Fibroblast growth factor receptor-bearing neurons in the CNS: identification by receptor-mediated retrograde transport.

Neurons that internalize and retrogradely accumulate acidic (aFGF) or basic (bFGF) fibroblast growth factor were identified by autoradiography after injections of 125 I-aFGF or 125I-bFGF into the adult rat central nervous system (CNS). Neuronal cell bodies within the lateral hypothalamus, pedunculpontine tegmental nucleus, laterodorsal tegmental nucleus, and the paracentral dorsal tegmental nucleus accumulated 125I-aFGF. Neurons in the hippocampus, subiculum, the centrolateral, paracentral, central medial, and parafascicular thalamic nuclei, the supramammillary nucleus, and substantia nigra compacta accumulated 125I-bFGF. The pattern of neuronal labeling with 125I-bFGF in adult rats was similar to that observed in newborn guinea pigs. No 125I-FGF labeling was observed in nerve growth factor (NGF) receptor-bearing neurons, including the basal forebrain cholinergic neurons. Time-course studies indicate that 125I-FGF was internalized at the terminals and retrogradely transported to the neuronal cell bodies. Neurons were retrogradely labeled either by injection of 125I-bFGF into the lateral ventricle or by injection into innervated target tissues. Co-injection of a 250-fold excess of unlabeled FGF with the 125I-FGF abolished the neuronal labeling. Co-injection of wheat germ agglutinin (WGA), which nonspecifically blocks binding of 125I-bFGF to its receptor, also prevented neuronal labeling. These studies demonstrate that specific neuronal populations within the CNS express functional receptors for aFGF and/or bFGF; in these neurons, aFGF and/or bFGF bind specifically to these receptors, are internalized and retrogradely transported to the neuronal soma in a manner analogous to NGF. The data indicate that FGF can provide trophic support to CNS neurons by both direct and indirect mechanisms.     

Effects of chronic paralysis with α-bungarotoxin on development of innervation

Chronic paralysis of the duck embryo with α-bungarotoxin increases the number of trochlear motor neurons that survive by decreasing the magnitude of embryonic cell death. The present study examined how such an increase in the motor neuron projection is accommodated at the peripheral target. The results indicate that the overall size of the duck superior oblique muscle was drastically reduced following chronic paralysis with α-bungarotoxin. The number of motor end-plates was reduced to about 25% of the control. Although the total number of end-plates was significantly reduced, their size was larger and they became innervated by far more nerve terminals than in the control. Thus, increased motor neuron survival occurred while the size of the periphery and the number of synaptic sites (end-plates) were greatly reduced. These observations suggest that the cause of embryonic cell death may not be related to the availability of adequate numbers of synapses with the periphery. They also suggest that the physiologic activity of the embryo may play a significant role in regulation of cell number in the developing nervous system.     

Target regulation of sympathetic sprouting in the rat hippocampal formation

Sympathetic fibers appear in the rat hippocampal formation after lesions of the medial septal nucleus. The distribution of sympathetic axons correlates most closely with the distribution of dentate granule cells and their axons. To test the hypothesis that granule cells are the targets of sympathetic fibers, neurotoxins were injected to destroy either the granule cells (colchicine) or the pyramidal cells (kainic acid) in the hippocampal formation 1 month prior to placing septal lesions. Sympathetic fibers appeared in animals with kainic acid lesions but not in regions lacking granule cells in animals treated with colchicine. These results support the hypothesis that the granule cells of the dentate gyrus are the targets of sprouting sympathetic fibers.     

Sprouting of sympathetic nerves in the absence of afferent input

One day before a medial septal lesion, the preganglionic afferents to the superior cervical ganglion were transected. We found that deafferented peripheral noradrenergic neurons appear in the hippocampal formation in response to the septal lesion in spite of loss of input from the central nervous system. This observation supports the hypothesis that sprouting is initiated by some factor derived from the target tissue.     

Production in vitro of a neurotropic substance from proliferative neurolemma-like cells.

The objective of this study was to better define the biological and chemical properties of a neurotropic substance capable of mediating recognition between neurolemma-like cells and neurites during growth. Using in vitro methods, we showed that it was a macromolecular protein apparently synthesized by proliferative cells and was responsible for attracting and promoting neurite outgrowth from chick embryo dorsal root ganglia. The substance appeared to be a normal component of the cell coat during the proliferative phase, and was secreted into the tissue culture medium. Fractionation of the culture fluid by gel filtration chromatography yielded three major fractions containing the biologically active component. The fraction that had the greatest nerve-growth-stimulating activity appeared in a small molecular fraction (mol. wt., 10,000 to 20,000), and was biologically similar to the mouse salivary gland nerve growth factor (NGF). The neurotropic substance appeared to play a central role in promoting and guiding axonal growth during normal development and regeneration of the peripheral nerve.     

Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP-regulated phosphoprotein, in the primate brain.

The localization of DARPP-32, a dopamine and cAMP-regulated phosphoprotein, has been studied in monkey brain by immunocytochemistry. This study indicates that DARPP-32 is enriched in neurons in regions receiving a dense dopamine input from the substantia nigra and ventral tegmental area. Thus, the majority of somata in the anterior olfactory area, nucleus accumbens, caudate nucleus, and putamen are immunoreactive for DARPP-32. In the caudate nucleus, immunoreactive spines receive asymmetric contacts from unlabeled axon terminals. Immunoreactive somata have diameters of 10-15 microns. In regions known to receive projections from these nuclei, immunoreactivity is confined to small puncta that represent axons and axon terminals. Regions in which immunoreactivity is present in puncta include the ventral pallidum, globus pallidus, and substantia nigra pars reticulata. Dopaminergic neurons themselves are not immunoreactive. Neurons containing moderate to weak immunoreactivity for DARPP-32 are observed in portions of the cerebral cortex, particularly in the temporal cortex (layer VI). DARPP-32-positive neurons are also present in the cerebellum, in the medial habenula, and in portions of the bed nucleus of the stria terminalis and amygdaloid complex. DARPP-32 immunoreactivity is also present in astrocytes in the subcortical white matter and in tanycytes in the arcuate nucleus and median eminence. DARPP-32 may be an effective marker for dopaminoceptive neurons in which the actions of dopamine on the D-1 dopamine receptor are mediated through cAMP and its associated protein kinase.     

NGF-producing transfected 3T3 cells: Behavioral and histological assessment of transplants in nigral lesioned rats

The rodent fibroblast clonal cell line, 3T3, was retrovirally transfected with the rat nerve growth factor (NGF) gene and selected for NGF synthesis. This study tested the hypothesis that transplanted 3T3 cells, transfected to secrete nerve growth factor (3T3^NGF+), change motor behavioral indices created by striatal denervation in a dose-dependent fashion. 3T3^NGF+ cells were transplanted into the lateral ventricle of rats following ipsilateral lesions of the substantia nigra pars compacta by stereotaxic injections of 6-hydroxydopamine (10 μg), an established lesion model. Control groups included vehicle injections and transplanted untransfected cells. The extent of the lesions was measured by determining rotational behavior before and two weeks after transplantation. Immediately prior to transplantation, cells were incubated with the fluorescent dye marker, Dil. To assess cell viability, whole brains were cryosectioned and examined for Dil-labeled 3T3 cells using fluorescent microscopy. The number Uf Dil-labeled profiles in five animals per group were counted in at least five noncontiguous sections per animal. From these data a statistically derived estimate of viable, transplanted 3T3 cells was obtained. The number of surviving transplanted cells correlated with the behavioral changes measured. The 3T3NGF + transplants reduced rotational behavior, while control 3T3 transplants exacerbated rotational behavior. Thus, while NGF delivery was found to be beneficial, it was apparent that naive 3T3 had detrimental effects. These results underscore the importance of making doseresponse measurements when attempting transplantbased modifications of CNS behavior. © 1995 Wiley-Liss, Inc.     

Alkylcatechols regulate NGF gene expression in astroglial cells via both protein kinase C-and cAMP-independent mechanisms

