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Ginsenosides are known to attenuate glutamate-induced cell injuries in vitro. We investigated the in vivo effect of ginsenosides on kainic acid (KA)-induced neurotoxicity in rat hippocampus using the methods of acid fuchsin (AF) staining and heat-shock protein-70 (HSP-70) immunoreactivity to detect neuronal death and stress, respectively. Pretreatment of ginsenosides (50 or 100 mg/kg for 7 days) via intraperitoneal (i.p.) administration significantly attenuated KA (10 mg/kg i.p.)-induced cell death by decreasing AF-positive neurons in both CA1 and CA3 regions of rat hippocampus compared with KA treatment alone. Pretreatment of ginsenosides (50 or 100 mg/kg for 7 days) via i.p. administration also significantly suppressed KA-induced induction of HSP-70 in both regions of rat hippocampus. These results show that ginsenosides are effective in protecting hippocampal CA1 and CA3 cells against KA-induced neurotoxicity.

Docosahexaenoic acid (C22:6, n-3), a major n-3 fatty acid of the brain, has been implicated in restoration and enhancement of memory-related functions. Because Alzheimer's disease impairs memory, and infusion of amyloid-beta (Abeta) peptide (1-40) into the rat cerebral ventricle reduces learning ability, we investigated the effect of dietary pre-administration of docosahexaenoic acid on avoidance learning ability in Abeta peptide-produced Alzheimer's disease model rats. After a mini-osmotic pump filled with Abeta peptide or vehicle was implanted in docosahexaenoic acid-fed and control rats, they were subjected to an active avoidance task in a shuttle avoidance system apparatus. Pre-administration of docosahexaenoic acid had a profoundly beneficial effect on the decline in avoidance learning ability in the Alzheimer's disease model rats, associated with an increase in the cortico-hippocampal docosahexaenoic acid/arachidonic acid molar ratio, and a decrease in neuronal apoptotic products. Docosahexaenoic acid pre-administration furthermore increased cortico-hippocampal reduced glutathione levels and glutathione reductase activity, and suppressed the increase in lipid peroxide and reactive oxygen species levels in the cerebral cortex and hippocampus of the Alzheimer's disease model rats, suggesting an increase in antioxidative defence. Docosahexaenoic acid is thus a possible prophylactic means for preventing the learning deficiencies of Alzheimer's disease.

1. The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba(2+)-sensitive K(+) channels in the endothelium-dependent component of urocortin-induced relaxation in the rat left anterior descending coronary artery. 2. Urocortin induced both endothelium-dependent and -independent relaxation with respective pD(2) of 8.64+/-0.03 and 7.90+/-0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10(-4) M N(G)-nitro-L-arginine methyl ester, 10(-5) M methylene blue or 10(-5) M ODQ, the urocortin-induced relaxation was similar to that observed in endothelium-denuded rings. L-Arginine (5x10(-4) M) antagonized the effect of N(G)-nitro-L-arginine methyl ester. 3. The relaxant response to urocortin was reduced in endothelium-intact rings preconstricted by 3.5x10(-2) M K(+) and abolished when extracellular K(+) was raised to 5x10(-2) M. Pretreatment with 10(-4) M BaCl(2) significantly inhibited urocortin-induced relaxation. Combined treatment with 10(-4) M BaCl(2) plus 10(-4) M N(G)-nitro-L-arginine methyl ester did not cause further inhibition. In urocortin (10(-8) M)-relaxed rings, BaCl(2) induced concentration-dependent reversal in vessel tone. Tertiapin-Q (10(-6) M) also attenuated urocortin-induced relaxation. In contrast, BaCl(2) did not alter urocortin-induced relaxation in endothelium-denuded rings. 4. In endothelium-denuded rings, hydroxylamine- and nitroprusside-induced relaxation was inhibited by 10(-4) M BaCl(2), but not by 10(-6) M tertiapin-Q. 5. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. 6. Taken together, the present results indicate that the urocortin-induced endothelium-dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba(2+)- or tertiapin-Q-sensitive K(+) channels. The urocortin-induced endothelium-dependent relaxation appears to be mediated by cyclic GMP-dependent mechanisms.

