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Morphine and enkephalin effects on hypothalamic glucoresponsive neurons
208
Brain Research, 185 (1980) 208-212 © Elsevier/North-Holland Biomedical Press
Morphine and enkephalin effects on hypothalamic glucoresponsive neurons
T. ONO, Y. OOMURA, H. NISHINO, K. SASAKI, K. MURAMOTO and 1. YANO Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-01 and ( Y. 0.) Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka 812 (Japan)
(Accepted October 4th, 1979) Key woras: morphine - - enkephalin -- bypotbalamus -- glucoresponsive neurons
Morphine has been shown to affect hypothalamic neuron activity in various ways depending on the nucleus involved3, 4. Systemic application increased neuronal activity in the ventromedial hypothalamic nucleus (VMH) and decreased it in the lateral hypothalamic area (LHA) 4, with 1.4-1.8 sec latency of the VMH effects behind the LHA changes. From this, it was inferred that the excitatory effect on VMH neurons may have been disinhibition due to release from LHA inhibition 4. Reliable data correlate LHA excitation and VMH inhibition with motivation to eat, and vice versa1,6,7; and lesion experiments associate these two centers with hypoand hyperphagia 1. Other data demonstrate effects on LHA and VMH activity by blood-borne metabolites and hormones which are in turn closely related to the nutritional state of the animalla, 14. An example of these data is inhibition of glucosesensitive LHA neurons and excitation of VMH glucoreceptor neurons by electroosmotic glucose application s-11. We report here, direct effects of morphine and enkephalin electrophoretically applied directly to individual glucose-sensitive LHA and glucoreceptor VMH neurons which are considered to be important in motivation and cessation of eating. Forty-two male Wistar rats (180-230 g) were used under anesthesia (mixture of 0.8 g/kg urethane and 80 mg/kg chloralose, i.p.). Subsequent maintenance doses were injected as necessary. Five barrelled glass micropipettes glued to single recording electrodes were used as previously described s-11. Each barrel was filled with one of the following materials: 0.2 M sodium chloride (pH 6.0), 1 M monosodium L-glutamate (pH 8.0), 0.4 M glucose (in 150 mM sodium chloride, pH 6.0), 50 mM morphine hydrochloride (pH 5.0), 50 mM methionine enkephalin (pH 4.0) and 50 mM levallorphan tartrate (pH 5.0) or 50 mM naloxone hydrochloride (in 150 mM sodium chloride, FH 5.5). The DC resistance of the pipettes ranged between 50 and 150 M f~, and that of the recording electrode was 10-35 M ft. Single unit discharges were recorded from LHA (A, 5.0-4.0; L, 1.5; H, - - 2 . 0 - --3.4) 5 and V M H (A, 5.0-4.0; L, 0.5: H, - - 3 . 0 - --3.8) 5 neurons. For application of a particular substance, a constant current
209 TABLE I
Summary of morphine effects on glucose-sensitive L H A and glucoreceptor V M H neurons E, excitation; I, inhibition; N, no effect; ( ) , sodium responsive.
