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The present study was done to determine the possible effects of endogenous adenosine, present in the extracellular fluid of the hippocampal slice, on pyramidal cells in the CA1 region using intracellular recording techniques. Administration of 5 microM of the adenosine receptor antagonist, 8-sulfophenyltheophylline (n=11), induced a depolarization (2.6+/-0.4 mV, mean+/-S.E.M.) with an increase in input resistance (6.7+/-2.1%) in pyramidal cells, and increased the amplitude of the excitatory postsynaptic potentials elicited by stimulation of Schaffer collateral afferents; 50 microM 8-sulfophenyltheophylline (n=68) produced a similar depolarization (3.4+/-1.7 mV) and an increase in input resistance (26+/-3.0%), but also produced spontaneous, synchronized giant excitatory postsynaptic potentials which could generate bursts of spikes. These effects lasted more than 10 min after washout. In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate receptor antagonist, and 50 microM D-2-amino-5-phosphonovalerate, an N-methyl-D-aspartate receptor antagonist, 50 microM 8-sulfophenyltheophylline (n=4) induced only depolarization (3.1+/-1.3 mV) and an increase in input resistance (23+/-3.8%). In the presence of 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione only, 50 microM 8-sulfophenyltheophylline (n=7) induced not only the depolarization with an increase in input resistance, but also the occurrence of small-amplitude (11+/-5.6 mV), fast rising, all-or-none, voltage-sensitive spikes of 2-3 ms duration, which were attributed to a dendritic origin. The latency of these dendritic spikes in response to stimulation of Schaffer collateral afferents lasted up to 21 ms. These dendritic spikes could generate one or more action potentials, depending on the resting membrane potential and the frequency of the dendritic spikes. In the presence of 50 microM 8-sulfophenyltheophylline plus 20 microM 6-cyano-7-nitro-quinoxaline-2,3-dione, 50 microM D-2-amino-5-phosphonovalerate blocked the spontaneous dendritic spikes (n=4). In the presence of 5 microM 8-sulfophenyltheophylline, 200 microM N-methyl-D-aspartate (n=5) increased the occurrence of dendritic spikes. These data indicate that adenosine present in the extracellular fluid of the hippocampal slice tonically inhibits not only (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-mediated synaptic transmission, but also voltage- and N-methyl-D-aspartate receptor-sensitive dendritic spikes. Endogenous adenosine acting on adenosine A(1) receptors is thus visualized as a control to prevent the genesis of synchronized giant excitatory postsynaptic potentials. In our experiments, blockade of this tonic activation of adenosine receptors appears to have altered the origins of action potentials and led to epileptiform firing in CA1 pyramidal cells.

In transgenic mice expressing ectopic substance P fibres in the spinal white matter, a normally innocuous mechanical stimulus induces hyperalgesia and allodynia which are reversed by substance P and N-methyl- d -aspartate receptor antagonists. This period of enhanced excitation is followed by a rebound overshoot in these animals. As previous evidence indicates opioid mechanisms in a similar rebound in normal animals, the present study was done to determine the effects of systemic administration of morphine and the opiate receptor antagonist, naloxone, on the stimulus-induced responses in the tail withdrawal reflex. Once baseline reaction times had been taken, different combinations of saline, naloxone and morphine were administered intraperitoneally to transgenic and control mice of either sex. A mechanical conditioning stimulus of 450 g was then applied to the tip of the tail for 2 s. This stimulus was innocuous in control mice given saline or naloxone, but provoked a nociceptive response in transgenic mice given these compounds. In control and transgenic mice, following morphine administration there was an antinociceptive effect. In control mice the subsequent mechanical stimulus had no effect. However, in transgenic mice the mechanical stimulus produced a further antinociception. Naloxone blocked the effect of morphine and the subsequent conditioning stimulus in both control and transgenic mice. The results indicate that while morphine is equally effective on the withdrawal reflex in both types of animal, in the transgenic mice morphine reveals an intrinsic, naloxone-sensitive antinociceptive mechanism. The data are interpreted to suggest that over-expression of substance P or some other factor in the spinal cord of transgenic mice is associated with the up-regulation or facilitation of an opiate-mediated intrinsic antinociceptive mechanism. This is a novel observation because the genetic manipulation in this transgenic mouse results in a transient over-expression of nerve growth factor during development that leads to the formation of ectopic primary afferent fibres in the spinal cord containing substance P. These fibres persist indefinitely after the nerve growth factor levels return to normal. Opioid mechanisms, which are likely of dorsal horn origin, do not fall under the direct influence of nerve growth factor mechanisms and therefore the intriguing possibility is raised that opioid mechanisms in the spinal cord are regulated at least in part by substance P-related mechanisms.

