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Endogenous opioids regulate dendritic growth and spine formation in developing rat brain
Brain Research, 416 (1987) 157-161 Elsevier
157
BRE 22372
Endogenous opioids regulate dendritic growth and spine formation in developing rat brain Kurt F. Hauser, Patricia J. McLaughlin and Ian S. Zagon Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033 (U.S.A.)
(Accepted 7 April 1987) Key words: Opioid; Development; Cerebellum; Cerebral cortex; Hippocampus; Dentate gyrus
Continuous blockade of endogenous opioid-opioid receptor interaction by opioid antagonists from birth to day 10 increased neuronal maturation in the rat brain. The lengths of oblique dendrites of pyramidal cells in the cerebral cortex and basilar dendrites of the hippocampus were increased from controls by 136 and 51%, respectively, whereas the concentrations of spines in these ceils were increased 183 and 69%, respectively. Total dendritic length of spiny branches of cerebellar Purkinje neurons was 65% greater than controis, and spine concentration of granule cells in the dentate gyrus was increased by 76%. Thus, endogenous opioids exert a remarkable influence on the timetable and magnitude of dendritic elaboration and spine formation, and serve as an important trophic influence in the regulation of neuro-ontogeny.
Endogenous opioid systems (i.e., endogenous opioids and opioid receptors) are involved in a wide variety of functions 1'2, including the regulation of nervous system development 18-27. Components of endogenous opioid systems are present in brain tissues during ontogeny 1°'11,16. Although the presence of some of these opioid systems may be indicative of adult neural function, evidence now points to the elaboration of endogenous opioids/opioid receptors that may be present only during neurodevelopment and are related to cell proliferation and differentiation 26'27. Experimental paradigms that have utilized opioid antagonists to interrupt the interaction of endogenous opioids and opioid receptors have been crucial in elucidating the principles of neuropeptide regulation of growth 19-25'27. Thus, when infant rats receive the potent antagonist naltrexone in a dosage regimen that produces a continuous or intermittent blockade of the interfacing of endogenous opioids and opioid receptors, somatic and neurobiological development is markedly altered 19-25. The effects on growth depend on the duration of opioid receptor
blockade 25. For example, continuous blockade during early postnatal life dramatically increases body and brain size of young rats, increases the number of brain cells, and accelerates the appearance of physical characteristics and spontaneous motor and sensorimotor behaviors 19-25. Given the extensive literature devoted to structure-function relationships in regard to neuro-ontogeny 5'7'15A7, it could be hypothesized that the establishment of neuronal organization and intricacy in the developing brain is dependent on endogenous opioid-opioid receptor interactions. To address the role of endogenous opioid systems in neuroplasticity, developing neurons in the cerebral cortex, hippocampus, and cerebellum were structurally examined in 10-day-old rats subjected to continuous opioid receptor blockade. Neuronal complexity, as evidenced by dendritic elaboration and/or synaptic development, was markedly more mature in the brains of these animals than in control counterparts. These results provide the first evidence that endogenous opioids are important regulators of neuronal growth and dendrit-
Correspondence: K.F. Hauser, Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
158 TABLE I Dendritic length and spine concentration of neurons from lOday-old rats receiving daily s.c. injections of sterile water (controls) or naltrexone (50 mg/kg) beginning at birth
Estimates of total dendritic lengths represent the number of intersections within a reticule containing concentric circles (4.2 ~m spacings; 630x magnification); 10-16 separate cells/group (3-4 cells/animals, 3-4 animals/group) were analyzed. Dendritic spine concentration4,6A3was determined along the midsegment length of third-order basilar dendrites (pyramidal cells), the mid-portion of terminal segments of oblique dendrites (pyramidal cells) and spiny branches (Purkinje cells), and along dendrites (granule cells) in intermediate and superficial layers at 1500x. Spine density is given as the number of spines/50/~m dendrite length; at least 20 cells/groups (50/~m/dendrite, 6 dendrites/cell, 4 cells/rat, 5 animals/group) were measured. Values are the mean _+S.E.M. Brain region-cell type
Hippocampus-pyramidal cells Basilar dendrites Length Spines Dentate gyrus-granule cells Length Spines (intermediate layer) Spines (superficiallayer) Cerebral cortex-pyramidal cells Basilar dendrites Length Spines Oblique dendrites Length Spines Cerebellum-Purkinje cells Smooth branches Length Spiny branchlets Length Spines
Control group
Naltrexone group
190 + 29 7.8 + 0.6
287 + 24* 13.2 + 1.5"**
228 + 11 21.3 + 1.9 36.2 _+2.2
225 + 1 37.4 + 2.4*** 62.1 + 3.7***
169 + 25 8.5 _+0.8
189 + 19 17.0 + 1.0"**
25 + 2 6.5 + 1.2
59 + 3** 18.4 + 3.7*
47 + 6
56 + 7
143 + 15 60.5 + 2.2
235 + 20** 61.2 + 5.1
*P < 0.05; **P < 0.01; ***P < 0.005.
