Population Of Dorsal Horn Neurons Research Articles

Glycinergic dysfunction in a subpopulation of dorsal horn interneurons in a rat model of neuropathic pain.

The development of neuropathic pain involves persistent changes in signalling within pain pathways. Reduced inhibitory signalling in the spinal cord following nerve-injury has been used to explain sensory signs of neuropathic pain but specific circuits that lose inhibitory input have not been identified. This study shows a specific population of spinal cord interneurons, radial neurons, lose glycinergic inhibitory input in a rat partial sciatic nerve ligation (PNL) model of neuropathic pain. Radial neurons are excitatory neurons located in lamina II of the dorsal horn, and are readily identified by their morphology. The amplitude of electrically-evoked glycinergic inhibitory post-synaptic currents (eIPSCs) was greatly reduced in radial neurons following nerve-injury associated with increased paired-pulse ratio. There was also a reduction in frequency of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSC) in radial neurons without significantly affecting mIPSC amplitude. A subtype selective receptor antagonist and western blots established reversion to expression of the immature glycine receptor subunit GlyRα2 in radial neurons after PNL, consistent with slowed decay times of IPSCs. This study has important implications as it identifies a glycinergic synaptic connection in a specific population of dorsal horn neurons where loss of inhibitory signalling may contribute to signs of neuropathic pain.

Tumor necrosis factor α sensitizes spinal cord TRPV1 receptors to the endogenous agonist N-oleoyldopamine

Modulation of synaptic transmission in the spinal cord dorsal horn is thought to be involved in the development and maintenance of different pathological pain states. The proinflamatory cytokine, tumor necrosis factor α (TNFα), is an established pain modulator in both the peripheral and the central nervous system. Up-regulation of TNFα and its receptors (TNFR) in dorsal root ganglion (DRG) cells and in the spinal cord has been shown to play an important role in neuropathic and inflammatory pain conditions. Transient receptor potential vanilloid 1 (TRPV1) receptors are known as molecular integrators of nociceptive stimuli in the periphery, but their role on the spinal endings of nociceptive DRG neurons is unclear. The endogenous TRPV1 receptor agonist N-oleoyldopamine (OLDA) was shown previously to activate spinal TRPV1 receptors. In our experiments the possible influence of TNFα on presynaptic spinal cord TRPV1 receptor function was investigated. Using the patch-clamp technique, miniature excitatory postsynaptic currents (mEPSCs) were recorded in superficial dorsal horn neurons in acute slices after incubation with 60 nM TNFα. A population of dorsal horn neurons with capsaicin sensitive primary afferent input recorded after the TNFα pretreatment had a basal mEPSC frequency of 1.35 ± 0.20 Hz (n = 13), which was significantly higher when compared to a similar population of neurons in control slices (0.76 ± 0.08 Hz; n = 53; P < 0.01). In control slices application of a low concentration of OLDA (0.2 uM) did not evoke any change in mEPSC frequency. After incubation with TNFα, OLDA (0.2 uM) application to slices induced a significant increase in mEPSC frequency (155.5 ± 17.5%; P < 0.001; n = 10). Our results indicate that TNFα may have a significant impact on nociceptive signaling at the spinal cord level that could be mediated by increased responsiveness of presynaptic TRPV1 receptors to endogenous agonists. This could be of major importance, especially during pathological conditions, when increased levels of TNFα and TNFR are present in the spinal cord.

Enkephalins, dynorphins, and β‐endorphin in the rat dorsal horn: An immunofluorescence colocalization study

To characterize neuronal pathways that release opioid peptides in the rat dorsal horn, multiple-label immunohistochemistry, confocal microscopy, and computerized co-localization measures were used to characterize opioid-containing terminals and cells. An antibody that selectively recognized beta-endorphin labeled fibers and neurons in the ventral horn as well as fibers in the lateral funiculus and lamina X, but practically no fibers in the dorsal horn. An anti-enkephalin antibody, which recognized Leu-, Met-, and Phe-Arg-Met-enkephalin, labeled the dorsolateral funiculus and numerous puncta in laminae I-III and V of the dorsal horn. An antibody against Phe-Arg-Met-enkephalin, which did not recognize Leu- and Met-enkephalin, labeled the same puncta. Antibodies against dynorphin and prodynorphin labeled puncta and fibers in laminae I, II, and V, as well as some fibers in the rest of the dorsal horn. Dynorphin and prodynorphin immunoreactivities colocalized in some puncta and fibers, but the prodynorphin antibody additionally labeled cell bodies. There was no co-localization of dynorphin (or prodynorphin) with enkephalin (or Phe-Arg-Met-enkephalin). Enkephalin immunoreactivity did not colocalize with the C-fiber markers calcitonin gene-related peptide (CGRP), substance P, and isolectin B4. In contrast, there was some colocalization of dynorphin and prodynorphin with CGRP and substance P, but not with isolectin B4. Both enkephalin and dynorphin partly colocalized with vesicular glutamate transporter 2, a marker of glutamatergic terminals. The prodynorphin-positive neurons in the dorsal horn were distinct from neurons expressing mu-opioid receptors, neurokinin 1 receptors, and protein kinase C-gamma. These results show that enkephalins and dynorphins are present in different populations of dorsal horn neurons. In addition, dynorphin is present in some C-fibers.

