Spinal Lamina Research Articles

Mechanical allodynia generated by stimulation of unmyelinated afferent nerve fibres

Mechanical allodynia generated by stimulation of unmyelinated afferent nerve fibres

Roles of BDNF in spinal neuroplasticity in cats subjected to partial dorsal ganglionectomy

This study investigated the role of brain-derived neurotrophic factor (BDNF) in neuroplasticity in cats subjected to the removal of dorsal root ganglia (DRG). Following partial ganglionectomy, the number of BDNF-positive varicosities from spared L6 DRG decreased significantly. This reduction was observed at 3 days post operation (dpo) in spinal lamina II of L3 and L5. Whereas the percentages of positive neurons for BDNF and its mRNA in spared L6 DRG at 10 dpo were significantly increased, and accumulated BDNF was seen on the DRG side of the ligated axons. Importantly, BDNF antibody neutralization in vivo results in a significant reduction in the number of varicosities in spinal lamina II, evidenced by BDNF and calcitonin gene-related peptide immunohistochemical staining. These findings suggested that peripheral-derived BDNF could play a critical role in spinal neuroplasticity in cats subjected to partial ganglionectomy. This may underlie the basis of molecular therapy depending on gene drug-like BDNF release.

Pharmacological enhancement of δ-subunit-containing GABA A receptors that generate a tonic inhibitory conductance in spinal neurons attenuates acute nociception in mice

The development of new strategies for the treatment of acute pain requires the identification of novel nonopioid receptor targets. This study explored whether δ-subunit-containing GABA ARs (δGABA ARs) in neurons of the spinal cord dorsal horn generate a tonic inhibitory conductance in vitro and whether δGABA AR activity regulates acute nociception. Whole-cell recordings revealed that δGABA ARs generate a tonic inhibitory conductance in cultured spinal neurons and lamina II neurons in spinal cord slices. Increasing δGABA AR function by applying the δGABA AR-preferring agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP) increased the tonic current and inhibited neuronal excitability in spinal neurons from wild-type (WT) but not δ subunit null-mutant ( Gabrd −/− ) mice. In behavioral studies, baseline δGABA AR activity did not regulate acute nociception; however, THIP administered intraperitoneally or intrathecally attenuated acute nociception in WT but not Gabrd −/− mice. In the formalin nociception assay, the phase 1 response was similar for WT and Gabrd −/− mice. In contrast, the phase 2 response, which models central sensitization, was greater in Gabrd −/− mice than WT. THIP administered intraperitoneally or intrathecally inhibited phase 1 responses of WT but not Gabrd −/− mice and had no effect on phase 2 responses of WT mice. Surprisingly, THIP reduced the enhanced phase 2 response in Gabrd −/− mice. Together, these results suggest that δGABA ARs in spinal neurons play a major physiological and pharmacological role in the regulation of acute nociception and central sensitization. Spinal δ-subunit-containing GABA A receptors were identified with electrophysiological methods and behavioral models as novel targets for the treatment of acute pain.

Leptin enhances NMDA-induced spinal excitation in rats: A functional link between adipocytokine and neuropathic pain

Recent studies have shown that leptin (an adipocytokine) played an important role in nociceptive behavior induced by nerve injury, but the cellular mechanism of this action remains unclear. Using the whole-cell patch-clamp recording from rat’s spinal cord slices, we showed that superfusion of leptin onto spinal cord slices dose-dependently enhanced N-methyl- d-aspartate (NMDA) receptor-mediated currents in spinal cord lamina II neurons. At the cellular level, the effect of leptin on spinal NMDA-induced currents was mediated through the leptin receptor and the JAK2/STAT3 (but not PI3K or MAPK) pathway, as the leptin effect was abolished in leptin receptor-deficient ( db/db) mice and inhibited by a JAK/STAT inhibitor. Moreover, we demonstrated in naïve rats that a single intrathecal administration of leptin enhanced spontaneous biting, scratching, and licking behavior induced by intrathecal NMDA and that repeated intrathecal administration of leptin elicited thermal hyperalgesia and mechanical allodynia, which was attenuated by the noncompetitive NMDA receptor antagonist MK-801. Intrathecal leptin also upregulated the expression of NMDA receptors and pSTAT3 within the rat’s spinal cord dorsal horn, and intrathecal MK-801 attenuated this leptin effect as well. Our data demonstrate a relationship between leptin and NMDA receptor-mediated spinal neuronal excitation and its functional role in nociceptive behavior. Since leptin contributes to nociceptive behavior induced by nerve injury, the present findings suggest an important cellular link between the leptin’s spinal effect and the NMDA receptor-mediated cellular mechanism of neuropathic pain. A functional link is demonstrated between leptin, an adipocytokine, and the cellular mechanisms of neuropathic pain via enhancement of function and expression of spinal N-methyl- d-aspartate receptors.

Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury.

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.

Follistatin-like 1 Suppresses Sensory Afferent Transmission by Activating Na+,K+-ATPase

Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.

Brain-derived neurotrophic factor from microglia: a molecular substrate for neuropathic pain

One of the most significant advances in pain research is the realization that neurons are not the only cell type involved in the etiology of chronic pain. This realization has caused a radical shift from the previous dogma that neuronal dysfunction alone accounts for pain pathologies to the current framework of thinking that takes into account all cell types within the central nervous system (CNS). This shift in thinking stems from growing evidence that glia can modulate the function and directly shape the cellular architecture of nociceptive networks in the CNS. Microglia, in particular, are increasingly recognized as active principal players that respond to changes in physiological homeostasis by extending their processes toward the site of neural damage, and by releasing specific factors that have profound consequences on neuronal function and that contribute to CNS pathologies caused by disease or injury. A key molecule that modulates microglia activity is ATP, an endogenous ligand of the P2 receptor family. Microglia expresses several P2 receptor subtypes, and of these the P2X4 receptor subtype has emerged as a core microglia-neuron signaling pathway: activation of this receptor drives the release of brain-derived neurotrophic factor (BDNF), a cellular substrate that causes disinhibition of pain-transmitting spinal lamina I neurons. Converging evidence points to BDNF from spinal microglia as being a critical microglia-neuron signaling molecule that gates aberrant nociceptive processing in the spinal cord. The present review highlights recent advances in our understanding of P2X4 receptor-mediated signaling and regulation of BDNF in microglia, as well as the implications for microglia-neuron interactions in the pathobiology of neuropathic pain.

Facilitation of μ-Opioid Receptor Activity by Preventing δ-Opioid Receptor-Mediated Codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane isa potential strategy to improve opioid analgesic therapies.

The somatostatin analogue octreotide inhibits capsaicin-mediated activation of nociceptive primary afferent fibres in spinal cord lamina II ( substantia gelatinosa)

Somatostatin (SST) in spinal cord has been linked with the inhibition of nociceptive neurotransmission in several experimental paradigms. The SST2 receptor (SSTR2) is the main SST receptor subtype in the superficial dorsal horn (DH) and is activated, besides to the naïve peptide, by the SST synthetic analogue octreotide (OCT). In the present work, we have studied the central effects of SSTR2 activation on capsaicin (CAP)-induced glutamate release in mouse DH. In neurons of the lamina II of DH, CAP (2 μM) induced a strong increase of mEPSC frequency that was significantly reduced (70%) by OCT. SSTR2 involvement was assessed by using the specific antagonist CYN 154806. No differences were observed between frequency increase in CAP alone vs. CAP in the presence of CYN 154806 + OCT. The effect of OCT was further investigated by studying c-fos expression in spinal cord slices. The CAP-induced increase in density of Fos immunoreactive nuclei in the superficial DH was strongly prevented by OCT. SSTR2a (a splicing variant of SSTR2) immunoreactivity was found in both pre- and post-synaptic compartments of laminae I–II synapses. By light and electron microscopy, SSTR2a was mainly localized onto non-peptidergic isolectin B4 (IB4)-positive primary afferent fibres (PAFs). A subset of them was also found to express the CAP receptor TRPV1. These data show that the SST analogue OCT inhibits CAP-mediated activation of non-peptidergic nociceptive PAFs in lamina II. Our data indicate that SSTR2a plays an important role in the pre-synaptic modulation of central excitatory nociceptive transmission in mouse.

Monosynaptic excitatory inputs to spinal lamina I anterolateral‐tract‐projecting neurons from neighbouring lamina I neurons

