- Email: [email protected]
Effects of narcotic opiates and serotonin on the electrical behavior of neurons in the guinea pig myenteric plexus
Pergamon Preee
Life Sciences Vol. 14, pp . 2299-2909 Printed in D.S .A .
EFFECTS OF NARCOTIC OPIATES AND SEROTONIN ON TAB ELECTRICAL BEHAVIOR OF NEDRONS IN THE WINEA PIG MYENTERIC PLE%OS Raymond Dingledine, Avram Goldstein and Joan &endig Departments of Pharmacology and Anesthesia, Stanford Dniversity, and Addiction Research Foundation, Palo Alto, California 94304 (Received in final form 21 May 1974) Sumary Action potentials Were recorded earacellularly from spoataaeouely firing neurone is the enteric plexus of the guinea pig ileum. Morphine, which inhibits acetylcholine release from the myeateric plazus, inhibited the spontaneous electrical activity of about half the calls studied, while serotonin elevated the firing rata of these calls . Daits not etiaulated by earotonia were not inhibited by morphine or lavorphaaol . Morphine also prevented the increase in firing rate cavand by serotonin. These affects of morphine were stereoepacific and blocked by nalozone, and are therefore considered to ba specific opiate effects . This study demonstrates opposing affects of narcotic opiates and serotonin on the electrical activity of serotoninoceptive neurons in the myenteric ple:ve. The myenteric plezvs of the guinea pig ileum contains a class of neurons that is sensitive to the narcotic drugs, evidenced by the observatioa that morphine inhibits contraction of the longitudinal muscle by depressing acetylcholine release from the plezus (1) . of opiate action on ayanteric neurone is unknown.
The site and nature
In addition to the well
recognized parasympathetic pathway in the myenteric plans, theta is evidence for the presence of adrenergic and earotoninergic fibers as well . Ezogenous earotonin causes a contraction of the longitudinal muscle that is blocked by atropine and ie
therefore probably mediated by acetylcholine
I release (2), while aorepinephrine and other catacholamines can depress electrically induced muscle contraction by inhibiting acetylcholine release from the plezus (3,4) .
Moreover, there ie histochamical doidence for a
dense network of catecholminnrgiâ sad serotoninargic fibers ter~natiag 2299
2300
Vol. 14, No . 11
Narcotic Effects on Neuronal Activity
on or near ganglion cells within the mgenteric plexus (5-9) .
The serotonin
induced contraction of the longitudinal muscle is antagonized by morphine (2,10) and by epinephrine (ß . Schulz, unpublished observation), suggesting that the opiates, serotonin sad catecholamines influence the longitudinal muscle through a common neural pathway.
Ae postulated elsewhere (11),
a synaptic arrangement is suggested whereby inhibitory catecholaminergic and excitatory serotoniaergic fibers impinge on the cholinergic motor neuron (Fig . 1) .
The complez neural circuitry proposed for the myenteric plexus
NE
lonpifudinal muscle
FIG. 1 Possible Neuronal Connectivity in the Iiyentaric Plezus Ezcitatory (+) synapses of serotoninergic (5HT) and cholinergic (ACh) neurones, as well as as inhibitory synapse (-) made by an noradrenergic (NE) neuron are represented . provides several sites at which morphine could exert its inhibitory effect on longitudinal muscle contraction .
For instance, the opiates could act
by blocking serotonin receptors or by stimulating an inhibitory neuron or its receptor .
Alternatively, opiates could act directly on the cholinergic
nerve terminal to block acetylcholine output, as suggested by Ehrenpreis
(12) .
A decision among the above possibilities might be obtained by examining the effects of opiates, serotonin and the catecholamines on the firing behavior
Vol. 14, äo . 11
Narcotic 8fiacts an NsurOaal Activity
of myeataric neurons .
2301
Thora has bass each steal interest in th~ nlnctro-
physiology of the myenteric plane (13-16), including a report by Sato et al . (17) xhich shared that aorphiaa can inhibit spontaneous firing of plexus neurons, and can prnvant a nicotine-induced increase in their electrical activity .
In this paper xe damonstratn as antagonistic interaction betxaea
narcotic opiates and snrotania on the firing behavior of neurons in the guinea pig myanteric plazas .
Methods Male guinea pigs, 400-500 g, xern decapitated, the ilas~ xas removed, and the longitudinal muscle xith attached =yentnric plazas vas stripped off by a modification of the sethod of Paton and tar (18) .
Cars xas taken
to obtain preparations aeseatially fraa of circular suede, so that the myeataric ganglia xern e:posad on tha surface of the longitudinal needs. IInder a dissecting micrwcope a 0 .8
~2
area of this preparation, contain-
ing ono or txo ganglia, vas outlined by light pressure with a #16 gauge needle, the tip of xhich had been filed flat .
This region xas then excised
sad planed out on a thin layer of silicone rubber (Sylgard #184 ; Dox Corning Corp .) in a 1 .1 ml bath . such preparations .
Muscle movement xas virtually absent in
This precaution is necessary to maintain good contact
betxeen electrode and neuron, sad to insure that a drug-induced change in unit firing pattern is not secondary to mechanical stiauletion of the ganglia caused by changes in needs tonuB.