Both Ca2+‐permeable and ‐impermeable AMPA receptors contribute to primary synaptic drive onto rat dorsal horn neurons

Blockade of Ca2+-permeable AMPA receptors in the rat spinal cord diminishes the development of hyperalgesia and allodynia associated with peripheral injury. Cobalt uptake studies reveal that Ca2+-permeable AMPA receptors are expressed by some substance P receptor-expressing (NK1R+) neurons in lamina I, as well as other neurons throughout the superficial dorsal horn. Selective elimination of NK1R+ neurons in lamina I and lamina III/IV of the dorsal horn also suppresses development of hyperalgesia and allodynia. These observations raise the possibility that Ca2+-permeable AMPA receptors contribute to excitatory synaptic drive onto the NK1R+ neurons associated with allodynia and hyperalgesia. The first synapse in the pain pathway is the glutamatergic excitatory drive from the primary afferent fibres onto dorsal horn neurons. Therefore, we tested whether Ca2+-permeable AMPA receptors are located on lamina I and lamina III/IV NK1R+ neurons postsynaptic to primary afferent fibres, using inward rectification and polyamine toxins for receptor identification. We examined three different populations of dorsal horn neurons; lamina I NK1R+ neurons, including projection neurons, and non-NK1R+ (NK1R-) neurons including interneurons, and lamina III/IV NK1R+ neurons, believed to contribute to the low-threshold mechanosensory pathway. The majority of synapses in all three groups had rectification indices less than 1.0 and greater than 0.4, indicating that the AMPA receptors at these synapses are a mixture of Ca2+-permeable and -impermeable forms. Lamina III/IV NK1R+ neurons and lamina I NK1R- neurons have a significantly higher proportion of postsynaptic Ca2+-permeable AMPA receptors than lamina I NK1R+ neurons. Thus synaptically positioned Ca2+-permeable AMPA receptors directly contribute to low-threshold sensory afferent drive into the dorsal horn, and can mediate afferent input onto interneurons such as GABAergic neurons. These receptors also contribute to high-threshold primary afferent drive onto NK1R+ neurons in the superficial dorsal horn, but do so less consistently.

Differential effects of spinal CNQX on two populations of dorsal horn neurons responding to colorectal distension in the rat

The present study examined the effect of a spinally administered excitatory amino acid antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 2.5, 5 μg) on responses of spinal dorsal horn neurons to graded intensities (20, 40, 60, 80 mmHg) of colorectal distention (CRD). Extracellular single unit recordings were made from 28 dorsal horn neurons in the L6–S2 spinal cord. Neurons excited by CRD were subclassified as short latency abrupt (SLA) neurons and short latency sustained (SLS) neurons. The response to graded intensities of CRD was dose-dependently attenuated in 9/17 SLA neurons (53%). The response to CRD was also dose-dependently attenuated in 8/11 SLS neurons (73%). The response to CRD in the remaining eight SLA neurons and three SLS neurons was not attenuated by CNQX. Comparing only neurons that were significantly attenuated by the CNQX, it was found that the magnitude of attenuation of the response to noxious CRD (80 mmHg) produced by 5 μg CNQX was significantly greater in SLA (63±6%) vs. SLS (40±6%) neurons. While CNQX produced a significant attenuation of the response to innocuous CRD (20 mmHg), there was no difference between the SLA and SLS neurons. The effects of CNQX on the response to somatic stimulation (touch, pinch) of the cutaneous receptive field of these 28 neurons were qualitatively examined in all neurons and quantitatively examined in nine neurons (five SLA and four SLS neurons). CNQX generally decreased the response to pinch or touch, even if CNQX did not attenuate the response to CRD. These results suggest that subpopulations of SLA and SLS neurons are differentially modulated by non-NMDA ionotropic excitatory amino acid receptors and that these neuronal subtypes contribute differently to visceral sensory processing. Furthermore, the lack of correlation between the effects of CNQX on visceral and somatic sensory processing in the same neuron underscores potential differences in processing of visceral and somatic pain.

