306
Brain Research, 503 (1991) 306-3l!~ © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.5(I ADONIS 000689939124902R
BRES 24902
Differential alterations of second messenger systems and cerebral glucose use following excitotoxic lesion of rat cerebral cortex K. Horsburgh and J. McCulloch Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Glasgow (U. K.)
(Accepted 23 July 1991) Key words: Ibotenate-lesion; Visual cortex; [3H]Forskolin binding; [3H]Phorbol 12,13 dibutyrate binding; [14C]-2-Deoxyglucose;
Quantitative autoradiography
Quantitative autoradiography of [3H]forskolin and [3H]phorb01 12,13 dibutyrate (PDBu) binding was examined 21 days after unilateral lesioning of the rat visual cortex using ibotenic acid. In the same animals, the functional deficit was assessed using quantitative [14C]-2deoxyglucose autoradiography. [3H]Forskolin binding was significantly reduced in each layer of the lesioned visual cortex by at least 40% compared to the contralateral hemisphere. Significant reductions in [3H]forskolin binding were observed in the superior colliculus (-15%) and dorsal lateral geniculate body (-12%) ipsilateral to the lesioned cortex. [aH]PDBu binding was significantly reduced in the lesioned visual cortex (layers V-VI) by 34%, compared with the control hemisphere. There were no significant alterations in [3H]DPBu binding in any other brain regions. Following ibotenate-induced lesioning of the visual cortex, glucose use was significantly reduced throughout the lesioned cortex by at least 25% with minor alterations in glucose use in the ipsilateral dorsal lateral geniculate body and superior colliculus. The present study highlights the relative robustness of [3H]PDBu binding compared to [3H]forskolin binding after excitotoxic damage to the cerebral cortex and suggests that [3H]forskolin binding sites are present on cortical efferent fibres. The binding of [3H]forskolin and [3H]phorbol 12,13 dibutyrate (PDBu) has proved to be a powerful approach to localise Gs-adenylate cyclase and protein kinase C, respectively, key components of second messenger systems in the central nervous system (CNS) 26'27. It has been demonstrated, using quantitative autoradiography, that [3H]forskolin and [3H]PDBu binding in cerebral ischaemia and in Alzheimer's disease display differential alterations in certain discrete brain areas 3'8A8'19. Glutamatergic mechanisms have been implicated in the pathophysiological progression of neurodegenerative disorders such as Alzheimer's disease 7 and are believed to play a central role in the aetiology of ischaemic brain damage 14. In order to interpret alterations in second messenger ligand binding sites in human disease, it may be fundamental that second messenger systems be investigated in simple neuronal pathways in animal brain. Biochemical and electrophysiological studies have implicated a major role for glutamate as a neurotransmitter in cortico-cortical and corticofugal pathways of the rat visual system 4'5'12. In the present study, we have sought to examine ligand binding to second messenger systems, using in vitro quantitative autoradiography, following excitotoxic lesion of a discrete region of the rat neocortex, (i.e. the primary visual cortex), to provide mechanistic
insight into second messenger ligand binding associated with well characterised glutamatergic pathways. Functional alterations within the lesioned cortex and its principal efferent projections were assessed in the same animals in vivo by measuring the rate of local cerebral glucose utilisation with [14C]-2-deoxyglucose autoradiography. The investigations were performed in adult, male, black-hooded, P V G rats (250-400 g). Anaesthesia was induced by 5% halothane and maintained by injection of H y p o n o v e l / H y p n o r m (3.3 mg/kg, i.p.). Ibotenic acid (10 mg/ml) was dissolved in phosphate-buffered saline (pH 7.4) and 1 ~l injected at a rate of 0.2/A/min into the right hemisphere at the level of the visual cortex (stereotaxic co-ordinates: -7.8 m m anterior from Bregma; -3.0 m m lateral to the right and -0.1 m m below dura). Sham-operated animals were treated with a procedure identical to that outlined above with the exception that phosphate-buffered saline only was injected into the visual cortex. Following surgery, the animals survived for 21 days, at which time autoradiographic measurements were recorded. The survival period of 21 days was chosen to allow recovery of animals to normal feeding and behavioural activity. Additionally, previous lesion studies have demonstrated neurotransmitter receptor and second mes-
Correspondence: K. Horsburgh. Present address: University of California, San Diego, School of Medicine, Department of Neurosciences, 0624, La Jolla, CA 92093, U.S.A.
