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Reversible hyperphagia and obesity following intracerebral microinjections of colchicine into the ventromedial hypothalamus of the rat
Brain Research, 153 (I 978) 99-107
99
~) Elsevier/North-Holland Biomedical Press
REVERSIBLE H Y P E R P H A G I A AND OBESITY F O L L O W I N G INTRACEREBRAL MICROINJECTIONS OF COLCHICINE INTO T H E VENTROMEDIAL H Y P O T H A L A M U S OF T H E RAT
DOUGLAS AVR1TH and GORDON J. MOGENSON Departments of Psychology and Physiology, The University of Western Ontario, London N6A 3K7, Ontario (Canada)
(Accepted January 4th, 1978)
SUMMARY Colchicine, a drug which produces a reversible inhibition ofintraaxonal transport and synaptic transmission, was used as a reversible neural blocker to investigate the role of the ventromedial hypothalamus (VMH) in the control of ingestive behavior and body weight regulation. Male Sprague-Dawley rats received intracranial microinjections of colchicine into the VMH. Volume and concentration of the colchicine solution were varied to assess specificity of action and dose-response relationship. When colchicine (2 and 4/~g) was microinjected bilaterally into the VMH, there was a dose-dependent increase in food and water intakes and body weight gain which lasted several days. The acute period of hyperphagia was followed by a marked depression in feeding which persisted until body weight was lowered to control levels. This suppression of feeding appeared to be a consequence of the preceding period of hyperphagia and obesity, since colchicine-treated rats which were pair-fed with controls to prevent obesity continued to maintain normal food intake and body weight gain when later fed ad libitum. The results of this study confirm the importance of the VMH in the long term regulation of feeding, and indicate that reversible neuronal blocking with colchicine is a useful technique for investigating the neural substrates of feeding and other behaviors.
INTRODUCTION Colchicine is an alkaloid compound which inhibits axoplasmic transport by disrupting the structural organization of microtubules that are involved in axonal transport:Lll,15,')2,27,28,36. The local application of colchicine to neurons is highly effective in blocking the proximodistal movement of dense-core vesicles containing neurotransmitters a,ll,l%aS, and enzymes associated with the synthesis and degradation of neurotransmitters ~,1°,17,19,41. Electrical stimulation of neurons treated with colchicine still
100 produces normal action potentials16,a°'aT; however, the postsynaptic responses are depressed3°, zl. This deficit in synaptic transmission is thought to result from the blockade of transported material required at the synaptic terminals 3°. Since the action ofcotchicine on microtubules 5,8, axonat transport4 and synaptic transmission zo is reversible, it seemed feasible to investigate whether this drug might be used to produce a reversible functional bockade o f neural systems and pathways. Our initial experiments on the nigrostriatal pathway (using rotational behaviour) and on the dorsal noradrenergic pathway (using brain self-stimulation) indicated that a reversible neural block 0f4-7 days' duration could readily and reliably be obtained by administer' ing colchicine to these pathways ~4. Since the disruption of neural activity in the ventromedial hypothalamus by electrolytic lesions 1,1s or by microinfusions of a local anesthetiO4,23, 4~ produces increased food intake and body weight gain, it was decided to see whether microinfusions of colchicine to this region of the brain would result in a reversible hyperphagia. In the first series of animals, 2 and 4 pg of colchicine were administered bilaterally. In a second series, the colchicine-treated animals either had food available ad libitum or were fed an amount equivalent to that received by operated control rats. This was done in order to investigate whether the suppression of food in, take which followed the period of hyperphagia is associated with a compensation for excess body weight gain or with supersensitivity or rebound activity in the 'satiety' neurons of the VMH. METHODS Male Sprague-Dawley rats weighing 140-t60 g were individually housed in wire cages (18 × 23 × l 8 cm) in a room illuminated from07.30 to 19.30 h. Room temperature was kept at 20 °C. A highly palatable synthetic high-fat diet was available from glass jars attached to the inside of the cages. Tap water was provided from inverted bottles with stainless steel drinking spouts. Food and water were available ad libitum unless stated otherwise. Body weight, food and water intakes were measured daily. Under sodium pentobarbital anesthesia (50 mg/kg) the rats were mounted in a stereotaxic frame. The rats received bilateral infusions of colchicine (2 or 4 #g) into the VMH, delivered in a vehicle of isotonic saline (0.2 or 1.0 #1). In order to minimize structural damage to the VMH nucleus, the cannula was lowered to a depth of 0.3 mm above the structure. The coordinates used according to the atlas of K6nig and Klippel ~1 were: A 4.62; L 0.6: V - - 3 . 1 mm. Control groups were given bilateral infusions of the saline vehicle alone, with the osmolality and p H of the saline adjusted to approximate the osmolality (330 mOsm) and pH (6.0) of the colchicine solutions. All infusions were made over l min and thereafter the cannula was kept in place for an additional I min before being withdrawn. After an adaptation period of 2 days, measures of food and water intake and body weight gain were obtained for a period of 1 week. On the 7th day all of the rats underwent stereotaxic surgery. In the first series of animals two doses of colehicmewere administered (2 and 4 #g) with 5 rats per group, and all quantities of colchicine were delivered in a 0.2 ~t volume of saline. As a control, 8 rats were bilaterally infused with sali-
