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Stereoscpecific actions of morphine on single neurones in the brain stem of the rat
Neuropharmocology.
1977. 16. 519-526.
Pergamon
Press.
Printed
I” Gt. Brilain.
STEREOSCPECIFIC ACTIONS OF MORPHINE ON SINGLE NEURONES IN THE BRAIN STEM OF THE RAT P. B. BRADLEY and G. J. BRAMWELL* Department
of Pharmacology
(Preclinical),
The Medical
(Accepted 10 February
School,
Birmingham
B15 2TJ
1977)
Summary-The effects of microiontophoretically applied morphine, levorphanol and dextrorphan were compared on the firing rate of spontaneously active brain stem neurones. Both morphine and levorphanol produced three different effects: excitation, excitation followed by long-lasting depression, or long-lasting depression; whereas dextrorphan produced only excitation or short-lasting depression. Depression produced by morphine and levorphanol, but not dextrorphan, was antagonized by naloxone or nalorpine, whereas excitation was unaffected or potentiated. It is concluded that long-lasting depression of brain stem neuronal firing produced by morphine or levorphanol represents a stereospecific action.
many experimental studies on the pharmacological actions of morphine and other narcotic analgesics, present knowledge concerning their central sites and mechanism of action in producing antinociception or dependence remains limited. Work in the unanaesthetised rabbit (Herz, Albus, Metjrs and Schubert, 1970; Vigouret, Teschemacher, Albus and Herz, 1973; Teschemacher, Schubert and Herz, 1973) has provided evidence that morphine exerts at least part of its antinoceptive effect through an action in the brain stem region. Thus, localized perfusion of the ventricular system caused antinociception when morphine was able to reach the anterior part of the 4th ventricle, but not when it was confined to the 3rd and lateral ventricles by a plug in the aqueduct. Furthermore, intraventricularly administered levallorphan antagonized the effect of systemically administered morphine. Subsequently it was shown that withdrawal effects could be induced in morphine-tolerant rabbits, if nalorphine was perfused through the 4th ventricle, but not when it was restricted to other parts of the ventricular system (Herz, Teschemacher, Albus and Zieglglnsberger, 1971). Further evidence that morphine exerts actions within the brain stem was provided by Bradley and Dray (1973, 1974), who demonstrated that neurones in the brain stem of urethane-anaesthetized rats were sensitive to microiontophoretically applied morphine. Moreover, these studies showed that morphine could cause excitation as well as depression of the activity of single neurones in this region. The purpose of the present study was to examine the pharmacology and in particular, the stereospecificity, of morphine-induced neuronal excitation and depression in the brain stem. This entailed determining the susceptibility of these two effects to anta-
Despite
gonism by the narcotic antagonist naloxone and the agonist-antagonist nalophine. In addition, the effects of another narcotic analgesic, namely levorphanol, were compared with those produced by morphine, as well as with those of its non-narcotic isomer, dextrorphan. Some of the results have been presented to the British Pharmacological Society (Bradley and Bramwell 1975). METHODS Male albino rats (Sprague-Dawley) were anaesthetized with urethane (125 g kg- ‘) and partially cerebellectomized by suction to expose the floor of the 4th ventricle. Five-barrelled glass micropipettes (Boakes, 1972) with overall tip diameter 6-10~ were used to record extra-cellularly the action potentials of single spontaneously active neurones, and to apply drugs in the immediate vicinity of the cells. Electrode penetrations were made O-3 mm rostra1 of the obex and up to 2 mm lateral of the midline, avoiding the midline. The neurones studied were in the reticular formation, mainly in the nucleus reticularis paramedianus and nucleus reticularis gigantocellularis. The histological localization of neurones excited by morphine in this region has already been published (Boakes, Bramwell, Briggs, Candy and Tempesta, 1974). To record extracellular neuronal action potentials, one barrel of the micropipette was filled with 4 M NaCl. Other barrels were filled with one or other of the following drug solutions; 0.026 M morphine hydrochloride, pH 4.8 (Macfarlan Smith); 0.013-0.026 M levorphanol tartrate, pH 4.5 (Roche); O.O13C).O26M dextrorphan tartrate, pH 4.5, or 0.0134.026 M dextrorphan base in dilute HCl at pH 4.0 (Roche); 0.014X).28 M naloxone hydrochloride pH 4.5 in 0.2 M NaCl (Endo Laboratories); 0.029 M nalorphine hydrochloride, pH 4.5 (Burroughs Wellcome); 0.28 M acetylcholine chloride (ACh), pH 5.0 (Sigma); 0.036 M 5-hy-
* Present address: The Pharmacology Laboratories Department of Pharmacy, University of Nottingham, University Park, Nottingham NC7 2RD. 519 N.P.16
7 x
,
520
P. B. BRADLEY and G. J. BRAMWELL
droxytryptamine
bimaleinate
(5-HT),
pH
ley. 1974). Thus. 113 neurones (305”) were excited by morphine (Fig. IA), 44 (12?;,) responded with longlasting depression (Fig. IB) and (3”/,) showed a biphasic response characterized by initial excitation followed by long-lasting depression (Fig. 2A, and B). The remaining 205 neurones (55”,,) either did not respond at all to morphine or else showed depression which paralleled the current application in onset and recovery. These short-lasting depressions were often eliminated by employing current balancing, or could be mimicked by a similar application of Na+, they were therefore treated as current effects.
4.0 (Koch-
Light).
