Increased responsiveness of rat dorsal horn neurons in vivo following prolonged intrathecal exposure to interferon-gamma.

Prolonged increases in the level of the pro-inflammatory cytokine interferon-gamma occur in the CNS during some disease states associated with persistent pain. Administration of interferon-gamma to both humans and rodents has produced pain or pain-related behavior but the underlying mechanisms are unknown. The present study examined the effects of repeated intrathecal administration of interferon-gamma on dorsal horn neuronal responses under in vivo conditions. In addition, behavioral effects of interferon-gamma treatment were studied. Intrathecal cannulae were implanted into anesthetized rats. Animals then received either 1000 U of recombinant rat interferon-gamma in 10 microl buffer intrathecally, repeated four times over 8 days, or similarly administered buffer (controls). Interferon-gamma-treated animals showed a significant reduction in paw withdrawal threshold to mechanical stimulation of the hind paw. Electrophysiological experiments were performed under halothane anesthesia. Extracellular recordings of spontaneous and evoked responses were obtained from dorsal horn neurons (n=64) in the lumbar spinal cord. There was a significantly higher proportion of spontaneously active neurons in the interferon-gamma-treated animals (50%) when compared with controls (19%). A significantly increased proportion of neurons from interferon-gamma-treated animals displayed afterdischarges following both innocuous and noxious mechanical stimulation of the receptive field (brush: 21% in interferon-gamma-treated, 3% in controls; pinch: 97% in interferon-gamma-treated, 50% in controls). Neurons from interferon-gamma-treated animals also showed significantly increased wind-up of action potentials in response to repeated electrical stimulation of the sciatic nerve at C-fiber strength at both 0.5 and 1 Hz. Paired-pulse inhibition, evoked through electrical stimulation of the cutaneous receptive field, was significantly decreased in neurons from interferon-gamma-treated animals at 50 and 100 ms inter-stimulus intervals. We propose that this demonstrated reduction in inhibition may underlie the enhanced excitatory responses. Such interferon-gamma-induced changes in evoked responses may contribute to persistent pain following damage or disease states in the nervous system.

Periaqueductal grey stimulation: an association between selective inhibition of dorsal horn neurones and changes in peripheral circulation.

In barbiturate-anaesthetized and paralysed cats, dorsal horn neurones were studied during electrical stimulation of the periaqueductal grey matter (PAG) and the midbrain ventral tegmentum (VT). Responses to impulses in unmyelinated primary afferents were selectively inhibited by stimulation in the PAG, whereas stimulation in the VT non-selectively reduced both these responses and those to innocuous cutaneous stimuli. Stimulation in the PAG but not the VT produced changes in peripheral circulation. This was observed as a rise in the levels of carbon dioxide in expired air, a rise in muscle temperature in the hind limb and a fall in skin temperature of the pinna or glabrous skin. The combination of suppression of spinal transmission of impulses related to pain and an increase in perfusion of muscles may be a mechanism appropriate to coping with a potentially injurious environment.

Bicuculline and spinal inhibition produced by dorsal column stimulation in the cat.

In barbiturate anaesthetized cats, dorsal column stimulation inhibited ascending volleys recorded in the antero-lateral spinal fasciculus from electrical stimulation of the contralateral tibial nerve and the excitation of neurones of the dorsal horn by noxious heating of the skin. The inhibition was non-selective. Intravenous bicuculline (0.2-0.6 mg/kg) reduced dorsal column induced inhibition of ascending volleys. Bicuculline but not strychnine, administered electrophoretically from micropipettes, reduced dorsal column induced inhibition of the excitation of dorsal horn neurones by noxious heat. These findings suggest that the inhibition studied was produced by release of gamma-aminobutyric acid. This amino acid may play a role in the clinical suppression of pain produced by dorsal column stimulation.

Brain-stem areas tonically inhibiting dorsal horn neurones: studies with microinjection of the GABA analogue piperidine-4-sulphonic acid.

In barbiturate anaesthetized cats, tonic inhibition of the excitation of lumbar dorsal horn neurones by impulses in unmyelinated primary afferents was measured by reversibly cooling the spinal cord at the thoraco-lumbar junction. Tonic inhibition was reduced by microinjection of the GABA analogue, piperidine-4-sulphonic acid (2.5 nM in 0.5 microliter) mainly at AP -7, L 2-5 and V -8 to -10. This area in the ventrolateral medulla is just ventral to the facial nucleus and has been shown to be important in cardiovascular control, particularly in relation to fear-defence reactions. It is proposed that tonic inhibition of the nociceptive responses of spinal neurones is part of such a reaction in response to the trauma of surgery. Since previous experiments had shown that the ventrolateral medulla was important in spinal inhibition produced by PAG stimulation, these experiments support the proposal that analgesia does not occur in isolation but is part of a complex behavioural response of an animal in a potentially injurious environment.

