Rat Brain-derived Neurotrophic Factor Research Articles

Biphasic change in BDNF gene expression following antidepressant drug treatment explained by differential transcript regulation

Brain-derived neurotrophic factor (BDNF) has been suggested as a possible target for the treatment of depression. The effect by antidepressant drugs on BDNF mRNA expression is, however, strictly dependent on both treatment duration and time after the last administration. The rat BDNF gene itself is complex and expresses four different mRNA isoforms which can be regulated by different signaling cascades. The aim of the present study was to test the hypothesis that the previously shown biphasic action by the antidepressant drugs on total BDNF expression is explained by differential BDNF transcript regulation. For this purpose, we used in situ hybridization with exon-specific oligo nucleotides for exon V (total BDNF mRNA), exon I (protein synthesis-dependent transcripts), exon III and exon IV (immediate early-gene like-transcripts). Following an acute injection, all three drugs tested: fluoxetine, desipramine and TCP decreased total BDNF mRNA (exon V) as well as exon IV mRNA, while no significant effect was recorded for exons I and III mRNAs. In contrast chronic administration of all three drugs resulted in increased expression of exon V- and exon I-containing transcripts (fluoxetine and TCP only) but no significant changes were recorded for exon III and IV mRNAs. Electroconvulsive shock administration showed up-regulation of all four BDNF mRNAs following a single shock, but after repeated administration increases were restricted to exons I- and V-containing transcripts. In summary, this study shows clear evidence of differential BDNF transcript regulation following acute and chronic antidepressant drug treatment.

Cytosolic branched chain aminotransferase (BCATc) mRNA is up-regulated in restricted brain areas of BDNF transgenic mice

Branched chain aminotransferase (BCAT) catalyzes the transamination of the essential branched chain amino acids (leucine, isoleucine and valine) with α-ketoglutarate. BCAT exists in two isoforms: one cytosolic (BCATc), mainly expressed in the nervous system, and the other mitochondrial (BCATm), present in a greater number of tissues. We previously showed that BCATc mRNA and protein expression in the dorsal lateral geniculate nucleus of the thalamus is up-regulated by exogenous administration of brain-derived neurotrophic factor (BDNF) following lesion of the visual cortex in newborn rats. Here, we analyzed the expression of BCATc mRNA in the brain of transgenic mice overexpressing the rat BDNF cDNA under the control of the α-calcium/calmodulin-dependent kinase II (αCaMKII) promoter. In these animals, BDNF is overexpressed in the telencephalon starting from the second postnatal week. RT-PCR and in situ hybridization experiments showed that BCATc mRNA is overexpressed in restricted regions of the cerebral cortex (parietal area) and hippocampus (hilus and CA3 pyramidal cell layer) of adult BDNF transgenic mice respect to wild-type animals. These differences between wt and BDNF mice were not detected in animals of 1 week of age. These results demonstrate that the expression of the BCATc gene in the brain is specifically regulated by BDNF in a time- and region-dependent fashion.

Human immunodeficiency virus type 1 glycoprotein gp120 reduces the levels of brain‐derived neurotrophic factor in vivo: potential implication for neuronal cell death

Neuronal loss has been observed in post mortem brains of patients with human immunodeficiency virus type 1 (HIV-1). Experimental evidence has implicated HIV-1-derived envelope glycoprotein 120 (gp120) in the neuronal cell death observed in these patients. However, the intrinsic mechanisms by which gp120 causes neurotoxicity are still unknown. We have recently shown that the neurotoxic effect of gp120 in vitro is reduced by brain-derived neurotrophic factor (BDNF). We therefore tested the hypothesis that low levels of BDNF render neurons more sensitive to gp120. Gp120 was injected acutely into the striatum of BDNF heterozygous mice and wild-type littermates. BDNF heterozygous mice exhibited more apoptotic neurons in the striatum than wild-type mice, suggesting that BDNF is neuroprotective also in vivo. Because several neurodegenerative disorders are characterized by lack of trophic support, we tested the hypothesis that gp120 may cause apoptosis by reducing BDNF expression. Gp120 was injected acutely in the rat striatum and BDNF levels determined by a two-site immunoassay at various times after the injection. Gp120 elicited a dramatic decrease in BDNF protein levels by 24 h. Reduced BDNF levels were still present at 4 days. Cellular localization of BDNF immunoreactivity revealed that gp120 decreases BDNF immunoreactivity mainly in neuronal processes. This effect of gp120 precedes the peak of caspase-3 activation and neuronal cell death. We propose that one of the mechanisms whereby gp120 causes neurotoxicity is a reduction of the neurotrophic factor environment crucial for cell survival.