It has been previously demonstrated that, in mouse astroglial cells and fibroblast cells in culture, alkylcatechols cause a rapid increase in the nerve growth factor (NGF) mRNA level followed by a marked increase in the amount of NGF released into the medium. To understand the mechanism of this alkylcatechol effect on NGF gene expression in astroglial cells, we examined the effects of protein kinases that influence intracellular signal transduction and of their inhibitors. The reagents to increase the intracellular content of cyclic AMP (cAMP) such as dibutyryl cyclic AMP (Bt₂cAMP), forskolin, or cholera toxin did not mimic alkylcatechol induction of NGF gene expression. Phorbol ester, a direct activator of protein kinase C (PKC), caused an increase in the NGF synthesis/secretion. The stimulatory effect of homocatechol (4-methylcatechol) on NGF synthesis was not completely inhibited by staurosporine, an inhibitor of PKC. The treatment of cells with homocatechol resulted in the translocation of PKC from cytosol to membrane-associated fractions, although the levels of the subcellular location of PKC were not correlated with the level of the induction of NGF gene expression. The concomitant administration of homocatechol (10^?4M) and PMA (10^?8?10^?6 M) evoked a drastic and prolonged increase in the NGF mRNA level, and also markedly increased the amounts of NGF secreted by the cells (approximately 150-fold). This synergism was inhibited in part by staurosporine, but the level of increase in NGF mRNA and in NGF protein was rather greater than that of activation of PKC. These results suggest that neither PKC-dependent pathway nor cAMP-dependent pathway is dominant in the stimulatory effect of alkylcatechol on NGF synthesis. © 1993 Wiley-Liss, Inc.     

Up- and down-expression of the dopamine transporter by plasmid DNA transfer in the rat brain

The functional role of the dopamine transporter (DAT) in central dopaminergic neurotransmission was assessed further by investigating the consequences on dopamine (DA) turn-over of up- and down-regulation of this protein induced by a non-viral gene transfer approach. For this purpose, expression plasmids containing the sense or antisense coding sequence of DAT complexed with the cationic polymer, polyethylenimine (PEI), were injected into the rat substantia nigra, the brain region containing the majority of DA cell bodies. Before in vivo injection, the efficacies of the different DNA constructs were assessed by transfection studies into LLC-PK1 cells. Stereotaxic administration of the sense plasmid complexed to PEI induced, 3 days later, a significant increase in the immunoautoradiographic labelling by anti-DAT antibodies of the substantia nigra and various DA projection areas. These effects were associated with a significantly enhanced capacity of striatal synaptosomes to take up [³H]-DA and lasted up to 14 days postinjection. In contrast, 7 days after intranigral administration of the antisense plasmid complexed to PEI, we observed a significant decrease of DAT immunolabelling in the substantia nigra and [³H]-DA uptake by striatal synaptosomes. Whereas DA turnover in the striatum was unaltered 3 days after intranigral administration of the sense plasmid, it was increased 7 days after intranigral administration of the antisense construct. These data indicate that non-viral transfer of the sense or antisense coding sequence of DAT can be used as a novel approach to induce long-term changes in central DA neurotransmission.     

Subthalamic projections to the globus pallidus: An electrophysiological study in the rat

The connections between the subthalamic nucleus (STN) and the globus pallidus (GP) were investigated electrophysiologically. By the use of stimulating electrode arrays it was determined that the predominant response of globus pallidus neurons to stimulation of the subthalamic nucleus was inhibition. A smaller proportion of short-latency orthodromic activation was also observed. Antidromic activation from the subthalamic nucleus was seen occasionally, although more laterally located in the pallidum. The results demonstrate the existence of reciprocal connections between these two regions and show that the predominant effect of subthalamic activation is inhibition of pallidal cells.     

Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligodeoxyribonucleotide probe

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines in neural tissues and adrenal medulla. To study the expression of the TH gene and its regulation in adult and developing neural tissues, we have synthesized an oligodeoxyribonucleotide probe (oligomer) that is specific for TH mRNA. Using Northern blot hybridization of polyadenylated RNAs from adrenal gland, brain stem, liver, and cerebral cortex with the 32P-labeled oligomer, a single TH mRNA of 1.9 kb was detected in adrenal gland and brain stem but not in liver and cerebral cortex. Using this TH-specific oligomer, TH mRNAs were localized to the chromaffin cells in the adrenal medulla and to catecholaminergic neurons in locus coeruleus and substantia nigra by in situ hybridization histochemistry. After reserpine administration, the intensity of hybridization signal was increased to threefold that of normal in sections of adrenal medulla and twofold that of normal in locus ceruleus. No difference in hybridization signal intensity was observed in the substantia nigra of normal and reserpine-treated animals. Use of this specific TH probe in in situ hybridization procedures represents a powerful approach to the study of regulation of TH gene expression at the cellular level.     