Recent immunohistological and imaging studies emphasize the crucial role of axonal injury in determining the extent of permanent neurological deficits in patients with multiple sclerosis. We have recently shown that human immune cells are capable of producing the neurotrophin brain-derived neurotrophic factor (BDNF), which can prevent axonal and neuronal damage after various pathological insults. BDNF imported into the CNS by immune cells would thus be an attractive candidate for mediating neuroprotective effects in multiple sclerosis. The aim of the present study was to perform a detailed immunohistochemical analysis of the expression of BDNF and its receptor truncated trkB tyrosine kinase receptor (gp145trkB) in a series of multiple sclerosis brain lesions. Our data show that various types of neurones throughout the brain are BDNF immunopositive in multiple sclerosis patients as well as in controls. Furthermore, in multiple sclerosis lesions, BDNF is primarily present in immune cells (T cells, macrophages/microglia) and reactive astrocytes. The number of BDNF immunopositive cells correlates with lesional demyelinating activity. The BDNF receptor gp145trkB is found in neurones in the immediate vicinity of multiple sclerosis plaques as well as in reactive astrocytes within the lesion, but not in immune cells. Our results demonstrate that both BDNF and gp145trkB are expressed in multiple sclerosis lesions. This suggests that BDNF and gp145trkB are involved in immune-mediated neuroprotective interactions in multiple sclerosis, and supports the concept that immune cells produce both damaging and protective factors in multiple sclerosis lesions.

Exogenous application of transforming growth factors-beta (TGF beta) family proteins, including glial cell line-derived neurotrophic factor (GDNF), neurturin, activin, and bone morphogenetic proteins, has been shown to protect neurons in many models of neurological disorders. Finding a tissue source containing a variety of these proteins may promote optimal beneficial effects for treatment of neurodegenerative diseases. Because fetal kidneys express many TGF beta trophic factors, we transplanted these tissues directly into the substantia nigra after a unilateral 6-hydroxydopamine lesion. We found that animals that received fetal kidney tissue grafts exhibited (1) significantly reduced hemiparkinsonian asymmetrical behaviors, (2) a near normal tyrosine hydroxylase immunoreactivity in the lesioned nigra and striatum, (3) a preservation of K(+)-induced dopamine release in the lesioned striatum, and (4) high levels of GDNF protein within the grafts. In contrast, lesioned animals that received grafts of adult kidney tissues displayed significant behavioral deficits, dopaminergic depletion, reduced K(+)-mediated striatal dopamine release, and low levels of GDNF protein within the grafts. The present study suggests that fetal kidney tissue grafts can protect the nigrostriatal dopaminergic system against a neurotoxin-induced parkinsonism, possibly through the synergistic release of GDNF and several other neurotrophic factors., (Copyright 2001 Academic Press.)

The following flavonoid compounds have been isolated and identified from the flowers of Asclepias svriaca L.: kaempferol, kaempferol 7-O-beta-glucoside, kaempferol 3-O-beta-galactopyranoside, kaempferol 3-O-beta-xylopyranosyl (1-->2)-beta-galactopyranoside, kaempferol 3-O-beta-glucopyranosyl (1-->2)-beta-galactopyranoside, isorhamnetin, isorhamnetin 7-O-beta-glucoside, isorhamnetin 3-O-beta-galactoside and isorhamnetin 3-O-beta-xylopyranosyl (1-->2)-beta-galactopyranoside. Their structures were established by conventional (acid, enzymatic hydrolysis and H2O2 oxidation) and spectral analysis (UV, 1H NMR, 13C NMR).

A high-performance liquid chromatographic method was developed for the determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31378, in human plasma and urine and in rat tissue homogenates. The method involved deproteinization of the the biological samples with 0.5 volumes of saturated Ba(OH)2, 0.5 volumes of 0.04 M ZnSO4 and 1 volume of acetonitrile. A 80-microl aliqout of the supernatant was injected onto a reversed-phase C18 column. The mobile phase, 50 mM triethylamine acetate : acetonitrile : tetrahydrofuran (65:30:5, v/v/v), was run at a flow rate of 1.0 ml/min. The column effluent was mornitored by a ultraviolet detector set at 310 nm. The retention time of KR-31378 was approximately 6.5 min. The detection limits of KR-31378 in human plasma and urine and rat tissue homogenates were 0.2, 0.5 and 0.5 microg/ml, respectively. The coefficients of variation (within-day and between-day) were below 13.6% for human plasma and urine and rat homogenates. No interferences from endogenous substances were found.

