CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus.

Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.

Nerve constriction in the rat: model of neuropathic, surgical and central pain.

In preparation for a series of electrophysiological experiments in a model of neuropathic pain, the present spinal reflex study was done to determine the optimal time after sciatic nerve constriction in the rat for tactile allodynia and to determine also the appropriate 'control' for the nerve constriction model. Therefore, this study focused on the magnitude and time course of change in paw withdrawal threshold following unilateral sciatic nerve constriction in the rat. Male Sprague-Dawley rats (375-425g) were used. Nerve constriction was done by placing a 2 mm polyethylene cuff (PE-90) around the left sciatic nerve (n=8). A second group of rats (n=8) received unilateral sham surgery and a third group (n=8) was unoperated. The ipsi- and contralateral hind paw withdrawal thresholds in each of the 3 groups were measured using von Frey hairs. In unoperated rats, the withdrawal threshold of each of the hind paws remained unchanged at approximately 50 g throughout the entire time course of the study, which lasted 145 days. However, in cuff-implanted rats, the withdrawal threshold of the nerve-injured hind paw decreased as soon as 1 day after surgery, reached as low as 1 to 2 g by 5 days and remained low throughout the test period. Threshold in sham-operated rats showed a bilateral decrease starting on days 1-3, which stabilised at about 30 g until about day 40, after which values returned gradually toward the unoperated withdrawal thresholds. In nerve-constricted rats the withdrawal threshold of the hind paw contralateral to the cuff followed the same change seen in sham-operated rats until about day 37, after which the withdrawal threshold matched that of the cuff-implanted hind paw. The data show that the cuff-induced sciatic nerve constriction produces a sustained hypersensitivity to normally innocuous tactile sensory input and that a relatively constant ipsilateral mechanical hyperalgesia can be found from days 5-27. It is also demonstrated that the contralateral hind paw and either hind paw in sham-operated rats are inappropriate as 'controls'. The data in this study suggest that three distinct types of allodynia are expressed. Ipsilateral allodynia may be representative of a model of neuropathic pain. The contralateral allodynia may be a model of central pain, as it likely arises from changes in central sensory processing. Allodynia in sham-operated rats was also expressed bilaterally and may be a model of long-term postoperative pain.

Mediation and modulation by eicosanoids of responses of spinal dorsal horn neurons to glutamate and substance P receptor agonists: results with indomethacin in the rat in vivo.

In view of the widespread use of non-steroidal anti-inflammatory drugs for treatment of inflammatory pain, we determined the effects of the non-steroidal anti-inflammatory drug, indomethacin, on dorsal horn neurons in the rat spinal cord in vivo. At 2.0-12.0 mg/kg (i.v.), indomethacin depressed the responses of spinal dorsal horn neurons to the effects of iontophoretic application of substance P, N-methyl-D-aspartate, quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. As indomethacin inhibits cyclo-oxygenase, these are the first data linking prostanoids and possibly arachidonic acid and other eicosanoids to the effects of substance P and glutamate in the spinal dorsal horn. As responses to iontophoretic application can be assumed to have been postsynaptic and as indomethacin had an effect generalized to all excitatory responses, we suggest a postsynaptic site for cyclo-oxygenase. We also suggest that elements in the cyclo-oxygenase signal transduction pathway may thus mediate at least some of the effects of substance P and glutamate receptor activation. Activation of the cyclo-oxygenase pathway in CNS neurons is Ca2- dependent, and activation of both N-methyl-D-aspartate and substance P receptors increases intracellular Ca2+. This led to the expectation that indomethacin would have a greater effect on responses to N-methyl-D-aspartate than to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, but the reverse was observed. Thus, in addition to a mediator role, we hypothesize that an element(s) of the cyclo-oxygenase pathway may regulate the efficacy of excitation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors and perhaps other membrane-bound receptors. The cyclo-oxygenase signal transduction pathway thus appears to play at least two major roles in regulation of sensory processing in the spinal cord. Therefore, non-steroidal anti-inflammatory drugs, via cyclo-oxygenase inhibition, may have multiple actions in control of spinal sensory mechanisms.

Mechanisms underlying antinociception provoked by heterosegmental noxious stimulation in the rat tail-flick test.

