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L-DOPA is the most commonly used treatment for symptomatic control in patients with Parkinson's disease. Unfortunately, most patients develop severe side-effects, such as dyskinesia, upon chronic l-DOPA treatment. The patophysiology of dyskinesia is unclear; however, involvement of serotonergic nerve fibers in converting l-DOPA to dopamine has been suggested. Therefore, potassium-evoked dopamine release was studied after local application of l-DOPA in the striata of normal, dopamine- and dopamine/serotonin-lesioned l-DOPA naive, and dopamine-denervated chronically l-DOPA-treated dyskinetic rats using in vivo chronoamperometry. The results revealed that local l-DOPA administration into normal and intact hemisphere of dopamine-lesioned l-DOPA naive animals significantly increased the potassium-evoked dopamine release. l-DOPA application also increased the dopamine peak amplitude in the dopamine-depleted l-DOPA naive striatum, although these dopamine levels were several-folds lower than in the normal striatum, whereas no increased dopamine release was found in the dopamine/serotonin-denervated striatum. In dyskinetic animals, local l-DOPA application did not affect the dopamine release, resulting in significantly attenuated dopamine levels compared with those measured in l-DOPA naive dopamine-denervated striatum. To conclude, l-DOPA is most likely converted to dopamine in serotonergic nerve fibers in the dopamine-depleted striatum, but the dopamine release is several-fold lower than in normal striatum. Furthermore, l-DOPA loading does not increase the dopamine release in dyskinetic animals as found in l-DOPA naive animals, despite similar density of serotonergic innervation. Thus, the dopamine overflow produced from the serotonergic nerve fibers appears not to be the major cause of dyskinetic behavior.

l-glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system (CNS) and is associated with motor behavior and sensory perception. While microdialysis methods have been used to record tonic levels of Glu, little is known about the more rapid changes in Glu signals that may be observed in awake rats. We have reported acute recording methods using enzyme-based microelectrode arrays (MEA) with fast response time and low detection levels of Glu in anesthetized animals with minimal interference. The current paper concerns modification of the MEA design to allow for reliable measures in the brain of conscious rats. In this study, we characterized the effects of chronic implantation of the MEA into the brains of rats. We were capable of measuring Glu levels for 7 days without loss of sensitivity. We performed studies of tail-pinch induced stress, which caused a robust biphasic increase in Glu. Histological data show chronic implantation of the MEAs caused minimal injury to the CNS. Taken together, our data show that chronic recordings of tonic and phasic Glu can be carried out in awake rats for up to 17 days in vivo allowing longer term studies of Glu regulation in behaving rats.

Neuroinflammation is a chronic event in neurodegenerative disorders. In the rat model of Parkinson's disease, including a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA), antioxidant treatment affects the inflammatory process. Despite a heavy accumulation of microglia early after the injury, dopamine nerve fibre regeneration occurs. It remains unclear why this heavy accumulation of microglia is found early after the lesion in antioxidant-treated animals, or even more, what is the origin of these microglia. In this study magnetic resonance imaging (MRI) was used to elucidate whether the inflammatory response was generated from the blood or from activated brain microglia. Superparamagnetic iron oxide (SPIO) nanoparticles were injected intravenously prior to a striatal 6-OHDA injection to tag phagocytes in the blood. Rats were fed either with bilberry-enriched or control diet. T2*-weighted MRI scans were performed 1 week after the lesion, and hypointense areas were calculated from T2*-weighted images, to monitor the presence of SPIO particles. The results revealed that feeding the animals with bilberries significantly promoted accumulation of blood-derived immune cells. Gadolinium-enhanced MRI demonstrated no difference in leakage of the blood-brain barrier independent of diets. To conclude, bilberry-enriched diet promotes an influx of periphery-derived immune cells to the brain early after injury.

The assembly process of α-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson’s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of α-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of α-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and followed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substantia nigra of normal and α-synuclein knock-out mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed significant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in α-synuclein knock-out mice injected with accelerator into the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson’s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

One obstacle with grafting of dopamine neurons in Parkinson's disease is the insufficient ability of the transplant to reinnervate the host striatum. Another issue is the prospective interaction between the donor fetal tissue and the adult astrocytes of the host. To study nerve fiber growth and its interaction with immature/mature astrocytes, ventral mesencephalic (VM) organotypic rat tissue cultures from embryonic days (E) 12, E14, and E18 were studied up to 35 days in vitro (DIV), and co-cultures of E14 VM tissue and mature green fluorescent protein (GFP)-positive astrocytes were performed. Generally, nerve fibers grew from the tissue slice either in association with a monolayer of migrated astroglia surrounding the tissue (glial-associated), or distal to the astroglia as non-glial-associated outgrowth. The tyrosine hydroxylase (TH)-positive glial-associated nerve fiber outgrowth reached a plateau at 21 DIV in E12 and E14 cultures. In E18 cultures, TH-positive neurons displayed short processes and migrated onto the astrocytes. While the non-glial-associated nerve fiber outgrowth dominated the E14 cultures, it was found absent in E18 cultures. The GFP-positive cells in the VM and GFP-positive astrocyte co-cultures were generally located distal to the monolayer of migrated fetal astrocytes, a few GFP-positive cells were however observed within the astrocytic monolayer. In those cases TH-positive neurons migrated towards the GFP-positive cells. Both the non-glial- and glial-associated nerve fibers grew onto the GFP-positive cells. Taken together, the glial-associated growth has limited outgrowth compared to the non-glial-associated nerve fibers, while none of the outgrowth types were hampered by the mature astrocytes.

Neuroinflammation plays a critical role in loss of dopamine neurons during brain injury and in neurodegenerative diseases. Diets enriched in foods with antioxidant and anti-inflammatory actions may modulate this neuroinflammation. The model of 6-hydroxydopamine (6-OHDA) injected into the dorsal striatum of normal rats, causes a progressive loss of dopamine neurons in the ventral mesencephalon. In this study, we have investigated the inflammatory response following 6-OHDA injected into the striatum of adult rats treated with diet enriched in blueberry or spirulina. One week after the dopamine lesion, a similar size of dopamine degeneration was found in the striatum and in the globus pallidus in all lesioned animals. At 1 week, a significant increase in OX-6- (MHC class II) positive microglia was found in animals fed with blueberry- and spirulina-enriched diets in both the striatum and the globus pallidus. These OX-6-positive cells were located within the area of tyrosine hydroxylase (TH) -negativity. At 1 month after the lesion, the number of OX-6-positive cells was reduced in diet-treated animals while a significant increase beyond that observed at 1 week was now present in lesioned control animals. Dopamine recovery as revealed by TH-immunohistochemistry was significantly enhanced at 4 weeks postlesion in the striatum while in the globus pallidus the density of TH-positive nerve fibers was not different from control-fed lesioned animals. In conclusion, enhanced striatal dopamine recovery appeared in animals treated with diet enriched in antioxidants and anti-inflammatory phytochemicals and coincided with an early, transient increase in OX-6-positive microglia.

The present study was designed to test whether chronic neuroleptic treatment, which is known to alter both expression and density of dopamine D(2) receptors in striatal regions, has effects upon function and binding level of the cannabinoid CB(1) receptor in the basal ganglia by using receptor autoradiography. As predicted, subchronic haloperidol treatment resulted in increased binding of (3)H-raclopride and quinpirole-induced guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) in the striatum when compared to that measured in control animals. This increased D(2) receptor binding and function after 3 days washout was normalized after a 2-week washout period. Effect of haloperidol treatment was studied for CB(1) receptor binding and CP55,940-stimulated [(35)S]GTPgammaS in the striatum, globus pallidus, and substantia nigra. (3)[H]CP55,940 binding levels were found in rank order from highest to lowest in substantia nigra > globus pallidus > striatum. Furthermore, subchronic haloperidol treatment resulted in elevated binding levels of (3)[H]CP55,940 in the striatum and the substantia nigra and CB(1) receptor-stimulated [(35)S]GTPgammaS bindings in the substantia nigra after 3 days washout. These increased binding levels were normalized at 1-4 weeks after termination of haloperidol treatment. Haloperidol treatment had no significant effect on CB(1) receptor or [(35)S]GTPgammaS binding levels in globus pallidus. The results help to elucidate the underlying biochemical mechanism of CB(1) receptor supersensitivity after haloperidol treatment.