101 ne (osmolality and pH adjusted to match those of the colchicine solution) in the same site. In a second series of animals after measures of food and water intakes and body weight gain were obtained for 1 week, 10 of the rats were bilaterally infused with colchicine (2 #g/1/~1 saline) into the VMH and other 5 rats received bilateral infusions of the saline vehicle alone (osmolality 330 mOsm, pH 6.0). Following surgery, the controls and 5 of the colchicine-treated rats were given equal amounts of food (17 g) daily. The remaining rats were allowed free access to food. The rats on the restricted diet were given approximately half portions of their food twice daily, at 10.00 and 19.00 h, in order to prevent the animals from consuming all of their food at one time. After 8 days of this regimen, the rats were given free access to food. Six weeks postoperatively all rats were perfused and frozen, frontal brain sections were stained with cresyl violet or thionine. An examination of the site and extent of the cannulae tracts was conducted using the atlases of K6nig and Klippe121 and Pellegrino and Cushman 29 as a guide. Overall differences between groups were evaluated using a two-way analysis of variance test with repeated measures on one factor. Following analysis of variance tests, the differences between all pairs of means were tested using the Newman-Keuls multiple comparison method. RESULTS Cannulae tracts were found to terminate 0.2-0.5 mm dorsal to the lateral portion of the VMH nucleus, except for 3 rats in which cannulae had penetrated into the VMH. Gliosis was present along the shaft of the cannulae and at the cannulae tips in both the colchicine-treated and control rats. Colchicine infusions into the VMH resulted in a dramatic but reversible increase in food and water intakes and body weight gain. Hyperphagia and hyperdipsia, as well as excessive body weight gain, were observed within 24 h (P < 0.01 for all cases). The hyperphagia continued in the 2 and 4 #g groups for 6 and 10 days (all Ps < 0.01) respectively, and significant weight gains persisted for 4 days in the 2/~g group and 5 days in the 4/zg group (both groups P < 0.0l for each day) (Fig. 1). It should be noted that the period of excess body weight gain closely corresponds with the period of hyperphagia and hyperdipsia (2 #g, days 1-5, P < 0.01 ; 4/~g, days 1-7, P < 0.01). The hyperphagia and hyperdipsia were followed by reduced food and water intakes below baseline levels for several days and a reduced rate of body weight gain. The results for the second series of animals are shown in Fig. 2. Body weight increased dramatically following the infusion of colchicine in those rats which were given free access to food and in which increased food intake could occur, and only these rats later became hypophagic (Fig. 2). Both the unrestricted and pair-fed colchicine-treated groups showed significant increases in water intake (Ps < 0.01, 24 h after surgery) even though their food intake and body weight gain remained comparable to controls at this time (Ps > 0.10), suggesting that colchicine was having a primary effect on water intake. By 48 h, the rats which had received colchicine and were given unrestricted access to
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Fig. I. Food and water intakes and cumulative body weigh[ gain (mean & S.E.M.) following bilateral microinjections of colchicine (2/~g or 4/*g/0.2/tl saline) into the VMH. The average of these measures for 6 days prior to surgery is designated by A. Microinjections ofcolchicine were made on Day 0.Similar increases in food intake, water intake and body weight gain were observed in another series ofl4 rats in which the volume of colchicine microinjected into the VMH was 1/~1. The difference was that the magnitude of the increases was approximately one-third less than those shown here. Estimates of extent of diffusion25 using malachite green indicated that a microinjection of 0.2/d was limited to the VMH, whereas a volume of t/,1 involved spread to the medial aspect of the lateral hypothalamus (perhaps thereby partially reversing the VMH hyperphagial). A group of 8 rats in which bilateral electrolytic lesions were made in the VMH showed hyperphagia and body weight increases equivalentto those of the rats in which 2/,g of colchicine was microinjected, and these changes were still observed after 35 days. There was also an increase in water intake, but the magnitude was considerably less (approximately one-half) than that of the colcbicine-treated animals. In a parallel series of experiments, colehicine microinjected into the substantia nigra bilaterally was followed by reduced food intake, water intake and body weight gain for several days, and similarly these changes were reversible 2.
food were overeating (P < 0.01), a n d their food intake a n d b o d y weight gains r e m a i n e d significantly greater t h a n either pair-fed groups for a n o t h e r 4 a n d 3 days respectively (all Ps < 0.01). These rats also d r a n k m o r e water t h a n controls for a t o t a l of 5 days following i n f u s i o n (Ps < 0.01 for each day). A l t h o u g h n o t statistically significant, the pair-fed colchicine-treated rats t e n d e d to gain m o r e weight t h a n controls d a r i n g the first 5 days following infusion. This effect is p r o b a b l y due to the significant increases in water intake (and p r e s u m a b l y water retention) which were observed in this g r o u p d u r i n g this period (days 1-5, Ps < 0.01).