A 25550nA current was usually employed for expelling drug ions and a retaining current of 15 nA was universally used to prevent unwanted leakage between applications. An electrode barrel containing 2M NaCl, or pontamine sky blue in acetate buffer, which was used for marking the position of recorded neurones (Boakes et ul.. 1974), was also used for current balancing in some experiments. RESULTS (a) Eflkts
ofmorphine
(h) Antugonism
on hruirl stern newones
Morphine was applied microiontophoretically with a current of 25-50 nA, for 6@120 set to 375 spontaneously active brain stem neurones and was found to produce effects similar to those reported previously (Bradley and Dray. 1973. 1974: Bramwell and Brad-
of morphine
pflects
Morphine-induced depression was completely or partially antagonized by nalorphine or naloxone on 20/24 occasions. an example of which is illustrated in Figure 1B. This antagonism was demonstrated in two ways. Firstly. nalorphine or naloxone appli-
n
L_
0
i-2
5
5
4
6
7-n-y
I4
16
23
24
25
26
____ 28 ~-29 30
27
31
min
0 Ash
40
NAL
A$h
Morp
I
f
.:I ll.J IO
0
::I*
$ 2
4
6
6
IO
II
I2
14
1, 15
I6
min
Fig. 1. Effects of microiontophoretically applied morphine on spontaneously firing brain stem neurones. The mean firing rate of the neurone (f, spikes/set) in successive 5sec epochs is plotted against time (min). Iontophoretic applications are indicated by the horizontal bars. A : reproducible excitations produced by regular applications of morphine (Morp: 50nA for 50sec) with 2min intervals between applications. Naloxone (25 nA) failed to antagonize morphine excitation during or following a 20 min application. B: Morphine (Morp: 50 nA) applied for 1: min produced long-lasting depression which was terminated by an application of naloxone (NAL: 25 nA). A subsequent application of morphine then failed to have any effect. The response to acetylcholine (ACh: 50nA) was unchanged.
521
Stereospecific actions of morphine
Morphine-induced excitation was not antagonized specifically by naloxone or nalorphine (12/12) (Fig. 1A) but potentiation of this effect sometimes occurred instead. However, on one occasion both morphineinduced and ACh-induced excitations were antagonized by nalorphine. In the case of the small number of neurones showing a biphasic response to morphine, only the depression was antagonized by naloxone (516) (Fig. 2B). This indicated that the individual components of the biphasic response showed the same susceptibility towards antagonism by naloxone as did simple depression or excitation. If a second morphine application was made during a period of morphineinduced depression, excitation was sometimes superimposed upon the depression. This phenomenon was observed both with neurones exhibiting a biphasic response initially (n = 5, see Fig. 2B) and also neurones which responded initially to morphine with long-last-
cations brought about a return of control firing, when applied during a period of depression and secondly, the antagonist reduced or completely prevented a subsequent morphine application from producing depression. This antagonism of morphine-induced depression was not due to spontaneous recovery from depression, or the manifestation of tachyphylaxis, for two reasons: (a) The morphine-induced depression studied was invariably prolonged and, where spontaneous recovery was permitted, control firing only rarely (2/37) returned within 2min of the end of the application (mean 9min). (b) The inability of morphine to cause depression after naloxone was not due to tachyphylaxis, since this phenomenon was not observed with morphine-induced depression (Bradley and Dray, 1974). Furthermore, the antagonistic action of naloxone was short lasting, so that control morphine responses returned after some time (Fig. 3A and B).
A Morp
ACh
J
NAL
ACh w
nn:
5 min
IO
3 ACh I
--
Morp
ACh
ACh _
Morp Pm
ubJ
ACh
:I
8
9
mm
Fig. 2. (continued over).
--
Morp
ACh
522
P. B. BRADLEY
ACh
LEV
ACh I
I
and G.J. BRAMWELL
5-HT
7
6
9
8
IO
II
ACh I
,r,
h;
I
4
NAL
ACh I
12
13
14
15
I6
min Fig. 2. Biphasic effects of morphine and levorphanol on spontaneously active brain stem neurones. A: Morphine (Morp: 50 nA) applied for 2 min produced a biphasic response, i.e. excitation followed by long-lasting depression. During the depression, ACh (50nA) still produced excitation. Recovery of firing was precipitated by a 2 min application of naloxone (NAL: 50nA). B: Morphine (Morp: 50 nA), applied for 1: min produced a biphasic response consisting of excitation during the application, followed by long-lasting depression when the application was terminated. During the depression not only did ACh (50nA) still produce excitation but so did a second timin application of morphine. A third 1: min application of morphine, within 3 min of naloxone-induced recovery (NAL: 50 nA for 1; min) produced only excitation. C: Levorphanol (LEV: 50 nA) applied for lf min produced excitation during the application, followed by long-lasting depression once the application was finished. During the depression both ACh (50 nA) and 5-hydroxytryptamine (5-HT: 50 nA) produced reversible excitations. The control firing rate was restored following a 2min application of naloxone (NAL: 50nA). ing
depression
(II = 4). It is not
spread this phenomenon tensively.