A transgenic mouse has been developed which, during development, over-expresses nerve growth factor under the control of a myelin basic protein promoter. These animals display an ectopic network of substance P-containing sensory fibers in the white matter of the spinal cord. To study the functional significance of this model to nociception, these mice were studied in a test measuring the latency to tail withdrawal from a noxious radiant heat stimulus. Baseline reaction times were significantly less in transgenic mice, suggesting thermal allodynia. A mechanical stimulus was then applied to the tip of the tail at either 450 g or 1 400 g for 2 s and tail withdrawal readings were taken for another 10 min. In control mice, the 450 g stimulus was without effect, suggesting that it is normally innocuous. In transgenic mice, this stimulus induced a transient decrease in withdrawal latency at 1 min. Thus, transgenic mice exhibited mechanical allodynia. The 1 400 g stimulus decreased withdrawal latency in both transgenic and control mice. However, the response was greater in transgenic mice, indicating that they exhibited mechanical hyperalgesia. The neurokinin-1 receptor antagonist CP-96,345, but not the inactive stereoisomer CP-96,344, administered subcutaneously 30 min before the 450 g stimulus, blocked the stimulation-induced allodynia in transgenic mice, and revealed a transient antinociception in transgenic and control mice. Ketamine, an N -methyl- d -aspartate receptor antagonist, given intraperitonally 10 min before 450 g stimulation, blocked the allodynia in transgenic mice. These results indicate that these transgenic mice display hyperalgesia and allodynia, and that these nociceptive responses are reversed by substance P and N -methyl- d -aspartate receptor antagonists.

The effects of novel substance P (NK-1) receptor antagonists LY303870 and LY306740, as well as LY306155, the enantiomer of LY303870, were tested on the responses of nociceptive spinal dorsal horn neurons to iontophoretically applied substance P and to peripheral noxious stimuli. The peripheral stimuli included noxious thermal and pinch stimuli applied to the cutaneous receptive field in the hind paw and stimulation of the superficial peroneal nerve with a train of high-intensity electrical stimuli. Extracellular recordings were obtained using multi-barrel electrodes from L4–L7 segments of the spinal cord in cats anaesthetized with α-chloralose and spinalized at the L1 level. The antagonists were given i.v. (0.5–4.0 mg/kg). Responses to substance P were inhibited by LY303870 and by LY306740 in a dose-related manner, but were not affected by LY306155. Responses to peripheral noxious thermal stimulation were inhibited in a dose-related manner by LY303870 and LY306740, and only at higher doses (2 mg/kg or more) by LY306155. Responses to pinch stimuli were inhibited by LY303870 and LY306740. LY306155 lacked consistent effects on pinch responses. LY303870 selectively inhibited the late component of the response to electrical stimulation of the superficial peroneal nerve. When these three drugs were tested against the responses of dorsal horn neurons to the excitatory amino acid, N-methyl- d -aspartate, the responses were unaffected. These data suggest that LY303870 and LY306740 pass from the circulation into the spinal cord where they antagonize dorsal horn neuronal responses to substance P and nociceptive inputs.

