EXPERIMENTAL

NEUROLOGY

52, 325-338 (1976)

Interaction of lontophoretically Responses of Interneurons JONATHAN Dcpartnmt

of Zoology,

0. DOSTROVSKY University Received

of

Toronto, March

Applied Morphine with in Cat Spinal Cord AND BRUCE POMERANZ Toronto,

Ontario, MSS

l lA1,

Canada

29, 1976

A previous study reported that morphine blocked excitation by glutamate and depression by glycine. The present study examines the interaction of naloxone, an antagonist of morphine and dextrorphan, an inactive stereoisomer, with the responses elicited by these two amino acid putative transmitters. Drugs were applied by microiontophoresis from multibarreled pipettes onto sensory interneurons in the lumbar spinal cord of spinal cats. Both naloxone and dextrorphan were found to reduce the excitation produced by glutamate in a manner similar to morphine, thereby ruling out this interaction as relevant to analgesia. Neither naloxone nor dextrorphan were capable of reducing glycine-induced depression. However, naloxone did not antagonize the morhine blockade of glycine depression and it is therefore unlikely that morphine’s interaction with glycine depression is relevant to morphine analgesia. The effects of iontophoretically applied morphine were also tested on the excitation of interneurons by cutaneous stimuli and were found to be small and varied. Neurons were excited by natural light and noxious tactile stimuli and by electrical cutaneous stimulation. Depression of naturally evoked responses was observed in 16% of neurons (13% were enhanced) and depression of electrically evoked responses in 9% of neurons (38% enhanced). Depression of lamina IV neurons was not observed. Although the results demonstrate a depression of responses of some spinal cord sensory interneurons by morphine, it is not thought that they support a direct action of morphine on these interneurons as the major site of its action in the production of analgesia.

INTRODUCTION There is no satisfactory explanation of how morphine and other narcotic analgesics produce analgesia. It is generally believed that analgesia 1 This work was supported by grants from the M.R.C. and N.R.C. of Canada. We wish to thank Professor P. D. Wall for his critical reading of this manuscript and hlr. W. Aldridge for his able technical assistance. Present address of J. Dostrovsky is Cerebral Functions Group, Department of Anatomy, University College London, London WClE 6BT, England. 325 Copyright 0 1976 by Academic Press,Inc. All

rights

of reproduction

in any form reserved.

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results from morphine’s action on neurons of the central nervous system (CNS) since studies (7, 16) have shown a lack of effect on peripheral nerves. Within the CNS it is not yet clear where and by what mechanism morphine acts. The spinal cord and central gray region of the midbrain have been the primary areas considered in recent studies on the site of action of morphine in analgesia (1, 3, 6, 11-14, 18, 23, 25). Although many recent experiments have produced evidence for an action of morphine in the spinal cord (1, 3, 13, 14, IS), other studies (11, 12, 17, 23, 25) do not support a major role for morphine at the spinal level. Recent work (22) has shown that presumably morphine interacts with the neuron membrane via a stereospecific narcotic receptor. Our earlier studies (9) showed that morphine applied iontophoretically reduced the excitation produced by glutamate and the depression produced by glycine. These effects have since been confirmd (3, 19). There is substantial evidence that glutamate and glycine are transmitters in the spinal cord (4, 15) and that dorsal horn neurons are involved in the transmission of nociceptive stimuli to higher brain levels. Thus it is important to investigate the interactions of morphine with glutamate and glycine in order to determine whether or not )they might be relevant to analgesia. The present study examined the interaction of morphine with glutamate excitation and glycine depression in greater detail and attempted to discover if they are related to morphine’s analgesic properties. This was done by comparing the interaction of the amino acids with morphine, with naloxone, a narcotic analgesic antagonist, and with dextrorphan, a narcotic stereoisomer with no analgesic properties. We also examined the effect of iontophoretically-applied morphine on the responses of dorsal horn neurons to noxious mechanical and electrical stimuli to determine whether or not morphine acts on these neurons to block nociceptive input to higher CNS levels. METHODS Experiments were performed on cats (2.5 to 4 kg) the spinal cords of which were transected at Cl and the brains destroyed under ether anesthesia, and which were mechanically respirated. A bilateral pneumo.thorax was performed to reduce respiratory movements and the animals paralyzed with gallamine triethiodide. Blood pressure was monitored and maintained above 65 mm Hg. Body and oil bath temperatures were kept at 37 f 0.5”C. Blood oxygen concentration was routinely measured. Multibarrel electrodes were made by gluing together seven or ten capillary tubes (Kimax 46485). This array was pulled on a Narashige Vertical puller and the combined tips broken to 3 to 7 pm (5 to 9 pm for lo-barrel electrodes). The electrodes were filled by centrifugation (2OOOg for 1 min)

