S.W.N. Thompson

The induction and maintenance of central sensitization is dependent on N-methyl-d-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states

&NA; Repetitive stimulation of small diameter primary afferent fibres produces a progressive increase in action potential discharge (windup) and a prolonged increase in the excitability of neurones in the spinal cord following the stimulus. Previous studies have demonstrated that windup is the consequence of the temporal summation of slow synaptic potentials and that the slow potentials and windup are reduced by pretreatment with N‐methyl‐d‐aspartic acid (NMDA) antagonists. We have now examined whether primary afferent induced hypersensitivity states in flexor motoneurones are also dependent on the activation of NMDA receptors and whether windup is a possible trigger for the production of the central hypersensitivity. Both a non‐competitive (MK‐801) and a competitive (D‐CPP) NMDA antagonist, at doses that did not modify the baseline reflex, reduced the facilitation of the flexor reflex produced by either brief electrical stimulation of the sural nerve (1 Hz for 20 sec at C‐fibre strength), or by the cutaneous application of the chemical irritant mustard oil. These antagonists also prevented windup from occurring in the motoneurones. When the the MK‐801 and the D‐CPP were administered once a state of central facilitation had been induced by prior treatment with mustard oil, they returned the facilitated reflex to its pretreatment level. These results indicate that NMDA receptors are involved in the induction and maintenance of the central sensitization produced by high threshold primary afferent inputs. Because central sensitization is likely to contribute to the post‐injury pain hypersenstivity states in man, these data have a bearing both on the potential role of NMDA antagonists for pre‐emptive analgesia and for treating established pain states.

Activity-Dependent Changes in Rat Ventral Horn Neurons in vitro; Summation of Prolonged Afferent Evoked Postsynaptic Depolarizations Produce a D-2-Amino-5-Phosphonovaleric Acid Sensitive Windup

The synaptic responses of lumbar ventral horn neurons including identified flexor motoneurons, to graded stimulation of peripheral nerves have been recorded in vitro in the young rat spinal cord‐hindlimb preparation. Single shock stimulation of low threshold myelinated afferents evoked short latency (< 20 ms) short duration (<1.0 s, 391 ± 42 ms n = 43 SEM) compositive mono‐ and polysynaptic potentials. Recruitment of both thinly myelinated (A delta) and unmyelinated (C) afferent fibres elicited a prolonged postsynaptic depolarization (> 1 s) in all cells. In the majority of cells (67.4%), this depolarization exceeded 4.0 s in duration (8.01 ± 0.4 s, n = 26, maximum 14 s). In the remainder, shorter responses were evoked (< 3.0 s, mean = 1.74 ± 0.4 s, n = 18). In those cells where the postsynaptic response to a single A delta or C fibre strength stimulus exceeded 4 s, low frequency (0.5–1.0 Hz) repetitive stimulation resulted in a temporal summation of the postsynaptic depolarizations, which generated a cumulatively increasing depolarization. This incrementing depolarization was sufficient in 33% of the cells to produce a progressive increase in spike discharge (windup). On cessation of the train of stimuli the depolarization decayed slowly (65 ± 27 s). The N‐methyl D‐aspartic acid (NMDA) receptor antagonist D‐2‐amino‐5‐phosphonovaleric acid (d‐APV) reduced the duration and amplitude of the prolonged postsynaptic depolarizations elicited by a single shock stimulation of small diameter afferents by 57% and 50% respectively. A smaller effect was produced on the low threshold afferent evoked early excitatory postsynaptic potentials (EPSP) (3% decrease in amplitude and 24% decrease in duration). In the presence of d‐APV the cumulatively incrementing depolarization produced by repetitive stimulation was substantially reduced and windup failed to occur. Activity‐dependent amplifications of primary afferent evoked responses in spinal neurons therefore involves a temporal summation of d‐APV sensitive prolonged postsynaptic depolarizations.