Several studies have shown that elevated plasma cholesterol levels (i.e. hypercholesterolemia) serve as a risk factor for late-onset Alzheimer's disease (AD). However, it remains unclear how hypercholesterolemia may contribute to the onset and progression of AD pathology. In order to determine the role of hypercholesterolemia at various stages of AD, we evaluated the effects of high cholesterol diet (5% cholesterol) in wild-type (WT; C57BL6) and triple-transgenic AD (3xTg-AD; Psen1, APPSwe, tauB301L) mice at 7, 14, and 20 months. The transgenic APP-Swedish/Dutch/Iowa AD mouse model (APPSwDI) was used as a control since these animals are more pathologically-accelerated and are known to exhibit extensive plaque deposition and cerebral amyloid angiopathy. Here, we describe the effects of high cholesterol diet on: (1) cognitive function and stress, (2) AD-associated pathologies, (3) neuroinflammation, (4) blood–brain barrier disruption and ventricle size, and (5) vascular dysfunction. Our data show that high dietary cholesterol increases weight, slightly impairs cognitive function, promotes glial cell activation and complement-related pathways, enhances the infiltration of blood-derived proteins and alters vascular integrity, however, it does not induce AD-related pathologies. While normal-fed 3xTg-AD mice display a typical AD-like pathology in addition to severe cognitive impairment and neuroinflammation at 20 months of age, vascular alterations are less pronounced. No microbleedings were seen by MRI, however, the ventricle size was enlarged. Triple-transgenic AD mice, on the other hand, fed a high cholesterol diet do not survive past 14 months of age. Our data indicates that cholesterol does not markedly potentiate AD-related pathology, nor does it cause significant impairments in cognition. However, it appears that high cholesterol diet markedly increases stress-related plasma corticosterone levels as well as some vessel pathologies. Together, our findings represent the first demonstration of prolonged high cholesterol diet and the examination of its effects at various stages of cerebrovascular- and AD-related disease.

Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson’s disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.

One of the drawbacks with fetal ventral mesencephalic (VM) grafts in Parkinson's disease is the limited outgrowth into the host striatum. In order to enhance graft outgrowth, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were administered by implantation of bioactive rods to the lateral part of the striatum to support grafted fetal VM implanted to the medial portion of the striatum. The polymer-based bioactive rods allow for a local secretion of neurotrophic factors over a time period of approximately 2 weeks. Moreover, glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-beta1 (TGFbeta1) were administered using the same technique. Concomitant administration of GDNF and TGFbeta1 was achieved by insertion of one GDNF and one TGFbeta1 rod. This was performed to investigate possible additive effects between GDNF and TGFbeta1. Rotational behavior, outgrowth from and nerve fiber density within the VM graft, and the number of TH-positive cells were studied. Functional compensation by reduction of rotational behavior was significantly enhanced in animals carrying bFGF and GDNF rods in comparison with animals carrying only VM graft. EGF and bFGF significantly increased the innervation density. Moreover, the nerve fiber density within the grafts was significantly enhanced by bFGF. Cell counts showed that a significantly higher number of TH-positive neurons was found in grafts treated with bFGF than that found in GDNF-treated grafts. An additive effect of TGFbeta1 and GDNF was not detectable. These results suggest that bioactive rods is a useful tool to deliver neurotrophic factors into the brain, and since bFGF was a potent factor concerning both functional, immunohistochemical and cell survival results, it might be of interest to use bFGF-secreting rods for enhancing the overall outcome of VM grafts into patients suffering from Parkinson's disease.

Adrenal chromaffin cells have been characterized by the ability to change the phenotype in response to neurotrophic factor stimulation. The adrenal gland expresses numerous trophic factors endogenously, but there is still a lack of knowledge as to how the adrenal medullary cells respond to these factors. Accordingly, we evaluated nerve fiber outgrowth and cell morphology, and measured catecholamine content in adult rat adrenal medullary tissue transplanted to the anterior chamber of the eye after exposure to neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF) compared with the effects after exposure to recombinant human nerve growth factor (rhNGF). The results show that rhNGF was the most potent factor in inducing neurite outgrowth from the grafted chromaffin cells. CNTF was also a powerful inducer of nerve fiber formation, while NT-4/5, GDNF, and bFGF were less potent. NT-3 did not produce neurite outgrowth above that seen in vehicle-treated eyes. Combining two neurotrophins, rhNGF and NT-3, reduced nerve fiber formation. Tyrosine hydroxylase (TH) immunohistochemistry revealed good cell survival in all grafts, and no morphological differences were detected with the different treatments. The adrenaline: noradrenaline: dopamine ratio was approximately 49%: 49%: 2%, independent of treatment, and the catecholamine content was equal irrespective of treatment. In conclusion, all neurotrophic factors used, except for NT-3, promoted neurite outgrowth from adult rat chromaffin transplants. Differences in outgrowth induced by the various trophic factors did not, however, change the catecholamine content in grafts when analyzed together with the graft-derived nerve plexus.

During development of the nigrostriatal dopamine system, a patchy and a diffuse type of striatal innervation pattern can be seen. It has been suggested that when fetal dopaminergic neurons, obtained from the ventral mesencephalon (VM), are grafted adjacent to mature striatal tissue, only the diffuse growth is induced. Intraocular grafting studies have indicated that the dopaminergic growth pattern might be influenced by the age of the target area, the lateral ganglionic eminence (LGE). In this study VM grafts were allowed to innervate LGE grafts of different ages. Fetal VM was implanted next to 2-wk-old or 26-day-old striatal in oculo grafts, and the resulting dopaminergic innervation of the striatal grafts was studied using tyrosine hydroxylase (TH) immunohistochemistry. In striatal grafts receiving innervation at the age of 2 wk in oculo, a patchy TH-immunoreactive growth pattern was found, while in striatal grafts innervated at the age of 26 days mainly the diffuse growth pattern was seen. This implies that grafted striatum reached maturity at approximately 1 mo of age. The age of the dopaminergic neurons at dissection and grafting was also studied concerning the ability to induce patchy growth into mature striatum. Thus, VM dissected from 13- and 18-mm fetuses was implanted to either 4-mo-old LGE (grafted in sequence) or to LGE from the same fetus (grafted simultaneously) as controls. TH-positive innervation of striatal tissue, evaluated 4 wk after implantation of VM, revealed a patchy growth pattern in LGE grafted simultaneously with 13- and 18-mm VM. However, when the striatum was mature at the time of innervation, diffuse growth was observed in striatum innervated by VM dissected from 13-mm fetuses. Interestingly, patchy growth was noted in striatal areas close to VM grafts when the dopaminergic neurons were derived from older fetuses (CRL 18 mm). Furthermore, potassium-induced dopamine release was greater in striatal grafts exhibiting the patchy growth than those showing the diffuse pattern of innervation. In conclusion, patchy dopaminergic growth can be induced in mature striatal tissue by grafting VM from older fetuses. Functionally, potassium-evoked dopamine release is enhanced in dopaminergic patches. These results have implications in terms of finding ways to induce patchy growth when grafting to the mature striatum of patients suffering from Parkinson's disease.