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TIME- doys Fig. 2. Food and water intakes and body weight gain (mean ± S.E.M.) following bilateral microinjections of colchicine (2 ffg/l / 4 saline) into the VMH. One of the colchicine-treated groups (designated with an asterisk) was pair-fed with the control group until day 8, and fed ad libitum thereafter. The average of the 3 measures for 6 days prior to surgery is designated by A. Microinjections of colchicine were made on Day 0.
Following the period of hyperphagia, the obese colchicine-treated rats reduced their food and water intakes and body weight gain below that of controls (days 7-14, Ps < 0.01) until their body weights had returned to control levels. On the other hand, when the pair-fed rats were given free access to food on day 8, they continued to maintain food and water intakes and body weight gains at a level similar to controls (all Ps > 0.10).
104 DISCUSSION The results of this study clearly demonstrate that bilateral microinjections of colchicine into the V M H produce increased food intake and body weight gains lasting several days. These changes are remarkably similar to those classically described following bilateral electrolytic lesions of the VMHTA s, with the major difference that after colchicine they are reversible. The observation of a dose-dependent relationship is in agreement with previous studies showing that the inhibitory action of colchicine on axoplasmic transport 16 and on synaptic transmission a0 is dose-dependent. The observation of reversibility of the effects of colchicine on food intake and body weight gain is consistent with the results of earlier experiments showing reversibility of the suppression of formalin-induced sodium intake with bilateral microinjections of colchicine into the medial amygdaloid nucleus 44. It has also been shown that postural asymmetry and circling behavior following administration of amphetamine, when colchicine is microinjected unilaterally into the substantia nigra, are reversible 24, as are the motor deficits, hypophagia and hypodipsia, which occur for 5 or 6 days after bilateral microinjections of colchicine into the substantia nigra 2. The hyperphagia initiated by microinjecting colchicine into the VMH was followed by several days of reduced food intake and body weight gain. The observations in colchicine-treated animals, pair-fed with controls, indicate that this was not the result of supersensitivity (or rebound activity) in 'satiety' neurons of the VMH. but rather a compensation to excess body weight gain. It has been suggested that the hypothalamus contributes to the control of food intake in relation to the 'set-point' for body weight regulation 20: the reduced food intake following lesions of the lateral hypothalamus has been attributed to a lowering of the set point for body weight regulationaL whereas increased food intake and body weight following lesions of the ventromedial hypothalamus has been attributed to elevating the set point 26. According to this view. lesions of the V M H elevate the set point permanently, while microinjections ofcolchicine elevate the set point temporarily, followed by a compensatory reduction in food intake for a few days, when the set point is lowered again after the colchicine block. A similar compensatory reduction of food intake has been observed when food intake and body weight were increased by force-feeding9 or by electrical stimulation of the lateral hypothalamus 39. Microinjections of colchicine into the VMH resulted in a reversible increase in water intake in adaition to reversible hyperphagia. Since the hyperdipsia was observed in the pair-fed rats, it is apparent that this excessive drinking is not the result of increased food intake. Colchicine-treated rats reduced urine output to the same magnitude as controls during a 24-h period of water deprivation (unpublished observations). Since electrolytic lesions of the VMH have been reported to result in water retention 43, it is possible that body fluid retention contributed to the substantial body weight gain observed following the administration of colchicine to the VMH. The use of colchicine as a reversible neural blocking technique offers investigators a unique approach for investigating the functions of discrete neural structures and pathways, permitting a comparison of the behavioral and physiological changes which occur
105 following the d i s r u p t i o n o f neural function with those observed following recovery from the blockade. The c o m p e n s a t o r y a d a p t a t i o n s o f the b r a i n to chronic necrosisl3, 40 a n d the irritation o f s u r r o u n d i n g tissue 33-35, which can c o m p l i c a t e the i n t e r p r e t a t i o n o f results o b t a i n e d after electrolytic lesions can be avoided. U s i n g colchicine, it is possible to detect effects o f the functional block, p e r h a p s subtle ones, that m i g h t be obscured by the t r a u m a t i c p o s t o p e r a t i v e effects of electrolytic lesions a n d / o r by the c o m p e n s a t o I y reactions o f the C N S to surgical insult 40. The h y p e r p h a g i a which occurs after colcbicine microinjections into the V M H can be r e p r o d u c e d in the same animal 24, an a d v a n t a g e over using local anesthetics, which lose their effect iveness when administered repeatedly a4. F u r t h e r m o r e , unlike neuronal b l o c k with procaine, which lasts a few minutes only 14,z3,42, the b l o c k i n g a c t i o n o f colchicine persists for several days. This long-lasting effect is p a r t i c u l a r l y useful in experiments which require observations over an extended t i m e period. ACKNOWLEDGEMENTS The research was s u p p o r t e d by a g r a n t from the N a t i o n a l Research Council o f Canada. The assistance o f B. Box and R. W o o d s i d e is acknowledged.
