Spinal somatostatin superfusion in vivo affects activity of cat nociceptive dorsal horn neurons: Comparison with spinal morphine

0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990IBRO

Neuroscience Vol. 34, No. 3, pp. 565-576, 1990 Printed in Great Britain

SPINAL SOMATOSTATIN SUPERFUSION IN VW0 AFFECTS ACTIVITY OF CAT NOCICEPTIVE DORSAL HORN NEURONS: COMPARISON WITH SPINAL MORPHINE J. SANDK~~HLER,Q.-G. Fu and C. HELMCHEN II. Physiologisches Institut, UniversitPt Heidelberg, Im Neuenheimer Feld 326, 69 Heidelberg, F.R.G. Abstract-A controversy exists concerning the role of the neuropeptide somatostatin for the transmission or inhibition of nociceptive information in the spinal cord. To better correlate electrophysiological effects of somatostatin at single cell level with results obtained with intrathecal injections of somatostatin in behaving animals and human pain patients we applied somatostatin to the spinal cord by controlled superfusion of the recording segment in vivo. The hypothesis of an opiod link and possible neurotoxic effects of somatostatin were also addressed. In cats deeply anaesthetized with pentobarbitone, halothane and nitrous oxide, extracellular recordings were made from 27 neurons located in laminae I-VI. All neurons responded to both innocuous mechanical and noxious radiant heat stimuli applied to the glabrous skin of the ipsilateral hindpaw. The dorsal surface of the spinal cord was superfused at the recording segment by means of a Perspex chamber (7 x 7 mm). Somatostatin superfusions at 1.2 PM had no effect on responses to noxious heat. Responses were, however, depressed by somatostatin at 61 PM to 59.7 + 5.1% of control and by somatostatin at 1.53 mM to 39.9 + 9.5% of control. This inhibition was not antagonized by the p-opiate antagonist naloxone applied to the spinal cord at concentrations of 2.7 mM, either together with somatostatin, or after the inhibition by somatostatin had fully developed. Neuronal responses were linear functions of the skin temperatures for stimulation intensities between 42°C and 52°C. The slopes of these stimulus response functions were reduced during somatostatin superfusion at 61 PM to 46.8 + 9.3% of control, without changing the temperature thresholds for responding (42.5 + 0.6”C). Somatostatin superfusion at 61 PM had no effect on the number of action potentials evoked by innocuous skin brushing, or by electrical stimulation of primary afferent A-fibres in cutaneous nerves. The amplitude of intraspinally recorded field potentials evoked by these electrical nerve stimuli was also unaffected by somatostatin. The inhibition of nociceptive spinal dorsal horn neurons by spinally administered morphine was assessed in eight experiments. Morphine reduced noxious heat-evoked responses to 42.1 k 9.6% of control at 0.3 mM and to 51.8 f 6.9% of control at 3.0 mM. The slopes of the stimulus-response functions were reduced by morphine at 0.3 mM to 53.1 k 11.3% of control, without changing the temperature thresholds (42.7”C). Naloxone superfusion (2.7 mM) reliably antagonized the inhibition by morphine. Brush-evoked responses were not, or much less, affected by spinal morphine. It is concluded that both, somatostatin and morphine selectively depress nociceptive responses of multireceptive dorsal horn neurons by a direct, spinal site of action, providing a neuronal basis for their antinociceptive effects in behaving animals and in pain patients. The effects of somatostatin and morphine are qualitatively and quantitatively similar, although the inhibition by morphine, but not by somatostatin, is mediated via spinal p-opiate receptors.

The role of the neuropeptide somatostatin in the spinal cord for the transmission of nociceptive information has been discussed controversially by investigators who have applied somatostatin intrathecally in pain patients or behaving laboratory animals, by iontophoresis in the vicinity of spinal nociceptive neurons in vivo, or in a superfusion bath to neurons in a spinal cord slice preparation. For a better correlation of the electrophysiological effects of somatostatin on the single cell level and the effects observed in behaving animals or patients we have for the first time applied somatostatin to the spinal cord in vivo by controlled superfusion which is most comparable to the intrathecal route of administration used in behavioural models of nociception. Abbreviafion:

SRF, stimulus-response

function.

