J. Palecek

Combined application of excitatory amino acids and substance P produces long-lasting changes in responses of primate spinothalamic tract neurons

Sensitization of dorsal horn neurons following injury may underlie the generation of secondary hyperalgesia and so the chemical basis of sensitization is now receiving considerable attention. The present study used microiontophoretic applications of excitatory amino acids (EAAs) and substance P (SP) to test their roles in the sensitization of primate spinothalamic tract (STT) neurons. Of 70 STT cells examined in laminae I-VI of the dorsal horn, 40 showed an increase in responses to one or more EAAs following their co-application with SP. The increased responses were usually specific to either N-methyl-D-aspartate (NMDA) or to the non-NMDA agonists, quisqualate (QUIS) or D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA). The enhancement of EAA responses was long-lasting (> 15 min) in 18 cases, was accompanied by similarly long-lasting increases in responses to mechanical stimulation of the receptive field in 14 cases and was accompanied by an increase in responses to either glutamate (Glu) or aspartate (Asp) in eleven cases. A global decrease in all EAA responses tested was produced in 26 other STT neurons. The inhibition, unlike the increases, was generalized to both NMDA and non-NMDA ligands, was long-lasting in only six cases and was never accompanied by a change in the responses to mechanical stimuli. The excitatory and inhibitory effects of SP on the responses to NMDA were uniformly reversed by the NK-1 receptor selective antagonist, CP96345. In contrast, only the inhibitory effects of SP on the responses to QUIS or AMPA were reversed by CP96345. The long-lasting enhancement of EAA responses by SP may follow the combined synaptic release of the natural ligands in vivo, resulting in the sensitization of dorsal horn neurons and secondary hyperalgesia. However, the reductions in EAA responses produced by SP are problematic for this hypothesis and need further elucidation.

The roles of pathways in the spinal cord lateral and dorsal funiculi in signaling nociceptive somatic and visceral stimuli in rats

&NA; The spinothalamic tract (STT) is a major ascending nociceptive pathway, interruption of which by cordotomy is used for pain relief, whereas the dorsal column (DC) pathway is usually not considered to be involved in pain transmission. However, recent clinical studies showed good relief of visceral pain in cancer patients after a DC lesion. Electrophysiological recordings in animals suggest that the analgesic effect is due to interruption of axons ascending from postsynaptic dorsal column (PSDC) neurons located in the vicinity of the central canal. In this behavioral study, we used a decrease in exploratory activity in rats after a noxious stimulus as an indicator of perceived pain, independent of withdrawal reflexes. Intradermal capsaicin injection almost abolished exploratory activity in naïve animals or in rats after a DC lesion, but did not change it in rats after ipsilateral dorsal rhizotomy or a lesion of the lateral funiculus on the side opposite to the injection. In contrast, a bilateral DC lesion counteracted the decrease in exploratory activity induced by noxious visceral stimuli for at least 180 days after the surgery. Although neurons projecting in both the STT and the PSDC path can be activated by noxious stimuli of cutaneous or visceral origin, our results suggest that the STT plays a crucial role in the perception of acute cutaneous pain and that the DC pathway is important for transmission of visceral pain.

Fos expression in spinothalamic and postsynaptic dorsal column neurons following noxious visceral and cutaneous stimuli

&NA; The spinothalamic tract (STT) has been classically viewed as the major ascending pathway for pain transmission while the dorsal column (DC) was thought to be involved primarily in signaling innocuous stimuli. Recent clinical studies have shown that limited midline myelotomy, which transects fibers in the DC, offers good pain relief in patients with visceral cancer pain. Experimental studies provided evidence that a DC lesion decreases the activation of thalamic neurons by visceral stimuli and suggested that this effect is due to transection of the axons of postsynaptic dorsal column (PSDC) neurons. In our study, Fos protein expression in retrogradely labeled STT and PSDC neurons in the lumbosacral enlargement in rats was used as an anatomical marker of enhanced activation to compare the role of these neurons in cutaneous and visceral pain. The noxious stimuli used were intradermal injection of capsaicin and distention of the ureter. Retrogradely labeled PSDC neurons were found in laminae III–IV and in the vicinity of the central canal. STT neurons were located in laminae I, III–VII and X. Ureter distention evoked Fos expression in PSDC and STT neurons located in all laminae in which retrogradely labeled cells were found, with the maximum in the L2 spinal segment. The Fos‐positive PSDC neurons represented a significantly higher percentage of the retrogradely labeled PSDC neurons (19.3±2.3% SEM) than of the STT Fos‐positive neurons (13.2±1.5% SEM). Intradermal capsaicin injection also evoked Fos expression in both PSDC and STT neurons, but with no significant difference between these two, when expressed as a percentage of the retrogradely labeled cells (11.6±2.9% SEM, 10.8±1.1% SEM). These results show that both PSDC and STT neurons are activated by cutaneous and visceral noxious stimuli. Their particular role in transmission and modulation of painful stimuli needs to be investigated further.

