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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.

Glial cell line-derived neurotrophic factor (GDNF) is a potent factor for the ventral mesencephalic dopamine neurons. However, studies on the Gdnf gene deleted (Gdnf(-/-)) mouse have been limited t ...

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.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3’UTR targeting may constitute a useful tool in analyzing gene function., Author Summary Intracranial delivery of GDNF has been attempted for Parkinson’s disease (PD) treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we utilize an innovative genetic approach by targeting the 3’UTR regulation of Gdnf in mice. Such animals express elevated levels of Gdnf exclusively in natively Gdnf-expressing cells, enabling dissection of endogenous GDNF functions in vivo. We show that endogenous GDNF regulates dopamine system development and function and protects mice in a rodent PD model without side effects associated with ectopic GDNF applications. Further, we report how GDNF levels regulate kidney development and identify microRNAs which control GDNF expression. Our study highlights the importance of correct spatial expression of GDNF and opens a novel approach to study gene function in mice.

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.

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.

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.

In animal models of Parkinson's disease, transplanted fetal mesencephalic dopaminergic neurons can innervate the dopamine-depleted host brain, but it is unclear why large portions of the host striatum are left uninnervated. During normal development, the dopaminergic innervation first occurs in the form of a dense patchy pattern in the striatum, followed by a widespread nerve fiber network. Using intraocular double grafts we have investigated dopaminergic growth patterns initiated when ventral mesencephalic grafts innervate striatal targets. The fetal lateral ganglionic eminence was implanted into the anterior eye chamber. After maturation in oculo, fetal ventral mesencephalon was implanted and placed in contact with the first graft. In other animals the two pieces of tissue were implanted simultaneously. Tyrosine hydroxylase (TH) immunohistochemistry revealed a pattern of dense TH-positive patches throughout the total volume of the striatal grafts in simultaneously transplanted cografts, while a widespread, less dense, pattern was found when mature striatal transplants were innervated by fetal dopaminergic grafts. To investigate which type or types of growth patterns that developed after grafting to striatum in situ of an adult host, fetal ventral mesencephalic tissue was implanted into the lateral ventricle adjacent to the dopamine-lesioned striatum. After maturation of the mesencephalic graft, the fetal lateral ganglionic eminence was implanted into the reinnervated part of the host striatum. TH immunohistochemistry revealed a few nerve fibers within the striatal graft and the growth pattern was of the widespread type. In conclusion, grafted dopaminergic neurons preferably innervate mature striatum with a widespread sparse nerve fiber network, while the innervation of the immature striatum occurs in the form of dense patches. Furthermore, when the patchy pattern is formed, the total volume of the striatal target is innervated while growth of the widespread type terminates prior to reaching distal striatal parts. Thus, the growth pattern seems essential to the final volume that is innervated. Once the widespread growth pattern is initiated, the presence of immature striatum does not change the dopaminergic growth pattern.

L-DOPA-induced dyskinesia is a common side effect developed after chronic treatment with 3,4-dihydroxyphenyl-L-alanine (L-DOPA) in Parkinson's disease. The biological mechanisms behind this side effect are not fully comprehended although involvement of dopaminergic, serotonergic, and glutamatergic systems has been suggested. The present study utilizes in vivo amperometry to investigate the impact from unilateral 6-hydroxydopamine lesions and L-DOPA (4 mg/kg, including benserazide 15 mg/kg) -induced dyskinetic behavior on striatal basal extracellular glutamate concentration and potassium-evoked glutamate release in urethane-anesthetized rats. Recordings were performed before and after local L-DOPA application in the striatum. In addition, effects from the 5-HT1A receptor agonist (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OHDPAT; 1 mg/kg) was assessed on glutamate release and on dyskinetic behavior. The results revealed a bilateral similar to 30% reduction of basal extracellular glutamate concentration and attenuated potassium-evoked glutamate release after a unilateral dopamine-depletion in L-DOPA naive animals. In dyskinetic subjects, basal glutamate concentration was comparable to normal controls, although potassium-evoked glutamate release was reduced to similar levels as in drug naive dopamine-lesioned animals. Furthermore, acute striatal L-DOPA administration attenuated glutamate release in all groups, except in the dopamine-lesioned striatum of dyskinetic animals. Co-administration of 8-OHDPAT and L-DOPA decreased dyskinesia in dopamine-lesioned animals, but did not affect potassium-evoked glutamate release, which was seen in normal animals. These findings indicate altered glutamate transmission upon dopamine-depletion and dyskinesia.