known
how
wide-
is as it was not studied
ex-
(c) EfSeects of lecorphunol und dextrorphan When levorphanol was applied to brain stem neurones with currents of 25_50nA, it produced effects qualitatively similar to those of morphine. Thus. 1l/84 cells were excited (Fig. 3C), IO/84 showed longlasting depression with a time course similar to morphine-induced depression (Fig. 3B) and 2/84 a biphasic response (see Table 1 and Fig. 2C). In addition, a further group of cells (31/84) showed reductions in spike height with levorphanol application and this made it impossible to assess precisely the effect on firing rate. These cells were therefore classified under ‘no effect’. Where a direct comparison between the effects of morphine and levorphanol on the same neurone was possible, it was found that the response to levorphanol tended to parallel that to morphine. Thus, no cell responded to morphine and levorphanol in opposite ways, though only @‘I5 cells excited by morphine were also excited by levorphanol. However. all IO cells depressed by morphine were also inhibited by levorphanol and these depressions of activity were antagonized by naloxone on 7/l occasions. Parallel experiments with dextrorphan on 83 neurons revealed that this isomer did not cause long-
lasting depression, nor excitation followed by depression (see Table 1). Instead, dextrorphan excited 8 neurones (Fig. 4B) and also produced short-lasting depression, i.e. in which recovery occurred within 2 min, on 12 occasions (Fig. 4A). This short-lasting depression was not accompanied by any obvious changes in spike height and was not antagonized by naloxone. In addition, dextrorphan reduced spike height on a further 30 occasions and these cells were included in ‘no effects’. Where direct comparisons were made of the effects of morphine and dextrorphan, it was found that the response to dextrorphan often did not parallel the
Table I. Comparison of the effects of morphine (Morp), levorphanol (Lev) and dextrorphan (Dex) T
TL
L
0
(YY$ 1376 (11) 10% (8)
(‘;;; 2”, (2)
12”,, (44) 12”” (10)
(i)
c:,
55”,:, (205) 730,, (61) 90”, (75)
Morp Lev Dex
The numbers in brackets represent the numbers of neurOnes. t = excitation. tJ = biphasic effect (excitation followed by depression), 1 = long-lasting depression. 0 = no effect.
523
Stereospecific actions of morphine
the same cell, during continuous current balancing. Of the 14 neurones so tested, morphine applied with a current of 25 nA for 90-180 set excited 2 cells, inhibited 7 cells and was without effect on 5. Levorphanol applied with the same current and for the same time, produced identical effects to those of morphine on the 9 cells affected by morphine (i.e. 2 excited and 7 inhibited) and had no action on the 5 cells unaffected by morphine. Furthermore, morphine and
response to morphine. Not only did dextrorphan excite fewer neurones (S/73 compared with 22/73 P < 0.05), but on two occasions dextrorphan was inhibitory when morphine was excitatory (Fig. 4A). (d) Comparison pf the effects of morphine, leuorphanol and dextrorphan, applied to the same neurone On some occasions it was possible to assess effects of morphine, levorphanol and dextrorphan, A
the on
Morp
NAL
min B
LEV
24
NAL
25
30 LEV
Morp
Fig. 3. Similarity between the effects of morphine and levorphanol on neuronal firing in the brain stem. A: Morphine (Morp. 25 nA) produced long-lasting depression following a l+ min application. Recovery was induced after li_ min by a 2 min application of naloxone (NAL: 25 nA). B: The same neurone as in ‘A’, 19 min after recovery from morphine depression, levorphanol (LEV: 25 nA), applied for limin also produced long-lasting depression and this was also reversed by a 2 min application of naloxone (NAL: 25 nA). N.B. Dextrorphan applied with the same current and for the same time as morphine, failed to affect the firing rate of this neurone. C: Excitation of a neurone by both morphine (Morp: 50 nA) and levorphanol (LEV: 50 nA).
524
P.B.
BRADLEY
and G.J.BRAMWELL
A
0 ACh _
I2
-
Morp
ACh m
3
4
-_
5
DEX
6
ACh
7
min
Morp
8
01l
DEX
63
64
65
66
min
Fig. 4. Comparison of the effects of morphine and dextrorphan on the firing rates of brain stem neurones. A: a 1 min application of morphine (Morp: 50nA) produced excitation but dextrorphan (DEX: 25 nA for 75 set) caused depression. B: a neurone excited by morphine (Morp: 50 nA for 75 set) and also by Dextrorphan (DEX: 25 nA for 1: min).
levorphanol-induced depressions were reversed or antagonized by naloxone on l/l occasions. In contrast, dextrorphan did not cause any naloxone-reversible depressions, though it did induce short-lasting depression on three occasions. Dextrorphan did, however, excite both of the neurones excited by morphine and levorphanol. (e) Effects of nalorphine
und naloxone
Both nalorphine and naloxone affected the firing rate of some brain stem neurones, without altering spike height or shape. This consisted of slowing of neuronal firing, which paralleled the applying current in its onset and recovery. This occurred 8/24 for nalorphine and 14/99 for naloxone. These short-lasting depressions sometimes delayed nalorphine or naloxone-induced recovery from long-lasting morphine depression until just after the end of the application. Long-lasting depressions with nalorphine or naloxone did not occur, but naloxone-induced excitation was seen on 6199 occasions.
DISCUSSION
The results presented here largely confirm previous findings on the effects of morphine on brain stem neurones (Bradley and Dray, 1973, 1974) although there are some differences in the frequency of occurrence of some of the responses, which may be explained as follows: Firstly, in the previous studies (Bradley and Dray, 1973, 1974) no distinction was made behveen short- and long-lasting depressions and both were included as ‘simple depressions’, thus increasing the proportion of depressant responses. In the present study, short-lasting depressions have been
included under ‘no effects’ as they often appeared to resemble current artefacts. This seems justified in the light of results of recent experiments (Bramwell and Bashir, unpublished observations) which have shown that short-lasting morphine depressions are not encountered when current balancing is rigorously maintained. Secondly, biphasic responses were not seen in the earlier studies (Bradley and Dray, 1973, 1974). Finally, in some experiments recordings were made from regions of the brain stem which had been found to contain greater numbers of morphine-sensitive neurones (Boakes et al., 1974). For this reason, the proportions of responses reported here are not necessarily representative of the brain stem as a whole. The difference in susceptibility to naloxone antagonism of morphine-induced neuronal excitation and inhibition, which had been observed previously (Bramwell and Bradley 1974) has been confirmed and extended by the results obtained with levorphanol. Moreover, morphine-induced long-lasting depression of neuronal firing exhibits the characteristics of a stereospecific effect, in that it is antagonized by naloxone and mimicked by levorphanol but not by dextrorphan. A similar stereospecificity has been demonstrated in the rat cerebral cortex (Satoh, Zieglglnsberger. Fries and Herz, 1974) although in the cat spinal cord it is the excitatory effect which appears to be stereospecific (Davies and Duggan, 1974). The identification of stereospecific morphine effects on single neurones is in keeping with the demonstration of stereospecific binding of various narcotic agonists and antagonists to brain homogenate fractions derived from cortex, spinal cord and brain stem (Goldstein, Lowney and Pal, 1971; Pert and Snyder, 1973; Kuhar, Pert and Snyder, 1973).