Somatostatin, a cyclic tetradecapeptide, is present in neurons throughout the central and peripheral nervous system and it has been proposed that somatostatin might function as a neurotransmitter or neuromodulator” with a possible opioid link.‘O The presence of somatostatin in small primary afferent nerve fibres,16 in neurons of the substantia gelatinosa of the spinal dorsal horn,” and in neurons of the midbrain periaqueductal gray projecting to the medullary nucleus raphC magnus which are putatively involved in mechanisms of endogeneous pain control,’ has given rise to many speculations about the role of somatostatin in nociception. Some authors have suggested that somatostatin may be involved in the transmission of impulses from nociceptors to spinal dorsal horn neurons.23 Others have proposed that somatostatin may mediate the 565

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inhibition of nociceptive neuronal responses32 and may cause analgesia in humans.’ There is also evidence that somatostatin may have severe neurotoxic effects resulting in neurological deficits when applied directly to the rat spinal cord.26 The present study was, therefore, undertaken to determine the effect of spinal somatostatin on spontaneous and evoked activity of multireceptive spinal dorsal horn neurons. Efforts were made to detect any neurotoxic effects of somatostatin. Parts of the present results have been published in preliminary form.36

EXPERIMENTAL

PROCEDURES

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A surgical level of anaesthesia was maintained throughout the course of the experiment by a continuous intravenous infusion of pentobarbitone (6612 mg/kg per h) and, if necessary, by periodically adding halothane (0.4-1.0 percentage volume) to the inhalation gas. Fractional endtidal carbon dioxide concentration, rectal temperature and the electrocardiogram were continuously monitored. The left hindpaw was embedded in paraffin wax and fixed to a holder with the pad upwards. The left posterior tibia1 nerve and the superficial peroneal nerve were dissected free and left in continuity for bipolar electrical stimulation with platinum hook-electrodes. A vertebral laminectomy was performed to expose the lumbar enlargement of the spinal cord. The dorsal surface of the spinal cord was superfused at the recording segment by means of a small Perspex chamber (Fig. lA), as described previously.‘* All other exposed nervous tissue was covered by warm paraffin oil.

Animal preparation

Recordings

Results were obtained from 25 adult female cats, weighing 2.3-3.5 kg. Experimental procedures are described in

Extracellular recordings were made from single dorsal horn neurons below the superfused spinal cord area, using tungsten microelectrodes. Electrical stimulation of the posterior tibia1 and superficial peroneal nerves at supramaximal strength for the activation of A-B-8 -fibres (Fig. 1B) and/or light mechanical probing of the glabrous skin of the ipsilatera1 hindpaw, were used as search stimuli. The excitatory receptive field was located and adequate mechanical excitatory stimuli such as brushing, touch, pressure, pinch and movement of hair follicles were determined. Only neurons which also responded to a standard noxious radiant heat stimulus (WC, 10 s, Fig. 1D) were considered further.

detail elsewhere,j’ in brief, cats were initially anaesthetized with sodium pentobarbitone, 40 mg/kg, given intraperitoneally. Catheters were inserted into a carotid artery, for continuous monitoring of the arterial blood pressure, into an external jugular vein, to measure central veneous pressure and to administer drugs, and into the trachea for controlled mechanical ventilation of the lungs with a gaseous mixture of 75% nitrous oxide and 25% oxygen. Pancuronium bromide (0.4 mg/kg) was given intravenously for muscle relaxation.

and stimulutions

Fig. 1. Schematic illustration of the experimental set-up, the stimulation and neuronal response characteristics. (A) A Perspex chamber which is suitably formed to fit to the surface of the cord dorsum at the lumbar enlargement was used for superfusion at the recording segment. The chamber was placed on a ring of silicon grease (Bayer AG, Leverkusen, F.R.G.) for secure sealing. This chamber contained about 200 ~1 of an isotonic saline solution which could be exchanged through polyethylene tubing within seconds without interfering with the stability of the single cell recording. Extracellular recordings were made underneath the superfused cord surface with tungsten microelectrodes. (B) Platinum hook electrodes were used for bipolar electrical nerve stimulation (anode distal) with monophasic square pulses (pulse width: 0.1 ms, 2.5 V) to excite all A-fibres. The oscilloscopic record of one neuron’s response to this stimulus is displayed at the top. (C) Innocuous brush stimuli were applied by electronically controlled horizontal movements of a soft tooth brush for 15 s. Each stimulus consisted of 10 consecutive sinusoidal movements with an amplitude of 14 mm and a cycle length of 1.5 s. (D) Radiant heat stimuli, each lasting 10 s, were given at preselected temperatures (42-52°C). A feed-back controlled quartz-halogen lamp was used. The light beam was focused on the glabrous skin of the foot- or toepad within the cutaneous receptive field of the selected neuron. The skin temperature during a standard 50°C stimulus is displayed underneath the peristimulus time histogram of one neuron’s response to this heat stimulus. The black horizontal bar indicates the period of skin heating.

