Ethanol consumption and serotonin-1A (5-HT1A) receptor function in heterozygous BDNF (+/-) mice.

Heterozygous brain-derived neurotrophic factor (BDNF) (+/-) mice display abnormalities in central serotonergic neurotransmission, develop decrements in serotonergic innervation of the forebrain, and exhibit enhanced intermale aggressiveness. As disturbances of serotonin neurotransmission are implicated in alcohol abuse and aggression, we have examined in BDNF (+/-) mice alcohol drinking behavior, as well as central 5-hydroxytryptamine (5-HT)1A receptor function at the level of 5-HT1A receptor-G protein interaction. BDNF (+/-) mice displayed increased ethanol intake in a two-bottle choice procedure. There was no difference in the preference ratio for non-alcoholic tastants (i.e. quinine or saccharin) between genotypes. In the brains of alcohol-naive mice, we measured [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (+/-)-8-hydroxy-2-dipropyl-aminotetralin hydrobromide (8-OH-DPAT; 1 microM). In BDNF (+/-) versus wild-type (WT) mice, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in the median raphe nucleus. There was a decrease in (+/-)8-OH-DPAT-stimulated [35S]GTP gamma S binding in the dorsal raphe, which did not reach statistical significance. In the hippocampus, 5-HT1A receptor-stimulated [35S]GTP gamma S binding was significantly attenuated in BDNF (+/-) mice. 5-HT1A receptor-stimulated [35S]GTP gamma S binding was attenuated in the anterior cingulate cortex and lateral septum, although these reductions did not reach statistical significance. 5-HT1A receptor number was not different between genotypes in any area of brain examined, suggesting that 5-HT1A receptor function, specifically the capacity of the 5-HT1A receptor to activate G proteins, is attenuated in BDNF (+/-) mice.

Dissection of NT3 functions in vivo by gene replacement strategy.

The development of the peripheral nervous system is governed in part by a family of neurotrophic factors that signal through Trk tyrosine kinase receptors. Neurotrophin 3 (NT3) ablation in mice causes a more severe neuronal phenotype than deletion of its receptor TrkC, suggesting that NT3 acts also through other non-preferred Trk receptors. To study the role of low-affinity ligand receptor interactions in vivo, we have replaced the Nt3 gene with the gene for brain-derived neurotrophic factor (BDNF), a TrkB ligand. As in NT3 and TrkC null mice, the proprioception system of these mutants failed to assemble. However, sensory fiber projections in the embryonic spinal cord suggest chemotropic effects of BDNF in vivo. In the dorsal root ganglia, the developmental dynamic of neuron numbers demonstrates that NT3 is required for activation of TrkB during neurogenesis and that TrkA is required during target tissue innervation. In the inner ear, the ectopic BDNF rescued the severe neuronal deficits caused by NT3 absence, indicating that TrkB and TrkC activate equivalent pathways to promote survival of cochlear neurons. However, specific increased innervation densities suggest unique functions for BDNF and NT3 beyond promoting neuronal survival. This mouse model has allowed the dissection of specific spatiotemporal Trk receptor activation by NT3. Our analysis provides examples of how development can be orchestrated by complex high- and low-affinity interactions between ligand and receptor families.

Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions in neuronal signaling.

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. Here, we report the identification of B-KSR1, a novel splice variant of murine KSR1 that is highly expressed in brain-derived tissues. B-KSR1 protein is detectable in mouse brain throughout embryogenesis, is most abundant in adult forebrain neurons, and is complexed with activated mitogen-activated protein kinase (MAPK) and MEK in brain tissues. Expression of B-KSR1 in PC12 cells resulted in accelerated nerve growth factor (NGF)-induced neuronal differentiation and detectable epidermal growth factor (EGF)-induced neurite outgrowth. Sustained MAPK activity was observed in cells stimulated with either NGF or EGF, and all effects on neurite outgrowth could be blocked by the MEK inhibitor PD98059. In B-KSR1-expressing cells, the MAPK-B-KSR1 interaction was inducible and correlated with MAPK activation, while the MEK-B-KSR1 interaction was constitutive. Further examination of the MEK-B-KSR1 interaction revealed that all genetically identified loss-of-function mutations in the catalytic domain severely diminished MEK binding. Moreover, B-KSR1 mutants defective in MEK binding were unable to augment neurite outgrowth. Together, these findings demonstrate the functional importance of MEK binding and indicate that B-KSR1 may function to transduce Ras-dependent signals that are required for neuronal differentiation or that are involved in the normal functioning of the mature central nervous system.