525
Stereospecific actions of morphine It is possible that morphine-induced depression and excitation of neuronal activity are the result of morphine acting on two different receptors, differing in stereospecificity. The presence of two receptors responsible for mediating the effects of morphine on neuronal firing in the brain stem is suggested by the ability of naloxone to antagonize the inhibitory phase. but not the excitatory phase, of biphasic responses. In addition. the ability to elicit morphine excitation during a period of morphine-induced depression further reinforces the concept that neuronal excitation and depression by morphine in the brain stem are brought about by two entirely different mechanisms. The relatively small proportion of neurones excited by levorphanol compared with morphine cannot be explained entirely in terms of sampling errors. Thus, although many more cells were tested with morphine than with levorphanol, a direct comparison of the effects of both compounds on 34 neurones showed that morphine was excitatory on many occasions when levorphanol was without effect. It is possible, however, that levorphanol excitation might have been masked on many occasions by non-specific effects. e.g. changes in spike height. The recent discovery of an endogenous morphinelike. substance in mammalian brain (Hughes, 1975; Kosterlitz and Hughes, 1975; Terenius and Wahlstrom, 1975; Pasternak, Goodman and Snyder, 1975) and its identification as enkephalin, consisting of two pentapeptides (Hughes et u!., 1975), provides an alternative explanation for the findings reported here, and this explanation requires only one receptor for morphine. Peripherally, enkephahn appears to have potent effects on opiate receptors (Hughes et al., 1975) and the results of preliminary studies in the rat brain stem indicate that this substance inhibits neurones which are sensitive to morphine, including many which are excited by morphine (Bradley. Briggs. Gayton and Lambert, 1976). Thus, the dual effects of morphine on brain stem neurones could be due to (a) an agonist action on enkephalin receptors, thus mimicking enkephalin depression of neuronal activity, and (b) occupation of enkephalin receptor sites, thus preventing the endogenously released substance from reaching its receptors, and this would result in excitation In this case. not only would naloxone not antagonize morphine excitation, but might even potentiate this excitation as well as producing excitation itself, all of which have been observed. Furthermore, the weak binding capacity of dextrorphan for the opiate receptor (Pert and Snyder, 1973) might lead to some excitation when competing for receptors, and in fact, dextrorphan did cause excitation on some occasions. A dual action of morphine on enkephalin receptors can also explain a number of other observations in the present experiments. For example. why excitation could be superimposed on morphineinduced depression and why excitation had a slow onset and rapid recovery. The fact that biphasic effects always consisted of excitation followed by
depression might have been due to the use of rather high expelling currents so that ‘competition’ (excitation) occurred first, followed by ‘mimicking’ (depression) when the local concentration was falling i.e. after switching off the applying current. Ack,lowledg~mrnrs~We wish to thank Endo Laboratories for supplying naloxone. This work was supported by the Medical Research Council. REFERENCES
Boakes, R. J. (1972). A simple device for the construction of multi-barrelled micropipettes. Br. J. Pharmuc. 45: 118P. Boakes, R. J., Bramwell. G. J., Briggs, 1.. Candy, J. M. and Tempesta. E. (1974). Localization with pontamine sky blue of neurones in the brainstem responding to microiontophoretically applied compounds. Neuropharmucoloy~ 13: 475479. Bradley, P. B. and Dray, A. (1973). Actions and interactions of microiontophoretically applied morphine with transmitter substances on brain stem neurones. Br. J. Pharmuc. 47: 642P. Bradlev. P. B. and Drav. A. (19741. Moruhine and Neurotransmitter substances: microiontophdretic study in the rat brain stem. Br. J. Phurmuc. SO: 47755. Bradley, P. B. and Bramwell, G. J. (1975). A stereospecitic action of morphine on brain stem neuronal activity: a microiontophoretic study. Br. .I. Pharmuc. 53: 462P. Bradley. P. B.. Briggs, I., Gayton, R. J. and Lambert, Lynn A. (1976). The effects of microiontophoretically applied Methianine-enkephalin on single neurones in the rat brain stem. Nuturr 261: 4255426. Brdmwek G. J. and Bradley. P. B. (1974). Actions and interactions of narcotic agonists and antagonists on brain stem neurones. Brain Rex 73: 1677170. Davies, J. and Duggan, A. W. (1974). Opiate agonisttantagonist effects on Renshaw cells and spinal interneurones. Nature 250: l&71. Goldstein. A., Lowney. L. 1. and Pal, B. K. (1971). Stereospecific and nonspecific interactions of the morphine congencr levorphanol in subcellular fractions of mouse brain. Proc. wt~ Acud. Sci. 68: 1742-1747. Herz, A.. Albus. K.. Metys. J. and Schubert. P. (1970). On the central sites for the antinociceptive action of morphine and fentanyl. Nruropharmacolog.v 9: 539-551. Herz. A., Teschemacher, H. J.. Albus, K. and Zieglglnsberger. W. (1971). Caudal brain stem structures mediating the precipitated abstinence syndrome. Proc. XXV Iur. Physiol. Corlg. p. 246. Hughes, J. (1975). Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine Bruirl Res. 88: 2955308. Hughes. J.. Smith, T. W.. Kosterlitz. H. W., Fothergill, L. A.. Morgan. B. A. and Morris, H. R. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258: 5777579. Kosterlitz. H. W. and Hughes, J. (1975). Some thoughts on the significance of enkephalin. the endogenous ligand. Lfi Sci. 17: 91-96. Kuhar. M. J.. Pert. C. B. and Snyder, S. H. (1973). Regional distribution of opiate receptor binding in monkey and human brain. Nature 245: 447450. Pasternak, G. W.. Goodman. R. and Snyder, S. H. (1975). An endogenous morphine-like factor in mammalian brain. I!& Sci. 16: 176551769. Pert. C. B. and Snvder. S. H. (1973). Oniate recentor: demonstration in nervous tissue. Scirnc; 179: 101’1~1014. Satoh, M., Zieglgansberger. W.. Fries. W. and Herz, A. (1974). Opiate agonist- antagonist interaction at cortical neurones of naive and tolerant rats. Brain Rex 82: 37% 3x3.