We investigated the enzymatic function, stability, and regional distribution of rat brain cytochrome P-450 (CYP) 2D1 activity. CYP2D1 is the homolog of human CYP2D6, a genetically variable enzyme that activates or inactivates many clinical drugs acting on the central nervous system (e.g., antidepressants, monoamine oxidase inhibitors, serotonin uptake inhibitors, and neuroleptics), drugs of abuse (e.g., amphetamine and codeine), neurotoxins (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3, 4-tetrahydroquinoline), and endogenous neurochemicals (e.g., tryptamine). The CYP2D family has been identified in rodent, canine, and primate brain. Conversion of dextromethorphan to dextrorphan by rat brain membranes was assayed by HPLC and was dependent on NADPH, protein concentration, and incubation time. Significant loss of activity was observed in some homogenizing buffers and after freezing of whole tissues or membrane preparations. Dextromethorphan (0.5-640 microM) metabolism was mediated by high- and low-affinity enzyme systems; K(m1) was 2.7 +/- 2.6 and K(m2) was 757 +/- 156 microM (n = 3 rats, mean +/- S.E.). The enzyme activity was significantly (p <.01) and stereoselectively inhibited by CYP2D1 inhibitors quinine and quinidine (not by CYP2C or CYP3A inhibitors), and by anti-CYP2D6 peptide antiserum (not by anti-CYP2C, -CYP2B, or -CYP3A antibodies). The enzymatic activity demonstrated significant brain regional variation (n = 10 regions, p <.001). These data characterize CYP2D1-mediated dextromethorphan metabolism in rat brain and suggest that localized metabolism of other CYP2D1 substrates (drugs, neurotoxins, and possibly endogenous compounds) within the brain will occur. In humans, CYP2D6 is genetically polymorphic; the variable expression of brain CYP2D6 may result in interindividual differences in central drug and neurotoxin metabolism, possibly contributing to interindividual differences in drug effects and neurotoxicity.

A rapid, simple and reliable high-performance liquid chromatography (HPLC) column-switching method with UV detection (270 nm) for the simultaneous determination of propentofylline and its metabolites in human and rat sera was developed. The method involves direct injection of serum onto an HPLC column, which contains a shielded hydrophobic stationary phase for the separation of analytes from proteins in serum, and then loading the analytes onto a short octadecylsilylated silica gel (ODS) column using a switching valve. Propentofylline and its three metabolites in serum were separated from the serum components within 30 min after the injection. The detection limits (S/N = 3) of analytes spiked in human and rat sera ranged from 0.08 to 0.57 nmol/mL, and the net volume of serum used was 20 microL. The relative standard deviations for within- and between-day variations using rat serum were less than 4.3 and 5.6%, respectively. The method was used to determine propentofylline and its main metabolites in rat serum after a single intravenous dose of propentofylline (5 mg/kg)., (Copyright 1999 John Wiley & Sons, Ltd.)

Several lines of evidence indicate that adult neurons remain dependent on neurotrophins and that changes in tissue expression of neurotrophins and/or their receptors may play a role in senile neurodegeneration. We have studied the expression of p75NTR, trkB and trkC, respectively, in lumbar motoneurons of young adult (2-3 months) and aged (30 months) rats subjected to sciatic transection using in situ hybridization and immunohistochemistry. Nonmanipulated age-matched animals were processed in parallel. In nonmanipulated aged rats, high levels of p75NTR could be seen in a number of motoneurons (10-15%), while in young adult animals no p75NTR could be detected. Seven days following sciatic axotomy, a conspicuous ipsilateral upregulation p75NTR was observed in young adult rats. Also in aged rats there was a marked ipsilateral increase in number of p75NTR expressing neurons ( approximately 100%). In comparison to young adult rats, aged rats showed a decreased expression of both trkB (5/6 animals) and trkC (6/6 animals). Furthermore, in response to sciatic transection, 3 out of 5 aged rats did not show an increased expression of trkB. In aged rats, axotomy did not induce any significant change in trkC expression. In the young adult rats, we recorded a side-to-side effect with lower values ipsilaterally, however, it cannot be excluded that this difference was caused by an upregulation in the contralateral motoneurons. Oligonucleotide probes against BDNF and NT3 mRNA showed only very few faintly positive neurons in both age groups. Our results indicate that the pattern of regulatory changes of NT receptors in response to axotomy is different in aged and young adult rats. The lack of covariation between p75NTR and trkB and trkC regulation in aged rats indicates a changed role for p75NTR in senescent motoneurons., (Copyright 1999 Elsevier Science B.V.)