Ia sos~e ezperiaeats the longi-
tudinal muscle xae partially i>.eobilisad xithout cutting by pinning out as area surrounding 1-3 ganglia.
Modified Rrabs-bicarbonate buffer vas con-
tinuously parfueed over the preparation by gravity flux, and removed from the bath by continuous aspiration . buffer xaa (in ~ :
The conpoaition of Rrebs-bicarbonate
NaCl, 118 ; KC1, 4.75 ; CaC1 2 , 2.54 ; ~2P04 , 1.19 ;
Mg50 4 , 1 .2 ; NaflC03 , 25 .0 ; glucose, 11 .0 ; choline chloride, 0 .02; pyrilanine
2302
Vol . 14, No . 11
Narcotic Effects on Neuronal Activity
molests, 125 nM ; bubbled in reservoir with 95% OZ/5X CO Z , pR 7.4 (19) . Tissue bath bath .
temperature was maintained at 36-37°C by a surrounding water
Drug solutions were prepared is separate reservoirs of buffer and
washed into the bath through a three way valve.
with this method a com-
plete change of bath fluid could be effected within 10-15 sec . A glass auction electrode of tip diameter 40-70 um was employed to record eatracellular action potentials .
Nerve action potentials could
easily be distinguished from muscle slow waves on the basis of waveform duration and sensitivity to lidocaine.
Nerve spikes were 1-4 meet in
duration, while muscle spikes were 20-30 meet .
Lidocaine (0 .1-1 mM) com-
pletely shut off nerve activity while having no effect or slightly increasing the frequency of muscle slw waves (cf. ref . 14) .
After amplification,
signals ware led to an audio monitor and a magnetic tape deck, and displayed on a dual-beam oscilloscope .
Signals ware also gated and fed into
a Schmitt trigger, the output of which was integrated and led to a polygraph for continuous representation of cell firing rate .
The output of the
Schmitt trigger was also displayed oa the second beam of the oscilloscope, and an effort was made to admit only the largest size spike into the Schmitt trigger for analysis of firing rate .
Gaaglie were sampled with
the suction electrode until a position was found in which a single wait was predominant .
Drug effects were measured as a change in unit firing rate .
A change of discharge frequency of about 2 spikes sec1 was usually the minimam detectable in our system.
Electrical activities of different unite were
distinguished by the shape sad amplitude of spike waveform . Drugs employed were morphine sulfate (Merck), levorphanol tartrate and deztrorphan tartrate (Hoffmann-La Roche), nalozone hydrochloride (Endo), serotonin crenciaine sulfate (ICN Nutritional Siochemicals), lidocaine hydrochloride moaohydrate (Astra Pharmaceutical Products, Inc .) .
Vol. 14, Dio. 11
Narcotic Effects on Neuronal Activity
2303
Results ltyantnric neurons displayed considerable spontaneous electrical activity .
We use the tars "spontaneous" to denote activity that occurred in the
abseaca of pharmacologic stimulation, at least part of which aay have been the result of mechanical etimulatioa provided to the ganglion by the auction electrode .
A total of 108 units from 35 preparations were examined .
The
neuron eapple was biased tward units with large spike applitudes and steady firing patterns .
Burst vans (13,17) were detected occasionally, but poet
units fired irregularly at rates of 1-15 spikes sac
1
.
IInits with stable
firing patterns were held routinely for at least 60-120 min, and eoaetimes up to 7 hr . Of those units azposed to 10~ M enrotonin, about half responded with an increased firing rate, a few with decreased firing rate, and the rest ware sot affected (Table 1) .
The ezcitation produced by serotonin was TABLE 1
Drug Effects on Spontaneous Firing of Myentnric Neurone Drug
Serotonia (106M)
IIaita reapanding withl + 0 56x
lOx
33X
Firing rata in presence of drug2 429x
Number of units tested
t 34x
87
Morphine (107M)
0
30
70
19 .7 t 6.3
23
Morphine (3 " 10 7~
0
64
36
11 .6 t 3.3
22
Levorphaaol (3 " 10 -7M)
5
84
11
10 .1 t 3 .2
19
Daztrorphaa (3 " 10 7M)
5
0
95
20
Nalaxona (108-3" 107~
0
0
T00
18
1.
"+" defined ae as increased firing rate, "" ae a decreased firing rate, sad "0" as no effect . Data presented as per cent of all vans tested .
2.
Drug induced firing rate, ae percent of epontaaeous firing rate preceding drug addition . Data refer calq to vans stipulated bq nerotonin or inhibited by morphine or levorphaaol. Data are mean t.3EM .
2304
Narcotic Effects on Net;ronal Activity
dose-dependent over a range of 10 7-10 range ~fidch
5
Vol . 14, No . 11
M, approximately the concentration
causes contraction of the longitudinal muscle-myenteric plexus
preparation (11) . lated by 10
6
The mean increase in firing rate of those units stimu-
M aerotonin was 12 .0 t 1 .2 spikes
sec1 (mean ± SBM; n-49) .