The non-NMDA antagonist CNQX prevents release of amino acids into the rat spinal cord dorsal horn evoked by sciatic nerve stimulation

Basal extracellular concentrations of 9 amino acids (AAs: aspartate, Asp; glutamate, Glu; asparagine, Asn; serine, Ser; glycine, Gly; threonine, Thr; alanine, Ala; taurine, Tau; and glutamine, Gln) were determined in the spinal cord dorsal horn of anesthetized rats using microdialysis and HPLC techniques. The concentrations of all measured AAs but Gln increased significantly (P < 0.05) during sciatic nerve stimulation at C-fiber strength. The concentration of Tau remained elevated following stimulation, while the other AAs returned to prestimulation values. Addition of the specific non-NMDA antagonist, CNQX, to the perfusing solution prevented the nerve stimulation-evoked AA release. Since the measured increases in extracellular AA concentrations are probably mainly due to activation of interneurons, these results suggest that blockade of non-NMDA receptors prevented activation of interneurons in the dorsal horn and support a major role of non-NMDA receptors at the first synapse of primary afferent fibers in the dorsal horn. Complete block of AA release and decreased basal levels of Glu after infusion of TTX into the dorsal horn also implies increased neuronal activity as the main source of higher AA levels during nerve stimulation.

The dorsal column pathway facilitates visceromotor responses to colorectal distention after colon inflammation in rats.

Recent clinical studies have demonstrated that a midline lesion of the dorsal columns (DC, limited midline myelotomy) reduces pain of visceral origin in patients with pelvic cancer. Animal experiments showed that a DC lesion leads to decreased activation of thalamic neurons by visceral stimuli, lowers the impact of noxious colon stimulation in behavioral tests and suggested that the effect is mediated mainly by postsynaptic DC neurons. In the present experiments we examined the effect of bilateral DC or ventrolateral (VL) spinal cord lesions on visceromotor reflex EMG activity evoked by graded colorectal distention (30, 60, 80 mmHg) under control conditions and after colon inflammation with mustard oil. The colon inflammation increased significantly the visceromotor responses so that the response to a 30 mmHg distention was larger than that produced by 80 mmHg before inflammation. The DC lesion did not affect the visceromotor reflex response under control conditions but reduced the increased responses after colon inflammation back to control levels and prevented the potentiation of the reflex responses by colon inflammation when performed before the inflammation. Our results suggest that the role of the DC pathway in transmission of visceral pain is augmented under inflammatory conditions when symptoms of visceral allodynia and hyperalgesia may be present. The VL lesions eliminated the visceromotor reflex, presumably by interrupting a facilitatory pathway that involves the brain stem.

Postsynaptic dorsal column neurons express NK1 receptors following colon inflammation

Recent clinical and experimental studies have suggested that the dorsal column pathway and specifically postsynaptic dorsal column neurons play an important role in the transmission of visceral pain. In our study we have mapped the distribution of postsynaptic dorsal column neurons in thoracic, lumbar and sacral spinal cord segments. The presence of immunoreactivity for neurokinin 1 receptors on these postsynaptic dorsal column neurons was examined under control conditions and after colon inflammation. The largest number of postsynaptic dorsal column neurons was found in the lumbar enlargement. They were mostly located in laminae III-IV, but depending on the spinal segment, about 7-15% of them were in the deep medial dorsal horn and in the central canal area. Under control conditions none of the 1438 postsynaptic dorsal column neurons examined expressed neurokinin 1 receptors. However, after induction of colon inflammation about 1.4% of the 2015 postsynaptic dorsal column neurons observed in the experimental group showed immunoreactivity for neurokinin 1 receptors. These neurons were preferentially found in the lower thoracic and lumbosacral spinal segments where they represented about 3-4% of the total population of postsynaptic dorsal column neurons examined. The de novo expression of neurokinin1 receptors on postsynaptic dorsal column neurons after colon inflammation suggests that substance P released from visceral primary afferents under inflammatory conditions could help produce central sensitization of these neurons.

The effect of phorbol esters on spinal cord amino acid concentrations and responsiveness of rats to mechanical and thermal stimuli.

