Developmental Brain Research, 61 (1991) 277-280 © 1991 Elsevier Science Publishers BN. All fights reserved. 0165-3806/91/$03.50 ADONIS 016538069160411Q

BRESD 60411

277

Preserved striatal tyrosine hydroxylase activity, assessed in vivo, following neonatal hypoxia-ischemia

Robert E. Burke 1 and A v i n o a m R e c h e s 2

t Department of Neurology, Columbia University, New York City, NY 10032 (U.S.A.) and 2Department of Neurology, Haddassah University Hospital, Jerusalem (Israel)

(Accepted 7 May 1991)

Key words: Striatum; Tyrosine hydroxylase; Hypoxia-ischemia; Neonatal; Dopamine; Autoreceptor

Previous in vitro studies have shown that biochemical indices of striatal dopaminergic systems are preserved following neonatal hypoxia-ischemia. There has been no previous assessment of these systems in vivo. Using the accumulation of striatal dopa following administration of a dopa decarboxylase inhibitor as an in vivo measure of tyrosine hydroxylase (TH) activity, we have found that baseline TH activity, and its regulation by both neuronal activity and presynaptic autoreceptors are also preserved following hypoxia-ischemia.

In spite of the importance of the basal ganglia in motor control, little is known of alterations in their neuroche- mical anatomy and function in the static motor encepha- lopathies of childhood. The striatum is frequently a site of major pathology following hypoxia-ischemia during development, and this pathology is often associated with dystonia ~4, an abnormality of motor control characterized by sustained, twisting involuntary movements.

There has been some study of nigrostriatal dopamin- ergic systems in an experimental rodent model of developmental hypoxic-ischemic injury. Johnston 4 has shown in this unilateral model that, at 3 weeks of age, there is a relative preservation of striatal tyrosine hydroxylase (TH) activity and dopamine re-uptake mea- sured in vitro. We have shown that these biochemical data have a morphologic correlate; there is an increase in the relative density of striatal TH-positive neuropil staining on the injured side 3. This increase in staining persists into adulthood. In spite of these morphologic and in vitro biochemical data indicating preservation of striatal dopaminergic fibers, it remains possible that there are functional alterations of dopamine metabolism in vivo following this injury. We sought to investigate this possibility using an in vivo pharmacologic paradigm for the assessment of striatal TH activity. In this paradigm, in vivo TH activity is assessed by measuring the accu- mulation of striatal dopa following systemic administra- tion of a centrally active inhibitor of dopa decarboxylase, such as R04-4602 (benserazide) 13 or NSD-10151°.

In addition, we have previously shown that although

presynaptic striatal dopaminergic markers are preserved following hypoxic-ischemic injury, there is, in immature rats, a 16% reduction in the Bma x for both [3H]SCH 23390-labeled D 1 and [3H]spiperone-labeled D 2 striatal receptors 5. While the D1 loss is reversed by subsequent maturation, the D 2 loss persists into adulthood 6. Al- though we originally proposed that the striatal D 2 receptor loss could probably be accounted for entirely by loss of intrinsic striatal neurons, we could not exclude the possibility that part of the loss was related to alterations at the level of the presynaptic D 2 autoreceptor. The in vivo pharmacologic paradigm utilizing a dopa decarbox- ylase inhibitor permits assessment of presynaptic D 2 autoreceptor function. In the presence of the anesthetic ~-butyrolactone (GBL), nerve impulse flow in the nigro- striatal pathway is interrupted, and striatal dopa levels (in the presence of R04-4602) increase markedly, presum- ably due to cessation of dopamine release with loss of feedback inhibition of TH activity via the autoreceptor 13. This increase in dopa induced by GBL can, in turn, be blocked by administration of the dopamine agonist apomorphine, by direct action at the D 2 autoreceptor 13. We and others have previously used this ability of apomorphine as an index of D 2 autoreceptor function in vivo 9A°. We have therefore used this paradigm to assess D 2 autoreceptor function, as well as endogenous striatal TH activity, following neonatal hypoxia-ischemia.

Female rats (Sprague-Dawley) 14-16 days pregnant on arrival were received from Charles River Laboratories (Wilmington, MA). The day of delivery was defined as

Correspondence: R.E. Burke, Department of Neurology, Columbia University, 630 West 168th, NYC, NY 10032, U.S.A.