Morphine, is concentration 1-3 " 10-7 M, depressed the spontaneous firing rate of many of the unite tested, and was never found to have any excitatory effects (Table 1) .
Morphine antagonized a serotonin-induced elevation in
firing rate in 15 of 16 units tasted, including some in which morphine did not itself depress spontaneous firing .
Pre-exposure of unite to 3 " 10-7 M
morphine for 60 sec. reduced serotonia-induced firing to 17 .9 t 5.7x (mean t SEM; n~10) of the rate in the presence of serotonin alone . eaawple is shown in Fig. 2a,b .
A typical
In contrast, inhibitory effects of serotonin
were never antagonized by morphine (4 units tested) . The opiate antagonist aalozone, in concentrations of 10 -8-3 " 10 -7 M, prevented the inhibitory effects of morphine on spontaneous firing in all 9 unite tested .
Naloaone, is 5 out of 6 cases, also blocked the antago-
nistic action of morphine on serotonin-induced excitation (Fig . 2b,d) . Morphine-induced depression of spontaneous firing rate and blockade of excitatory serotonia effects were atereospecific .
In all 16 unite the fir-
ing rates of which were depressed by 3" 10 -7 M levorphanol, the eaantiomer deatrorphaa (which ie pharnacologiaally inert in the longitudinal muecleFIG . 2 Effects of Serotonia and Morphine on the Firing Rate of a Myenteric Neuron Drugs were added to the chamber in the order shown by a-d, 15 min being allowed between each test . Serotoain had an excitatory effect on this unit (a,c), while morphine depressed vast firing rate and reduced the excitatory eerotonia effect (b) . Nalozone, while having no effect of it~ owe, antagonized both the inhibitory effect of morphine, and its ability to reduce esrotonin-induced excitation (d) . Spike patterae of this wait are shown before any drug is added (1), in the presence of serotonin (2), and in the presence of both morphine and aerotonin (3) . Photographs 1-3 were taken from magnetic tape . The output of the Schmitt trigger appears ae a dot -6 above each spike that_s~aa counted is firing-;ate aaalysie . Drugs are 10 M serotonia (SLIT), 3" 10 M morphine and 3 " 10 M naloaone .
Narcotic 8ff~cts on Neuronal Activity
Vol . 14, No . 11
t
N,
â
N
N
b
O
p
J9~ +~ C3Nid!
2305
2306
Vol . 14, No . 11
Narcotio Sffecta on Neuronal Activity
myenteric plexus preparation) was without effect at the same concentration (Table 1) .
Moreover, the excitatory effects of serotoain were partially or
completely blocked by levorphaaol, but not by deatrorphaa, in 9 of 10 units tested .
Nalozone reversed the inhibitory effect of levorphanol on unit fir-
ing rate in all 12 cases tested .
Except for one cell in which the firing
rate was elevated by eerotonin, levorphanol aad deztrorphan, no effects of dextrorphan or nalozone alone were ever detected (Table 1) . Opiate sensitivity was associated with an excitatory response to eerotonin in a majority of aeurons .
Of the units tested with both serotoain
aad levorphaaol, all 10 unite excited by 10
-6
M eerotonin were inhibited by
3" 10 -~ M levorphaaol ; conversely, the one unit that was not affected by eerotonin also was not affected by levorphanol .
Similarly, in units ex-
posed to bath eerotonin and morphine, 3 " 10-~,M morphine inhibited 7 of 11 units excited by eerotonin, aad had no effect on 4 of 5 units unaffected by eerotonin. (10-~ M)
This relationship was sot observed at a lower concentration
of morphine (Table 2) . TABLE 2
Correlation Between Effects of Serotonin and Morphine or Levorphanol on Spontaneous Firing of Myanteric Neurone -6 10 M eerotonin
excitation no effect, or inhibition
3 " 10 - ~ M levorphanol
3" 10 -~ M morphine
inhib .
no effect
inhib .
10
0
7
0
1
1
10 ~ M morphine inhib .
no effect
4
1
6
4
1
5
no effect
The response of unite to eerotonin and either morphine or levorphanol was tested . The number of units exhibiting each of the four possible com"Excitation" is defined as binations of response to the drugs are shown. an increased firing rate, "inhibition" as a decreased firing rate .
Vol . 14, No . 11
2307
Narcotic 8ffects on Neuronal Activity
Burst units (13,17) were infrequently observed, and seemed to be rather insensitive to morphine sad sarotonin, although no systematic survey vas undnrtakea.
Diecusaion As a logical eztaasion of work characterizing the action of narcotics is the in vitro preparation of the longitudinal muscle with attached fnyenteric pleuue (11,19) ve have attemped to investigate the neuronal basis for narcotic action on the myenteric plezue .
Morphine, which depresses
output of acetylcholiae from the myentaric pleas, inhibited the spontaneous electrical activity of many of the neurone studied. with the observation of Sato et al . (17) .
This agrees
Korphine also antagonized the
excitatory effects of sarotonin in virtually every case .
Morphine-induced
inhibition of epantaaeous firing and aatagoniam of serotonin-induced excitation were stereoepecific and blocked by aaloxone, and on this basis are assumed to be specific opiate affects .