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P. B. BRADLEY and G. J. BRAMWELL
Terenius, L. and Wahlstrom, A. (1975). Morphine-like ligand for opiate receptors in human C.S.F. Life Sci. 16: 175991764. Teschmecher, H.. Schubert, P. and Herz, A. (1973). Autoradiographic studies concerning the supraspinal site of the antinociceptive action or morphine when inhibiting
the hind limb reflex in rabbits. Nemopharmacology 12: 1233131. Viqouret, J., Teschemacher. Hj., Albus, R. and Herz. A. (1973). Differentiation between spinal and supraspinal sites of morphine when inhibiting the hindleg flexor in rabbits Neuropharmacology 12: I1 l-121.
1977. 16. 519-526.
Pergamon
Press.
Printed
I” Gt. Brilain.
STEREOSCPECIFIC ACTIONS OF MORPHINE ON SINGLE NEURONES IN THE BRAIN STEM OF THE RAT P. B. BRADLEY and G. J. BRAMWELL* Department
of Pharmacology
(Preclinical),
The Medical
(Accepted 10 February
School,
Birmingham
B15 2TJ
1977)
Summary-The effects of microiontophoretically applied morphine, levorphanol and dextrorphan were compared on the firing rate of spontaneously active brain stem neurones. Both morphine and levorphanol produced three different effects: excitation, excitation followed by long-lasting depression, or long-lasting depression; whereas dextrorphan produced only excitation or short-lasting depression. Depression produced by morphine and levorphanol, but not dextrorphan, was antagonized by naloxone or nalorpine, whereas excitation was unaffected or potentiated. It is concluded that long-lasting depression of brain stem neuronal firing produced by morphine or levorphanol represents a stereospecific action.
many experimental studies on the pharmacological actions of morphine and other narcotic analgesics, present knowledge concerning their central sites and mechanism of action in producing antinociception or dependence remains limited. Work in the unanaesthetised rabbit (Herz, Albus, Metjrs and Schubert, 1970; Vigouret, Teschemacher, Albus and Herz, 1973; Teschemacher, Schubert and Herz, 1973) has provided evidence that morphine exerts at least part of its antinoceptive effect through an action in the brain stem region. Thus, localized perfusion of the ventricular system caused antinociception when morphine was able to reach the anterior part of the 4th ventricle, but not when it was confined to the 3rd and lateral ventricles by a plug in the aqueduct. Furthermore, intraventricularly administered levallorphan antagonized the effect of systemically administered morphine. Subsequently it was shown that withdrawal effects could be induced in morphine-tolerant rabbits, if nalorphine was perfused through the 4th ventricle, but not when it was restricted to other parts of the ventricular system (Herz, Teschemacher, Albus and Zieglglnsberger, 1971). Further evidence that morphine exerts actions within the brain stem was provided by Bradley and Dray (1973, 1974), who demonstrated that neurones in the brain stem of urethane-anaesthetized rats were sensitive to microiontophoretically applied morphine. Moreover, these studies showed that morphine could cause excitation as well as depression of the activity of single neurones in this region. The purpose of the present study was to examine the pharmacology and in particular, the stereospecificity, of morphine-induced neuronal excitation and depression in the brain stem. This entailed determining the susceptibility of these two effects to anta-
Despite
gonism by the narcotic antagonist naloxone and the agonist-antagonist nalophine. In addition, the effects of another narcotic analgesic, namely levorphanol, were compared with those produced by morphine, as well as with those of its non-narcotic isomer, dextrorphan. Some of the results have been presented to the British Pharmacological Society (Bradley and Bramwell 1975). METHODS Male albino rats (Sprague-Dawley) were anaesthetized with urethane (125 g kg- ‘) and partially cerebellectomized by suction to expose the floor of the 4th ventricle. Five-barrelled glass micropipettes (Boakes, 1972) with overall tip diameter 6-10~ were used to record extra-cellularly the action potentials of single spontaneously active neurones, and to apply drugs in the immediate vicinity of the cells. Electrode penetrations were made O-3 mm rostra1 of the obex and up to 2 mm lateral of the midline, avoiding the midline. The neurones studied were in the reticular formation, mainly in the nucleus reticularis paramedianus and nucleus reticularis gigantocellularis. The histological localization of neurones excited by morphine in this region has already been published (Boakes, Bramwell, Briggs, Candy and Tempesta, 1974). To record extracellular neuronal action potentials, one barrel of the micropipette was filled with 4 M NaCl. Other barrels were filled with one or other of the following drug solutions; 0.026 M morphine hydrochloride, pH 4.8 (Macfarlan Smith); 0.013-0.026 M levorphanol tartrate, pH 4.5 (Roche); O.O13C).O26M dextrorphan tartrate, pH 4.5, or 0.0134.026 M dextrorphan base in dilute HCl at pH 4.0 (Roche); 0.014X).28 M naloxone hydrochloride pH 4.5 in 0.2 M NaCl (Endo Laboratories); 0.029 M nalorphine hydrochloride, pH 4.5 (Burroughs Wellcome); 0.28 M acetylcholine chloride (ACh), pH 5.0 (Sigma); 0.036 M 5-hy-
* Present address: The Pharmacology Laboratories Department of Pharmacy, University of Nottingham, University Park, Nottingham NC7 2RD. 519 N.P.16
7 x
,
520
P. B. BRADLEY and G. J. BRAMWELL
droxytryptamine
bimaleinate
(5-HT),
pH
ley. 1974). Thus. 113 neurones (305”) were excited by morphine (Fig. IA), 44 (12?;,) responded with longlasting depression (Fig. IB) and (3”/,) showed a biphasic response characterized by initial excitation followed by long-lasting depression (Fig. 2A, and B). The remaining 205 neurones (55”,,) either did not respond at all to morphine or else showed depression which paralleled the current application in onset and recovery. These short-lasting depressions were often eliminated by employing current balancing, or could be mimicked by a similar application of Na+, they were therefore treated as current effects.