Mo~h~e E
1
N
0 22 10
1 5 12
2 0 0
5 0 14
LHA Glucose (n = 56)
E 1 N
5 28 23
E I N
24 0 19
4 (3) 1 1
VMH
Glucose (n = 43)
17 0 5
of appropriate polarity and strength was passed through the pipetteS, 9. Only those neurons which were excited by glutamate were further analyzed. The effect, if any, of electric current alone was differentiated from that of the applied substance by using the criteria of Curtis and KoizumiL Observed effects were attributed to glucose, morphine or enkephalin when a neuron responded to those chemicals and not to sodium or to negative current. Electrode positions were determined histologically. Of 120 neurons tested, 56 were identified as LHA, and 43 as VMH neurons, The remaining 21 were located outside of the LHA and VMH. Results obtained are summarized in Table I. In the LHA, 56 neurons were tested with both glucose and morphine. Of these, 28 were inhibited, 5 were excited, and 23 were not affected by glucose. Of the 28 neurons which were inhibited by glucose, 1 was excited, 22 were inhibited, and 5 were not affected by morphine. Of the 5 neurons which were excited by glucose, 4 were excited by morphine, and 3 of these were found to also respond to either current or sodium. Of 23 neurons which did not respond to glucose, 1 was excited, 10 were inhibited, and 12 were not affected by morphine. Glucose administered to 43 neurons in the V M H excited 24 and did not affect 19. Of the 24 neurons which were excited by glucose, 17 were excited, 2 were inhibited, and 5 were unaffected by morphine. Of the 19 neurons which were unaffected by glucose, 5 were excited, and the other 14 were unaffected by morphine. None of these neurons responded to sodium. Fig. 1 shows the similarity of effects produced by glucose and morphine on LHA (A), and VMH (B) neurons. These same neurons were not affected by either sodium (not shown) or --80 nA. Glucose and morphine produced similar latency, magnitude and dose effects, as shown. Duration of the morphine effect was about double that of the glucose effect, but magnitude, dose-response and latency (3-10 sec) appeared to be nearly alike for both glucose and morphine. In order to determine the significance of the difference between glucose and morphine effects in the LHA and VMH, the data were analyzed by means of Fisher's
210 A Gluc+30
g
Gluc+50
6
4
Gluc+. I0
Gluc+_30
Gluc+50
M+I0
6
M+1..__0
M+20
8
M+30
M+§0
10
M+50
1"2
M+80
M+80
14 min
M -80
10
,
Fig. 1. Effects of glucose and morphine on glucose-sensitive LHA neurons (A) and glucoreceptor VMFI neurons (B). Abscissa: time; ordinates: neuronal discharge in pulses/see; overbars: duration of electrophoretic application of indicated factor at current specified by arabic numeral (nA). Glue, Glucose; M, Morphine. Note dose-dependentinhibition in LHA and excitation in VMH. LHA glucosesensitive and VMH glucoreceptor neurons did not respond to sodium.
Exact Test. Both glucose and morphine had quite different effects in the LHA and VMH (inhibition and excitation, respectively, P < 0.001). A morphine-like peptide, enkephalin (abundant in the hypothalamus, limbic system, etc.), produced almost the same effects as morphine. Enkephalin was applied to 14 of 22 LHA neurons which were inhibited by glucose and morphine; 2 were excited, 10 were inhibited, and 2 were not affected. A typical example is shown in Fig. 2A. Of 11 VMH neurons which increased in firing rate in response to glucose and morphine, 10 also increased and 1 was not affected by enkephalin (Fig. 2B). Morphine antagonists, naloxone and levallorphan, blocked the inhibition induced by morphine in LHA neurons and the excitation of VMH neurons, but did not block the effects of glucose on these same neurons (Fig. 2A and B). Effects of morphine and enkephalin which were blocked completely by simultaneous application of naloxone (q- 10-+30 nA), reappeared after cessation of the naloxone application. The results reported here agree in general with those reported by Kerr et al. 4, but further point out that the morphine and enkephalin effects on neurons associated with feeding are direct, not secondary. Yanaura et al. concluded that morphine has no effect on food intake, although the data charted in their Fig. 2 do not appear to support that conclusion 15. Risner and Khavari lz attributed decreased food intake after morphine administration to taste, but the data from the present experiments also indicate morphine depression of CNS feeding motivation.
211 N..x.,.+..~!..........................................
A Gluc'C'30
'°t
I
,i
Enk*30
M+IQ
Na÷$e
CI-30
Enk÷30
M÷3_..e
r
I
I
la
u e e
e
6
i
i
i
i
1"o
(z
1~
I Glue+30 .
20'
.
.
