Klaus D. Beck

Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal granule and striatal parvalbumin-containing neurons

Homozygous Igf1-/- mice at 2 months of age had reduced brain weights, with reductions evenly affecting all major brain areas. The gross morphology of the CNS was normal, but the size of white matter structures in brain and spinal cord was strongly reduced, owing to decreased numbers of axons and oligodendrocytes. Myelinated axons were more strongly reduced in number than unmyelinated axons. The volume of the dentate gyrus granule cell layer was reduced in excess of the decrease in brain weight. Among populations of calcium-binding protein-containing neurons, there was a selective reduction in the number of striatal parvalbumin-containing cells. Numbers of mesencephalic dopaminergic neurons, striatal and basal forebrain cholinergic neurons, and spinal cord motoneurons were unaffected. Cerebellar morphology was unaltered. Our findings suggest cell type- and region-specific functions for IGF-I and emphasize prominent roles in axon growth and maturation in CNS myelination.

The nature of the trophic action of brain-derived neurotrophic factor, des(1-3)-insulin-like growth FACTOR-1, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture

Brain-derived neurotrophic factor, basic fibroblast growth factor and des(1-3)-insulin-like growth factor-1, a brain specific form of insulin-like growth factor-1, were analysed, in the rat, for their influence on survival, morphological growth, and transmitter-specific differentiation of dopaminergic neurons in vitro. Brain-derived neurotrophic factor, des-insulin-like growth factor-1, and basic fibroblast growth factor were found to differentially regulate development of dopaminergic cells. Brain-derived neurotrophic factor stimulated survival, the formation of primary neurites and dopamine uptake activity. des-Insulin-like growth factor-1 was most effective in promoting survival, stimulated dopamine uptake less effectively than brain-derived neurotrophic factor and did not alter the morphology of dopaminergic cells. Basic fibroblast growth factor produced comparatively mild increases in survival and dopamine uptake, and slightly reduced neurite growth of the cells. None of the factors stimulated the expression of the tyrosine hydroxylase gene. These findings suggest that (i) effective growth factors may stimulate different, but partially overlapping, molecular pathways during developmental differentiation, (ii) none of the factors stimulates dopaminergic cell differentiation comparable to the pronounced trophic action of nerve growth factor on peripheral sympathetic or basal forebrain cholinergic neurons, and (iii) localization and effects of none of the factors are compatible with a role as target-derived survival-regulating neurotrophic factor.

K‐252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin‐3

Abstract: K‐252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K‐252b was found to prevent trophic actions of two other neurotrophins, brain‐derived neurotrophic factor, and neurotrophin‐3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 μM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1‐100 nM, K‐252b selectively enhanced the trophic action of neurotrophin‐3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC 12 cells, K‐252b potentiated the neurotrophin‐3‐induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K‐252a, K‐252b, and staurosporine, only the first two also mediated neurotrophin‐3 potentiation. These findings indicate that K‐252b generally and selectively potentiates the neurotrophic action of neurotrophin‐3 and suggest that this action involves trk‐type neurotrophin receptors.

Expression of neurotrophin and trk receptor genes in adult rats with fimbria transections : effect of intraventricular nerve growth factor and brain-derived neurotrophic factor administration

The expression of the specific trk receptors for nerve growth factor and brain-derived neurotrophic factor (trkA and trkB) has been assayed by messenger RNA in situ hybridization in adult rats with partial fimbrial transections along with intraventricular treatment of nerve growth factor or brain-derived neurotrophic factor. In the forebrain, specific hybridization labeling for trkA messenger RNA showed an identical pattern to that of choline acetyltransferase messenger RNA, supporting the view that trkA expression is confined to the cholinergic population in the basal forebrain and the cholinergic interneurons in the striatum. After partial unilateral transections of the fimbria there was a progressive loss of choline acetyltransferase and trkA messenger RNA expression in the septal region ipsilateral to the lesion. Daily intraventricular administration of brain-derived neurotrophic factor or nerve growth factor partially prevented the lesion-induced decrease in the levels of both messengers, the latter being more effective than the former. Grain count analysis of individual cells was used to test whether the two factors upregulated choline acetyltransferase or trkA expression in individual cells surviving the lesion. Brain-derived neurotrophic factor treatment failed to induce any change in the levels of both messengers per neuron in the septal area. In contrast, daily intraventricular administration of nerve growth factor upregulated both choline acetyltransferase and trkA messenger RNA expression in individual neurons. This upregulation was evident on ipsilateral and contralateral sides, suggesting that nerve growth factor is able to upregulate these markers in intact and injured cholinergic cells in the basal forebrain. Similar to the situation in the septum, brain-derived neurotrophic factor did not upregulate choline acetyltransferase or trkA expression in the striatum. However, nerve growth factor administration strongly upregulated choline acetyltransferase messenger RNA expression by individual cholinergic neurons of the striatum. A medial to lateral gradient decrease in this upregulation was detected in the striatum ipsilateral to the side of administration, suggesting a limited diffusion of the nerve growth factor protein from the ventricle into brain parenchyma. In contrast to the strong effect on choline acetyltransferase expression, nerve growth factor treatment was ineffective in altering trkA messenger RNA in the striatum. The contrasting findings between septum and striatum suggest different regulatory mechanisms for trkA messenger RNA expression in the two cholinergic populations. Since nerve growth factor was found to upregulate the expression of its trkA receptor, we tested whether brain-derived neurotrophic factor administration had similar effects on the regulation of its trkB receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

BDNF and trkB mRNA expression in the hippocampal formation of aging rats.