4.0 (Koch-
Light).
A 25550nA current was usually employed for expelling drug ions and a retaining current of 15 nA was universally used to prevent unwanted leakage between applications. An electrode barrel containing 2M NaCl, or pontamine sky blue in acetate buffer, which was used for marking the position of recorded neurones (Boakes et ul.. 1974), was also used for current balancing in some experiments. RESULTS (a) Eflkts
ofmorphine
(h) Antugonism
on hruirl stern newones
Morphine was applied microiontophoretically with a current of 25-50 nA, for 6@120 set to 375 spontaneously active brain stem neurones and was found to produce effects similar to those reported previously (Bradley and Dray. 1973. 1974: Bramwell and Brad-
of morphine
pflects
Morphine-induced depression was completely or partially antagonized by nalorphine or naloxone on 20/24 occasions. an example of which is illustrated in Figure 1B. This antagonism was demonstrated in two ways. Firstly. nalorphine or naloxone appli-
n
L_
0
i-2
5
5
4
6
7-n-y
I4
16
23
24
25
26
____ 28 ~-29 30
27
31
min
0 Ash
40
NAL
A$h
Morp
I
f
.:I ll.J IO
0
::I*
$ 2
4
6
6
IO
II
I2
14
1, 15
I6
min
Fig. 1. Effects of microiontophoretically applied morphine on spontaneously firing brain stem neurones. The mean firing rate of the neurone (f, spikes/set) in successive 5sec epochs is plotted against time (min). Iontophoretic applications are indicated by the horizontal bars. A : reproducible excitations produced by regular applications of morphine (Morp: 50nA for 50sec) with 2min intervals between applications. Naloxone (25 nA) failed to antagonize morphine excitation during or following a 20 min application. B: Morphine (Morp: 50 nA) applied for 1: min produced long-lasting depression which was terminated by an application of naloxone (NAL: 25 nA). A subsequent application of morphine then failed to have any effect. The response to acetylcholine (ACh: 50nA) was unchanged.
521
Stereospecific actions of morphine
Morphine-induced excitation was not antagonized specifically by naloxone or nalorphine (12/12) (Fig. 1A) but potentiation of this effect sometimes occurred instead. However, on one occasion both morphineinduced and ACh-induced excitations were antagonized by nalorphine. In the case of the small number of neurones showing a biphasic response to morphine, only the depression was antagonized by naloxone (516) (Fig. 2B). This indicated that the individual components of the biphasic response showed the same susceptibility towards antagonism by naloxone as did simple depression or excitation. If a second morphine application was made during a period of morphineinduced depression, excitation was sometimes superimposed upon the depression. This phenomenon was observed both with neurones exhibiting a biphasic response initially (n = 5, see Fig. 2B) and also neurones which responded initially to morphine with long-last-
cations brought about a return of control firing, when applied during a period of depression and secondly, the antagonist reduced or completely prevented a subsequent morphine application from producing depression. This antagonism of morphine-induced depression was not due to spontaneous recovery from depression, or the manifestation of tachyphylaxis, for two reasons: (a) The morphine-induced depression studied was invariably prolonged and, where spontaneous recovery was permitted, control firing only rarely (2/37) returned within 2min of the end of the application (mean 9min). (b) The inability of morphine to cause depression after naloxone was not due to tachyphylaxis, since this phenomenon was not observed with morphine-induced depression (Bradley and Dray, 1974). Furthermore, the antagonistic action of naloxone was short lasting, so that control morphine responses returned after some time (Fig. 3A and B).
A Morp
ACh
J
NAL
ACh w
nn:
5 min
IO
3 ACh I
--
Morp
ACh
ACh _
Morp Pm
ubJ
ACh
:I
8
9
mm
Fig. 2. (continued over).