. Gluc÷3q)
M+30
Enk+30
0
1'6
18
2"0
22
2"4
26
2"8
3"0 min
N.x.~.3.O................................................................................ . Gluc*30
Gluc ÷30
u
CL
=. s ci
0'
Fig. 2. Effects of glucose, morphine, enkephalin and naloxone on glucose-sensitive LHA neurons (A) and glucoreceptor V M H neurons (]3). Enk, enkepbalin; Nx, Naloxone. Morphine and enkephalin have almost the same effects as glucose in both LHA (inhibition) and V M H (excitation). Naloxone completely blocks effect of morphine and enkephalin but has no effect on glucose. Effects attributed to specific chemicals since sodium ( + 3 0 nA) had no effect on this neuron.
In conclusion, morphine and enkephalin have been shown to have reciprocal effects directly on LHA glucose-sensitive and VMH glucoreceptor neurons (inhibition and excitation, respectively) in naive rats. This suggests that they could affect feeding behavior by their effects on these specific structures, the LHA and VMH. We thank Prof. A. Simpson, Showa University, for help in manuscript preparation. This work was supported in part by Ministry of Education, Science and Culture, Japan, Grants-in-Aid for Scientific Research 257027, 221216, 212110 and 311408, and Naito Research Foundation Grant 1977.
212 REFERENCES 1 Anand, B. K. and Brobeck, J. R., Hypothalamic control of food intake in rats and cats, Yale J. Biol. Med., 24 (1951) 123-140. 2 Curtis, D. R. and Koizumi, K., Chemical transmitter substances in brain stem of cat, J. NeurophysioL, 24 (1961) 80-90. 3 Eidelberg, E. and Bond, M. L., Effects of morphine and antagonists on tgvpothalamic cell activity, Arch. int. Pharmacodyn., 196 (1972) 16-24. 4 Kerr, F. W. L., Triplett, J. N., Jr. and Beeler, G. W., Reciprocal (push-pull) effects of morphine on single units in the ventromedian and lateral hypothalamus and influences in other nuclei : with a comment on methadone effects during withdrawal from morphine, Brain Research, 74 (1974) 81-103. 5 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain andLower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 6 Miller, N. E., Motivational effects of brain stimulation and drugs, Fed. Proc., 19 (1960) 846-854. 7 0 n o , T., Oomura, Y., Sugimori, M~, Nakamura, T., Sbimizu N., Kita, H. and Ishibashi, S., Hypothalamic unit activity related to lever pressing and eating in chronic monkey. In D. Novin, W. Wyrwicka and G. A. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1976, pp. 159-170. 8 0 o m u r a , Y., Significance of glucose, insulin and free fatty acid on the hypotbalamic feeding and satiety neurons. In D. Novin, W. Wyrwicka and G. A. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1976, pp. 145-157. 9 0 o m u r a , Y., Ohta, M., Kita, S., lshibashi, S. and Okajima, T., Hypothalamic neurons response to glucose, phlorizin and cbolecystokinin. In R. W. Ryall and J. S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Neurons System, Elsevier/North-Holland Biomedical Press, Amsterdam, New York, 1978, pp. 120-123. 10 Oomura, Y., Ono, T., Ooyama, H. and Wayner, M. J., Glucose and osmosensitive neurons of the rat hypothalamus, Nature (Lond.), 222 (1969) 282-284. 11 Oomura, Y., Ooyama, H., Sugimori, M., Nakamura, T. and Yamada, Y., Glucose inhibitionof the glucose-sensitive neuron in the rat lateral hypothalamus, Nature (Lond.), 247 (1974) 284--286. 12 Risner, M. E. and Khavari, K. A., Morphine dependence in rats produced after five days of ingestion, Psychopharmacologia (BerL), 28 (1973) 51~2. 13 Simson, P. C. and Booth, D. A., Subcutaneous release of amino acid loads on food and water intakes in the rat, PhysioL Behav., 11 (1973) 329-336. 14 Steffens, A. B., Influence of reversible obesity on eating behavior, food glucose, and insulin in the rat, Amer. J. PhysioL, 228 (1975) 1738-1744. 15 Yanaura, S., Tagashira, E. and Suzuki, T., Physical dependence on morphine, phenobarbital and diazepam in rats by drug-admixed food ingestion, Jap. J. PharmacoL, 25 (1975) 453-463.