ic spine formation. Newborn S p r a g u e - D a w l e y rats, r e a r e d in litters of 8 pups p e r m o t h e r , were given daily s.c. injections of naltrexone (50 mg/kg) or sterile water and sacrificed on postnatal day 10. O n day t0, the naltrexonet r e a t e d animals weighed 16.5% m o r e than the controls (19.6 + 0.28 g). Brains were stained with the G o l g i - K o p s c h m e t h o d 3'5 and analyzed quantitatively for dendritic length and spine concentration 4-6. All neurons of a p p r o p r i a t e type and location, that were c o m p l e t e l y i m p r e g n a t e d by the Golgi technique, were examined; it was assumed that n e u r o n s were r a n d o m l y impregnated. O n l y cells with s o m a t a
within the center of the section thickness, and cell processes unobscured, were utilized. To avoid errors incurred by potential gradients of m a t u r a t i o n within a brain region, neurons were studied within highly circumscribed and rigorously defined anatomical landmarks, and comparisons were made between matched sections. Pyramidal cells from h i p p o c a m p a l area C A I , and granule cells from the s u p r a p y r a m i d a l layer of the dentate gyrus, were examined from the two serial sections in the coronal plane immediately posterior (i.e. within 266 ~ m ) to the caudal b o r d e r of the corpus callosum (i.e. where it becomes interhemispheric). Pyramidal cells from the m o t o r portion of the frontoparietal cortex, layer I l i a . were analyzed in the two serial sections i m m e d i a t e l y anterior to the rostral b o r d e r of the interhemispheric portion of the corpus callosum. Purkinje cells were evaluated midway between gyri and sutci of lobule VIII of the cerebellum. All slides were c o d e d so that the observer was unaware of group identification. Selective changes in dendritic morphology were r e c o r d e d in every brain region e x a m i n e d (Table I). In rats receiving naltrexone, the pyramidal neurons in the hippocampal formation were increased 51% from controls in estimates of basilar dendritic length (Fig. I A ) , along with a 69% increase in the concentration of dendritic spines. The dendrites of naltrexonetreated animals not only contained more spines than controls, but they often a p p e a r e d to be of larger caliber (Fig. 1B). The numerous varicosities found in dendrites of control animals, assumed to be a characteristic of immaturitv a4. were infrequently o b s e r v e d in 10-day-old rats chronically injected with an opioid antagomst (Fig. 1B). Estimates of dendritic length for granule cells in the d e n t a t e gyrus were c o m p a r able for naltrexone-treated and control rats. but the n u m b e r of spines on granule cells projecting into the superficial and intermediate layers of the stratum moleculare was increased 7 2 - 7 6 ~ m rats receiving naltrexone. Pyramidal neurons in the cerebral cortex of rats given naltrexone did not differ in estimates of basilar dendritic length, but did show a m a r k e d increase in oblique dendritic length (136% of control levels). The concentration of spmes increased dramatically on both basilar and oblique dendrites of pyramidal neurons, being 100 and 183~;k. respectively, of control values In the cerebellum, the length of Purkin ie cell dendrites was 64% greater than control
159
A
jf ./
,
B
,r
jr
j
C
CONTROL
NALTREXONE
?ig. 1. Camera lucida drawings (A, C) and photomicrographs (B) of Golgi-Kopsch-stained preparations from 10-day-old rats chroni:ally receiving sterile water (control) or 50 mg/kg naltrexone. Pyramidal cells from region CA 1 of the hippocampal formation (A) in laltrexone-treated rats had a notable increase in dendritic arborization, with the length of oblique dendrites (along the main dendritic ;haft) and basilar dendrites (extending from the cell soma) often considerably longer than controls. Upon closer inspection, basilar (C) md oblique dendrites of the pyramidal cells from rats receiving naltrexone contained more spines (arrow), and were usually of larger Jiameter. Unlike the controls, constrictions along the dendritic shaft were infrequently observed in naltrexone-treated rats. Purkinje :ells (C) in the cerebellum (lobule VIII) of animals chronically given naltrexone exhibited a dendritic expanse that was considerably ~ider and larger than that of control specimens, and branching of the dendrites was often more complex. Bars: A = 50pm; B = 5pm; 2 = 25 urn.