307 senger system alterations at this time point following glutamatergic denervation 2'9'17. Local rates of glucose utilisation were measured according to the method described by Sokoloff and colleagues 22. At the end of this procedure, the animal was sacrificed, the brain rapidly removed and frozen in isopentane (-42 °C). From each brain, 20 /~m thick coronal sections were cut using a cryostat microtome. One section in 8 was picked onto a thin glass coverslip and dried immediately on a hotplate (70 °C) prior to preparation for deoxyglucose autoradiograms. Of the remaining 7 sections, 6 of these were thaw-mounted onto gelatin-coated subbed slides and allowed to dry at room temperature for 1 h in preparation for ligand binding. The last section was picked onto a glass slide for histological staining using Cresyl violet. For autoradiographic studies of [3H]forskolin binding, sections were incubated at room temperature for 20 min in 50 mM Tris-acetate (pH 7.7), 10 mM MgCI 2 to which 20 nM [3H]forskolin (New England Nuclear) was added. Non-specific binding was determined in the presence of 20 /~M unlabelled forskolin (Sigma). Sections were washed twice for 1 min in buffer (4 °C) and immediately dried under a cold stream of air. [3H]PDBu binding was performed in adjacent sections to those used for [3H]forskolin binding. Sections were incubated for 90 min at room temperature in 50 mM Tris-HCl (pH 7.7), 1 mM CaC12 containing 2.5 nM [3H]PDBu (New England Nuclear) after which sections were washed twice for 2 min in buffer (4 °C) and dried. Non-specific binding was defined in the presence of 1/~M unlabelled PDBu (Sigma). Non-specific binding also provided confirmation of the elution of [14C]2. Slides were apposed to LKB Ultrofilm for 3 weeks and 1 week along with tritium standards (Amersham [3H]-microscale) to generate forskolin and PDBu autoradiograms, respectively. Analysis of ligand binding autoradiograms and deoxyglucose autoradiograms was performed using a computer-assisted image analysis system (Quantimet 970, Cambridge). Characterisation of visual cortex lesions. Microscopic examination of Cresyl violet-stained brain sections revealed that infusion of ibotenic acid into the right hemisphere of the rat visual cortex resulted in selective loss of neuronal cell bodies (Fig. 1). Within the cortex, the lesion site was well defined in each rat brain being confined to area 17 of the visual cortex and extending to all cortical layers. There was no histological evidence of ibotenate damage in adjacent brain areas such as the superior colliculus. Twenty-one days after lesioning there was an abundance of glial elements surrounding the lesion and to a greater extent in the superficial layers of cortex. In sham-treated rats, there was minimal histological evidence of cortical disruption at the injection site, and there was no loss of neuronal cell bodies with saline
as the injectate as compared to ibotenic acid. [3H]Forskolin binding. In the sham-treated animals, there was no significant alteration in [3H]forskolin binding between the ipsilateral (lesioned) hemisphere compared to the contralateral intact hemisphere (Table I). Following ibotenate lesion of the visual cortex, [3H]forskolin binding was significantly reduced in each cortical layer of the lesioned hemisphere of visual cortex (by at least 40%), (Fig. 1, Table I). Significant reductions in [3H]forskolin binding were observed in the ipsilateral superior colliculus (15%) and the ipsilateral dorsal lateral geniculate body (12%). There were no significant alterations in [3H]forskolin binding in non-visual structures. [3H]Forskolin binding was unchanged in each brain region in the contralateral hemisphere of the lesioned group compared to the sham-treated group (ANOVA). [3H]PDBu binding. [aH]PDBu binding was asymmetrical between both hemispheres of the sham-treated animals in all structures (Table I). After cortical ibotenic acid injections, the only significant alteration in [3H]PDBu binding was observed in layers V - V I of the lesioned visual cortex which was reduced by 34% compared to the intact hemisphere (Fig. 1, Table I). There was no significant difference in [aH]PDBu binding in all other visual structures or non-visual structures in the lesioned hemisphere compared to the intact hemisphere. [aH]PDBu binding was unaltered in the intact hemisphere of the lesioned compared to sham group (ANOVA). Local cerebral glucose utilisation. In sham-treated animals, injection of buffered saline into the visual cortex caused a small (6-10%) reduction in glucose utilisation in the vicinity of the injection. There were no significant alterations in glucose use in any other region in the sham group. Following infusion of ibotenate into the visual cortex, glucose use was markedly reduced in each layer of the lesioned cortex by at least 25% compared to the intact contralateral cortex (Fig. 1, Table I). In the ipsilateral dorsal lateral geniculate body, a small, significant reduction in glucose use was demonstrated post-lesion. Although there was a reduction in glucose use in the ipsilateral superior colliculus, this did not achieve statistical significance. In every other structure examined, there were no significant inter-hemispheric differences in glucose use. There was no alteration in glucose use in the intact hemisphere of the lesioned group compared to the sham group (ANOVA). Ibotenic acid has been widely used as a neurotoxin in morphological and functional analysis of the CNS 21. Intracerebral injection of ibotenic acid into the visual cortex produced a discrete loss of nerve cell bodies and dendrites which was not observed in the sham-treated
308
A
VC
cr
HISTOLOGY vc
B
VC
~#,.,
G L U C O S E USE
~.,..
