John L. Hudson

GLIAL CELL LINE- DERIVED NEUROTROPHIC FACTOR REVERSES TOXIN-INDUCED INJURY TO MIDBRAIN DOPAMINERGIC NEURONS IN VIVO

Fischer 344 rats were unilaterally injected into the medial forebrain bundle with 6-hydroxydopamine (6-OHDA). Apomorphine-induced rotational behavior was used to select animals whose rotation exceeded 300 turns/h, corresponding to greater than 95% dopamine (DA) depletion in the ipsilateral striatum. Four weeks later, glial cell line-derived neurotrophic factor (GDNF) or vehicle was injected intranigrally ipsilateral to the lesion (0.1-100 micrograms). The highest dose of GDNF tested produced a marked decrease in rotational behavior. This dose also produced levels of DA in the ipsilateral substantia nigra (SN) which were not statistically different from the contralateral side. Vehicle-treated animals showed a marked DA depletion in the ipsilateral SN. These results demonstrate neurochemical and behavioral improvements in unilaterally DA-lesioned rats following intranigral administration of GDNF, suggesting that GDNF may develop into a useful therapy for Parkinsons disease.

Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinsons disease, controversy exists concerning the use of apomorphine- or D-amphetamine-induced rotations as reliable indicators of nigrostriatal dopamine depletion. Our objective was to evaluate which, if either, drug-induced behavior is more predictive of the extent of nigrostriatal dopamine depletion. Fischer 344 and Sprague-Dawley rats were unilaterally injected with 9 micrograms/4 microliters/4 min 6-hydroxydopamine into the medial forebrain bundle. The animals were behaviorally tested with apomorphine (0.05 mg/kg, s.c.) and D-amphetamine (5.0 mg/kg, s.c.). Following testing, the brains were removed and the right and left striata, substantia nigra and ventral tegmental area were dissected free and quickly frozen at -70 degrees C for analysis of catecholamine content by high performance liquid chromatography coupled with electrochemical detection. Our results indicate that an animal which has greater than a 90% depletion of dopamine in the striatum might not rotate substantially on apomorphine, without a concomitant depletion of > 50% of the DA content in the corresponding substantia nigra. No correlations were seen involving depletions of the ventral tegmental area and the extent of the lesions to the striatum. Submaximally lesioned (75-90% depleted) rats were found to rotate on D-amphetamine but not on apomorphine. In addition, control rats that did not receive lesions were often seen to rotate extensively on D-amphetamine. We therefore conclude that maximal lesions of the striatum and substantia nigra are required to generate rotations demonstrable with low dose apomorphine but not with D-amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.

A 16-channel automated rotometer system for reliable measurement of turning behavior in 6-hydroxydopamine lesioned and transplanted rats.

Unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway in rats result in a massive dopamine (DA) denervation of the ipsilateral striatum. Such animals have proven extremely useful as a model for the study of Parkinsons disease, an idiopathic neurodegenerative disorder of humans. Extensive unilateral DA disruption leaves the rat relatively normal in motor behavior; however, the extent of the lesion can be documented by drug-induced rotational behavior. When given an injection of a dopamine agonist, such as apomorphine or d-amphetamine, a lesioned animal will manifest rotational behavior; the number of turns correlates with the degree of unilateral denervation. In order to identify, for various studies, large numbers of animals with specific levels of denervation, the necessity of an automated and reliable rotational counting system (rotometer) becomes obvious. We have developed such a device that allows up to 16 rats to be tested concurrently with one inexpensive computer. This system is more reliable than, and certainly preferable to, more tedious methods such as videotaping and subsequent manual analysis or various other mechanical systems. Plexiglass, formed into large bowls, serve as the rotometer chambers. We have designed a simple, inexpensive, and accurate counting head that can be manufactured from readily available parts and that is very sturdy and reliable. This, together with a thoracic harness, completes the rotometer assembly. The rotational data, from up to 16 separate channels, is collected by a single-chip microprocessor and sent on a serial line to an IBM-type or Macintosh host computer. There, it is graphically displayed on line and subsequently saved to disk with a novel acquisition program. Files generated are in code readable by most spreadsheet software currently available. Therefore, rotational data can be imported to a number of different spreadsheets and macros used for analysis. In summary, the multiple-channel automation for monitoring turning behavior in rats, described here, is a simple, inexpensive and effective system for accurate and rapid data acquisition and analysis.