160 levels (Fig. 1C). This was accompanied by an increase of similar magnitude in the number of spiny branches. However, there were no changes in spine concentration, or the number of smooth branches. The results of this study show that interruption of endogenous opioid-opioid receptor interaction by administration of an opioid antagonist has a profound effect on neuronal growth and the expression of dendritic spines, with the net effect being an increase in the content of dendritic spines in neurons. Since 4 different cell types in 4 different brain regions were markedly influenced, it appears that opioid action is not restrictive in scope nor dependent on the stage of neuro-ontogeny. However, the effects were selective as to cell type and brain region. The increase in neuronal growth also may not depend on the expression of complex behavior since the animals utilized for study were 10 days of age. For example, eye opening normally does not occur until days 15-16, while the median age of walking occurs on day 12 (ref. 22). The function subserved by dendritic spines is still only suggestive 4. Ramon y Caja114 hypothesized that spines served to increase the receptive area of a dendrite and, indeed, 85% of the synapses on a spiny cortical neuron occur on spines t2. The dendritic spine may be the first site of active integration of the afferent supply, and may play an important role in the quality and quantity of input to a neuron 4. Our results demonstrate acceleration in the acquisition of dendritic spines in animals with chronic opioid receptor blockade. Interestingly, the ontogeny of gross motor development and the maturation of both simple and complex sensory motor behaviors have been explored in rat pups treated with 50 mg/kg naltrexone 22, and these young rats have an accelerated
1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khatchaturian, H. and Walker, J.M., Endogenous opioids - - biology and function, Annu. Rev. Neurosci., 7 (1984) 223-255. 2 Beaumont, A. and Hughes, J., Biology of opioid peptides, Annu. Rev. Pharmacol. Toxicol., 19 (1979) 245-267. 3 Colonnier, M., The tangential organization of the visual cortex, J. Anat. (London), 98 (1964) 327-344. 4 Connor, J.R. and Diamond, M.C., A comparison of dendritic spine number and type on pyramidal neurons of the visual cortex of old adult rats from social or isolated environments, J. Comp. Neurol., 210 (1982) 99-106. 5 Floeter, M.K. and Greenough, W.T., CerebeUar plasticity: modification of Purkinje celt structure by differential rearing in monkeys, Science, 206 (1979) 227-229.
course of behavioral ontogeny. These findings wou support earlier contentions of a relationship betwe~ structure of the nervous system and functional inte actions 5'7'15,17, and indicate the importance of e dogenous opioid systems in the emergence of bra cytoarchitecture and behavior; Of course, ctarific tion of the relationship of endogenous opioids al synaptogenesis awaits electron microscopic analysi Moreover, we cannot conclude that the total numb, of spines in adults will be different in the stimulate animals. In conclusion, the present report has investigat~ the repercussion of unbalancing the equilibrium b tween endogenous opioids and opioid receptors the developing rat brain. It appears that this relatio: ship governs, either by directly influencing deve oping neurons or indirectly through informatk (e.g., afferent fibers, neurotransmitters) gained fro other neural elements, neuronal differentiation ar the formation of postsynaptic structures (i.e. dendri ic spines). This would suggest that neurons in the d, veloping nervous system appear t o be plastic ar modifiable by endogenous opioid-opioid recept~ interactions. Clinical implications of this finding aJ unclear, however, alterations of endogenous opioJ systems during early life could have profound impo tance in the subsequent appearance and behavior neuronal networks 18. Indeed, disorganization of tt developing nervous system resulting from impai ment of endogenous opioid systems could be the eric logical basis of self-injurious behavior 9 and autisn attributed to opioid dysfunction in children, This work was supported by NIH Grants NS-205£ and NS-20623 to I.S.Z.