These data, together with the obser-
vation that serotonia and the opiates had opposite effects on many of the unite studied, corroborate previous suggestions that morphine may depress acetylcholine release from the myeateric plexus by inhibiting the firing of serotoninoceptive neurone (11) . or through as iatarneuron .
This inhibition may be effected directly
If the proposed modal for neuronal connectivity
in the pleas ie correct (Fig . 1), the eerotoainoceptive element would be the cholinargic motor neuron . Although the ezperiaental techniques used in the present study do not peradt functional identification of the neurone, it seems that at least three types of spontaneously firing units are present in the myenteric ple.:us, based on firing pattern and pharmacologic properties :
burst units,
irregularly firing single-spike waits that were uareeponeiva to serotonin and norphiae, and single spike vane with spontaneous firing rate elevated by
Narcotic Effects on Neuronal Activity
2308
Vol . 14, No . 11
serotonin and generally depressed by narcotics . Sato et al . (17) (3 "10
-5
found that a very high concentration of morphine
M) antagonizes an excitatory effect of nicotine on myenteric neur-
one, arguing against a specific interaction between morphine and the sero tonin receptor .
However, they did not demonstrate stereospecificity or
nalozone blockade of this morphine effect, sad their findings should be confirmed with a lower concentration of morphine to reduce the possibility of canductioa blockade .
In the snail Helix pomatia morphine, without itself
affecting membrane potential or resistance, has been shown (20) to reduce the depolarizing effect of serotonin applied by ioatophoresis to as identified neuron is the buccal ganglion, and to antagonize synaptic transmission from the giant serotonin-containing neurone of the cerebral ganglion to these buccal cells, arguing for a close relationship between morphine and serotonin on buccal cells . Our results do demonstrate narcotic effects on the electrical behavior of neurone within the myenteric plexus, making it unnecessary to postulate that opiates act directly on cholinergic nerve terminals at the longitudinal muscle (18) .
Further progress in localizing the site of narcotic action
in the myenteric plexus will depend on exnmi*,i ng interactions between morphine and other agents that are known to modify the electrical behavior of myenteric neurone .
Ackaowledgemente We thank Lloyd Gano for technical advice concerning the integrator . Dingledine ie a National Science Foundation predoctoral fellw .
R.
This in-
vestigation was supported by research great DA00026 from National Institute on Drug Abuse and GM12527 from National Institute of General Medical Sciences .
Vol,
14, No . 11
Narcotic Blfects on Neuronal Activity
2309
ßaferences 1.
W.D .M . PATON, Brit . J. Fharmcol . 12, 119-127 (1957) .
2.
G.H . GADDUM and Z.P . PI~~R4~-T~ I, Brit . J. Pharaacol . 12, 323-328 (1957) .
3.
W.D .M . PATON and 8.5 . VIZI, Brit . J. Pharnacol. 35 , 10-28 (1969) .
4.
H.W . &OSTEßLITZ, ß.J . LYDON and A.J . WATT, Brit . J. Pharmacol . _, 398-413 (1970) .
5.
R.A . NOßBE&G, Iat . J. Neuropharsacol . 3, 379-382 (1964) .
6.
J.B . ßEAD and G. BDHNSTOCR, COmp . Biochea . Phy~iol. ~, 505-517 (1968) .
7.
ß.G . HOBINSON and M.D . GER.4HON, J. Pharsacol. E:>p, Than . 178, 311-324 (1971) .
8.
L.L . ßOSS and M.D . GSRSHON, J. Cell Hiol . 5= , 220a (1972) .
9.
M. COSTA and J .B . FIIHNESS, Hiatochem. J. 5, 343-349 (1973) .
10 .
ß. SCHIILZ and C. CAßTWHIGHT, J. Pharsacol. Esp . Ther . In press (1974) .
11 .
ß. SCHIILZ and A. OOLDSTEIN, Nature 244, 168-170 (1973) .
12 .
S. EHREI~ßEIS, I . LIGHT and G.H . SCHONBUCH, ûea of the electricallystimulated guinea pig ileum to study potent aaalgesica, is Addiction :
E~erineatal Pharaacology , J.H . Siagh, L.H . Millerand, sad
H. Lal, eds., Future Pub . Co ., Mrnint Rieco, N.Y . (1972) . 13 .
J:D . WOOD, Am . J . Phyeiol. 219, 159-169 (1970) .
14 .
H. OHRAWA and C .L . PßOSSEß, Aa . J. Physiol. 222, 1420-1426 (1972),
15 .
S . NISHI and ß.A . NOßTH, J . $hyeiol. (Loads ) 231, 471-491 (1973) .
16 .
G .D .S . HIßST, M.E . HOLMAN and I. SPENCB, J. Physiol. 236, 303-326 (1974) .
17 .
T . SATO, I. TARAYANAGI and R. TASAGI, Jap. J. Pharuacol . 23, 665-671 (1973) .
18 .
W.D .M . PATON and M.A . ZAß, J . Physiol. 194, 13-33 (1968) .
19 .
A. GOLDSTEIN and ß. SCHIILZ, Brit . J. Pharmacol. 48, 655-666 (1973) .
20 .