A high-performance liquid chromatographic determination of a neuronal cell protective compound, propentofylline [3-methyl-1-(5-oxohexyl)-7-propyl-7H-purine-2(3H),6(1H)-dione] was performed combining a microdialysis technique with peroxyoxalate chemiluminescence (PO-CL) detection. The microdialysate was subjected to a fluorescent derivatization of propentofylline with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) without further cleanup, because the membrane used in the microdialysis excluded high-molecular-mass proteins. The proposed method showed a good linearity in the determination range of 0.031 to 1.25 ng/injection; y (microV)=4234 x (ng)- 13.43, r=0.9993 (y=peak height and x=amount of propentofylline). The very low determination limit of 0.031 ng/injection was ca. 200 times more sensitive than that of HPLC-UV determination. The HPLC-PO-CL method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus after a single oral administration (25 mg/kg). Propentofylline showed its maximum extracellular fluid (ECF) concentration of 125.1+/-15.1 ng/ml (mean+/-SD, n=3) at 0.33 h after administration.

The role of neurotrophic factors and their therapeutic potential have been investigated in various neurodegenerative disorders. In neurodegeneration associated with human immunodeficiency virus (HIV) infection, neuronal function and survival may be affected by abnormal neurotrophic regulation involving HIV-infected microglia and reactive astrocytes. To characterize the cellular localization of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor, trkB, proteins in HIV-1 encephalitis, we examined post-mortem brains from patients with acquired immunodeficiency syndrome and brains from non-HIV-infected controls. Using double immunofluorescent confocal microscopy, we found that BDNF immunoreactivity was distributed in neocortical neuronal perikarya and neuritic processes, while in the striatum only neurites were BDNF-immunoreactive. Additionally, the striatum with HIV infection was characterized by BDNF immunoreactivity in infiltrating activated microglia/macrophages and multinucleated giant cells. Catalytic trkB receptor immunoreactivity was observed in neuronal perikarya in the neocortex and striatum, as well as in reactive astrocytes within HIV-infected regions. Our findings suggest that expression of BDNF by activated microglia in HIV-1 encephalitis may affect neuronal survival and astroglial response through corresponding trkB receptors.

Numerous studies have suggested that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic molecule. We show now on a variety of cultured neurons including peripheral autonomic, sensory, and CNS dopaminergic neurons that GDNF is not trophically active unless supplemented with TGF-beta. Immunoneutralization of endogenous TGF-beta provided by serum or TGF-beta-secreting cells, as e.g., neurons, in culture abolishes the neurotrophic effect of GDNF. The dose-response relationship required for the synergistic effect of GDNF and TGF-beta identifies 60 pg/ml of either factor combined with 2 ng/ml of the other factor as the EC50. GDNF/TGF-beta signaling employs activation of phosphatidylinositol-3 (PI-3) kinase as an intermediate step as shown by the effect of the specific PI-3 kinase inhibitor wortmannin. The synergistic action of GDNF and TGF-beta involves protection of glycosylphosphatidylinositol (GPI)-linked receptors as shown by the restoration of their trophic effects after phosphatidylinositol-specific phospholipase C-mediated hydrolysis of GPI-anchored GDNF family receptor alpha. The biological significance of the trophic synergism of GDNF and TGF-beta is underscored by colocalization of the receptors for TGF-beta and GDNF on all investigated GDNF-responsive neuron populations in vivo. Moreover, the in vivo relevance of the TGF-beta/GDNF synergism is highlighted by the co-storage of TGF-beta and GDNF in secretory vesicles of a model neuron, the chromaffin cell, and their activity-dependent release. Our results broaden the definition of a neurotrophic factor by incorporating the possibility that two factors that lack a neurotrophic activity when acting separately become neurotrophic when acting in concert. Moreover, our data may have a substantial impact on the treatment of neurodegenerative diseases.

The expression of the neurotrophic factor BDNF increases during learning-related events and is decreased in the hippocampus of Alzheimer's Disease patients, suggesting that it plays a role in learning, memory, and/or age-related memory deficits. We examined the expression of BDNF and its high affinity receptor, trkB, in young and aged Sprague-Dawley rats. BDNF and trkB mRNA were measured by semi-quantitative in situ hybridization and BDNF protein was measured by ELISA. Significant decreases with age were detected for BDNF mRNA in the pons, BDNF protein in the midbrain, and trkB mRNA in many areas of the brain. Rats were evaluated on the Morris water maze before sacrifice so that BDNF and trkB levels could be related to cognitive status. Regression analyses revealed that decreased trkB mRNA in the pons significantly predicted impaired memory performance in aged rats. These results suggest that decreases in trkB mRNA with age are more widespread than decreases in BDNF, and that BDNF decreases are restricted to more caudal brain regions., (Copyright 1998 Elsevier Science B.V.)

