Activation of p38 mitogen-activated protein kinase is required for in vivo brain-derived neurotrophic factor production in the rat hippocampus.

Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation.

Activity-dependent change in the protein level of brain-derived neurotrophic factor but no change in other neurotrophins in the visual cortex of young and adult ferrets.

Neurotrophins are suggested to play a role in activity-dependent plasticity of visual cortex during the critical period of postnatal development. Thus, the concentration of neurotrophins in the cortex is expected to change with development and/or with alteration in neuronal activities. To test this, we measured protein levels of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 in visual cortex of young (postnatal day 38-46, at the peak of the critical period) and adult ferrets with two-site enzyme-immunoassay systems. Measurements were carried out also in somatosensory cortex, hippocampus and cerebellum as control. With development the level of brain-derived neurotrophic factor did not significantly change, while those of the other neurotrophins changed in the visual cortex. A blockade of visual inputs for 24 h by an injection of tetrodotoxin into both eyes significantly decreased brain-derived neurotrophic factor protein level in the visual cortex, but not in the other regions in both young and adult ferrets. On the other hand, no significant decrease was seen in the protein level of the other neurotrophins in the visual cortex of young and adult ferrets. A monocular injection of tetrodotoxin in young ferrets resulted in the reduction of brain-derived neurotrophic factor by approximately half that by binocular injection. The degree of the decrease in the contralateral cortex to the injected eye was significantly larger than that in the ipsilateral cortex, reflecting that the contralateral eye is dominantly represented in the cortex in ferrets. Blockade of cortical neuronal activities by a GABA(A) receptor agonist led to a remarkable reduction of brain-derived neurotrophic factor protein in the visual cortex. These results suggest that the level of brain-derived neurotrophic factor protein in visual cortex is regulated by activities of cortical neurons.

Selective localization of G protein gamma5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain.

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, alpha, beta and gamma, are highly expressed in the brain. The Ggamma5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Ggamma5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Galphai2 subunit colocalized with Ggamma5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Ggamma5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Ggamma5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.

Distribution of yieldin, a regulatory protein of the cell wall yield threshold, in etiolated cowpea seedlings.

We examined the distribution and the immunohistochemical localization of yieldin in etiolated cowpea seedlings with an anti-yieldin antibody. An immunoblotting analysis revealed that the yieldin was located in the aerial organs (plumule, epicotyl and hypocotyl) but not in the roots. The intensity of the yieldin signal in the hypocotyls was highest in the apical pre-elongation region (the hook region) and decreased toward the elongated mature base indicating that the yieldin disappeared with the ceasing of cell elongation. Tissue-print immunoblotting analysis using hypocotyls in different germination stages supports this view because the apical yieldin-rich regions, just beneath the cotyledonary node (the hook and rapidly elongating regions), acropetally migrated together with hypocotyl elongation. Immunohistochemical microscopy demonstrated that yieldin was localized in the cell walls of the cortex and epidermis of the germ axes. The present results are consistent with the view that yieldin participates in the regulation of cell wall yielding during elongation growth.

Identification and molecular cloning of a novel brain-specific receptor protein that binds to brain injury-derived neurotrophic peptide. Possible role for neuronal survival.

Brain injury-derived neurotrophic peptide (BINP) is a synthetic 13-mer peptide that supports neuronal survival and protects hippocampal neurons in primary cultures from cell death caused by glutamate. We have developed a monoclonal antibody named mAb 6A22 against the 40-kDa BINP-binding protein, p40BBP. mAb 6A22 inhibits binding between BINP and rat brain synaptosomes and abolishes the protective effect of BINP. The antigen of mAb 6A22 should be the BINP-binding protein that mediates the neuroprotective action of BINP. Using an expression cloning approach with mAb 6A22, we isolated a cDNA encoding a novel receptor protein that shows binding activity of BINP. COS7 cells transfected with the cloned cDNA show binding of BINP and cell surfaces that are stained by 6A22. The mRNA for p40BBP is specific for the rat brain and is increased after birth. From immunohistochemical studies using mAb 6A22, p40BBP increased after kainic acid treatment in rat hippocampal neurons.