Spinal lamina I receives nociceptive primary afferent input to project through diverse ascending pathways, including the anterolateral tract (ALT). Large projection neurons (PNs) form only a few per cent of the cell population in this layer, and little is known about their local input from other lamina I neurons. We combined single-cell imaging in the isolated spinal cord, paired recordings, 3-D reconstructions of biocytin-labelled neurons and computer simulations to study the monosynaptic input to large ALT-PNs from neighbouring (somata separated by less than 80 μm) large lamina I neurons. All 11 connections identified were excitatory. We have found that an axon of a presynaptic neuron forms multiple synapses on an ALT-PN, and both Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors are involved in transmission. The monosynaptic EPSC latencies (1-12 ms) are determined by both post- and presynaptic factors. The postsynaptic delay, resulting from the electrotonic EPSC propagation in the dendrites of an ALT-PN, could be 4 ms at most. The presynaptic delay, caused by the spike propagation in a narrow highly branched axon of a local-circuit neuron, can be about 10 ms for neighbouring ALT-PNs and longer for more distant neurons. In many cases, the EPSPs evoked by release from a lamina I neuron were sufficient to elicit a spike in an ALT-PN. Our data show that ALT-PNs can receive input from both lamina I local-circuit neurons and other ALT-PNs. We suggest that lamina I is a functionally interconnected layer. The intralaminar network described here can amplify the overall output from the principal spinal nociceptive projection area.

Role of M3 muscarinic acetylcholine receptor in glycinergic neurotransmitter release in spinal lamina Ineurons in rats

Objective To evaluate the role of M3 muscarinic acetylcholine receptor (M3 mAChR) in the release of glycinergic neurotransmitter by using oxotremorine-M (Oxo-M: a nonselective mAChR agonist) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP: a highly selective M3mAChR antagonist). Methods Twenty male 3-4 weeks old SD rats weighing 160-180 g after successful intrathecal catheterization were randomized into 2 groups (n = 10 each): normal saline group (group NS) and pertussis toxin (group PTX).Pertussis toxin 1.5 μg/10 μl was injected IT in group PTX, while in group NS normal saline 10 μl was injected IT instead. The animals were killed at day 7 after injection. The spinal cords were removed and sliced and placed in artificial CSF. Glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were measured in spinal lamina Ⅱneurons using whole-cell voltage-clamp technique. Five minutes after sealing, Oxo-M (final concentration 3 μ mol/L) was added. Oxo-M was then completely washed out 3 min later and 4-DAMP (final concentration 25 nmol/L) was added after 5 min of stabilization. In the presence of 4-DAMP, Oxo-M (final concentration 3 μmol/L) was added again 3 min later. sIPSCs were recorded before addition of Oxo-M (T1), 3 min after addition of Oxo-M (T2), 3 min after addition of 4-DAMP (T3), 3 min after the second addition of Oxo-M (T4). Results Compared with the baseline value at T1 , Oxo-M significantly increased the frequency of glycinergic sIPSCs at T2without changing the amplitude at T2-4 in both groups. The frequency of sIPSCs was significantly lower at T4 than at T2 in both groups (P ＜ 0.05 or 0.01). There was no significant difference in both frequency and amplitude of glycinergic sIPSCs between the two groups. Conclusion M3 mAChR plays a predominant role in the release of glycinergic transmitter in the spinal lamina Ⅱ neurons in rats. Key words: Receptor, muscarinic M3; Glycine; Neurons; Neurotransmitter agents; Spinal cord

Neurochemical properties of enkephalinergic neurons in lumbar spinal dorsal horn revealed by preproenkephalin–green fluorescent protein transgenic mice

Enkephalin (ENK) has been implicated in nociceptive transmission in the spinal cord while its functional role is not clear because of difficulties in ideally visualizing ENKergic neurons. We thus developed preproenkephalin-green fluorescent protein transgenic mice to better identify ENKergic neurons. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) together with immunohistochemistry and in situ hybridization were first employed to confirm the successful transgenic manipulation and its application in showing spinal ENKergic neurons. The proportions of ENKergic neurons in the spinal cord laminae I, II, III and IV-VI among dorsal horn neurons were 15.8 +/- 3.1%, 39.5 +/- 3.3%, 11.8 +/- 1.9% and 10.7 +/- 2.1%, respectively. Double labeling with other molecules was then performed to further clarify the neurochemical properties of spinal ENKergic neurons. GABA was found to exist in 42.9 +/- 2.8% of ENKergic neurons that were mainly located in lamina I-III. The proportions of parvalbumin-, calretinin-, calbindin- and neuronal nitric oxide synthase-positive cells among the ENKergic neurons were 5.2 +/- 0.7%, 42.6 +/- 2.3%, 25.8 +/- 2.2% and 11.1 +/- 1.6%, respectively. Compared with previously findings obtained with ENK antibody labeling, this line of newly generated mice can be a reliable tool for the study of specific spinal ENKergic neuronal population.

Skeletal Site-Related Variation in Human Trabecular Bone Transcriptome and Signaling

BackgroundThe skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored.MethodologyTo address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina.Principal FindingsIn the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling.Conclusions and SignificanceThis study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified.