MPTP-Induced Hemiparkinsonism in Nonhuman Primates 6–8 Years after a Single Unilateral Intracarotid Dose

Five female adultMacaca nemestrinamonkeys, given a unilateral intracarotid (ic) infusion of 2.3–3.5 mg of MPTP-HCl, were studied for 6–8 years. Two to 3 days after MPTP, the animals developed hemiparkinsonism characterized by rigidity and flexed posture of the arm contralateral to the side of infusion with episodes of tremor, circling ipsilateral to the lesioned side, a slight balance disturbance, and stooped posture. Rating of parkinsonian features 4 months after ic infusion, and yearly thereafter, did not show any statistically significant changes. The animals maintained their usual appetite and body weight increased normally. Each animal responded to -DOPA methyl ester with decreased parkinsonian signs and symptoms and increased contralateral turning. In contrast, after control vehicle administration, the animals continued to have the same parkinsonian signs and predominant ipsilateral turns. In three of the five monkeys, contralateral turns after vehicle significantly increased after 6–8 years. Unilateral intracarotid MPTP induced asymmetric motor behavior that remained stable after 6–8 years. Animals that showed an increased frequency of contralateral circling after control vehicle showed a decrease in contralateral turns after -DOPA methyl ester, suggesting neuroplastic changes over the years.     

Effects of lesions in some basal ganglia nuclei and efferent projections on tremorine-induced limb rigidity in rats

A mechanical apparatus was used to measure in rats the effects of some unilateral basal ganglia lesions on the hind-limb rigidity produced by the cholinomimetic compound, tremorine. A globus pallidus lesion reduced the resting tone and greatly increased rigidity in the contralateral leg. Lesions in the entopeduncular, subthalamic, and accumbens nuclei had no effects on rigidity, although the entopeduncular nucleus lesion reduced resting limb tone. The role of the globus pallidus in rigidity is significant in relation to work in which striatal lesions abolished tremorine-induced rigidity. Some brain regions that receive basal ganglia efferent fibers received a lesion unilaterally. Lesions in the substantia nigra, red nucleus, or pedunculopontine nucleus had no effect on tremorine-induced rigidity. A habenular lesion significantly reduced rigidity in both hind legs. Very pronounced reductions in rigidity occurred after midbrain lesions involving the central periaqueductal grey region and the superior colliculus. The basal ganglia output pathways that may mediate tremorine rigidity are discussed, together with the possibility that the superior colliculus, which has many muscarinic receptors, may be influenced directly by tremorine.     

Glial reaction to nerve growth factor in the regenerating optic nerve of the newt (Triturus viridescens)

Nerve growth factor (NGF), administered as a single intraocular injection at the time of nerve transection, elicited cellular hyperplasia and hypertrophy in the astrocyte-like glial cell population of the regenerating newt (Triturus viridescens) optic nerve at 14 days postlesion. More specifically, there was a significant dose-dependent increase in the number of glial cells in response to various NGF concentrations [2 to 2000 biologic units (BU)], which correlated directly with the dose-dependent rise in the neuronal (regenerating axons) area of 14-day regenerating nerve cross sections. We refer to this phenomenon (i.e., cell hyperplasia) as a NGF dose-dependent glial cell response. Quantitation of the astrocyte-like cell perikaryal area and nuclear area, and calculation of cell: nuclear ratios indicated that cell hypertrophy was elicited by the 2000-BU dose of NGF, but not by lower NGF concentrations (i.e., 2 to 200 BU). Corroborative ultrastructural observations were even more revealing. Not only did electron micrographs verify cellular hypertrophy in the astrocyte-like glial cells, but also they revealed hypertrophy of cell processes and a massive increase in the number of microfilaments in response to the 2000-BU NGF treatment. We speculate that these phenomena may represent an NGF dose-independent glial cell response.     

Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): Effect of nerve growth factor antiserum

At 14 days after transection those regenerating newt (Triturus viridescens) optic nerves receiving anti-nerve growth factor treatment were easily distinguished from regenerating controls. Quantitative analysis revealed that antiserum treatment significantly reduced nerve diameter and cross-sectional areas compared to the control groups. Quantitation from electron microscope montages of nerve cross sections revealed similar results. In addition, antiserum treatment significantly reduced the area of regenerating axon fascicles per nerve cross section compared to the control groups. Most significantly, the mean number of regenerating axons per nerve decreased more than 50% in the antiserum-treated group. Regenerating axons appeared normal in all three groups. Axons were filled with clear cytoplasm containing neurofilaments, neurotubules, and an occasional mitochondrion. Axon density was not significantly different among the three groups and axon diameters were similar from 0.1 to 0.8 μm. Distention of glial cell processes surrounding fascicles of axons and increased intra- and extracellular debris may indicate an alteration of glial cell activity in the antiserum group. Many of the 14-day antiserum-treated nerves have the appearance of an untreated transected optic nerve 6 to 10 days after lesion.     