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 Avrith, D., Hass, H. L. and Mogenson, G., Behavioural and electrophysiologicalchanges following microinjections of colchicine in the substantia nigra, Proe. X X V I I Int. Congr. Physiol. Sci., Paris, 13 (1977) 39 (abstract 97). 3 Banks, P., Mayor, D., Mitchell, M. and Tomlinson, D., Studies on the translocation of noradrenaline-containing vesicles in post-ganglionic sympathetic neurons in vitro. Inhibition of movement by colchicine and vinblastine and evidence for the involvement of microtubules, J. Physiol. (Lond.), 216 (1971) 625-639. 4 Boesch, J., Marko, P. and Cuenod, M., Effects of colchicine on axonal transport of proteins in the pigeon visual pathways, NeuroOiology, 2 (1972) 123-132. 5 Borisy, G. C. and Taylor, E. W., The mechanism of action of colchicine, J. CellBiol., 34 (1967) 525 533. 6 Brimijoin, S., Transport and turnover of dopamine-fl-hydroxylasein sympathetic nerves of the rat, J. Pharmacol. exp. Ther., 178 (1972) 417-424. 7 Brobeck, J. R., Tepperman, J. and Long, C. N. H., Experimental hypothalamic hyperphagia in the albino rat, Yale J. biol. Med., 15 (1943) 831-853. 8 Bunge, R. and Bunge, M., Electron microscopic observations on colchicine-induced changes in neural cytoplasm, Anat. Rec., 160 (1968) 323. 9 Cohn, C. and Joseph, D., Influence on body weight and body fat on appetite of normal lean and obese rats, Yale J. biol. Med., 34 (1962) 598-607. 10 Coyle, J. and Wooten, G. F., Rapid axonal transport of tyrosine hydroxylase and dopamine-flhydroxylase, Brain Research, 44 (1972) 70t-704. 1 l Dahlstr6m, A.,Effectofcolchicineontransport ofamine storage granules in sympathetic nerves of rat, Europ. J. Pharmacol., 5 (I 968) 111-113. 12 Dahlstr6m, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. 13 Dawson, R. G., Recovery of function: implications for theories of brain function, Behav. Biol., 8 (1973) 439-460.
106 14 Epstein, A. N., Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injections, ,4mer. J. Physiol., 199 (I 960) 969-974. 15 Fernandez, H. L., Huneeus, F. C. and Davidson. P. F.. Studies on the mechanisms of axoplasmic transport in the crayfish nerve cord. J. Neurobiol.. 1 (197131 395-400. 16 Fink. B. R., Byers, M. and Middaugh, M., Dynamics ofcolchicine effects on rapid axonal transpert and axonal morphology, Brain Research, 56 (1973) 299-311. t7 Frizell. M., Hassetgran, P. and Sj6strand, J.. Axoplasmic transport of acetytcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit, Exp. Brain Res.. It) (19701 526-531 18 Hetherington. A. W. and Ranson. S. W.. H ypothalamic lesions and adiposity in the rat. Anat. R e c . 78 (19401 149-172. 19 Jackson, P. and Diamond, J.. Colchicine block of cholinesterase transport in rabbit sensory nerves without interference with the long-term viability of the axons, Brain Research. 130 (/977) 579-584. 20 Kennedy, G. C., Food intake energy balance and growth. Brit. reed. Bull.. 22 (1966) 216-220. 21 K6nig, J. F. and Klippel, R. A., The Rat Brain: a Stereotaxie ,4tlas o/the Forebrain and Lower Parts o] the Brain Stem, Williams and Wilkins. Baltimore, 1963. 22 Kreutzberg, G. W., Neuronal dynamics and axonal flow. IV. Blockage ofintraaxcmal transport by colchicine. Proc. nat. Aead. Sci. ~Wash.,. 62 (1969) 722-728. 