Glutamate-induced apoptosis results in a loss of striatal neurons in the parkinsonian rat

Pergamon

0306-4522(94)00389-O

Neuroscience Vol. 63, No. I, pp. l-5, 1994 Elsevier Science Ltd Copyright 0 1994 IBRO Printed in Great Britain. All rights reserved 0306.4522/94 $7.00 + 0.00

Letter to I\leuroscience GLUTAMATE-INDUCED APOPTOSIS RESULTS IN A LOSS OF STRIATAL NEURONS IN THE PARKINSONIAN RAT I.

J. MITCHELL, S. LAWSON, B. MOSER, S. M. LAIDLAW, A. J. COOPER, G. WALKINSHAW a nd C. M. WATERS* G.38, Stopford Building, Oxford R.oad, Manchester Ml3 9pT, U.K.

The motor symptoms of Parkinson’s diiase are caused by an increase in activity of striatal neurons which project to the globus pallidus.” The discharge activity of these striatal cells is normally regulated by a balance between an inhibitory nigral dopamiw input and an excitatory cortical glutamate input.” The loss of nigrostriatal dopamine in Parkinson’s disease allows the cortical glutamatergic input to dominate (see Fig. 1). Pharmacological or surgical manipulations which redress this imbalauce in activity in the striatum, or prevent its propagation throughout the basal ganglia, alleviate thcmotor symptoms of Parkinsonism.‘A’4We presentevidence to suggest the existence of an endogenous mechauism which compensates for the striatal imbalance during the early stages of Parkinsonism. In the rat rendered parkinsonian by systemic administration of reserpiue, selective deletion of striatal neurons was observed. The dying striatal neuronsexhibited all of the morphological nod biochemical hallmarks of apoptosis. This apoptotic cell death was blocked by either administration of glutamate antagonists or decortication. Our data demonstrate that unchecked endogeuous glutamate can iuduce apoptosis of striatal projection neurous in uiuo. This observation may have relevance to the neurophysiologicalmechanisms which maintain the balance of neural activity within the CNS and to the pathology of neurological diseases. Apoptosis whereby

is an active

individual

swiftly phagocytosed neighbouring has

been

without

cells. 30 To

most

studied

system where it appears organisation it

has

underlie

of neuronal

been the

physiological

cells are selectively

speculated aetiology

and

effect upon

apoptosis

of neurons

in the

developing

to play a crucial

nervous role in the

networks.9s20 More recently that

apoptosis

may

neurodegenerative

also

diseases.‘5V’6X” There is however little direct evidence to support this hypothesis and the results from in vitro studies

remain

of

equivocal.‘0~“J3

*To whom correspondence

glutamate (+)

mechanism deleted

deleterious

date

The current experiments were designed to test the hypothesis that changes in the balance of neural activity within the adult CNS can induce an active programme of neuronal death. Rats were treated with reserpine in order to induce a temporary parkinsonian syndrome. ‘L’ Reserpine treatment results in a reversible but profound depletion of the dopamine content of the nigrostriatal pathway.‘,‘,’ The dopaminergic neurons themselves, however, are left intact. Brain tissue from these animals was processed in order to identify apoptotic cells.

should be addressed.

enkephalin

GLOBUS PALLIDUS

Fig. 1. Topography of synaptic inputs to the medium spiny striatal output neuron. The striatal neuron illustrated is enkephalinergic, projects to the globus pallidus and receives two major afferents. Glutamatergic inputs from the cerebral cortex terminate on distal dendritic spines and arc excitatory. The inhibitory dopaminergic input neurons from the substantia nigra contact the cell body, dendritic shaft and proximal spines.24 Nigrostriatal dopamine thus functions to regulate the flow of information from cortical areas through the striatum to basal ganglia output structures. Reductions in nigrostriatal dopamine abolish the inhibitory control over the excitatory glutamate. We demonstrate that excess glutamate transmission induces apoptosis of these neurons.

1. J. Mitchell

Fig. 2.-Caption

et ul.