The nicotinic cholinergic antagonist alpha-bungarotoxin (alpha-BT) binds throughout the rat hippocampal formation. The binding is displaceable by d-tubocurarine. The most heavily labeled cells are GABA- containing interneurons in the dentate and in Ammon's horn. These neurons have several different morphologies and contain several neuropeptides. alpha-BT-labeled interneurons in the dentate are small cells between the granular and molecular layers that often contain neuropeptide Y. alpha-BT-labeled interneurons in CA1 are medium-sized interneurons, occasionally found in stratum pyramidale, but more often found in stratum radiatum and stratum lacunosum moleculare. These neurons often contain cholecystokinin. The largest alpha-BT-labeled interneurons are found in CA3, in both stratum radiatum and stratum lucidum. These neurons are multipolar and frequently are autofluorescent. They often contain somatostatin or cholecystokinin. These large interneurons have been found to receive medial septal innervation and may also have projections that provide inhibitory feedback directly to the medial septal nucleus. The cholinergic innervation of the hippocampus from the medial septal nucleus is under the trophic regulation of NGF and brain-derived neurotrophic factor, even in adult life. Expression of mRNA for both these factors is increased in CA3 and the dentate after intraventricular administration of alpha-BT, but not after administration of the muscarinic antagonist atropine. alpha-BT-sensitive cholinergic receptors on inhibitory interneurons may be critical to medial septal regulation of the hippocampal activity, including the habituation of response to sensory input.

Chronic implantation of neurotransmitter measuring devices is essential for awake, behavioral studies occurring over multiple days. Little is known regarding the effects of long term implantation on surrounding brain parenchyma and the resulting alterations in the functional properties of this tissue. We examined the extent of tissue damage produced by chronic implantation of either ceramic microelectrode arrays (MEAs) or microdialysis probes. Histological studies were carried out on fixed tissues using stains for neurons (cresyl violet), astrocytes (GFAP), microglia (Iba1), glutamatergic nerve fibers (VGLUT1), and the blood-brain barrier (SMI-71). Nissl staining showed pronounced tissue body loss with microdialysis implants compared to MEAs. The MEAs produced mild gliosis extending 50-100 microm from the tracks, with a significant change in the affected areas starting at 3 days. By contrast, the microdialysis probes produced gliosis extending 200-300 microm from the track, which was significant at 3 and 7 days. Markers for microglia and glutamatergic fibers supported that the MEAs produce minimal damage with significant changes occurring only at 3 and 7 days that return to control levels by 1 month. SMI-71 staining supported the integrity of the blood-brain barrier out to 1 week for both the microdialysis probes and the MEAs. This data support that the ceramic MEA's small size and biocompatibility are necessary to accurately measure neurotransmitter levels in the intact brain. The minimal invasiveness of the MEAs reduce tissue loss, allowing for long term (>6 month) electrochemical and electrophysiological monitoring of brain activity.

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.

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.

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.

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3–21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 μm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.

During the past two decades several revisions of the concepts underlying interneuronal communication in the central nervous system have been advanced. We propose here to classify communicational phenomena between cells of the central neural tissue under two general frames: "wiring" and "volume" transmission. "Wiring" transmission is defined as intercellular communication occurring through a well-defined connecting structure. Thus, wiring transmission is characterized by the presence of physically identifiable communication channels within the neuronal and/or glial cell network. It includes synaptic transmission but also other types of intercellular communication through a connecting structure (e.g., gap junctions). "Volume" transmission is characterized by signal diffusion in a three-dimensional fashion within the brain extracellular fluid. Thus, multiple, structurally often not well characterized extracellular pathways connect intercommunicating cells. Volume transmission includes short- (but larger than synaptic cleft, i.e. about 20 nm) and long-distance diffusion of signals through the extracellular and cerebrospinal fluid. It must be underlined that the definitions of wiring and volume transmission focus on the modality of transmission and are neutral with respect to the source and target of the transmission, as well as type of informational substance transmitted. Therefore, any cell present in the neural tissue (neurons, astroglia, microglia, ependyma, tanycytes, etc.) can be a source or a target of wiring and volume transmission. In this paper we discuss the basic definitions and some distinctive characteristics of the two types of transmission. In addition, we review the evidence for different types of intercellular communication besides synaptic transmission in the central nervous system during phylogeny, and in vertebrates in physiological and pathological conditions.

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.