Ciliary neurotrophic factor (CNTF) has been implicated in the development, survival, and maintenance of a broad range of neurons and glia in the peripheral nervous system and the CNS. Evidence also suggests that CNTF may affect development of cells outside the nervous system. We have found that functional CNTF and its receptor are expressed in developing embryonic chick heart and may be involved in parasympathetic synapse formation. CNTF and CNTF receptor mRNA levels were highest at embryonic day 11 (E11)-E13, the period of parasympathetic innervation in chick atria. Levels of atrial CNTF receptor mRNA were fourfold greater at E13 than at E6 and at E13 were 2.5-fold higher in atria than in ventricle, corresponding to the higher degree of parasympathetic innervation occurring in atria. Treatment of isolated atria or cultured atrial myocytes with recombinant human or avian CNTF resulted in the tyrosine phosphorylation and nuclear translocation of the signal transducer and activator of transcription STAT3. The developmental increase in atrial CNTF receptor mRNA was enhanced by stimulating muscarinic receptors with carbachol in ovo and was inhibited by blocking muscarinic cholinergic receptors with atropine. Treatment of cultured atrial myocytes with CNTF resulted in a twofold increase in the levels of muscarinic receptors. Thus, CNTF was able to regulate a key component of parasympathetic synapses on atrial myocytes. These results suggest a postsynaptic role for CNTF in the onset of parasympathetic function in the developing heart and provide new clues to molecular mechanisms directing synapse formation at targets of the autonomic nervous system.

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7-T10, disappear. Administration of NT-4 in gelfoams (6 microgram) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7-T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (-/-) and trkC (-/-) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (-/-) and trkC (-/-) mice. In addition, electron microscopic analyses showed that trkB (-/-) and trkC (-/-) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (-/-) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

Systemic administration of kainic acid (KA) at convulsant doses results in irreversible cell damage and neuron loss in the hilus of the dentate gyrus and in the CA1 area of the hippocampus. This is followed by reactive astrocytosis in these regions, and sprouting of mossy fibers into the molecular layer of the dentate gyrus. Since trophic factors are probably implicated in the cellular responses to the excitotoxic insult, and early induction of BDNF and TrkB mRNAs has been observed following KA injection, the present study examines BDNF, full-length and truncated TrkB protein expression in the hippocampus, as revealed by immunohistochemistry, up to 30 days following KA administration to adult rats. Reduction in BDNF and full-length TrkB immunoreactivity preceding neuron loss is observed in the damaged areas. However, transient increase in BDNF immunoreactivity is observed in surviving CA1 neurons and in granule cells of the dentate gyrus. In contrast, full-length TrkB immunoreactivity progressively increases in the molecular layer of the dentate gyrus up to day 30 following KA administration. A second peak in BDNF immunoreactivity is observed in reactive astrocytes, as revealed with double-labeling immunohistochemistry to BDNF and GFAP, in the plexiform layers of CA1 and, to a lesser degree, in the molecular layer of the dentate gyrus. In addition, strong truncated TrkB immunoreactivity is found in reactive astrocytes, as revealed with double-labeling immunohistochemistry to truncated TrkB and GFAP, in the same regions. These results, in concert with previous observations in the same model of hippocampal damage, suggest that BDNF participates in the early response to excitotoxic damage, and that expression of full-length TrkB at strategic sites in the molecular layer of the dentate gyrus has a role in the regenerative response linked to mossy fiber sprouting. Interestingly, delayed expression of BDNF and truncated TrkB in reactive astrocytes may act as negative regulators of neurite growth in devastated regions, such as the CA1 area, which are impoverished of putative postsynaptic sites., (Copyright 1998 Elsevier Science B.V.)

