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Histochemical effects of kainic acid on neostriatal dopamine and acetylcholinesterase
European Journal of Pharmacology, 50 (1978) 287--289 © Elsevier/North-Holland Biomedical Press
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Rapid c o m m u n i c a t i o n
HISTOCHEMICAL EFFECTS OF KAINIC ACID ON NEOSTRIATAL DOPAMINE AND ACETYLCHOLINESTERASE LARRY L. BUTCHERand RICHARD C. ROGERS Department of Psychology and Brain Research Institute, University of California, 405 Hilgard Avenue, Los Angeles, California 90024, U.S.A. Received 20 June 1978, accepted 22 June 1978
Stereotaxic injections of kainic acid into the caudate-putamen complex have been reported to produce long-term reductions in biochemically assessed cholinergic and GABAergic indices while leaving dopamine, dopamine uptake, and tyrosine hydroxylase unchanged or slightly increased (Schwarcz and Coyle, 1977). Since neuronal somata containing GABA are apparently found in the candate-putamen nucleus (see Schwarcz and Coyle, 1977) and since acetylcholine and other substances related to cholinergic function are associated with neostriatal interneutons (Butcher and Butcher, 1978), whereas dopamine is contained within axons and axon terminals deriving from the mesencephalon, one explanation for the observed biochemical findings is that kalnic acid preferentially affects cell bodies (Schwarcz and Coyle, 1977). In this report we present histologic and histochemical data that urge closer scrutiny of this conjecture. Female Sprague-Dawley rats weighing 200--300 g were used. They were anesthetized with sodium methohexital, 50 mg/kg i.p., and placed in a stereotaxic instrument. Kainic acid (Sigma Chemical Co.; St. Louis, MO; U.S.A.), dissolved in 0.9% saline, was infused unilaterally into the caudate-putamen nucleus according to the procedures outlined in Schwarcz and Coyle (1977). Total volume of infusion was 1 gl delivered over a 4 min period. The amount of kalnic acid applied was 2, 3, 6 or I0 ~g. At various times after kainic acid adminis-
tration or after infusion of 1 #I saline alone, the rats were sacrificed.Some animals' brains were processed for catecholamines according to the glyoxylic acid procedure of the la Torre and Surgeon (1976). Other rats were injected with 0.Smg/kg bis(1-methylethyl)phosphorofluoridate (DFP) at different times prior to death; their brains were subsequently processed for acetylcholinesterase(ACHE, E C 3.1.1.7) according to the pharmaco.histochemical regimen described in Butcher et al. (1975). This protocol demonstrates local~ircult, AChE~ontaining neurons in the neostriatum especially well. The brains of a few animals injected with D F P were processed for both catecholamines (De la Torte and Surgeon, 1976) and, on alternate histologic sections, A C h E (Butcher et al.,1975). Some sections were also stained with thionin. Similar to tissuedamage produced by intracerebral infusion of other cytotoxins (see Butcher, 1975), different zones of pathology, occurring successively in relation to the cannula, could be discerned after intrastriatal administration of kainic acid (fig.1B): (1)a zone (Z) of complete destruction of neuronal, glial, and vascular elements due to tissue displacement by the injection cannula (Z1 in fig. 1B), (2) a region of extensive, virtually total loss of neuronal elements regardless of their chemical characteristics(Z2 in fig.1B; compare with fig.1A), and (3) an area in which different subcellular constituents of neurons were differentially affected (Z3 in fig.1B; compare with fig.1D). This region, Z3,
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Fig. 1. AChE (B) and dopamine (D) in the caudate-putamen (CP) nucleus 15 days (B) or 48 h (D) after unilateral intrastriatal infusion of 3 pg/1 pl kainic acid. Non-infused sides of the brain are shown for AChE and dopamine in A and C, respectively. In A and B rat was administered 0.8 mg/kg DFP 12 h prior to euthanasia. Erythrocytes delimit zone-1 pathology (Z1) in B. Z2 = zone-2 pathology, Z3 = zone-3 pathology, fb = fiber bundle perforating CP. Black-on-white arrows in Z3 (B) point to AChE somata displaying decreased staining intensities; black arrows (B) show additional AChE-staining neuronal elements. D depicts staining in Z3. Scale in A is 300 pm and applies also to B. Scale in C is 50 pm and applies also to D.