In Vivo Electrochemical Studies of Dopamine Overflow and Clearance in the Striatum of Normal and MPTP-Treated Rhesus Monkeys

Abstract: Rapid chronoamperometric recordings, using Nafion‐coated carbon‐fiber electrodes (30–90 µm o.d.), were used to investigate overflow and uptake of dopamine (DA) in the striatum of normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated rhesus monkeys. The monkeys were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging‐guided sterile stereotaxic procedures were used for implantations of the electrochemical electrodes coupled with single‐barrel micropipettes that were used to apply potassium or DA locally. Potassium evoked a robust overflow of DA‐like electrochemical signals into the brain extracellular space in the unlesioned or normal putamen and caudate nucleus of the rhesus monkeys. In contrast, potassium did not produce any detectable changes (> 97% depletion) of DA in the MPTP‐lesioned striatum. In addition, the diffusion/clearance of locally applied DA was markedly altered in the lesioned caudate nucleus and putamen compared with unlesioned striatum. Cell counts of the number of residual tyrosine hydroxylase‐positive neurons in MPTP‐treated monkeys, in conjunction with whole‐tissue levels of DA and its metabolites, showed that the MPTP lesions produced extensive damage of the nigrostriatal DA system. These data indicate that residual dopaminergic fibers remaining after MPTP lesions are dysfunctional and have a greatly diminished capacity for high‐affinity DA uptake.

Allogeneic grafts of fetal dopamine neurons: immunological reactions following active and adoptive immunizations

To define the importance of adoptive sensitization and duration of graft residence on transplant alloimmunization, behavioral and histochemical parameters were examined in unilaterally 6-OHDA-lesioned F344 rat hosts which received fetal ventral mesencephalic (VM) grafts from Wistar-Furth (WF) donors. In all animals which showed increased rotations after alloimmunization, increased numbers of T cell receptor (TcR) positive, CD8+ lymphocytes were detected in the grafts. In addition, an increased density of class I MHC antigens was seen in the graft and in the adjacent host brain. Lesser numbers of CD4+, CD11b+, and MHCII+ positive elements were also seen. Perivascular cuffing was often found in actively immunized animals. An increase in TcR+ and MHC class I+ elements was also seen in animals only adoptively immunized. The tyrosine hydroxylase positive graft area was also markedly reduced in actively immunized animals and the extent of reduction correlated with the number of cells used for immunization. These studies indicate that established allografts can evade rejection as long as host lymphocytes are not activated against graft alloantigens. In addition, increasing graft residence time in the host and adoptive immunization render the graft more susceptible to subsequent rejection.

Microglial Cell Responses to Fetal Ventral Mesencephalic Tissue Grafting and to Active and Adoptive Immunizations

Microglia express cytokines, major histocompatibility (MHC) loci, and several other immunologically important constituents. The aim of this study was to detect immunological responses of microglial cells following allogeneic dopaminergic transplantation using active and adoptive immunizations. Adult inbred Fisher 344 (F344 RT1) rats were unilaterally dopamine (DA) depleted in striatum by injection of 6-hydroxydopamine. The degree of degeneration was assessed by recording the rotational response to apomorphine. Fetal ventral mesencephalic tissue containing DA neuroblasts from Wistar-Furth (WF, RT1u) rat donors (9-12 mm CRL) were later implanted in striatum on the lesioned side. Lymph nodes and spleen cells were collected aseptically, resuspended, and diluted for isovolumetric injections. Animals selected for active immunization were injected intraperitoneally with varying amounts of WF lymphocytes. Animals selected for adoptive immunization (transferred immunity) were intraperitoneally injected with 10(8) F344 lymphocytes prepared from animals actively immunized 3 weeks previously. Monoclonal antibodies against CD4 (OX38), CD8 (OX8), CD11b (OX42), MHC class I (OX18), monomorphic MHC class II (OX-6), and ED1 and polyclonal antibodies against tyrosine hydroxylase (TH) were used for immunohistochemistry. We found that the degree of ED1-positive cell proliferation was well correlated to the immunization patterns. Groups that were actively immunized with or without prior adoptive immunization had a larger amount of reactive microglial proliferation. ED1 immunohistochemistry revealed patterns of immunolabeling of engrafted areas: 8-12 weeks after grafting in nonimmunized and adoptively immunized groups reactive microglial proliferation occurred only at the graft periphery. Active and adoptive + active immunization led to ED1-IR within the grafts themselves. At early stages nonimmunized groups had an ED1 pattern which was partially inside the grafts. At early time points nonimmunized groups contained ameboid microglial cells within the grafts which disappeared at later stages and were absent in the immunized groups. ED1-positive ameboid microglial cells within the grafts may be of graft origin and constitute a part of a continued normal development of the fetal tissue.