F O R S K O L I N BINDING
F LJUU
UlllUII'~II~,,,ql
Fig. 1. Representative autoradiograms of (B) glucose use, (C) [3H]forskolin binding and (D) [3H]PDBu binding, in adjacent sections, following unilateral lesion of the visual cortex with ibotenic acid. In the same animal, histological verification of a loss of neuronal cell bodies in the visual cortex is shown (A). Note the reduction in glucose use (B) in the lesioned (left) visual cortex (VC). [3H]Forskolin binding (C) is reduced in the lesioned hemisphere of VC and superior colliculus (SC). [3H]PDBu binding is reduced in VC but unaltered in SC (D).
group treated with saline, as verified histologically. The lesion was reproducible, confined to visual cortex (area 17) where it extended from superficial layers to deep layers. The reductions in [3H]forskolin binding sites in the lesioned hemisphere of visual cortex, dorsal lateral geniculate body and superficial layers of superior colliculus would appear to indicate that a major proportion of [3H]forskolin binding sites are associated with intrinsic cortical neurons and corticofugal fibres. These findings are consistent with a presynaptic localisation of [3H]forskolin binding sites on glutamatergic terminals in the superior colliculus and lateral geniculate body. In these regions, there is evidence that [3H]forskolin binding sites are located also on glutamatergic fibres of retinal efferents 9. [3H]PDBu binding to protein kinase C (PKC) was minimally altered after visual cortex lesion compared to [3H]forskolin binding. PKC is localised in astrocytes as well as neurones t5'16. Since vigorous gliosis accompanies cortical lesion as verified histologically and from previous observations 13 it is possible that the lack of asym-
metry in [3H]PDBu binding in the superficial layers of visual cortex was masked by an increase in astrocyte proliferation which is seen to a lesser extent in deep cortical layers. A loss of [3H]PDBu binding in response to ibotenate-induced lesion of the visual cortex would be consistent with immunocytochemical localisation of PKC on intrinsic neurons 23. Ablation of the visual cortex markedly reduces the levels of glutamate, in the ipsilateral dorsal lateral geniculate body and superior colliculus 12. Glutamate receptors are coupled to PKC, thus alterations in the levels of [3H]PDBu binding in these subcortical visual areas might be expected. However, denervation of the glutamatergic pathway from frontal cortex to the striatum is associated with a potentiation of excitatory amino acid-induced stimulation of PI hydrolysis ~7. This may be indicative of an upregulation of PKC following denervation. Whether this applies for [3H]PDBu binding following denervation of a major glutamatergic pathway in the visual system is unclear. It must be noted that the present studies provide greater insight into the anatomical localisation of
309 TABLE I
Visual structures Visual cortex II/III Visual cortex IV Visual cortex V/VI Superior colliculius (superficial layer) Dorsal lateral Geniculate body Pretectal nuclei Lateral post. nucleus Non-visual structures Medial geniculate Lateral habenula Hippocampus (Molecular layer) Parietal cortex IV Caudate Putamen Thalamus
[3H]Forskolin binding (pmol/g tissue)
[3H]PDBu binding (pmol/g tissue)
Glucose utilisation (l~mol/l OO/min)
Sham
Lesion
Sham
Lesion
Sham
Lesion
Ipsi./Contra.
lpsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
64-+4/73-+7 69-+5/77-+7 61-+4/63-+4
44-+6/74-+4** 429_+24/476-+44 380_+11/405-+36 79+10/84---9t 45-+6/77-+3*** 314-+18/315---23 239-+6/286-+26 87---8/95+9* 34-+5/63-+3"* 311---14/315+24 185+_14/279+19" 75-+7/83-+3t
63-+10/83+-7"* 63-10/95-+8'** 59-+9/82-+8"*
74-+5/75-+6
70-+-4/82-+4"** 289-+16/291-+18
84+8/91---8
49-+3/49-+4 34-+3/34-+3 59---3/61-+3
46-+2/52-+2*** 187_+14/187+14 181-+11/180-+4 93-+6/92-+6 37-+2/38-+1 124-+11/126-+9 136±16/133-+13 83-+5/83-+6 61---3/64-+3 212-+14/208-+12 228+10/230+10 86-+7/87-+8
91-+11/94-+11" 83---8/86-+9 86-+10/88---10
48+4/51---3 32-+2/33---2
46-+2/49---2 41-+3/40+-2
97-+10/101-+10 96±6/97-+7
92-+10/97-+12 95-+9/98-+10
72-+5/70---5 91-+7/92--+8 67---5/68-+4 78-+4/78-+5
72---8/72---8 82-+9/83-+6 66-+4/67-+4 85+5/83-+5
262-+26/260-+25 91---9/93+11
194+13/197-+11 182±16/180-14 104---17/98-+17 124± 13/110-+13
85-+9/75-+7 77-+6/79-+6 270---15/281---22 280±16/294-+19 80-+7/78---7 81-+4/81-+5 284---35/257---36 308±28/314-+28 131-+8/135-+10 140-+8/144-+12 258-+12/263-+19 252-+26/260-+23 63-+3/65-+4 64_+4/64-+3 229-+8/277+9 222-+12/223_+13
Data are presented as mean --- S.E.M. (n = 6). Statistical differences were evaluated using paired t-test tp < 0.05, *P < 0.01, **P < 0.005, ***P < 0.001 significant difference between contralateral and ipsilateral hemisphere. F1.94 (P < 0.5) for all structures in contralateral hemisphere between sham and lesion animals by ANOVA. Roman numerals indicate the cortical layer examined.