e x t e n d e d to u n a f f e c t e d tissue a n d d i s p l a y e d d i m i n i s h i n g degrees o f p a t h o l o g y w i t h increasing d i s t a n c e f r o m t h e c a n n u l a t r a c t (fig. 1B; c o m p a r e w i t h fig. 1A). With t h e passage o f t i m e Z2 and t h e m a r g i n s o f Z1 e x h i b i t e d e x t e n s i v e gliosis. I n t r a s t r i a t a l i n j e c t i o n o f saline r e s u l t e d o n l y in t h e a p p e a r a n c e o f Z1. T h e size o f Z2 i n c r e a s e d w i t h increasing a m o u n t s o f kalnic acid infused, w i t h 6 a n d 10 pg p r o d u c i n g p r o m i n e n t c a v i t a t i o n . A f t e r i n f u s i o n o f 2 or 3 gg kainic acid, t h e largest z o n e o f p a t h o l o g y was Z3, w h i c h e x t e n d e d
a p p r o x i m a t e l y 1.2 m m f r o m t h e c a n n u l a t r a c t (fig. 1B); Z2 in t h e s e rats e n c o m p a s s e d a region r o u g h l y 0.17 m m f r o m t h e o u t e r b o r d e r o f Z l (fig. 1B). F r o m 48 h t o 15 d a y s a f t e r 2 or 3 ~g kainic acid, A C h E - c o n t a i n i n g n e u r o n a l s o m a t a and t h e i r p r o x i m a l p r o c e s s e s in Z3 n o l o n g e r stained f o r t h e e n z y m e (fig. 1B), e x c e p t f o r a f e w n e u r o n s at the o u t e r regions o f Z3 t h a t displayed diminished activity (black-on-white a r r o w s ; fig. 1B). O c c a s i o n a l l y , A C h E - s t a i n i n g e l e m e n t s w e r e o b s e r v e d closer t o t h e c a n n u l a
HISTOCHEMICAL EFFECTS OF KAINIC ACID
tract (black arrows; fig. 1B), and some background AChE activity remained in Z3 (compare with Z2 in fig. 1B and with fig. 1A). At the same doses of kainic acid, 2 or 3/~g, a n d at short intervals (~< 48 h) after intrastriatal infusion of the cytotoxin, tissue elements in Z3 most probably containing dopamine, although reduced in number, appeared more intensely fluorescent (fig. 1D; compare with non-injected caudate-putamen complex in fig. 1C). 13 days later, histochemically assessed levels in these fluorescent fibers and/or axon terminals declined to subnormal levels. Our histochemical observations that a cholinergic substance (ACHE) associated with neostriatal interneurons decreases in Z3 after exposure to kainic acid while dopamine levels in a subtotal population of neuronal elements in that same region first increase and then decrease over a 15
289
than initially suspected. Like other intracerebrally administered "magic bullets" (see Butcher, 1975), kainic acid must be used with caution and with full awareness of its various histochemical, neuropathologic and biochemical effects.
Acknowledgements This work was supported by U S P H S grant NS10928 to L.L.B. We thank Ken Hirabayashi for his excellent technical assistance.
References Butcher, L.L., 1975, Degenerative processes after punctate intracerebral administration of 6-hydroxydopamine, J. Neural Transm. 37, 189. Butcher, L.L., K. Talbot and L. Bilezikjian, 1975, Acetylcholinesterase in dopamine-containing regions of the brain, J. Neural Transm. 37, 127. Butcher, S.G. and L.L. Butcher, 1978, Acetylcholine levels and synthesis in the neostriatum after lesions in the cerebral cortex, thalamus, and globus pallidus, in: Cholinergic-monoaminergic Interactions in the Brain, ed. L.L. Butcher (Academic Press, N e w York, San Francisco, London) p. 109. De la Torre, J.C. and J.W. Surgeon, 1976, A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The S P G method, Histochemistry 49, 81. Schwarcz, R. and J.T. Coyle, 1977, Striatal lesions with kainic acid: Neurochemical characteristics, Brain Res. 127, 235.