Physiological studies were conducted to examine the effects of noxious stimulation of one hindpaw or one forepaw on the latency of the withdrawal reflex in the tail-flick test in lightly anesthetized spinally intact or transected rats. Male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, the effects of various noxious stimuli applied to remote body regions were assessed. The noxious stimuli included unilateral or bilateral hindpaw or unilateral forepaw thermal (immersion in water at 55 degrees C for 90 s), unilateral or bilateral chemical (subcutaneous hindpaw injection of 50 microliters of 5% formalin) and unilateral or bilateral mechanical (pinch with clamp exerting a force of 14.75 or 27 N) stimulation. Bilateral chemical and thermal, and unilateral thermal stimulation induced an antinociceptive response, consisting of an increase in tail-flick latency, peaking at 30 s after stimulation. Recovery to baseline levels occurred over the next 3-6 min. The antinociceptive effect of noxious thermal stimulation was attenuated or absent in chronically spinalized animals (T6/7) following hindpaw or forepaw immersion, respectively. Noxious mechanical stimulation had no effect on tail-flick latency. The data provide evidence that a noxious thermal or chemical stimulus produces a heterosegmental antinociceptive effect which is mediated in part via a supraspinal mechanism and in part via a local spinal mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands.

The effect of testing surface on the rat hind paw withdrawal threshold in the von Frey hair test is investigated in this study. The data indicate that wire mesh, which is typically used to apply von Frey hairs, may have an effect on the paw withdrawal threshold. For example, in control rats tested on the wire mesh, variability in the withdrawal threshold was observed between the left and the right hind paws (51.04+/-12.29 and 64.31+/-9.37 g, respectively) and on different days of testing (35.24+/-9.54 and 45.83+/-12.97 g for the left and right hind paws, respectively, 7 days later). In an attempt to reduce this variability, a customized platform was used to measure the von Frey hair-induced paw withdrawal in the rat. It consists of an opaque, flat-surfaced plastic platform with holes through which von Frey hairs are inserted and applied to the plantar surface of the paw. In control rats tested with von Frey hairs using this customized platform, variability in the paw withdrawal thresholds between the left and right hind paws in single rats over time as well as between different rats was reduced (49.86+/-6.97 and 49.29+/-6.56 g for the left and right hind paws, respectively, on day 0; 48.29+/-5.82 and 53.00+/-4.59 g for the left and right hind paws, respectively, 7 days later). Furthermore, in rats in which a 2 mm polyethylene cuff was used to constrict the left common sciatic nerve, the ipsilateral as well as the contralateral hind paw withdrawal thresholds were decreased (2.45+/-0.65 and 26.09+/-5.86 g, respectively, 7 days later). In similar rats tested on the wire mesh, the ipsilateral but not the contralateral paw withdrawal threshold decreased (12.80+/-2.21 and 65.00+/-10.28 g, respectively, at 7 days). The data suggest that the flat surface and opaque properties of the customized platform enable accurate, reliable and repeatable measurements of ipsilateral and contralateral paw withdrawal threshold using von Frey hairs in normal and nerve-injured rats.

Noxious peripheral stimulation produces antinociception mediated via substance P and opioid mechanisms in the rat tail-flick test.

Physiological experiments were run to examine the effects of noxious thermal stimulation of one hindpaw on the tail-flick reflex in the lightly anesthetized rat. Male Sprague-Dawley rats were anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail-flick test, either a non-noxious or a noxious stimulus was applied which consisted of immersion of one hindpaw in water at 40, 45, 50 or 55 degrees C for 1.5 min. After immersion, tail-flick readings were taken at 3-min intervals for at least 16 min. Paw immersion in water at 55 degrees C induced an antinociceptive response, consisting of an increase in the reaction time, at 0.5 min after immersion. Recovery to baseline levels occurred over the next 3-6 min. Immersion at lower temperatures provoked smaller antinociceptive responses, except at 40 degrees C, where readings remained around the baseline values. The increase in reaction time in response to immersion at 55 degrees C was attenuated or blocked by the novel, nonpeptide substance P (NK-1) receptor antagonist, CP-96,345, administered s.c. 30 or 60 min, respectively, prior to paw immersion. Similar injection of CP-96,344, the inactive stereoisomer, had no effect on the response, while another NK-1 receptor antagonist, CP-99,994, also attenuated the antinociceptive effect of the immersion. The increase in reaction time induced by immersion at 55 degrees C was absent in animals treated neonatally with capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Meloxicam selectively depresses the afterdischarge of rat spinal dorsal horn neurones in response to noxious stimulation.

