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On the specificity of 6-hydroxydopamine-induced degeneration of central noradrenaline neurons after intracerebral injection
reroscience .bQtt~rs, l(l975) 6 ~~~er~No~~-~oll~d,
35-39 Amsterdam - Printed in The Netherlamb
P. LIDBRINK and G. JONSSON Departmentof Histology, Karotinska Institute& S-104 01 Stockholm
techniques. The 6 ow waves and synchro
60 (Sweden)
36
frequently used method of 6-OHDA administration for functional studies on central catecholamine neurons is the local intracerebral injection technique. However, the results reported on the use of S i s administration route are rather inconsistent and the views as to the specificity of 6~)HDA are very confludng and contradictory [1,2,4,11,13]. The present study was undertaken to evaluate the specificity of a 6 ~ H D A induced lesion of a noradrenergic neuron system after local intracerebral injection. The aim was to obtain animals in which the noradrenaline (NA) neurons would remain intact after the 6~)HDA injection. This has previously been shown possible in animals given intraventricular 6~)HDA after p r ~ t.-iptyline pretreatment [3]. The drug in all probability antagonizes the effects of 6~)HDA by competitive inhibition of the neuronal uptake of 6 ~ H D A [ 5]. On the other hand, it seems reasonable to assume that effects not related to uptake into NA neurons ('non-specific' effects) after intracerebral injections of 6-OHDA would be equal whether the animal had been pr-..treated with an uptake blocker or not. The N A neuron system investigated was that originating from the locus coeculeus projecting to the cerebral cortex [ 14]. The axons of this system make up a fairly well defined pathway in the mesencephalon, the so-called dorsal N A bundle. These axons are easily available for local application of 6~)HDA which produces an almost complete NA denervation in the cerebral cortex [9]. Such a NA denervation has also been shown to be causally related to typical changes in EEG waking in the acute phase [7]. Thus, 6-OHDAinduced lesions of the dorsal N A bundle were carried out, changes in EEG and EMG recorded and the lesions and the degree of nerve terminal degeneration in the cerebral cortex evaluated with histo- and neurochemical techniques and correlated in each animal. Male Sprague-Dawley rats (150--160 g) were used. Half of the animals were pretreated with protriptyline (Concordin®, 25 mg/kg i.p.) 30 rain before being operated on. The lesions of the dorsal NA bundle were effected by stereotaxic injections of 6-OHDA as described by Ungerstedt [ 14]. Each animal received a bilateral injection of 8/~g 6-OHDA dissolved in 4 ~1 isotonic NaCI containing 0.2 mg/ml ascorbic acid in the dorsomedial tegmentum of the anterior mesencephalon (for details, see ref. 7). A control group received the vehicle only. The coordinates were: A, 0.6 ram; L, 1.3 mm and V, 0.5 mm according to the stereotaxic atlas of Kt~nig and Klippel [6]. The location of the injected solution was found to be just dorsolateral of the dorsal NA bundle. After the injection, electrodes for EEG and EMG registrations were implanted as previously described [7 ]. The animals were recorded on days 2--4 after the operation, each session being of 4 h duration. Four different states of activity were distinguished (see ref. 7) and the mean duration of each state per session was calculated for each animal. The effectiveness of the lesion was evaluated after 15 days, when the anterograde nerve terminal degeneration is known to be complete [9]. Slices from the cerebral cortex (neocortex) were prepared and incubated in vitro in a Krebs-Ringer bicarbonate buffer
37
(pH 7.4) containing 0.05/zM [3H]NA (specific activity: 15 Ci/mmole, Radiochemical Centre, Amersham) for 5 min at +37°C [9]. For fluorescence h~tochemical analysis according to Falck and Hillarp, smears from the cerebra1 cortex were also prepared [8 ] and sections from the mesencephalon were stained in cresyl viole~ to be able to analyze the site of injection for possible general tissue damage. % -q. "~5o, ,q
ILl F-
Q.
D
SALINE
Q
SALINE • PROTRIPTYLINE
Q
6-OH'OA
Q
6-OH-DA • PROTRIPTYLINE
4.
nionnono W ,t. K I N G
SLOW SLEEP 1
SLOW SLEEP 2
PARADOXICALSL.
