Herpes simplex virus encephalitis

Herpes simplex virus encephalitis

Journal of the Neurological Sciences, 1985, 71:325-337 325 Elsevier JNS 2585 Herpes Simplex Virus Encephalitis Neuroanatomical and Neurochemical Se...

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Journal of the Neurological Sciences, 1985, 71:325-337

325

Elsevier JNS 2585

Herpes Simplex Virus Encephalitis Neuroanatomical and Neurochemical Selectivity S.P. Neeley, A.J. Cross*, T.J. Crow, J.A. Johnson and G.R. Taylor Division of Psychiatry, Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ (U.K.)

(Received 11 February, 1985) (Revised, received 18 July, 1985) (Accepted 24 July, 1985)

SUMMARY Intracerebral infection o f mice with H S V - I was found to produce a 2-3-fold increase in dopamine and serotonin metabolism in cortex, striatum, diencephalon and brain stem. Neurochemical markers o f G A B A and acetylcholine neurones, and neurotransmitter receptor binding sites were unchanged. The immunohistochemical distribution o f virus antigen revealed high levels o f immunoreactivity in s. nigra, ventral tegmental area, locus coeruleus and dorsal raphe nucleus, whilst other areas o f brain stem were clear of virus antigen. The changes in monoamine metabolism observed in experimental H S V encephalitis may be related to the concentration o f virus in monoamine neurones.

Key words: D o p a m i n e - Encephalitis - H e r p e s s i m p l e x virus - Serotonin

INTRODUCTION Certain viruses are able to infect different cell types within the C N S , or even particular sub-populations o f neurones in specific brain regions (reviewed by J o h n s o n 1980, 1982). Such differential sensitivity of neuronal populations to some viruses may * Present address: Department of Physiology,University of Manchester, Manchester M139PT, U.K. Abbreviations: CAT --- choline acetyltransferase; DHA = dihydroalprenolol, DOPAC = dihydroxyphenylacetic acid; GABA = ?-aminobutyric acid; GAD = glutamic acid decarboxylase; 5-HIAA = 5hydroxy-indoleacetic acid; HSV = herpes simplex virus; 5-HT = 5-hydroxytryptamine; HVA = homovanillic acid; LSD = lysergic acid diethylamide; MHPG = methoxyhydroxyphenyglycol. 0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

326 be determined in part by the presence of specific viral receptors (Lonberg-Holm and Phillipson 1981) and by the route of virus entry into the brain once infected. It has also been suggested that some neuronal populations are more permissive to viral replication. Virus infection of the central nervous system (CNS) has a variety of consequences ranging from cell lysis to functional changes in the absence of observed histopathological abnormalities (Oldstone et al. 1982). There have however been few studies of the neurochemical sequelae of CNS virus infections: our understanding of the ability of viruses to interact with neurochemically discrete systems thus remains limited. Herpes simplex virus type 1 (HSV-1) infection of the CNS in mice provides a useful experimental model for studying the pathogenesis of such infections. Moreover this highly neurotropic virus is of particular clinical interest as HSV-1 is a common human pathogen and may be the most frequent cause of fatal encephalitis in man. The ability of herpes viruses to become latent in neural tissue and subsequently to re-activate raises the possibility that these viruses could be involved in chronic recurring diseases of the nervous system in man. Previous studies of HSV encephalitis in experimental animals have concentrated either on the spread of virus in the CNS (Kristensson et al. 1982; Anderson and Field 1983; Tomlinson and Esiri 1983) or on neurochemical changes in whole brain (Lycke et al. 1968, 19770, 1972). There have however been no studies on the relationship between the infection of specific neurones by HSV and the neurochemical consequences of such infection. In the present study to selectively infect those cells which are particularly sensitive to H SV-1, virus was injected intracerebrally in a large volume ofinnoculum. The effects of the encephalitis on the concentrations of the monoamines serotonin and dopamine, their metabolites and receptors and on markers of acetylcholine and 4-aminobutyric acid containing neurones were examined in different brain regions. In addition, virus distribution was studied using infectivity assays and immunohistochemistry of viral antigen. We have related the neurochemical consequences of HSV encephalitis with the distribution of HSV within the brain. MATERIALS AND METHODS Virus growth and assay Herpes simplex virus type 1 (Justin strain), a clinical isolate of low passage history was provided by Dr. R. W. Honess (NIMR, Mill Hill, London) as a frozen tissue culture supernatant. Virus stocks were grown in Vero cell monolayers in Eagles MEM supplemented with 6~o new born calf serum. Monolayers were infected at a multiplicity of 10-20 plaque forming units per cell. When extensive cytopathic effect developed, the medium was removed and clarified by centrifugation at 3 000 x g for 10 min. The supernatant was stored over liquid nitrogen in 50~ glycerol until use. Viral infectivity was estimated in brain samples by a plaque assay (Russell 1962). Brain homogenates were centrifuged at 12000 x g for 20 s and the supernatant diluted 1 : 2 with Eagles MEM with 4% new born calf serum added. Ten-fold serial dilutions of this preparation were applied in duplicate to Vero cell monolayers for 30 rain at 37 ° C, after which Eagles MEM with pooled human serum (10~) was applied. After 4 days