278

postnatal day 1. On day 7 or 8, the pups underwent a permanent left carotid ligation under metofane anesthe- sia as previously described 2. Ligation alone does not produce tissue injury 4. In the afternoon of the same day, pups were exposed to humidified 8% oxygen (balance nitrogen) in a plastic chamber partially submerged in a 37 °C water bath for 3 h. After behavioral recovery, pups were returned to the dam. Two controls were performed: age-matched normal control litters, and within each hypoxia-ischemia (H/I) litter, some pups underwent a sham operation, without carotid ligation, but with expo- sure to hypoxia.

At 3 weeks, H/I-exposed pups and controls were submitted to one of two protocols. In the first, they were injected with NSD-1015 (Sigma) 100 mg/kg i.p. and sacrificed after 30 min. In the second protocol, half of the pups were pretreated with apomorphine 0.5 mg/kg 40 min prior to sacrifice, the others were not. All pups were then given GBL (400 mg/kg) and NSD 35 and 30 min, respectively, prior to sacrifice. Brains were rapidly removed and placed in a rat brain matrix (Harvard) in chilled saline. A 2-mm coronal section encompassing the striata was then cut, placed on a chilled plate, and the striata were dissected bilaterally by 3 mm punch. Striatal dopa levels were determined by HPLC as previously described 8 and expressed as ng/mg wet weight tissue.

Data were analyzed by one-way analysis of variance (ANOVA) for 3 or more groups or by t-test for two group comparisons, using a significance level of 0.05.

The effect of NSD on striatal dopa accumulation in pups subjected to hypoxia-ischemia and controls is shown in Table I. As previously reported 1°, NSD injection resulted in measurable levels of striatal dopa, which is otherwise undetectable. There was no difference between striata subjected to hypoxia-ischemia and the contrala-

TABLE i

The effect of neonatal hypoxia-ischemia on NSD-induced striatal dopa accumulation

At 3 weeks of age, pups subjected to hypoxia-ischemia or controls were injected with NSD-1015 and sacrificed 30 rain later. Dopa levels are expressed as rig/rag wet weight tissue (mean + S.E.M.). There was no difference among these groups in dopa accumulation (P = 0.1, n.s, ANOVA). Exp, experimental (ligated) side of pups subjected to hypoxia; Cn, contralateral (non-ligated) side.

Normal Sham-operated Hypoxia- controls controls ischemia (n = 12) (n = 10) (n = 15)

Left Right Left Right Left Right (Exp) (Cn)

1.62 + 1.63 + 1.74 __+ 1.47 + 2.02 + 1.79 + 0.1 0.1 0.2 0.1 0.2 0.1

teral control (non-ligated) side within the same animals or in comparison to either side of sham-operated or normal controls (P = 0.1, n.s., ANOVA). We ~ and others 4 have previously noted marked variability in the extent of striatal injury in this model. In some pups, in spite of the unilateral carotid ligation and exposure to hypoxia, there is no gross or microscopic injury to the hemisphere, whereas in other pups gross hemispheric (and striatal) shrinkage is apparent. If we confined our analysis of the data in Table I to those 5 H/I pups which showed gross shrinkage, there still was no difference between the injured side and the contralateral control: Experimental (L) 2.22 + 0.3; Contralateral Control (R) 1.79 +_ 0.1 (P = 0.3, n.s., paired t-test).

The ability of apomorphine to reverse GBL-induced increases in striatal dopa levels in NSD-treated pups is shown for normal and H/I animals in Fig. I. In normal pups, administration of GBL alone led to increased levels of striatal dopa, as reported l°'z3. In pups subjected to

hypoxia-ischemia, there likewise was an increase in striatal dopa levels following GBL, both on the experi- mental (ligated) side and the contralateral control (non- ligated) side. There was no difference among these 3 conditions in the level of striatal dopa following GBL (P = 0.2, n.s., ANOVA). As shown in Fig. 1., apomorphine pretreatment reversed GBL-induced increases in normal pups and in pups subjected to hypoxia-ischemia on both

NORMAL HYPOXIA ! ISGHEMIA

(L*R) LEFT RIGHT (EXPERIMENTAL) (CONTROL)

I t o ~ p , , N o m ~ , N o A P O ( N - , O / 4.0 * * * • Group 2, No,,~l, APO (N - 11 )

~ Geoup 3, HP;NO APO (N = 6) ~' 3.0 [ iS Group4, H/I:APO(N-4)