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327

(8). The center barrel of each electrode was filled with 4 M NaCl and was used for recording action potentials. Two other barrels were filled with 1 M NaCl and were used for current neutralization and curent controls. The other barrels were filled with some of the following solutions: glutamate (0.5 M, pHS), aspartate (0.5 M, pHS), glycine (0.5 M, pH4), GABA (0.5 M, pH4), morphine sulphate (70 mM ) , naloxone hydrochloride (130 mM), levorphanol tartrate (20 mM in 165 mM NaCl), dextrorphan tartrate (20 mM in 165 mM NaCl), glutamate diethylester (0.5 M, pH4) and strychnine sulphate (10 mM in 165 mM NaCl). Drugs were ejected by an S-channel iontophoresis polarizer, which incorporated a current neutralizing circuit which produced a current through one NaCl barrel equal to the algebraic sum of the currents in all the other barrels, but of opposite polarity, thereby producing at all Itimes a net current of zero between the electrode tip and ground (the preparation). The current control barrel (the other NaCl barrel) was always tested to ensure that there were no residual current effects. A retaining current of 10 to 15 nA was applied to each drug-containing barrel to prevent diffusion out of the electrode. In a few cases, drugs were ejected by applying pressure to the barrel (using a plastic tube connected to a syringe). This was used as an additional control when testing the interaction of two drugs ejected with opposite polarity (to ensure that the effect was not due to one drug being attracted up the barrel containing the other drug). Neural activity was amplified and displayed by conventional methods. Neuronal firing rate was monitored with an epochal ratemeter and displayed on a storage oscilloscope. A Fabriteck 1072 on-line computer was used for performing poststimulus time histograms. The electrode was advanced into the dorsal horn of the Sl-L6 region of the spinal cord. Cells recorded with action potentials of over 100 PV were studied. They were identified as being in lamina IV, V, or elsewhere primarily by their physiological responses to probing the skin of the hind leg, but also by their depth as determined from the micromanipulator reading. In five cats the electrode position was determined by observing the tip of the cut electrode or a dye mark (Niagara sky blue 3%) in clear thick sections of spinal cord. RESULTS Glutamate, glycine, and GABA were all found to have potent effects on dorsal horn neurons, as has been reported by others, and the interaction of the opiates with these effects were tested. Acetylcholine, noradrenaline, dopamine, and serotonin were found to have only weak and variable effects which precluded further investigation of their interaction with opiates.

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Interaction

AND

of Opiates with

POMERANZ

Glutavnate Excitation

Effects of Morphine. Glutamate is a very potent excitatory agent. Iontophoretic application of morphine was founld to reduce the effectiveness of glutamate to produce excitation in 78% (146 of 188) of the neurons tested. Excitation by aspartate was similarly reduced (43 of 53 neurons). In most cases morphine ejection currents of more than 100 nA were necessary to produce reductions of more than 50% in the glutamate-induced firing. In Fig. lA, top row, a ratemeter recording shows how applications of morphine (140 nA) blocks the excitation produced by pulses of glutamate (25 nA). Frequently, when morphine was applied for any length of time (20 to 120 set depending on the cell) the action potential size and shape would deteriorate. However it was usually possible to demonstrate antagonism of glutamate before the action potential size decreased. Even when the action potential size decreased, the spontaneous rate and response to synaptic activation rarely changed, indicating that the overall excitability of the neuron was unchanged. Although ,the standard controls for current artifacts were performed and proved negative, it was nevertheless felt that some of the results could have been due to a technical artifact which was difficult to control for.2 Therefore additional tesits were performed to ensure that the effects were not due to an artifact. Pressure injection of morphine reduced glutamate excitation in four of 11 units tested (two units were enhanced). It was also possible to demonstrate the reduction of glutamate excitation when morphine was applied before, but not during glutamate ejection (so that both drugs were never ejected at the same time). However, it was not possible to demonstrate a redutcion of glutamate sensitivity by iv injections of morphine (10 mg/kg). Comparisons of the application of morphine and glutamate diethylester, a proposed glutamate antagonist, revealed that both agents reduced glutamate excitation by approximately the same extent although there were cases when

one or the other

agent

was more

effective.