Fig.1. Effect of protTiptyline pretreatment on changes in EEC waking after ,dorsal noradrenaline bundle lesion. The animals were given protriptyEne (25 mg/kg i.p.) 30 min before they were injected intracerebrally with 6-OHDA (8 . g / 4 pl) close to the dorsal NA bundle in the antetdor mesencephalon. Controls received saline only. Electrodes were implanted and 2--4 dsys after the operation the animals were recorded for EEG and EMG activity. The recorded time (3 sessiop~s, each of 4 h) was divided into 4 activity states: waking: low voltage fast cortical activity and a high variable muscle activity; slow sleep 1: high voltage slow waves on a background of fast activity and a more regular muscle activity; slow sleep 2: continous high voltage waves and reduced muscle activity; paradoxical sleep: low voltage fast activity and inhibition of muscle activity. The duration of each state was expressed as per cent of the total recording time.
In agreement "with previous studies [7] acutely after the 6 - O H D A administration there was a marked decrease in E E G waking (characterized by low voltage fastactivity,desynchronized activity)with a corresponding increase in the periods of high voltage slow waves and synchronized activity (Fig. 1). From the neuro- and histochemical analyses of these animals it was found that the N A denervation in the cerebral cortex was almost complete, as reflected by a considerable reduction in the in vitro uptake of [ZH]NA (65--70% reduction) and an almost complete disappearance of N A nerve terminals evaluated by fluorescence his~x)chemistry(see also ref. 9). Injection of the solvent alone (omitting 6~)HDA) did not produce any significant effects in EEG, [3H]NA uptake or the number of N A nerve terminals seen in the fluorescence microscope. Pretreatment of the animals with the potent N A 'membrane pump' blocker, protriptyline,30 rain before the 6 ~ ) H D A injection completely counteracted the changes in E E G as well as the changes seen in [3H]NA uptake wnd number of N A nerve terminals in the cerebral cortex. Protriptyline itself had no significant effects on the parameters recorded.
The present results clearly show that blockade of the axonal 'membrane pump' mechanism of NA neurons by protriptyline prevents the 6-OHDAinduced anterograde degeneration of the NA nerve terminals in the cerebral cortex. The present d e g e n e r a t e action of 6.OHDA must consequently be associated with its specific neurotoxicity elicited by an uptake and accumulation of 6-OHDA in the NA axo'ns of the dorsal bundle. Already the choice of the placement of the 6~)HDA injection (dorsolateral to the NA axons) would indicate a specific neurotoxic action, unless the injection would cause a rather large umq3eciflc tism~ &mmge at the injection site. Such extensive damage was never observed when analyzing the injection site with conventional histological techniques, but only very limited unspecific tissue necrosis was noticed. In the presevt study it was found that 'membrane pump' blockade with protriptyline also prevented the changes in EEG associated with the 6-OHDA injection. Thus, blockade of the inh'aneu~onal accumulation of 6-OHDA prevents both the.degeneration of the NA nerve terminals and the 6-OHDA-induced changes in EEG. Therefore, it may be concluded that the present injection technique for 643HDA produces a specific denervation of NA nerve terminals in the cerebral cortex, equally well from the morphological, neurochemical and functional points of view. This does not mean that 6-OHDA produces a specific lesion in all experimental situations, but rather that it is possible to obtain a ~elective denervation of catecholamine neurons after intracerebral 6-OHDA injection, when properly used.
ACKNOWLEDGEMENTS The present study has been supported by research grants from the Swedish Medical Research Council (04X-2295) and the Karolinska Instituter, REFERENCES 1 Agid, i,., Javoy, F., Glowinski, J., Bouvet, D., and Sotelo, C., ln|eetion of 6-hydr(,xydopamine into the s~bstantia nigra of the rat. IL Diffusion and specificity, Brain Res., 58 (1973} 291--301. 2 Butcher, L.L., E~tgate, S.M., and Hodge, G.K., Evidence that punetate intracerebml administration of 6-hydroxydop~mjne faill to produce selective neuronal degeneration, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 285 (1974) 31--70. 3 Evetts, K.O., and Iversen, L.L., Effects of protriptYline on t~.e depletion of catecholamines induced by 6~hydroxydopamine in the brain of the rat, J. Pharm. Pharmacol., 22 (1970) 540--543. 4 H~kfelt, T., aud Ungerstedt, U., Specificity of 6-hydroxydopamine induced degeneration of central monoamine neurons: an electron and fluorescence microscopic study with special reference to intracerebral injection of the nigro-striatal dopamine system, Brain Res., 60 (1973) 269-297. 5 Jonsson, G., and Sachs, Ch., Effects of 6-hydroxydopamlne on the uptake and storage of noradrenaline in sympathetic adrenergic neurons. Europ. J. Pharmacol., 9 (1970) 141--155.