Multisegmental Aδ- and C-Fiber Input to Neurons in Lamina I and the Lateral Spinal Nucleus

Spinal lamina I and the lateral spinal nucleus (LSN) receive and integrate nociceptive primary afferent inputs to project through diverse ascending pathways. The pattern of the afferent supply of individual lamina I and LSN neurons through different segmental dorsal roots is poorly understood. Therefore, we recorded responses of lamina I and LSN neurons in spinal segments L4 and L3 to stimulation of six ipsilateral dorsal roots (L1-L6). The neurons were viewed through the overlying white matter in the isolated spinal cord preparation using the oblique infrared LED illumination technique. Orientation of myelinated fibers in the white matter was used as a criterion to distinguish between the LSN and lamina I. Both types of neurons received mixed (monosynaptic and polysynaptic) excitatory Adelta- and C-fiber input from up to six dorsal roots, with only less than one-third of it arising from the corresponding segmental root. The largest mixed input arose from the dorsal root of the neighboring caudal segment. Lamina I and LSN neurons could fire spikes upon the stimulation of up to six different dorsal roots. We also found that individual lamina I neurons can receive converging monosynaptic Adelta- and/or C-fiber inputs from up to six segmental roots. This study shows that lamina I and LSN neurons function as intersegmental integrators of primary afferent inputs. We suggest that broad monosynaptic convergence of Adelta- and C-afferents onto a lamina I neuron is important for the somatosensory processing.

Potentiation of Excitatory Transmission in Substantia Gelatinosa Neurons of Rat Spinal Cord by Inhibition of Estrogen Receptor Alpha

BackgroundIt has been shown that estrogen is synthesized in the spinal dorsal horn and plays a role in modulating pain transmission. One of the estrogen receptor (ER) subtypes, estrogen receptor alpha (ERα), is expressed in the spinal laminae I-V, including substantia gelatinosa (SG, lamina II). However, it is unclear how ERs are involved in the modulation of nociceptive transmission.ResultsIn the present study, a selective ERα antagonist, methyl-piperidino-pyrazole (MPP), was used to test the potential functional roles of spinal ERα in the nociceptive transmission. Using the whole-cell patch-clamp technique, we examined the effects of MPP on SG neurons in the dorsal root-attached spinal cord slice prepared from adult rats. We found that MPP increased glutamatergic excitatory postsynaptic currents (EPSCs) evoked by the stimulation of either Aδ- or C-afferent fibers. Further studies showed that MPP treatment dose-dependently increased spontaneous EPSCs frequency in SG neurons, while not affecting the amplitude. In addition, the PKC was involved in the MPP-induced enhancement of synaptic transmission.ConclusionsThese results suggest that the selective ERα antagonist MPP pre-synaptically facilitates the excitatory synaptic transmission to SG neurons. The nociceptive transmission evoked by Aδ- and C-fiber stimulation could be potentiated by blocking ERα in the spinal neurons. Thus, the spinal estrogen may negatively regulate the nociceptive transmission through the activation of ERα.

Effect of muscarinic acetylcholine receptor agonist on glycinergic spontaneous inhibitory postsynaptic currents and micro-inhibitory postsynaptic currents in spinal lamina II neurons of rats

Objective To investigate the effect of the mascarinic acetylcholine receptor (mAChR) agonist Oxo-tremorine-M (Oxo-M) on glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) and micro-inhibitory postaynaptic currents (mIPSCs) in lamina Ⅱ neurons in the spinal cord of rats. Methods Glycinergic IPSCs (sIPSCs and mIPSCs) in lamina Ⅱ neurons of spinal slices were recorded using the whole-cell voltage-clamp method. The non-selective mAchR ngonist Oxo-M was applied through bath perfusian. The effects of Oxo-M 1, 3, 5 and 10 μmol/L on sIPSCs and mIPSCs were examined. Results Oxo-M at the concentrations of 3-10 μmol/L significantly increased the frequency of sIPSCs without changing the amplitude in 16 lamina Ⅱ neurons tested. Interestingly, when the concentration of Oxo-M was increased to 10 μmol/L, the potentiating effect of Oxo-M on the frequency of slPSCs was decreased as compared with 3 μmol/L Oxo-M in the above 16 neurons. The slPSCs were completely abolished by 2 μmol/L strychnine. Atropine, the specific mAChR antagonist, completely blocked the effect of Oxo-M on the frequency of sIPSCs. In 9 additional lamina Ⅱ neurons, 1-10 μmol/L oxo-M failed to alter significantly the frequency and amplitude of glycinergic mIPSCs. Conclusion Activation of mAChRs in the somatodendritic site of glycinergic interneurous increases the synaptic glycine input to spinal dorsal horn neurons, but not in a dose-dependent manner. Key words: Cholinergic agonists; Receptors,cholinergic; Glycine; Spinal cord

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA(A) receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co-release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR alpha3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR alpha3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR alpha1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR alpha1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin-E-prostanoid type 2 (EP2) receptor-protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.