The 5-HT (5-hydroxytryptamine) system has been assigned a key role in the development of 3,4-dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia, mainly due to 5-HT neuronal ability to decarboxylate l-DOPA into dopamine. Nevertheless, knowledge of l-DOPA-induced events that could lead to development of dyskinesias are limited and therefore the present work has evaluated (i) the role of the 5-HT system in l-DOPA-derived dopamine synthesis when dopamine neurons are present, (ii) l-DOPA-induced effects on striatal dopamine release and clearance, and on 5-HT nerve fiber density, and (iii) the behavioral outcome of altered 5-HT transmission in dyskinetic rats. Chronoamperometric recordings demonstrated attenuated striatal l-DOPA-derived dopamine release (∼30%) upon removal of 5-HT nerve fibers in intact animals. Interestingly, four weeks of daily l-DOPA treatment yielded similar-sized dopamine peak amplitudes in intact animals as found after a 5-HT-lesion. Moreover, chronic l-DOPA exposure attenuated striatal 5-HT nerve fiber density in the absence of dopamine nerve terminals. Furthermore, fluoxetine-induced altered 5-HT transmission blocked dyskinetic behavior via action on 5-HT1A receptors. Taken together, the results indicate a central role for the 5-HT system in l-DOPA-derived dopamine synthesis and in dyskinesia, and therefore potential l-DOPA-induced deterioration of 5-HT function might reduce l-DOPA efficacy as well as promote the upcoming of motor side effects.

New strategies have recently been developed where infusion of neurotrophic factors into the brain can rescue different populations of neurons. Infusion of nerve growth factor (NGF) has been used in combination with transplants of chromaffin tissue to the striatum in the rat model of Parkinson's disease as well as to patients suffering from Alzheimer's disease. In this study we have evaluated the distribution of recombinant human NGF (rhNGF) in different brain areas and evaluated morphological and electrophysiological effects after continuous infusion for 2 weeks of rhNGF (500 micrograms/ml) into the striatum of normal rats. One week after termination of rhNGF infusion, NGF levels in the infused striata were 10-fold increased while in contralateral striata normal levels were found. Extracellular recordings from striatal neurons revealed a significantly decreased spontaneous firing rate (0.76 +/- 0.07 Hz) in rats infused with rhNGF compared to vehicle-infused control animals (1.36 +/- 0.16 Hz). Local application of rhNGF during recordings showed no direct inhibitory effect of NGF on neuronal discharge rate. Immunohistochemistry, using antibodies against acetyl cholinesterase (AChE) and glial fibrillary acidic protein (GFAP), revealed a 38.7 +/- 7.0% increase in optical density of AChE immunoreactivity close to the NGF source and an increase in GFAP-positive profiles that was restricted close to the implanted dialysis fibre. In situ hybridization showed an increase in mRNAs for choline acetyltransferase, trkA, p75 and muscarinic m2 receptor in the large neurons of rhNGF-infused striatum. Messenger RNAs for m1 and m4 receptors in striatal neurons were not changed. Thus, chronic infusion of rhNGF into the striatum caused a cholinergic hyperinnervation and reduced spontaneous activity of striatal neurons.

Long-term survival of fetal ventral mesencephalic grafts implanted into dopamine-depleted rats was studied. There was a reduction in apomorphine-induced rotations, which reached a maximum 3 months post-grafting. Striatal neuronal discharge rate was increased on the intact side of the aged grafted animals when compared with young adult striatum. Ipsilateral to the lesion, proximal to the graft, where the dopamine nerve terminal density was high but still much lower than that seen on the intact side, the firing rate was significantly lower than that measured in the intact side of the aged host. In conclusion, the increased firing rate seen in striatum after dopamine depletion is normalized by ventral mesencephalic grafts and does not show the age-related increase seen in 2-year-old rats.

Fetal ventral mesencephalic grafts have been used as a tool to counteract the symptoms of Parkinson's disease. In this study human fetal ventral mesencephalic xenografts were implanted into the lateral ventricle of unilaterally dopamine-depleted immunosuppressed rats. Rotational behavior elicited by low doses of apomorphine, host striatal dopamine receptor binding, and mRNA levels were investigated. Rotational behavior was reduced beginning 2 months after grafting. After 4 months only a small number of rotations, lasting approximately 30 min, were recorded. Seven months after transplantation, the rotational behavior was completely eleminated. Dopamine D2 receptor binding revealed significantly increased levels in sham-operated 6-hydroxydopamine- (6-OHDA) lesioned control striata. These increased levels decreased, and although still significantly higher at 4 months, normalized at the survival time of 7 months postgrafting. Regional differences were still obvious at 7 months in the dorsolateral quadrant of dorsal striatum. Dopamine D2 receptor mRNA revealed significantly increased levels in the lateral aspects of 6-OHDA-lesioned control striata, reversing by 4 months postgrafting. The D1 receptor binding revealed a moderately reduced signal in striata of lesioned animals. After grafting, this reduction became significantly lower than that seen in the control side, with a continous decrease over time. The same pattern was detected using in situ hybridization for dopamine D1 receptor mRNA, that is, moderate decreases after dopamine depletion and a significant decrease in the dorsomedial part of dorsal striatum 7 months postgrafting. Dopamine D3 receptor binding was increased after dopamine depletion, but reversed already by 4 months postgrafting. Taken together, human ventral mesencephalic xenografts are able to completely reverse apomorphine-induced rotational behavior, provided the grafts are left in vivo for a sufficient time. The increased striatal D2 receptors are reversed after grafting, but the human xenograft further suppressed the D1 receptor subtype both at binding and at mRNA levels. There was no strict correlation in the time courses of dopamine receptor changes and reduction of rotational behavior.

The human-to-rat xenograft approach offers possibilities to study aspects of primate cortex development and function without monkeys. Human fetal cortical tissue was grafted to prepared cortical cavities of immunosuppressed host rats. Fetal tissue fragments were collected after routine low-pressure vacuum aspiration abortions performed in the first trimester of gestation. Human derived neurons and human nerve fiber outgrowth were visualized by immunohistochemistry with antibodies against human neurofilament protein 70 kD (hNFP70). Ingrowth from rat host striatum or cortex into the grafts was analyzed by immunohistochemistry with antibodies against tyrosine hydroxylase. Astrocytes were evaluated by immunohistochemistry with antibodies against glial fibrillary acidic protein. The grafts grew into different sizes (1-10 mm in diameter) and contained large numbers of hNFP70-positive nerve fibers. All grafts gave rise to outgrowth of hNFP70-positive fibers into the host with partly a cortical layering; layers III and IV received a majority of the human fibers. In several cases, the graft-derived nerve fibers entered the host brain at restricted areas, while there was no crossing over of nerve fibers at the rest of the graft-host interface. Tyrosine hydroxylase-positive fibers were usually not abundant in the grafts. Interestingly, cases of massive ingrowth occurred from host striatum into the graft in a pattern suggesting "permissive sites" at the graft-host interface in the same way as outgrowth from graft to host was found. Additionally, tyrosine hydroxylase-immunoreactive fibers from host cortex were found to grow into the transplant. Glial fibrillary acidic protein immunoreactivity was increased at the interfaces between graft and host cortex or host striatum. Immunohistochemistry using antibodies against rat IgG indicated the presence of rat IgG within the grafts, and in bordering areas of host brain, possibly indicating a defective graft-host barrier. Taken together, these results show that human cortical tissue pieces grafted to cortical cavities of immunosuppressed rats survive grafting and develop, and that reciprocal nerve fiber growth between grafts and hosts occur. Human cortical neurons can grow into the rat host brain in a pattern which is partly determined by host cortical architecture.