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Innocuous mechanical skin stimuli were applied with a soft tooth brush (Fig. 1C). Once stable heat-evoked responses were established, i.e. responses which did not differ by more than k 15%from the mean the contents of the superfusion chamber was exchanged for the appropriate drug solution and heat-evoked responses were again determined for up to 1h before drugs were removed from the cord surface by multiple flushings with isotonic saline solution. If not stated otherwise, only one neuron and one drug were tested per cat. At the conclusion of the experiments cats were killed with an overdose of pentobarbitone given intravenously.

Data analysis Spontaneous activity was analysed during the first 5 s of the recording period of 60s and is expressed as the mean number of action potentials per second. Brush- and heat-

evoked responses were analysed as total number of action potentials in 25 s, beginning with the onset of the stimulus, and corrected for spontaneous activity. Mean responses immediately before drug application served as control values for comparison with mean responses 20 to 30min after beginning of the superfusion. Responses during superfusion are expressed as a percentage of control values. Means are given with their standard errors (S.E.M.). Statistical comparisons were made using Student’s two-tailed t-test for paired or unpaired data. P % 0.05 was considered significant.

RESULTS

Unit sample Results were obtained from 27 neurons located in laminae I-VI of the lumbar spinal dorsal horn.35 All neurons included in this study have excitatory

receptive fields at one or more toes of the ipsilateral hindpaw. One-third of the neurons also received excitatory afferent input from hair follicle receptors. All neurons responded to noxious radiant heating of the glabrous skin within the receptive field as well as to innocuous mechanical skin stimuli such as brushing with a soft tooth brush. All neurons also responded to stimulation of the ipsilateral superficial peroneal and/or posterior tibia1 nerves at strength supramaximal for the activation of A-/I-fibres and, with a late (> 100 ms) discharge, to electrical stimuli supramaximal for the activation of A-6-C-fibres. Thus, neurons were typical multireceptive,’ wide dynamic range*’ or class 2’j neurons. No attempt was made to differentiate spinal interneurons from neurons of orgin of long ascending tract fibres. Spinal superfusion with somatostatin Inhibition of noxious heat-evoked responses by spinal somatostatin. Somatostatin applied to the dorsal surface of the spinal cord at the recording segment depressed noxious heat-evoked responses when given at concentrations of 61 PM or 1.53 mM, but had no effect at a concentration of 1.2 pM (Fig. 2). Inhibition by somatostatin typically began 12-15 min after the onset of the superfusion and reached maximum effect 2&30 min after the onset. This inhibition was partially reversed 35 min after the removal of somatostatin from the cord surface.

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Fig. 2. Summary of the effect of spinal somatostatin (SOM) on noxious heat-evoked responses of spinal dorsal horn neurons. Mean noxious heat-evoked responses of four to eight dorsal horn neurons are expressed as a percentage of control values and plotted vs time. Vertical lines represent S.E.M. In A to C hatched vertical lines indicate the beginning of the superfusion with somatostatin at 1.2, 61 PM or 1.53 mM, respectively. In D vertical bars represent mean heat-evoked activity in percentage of control values pooled 2&30min after the onset of the superfusion with somatostatin.