Utilizing a specific polyclonal antibody against a peptide unique for brain-derived neurotrophic factor (BDNF), we investigated the regional and temporal profiles of immunoreactivity of the BDNF protein in the rat hippocampus after transient forebrain ischemia. The pattern of immunoreactivity for the BDNF receptor (TrkB) was also examined and compared with that for BDNF. In the early phase after ischemia, we observed a distinct regional difference in immunoreactivity between the pyramidal cell layer and the stratum radiatum of the CA1 subfield. In the pyramidal cell layer, there was a rapid and transient increase in the positive immunostaining for both BDNF and TrkB. By contrast, in the stratum radiatum there was a marked decrease in BDNF immunoreactivity, but not one in that of TrkB. One week after ischemia, high immunoreactivity for both BDNF and TrkB was observed in the reactive astrocytes in the dendritic field of the CA1 subfield. These findings suggest that a transport of BDNF from the neuronal soma to the dendrites of the stratum radiatum might be ceased after the ischemic insult. Thus, a dysfunctional autocrine mechanism of BDNF within the CA1 neuron may be involved in the pathogenesis of selective neuronal damage after ischemia.

Expression of TrkB receptors were studied in the cerebral cortex of normal rats and young rats with hypoxic/ischemic injury. TrkB expressing cells were present in the piriform cortex at birth and increased in number with age, and were finally present in the entire cerebral cortex. Density of TrkB cells reached adult levels at P30. They were morphologically regarded as pyramidal neurons and interneurons. Hypoxic/ischemic injury induced a tentative increase of full-length TrkB receptors. A novel appearance of TrkB expressing neurons and enhanced immunostaining on both cell soma and dendrites were observed in the peri-infarct areas and increased number of TrkB expressing neurons were detected in the contralateral cortex after carotid artery ligation. This increase was no longer evident after 48 h of hypoxia. Double immunostaining using antiserum against GFA or OX-42 revealed no co-localization of TrkB receptors and these molecules, while there were only slight co-localization of TrkB and calbindin-D28k molecules. The altered levels in responses to injury indicate that TrkB may play a crucial role in the early protective mechanism of the neurons with hypoxic/ischemic injury through ligands BDNF and/or NT-4/5., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Alteration of insulin-like growth factor-1 (IGF-1) receptor and its mRNA after long-term adrenalectomy (ADX) was studied in the hippocampus by in vitro receptor autoradiography and in situ hybridization histochemistry, respectively. Significantly, decreased levels of IGF-1 receptor and its mRNA was noted in the dentate and CA1-CA4 regions of the hippocampus of the ADX animals, suggesting that the level and expression of IGF-1 receptors in the hippocampus is influenced by adrenal hormones., (Copyright 1998 Elsevier Science B. V. All rights reserved.)

The interaction of ethanol and neurotrophin-mediated cell survival was examined in primary cultures of cortical neurons. Cells were obtained from rat fetuses on gestational day 16 and maintained in a medium supplemented with either 10% or 1.0% fetal calf serum (FCS). Exogenous nerve growth factor (NGF; 20 ng/ml), brain-derived neurotrophic factor (BDNF; 20 ng/ml) or neurotrophin 3 (NT-3; 20 ng/ml) was added to the cultures alone, or in combination with ethanol (400 mg/dl). The number of viable neurons was determined after a 48 h treatment with a growth factor and/or ethanol. The effects of ethanol on the expression of high affinity neurotrophin receptors (trkA, trkB, and trkC) and the low-affinity receptor (p75), were analyzed using Western immunoblots. In untreated cultures, 22.7% and 26.3% of the cells raised in a medium containing 10% and 1.0% FCS, respectively, were lost. Only NGF prevented the death of the cultured cortical neurons. Ethanol was toxic; it caused a 23.5% and 16.7% loss of cells (for cells grown in a medium containing 10% and 1.0% FCS, respectively) beyond that occurring 'naturally' in an untreated culture. Ethanol completely blocked the NGF-mediated cell survival. In general, BDNF and NT-3 did not offset the toxic effect of ethanol. Immunoblotting studies showed that the expression of p75 was significantly (p < 0.05) lower (40%) in ethanol-treated cultures, but ethanol did not affect trk expression. Thus, ethanol has specific effects upon NGF-mediated cell survival and the effects on the low affinity receptor imply that p75 specifically plays an important role in NGF signaling.