6 Globus, A. and Scheibel, A.B.. The effect of visual dep: vation on cortical neurons: a Golgi study, Exp. Neurol., (1967) 331-345. 7 Hebb, D.O., The Organization of Behavior, Wiley, Nc York, 1949. 8 Herman, B,H., Hammock, M.K., Egan J., Feinstein, ( Chatoor, I,, Boeckx, R., Zelnick, N., Jack, R. and Rose quist, J., Naltrexone induces dose-dependent decreases self-injurious behavior, Soc: Neurosci. Abstr., 11 (198 468. 9 Herman, B.H., Hammock, M.K., Arthur-Smith, ,~ Egan, J., Chatoor, I., Zelnick, N., Corradine,iM., App4 gate, K., Boeckx, R.L, and Sharp, S:D.; Role of opie peptides in autism: effects of acute administration of ni
161 trexone, Soc. Neurosci. Abstr., 12 (1986) 1172. 10 Marzullo, G. and Friedhoff, A.J., Opiate binding and cerebroside sulfates in the brain and lung of developing chicks. In E.L. Way (Ed.), Endogenous and Exogenous Opiate Agonists and Antagonists, Pergamon, Elmsfort, 1980, pp. 45-50. ll Maseda, C.E.G., Aguade, E.G., Mena, M.A. and De Yevenes, J.G., Ontogenic development of/3-endorphin immunoreactivity in rat brain regions, Neurosci. Lett., Suppl. 14 (1983) $237. 12 Peters, A. and Feldman, M.L., Projection of lateral geniculate nucleus to area 17 of rat cerebral-cortex. I. General description, J. Neurocytol., 5 (1976) 63-84. 13 Pysh, J.J. and Weiss, G.M., Exercise during development induces an increase in Purkinje-cell dendritic tree size, Science, 206 (1979) 230-232. 14 Ram6n y Cajal, S., Histologie du Syst~me Nerveus de l'Homme et des Vertebrates, C.S.I.C., Madrid, reprinted 1952-1955. 15 Schapiro, S. and Vukovich, K.R., Early experience effects on cortical dendrites: a proposed model for development, Science, 158 (1970) 292-294. 16 Tsang, D., Ng, S.C, Ho, K.P. and Ho, W.K.K., Ontogenesis of opioid binding sites and radioimmunoassayable/3-endorphin and enkephalin in regions of rat brain, Dev. Brain Res., 5 (1982) 257-261. 17 Volkmar, F.R. and Greenough, W.T., Rearing complexity affects branching of dendrites in the visual cortex of the rat, Science, 176 (1972) 1445-1447. 18 Zagon, I.S., Opioids and development: new lessons from
old problems, N1DA Res. Monogr., 60 (1985) 58-77. 19 Zagon, I.S. and McLaughlin, P.J., Increased brain size and cellular content in infant rats treated with an opiate antagonist, Science, 221 (1983) 1179-1180. 20 Zagon, I.S. and McLaughlin, P.J., Naltrexone modulates growth in infant rats, Life Sci., 33 (1983) 2449-2454. 21 Zagon, I.S. and McLaughlin, P.J., Naltrexone modulates body and brain development in rats: a role for endogenous opioids in growth, Life Sci., 35 (1984 i 2057-2064. 22 Zagon, I.S. and McLaughlin, P.J., Naltrexone's influence on behavioral development, Pharmacol. Biochem. Behav., 22 (1985) 441-448. 23 Zagon, I.S. and McLaughlin, P.J., Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies, J. Neurosci., 6 (1986) 1424-1432. 24 Zagon, I.S. and McLaughlin, P.J., Opioid antagonist-induced modulation of cerebral and hippocampal development: histological and morphometric studies. Dev. Brain Res., 28 (1986) 233-246. 25 Zagon, I.S. and McLaughlin, P.J., Endogenous opioids regulate cell proliferation in the developing rat brain, Brain Research, 412 (1987) 68-72. 26 Zagon, I.S. and McLaughlin, P.J., /~-Funaltexamine (flFNA) and the regulation of body and brain development in rats, Brain Res. Bull., 17 (1986) 5-9. 27 Zagon, I.S., Rhodes, R.E. and McLaughlin, P.J., Localization of enkephalin immunoreactivity in germinative cells of the developing rat cerebellum, Science, 227 (1985) 1049-1051.
157
BRE 22372
Endogenous opioids regulate dendritic growth and spine formation in developing rat brain Kurt F. Hauser, Patricia J. McLaughlin and Ian S. Zagon Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033 (U.S.A.)