23 Larkin. R. P., Effects of ventromedial hypothalamic procaine injections on feeding, lever pressing and other behaviours in rats, J. comp. Physiol., 80 ~19751 1100-1108. 24 Mogenson, G. J. and Avrith. D. B.. The use ofcolchicine as a reversible neural blocker to investigate the control of food intake and body weight regulation, Katsuki, Y. and Oomura. Y. (Eds.), Symposmm on Food Intake and Chemical Senses, University of Tokyo Press, Tokyo, 1977. pp. 387-397. 25 Myers, R. D.. Handbook o / D r u g and Chemical Stimulation o f the Brain. Van Nostrand-Reinhold. New York. 1974. 26 Nisbett, R. E.. Hunger. obesity and the ventromedial hypothalamus. P~w'hol. leer., 79 ~1972~ 433 453. 27 Norstrom, A., Hansson. H. A. and Sj6strand, J.. Effects ofcolchicine on axonal transport and ultrastructure of the hypothalamo-neurohypophyseal of the rat. Z . Zell]brseh.. 113 (1971 ~ 271-293. 28 Paulson. J C. and McClure. W. O.. Microtubules and axoplasmic transport. Brain Research. 73 (1974) 333--337. 29 Pelligrino, C. and Cushman. A. J.. A Stereotaxk ~Atlas o f the Rat Bra#t, Appelton-Century-Crofts, New York, 1967. 30 Perisic, M. and Cuenod, M., Synaptic transmission depressed by colchicine blockade of axoplasmic flow, Science, 1975 (19721 1140-1142. 31 Pitar. G. and Landmesser. L.. Axotomy mimicked by localized colchicine application, Science. 177 (1972) 1116-1118. 32 Powley, T. L. and Keesey, R. E.. Relationship of body weight to the lateral hypothalamic feeding syndrome, J. comp. physiol. Psychol., 70 (1970) 25-36. 33 Rabin, B. M. and Smith, C. J., Behavioural comparison of the effectiveness of irritative and nonirrttative lesions in producing hypothalamic hyperphagia, Physiol. Behav.. 3 (1968/ 417-420. 34 Reynolds, R. W., Ventromedial hypothalamus lesions without hyperphagia, ,4met. J. Physiol., 204 (1963) 60--62. 35 Rolls, B. J., Drinking by rats after irritative lesions in the hypothalamus, PhysioL Behav., 5 (19701 1385-1393. 36 Samson, F. E., Mechanism of axoplasmic transport, J. Neurobiol., 2 (19711 347-360. 37 Schubert, P., Kreutzberg, G. W. and Lux, H. D.. Neuroplasmic transport in dendrites: effects of colchicine on morphology and physiology of motorneurones in the cat. Brain Research. 47 (19711 331-343. 38 Sj6strand, J.. Frizell, M. and Hasselgren, P. O., Effects of colchicine on axonal transport in peripheral nerves, J. Neurochem., 17 (1970) 1563-1570. 39 Steffens, A. B., Influence of reversible obesity on eating behavior, blood glucose, and insulin in the rat, ,4mer. J. PhysioL, 228 (19751 1738-1744. 40 Stein, D. G., Rosen, J. J. and Butters, N., Plastieity and Recovery o f Function in the Central Nervous System, Academic Press, New York, 1974. 41 Thoenen, H., Mueller, R. A. and Axelrod, J., Phase difference in the indication of tyrosine-t;hydroxylase in cell body and nerve terminals of sympathetic neurons, Proc. nat. Acad. Sei. " Was'h. ), 65 (19701 58-62.
107 42 Wagner, J. W. and De Groot, J., Changes in feeding behaviour after intracerebral injections in the rat, Amer. J. Physiol., 204 (1963) 483-487. 43 Wishart, T. B. and Walls, E. K., Water intoxication death following hypothalamic lesions in the rat, Physiol. Behav., 15 (1975) 377-379. 44 Zolovick, A., Avrith, D. and Jalowiec, J., Reversible colchicine-induced disruption of amygdaloid function in sodium appetite, in preparation.