Ex Vivo Adenoviral Vector-Mediated Neurotrophin Gene Transfer to Olfactory Ensheathing Glia: Effects on Rubrospinal Tract Regeneration, Lesion Size, and Functional Recovery after Implantation in the Injured Rat Spinal Cord

The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or bacterial marker protein beta-galactosidase (LacZ) and subsequently implanted into adult Fischer rats directly after unilateral transection of the dorsolateral funiculus. Implanted animals received a total of 2 x 105 OEG that were subjected to transduction with neurotrophin-encoding AdV vector, AdV-LacZ, or no vector, respectively. At 4 months after injury, lesion volumes were smaller in all OEG implanted rats and significantly reduced in size after implantation of neurotrophin-encoding AdV vector-transduced OEG. All OEG grafts were filled with neurofilament-positive axons, and AdV vector-mediated expression of BDNF by implanted cells significantly enhanced regenerative sprouting of the rubrospinal tract. Behavioral analysis revealed that OEG-implanted rats displayed better locomotion during horizontal rope walking than unimplanted lesioned controls. Recovery of hind limb function was also improved after implantation of OEG that were transduced with a BDNF- or NT-3-encoding AdV vector. Hind limb performance during horizontal rope locomotion did directly correlate with lesion size, suggesting that neuroprotective effects of OEG implants contributed to the level of functional recovery. Thus, our results demonstrate that genetic engineering of OEG not only resulted in a cell that was more effective in promoting axonal outgrowth but could also lead to enhanced recovery after injury, possibly by sparing of spinal tissue.

Pathways to care in early psychosis

Recent studies have suggested that first and second generation antipsychotics (FGAs and SGAs) have different neuroprotective effects. However, the molecular mechanisms of SGAs are not fully understood, and investigations into changes in intracellular signaling related to their neuroprotective effects remain scarce. In the present study, we compared the SGA aripiprazole with the FGA haloperidol in SH-SY5Y human neuroblastoma cells via brain-derived neurotrophic factor (BDNF)-mediated signaling, notably BDNF, glycogen synthase kinase-3β (GSK-3β), and B cell lymphoma protein-2 (Bcl-2). We examined the effects of aripiprazole (five and 10 μM) and haloperidol (one and 10 μM) on BDNF gene promoter activity in SH-SY5Y cells transfected with a rat BDNF promoter fragment (− 108 to + 340) linked to the luciferase reporter gene. The changes in BDNF, p-GSK-3β, and Bcl-2 levels were measured by Western blot analysis. The haloperidol was not associated with a significant difference in BDNF promoter activity. In contrast, aripiprazole was associated with increased BDNF promoter activity only with a dose of 10 μM (93%, p < 0.01). Treatment with aripiprazole at 10 μM increased the levels of BDNF by 85%, compared with control levels (p < 0.01), whereas haloperidol had no effect. Moreover, cells treated with aripirazole effectively increased the levels of GSK-3β phosphorylation and Bcl-2 at doses of five and 10 μM (30% and 58% and 31% and 80%, respectively, p < 0.05 or p < 0.01). However, haloperidol had no effects on p-GSK-3 β and Bcl-2 expression. This study showed that aripiprazole, but not haloperidol, appeared to offer neuroprotective effects on human neuronal cells. The actions of signaling systems associated with BDNF may represent key targets for both aripiprazole and haloperidol, but the latter may be associated with distinct effects. These differences might be related to the different therapeutic effects of FGAs and SGAs in patients with schizophrenia.