BDNF promotes the regenerative sprouting, but not survival, of injured serotonergic axons in the adult rat brain.

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the adult brain and can prevent the severe loss of cortical 5-HT axons caused by the neurotoxin p-chloroamphetamine (PCA). However, it has not been determined whether BDNF promotes the survival of 5-HT axons during PCA-insult or facilitates their regenerative sprouting after injury. We show here that BDNF fails to protect most 5-HT axons from PCA-induced degeneration. Instead, chronic BDNF infusions markedly stimulate the sprouting of both intact and PCA-lesioned 5-HT axons, leading to a hyperinnervation at the neocortical infusion site. BDNF treatment promoted the regrowth of 5-HT axons when initiated up to a month after PCA administration. The sprouted axons persisted in cortex for at least 5 weeks after terminating exogenous BDNF delivery. BDNF also encouraged the regrowth of the 5-HT plexus in the hippocampus, but only in those lamina where 5-HT axons normally ramify. In addition, intracortical BDNF infusions induced a sustained local activation of the TrkB receptor. The dose-response profiles for BDNF to stimulate 5-HT sprouting and Trk signaling were remarkably similar, suggesting a physiological link between the two events; both responses were maximal at intermediate doses of BDNF but declined at higher doses ("inverted-U-shaped" dose-response curves). Underlying the downregulation of the Trk signal with excessive BDNF was a decline in full-length TrkB protein, but not truncated TrkB protein or TrkB mRNA levels. Thus, BDNF-TrkB signaling does not protect 5-HT neurons from axonal injury, but has a fundamental role in promoting the structural plasticity of these neurons in the adult brain.

Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities.

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the central nervous system. However, the role of endogenous BDNF in the development and function of these neurons has not been established in vivo because of the early postnatal lethality of BDNF null mice. In the present study, we use heterozygous BDNF(+/-) mice that have a normal life span and show that these animals develop enhanced intermale aggressiveness and hyperphagia accompanied by significant weight gain in early adulthood; these behavioral abnormalities are known to correlate with 5-HT dysfunction. Forebrain 5-HT levels and fiber density in BDNF(+/-) mice are normal at an early age but undergo premature age-associated decrements. However, young adult BDNF(+/-) mice show a blunted c-fos induction by the specific serotonin releaser-uptake inhibitor dexfenfluramine and alterations in the expression of several 5-HT receptors in the cortex, hippocampus, and hypothalamus. The heightened aggressiveness can be ameliorated by the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that endogenous BDNF is critical for the normal development and function of central 5-HT neurons and for the elaboration of behaviors that depend on these nerve cells. Therefore, BDNF(+/-) mice may provide a useful model to study human psychiatric disorders attributed to dysfunction of serotonergic neurons.

Anterograde transport of endogenous brain-derived neurotrophic factor in hippocampal mossy fibers.

Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) are assumed to provide trophic support via a target-derived, retrograde mechanism of action. However, recent studies suggest that neurotrophic factors can act in an autocrine fashion and perhaps even in an anterograde direction similar to neurotransmitters. To further explore this hypothesis, we compared the neuroanatomical pattern of BDNF mRNA and protein in response to electroconvulsive seizures (ECS) or kainic acid-induced seizure activity. Using in situ hybridization, we found that chronic ECS induced BDNF mRNA predominantly in the granule neurons of the dentate gyrus. However, immunohistochemistry with an anti-BDNF antibody revealed that ECS increased endogenous BDNF protein in the mossy fibers, which are composed of axons projecting from the granule neurons of the dentate gyrus to the CA3 pyramidal layer of the hippocampus. Kainic acid administration (10 mg/kg, i.p., once) was used to lesion CA3 neurons selectively, as these are a possible retrograde source of BDNF protein in mossy fibers. Three weeks later, a prolonged elevation of BDNF mRNA in granule neurons, but not elsewhere in hippocampus, was accompanied by an increase in BDNF protein in the mossy fibers. These results suggest that BDNF was transcribed and translated in granule neuron cell bodies but transported in an anterograde direction to provide trophic support of CA3 pyramidal neurons.