Brain Research, 185 (1980) 208-212 © Elsevier/North-Holland Biomedical Press
Morphine and enkephalin effects on hypothalamic glucoresponsive neurons
T. ONO, Y. OOMURA, H. NISHINO, K. SASAKI, K. MURAMOTO and 1. YANO Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-01 and ( Y. 0.) Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka 812 (Japan)
(Accepted October 4th, 1979) Key woras: morphine - - enkephalin -- bypotbalamus -- glucoresponsive neurons
Morphine has been shown to affect hypothalamic neuron activity in various ways depending on the nucleus involved3, 4. Systemic application increased neuronal activity in the ventromedial hypothalamic nucleus (VMH) and decreased it in the lateral hypothalamic area (LHA) 4, with 1.4-1.8 sec latency of the VMH effects behind the LHA changes. From this, it was inferred that the excitatory effect on VMH neurons may have been disinhibition due to release from LHA inhibition 4. Reliable data correlate LHA excitation and VMH inhibition with motivation to eat, and vice versa1,6,7; and lesion experiments associate these two centers with hypoand hyperphagia 1. Other data demonstrate effects on LHA and VMH activity by blood-borne metabolites and hormones which are in turn closely related to the nutritional state of the animalla, 14. An example of these data is inhibition of glucosesensitive LHA neurons and excitation of VMH glucoreceptor neurons by electroosmotic glucose application s-11. We report here, direct effects of morphine and enkephalin electrophoretically applied directly to individual glucose-sensitive LHA and glucoreceptor VMH neurons which are considered to be important in motivation and cessation of eating. Forty-two male Wistar rats (180-230 g) were used under anesthesia (mixture of 0.8 g/kg urethane and 80 mg/kg chloralose, i.p.). Subsequent maintenance doses were injected as necessary. Five barrelled glass micropipettes glued to single recording electrodes were used as previously described s-11. Each barrel was filled with one of the following materials: 0.2 M sodium chloride (pH 6.0), 1 M monosodium L-glutamate (pH 8.0), 0.4 M glucose (in 150 mM sodium chloride, pH 6.0), 50 mM morphine hydrochloride (pH 5.0), 50 mM methionine enkephalin (pH 4.0) and 50 mM levallorphan tartrate (pH 5.0) or 50 mM naloxone hydrochloride (in 150 mM sodium chloride, FH 5.5). The DC resistance of the pipettes ranged between 50 and 150 M f~, and that of the recording electrode was 10-35 M ft. Single unit discharges were recorded from LHA (A, 5.0-4.0; L, 1.5; H, - - 2 . 0 - --3.4) 5 and V M H (A, 5.0-4.0; L, 0.5: H, - - 3 . 0 - --3.8) 5 neurons. For application of a particular substance, a constant current
209 TABLE I
Summary of morphine effects on glucose-sensitive L H A and glucoreceptor V M H neurons E, excitation; I, inhibition; N, no effect; ( ) , sodium responsive.