The effect of conduction block on the spinal release of immunoreactive-neuropeptide Y (ir-NPY) in the neuropathic rat.

Peripheral nerve injury may result in significant changes in neuropeptide production and the development of neuropathic pain behaviour. Rats with a chronic constriction injury of one sciatic nerve were used to study the spinal release of immunoreactive neuropeptide Y (ir-NPY), using the antibody-coated microprobe technique. Previous work has shown an increase in NPY synthesis by large to medium-sized primary afferent neurones, as well as a new area of ir-NPY release in the deep dorsal horn on the side of nerve injury, when compared to uninjured rats. The stimulus for spontaneous ir-NPY release was unclear, but may have been due to ectopic neuronal discharges developing after nerve injury. This study used local anaesthetic to block all electrical input from the injured nerve. No change was found in the new zone of spontaneous release of ir-NPY in the deep dorsal horn ipsilateral to nerve injury. It appears therefore, that ir-NPY is released from the central termination of primary afferent neurones, without regulation from neuronal activity in the primary afferent neurones themselves.

Clonidine and the hyper-responsiveness of dorsal horn neurones following morphine withdrawal in the spinal cat.

In barbiturate-anaesthetized spinal cats, exaggerated responses of dorsal horn neurones to impulses in unmyelinated primary afferents, were produced by administering naloxone after iontophoretic administration of morphine in the substantia gelatinosa. Clonidine, given both intravenously (5-10 micrograms/kg) and iontophoretically into the substantia gelatinosa, reduced cell responses to pre-naloxone values. The action of clonidine was reversed by idazoxan. This spinal action of clonidine may be an important component in the suppression of the opiate withdrawal syndrome observed in the whole animal.

Idazoxan blocks the action of noradrenaline but not spinal inhibition from electrical stimulation of the locus coeruleus and nucleus Kolliker-Fuse of the cat.

The effects of idazoxan, a specific alpha 2-adrenoceptor antagonist, on spinal inhibition by administration of noradrenaline and from electrical stimulation of the dorsolateral pons were studied in 26 cats anaesthetized with sodium pentobarbitone. The excitation of dorsal horn neurons of the spinal cord by noxious heating of the skin or by impulses in unmyelinated primary afferent fibres was markedly reduced when noradrenaline was administered microelectrophoretically in the substantia gelatinosa or near cell bodies. Electrical stimulation of the region of the locus coeruleus selectively inhibited spinal nociceptive transmission when dorsal horn neurons were excited by noxious and non-noxious stimuli. In contrast to stimulation of the locus coeruleus, stimulation of the nucleus Kolliker-Fuse produced non-selective inhibition of both nociceptive and non-nociceptive responses of dorsal horn neurons of the spinal cord. Microelectrophoretic ejection of idazoxan reduced or abolished noradrenaline-induced inhibition with 19 of 20 neurons. This antagonist did not alter inhibition from stimulation of the locus coeruleus and the nucleus Kolliker-Fuse, regardless of whether it was administered microelectrophoretically (11 neurons), systemically (3 neurons) or topically (4 neurons). The results suggest that alpha 2-adrenoceptors do not mediate inhibition of spinal nociceptive transmission from electrical stimulation of the locus coeruleus and the nucleus Kolliker-Fuse. Other possibilities for the failure of idazoxan to modify inhibition from such stimulation are discussed.

Thalamic neuronal activity in rats with mechanical allodynia following contusive spinal cord injury.

Pain and allodynia following spinal cord injury are poorly understood and difficult to treat. Since there is evidence that supraspinal mechanisms are important in such pain, we have studied the role of the thalamus in an experimental model of spinal injury. Extracellular recordings were obtained from neurones of the thalamic nucleus ventralis postero-lateralis (VPL) in normal rats and those which had sustained a contusive spinal cord injury to the thoraco-lumbar junction 7 days previously. Behavioural testing with von Frey hairs established that 11 spinally injured rats showed exaggerated vocal responses to normally innocuous mechanical stimulation (allodynia) whereas eight were non-allodynic. Thalamic VPL neurones in spinally injured rats (both allodynic and non-allodynic) exhibited a dysrhythmia in that a significantly higher proportion fired spontaneously in an oscillatory mode when compared with neurones in uninjured rats. Thus this dysrhythmia was linked to spinal injury, not to allodynia. The evoked responses of VPL thalamic neurones to brushing the skin, however, were significantly elevated in allodynic rats when compared with those in uninjured rats and neuronal afterdischarges to these stimuli (which were absent in uninjured rats) were more common in allodynic than in non-allodynic rats. We have previously reported that a proportion of spinal neurones in allodynic spinally injured rats show increased evoked responses and afterdischarges following brushing the skin and hence the enhanced thalamic responses may reflect a greater spinal input. In view of the increasing evidence that thalamo-cortical rhythmical firing is linked to sensorimotor and cognitive brain functions, we propose that pain following brushing the skin results from an exaggerated spinal input being processed by a dysrhythmic thalamus. Thus both spinal and thalamic mechanisms may be important in the genesis of pain and allodynia following spinal cord injury.