327 plaques were stained using neutral red in culture medium (80/~g/ml) for 4-6 h. Virus titres were calculated and expressed as plaque-forming units (pfu)/mg protein.

Experimental protocol Swiss outbred (to) mice 4-5 weeks old were injected intracerebrally with 30 #1 of the virus preparation diluted to 10 mouse LD50 units per injection in Eagles MEM. Control mice were given identical injections of Eagles MEM alone. On day 5 p.i. mice were killed, and their brains were removed and dissected free-hand into cerebellum, brain stem, hippocampus, striatum, cortex and diencephalon (i.e. remainder). These portions were stored at - 40 ° C until used in neurochemical analysis. For immunohistochemical staining mice were perfused through the heart under Penthrane anaesthesia with isotonic saline followed by 4 % formalin. Brains were then removed and post-fixed in a solution of 4~o formalin.

Neurochemical analysis High affinity ligand binding to neurotransmitter receptors was performed using established techniques. Tissues were homogenised in 40 vol of 50 mM Tris/HCl, pH 7.4, using an Ultra-Turrax and centrifuged at 10 000 x g for 10 min. The pellet was resuspended in the original volume of buffer and aliquots of this preparation were used in ligand binding assays. For [3H]serotonin binding the membrane preparation was washed a further 2 times, and incubated at 37 °C for 15 min prior to the binding assay. For [3H]muscimol binding membranes were washed a further five times by repeated centrifugation and freeze thawing. All other binding assays used the initial crude membrane preparation. Ligand binding assays were as follows: for dopamine D2 receptors [3H]spiperone (0.5 nM) was used as ligand, and non-specific binding defined with 1/~M ( + ) butaclamol, serotonin S1 receptors with 4 nM [3H]serotonin and 1/~M LSD, serotonin $2 receptors with 1 nM ketanserin and 1/~M LSD, muscarinic cholinergic receptors with 1 nM [3H]N methylscopolamine and 1/~M atropine, GABA receptors with 5 nM [3H]muscimol and 10 #M GABA, ~l-adrenergic receptors with 2 n M [3H]WB4101 and 1 #M phentolamine, ~2-adrenergic receptors with 2 n M [3H]rauwolscine and 1 #M phentolamine, ]~-adrenergic receptors with 2 nM [3H]dihydroalprenolol and 1 #M propranolol, and benzodiazepine receptors with 1 nM [3H]flunitrazepam and 1/~M clonazepam. All binding assays were performed in a total volume of 200/~1 in 96-well microtitre plates. After incubation bound ligand was separated by rapid fdtration over Whatman GFB glass-fibre paper using a cell-harvester as previously described (Hall and Thor 1979). Dopamine uptake was determined on crude synaptosomal fractions as described previously (Horn et al. 1974). Monoamine and monoamine metabolite concentrations were assessed using high-pressure liquid chromatography. Tissues were homogenised in distilled water and stored frozen at - 40 °C until required. Immediately before assay, samples were thawed and acidified with perchloric acid to a final concentration of 0.8 M. After centrifugation at 10000 x g for 2 min, the supernatant was injected directly onto a 10-cm Hypersil ODS reverse-phase HPLC column. The eluting buffer employed was 0.1 M sodium

328 phosphate buffer containing 15 ~ methanol and 25 mM pentane sulphonic acid, final pH 3.5, at a flow-rate of 1.0 ml/min. Electrochemical detection was used as described previously (Cross and Joseph 1981). Using this system the monoamines serotonin and dopamine, and the acidic metabolites 5-hydroxyindole acetic acid (5-HIAA), dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) could be quantitatively assessed. In addition the noradrenaline metabolite methoxyhydroxyphenyl ethylene glycol (MHPG) could be quantified in cerebral cortex. The activities of glutamic acid decarboxylase, and marker of GABA-containing neurones, and choline acetyltransferase a marker of cholinergic neurones were assessed using radiometric techniques (Fonnum 1969; Waddington and Cross 1978). Protein was measured using a phenol reagent technique (Sutherland et al. 1949).