99
~) Elsevier/North-Holland Biomedical Press
REVERSIBLE H Y P E R P H A G I A AND OBESITY F O L L O W I N G INTRACEREBRAL MICROINJECTIONS OF COLCHICINE INTO T H E VENTROMEDIAL H Y P O T H A L A M U S OF T H E RAT
DOUGLAS AVR1TH and GORDON J. MOGENSON Departments of Psychology and Physiology, The University of Western Ontario, London N6A 3K7, Ontario (Canada)
(Accepted January 4th, 1978)
SUMMARY Colchicine, a drug which produces a reversible inhibition ofintraaxonal transport and synaptic transmission, was used as a reversible neural blocker to investigate the role of the ventromedial hypothalamus (VMH) in the control of ingestive behavior and body weight regulation. Male Sprague-Dawley rats received intracranial microinjections of colchicine into the VMH. Volume and concentration of the colchicine solution were varied to assess specificity of action and dose-response relationship. When colchicine (2 and 4/~g) was microinjected bilaterally into the VMH, there was a dose-dependent increase in food and water intakes and body weight gain which lasted several days. The acute period of hyperphagia was followed by a marked depression in feeding which persisted until body weight was lowered to control levels. This suppression of feeding appeared to be a consequence of the preceding period of hyperphagia and obesity, since colchicine-treated rats which were pair-fed with controls to prevent obesity continued to maintain normal food intake and body weight gain when later fed ad libitum. The results of this study confirm the importance of the VMH in the long term regulation of feeding, and indicate that reversible neuronal blocking with colchicine is a useful technique for investigating the neural substrates of feeding and other behaviors.
INTRODUCTION Colchicine is an alkaloid compound which inhibits axoplasmic transport by disrupting the structural organization of microtubules that are involved in axonal transport:Lll,15,')2,27,28,36. The local application of colchicine to neurons is highly effective in blocking the proximodistal movement of dense-core vesicles containing neurotransmitters a,ll,l%aS, and enzymes associated with the synthesis and degradation of neurotransmitters ~,1°,17,19,41. Electrical stimulation of neurons treated with colchicine still
100 produces normal action potentials16,a°'aT; however, the postsynaptic responses are depressed3°, zl. This deficit in synaptic transmission is thought to result from the blockade of transported material required at the synaptic terminals 3°. Since the action ofcotchicine on microtubules 5,8, axonat transport4 and synaptic transmission zo is reversible, it seemed feasible to investigate whether this drug might be used to produce a reversible functional bockade o f neural systems and pathways. Our initial experiments on the nigrostriatal pathway (using rotational behaviour) and on the dorsal noradrenergic pathway (using brain self-stimulation) indicated that a reversible neural block 0f4-7 days' duration could readily and reliably be obtained by administer' ing colchicine to these pathways ~4. Since the disruption of neural activity in the ventromedial hypothalamus by electrolytic lesions 1,1s or by microinfusions of a local anesthetiO4,23, 4~ produces increased food intake and body weight gain, it was decided to see whether microinfusions of colchicine to this region of the brain would result in a reversible hyperphagia. In the first series of animals, 2 and 4 pg of colchicine were administered bilaterally. In a second series, the colchicine-treated animals either had food available ad libitum or were fed an amount equivalent to that received by operated control rats. This was done in order to investigate whether the suppression of food in, take which followed the period of hyperphagia is associated with a compensation for excess body weight gain or with supersensitivity or rebound activity in the 'satiety' neurons of the VMH. METHODS Male Sprague-Dawley rats weighing 140-t60 g were individually housed in wire cages (18 × 23 × l 8 cm) in a room illuminated from07.30 to 19.30 h. Room temperature was kept at 20 °C. A highly palatable synthetic high-fat diet was available from glass jars attached to the inside of the cages. Tap water was provided from inverted bottles with stainless steel drinking spouts. Food and water were available ad libitum unless stated otherwise. Body weight, food and water intakes were measured daily. Under sodium pentobarbital anesthesia (50 mg/kg) the rats were mounted in a stereotaxic frame. The rats received bilateral infusions of colchicine (2 or 4 #g) into the VMH, delivered in a vehicle of isotonic saline (0.2 or 1.0 #1). In order to minimize structural damage to the VMH nucleus, the cannula was lowered to a depth of 0.3 mm above the structure. The coordinates used according to the atlas of K6nig and Klippel ~1 were: A 4.62; L 0.6: V - - 3 . 1 mm. Control groups were given bilateral infusions of the saline vehicle alone, with the osmolality and p H of the saline adjusted to approximate the osmolality (330 mOsm) and pH (6.0) of the colchicine solutions. All infusions were made over l min and thereafter the cannula was kept in place for an additional I min before being withdrawn. After an adaptation period of 2 days, measures of food and water intake and body weight gain were obtained for a period of 1 week. On the 7th day all of the rats underwent stereotaxic surgery. In the first series of animals two doses of colehicmewere administered (2 and 4 #g) with 5 rats per group, and all quantities of colchicine were delivered in a 0.2 ~t volume of saline. As a control, 8 rats were bilaterally infused with sali-
