Absence of hippocampal mossy fiber sprouting in transgenic mice overexpressing brain-derived neurotrophic factor.

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2--3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus.

Neurotrophin-4/5 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons.

Ca(2+) currents and their modulation by neurotrophin-4/5 were studied in cultured mesencephalic neurons. Tyrosine hydroxylase-positive neurons consistently had larger somas than tyrosine hydroxylase-negative neurons. Neurons with larger somas were therefore targeted for recording. In both control and neurotrophin-4/5-treated cultured neurons, isolation of Ca(2+) currents in cultured mesencephalic neurons revealed prominent low- and high-voltage-activated currents. These currents were separable based upon their voltage dependence of activation, the response to replacement of Ca(2+) with Ba(2+) and the response to Ca(2+) channel blockers. Replacement of Ca(2+) with Ba(2+) resulted in a slight reduction of low-voltage-activated currents and a significant enhancement of high-voltage-activated currents. Cd(2+) blocked a larger fraction of the high-voltage-activated current than Ni(2+). The synthetic conotoxins SNX-124 and SNX-230 selectively blocked high-voltage-activated currents. Morphological analysis of mesencephalic cultures pretreated with neurotrophin-4/5 revealed an increase in soma size and dendritic length in tyrosine hydroxylase-positive neurons. In agreement with the neurotrophin-4/5 induction of growth, neurotrophin-4/5 also increased cell capacitance in whole-cell recordings. Neurotrophin-4/5 significantly enhanced both low- and high-voltage-activated currents, but normalization for changes in capacitance revealed only a significant increase in high-voltage-activated current density. This study demonstrates the existence of low-voltage-activated and multiple classes of high-voltage-activated calcium currents in cultured mesencephalic neurons. Morphological and physiological data demonstrate that the increases in calcium currents due to neurotrophin-4/5 pretreatment are associated with somatodendritic growth, but an increase in high-voltage-activated Ca(2+) channel expression also occurred.

Metabolic compromise with systemic 3-nitropropionic acid produces striatal apoptosis in Sprague-Dawley rats but not in BALB/c ByJ mice.

Metabolic compromise with systemic 3-nitropropionic acid (3-NP) results in the degeneration of striatal cells, mimicking the pathology of Huntington's disease (HD). Here we show that 10-week- and 8-month-old BALB/c ByJ mice show an unexpected striatal resilience to single and multiple systemic injections of 3-NP, while Sprague-Dawley rats are vulnerable, albeit in a variable manner. Identification of lesions was made by staining of DNA fragmentation with terminal deoxytransferase-mediated dUTP-biotin nick-end labeling (TUNEL) and hematoxylin/eosin, 1-10 days after injection. Quantitative imaging of histochemistry for succinate dehydrogenase (SDH) activity, the target of 3-NP inhibition, revealed that vulnerable rats reached maximal inhibition in brain at 1 day after 3-NP, whereas mice and resilient rats took 7 days to reach maximal inhibition. All groups of animals reached similar maximal decreases in SDH activity in striatum and cortex. Remarkably, only the fast decline in SDH activity seen in vulnerable rats was associated with TUNEL labeling. In addition, vulnerable rats developed a region within striatum where SDH activity was fully depleted and a similarly depleted region in CA1 hippocampus. While mice did not develop this region in striatum, some developed one in CA1. These regions of SDH depletion in both structures were associated with widespread TUNEL staining, with maximal labeling at 3 days after 3-NP. The existence of an animal strain resilient to 3-NP suggests that there are mediating factors involved in the preferential vulnerability of striatum to metabolic lesioning. The identification of these factors could provide strategies for therapeutic intervention in HD.

Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning in Stargazer mutant mice.

In the spontaneous ataxic mutant mouse stargazer, there is a selective reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. BDNF protein levels in the cerebellum are reduced by 70%. Despite normal levels of full-length and truncated TrkB receptor, constitutive and neurotrophin-4/5-induced tyrosine phosphorylation was significantly reduced in several signal transduction molecules, including phospholipase-Cgamma1, erk1, and erk2. Morphological examination revealed an increased number of external granule cells at postnatal day 15 and the presence of abnormal neurons resembling immature granule cells in the adult. These abnormalities are associated with a severe impairment in the acquisition of classical eyeblink conditioning, indicating cerebellar malfunction. Our data suggest that normal BDNF expression and TrkB signal transduction in the cerebellum are necessary for learning and plasticity in this model.

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Laminin-alpha2 chain-like antigens in CNS dendritic spines.

The laminin-alpha2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-alpha2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-alpha2 chain G-domain localized laminin-alpha2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-alpha2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-alpha2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-alpha2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-alpha2 chain and/or laminin-alpha2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-alpha2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

Elevated mRNA expression of brain-derived neurotrophic factor in retinal ganglion cell layer after optic nerve injury.