Amyloid precursor protein potentiates the neurotrophic activity of NGF.

Cortical amyloid precursor protein (APP) is induced and secreted in response to subcortical lesions of cholinergic innervation. To understand the physiological role of the induced APP, we have characterized its neurotrophic activity on PC12 cells. Highly purified human APP751 (50-1000 pM) induced outgrowth of neurites. The neurotrophic activity was inhibited by an antibody that was directed to the C-terminal portion of the secreted APP but not by an antibody directed to the KPI domain. The neurotrophic activity of APP was independent of the TrkA NGF receptor because neither phospholipase C-gamma1 nor TrkA exhibited tyrosine phosphorylations with APP treatment. Furthermore, APP stimulated neurite outgrowth from PC12 cells lacking TrkA receptors. At lower concentrations (10-50 pM), APP synergistically potentiated the neurotrophic effects of NGF when added with NGF or before NGF as a priming pretreatment. These results implicate APP, a rapidly induced protein in the injured cortex, as a potentiating agent that may render compromised neurons more responsive to low levels of NGF or other neurotrophins.

Amyloid precursor protein requires the insulin signaling pathway for neurotrophic activity.

Picomolar concentrations of purified amyloid precursor protein (APP) potentiate the neurotrophic activity of suboptimal concentrations of NGF on PC12 cells. To understand the molecular basis for this potentiation, we have characterized the signal transduction pathway used by APP for its neurotrophic activity. APP stimulated the tyrosine phosphorylation of a number of proteins including insulin receptor substrate-1 (IRS-1). Incubation of naive cells with antisense oligonucleotides to IRS-1 mRNA resulted in a dramatic reduction of IRS-1 levels and inhibition of APP stimulated neurite outgrowth. Phosphotidylinositol 3-kinase became associated with IRS-1 and activated upon APP stimulation. Extracellular signal-regulated kinase (ERK 1 and ERK 2) phosphorylation was detected by both immunoblot analysis and immunocytochemistry using antibodies directed to their phosphorylated (and hence, activated) form. There was also an elevation of ERK kinase activity. The potentiation of NGF activity was reflected in a correspondingly synergistic elevation of tyrosine phosphorylated ERK. The pattern of signal transduction targets indicates that APP potentiated the neurotrophic effects of NGF via the activation of the IRS-1 signaling pathway.

Neuronal regeneration enhances the expression of the immunophilin FKBP-12.

Immunophilins are a group of proteins that serve as receptors for the immunosuppressant drugs cyclosporin A and FK506. The immunophilin designated FK-506 binding protein-12 (FKBP-12) is concentrated more than 10 times higher in the brain than in immune tissues. The complex of FK506 and FKBP-12 inhibits the calcium activated phosphatase, calcineurin, increasing phosphorylated levels of calcineurin substrates with growth associated protein-43 (GAP-43), being most prominent in the brain. We now demonstrate an association of FKBP-12 with neuronal regeneration and GAP-43 disposition. Facial nerve crush markedly augments expression of FKBP-12 mRNA in the facial nucleus with a time course paralleling changes in GAP-43 mRNA. Following sciatic nerve lesions, similar increases in FKBP-12 mRNA occur in lumbar motor neurons and dorsal root ganglia neuronal cells. Increased FKBP-12 expression appears linked to regeneration rather than degeneration as facial nerve lesions elicited by ricin injection, which produce neuronal death without regeneration, fail to augment FKBP-12 expression in the facial nucleus. The time course for accumulation of FKBP-12 in sciatic nerve segments following nerve crush indicates rapid axonal transport at a rate similar to GAP-43.