Subpopulations of GABAergic and non-GABAergic rat dorsal horn neurons express Ca2+-permeable AMPA receptors.

Subpopulations of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors that are either permeable or impermeable to Ca2+ are expressed on dorsal horn neurons in culture. While both mediate synaptic transmission, the Ca2+ -permeable AMPA receptors provide a Ca2+ signal that may result in a transient change in synaptic strength [Gu, J.G., Albuquerque, C., Lee, C.J. & MacDermott, A.B. (1996) Nature, 381, 793]. To appreciate the relevance of these receptors to dorsal horn physiology, we have investigated whether they show selective expression in identified subpopulations of dorsal horn neurons. Expression of Ca2+-permeable AMPA receptors was assayed using the kainate-induced cobalt loading technique first developed by Pruss et al. [Pruss, R.M., Akeson, R.L., Racke, M.M. & Wilburn, J.L. (1991) Neuron, 7, 509]. Subpopulations of dorsal horn neurons were identified using immunocytochemistry for gamma-aminobutyric acid (GABA), glycine, substance P receptor (NK1 receptor) and the Ca2+-binding proteins, calretinin and calbindin D28K. We demonstrate that, in dorsal horn neurons in culture, kainate-induced cobalt uptake is selectively mediated by Ca2+-permeable AMPA receptors, and that a majority of GABA and NK1 receptor-expressing neurons express Ca2+-permeable AMPA receptors. GABAergic dorsal horn neurons are important in local inhibition as well as in the regulation of transmitter release from primary afferent terminals. NK1 receptor-expressing dorsal horn neurons include many of the projection neurons in the nociceptive spino-thalamic pathway. Thus, we have identified two populations of dorsal horn neurons representing important components of dorsal horn function that express Ca2+-permeable AMPA receptors. Furthermore, we show that several subpopulations of putative excitatory interneurons defined by calretinin and calbindin expression do not express Ca2+-permeable AMPA receptors.

Midbrain periaqueductal gray (PAG) inhibits nociceptive inputs to sacral dorsal horn nociceptive neurons through alpha2-adrenergic receptors.

Modulation of sacral spinal dorsal horn neurons by the ventrolateral PAG was studied by extracellular recording combined with microiontophoretic applications of alpha-adrenergic agonists or antagonists. Bicuculline (BIC, 15 ng) microinjected into the ventrolateral PAG produced a consistent inhibition of the responses of nociceptive dorsal horn neurons. After PAG-BIC applications, the total number of spikes per heat stimulation period was significantly decreased to a mean of 37 +/- 19% (n = 8) of the pre-BIC control. Local iontophoresis of the selective alpha2-adrenoceptor antagonists idazoxan or yohimbine but not the selective alpha1 antagonist benoxathian significantly reversed PAG-BIC-evoked inhibition. At low ejection currents, clonidine, an alpha2-adrenoceptor agonist, markedly reduced noxious heat-evoked responses but had no consistent action on the responses to iontophoresed excitatory amino acids [EAA; N-methyl--aspartate (NMDA) or kainic acid]. At ejection currents higher than required to block descending inhibition, idazoxan potentiated responses to both heat and EAA iontophoresis. At higher ejection currents, EAA responses were inhibited by clonidine. This indicates that both presynaptic and postsynaptic alpha2 receptors are capable of inhibiting the recorded neurons. Activation of the alpha1 adrenoceptors by iontophoresis of methoxamine often led to a marked increase in the responses to kainic acid and, to a lesser extent, to NMDA iontophoresis or noxious heat. Together with previously reported work, the current experiments demonstrate that PAG neurons inhibit nociceptive dorsal horn neurons primarily through an indirect alpha2 adrenoceptor mechanism. In this same population of dorsal horn neurons, norepinephrine has a direct alpha1-mediated excitatory effect.

Sympathetically correlated activity of dorsal horn neurons in spinally transected rats.