Grafting of human fetal tissue fragments has been used successfully in experimental and clinical trials. The development of techniques to store human fetal tissue fragments for longer time periods would allow to establish temporary tissue banks. We dissected several human cortical tissue fragments from one fetus and tested different storage conditions (cooling, freezing, culturing). After storage, the tissue fragments were transplanted into cavities in the cortex of host rats and the volume of the surviving grafts calculated. We report that human cortical tissue fragments grafted immediately after dissection (control group) or grafted after storage for 3 h in cryopreservation medium at room temperature survived grafting and resulted in graft sizes of 102 +/- 26 mm3 and 242 +/- 210 mm3, respectively, however, statistically not different. When the human cortical tissue fragments were slowly frozen and stored for 1 wk and/or when the fragments were cultured for 1 week in culture medium using a roller tube technique, grafts did not survive under our conditions. However, when the human cortical tissue fragments were stored for 1 week at +4 degrees C in cryopreservation medium, the graft size (48 +/- 24 mm3) was reduced but statistically not different from the control group. We conclude that human cortical tissue fragments can be stored at +4 degrees C for at least 1 wk without major loss of ability to survive grafting.

The ability of grafted fetal ventral mesencephalic dopaminergic (DAergic) neuroblasts to reinnervate the unilaterally DA denervated rat striatum and improve motoric asymmetry has been well documented in several laboratories. The importance of host target specificity, and catecholamine (CA) neurotransmitter species, in the ability of grafts to ameliorate rotational responses to apomorphine and to affect electrophysiological characteristics of striatal neurons has not been systematically studied. We unilaterally lesioned Sprague-Dawley rats with 6-hydroxydopamine (6-OHDA) and verified the lesions using apomorphine (0.05 mg/kg, s.c.)-induced rotational behavior. Some of the animals subsequently received, intrastriatally, either DA neuroblasts from ventral mesencephalon that normally innervate the striatum, or from arcuate nucleus that do not. Additionally, two other groups were included that received either a CAergic graft from the noradrenergic nucleus locus coeruleus or a graft of cerebral cortex, which normally projects to the striatum but does not contain CAergic neurons. Only the fetal ventral mesencephalic grafts were able to reduce apomorphine- induced rotations and normalize striatal cell firing rates; striatal cell firing rates with ventral mesencephalic grafts were 1.43 Hz +/- 0.22, with arcuate nucleus grafts were 6.03 +/- 0.73, with locus coeruleus grafts were 4.71 +/- 0.74, and with cerebral cortex grafts were 4.36 +/- 0.45. Moreover, only the ventral mesencephalic grafts produced a dense tyrosine hydroxylase (TH)-immunoreactive nerve terminal network in the striatum; in contrast, the arcuate nucleus grafts did not reinnervate the striatum. In locus coeruleus grafted striata, few very long TH-positive axons were seen. We thus conclude that target specificity and neurotransmitter type are critically important in the ability of a graft to functionally reinnervate the 6- OHDA denervated striatum.

Human fetal mesencephalic tissue obtained from elective first-trimester abortions was grafted to 6-hydroxydopamine-denervated striatum of athymic (nude) rats. After 3–6 months, the transplants were evaluated by light and electron microscopy using antibodies against tryosine hydroxylase (TH), human specific Thy-1 (Thy-1), 5-hydroxytryptamine (5- HT), and laminin. In vivo chronoamperometric studies of K+-induced release of electroactive species were done prior to the histochemical evaluations. At the light microscopical level, Thy-1-immunoreactivity was evenly distributed throughout the entire transplants. Thy-1- immunoreactive nerve fibers were observed radiating from the graft into the host striatum. In sections that were double-stained with antibodies against Thy-1 and TH, such nerve fibers contained both markers. Also 5- HT-immunoreactive cells were found in the grafts with processes both in the grafts and radiating into host neuropil. Laminin immunohistochemistry showed an even distribution of capillaries in the graft with less density than in host brain, suggesting immaturity of graft tissue. At the ultrastructural level, TH-immunoreactive axons made symmetric contacts with unlabeled dendritic shafts and dendritic spines within the host brain. A few asymmetric contacts with TH- immunoreactive axons were seen. 5-HT-immunoreactive terminals made both symmetric and asymmetric contacts with unlabeled dendritic shafts and spines. In vivo chronoamperometry using local application of K+ revealed average signals that were lower on the transplanted side than in control striatum. However, close to the grafts significant amounts of the K+-evoked signal amplitudes were as large as 1.3 microM, and the ratio of the reduction to oxidation currents suggested release of a mixture of dopamine and 5-HT. Taken together, this study shows that human fetal mesencephalic tissue pieces survive grafting into nude rats, develop normal vascularization, and express coexistence of TH- and Thy-1-immunoreactivity. Human TH- and 5-HT-immunoreactive nerve fibers form synapses in host striatum and release monoamine neurotransmitters.