second messenger ligand binding sites in cortical and sub-cortical projection areas rather than insight into the functional significance of second messenger systems in visual physiology. Functional m a p p i n g using the [14C]-2-deoxyglucose technique did not provide evidence of any major alterations in activity in visual pathways following cortical lesion, in contrast to previous reports 24'25. The basis for the discrepancies lies in the p r o c e d u r e s for lesioning the visual cortex. In the present study, a discrete lesion of cortex was p r o d u c e d by focal injection of neurotoxin c o m p a r e d to ablation of visual cortex by suction 24 or electrolytic lesion 25. These latter two procedures would ensure a m o r e gross lesion of the visual cortex and destruction of nerve terminals and fibres en passage in addition to cell bodies. Timing of glucose use m e a s u r e m e n t following visual cortex lesions, 21 days in the present study c o m p a r e d to one day or less previously 24'25 m a y
tures 6 and reactive gliosis 13. The minimal alterations in glucose use in cortical projection areas m a y be due to a functional reorganisation within the visual pathways. Following unilateral orbital enucleation, recovery in functional activity in the d e n e r v a t e d superior colliculus involves an increase in cortical control over metabolic activity24. In o t h e r experimentally-lesioned animal models a metabolic recovery with increasing survival time is seen in areas which have shown alterations in glucose use previously at earlier time points 14'2°.
also be crucial. A b l a t i o n of the visual cortex is associated with axonal sprouting in subcortical visual struc-
The present data provide, with the most conservative interpretation, evidence of the anatomical localisation of second messenger ligand binding sites within the cortex and in cortical efferent projections to the thalamus and mid-brain. The relative stability of [3H]PDBu binding c o m p a r e d to [3H]forskolin binding following excitotoxic cortical lesions was readily d e m o n s t r a b l e and the alterations in extracortical binding suggests that [3H]forskolin binding sites are principally located on cortical efferent fibres.
1 Baughman, R.W. and Gilbert, C.D., Aspartate and glutamate as possible neurotransmitters in the visual cortex, J. Neurosci., 1 (1981) 427-439. 2 Chalmers, D.T. and McCulloch, J., Alterations in neurotransmitter receptors and glucose use after unilateral orbital enucleation, Brain Research, 540 (1991) 243-254.
3 Dewar, D., Horsburgh, K., Graham, D.I. and McCulloch, J., Selective alterations of high affinity [3H]forskolin binding sites in Alzheimer's disease: a quantitative autoradiographic study, Brain Research, 511 (1990) 241-248. 4 Fosse, V.M., Iversen, E. and Fonnum, E, A bioluminescence method for the measurement of L-glutamate: applications to the
310
5
6
7 8
9
10
11
12
13
14
15
16
study of changes in the release of L-glutamate from lateral geniculate nucleus and superior colliculus after visual cortex ablation in rats, J. Neurochem., 47 (1986) 340-349. Fosse, V.M. and Fonnum, E, Biochemical evidence for glutamate and/or aspartate as neurotransmitters in fibers from the visual cortex from the lateral posterior thalamic nucleus (pulvinar) in rats, Brain Research, 400 (1987) 219-224. Goodman, D.C. and Horel, J.C., Sprouting of optic tract projections in the brain stem of the rat, J. Comp. Neurol., 127 (1966) 71-88. Greenamyre, J.T., The role of glutamate in neurotransmission and in neurologic disease, Arch. Neurol., 43 (1986) 1058-1063. Horsburgh, K., Dewar, D., Graham, D.I. and McCulloch, J., Autoradiographic imaging of [3H]-phorbol 12,13 dibutyrate binding to protein kinase C in Alzheimer's disease, J. Neurochem., 56 (1991) 1121-1129. Horsburgh, K. and McCulloch, J., Alterations of functional glucose use and ligand binding to second messenger systems following unilateral orbital enucleation, Brain Research, (1991) in press. Kasamatsu, T., Pettigrew, J.D. and Ary, M., Cortical recovery from effects of monocular deprivation: acceleration with norepinephrine and suppression with 6-hydroxydopamine, J. Neurophys., 45 (1981) 254-266. London, E.D., McKinney, M., Dam, M., Ellis, A. and Coyle, J.T., Decreased cortical glucose utilisation after ibotenate lesion of the rat ventromedial globus pallidus, J. Cereb. Blood Flow Metab., 3 (1984) 381-390. Lund-Karlsen, R. and Fonnum, E, Evidence for glutamate as a neurotransmitter in the cortico fugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Research, 151 (1978) 457-467. Mathewson, A.J. and Berry, M., Observations on the astrocyte response to a cerebral stab wound in adult rats, Brain Research, 327 (1985) 61-69. McCulloch, J., Bullock, R. and Teasdale, G.M., Excitatory amino acid antagonists: opportunities for the treatment of ischaemic brain damage in man. In B. Meldrum (Ed.), Excitatory Amino Acids, Blackwell, U.K., 1991, pp. 287-326. Mobley, P.L., Scott, S.L. and Cruz, E.G., Protein kinase C in astrocytes: a determinant of cell morphology, Brain Research, 398 (1986) 366-369. Neary, J.T., Norenberg, L.B. and Norenberg, M.D., Calciumactivated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes, Brain Research, 385