39 6 K~nig, J.F.R., and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, B~itimore, Md., 1963. ? Lidbrink, P., The effect of lesions of ascending noradrenaline pathways on sleep and waking in the rat, Brain Res., 74 (1974) 19--40. 8 IAdbrink, P., and Jonsson, G., Semiquantitative estimation of formaldehyde-induced fluorescence of noradrenaline nerve terminals, J. Histochem. Cytochem., 19 (1971) 747--757. 9 Lidbrln~, P., and Jonsson, G., Noradrenaline nerve terminals in the cerebral corLex: effects on noradrenaline uptake and storage following axonal lesion with 6-hydroxydopamine, J. Neurochem., 22 (1974) 617---626. 10 Malmfors, T., and Thoenen, H., 6-Hydroxydopamine ~nd Catecholamine Neurons, Elsevier, North-Holland, Amsterdam, 1971. 11 Poirier, L.J., Langlier, P., Roberge, A~, Boucher, R., and Kitsikis, A., Non-specific histopathologicai changes induced by the intracerebral injection of 6-hydroxydopamine (6-OH-DA), J. neurol. ScL, 16 (1972) 401--416. 12 Sachs, Ch., and Jonsson, G., Mechanisms of action of 6-hydroxydopamine, Biochem. Pharmaeol., 24 (1975) 1--8. 13 Sotelo, C., Javoy, F., Agid, Y., and Glowinski, J., Injection of 6-hydroxydopamine in the substantia nigra of the rat. I. Morphological study, Brain Res., 58 (1973) 269--290. 14 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol, scan&, Suppl. 367 (1971) 1--48.
35-39 Amsterdam - Printed in The Netherlamb
P. LIDBRINK and G. JONSSON Departmentof Histology, Karotinska Institute& S-104 01 Stockholm
techniques. The 6 ow waves and synchro
60 (Sweden)
36
frequently used method of 6-OHDA administration for functional studies on central catecholamine neurons is the local intracerebral injection technique. However, the results reported on the use of S i s administration route are rather inconsistent and the views as to the specificity of 6~)HDA are very confludng and contradictory [1,2,4,11,13]. The present study was undertaken to evaluate the specificity of a 6 ~ H D A induced lesion of a noradrenergic neuron system after local intracerebral injection. The aim was to obtain animals in which the noradrenaline (NA) neurons would remain intact after the 6~)HDA injection. This has previously been shown possible in animals given intraventricular 6~)HDA after p r ~ t.-iptyline pretreatment [3]. The drug in all probability antagonizes the effects of 6~)HDA by competitive inhibition of the neuronal uptake of 6 ~ H D A [ 5]. On the other hand, it seems reasonable to assume that effects not related to uptake into NA neurons ('non-specific' effects) after intracerebral injections of 6-OHDA would be equal whether the animal had been pr-..treated with an uptake blocker or not. The N A neuron system investigated was that originating from the locus coeculeus projecting to the cerebral cortex [ 14]. The axons of this system make up a fairly well defined pathway in the mesencephalon, the so-called dorsal N A bundle. These axons are easily available for local application of 6~)HDA which produces an almost complete NA denervation in the cerebral cortex [9]. Such a NA denervation has also been shown to be causally related to typical changes in EEG waking in the acute phase [7]. Thus, 6-OHDAinduced lesions of the dorsal N A bundle were carried out, changes in EEG and EMG recorded and the lesions and the degree of nerve terminal degeneration in the cerebral cortex evaluated with histo- and neurochemical techniques and correlated in each animal. Male Sprague-Dawley rats (150--160 g) were used. Half of the animals were pretreated with protriptyline (Concordin®, 25 mg/kg i.p.) 30 rain before being operated on. The lesions of the dorsal NA bundle were effected by stereotaxic injections of 6-OHDA as described by Ungerstedt [ 14]. Each animal received a