Microinjection of neuropeptide Y into the superficial dorsal horn reduces stimulus-evoked release of immunoreactive substance P in the anaesthetized cat.

In barbiturate anaesthetized spinal cats, antibody microprobes were used to measure release of immunoreactive substance P in the superficial dorsal horn following electrical stimulation of unmyelinated primary afferents of the ipsilateral tibial nerve. Prior microinjection of neuropeptide Y (0.2-0.6 microliters of 10(-5) mol/l solution) in the region of the substantia gelatinosa reduced the evoked release of immunoreactive substance P for up to 40 min. Microinjection of similar volumes of phosphate-buffered saline at similar sites was without effect. This action of neuropeptide Y could contribute to analgesia, particularly if this neuropeptide is co-released with noradrenaline from axon terminals in the superficial dorsal horn.

Spontaneous release of immunoreactive neuropeptide Y from the central terminals of large diameter primary afferents of rats with peripheral nerve injury.

Microprobes bearing immobilized antibodies to the C-terminus of neuropeptide Y were used to measure the release of this neuropeptide in the spinal cords of rats with a unilateral peripheral neuropathy and in sham-operated animals. All neuropathic animals showed the characteristic behavioural syndrome and were studied at 14 days postsciatic nerve loose-ligation. An extensive spontaneous release of immunoreactive neuropeptide Y was detected in the spinal cords of the neuropathic rats and, compared to sham-operated rats, a new zone of release was found in the deep dorsal horn. Electrical stimulation of large diameter primary afferents proximal to the nerve ligature produced widespread release of neuropeptide Y in the dorsal horn which persisted for up to 1 h poststimulation. It is possible that ectopic impulses arising in the injured nerve were responsible for the spontaneous central release of neuropeptide Y and this neuropeptide may play a role in the central response to peripheral nerve injury.

Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes.

In barbiturate anaesthetized spinal cats antibody microprobes were used to examine release of immunoreactive neurokinin A following cutaneous thermal and mechanical stimulation. In the absence of peripheral stimuli, microprobes detected a diffuse basal presence of immunoreactive neurokinin A. Noxious mechanical and to a lesser extent noxious thermal stimuli increased the levels of immunoreactive neurokinin A diffusely throughout the dorsal horn which, in many cases, spread into the adjacent white matter. These diffuse stimulus-evoked increases contrast with previous experiments where the same stimuli produced discrete focal increases in levels of immunoreactive substance P. Evidence was obtained that released immunoreactive neurokinin A persisted in the spinal cord for at least 30 min beyond the period of stimulation. Neurokinin A needs consideration as the agent responsible for the long-lasting increases in excitability of some spinal neurons found by several laboratories to follow a brief input from unmyelinated primary afferents.

Afferent volley patterns and the spinal release of immunoreactive substance P in the dorsal horn of the anaesthetized spinal cat.

Microprobes bearing immobilized antibodies to the C-terminus of substance P were used to measure release of this neuropeptide in the spinal cord of the anaesthetized spinal cat in response to peripheral nerve stimulation. Release of substance P was just detectable in laminae I, II with 150 stimuli (0.5 Hz, 5 min) and was near maximal with 300 stimuli. Using two periods of stimulation of 10 min separated by 15 min, greater levels of substance P were detected during the second period. Fifteen to 25 min after two periods of peripheral nerve stimulation levels of substance P detected by microprobes were still elevated above those present prior to stimulation. Stimulation with bursts of three impulses when delivering a fixed number of stimuli resulted in detection of increased levels of substance P at sites adjacent to the areas of maximal release. The results suggest that maximal release of substance P from the central terminals of primary afferent fibres occurs with relatively few impulses and at low frequencies in agreement with what is known of release from the peripheral terminals of these fibres.