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's 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)

Members of the fibroblast growth factor (FGF) family have earlier been shown to exert potent trophic effects on cells of both the central and peripheral nervous system. The presence of FGF-1 and -2 (FGF-1, acidic FGF; FGF-2, basic FGF) has recently been demonstrated in the dopaminergic cells of substantia nigra in rat and FGF-2 has been shown to be able to increase survival and promote neurite outgrowth of cultured mesencephalic neurons. In the present study, we have investigated possible trophic effects of FGF-1 and FGF-2 on developing rat ventral mesencephalon of different fetal stages by utilizing the in vivo method of intraocular transplantation to sympathetically denervated hosts. Survival and growth of developing grafts after growth factor treatment was followed in oculo. The Falck-Hillarp technique was used for evaluation of catecholaminergic fiber outgrowth into the host iris in whole-mount preparations. FGF-2 significantly increased the volume of the mesencephalic grafts when compared to grafts treated with the vehicle alone. The mean volume of FGF-1-treated grafts was larger than that of control grafts, but this difference was not statistically significant. FGF-1 significantly increased the area of outgrowth of dopaminergic fibers into the host iris without a corresponding increase in the number of dopaminergic neurons, as evaluated by TH immunohistochemistry. FGF-2 had no effect on dopaminergic fiber outgrowth on grafted E14 ventral mesencephalon but it did have a significant effect on fiber outgrowth from E15 and E16 grafts. Moreover, the FGF-2 treated E16 grafts contained a larger number of dopaminergic neurons as compared to controls. These findings suggest that both FGF-1 and FGF-2 may exert trophic effects on dopaminergic neurons of the ventral mesencephalon in vivo. The two FGF's enhance the outgrowth of histochemically verified catecholamine-containing nerve fibers from grafted substantia nigra neurons with factor specific patterns.

Human fetal brain tissue was obtained from first-trimester elective abortions of two women who also has schizophrenia. Portions of the embryonic hippocampus or cerebral cortex were transplanted into the anterior eye chamber of immunologically compromised athymic nude rats. In this environment, embryonic brain tissue derived from normal women generally continues organotypic growth and development for many months. Although initial survival after transplantation was normal, the tissue derived from schizophrenic women manifested less robust growth. However, cells in the transplants showed typical neuronal differentiation, with development of different neuronal types, such as pyramidal cells, granule cells, and gamma-aminobutyric acid (GABA)-containing interneurons. Rhythmic electrical activity was also observed, indicative of some local synaptic organization. The presence of messenger RNA (mRNA) for brain-derived neuronotropic factor (BDNF) was observed using in situ hybridization. The reason for the decreased rate of growth of these transplants remains unknown and the significance of the finding cannot be assessed from only two fetuses. However, these preliminary findings suggest that fetal transplants may be a useful model system for the detection of developmental pathogenic processes in the expression and transmission of schizophrenia.

Fetal human brainstem tissue including the nucleus locus coeruleus was transplanted to the anterior eye chamber of athymic nude rats. Most transplants survived and grew in the anterior chamber of the eye. After 9–15 months, the host animals were anesthetized and electrophysiological or in vivo electrochemical recordings were performed. The brainstem transplants contained spontaneously active neurons with regular single-spike firing patterns. The neurons responded to ipsilateral light stimulation with an increase in firing rate and to the α 2 -receptor agonist clonidine with significantly decreased firing rates. In vivo electrochemical studies demonstrated reproducible noradrenergic overflow after local application of potassium. Immunohistochemical evaluation of the brainstem transplants showed an abundance of tyrosine hydroxylase-positive neurons and neurites in all transplants and a dense network of neurofilament-, synapsin-, and glial fibrillary acidic protein-positive profiles throughout the grafts. Taken together, the present physiological and histochemical data indicate that it is possible to obtain transplants containing a specific monoaminergic population within the brainstem from human fetal fragments and to maintain these transplants in oculo in athymic nude rats for at least 15 months, during which time noradrenergic neurons develop.

Supplementation of antioxidants to the diet has been proved to be beneficial in aging and after brain injury. Furthermore, it has been postulated that the locus coeruleus promotes survival of dopamine neurons. Thus, this study was performed to elucidate the effects of a blueberry-enriched diet on fetal ventral mesencephalic tissue in the presence or absence of locus coeruleus utilizing the in oculo grafting method. Sprague-Dawley rats were given control diet or diet supplemented with 2% blueberries, and solid tissue pieces of fetal locus coeruleus and ventral mesencephalon were implanted as single and co-grafts. The results revealed that the presence of locus coeruleus tissue or the addition of blueberries enhanced the survival of ventral mesencephalic tyrosine hydroxylase (TH)-positive neurons, whereas no additive effects were observed for the two treatments. The density of TH-positive nerve fibers in ventral mesencephalic tissue was significantly elevated when it was attached to the locus coeruleus or by blueberry treatment, whereas the innervation of dopamine-beta-hydroxylase-positive nerve fibers was not altered. The presence of locus coeruleus tissue or bluberry supplementation reduced the number of Iba-1-positive microglia in the ventral mesencephalic portion of single and co-grafts, respectively, whereas almost no OX6 immunoreactivity was found. Furthermore, neither the attachment of ventral mesencephalic tissue nor the addition of blueberries improved the survival of TH-positive neurons in the locus coerulean grafts. To conclude, locus coeruleus and blueberries are beneficial for the survival of fetal ventral mesencephalic tissue, findings that could be useful when grafting tissue in Parkinson's disease.

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia often develops as a side effect of chronic l-DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l-DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l-DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l-DOPA naive animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l-DOPA treatment, independent on dyskinetic behavior. Acute l-DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l-DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l-DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l-DOPA replacement therapy.

Peripheral nerve "bridges" demonstrate the ability to facilitate axonal growth and regenerate adult and fetal central nervous system tissue. The purpose of this study was to determine if co-grafted peripheral nerve tissue could enhance the ability of fetal dopamine (DA) cell transplants to reinnervate host striatum that had been denervated unilaterally. Male Fisher-344 rats were unilaterally lesioned with 6-hydroxydopamine to eliminate the nigrostriatal DA pathway. A total of 31 rats demonstrated a pattern of rotation indicative of a greater than 98% depletion in DA. Rats were kept as nongrafted controls (n = 6), grafted with sciatic nerve (PN) minces (n = 6), grafted with fetal ventral mesencephalon (VM; n = 10), or co-grafted with VM and PN minces (n = 9). All groups were then tested for changes in apomorphine-induced rotational behavior. The PN control group showed no significant differences in rotation when compared to pregrafting levels and to the lesioned nongrafted group. Both the VM-grafted group and the VM-PN co-grafted group showed significant (P less than 0.01, one-way ANOVA) decreases in rotations beginning at 1.5 weeks postgrafting. There was a progressive decrease in rotations up to 12 weeks, the last test point examined. Interestingly, the co-graft group revealed a significantly greater decrease in rotation (P less than 0.05) than the VM group beginning at 5 weeks and continuing out to the 12-week test point. Histological and immunocytochemical studies showed good survival of both PN and VM grafts. The augmented recovery could not be accounted for by increased DA cell survival or host brain DA reinnervation in the co-graft group. Taken together, these findings suggest that PN tissue enhances the ability of fetal VM grafts to reinnervate host brain.

Solid pieces of human fetal mesencephalic tissue were grafted to the lateral ventricle adjacent to dopamine-depleted striata of rats immunosuppressed with cyclosporin A. Apomorphine-induced rotations were performed before and at monthly intervals after grafting. Reductions in rotations were seen at 2 months postgrafting and these reductions progressively increased. Spontaneously active dopaminergic cells were found within the grafts using extracellular single-unit recording techniques. Recordings of striatal cells ipsilateral to the graft revealed “normal” firing rates compared to those of neurons in the control striatum. In response to the local application of the dopamine antagonist cis-flupenthixol, both the dopaminergic and striatal neurons showed dose-dependant excitations. Potassium-evoked releases of electroactive species ipsilateral to the fetal human graft, measured using high-speed in vivo electrochemistry, revealed response amplitudes that were similar to control striatum when an electrode was placed adjacent to the graft; distal to the graft the responses showed smaller amplitudes but prolonged time courses. Much greater levels of dopamine and serotonin were detected in the grafts, compared to in normal rat substantia nigra, as measured with HPLC coupled to a 16-channel electrochemical array detector. Immunocytochemical studies using antibodies against tyrosine hydroxylase (TH), revealed not only TH-positive cells within the graft, but also a few positive neurons that migrated into the host striatum. Numerous TH-immunoreactive fibers penetrated into striatum and reinnervated its total volume. Taken together, these data suggest that intraventricular graft placement may be a highly efficacious technique for studying fetal brain tissues in terms of maturation, reinnervation, and function.