--
Morp
ACh
522
P. B. BRADLEY
ACh
LEV
ACh I
I
and G.J. BRAMWELL
5-HT
7
6
9
8
IO
II
ACh I
,r,
h;
I
4
NAL
ACh I
12
13
14
15
I6
min Fig. 2. Biphasic effects of morphine and levorphanol on spontaneously active brain stem neurones. A: Morphine (Morp: 50 nA) applied for 2 min produced a biphasic response, i.e. excitation followed by long-lasting depression. During the depression, ACh (50nA) still produced excitation. Recovery of firing was precipitated by a 2 min application of naloxone (NAL: 50nA). B: Morphine (Morp: 50 nA), applied for 1: min produced a biphasic response consisting of excitation during the application, followed by long-lasting depression when the application was terminated. During the depression not only did ACh (50nA) still produce excitation but so did a second timin application of morphine. A third 1: min application of morphine, within 3 min of naloxone-induced recovery (NAL: 50 nA for 1; min) produced only excitation. C: Levorphanol (LEV: 50 nA) applied for lf min produced excitation during the application, followed by long-lasting depression once the application was finished. During the depression both ACh (50 nA) and 5-hydroxytryptamine (5-HT: 50 nA) produced reversible excitations. The control firing rate was restored following a 2min application of naloxone (NAL: 50nA). ing
depression
(II = 4). It is not
spread this phenomenon tensively.
known
how
wide-
is as it was not studied
ex-
(c) EfSeects of lecorphunol und dextrorphan When levorphanol was applied to brain stem neurones with currents of 25_50nA, it produced effects qualitatively similar to those of morphine. Thus. 1l/84 cells were excited (Fig. 3C), IO/84 showed longlasting depression with a time course similar to morphine-induced depression (Fig. 3B) and 2/84 a biphasic response (see Table 1 and Fig. 2C). In addition, a further group of cells (31/84) showed reductions in spike height with levorphanol application and this made it impossible to assess precisely the effect on firing rate. These cells were therefore classified under ‘no effect’. Where a direct comparison between the effects of morphine and levorphanol on the same neurone was possible, it was found that the response to levorphanol tended to parallel that to morphine. Thus, no cell responded to morphine and levorphanol in opposite ways, though only @‘I5 cells excited by morphine were also excited by levorphanol. However. all IO cells depressed by morphine were also inhibited by levorphanol and these depressions of activity were antagonized by naloxone on 7/l occasions. Parallel experiments with dextrorphan on 83 neurons revealed that this isomer did not cause long-
lasting depression, nor excitation followed by depression (see Table 1). Instead, dextrorphan excited 8 neurones (Fig. 4B) and also produced short-lasting depression, i.e. in which recovery occurred within 2 min, on 12 occasions (Fig. 4A). This short-lasting depression was not accompanied by any obvious changes in spike height and was not antagonized by naloxone. In addition, dextrorphan reduced spike height on a further 30 occasions and these cells were included in ‘no effects’. Where direct comparisons were made of the effects of morphine and dextrorphan, it was found that the response to dextrorphan often did not parallel the
Table I. Comparison of the effects of morphine (Morp), levorphanol (Lev) and dextrorphan (Dex) T
TL
L
0
(YY$ 1376 (11) 10% (8)
(‘;;; 2”, (2)
12”,, (44) 12”” (10)
(i)
c:,
55”,:, (205) 730,, (61) 90”, (75)
Morp Lev Dex
The numbers in brackets represent the numbers of neurOnes. t = excitation. tJ = biphasic effect (excitation followed by depression), 1 = long-lasting depression. 0 = no effect.
523
Stereospecific actions of morphine
the same cell, during continuous current balancing. Of the 14 neurones so tested, morphine applied with a current of 25 nA for 90-180 set excited 2 cells, inhibited 7 cells and was without effect on 5. Levorphanol applied with the same current and for the same time, produced identical effects to those of morphine on the 9 cells affected by morphine (i.e. 2 excited and 7 inhibited) and had no action on the 5 cells unaffected by morphine. Furthermore, morphine and
response to morphine. Not only did dextrorphan excite fewer neurones (S/73 compared with 22/73 P < 0.05), but on two occasions dextrorphan was inhibitory when morphine was excitatory (Fig. 4A). (d) Comparison pf the effects of morphine, leuorphanol and dextrorphan, applied to the same neurone On some occasions it was possible to assess effects of morphine, levorphanol and dextrorphan, A
the on
Morp
NAL
min B
LEV
24
NAL
25
30 LEV
Morp
Fig. 3. Similarity between the effects of morphine and levorphanol on neuronal firing in the brain stem. A: Morphine (Morp. 25 nA) produced long-lasting depression following a l+ min application. Recovery was induced after li_ min by a 2 min application of naloxone (NAL: 25 nA). B: The same neurone as in ‘A’, 19 min after recovery from morphine depression, levorphanol (LEV: 25 nA), applied for limin also produced long-lasting depression and this was also reversed by a 2 min application of naloxone (NAL: 25 nA). N.B. Dextrorphan applied with the same current and for the same time as morphine, failed to affect the firing rate of this neurone. C: Excitation of a neurone by both morphine (Morp: 50 nA) and levorphanol (LEV: 50 nA).
524
P.B.