Eflects of Naloxone. Naloxone, a drug which antagonizes the analgesic properties of morphine, was tested on nine neurons and found to reduce glutamate excitation in five. In Fig. lA, line 2, it is seen that naloxone (120 nA) caused a reduction of glutamate excitation which is not mims Glutamate and morphine ions are of opposite polarity and the transport number of morphine is lower than for glutamate or the control ion Na+. Thus it is possible that some glutamate ions could be attracted into the morphine barrel when both glntamate and morphine are being ejected concurrently, thus causing a decrease in glntamate concentration in the electrode tip vicinity. This effect might not occur when the Na+ control is tested due to the difference in transport numbers (8).

MORPHINE

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INTERNEURONS

B 0 S’ iillkA&F

0

FIG. 1. Blockade of glutamate-indured excitation by opiates. In these ratemeter recordings (and those in Figs. 2 and 3) the top line of each pair is the ratemeter record representing the rate of firing of a single neuron. The line immediately beneath the record is a marker which indicates, by means of an upward step, when drugs are being ejected. When two drugs are ejected concurrently, the duration of ejection of one of them is represented by a dashed line. For clarity all the records have been retouched by hand from the original dot display of the ratemeter to look like bar histograms. FIG. IA. The record in line 1 shows that morphine (M) ejected at 140 nA (indicated by dashed line in center of record) blocks excitation produced by repetitive ejection of glutamate (G) 25 nA at times indicated in lower trace (first application is marked by a letter G). Line 2 shows a similar effect when naloxone (N) 120 nA is applied and line 3 shows that a control current of sodium ions (Na) 120 nA has minimal effect on glutamate excitation. (Note that sensitivity of the neuron to glutamate has decreased slightly and the spontaneous rate has increased gradually as the testing progressed.) FIG. 1B. The record from a different interneuron which is excited by continuous ejection of glutamate shows that application of levorphanol (L) 100 nA and dextrorphan (D) 100 nA but not a current control (Na) 160 nA blocked the excitation of the neuron by glutamate (the neuron was not spontaneously active).

icked by ejection (line 3).

of sodium

ions

(120 nA) from

the NaCl control

Efects of Levorphanol and Dextrorphan. The narcotic phanol,

and

its

inactive

stereoisomer,

dextrorphan,

were

barrel

analgesic levortested on the

330

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excitation produced by glutamate. These two drugs were found to be much more potent in causing a decrease in the neuronal action potential amplitude than morphine and this effect prevented the use of high ejection currents (greater than 50 to 100 nA) or prolonged ejection periods. Both levorphanol and dextrorphan were found to reduce the glutamate excitation of five cells to the same extent and had no effect on the other five neurons tested (except for causing action potential size reduction at high ejection currents). In Fig. 1B excitation caused by a continuous application of glutamate is greatly reduced by application of levorphanol (100 nA) and dextrorphan (100 nA) but not by sodium ions (160 nA). Interaction

of Opiates with

Glycine and GABA

Efects of Morphine. Glycine and GABA are both potent inhibitory agents. Morphine was found consistently to reduce or block the inhibitory effects of glycine. Antagonism was observed in 95% (147/155) of the neurons. Currents of the order of SO nA for 10 sec. were generally sufficient to demonstrate complete blockade of glycine inhibition, although neuron sensitivity varied greatly. In these experiments, the amount of glycine released was chosen as the minimum current necessary to produce complete blockade of the spontaneous rate or the naturally evoked firing. Higher glycine currents required larger morphine currents to produce antagonism. Conversely, antagonism by morphine could be overcome by increasing the glycine current. The antagonism of glycine could always be demonstrated at morphine current levels which did not reduce the size or shape of the action potentials. The inhibitory effects of GABA were much less readily antagonized by morphine. However, with high ejection currents of morphine, weak antagonism of the depressant effects of GABA was observed in 50% (21/42) of the neurons. In most cases in which glycine and GABA were tested on the same neuron, morphine currents causing complete blockade of glycine depression had no effect on the depression produced by GABA. Nevertheless, on a few cells morphine was almost equally effective in blocking both amino acid effects. The potency of morphine’s blockade of glycine depression was compared with that of strychine, a specific antagonist of glycine. Strychnine was found to consistently block the effect of glycine at currents which were 0.2 to 0.3 times those needed to obtain the same effect with morphine. The blockade by strychine usually took longer to wear off than that of morphine, frequently taking up to 60 set and sometimes even longer compared with approximately 10 set for morphine. Figure 2A shows a ratemeter record of a spontaneously firing neuron with a rate depressed by application of