2,0

1.0 I

APO + ~" - +

Fig. 1. The effect of hypoxic-ischemic injury on striatal dopa accumulation in the presence of NSD (100 mgtl~) and GBL (400 mg/kg), with or without apomorphine (0.5 rag/kg) pretreatment. Four groups of rats were studied: Group 1, normal rats without apomorphine; Group 2, normal rats with apomorphine; Group 3: hypoxia-ischemia without apomorphine; Group 4: hypoxia-iscbemia with apomorphine. In the normal rats, data from the r ightand left striata have been combined. In the normal rats, apomorphinc pretreatment led to a significant decrease (P = 0.001, t-test) in striatal dopa. Among animals exposed to hypoxia-ischemia, apo. morphine also led to a significant decrease in dopa levels on both the experimental (P = 0.002, t-test) and control sides (P = 0.004). There was no difference in dopa levels among the non-apomor- phine-treated animals (P = 0.2, ANOVA), or among the apomor- phine-treated animals (P = 0.9, ANOVA). *** P = 0.001, Group 1 vs Group 2; ** P = 0.004, Group 3 (Experimental side) vs Group 4 (Experimental side); * P = 0.002, Group 3 (Control side) vs Group 3 (Control side).

ligated and control sides. Levels of striatal dopa following apomorphine pretreatment were not significantly differ-

ent among the groups (P = 0.9, n.s., ANOVA). When the effect of apomorphine is expressed as a percent reduction of dopa, in relation to mean dopa values for non-apomorphine-treated pups, there remains no signifi- cant difference among them: Normais 46.1 + 4.2 (%); H/I Experimental (ligated) side: 49.6 _+ 6.4 (%); H/I Contralateral (non-ligated) side: 59.5 +_ 4.1 (%) (P = 0.2, n.s., ANOVA).

The accumulation of striatal dopa after administration of a centrally active dopa decarboxylase inhibitor is considered to be an index of in vivo TH activity ~3. The validity of this concept is supported by the ability of both neuroleptics and blockade of nigrostriatal impulse flow (by administration of GBL) to induce an increase in striatal dopa accumulation in the presence of R04-460213. Therefore, the ability of hypoxia-ischemia-injured striata to accumulate dopa to the same degree as controls in our study indicates a preservation of TH activity in vivo. This result confirms and extends Johnston's previous result, demonstrating relative preservation of striatal TH activity in vitro, following hypoxic-ischemic injury 4. This result is also compatible with earlier biochemical results indicat- ing preservation of striatal dopamine re-uptake 4, [3H]- mazindol binding 5 and striatal dopamine levels 5. These biochemical data are, in turn, compatible with the morphologic demonstration of relative preservation of TH-positive fiber staining in the hypoxia-ischemia-in- jured striatum 3. The preservation of TH activity and its regulation in vivo in these injured striata is further indicated by an increase of striatal dopa comparable to control values following GBL administration.

The ability of apomorphine to inhibit GBL-induced increases in striatal dopa levels has been interpreted as an effect at a dopaminergic terminal autoreceptor, which has been pharmacologically defined as the D 2 subtype 13. The

identity of autoreceptors as the D2 subtype is supported by recent in situ hybridization studies demonstrating binding of a selective D 2 probe within the substantia nigra H. It is possible, however, that at least some portion

1 Bannon, M.J., Bunney, E.B., Zigun, J.R., Skirboll, L.R. and Roth, R.H., Presynaptic dopamine receptors: insensitivity to kainic acid and the development of supersensitivity following chronic haloperidol, Naunyn Schmiedeberg's Arch. Pharmacol., 312 (1980) 161-165.

2 Burke, R.E. and Karanas, A.L., Quantitative morphologic analysis of striatal cholinergic neurons in perinatal asphyxia, Ann. Neurol., 27 (1990) 81-88.

3 Burke, R.E., Kent, J., Kenyon, N. and Karanas, A.L., Unilateral hypoxic-ischemic injury in neonatal rat results in a persistent increase in the density of striatal tyrosine hydroxylase immunoperoxidase staining, Dev. Brain Res., (1991) 171-179.