This study examined the effects of the cyclooxygenase-2 (COX-2) inhibitor, meloxicam, on responses of spinal dorsal horn neurones to synaptic inputs in vivo. Experiments were run using male Sprague-Dawley rats (350-375 g) anesthetized with Na-pentobarbital (50 mg/kg, i.p.) and acutely spinalized at T9. Spinal segments L1-4 were exposed for extracellular single unit recording of on-going and stimulation-evoked activity of eight wide dynamic range neurones. On-going activity was unaffected by meloxicam (0.1 mg/kg, i.v.). However, responses to noxious mechanical stimulation (21 N for 3 s) of the cutaneous receptive field of the hind paw were depressed by meloxicam in particular the afterdischarge (34.50 +/- 6.06% inhibition) in all eight neurones studied. The brief initial discharge in response to stimulation was depressed less (15.31 +/- 4.89% inhibition, n = 7/8). The data indicate that the percent inhibition of the afterdischarge was significantly greater than that of the initial discharge (P < 0.05). Given the selectivity of meloxicam for COX-2, the data suggest that COX-2 may be involved in mediating and/or modulating excitatory effects of nociceptive inputs to dorsal horn neurones, in particular the prolonged stimulation-evoked afterdischarge.

Cellular mechanisms of hyperalgesia and spontaneous pain in a spinalized rat model of peripheral neuropathy: changes in myelinated afferent inputs implicated.

Various hypotheses have been proposed to account for the mechanical hyperalgesia and spontaneous pain seen in animal models of peripheral neuropathy. The purpose of the present study was to determine whether there exists a spinal neuronal correlate to these properties. An experimental neuropathy was induced in male Sprague-Dawley rats by placing a 2-mm PE-90 polyethylene cuff around the sciatic nerve. All rats were subsequently confirmed to exhibit mechanical allodynia in the von Frey test. After induction of anaesthesia with pentobarbital and acute spinalization at T9, electrophysiological experiments were performed, recording extracellular single unit activity from ipsi- and contralateral wide dynamic range dorsal horn neurons in spinal segments L1-4. On-going activity was greater in short-term (11-22 days after cuff implantation) and long-term (42-52 days) cuff-implanted rats; 38 spikes/s in short-term versus 19 spikes/s in controls; 29 spikes/s in long-term ipsi- and contralateral neurons. Receptive fields in controls were always restricted, but in almost all cuff-implanted rats extended over the whole hind paw. Responses to noxious mechanical (pinch) and noxious heat stimulation of the cutaneous receptive field in controls consisted of the typical fast initial discharge followed by an afterdischarge. In all neurons from cuff-implanted rats the initial discharge resembled that in controls. However, the afterdischarge, particularly that in response to noxious pinch, was markedly greater in both magnitude and duration. It is suggested that the greater on-going discharge is the cellular correlate of spontaneous pain, and the potentiation of the afterdischarge in response to noxious stimulation is the correlate of hyperalgesia. Given that acutely spinalized rats were tested, only peripheral and/or spinal mechanisms can be considered to explain these data. Considering all the data, it can be concluded that there is a greater change in fibres mediating noxious mechanical than noxious thermal inputs. Among different hypotheses, the one with which the present data are most compatible is that which proposes that chronic nerve injury or inflammation induces phenotypic changes predominantly in myelinated afferents. There may be a redistribution of membrane-bound ion channels, predominantly sodium channels, which leads to ectopic activity and thus spontaneous discharge of dorsal horn neurons. With regard to mechanical stimulation-evoked synaptic input, the central terminals of myelinated afferents expand into regions of the spinal cord which normally receive their predominant input from unmyelinated nociceptive afferents. This may be coupled with a change in these myelinated afferents so that they now synthesize and release peptides, primarily substance P, from their central terminals with the result that the effects of their chemical mediators of synaptic transmission add to the effects of nociceptive inputs leading to exaggerated responses to painful stimuli, thus the basis of clinical hyperalgesia.

Noxious thermal and chemical stimulation induce increases in 3H-phorbol 12,13-dibutyrate binding in spinal cord dorsal horn as well as persistent pain and hyperalgesia, which is reduced by inhibition of protein kinase C.