The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin.

The immunosuppressant drugs cyclosporin A and FK506 bind to small, predominantly soluble proteins cyclophilin and FK506 binding protein, respectively, to mediate their pharmacological actions. The immunosuppressant actions of these drugs occur through binding of cyclophilin-cyclosporin A and FK506 binding protein-FK506 complexes to the calcium-calmodulin-dependent protein phosphatase, calcineurin, inhibiting phosphatase activity. Utilizing immunohistochemistry, in situ hybridization and autoradiography, we have localized protein and messenger RNA for FK506 binding protein, cyclophilin and calcineurin. All three proteins and/or messages exhibit a heterogenous distribution through the brain and spinal cord, with the majority of the localizations being neuronal. We observe a striking co-localization of FK506 binding protein and calcineurin in most brain regions and a close similarity between calcineurin and cyclophilin. FK506 binding protein and cyclophilin localizations largely correspond to those of calcineurin, although cyclophilin is enriched in some brain areas that lack calcineurin. The dramatic similarities in localization of FK506 binding proteins and cyclophilins with calcineurin suggest related functions.

Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia.

The immunosuppressant drug FK506 acts by binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca(2+)-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our findings in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-associated protein GAP43, a calcineurin substrate that regulates neurite extension, we examined effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increasing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory ganglia by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at subnanomolar concentrations, suggesting therapeutic application in diseases involving neural degeneration.

Dissociation of nitric oxide generation and kainate-mediated neuronal degeneration in primary cultures of rat cerebellar granule cells.

In the presence of physiological concentrations of Mg2+ and in glycine-free buffer, the relationship between KA-mediated generation of NO and neurotoxicity in cultures of cerebellar granule cells of the rat was examined. The neuronal damage elicited by KA was not dependent on the presence of L-arginine, a precursor of NO, since neither the potency nor magnitude of KA-mediated cell death was altered in either the absence or presence of exogenously applied L-arginine. Similarly, with the exception of 4-hydroxy-azobenzene-4'-sulfonic acid, disodium salt dihydrate (HBS), the salt associated with NG-monomethyl-L-arginine (di-(p-hydroxyazobenzene-p'-sulfonate) (MA(HBS)), treatment with several different competitive NO synthetase inhibitors did not provide protection against the toxicity of KA. However, the ability of KA to induce neuronal damage was significantly decreased in cerebellar granule cells treated with either HBS or alpha-tocopherol (VE). On the basis of these results, it is concluded that the generation of free radicals may be involved in the process of KA-elicited neuronal death in cultures of cerebellar granule cells but that this is unrelated to the synthesis of NO. This conclusion agrees with both in vivo and in vitro studies, implicating the involvement of free radicals in non-NMDA mediated neuronal damage.

Developmental time course and ionic dependence of kainate-mediated toxicity in rat cerebellar granule cell cultures.

The mechanisms associated with the neurotoxic response caused by kainate (KA) were examined in cerebellar granule cell cultures. Under the conditions studied, millimolar concentrations of quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, glutamate and N-methyl-D-aspartate did not cause significant cytolysis. In contrast, KA induced complete cell death, which was antagonized by 6,7-dinitroquinoxaline-2,3-dione, quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and glutamate. This neurotoxic effect was dependent on the dose of KA and the age of the cultures. Two separate components of KA-induced neurotoxicity were observed and differentiated according to morphological changes, time of onset and ionic dependence. For acute neurotoxicity, release of lactate dehydrogenase measured after 30 min of KA exposure, became apparent between 8 and 11 days in culture and was dependent on both Cl- and Na+. However, vulnerability to acute toxicity did not correlate with [3H]KA receptor expression with receptor-mediated Cl- influx. On the other hand, delayed toxicity, as determined by lactate dehydrogenase release 24 hr after KA exposure, was dependent on Cl-. This delayed neurotoxicity induced by KA shares time course features with N-methyl-D-aspartate-mediated toxicity. Yet in contrast to studies reported for N-methyl-D-aspartate, glutamate was ineffective as an agonist, measured by its ability to elicit a neurotoxic response, and the KA delayed response did not appear to be dependent upon the presence of extracellular Ca++, during the exposure to KA.