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons.

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels.

A high level of hippocampal brain-derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (gamma-hexachlorocyclohexane); and L-type voltage-dependent calcium channel agonist, BAY-K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY-K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12--24 h, 8--16 h and 6 h following administration of kainic acid, PTZ and BAY-K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY-K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY-K 8644, but not PTZ, effects. omega-Conotoxin GVIA and DCG-IV partially blocked kainic acid-induced enhancement of BDNF, indicating involvement of L-type and N-type voltage-dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in D-myo-inositol-1,4,5-triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY-K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant-dependent stimulation of BDNF production and involvement of region-specific voltage-dependent calcium channels.

Neurotrophin-3 controls proliferation of granular precursors as well as survival of mature granule neurons in the developing rat cerebellum.

Levels of neurotrophin-3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin-3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin-3 into the lateral ventricle of 7.5-day-old rats. The resultant depletion of neurotrophin-3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin-3 included reduced incorporation of bromode-oxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin-3 protein in the posterior region, including the central lobe, was about four- and threefold higher than that in the anterior region of the cerebellum of 9.5- and 30-day-old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin-3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin-3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.

Functional regulation of tactile sense by brain-derived neurotrophic factor in adult rats during acute inflammation.

Nerve growth factor is present in skin in limiting amounts and is known to regulate the plasticity and the sensitivity of nociceptive neurons. Recently, knock-out mouse studies showed that neurotrophin-3 and brain-derived neurotrophic factor are required for the postnatal survival and functional maturation, respectively, of tactile sensory neurons. However, the roles of neurotrophin-3 and brain-derived neurotrophic factor in adult sensory neurons have not been clarified. Here, we report an unexpected and marked acute loss of tactile sense in the rat hind paw after adjuvant-induced inflammation. This loss was shown to be closely correlated with decreases in the expression of brain-derived neurotrophic factor, and to a lesser extent of neurotrophin-3 in the inflamed skin. Administration of brain-derived neurotrophic factor, but not neurotrophin-3, after inflammation accelerated the recovery of tactile sense. These results suggested a role of brain-derived neurotrophic factor in the physiological regulation of tactile sense in adulthood.

Inhibition of adjuvant-induced inflammatory hyperalgesia in rats by local injection of neurotrophin-3.

The induction of nerve growth factor (NGF) in inflammatory tissue has been shown to be involved in hyperalgesia. In the present study, the role of neurotrophin-3 (NT-3) in the regulation of inflammatory hyperalgesia was analyzed. Inflammatory hyperalgesia was induced by intraplantar injection of complete Freund's adjuvant (CFA) to the rat hind paw. NT-3 levels in the plantar skin were much higher than NGF levels (1.24 and 0.14 ng/g tissue, respectively) before CFA injection, but decreased significantly 6 h to 48 h after the injection while NGF was markedly induced at 6 h but decreased thereafter. When 1 microg of NT-3 was locally injected at 5 h after CFA injection at the time NT-3 levels decreased, hyperalgesia was reversed transiently but specifically. These results suggest an inhibitory role of NT-3 in the regulation of pain sensitivity.

Brain-derived neurotrophic factor, nerve growth and neurotrophin-3 selected regions of the rat brain following kainic acid-induced seizure activity.

Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12-24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (approximately 10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (approximately 10-fold) and 8.2 ng/g in the olfactory bulb (approximately 16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.

Responses of heat shock proteins hsp27, alphaB-crystallin, and hsp70 in rat brain after kainic acid-induced seizure activity.

