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
566
J.
SANDK~~HLER~~
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
al
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.
Spinal
antinociception
by somatostatin
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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Time during superfusion K
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Time during superfusion
1.2
pM
61
153mM
Concentration of SOM in spmol superfusote
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.
568
J. SANDK~~HLER et ul
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 0.05, n = 4).
C
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Spinal SOM l61p&jJ
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Control
After
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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.
J. SANDK~~HLER et al
570
A
Total no of Imp/ 25s 2500 -
Control
Spinal IO.3
Sptnol
Morphlne
I
mM
(2.7
-15
5
Sollne
Naloxone
25
45
65
nmolcslkgl
85
105 Time
Spat-al
-Skin
heating.
LA
50%
-
Noloxone
Morphma
l
I.V.
125 Imin)
Iv
n
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.
571
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).
A
K Confrol 120
Spinal
Morphine
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
10.3 mM
I
Spin01 Naloxone
30
B
100
% Control
Spinal P ._
z
8o
Morphine (3.0 mM1
I I Spinal Naloxone , 12.7mMl I i
I.0
60
Ttmo 01 sptnot
suporfvsron
60 I mtn)
(n&l
*
11 Time of spinal
23 17 superfusion lminl
29
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.
572
J.
SANDK~HLEK
rt ul.
B
60 -
LO -
20 -
0
20
LO
60
80
Response
100
0
120
to innocuous
skin
20
brushing
LO
60
I % Control
80
100
120
I
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.
effect of somatostatin was not antagonized p-opiate receptor antagonist naloxone.
by the
Spinul supe
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
566
J.
SANDK~~HLER~~
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
al
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.
Spinal
antinociception
by somatostatin
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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or morphine
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% 20
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0
10
20
30
LO
50
60 mm
Time during superfusion K
col*-10
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’ 20
Tie
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8 LO
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50 mm
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during superfusion
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of control
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n=8
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-10
0
m
10
’ 20
m
30
’ LO
a
50
8
60 mm
Time during superfusion
1.2
pM
61
153mM
Concentration of SOM in spmol superfusote
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.
568
J. SANDK~~HLER et ul
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 0.05, n = 4).
C
.E NO. Of mp”lseS
Spinal SOM l61p&jJ
PT-Stim 26 ml” >
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-
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-
D
Control
After
spinal
SOW 161 mY1
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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.
J. SANDK~~HLER et al
570
A
Total no of Imp/ 25s 2500 -
Control
Spinal IO.3
Sptnol
Morphlne
I
mM
(2.7
-15
5
Sollne
Naloxone
25
45
65
nmolcslkgl
85
105 Time
Spat-al
-Skin
heating.
LA
50%
-
Noloxone
Morphma
l
I.V.
125 Imin)
Iv
n
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).
A
K Confrol 120
Spinal
Morphine
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
10.3 mM
I
Spin01 Naloxone
30
B
100
% Control
Spinal P ._
z
8o
Morphine (3.0 mM1
I I Spinal Naloxone , 12.7mMl I i
I.0
60
Ttmo 01 sptnot
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(n&l
*
11 Time of spinal
23 17 superfusion lminl
29
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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SANDK~HLEK
rt ul.
B
60 -
LO -
20 -
0
20
LO
60
80
Response
100
0
120
to innocuous
skin
20
brushing
LO
60
I % Control
80
100
120
I
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.
effect of somatostatin was not antagonized p-opiate receptor antagonist naloxone.
by the
Spinul supe