Photic entrainment of mammalian circadian rhythms occurs because the pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus is endowed with a rhythmic sensitivity to photic signals conveyed by the retinohypothalamic tract. Since brain-derived neurotrophic factor (BDNF) has been implicated in the functional modulation of other retinal targets, the rat SCN was examined for expression and cellular distribution of this neurotrophin and TrkB, the tyrosine kinase receptor that preferentially binds BDNF. The rat SCN was found to express the mature BDNF peptide and mRNA by Western blotting, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) analyses. BDNF-immunoreactivity and hybridization signal for its mRNA were coextensively localized within a number of SCN cells throughout the rostrocaudal axis of each nucleus. In addition, some cells intercalated within the optic chiasm were distinguished by expression of BDNF immunoreactivity or mRNA. Immunostaining for the TrkB receptor was also evident in the SCN within terminals or fibers predominantly located along the SCN/optic chiasm interface and within scattered perikarya near the medial border of each nucleus. Combined in situ hybridization and immunocytochemical analysis revealed that BDNF mRNA-expressing cells within the ventrolateral SCN were often closely apposed to TrkB-positive fibers extending from the optic chiasm. These findings raise the possibility that target-derived interactions between BDNF and TrkB receptors could play a role in the circadian modulation of SCN pacemaker sensitivity to photic input transmitted by the retinohypothalamic tract., (Copyright 1998 Academic Press.)

Retinal ganglion cells (RGC) are supported by brain-derived neurotrophic factor (BDNF), but it is not known if BDNF acts as a target-derived factor or as an afferent or autocrine trophic factor. Here we demonstrate that BDNF mRNA is expressed in the retinorecipient layer of the chick optic tectum as well as in the inner nuclear layer and ganglion cell layer of the retina. Amacrine cells rather than RGC were the main source of BDNF mRNA in the ganglion cell layer, as determined by in situ hybridization that was combined with retrograde labeling of RGC and destruction of RGC by optic stalk transection, followed by quantitative RT-PCR. Cells in the ganglion cell layer as well as the retinorecipient layers of the optic tectum were BDNF-immunolabeled. After injections into the tectum, radio-iodinated BDNF was transported to the retina where autoradiographic label accumulated in the inner plexiform and ganglion cell layers. After intraocular injection, iodinated BDNF accumulated in these same retinal layers and correlated with the distribution of p75 neurotrophin receptor protein. The majority of cross-linked receptor-bound BDNF in the retina immunoprecipitated with p75 antibodies. No difference in the intensity of BDNF immunolabel was observed in the experimental retina or tectum after optic stalk transection, indicating that most of the BDNF in the RGC was not derived from the optic tectum. These data indicate that a substantial fraction of the BDNF in the ganglion cell layer is derived from local sources, afferents within the retina, rather than from the optic tectum via retrograde transport.

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.

In eukaryotes, protein de novo synthesis is mainly under the control of transcription factors at the level of gene transcription in cell nuclei. Gel retardation electrophoresis was employed for determination of DNA-binding activity of the transcription factor activator protein-1 (AP1), which is a dimer between c-Fos and c-Jun protein families. Binding of a radiolabeled double-stranded oligonucleotide probe for AP1 was rapidly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of gerbils with forebrain ischemia for 5 min. Similarly marked potentiation was seen in the thalamus and the striatum, but not in the frontal cortex, following the recirculation of blood supply. The potentiation was transient in the vulnerable CA1 subfield, but was rather persistent in the thalamus and the striatum in addition to the resistant CA3 subfield and dentate gyrus. However, administration of the neuroprotective drug bifemelane (10 to 20 mg/kg, i.p.) resulted in prolongation of the potentiation of AP1 binding in the CA1 subfield up to 6 hr after ischemia, without significantly affecting that in other central structures. Limited proteolysis revealed that bifemelane induced expression of the AP1 consisting of constructive proteins different from those expressed in control animals in the CA1 subfield. These results suggest that bifemelane may protect neuronal cells against ischemic injuries through molecular mechanisms associated with prolongation of the potentiation of AP1 binding in the vulnerable CA1 subfield after ischemia.

Recent findings have focused attention on the role of apoptosis in neurodegenerative diseases, however, the apoptotic process in child-onset brain disorders has been little investigated. Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are hereditary disorders characterized by impaired DNA repair and neurodegeneration. We investigated apoptotic cell death in the cerebellum of five cases of XP group A (XPA), four cases of CS, and twelve controls, using TdT-mediated DIG-dUTP nick-end labeling (TUNEL) and immunohistochemical staining for bcl-2, bcl-x, p53, bax, BDNF and Trk B. The TUNEL-positive cells were found in the granule cells of the cerebellar cortex of two patients with XPA and two patients with CS, whereas such cells were not detected in the cerebellar cortex in controls. Upregulation of bcl-2 or BDNF was not observed, and bcl-x expression was not altered. Some patients showed nuclear expression of p53 in the granule cells and/or molecular layer, bax-positive glial cells in the cerebellar white matter, and a few Trk B-positive cells in the granular layer. These findings suggest that apoptotic cell death can be involved in the cerebellar degeneration in patients with hereditary defects in DNA repair mechanisms.