A combination of intracellular electrophysiological recording and injection of horseradish peroxidase with ultrastructural immunocytochemistry was used to investigate the synaptic interplay between substance P- and enkephalin-immunoreactive axonal boutons and three types of functionally characterized dorsal horn neurons in the cat spinal cord. The dorsal horn neurons were classified as nociceptive specific, wide dynamic range and non-nociceptive based on their responses to innocuous and noxious stimuli. Most of the nociceptive neurons (either nociceptive specific or wide dynamic range) contained enkephalin immunoreactivity, but none of the non-nociceptive neurons were positive for enkephalin. Three types of immunoreactive boutons were found in contact with the functionally characterized dorsal horn neurons. These boutons were positive for either substance P, enkephalin, or substance P+enkephalin. Quantitative analysis revealed that the percentages of substance P-immunoreactive boutons apposed to the cell bodies, proximal dendrites and distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons. Furthermore, the percentages of substance P+enkephalin-immunoreactive axonal boutons apposed to the distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons and the percentages of enkephalin-immunoreactive boutons apposed to the cell bodies and proximal dendrites of nociceptive neurons were significantly higher than in non-nociceptive neurons. Finally, neither enkephalin-immunoreactive nor substance P+enkephalin-immunoreactive boutons were ever seen presynaptic to substance P-immunoreactive boutons. These results provide evidence of an anatomical substrate within the dorsal horn for the interaction of substance P-mediated with enkephalin-mediated mechanisms. The data support the idea that the modulation of nociceptive input in the dorsal horn by enkephalinergic neurons occurs mainly via a postsynaptic mechanism, and thus suggest that dorsal horn enkephalinergic neurons participate in a local inhibitory feedback loop in a distinct pathway from the previously postulated opioid-mediated depression of substance P release from primary afferent terminals.

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)

Extracellular and intracellular studies were undertaken to test the effects of the non-peptide, substance P (NK-1) receptor antagonists CP-96,345 and CP-99,994, and of CP-96,344, the inactive enantiomer of CP-96,345, on the responses of spinal dorsal horn neurons to peripheral noxious and non-noxious cutaneous stimuli in spinalized cats anesthetized with α-chloralose. The effect of these agents on the response of dorsal horn neurons to iontophoretic application of substance P was also tested in extracellular studies. The substance P-induced slow, prolonged discharge of dorsal horn neurons was blocked by administration (0.5 mg/kg, i.v.) of CP-96,345 ( n = 10) or CP-99,994 ( n = 9), but was unaffected by CP-96,344 ( n = 9). The response of substance P-sensitive neurons to noxious thermal stimulation of the cutaneous receptive field, especially the late afterdischarge phase, was also significantly inhibited by CP-96,345 ( n = 10) and by CP-99,994 ( n = 7). The response of such neurons to noxious pinch stimulation of the receptive field was also significantly inhibited by CP-96,345 ( n = 7) and CP-99,994 ( n = 8), but the response of three other substance P-sensitive neurons to pinch was unaffected by CP-96,345. CP-96,344 did not affect the response of any neuron tested to either of these noxious stimuli (noxious thermal, n = 7; pinch, n = 6). The response to hair afferent stimulation was unaffected by any of these compounds (CP-96,345, n = 16; CP-96,344, n = 5; CP-99,994, n = 6). In intracellular studies, the effect of these antagonists was tested on responses of dorsal horn neurons to noxious pinch stimulation or to a train of high intensity electrical stimulation of the superficial peroneal nerve. Both stimuli produced an initial fast depolarization followed by a slow and prolonged depolarization with corresponding discharge patterns. CP-96,345 ( n = 3) and CP-99,994 ( n = 6) selectively blocked the late, slow components of the stimulus-evoked response without affecting the early components. Responses to single electrical pulses of the same intensity and duration were not affected. CP-96,344 did not affect any of the responses tested ( n = 5). The data indicate that nociceptive responses of a subset of spinal dorsal horn cells are selectively blocked by the NK-1 receptor antagonists, CP-96,345 and CP-99,994, thus confirming the involvement of NK-1 receptors in these responses. As CP-96,344 did not affect any of the responses, it is likely that some of the properties common to CP-96,345 and CP-96,344, such as blockade of calcium channels, may not play a significant role in the selective antagonism of nociceptive responses by these non-peptide antagonists.