287
Rapid c o m m u n i c a t i o n
HISTOCHEMICAL EFFECTS OF KAINIC ACID ON NEOSTRIATAL DOPAMINE AND ACETYLCHOLINESTERASE LARRY L. BUTCHERand RICHARD C. ROGERS Department of Psychology and Brain Research Institute, University of California, 405 Hilgard Avenue, Los Angeles, California 90024, U.S.A. Received 20 June 1978, accepted 22 June 1978
Stereotaxic injections of kainic acid into the caudate-putamen complex have been reported to produce long-term reductions in biochemically assessed cholinergic and GABAergic indices while leaving dopamine, dopamine uptake, and tyrosine hydroxylase unchanged or slightly increased (Schwarcz and Coyle, 1977). Since neuronal somata containing GABA are apparently found in the candate-putamen nucleus (see Schwarcz and Coyle, 1977) and since acetylcholine and other substances related to cholinergic function are associated with neostriatal interneutons (Butcher and Butcher, 1978), whereas dopamine is contained within axons and axon terminals deriving from the mesencephalon, one explanation for the observed biochemical findings is that kalnic acid preferentially affects cell bodies (Schwarcz and Coyle, 1977). In this report we present histologic and histochemical data that urge closer scrutiny of this conjecture. Female Sprague-Dawley rats weighing 200--300 g were used. They were anesthetized with sodium methohexital, 50 mg/kg i.p., and placed in a stereotaxic instrument. Kainic acid (Sigma Chemical Co.; St. Louis, MO; U.S.A.), dissolved in 0.9% saline, was infused unilaterally into the caudate-putamen nucleus according to the procedures outlined in Schwarcz and Coyle (1977). Total volume of infusion was 1 gl delivered over a 4 min period. The amount of kalnic acid applied was 2, 3, 6 or I0 ~g. At various times after kainic acid adminis-
tration or after infusion of 1 #I saline alone, the rats were sacrificed.Some animals' brains were processed for catecholamines according to the glyoxylic acid procedure of the la Torre and Surgeon (1976). Other rats were injected with 0.Smg/kg bis(1-methylethyl)phosphorofluoridate (DFP) at different times prior to death; their brains were subsequently processed for acetylcholinesterase(ACHE, E C 3.1.1.7) according to the pharmaco.histochemical regimen described in Butcher et al. (1975). This protocol demonstrates local~ircult, AChE~ontaining neurons in the neostriatum especially well. The brains of a few animals injected with D F P were processed for both catecholamines (De la Torte and Surgeon, 1976) and, on alternate histologic sections, A C h E (Butcher et al.,1975). Some sections were also stained with thionin. Similar to tissuedamage produced by intracerebral infusion of other cytotoxins (see Butcher, 1975), different zones of pathology, occurring successively in relation to the cannula, could be discerned after intrastriatal administration of kainic acid (fig.1B): (1)a zone (Z) of complete destruction of neuronal, glial, and vascular elements due to tissue displacement by the injection cannula (Z1 in fig. 1B), (2) a region of extensive, virtually total loss of neuronal elements regardless of their chemical characteristics(Z2 in fig.1B; compare with fig.1A), and (3) an area in which different subcellular constituents of neurons were differentially affected (Z3 in fig.1B; compare with fig.1D). This region, Z3,
288
L.L. BUTCHER, R.C. ROGERS
Fig. 1. AChE (B) and dopamine (D) in the caudate-putamen (CP) nucleus 15 days (B) or 48 h (D) after unilateral intrastriatal infusion of 3 pg/1 pl kainic acid. Non-infused sides of the brain are shown for AChE and dopamine in A and C, respectively. In A and B rat was administered 0.8 mg/kg DFP 12 h prior to euthanasia. Erythrocytes delimit zone-1 pathology (Z1) in B. Z2 = zone-2 pathology, Z3 = zone-3 pathology, fb = fiber bundle perforating CP. Black-on-white arrows in Z3 (B) point to AChE somata displaying decreased staining intensities; black arrows (B) show additional AChE-staining neuronal elements. D depicts staining in Z3. Scale in A is 300 pm and applies also to B. Scale in C is 50 pm and applies also to D.