PURPOSE: Recent studies show that exogenous brain-derived neurotrophic factor (BDNF) can promote retinal ganglion cell survival in vivo and in vitro. BDNF is expressed by a subpopulation of cells in the ganglion cell layer (GCL). To investigate whether endogenous BDNF may play a role in neuronal protection after ganglion cell trauma, BDNF expression in the retina was examined after optic nerve (ON) injury. METHODS: The optic nerve in Sprague-Dawley rats was crushed intraorbitally posterior to the optic disc. For controls, the optic nerve on the opposite side in each animal was similarly exposed but was not crushed. After intervals of 6 hours to 6 weeks, eye tissues were processed for in situ hybridization, Northern blot, and RNase protection assay using radiolabeled rat riboprobes. RESULTS: After ON injury, BDNF expression was significantly elevated in cells restricted to the GCL, and more cells demonstrated expression of BDNF than were observed in the controls. Elevated BDNF expression was first observed at 24 hours, peaked at 48 hours, and declined to the basal level 2 weeks after ON injury. Quantitative analysis showed a fivefold to sixfold increase in the number of BDNF-positive cells and a 54% increase in BDNF signal intensity in individual cells in the GCL 48 hours after ON injury. In control retinas without ON injury, BDNF expression was localized to some cells in the GCL, as was observed in normal eyes without surgery. Northern blot and RNase protection assay demonstrated a 38% elevation in BDNF expression above control levels 48 hours after ON injury. CONCLUSIONS: These results indicate that cells in the GCL can upregulate gene expression of BDNF in response to ganglion cell axonal injury and suggest that endogenous BDNF may contribute to a natural neuroprotective process after ON injury.

Axotomized septal cholinergic neurons rescued by nerve growth factor or neurotrophin-4/5 fail to express the inducible transcription factor c-Jun.

The inducible transcription factor c-Jun increases in neurons in response to axotomy by unknown mechanisms, and it has been postulated that c-Jun may regulate genes involved in promoting either degeneration or regeneration of axotomized neurons. In this report, we investigated the effect of daily or twice daily intraventricular administration of the neurotrophins nerve growth factor or neurotrophin-4/5 on the decrease in choline acetyltransferase expression and the increase in c-Jun expression in rat medial septum/diagonal band neurons three, seven and 14 days following unilateral, complete, fornix fimbria lesion. We also examined whether medial septum/diagonal band neurons might die by apoptosis within two weeks of fornix fimbria lesion using terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling. Our results show that both nerve growth factor and neurotrophin-4/5 maintain the phenotype of basal forebrain cholinergic neurons following axotomy. Furthermore, using double-labelling immunofluorescence, we found that while c-Jun was expressed in cholinergic neurons in control-treated rats seven days following fornix fimbria lesion, cholinergic neurons rescued by either nerve growth factor or neurotrophin-4/5 in neurotrophin-treated rats failed to express c-Jun. At no time-point (three, seven or 14 days post-axotomy) did any neurons in the medial septum/diagonal band stain positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling, suggesting that medial septum/diagonal band neurons do not undergo apoptosis within the first two weeks following axotomy at the time-points observed by us. Therefore, these results show that both nerve growth factor and neurotrophin-4/5 rescue the phenotype of axotomized cholinergic neurons and that these rescued neurons fail to express c-Jun in response to axotomy. In addition, since neither nerve growth factor nor neurotrophin-4/5 induced c-Jun in medial septum/diagonal band cholinergic neurons, it seems unlikely that the neurotrophic effects of nerve growth factor and neurotrophin-4/5 on cholinergic neurons are mediated via c-Jun expression. Furthermore, since axotomy failed to increase terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling in septal neurons, it appears unlikely that c-Jun expression in these axotomized neurons is related to neuronal degeneration via apoptosis.

Ligand-induced down-regulation of Trk messenger RNA, protein and tyrosine phosphorylation in rat cortical neurons.

Chronic exposure of brain neurons to nerve growth factor in vitro and in vivo results in increased levels of the nerve growth factor receptor TrkA. In contrast, in the present study, we have found that chronic exposure of rat embryonic cortical neurons to brain-derived neurotrophic factor (BDNF) leads to a pronounced reduction of the levels of protein and messenger RNA for the full-length but not the truncated BDNF receptor TrkB. Similar effects were observed with the other TrkB ligands neurotrophin-3 and neurotrophin-4/5. After pretreatment with BDNF, neurotrophin-3 or neurotrophin-4/5, subsequent tyrosine phosphorylation responses of the remaining Trks to the same factors were greatly reduced. Three days exposure of rat embryonic cortical neurons to BDNF induced an absolute refractory period of several hours, with no subsequent response to the same factor. Similar but less pronounced refractory effects were observed with neurotrophin-3 and neurotrophin-4/5. Our results suggest a negative regulatory effect of BDNF and other TrkB ligands on TrkB receptors. Down-regulation of the TrkB response by its ligands might play a role in the control of BDNF action during early development, when BDNF levels significantly increase. Our findings are also of potential clinical relevance, since the possibility of ligand-induced down-regulation of the receptor response needs to be addressed when considering BDNF or other neurotrophins for the therapy of neurodegeneration.

Cloning/brain localization of mouse glutamylcysteine synthetase heavy chain mRNA.

Glutathione (GSH) is considered the primary molecule responsible for peroxide removal from the brain. Inhibition of its rate-limiting synthetic enzyme, glutamylcysteine synthetase (GCS), results in morphological damage to both cortical and nigral neurons in rodents. Here, we report cloning of the catalytic heavy chain GCS mRNA from mouse and its localization in the murine brain. Heavy chain GCS appears to be localized in glial populations in the hippocampus, cerebellum and olfactory bulb, with lower levels of expression in the cortex and substantia nigra. Variations in GCS levels and subsequent GSH synthesis may explain differences in susceptibility to neuropathology associated with oxidative stress noted in these various brain regions.