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The mean, heat-evoked responses of eight neurons were reduced 20-30 min after the beginning of superfusion with 61 nM from 1403 f 187imp per 25 s to 59.7*5.1% (t =7.90, P
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somatostatin (6 1 p M). Neither the mean time course, nor the efficacy of inhibition was changed during p-opiate receptor blockage (Fig. 4A and B). Inhibition by somatostatin alone, as compared to the inhibition by somatostatin plus naloxone (numbers in parentheses) was to 62.5 + 4.8% (68.0 f 5.8%) at 15-25 min, to 55.6 f 4.4% (59.0 f 7.6) at 26-35 min and to 57.8 f 5.6% (53.4 + 8.0%) at 3645 min after the beginning of the superfusion (see Fig. 4B). Spinal somatostatin does not affect responses to innocuous stimuli. The effects of spinal somatostatin (61 PM) on responses evoked by innocuous brushing of the skin or electrical stimulation of cutaneous nerves, supramaximal for the activation of A-fibres, were assessed for 13 neurons. During somatostatin superfusion mean brush-evoked responses of six dorsal horn neurons were not significantly different (104.2 + 4.0%) from control responses before somatostatin. The responses of the same and two additional neurons to noxious skin heating were, however, inhibited during somatostatin superfusion to 61.2 & 4.4% of control (t = 8.82, P 5 0.01, n = 8). See Fig. 5A and B for a typical example and Fig. 8A for a summary of the results. The effect of spinal somatostatin (61 PM) on dorsal horn neuronal responses to electrical stimulation of primary afferent A-fibres was determined in seven experiments. The mean number of action potentials which were evoked at short (I 20 ms) latencies by six

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Fig. 3. The inhibition of noxious heat-evoked reponses by spinal somatostatin (SOM) is not antagonized by blockage of p-opiate receptors. In A a typical example of one neuron’s response to radiant skin heating (52°C 10s) is plotted as total number of impulses in 25s vs time (0-O). The spinal cord was superfused at the recording segment with somatostatin at 61 PM (first vertical line) and heat-evoked activity was progressively depressed. Exchange of pool content with naloxone at 2.7 mM (second vertical line) did not readily antagonize this inhibition. Spontaneous activity is indicated by circles and connected by a dashed line, right hand scale. Representative responses (0 in A) are plotted as peristimulus time histograms (bin width: 1 s) in B.

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during p-opiate receptor blockage. Mean responses of eight neurons (m) are expressed as a percentage of control values and plotted vs time before and during spinal superfusion with somatostatin at 61 PM. Neither the magnitude nor the time course of inhibition by somatostatin was different in five other neurons (0) when naloxone at 2.7 mM was added to the superfusate together with somatostatin at mean heat-evoked responses 15-25, 2635 or 3645 min after the onset of superfusion with (61 PM, n = 8) or with somatostatin (61 PM) plus naloxone (2.7 PM, n = 5) are plotted as vertical lines indicate S.E.M. (0 somatostatin, (n = 8) q somatostatin + naloxone,

consecutive electrical nerve stimuli, supramaximal for the activation of A-fibres, were compared for each of the seven neurons before and during superfusion of the spinal cord with somatostatin. These responses were not significantly altered during the 60-min periods of somatostatin superfusion as illustrated in Fig. SC. Field potentials evoked by electrical stimulation

61 PM. In B somatostatin vertical bars, n = 5).

of the superficial peroneal or posterior tibia1 nerves at strengths supramaximal for the activation of A-fibres were recorded 1695 f 182 pm below the cord surface of the lower lumbar cord. The mean amplitude of the negative deflection was 69.4 + 13.5 PV before somatostatin and 96.8 f 4.6% of these controls during superfusion with somatostatin (t = 0.71, P > 0.05, n = 4).

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Fig. 5. Inhibition by spinal somatostatin (SOM) is relatively selective for noxious vs non-noxious-evoked responses. Representative heat- and brush-evoked responses of one spinal dorsal horn neuron are illustrated as peristimulus time histograms. Periods of stimulation are indicated by horizontal bars. In A, a control heat-evoked response before and progressively depressed responses 23 and 26min after beginning of the superfusion with somatostatin at 61 PM are depicted. Brush-evoked responses 18 and 28 min after beginning of the same superfusion were, however, not different from control. The number of action potentials in response to electrical A-fibre stimulation of the superficial peroneal (SP) or posterior tibia1 nerves (PT) are illustrated in C for each of seven neurons before (left hand) and during (right hand) superfusion with somatostatin at 61 PM. Part D illustrates oscilloscopic records of field potentials, evoked by electrical stimulation of the posterior tibia1 nerve, recorded 1244pm below the surface of the fifth lumbar spinal segment. Records before and 25 or 40min after the onset of the superfusion with somatostatin at 61 PM are shown.