Long duration ventral root potentials in the neonatal rat spinal cord in vitro; the effects of ionotropic and metabotropic excitatory amino acid receptor antagonists

Long duration, primary afferent evoked ventral root potentials (VRPs) have been recorded in vitro from hemisected spinal cords prepared from 8-12-day-old rat pups. Single shock stimulation of a dorsal root at stimulus strengths sufficient to recruit C/group IV afferent fibres evoked a long duration (11.9 +/- 1.2 s) ipsilateral VRP in all preparations. This long duration VRP consisted of two components, (i) a slow wave, time to peak 137.0 +/- 5.1 ms, the amplitude of which was reduced to 8.7% of mean control value in the presence of the N-methyl-D-aspartate (NMDA) antagonist D-AP5 (40 microM), (ii) a prolonged wave with a time to peak of 2.0 +/- 0.2 s which was partially resistant to D-AP5 (40 microM). Both the slow and the prolonged waves were unaffected following superfusion with the metabotropic excitatory amino acid (EAA) receptor antagonist L-AP3 (100-200 microM). Low frequency (1-10 Hz) repetitive stimulation (20 s duration) of high threshold dorsal root afferents evoked a temporal summation of synaptic activity which generated a progressively depolarizing VRP. This cumulative VRP was graded with frequency of stimulation (0.89 +/- 0.13 to 1.25 +/- 0.19 mV). The cumulative VRP was followed by a post-stimulus depolarization which outlasted the period of repetitive stimulation by tens of seconds (47.6 +/- 8.4 to 91.2 +/- 19.9 s). In the presence of AP5 the amplitude of the cumulative VRP was depressed to 54.5 +/- 11.5% of control values when low frequency (1.0 Hz) stimulation was used. The proportion of the cumulative VRP resistant to D-AP5 increased as the frequency of stimulation was increased to 10 Hz. The decay time of the post-stimulus depolarization was unaffected by AP5. Neither the amplitude nor the post-stimulus depolarization of the cumulative VRP was affected by 200 microM L-AP3. It is suggested that both an AP5 sensitive and AP5 insensitive potential contribute to the long duration VRP evoked in the neonatal rat spinal cord following single shock high threshold afferent stimulation. Moreover, the AP5 insensitive prolonged depolarization is manifest following sustained low frequency stimuli and higher frequency inputs.

An intracellular analysis of amino acid induced excitations of deep dorsal horn neurones in the rat spinal cord slice

The rat spinal cord slice preparation has been used to investigate the sensitivity of deep dorsal horn neurones to the excitatory amino acids N-methyl-D-aspartate (NMDA), quisqualate and L-glutamate. Intracellular recordings were made from 44 neurones in laminae III-VI of 14- to 16-day rats. Superfusion of quisqualate (30 microM) excited all neurones, NMDA (50 microM) excited 72% and L-glutamate (0.5-1 mM) 63% of the neurones. Depolarizations were retained after tetrodotoxin but with a reduced amplitude. The NMDA antagonist D-aminophosphonovalerate (D-APV, 10 microM) reduced NMDA and L-glutamate depolarizations by 66% and by 40%, respectively, while the quisqualate responses were enhanced by 27%. Dorsal root stimulation elicited two main patterns of activity; short-latency single/double spikes followed by subthreshold excitatory postsynaptic potentials (EPSPs) or a burst of spikes rising from a long duration composite EPSP. D-APV reduced the long-latency components of the first type and reduced the amplitude and duration of the composite EPSP of the second. These results support a specialized role for NMDA receptors in synaptic transmission in the dorsal horn.

Nerve growth factor induces mechanical allodynia associated with novel A fibre-evoked spinal reflex activity and enhanced neurokinin-1 receptor activation in the rat.

