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.

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.

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection.

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.

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.

Responses of spinal neurones to cutaneous and dorsal root stimuli in rats with mechanical allodynia after contusive spinal cord injury.

The firing of neurones in spinal segments adjacent to a contusive T13 spinal cord injury was characterised in anaesthetised rats. Three groups of rats were examined: (1) allodynic spinally injured, (2) non-allodynic spinally injured and (3) normal, uninjured. Spinal cord field potentials evoked by electrical dorsal root stimulation and the responses of 207 dorsal horn neurones to mechanical stimuli applied to the skin were studied. Within the lesioned spinal segment few active neurones were encountered and field potentials were absent. Depolarising field potentials recorded rostral to the lesion were reduced in both allodynic and non-allodynic animals compared to uninjured controls, while those recorded in caudal segments were enhanced in allodynic animals. Neuronal recordings revealed that allodynia was associated with exaggerated responses, including afterdischarges, to innocuous and noxious mechanical stimuli in a proportion of wide dynamic range, but not low threshold, neurones. These changes were observed both rostral and caudal to the site of injury. The results suggest that an increased responsiveness of some dorsal horn neurones in segments neighbouring a contusive spinal cord injury may contribute to the expression of mechanical allodynia. It is proposed that a relative lack of inhibition underlies altered cell responses.

Primary afferent-evoked release of immunoreactive galanin in the spinal cord of the neuropathic rat.

We have determined if peripheral nerve stimulation altered the increased spontaneous release of immunoreactive (ir)-galanin that is found in the superficial dorsal horn of the spinal cord of neuropathic rats. Using the antibody microprobe technique to study the localized sites of ir-galanin release in vivo, we found that high intensity electrical stimulation of the injured nerve resulted in a further increase in ir-galanin release in the superficial dorsal horn, with no significant persistence of ir-galanin after release. Release of ir-galanin at stimulus strengths sufficient to activate C fibres, in an area of the spinal cord thought to be concerned with nociceptive transmission, indicates a possible role for this peptide in the spinal modulation of pain after peripheral nerve injury.

The release of beta-endorphin and the neuropeptide-receptor mismatch in the brain.

Microprobes bearing immobilized antibodies to the carboxy-terminus of beta-endorphin were used to study the release of beta-endorphin in the urethane anaesthetized rat following electrical stimulation of the ipsilateral arcuate nucleus. The microprobes were inserted through the cerebral hemisphere, the superior colliculus and the midbrain periaqueductal grey. Since such microprobes detect extracellular molecules along their entire length they give information on the persistence and spread of compounds following release. Little immunoreactive-beta-endorphin was detected in the areas of brain sampled during electrical stimulation of arcuate nucleus but a remarkable spread throughout the midbrain and cerebral cortex occurred within 30 min of the cessation of stimulation. The results suggest that although beta-endorphin-containing fibres are absent in many parts of the brain, this neuropeptide can access receptors in these sites and it is not necessary for release to be directly adjacent to opiate receptors. As such it is important evidence supporting the hypothesis of volume transmission as a means of neuronal communication. The results also suggest that an important mechanism of the transport of beta-endorphin is the cerebrospinal fluid.

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.

The effect of a peripheral mononeuropathy on immunoreactive (ir)-galanin release in the spinal cord of the rat.

The pattern of ir-galanin release in the spinal cord of rats with a peripheral mononeuropathy was studied. On the side of the cord ipsilateral to the nerve injury enhanced ir-galanin release was found in the superficial dorsal horn. It is probable that, after nerve injury, some primary afferent neurons spontaneously release galanin from their central terminals.

The release of immunoreactive neuropeptide Y in the spinal cord of the anaesthetized rat and cat.

The release of immunoreactive (ir-) neuropeptide Y (NYP) was studied in the anaesthetized rat and cat by means of microprobes bearing immobilized antibodies to the C terminus of NPY. An extensive basal release of ir-NYP was detected throughout the dorsal and upper ventral horn of the rat. This spontaneous release was not significantly altered by sectioning the spinal cord at the thoraco-lumbar junction nor by electrical stimulation of peripheral nerves. Since NPY is virtually absent in primary afferents it is probable that spontaneous release within the spinal cord comes from active NPY-containing intrinsic spinal neurones. In the spinal cat spontaneous release of ir-NPY was detected in the mid-dorsal horn and this was unaltered by peripheral noxious thermal or noxious mechanical stimuli. As in the rat, release from intrinsic spinal neurones is most probable. The extensive spontaneous release of ir-NPY in both species suggests a widespread role in spinal cord function.

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.

Bilaterally enhanced dorsal horn postsynaptic currents in a rat model of peripheral mononeuropathy.