(Accepted 7 April 1987) Key words: Opioid; Development; Cerebellum; Cerebral cortex; Hippocampus; Dentate gyrus
Continuous blockade of endogenous opioid-opioid receptor interaction by opioid antagonists from birth to day 10 increased neuronal maturation in the rat brain. The lengths of oblique dendrites of pyramidal cells in the cerebral cortex and basilar dendrites of the hippocampus were increased from controls by 136 and 51%, respectively, whereas the concentrations of spines in these ceils were increased 183 and 69%, respectively. Total dendritic length of spiny branches of cerebellar Purkinje neurons was 65% greater than controis, and spine concentration of granule cells in the dentate gyrus was increased by 76%. Thus, endogenous opioids exert a remarkable influence on the timetable and magnitude of dendritic elaboration and spine formation, and serve as an important trophic influence in the regulation of neuro-ontogeny.
Endogenous opioid systems (i.e., endogenous opioids and opioid receptors) are involved in a wide variety of functions 1'2, including the regulation of nervous system development 18-27. Components of endogenous opioid systems are present in brain tissues during ontogeny 1°'11,16. Although the presence of some of these opioid systems may be indicative of adult neural function, evidence now points to the elaboration of endogenous opioids/opioid receptors that may be present only during neurodevelopment and are related to cell proliferation and differentiation 26'27. Experimental paradigms that have utilized opioid antagonists to interrupt the interaction of endogenous opioids and opioid receptors have been crucial in elucidating the principles of neuropeptide regulation of growth 19-25'27. Thus, when infant rats receive the potent antagonist naltrexone in a dosage regimen that produces a continuous or intermittent blockade of the interfacing of endogenous opioids and opioid receptors, somatic and neurobiological development is markedly altered 19-25. The effects on growth depend on the duration of opioid receptor
blockade 25. For example, continuous blockade during early postnatal life dramatically increases body and brain size of young rats, increases the number of brain cells, and accelerates the appearance of physical characteristics and spontaneous motor and sensorimotor behaviors 19-25. Given the extensive literature devoted to structure-function relationships in regard to neuro-ontogeny 5'7'15A7, it could be hypothesized that the establishment of neuronal organization and intricacy in the developing brain is dependent on endogenous opioid-opioid receptor interactions. To address the role of endogenous opioid systems in neuroplasticity, developing neurons in the cerebral cortex, hippocampus, and cerebellum were structurally examined in 10-day-old rats subjected to continuous opioid receptor blockade. Neuronal complexity, as evidenced by dendritic elaboration and/or synaptic development, was markedly more mature in the brains of these animals than in control counterparts. These results provide the first evidence that endogenous opioids are important regulators of neuronal growth and dendrit-
Correspondence: K.F. Hauser, Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
158 TABLE I Dendritic length and spine concentration of neurons from lOday-old rats receiving daily s.c. injections of sterile water (controls) or naltrexone (50 mg/kg) beginning at birth
Estimates of total dendritic lengths represent the number of intersections within a reticule containing concentric circles (4.2 ~m spacings; 630x magnification); 10-16 separate cells/group (3-4 cells/animals, 3-4 animals/group) were analyzed. Dendritic spine concentration4,6A3was determined along the midsegment length of third-order basilar dendrites (pyramidal cells), the mid-portion of terminal segments of oblique dendrites (pyramidal cells) and spiny branches (Purkinje cells), and along dendrites (granule cells) in intermediate and superficial layers at 1500x. Spine density is given as the number of spines/50/~m dendrite length; at least 20 cells/groups (50/~m/dendrite, 6 dendrites/cell, 4 cells/rat, 5 animals/group) were measured. Values are the mean _+S.E.M. Brain region-cell type
Hippocampus-pyramidal cells Basilar dendrites Length Spines Dentate gyrus-granule cells Length Spines (intermediate layer) Spines (superficiallayer) Cerebral cortex-pyramidal cells Basilar dendrites Length Spines Oblique dendrites Length Spines Cerebellum-Purkinje cells Smooth branches Length Spiny branchlets Length Spines
Control group
Naltrexone group
190 + 29 7.8 + 0.6
287 + 24* 13.2 + 1.5"**
228 + 11 21.3 + 1.9 36.2 _+2.2
225 + 1 37.4 + 2.4*** 62.1 + 3.7***
169 + 25 8.5 _+0.8
189 + 19 17.0 + 1.0"**
25 + 2 6.5 + 1.2
59 + 3** 18.4 + 3.7*
47 + 6
56 + 7
143 + 15 60.5 + 2.2
235 + 20** 61.2 + 5.1
*P < 0.05; **P < 0.01; ***P < 0.005.