We determined the changes in the levels of the mammalian small heat shock protein of 25-28 kDa (hsp27) and the hsp alphaB-crystallin in various regions of rat brain after kainic acid-induced seizure activity by means of specific immunoassays. The levels of hsp27 in the hippocampus and entorhinal cortex were markedly increased and reached a maximum (1.5-2 microg/mg of protein) 2-4 days after the seizure. The levels of hsp27 in these regions were considerably high even 10 days after the seizure. A marked increase in levels of mRNA for hsp27 was also observed in the hippocampus of rats 1-2 days after the seizure. A severalfold increase in the levels of alphaB-crystallin was observed in the hippocampus and entorhinal cortex of rats 2 days after the seizure. However, the maximum levels were <50 ng/mg of protein. The levels of protein sulfhydryl group and glutathione were significantly reduced in the hippocampus of rats at 24 h after the seizure, which might have enhanced the expressions of hsp27 and alphaB-crystallin. The expression of inducible mammalian hsp of 70 kDa (hsp70) was also enhanced in the hippocampus of rats after the seizure, as detected by western and northern blotting analyses. Immunohistochemically, an intensive staining of hsp27 was observed in both glial cells and neurons in the hippocampus, piriform cortex, and entorhinal cortex of rats with kainic acid-induced seizure. However, in the cerebellum, where the receptors for kainic acid are also rich, hsp27 was barely induced in the same rats. This might be due to high levels of the cerebellar calcium-binding proteins parvalbumin and 28-kDa calbindin-D, which might have a protective effect against the kainic acid-inducible damage.

Brain-derived neurotrophic factor prevents low-frequency inputs from inducing long-term depression in the developing visual cortex.

Brain-derived neurotrophic factor (BDNF) is reported to enhance synaptic transmission and to play a role in long-term potentiation in hippocampus and neocortex. If so, a shortage or blockade of BDNF might lead to another form of synaptic plasticity, long-term depression (LTD). To test this possibility and to elucidate mechanisms if it is the case, EPSCs evoked by test stimulation of layer IV were recorded from layer II/III neurons in visual cortical slices of young rats in the whole-cell voltage-clamp mode. LTD was induced by low-frequency stimulation (LFS) at 1 Hz for 10-15 min if each pulse of the LFS was paired with depolarization of neurons to -30 mV but was not induced if their membrane potentials were kept at -70 mV. Such an LTD was blocked by exogenously applied BDNF, probably through presynaptic mechanisms. Suppression of endogenous BDNF activity by the anti-BDNF antibody or an inhibitor for BDNF receptors made otherwise ineffective stimuli (LFS without postsynaptic depolarization) effective for LTD induction, suggesting that endogenous BDNF may prevent low-frequency inputs from inducing LTD in the developing visual cortex.

An enhanced conversion from tightly bound to loosely bound form of NGF in selected regions of brains from male mice.

Most of the nerve growth factor (NGF) protein in the rat and mouse brain is readily extractable in the presence of guanidine hydrochloride as is the case of brain-derived neurotrophic factor. In the present study, we measured amounts of NGF that could be extracted in the presence and absence of 1 M guanidine hydrochloride from various regions of the brains of male and female mice. About 14% of the total NGF in the hippocampus from female mice at 4 months of age could be extracted without 1 M guanidine hydrochloride (designated loosely bound NGF; about 32% in the rat hippocampus) and the remainder only in its presence (designated tightly bound NGF). The molecular masses of the NGF-immunoreactive protein in both cases were approximately 14 kDa. There were significant differences in respective concentrations of total NGF (the loosely bound plus tightly bound NGF) in the hypothalamus and hypophysis, but not in other brain regions, between male and female mice at 4 months of age. However, levels of loosely bound NGF in the cerebellum and olfactory bulb from males were significantly higher than those in the same regions from females. This difference resulted in two-fold higher ratios of the concentrations of loosely bound to total NGF in males as compared to females. On the other hand, the ratio in the hypophysis was close to unity in both sexes. The concentrations of loosely bound NGF in the hippocampus and cerebral cortex decreased slightly with age in both males and females. Levels of loosely bound NGF increased significantly from 2 to 12 months after birth in the whole brain, olfactory bulb, cerebellum, hypothalamus and hypophysis to a greater extent in males than in females. Thus, it is suggested that high ratios of loosely bound to total NGF in selected regions of brains from male mice are due to an enhanced conversion from tightly to loosely bound form, which is considered to be regulated by androgens (see Brain Res. 322, 112-117, 1990). They may also influence the total NGF expression.