Mouse 3T3 fibroblasts were genetically modified by transfection with a mammalian expression vector containing the rat beta-nerve growth factor (NGF) gene. The transfected cell line, designated 3E, contains several hundred copies of the rat NGF gene and secretes high levels of biologically active NGF. Pieces of collagen gel containing the NGF-secreting 3E cells were grafted to the brains of unilaterally fimbria-fornix-lesioned rats. Grafts of the genetically modified NGF-producing cells rescued axotomized basal forebrain cholinergic neurons and significantly reduced cholinergic cell death in the medial septum as compared with rats treated with grafts of the parental 3T3 cells. Grafted fibroblast cells were detected, and rescue effects were noted up to 6 weeks after grafting. Local effects of NGF secreted by grafted cells were also seen at the gel-brain border in the form of sprouting acetylcholinesterase immunoreactive host cortical fibers. We suggest that implantation of genetically modified cells producing NGF may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease.

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used "roller-drum" organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in "roller-drum" cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.

Glial cell line-derived neurotrophic factor (GDNF) is potent for survival and promotion of nerve fibers from midbrain dopamine neurons. It is also known to exert different effects on specific subpopulations of dopamine neurons. In organotypic tissue cultures, dopamine neurons form two diverse nerve fiber growth patterns, targeting the striatum differently. The aim of this study was to investigate the effect of GDNF on the formation of dopamine nerve fibers. Organotypic tissue cultures of ventral mesencephalon of gdnf gene-deleted mice were studied. The results revealed that dopamine neurons survive in the absence of GDNF. Tyrosine hydroxylase immunoreactivity demonstrated, in gdnf knockout and wildtype cultures, nerve fiber formation with two separate morphologies occurring either in the absence or the presence of astrocytes. The outgrowth that occurred in the absence of astrocytes was unaffected by gdnf deletion, whereas nerve fibers guided by the presence of astrocytes were affected in that they reached significantly shorter distances from the gdnf gene-deleted tissue slice, compared to those measured in wildtype cultures. Treatment with GDNF reversed this effect and increased nerve fiber density independent of genotype. Furthermore, migration of astrocytes reached significantly shorter distances from the tissue slice in GDNF knockout compared to wildtype cultures. Exogenous GDNF increased astrocytic migration in gdnf gene-deleted tissue cultures, comparable to lengths observed in wildtype tissue cultures. In conclusion, cultured midbrain dopamine neurons survive in the absence of GDNF, and the addition of GDNF improved dopamine nerve fiber formation — possibly as an indirect effect of astrocytic stimulation.

Brain-derived neurotrophic factor (BDNF) expression is strongly regulated by adrenocorticosteroids via activated gluco- and mineralocorticoid receptors. Four separate promoters are located upstream of the BDNF noncoding exons I to IV and may thus be involved in adrenocorticosteroid-mediated gene regulation. In adrenalectomised rats, corticosterone (10 mg/kg s.c.) induces a robust down-regulation of both BDNF mRNA and protein levels in the hippocampus peaking at 2-8 h. To study the role of the individual promoters in the corticosterone response, we employed exon-specific riboprobe in situ hybridisation as well as real-time polymerase chain reaction (PCR) in the dentate gyrus. We found a down-regulation, mainly of exon IV and the protein-coding exon V, in nearby all hippocampal subregions, but exon II was only down-regulated in the dentate gyrus. Exon I and exon III transcripts were not affected by corticosterone treatment. The results could be confirmed with real-time PCR in the dentate gyrus. It appears as if the exon IV promoter is the major target for corticosterone-mediated transcriptional regulation of BDNF in the hippocampus.

Extracellular electrophysiological recordings were used to study the firing rate of striatal neurons before and up to 4 h after intrastriatal c-fos antisense oligonucleotide injections in urethane-anesthetized rats. A four-fold increase from baseline neuronal activity was observed between 1 and 3 h upon antisense treatment, but not after control oligonucleotide injections. We conclude that, under urethane anesthesia, which here does not affect c-fos expression in the striatum by itself, neuronal activity appears to be tonically suppressed by basal striatal c-fos expression.

Nurr1 is an orphan nuclear receptor belonging to the family of evolutionary conserved steroid/thyroid hormone receptors. It has been shown that Nurr1 is required for development of ventral mesencephalic dopaminergic cells in vivo and that Nurr1 regulates the tyrosine hydroxylase (TH) gene. The aim of this study was to investigate the possibility of finding ventral mesencephalic TH-positive neurons in Nurr1 deficient tissue when developed in the presence of wild type (WT) striatum. Therefore, fetal ventral mesencephalic tissue from embryonic day (E) 9.5–10.5 fetuses from Nurr1 mutant mice was co-cultured with lateral ganglionic eminence (LGE) from WT fetuses using the ‘roller-drum’ culture technique. TH-immunohistochemistry revealed similar number of positive neurons in WT, heterozygous, and Nurr1 deficient tissue, respectively. When ventral mesencephalon, dissected from E10.5 fetuses, was cultured alone without the presence of LGE, significantly more TH-immunoreactive neurons were found in WT and Nurr1 +/− than that seen in Nurr1 −/− cultures. In single ventral mesencephalic cultures dissected from E15.5, TH-positive neurons were found in all tissue cultures derived from knockout animals. Interestingly, the formation of TH-positive nerve fiber bundles was obvious in WT cultures while not observed in cultures of knockout tissue. When ventral mesencephalon was cultured alone in serum-free medium, almost no TH-positive neurons were found in cultures of knockout tissue. The addition of the growth factors epidermal growth factor and fibroblast growth factor-8 did not induce TH-immunoreactivity in serum-free Nurr1 −/− tissue cultures. In conclusion, TH-positive neurons may be generated in ventral mesencephalic tissue of Nurr1 deficient mice, suggesting that Nurr1 is not required for TH gene expression in ventral midbrain in vitro.

Neurotrophic factors, such as nerve growth factor (NGF), have been shown to promote the differentiation of neural crest neuroblasts into sympathetic neurons, whereas glucocorticoids promote the endocrine phenotype of adrenal medullary chromaffin cells. This pluripotency is preserved to some extent in adult chromaffin cells, with NGF and other neurotrophic factors influencing the differentiation of these cells. In this study, the effects of glial cell line-derived neurotrophic factor (GDNF) on explanted chromaffin tissue have been investigated. The localization of mRNAs corresponding to the two components of the GDNF receptor, GDNF family receptor alpha 1 (GFRalpha1) and Ret, were demonstrated in adult adrenal medullary ganglion cells. GFRalpha1 mRNA was expressed in explanted chromaffin tissue at levels dependent on the presence of serum in the medium but decreased on the addition of blocking antibodies against transforming growth factor beta (TGFbeta). However, TGFbeta1 (1 ng/ml) did not upregulate GFRalpha1 mRNA expression when added to serum-free medium. GDNF induced neurite formation from chromaffin cells, as measured by the ratio of neurite-bearing versus total number of chromaffin cells in primary cultures of adult adrenal medulla. The most potent dose inducing neurites from chromaffin cells was 100 ng/ml GDNF. However, this dose was not as efficient as that seen when chromaffin cells were stimulated with NGF (100 ng/ml). Thus, adrenal medullary cells express mRNAs for the GDNF receptor components Ret and GFRalpha1, increase their expression upon being cultured in serum-containing medium and respond to GDNF treatment with an increase in the number of cells that develop nerve processes.