Immunoperoxidase staining Formalin-fixed brains from 6 infected and 6 control mice were paraffin-embedded and 12-#m sections were cut at 100-/~m levels in the coronal plane. The sections were deparaffinized and stained by the PAP method (Sternberger 1979). Briefly, the sections were incubated in (1)rabbit antiserum to HSV-1 (Maclntyre VR3, Dako), diluted to 1:500 in PBS containing 5~o normal swine serum and 0.1~ Triton-X I00 overnight at room temperature; (2)swine antirabbit lgG (Dako), diluted to 1:100 in PBS containing 0.1~o Triton-X 100 for 30 min at room temperature; and (3) PAP (Dako), diluted to 1 : 100 in PBS containing 0.1 ~ Triton-X 100 for 30 min at room temperature. The sections were washed 3 times in PBS after each incubation and were developed by incubation with diaminobenzidine (Sigma) and hydrogen peroxide for 5 min. Sections were counterstained with haematoxylin. Controls were performed using sections from infected animals with rabbit antiserum to HSV-1 which had been preadsorbed with HSV-1 antigen.

Statistical analysis For neurochemical markers for a given brain area, control and HSV-I infected group means were compared using Student's t-test (two-tail probability) when variance in the two groups was comparable, and the Mann-Whitney U-test (2-tailed) in cases of unequal variance. Analysis of variance for repeated measures was performed on log-transformed data from monoamine and monoamine metabolite levels from all brain areas examined using the BMDP2V program (Jennrich and Sampson 1977). This analysis was used to test for significant interaction between the substance measured and brain area with respect to treatment with HSV-1, e.g. whether levels of some substances change more in certain brain areas than in others as a result of infection. RESULTS

Neurochemistry (1) Acetylcholine and GABA The binding of [3H]N-methyl scopolamine, [3H]muscimol and [3H]flunitrazepam in HSV- 1-infected mouse brain did not differ from control levels for any brain

329 TABLE 1 ACETYLCHOLINE AND 7-AMINOBUTYRIC ACID MARKERS 1N HSV-I-INFECTED MICE Values expressed as percentage of control group + SEM; n = 7-10 for both groups. Brain region

Choline acetyltransferase

Glutamate decarboxylase

[3H]NMS binding

[3H]muscimol binding

[3H]FNZ binding

Cortex Striatum Hippocampus Diencephalon Brain stem Cerebellum

108 + 105 + 114 + 94 + 130 + 111 +

83 + 6 93 + 8 ND 94 + 6 99 + 5 118 + 8

102 + 4 112 + 3 ND 90 _+5 102 + 5 103

ND ND ND 108 + 11 133 + 14 78 + 5

ND ND ND 99 + 4 96 +_3 95 + 3

7 5 8 7 12 4

ND = not determined.

area examined (Table 1). Similarly, cholineacetyltransferase and glutamic acid decarboxylase activities were unchanged in HSV-infected mice (Table 1).

(2) Dopamine The binding o f [3H]spiperone to dopamine receptors in striatum of HSV-1infected mouse brain did not differ from control levels (Table 2). The rate o f uptake o f dopamine in striatum o f HSV-l-infected mouse brain was not significantly different from that o f controls (control 12.0 + 1.2; infected 15.2 + 0.9 pmol/mg protein/10 min). HSV-1 infection did result in large increases in the amount o f the D A metabolite homovanillic acid present in cortex, striatum, diencephalon and brain stem (Table 2). The increase over control levels was approximately the same in cortex, diencephalon and brain stem (3-fold), and was significantly greater than that in striatum (F(3,42) = 1.82, P < 0.05). The D A metabolite ( D O P A C ) was increased by a small but significant amount in the cortex o f infected animals, but was not different from controls in other brain areas (Table 2). The amount of D A present in HSV-l-infected brain was not different from control levels in cortex, striatum, diencephalon, brain stem (Table 2).