bilateral injection of 8/~g 6-OHDA dissolved in 4 ~1 isotonic NaCI containing 0.2 mg/ml ascorbic acid in the dorsomedial tegmentum of the anterior mesencephalon (for details, see ref. 7). A control group received the vehicle only. The coordinates were: A, 0.6 ram; L, 1.3 mm and V, 0.5 mm according to the stereotaxic atlas of Kt~nig and Klippel [6]. The location of the injected solution was found to be just dorsolateral of the dorsal NA bundle. After the injection, electrodes for EEG and EMG registrations were implanted as previously described [7 ]. The animals were recorded on days 2--4 after the operation, each session being of 4 h duration. Four different states of activity were distinguished (see ref. 7) and the mean duration of each state per session was calculated for each animal. The effectiveness of the lesion was evaluated after 15 days, when the anterograde nerve terminal degeneration is known to be complete [9]. Slices from the cerebral cortex (neocortex) were prepared and incubated in vitro in a Krebs-Ringer bicarbonate buffer
37
(pH 7.4) containing 0.05/zM [3H]NA (specific activity: 15 Ci/mmole, Radiochemical Centre, Amersham) for 5 min at +37°C [9]. For fluorescence h~tochemical analysis according to Falck and Hillarp, smears from the cerebra1 cortex were also prepared [8 ] and sections from the mesencephalon were stained in cresyl viole~ to be able to analyze the site of injection for possible general tissue damage. % -q. "~5o, ,q
ILl F-
Q.
D
SALINE
Q
SALINE • PROTRIPTYLINE
Q
6-OH'OA
Q
6-OH-DA • PROTRIPTYLINE
4.
nionnono W ,t. K I N G
SLOW SLEEP 1
SLOW SLEEP 2
PARADOXICALSL.
Fig.1. Effect of protTiptyline pretreatment on changes in EEC waking after ,dorsal noradrenaline bundle lesion. The animals were given protriptyEne (25 mg/kg i.p.) 30 min before they were injected intracerebrally with 6-OHDA (8 . g / 4 pl) close to the dorsal NA bundle in the antetdor mesencephalon. Controls received saline only. Electrodes were implanted and 2--4 dsys after the operation the animals were recorded for EEG and EMG activity. The recorded time (3 sessiop~s, each of 4 h) was divided into 4 activity states: waking: low voltage fast cortical activity and a high variable muscle activity; slow sleep 1: high voltage slow waves on a background of fast activity and a more regular muscle activity; slow sleep 2: continous high voltage waves and reduced muscle activity; paradoxical sleep: low voltage fast activity and inhibition of muscle activity. The duration of each state was expressed as per cent of the total recording time.
In agreement "with previous studies [7] acutely after the 6 - O H D A administration there was a marked decrease in E E G waking (characterized by low voltage fastactivity,desynchronized activity)with a corresponding increase in the periods of high voltage slow waves and synchronized activity (Fig. 1). From the neuro- and histochemical analyses of these animals it was found that the N A denervation in the cerebral cortex was almost complete, as reflected by a considerable reduction in the in vitro uptake of [ZH]NA (65--70% reduction) and an almost complete disappearance of N A nerve terminals evaluated by fluorescence his~x)chemistry(see also ref. 9). Injection of the solvent alone (omitting 6~)HDA) did not produce any significant effects in EEG, [3H]NA uptake or the number of N A nerve terminals seen in the fluorescence microscope. Pretreatment of the animals with the potent N A 'membrane pump' blocker, protriptyline,30 rain before the 6 ~ ) H D A injection completely counteracted the changes in E E G as well as the changes seen in [3H]NA uptake wnd number of N A nerve terminals in the cerebral cortex. Protriptyline itself had no significant effects on the parameters recorded.