Mo~h~e E
1
N
0 22 10
1 5 12
2 0 0
5 0 14
LHA Glucose (n = 56)
E 1 N
5 28 23
E I N
24 0 19
4 (3) 1 1
VMH
Glucose (n = 43)
17 0 5
of appropriate polarity and strength was passed through the pipetteS, 9. Only those neurons which were excited by glutamate were further analyzed. The effect, if any, of electric current alone was differentiated from that of the applied substance by using the criteria of Curtis and KoizumiL Observed effects were attributed to glucose, morphine or enkephalin when a neuron responded to those chemicals and not to sodium or to negative current. Electrode positions were determined histologically. Of 120 neurons tested, 56 were identified as LHA, and 43 as VMH neurons, The remaining 21 were located outside of the LHA and VMH. Results obtained are summarized in Table I. In the LHA, 56 neurons were tested with both glucose and morphine. Of these, 28 were inhibited, 5 were excited, and 23 were not affected by glucose. Of the 28 neurons which were inhibited by glucose, 1 was excited, 22 were inhibited, and 5 were not affected by morphine. Of the 5 neurons which were excited by glucose, 4 were excited by morphine, and 3 of these were found to also respond to either current or sodium. Of 23 neurons which did not respond to glucose, 1 was excited, 10 were inhibited, and 12 were not affected by morphine. Glucose administered to 43 neurons in the V M H excited 24 and did not affect 19. Of the 24 neurons which were excited by glucose, 17 were excited, 2 were inhibited, and 5 were unaffected by morphine. Of the 19 neurons which were unaffected by glucose, 5 were excited, and the other 14 were unaffected by morphine. None of these neurons responded to sodium. Fig. 1 shows the similarity of effects produced by glucose and morphine on LHA (A), and VMH (B) neurons. These same neurons were not affected by either sodium (not shown) or --80 nA. Glucose and morphine produced similar latency, magnitude and dose effects, as shown. Duration of the morphine effect was about double that of the glucose effect, but magnitude, dose-response and latency (3-10 sec) appeared to be nearly alike for both glucose and morphine. In order to determine the significance of the difference between glucose and morphine effects in the LHA and VMH, the data were analyzed by means of Fisher's
210 A Gluc+30
g
Gluc+50
6
4
Gluc+. I0
Gluc+_30
Gluc+50
M+I0
6
M+1..__0
M+20
8
M+30
M+§0
10
M+50
1"2
M+80
M+80
14 min
M -80
10
,
Fig. 1. Effects of glucose and morphine on glucose-sensitive LHA neurons (A) and glucoreceptor VMFI neurons (B). Abscissa: time; ordinates: neuronal discharge in pulses/see; overbars: duration of electrophoretic application of indicated factor at current specified by arabic numeral (nA). Glue, Glucose; M, Morphine. Note dose-dependentinhibition in LHA and excitation in VMH. LHA glucosesensitive and VMH glucoreceptor neurons did not respond to sodium.
Exact Test. Both glucose and morphine had quite different effects in the LHA and VMH (inhibition and excitation, respectively, P < 0.001). A morphine-like peptide, enkephalin (abundant in the hypothalamus, limbic system, etc.), produced almost the same effects as morphine. Enkephalin was applied to 14 of 22 LHA neurons which were inhibited by glucose and morphine; 2 were excited, 10 were inhibited, and 2 were not affected. A typical example is shown in Fig. 2A. Of 11 VMH neurons which increased in firing rate in response to glucose and morphine, 10 also increased and 1 was not affected by enkephalin (Fig. 2B). Morphine antagonists, naloxone and levallorphan, blocked the inhibition induced by morphine in LHA neurons and the excitation of VMH neurons, but did not block the effects of glucose on these same neurons (Fig. 2A and B). Effects of morphine and enkephalin which were blocked completely by simultaneous application of naloxone (q- 10-+30 nA), reappeared after cessation of the naloxone application. The results reported here agree in general with those reported by Kerr et al. 4, but further point out that the morphine and enkephalin effects on neurons associated with feeding are direct, not secondary. Yanaura et al. concluded that morphine has no effect on food intake, although the data charted in their Fig. 2 do not appear to support that conclusion 15. Risner and Khavari lz attributed decreased food intake after morphine administration to taste, but the data from the present experiments also indicate morphine depression of CNS feeding motivation.
211 N..x.,.+..~!..........................................
A Gluc'C'30
'°t
I
,i
Enk*30
M+IQ
Na÷$e
CI-30
Enk÷30
M÷3_..e
r
I
I
la
u e e
e
6
i
i
i
i
1"o
(z
1~
I Glue+30 .
20'
.
.