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Fig. 6. Characteristics of inhibition by spinal morphine Data derived from one neuron arc illustrated. In A, evoked responses are expressed as total number of impulses and plotted vs time. Vertical lines indicate exchange of spinal pool content with superfusates containing normal saline (Control) or morphine (0.3 mM), or the intravenous injection of naloxone (2.7 nmol/kg body weight). Noxious heat-evoked responses (O--O) were strongly suppressed in this neuron 28 to 84min after beginning of the superfusion. This depression was partially reversed by intravenous injection of naloxone. Representative responses (0 in A) are plotted as peristimulus time histograms in part B. Non-noxious brush-evoked responses (A) were, however, not affected by spinal morphine. Mean peristimulus time histograms of two to three brush-evoked responses are shown in part C. Spontaneous activity (O- --O, right hand scale) was low and apparently unaffected by somatostatin or naloxone.

Effect of spinal somatostatin on spontaneous activity. The spontaneous activity of the dorsal horn neurons examined here was low under these particular experimental conditions; this feature is evident in the peristimulus time histograms in Figs 3, 5 and 6. Mean spontaneous activity of eight neurons was 3.1 f 0.7 imp/s in the absence of somatostatin and 3.9 f 1.4 imp/s during spinal somatostatin superfusion (61 PM). In four other experiments spontaneous activity was 1.5 + 0.5 imp/s before and 2.0 k 1.1 imp/s during somatostatin superfusion at 1.53 mM. None of these differences is statistically significant. Spinal somatostatin affects intensity coding for noxious skin heating. Throughout the temperature range employed (42252°C) heat-evoked responses were monotonic linear functions of the stimulus intensity, stimulus-response function (SRF).45 These SRFs were characterized by their extrapolated intercepts with the abscissa, i.e. the temperature thresholds for a response, and by their slopes, which reflect the increase in the number of action

potentials per degree increase in the intensity of the skin heating. Control SRFs in the absence of somatostatin revealed a mean temperature threshold of 42.5 k 0.6C (n = 4) and a slope of 61.6 f 16.9 imp per 25 s per “C. The SRFs of the same neurons were again determined 25 to 45 min after beginning of the superfusion and revealed a mean temperature threshold of 42.0 f 0.5”C, which is not significantly different from control. The slopes of the SRFs were, however, significantly reduced to 34.6 k 10.3 imp per 2.5 s per ‘C, i.e. to 46.8 k 9.3% of control (t = 5.75, P 5 0.01, n = 4), see Fig. 9A. Spinal superfusion

with morphine

In nine cats morphine was added to the superfusate in concentrations of 0.3 or 3.0mM while recording from nine multireceptive neurons. Noxious heatevoked responses were reduced to 42.1 f 9.6% of control by morphine at 0.3 mM (n = 4) and to 51.8 + 6.9% of control by morphine at 3.0 mM (n = 5). This difference is not statistically significant.

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Spinal antinociception by somatostatin or morphine The intravenous injection of naloxone (2.7 nmol/kg body weight) partially antagonized this inhibition (Fig. 6). This response of this neuron to noxious heat was depressed maximally to 10.4% of control 28 min after the onset of the superfusion with 0.3 mM morphine. Responses recovered only incompletely 8 min after the intravenous injection of naloxone. In contrast, the spinal application of naloxone (2.7mM) fully antagonized the inhibition by morphine (0.3 mM) in all five neurons tested (Fig. 7B). The time course of inhibition by spinal morphine and the antagonism by spinal naloxone is illustrated for one typical example in Fig. 7A. The inhibition by spinal morphine (0.3 or 3.0 mM) was relatively selective for nociceptive responses, which were depressed to 48.7 i: 5.9% of control vs innocuous, brush-evoked responses, which were depressed to 83.6 + 9.4% of control (n = 9, Fig. 8B). This difference in efficacy is statistically significant (P 50.01).