G.A . C OTI'attr .T . and J.B . MACON, J . Physiol . 236, 425-464 (1974) .
Life Sciences Vol. 14, pp . 2299-2909 Printed in D.S .A .
EFFECTS OF NARCOTIC OPIATES AND SEROTONIN ON TAB ELECTRICAL BEHAVIOR OF NEDRONS IN THE WINEA PIG MYENTERIC PLE%OS Raymond Dingledine, Avram Goldstein and Joan &endig Departments of Pharmacology and Anesthesia, Stanford Dniversity, and Addiction Research Foundation, Palo Alto, California 94304 (Received in final form 21 May 1974) Sumary Action potentials Were recorded earacellularly from spoataaeouely firing neurone is the enteric plexus of the guinea pig ileum. Morphine, which inhibits acetylcholine release from the myeateric plazus, inhibited the spontaneous electrical activity of about half the calls studied, while serotonin elevated the firing rata of these calls . Daits not etiaulated by earotonia were not inhibited by morphine or lavorphaaol . Morphine also prevented the increase in firing rate cavand by serotonin. These affects of morphine were stereoepacific and blocked by nalozone, and are therefore considered to ba specific opiate effects . This study demonstrates opposing affects of narcotic opiates and serotonin on the electrical activity of serotoninoceptive neurons in the myenteric ple:ve. The myenteric plezvs of the guinea pig ileum contains a class of neurons that is sensitive to the narcotic drugs, evidenced by the observatioa that morphine inhibits contraction of the longitudinal muscle by depressing acetylcholine release from the plezus (1) . of opiate action on ayanteric neurone is unknown.
The site and nature
In addition to the well
recognized parasympathetic pathway in the myenteric plans, theta is evidence for the presence of adrenergic and earotoninergic fibers as well . Ezogenous earotonin causes a contraction of the longitudinal muscle that is blocked by atropine and ie
therefore probably mediated by acetylcholine
I release (2), while aorepinephrine and other catacholamines can depress electrically induced muscle contraction by inhibiting acetylcholine release from the plezus (3,4) .
Moreover, there ie histochamical doidence for a
dense network of catecholminnrgiâ sad serotoninargic fibers ter~natiag 2299
2300
Vol. 14, No . 11
Narcotic Effects on Neuronal Activity
on or near ganglion cells within the mgenteric plexus (5-9) .
The serotonin
induced contraction of the longitudinal muscle is antagonized by morphine (2,10) and by epinephrine (ß . Schulz, unpublished observation), suggesting that the opiates, serotonin sad catecholamines influence the longitudinal muscle through a common neural pathway.
Ae postulated elsewhere (11),
a synaptic arrangement is suggested whereby inhibitory catecholaminergic and excitatory serotoniaergic fibers impinge on the cholinergic motor neuron (Fig . 1) .
The complez neural circuitry proposed for the myenteric plexus
NE
lonpifudinal muscle
FIG. 1 Possible Neuronal Connectivity in the Iiyentaric Plezus Ezcitatory (+) synapses of serotoninergic (5HT) and cholinergic (ACh) neurones, as well as as inhibitory synapse (-) made by an noradrenergic (NE) neuron are represented . provides several sites at which morphine could exert its inhibitory effect on longitudinal muscle contraction .
For instance, the opiates could act
by blocking serotonin receptors or by stimulating an inhibitory neuron or its receptor .
Alternatively, opiates could act directly on the cholinergic
nerve terminal to block acetylcholine output, as suggested by Ehrenpreis
(12) .
A decision among the above possibilities might be obtained by examining the effects of opiates, serotonin and the catecholamines on the firing behavior
Vol. 14, äo . 11
Narcotic 8fiacts an NsurOaal Activity
of myeataric neurons .
2301
Thora has bass each steal interest in th~ nlnctro-
physiology of the myenteric plane (13-16), including a report by Sato et al . (17) xhich shared that aorphiaa can inhibit spontaneous firing of plexus neurons, and can prnvant a nicotine-induced increase in their electrical activity .
In this paper xe damonstratn as antagonistic interaction betxaea
narcotic opiates and snrotania on the firing behavior of neurons in the guinea pig myanteric plazas .
Methods Male guinea pigs, 400-500 g, xern decapitated, the ilas~ xas removed, and the longitudinal muscle xith attached =yentnric plazas vas stripped off by a modification of the sethod of Paton and tar (18) .
Cars xas taken
to obtain preparations aeseatially fraa of circular suede, so that the myeataric ganglia xern e:posad on tha surface of the longitudinal needs. IInder a dissecting micrwcope a 0 .8
~2
area of this preparation, contain-
ing ono or txo ganglia, vas outlined by light pressure with a #16 gauge needle, the tip of xhich had been filed flat .
This region xas then excised
sad planed out on a thin layer of silicone rubber (Sylgard #184 ; Dox Corning Corp .) in a 1 .1 ml bath . such preparations .
Muscle movement xas virtually absent in
This precaution is necessary to maintain good contact
betxeen electrode and neuron, sad to insure that a drug-induced change in unit firing pattern is not secondary to mechanical stiauletion of the ganglia caused by changes in needs tonuB.