(3) 5-Hydroxytryptamine The binding of [ 3 H ] 5 - H T and [3H]ketanserin to serotonin receptors in the cortex o f HSV-l-infected mouse brain did not differ from control levels (Table 3). HSV-1 infection resulted in increases in the amount of the 5 - H T metabolite 5-hydroxyindoleacetic acid ( 5 - H I A A ) in cortex, striatum, brain stem and diencephalon. There were no significant differences in the increases over controls between the different brain areas (Table 3). The amount o f 5 - H T present in HSV-l-infected brain was not different from control levels in any brain area.

330 TABLE 2 D O P A M I N E M A R K E R S IN C O N T R O L A N D I N F E C T E D M O U S E B R A I N Values are e x p r e s s e d as a ng/mg protein and b fmol/mg protein; m e a n + S E M ; n = 10 for both groups.

Brain area

Control

Infected

DA a

Cortex Striatum Diencephalon Brain stem

1.8 27.2 1.1 1.4

_+ 0.9 _+ 1.9 _+ 0.1 + 0.1

2.0 27.7 1.2 1.2

_+ 0.2 + 1.5 + 0.1 _+ 0.1

DOPAC

Cortex Striatum Diencephalon Brain stem

1.2 9.0 1.5 0.9

+ 0.06 + 0.6 _+ 0.1 _+ 0.03

1.7 9.3 1.8 0.5

+ 0.1" + 0.9 _+ 0.2 _+ 0.06

HVA

Cortex Striatum Diencephalon Brain stem

2.5 23.4 2.5 0.7

+ 0.1 _+ 1.3 + 0.2 _+ 0.06

8.5 53.1 7.9 2.3

_+ 1.4"* + 6.0** _+ 1.0"* + 0.3**

[3H]Spiperone binding b (D2 receptors)

Striatum

153 + 14

149 + 10

* P < 0.05; ** P < 0.001.

TABLE 3 SEROTONIN MARKERS IN CONTROL AND INFECTED MOUSE BRAIN Values e x p r e s s e d as " ng/mg protein and b fmol/mg protein; m e a n _+ S E M ; n = 10 in both groups.

Brain area

Control

Infected

5-HT a

Cortex Striatum Diencephalon Brain stem

0.5 1.6 0.9 0.4

+ 0.03 + 0.2 + 0.1 _+ 0.03

0.6 1.2 1.1 0.9

+ 0.06 _+ 0.1 + 0.06 _+ 0.03

5-HIAA

Cortex Striatum Diencephalon Brain stem

1.6 2.7 4.4 3.1

_+ 0.2 + 0.1 + 0.2 + 0.2

2.9 5.0 7.3 4.9

+ 0.3* _+ 0.5* + 0.6* + 0.3*

[3H]5-HT binding b (SI receptors)

Cortex

71

+ 2

60

+ 2

[3H]Ketanserin binding b ($2 receptors)

Cortex

94

+ 3

95

_+2

* P < 0.01.

331 TABLE 4 NORADRENALINE MARKERS IN CORTEX OF CONTROL AND INFECTED MICE Values expressed as a ng/mg protein and b fmol/mg protein; mean + SEM of 10 animals.

Noradrenalinea MHPGa [3H]WB4101 bindingb (~tl-adrenoceptor) [3H]Rauwolscine bindingb [3H]DNA bindingb (fl-adrenoceptor)

Control

Infected

17.8 _+ 1.6 0.4 + 0.1 109 + 16

17.5 + 1.1 0.4 + 0.1 97 +_8

13 + 2 41 + 4

13 + 1 45 + 3

(4) Noradrenaline The binding of[3H]WB4101, [3H]rauwolscine and [ 3H ]dihydroalprenolol to ~1-, ~2- and fl-adrenergic receptors in the cortex of HSV-l-infected mouse brain did not differ from control levels (Table 4). The concentrations of the noradrenaline metabolite ( M H P G ) present in cortex of HSV-1-infected brain were not significantly different from control levels (Table 4).

Infectivity assays The mean values for infectivity levels (expressed as pfu/#g protein) in the HSV-l-infected brains were: cortex 0.89 + 0.22, striatum 1.74 + 0.59, diencephalon 1.63 + 0.63 and brain stem 1.84 + 0.65. There were no significant differences in levels of virus infectivity detected in any of these brain areas, and there were no significant relationships between levels of virus detected in brains of individual animals and the magnitude of neurochemical changes.