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor beta (TGFbeta) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFbeta may influence NGF-induced neuronal transformation and regulation of NGF, TGFbeta1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFbeta1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFbeta luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFbeta1 and TGFbeta1 mRNA were detected. When grafts instead were treated with TGFbeta1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TbetaRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TbetaRII mRNA levels was detected after transplantation or after TGFbeta1 treatment. Instead a reduction of TbetaRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFbeta1. NGF treatment resulted in a robust innervation of the host irides. TGFbeta1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFbeta1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFbeta1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TbetaRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFbeta1 were found on NGF-induced neurite outgrowth, it was found that TGFbeta1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.

Grafting of ventral mesencephalic tissue in Parkinson's disease results in a partial dopaminergic reinnervation of host brain and dopamine agonist-induced rotational behavior is not completely reversed. To study a possible malfunction of the grafts, extracellular recordings with local applications of quinpirole were utilized and the neurophysiological results showed that a normalization of the upregulated dopamine D2 receptor supersensitivity occurred in reinnervated areas of the host striatum as well as in noninnervated areas remote from the graft innervation. Furthermore, the inhibitory effects on striatal nerve cell firing rate by the D1 receptor agonist SKF 81297 were not different in noninnervated or reinnervated areas of the striatum compared to the control side as seen from the dose-response curves. However, spontaneous striatal neuronal firing was significantly upregulated in noninnervated areas, while it was normalized in areas reached by graft-derived nerve fibers. Dual-probe microdialysis studying potassium-evoked glutamate release revealed that there was no difference in extracellular glutamate levels measured within or lateral to graft dopamine reinnervation. Thus, the upregulated spontaneous activity was not due to a difference in extracellular glutamate levels. The remaining rotational behavior seen after grafting was studied and recordings were performed in the striatum following systemic injection of the D1/D2 agonist apomorphine. The results revealed that apomorphine at the dose used to elicit turning behavior (0.05 mg/kg) still affected striatal neurons in noninnervated areas, while no effect was detected in reinnervated areas and in the intact side. However, a lower dose of apomorphine (0.005 mg/kg) showed no effects on striatal firing in graft reinnervated striata but only after dopamine depletion. In conclusion, the D2 supersensitivity is downregulated in graft-reinnervated striatum as well as in striatal areas lateral to the reinnervation when using selective D2 agonists, but the downregulation is not completely normalized when studying combined effects of D1/D2 agonists. Furthermore, the striatal neurons were firing significantly faster in noninnervated areas compared to reinnervated areas of graft-reinnervated striatum, which was most likely not due to changes in the glutamatergic input.

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.

The lazaroid U-74006F enhances survival of grafted ventral mesencephalic neurons. In this study the intraocular grafting model was used and survival and outgrowth from fetal ventral mesencephalic grafts treated with U-74006F was evaluated in nigrostriatal co-grafts. Fetal lateral ganglionic eminence was implanted into the anterior eye chamber and left to mature. Fetal ventral mesencephalon was then implanted and the eyes were treated with U-74006F. The lazaroid treatment enhanced survival of tyrosine hydroxylase (TH)-positive neurons, but did not enhance TH-positive nerve fiber growth into the striatal portions of the co-grafts. However, a marked increase in nerve fiber formation was found within the ventral mesencephalic grafts. In conclusion, increased cell survival enhanced nerve fiber formation within the ventral mesencephalic portion of the co-graft and not, as expected, in the striatal part.

During development, dopaminergic neurons innervate the developing striatal target, forming two different growth patterns: a patchy growth and a diffuse growth. Chronic treatment with the dopamine antagonist haloperidol increase the neurotrophic activity in striatum, but it is not known how this trophic activity influences different patterns of dopaminergic growth. In this paper we have studied dopaminergic growth patterns by evaluating tyrosine hydroxylase (TH)-positive growth from mature midbrain dopaminergic neurons innervating grafted fetal lateral ganglionic eminence (LGE) or fetal cortical tissue implanted into the dorsal striatum. Host dopaminergic neurons innervated LGE grafts with a patchy growth pattern, leaving large portions of the striatal graft noninnervated, and cortical grafts with diffuse, nonpatchy growth, evenly distributed over the total volume of the graft. Both types of growth patterns were enhanced over time, albeit the most pronounced change was found in the nonpatchy pattern in the cortical grafts. When fetal LGE or cortical tissues were transplanted into the dorsal striatum of chronically haloperidol-treated animals, the nonpatchy growth into fetal cortical grafts was enhanced while the patchy growth into fetal striatal tissue was not. Taken together, the results suggest that both the patchy and diffuse growth patterns may be induced from adult midbrain dopaminergic neurons depending on the target of innervation, and that increased striatal trophic activity due to blockade of the dopaminergic input stimulates growth from a subpopulation of dopaminergic neurons that produce the nonpatchy growth.

Aging differentially affects receptor function. In the present electrophysiological study we compared neuronal responsiveness to locally applied dopamine D1 and D2 receptor agonist in the striatum of female Fischer 344 rats aged 3 and 26–27 months. In a subgroup of the old rats, the nigrostriatal dopamine bundle was destroyed unilaterally with 6-hydroxydopamine (6-OHDA) to assess receptor plasticity in response to denervation. Spontaneous firing rate of striatal neurons was higher in aged compared to young rats. Higher doses of the D1 agonist SKF 38393 or the D2 agonist quinpirole were required to elicit a 50% change in firing rate in aged compared to young rats. No difference with SKF 38393 or quinpirole was detected between 6-OHDA denervated and control (nonlesioned) striatum in aged rats. Supersensitivity to D2 agonists has been reported following 6-OHDA lesions in young rats. These observations suggest that D2 receptors in aged rat striatum might not be as plastic as in younger rats.

The search for trophic factors that can support injured dopaminergic neurons and can enhance dopaminergic graft survival and outgrowth for therapeutic uses in Parkinson's disease has lately focused on members of the transforming growth factor (TGF) beta super-family. In this paper we have studied the effects of a member of the TGB beta family, glial cell line-derived neurotrophic factor (GDNF), on immature and mature ventral mesencephalic tissue grafted to the anterior chamber of the eye. The results confirm that GDNF increases survival of TH-positive neurons and enhances TH-immunoreactive nerve fiber formation when the grafts are treated during their development. The distribution of nerve terminals is densest within the area of TH-immunoreactive neurons and at the surface of the grafts. However, there is no change in the number of calcium-binding protein (CaBP)-positive neurons, suggesting that the subpopulation of TH-positive neurons that is increased are the CaBP-negative neurons of the ventral tier of pars compacta. Terminals from those neurons form the striatal patches during normal development. When the grafts are treated with GDNF after maturation, no change in TH-positive cell survival is seen but an increase of nerve terminals is still found within the cell dense area of the graft. Potassium-evoked dopamine release, measured using in vivo chronoamperometry, revealed significantly increased extracellular overflow in transplants treated with GDNF during development. The dopamine uptake blocker nomifensine significantly increased the time for clearance of the released dopamine. These data suggest that GDNF treatment of immature grafts enhances survival of TH-positive neurons, which would have innervated the striatal patches, and also increases TH-immunoreactive nerve fiber formation and dopamine release. Furthermore, GDNF treatment of mature grafts also increases dopamine fiber formation within the TH-positive neuronal area, indicating that adult dopaminergic neurons are also responsive to this agent.

In oculo transplantation provides a powerful tool to study development and gene expression of isolated brain regions. In this study we grafted striatal and mesencephalic brain tissue to the anterior eye chamber and allowed it to survive for 2 and 6 weeks. Striatal or mesencephalic pieces were either grafted alone (single grafts) or together in close connection (co-grafts). As a control normal adult untreated rats were analyzed at the striatal and hippocampal level. Using non-radioactive in situ hybridization with digoxigenin-labeled riboprobes we detected preprotachykinin-A mRNA, a neuropeptide marker for striatal neurons. We report that adult normal rats show a strong expression of preprotachykinin-A mRNA in the striatum, medial habenula and piriform cortex, verifying the specificity of the method. Mesencephalic in oculo grafts did not reveal any staining for preprotachykinin-A mRNA. In single striatal grafts only a very weak expression of preprotachykinin-A mRNA was found at both time points investigated. Co-grafts grown for 2 weeks were not different from single striatal grafts, however, when striatum was grown together with ventral mesencephalon for 6 weeks the level of preprotachykinin-A mRNA was strong and near normal adult levels. We conclude that the mesencephalic dopaminergic innervation to the striatum might be a potent stimulus to neurons expressing preprotachykinin-A mRNA.