Two types of brain chondroitin sulfate proteoglycan: their distribution and possible functions in the rat embryo.

The distribution of neurocan-like and 6B4 proteoglycan-like immunoreactivities in the rat embryo was investigated from gestational days 10.5-15.5 with monoclonal antibody 1G2 or 6B4 that immunoreacted with neurocan and 6B4 proteoglycan, respectively. In the brain region, the leptomeningeal layer in the myelencephalon, metencephalon, diencephalon or telencephalon was first stained with monoclonal antibody 1G2 at embryonic day 12.5. In the spinal cord, monoclonal antibody 1G2 stained the regions corresponding to the boundary caps (designated the boundary caps) after embryonic day 11.5 and the roof plate after embryonic day 12.5. The intensity of staining in the boundary caps reached a maximum at embryonic day 13.5, at around the time when the axons from the dorsal root ganglia reach this region. However, the points of contact of the axons with the boundary caps were hardly stained. By contrast, the roof plate was most strongly and widely stained at embryonic day 14.5, at around the time when the axons enter the spinal cord. Western blotting of preparations from the spinal cord that included the boundary caps revealed the presence of neurocan in this region. Thus, it is likely that neurocan serves as a barrier molecule to regulate the direction of axonal growth from the dorsal root ganglia. By contrast, in addition to staining of the future brain and spinal cord, monoclonal antibody 6B4 stained the trigeminal and sympathetic ganglia in the rat embryo on and after embryonic day 12.5, as well as the vestibular, facial and dorsal root ganglia after embryonic day 12.5. In studies in tissue culture, monoclonal antibody 6B4 prevented the inhibitory effects of 6B4 proteoglycan on the proliferation of PC12D cells. No immunostaining with monoclonal antibody 6B4 was observed in cells that had incorporated bromodeoxyuridine in vivo. Possible functions of 6B4 proteoglycan in the rat embryo are discussed.

Age-related changes in levels of brain-derived neurotrophic factor in selected brain regions of rats, normal mice and senescence-accelerated mice: a comparison to those of nerve growth factor and neurotrophin-3.

Age-related changes in the levels of brain-derived neurotrophic factor (BDNF) in selected regions of brains from rats, normal mice and senescence-accelerated mice were compared to those of nerve growth factor (NGF) and neurotrophin-3 (NT-3). The concentration of BDNF increased with age in the rat hippocampus while it decreased in the rat cerebral cortex. The level of BDNF in the hippocampus from aged rats was about 260%, of that in the same region from young adult rats. A strong staining with antibodies specific for BDNF was observed in the hilus of the dentate gyrus in the hippocampus from aged rats. By contrast, BDNF levels were significantly lower in four brain regions from aged rats as compared to young adult rats (30, 56, 52 and 52%, lower in the septum, cerebral cortex, cerebellum and striatum, respectively). Patterns of age-related changes in the level of BDNF in the mouse hippocampus. cerebral cortex, cerebellum and olfactory bulb were similar to those in the respective regions from rats. In rats, the concentration of NGF decreased with age in the cerebral cortex but remained unchanged in the hippocampus, cerebellum and olfactory bulb. In mice, levels of NGF increased in all four brain regions from 1 to 18 months after birth. The concentrations of NT-3 increased and decreased with age in the rat cerebral cortex and cerebellum, respectively, while minimal changes were observed in the rat hippocampus and olfactory bulb as was also true in mice. In senescence-accelerated mice with memory disturbances, no marked increases in levels of NGF and BDNF in the hippocampus and in the level of NT-3 in the cerebral cortex were found. Thus, increases in levels of BDNF and NT-3 occurred in the murine hippocampus and cerebral cortex, respectively, during normal aging, but not during aging of mice with pathological changes.

G protein gamma subunits coimmunoprecipitated with antibodies against alpha subunits: identification of major isoforms in cultured cells by silver stain and immunoblotting with conventional transfer procedure.