ic spine formation. Newborn S p r a g u e - D a w l e y rats, r e a r e d in litters of 8 pups p e r m o t h e r , were given daily s.c. injections of naltrexone (50 mg/kg) or sterile water and sacrificed on postnatal day 10. O n day t0, the naltrexonet r e a t e d animals weighed 16.5% m o r e than the controls (19.6 + 0.28 g). Brains were stained with the G o l g i - K o p s c h m e t h o d 3'5 and analyzed quantitatively for dendritic length and spine concentration 4-6. All neurons of a p p r o p r i a t e type and location, that were c o m p l e t e l y i m p r e g n a t e d by the Golgi technique, were examined; it was assumed that n e u r o n s were r a n d o m l y impregnated. O n l y cells with s o m a t a
within the center of the section thickness, and cell processes unobscured, were utilized. To avoid errors incurred by potential gradients of m a t u r a t i o n within a brain region, neurons were studied within highly circumscribed and rigorously defined anatomical landmarks, and comparisons were made between matched sections. Pyramidal cells from h i p p o c a m p a l area C A I , and granule cells from the s u p r a p y r a m i d a l layer of the dentate gyrus, were examined from the two serial sections in the coronal plane immediately posterior (i.e. within 266 ~ m ) to the caudal b o r d e r of the corpus callosum (i.e. where it becomes interhemispheric). Pyramidal cells from the m o t o r portion of the frontoparietal cortex, layer I l i a . were analyzed in the two serial sections i m m e d i a t e l y anterior to the rostral b o r d e r of the interhemispheric portion of the corpus callosum. Purkinje cells were evaluated midway between gyri and sutci of lobule VIII of the cerebellum. All slides were c o d e d so that the observer was unaware of group identification. Selective changes in dendritic morphology were r e c o r d e d in every brain region e x a m i n e d (Table I). In rats receiving naltrexone, the pyramidal neurons in the hippocampal formation were increased 51% from controls in estimates of basilar dendritic length (Fig. I A ) , along with a 69% increase in the concentration of dendritic spines. The dendrites of naltrexonetreated animals not only contained more spines than controls, but they often a p p e a r e d to be of larger caliber (Fig. 1B). The numerous varicosities found in dendrites of control animals, assumed to be a characteristic of immaturitv a4. were infrequently o b s e r v e d in 10-day-old rats chronically injected with an opioid antagomst (Fig. 1B). Estimates of dendritic length for granule cells in the d e n t a t e gyrus were c o m p a r able for naltrexone-treated and control rats. but the n u m b e r of spines on granule cells projecting into the superficial and intermediate layers of the stratum moleculare was increased 7 2 - 7 6 ~ m rats receiving naltrexone. Pyramidal neurons in the cerebral cortex of rats given naltrexone did not differ in estimates of basilar dendritic length, but did show a m a r k e d increase in oblique dendritic length (136% of control levels). The concentration of spmes increased dramatically on both basilar and oblique dendrites of pyramidal neurons, being 100 and 183~;k. respectively, of control values In the cerebellum, the length of Purkin ie cell dendrites was 64% greater than control
159
A
jf ./
,
B
,r
jr
j
C
CONTROL
NALTREXONE
?ig. 1. Camera lucida drawings (A, C) and photomicrographs (B) of Golgi-Kopsch-stained preparations from 10-day-old rats chroni:ally receiving sterile water (control) or 50 mg/kg naltrexone. Pyramidal cells from region CA 1 of the hippocampal formation (A) in laltrexone-treated rats had a notable increase in dendritic arborization, with the length of oblique dendrites (along the main dendritic ;haft) and basilar dendrites (extending from the cell soma) often considerably longer than controls. Upon closer inspection, basilar (C) md oblique dendrites of the pyramidal cells from rats receiving naltrexone contained more spines (arrow), and were usually of larger Jiameter. Unlike the controls, constrictions along the dendritic shaft were infrequently observed in naltrexone-treated rats. Purkinje :ells (C) in the cerebellum (lobule VIII) of animals chronically given naltrexone exhibited a dendritic expanse that was considerably ~ider and larger than that of control specimens, and branching of the dendrites was often more complex. Bars: A = 50pm; B = 5pm; 2 = 25 urn.