The burst-like firing of spinal neurons in rats with peripheral inflammation is reduced by an antagonist of N-methyl-D-aspartate.

Ankle inflammation was induced in rats by subcutaneous injection of complete Freund's adjuvant and the firing properties of spinal neurons receiving afferent input from the inflamed areas were studied four to six days later. Comparable neurons in normal rats were also studied. In normal animals the response of neurons to ankle compression consisted of a brief burst of action potentials followed by sustained firing during stimulus application. On cessation of the stimulus there was no prolonged afterdischarge. In rats with an inflamed ankle, compression of the ankle produced firing while the stimulus was applied, but with 17 of 22 neurons there was a prolonged (219 +/- 55 s) post-stimulus afterdischarge. All neurons studied in rats with peripheral inflammation fired with intermittent bursts of action potentials, particularly during the afterdischarge and spontaneous firing. The N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonopentanoate was ejected microiontophoretically near the cells studied. The major effect was a near abolition of bursts present in spontaneous firing and post-stimulus afterdischarges with a lesser reduction in firing during stimulus application. Effects on afterdischarge duration were variable. Since firing in bursts is known to increase transmitter release at some sites in the brain, it is proposed that when the relevant spinal neurons fire in bursts, additional intraspinal pathways are recruited and this contributes to the expanded receptive fields of neurons and possibly to the enhanced pain experienced by manipulation of inflamed peripheral tissues.

Release of immunoreactive galanin in the spinal cord of rats with ankle inflammation: studies with antibody microprobes.

Antibody microprobes bearing antibodies to the carboxy-terminus of rat galanin were inserted into the spinal cords of anaesthetized normal rats and those in which ankle inflammation had been induced by the unilateral subcutaneous injection of Freund's adjuvant four to six days previously. In normal rats, a basal presence of immunoreactive galanin was detected in the dorsal horn. Similar levels of immunoreactive galanin were found in the dorsal horn of both sides of the spinal cord in animals with unilateral ankle inflammation. Flexing the ankle or compressing the foot in normal rats failed to alter levels of immunoreactive galanin detected by microprobes. In animals with ankle inflammation, prolonged periods of ankle flexion did release immunoreactive galanin in the ipsilateral dorsal horn. Subsequent noxious ankle compression in these animals did not increase but rather decreased immunoreactive galanin in the dorsal horn to below basal levels. The reason for this decrease is unknown but it may represent an inhibition of release or a depletion of spinal stores of galanin.

Methyl xanthines, adenosine 3',5'-cyclic monophosphate and the spinal transmission of nociceptive information.

1 In spinal cats anaesthetized with either alpha-chloralose or sodium pentobarbitone, a study was made of the effects of adenosine 3',5'-cyclic monophosphate (cyclic AMP), mono- and di-butyryl cyclic AMP and the methyl xanthines, theophylline and isobutyl methyl xanthine (IBMX), on the responses of neurones of laminae I, IV and V to noxious and innocuous skin stimuli. The compounds were administered from micropipettes positioned in the substantia gelatinosa. IBMX was also given intravenously.2 When administered in the substantia gelatinosa, neither cyclic AMP, its butyryl derivatives, nor the methyl xanthines had any effect on the excitation of neurones of spinal laminae IV and V by noxious heating of the skin or deflection of hairs. When the nociceptive responses of cells had been reduced by electrophoretic morphine, methyl xanthines and cyclic AMP failed to modify the effects of morphine on these deeper neurones. Electrophoretically administered naloxone reversed the effect of morphine.3 Intravenously administered IBMX (1 to 2 mg/kg) produced large transient increases in the firing rate of both C fibres and the excitation of dorsal horn neurones by noxious heating of the skin. These increases coincided with decreases in the mean systemic blood pressure, and probably resulted from increased temperatures being attained in the dermis by each noxious stimulus. When dorsal horn neurones were activated by electrical stimulation of the tibial nerve by a stimulus adequate to excite C fibres, intravenous IBMX produced a small or no increase in the number of spikes per stimulus.4 These results in the spinal cord do not support the hypothesis that the inhibition of synthesis of cyclic AMP is relevant to the analgesic action of morphine in mammals.

Acetylcholine-sensitive cells in the caudal medulla of the rat: distribution, pharmacology and effects of pentobarbitone.