&NA; A single dose of nerve growth factor (NGF, 1 &mgr;g/g, i.p.) administered to rats aged between postnatal days (PND) 12 and 14 resulted in a behavioural hypersensitivity of the hindlimb flexion withdrawal reflex to mechanical stimuli which developed 2 h after NGF and remained significant for 24 h. Heat hyperalgesia occurred some 4 h following NGF injection and lasted for 24 h. Isolated spinal cords were prepared from animals treated with NGF and were maintained in vitro for physiological and pharmacological analysis of lumbar spinal reflex activity. Repetitive, low‐frequency group I/II A&bgr;‐fibre stimulation evoked a novel wind‐up response after NGF injection similar to that produced by C‐fiber group III/ IV stimulation in normal animals. Theneurokinin‐1 (NK1) receptor antagonist RP67580 reduced the C fiber‐evoked responses following NGF treatment but not in naive preparations. The novel A&bgr; fiber‐evoked wind‐up response was also reduced by RP67580. The NGF‐induced changes in NK1 receptor responses occurred in the absence of any detectable changes in either spinal cord NK1 receptor dose‐response relationships or NK1 receptor mRNA levels. These findings are likely to be related to the behavioural allodynia observed in the present study and to central excitability changes observed after chronic inflammation where NGF levels are increased.

The responses recorded in vitro of deep dorsal horn neurons to direct and orthodromic stimulation in the young rat spinal cord

The electrophysiological properties of 87 neurons in the deep dorsal horn (laminae III-VI) of the rat spinal cord have been investigated in vitro. Two preparations have been used; the transverse spinal cord slice preparation from the third or fourth lumbar segments of 14-16-day-old rats (71 cells) and a hemisected lumbar spinal cord preparation from 10-12-day-old rats (16 cells). The input impedances (range 11-128 M omega), membrane potentials (-67 +/- 8 mV S.D.), action potential amplitude (77 +/- 11.8 mV) duration (1.4 +/- 0.5 ms) and afterpotentials, were effectively identical in the neurons recorded from the two preparations. Neurons in both preparations when activated with long-duration (1-2 s) outward current pulses showed a single steady-state firing range with little adaptation of firing frequency or action potential amplitude. This pattern of responses was unaffected by changing the membrane potential. Orthodromic synaptic activity could be elicited in the neurons by stimulating either the small dorsal root remnants in the slice or the dorsal roots in the hemisected spinal cord. The responses evoked by single stimuli of increasing intensity varied in different neurons in both preparations. The commonest response (32/62) consisted of a short-latency, short-duration composite excitatory postsynaptic potential which generated one or two spikes with no further spiking activity at longer latency when the stimulus intensity was increased beyond threshold. In 20 neurons, graded stimulation produced a graded response with recruitment, at high intensities, of a discharge of action potentials lasting several hundred milliseconds. A small number of cells (4) responded to the single stimulus with a train of action potentials lasting several seconds. Stimulating adjacent dorsal roots in the hemisected cord preparation could evoke quite different responses from the neurons. The heterogeneity of the types of orthodromic responses obtained in both preparations, in spite of the almost uniform intrinsic membrane properties, is likely to reflect differences in the strength, location and type of afferent and interneuronal input to different dorsal horn cells.

Metabotropic glutamate receptor activation contributes to nociceptive reflex activity in the rat spinal cord in vitro.

The contribution of metabotropic glutamate receptor activation to the spinal segmental reflex response evoked at high-intensity electrical stimulation suggesting a role in nociception, has been examined in an in vitro preparation of neonatal rat spinal cord. Segmental reflex responses were recorded as a ventral root depolarization evoked following drug perfusion to the spinal cord or by electrical activation of high-threshold nociceptive afferent fibres. Superfusion of the selective metabotropic glutamate receptor agonist, (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD], to the spinal cord produced a dose-dependent, reversible ventral root depolarization (EC50 = 58 +/- 7 microM; n = 4), which was antagonized by the selective metabotropic glutamate receptor antagonist, (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; IC50 = 243 +/- 61 microM; n = 4). MCPG, over the same concentration range (10 microM-5.0 mM) did not affect N-methyl-D-aspartate-induced ventral root depolarizations. In contrast, the specific N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) reduced N-methyl-D-aspartate-evoked ventral root depolarization but did not affect the depolarization evoked by (1S,3R)-ACPD, thus indicating the specificity of the antagonists for these aggregate responses. MCPG significantly reduced the prolonged phase of the single shock C-fibre-evoked ventral root depolarization (IC50 = 2.9 +/- 0.2 mM; n = 3-5). Low frequency high intensity stimulation of the dorsal root evoked a wind-up response, the amplitude of which was attenuated by both D-AP5 and MCPG in a dose-dependent manner. The ventral root depolarization evoked by capsaicin application (1.0 microM, 30 s) was blocked by both MCPG (IC50 = 809 +/- 35 microM; n = 4) and D-AP5 (IC50 = 143 +/- 43 microM; n = 4). These data suggest that both D-AP5 and MCPG reduced C-fibre-induced ventral root responses. In addition to N-methyl-D-aspartate receptor, metabotropic glutamate receptor activation appears to be involved in the generation of the segmental spinal reflex evoked by high-intensity stimulation in the neonatal rat spinal cord in vitro.