The purpose of this study was to investigate the effects of nerve growth factor (NGF) conjugated to a monoclonal transferrin receptor antibody (OX-26) on septal transplants in oculo. Three different doses of OX-26-NGF conjugate (0.3, 3, and 50 micrograms/injection) were injected into the tail vein of young adult hosts 2, 4, and 6 weeks following intraocular transplantation of fetal forebrain tissue containing septal nuclei. Intravenous injections of OX-26 alone, NGF alone, and saline served as controls. An increase in intraocular tissue growth, as well as an increase in the intensity of immunoreactivity for p75 receptors and acetylcholinesterase, was observed following peripheral OX-26-NGF administration at the two highest doses tested. In addition, aged host rats with 16-month-old intraocular septal grafts were injected intravenously with OX-26 or OX-26-NGF (10 micrograms NGF/injection) every 2 weeks until the transplants were 24 months old. The intensity of choline acetyltransferase-like (ChAT) staining appeared to be greater and the cell bodies were larger with more processes in aged transplants in hosts treated with the OX-26-NGF conjugate than in aged OX-26-treated subjects. The present results suggest that peripheral OX-26-NGF can deliver biologically active NGF across the blood-brain barrier and have dose-dependent positive effects on both aged and developing cholinergic neurons in septal transplants.

Neuroinflammation is found both in the brain of humans suffering from Parkinson's disease and in animal models of disease. It is suggested to be involved in the pathogenesis of the disease. In the present study, in order to study the effects of antioxidants on neuroinflammation, microglial phenotypes were evaluated in rats fed with diets containing bilberries, blueberries, or crowberries at 1 and 4 weeks following striatal injection of 6-hydroxydopamine. The dopamine innervation was visualized using antibodies raised against tyrosine hydroxlase (TH) in the striatum and in the globus pallidus. One week post-lesion, the expression of Iba1-positive cells, a general microglial marker, was significantly increased in the striatum of all animals fed with antioxidant-enriched diets compared to control-diet fed animals, while the diameter of the TH-negative zone was similar in all animals. At four weeks post-lesion, the Iba1-positive microglia was significantly reduced in animals fed with antioxidant-enriched diets. The diameter of the TH-negative zone was significantly reduced in animals fed bilberry and crowberry. The expression and distribution of ED1-positive cells was similar to that of Iba1-positive cells found in the lesioned areas. A cell division marker Ki67 revealed that few microglia were proliferating in crowberry-treated animals. Otherwise dividing cells were associated with blood capillary cells. Although the antioxidant level should be equal in the entire brain, no regeneration was found in globus pallidus, suggesting the mechanism promoting regeneration in the striatum is not effective in the globus pallidus. In conclusion, diets rich in bilberries and crowberries and with high contents of antioxidants stimulate an early phase of accumulation of reactive migroglia that fades at longer time points i.e. promotes regeneration of the striatal dopamine system.

In the present study the hypothesis has been tested if the dopamine releasing drug d -amphetamine via volume transmission can, at least partly, restore dopamine communication in the dopaminergically denervated neostriatum of rats. The experimental model used, has been a unilateral 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons, based on nigral microinjections of this neurotoxin. Studies on c-fos like immunoreactivity after systemic d -amphetamine treatment demonstrated a wide-spread appearance of c-fos like immunoreactive neuronal nuclear profiles within the neostriatum on both the unlesioned and denervated side. In the unlesioned neostriatum a peak density of c-fos like immunoreactive profiles was found within the central part of the neostriatum, while on the denervated side the distribution pattern of c-fos like immunoreactive profiles peaked medially and gradually declined in a lateral direction. The microdialysis experiments demonstrated, after systemic d-amphetamine treatment, a marked and sustained increase of extracellular dopamine levels in the neostriatum on the unlesioned side, while no increases in the extracellular dopamine levels were observed on the dopaminergically denervated neostriatum. In the electrophysiological experiments, systemic d -amphetamine treatment produced an inhibition of the neuronal activity on the denervated side which showed a significant increase in basal discharge rate compared with the recordings obtained from the striata on the unlesioned side. The present immunocytochemical microdialysis and electrophysiological analysis provides evidence that in the unilaterally markedly dopamine depleted neostriatum with clearcut signs of dopamine receptor supersensitivity (rotational behaviour results), dopamine transmission may be partly restored via systemic d -amphetamine treatment through the release of dopamine, predominantly from the unlesioned neostriatum, which may diffuse into the cerebrospinal fluid to reach the contralateral dopaminergically denervated neostriatum.

It has previously been shown that the chromaffin cells of the adrenal medulla respond to nerve growth factor (NGF) with neurite outgrowth and increased cell survival in tissue culture or after grafting. In the present study we evaluated the dose dependency in neurite outgrowth from chromaffin tissue to recombinant human NGF (rhNGF). Therefore, pieces of adrenal medullary tissue from adult rat were grafted into the anterior chamber of the eye of previously sympathectomized recepients. Survival time was 4 weeks. At grafting and at Days 7, 14, and 21 postgrafting, the eyes were injected with 5 microliters of rhNGF at concentrations of 10, 30, 60, 100, 150, and 200 micrograms/ml, or with a control solution. All grafts, including the controls, survived well and became vascularized. At the low doses of rhNGF, 10 and 30 micrograms/ml, a small area of the irides was reinnervated and the density of the nerve fiber network was low. The maximal response was obtained at 100 micrograms rhNGF/ml. Using larger concentrations of 150 and 200 micrograms rhNGF/ml, the density of the nerve fiber network did not change, but the reinnervated area of the irides was significantly decreased compared to the outgrowth seen in irides treated with 100 micrograms/ml. In conclusion, adult rat chromaffin tissue responds to rhNGF in a dose-dependent manner. However, at the highest doses used, the outgrowth area was suboptimal, although nerve fiber density was maximal. These results indicate that to obtain maximal effects, the dose of NGF is critical.

Glial cell line-derived neurotrophic factor (GDNF) is a novel member of the transforming growth factor-beta superfamily with potent trophic effects on dopamine neurons. Kainate-induced epileptic seizures have been shown to induce gene expression of trophic factors, particularly members of neurotrophin or fibroblast growth factor families, in the hippocampus. In this study, we examined the effects of kainate (12 mg/kg, i.p.)-induced epileptic seizures on the expression of the novel neurotrophic factor GDNF in the hippocampus. While GDNF messenger RNA was not detected during development or in normal adult rats in the hippocampus, kainate-induced epileptic seizures markedly increased GDNF messenger RNA in scattered neurons in the dentate granule layer 3 h after injection. Six hours after kainate almost all dentate granule cells and expressed GDNF messenger RNA. The increase in GDNF messenger RNA in the dentate granule layer returned almost to control levels 24 h after kainate; however, there was still expression of GDNF messenger RNA in the hilus/CA4 and also in pyramidal neurons in areas CA1-CA3. We conclude that GDNF messenger RNA is regulated, in part, via glutamate-mediated excitation and may play a role in long-lasting structural and/or functional reorganization in the hippocampal formation.