. Gluc÷3q)
M+30
Enk+30
0
1'6
18
2"0
22
2"4
26
2"8
3"0 min
N.x.~.3.O................................................................................ . Gluc*30
Gluc ÷30
u
CL
=. s ci
0'
Fig. 2. Effects of glucose, morphine, enkephalin and naloxone on glucose-sensitive LHA neurons (A) and glucoreceptor V M H neurons (]3). Enk, enkepbalin; Nx, Naloxone. Morphine and enkephalin have almost the same effects as glucose in both LHA (inhibition) and V M H (excitation). Naloxone completely blocks effect of morphine and enkephalin but has no effect on glucose. Effects attributed to specific chemicals since sodium ( + 3 0 nA) had no effect on this neuron.
In conclusion, morphine and enkephalin have been shown to have reciprocal effects directly on LHA glucose-sensitive and VMH glucoreceptor neurons (inhibition and excitation, respectively) in naive rats. This suggests that they could affect feeding behavior by their effects on these specific structures, the LHA and VMH. We thank Prof. A. Simpson, Showa University, for help in manuscript preparation. This work was supported in part by Ministry of Education, Science and Culture, Japan, Grants-in-Aid for Scientific Research 257027, 221216, 212110 and 311408, and Naito Research Foundation Grant 1977.
212 REFERENCES 1 Anand, B. K. and Brobeck, J. R., Hypothalamic control of food intake in rats and cats, Yale J. Biol. Med., 24 (1951) 123-140. 2 Curtis, D. R. and Koizumi, K., Chemical transmitter substances in brain stem of cat, J. NeurophysioL, 24 (1961) 80-90. 3 Eidelberg, E. and Bond, M. L., Effects of morphine and antagonists on tgvpothalamic cell activity, Arch. int. Pharmacodyn., 196 (1972) 16-24. 4 Kerr, F. W. L., Triplett, J. N., Jr. and Beeler, G. W., Reciprocal (push-pull) effects of morphine on single units in the ventromedian and lateral hypothalamus and influences in other nuclei : with a comment on methadone effects during withdrawal from morphine, Brain Research, 74 (1974) 81-103. 5 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain andLower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 6 Miller, N. E., Motivational effects of brain stimulation and drugs, Fed. Proc., 19 (1960) 846-854. 7 0 n o , T., Oomura, Y., Sugimori, M~, Nakamura, T., Sbimizu N., Kita, H. and Ishibashi, S., Hypothalamic unit activity related to lever pressing and eating in chronic monkey. In D. Novin, W. Wyrwicka and G. A. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1976, pp. 159-170. 8 0 o m u r a , Y., Significance of glucose, insulin and free fatty acid on the hypotbalamic feeding and satiety neurons. In D. Novin, W. Wyrwicka and G. A. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1976, pp. 145-157. 9 0 o m u r a , Y., Ohta, M., Kita, S., lshibashi, S. and Okajima, T., Hypothalamic neurons response to glucose, phlorizin and cbolecystokinin. In R. W. Ryall and J. S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Neurons System, Elsevier/North-Holland Biomedical Press, Amsterdam, New York, 1978, pp. 120-123. 10 Oomura, Y., Ono, T., Ooyama, H. and Wayner, M. J., Glucose and osmosensitive neurons of the rat hypothalamus, Nature (Lond.), 222 (1969) 282-284. 11 Oomura, Y., Ooyama, H., Sugimori, M., Nakamura, T. and Yamada, Y., Glucose inhibitionof the glucose-sensitive neuron in the rat lateral hypothalamus, Nature (Lond.), 247 (1974) 284--286. 12 Risner, M. E. and Khavari, K. A., Morphine dependence in rats produced after five days of ingestion, Psychopharmacologia (BerL), 28 (1973) 51~2. 13 Simson, P. C. and Booth, D. A., Subcutaneous release of amino acid loads on food and water intakes in the rat, PhysioL Behav., 11 (1973) 329-336. 14 Steffens, A. B., Influence of reversible obesity on eating behavior, food glucose, and insulin in the rat, Amer. J. PhysioL, 228 (1975) 1738-1744. 15 Yanaura, S., Tagashira, E. and Suzuki, T., Physical dependence on morphine, phenobarbital and diazepam in rats by drug-admixed food ingestion, Jap. J. PharmacoL, 25 (1975) 453-463.