As excitatory amino acid receptors have been implicated in nociceptive sensory transmission, the principal objective of the present study was to investigate the effects of various excitatory amino acid antagonists on naturally evoked responses in spinal dorsal horn neurons. Extracellular single unit activity was recorded from functionally identified, spinal dorsal horn neurons in unanesthetized, decerebrated cats and in alpha-chloralose-anesthetized cats. The tests included iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV), the non-N-methyl-D-aspartate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and kynurenate, and also the intravenous administration of the N-methyl-D-aspartate receptor antagonist, ketamine. In addition, attempts were made to determine the effects on these neurons of iontophoretic application of the excitatory amino acid agonists, L-glutamate, N-methyl-D-aspartate, quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate. Marked differences were noted in the actions of agonists and antagonists between the responses observed in the unanesthetized, decerebrated and the anesthetized animals. In decerebrated cats, responses to hair afferent stimulation were blocked by kynurenate, 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were attenuated by 2-amino-5-phosphonovaleric acid and in one unit also by ketamine. Neither 6-cyano-7-nitroquinoxaline-2,3-dione nor kynurenate affected the responses to noxious thermal stimulation. The proportion of cells responding to the agonists were: N-methyl-D-aspartate 24/27 (89%), quisqualate 12/13 (92%) and domoate 6/7 (86%). In chloralose-anesthetized cats, responses to hair afferent stimulation were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate but not by 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were not affected by any of these antagonists, while the response to non-noxious thermal stimulation was blocked by 2-amino-5-phosphonovaleric acid, ketamine and kynurenate in the one neuron studied. The proportion of cells excited by the agonists differed from those observed in decerebrated cats: N-methyl-D-aspartate 9/32 (28%), quisqualate 50/54 (93%), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate 19/23 (83%) and domoate 17/38 (45%). Application of the putative endogenous excitatory amino acid precursor N-acetyl-aspartyl-glutamate (NAAG) did not elicit a response in any of the neurons studied.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of subcutaneous administration of the non-peptide NK-1 (substance P) receptor antagonist, CP-96,345, and its stereoisomer, CP-96,344, were tested in three nociceptive paradigms in the rat. In the first paradigm, tail flick responses were monitored before and after intrathecal administration of substance P (6.5 nmol) in rats pretreated subcutaneously with saline, CP-96,344 (5 mg/kg) or CP-96,345 (5 mg/kg). In the control groups, pretreated with saline (n = 6) or with CP-96,344 (n = 5), substance P reduced the tail flick reaction time at 1 min after administration to 38.3 ± 5.1 (mean ±S.E.M.) and 32.1 ± 7.7% of the mean baseline value, respectively. In contrast, in the group pretreated with CP-96,345 (n = 6) the reaction time following administration of substance P was 98.8 ± 3.3% of the baseline reaction time; this value was not significantly different from the baseline value of this group, indicating a block (P < 0.01) of the substance P-induced facilitation of the tail flick response. In the second paradigm, rats were anesthetized with a mixture of chloral hydrate (120 mg/kg, i.p.) and sodium pentobarbital (20 mg/kg, i.p.), and the effects were determined on tail flick reaction time of a sustained noxious cutaneous stimulation, immersing the tip of the tail in hot water at 55°C. In the groups of rats pretreated with saline (n = 4) or with CP-96,344 (n = 7 ), this noxious stimulus produced a transient decrease in reaction time to 62–74% of the baseline value. However, in the group given CP-96,345 (n = 6), the reaction time was 97.4 ± 13.7% of the baseline reaction time, again indicating a block of the transient response and the involvement of NK-1 receptors. In the third paradigm, nociceptive scores were determined after subcutaneous injection of 50 μl of 2.5% formalin into one hind paw. The first phase of the response to this noxious chemical stimulus was similar in rats pretreated with saline, with CP-96,344 and with CP-96,345. The second phase was also unaffected in rats pretreated with saline (n = 10), with 20 mg/kg of CP-96,344 (n = 8) or with 5 mg/kg of CP-96,345 (n = 9). However, in the groups given 10 (n = 8) or 20 (n = 6) mg/kg of CP-96,345, there was a significant reduction in this second phase (P < 0.05−0.01). indicating an involvement of NK-1 receptors in evoking this second phase. The results indicate the efficacy of a systemically administered NK-1 receptor antagonist in blocking substance P-induced facilitation of a spinal nociceptive reflex and the stereospecificity of the actions of CP-96,345 in nociceptive tests. The data support the involvement of substance P or a related agent, acting via NK-1 receptors, in nociceptive responses to sustained noxious thermal and noxious chemical stimuli. NK-1 receptor antagonists may, therefore, be useful in the clinical treatment of pain originating from burns or lacerations of the skin or from inflamed tissue.