Studies of dopamine (DA) clearance and diffusion were carried out after unilateral destruction of striatal DA afferents using intraparenchymal injection of 6-hydroxydopamine (6-OHDA). Rats were screened for the extent of DA depletion by testing for contralateral rotations induced by 0.05 mg/kg of apomorphine. Exogenous DA clearance and diffusion were determined using rapid (5 Hz) in vivo chronoamperometry with Nafion-coated carbon-fiber electrodes. Local applications of DA by pressure ejection from micropipettes into the lesioned vs. nonlesioned striata, with dose adjusted to elicit roughly equivalent amplitudes of electroactive signals, manifested a much prolonged clearance time on the lesioned side. A second protocol involved administration of equal volumes (amounts) of DA into lesioned vs. nonlesioned striata. In such cases, a volume of DA which manifested no detectable signal on the intact side produced a pronounced electrochemical signal on the lesioned side. When nomifensine, a high-affinity DA uptake inhibitor, was locally applied into the intact striatum, a subsequent ejection of DA produced a much larger signal than when the same volume was given before nomifensine. Very little or no effects of nomifensine were seen in 6-OHDA-lesioned striata. Taken together, these data indicate a much prolonged clearance time of DA in 6-OHDA-denervated striata. Moreover, the data also suggest that the primary mechanism underlying this effect is the loss of high affinity neuronal uptake. Thus, such changes in DA clearance may help account for some of the well documented compensatory phenomena which occur after DA-depleting lesions in animals and in man.

The potential role of glial cell line-derived neurotrophic factor (GDNF) as a trophic molecule for midbrain dopamine neurons was examined using two different approaches: in situ hybridization and intraocular transplantation. The presence of mRNA for GDNF was noted in striatal and ventral limbic dopaminergic target areas in the developing (E20-P7) rat, but not the adult rat. Signals were also found in nondopaminergic areas during maturation, such as the cerebellar anlage, spinal cord, and thalamus. Lesions of the nigrostriatal pathway in neonatal or adult rats, using 6-hydroxydopamine injected into the medial forebrain bundle, did not elicit upregulation of mRNA for GDNF. Grafts of fetal ventral mesencephalon in the anterior eye chamber were exposed to repeated injections of GDNF, which elicited a marked and dose-dependent increase in transplant volume. A low (0.1 microgram/eye) and high (1 microgram/eye) dose of GDNF both led to a somewhat larger mean area of dopamine fiber outgrowth into host irides. In the transplants, cell counts of tyrosine hydroxylase (TH)-immunoreactive neurons revealed a doubling of cell numbers in the low-dose group and about four times as many cells in the high-GDNF-dose group compared to controls. Moreover, the density of TH-immunoreactive nerve fibers was markedly and significantly higher in transplants treated with the high GDNF dose. Since the volumes of these transplants were also larger, the total amount of both TH-positive cells and TH-positive nerve fibers was many-fold greater in the high-GDNF group than that in the controls. Taken together, these data support the concept that GDNF functions as a dopaminotrophic factor in vivo.

Two patients with advanced Parkinson's disease were followed for 6 months before, and 18 months after, receiving stereotaxic grafts of fetal mesencephalic tissue from aborted human fetuses. Parameters studied included a series of standardized tests of movement, response to levodopa, electrophysiological recording of the motor readiness potential, and positron emission tomography (PET) with ligands based upon levodopa and upon the dopamine reuptake inhibitor nomifensine. The patients each received stereotaxic implantation of ventral mesencephalic tissue containing midbrain dopamine neurons from aborted human fetuses of 8 to 10 weeks gestational age into the caudate and putamen of one hemisphere. Throughout their 18-month course, the patients were treated with cyclosporine, azathioprine, and glucocorticoids to minimize the risk of graft rejection. There were no significant complications from the procedure, but there was also no major change in their assessment of impairment on the Hoehn and Yahr scale. However, significant changes were observed in clinical, electrophysiological, and PET measures. Changes in these parameters, apparent at 6 months postoperatively, were described in detail in a previous report. The purpose of this present report is to provide follow-up data from the subsequent year with an emphasis on longitudinal evaluation methodology. Standardized clinical testing showed a small but long-term improvement in the first of the two patients. Following the operation, she was able to walk in "off" periods, which she had not been able to do preoperatively. This improvement was accompanied by increased walking speed and reduction in the time necessary to perform a series of pronation and supination movements using both hands. Although these improvements have continued throughout the postoperative period, they have not alleviated her basic neurological impairment. The second patient showed similar improvement during the first 6 months; she then reverted to her preoperative status at the end of the 18-month follow-up period. The electrophysiological recordings were consistent with the clinical findings. Both patients had significant changes in the motor readiness (bereitschafts) potential amplitude, which was greatest 5 to 7 months postoperation. The amplitude of the potential declined subsequently for both patients, but remained significantly elevated over the preoperative baseline for patient 1. The analysis of the PET scans was somewhat compromised by technical problems in the preoperative scans. However, they are also consistent with the clinical data. In comparisons of the operated and the unoperated sides, fluoro-dopa showed increased uptake in the caudate nucleus of patient 1 at 6 months and at 13 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological recordings were performed on caudate neurons in rats with dopamine (DA) depleted striatum in combination with pertussis toxin (PT) lesions. Pertussis toxin inactivates the G protein coupled to D2 receptors. DA depletions were performed by unilateral injections of 6-hydroxydopamine (6-OHDA). After the 6-OHDA lesion, rats were challenged with low doses of apomorphine. When a double peak rotational pattern was stable over repeated rotational tests, PT was injected into striatum ipsilateral to the DA depleted side. Two days after the PT injections extracellular recordings with local applications of the D1 agonist SKF 38393 and the D2 agonist N-0437 were performed. Spontaneous firing rates, measured before drug application, were elevated in animals with both 6-OHDA and 6-OHDA/PT combination of lesions. In rats with only 6-OHDA lesions, a supersensitivity to N-0437 was observed, while no significant change in response to the D1 agonist was detected. Recordings from caudate neurons in rats with a combination of 6-OHDA and PT resulted in no response to the D2 agonist. However, a subsensitivity to the D1 agonist was detected and only 60% of neurons were inhibited by SKF 38393. Taken together, these data suggest an interaction between the D1 and D2 receptors, which is revealed only after an upregulation of the D2 receptors and subsequent blockade of D2 mediated effects.

The purpose of the present study was to investigate the viability, growth characteristics and neuropeptide expression of intraocular superior cervical ganglia (SCG) grafts from neonatal (1-3 d), mature (4-5 months) and aged (20-24 months) rats. In vivo measurements, Falck-Hillarp histochemistry using iris whole mounts to assess catecholamine fiber outgrowth and immunocytochemical localization of tyrosine hydroxylase (TH-), neuropeptide Y (NPY-), leu-enkephalin (ENK-) and calcitonin gene-related peptide (CGRP-) like immunoreactivity (LI) were used. Measurements indicated a marked decrease in volume during the first week after grafting and a more gradual decrease thereafter. This was most evident in newborn SCG. With prolonged survival time, the newborn ganglia demonstrated more varicose nerve terminals and increased catecholamine fiber outgrowth and arborization. Extensive and complex outgrowth of catecholamine fibers with varicose nerve terminals occurred more rapidly with mature and aged ganglia. In situ, all ganglion cell bodies and fibers demonstrated TH-LI. Localization of TH-LI after grafting indicated an increase in fiber density and a decrease in cell body density of 65%, 40% and 40% in newborn, mature and aged ganglia respectively. NPY-LI in cell bodies had a perinuclear fluorescence pattern consistent with localization in the Golgi apparatus. Grafting of newborn, mature and aged SCG resulted in a 20%, 20% and 35% decrease respectively of cell bodies containing NPY-LI. A concommitant increase in fiber diameter, fluorescence intensity and extent of arborization was observed. The characteristic distribution of ENK-LI in cell bodies and axons in mature and aged ganglia was not affected by grafting. However, there was a greater than 50% reduction in the number of cell bodies expressing ENK-LI. CGRP-LI, localized in fibers and axon terminals in SCG in situ, was not identified after grafting. In summary, we have demonstrated that SCG from all age groups form extensive fiber networks and continue neuropeptide expression after intraocular grafting. This was seen best in mature and aged donors and may suggest a role for SCG transplants in the replacement of monoaminergic neurons in the CNS.