Surface compound potentials were recorded from the surgically exposed lumbar spinal cord in anaesthetized rats which had had one sciatic nerve loosely ligatured 12-15 days previously, resulting in unilateral allodynia and hyperalgesia, as assessed behaviourally. These cord dorsum potentials were recorded in response to electrical stimulation of the ligatured and non-ligatured sciatic nerve, respectively, on both the ipsi- and contralateral side with respect to the stimulated nerve. Compared to potentials produced by stimulation of the non-ligatured sciatic nerve, electrical stimulation of large diameter fibres proximal to the ligatures resulted in a smaller afferent fibre input arriving at the spinal cord. However, larger net postsynaptic currents in the contralateral dorsal horn and a larger net postsynaptic current per unit of afferent fibre input were found in the ipsilateral and contralateral spinal cord. Such changes may result from structural changes or increased synaptic efficacy in the dorsal horn following peripheral nerve injury.

Spinal release of immunoreactive dynorphin A(1-8) with the development of peripheral inflammation in the rat.

Microprobes bearing immobilised antibodies to dynorphin A(1-8) were used to study the basal and evoked release of this prodynorphin derived peptide in the spinal cord of urethane anaesthetised normal rats and those with a peripheral inflammation. In the absence of any active peripheral stimulus the antibody microprobes detected immunoreactive (ir)-dynorphin A(1-8) in two areas (lamina I and laminae IV-V) in the dorsal horn of the spinal cord of normal rats. With the development of unilateral ankle inflammation over 3 to 5 days following subcutaneous injections of Freund's complete adjuvant, a basal presence of ir-dynorphin A(1-8) was found in both the dorsal and ventral horn regions of both sides of the spinal cord. Lateral compression of the ankles of the normal animals did not release ir-dynorphin A(1-8) during the period of stimulation, but this neuropeptide was detected in increased amounts in the ventral horn following the stimulus. By contrast, compression of inflamed ankles produced elevated levels of ir-dynorphin A(1-8) during the period of stimulus application at three major sites in the ipsilateral spinal grey matter. The largest peak was in the deep dorsal horn/upper ventral horn (laminae VI-VII), with further sites of significant release in the mid dorsal horn (laminae II-V) and the lower ventral horn. The observation that ir-dynorphin A(1-8) is physiologically released in the ventral and deep dorsal in addition to the superficial dorsal horn of the rat suggests an involvement of dynorphins in several aspects of spinal function.

In vivo detection of immunoreactive neurokinin A release within rat substantia nigra and its dependency on a dopaminergic input.

In the striatum, the tachykinin peptide neurokinin A (NKA) is thought to coexist with substance P in the gamma-aminobutyric acid-containing spiny neurones which project to the substantia nigra. We have used in vivo antibody-coated microprobes to directly monitor the release of NKA-like immunoreactivity (NKA-LI) within substantia nigra during various pharmacological manipulations. The data clearly illustrates a basal or resting extracellular presence of NKA-LI restricted to substantia nigra reticulata which was found to be largely dependent on a dopaminergic input. Acute administration of haloperidol (0.1-0.2 mg/kg i.p.) considerably reduced this basal NKA-LI whereas depot administration (14 mg/kg i.m. released over 2 weeks) produced a less substantial reduction. Lesion of nigro-striatal dopamine neurones with the neurotoxic agent 6-hydroxydopamine produced significant reductions in the nigral NKA-LI detected. However, d-amphetamine administration (4 mg/kg i.p.) did not alter the pattern of NKA-LI release for up to 4 h posttreatment. These results indicate that changes in peptide mRNA levels do not necessarily reflect changes in peptide release and suggest that NKA may be the more physiologically relevant tachykinin within the substantia nigra of the rat.

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.

Probing the brain and spinal cord with neuropeptides in pathways related to pain and other functions.

The principles involved in the fabrication and use in vivo of antibody microprobes are described. These devices have shown that immunoreactive (ir)-substance P and ir-neurokinin A are released in the region of the substantia gelatinosa of the spinal cord when impulses arrive in nociceptors. Particularly with ir-neurokinin A, rapid inactivation does not appear to occur, resulting in the released neuropeptides accessing sites relatively remote from sites of release. Microprobes have also provided evidence that the sites accessed by ir-substance P are controlled by spinal cord peptidases and that peptidase inhibition by the endogenous neuropeptide calcitonin gene-related peptide expands the distribution of sites reached. Inflammatory joint disease results in a relatively massive central release of ir-substance P when the damaged joints are flexed or compressed. Antibody microprobe studies of the spinal release of ir-galanin have favored release from intrinsic spinal neurons rather than from primary afferent terminals following peripheral noxious stimuli. Immunoreactive-somatostatin was found to be released following noxious thermal but not noxious mechanical peripheral stimuli but it is uncertain whether this results from release predominantly from primary afferents or intrinsic spinal neurons. Studies using antibody microprobes inserted into the brain have detected the release of ir-substance P in the ventral region of the striatum following administration of amphetamine. Microprobes have also followed peptide release from striatal terminals in substantia nigra and have provided evidence of a basal presence of ir-neurokinin A but not of substance P. Depletion of the dopamine input to the striatum, or blockade of dopamine receptors, caused considerable reduction of ir-neurokinin A released within the substantia nigra.

Lack of effect of microinjection of noradrenaline or medetomidine on stimulus-evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes.

1. Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C-terminus-directed antibodies to substance P. 2. Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 microliters and a concentration of 10(-3) M. 3. In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C-fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus-evoked release. 4. In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.