Brain Research, 169 (1979) 401-405 ~(3Elsevier/North-Holland Biomedical Press


Alteration in tyrosine hydroxylase, glutamic acid decarboxylase and choline acetyltransferase in basal ganglia following herpes simplex virus inoculation in rat neostriatum



Department of Physiology, Ehime University School of Medicine, Onsen-gun, Ehime 791-02 (Japan) ; (I.J.B. and C.H.M.) Department of Neurology and (LJ.B.) Department of Pharmacology, UCL.4 School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)

(Accepted February 22nd, 1979)

In a previous report 3, we described that inoculation of herpes simplex virus (HSV) in the neostriatum of rats induces typical morphological alterations of neurons in certain non-contiguous but related structures. Three to 4 days after such inoculation, virus particles in many nerve cell somas could be found in the ipsilateral substantia nigra, ipsi- and contralateral cerebral cortex and in the midbrain raph~ nuclei. These facts suggested retrograde axonal transport of the virus. Some of the neurons were also affected in the inoculated neostriatum, particularly the rare large-sized neurons. The main morphological change in the inoculated striatum consisted of massive destruction of nerve terminals. Experimental animals showed myoclonic seizures of the forelimbs (either ipsilateral or contralateral), the face, the neck and axial musculature. They also showed increased startle responses. Their muzzles and periocular regions became moist. These changes started by the third day after virus inoculation and progressively worsened until death by the fourth to sixth day. The clinical course together with the aforementioned morphological findings suggest an initial selective infection which later becomes generalized. In order to confirm the initial selective degenerative involvement of neurons and to analyze the extent quantitatively, we studied the activity of enzymes relating to the synthesis of putative transmitter substances in some of the involved neuronal systems. These include dopaminergic nigrostriatal neuronsZ,Z,7,11,18, GABAergic strionigral neurons t°.lz,t3,19,zz and two neostriatal interneuronal systems, one of which appears to be cholinergic 16 and the other GABAergic. The synthesizing enzymes for dopamine, GABA and acetylcholine, namely tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and choline acetyltransferase (CHAc), respectively, were analyzed. Preparation of HSV used in the present experiment was the same as that employed in a previous morphological studya. Three control preparations were used: (1) tissue * To whom correspondence should be addressed.

402 from normal rats; (2) tissue from contralateral sites in virus-infected rats and (3) tissue from animals inoculated with virus solution treated with ultraviolet irradiation in order to inactivate the virus. One microliter containing 10,0(0100,000 plaque-forming units of living or inactivated virus was injected stereotaxically through a Hamilton syringe, which had a glass needle tip 40-70/~m in diameter, into the left neostriatum. Stereotaxic coordinates were anterior 0.5 mm from the bregma, lateral 3 mm and depth 5 ram. The inoculated animals were housed individually and had free access to water and commercially supplied standard diet. On the second, third, fourth or fifth day after the virus inoculation, they were decapitated and whole brains were removed and kept in a chilled saline solution for 5-6 rain. Portions of the neostriatum, the pallidum, the substantia nigra and the cerebral cortex of both sides were dissected out using small pieces of razor blades. Tissue specimens thus dissected were weighed and homogenized with 0.1 ml of 0.1 ~ Triton X-100 solution. The activity of ChAc of the tissues was analyzed by a kalignost extraction of radioactive acetylcholine formed by a reaction of choline with [l-14C]acetyl coenzyme A S(Radiochemical Center, specific activity: 58 mCi/mmol). G A D activity was assayed by a radioactive 14CO2 trapping method using [l14C]L-glutamic acid (Radiochemical Center, specific activity: 50 mCi/mmol) 1. The activity of T H was determined by a radioactive 14CO2 trapping method using [1-14C] ktyrosine ~°,21(Radiochemical Center, specific activity: 57 mCi/mmol). The analysis o f ChAc, G A D and T H on each bit of tissue was done in duplicate and the mean of the two trials used in subsequent calculations. If the results of the duplicate analyses were not reasonably close, the results were discarded. Experimental animals receiving living HSV into one striatum showecl practically no alteration of their behavior on the first or second day after inoculation. Some of the animals exhibited abnormal behavior by the third day and all did by the fourth day (see above and the previous report3). A few experimental rats died on the fourth day, but most survived until the fifth or occasionally sixth day. By the fourth day the injection site in the neostriatum could be identified as a 2 mm diameter whitish area. In this area and also rostral and caudal to it the main morphological change was a massive destruction of nerve terminals. Relatively few neuron somas were affected:~ The ChAc activity progressively decreased in the ipsilateral neostriatum, relative to the contralateral side, after the HSV inoculation, namely 16 ~o loss at the third day, 36 ~o at the fourth day and 42 ~ at the fifth day (mean of 4-6 experiments each). These differences were significant by the fourth and fifth day (see Table 1). No significant change in ChAc activity was observed in the contralateral neostriatum when compared to either tissue from normal rats or from rats injected with inactivated virus. When 4 mg samples of the ipsilateral neostriatum were analyzed, the ChAc activity had decreased 38 ~ , 23 ~ and 17 ~ in tissue samples taken close to, rostrally (that part rostral to the anterior commissure) and caudal (where the neostriatum is adjacent to the pallidum) to the injection site, respectively (mean of 4 experiments at the fourth day at each injection site). ChAc activity was unchanged in the ipsilateral or contralateral pallidum, substantia nigra or cerebral cortex. G A D activity was lowered in the ipsilateral neostriatum, pallidum and substantia nigra, leaving that in the cerebral cortex unchanged. The time courses of the neo-