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Spinal morphine affected the intensity coding of spinal neurons for noxious heat (Fig. 9B). The slopes of the SRFs were reduced by morphine at 0.3 mM from 92.2 k 12.4imp per 25 s per “C to 46.3f 15.4imp per 25s per “C, i.e. to 53.1 + 11.3% of control (t = 4.15, P I 0.05, n = 3). However, the temperature thresholds for the response were not affected by morphine (42.7 f 0.7”C). DKSCUSSION

The present results show that spinal somatostatin su~rfusions in uivo may selectively depress responses of multireceptive spinal neurons to noxious stimuli which may explain the antinociception by somatostatin when applied to the cord via a similar topical route to behaving animals or to pain patients. The inhibition by somatostatin is qualitatively and quantitatively similar to the effect of intrathecal morphine. But in contrast to the inhibition by morphine the

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Fig. 7. Inhibition by spinal morphine is naloxone reversible. Heat-evoked responses of two different spinal dorsal horn neurons in different cats are plotted in A vs time. Spinal superfusion with morphine (0.3 mM) was begun at time zero (dashed vertical line) and depressed responses to 20.4% of control. In one experiment (0) the pool content was exchanged (1) and naloxone was applied to the cord at 2.7 mM and fully reversed the inhibition by morphine. (B) Mean heat-evoked responses of five dorsal horn neurons are plotted with their standard errors during morphine superfusion and five to 29 min after removal of morphine from the cord surface and superfusion with nafoxone (2.7 mM). Asterisks indicate significant reduction from control (i.e. pre-morphine) values.

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Fig. 8. The depression by spinal somatostatin (SOM) or spinal morphine is relatively selective for nociceptive response. Each data point represents evoked responses of one neuron during spinal superfusion with somatostatin (61 PM) (A) or morphine (0, 0.3 mM, n , 3.0 mM), (B) in percentage of control values before superfusion. Heat-evoked responses are plotted on the ordinate vs brush-evoked responses on the abscissa. The diagonal lines indicate equal inhibition of heat- and brush-evoked responses.

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ma1 effect (2 20 min) suggests that the diffusion into the cord may be relatively slow. We do not believe that a severe diffusion barrier existed in the present experiments, however, since the rather hydrophilic compound morphine hydrochloride was shown to modulate dorsal horn neuronal activity receptor specifically when applied via the same topical route. In an in vitro slice preparation of the rat spinal cord somatostatin produced a reversible hyperpolarization and a reduction of synaptic activity when applied directly onto the neurons under study at concentrations between I and 60 ,nM.” Comparable concentrations within the spinal cord may be gained in the present study during somatostatin superfusion at 61 PM.

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Fig. 9. Spinal somatostatin and morphine affect intensity coding for noxious heat similarly. Responses to graded noxious skin heating, expressed as a percentage of responses to a 52°C heat stimulus, plotted on the ordinate, were linear functions of the stimulation intensity which is plotted on the abscissa @RF). Linear regression lines are shown for corresponding data points. Control SRFs (0) were determined before superfusion for comparison with SRFs of the same neurons measured 2G50 min after the beginning of the superfusion with somatostatin at 61 PM (0 in A) or morphine at 3.0 mM (A in B).

Spinal antin~iception by somatostatin or morphine to cause severe neurological and histological impairments such as flaccid paralysis, loss of anal sphincter reflexes, inflammatory cell reaction, necrosis and cell death.26929943 Somatostatin doses causing motor impai~~nt for 30min to 48 h ranged between 3.1 nmol (in 15 ~1) and 6.1 nmol (in 10 PI), i.e. at concentrations of 200 and 6lOpM, respectively. In one study4’ intrathecal injections of 6.1 nmol in 10 ~1 (610 PM) had no effect on motor performance of rats, while 15.3 nmol (1.53 mM) produced a reversible hindlimb paralysis. Somatostatin doses between 18.3 nmol (in 10~1~~) and 25nmol (in lS@6) produced marked inflammatory cell reaction and extensive neuronal necrosis. In the same reports26,2g.43 somatostatin had apparently no neurotoxic effects when applied at doses of 1 nmol or less, i.e. at concentrations of 100pM or less. The non-toxic doses may be considerably higher in species other than rats since Mollenholt et ~1.~~ reported that somatostatin at doses more than 10 times higher, on a body weight basis, than those producing toxic effects in rats had no toxic effects when given to mice. Further, no neurological deficits were reported when a bolus of 153 nmol or a continuous infusion of l&SOpg/h of somatostatin was injected intrathecally in humans.’ The concentrations of somatostatin could not be determined from their report. If somatostatin, at the concentrations employed here, had caused unspecific membrane alterations, cell death or necrosis, one would expect that all electrical activity of the damaged neurons such as spontaneous activity, noxious and non-noxiousevoked responses as well as postsynaptic potentials would be attenuated or abolished. However, somatostatin given at a concentration of 61 pM depressed noxious heat-evoked activity only leaving brushevoked responses, responses to electrical stimulation of primary afferent A-fibres and spontaneous activity unaffected; i.e. the effect was selective. We also measured the amplitude of the negative deflection of intraspinally recorded field potentials in response to electrical nerve stimulation. This potential, reflecting the summation of postsynaptic potentials of dorsal horn neurons, was also unaffected by spinal somatostatin. Clearly, dorsal horn cells were still capable of producing postsynaptic potentials and action potentials, and this argues strongly against any severe unspecific membrane- or receptor”m~iated neurotoxic effects of somatostatin.26 Gross histological examination of some of our superfused spinal cord segments also did not reveal any apparent abnormalities such as inflammatory cell reaction. In conclusion, somatostatin is undoubtedly capable of causing severe neurotoxic effects when applied to the rat spinal cord at concentrations of 600 PM or more. This neurotoxicity may vary considerably from species to species, with the rat apparently being most vulnerable. At concentrations up to 100 PM, however, intrathccally administered