Colocalization of prostaglandin F2α receptor FP and prostaglandin F synthase-I in the spinal cord

Prostaglandin F(2alpha) is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F(2alpha) binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F(2alpha) synthesized in the neuronal somata and dendrites exert an autocrine action there.

FORMALIN‐INDUCED INCREASE IN P2X3 RECEPTOR EXPRESSION IN DORSAL ROOT GANGLIA: IMPLICATIONS FOR NOCICEPTION

1. ATP-gated P2X receptors in nociceptive sensory neurons participate in the transmission of pain signals from the periphery to the spinal cord. The effect of formalin on the expression of P2X(3) receptors in dorsal root ganglia (DRG) was characterized using molecular and immunological approaches and the patch-clamp technique. 2. Adult Sprague-Dawley rats were injected with 100 microL of 5% formalin in the planar surface of the hindpaw and were killed 30 min and 1, 3, 6, 12, 24 and 48 h later for in vitro analyses. The expression and distribution of P2X(3) receptors in the lumbar spinal cord and in L5/L6 DRG were examined; 24 and 48 h after formalin injection, currents in neurons were examined using whole-cell patch-clamp recording. 3. Western blots showed that anti-P2X(3) antibody recognized a major monomer of approximately 64 kDa in DRG. Immunoreactivity for P2X(3) receptors was detected predominantly in the cytoplasm and plasma membrane of small (< 25 microm) and middle-sized (25-50 microm) DRG neurons. Expression of the P2X(3) transcript in the DRG was unchanged 30 min and 1 h after formalin injection, but increased after 12 h. There was no distinct change in P2X(3) immunostaining of the spinal cord lamina at 30 min or 1 h after injection, but after 24 h P2X(3) labelling increased. At 24 h after the formalin injection, currents in isolated small and middle-sized DRG neurons were increased by 1 micromol/L alpha,beta-methylene-ATP. These currents were completely inhibited by 1 micromol/L A-317491, a potent and selective P2X(3) receptor antagonist. 4. These data suggest that formalin injection leads to early upregulation of P2X(3) expression in the spinal cord and DRG and that this may be one of the mechanisms giving rise to nociception.

Direct excitation of spinal GABAergic interneurons by noradrenaline

Endogenous pain control is, in part, mediated by descending inhibition of spinal nociception via spinal release of noradrenaline. Antinociception by activation of descending noradrenergic fibres has partially been attributed to the direct inhibition of nociceptive spinal neurons. Here, we tested the alternative hypothesis: the direct excitation of inhibitory spinal interneurons by noradrenaline. Transverse lumbar spinal cord slices were obtained from adult mice expressing enhanced green fluorescent protein (EGFP) in GABAergic neurons under control of the GAD67 promoter. Recordings were made from a total of 113 EGFP-expressing neurons and non-EGFP-expressing neurons in spinal laminae II and III with the perforated patch–clamp technique. In lamina II, where mainly nociceptive afferents terminate, noradrenaline (20μM) depolarised significantly more EGFP-labelled (41%) than non-EGFP-labelled GABAergic neurons (4%). In contrast, noradrenaline hyperpolarised significantly more non-EGFP-labelled (46%) than EGFP-labelled GABAergic neurons (20%). In lamina III, where low threshold afferents terminate, EGFP-labelled neurons were never depolarised but either hyperpolarised (25%) or not affected (75%) by noradrenaline. Depolarisations of EGFP-labelled lamina II neurons were mimicked by the α1-adrenoceptor agonist phenylephrine (10–20μM) and abolished by the α1-adrenoceptor antagonist prazosin (2μM). Hyperpolarisations of EGFP- and non-EGFP-labelled neurons were abolished by the α2-adrenoceptor antagonist yohimbine (2μM). These results show that noradrenaline directly excites inhibitory (GABAergic) lamina II interneurons in addition to its inhibitory effect on (putatively excitatory) interneurons in superficial spinal dorsal horn. Both effects of noradrenaline constitute a synergism in descending inhibition of nociceptive information in the spinal dorsal horn.