Ia sos~e ezperiaeats the longi-
tudinal muscle xae partially i>.eobilisad xithout cutting by pinning out as area surrounding 1-3 ganglia.
Modified Rrabs-bicarbonate buffer vas con-
tinuously parfueed over the preparation by gravity flux, and removed from the bath by continuous aspiration . buffer xaa (in ~ :
The conpoaition of Rrebs-bicarbonate
NaCl, 118 ; KC1, 4.75 ; CaC1 2 , 2.54 ; ~2P04 , 1.19 ;
Mg50 4 , 1 .2 ; NaflC03 , 25 .0 ; glucose, 11 .0 ; choline chloride, 0 .02; pyrilanine
2302
Vol . 14, No . 11
Narcotic Effects on Neuronal Activity
molests, 125 nM ; bubbled in reservoir with 95% OZ/5X CO Z , pR 7.4 (19) . Tissue bath bath .
temperature was maintained at 36-37°C by a surrounding water
Drug solutions were prepared is separate reservoirs of buffer and
washed into the bath through a three way valve.
with this method a com-
plete change of bath fluid could be effected within 10-15 sec . A glass auction electrode of tip diameter 40-70 um was employed to record eatracellular action potentials .
Nerve action potentials could
easily be distinguished from muscle slow waves on the basis of waveform duration and sensitivity to lidocaine.
Nerve spikes were 1-4 meet in
duration, while muscle spikes were 20-30 meet .
Lidocaine (0 .1-1 mM) com-
pletely shut off nerve activity while having no effect or slightly increasing the frequency of muscle slw waves (cf. ref . 14) .
After amplification,
signals ware led to an audio monitor and a magnetic tape deck, and displayed on a dual-beam oscilloscope .
Signals ware also gated and fed into
a Schmitt trigger, the output of which was integrated and led to a polygraph for continuous representation of cell firing rate .
The output of the
Schmitt trigger was also displayed oa the second beam of the oscilloscope, and an effort was made to admit only the largest size spike into the Schmitt trigger for analysis of firing rate .
Gaaglie were sampled with
the suction electrode until a position was found in which a single wait was predominant .
Drug effects were measured as a change in unit firing rate .
A change of discharge frequency of about 2 spikes sec1 was usually the minimam detectable in our system.
Electrical activities of different unite were
distinguished by the shape sad amplitude of spike waveform . Drugs employed were morphine sulfate (Merck), levorphanol tartrate and deztrorphan tartrate (Hoffmann-La Roche), nalozone hydrochloride (Endo), serotonin crenciaine sulfate (ICN Nutritional Siochemicals), lidocaine hydrochloride moaohydrate (Astra Pharmaceutical Products, Inc .) .
Vol. 14, Dio. 11
Narcotic Effects on Neuronal Activity
2303
Results ltyantnric neurons displayed considerable spontaneous electrical activity .
We use the tars "spontaneous" to denote activity that occurred in the
abseaca of pharmacologic stimulation, at least part of which aay have been the result of mechanical etimulatioa provided to the ganglion by the auction electrode .
A total of 108 units from 35 preparations were examined .
The
neuron eapple was biased tward units with large spike applitudes and steady firing patterns .
Burst vans (13,17) were detected occasionally, but poet
units fired irregularly at rates of 1-15 spikes sac
1
.
IInits with stable
firing patterns were held routinely for at least 60-120 min, and eoaetimes up to 7 hr . Of those units azposed to 10~ M enrotonin, about half responded with an increased firing rate, a few with decreased firing rate, and the rest ware sot affected (Table 1) .
The ezcitation produced by serotonin was TABLE 1
Drug Effects on Spontaneous Firing of Myentnric Neurone Drug
Serotonia (106M)
IIaita reapanding withl + 0 56x
lOx
33X
Firing rata in presence of drug2 429x
Number of units tested
t 34x
87
Morphine (107M)
0
30
70
19 .7 t 6.3
23
Morphine (3 " 10 7~
0
64
36
11 .6 t 3.3
22
Levorphaaol (3 " 10 -7M)
5
84
11
10 .1 t 3 .2
19
Daztrorphaa (3 " 10 7M)
5
0
95
20
Nalaxona (108-3" 107~
0
0
T00
18
1.
"+" defined ae as increased firing rate, "" ae a decreased firing rate, sad "0" as no effect . Data presented as per cent of all vans tested .
2.
Drug induced firing rate, ae percent of epontaaeous firing rate preceding drug addition . Data refer calq to vans stipulated bq nerotonin or inhibited by morphine or levorphaaol. Data are mean t.3EM .
2304
Narcotic Effects on Net;ronal Activity
dose-dependent over a range of 10 7-10 range ~fidch
5
Vol . 14, No . 11
M, approximately the concentration
causes contraction of the longitudinal muscle-myenteric plexus
preparation (11) . lated by 10
6
The mean increase in firing rate of those units stimu-
M aerotonin was 12 .0 t 1 .2 spikes
sec1 (mean ± SBM; n-49) .
Morphine, is concentration 1-3 " 10-7 M, depressed the spontaneous firing rate of many of the unite tested, and was never found to have any excitatory effects (Table 1) .