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somatostatin consistently had no toxic effects in any of the species, including cats tested so far. is somatostatin an inhibitory neurotransmitter in the spinai cord? Two hypotheses have been proposed: somatostatin might mediate or facilitate the transmission of nociceptive information in the spinal dorsal horn. This proposal is supported by (1) the presence of somatostatin in small primary afferent nerve fibres16 some of which might convey information from nociceptors;2,6 (2) the release of somatostatin in the spinal cord upon cutaneous noxious thermal stimulation,2”*29a(3) behavioural responses to intrathecally applied somatostatin, such as caudally directed bites and scratching, which have been interpreted as indicative of pain perception,39,43 and (4) the facilitation of nociceptive motor reflexes following intrathecal somatostatin.43 Alternatively, somatostatin may depress but not mediate nociception in the spinal cord, as (1) somatostatin is not detected in the majority of primary afferent A-6 -C-fibres which transmit impulses from cutaneous nociceptors;24 (2) somatostatin is present in neurons of the substantia gelatinosa,‘s~22 a region which is believed to play a key role in the modulation of nociceptive messages.’ This spinal distribution of somatostatin corresponds remarkably to the distribution of somatostatin receptors;“4 (3) somatostatin is present in neurons descending from the brainstem to the spinal cord,*’ some of which might be involved in endogeneous, descending pain controlling systems, see Refs 4 and 13 for recent reviews; (4) somatostatin is a predominantly inhibitory agent3’ and hyperpolarizes dorsal horn neuronq3” (5) nociceptive spinal neurons are inhibited by ionophoretically applied somatostatin;32 (6) intrath~ally applied somatostatin may inhibit motor reflexes in response to noxious stimuli, in a dose-dependent manner;26s29(7) somatostatin has been shown to be analgesic when given systemically to patients with cluster headache,40 or when given intrathecally to patients with cancer pain.’ The present study was designed to test these hypotheses. If somatostatin mediates or facilitates the transmission of nociceptive information in the spinal dorsal horn it would be expected that superfusion of the spinal cord with somatostatin would enhance the background activity of spinal dorsal horn neurons with known excitatory afferent input from nociceptors. Somatostatin might then also enhance spinal neuronal responses to impulses in primary afferent nociceptors and/or shift neuronal thresholds for nociceptive responses to lower stimulation intensities. However, neither the background activity of multireceptive dorsal horn neurons was altered during somatostatin superfusion, nor were noxious heatevoked responses facilitated, or thresholds for a response shifted to lower temperatures. This lack of effect is probably not caused by an inappropriate