MORPHINE

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glycine (25 nA) and GABA (65 nA). Application of morphine (80 nA) (top line) blocks the depression produced by glycine but not of GABA. The second line shows that ejection of strychine 20 nA also blocks the depression produced by glycine but not by GABA whereas application of a control current of sodium ions (80 nA) has no effect on either depression. Morphine applied by pressure injeotion through the micropipette was found to block the depression of glycine on seven of nine neurons (in four cats) as effectively as when ejected from the same barrel by iontophoresis; pressure injection of NaCl from the control barrel had no effect on those neurons. In nine cats, morphine was injected intravenously or intraarterially at a concentration of up to 8 mg/kg and its effect on glycine depression (iontophoretically applied) tested. On six of the cells no effect on the depression of glycine was observed, while on the remaining five neurons a weak reduction was seen. Naloxone Effects. Naloxone did not affect the depression produced by glycine in 79% (n = 38) of neurons tested even at currents significantly

FIG. 2. Morphine blockade of depression by glycine. The ratemeter record shows the depression of spontaneous activity of a lamina V neuron produced by iontophoresis of glycine 25 nA and GABA 65 nA. The first applications of glycine and GABA are indicated by GL and GA, respectively. (GABA application is differentiated from the glycine ejection marker by a lower step and an arrowhead over the marker.) During morphine (M) 80 nA ejection (indicated by dashed line) in line 1 and strychnine (S) 20 nA, depression caused by glycine was abolished but not the depression by GABA. Application of a control current Na+ 80 nA had no effect on either depression,

332

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Bll -

GL

__________..__

L- . . . . . . . . . . . . ..--m

FIG. 3. Effects of opiates on glycine-induced depression. A. Ratemeter records showing firing of a lamina V neuron in response to pressure applied to foot. Glycine ejected (5 nA) repetitively (first instance indicated by GL) depresses this response. Morphine (M) ejected (48 nA) blocks the glycine-induced depression (line l), whereas an equal current (48 nA) of naloxone (N) (line 2) has no effect on firing rate or glycine depression. Line 3 shows blockade of glycine depression by strychnine (S) 8 nA. (The firing of the neuron stops before the end of the ratemeter record because the pressure stimulus was removed.) FIG. 3B. Ratemeter recordings showing a neuron excited by light pressure applied to the foot. Depression produced by repetitive application of glycine (G) 3 nA is blocked by levorphanol (L) 3 nA (line 1) but not by an equal current (30 nA) of dextrorphan (D) (line 2).

higher than those producing an effect with morphine. On the few cells in which naloxone did show some slight reduction of glycine depression, high currents were always necessary and the action potential amplitudes had greatly deteriorated. Naloxone did not antagonize morphine’s blockade of glycine depression when applied by currents of the same magnitude as morphine. It was not possible to apply naloxone with high current,s because the total current of glycine, morphine, and naloxone (which are all of the same polarity) was so high as to cause severe deterioration of the action potential. In Fig. 3A the lack of effect of naloxone (48 nA) on

MORPHINE

glycine glycine

(5 nA) depression depression produced

ON

SPINAL

is contrasted by morphine

Levorphnnol and Dextrorjhan

CORD

333

INTERNEURONS

(2nd

line)

with

the blockade

of

(48 nA) on the same neuron.

Efects. Levorphanol

blockade of glycine depression was observed in 10 out of 21 cells (in three cats). In all cases in which antagonism of glycine by levorphanol was observed (ten cells), dextrorphan, applied at the same current or at higher currents (up to twice the levorphanol ejecting current) and for longer periods, never produced a reduction of glycine depression. In all these cases high ejection currents of dextrorphan produced the same reduction in action potential size as an equivalent dose of levorphanol. Figure 3B shows how the depression produced by application of glycine (3 nA) was blocked by levorphanol (30 nA) (line 1) but not by dextrorphan (30 nA) (line 2).