(1986) 420-424. 17 Nicoletti, E, Wroblewski, J.T., Alho, H., Eva, C., Fadda, E. and Costa, E., Lesions of putative glutaminergic pathways potentiate the increase of inositol phospholipid hydrolysis elicited by excitatory amino acids, Brain Research, 436 (1987) 103-112. 18 Onodera, H., Araki, T. and Kogure, K., Protein kinase C activity in the rat hippocampus after forebrain ischaemia: autoradiographic analysis by [3H]-phorbol 12,13 dibutyrate, Brain Research, 481 (1989) l-7. 19 Onodera, H. and Kogure, K., Mapping second messenger systems in the rat hippocampus after transient forebrain ischaemia: in vitro [3H]forskolin and [3H]inositol 1,4,5 trisphosphate binding, Brain Research, 487 (1989) 343-347. 20 Orzi, F., Diana, G., Casamenti, E, Palombo, E. and Fieschi, C., Local cerebral glucose utilisation following unilateral and bilateral lesions of the nucleus basalis magnocellularis in the rat, Brain Research, 462 (1988) 99-103. 21 Schwartz, R., H6kfelt, T., Fuxe, K., Jonsson, G., Goldstein, M. and Terenius, L., Ibotenic acid-induced neuronal degeneration: a morphological and neurochemical study, Exp. Brain Res., 37 (1979) 199-216. 22 Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization theory, procedure and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. 23 Stichel, C.C. and Singer, W., Localization of isoenzymes II/III of protein kinase C in the rat visual cortex (area 17), hippocampus and dentate gyrus, Exp. Brain Res., 72 (1988) 443-449. 24 Thurlow, G.A. and Cooper, R.M., Increased dependence of superior colliculus metabolic activity on visual cortex after eye enucleation, Exp. Neurol., 90 (1985) 594-600. 25 Toga, A.W. and Collins, R.C., Metabolic response of optic centers to visual stimuli in the albino rat: anatomical and physiological considerations, J. Comp. Neurol., 199 (1981) 443-464. 26 Worley, P.E, Baraban, J.M., De Souza, E.B. and Snyder, S.H., Mapping second messenger systems in the brain: differential localization of adenylate cyclase and protein kinase C, Proc. Natl. Acad. Sci. U.S.A., 3 (1986) 4053-4057. 27 Worley, P.E, Baraban, J.M. and Snyder, S.H., Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding, J. Neurosci., 6 (1986) 199-207.
Brain Research, 503 (1991) 306-3l!~ © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.5(I ADONIS 000689939124902R
BRES 24902
Differential alterations of second messenger systems and cerebral glucose use following excitotoxic lesion of rat cerebral cortex K. Horsburgh and J. McCulloch Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Glasgow (U. K.)
(Accepted 23 July 1991) Key words: Ibotenate-lesion; Visual cortex; [3H]Forskolin binding; [3H]Phorbol 12,13 dibutyrate binding; [14C]-2-Deoxyglucose;
Quantitative autoradiography
Quantitative autoradiography of [3H]forskolin and [3H]phorb01 12,13 dibutyrate (PDBu) binding was examined 21 days after unilateral lesioning of the rat visual cortex using ibotenic acid. In the same animals, the functional deficit was assessed using quantitative [14C]-2deoxyglucose autoradiography. [3H]Forskolin binding was significantly reduced in each layer of the lesioned visual cortex by at least 40% compared to the contralateral hemisphere. Significant reductions in [3H]forskolin binding were observed in the superior colliculus (-15%) and dorsal lateral geniculate body (-12%) ipsilateral to the lesioned cortex. [aH]PDBu binding was significantly reduced in the lesioned visual cortex (layers V-VI) by 34%, compared with the control hemisphere. There were no significant alterations in [3H]DPBu binding in any other brain regions. Following ibotenate-induced lesioning of the visual cortex, glucose use was significantly reduced throughout the lesioned cortex by at least 25% with minor alterations in glucose use in the ipsilateral dorsal lateral geniculate body and superior colliculus. The present study highlights the relative robustness of [3H]PDBu binding compared to [3H]forskolin binding after excitotoxic damage to the cerebral cortex and suggests that [3H]forskolin binding sites are present on cortical efferent fibres. The binding of [3H]forskolin and [3H]phorbol 12,13 dibutyrate (PDBu) has proved to be a powerful approach to localise Gs-adenylate cyclase and protein kinase C, respectively, key components of second messenger systems in the central nervous system (CNS) 26'27. It has been demonstrated, using quantitative autoradiography, that [3H]forskolin and [3H]PDBu binding in cerebral ischaemia and in Alzheimer's disease display differential alterations in certain discrete brain areas 3'8A8'19. Glutamatergic mechanisms have been implicated in the pathophysiological progression of neurodegenerative disorders such as Alzheimer's disease 7 and are believed to play a central role in the aetiology of ischaemic brain damage 14. In order to interpret alterations in second messenger ligand binding sites in human disease, it may be fundamental that second messenger systems be investigated in simple neuronal pathways in animal brain. Biochemical and electrophysiological studies have implicated a major role for glutamate as a neurotransmitter in cortico-cortical and corticofugal pathways of the rat visual system 4'5'12. In the present study, we have sought to examine ligand binding to second messenger systems, using in vitro quantitative autoradiography, following excitotoxic lesion of a discrete region of the rat neocortex, (i.e. the primary visual cortex), to provide mechanistic