concentration of somatostatin, as a wide range from 1.2 FM to 1.53mM somatostatin was tested. Thus, our data do not support the notion that somatostatin plays a major role in the transmission or facilitation of nociceptive information in the spinal dorsal horn. This conclusion is further supported by recent results (Sandkiihler et al., unpublished observations), showing that heterosegmental superfusion of the cat spinal cord with somatostatin fails to mimic the effect of peripheral noxious stimulation involving the same cervical spinal segment. If somatostatin is a neurotransmitter at primary afferent nociceptor terminals, it would be expected that both cervical somatostatin superfusion and heterosegmental natural noxious stimulation would have an inhibitory effect on nociceptive responses of multireceptive lumbar spinal doral horn neurons, a phenomenon which has been termed “diffuse noxious inhibitory control”.25 But somatostatin superfusions were ineffective in these experiments. At concentrations of 61 PM somatostatin reduced noxious heat-evoked responses of spinal dorsal horn neurons while brush-evoked responses, or responses to electrical stimulation of primary afferent A-fibres were unaffected. This is consistent with the finding that ionophoretic application of somatostatin also depresses nociceptive responses of rat spinal dorsal horn neurons.r2 This selective depression of nociceptive spinal dorsal horn neurons may be relevant to the inhibition of spinally mediated motor retlexes in responses to noxious stimuli26.2y and the analgesia observed following intrathecal injections of somatostatin in humans.’ However, it is crucial to establish that inhibitory and analgetic effects are not due to unspecific neurotoxic effects which are clearly relevant at high somatostatin doses, at least when given to rats.‘6.29,4’ Under physiological conditions somatostatin may be released from substantia gelatinosa neurons18.22 to trigger segmental mechanisms of antinociception as described by Melzack and Wall.” Somatostatin may also be released from bulbospinal neurons2’ which are descending through various aspects of the spinal cord white matter37 mediating the descending inhibition of nociceptive neurons.’ Finally, somatostatin may be released from ascending or descending spinal interneurons4’ involved in propriospinal mechanisms of antinociception which have only been characterized recently (Sandktihler ef al., unpublished observations). Eflects of spinal morphine

It is well established that antinociception by the classical p-opiate receptor agonist morphine is in part due to a direct spinal site of action: in spinalized animals intravenous morphine depresses nociceptive dorsal horn neurons (e.g. Ref. 5). Morphine, applied topically to the spinal cord at the recording segment by means of an agar-agar “po01”‘~.‘~ or through pipette ejection I2 has also been shown to depress noxious heat- or pinch-evoked responses of multi-

receptive dorsal horn neurons with little or no effect on responses to electrical activation A-P-fibres and innocuous mechanical skin stimuli. Injections of morphine through catheters inserted into the intrathecal lumbar space also depresses C-fibre evoked responses in ascending axons of spinal multireceptive neurons.20 In the present study controlled superfusion of the spinal cord at the recording segment with morphine also depressed noxious heat-evoked responses (to about 50% of control), while innocuous brushevoked responses were largely unaffected. This inhibition by morphine was mediated via p-opiate receptors, as it was readily antagonized by naloxone (see also Refs 17, 19 and 20). Morphine applied directly onto the spinal cord surface reduced the slopes of the SRFs without a change in threshold, as did systemic or supraspinal morphine in our previous workI suggesting that basic inhibitory mechanisms in the spinal cord are similar. Comparison ~~h~~jt~on

of somatostatin and morphine-induced

Evidence has been presented that some somatostatin effects may be mediated through an opiate link, as somatostatin and somatostatin analogues may bind to opiate receptors.‘“,“.42 To test whether the inhibition by spinai somatostatin in the present report shares common mechanisms with the inhibition by morphine, the classical p-opiate receptor agonist, we have quantitatively compared their effects. Both somatostatin and morphine failed to completely abolish noxious heatevoked responses even at concentrations 10 times higher than those producing a 50% inhibition. Other authors have also found that spinal morphine does not completely suppress nociceptive responses of spinal dorsal horn neurons.‘2.‘7.2o The inhibition by somatostatin and by morphine was relatively selective for noxious vs non-noxiousevoked responses and somatostatin and morphine affected the intensity coding for noxious heat similarly. Thus several lines of evidence suggest that somatostatin and morphine actions might involve similar inhibitory mechanisms in the spinal dorsal horn, providing a neuronal basis for their antinociceptive and analgetic effects when applied intrathetally to awake, unrestrained animals or to human pain patients. However, morphine- but not somatostatin-induced inhibition in the spinal cord is mediated through p-opiate receptors. thank Anna Raccioppoli for technical assistance, Almuth E. Manisali for illustrations and Roland Lux and Wolfgang Salz for computer programming and Drs D. Baliantyne and J. D. Leah for reading the Ackno~(edgeme~ts--We

manuscript. Somatostatin was a gift of Drs J. Chrubasik and E. Wiinsch. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn) to Prof. M. Zimmermann and to J.S.

Spinal

antinociception

by somatostatin

or morphine

575

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