Conditioned apomorphine-induced turning in 6-OHDA-lesioned rats

Apomorphine-induced turning has been used to evaluate the extent of unilateral nigrostriatal denervation after 6-hydroxydopamine (6-OHDA) lesions and subsequent functional striatal reinnervation by catecholaminergic grafts. It has been noted that the pregraft rotational pattern is usually double peaked and that fetal ventral mesencephalic grafts or dopaminergic drugs will alter the second peak but leave the first relatively unchanged. We hypothesized that the first peak may be the result of factors extrinsic to the nigrostriatal dopamine system, specifically a conditioned turning response, and would, therefore, be unperturbed by the above treatments which increase dopaminergic (DA) inputs. This was investigated by injecting 6-OHDA, unilaterally, into the nigrostriatal pathway of several groups of young Fisher 344 rats. One experimental group was repeatedly tested with 0.05 mg/kg apomorphine and the rotations quantified. A second group received similar injections of apomorphine but were prevented from rotating. Vehicle control animals were also studied for both of the above experimental groups. Subsequent to the above treatment, all animals were tested unrestrained repeatedly on apomorphine. Our results support the conditioned response hypothesis in that the first peak is not present with the initial unrestrained apomorphine behavioral trial but is present upon the second and subsequent unrestrained trials. Moreover, the restrained but apomorphine-injected rats, as well as the control animals, manifest no first peak upon their first freely moving apomorphine test; the second and subsequent unrestrained apomorphine trials, in these groups, do manifest a first peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial effects of intraventricularly administered BMP-7 following a striatal 6-hydroxydopamine lesion

The present study was undertaken to investigate the effects of bone morphogenetic protein-7 (BMP-7), also named osteogenic protein-1 (OP-1), on the progression of a striatal 6-hydroxydopamine (6-OHDA) lesion. BMP-7, a member of the transforming growth factor-beta (TGF-beta) superfamily of proteins, has been shown to have protective effects in other animal models of neuronal damage. In this study, male Fischer 344 rats received striatal 6-OHDA lesions followed 1 week later by an intraventricular dose of BMP-7. No significant effect of BMP-7 treatment on spontaneous locomotor activity was observed, however BMP-7 significantly increased the density of tyrosine hydroxylase (TH) immunoreactivity (TH-ir) in the substantia nigra (SN) pars compacta, in the lesioned hemisphere [31.7+/-5.2 (optical density (O.D.) arbitrary units) control vs. 50.2+/-4.3 O.D. BMP-7-treated; p<0.05]. Interestingly, BMP-7 significantly increased TH-ir in the SN of the non-lesioned hemisphere (pars reticulata: 14.8+/-1.19 O.D. control vs. 36+/-2.6 O.D. BMP-7-treated, p<0.05; pars compacta: 29.0+/-4.9 O.D. control vs. 64.4+/-6.9 O.D. BMP-7-treated, p<0.001). A significant increase in DA concentration in the contralateral, non-lesioned hemisphere was also noted (113.2 ng/g control vs. 198.2 ng/g BMP-7-treated, p<0.01). In contrast to other intraventricularly administered neurotrophic factors, BMP-7 was not associated with an increase in the sensitivity to pain. These results suggest that BMP-7 is able to act as a dopaminotrophic agent without unwanted side effects and as such may be a useful pharmacological tool in the treatment of Parkinsons disease in humans.

Chronic Treatment with Levodopa and/or Selegiline Does Not Affect Behavioral Recovery Induced by Fetal Ventral Mesencephalic Grafts in Unilaterally 6-Hydroxydopamine-Lesioned Rats

It has been suggested that levodopa (L-dopa), a dopamine precursor used to treat Parkinsons disease, may be toxic to grafted fetal neuroblasts; if so, the use of the monoamine oxidase B inhibitor selegiline might prevent such toxicity. We randomly assigned 30 unilaterally 6-hydroxydopamine-lesioned male Sprague-Dawley rats, whose lesions were verified with low-dose apomorphine-induced rotations, to one of five treatment groups: (i) L-dopa methyl ester (125 mg/kg/day) with benserazide (a peripheral decarboxylase inhibitor; 25 mg/kg/day), (ii) L-dopa methyl ester with benserazide and selegiline (L-deprenyl; 0.5 mg/kg/day), (iii) selegiline only, (iv) and (v) vehicle (ascorbate in normal saline) only. After 2 weeks of twice-daily ip injections, the rats received fetal ventral mesencephalic grafts into the lesioned striatum; one vehicle group received sham grafts. Drug therapy was continued for 2 1/2 months post grafting. At 1 month after grafting, and every 2 weeks thereafter, the rats were tested using low-dose apomorphine-induced rotation. A 70% decrease in rotations among all grafted groups, relative to the shams, was found. No statistical differences among groups receiving various drug therapies were seen in behavior or in counts or dimensions of tyrosine hydroxylase-positive cells. We therefore conclude that, in the unilaterally lesioned rat model of Parkinsons disease, there is no adverse effect of L-dopa nor any significant effect of selegiline, either alone or coadministered with L-dopa, on behavioral recovery induced by fetal ventral mesencephalic grafts.