradoxical excitation.ConclusionReduction of Eanion dramatically compromises the inhibitory control of firing rate and, if compensation fails, is likely to contribute to the allodynia and hyperalgesia associated with neuropathic pain. These data help explain the relative intractability of neuropathic pain and illustrate how it is important to choose therapies not only based on disease mechanism, but based on quantitative understanding of that mechanism.

Effects of electro-acupuncture on the expression of c-jun and c-fos in spared dorsal root ganglion and associated spinal laminae following removal of adjacent dorsal root ganglia in cats

This study evaluated the plastic changes of c-jun and c-fos in the right sixth lumbar dorsal root ganglion (L 6 DRG), Rexed’s lamina II in representative spinal segments L 3, L 5, and L 6 and in the nucleus dorsalis (ND) at L 3 segments after electro-acupuncture (EA) in cats subjected to removal of L 1–L 5 and L 7–S 2 DRG. Following dorsal root ganglionectomy, there was a significant increase in the density of c-jun immunoreactivity in the neurons and glia in spinal lamina II and in the ND; there was also marked elevation in the expression of c-fos in ND. In both cases there was no change in the c-jun and c-fos immunoreactivity in the DRG. After EA in the operated animals, there was an up-regulation in the expression of c-jun in the L 6 DRG and the associated spinal lamina II; however, increased c-fos expression was detected only in the L 6 DRG. Western blot and RT-PCR were also performed to quantitatively explore the mRNA and protein expression changes in the spinal dorsal horn and associated DRG. Following partial deafferentation, there was a significant increase in the protein level of both c-jun and c-fos in the dorsal horn, while, in both cases there was no change in c-jun and c-fos protein and mRNA in the DRG. After EA in the operated animals, both c-jun protein and its mRNA in the L 6 DRG as well as the associated dorsal horn of L6 spinal segment were upregulated, but increased c-fos protein and its mRNA was observed only in the L 6 DRG. These findings suggested that c-jun and c-fos might be related to the acupuncture promoted spinal cord plasticity as reported previously.

Relationship between tonic inhibitory currents and phasic inhibitory activity in the spinal cord lamina II region of adult mice.

Phasic and tonic inhibitions are two types of inhibitory activities involved in inhibitory processing in the CNS. In the spinal cord dorsal horn, phasic inhibition is mediated by both GABAergic and glycinergic inhibitory postsynaptic currents. In contrast to phasic inhibitory currents, using patch-clamp recording technique on spinal cord slices prepared from adult mice we revealed that tonic inhibitory currents were mediated by GABAA receptors but not by glycine receptors in dorsal horn lamina II region. We found that there was a linear relationship (r = 0.85) between the amplitude of tonic inhibitory currents and the frequency of GABAergic inhibitory postsynaptic currents. Analysis of charge transfer showed that the charges carried by tonic inhibitory currents were about 6 times of charges carried by phasic inhibitory currents. The prominent charge transfer by tonic inhibitory currents and their synaptic activity dependency suggest a significant role of tonic inhibition in sensory processing.

Altered discharges of spinal wide dynamic range neurons and down-regulation of glutamate transporter expression in rats with paclitaxel-induced hyperalgesia

Changes in the signaling of wide dynamic range neurons and the expression of glutamate transporters in the lumbar spinal dorsal horn of rats with Taxol-induced hyperalgesia are detailed in this report. Deep spinal lamina neurons have significantly increased spontaneous activity and after-discharges to noxious mechanical stimuli, increased responses to both skin heating and cooling, and increased after-discharges and abnormal windup to transcutaneous electrical stimuli. The expression of glutamate transporter proteins in the dorsal horn is decreased at the time point corresponding to the physiological changes. These results suggest a state of increased excitability develops in spinal pain-signaling neurons as a consequence of decreased glutamate clearance. These changes in dorsal horn neurobiology likely in turn contribute to the hyper-responsiveness to sensory stimuli seen in animals treated with Taxol and may play a role in the pain seen in cancer patients receiving Taxol.

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (E(anion)) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (gamma-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in E(anion). Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the E(anion) shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and E(anion). Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia-neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.

Differential Maturation of GABA Action and Anion Reversal Potential in Spinal Lamina I Neurons: Impact of Chloride Extrusion Capacity

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.

Expression of A‐type K+ channel α subunits Kv4.2 and Kv4.3 in rat spinal lamina II excitatory interneurons and colocalization with pain‐modulating molecules

Voltage-gated K(+) channel alpha subunits Kv 4.2 and Kv 4.3 are the major contributors of somatodendritic A-type K(+) currents in many CNS neurons. A recent hypothesis suggests that Kv 4 subunits may be involved in pain modulation in dorsal horn neurons. However, whether Kv 4 subunits are expressed in dorsal horn neurons remains unknown. Using immunohistochemistry, we found that Kv 4.2 and Kv 4.3 immunoreactivity was concentrated in the superficial dorsal horn, mainly in lamina II. Both Kv 4.2 and Kv 4.3 appeared on many rostrocaudally orientated dendrites, whereas Kv 4.3 could be also detected from certain neuronal somata. Kv 4.3(+) neurons were a subset of excitatory inerneurons with calretinin(+)/calbindin(-)/PKCgamma(-) markers, and a fraction of them expressed micro-opioid receptors. Kv 4.3(+) neurons also expressed ERK 2 and mGluR 5, which are molecules related to the induction of central sensitization, a mechanism mediating nociceptive plasticity. Together with the expression of Kv 4.3 in VR 1(+) DRG neurons, our data suggest that Kv C4 subunits could be involved in pain modulation.

Polysialic acid-induced plasticity reduces neuropathic insult to the central nervous system

Under chronic conditions of neuropathic pain, nociceptive C terminals are lost from their target region in spinal lamina II, leading to reduced thermal hyperalgesia. This region of the spinal cord expresses high levels of polysialic acid (PSA), a cell surface carbohydrate known to weaken cell-cell interactions and promote plasticity. Experimental removal of PSA from the spinal cord exacerbates hyperalgesia and results in retention of C terminals, whereas it has no effect on plasticity of touch Abeta fibers and allodynia. We propose that expression of PSA at this stress pathway relay point could serve to protect central circuitry from chronic sensory overload.