The present results clearly show that blockade of the axonal 'membrane pump' mechanism of NA neurons by protriptyline prevents the 6-OHDAinduced anterograde degeneration of the NA nerve terminals in the cerebral cortex. The present d e g e n e r a t e action of 6.OHDA must consequently be associated with its specific neurotoxicity elicited by an uptake and accumulation of 6-OHDA in the NA axo'ns of the dorsal bundle. Already the choice of the placement of the 6~)HDA injection (dorsolateral to the NA axons) would indicate a specific neurotoxic action, unless the injection would cause a rather large umq3eciflc tism~ &mmge at the injection site. Such extensive damage was never observed when analyzing the injection site with conventional histological techniques, but only very limited unspecific tissue necrosis was noticed. In the presevt study it was found that 'membrane pump' blockade with protriptyline also prevented the changes in EEG associated with the 6-OHDA injection. Thus, blockade of the inh'aneu~onal accumulation of 6-OHDA prevents both the.degeneration of the NA nerve terminals and the 6-OHDA-induced changes in EEG. Therefore, it may be concluded that the present injection technique for 643HDA produces a specific denervation of NA nerve terminals in the cerebral cortex, equally well from the morphological, neurochemical and functional points of view. This does not mean that 6-OHDA produces a specific lesion in all experimental situations, but rather that it is possible to obtain a ~elective denervation of catecholamine neurons after intracerebral 6-OHDA injection, when properly used.
ACKNOWLEDGEMENTS The present study has been supported by research grants from the Swedish Medical Research Council (04X-2295) and the Karolinska Instituter, REFERENCES 1 Agid, i,., Javoy, F., Glowinski, J., Bouvet, D., and Sotelo, C., ln|eetion of 6-hydr(,xydopamine into the s~bstantia nigra of the rat. IL Diffusion and specificity, Brain Res., 58 (1973} 291--301. 2 Butcher, L.L., E~tgate, S.M., and Hodge, G.K., Evidence that punetate intracerebml administration of 6-hydroxydop~mjne faill to produce selective neuronal degeneration, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 285 (1974) 31--70. 3 Evetts, K.O., and Iversen, L.L., Effects of protriptYline on t~.e depletion of catecholamines induced by 6~hydroxydopamine in the brain of the rat, J. Pharm. Pharmacol., 22 (1970) 540--543. 4 H~kfelt, T., aud Ungerstedt, U., Specificity of 6-hydroxydopamine induced degeneration of central monoamine neurons: an electron and fluorescence microscopic study with special reference to intracerebral injection of the nigro-striatal dopamine system, Brain Res., 60 (1973) 269-297. 5 Jonsson, G., and Sachs, Ch., Effects of 6-hydroxydopamlne on the uptake and storage of noradrenaline in sympathetic adrenergic neurons. Europ. J. Pharmacol., 9 (1970) 141--155.
39 6 K~nig, J.F.R., and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams and Wilkins, B~itimore, Md., 1963. ? Lidbrink, P., The effect of lesions of ascending noradrenaline pathways on sleep and waking in the rat, Brain Res., 74 (1974) 19--40. 8 IAdbrink, P., and Jonsson, G., Semiquantitative estimation of formaldehyde-induced fluorescence of noradrenaline nerve terminals, J. Histochem. Cytochem., 19 (1971) 747--757. 9 Lidbrln~, P., and Jonsson, G., Noradrenaline nerve terminals in the cerebral corLex: effects on noradrenaline uptake and storage following axonal lesion with 6-hydroxydopamine, J. Neurochem., 22 (1974) 617---626. 10 Malmfors, T., and Thoenen, H., 6-Hydroxydopamine ~nd Catecholamine Neurons, Elsevier, North-Holland, Amsterdam, 1971. 11 Poirier, L.J., Langlier, P., Roberge, A~, Boucher, R., and Kitsikis, A., Non-specific histopathologicai changes induced by the intracerebral injection of 6-hydroxydopamine (6-OH-DA), J. neurol. ScL, 16 (1972) 401--416. 12 Sachs, Ch., and Jonsson, G., Mechanisms of action of 6-hydroxydopamine, Biochem. Pharmaeol., 24 (1975) 1--8. 13 Sotelo, C., Javoy, F., Agid, Y., and Glowinski, J., Injection of 6-hydroxydopamine in the substantia nigra of the rat. I. Morphological study, Brain Res., 58 (1973) 269--290. 14 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol, scan&, Suppl. 367 (1971) 1--48.