Leukemia inhibitory factor induces mechanical allodynia but not thermal hyperalgesia in the juvenile rat.

Systemic administration or local injection to the rat hindpaw of leukemia inhibitory factor induced a prolonged, dose dependent, mechanical hypersensitivity of the hindpaw flexion withdrawal reflex. Mechanical stimuli which were innocuous prior to leukemia inhibitory factor administration, evoked a rapid hindpaw withdrawal reflex indicative of mechanical allodynia. Pre-administration of anti-leukemia inhibitory factor antibodies prevented this behavioural hypersensitivity. Hindpaw sensitivity to a noxious thermal stimulus was unaffected by leukemia inhibitory factor administration. Anti-leukemia inhibitory factor had no effect upon hindpaw withdrawal thresholds when injected alone nor influenced the mechanical hypersensitivity produced by a subcutaneous injection of nerve growth factor. Injection of the closely related cytokine ciliary neurotrophic factor did not affect mechanical or thermal reflex withdrawal thresholds. Elevation of the neuroactive cytokine leukemia inhibitory factor following peripheral nerve injury may be a contributory factor to the pathogenesis of neuropathic pain.

The postnatal development of the ventral root reflex in the rat; a comparative in vivo and in vitro study.

The ventral root reflex (VRR) and its postnatal development has been studied in vivo and in an isolated spinal cord preparation from birth up to 14 days. At postnatal days 0, 7 and 14 the VRR typically consists of an early highly synchronized peak (VRR1) and a later lower amplitude peak (VRR2). In some cases, especially at day 14 in vivo, there is a longer latency asynchronous wave (VRR3). As the age of the animal increases the latencies of the first two components VRR1 and VRR2 progressively decrease mainly due to the decrease in the central delay occurring over this period. Consideration of the central delays for VRR1 and VRR2 recorded in vivo and in vitro indicates that these waves are unlikely to be elicited by C-fibre activation and a long latency C-evoked reflex discharge equivalent to that of the adult could not be reliably recorded. Up to day 7 the appearance of the VRR was similar in both preparations but diverged in the second week of life.

No evidence for contribution of nitric oxide to spinal reflex activity in the rat spinal cord in vitro

The effects of nitric oxide (NO) synthase inhibition, NO generation and an N-methyl-D-aspartic acid (NMDA) receptor antagonist upon spinal reflex responses evoked by electrical activation of high threshold afferent fibres and brief application of NMDA have been compared in an in vitro preparation of the neonatal rat spinal cord. Reflex responses of spinal cords prepared from naive animals and those exhibiting a behavioural hyperreflexia following UV irradiation of the left hindpaw have been compared. C-fibre evoked and NMDA induced ventral root potential responses were significantly reduced by the selective NMDA receptor antagonist D-AP5 (40 microM) but completely unaffected by application of 7-nitroindazole (30 microM), NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) or sodium nitroprusside (50 microM) either in hyperalgesic or naive animals. In vivo behavioural experiments performed upon age-matched rat pups showed that reflex sensitivity was significantly reduced following administration of L-NAME (30 mg kg-1). The present study has failed to provide evidence that NO is involved in nociceptive spinal reflex activity measured in vitro. In contrast, an NO synthase inhibitor was shown to influence nociceptive reflex responses observed in vivo. We suggest it is possible that NO participates in post-injury induced hyperreflexia at sites other than directly upon spinal neurones.