insight into second messenger ligand binding associated with well characterised glutamatergic pathways. Functional alterations within the lesioned cortex and its principal efferent projections were assessed in the same animals in vivo by measuring the rate of local cerebral glucose utilisation with [14C]-2-deoxyglucose autoradiography. The investigations were performed in adult, male, black-hooded, P V G rats (250-400 g). Anaesthesia was induced by 5% halothane and maintained by injection of H y p o n o v e l / H y p n o r m (3.3 mg/kg, i.p.). Ibotenic acid (10 mg/ml) was dissolved in phosphate-buffered saline (pH 7.4) and 1 ~l injected at a rate of 0.2/A/min into the right hemisphere at the level of the visual cortex (stereotaxic co-ordinates: -7.8 m m anterior from Bregma; -3.0 m m lateral to the right and -0.1 m m below dura). Sham-operated animals were treated with a procedure identical to that outlined above with the exception that phosphate-buffered saline only was injected into the visual cortex. Following surgery, the animals survived for 21 days, at which time autoradiographic measurements were recorded. The survival period of 21 days was chosen to allow recovery of animals to normal feeding and behavioural activity. Additionally, previous lesion studies have demonstrated neurotransmitter receptor and second mes-
Correspondence: K. Horsburgh. Present address: University of California, San Diego, School of Medicine, Department of Neurosciences, 0624, La Jolla, CA 92093, U.S.A.
307 senger system alterations at this time point following glutamatergic denervation 2'9'17. Local rates of glucose utilisation were measured according to the method described by Sokoloff and colleagues 22. At the end of this procedure, the animal was sacrificed, the brain rapidly removed and frozen in isopentane (-42 °C). From each brain, 20 /~m thick coronal sections were cut using a cryostat microtome. One section in 8 was picked onto a thin glass coverslip and dried immediately on a hotplate (70 °C) prior to preparation for deoxyglucose autoradiograms. Of the remaining 7 sections, 6 of these were thaw-mounted onto gelatin-coated subbed slides and allowed to dry at room temperature for 1 h in preparation for ligand binding. The last section was picked onto a glass slide for histological staining using Cresyl violet. For autoradiographic studies of [3H]forskolin binding, sections were incubated at room temperature for 20 min in 50 mM Tris-acetate (pH 7.7), 10 mM MgCI 2 to which 20 nM [3H]forskolin (New England Nuclear) was added. Non-specific binding was determined in the presence of 20 /~M unlabelled forskolin (Sigma). Sections were washed twice for 1 min in buffer (4 °C) and immediately dried under a cold stream of air. [3H]PDBu binding was performed in adjacent sections to those used for [3H]forskolin binding. Sections were incubated for 90 min at room temperature in 50 mM Tris-HCl (pH 7.7), 1 mM CaC12 containing 2.5 nM [3H]PDBu (New England Nuclear) after which sections were washed twice for 2 min in buffer (4 °C) and dried. Non-specific binding was defined in the presence of 1/~M unlabelled PDBu (Sigma). Non-specific binding also provided confirmation of the elution of [14C]2. Slides were apposed to LKB Ultrofilm for 3 weeks and 1 week along with tritium standards (Amersham [3H]-microscale) to generate forskolin and PDBu autoradiograms, respectively. Analysis of ligand binding autoradiograms and deoxyglucose autoradiograms was performed using a computer-assisted image analysis system (Quantimet 970, Cambridge). Characterisation of visual cortex lesions. Microscopic examination of Cresyl violet-stained brain sections revealed that infusion of ibotenic acid into the right hemisphere of the rat visual cortex resulted in selective loss of neuronal cell bodies (Fig. 1). Within the cortex, the lesion site was well defined in each rat brain being confined to area 17 of the visual cortex and extending to all cortical layers. There was no histological evidence of ibotenate damage in adjacent brain areas such as the superior colliculus. Twenty-one days after lesioning there was an abundance of glial elements surrounding the lesion and to a greater extent in the superficial layers of cortex. In sham-treated rats, there was minimal histological evidence of cortical disruption at the injection site, and there was no loss of neuronal cell bodies with saline
as the injectate as compared to ibotenic acid. [3H]Forskolin binding. In the sham-treated animals, there was no significant alteration in [3H]forskolin binding between the ipsilateral (lesioned) hemisphere compared to the contralateral intact hemisphere (Table I). Following ibotenate lesion of the visual cortex, [3H]forskolin binding was significantly reduced in each cortical layer of the lesioned hemisphere of visual cortex (by at least 40%), (Fig. 1, Table I). Significant reductions in [3H]forskolin binding were observed in the ipsilateral superior colliculus (15%) and the ipsilateral dorsal lateral geniculate body (12%). There were no significant alterations in [3H]forskolin binding in non-visual structures. [3H]Forskolin binding was unchanged in each brain region in the contralateral hemisphere of the lesioned group compared to the sham-treated group (ANOVA). [3H]PDBu