Morphine antagonized a serotonin-induced elevation in
firing rate in 15 of 16 units tasted, including some in which morphine did not itself depress spontaneous firing .
Pre-exposure of unite to 3 " 10-7 M
morphine for 60 sec. reduced serotonia-induced firing to 17 .9 t 5.7x (mean t SEM; n~10) of the rate in the presence of serotonin alone . eaawple is shown in Fig. 2a,b .
A typical
In contrast, inhibitory effects of serotonin
were never antagonized by morphine (4 units tested) . The opiate antagonist aalozone, in concentrations of 10 -8-3 " 10 -7 M, prevented the inhibitory effects of morphine on spontaneous firing in all 9 unite tested .
Naloaone, is 5 out of 6 cases, also blocked the antago-
nistic action of morphine on serotonin-induced excitation (Fig . 2b,d) . Morphine-induced depression of spontaneous firing rate and blockade of excitatory serotonia effects were atereospecific .
In all 16 unite the fir-
ing rates of which were depressed by 3" 10 -7 M levorphanol, the eaantiomer deatrorphaa (which ie pharnacologiaally inert in the longitudinal muecleFIG . 2 Effects of Serotonia and Morphine on the Firing Rate of a Myenteric Neuron Drugs were added to the chamber in the order shown by a-d, 15 min being allowed between each test . Serotoain had an excitatory effect on this unit (a,c), while morphine depressed vast firing rate and reduced the excitatory eerotonia effect (b) . Nalozone, while having no effect of it~ owe, antagonized both the inhibitory effect of morphine, and its ability to reduce esrotonin-induced excitation (d) . Spike patterae of this wait are shown before any drug is added (1), in the presence of serotonin (2), and in the presence of both morphine and aerotonin (3) . Photographs 1-3 were taken from magnetic tape . The output of the Schmitt trigger appears ae a dot -6 above each spike that_s~aa counted is firing-;ate aaalysie . Drugs are 10 M serotonia (SLIT), 3" 10 M morphine and 3 " 10 M naloaone .
Narcotic 8ff~cts on Neuronal Activity
Vol . 14, No . 11
t
N,
â
N
N
b
O
p
J9~ +~ C3Nid!
2305
2306
Vol . 14, No . 11
Narcotio Sffecta on Neuronal Activity
myenteric plexus preparation) was without effect at the same concentration (Table 1) .
Moreover, the excitatory effects of serotoain were partially or
completely blocked by levorphaaol, but not by deatrorphaa, in 9 of 10 units tested .
Nalozone reversed the inhibitory effect of levorphanol on unit fir-
ing rate in all 12 cases tested .
Except for one cell in which the firing
rate was elevated by eerotonin, levorphanol aad deztrorphan, no effects of dextrorphan or nalozone alone were ever detected (Table 1) . Opiate sensitivity was associated with an excitatory response to eerotonin in a majority of aeurons .
Of the units tested with both serotoain
aad levorphaaol, all 10 unite excited by 10
-6
M eerotonin were inhibited by
3" 10 -~ M levorphaaol ; conversely, the one unit that was not affected by eerotonin also was not affected by levorphanol .
Similarly, in units ex-
posed to bath eerotonin and morphine, 3 " 10-~,M morphine inhibited 7 of 11 units excited by eerotonin, aad had no effect on 4 of 5 units unaffected by eerotonin. (10-~ M)
This relationship was sot observed at a lower concentration
of morphine (Table 2) . TABLE 2
Correlation Between Effects of Serotonin and Morphine or Levorphanol on Spontaneous Firing of Myanteric Neurone -6 10 M eerotonin
excitation no effect, or inhibition
3 " 10 - ~ M levorphanol
3" 10 -~ M morphine
inhib .
no effect
inhib .
10
0
7
0
1
1
10 ~ M morphine inhib .
no effect
4
1
6
4
1
5
no effect
The response of unite to eerotonin and either morphine or levorphanol was tested . The number of units exhibiting each of the four possible com"Excitation" is defined as binations of response to the drugs are shown. an increased firing rate, "inhibition" as a decreased firing rate .
Vol . 14, No . 11
2307
Narcotic 8ffects on Neuronal Activity
Burst units (13,17) were infrequently observed, and seemed to be rather insensitive to morphine sad sarotonin, although no systematic survey vas undnrtakea.
Diecusaion As a logical eztaasion of work characterizing the action of narcotics is the in vitro preparation of the longitudinal muscle with attached fnyenteric pleuue (11,19) ve have attemped to investigate the neuronal basis for narcotic action on the myenteric plezue .
Morphine, which depresses
output of acetylcholiae from the myentaric pleas, inhibited the spontaneous electrical activity of many of the neurone studied. with the observation of Sato et al . (17) .
This agrees
Korphine also antagonized the
excitatory effects of sarotonin in virtually every case .
Morphine-induced
inhibition of epantaaeous firing and aatagoniam of serotonin-induced excitation were stereoepecific and blocked by aaloxone, and on this basis are assumed to be specific opiate affects .