160 levels (Fig. 1C). This was accompanied by an increase of similar magnitude in the number of spiny branches. However, there were no changes in spine concentration, or the number of smooth branches. The results of this study show that interruption of endogenous opioid-opioid receptor interaction by administration of an opioid antagonist has a profound effect on neuronal growth and the expression of dendritic spines, with the net effect being an increase in the content of dendritic spines in neurons. Since 4 different cell types in 4 different brain regions were markedly influenced, it appears that opioid action is not restrictive in scope nor dependent on the stage of neuro-ontogeny. However, the effects were selective as to cell type and brain region. The increase in neuronal growth also may not depend on the expression of complex behavior since the animals utilized for study were 10 days of age. For example, eye opening normally does not occur until days 15-16, while the median age of walking occurs on day 12 (ref. 22). The function subserved by dendritic spines is still only suggestive 4. Ramon y Caja114 hypothesized that spines served to increase the receptive area of a dendrite and, indeed, 85% of the synapses on a spiny cortical neuron occur on spines t2. The dendritic spine may be the first site of active integration of the afferent supply, and may play an important role in the quality and quantity of input to a neuron 4. Our results demonstrate acceleration in the acquisition of dendritic spines in animals with chronic opioid receptor blockade. Interestingly, the ontogeny of gross motor development and the maturation of both simple and complex sensory motor behaviors have been explored in rat pups treated with 50 mg/kg naltrexone 22, and these young rats have an accelerated
1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khatchaturian, H. and Walker, J.M., Endogenous opioids - - biology and function, Annu. Rev. Neurosci., 7 (1984) 223-255. 2 Beaumont, A. and Hughes, J., Biology of opioid peptides, Annu. Rev. Pharmacol. Toxicol., 19 (1979) 245-267. 3 Colonnier, M., The tangential organization of the visual cortex, J. Anat. (London), 98 (1964) 327-344. 4 Connor, J.R. and Diamond, M.C., A comparison of dendritic spine number and type on pyramidal neurons of the visual cortex of old adult rats from social or isolated environments, J. Comp. Neurol., 210 (1982) 99-106. 5 Floeter, M.K. and Greenough, W.T., CerebeUar plasticity: modification of Purkinje celt structure by differential rearing in monkeys, Science, 206 (1979) 227-229.
course of behavioral ontogeny. These findings wou support earlier contentions of a relationship betwe~ structure of the nervous system and functional inte actions 5'7'15,17, and indicate the importance of e dogenous opioid systems in the emergence of bra cytoarchitecture and behavior; Of course, ctarific tion of the relationship of endogenous opioids al synaptogenesis awaits electron microscopic analysi Moreover, we cannot conclude that the total numb, of spines in adults will be different in the stimulate animals. In conclusion, the present report has investigat~ the repercussion of unbalancing the equilibrium b tween endogenous opioids and opioid receptors the developing rat brain. It appears that this relatio: ship governs, either by directly influencing deve oping neurons or indirectly through informatk (e.g., afferent fibers, neurotransmitters) gained fro other neural elements, neuronal differentiation ar the formation of postsynaptic structures (i.e. dendri ic spines). This would suggest that neurons in the d, veloping nervous system appear t o be plastic ar modifiable by endogenous opioid-opioid recept~ interactions. Clinical implications of this finding aJ unclear, however, alterations of endogenous opioJ systems during early life could have profound impo tance in the subsequent appearance and behavior neuronal networks 18. Indeed, disorganization of tt developing nervous system resulting from impai ment of endogenous opioid systems could be the eric logical basis of self-injurious behavior 9 and autisn attributed to opioid dysfunction in children, This work was supported by NIH Grants NS-205£ and NS-20623 to I.S.Z.
6 Globus, A. and Scheibel, A.B.. The effect of visual dep: vation on cortical neurons: a Golgi study, Exp. Neurol., (1967) 331-345. 7 Hebb, D.O., The Organization of Behavior, Wiley, Nc York, 1949. 8 Herman, B,H., Hammock, M.K., Egan J., Feinstein, ( Chatoor, I,, Boeckx, R., Zelnick, N., Jack, R. and Rose quist, J., Naltrexone induces dose-dependent decreases self-injurious behavior, Soc: Neurosci. Abstr., 11 (198 468. 9 Herman, B.H., Hammock, M.K., Arthur-Smith, ,~ Egan, J., Chatoor, I., Zelnick, N., Corradine,iM., App4 gate, K., Boeckx, R.L, and Sharp, S:D.; Role of opie peptides in autism: effects of acute administration of ni
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