binding. [aH]PDBu binding was asymmetrical between both hemispheres of the sham-treated animals in all structures (Table I). After cortical ibotenic acid injections, the only significant alteration in [3H]PDBu binding was observed in layers V - V I of the lesioned visual cortex which was reduced by 34% compared to the intact hemisphere (Fig. 1, Table I). There was no significant difference in [aH]PDBu binding in all other visual structures or non-visual structures in the lesioned hemisphere compared to the intact hemisphere. [aH]PDBu binding was unaltered in the intact hemisphere of the lesioned compared to sham group (ANOVA). Local cerebral glucose utilisation. In sham-treated animals, injection of buffered saline into the visual cortex caused a small (6-10%) reduction in glucose utilisation in the vicinity of the injection. There were no significant alterations in glucose use in any other region in the sham group. Following infusion of ibotenate into the visual cortex, glucose use was markedly reduced in each layer of the lesioned cortex by at least 25% compared to the intact contralateral cortex (Fig. 1, Table I). In the ipsilateral dorsal lateral geniculate body, a small, significant reduction in glucose use was demonstrated post-lesion. Although there was a reduction in glucose use in the ipsilateral superior colliculus, this did not achieve statistical significance. In every other structure examined, there were no significant inter-hemispheric differences in glucose use. There was no alteration in glucose use in the intact hemisphere of the lesioned group compared to the sham group (ANOVA). Ibotenic acid has been widely used as a neurotoxin in morphological and functional analysis of the CNS 21. Intracerebral injection of ibotenic acid into the visual cortex produced a discrete loss of nerve cell bodies and dendrites which was not observed in the sham-treated
308
A
VC
cr
HISTOLOGY vc
B
VC
~#,.,
G L U C O S E USE
~.,..
F O R S K O L I N BINDING
F LJUU
UlllUII'~II~,,,ql
Fig. 1. Representative autoradiograms of (B) glucose use, (C) [3H]forskolin binding and (D) [3H]PDBu binding, in adjacent sections, following unilateral lesion of the visual cortex with ibotenic acid. In the same animal, histological verification of a loss of neuronal cell bodies in the visual cortex is shown (A). Note the reduction in glucose use (B) in the lesioned (left) visual cortex (VC). [3H]Forskolin binding (C) is reduced in the lesioned hemisphere of VC and superior colliculus (SC). [3H]PDBu binding is reduced in VC but unaltered in SC (D).
group treated with saline, as verified histologically. The lesion was reproducible, confined to visual cortex (area 17) where it extended from superficial layers to deep layers. The reductions in [3H]forskolin binding sites in the lesioned hemisphere of visual cortex, dorsal lateral geniculate body and superficial layers of superior colliculus would appear to indicate that a major proportion of [3H]forskolin binding sites are associated with intrinsic cortical neurons and corticofugal fibres. These findings are consistent with a presynaptic localisation of [3H]forskolin binding sites on glutamatergic terminals in the superior colliculus and lateral geniculate body. In these regions, there is evidence that [3H]forskolin binding sites are located also on glutamatergic fibres of retinal efferents 9. [3H]PDBu binding to protein kinase C (PKC) was minimally altered after visual cortex lesion compared to [3H]forskolin binding. PKC is localised in astrocytes as well as neurones t5'16. Since vigorous gliosis accompanies cortical lesion as verified histologically and from previous observations 13 it is possible that the lack of asym-
metry in [3H]PDBu binding in the superficial layers of visual cortex was masked by an increase in astrocyte proliferation which is seen to a lesser extent in deep cortical layers. A loss of [3H]PDBu binding in response to ibotenate-induced lesion of the visual cortex would be consistent with immunocytochemical localisation of PKC on intrinsic neurons 23. Ablation of the visual cortex markedly reduces the levels of glutamate, in the ipsilateral dorsal lateral geniculate body and superior colliculus 12. Glutamate receptors are coupled to PKC, thus alterations in the levels of [3H]PDBu binding in these subcortical visual areas might be expected. However, denervation of the glutamatergic pathway from frontal cortex to the striatum is associated with a potentiation of excitatory amino acid-induced stimulation of PI hydrolysis ~7. This may be indicative of an upregulation of PKC following denervation. Whether this applies for [3H]PDBu binding following denervation of a major glutamatergic pathway in the visual system is unclear. It must be noted that the present studies provide greater insight into the anatomical localisation of
309 TABLE I
Visual structures Visual cortex II/III Visual cortex IV Visual cortex V/VI Superior colliculius (superficial layer) Dorsal lateral Geniculate body Pretectal nuclei Lateral post. nucleus Non-visual structures Medial geniculate Lateral habenula Hippocampus (Molecular layer) Parietal cortex IV Caudate Putamen Thalamus
[3H]Forskolin binding (pmol/g tissue)
[3H]PDBu binding (pmol/g tissue)
Glucose utilisation (l~mol/l OO/min)
Sham
Lesion
Sham
Lesion
Sham
Lesion
Ipsi./Contra.
lpsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
Ipsi./Contra.
64-+4/73-+7 69-+5/77-+7 61-+4/63-+4
44-+6/74-+4** 429_+24/476-+44 380_+11/405-+36 79+10/84---9t 45-+6/77-+3*** 314-+18/315---23 239-+6/286-+26 87---8/95+9* 34-+5/63-+3"* 311---14/315+24 185+_14/279+19" 75-+7/83-+3t
63-+10/83+-7"* 63-10/95-+8'** 59-+9/82-+8"*
74-+5/75-+6
70-+-4/82-+4"** 289-+16/291-+18
84+8/91---8
49-+3/49-+4 34-+3/34-+3 59---3/61-+3
46-+2/52-+2*** 187_+14/187+14 181-+11/180-+4 93-+6/92-+6 37-+2/38-+1 124-+11/126-+9 136±16/133-+13 83-+5/83-+6 61---3/64-+3 212-+14/208-+12 228+10/230+10 86-+7/87-+8
91-+11/94-+11" 83---8/86-+9 86-+10/88---10
48+4/51---3 32-+2/33---2
46-+2/49---2 41-+3/40+-2
97-+10/101-+10 96±6/97-+7
92-+10/97-+12 95-+9/98-+10
72-+5/70---5 91-+7/92--+8 67---5/68-+4 78-+4/78-+5
72---8/72---8 82-+9/83-+6 66-+4/67-+4 85+5/83-+5
262-+26/260-+25 91---9/93+11
194+13/197-+11 182±16/180-14 104---17/98-+17 124± 13/110-+13
85-+9/75-+7 77-+6/79-+6 270---15/281---22 280±16/294-+19 80-+7/78---7 81-+4/81-+5 284---35/257---36 308±28/314-+28 131-+8/135-+10 140-+8/144-+12 258-+12/263-+19 252-+26/260-+23 63-+3/65-+4 64_+4/64-+3 229-+8/277+9 222-+12/223_+13
Data are presented as mean --- S.E.M. (n = 6). Statistical differences were evaluated using paired t-test tp < 0.05, *P < 0.01, **P < 0.005, ***P < 0.001 significant difference between contralateral and ipsilateral hemisphere. F1.94 (P < 0.5) for all structures in contralateral hemisphere between sham and lesion animals by ANOVA. Roman numerals indicate the cortical layer examined.
second messenger ligand binding sites in cortical and sub-cortical projection areas rather than insight into the functional significance of second messenger systems in visual physiology. Functional m a p p i n g using the [14C]-2-deoxyglucose technique did not provide evidence of any major alterations in activity in visual pathways following cortical lesion, in contrast to previous reports 24'25. The basis for the discrepancies lies in the p r o c e d u r e s for lesioning the visual cortex. In the present study, a discrete lesion of cortex was p r o d u c e d by focal injection of neurotoxin c o m p a r e d to ablation of visual cortex by suction 24 or electrolytic lesion 25. These latter two procedures would ensure a m o r e gross lesion of the visual cortex and destruction of nerve terminals and fibres en passage in addition to cell bodies. Timing of glucose use m e a s u r e m e n t following visual cortex lesions, 21 days in the present study c o m p a r e d to one day or less previously 24'25 m a y
tures 6 and reactive gliosis 13. The minimal alterations in glucose use in cortical projection areas m a y be due to a functional reorganisation within the visual pathways. Following unilateral orbital enucleation, recovery in functional activity in the d e n e r v a t e d superior colliculus involves an increase in cortical control over metabolic activity24. In o t h e r experimentally-lesioned animal models a metabolic recovery with increasing survival time is seen in areas which have shown alterations in glucose use previously at earlier time points 14'2°.
also be crucial. A b l a t i o n of the visual cortex is associated with axonal sprouting in subcortical visual struc-
The present data provide, with the most conservative interpretation, evidence of the anatomical localisation of second messenger ligand binding sites within the cortex and in cortical efferent projections to the thalamus and mid-brain. The relative stability of [3H]PDBu binding c o m p a r e d to [3H]forskolin binding following excitotoxic cortical lesions was readily d e m o n s t r a b l e and the alterations in extracortical binding suggests that [3H]forskolin binding sites are principally located on cortical efferent fibres.
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