We have previously reported a vibration-induced, adenosine-mediated inhibition of nociceptive dorsal horn neurons in the cat spinal cord. The present study was conducted to investigate the mechanisms of this inhibition.In vivo intracellular recording was obtained from dorsal horn neurons in the lower lumbar segments of the anaesthetized cat. Vibration (80–250 Hz for 2–3 s every 15–20 s) was applied to the glabrous skin of the toes of the hind foot using a feedback-controlled mechanical stimulator. In 32 of 43 neurons tested, vibration produced a pronounced hyperpolarization of the membrane potential. This hyperpolarization peaked at −10 mV and decayed throughout the period of the application of vibration. It was associated with a decrease in membrane resistance, had a reversal potential negative to the resting membrane potential and was Cl− -independent, suggesting that it was due to an increase in a K+ conductance, properties typical of the response to adenosine. This inhibitory postsynaptic potential was unaffected by intravenous administration of bicuculline, strychnine and naloxone but was blocked by iontophoretic administration of 8-sulphophenyltheophylline, a P1-purinergic receptor antagonist. These results confirm our previous finding that vibration-induced inhibition of nociceptive dorsal horn neurons is mediated via the release of an endogenous purine compound and further suggests that this inhibition involves a postsynaptic inhibitory mechanism.

The mammalian tachykinins, substance P and neurokinin A, and the non-mammalian tachykinin, physalaemin, were tested on functionally identified dorsal horn neurones in vivo . The experiments were done on cats which were anaesthetized with sodium pentobarbital or were anaemically decerebrated. Extracellular single-unit recordings were made in the lumbar spinal cord and the tachykinins were applied by iontophoresis. Each neurone was classified functionally as wide dynamic range, non-nociceptive, nociceptive specific or proprioceptive. The response to tachykinin application was determined for each neurone. Application of each of the tachykinins evoked a characteristic excitatory response which was delayed in onset, slow in developing and prolonged: physalaemin excited 99/131 neurones tested, neurokinin A excited 45/63 neurones and substance P excited 32/49 neurones. With two neurones physalaemin evoked a depression of the rate of firing, which may have been caused indirectly by excitation of a neighbouring neurone. Such depression was not elicited by either substance P or by neurokinin A. Physalaemin had a preferential excitatory effect on nociceptive neurones evoking excitation of 76/94 nociceptive neurones compared with 12/23 non-nociceptive neurones (χ 2 = 7.9, 1d.f., P = 0.005). Substance P also caused a preferential excitation, with 30/40 nociceptive neurones being excited while all of the non-nociceptive neurones ( n = 7) were unaffected (χ 2 = 11.5, 1d.f., P = 0.0007). In contrast, neurokinin A failed to have a preferential effect; 32/46 nociceptive and 9/10 non-nociceptive neurones were excited (χ 2 = 1.0, 1d.f., P = 0.40). Comparing the proportions of nociceptive neurones excited by the different tachykinins indicated that this type of neurone was not differently sensitive to any of the three peptides (χ 2 = 3.2, 2 d.f., P = 0.20). On the other hand, non-nociceptive neurones were preferentially excited by neurokinin A and physalaemin compared with substance P (χ 2 = 13.4, 2 d.f., P = 0.001). With regard to the endogenous tachykinins the results of this study may be interpreted in the following ways. The differential excitatory effect of substance P on nociceptive neurones supports the proposed role for this peptide in the transmission specifically of nociceptive inputs at the first afferent synapse. On the other hand, as neurokinin A excited non-nociceptive as well as nociceptive neurones, there may be a functional role for neurokinin A distinct from that of substance P.