The betagamma subunits of G proteins were coimmunoprecipitated with antibodies against various alpha subunits, and analyzed by silver stain and immunoblotting with conventional transfer procedure and membrane-blocking buffer containing 2% BSA. Multiple isoforms of gamma were coimmunoprecipitated with no significant difference in form or ratio among the antibodies against alpha subunits used, suggesting antibodies against any alpha subunit could coimmunoprecipitate all forms of gamma. Therefore, this method was applicable to analyze gamma subunits in various cells, especially to clarify what forms of gamma subunits are major components. The major isoforms were: gamma5 in C6, NG108-15, HeLa, HEK293, and F9 cells; gamma12 in Swiss 3T3 and BRL-3A cells; and gamma3 in PC12 cells. In addition to most gamma subunits identified, unidentified gamma subunits were present in PC12, NG108-15, and BRL-3A cells. Furthermore, the method was applied to examine changes of isoforms of gamma during differentiation of HL-60 cells. Undifferentiated cells mainly contained gamma5, but retinoic acid treatment of cells replaced most gamma5 with gamma2. Thus, this method is useful to determine the major isoforms which seem to be the more important in cells.

High potassium enhances secretion of neurotrophic factors from cultured astrocytes.

Elevation of extracellular potassium concentration ([K+]o) in the central nervous system (CNS), which is observed such after physiological stimuli and during ischemia, is known to be regulated by astrocytes. We suspected that in response to increased [K+]o, astrocytes might secrete some neurotrophic factor(s) to promote the survival of active and/or ischemically damaged neurons. In the present study, we examined neurotrophic activity contained in HK-ACM, i.e., astrocyte-conditioned medium (ACM) obtained after culturing astrocytes in 40 mM potassium-containing medium (HK medium). Addition of HK-ACM to basal forebrain cultures from postnatal 2-week-old (P2w) rats increased both the choline acetyltransferase (ChAT) activity (4.40-fold) and the number of ChAT-positive neurons (2.01-fold) as compared with non-conditioned HK medium. On the other hand, the neurotrophic effects of LK-ACM, i.e., ACM collected after culturing astrocytes in 4 mM potassium-containing medium (LK medium), were much weaker (2.85- and 1.41-fold for ChAT activity and number of ChAT-positive neurons, respectively) than those of HK-ACM. The neurotrophic effects of ACMs increased in a manner dependent on potassium concentration and on astrocyte culture time. Addition of an antibody against nerve growth factor (NGF) neutralized the neurotrophic effects of HK- and LK-ACMs. Direct quantification of NGF protein in ACMs by the two-site ELISA method demonstrated that a high concentration of potassium enhanced NGF secretion from cultured astrocytes. These results suggested that astrocytes secrete NGF in response to [K+]o elevation in the CNS.

Distribution of brain-derived neurotrophic factor in rats and its changes with development in the brain.

A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that approximately 50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of approximately 14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.

A possible role of the nestin protein in the developing central nervous system in rat embryos.

Rat embryos at the head-hold stage (Slc:SD strain; 9.5 days of gestation) were cultured for 48 h in rat serum with the anti-nestin peptide antiserum. The antiserum identified a single band in Western blots of the tissue extracts from rat embryos and stained the cells from the neural tube, migrating neural crest, and somites immunohistochemically. The antiserum-treated embryos appeared to develop normally for the most part. However, histological observation disclosed that the ventral portion of the neural tube was deformed. The cells in the deformed portion did not show the elongated shape but were round. These round cells tended to crowd near the ventricular surface, and a gap was observed between the original pial surface and cells arranged in the most pialward region. The penetration of the anti-nestin peptide antibody into the embryos from the culture medium was confirmed by visualization of the penetrated antibody using biotinylated anti-rabbit IgG antibody raised in goats and Texas red-conjugated streptavidin. These results indicate that the nestin protein plays an important role in the organization or the maintenance of neuroepithelial cells of the elongated shape spanning the neural tube from the luminar to the pial side.