opposire

Glutamate-induced

apoptosis in parkinsonian

Cells undergoing apoptosis show a specific morphology which includes, cell shrinkage, cell membrane blebbing and chromatin condensation.30 Furthermore the DNA of apoptotic cells is cleaved into fragments of regular size.‘*,30Vibrotome cut brain sections were prepared with a silver stain and used to determine the distribution and morphological appearance of any dying cells. Following the administration of reserpine, numerous dying cells demonstrating a typical apoptotic morphology were observed in the striatum. (see Fig. 2A). At both light and electron microscopic level the dying cells appeared shrunken in volume, detached from their neighbours and had densely stained fragmented nuclei, (electron microscopic data not shown). These apoptotic-like cells were not homogeneously distributed throughout the striatum but tended to lie dorsomedially in a crescent shaped area within the rostra] striatum (Fig. 3). In contrast, very few dying cells, were seen in the striatum of the vehicle injected rats, (approximately 10% of the total number of the apoptotic cells observed in the treated animals). Similarly, only occasional dying cells were observed in other brain areas examined in the reserpine treated rats (including the substantia nigra, globus pallidus and cerebral cortex). DNA fragmentation is a key biochemical feature of cells undergoing apoptosis.“*‘” An in situ end-labelling method, which detected the “free” ends of fragmented DNA, was used to establish that the cells with the morphological appearance of apoptosis also satisfied this biochemical criteria (see Fig. 2B). In order to determine whether dying cells were striatal projection neurons and not interneurons or

rat

3

glial cells, a colloidal gold-based retrograde tracer was injected into the globus pallidus, a projection area of the striatum.24 This procedure resulted in the retrograde labelling of large numbers of striatal neuronal cell bodies. Occasional striatal cells in the reserpine treated animals were observed to be both retrogradely labelled with colloidal gold, and to exhibit condensed chromatin charactenstic of apoptotic cell death. (see Fig. 2C). These instances of double labelling imply that at least some of the dying cells seen in the striatum following reserpine administration were striatal projection neurons. All striatal projection neurons receive both a dopaminergic input from the substantia nigra and an excitatory amino acid input from the cortex24 (see Fig. 1). A reduction in nigrostriatal dopamine levels. such as that induced by reserpine, results in an elevation of excitatory amino acid-mediated corticostriatal transmission.6.24 High levels of excitatory amino acid release are known to bc neurotoxic’ and have been shown to induce apoptosis in cultured rat neocortical neurons.15 We therefore attempted to block the reserpine-induced striatal apoptosis by two separate approaches. We firstly blocked glutamatergic transmission using the N-methyl-D-aspartate antagonist ketamine.26 Secondly, we surgically removed part of the cerebral cortex which contributes to the corticostriatal glutamatergic pathway. Histological analysis of the lesion site showed a partial ablation of the media1 frontal and parietal lobes and of the cingulate cortex. Both procedures resulted in a significant reduction in the number of reserpine induced apoptotic striatal cells, (see Table I), though neither procedure completely abolished the apoptosis. This

Fig. 2. 2%3008 Sprague-Dawley rats, lightly anaesthetized with halothane, received either a single injection of reserpine (5 mg/kg s.c., in 0 1% acetic acid), or a control injection of the vehicle. The rats were transcardially perfused under deep anaesthesia 18 h after the reserpine/vehicle injection when the parkinsonian symptoms were at their maximum. Vibrotome sections (70 pm) were taken throughout the striatum and a 1 in 4 series processed using a modification of lone’s silver/methenamine method2a and were counter stained with cresyl violet. Systemic administration of reserpine was associated with the appearance of dying cells with the morphological features of apoptotic cells in silver stained sections of the striatum as shown in A. The cells appeared to have shrunk in volume and had densely stained fragmented nuclei. The appearance of the nuclei varied from a single darkly stained sphere which was sharply delineated to two or three similarly stained spheres. Significantly more apoptotic striatal cells were seen following reserpine injection than following vehicle injection (see Table 1). An in situ nick translation method was used to detect cells which had fragmented DNA.2’“* Free floating sections were preincubated with 20pg/ml proteinase K in 10mM Tris-HCl for 15min and then washed. The sections were then incubated at 37” for I h in 50mM Tris, IOmM MgSOa, pH 7.2, containing SOpg/ml bovine serum albumin, 1 pl digoxigenindNTP (Boehringer Ma&he&); 1pl Kornberg-polymerax (5 units,/pl) (Boehringer). The reaction was stopped __ using 20 mM Tris-HCl/2 mM EDTA (pH _ 7.4) for 10 min and the sections washed with Tris bufferd saline. Digoxigenin labelled DNA was detected using peroxidase conjugated anti-digoxigenin antibodies and revealed with 3,3-diaminobenzidine. Cells with fragmented DNA appeared as darkly staining cells. A typical cell is shown at low magnification in B +nd at higher magnification in inset. Some of the apoptotic cells were labelled following the retrograde transport of a neuroanatomical tracer which was stereotaxically injected into the globus pallidus at least three weeks prior to the administration of reserpine. WGAapo horseradish peroxidase Au (6&100 ~1) in 0.05 M Tris buffer @H 7.4) was stereotaxically injected into the globus pallidus. After quenching free aldehyde groups with ammonium ions the brain sections were permeabilized with 0.2% triton in phosphate buffered saline and the gold signal silver intensified. An apoptotic cell is shown in C which can be distinguished by its condensed chromatin and halo-like appearance due to its loss of contact with neighbouring cells. This cell is also labelled with retrogradely transported gold (small dark dots). Other striatal pro&ion neurons in the field are also gold labelled. Dying-cells w&e not observed follbwing control in-&ions of WGAapo horseradish peroxidase Au into the animals which did not receive an injection of reserpine. Scale bars = 5 pm.