e x t e n d e d to u n a f f e c t e d tissue a n d d i s p l a y e d d i m i n i s h i n g degrees o f p a t h o l o g y w i t h increasing d i s t a n c e f r o m t h e c a n n u l a t r a c t (fig. 1B; c o m p a r e w i t h fig. 1A). With t h e passage o f t i m e Z2 and t h e m a r g i n s o f Z1 e x h i b i t e d e x t e n s i v e gliosis. I n t r a s t r i a t a l i n j e c t i o n o f saline r e s u l t e d o n l y in t h e a p p e a r a n c e o f Z1. T h e size o f Z2 i n c r e a s e d w i t h increasing a m o u n t s o f kalnic acid infused, w i t h 6 a n d 10 pg p r o d u c i n g p r o m i n e n t c a v i t a t i o n . A f t e r i n f u s i o n o f 2 or 3 gg kainic acid, t h e largest z o n e o f p a t h o l o g y was Z3, w h i c h e x t e n d e d
a p p r o x i m a t e l y 1.2 m m f r o m t h e c a n n u l a t r a c t (fig. 1B); Z2 in t h e s e rats e n c o m p a s s e d a region r o u g h l y 0.17 m m f r o m t h e o u t e r b o r d e r o f Z l (fig. 1B). F r o m 48 h t o 15 d a y s a f t e r 2 or 3 ~g kainic acid, A C h E - c o n t a i n i n g n e u r o n a l s o m a t a and t h e i r p r o x i m a l p r o c e s s e s in Z3 n o l o n g e r stained f o r t h e e n z y m e (fig. 1B), e x c e p t f o r a f e w n e u r o n s at the o u t e r regions o f Z3 t h a t displayed diminished activity (black-on-white a r r o w s ; fig. 1B). O c c a s i o n a l l y , A C h E - s t a i n i n g e l e m e n t s w e r e o b s e r v e d closer t o t h e c a n n u l a
HISTOCHEMICAL EFFECTS OF KAINIC ACID
tract (black arrows; fig. 1B), and some background AChE activity remained in Z3 (compare with Z2 in fig. 1B and with fig. 1A). At the same doses of kainic acid, 2 or 3/~g, a n d at short intervals (~< 48 h) after intrastriatal infusion of the cytotoxin, tissue elements in Z3 most probably containing dopamine, although reduced in number, appeared more intensely fluorescent (fig. 1D; compare with non-injected caudate-putamen complex in fig. 1C). 13 days later, histochemically assessed levels in these fluorescent fibers and/or axon terminals declined to subnormal levels. Our histochemical observations that a cholinergic substance (ACHE) associated with neostriatal interneurons decreases in Z3 after exposure to kainic acid while dopamine levels in a subtotal population of neuronal elements in that same region first increase and then decrease over a 15
289
than initially suspected. Like other intracerebrally administered "magic bullets" (see Butcher, 1975), kainic acid must be used with caution and with full awareness of its various histochemical, neuropathologic and biochemical effects.
Acknowledgements This work was supported by U S P H S grant NS10928 to L.L.B. We thank Ken Hirabayashi for his excellent technical assistance.
References Butcher, L.L., 1975, Degenerative processes after punctate intracerebral administration of 6-hydroxydopamine, J. Neural Transm. 37, 189. Butcher, L.L., K. Talbot and L. Bilezikjian, 1975, Acetylcholinesterase in dopamine-containing regions of the brain, J. Neural Transm. 37, 127. Butcher, S.G. and L.L. Butcher, 1978, Acetylcholine levels and synthesis in the neostriatum after lesions in the cerebral cortex, thalamus, and globus pallidus, in: Cholinergic-monoaminergic Interactions in the Brain, ed. L.L. Butcher (Academic Press, N e w York, San Francisco, London) p. 109. De la Torre, J.C. and J.W. Surgeon, 1976, A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: The S P G method, Histochemistry 49, 81. Schwarcz, R. and J.T. Coyle, 1977, Striatal lesions with kainic acid: Neurochemical characteristics, Brain Res. 127, 235.