In mammals with an intact neuraxis, most sympathetic nerve activity is generated by brain stem systems. Therefore these systems have attracted much more attention than spinal systems that generate excitatory inputs to sympathetic preganglionic neurons. The purpose of this study was to determine whether, within hours of C1 spinal cord transection, spinal dorsal horn neurons (DHNs) play a role in generating sympathetic nerve activity. Experiments were conducted in chloralose-anesthetized rats. We recorded renal sympathetic nerve activity (RSNA) in the left renal nerve, and we recorded the activity of neurons located in the left dorsal horn at T2, T8, T10, T13, and L2. We also recorded the activity of neurons in the right dorsal horn at T10. The somatic fields and cutaneous modalities of most neurons were determined. Spike-triggered averaging was used to determine relationships between the ongoing activity of DHNs and ongoing RSNA. In the left dorsal horn, bursts of ongoing activity of 16% of DHNs at T8 and 43% of DHNs at T10 were positively correlated with bursts of ongoing RSNA at latencies of 59 +/- 8 (SE) ms. At no other level on the left side, nor in the T10 segment on the right side, was the activity of DHNs correlated with RSNA. DHNs with activity correlated with RSNA were located only in dorsal horn laminae III-V. Deeper laminae were not investigated in these experiments. The activity of all sympathetically correlated DHNs exhibited bursts of action potentials with interspike intervals of < 10 ms. All but one of the sympathetically correlated DHNs exhibited wide-dynamic-range modalities. The modalities of sympathetically uncorrelated neurons were more heterogeneous. Brief (5-10 s) noxious cutaneous stimulation of mid- and lower thoracic dermatomes on the left side excited all sympathetically correlated DHNs and simultaneously increased RSNA. The excitatory cutaneous fields of sympathetically correlated neurons were circumscribed by the excitatory fields for RSNA. The excitatory cutaneous fields of some sympathetically uncorrelated DHNs extended beyond the excitatory fields for RSNA. Noxious cutaneous stimulation of the extremities on the left side that decreased RSNA simultaneously decreased the activity of all sympathetically correlated DHNs. These data provide electrophysiological evidence that, in spinally transected rats, a population of DHNs may generate or convey excitatory input to renal sympathetic preganglionic neurons.

Colocalization of Fos- and glucocorticoid receptor-immunoreactivities is present only in a very restricted population of dorsal horn neurons of the rat spinal cord after nociceptive stimulation

Immunocytochemical techniques were used to study the colocalization of proto-oncogene c-fos protein (Fos)-like immunoreactivity activity and of glucocorticoid receptor immunoreactivity in the spinal cord dorsal horn of adult male rats after nociceptive stimulation. About 5–10% of the stimulus evoked Fos-immunoreactive profiles colocalized glucocorticoid receptor immunoreactivity. The results suggest the possibility of a corticosteroid action in a small neuronal population of the dorsal horn implicated in processing nociceptive inputs.

Activity evoked by A- and C-afferent fibers in rat dorsal horn neurons and its relation to a flexion reflex.

The responses of 56 neurons recorded in the lumbosacral spinal cord of halothane-anesthetized rats were studied following the application of mechanical stimuli to the skin on the lateral aspect of the paw or electrical stimulation of the sural nerve. Only neurons driven by A- and C-fiber stimulation were considered. The evoked activity in a nerve supplying flexor muscles, the common peroneal nerve, was also recorded to evaluate possible relations between neuronal events and reflex discharges. To quantify the reflex output we also recorded the activity of 12 motoneurons. Four different populations of dorsal horn neurons activated by C-fibers could be distinguished. The neurons were classified on the basis of their responses to mechanical stimuli and of their location in the dorsal horn. Class 1 neurons were driven by nonnoxious stimulation only. Neurons driven by nonnoxious stimuli and noxious stimuli were denoted class 2S (superficial to the location of the maximal A-beta-fiber-evoked field potentials) or class 2D (deep to the same potential). Class 3 neurons were driven by noxious stimuli only. The functional characteristics of these four classes of neurons differed in many respects. The latency for the A-beta-fiber-evoked discharge was, on average, 2 ms longer in class 2S than in class 2D neurons, indicating a polysynaptic A-beta input to the former class of neurons. The C-fiber-evoked neuronal discharge often showed time-locked peaks of activity during the interval 120-170 ms. Such peaks of activity occurred, in general, later in class 2D neurons (mean, 157 ms) than in class 2S (mean, 137 ms) or in class 3 (mean, 140 ms), suggesting that the different classes received C-fiber input via partially different routes. The responses to repeated C-fiber stimulation also differed markedly among the four classes. After 16 single electrical stimulations (100 T (T = threshold strength for activating A-beta-afferents), 1 Hz), the C-fiber-evoked discharge in class 2D neurons was increased by 196%, whereas the corresponding value for those in classes 2S, 3, and 1 was 41, 24, and 38%, respectively. Ten of 14 class 2D neurons showed a simultaneous increase of the A-fiber-evoked discharge, indicating an increased excitability of these neurons after repeated impulses in C-fiber afferents. An early reflex discharge (latency, 6-10 ms) was evoked in the common peroneal nerve by electrical stimulation of the sural nerve.(ABSTRACT TRUNCATED AT 400 WORDS)