Eflects of Morphine on Spontaneous Rate and Evoked Responses Iontophoretically-applied effects. The spontaneous

morphine did not in general produce large rate of 16% (7/45) of the neurons tested was

FIG. 4. Effect of morphine on synaptic activation of neurons. These records show poststimulus time histograms of the occurrence of firing of a single neuron to electrical stimulation of the skin. Each record is the average of 20 successive stimuli. FIG. 4A. These poststimuIus histograms demonstrate enhancement of the response of a lamina IV neuron excited by electrical stimulation (60 V ; 0.5 msec) of the skin of the foot. Line 1 is the control response, line 2 the response after 2 min of ejection of morphine (80 nA) and line 3 recovery of the response 1 min after cessation of morphine ejection. The stimulus was delivered at the beginning of the trace. (Vertical scale is counts per bin; bin width 1 msec.) FIG. 4B. This poststimulus histogram of the response of a lamina VI or VII neuron to electrical stimulation (60 V, 0.5 msec) shows a depression of response after 2 min of morphine ejection at 100 nA (line 2). Line 1 is the control response. The response

recovered after were delivered

5 min (not shown). Arrow heads mark to skin. (Vertical marker is counts/bin;

time when bin width

electrical 200 ps.)

stimuli

334

DOSTROVSKY

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POMERANZ

decreased by morphine. The decrease in spontaneous rate was usually 20% to 50% ‘of normal. Classification of these data according to the type of neuron revealed that the spontaneous rate of lamina IV neurons never decreased, in contrast to decreases for neurons of lamina V and other laminae (I, VI, VII). The spontaneous rate of 20% of the neurons showed small increases as a result of iontophoretic ejection of morphine. There was no significant correlation between these effects and the cells’ lamina. Morphine was tested on the responses of neurons to natural and electrical stimulation of skin. The effects were usually small. It was found difficult to test the effects of morphine Non naturally evoked responses to noxious pressure due to the great variability of the responses. Therefore, highintensity electrical stimulation of the skin, in the receptive field, with a pair of needle electrodes was used as an alternative. In many cases poststimulus histograms were obtained showing the average response (from 20 or more successive stimuli) before, during, and after morphine application. When natural stimulation was used (brushing the receptive field or application of pressure using calibrated and modified crocodile clips) a decrease of response was seen in 16% (ten of 61) of neurons tested and enhancement of the response in 13% (S/61) of neurons. When electrical stimulation was used, a decrease in response was observed in 9% (4/47) o f neurons. Analysis of these data and an enhancement in 38% (18/47) by type of neuron revealed that the responses of lamina IV neurons were never depressed, whereas 14% of lamina V neuron responses were depressed by morphine. The percentage of neurons having responses that were enhanced by morphine did not reveal lamina specificity. Figure 4A shows a poststimulus histogram demonstrating an enhancement of the response to electrical stimulation of the receptive field. The top line shows the control response, the second line the response after 2 min of morphine ejection (100 “A), and line 3 shows recovery of response after 1 min. Figure 4B shows a decrease in the response after 2 min application of morphine (line 2). Line 1 is the control response. DISCUSSION These experiments show that the blockade by morphine of glutamate excitation and glycine depression is not antagonized by naloxone and these amino acids are therefore presumably not involved in mediating the do not analgesia produced by morphine. In addition, the experiments provide strong evidence for an action of morphine on spinal cord dorsal horn neurons, although a partial action here appears possible. Since naloxone antagonizes the analgesia produced by morphine, any morphine effect related to analgesia should be antagonized by naloxone.