Quantitative in situ hybridization and northern blot analysis techniques were used to determine the topographical distribution and levels of mRNA coding for brain-derived neurotrophic factor (BDNF) and the tyrosine receptor kinase (trkB) mRNA in the hippocampal formation of two strains of male rat during aging. Age did not change the prevalence or regional distribution patterns of BDNF or trkB mRNA in the hippocampal formation throughout the lifespan of male Sprague-Dawley rats. There also were no significant differences in the prevalence or topographical distribution patterns of trkB mRNA transcripts during aging. Northern blot analysis of hippocampal RNA from male Fischer 344 confirmed that neither BDNF mRNA nor trkB mRNA levels changed with age. These findings suggest that age-related neurodegenerative changes, including the atrophy of the cholinergic septo-hippocampal pathway, are not the result of changes in hippocampal BDNF or trkB mRNA expression. Moreover, the prevalence and distribution of synaptosomal-associated protein 25 (SNAP-25), a neuron-specific protein located in synaptic terminals and a putative marker of synaptic integrity, did not change with age. These findings indicate that altered hippocampal synaptic plasticity which occurs in the aged rat brain is not a reflection of changes in the expression of BDNF or trkB receptor mRNA.

Rapid Phosphorylation of Phospholipase Cγ 1 by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Cultures of Embryonic Rat Cortical Neurons

Abstract: Phospholipase Cγ1 (PLC‐γ1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5‐bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron‐like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC‐γ1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) on phosphorylation of PLC‐γ1 in primary cultures of embryonic rat brain cells. BDNF and NT‐3 stimulated the phosphorylation of PLC‐γ1, followed by hydrolysis of PI. The stimulation of PLC‐γ1 phosphorylation occurred within 20 s after addition of BDNF or NT‐3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT‐3, with τ25 ng/ml. Phosphorylation of PLC‐γ1 by BDNF and NT‐3 was found in cultures from all major brain areas. K‐252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk‐type neurotrophin receptors, prevented the BDNF‐ and NT‐3‐stimulated phosphorylation of PLC‐γ1. Receptors of the trk type were coprecipitated with anti‐PLC‐γ1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT‐3 seems to be neuron specific because no phosphorylation of PLC‐γ1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT‐3, and they indicate that the transduction mechanism of BDNF and NT‐3 in the brain involves rapid phosphorylation of PLC‐γ1 followed by PI hydrolysis.

Chronic intranigral administration of brain-derived neurotrophic factor produces striatal dopaminergic hypofunction in unlesioned adult rats and fails to attenuate the decline of striatal dopaminergic function following medial forebrain bundle transection

The present study determined the effects of chronic intranigral injections of recombinant human brain-derived neurotrophic factor (1 micrograms) every second day for 19 days on the functional capacity of dopaminergic neurons of the nigrostriatal pathway of unlesioned adult rats. In animals chronically treated with brain-derived neurotrophic factor, we observed amphetamine (5 mg/kg)-induced circling behavior directed toward the neurotrophin-injected side (33 turns/5 min). The behavioral asymmetry was paralleled by reductions of striatal [3H]dopamine uptake (27%), tyrosine hydroxylase activity (68%), dopamine content (36%) and [3H]mazindol binding site density (35%) on the same side as brain-derived neurotrophic factor treatment. While chronic injections of brain-derived neurotrophic factor produced a modest decrease in the number of tyrosine hydroxylase-positive cell bodies in the vicinity of the injection site, a similar reduction in cell number was observed in animals injected with a control protein, cytochrome c. However, in contrast to the animals treated with brain-derived neurotrophic factor, rats treated with the control protein showed no amphetamine-induced circling behavior, and there were no significant reductions in neurochemical parameters of striatal dopaminergic function. Lastly, we found that in brain-derived neutrophic factor-injected animals there was a 30% decrease of tyrosine hydroxylase messenger RNA levels in the ventral mesencephalon. We also determined the effects of brain-derived neurotrophic factor treatment on animals with transections of the medial forebrain bundle. Medial forebrain bundle-lesioned animals challenged with amphetamine circled (55 turns/5 min) ipsilateral to the lesioned side. The medial forebrain lesions decreased the following markers of striatal dopaminergic function: [3H]opamine uptake (65%), tyrosine hydroxylase activity (79%), dopamine content (80%) and [3H]mazindol binding site density (52%), induced a pronounced loss of tyrosine hydroxylase-positive cell bodies within the substantia nigra and also reduced tyrosine hydroxylase messenger RNA levels. Chronic intranigral brain-derived neurotrophic factor treatment did not attenuate nor did it exacerbate the medial forebrain bundle lesion-induced decreases of dopaminergic parameters in either the substantia nigra or striatum. The results of the present study indicate that chronic intranigral administration of brain-derived neurotrophic factor to normal adult rats induces a dopaminergic hypofunction in the striatum which is manifested behaviorally by amphetamine-induced rotations. The brain-derived neurotrophic factor-induced striatal function is not the result of significant cell loss at the levels of the substantia nigra, but seems to be related to brain-derived neurotrophic factor-induced down-regulation of dopaminergic-specific proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Intrastriatal infusion of nerve growth factor after quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA

Excitotoxic striatal lesions induced by quinolinic acid, a model for Huntingtons disease, were used to test for neuroprotective actions of nerve growth factor on striatal cholinergic and GABAergic neurons. Expressions of the trkA receptor for nerve growth factor, choline acetyltransferase and glutamate decarboxylase were analysed by messenger RNA in situ hybridization in adult rats following quinolinic acid lesion (150 nmol) and daily striatal administration of nerve growth factor (1 microgram) or control protein (cytochrome C) for one week. One week after toxin administration, the numbers of cells expressing trkA or choline acetyltransferase messenger RNAs were decreased when compared with unlesioned animals. Moreover, the surviving cells showed a strong down-regulation of these messenger RNAs as deduced from grain count analysis of sections processed for emulsion autoradiography. Daily intrastriatal nerve growth factor administration for one week completely prevented the reduction in the number of cells expressing either of the two markers. Nerve growth factor treatment increased the cellular expression of choline acetyltransferase messenger RNA three times above control levels and restored the levels of trk A messenger RNA expression to control levels. In contrast to the protective effects on cholinergic cells, nerve growth factor treatment failed to attenuate the quinolinic acid-induced decrease in glutamate decarboxylase messenger RNA levels. Optical density measurements of the entire striatum on autoradiographs of brain sections from quinolinic acid-lesioned animals revealed a reduction of the glutamate decarboxylase messenger RNA-specific hybridization signal, which was unaltered by infusion of nerve growth factor or control protein. Our findings strongly suggest that in both the intact and the quinolinic acid-lesioned adult rat striatum, nerve growth factor action is confined to trk A-expressing cholinergic neurons. Striatal glutamate decarboxylase messenger RNA-expressing GABAergic neurons which degenerate in Huntingtons disease are not responsive to nerve growth factor.

Differential regulation of catalytic and non-catalytictrkB messenger RNAs in the rat hippocampus following seizures induced by systemic administration of kainate

Ribonuclease protection analysis and quantitative in situ hybridization histochemistry were used to investigate the coordination and regional expression of catalytic and non-catalytic trkB messenger RNAs in the adult rat hippocampus following systemic kainate-induced seizures. Changes in trkB expression were compared with the messenger RNA expression of its neurotrophic ligands, brain-derived neurotrophic factor and neurotrophin-3. TrkB messenger RNA expression was increased in the dentate granule cells at 1-4 h following the onset of seizures, and returned to control levels 16-24 h thereafter. In addition, seizures also induced expression of trkB messenger RNA in putative non-neuronal cells at four to seven days in the molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1 region. Hybridization with probes specific for the non-catalytic trkB receptor and the catalytic trkB receptor revealed that the increases at four and seven days in the molecular layers of the hippocampus reflected an up-regulation of only the non-catalytic form of the receptor. Furthermore, the neuronal increases observed 1-4 h were due to an up-regulation of both trkB TK- and trkB TK+ messenger RNAs. It was established that systemic administration of kainate increased brain-derived neurotrophic factor messenger RNA levels in the pyramidal and granule cell regions of the hippocampus 1-4 h following the onset of behaviorally manifested seizure activity. Early changes in neuronal expression of trkB TK- and trkB TK+ messenger RNA paralleled changes in brain-derived neurotrophic factor messenger RNA in the dentate granule cell and CA1 pyramidal cell layers, but not in the CA3 subregion. These data suggest that concomitant regulation of brain-derived neurotrophic factor and its cognate receptor may play a role in the selective vulnerability of hippocampal subregions to kainate-induced neuropathology. Furthermore, these data suggest a dual function for trkB receptor expression in the hippocampus following kainate-induced seizures, possibly related to both the plastic and degenerative consequences of seizure induction by kainate.

6-Hydroxydopamine lesions reduce BDNF mRNA levels in adult rat brain substantia nigra.

Expression of brain-derived neurotrophic factor (BDNF) in the ventral mesencephalon has been assayed by mRNA in situ hybridization in adult rats with unilateral injections of 6-hydroxydopamine in the substantia nigra. On contralateral control sides, a specific hybridization signal was detected in the substantia nigra pars compacta (A9), ventral tegmental area (A10) and substantia nigra pars lateralis (A8). Cellular levels of BDNF mRNA were lower than those in BDNF expressing cortical and hippocampal neurons. The 6-hydroxydopamine injections completely abolished BDNF mRNA labeling in the pars compacta of the substantia nigra, whereas many labeled neurons remained in the ventral tegmental area and pars lateralis. The results strongly suggest that BDNF is expressed by nigral dopaminergic neurons.