Up-regulation of brain-derived neurotrophic factor expression by brimonidine in rat retinal ganglion cells.

Brimonidine tartrate ophth, an alpha(2)-adrenergic agonist, is widely used as an antiglaucoma agent for lowering intraocular pressure. Recent studies suggest that brimonidine may be neuroprotective for retinal ganglion cells (RGCs) following optic nerve crush injury. Brain-derived neurotrophic factor (BDNF), a potent neuroprotective factor present in the RGCs, promotes RGC survival in culture and following optic nerve injury. We tested the hypothesis that a possible mechanism of brimonidine neuroprotection is through up-regulation of endogenous BDNF expression in the RGCs. A single dosage of brimonidine tartrate ophth solution (0.85-34 microM) was injected intravitreally into Sprague-Dawley rat eyes. The fellow eyes of each animal were injected with balanced salt solution (BSS) and used as control eyes. To determine BDNF messenger RNA expression, animal eyes were enucleated and processed for in situ hybridization, or retinas were isolated and processed for Northern blot analysis using rat BDNF radiolabeled riboprobes. In the control eyes injected with saline, BDNF was present in a minority of the RGCs. Two days after brimonidine injection, the number of BDNF-positive RGCs was increased from 55% to 166%, depending on brimonidine concentrations, when compared with those in the controls. In addition, the BDNF signal intensities in individual RGCs were elevated 50% in brimonidine-injected eyes compared with control eyes. Northern blot revealed a 28% increase of BDNF expression in the brimonidine group compared with the controls (P <.003). No significant difference was observed in BDNF receptor, trk B, expression between brimonidine, or BSS control groups. A single dose of a low concentration of intravitreal brimonidine is sufficient to significantly increase endogenous BDNF expression in RGCs. These results suggest that brimonidine neuroprotection may be mediated through up-regulation of BDNF in the RGCs. The BDNF should be further investigated regarding its role in the neuroprotective effects reported with brimonidine. Brimonidine may be (potentially) used clinically as a neuroprotective agent in optic neuropathy, including glaucoma, and ischemic and traumatic optic neuropathy.

Brain-derived neurotrophic factor gene organization and transcription in the zebrafish embryo

The gene encoding zebrafish brain-derived neurotrophic factor (BDNF) was cloned from a PAC genomic DNA library. The entire transcription unit was contained in two independently isolated clones that together encompass 120 kb of genomic DNA. The intron/exon organization of the zebrafish gene was found to be identical to that of the mammalian gene but only one promoter has so far been identified. The associated 5′ exon is 67% identical to exon 1c of the rat BDNF gene. A search of the 5′ flank of the cloned promoter for sequence similarities with known transcription factor binding sites revealed potential AP-1, CREB, and SP1 binding sites. Fusion constructs containing the cloned promoter and 1.7 kb of 5′ flank and an enhanced green fluorescent protein reporter that becomes membrane-anchored were injected into 1–8 cell stage embryos. Expression was seen in notochord, muscle, epithelial and endothelial cells of the 1-day-old embryo in consonance with the endogenous gene. These results demonstrate that the cloned promoter mediates cell-specific expression.

Differential usage of multiple brain-derived neurotrophic factor promoter in rat dorsal root ganglia following peripheral nerve injuries and inflammation