4 Johnston, M.V., Neurotransmitter alterations in a model of perinatal hypoxic-ischemic brain injury, Ann. Neurol., 13 (1983)

279

of dopamine autoreceptors are of the D 3 subtype, recently defined by molecular probes H. We have shown that following hypoxic-ischemic injury, apomorphine is

able to suppress GBL-induced dopa accumulation in injured striata as well as controls, examined either in absolute or relative values. This finding indicates that the

D 2 (and/or D3) autoreceptor-mediated regulation of TH activity is intact in these striata. This finding supports our original hypothesis that persistent decreases in the Bmax for striatal D 2 binding observed in this model are predominantly due to receptor loss accompanying loss of intrinsic striatal neurons, rather than loss (or down- regulation) at the level of the presynaptic dopamine

terminal. These data, as well as earlier in vitro studies, indicate

that dopaminergic terminals are spared following striatal hypoxic-ischemic injury. In this respect, this injury is like that induced by excitatory amino acids in which axons are spared. It has previously been shown that the regulation of striatal TH activity via autoreceptors persists after a kainic acid lesion 1.

The functional significance of this preservation of dopaminergic systems, in terms of dopamine regulation of striatal neural elements is unknown. We do not know whether there is a normal or increased amount of dopamine released into the extracellular space of these injured striata. It seems possible, however, in view of the preservation of these dopaminergic systems, that there may be a excessive amount of dopamine relative to the number of surviving intrinsic striatal neurons. If such is the case, this abnormality may be relevant to the abnormalities of motor control, such as dystonia, ob- served following hypoxic-ischemic injury to the striatum. It is also possible that such an abnormality may be relevant to the progression of clinical signs observed following 'static' hypoxic-ischemic brain injury 12.

R.E.B. was supported by NINDS R29NS26836, the Parkinson's Disease Foundation and by the United Cerebral Palsy Research and Education Foundation Inc. We are grateful to Eileen Janec and Vernice Jackson-Lewis for superb technical assistance. We are grateful to Ms. Joyce Rodriguez for typing the manuscript.

511-518. 5 Przedborski, S., Kostic, V., Jackson-Lewis, V., Cadet, J.L. and

Burke, R.E., Effect of unilateral perinatal hypoxic-ischemic injury in rat on dopamine D 1 and D 2 receptors and uptake sites: a quantitative autoradiographic study, J. Neurochem., in press.

6 Kostic, V., Przedborski, S., Jackson-Lewis, V., Cadet, J.L. and Burke, R.E., Effect of perinatal hypoxic-ischemic brain injury on striatal dopamine uptake sites and D~ and D 2 receptors in adult rats, Neurosci. Lett., in press.

7 Nowycky, M.C. and Roth, R.H., Presynaptic dopamine recep- tors. Development of supersensitivity following treatment with fluphenazine decanoate, Naunyn-Schmiedeberg's Arch. Pharrna- col., 300 (1977) 247-254.

8 Reches, A., Jiang, D. and Fahn, S., Catechol-o-rnethyl-trans-

280

ferase inhibition by U-0521 increases striatal utilization of levodopa, Naunyn-Schmiedeberg's Arch. Pharmacol. , 320 (1982) 34-37.

9 Reches, A., Burke, R.E., Kuhn, C.M., Hassan, M., Jackson, V.R. and Fahn, S., Tetrabenazine, an amine-depleting drug, also blocks dopamine receptors in rat brain, Z Pharm. Exp. Ther., 225 (1983) 515-521.

10 Reches, A., Wagner, H.R., Jackson-Lewis, V. and Fahn, S., Presynaptic inhibition of dopamine synthesis in rat striatum: effects of chronic dopamine depletion and receptor blockade, Brain Research, 347 (1985) 346-349.

11 Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. and Schwartz, J.-C., Molecular cloning and characterization of a

novel dopamine receptor (D3) as a target for neuroleptics, Nature, 347 (1990) 146--151.

12 Saint Hilaire, M.-H., Burke, R.E., Bressman, S.B., Brin, M.B. and Fahn, S., Delayed onset dystonia due to perinatal or early childhood asphyxia, Neurology, 41 (1991) 216-222.

13 Waiters, J.R. and Roth, R.H., Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors, Naunyn-Schmiedeberg's Arch. Pharmacol., 296 (1976) 5-14.

14 Zeman, W. and Whitlock, C.C., Symptomatic dystonias. In P.J. Vinken and G.W. Bruyn (Eds.), The Handbook of Clinical Neurology, Vol. 6, Elsevier, Amsterdam, 1968, pp. 544-566.