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca2+) levels induced by BDNF using a fluorescence Ca2+ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca2+ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca2+ in only selective proportion of resting neurons without Ca2+ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca2+ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-D-aspartate (NMDA) channel-gated Ca2+ influx via TrkB and regulates the frequency and the amplitude of Ca2+ oscillations. BDNF may have the potential to modulate spontaneous Ca2+ oscillations to regulate neuronal plasticity in developing hippocampal neurons.

During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al, 1991; Jacobson, 1991). At this time (E15-E21), reverse transcriptase-polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high-affinity receptor, TrkB. Immunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15-E21) using a quantitative in vitro chemotaxis assay. High-affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT-3, a low-affinity TrkB ligand, only stimulated significant migration at high concentrations (> or =100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF-induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+-chelator, BAPTA-AM, suggesting that BDNF-induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+-dependent mechanisms. BDNF-stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.

By virtue of its known segregated distribution of cell types, their known neurotransmitters and neurophysiologic properties, the cochlear nucleus is an excellent model and provides the opportunity to study the relation between neurotrophins and their receptors along with the functional properties of the adult cochlear nucleus. To investigate the potential role of neurotrophins in the mature cochlear nucleus, we determined the expression of the three major neurotrophin tyrosine kinase receptors (Trk) in the adult rat ventral cochlear nucleus, as revealed by antibodies against the full-Trk proteins. A qualitative and a cartographic analysis showed a widespread distribution of the three Trk receptors throughout the nucleus. Immunostaining was mainly restricted to neurons as shown by the lack of double immunostaining with specific markers for glial cells. However, we observed variability in immunostaining for given receptors. Three classes of cells were distinguished by their specificity for Trk receptors. The first one was a cell population that stained for TrkA or TrkB. This population characterizes the majority of small and small round neurons and fusiform cells. The second group consists of TrkC-immunolabeled cells and comprises the majority of spherical, globular, granule and small multipolar cells. The third group consists of cells that seem to be immunopositive for all three Trk receptors. This group includes in part multipolar, giant and octopus cells. A possible correlation between Trk expression and cell functional properties is suggested: TrkA- and TrkB-immunoreactive cells could include inhibitory neurons while cells stained for TrkC could include excitatory neurons. The abundant and widespread neuronal distribution of signal-transducing forms of TrkA, TrkB and TrkC predicts that their cognate ligands may exert significant effects on a large proportion of neurons within the mature ventral cochlear nucleus.

Neurotrophin-like immunoreactivity was studied in post-mortem motor cerebral cortex from patients with Amyotrophic Lateral Sclerosis (ALS) and controls. Neurotrophin-4/5 immunoreactivity was seen in small-(12-25 microm), medium-(26-39 microm), and large-(> 40 microm), neurones, neurotrophin-3 was seen in medium and small neurones, while brain-derived neurotrophic factor was restricted to small neurones. No difference in number or intensity of immunostained neurones was found between ALS and controls.

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta (TGFbeta) family, is a protein that is essential for the survival of dopaminergic, motor, and peripheral neurons. To facilitate its study, we and others have developed sensitive (low pg/ml) enzyme-linked immunosorbant assays (ELISA) to quantitate endogenous concentrations of GDNF, along with neurotrophin-3 (NT-3) and nerve growth factor (NGF). However, endogenous tissue levels of GDNF in adult animals are not readily detected by ELISA and do not correlate well with message RNA. Based upon previously described methods for the extraction of TGFbeta from tissue samples, we have developed an acid-treatment procedure to allow the quantification of total endogenous GDNF. This procedure also was evaluated for use when measuring total endogenous levels of NT-3 and NGF from biological samples. The acid-treatment procedure increases the detectable amounts of GDNF, NT-3, and NGF in all tissue samples and most of the serum samples tested. Moreover, these values were as much as 35 times greater than those detected using traditional extraction buffers. Such elevated concentrations likely resulted from the acid treatment promoting the dissociation of ligands from receptors or binding proteins, thereby making more of the analyte available to be measured in the ELISA. These findings indicate that appropriate sample treatment is essential for the measurement of total endogenous neurotrophic factors.

Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.