4

I. J. Mitchell ct ul

Fig. 3. Distribution of apoptotic striatal cells 18 h after systemic administration of reserpine. Cumulative cell plots from six reserpine treated animals at a single level within the rostra1 striatum are shown in the section on the left. The pattern of apoptotic cells was consistent for all animals examined and formed a rim around the dorsomedial striatum. The section on the right shows equivalent plots for control animals treated with saline Scale bar = 1 mm.

Table

1. Numbers

(A) Saline Reserpine/saline Reserpine/ketamine (B) ReserDineinormal Reser$ne/decorticate

of apoptotic striatal reserpine treatment Mean no. of 7.0 109.8 58.6

cells following

apoptotic cells f 0.98 + 12.9**** f 9.91”’

58.4 k 3.12 43.0 * 3.30*

Table l(A) shows the mean (and S.E.), number of apoptotic striatal cells counted over five sections (70hm) evenly spaced through the rostra1 striatum. Three groups of animals are shown. The first group (N = 6) received a systemic injection of saline. The second group (N = 6) received a systemic injection of reserpine followed by hourly saline injection for 6 h. The third group (N = 5) received a systemic injection of reserpine followed by injections of the N-acetyl-D-aspartate antagonist, ketamine (300 mg/kg, i.p.) supplemented hourly for 6 h. At 6 h the animals were killed and the brains processed as detailed in Fig. 2. Reserpine treatment showed a significant increase in the number of striatal apoptotic cells compared with saline injection, (P < 0.0001). Co-administration of ketamine reduced the number of apoptotic cells significantly, (P i 0.01). (ANOVA followed by Scheffe’s multiple f-test). Table 1(B) shows the number of striatal apoptotic cells in a group of four animals which received a partial unilateral parietal and cingulate cortical aspiration 3 weeks prior to reserpine administration. Animals were killed 18 h after reserpine injection. The mean number (and S.E.) of striatal apoptotic cells seen in five sections taken through the rostra1 striatum on the non decorticated side (reserpine normal) are compared with the number on the decorticated side (reserpine decorticate). Significantly fewer apoptotic cells were seen following the decortication (P < 0.05, paired c-test).

may be explained in part because AMPA mediated glutamatergic transmission was not antagonized by ketamine and the decortication would have spared a substantial cortical glutamatergic input to the striaturn. These data therefore indicate reserpine induced apoptosis is dependent upon the integrity of the cortical excitatory amino acid input to the striatum. It is well recognized that there is tremendous plasticity within the dopaminergic innervation of the striatum. Up to 80% of nigrostriatal dopamine can be lost before the behavioural symptoms of parkinsonism are seen. 31 Apoptosis of striatal neurons, which results from depletion of the dopamine content of the nigrostriatial pathway, may be viewed as an adaptive process which contributes to the delayed manifestation of parkinsonism in animal models. The neurons that behave in the most abnormal manner. that is those continuously excited by excitatory amino acid; are selectively deleted. The imbalance of neural activity within the circuitry of the basal ganglia is thus reduced. S&h a mechanism may also operate in Parkinson’s disease in humans where a limited secondary degeneration of striatal projection neurons occurs.12,25,29The aim of future experiments will be to determine whether this cell loss is indeed an adaptive response or whether it contributes to the manifestation of the clinical symptoms. Accumulating evidence suggests that a range of compounds (including MK-801, methamphetamine and MPP +), mediate their toxic effects indirectly via sysstimulation of endogenous glutamatergic tems.5J9.22x23 Our data demonstrate that it is possible for neurons in the adult brain to undergo apoptosis in response to endogenous excitotoxins and presumably also in response to exogenous toxic insults. This

Glulamate-induced

apoptosis in parkinsonian

in turn heightens the probability of programmed ceil death

playing

a role in neurodegenerative

ditions such as Huntington’s

disease.

con-

disease and Parkinson’s

rat

5

Acknowledgements-The financial support of The Wellcome Trust and the Medical Research Council is gratefully acknowledged. We thank Dr D. Bristow, Prof. J. Hickman and Prof. N, Rothwell for their critical discussion of the manuscript

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(Accepted I August 1994)