MORPHINE

ON

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111 addition, a stereoisomer of narcotic analgesics which is devoid of analgesic effects (such as dextrorphan) should not give rise to the change brought about by morphine. Since blockade of glutamate excitation by morphine was mimicked by both naloxone and dextrorphan, the effect cannot be related to morphine analgesia. Our inability to demonstrate antagonism of m’orphine’s blockade of glycine depression by naloxone, despite the lack of effect by dextrorphan, casts serious doubt as to the relevance of morphine’s interaction with glycine depression in producing analgesia. It is possible that not enough naloxone was applied to observe antagonism of the morphine blockade. Nevertheless this is probably not the case because others (3, 6, 14) have reported being able to antagonize other effects of morphine using lower doses of naloxone than of morphine. A recent preliminary communication of Zeiglgansberger and Satoh (26) has reported that naloxone antagonized the morphine blockade of glutamate-induced depolarization. They used intracellular recordings and thus were able to observe the effect of morphine on membrane depolarization. It is possible that the naloxone-sensitive effect that they observed was not observable in our experiments due to the greater amounts of glutamate needed to cause the neuron to fire, which consequently required a greater amount of morphine to yield a reasonably large effect. This would suggest the existence of a naloxone-antagonizable block of glutamate excitation by morphine at low concentrations and a nonspecific blockade at higher morphine concentrations. Morphine was found to depress the responses of some sensory neurons, as might have been expected. However, the effects even when limited only to lamina V neurons were generally small and infrequent in contrast to a few studies (13, 18) using intravenous administration of morphine. Moreover, we observed a significant proportion of neurons the responses of which were actually enhanced by iontophoretic application of morphine. These latter effects may well be due to the ability of morphine to block glycine depression because strychnine also enhances dorsal horn neuron responses [our unpublished results and (10) J. Enhancement is probably not apparent when morphine is applied systemically because the concentration of morphine is lower. Unfortunately it is not possible to determine the concentration of a drug at the neuron membrane when it is applied iontophoretically. This response-enhancing effect of morphine when applied iontophoretically could well be masking an additional depressant effect of the drug, In addition, systemically applied morphine could be acting on distant sites, thus affecting the neuron being monitored, via descending or other pathways. Possible sites of action of morphine producing such an effect could well be in the substantia gelatinosa (having cells to’o small to record from) and brain stem structures. In addition, systemically applied morphine might have an apparently greater effect

336

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on single neurons than local application of the drug due to a diffuse effect on a large number of interacting neurons and/or terminals. Calvillo and co-workers (3) have recently reported a study using iontophoretically applied morphine onto dorsal horn neurons and have shown that morphine depresses the responses of many neurons to noxious stimuli. The larger effects obtained in their study might be due to their different stimulus and restricted population of neurons. A noxious heat stimulus would excite small-diameter A6 and C fibers (2) without affecting low-threshold fibers. Our mechanical and electrical stimuli excited low- threshold fibers as well. Assuming morphine affects only the smaller fiber activation (18), the effects of morphine in our situation might be masked by the powerful excitation of the neuron by low-threshold afferents. In addition, our stimuli are more likely to activate inhibitory pathways leading to enhancement of responses by morphine and further masking of any depressant effect. We feel that our results do not favor ‘lamina V neurons as the sole site of action of morphine in producing analgesia, although a partial action probably exists. The concentration of morphine achieved in these iantophoretic application studies is, as mentioned, not known. However, due to the existence of enhancement of responses by morphine which is not s&n in systemic studies and the weak effects achieved even when maximal doses of morphine are applied (those which are just subthreshold for producing spike deterioration by membrane anesthetic effects (5) >, it is unlikely that morphine analgesia could be mediated entirely by an action on spinal cord sensory neurons. Recent studies (11, 12, 20, 23) have shown marked analgesia by microinjection of morphine and electrical stimulation at nonspinal sites. In addition, studies (21) have shown strong inhibition of lamina V neur,ons by electrical stimulation of supraspinal centers near or identical to those ‘sensitive to morphine. A study (25) on depletion of serotonin-containing descending pathways has demonstrated a concurrent decrease in morphine’s ability to produce analgesia. Finally, studies (17) on the distribution of opiate receptors in the brain reveal only a low concentration in the spinal cord. REFERENCES 1. BESSON, J. M., M. C. WYON-MAILLARD, J. M. BENOIST, C. CONSEILLER, and K. F. HAMANN. 1973.Effects of phenoperidine on lamina V cells in the cat

dorsalhorn. J.

Pharmacol.

Exp.

Ther.

187:

239-245.

2. BURGESS, P. R., and E. R. PERL. 1973. Cutaneous’mech&oreceptors and nociceptors, pp. 29-78,.In “Handbook of Sensory Physiology II.” A. Iggo [Ed.]. Springer-VFrlag, Berlin. 3. CALVILLO, O., J. L. HENRY, and R. S. NEUMAN. 1974. Effects of morphine and naloxone on dorsal horn neurones in the cat. Canad. J. Physiol. Pharmacol. 52 : 1207-1211.