Naloxone was administered in intravenous doses of 0.1–0.6 mg/kg in the spinal cat while recording extracellular spike activity from single, nociceptive dorsal horn units in lumbar segments 5–7 in the spinal transected cat. It was found that while naloxone increased on-going activity and excitatory responses to noxious radiant heat stimuli when it was administered during the day and early evening (12:30–20:00h) there was no observable effect when it was administered during the early hours of the morning (01:20–02:20 h). The one case in which early morning administration provoked an ‘excitation’ was in a cat without a regular rhythm. Units tested were in Rexed's laminae I, III, IV and V. These results suggest that an endogenous opioid factor is released into the circulation in amounts which vary on a diurnal cycle and that this factor crosses the blood-brain barrier at the spinal level to exert an inhibitory effect on transmission specifically in nociceptive pathways at the spinal level.

Administration of 10 micrograms of substance P intrathecally to the spinal T9 level of the adult rat, anaesthetized with urethane, provoked an increase in free catecholamines in plasma taken from the inferior vena cava. Adrenaline levels at 1 min after administration were 154.8 +/- 10.8% (mean +/- SE; n = 11) of preadministration levels and noradrenaline levels were 153.5 +/- 11.8% of preadministration levels. Differences between the values of free catecholamines in animals given substance P vs those given vehicle only were statistically significant at 1 and 10 min postinjection, but not at 30 min. Administration of a substance P analogue with central antagonistic properties 15 min before substance P was given prevented expression of the effects of substance P. These results suggest that substance P may be an excitatory chemical mediator of synaptic transmission in spinal pathways controlling adrenal medullary output. Thus dysfunction of substance P mechanisms may underlie some animal models of hypertension and may be involved in some cases of essential hypertension in man as well as in autonomic dysfunction associated with some neurological entities.

Nociceptive neurons in the dorsal horn of the cat spinal cord are depressed by vibration applied to the ipsilateral hind limb. The present study investigated the pharmacological properties of this depression because of the possibility that it represents the neural basis at the spinal level for the analgesic effects of vibration in humans. Experiments were done in cats anesthetized with sodium pentobarbital and acutely spinalized at the first lumbar level. Extracellular recordings were made from nociceptive neurons in the lower lumbar segments. The depression of these neurons induced by vibration to the hindlimb was attenuated by administration of the P 1 -purinergic (adenosine) receptor antagonist, caffeine (20–60 mg/kg i.v.); the maximum attenuation was 100%. Effects of caffeine began within 2 min after the start of injection (1–3 min injection period), were greatest in the 10 min period after the end of injection and lasted for up to 2 hr. Importantly, another P 1 -purinergic receptor antagonist, which does not cross the blood-brain barrier, 8-sulphophenyltheophylline (8–16 mg/kg), had no effect on the depression when given intravenously ( n = 5 ); however, when administered by iontophoresis 8-sulphophenyltheophylline blocked the depression in 2 of 6 units. Dipyridamole (1.0–2.0 mg/kg i.v.), an inhibitor of adenosine uptake, potentiated the depression in 2 of 5 cases. These results prompt us to suggest that depression induced by vibration may be mediated by adenosine via the activation of P 1 -purinergic receptors. On the other hand, the GABA A antagonist, bicuculline, failed to attenuate vibration-induced depression when administered either intravenously (0.2–0.4 mg/kg; n = 5 ) or by iontophoresis ( n = 10 ) and the glycine antagonist, strychnine (0.2–0.6 mg/kg; n = 3 ) and the opiate antagonist, naloxone (0.1–0.4 mg/kg; n = 4 ) were similarly ineffective. These findings suggest that vibration-induced depression of these units occurs without involvement of bicuculline-sensitive GABA receptors, strychnine-sensitive glycine receptors and naloxone-sensitive opiate receptors. In view of the fact that vibration-induced depression is evoked synaptically, this study is the first to demonstrate in the central nervous system a synaptic response which is mediated by adenosine. In addition, we suggest that the analgesic effects of vibration in humans may be mediated at the spinal level by activation of P 1 -purinergic receptors.