1. The distribution of cholinoceptive and non-cholinoceptive cells in various nuclei of the caudal medulla of the rat is described. 2. The nature of the responses of cells of the paramedian reticular nucleus and of the perihypoglossal nuclei to electrophoretically applied acetylcholine (ACh) was investigated. 3. In unanaesthetized decerebrate preparations ACh responses were usually of a "fast onset-fast offset" nature. Dihydro-beta-erythroidine was a more effective antagonist than atropine. 4. In rats anaesthetized with barbiturate nearly all the tach responses showed a slower onset and prolonged action. Atropine was the more effective antagonist. 5. The synaptic responses of cells of the paramedian reticular and perihypoglossal nuclei to stimulation of glossopharyngeal, superior laryngeal, lingual and hypoglossal nerves were investigated. It is concluded that ACh does not mediate the responses at the level of these nuclei.

Effect of etorphine, morphine and diprenorphine on neurones of the cerebral cortex and spinal cord of the rat.

1. The actions of etorphine, morphine and diprenorphine were investigated on neurones of the cerebral cortex and spinal cord of rats anaesthetized with pentobarbitone.2. In the cerebral cortex, intravenous etorphine increased the latency of the primary evoked response to a peripheral nerve stimulus and suppressed the rhythmical after-discharge. Diprenorphine reversed this effect. These actions were demonstrated on both field potentials and unit firing.3. Morphine had no effect on the primary response but the frequency of after-discharge bursts was reduced and there was an increase in firing between bursts.4. In the cerebral cortex, electrophoretically applied etorphine reduced after-discharges when applied for long periods but had no effect on the depressant actions of glycine and gamma-aminobutyric acid (GABA) nor on the excitant action of acetylcholine and L-glutamate. Similarly there was no alteration by etorphine of the effects of glycine, GABA and L-glutamate on spinal cord neurones.5. It is concluded that etorphine may act pre-synaptically in the cerebral cortex.

Morphine and supraspinal inhibition of spinal neurones: evidence that morphine decreases tonic descending inhibition in the anaesthetized cat.

1 A study was made in cats anaesthetized with barbiturate or alpha-chloralose, of the excitation of dorsal horn neurones by impulses in unmyelinated (C) primary afferent fibres of the tibial nerve. 2 Block of conduction in the first lumbar segment by cooling produced large increases in the number of action potentials evoked by C fibre afferents in neurones of more caudal segments. 3 Morphine (0.3 to 1.0 mg/kg) reduced the excitation of neurones by C fibre afferents and also reduced the increase produced by blocking conduction in the spinal cord. Naloxone (0.1 to 0.3 mg/kg) reversed these effects of morphine. 4 This decrease in descending inhibition supports findings in the decerebrate cat but gives no support to the hypothesis that an important component of morphine analgesia is an activation of descending inhibitory pathways.

Enkephalins and dorsal horn neurones of the cat: effects on responses to noxious and innocuous skin stimuli.

1. In spinal cats anaesthetized with alpha-chloralose, a study was made of the effects of methionine enkephalin and methionine enkephalin amide on the responses of neurones of spinal laminae IV and V to noxious and innocuous skin stimuli. The enkephalins were ejected from micropipettes either in the region of cell bodies or in the substantia gelatinosa. 2. Administered near cell bodies the enkephalins reduced spontaneous firing and cell responses to both types of skin stimuli. These effects were antagonized by naloxone when administered near cell bodies but not when given intravenously in doses (0.3-0.6 mg/kg) more than adequate to antagonize analgesic doses of morphine. 3. Administered in the substantia gelatinosa the enkephalins were more selective in their action. The predominant effect was a reduction in nociceptive responses with little effect on non-nociceptive responses although spontaneous firing was commonly reduced. Naloxone administered either in the substantia gelatinosa or intravenously (0.1-0.3 mg/kg) reversed these effects of the enkephalins.

Analgesic doses of morphine do not reduce noxious stimulus-evoked release of immunoreactive neurokinins in the dorsal horn of the spinal cat.

1. Antibody microprobes were used to detect immunoreactive neurokinin A release in the dorsal spinal cord of barbiturate-anaesthetized spinal cats. 2. Noxious mechanical stimulation of the ipsilateral hind paw and electrical stimulation (suprathreshold for unmyelinated primary afferent fibres) of the ipsilateral tibial nerve evoked immunoreactive neurokinin A release. 3. Systemic morphine, 5 mg kg-1, i.v., did not block immunoreactive neurokinin A release in response to these stimuli. 4. Subsequent naloxone administration, 0.5 mg kg-1, i.v., did not alter this stimulus-evoked release. 5. Basal levels of immunoreactive neurokinin A were unaltered by morphine or naloxone. 6. These results suggest that the analgesic effects of morphine at the spinal cord level are not brought about by activation of presynaptic opiate receptors on neurokinin A containing afferent terminals.