There is now mounting evidence supporting the hypothesis that pathological perceptual disorders described as secondary hyperalgesia and allodynia may be due to sensitization of spinal cord dorsal horn neurons. Protein kinase C (PKC) is thought to be one of the factors in the cascade of events leading from peripheral tissue damage to the sensitization of central neurons. In our experiments, we have used local microdialysis administration of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate PKC in the spinal cord dorsal horn in awake rats. In behavioral tests the responsiveness of the animals to von Frey filaments (1-1200 mN) and to heat stimuli applied to the hindpaws was tested. Thirty minutes after the TPA infusion the threshold for the paw withdrawal response was significantly decreased (from 160 to 6 mN) and the responses to suprathreshold stimuli were more robust. An increased mechanical sensitivity was no longer present when tested 1.5 and 5 h after the TPA application was terminated. When heat stimuli were tested, the TPA infusion resulted in a significantly prolonged time during which the animals held their hindpaws above the supporting surface after the heat stimulus (0.5 and 1.5 h after TPA), and in a decreased threshold for the heat stimulus (latency of withdrawal) 5 h after TPA. HPLC analysis of the perfusate obtained by microdialysis in vivo showed a significant increase in the extracellular levels of aspartate, glutamate, glycine and taurine, and a decrease of the glutamine level during TPA infusion. The levels of asparagine, serine, threonine and alanine did not change. Application of the inactive phorbol ester (alpha-TPA) did not evoke any change from the control values either in the AA concentrations or in the behavioral tests. Our results suggest that activation of PKC in the spinal cord evokes mechanical allodynia and thermal hyperalgesia and provides further evidence that PKC is involved in the process of the modulation of nociceptive information at the spinal cord level.

The effect of trans-ACPD, a metabotropic excitatory amino acid receptor agonist, on the responses of primate spinothalamic tract neurons

&NA; The responses of primate spinothalamic tract (STT) neurons to innocuous and noxious mechanical stimuli applied to the skin can be enhanced for more than an hour following prolonged noxious stimulation. This increased responsiveness is thought to reflect sensitization of dorsal horn neurons and may help account for secondary hyperalgesia and mechanical allodynia. The proposal that central sensitization is due to the activation of second messenger systems was tested in this study by examining the effect of trans‐ACPD (trans‐D,L‐1‐amino‐1, 3‐cyclopentanedicarboxylic acid), an agonist of metabotropic excitatory amino acid (EAA) receptors, introduced into the dorsal horn by microdialysis. A low dose of trans‐ACPD resulted in an increase in the responses of STT cells to an innocuous mechanical stimulus (BRUSH), but no increase in the responses to noxious mechanical and thermal stimuli or in the excitation produced by iontophoretically applied EAAs. A high dose of trans‐ACPD caused a transient increase in background activity, but no change in the responsiveness of spinothalamic cells to any of the test stimuli. It is concluded that low doses of trans‐ACPD can selectively enhance transmission through interneuronal pathways mediating tactile inputs to spinothalamic cells.

The effect of a kainate GluR5 receptor antagonist on responses of spinothalamic tract neurons in a model of peripheral neuropathy in primates.

&NA; The responses of antidromically identified spinothalamic tract (STT) neurons to mechanical and thermal stimuli were compared in anesthetized normal and neuropathic monkeys before and after administration of a GluR5 kainate receptor antagonist (LY382884) into the spinal cord dorsal horn through a microdialysis fiber. Peripheral neuropathy was induced by tight ligation of the L7 spinal nerve 13–15 days prior to the experiment. STT neurons recorded in the animals with neuropathy showed increased responsiveness to weak mechanical stimuli and to heating and cooling of the skin compared to STT cells in normal animals. In both normal and the neuropathic monkeys the responses of the STT neurons to mechanical and thermal stimuli were attenuated by LY382884 application in a concentration‐dependent manner. Intraspinal application of LY382884 in the neuropathic animals led to a potent reduction of those responses of the STT neurons that were aggravated by the peripheral neuropathy (weak mechanical, heat and innocuous cooling stimuli). These results suggest that kainate receptors are involved in synaptic activation of STT cells in the normal state and may also play an important role in pathological pain states such as peripheral neuropathy in primates. Kainate receptor antagonists could thus be useful for the treatment of certain forms of allodynia and hyperalgesia.

Does sensitization of responses to excitatory amino acids underlie the psychophysical reports of two modalities of increased sensitivity in zones of secondary hyperalgesia

S ensitization, or enhancement of the responses of dorsal horn neurons to afferent stimuli, is emerging as one mechanism for the production of secondary hyperalgesia. The focus article by Dr. Wilcox does a wonderful job of highlighting the complexity of the chemical bases for sensitization of dorsal horn neurons. In general, sensitization of the responses of sensory neurons in the spinal cord appears to fall into two broad classes: that produced by loss of normal inhibitory processes, disinhibition,~5,2s; and that produced by an increase in the efficacy of excitatory pathways, facil i tation? ,3, 6-11,14.21,24 Both of these processes, which presumably result in an increase in the activity of projection neurons, potentially involve multiple mechanisms. We would like to raise a few additional points concerning enhancement of excitatory pathways in the dorsal horn after peripheral injury or inflammation. Dr. Wilcox reviews many studies in his focus article that show that sensitization of neurons in the dorsal horn appears to require the actions of excitatory amino acids, primarily glutamate, but also aspartate, and the neurokinin peptide, substance P (SP). The excitatory amino acids and the neuropeptides seem