Immunohistochemistry All of the 6 HSV-l-infected brains stained immunohistochemically for HSV antigen showed similar patterns of antigen distribution. Antigen-positive cells in frontal cortex and striatum showed a diffuse pattern of distribution, with positive staining in both neurons and glia. This diffuse distribution of antigen-positive cells often continued through the thalamus and anterior hypothalamic areas. The distribution of antigenpositive cells occasionally appeared heavier on the injection side, particularly at levels nearer to the injection site. In general, however, the diffuse distribution of antigen positive cells seen in telencephalic and anterior diencephalic areas was fairly equal on both sides of the brain. Certain forebrain structures consistently showed a high concentration of heavily stained antigen-positive cells. These areas include the cingulate cortex, the septum, the pyramidal and granule cell layers of the hippocampus, the amygdala, and the internal and external cell layers of the entorhinal cortex (Fig. 1). Antigen-positive cells in these areas were predominantly large neurones, although gila and neuropil were sometimes

Fig. 1. Distribution of HSV antigen in forebrain sections of infected mouse brain. A: Low magnification at the level of the hippocampus demonstrating a high density of antigenpositive cells in hippocampal pyramidal layer and entorrhinal cortex. B: Antigen-positive pyramidal neurones in hippocampus. C: Distribution of positive staining in cingulate cortex. D: positive neurones in anterior hypothalamus. All sections counterstained with haematoxylin.

333 stained as well. The intensity of staining and distribution of antigen-positive cells in these structures generally appeared equivalent on both sides of the brain. In sections taken from more posterior levels in the infected brains, the number of diffusely located antigen-positive cells decreased noticeably. From the level of the posterior hypothalamus back, antigen-positive cells were only found in highly localised groups (Fig. 2). While the posterior diencephalon and the whole of the brain stem were mainly clear of antigen-positive cells in all 6 brains, large numbers of antigen-positive cells were always found in the substantia nigra, pars compacta-ventral tegmental area, the dorsal raphe nucleus, and nucleus locus coeruleus. The antigen-positive cells in these areas were primarily large multipolar neurones (Fig. 2). Staining of glia and surrounding neuropil was slight. Treatment of control brain sections with antiserum against H SV antigen produced no antigen-positive staining of any type. Pre-adsorbing the HSV anti-serum with HSV-l-infected PC 12 cells eliminated antigen-positive staining in sections from infected brain. The ability of the anti-sera to produce positive staining in sections from infected brain was retained after pre-adsorption with uninfected PC 12 cells. DISCUSSION The results of the present study provide evidence that HSV-1 can interact selectively with specific groups of neurones and induce neurochemical changes which are selective to these neurones. In the present experimental model HSV-1 encephalitis increased the concentrations of dopamine and serotonin metabolites, and in the brain stem virus antigen was localised to those regions known to contain the cell bodies of ascending dopamine and serotonin neurones. The changes in monoamine metabolite concentrations are unlikely to be due to non-specific factors associated with the morbid state of the infected mice. Thus in mice with encephalitis resulting from either scrapie or Theiler's virus infection, no increase in brain monoamine metabolites has been observed (unpublished observations). The pattern of changes in monoamine metabolite concentrations varies markedly with the viral agent used to induce encephalitis in mice (Lycke et al. 1970). It seems unlikely therefore that changes in monoamine systems are secondary to the encephalitis per se, but rather that they result from virus-induced changes in neuronal function. This conclusion is strengthened by the observation that neurochemical changes observed in other experimental models of virus-induced encephalitis were not found in the present HSV model. Thus infection of mice with Venezuelan equine encephalitis virus reduces activities of choline acetyltransferase and glutamic acid decarboxylase (Bonilla et al. 1982) and infections with lymphocytic choriomeningitis virus are characterised by losses of CAT activity (Oldstone et al. 1977). Rabies virus infection of mice has been shown to reduce ligand binding to muscarinic cholinergic receptors (Tsuang 1982). None of these changes were observed in the present study. As previously shown in whole brain (Lycke et al. 1970, 1972) whilst the concentrations of 5-HIAA and HVA were increased the concentrations of the parent amines were unchanged in infected mice. This pattern of changes is consistent with an increase

o

6

Zig. 2. Distribution of HSV antigen in posterior brain sections. A: Low magnification through substantia nigra, ventral tegmental area. B: Antigen positive neurones in ventral tegmental area. C: Low magnification through ~rain stem, showing positive neurones in locus coeruleus and dorsal raphe nucleus. D: Antigen staining in large neurones in locus coeruleus.