The presence of nerve growth factor (NGF) mRNA and protein in the rat central nervous system is documented. Blot-hybridization analysis showed an abundance of NGF mRNA in the hippocampus, cerebral cortex, and olfactory bulb. Enzyme immunoassay confirmed significant levels of a NGF-like protein in the hippocampus and cerebral cortex. Bioassay of a NGF-like immunoaffinity-purified protein from these regions was physiologically indistinguishable from NGF. Immunohistochemistry revealed a widespread distribution of NGF-like reactivity in the adult brain, preferentially in fiber tracts. NGF mRNA accumulation began at birth, with adult levels reached 3 weeks postnatally. Enzyme immunoassay detected the presence of a NGF-like protein in the embryonic rat brain. Postnatally, the level of NGF-like protein reached a maximum at 3 weeks. Additionally, a distinct fetal form of NGF may exist.

Intracerebral allografts of fetal neurons have been studied in both rodents and nonhuman primates. Such research has been directed towards problems in developmental neurobiology and in animal models of neurological diseases. Whether intracerebrally transplanted human fetal neurons are capable of forming synapses and releasing neurotransmitters are key questions in any application of this approach to human brain development and dysfunction. We studied these questions by examining the immunocytochemical and in vivo electrochemical properties of xenografts of human mesencephalic dopaminergic neurons placed into athymic "nude" rats. The transplanted neurons survive, continue to express human-specific Thy-1 immunoreactivity, and extend neuronal processes into the host brain where morphologically identifiable synapses form. Potassium-evoked release of monoamines occurs in the vicinity of the graft but is absent in more remote areas of the host neuropil. These results indicate that human fetal tissue fragments can provide a source of viable neuroblasts for transplantation. Further, synapses form between pre- and postsynaptic elements expressing different species-specific cell surface markers; thus, these markers do not play a determining role in synaptogenesis.

Human fetal tissue fragments from cortex cerebri and cerebellum were grafted to the anterior chamber of the eye of adult athymic nude rats. The grafts were obtained from tissue fragments recovered after elective routine abortions, performed in weeks 8-11 of gestation. Both cerebellar and cortex cerebri grafts survived and developed in the anterior chamber of the eye for 1-4 months. The transplants slowly became vascularized from the host iris. The grafts developed blood vessels with laminin-immunoreactive walls and contained relatively high amounts of glial fibrillary acidic protein- and neurofilament-immunoreactivity in the neuropil after 4 months in oculo. Recordings of extracellular action potentials from the grafts revealed spontaneously active neurons with action-potential waveforms similar to those observed in immature rodents. Morphologically, the grafts showed no signs of rejection. Clusters and bands of large neurons resembling Purkinje cells and dense aggregates of smaller granule-like cells could be found in the cerebellar grafts. Large neurons were also seen in the cortex grafts. Taken together, these data suggest that the athymic rat may serve as a useful tool for studies of central nervous system tissue from otherwise immunologically incompatible species.

The transcription of neurotrophic factors, i.e., basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF) is regulated by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation despite the lack of a classical glucocorticoid response element in their promoter region.A time course for corticosterone (10 mg/kg, s.c.) in adrenalectomized rats revealed a peak hormone effect at the 4 hr time interval forbFGF(110–204% increase),BDNF(53–67% decrease),GR(53–64% decrease), andMR(34–56% decrease) mRNA levels in all hippocampal subregions usingin situhybridization.c-fosmRNA levels were affected exclusively in the dentate gyrus after 50 min to 2 hr (38–46% decrease).Furthermore, it was evaluated whether corticosterone regulation of these genes depends on interactions with the transcription factor complex activator protein-1.c-fosantisense oligodeoxynucleotides were injected into the dorsal hippocampus of adrenalectomized rats. Corticosterone was given 2 hr later, and the effects on gene expression were measured 4 hr later. In CA1, antisense treatment significantly and selectively enhanced the hormone action on the expression ofbFGF(44% enhanced increase) andBDNF(38% enhanced decrease) versus control oligodeoxynucleotide treatment. In addition, an upregulation ofc-fosexpression (89% increase) was found. There were no effects ofc-fosantisense on hippocampalGRandMRexpression. Thus it seems that a tonicc-fosmechanism exists within CA1, which reducesGR- andMR-mediated effects on expression ofbFGFandBDNF.