101 ne (osmolality and pH adjusted to match those of the colchicine solution) in the same site. In a second series of animals after measures of food and water intakes and body weight gain were obtained for 1 week, 10 of the rats were bilaterally infused with colchicine (2 #g/1/~1 saline) into the VMH and other 5 rats received bilateral infusions of the saline vehicle alone (osmolality 330 mOsm, pH 6.0). Following surgery, the controls and 5 of the colchicine-treated rats were given equal amounts of food (17 g) daily. The remaining rats were allowed free access to food. The rats on the restricted diet were given approximately half portions of their food twice daily, at 10.00 and 19.00 h, in order to prevent the animals from consuming all of their food at one time. After 8 days of this regimen, the rats were given free access to food. Six weeks postoperatively all rats were perfused and frozen, frontal brain sections were stained with cresyl violet or thionine. An examination of the site and extent of the cannulae tracts was conducted using the atlases of K6nig and Klippe121 and Pellegrino and Cushman 29 as a guide. Overall differences between groups were evaluated using a two-way analysis of variance test with repeated measures on one factor. Following analysis of variance tests, the differences between all pairs of means were tested using the Newman-Keuls multiple comparison method. RESULTS Cannulae tracts were found to terminate 0.2-0.5 mm dorsal to the lateral portion of the VMH nucleus, except for 3 rats in which cannulae had penetrated into the VMH. Gliosis was present along the shaft of the cannulae and at the cannulae tips in both the colchicine-treated and control rats. Colchicine infusions into the VMH resulted in a dramatic but reversible increase in food and water intakes and body weight gain. Hyperphagia and hyperdipsia, as well as excessive body weight gain, were observed within 24 h (P < 0.01 for all cases). The hyperphagia continued in the 2 and 4 #g groups for 6 and 10 days (all Ps < 0.01) respectively, and significant weight gains persisted for 4 days in the 2/~g group and 5 days in the 4/zg group (both groups P < 0.0l for each day) (Fig. 1). It should be noted that the period of excess body weight gain closely corresponds with the period of hyperphagia and hyperdipsia (2 #g, days 1-5, P < 0.01 ; 4/~g, days 1-7, P < 0.01). The hyperphagia and hyperdipsia were followed by reduced food and water intakes below baseline levels for several days and a reduced rate of body weight gain. The results for the second series of animals are shown in Fig. 2. Body weight increased dramatically following the infusion of colchicine in those rats which were given free access to food and in which increased food intake could occur, and only these rats later became hypophagic (Fig. 2). Both the unrestricted and pair-fed colchicine-treated groups showed significant increases in water intake (Ps < 0.01, 24 h after surgery) even though their food intake and body weight gain remained comparable to controls at this time (Ps > 0.10), suggesting that colchicine was having a primary effect on water intake. By 48 h, the rats which had received colchicine and were given unrestricted access to
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Fig. I. Food and water intakes and cumulative body weigh[ gain (mean & S.E.M.) following bilateral microinjections of colchicine (2/~g or 4/*g/0.2/tl saline) into the VMH. The average of these measures for 6 days prior to surgery is designated by A. Microinjections ofcolchicine were made on Day 0.Similar increases in food intake, water intake and body weight gain were observed in another series ofl4 rats in which the volume of colchicine microinjected into the VMH was 1/~1. The difference was that the magnitude of the increases was approximately one-third less than those shown here. Estimates of extent of diffusion25 using malachite green indicated that a microinjection of 0.2/d was limited to the VMH, whereas a volume of t/,1 involved spread to the medial aspect of the lateral hypothalamus (perhaps thereby partially reversing the VMH hyperphagial). A group of 8 rats in which bilateral electrolytic lesions were made in the VMH showed hyperphagia and body weight increases equivalentto those of the rats in which 2/,g of colchicine was microinjected, and these changes were still observed after 35 days. There was also an increase in water intake, but the magnitude was considerably less (approximately one-half) than that of the colcbicine-treated animals. In a parallel series of experiments, colehicine microinjected into the substantia nigra bilaterally was followed by reduced food intake, water intake and body weight gain for several days, and similarly these changes were reversible 2.
food were overeating (P < 0.01), a n d their food intake a n d b o d y weight gains r e m a i n e d significantly greater t h a n either pair-fed groups for a n o t h e r 4 a n d 3 days respectively (all Ps < 0.01). These rats also d r a n k m o r e water t h a n controls for a t o t a l of 5 days following i n f u s i o n (Ps < 0.01 for each day). A l t h o u g h n o t statistically significant, the pair-fed colchicine-treated rats t e n d e d to gain m o r e weight t h a n controls d a r i n g the first 5 days following infusion. This effect is p r o b a b l y due to the significant increases in water intake (and p r e s u m a b l y water retention) which were observed in this g r o u p d u r i n g this period (days 1-5, Ps < 0.01).