~ ~ , ~ - ~ ~7,~~¸"

~9

335 in the synthesis and release of serotonin and dopamine as f'trst suggested by Lycke et al. (1972). The mechanism by which H SV encephalitis increases monoamine turnover is not clear. It has been suggested that this increase may be a consequence of post-synaptic receptor blockade and subsequent pre-synaptic activation via postulated neuronal feedback loops (Roos and Lycke 1974). The present results are not consistent with this suggestion as monoamine receptor binding levels were unchanged. Moreover we have observed a greater increase in HVA concentration in cerebral cortex compared with striatum, whereas the reverse occurs after dopamine receptor blockade (Westerink and Korf 1975). Destruction or interference with the function of GABA or acetylcholine containing neurones, which are known to interact with monoamine neurones (Straughan and James 1979) might also explain the changes. However, markers of these neurones were unchanged. The results differ from those ofBak et al. (1977) who observed marked reductions in GAD and CAT activity in rat striatum after stereotaxic injection of H SV-1. However, the injection of small volumes containing large amounts of virus as in the study of Bak et al. (1977) is likely to result in infection of cells which are less susceptible to the small amount of virus in the large injection volume used in the present study. This technique was used in the present study in an attempt to infect only those cells which are particularly sensitive to HSV-1. The findings do not however eliminate the possibility that the increases we have observed in HVA and 5-HIAA concentrations are secondary to changes in other neuronal populations. The anatomical studies suggest that HSV-1 has a particular affinity for neurones located in or associated with limbic structures such as the hippocampus and septum and also for neurones in or near several nuclei known to contain monoaminergic cell bodies. Indeed, one of the most consistent findings was the extremely high concentration of antigen in the ventral tegmental area and substantia nigra pars compacta (DA cell-containing area). The dorsal raphe nucleus (5-HT cell-containing area) and the locus coeruleus (NA-containing area). This fmding was the more striking as these structures were often the only areas in the section to show high concentrations of antigen. A retrograde transport mechanism could explain the concentrations of antigenpositive cells in monoamine-containing nuclei. Dopamine, noradrenaline and 5-HTcontaining neurones project to the forebrain sites in which extensive HSV-1 infection occurs (Lindvall and BjOrklund 1978, 1983; Moore and Bloom 1979; Steinbusch and Nieuwenhuys 1983). HSV-1 receptors are located on nerve terminals and not on neuronal cell bodies (Vahlne et al. 1978; Ziegler and Pozos 1981). A number of studies indicate that HSV-1 infection of both peripheral (Kristensson et al. 1971, 1982; Cook and Stevens 1973) and central neurones (Bak et al. 1977; Kristensson et al. 1982) involves the intra-axonal transport of virus from nerve terminals to neuronal cell bodies. In areas such as cerebral cortex and hippocampus, infection of these neurones might result from early exposure to virus of their terminal projections perhaps because of their proximity to ventricular spaces. Thus the selective infection of these neurones may be due to transport of virus from forebrain areas to brainstem cell bodies via retrograde

336 axonal transport. While this mechanism

need not be the only factor determining

virus-cell selectivity it c a n p r o v i d e a n e x p l a n a t i o n for t h e d i s t r i b u t i o n o f i n f e c t e d cells f o u n d in t h e p r e s e n t s t u d y . These findings leave open the mechanism by which virus infection produces an i n c r e a s e in m o n o a m i n e t u r n o v e r . H o w e v e r , t h e p r e s e n t m o d e l s u g g e s t s a s e q u e n c e o f e v e n t s w h e r e b y a p a t h o l o g i c a l p r o c e s s w h i c h is initially e s t a b l i s h e d in f o r e b r a i n a r e a s m i g h t d i s r u p t n e u r o n e s in t h e b r a i n s t e m as a r e s u l t o f t h e i r a n a t o m i c a l c o n n e c t i o n s . It h a s b e e n n o t e d p r e v i o u s l y ( R o s s o r

1982) t h a t t h e s e n e u r o n e s f o r m p a r t o f t h e

i s o d e n d r i t i c c o r e w h i c h m a y c o n s t i t u t e a s t r u c t u r e o f c o m m o n p a t h o l o g y in P a r k i n s o n ' s disease and Alzheimer-type dementia.

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