Contra Ipsi Contra lpsi Contra Ipsi

Contra lpsi Contra Ipsi Contra Ipsi

0.80, 1.00 0.75, 0.89 51.4,44.6 46.1,45.8

2.0, 2.0 2.8 31.0, 25.1 25.3

6.2, 6.2 9.4, 5.2 11.3,16.5 13.9,18.0

0.97 0.84 47.3 40.7

44i ±

0.31 0.12 5.9 3.2

2.18 ± 0.15 2.54 ± 0.85 26.6 ± 9.7 25.1±5.2

(4) (4) n.s. (4) (4)

(3) (3) n.s. (3) (3) n.s.

6.77 ± 1.40 (4) 5.67 4- 1.26 (4) 1 3 . 2 ± 1 . 3 (4) 1 1 . 7 ± 1 . 6 (4)

3rd day

4± ± ± 4± ± ± q4± 4-

0.89 0.99 52.7 37.3 0.24 0.16 1.81 1.66 13.6 13.8 0.006 0.006

(5) (4) (7) (7) (6) (6)

0.21 (4) 0.20 (4) 2.6 (4) 2.7 (4) 0.001 (6) 0.001 (6)

0.20 (6) 0.21 (6) 9.1 (8) 7.4 (8) 0.03 (6) 0.02(6)*

2.60 ± 0.14 2.61 ± 0.53 27.9 ± 3.6 19.8±2.5 0.06 ± 0.02 0.05 ± 0.02

7.13 ± 1.25 (6) 4.59 ± 0.47 (6)* 15.3±1.16(9) 8.9±1.49(8)* 0.42 i 0.05 (6) 0.29 ± 0.02 (6)*

4th day

* Difference from the contralateral value is significant at < 0.05.

Sub, Nigra ChAc Contra lpsi GAD Contra lpsi TH Contra ] psi Cortex ChAc Contra Ipsi GAD Contra Ipsi TH Contra Ipsi



Pallidum ChAc



Striatum ChAc

2nd day

Front rats inoculated with H S V into ±psistriatum

0.90 0.90 39.8 26.0

44± ±

0.26 0.19 6.4 3.2

(4) (4) n.s. (4) (4)*

2.83 ± 0.43 (4) 2.51 ± 0.30 (4) n.s. 26.5 i 2.7 (4) 11.4±4.1(4)*

6.85 ± 0.4 (4) 3.96 ± 0.06 (4)* 1 3 . 3 ± 0 . 7 (4) 7.14±0.67(4)*

5th day




0.78 0.94 42.4 41.6 n.d.