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D. R., and G. A. R. JOHNSTON. 1974. Amino acid transmitters in the mammalian central nervous system. Ergeb. Physiol. 69 : 97-l%. 5. CURTIS, D. R., and J. W. PHILLIS. 1960. The action of procaine and atropine on spinal neurones. J. Physiol. (Land.) 153: 17-34. 6. DAVIES, J. and A. W. DUGAN. 1974. Opiate agonist-antagonist effects on Renshaw cells and spinal interneurones. Natare 250: 70-71. 7. DOMINO, E. F. 1968. Effects of narcotic analgesics on sensory input activating system and motor output. Rcs. Publ. Assoc. Res. New. Merit. Dis. 46: 117147. 8. DOSTROVSKY, J. 0. 1974. Microiontophoretic studies of Izeztrotratts~~~lzitters alad morphine 01% cat s/&al cord sensory internczrrones. Ph.D. thesis, University of Toronto. 9. DOSTROVSKY, J. O., and B. POMERANZ. 1973. Morphine blockade of amino acid putative transmitters on cat spinal cord sensory interneurones. Nature (Neze CURTIS,

Biol.)

246 : 222-224.

10. GAME, C. J. A., and D. LODGE. 1975. The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afferents in the cat. Exj. Brain

11.

HERZ,

12.

JACQUET,

Res. 23 : 75-M

A., K. ALBUS, J. METYS, P. SCHUBERT, and H. TESCHEMACHER. 1970. On the central sites for the antinociceptive action of morphine and fentanyl. Nellro-

pharmacology

9 : 539-551.

Y., and A. LAJTHA. 1974. Paradoxical effects after microinjection of morphine in the periaqueductal grey matter in the rat. Science 185: 1055-1057. 13. KITAHATA, L. M., Y. KOSAKA, T. TAUB, and W. F. COLLINS. 1972. Laminaspecific suppression of dorsal horn unit activity by morphine sulphate. Fed. Proc. 32: 693 (Abst.) 14. KRIVOY, W., D. KROEGER, and E. ZIMMERMANN. 1973. Action of morphine on the segmental reflex of the decerebrate-spinal cat. Brit. J. Pharmacol. 47: 4.57-164. 15. KRNJEVIC, K. 1974. Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54: 418-540. 16. KOSTERLITZ, H. W., and D. I. WALLIS. 1964. The action of morphine-like drugs on impulse transmission in mammalian nerve fibers. Brit. J. Pharmacol. 22: 499-s 10. 17. KUHAR, M. J., C. B. PERT, and S. H. SNYDER. 1973. Regional distribution of opiate receptor binding in monkey and human brain. Nature (Lo&n) 245: 447-450. 18. LEBARS, D., D. MENETREY, C. CONSEILLER, and J. M. BESSON. 1975. Depressive effects of morphine on lamina V cells activated in the dorsal horn of the spinal cat. Brain Res. 98: 261-278. 19. LODGE, D., P. M. HEADLY, A. W. DUCGAN, and T. J. BISCOE. 1974. The effects of morphine, etorphine and sinomenine on the chemical sensitivity and synaptic responses of Renshaw cells and other spinal neurones in the rat. Eur. J. Pharmacol. 26 : 277-284. 20. MAYER, D. J., and J. C. LIEBESKIND. 1973. Pain reduction by focal electrical stimulation of the brain: An anatomical and behayiouraf analysis. Brain Rcs. 68: 73-93. 21. OLIVERAS, J. L., J. M. BESSON, G. GUILBAUD, and J. C. LIEBE~KIND. 1974. Behavioural and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat. Exp. Brain Res. 20: 32-44. 22. PERT, C. B., and S. H. SNYDER. 1973. Opiate receptor:demonstration in nervous tissue. Science 179 : 1011-1014.

338

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and T. YAKSH. 1974. Sites of morphine induced analgesia in the primate brain : Relation to pain pathways, Brain Res. 80: 135-140. 24. TERENIUS, L. 1973. Stereospecific interaction between narcotic analgesic and a synaptic plasma membrane fraction of rat cerebral cortex. Acta Pharwmcol. Toxicol. 32 : 317-320. 25. VOGT, M. 1974. The effect of lowering the 5-hydroxytryptamine content of the rat spinal cord on analgesia produced by morphine. J. Physiol. (Land.) 236: 483-498. 26. ZIEGLGANSBERGER, W., and M. SATOH. 1975. The mechanism of inhibition by morphine on spinal neurons of the cat. Exp. Brain Res. (Suppl.) 23 (Abstr.) : 23. PERT, A.,

444.