We have previously suggested that protein kinase C (PKC) contributes to persistent pain in the formalin test. This study compared the effects of pharmacological inhibition of PKC with either GF 109203X or chelerythrine on persistent pain following noxious chemical stimulation with its effects on mechanical hyperalgesia, which develops in the hindpaw contralateral to an injury produced by noxious thermal stimulation. Furthermore, we have assessed changes in membrane-associated PKC in spinal cord in response to both noxious chemical and thermal stimulation. Nociceptive responses, to a hindpaw injection of 50 microliters of 2.5% formalin, and flexion reflex thresholds, to mechanical stimulation (Randall-Selitto test) in the hindpaw contralateral to a thermal injury (15 sec immersion in water at 55 degrees C), were assessed following intrathecal injection of PKC inhibitors (GF 109203X or chelerythrine). Changes in the levels of membrane-associated PKC, as assayed by quantitative autoradiography of the specific binding of 3H-phorbol-12,13-dibutyrate (3H-PDBu) in spinal cord sections, were assessed in rats after noxious chemical (50 microliters of 5.0% formalin) and noxious thermal (90 sec immersion in water at 55 degrees C) stimulation. Inhibitors of PKC (GF 109203X, chelerythrine), produced significant reductions of nociceptive responses to 2.5% formalin, as well as a significant reduction in the mechanical hyperalgesia in the hindpaw contralateral to a thermal injury. In addition, both noxious chemical and thermal stimulation produced significant increases in specific 3H-PDBu binding in the dorsal horn of the lumbar spinal cord, likely reflecting alterations in membrane-associated PKC. The results provide both pharmacological and anatomical evidence that persistent pain produced by chemical stimulation with formalin and mechanical hyperalgesia in the hindpaw contralateral to a thermal injury are influenced by the translocation and activation of PKC in spinal cord dorsal horn neurons.

Evidence for tonic activation of NK-1 receptors during the second phase of the formalin test in the Rat.

Behavioral, electrophysiological, and autoradiographic experiments were done to study the second nociceptive phase in the formalin test. In initial experiments, this second phase was attenuated by 1-10 mg of the NK-1 receptor antagonist CP-99,994, given subcutaneously 10, 30, or 60 min before formalin (n = 8-10) and by 20 microgram given intrathecally 20 min after formalin (n = 13); the inactive isomer CP-100,263 was ineffective. In electrophysiological experiments on single dorsal horn neurons in vivo, the excitatory responses to subcutaneous formalin injection (50 microliter, 2.5%) were attenuated by subsequent intravenously administration of the NK-1 receptor antagonist CP-96,345 (0.5 mg/kg; n = 8), given 35-40 min after formalin, but not by the inactive enantiomer CP-96,344 (0.5 mg/kg; n = 9). Finally, autoradiographic binding of exogenous [(125)I]BH-substance P in the lumbar cord was reduced at 5 and 25 min after formalin (50 microliter, 1 or 5%), with an intermediate level of reduction at 12 min. These data are interpreted as evidence that the second phase of nociceptive scores in the formalin test is attributable at least partially to tonic activation of NK-1 receptors at the spinal level, whether because of a temporally limited release of substance P, for example only during the first phase, but a slow removal or breakdown of substance P, or, more likely, because of tonic release from primary afferents throughout the second phase. Irrespective of the mechanism, it can be concluded that at least some of the persistent nociceptive effects associated with peripheral inflammation, or at least those provoked by subcutaneous injection of formalin, are mediated via continuous activation of NK-1 receptors at the level of the spinal dorsal horn; this may relate directly to mechanisms underlying prolonged nociceptive pains in humans.

mu-, delta- and kappa-opiate receptors mediate antinociception in the rat tail flick test following noxious thermal stimulation of one hindpaw.

Experiments were performed to investigate the possible involvement of spinal mu-, delta- and kappa-opiate receptors in mediating the antinociceptive effects of noxious thermal stimulation of one hindpaw on the tail flick reflex in the rat. Male Sprague-Dawley rats were implanted with chronic intrathecal catheters to the lumbar level of the spinal cord. After 5 to 7 days, they were lightly anesthetized with an i.p. injection of a mixture of Na-pentobarbital (20 mg/kg) and chloral hydrate (120 mg/kg). After baseline readings were taken in the tail flick test a conditioning noxious thermal stimulus, which consisted of immersion of one hindpaw in water at 55 degrees C for 90 sec, was applied and the effects on the latency of the tail withdrawal reflex were studied over the next 30 min. In animals pretreated with CSF intrathecally 10 min before the stimulus, an increase in tail flick reaction time was observed peaking at 30 sec after the stimulus. This response was attenuated in a dose-related manner by preadministration of the specific mu-opiate receptor antagonist, beta-funaltrexamine, the specific delta-opiate receptor antagonist, H-Tyr-Tic psi[CH2NH]-Phé-Phe-OH or the specific kappa-opiate receptor antagonist, nor-binaltorphimine. The data show that the antinociceptive effect on the tail withdrawal reflex from a brief noxious thermal stimulus is provoked heterosegmentally by the noxious conditioning stimulus to the hindpaw and is mediated by the endogenous release of ligands that bind to mu-, delta- and kappa-opiate receptors in the spinal cord.