BRADLEY
and G.J.BRAMWELL
A
0 ACh _
I2
-
Morp
ACh m
3
4
-_
5
DEX
6
ACh
7
min
Morp
8
01l
DEX
63
64
65
66
min
Fig. 4. Comparison of the effects of morphine and dextrorphan on the firing rates of brain stem neurones. A: a 1 min application of morphine (Morp: 50nA) produced excitation but dextrorphan (DEX: 25 nA for 75 set) caused depression. B: a neurone excited by morphine (Morp: 50 nA for 75 set) and also by Dextrorphan (DEX: 25 nA for 1: min).
levorphanol-induced depressions were reversed or antagonized by naloxone on l/l occasions. In contrast, dextrorphan did not cause any naloxone-reversible depressions, though it did induce short-lasting depression on three occasions. Dextrorphan did, however, excite both of the neurones excited by morphine and levorphanol. (e) Effects of nalorphine
und naloxone
Both nalorphine and naloxone affected the firing rate of some brain stem neurones, without altering spike height or shape. This consisted of slowing of neuronal firing, which paralleled the applying current in its onset and recovery. This occurred 8/24 for nalorphine and 14/99 for naloxone. These short-lasting depressions sometimes delayed nalorphine or naloxone-induced recovery from long-lasting morphine depression until just after the end of the application. Long-lasting depressions with nalorphine or naloxone did not occur, but naloxone-induced excitation was seen on 6199 occasions.
DISCUSSION
The results presented here largely confirm previous findings on the effects of morphine on brain stem neurones (Bradley and Dray, 1973, 1974) although there are some differences in the frequency of occurrence of some of the responses, which may be explained as follows: Firstly, in the previous studies (Bradley and Dray, 1973, 1974) no distinction was made behveen short- and long-lasting depressions and both were included as ‘simple depressions’, thus increasing the proportion of depressant responses. In the present study, short-lasting depressions have been
included under ‘no effects’ as they often appeared to resemble current artefacts. This seems justified in the light of results of recent experiments (Bramwell and Bashir, unpublished observations) which have shown that short-lasting morphine depressions are not encountered when current balancing is rigorously maintained. Secondly, biphasic responses were not seen in the earlier studies (Bradley and Dray, 1973, 1974). Finally, in some experiments recordings were made from regions of the brain stem which had been found to contain greater numbers of morphine-sensitive neurones (Boakes et al., 1974). For this reason, the proportions of responses reported here are not necessarily representative of the brain stem as a whole. The difference in susceptibility to naloxone antagonism of morphine-induced neuronal excitation and inhibition, which had been observed previously (Bramwell and Bradley 1974) has been confirmed and extended by the results obtained with levorphanol. Moreover, morphine-induced long-lasting depression of neuronal firing exhibits the characteristics of a stereospecific effect, in that it is antagonized by naloxone and mimicked by levorphanol but not by dextrorphan. A similar stereospecificity has been demonstrated in the rat cerebral cortex (Satoh, Zieglglnsberger. Fries and Herz, 1974) although in the cat spinal cord it is the excitatory effect which appears to be stereospecific (Davies and Duggan, 1974). The identification of stereospecific morphine effects on single neurones is in keeping with the demonstration of stereospecific binding of various narcotic agonists and antagonists to brain homogenate fractions derived from cortex, spinal cord and brain stem (Goldstein, Lowney and Pal, 1971; Pert and Snyder, 1973; Kuhar, Pert and Snyder, 1973).
525
Stereospecific actions of morphine It is possible that morphine-induced depression and excitation of neuronal activity are the result of morphine acting on two different receptors, differing in stereospecificity. The presence of two receptors responsible for mediating the effects of morphine on neuronal firing in the brain stem is suggested by the ability of naloxone to antagonize the inhibitory phase. but not the excitatory phase, of biphasic responses. In addition. the ability to elicit morphine excitation during a period of morphine-induced depression further reinforces the concept that neuronal excitation and depression by morphine in the brain stem are brought about by two entirely different mechanisms. The relatively small proportion of neurones excited by levorphanol compared with morphine cannot be explained entirely in terms of sampling errors. Thus, although many more cells were tested with morphine than with levorphanol, a direct comparison of the effects of both compounds on 34 neurones showed that morphine was excitatory on many occasions when levorphanol was without effect. It is possible, however, that levorphanol excitation might have been masked on many occasions by non-specific effects. e.g. changes in spike height. The recent discovery of an endogenous morphinelike. substance in mammalian brain (Hughes, 1975; Kosterlitz and Hughes, 1975; Terenius and Wahlstrom, 1975; Pasternak, Goodman and Snyder, 1975) and its identification as enkephalin, consisting of two pentapeptides (Hughes et u!., 1975), provides an alternative explanation for the findings reported here, and this explanation requires only one receptor for morphine. Peripherally, enkephahn appears to have potent effects on opiate receptors (Hughes et al., 1975) and the results of preliminary studies in the rat brain stem indicate that this substance inhibits neurones which are sensitive to morphine, including many which are excited by morphine (Bradley. Briggs. Gayton and Lambert, 1976). Thus, the dual effects of morphine on brain stem neurones could be due to (a) an agonist action on enkephalin receptors, thus mimicking enkephalin depression of neuronal activity, and (b) occupation of enkephalin receptor sites, thus preventing the endogenously released substance from reaching its receptors, and this would result in excitation In this case. not only would naloxone not antagonize morphine excitation, but might even potentiate this excitation as well as producing excitation itself, all of which have been observed. Furthermore, the weak binding capacity of dextrorphan for the opiate receptor (Pert and Snyder, 1973) might lead to some excitation when competing for receptors, and in fact, dextrorphan did cause excitation on some occasions. A dual action of morphine on enkephalin receptors can also explain a number of other observations in the present experiments. For example. why excitation could be superimposed on morphineinduced depression and why excitation had a slow onset and rapid recovery. The fact that biphasic effects always consisted of excitation followed by
depression might have been due to the use of rather high expelling currents so that ‘competition’ (excitation) occurred first, followed by ‘mimicking’ (depression) when the local concentration was falling i.e. after switching off the applying current. Ack,lowledg~mrnrs~We wish to thank Endo Laboratories for supplying naloxone. This work was supported by the Medical Research Council. REFERENCES
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