Drug-free evaluation of rat models of parkinsonism and nigral grafts using a new automated rotarod test

A variety of tests are available for the evaluation of behavioural deficits in rat models of hemiparkinsonism; many, however, are of limited applicability or insufficiently objective. The drug-induced turning behaviour test is widely used. A disadvantage of this test is that the use of drugs may lead to misleading results. Here, we describe a drug-free rotarod test that was used to evaluate the effects of unilateral 6-hydroxydopamine lesions, nigral grafts, and subrotational doses of apomorphine. The rotarod unit was automated and interfaced to a personal computer allowing automatic recording of the time that each rat was able to stay on the rod at different rotational speeds (i.e., progressively increasing the difficulty of the task). A combination of lesion-induced deficits resembling those of Parkinson's disease appears to be involved in falling from the rod. The test shows high effectiveness for identifying rats with maximal dopaminergic lesions, but is also effective for identifying partial lesions. Rotarod performance profiles were useful for investigating the effects of intrastriatal nigral grafts, since low rotation speeds revealed differences from lesioned rats (i.e., improvements) while higher speeds revealed differences from normal rats (i.e., remaining deficits and partial lesions). The test was effective regardless of whether rats were trained on the rod before lesion, after lesion, or after grafting. Injections of apomorphine (0.0125 and 0.0250 mg/kg) did not induce consistent improvements. These results indicate that the rotarod test is a useful drug-free procedure for overall evaluation of basic motor abilities in rat models of parkinsonism and treatment-induced changes.     

神经生长因子对大鼠脑缺血再灌注海马神经细胞损伤的影响

目的 :观察神经生长因子 (NGF)对脑缺血再灌注后海马CA1区神经细胞损伤的影响。方法 :采用大鼠脑缺血再灌注模型 ,在光镜和透射电镜下观察NGF治疗组和缺血再灌组动物脑缺血 3 0min再灌注 2 4h和 72h时海马组织学及超微结构的改变。结果 :在缺血 3 0min再灌注 2 4h和 72h时 ,NGF治疗组海马CA1区神经细胞的结构损伤与缺血再灌组比较显著减轻 ;NGF可以减轻海马迟发性神经细胞死亡 (DND)性损伤。结论 :NGF对缺血再灌注所致的神经细胞损伤可能具有一定的保护作用     

Parkin expression in the adult mouse brain

Mutations in a protein designated Parkin were shown to be involved in the pathogenesis of autosomal recessive juvenile parkinsonism. Nothing is known about its regional and subcellular distribution in the mouse. In order to elucidate the Parkin mRNA and protein distribution in the adult mouse, the mouse cDNA was cloned and polyclonal antisera were generated against the N-terminal part of mouse Parkin. The antibodies were shown to be specific using Western blot analysis, immunostaining of cells transfected with mouse Parkin and pre-absorption tests. The Parkin protein expression profile was studied using immunohistochemistry and Western blot analysis and was compared with that of the mRNA yielded by in situ hybridization and RT-PCR analysis. Parkin protein was widely distributed in all subdivisions of the mouse brain. Low levels were found in the telencephalon and diencephalon, while the brainstem contained a large number of cells heavily expressing Parkin. Ultrastructural analysis and double immunohistochemistry revealed that the majority of Parkin-expressing cells were neurons, while only single glial cells exhibited immunostaining. The protein was distributed nonhomogeneously throughout the entire cytoplasm. A subpopulation of Parkin-immunopositive cells displayed speckled immunodeposits in the nucleus. Dopaminergic cells of the substantia nigra pars compacta exhibited high levels of Parkin mRNA but no Parkin protein, while the striatum contained immunopositive profiles but no mRNA signals. Our data indicate that Parkin is neither restricted to a single functional system nor associated with a particular transmitter system. The speckled nuclear distribution of Parkin immunoreactivity strongly suggests a role for Parkin in gene expression.     

Specific pathophysiological functions of JNK isoforms in the brain

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c-JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6-hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment-specific expression of JNK isoforms with 46-kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6-hydroxydopamine was transiently improved in JNK3 ko and c-JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c-JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid-induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c-Jun irrespective on the experimental set-up. This comprehensive study provides novel insights into the context-dependent physiological and pathological functions of JNK isoforms.