These data, together with the obser-
vation that serotonia and the opiates had opposite effects on many of the unite studied, corroborate previous suggestions that morphine may depress acetylcholine release from the myeateric plexus by inhibiting the firing of serotoninoceptive neurone (11) . or through as iatarneuron .
This inhibition may be effected directly
If the proposed modal for neuronal connectivity
in the pleas ie correct (Fig . 1), the eerotoainoceptive element would be the cholinargic motor neuron . Although the ezperiaental techniques used in the present study do not peradt functional identification of the neurone, it seems that at least three types of spontaneously firing units are present in the myenteric ple.:us, based on firing pattern and pharmacologic properties :
burst units,
irregularly firing single-spike waits that were uareeponeiva to serotonin and norphiae, and single spike vane with spontaneous firing rate elevated by
Narcotic Effects on Neuronal Activity
2308
Vol . 14, No . 11
serotonin and generally depressed by narcotics . Sato et al . (17) (3 "10
-5
found that a very high concentration of morphine
M) antagonizes an excitatory effect of nicotine on myenteric neur-
one, arguing against a specific interaction between morphine and the sero tonin receptor .
However, they did not demonstrate stereospecificity or
nalozone blockade of this morphine effect, sad their findings should be confirmed with a lower concentration of morphine to reduce the possibility of canductioa blockade .
In the snail Helix pomatia morphine, without itself
affecting membrane potential or resistance, has been shown (20) to reduce the depolarizing effect of serotonin applied by ioatophoresis to as identified neuron is the buccal ganglion, and to antagonize synaptic transmission from the giant serotonin-containing neurone of the cerebral ganglion to these buccal cells, arguing for a close relationship between morphine and serotonin on buccal cells . Our results do demonstrate narcotic effects on the electrical behavior of neurone within the myenteric plexus, making it unnecessary to postulate that opiates act directly on cholinergic nerve terminals at the longitudinal muscle (18) .
Further progress in localizing the site of narcotic action
in the myenteric plexus will depend on exnmi*,i ng interactions between morphine and other agents that are known to modify the electrical behavior of myenteric neurone .
Ackaowledgemente We thank Lloyd Gano for technical advice concerning the integrator . Dingledine ie a National Science Foundation predoctoral fellw .
R.
This in-
vestigation was supported by research great DA00026 from National Institute on Drug Abuse and GM12527 from National Institute of General Medical Sciences .
Vol,
14, No . 11
Narcotic Blfects on Neuronal Activity
2309
ßaferences 1.
W.D .M . PATON, Brit . J. Fharmcol . 12, 119-127 (1957) .
2.
G.H . GADDUM and Z.P . PI~~R4~-T~ I, Brit . J. Pharaacol . 12, 323-328 (1957) .
3.
W.D .M . PATON and 8.5 . VIZI, Brit . J. Pharnacol. 35 , 10-28 (1969) .
4.
H.W . &OSTEßLITZ, ß.J . LYDON and A.J . WATT, Brit . J. Pharmacol . _, 398-413 (1970) .
5.
R.A . NOßBE&G, Iat . J. Neuropharsacol . 3, 379-382 (1964) .
6.
J.B . ßEAD and G. BDHNSTOCR, COmp . Biochea . Phy~iol. ~, 505-517 (1968) .
7.
ß.G . HOBINSON and M.D . GER.4HON, J. Pharsacol. E:>p, Than . 178, 311-324 (1971) .
8.
L.L . ßOSS and M.D . GSRSHON, J. Cell Hiol . 5= , 220a (1972) .
9.
M. COSTA and J .B . FIIHNESS, Hiatochem. J. 5, 343-349 (1973) .
10 .
ß. SCHIILZ and C. CAßTWHIGHT, J. Pharsacol. Esp . Ther . In press (1974) .
11 .
ß. SCHIILZ and A. OOLDSTEIN, Nature 244, 168-170 (1973) .
12 .
S. EHREI~ßEIS, I . LIGHT and G.H . SCHONBUCH, ûea of the electricallystimulated guinea pig ileum to study potent aaalgesica, is Addiction :
E~erineatal Pharaacology , J.H . Siagh, L.H . Millerand, sad
H. Lal, eds., Future Pub . Co ., Mrnint Rieco, N.Y . (1972) . 13 .
J:D . WOOD, Am . J . Phyeiol. 219, 159-169 (1970) .
14 .
H. OHRAWA and C .L . PßOSSEß, Aa . J. Physiol. 222, 1420-1426 (1972),
15 .
S . NISHI and ß.A . NOßTH, J . $hyeiol. (Loads ) 231, 471-491 (1973) .
16 .
G .D .S . HIßST, M.E . HOLMAN and I. SPENCB, J. Physiol. 236, 303-326 (1974) .
17 .
T . SATO, I. TARAYANAGI and R. TASAGI, Jap. J. Pharuacol . 23, 665-671 (1973) .
18 .
W.D .M . PATON and M.A . ZAß, J . Physiol. 194, 13-33 (1968) .
19 .
A. GOLDSTEIN and ß. SCHIILZ, Brit . J. Pharmacol. 48, 655-666 (1973) .
20 .
G.A . C OTI'attr .T . and J.B . MACON, J . Physiol . 236, 425-464 (1974) .