The brain-derived neurotrophic factor (BDNF) may act as either an autocrine or paracrine survival factor for the dorsal root ganglion (DRG) neurons and may also serve as a neurotransmitter or neuromodulator within the dorsal horn of the spinal cord. The rat BDNF gene consists of four short 5′ exons linked to separate promoters and one 3′ exon encoding the mature BDNF protein. An exon-specific reverse transcription–polymerase chain reaction analysis was used to study the differential utilization of multiple promoters in the DRG following unilateral sciatic axotomy, dorsal rhizotomy and peripheral inflammation. The exon I transcript showed the highest induction rate with the ipsilateral expression elevated 4.3–5.8 times that of contralateral expression. Both exon II and III mRNAs showed a smaller increase 1 day after the three kinds of stimuli. In addition, exon IV mRNA transcription increased slightly only after rhizotomy, but not after axotomy and peripheral inflammation after 1 day. Furthermore, the elevated exon I mRNA levels 1 day after rhizotomy were sustained for up to 7 days. In contrast, those of the exon I mRNA after axotomy had declined 2.8 times the control level after 7 days. These findings suggest that the promoter linked to exon I may provide a major regulatory point of BDNF mRNA expression by peripheral nerve injuries and inflammation. In addition, both exon I and IV mRNA expression may show different temporal activation patterns according to the types of injury.

Nuclear factor kappaB is a critical determinant in N-methyl-D-aspartate receptor-mediated neuroprotection.

The role of a nuclear factor kappaB (NF-kappaB) in NMDA receptor-mediated neuroprotection is not known. A candidate sequence from the 5' flanking region of exon 3 of the rat brain-derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 microM NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK-801. Anti-p65 antibody or anti-p50 antibody 'supershifted' the DNA binding activity, suggesting that the DNA-protein complex was composed of p65 and p50 subunits. NMDA receptor-mediated neuroprotection was blocked when cerebellar neurons were transfected with a double-stranded oligonucleotide containing the BDNF gene NF-kappaB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double-stranded NF-kappaB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF-kappaB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3-specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3-specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF-kappaB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF-kappaB DNA binding activity. Taken together, these data support a mechanism where NF-kappaB plays a critical role in NMDA-mediated neuroprotection.

Human neuroblastomas with unfavorable biologies express high levels of brain-derived neurotrophic factor mRNA and a variety of its variants

The expression of human brain-derived neurotrophic factor (BDNF) was investigated in 16 primary human neuroblastomas with favorable biologies, 15 with unfavorable biologies, and in human neuroblastoma cell lines. We demonstrated higher expressions of human BDNF mRNA in neuroblastomas with unfavorable biologies and with N-myc amplification than in those with favorable biologies. For the first time we revealed the composition of splice variants of human BDNF mRNA and analyzed their expression in neuroblastomas by reverse transcription polymerase chain reaction (RT-PCR). Interestingly, human BDNF mRNA consisted of at least six isoforms, four isoforms resembling those of rat BDNF mRNA, a human-specific isoform and a new isoform. The expression of four isoforms were more prominent in tumors with unfavorable biologies than in those with favorable biologies ( P<0.05). As previously we had reported, over 80% of the primary tumors expressed either the full-length form of BDNF receptor, TRKB, or a truncated form of TRKB lacking the tyrosine kinase domain. The full-length TRKB was predominantly detected in tumors with unfavorable biologies, and the truncated one in those with favorable biologies. These results suggest that an autocrine and/or paracrine mechanism involving BDNF may stimulate signal transduction via TRKB receptors rich in neuroblastomas with unfavorable biologies, resulting in an aberrant survival of tumor cells.

Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction

Chronic delivery of anti-nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat brain-derived neurotrophic factor cDNA (BDNF), and the BDNF-synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33°C), when the cells were proliferating, and during differentiation at non-permissive temperature (39°C) in vitro. The bio-active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio-activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2–7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2–3 weeks after transplant and the anti-nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti-nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such ‘biologic minipumps’ can be developed for safe use in humans.