The present investigation was prompted by previous studies in our laboratory which have indicated that tachykinins enhance depressant effects of purincs and that the purine adenosine mediates a vibration-induced depression of nociceptive dorsal horn neurons. Extracellular recordings were made from single nociceptive neurons in the lower lumbar segments of anaesthetized cats. Vibration (80 Hz; 2.5–3.5 s every 20–25 s) was applied to the hindlimb using a feedback-controlled mechanical stimulator. The tachykinins physalaemin, substance P and neurokinin A were administered by iontophoresis. Physalaemin was tested on vibration-induced responses of 29 neurons: each neuron was excited by this tachykinin. To control for possible changes in the response to vibration caused by the excitation Per. Sc statistical comparisons were made of the vibration-induced responses during excitation by tachykinins and during excitation by glutamate. In 16 cases the magnitude of the vibration-induced depression was significantly greater during the excitation caused by physalaemin. With the remaining neurons the response to vibration failed to differ during the excitation by physalaemin compared with the excitation by glutamate. In four of the 16 cases subthreshold applications of vibration caused depression after administration of physalaemin. The P 1 -purinergic (adenosine) antagonist, caffeine, was administered in three cases where vibration caused depression only with application of physalaemin In each of these cases the depression was revcrsibly blocked by caffeine (10 or 40mgkg 1 i.V.). The magnitude of vibration-induced depression was significantly increased during excitation by neurokinin A (5/14 neurons) or by substance P (19 neurons). From the results of the present study we suggest that tachykinins enhance the vibration-induced depression. This enhancement may be due to enhanced depression by adenosine. The possibility is raised that tachykinins released endogenously may increase the sensitivity of nociceptive neurons to the depressant effects of vibration.

A study was done of the effects of iontophoretic application of adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) on functionally identified neurones in the spinal dorsal horn of the cat. AMP depressed nearly two-thirds of the 32 neurones tested regardless of functional type; the remainder were unaffected. ATP, on the other hand, had three types of effect: depression, excitation and a biphasic effect which consisted of excitation followed by depression. A significant difference was found when a comparison was made of the frequency of occurrence of each of these three types of effect in the samples of non-nociceptive (n = 18) and of wide dynamic range neurones (n = 42): of non-nociceptive neurones 61% were excited, 11% were depressed, 6% had a biphasic response and 22% were unaffected; of wide dynamic range neurones 45% had a biphasic response, 19% were depressed, 14% were excited and 21% were unaffected (chi 2 = 16.2, P less than 0.005). The depressant effects of both AMP and ATP and the depressant phase of the biphasic effect of ATP seem to be mediated through activation of P1-purinergic receptors because these effects were blocked by theophylline, a P1-purinergic antagonist [Burnstock (1978) In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, pp. 107-118]. Thus the biphasic effect appears to consist of excitatory and depressant responses in the same neurone. The differential effects of ATP on non-nociceptive vs wide dynamic-range neurones are similar to the differential effects on these neurones observed during activation of low-threshold primary afferents. This similarity, together with evidence that ATP can be released from primary afferent neurones [Holton and Holton (1954) J. Physiol., Lond. 126, 124-140; Holton (1959) J. Physiol., Lond. 145, 494-504], prompts us to suggest that ATP may be a chemical mediator of effects of low-threshold primary afferent inputs in the spinal dorsal horn.