± 444-

0.08 0.25 1.5 4.3

2.19 ± 0.37 2.16 ± 0.50 30.3 5_ 3.4 25.4±1.4 n.d.

7.55 4- 0.73 6.68 4- 1.40 14.8±2.6 13.6±1.4 n.d.

(4) (4) (4) (3)

(3) (3) n.s. (3) (3) n.s.

(4) (4) n.s. (5) (5) n.s.

Front rats inoculated with inactive virus

0.006 4- 0.001 (6)

14.0 ~_ 2.3 (4)

1.69:9 0.24 (4)

0.27 ~- 0.03 (6)

0.06 4- 0.02 (6)

0.48 ± 0.03 (6)

From normal rats

Values are expressed as mmol product formed/kg wet weight/h. Number of experiments are shown in parentheses. Each value is the mean of duplicate determinations. Values from experiments using inactivated HSV (±psi- as well as contralateral) and from experiments without inoculation are also presented. n.d. : not determined.



404 striatal and nigral G A D decreases were similar to that of ChAc, with a progressive decrease from the third to the fifth day. By the fourth day a significant decrease in T H activity was clearly shown both in the neostriatum and the substantia nigra of ipsilateral side to the HSV inoculation. TH was not examined on other days. It is clearly demonstrated that on all occasions when HSV inactivated with ultraviolet light was injected, no alteration of enzyme activity was noted, a finding which indicated a specific biological action of living virus on the neurons. Based on the present biochemical results, the following considerations may be useful. There is a significant fall in T H activity in both the ipsilateral neostriatum, close to the site of injection of HSV, and in the ipsilateral substantia nigra. T H , the rate-limiting enzyme in dopamine biosynthesis, is contained in nerve terminals and cell bodies x5,17,2° of catecholaminergic neurons. The loss of T H activity in the striatum fits well with the severe nerve terminal destruction observed morphologically '~. The loss of this enzyme activity and the presence of sick, virus-containing neurons in the pars compacta of the substantia nigra indicate that this region was infected by retrograde axonal spread of the virus a and this in turn may be correlated with the decreased level of T H in the substantia nigra. The marked decrease of ChAc activity in the striatum also fits well with the massive nerve terminal destruction in the same region s and with the view that at least one class of striatal interneurons are cholinergic. However. this has to be reconciled with the HSV-infected neurons in the striatum being confined to a significant number of the rare large neurons and to relatively few of the medium-sized neurons. It is unknown whether either of these neuron types are cholinergic since the cell bodies of cholinergic neurons have not been conclusively identified. GABAergic neurons projecting to the substantia nigra represent at least 30 ~/~,of striatal neurons, are believed to be medium-sized neurons 3,4,6,1° and some at least have extensive axon collaterals arborizing within the striatum 14 and probably also axon collaterals to the pallidumg,2L Therefore, it is not surprising to find a significant reduction in G A D in the striatum itself. This too, fits with the massive nerve terminal destruction seen there, although the relative sparing of striatal cell bodies is puzzling. The low G A D in the substantia nigra correlates with nerve terminal degeneration around dendrites in the zona reticulata z, the low G A D in the pallidum confirms recent work by Fonnum et alP. Whether this is from HSV involvement of the nerve terminals by anterograde invasion or is secondary to cell body destruction in the striatum is not known. W o r k to clarify this is now in progress. It may be concluded that stereotaxic inoculation of herpes simplex virus into the rat neostriatum causes ipsilateral decrease in TH and G A D in the striatum and substantia nigra, and ChAc in the ipsilateral striatum. While there are some unanswered questions, this work fits reasonably well with parallel morphological studies z,4. Miss Laurie Diamond is acknowledged for her skillful technical assistance. This study was done with the support of the Margaret Bundy Scott Charitable Trust and the Murdy Foundation.

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Alteration in tyrosine hydroxylase, glutamic acid decarboxylase and choline acetyltransferase in basal ganglia following herpes simplex virus inoculation in rat neostriatum.

Brain Research, 169 (1979) 401-405 ~(3Elsevier/North-Holland Biomedical Press 401 Alteration in tyrosine hydroxylase, glutamic acid decarboxylase an...
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