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TIME- doys Fig. 2. Food and water intakes and body weight gain (mean ± S.E.M.) following bilateral microinjections of colchicine (2 ffg/l / 4 saline) into the VMH. One of the colchicine-treated groups (designated with an asterisk) was pair-fed with the control group until day 8, and fed ad libitum thereafter. The average of the 3 measures for 6 days prior to surgery is designated by A. Microinjections of colchicine were made on Day 0.
Following the period of hyperphagia, the obese colchicine-treated rats reduced their food and water intakes and body weight gain below that of controls (days 7-14, Ps < 0.01) until their body weights had returned to control levels. On the other hand, when the pair-fed rats were given free access to food on day 8, they continued to maintain food and water intakes and body weight gains at a level similar to controls (all Ps > 0.10).
104 DISCUSSION The results of this study clearly demonstrate that bilateral microinjections of colchicine into the V M H produce increased food intake and body weight gains lasting several days. These changes are remarkably similar to those classically described following bilateral electrolytic lesions of the VMHTA s, with the major difference that after colchicine they are reversible. The observation of a dose-dependent relationship is in agreement with previous studies showing that the inhibitory action of colchicine on axoplasmic transport 16 and on synaptic transmission a0 is dose-dependent. The observation of reversibility of the effects of colchicine on food intake and body weight gain is consistent with the results of earlier experiments showing reversibility of the suppression of formalin-induced sodium intake with bilateral microinjections of colchicine into the medial amygdaloid nucleus 44. It has also been shown that postural asymmetry and circling behavior following administration of amphetamine, when colchicine is microinjected unilaterally into the substantia nigra, are reversible 24, as are the motor deficits, hypophagia and hypodipsia, which occur for 5 or 6 days after bilateral microinjections of colchicine into the substantia nigra 2. The hyperphagia initiated by microinjecting colchicine into the VMH was followed by several days of reduced food intake and body weight gain. The observations in colchicine-treated animals, pair-fed with controls, indicate that this was not the result of supersensitivity (or rebound activity) in 'satiety' neurons of the VMH. but rather a compensation to excess body weight gain. It has been suggested that the hypothalamus contributes to the control of food intake in relation to the 'set-point' for body weight regulation 20: the reduced food intake following lesions of the lateral hypothalamus has been attributed to a lowering of the set point for body weight regulationaL whereas increased food intake and body weight following lesions of the ventromedial hypothalamus has been attributed to elevating the set point 26. According to this view. lesions of the V M H elevate the set point permanently, while microinjections ofcolchicine elevate the set point temporarily, followed by a compensatory reduction in food intake for a few days, when the set point is lowered again after the colchicine block. A similar compensatory reduction of food intake has been observed when food intake and body weight were increased by force-feeding9 or by electrical stimulation of the lateral hypothalamus 39. Microinjections of colchicine into the VMH resulted in a reversible increase in water intake in adaition to reversible hyperphagia. Since the hyperdipsia was observed in the pair-fed rats, it is apparent that this excessive drinking is not the result of increased food intake. Colchicine-treated rats reduced urine output to the same magnitude as controls during a 24-h period of water deprivation (unpublished observations). Since electrolytic lesions of the VMH have been reported to result in water retention 43, it is possible that body fluid retention contributed to the substantial body weight gain observed following the administration of colchicine to the VMH. The use of colchicine as a reversible neural blocking technique offers investigators a unique approach for investigating the functions of discrete neural structures and pathways, permitting a comparison of the behavioral and physiological changes which occur
105 following the d i s r u p t i o n o f neural function with those observed following recovery from the blockade. The c o m p e n s a t o r y a d a p t a t i o n s o f the b r a i n to chronic necrosisl3, 40 a n d the irritation o f s u r r o u n d i n g tissue 33-35, which can c o m p l i c a t e the i n t e r p r e t a t i o n o f results o b t a i n e d after electrolytic lesions can be avoided. U s i n g colchicine, it is possible to detect effects o f the functional block, p e r h a p s subtle ones, that m i g h t be obscured by the t r a u m a t i c p o s t o p e r a t i v e effects of electrolytic lesions a n d / o r by the c o m p e n s a t o I y reactions o f the C N S to surgical insult 40. The h y p e r p h a g i a which occurs after colcbicine microinjections into the V M H can be r e p r o d u c e d in the same animal 24, an a d v a n t a g e over using local anesthetics, which lose their effect iveness when administered repeatedly a4. F u r t h e r m o r e , unlike neuronal b l o c k with procaine, which lasts a few minutes only 14,z3,42, the b l o c k i n g a c t i o n o f colchicine persists for several days. This long-lasting effect is p a r t i c u l a r l y useful in experiments which require observations over an extended t i m e period. ACKNOWLEDGEMENTS The research was s u p p o r t e d by a g r a n t from the N a t i o n a l Research Council o f Canada. The assistance o f B. Box and R. W o o d s i d e is acknowledged.
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