The noradrenergic neurotoxin DSP-4 eliminates the coeruleospinal projection but spares projections of the A5 and A7 groups to the ventral horn of the rat spinal cord.

Systemic administration of the noradrenergic neurotoxin DSP-4 results in a complete loss of staining of noradrenergic (NA) axons in the dorsal horn and intermediate zone of the rat spinal cord. NA axon staining in the ventral horn and in the intermediolateral cell column is only slightly decreased by the drug treatment. We have taken advantage of this differential effect of DSP-4 on NA axons to determine the location and number of cells that give rise to NA axons in the ventral horn and the intermediolateral cell column. Retrograde transport of the fluorescent tracer True blue was combined with dopamine-beta-hydroxylase immunohistochemistry 2 weeks after treatment of rats with 50 mg/kg of DSP-4. Compared with controls, the drug treatment resulted in a more than 90% decrease in the number of retrogradely labeled NA neurons in the locus coeruleus and an only 30-50% reduction in the number of retrogradely labeled NA cells in the A5 and A7 groups. The results reveal different sites of termination in the spinal cord of NA axons originating in the LC and in NA cells of the A5 and A7 groups: the LC distributes fibers mainly to the dorsal horn and the intermediate zone, while NA cells of the A5 and A7 groups project to motoneurons of the ventral horn and the intermediolateral cell column.

Neurotoxic effects of p-chloroamphetamine on the serotoninergic innervation of the trigeminal motor nucleus: a retrograde transport study.

The rat forebrain receives projections from both dorsal and median raphe nuclei. It has recently been shown that serotoninergic axons arising from the dorsal raphe nucleus, but not those from the median raphe nucleus, degenerate following systemic administration of p-chloroamphetamine (PCA). The present study was conducted to determine (i) whether the motor nucleus of the trigeminal nerve is innervated by overlapping projections from multiple serotonin cell groups and (ii) whether a particular subset of serotoninergic axon terminals in the trigeminal motor nucleus are sensitive to the neurotoxic effects of PCA. Retrograde transport was used in combination with immunofluorescence to identify the serotonin-positive cells that project to the trigeminal motor nucleus both in control rats and in rats previously treated with PCA. In untreated rats, an average of 95 retrogradely labeled serotonin-positive neurons were found in the dorsal raphe nucleus, 135 in the nucleus raphe obscurus, 132 in the nucleus raphe pallidus and 63 in the ventrolateral medulla. After treatment with PCA, there was a marked decrease (-77%) in the number of retrogradely labeled serotoninergic neurons in the dorsal raphe nucleus, whereas the number of labeled neurons was unchanged in the raphe obscurus and raphe pallidus. These results demonstrate that PCA selectively lesions serotonin axon terminals arising from the dorsal raphe nucleus, while sparing projections from the raphe obscurus and raphe pallidus to the trigeminal motor nucleus. This conclusion is in agreement with previous findings that in the forebrain only axons from the dorsal raphe are vulnerable to PCA. The data provide further evidence that serotoninergic axons originating in the dorsal raphe nucleus differ from other serotoninergic axons in their pharmacological properties and that the dorsal raphe may contain a functionally unique subset of serotonin neurons.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study.

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

Distribution of locus coeruleus axons in the rat spinal cord: a combined anterograde transport and immunohistochemical study.

The distribution of locus coeruleus axons in the rat spinal cord was studied by anterograde transport of Phaseolus vulgaris leucoagglutinin in combination with dopamine-beta-hydroxylase immunohistochemistry. Locus coeruleus axons were observed primarily in the superficial laminae of the dorsal horn. Few locus coeruleus fibers were seen in the vicinity of motoneuron pools or in the intermediolateral cell column of the thoracic spinal cord. The results of this study suggest a selective action of the coeruleo-spinal projection upon sensory inputs to the spinal cord.