Recent studies have implicated glutamate and substance P in synaptic transmission in the nuclei tractus solitarii and in central regulation of cardiorespiratory functions. Consequently, in chloralose-anaesthetized cats that were artificially ventilated, we examined the effects of the microiontophoretic application of both chemicals (and the substance P homologue, eledoisin-related peptide) on single neurones of the nuclei tractus solitarii implicated in the control of respiration and respiratory tract reflexes. These neurones were functionally identified as either respiratory neurones or presumed reflex interneurones, and showed functional properties comparable to those previously documented for each of these two types. The iontophoretic application of glutamate produced an excitation of rapid onset in 23 or 25 reflex interneurones tested, but the respiratory neurones showed a differential sensitivity: one type (n = 32) was “glutamate-sensitive” and showed rapid excitation with glutamate applications of less than 30 nA, the other type of respiratory neurone (n = 26) was termed “glutamate-insensitive” since it either showed excitation only with applications of 60 nA or more or showed no response even with currents up to 94 nA. Each neurone studied was clearly of one type or the other. Glutamate could increase the number of spikes per rhythmic burst and the burst duration of respiratory neurones, it facilitated evoked activity in the reflex interneurones and in those respiratory neurones having a superior laryngeal nerve or vagus nerve afferent input, and the magnitude of the excitatory responses to glutamate varied directly with the amount of ejecting current. Substance P and eledoisin-related peptide also had exeitatory effects on respiratory neurones and reflex interneurones, but compared with glutamate-induced effects the excitation was slower in onset and more prolonged in after-discharge. Both rhythmic and evoked activity could be facilitated, and the magnitude of the effect varied directly with the magnitude of the ejecting current. In showing that both glutamate and substance P (and its analogue, eledoisin-related peptide) have excitatory effects on the activity of respiratory neurones and reflex interneurones, this study provides evidence suggesting that these neurones have receptors for these neural chemicals, supportive of a role for each chemical in the regulation of respiration and respiratory tract reflexes.

We have shown previously that intrathecal administration of substance P to the lower thoracic vertebral level increases sympathetic output and increases the adrenal output of catecholamines. As opioid peptides are co-released with catecholamines from the adrenal medullae, experiments were done to determine whether the intrathecal administration of substance P to the eighth thoracic vertebral level would alter reaction time in the tail-flick test. Intrathecal administration of substance P (6.5 nmoles in artificial cerebrospinal fluid) in the awake restrained rat increased the reaction time at 1 and 6 min after injection to about 130–140% of the preadministration values; reaction time returned to preadministration values by 11 min. Similar administration of cerebrospinal fluid was without effect. Administration of 6.5 nmoles of thyrotropin-releasing hormone or oxytocin, peptides which also increase sympathetic output, failed to mimic the effects of substance P. The substance P-induced increase in reaction time was absent in rats which had been medullectomized and in rats pretreated with naloxone (10mg/kg). Pretreatment with 10 mg/kg of either phentolamine or the quaternary opiate antagonists, nalorphine methochloride and naloxone methobromide, had no effect on the substance P-induced increase in reaction time. These results suggest that substance P given intrathecally to the eighth thoracic vertebral level may activate spinal sympathetic neurons to the adrenal medullae to cause the release of an opioid into the circulation. This circulating opioid may in turn play a role in the regulation of the tail-flick reflex by a centrally-mediated depression.

Intravenous doses of 0.05–0.15 mg/kg of naloxone caused an increase in the spontaneous discharge rate of single dorsal horn units in lumbar spinal segments 5–7 in the spinal cat. This effect was seen with all of 21 units responding to noxious cutaneous stimulation. In contrast, the 7 units classified as non-nociceptive cutaneous were all unaffected by naloxone, even at doses of 0.4 mg/kg. Of two proprioceptive units, one was excited by naloxone. Naloxone also increased the response of nociceptive units to noxious radiant heat applied to the skin. Section of the ipsilateral dorsal roots of segments L4-S1 failed to abolish the excitatory effect of naloxone on the spontaneous activity of nociceptive units. Responses to naloxone were observed in laminae I, IV, V and VI. These results suggest that the effects of naloxone are not general and that nociceptive units in particular are excited. They also demonstrate that this effect occurs within the central nervous system and that its action is at the spinal level. It is suggested that the algesic effects of naloxone reported by others may be due, at least in part, to this action of naloxone in the spinal cord. While the results of this study support an hypothesis that an endogenous opioid agonist is being released continuously, and it is the prevention of the action of this endogenous substance on ‘opiate receptors’ at the spinal level which is observed as an excitation, it is also possible that naloxone may have an action at the spinal level independently of the actions of another agent, and that perhaps this action is on non-opiate receptors. A third possibility, raised from other recent work, is that the excitatory effects of naloxone may have been due to an antagonism of the effects of γ-aminobutyrate in the spinal cord.