Brain-derived Neurotrophic Factor Expression in VivoIs under the Control of Neuron-restrictive Silencer Element

Neuron-restrictive silencer element (NRSE) has been identified in multiple neuron-specific genes. This element has been shown to mediate repression of neuronal gene transcription in nonneuronal cells. A palindromic NRSE (NRSEBDNF) is present in the proximal region of brain-derived neurotrophic factor (BDNF) promoter II. Using in vitro binding assays, we establish that the upper half-site is largely responsible for the NRSEBDNF activity. To delineate the in vivorole of NRSE in the regulation of rat BDNF gene, promoter constructs with intact and mutated NRSEBDNF were introduced into transgenic mice. Our data show that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung,i.e. in the tissues in which the intact reporter gene and endogenous BDNF mRNAs are expressed. Mutation of NRSEBDNF did not lead to the ectopic activation of the reporter gene in any other nonneural tissues. In the brain, NRSEBDNF is involved in the repression of basal and kainic acid-induced expression from BDNF promoters I and II in neurons. However, NRSEBDNF does not control the activity of the BDNF gene in nonneuronal cells of brain.

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter γ-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1–8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Multiple promoters direct stimulus and temporal specific expression of brain-derived neurotrophic factor in the somatosensory cortex

Neuronal activity rapidly induces expression of brain-derived neurotrophic factor (BDNF) in the adult rat cortex. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5′ exon (I–IV) and a common 3′ exon (V) that encodes the mature BDNF protein. The present study used an exon-specific RT-PCR analysis to determine the time course of the induction from both seizures and whisker stimulation. Our data show that specific promoters are utilized at different stages of the activity-dependent induction of the BDNF gene. Furthermore, the data show a differential utilization of the four promoters following a specific stimulus.

Transcript-specific effects of adrenalectomy on seizure-induced BDNF expression in rat hippocampus

Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5′ exon (I–IV) and a common 3′ exon (V) that codes for BDNF protein. Exon-specific in situ hybridization was used to determine if adrenalectomy has differential effects on basal and activity-induced BDNF transcript expression in hippocampus. Adrenalectomy alone had only modest effects on BDNF mRNA levels with slight increases in exon III-containing mRNA with 7–10-day survival and in exon II-containing mRNA with 30-day survival. In the dentate gyrus granule cells, adrenalectomy markedly potentiated increases in exon I and II cRNA labeling, but not increases in exon III and IV cRNA labeling, elicited by one hippocampal afterdischarge. Similarly, for the granule cells and CA1 pyramidal cells, hilus lesion (HL)-induced recurrent limbic seizures elicited greater increases in exon I and II cRNA hybridization in adrenalectomized (ADX) as compared to adrenal-intact rats. In this paradigm, adrenalectomy modestly potentiated the increase in exon III-containing mRNA in CA1 but had no effect on exon IV-containing mRNA content. These results demonstrate that the negative effects of adrenal hormones on activity-induced BDNF expression are by far the greatest for transcripts containing exons I and II. Together with evidence for region-specific transcript expression, these results suggest that the effects of stress on adaptive changes in BDNF signalling will be greatest for neurons that predominantly express transcripts I and II.

Localization of putative calcium-responsive regions in the rat BDNF gene

Brain-derived neurotrophic factor (BDNF) has potent trophic and protective actions on CNS neurons, including mesencephalic dopaminergic neurons, ventral forebrain cholinergic neurons and spinal motor neurons. To evaluate the effects of calcium and other second messengers on BDNF gene transcription, C6 glioma cells were treated for 4 h with the calcium ionophore A23187, forskolin + isobutyl-methyl-xanthine (IBMX), or the phorbol ester, 12– O-tetradecanoyl-phorbol-13-acetate. Semi-quantitative RT-PCR analysis revealed that A23187 treatment increased BDNF transcripts containing the protein coding exon by 4.4–6.4-fold. Alternate BDNF transcripts were elevated to varying degrees after treatment with this ionophore and a subset of these transcripts was elevated following forskolin + IBMX treatment. When co-incubated with the RNA polymerase inhibitor, actinomycin D, A23187-induced increases were reduced or abolished, suggesting that calcium-mediated regulation of BDNF mRNA expression occurs at transcriptional as well as post-transcriptional levels. Transient transfection experiments employing reporter constructs containing serial 5′ deletions of alternate BDNF promoters suggested that A23187-induced elevations in BDNF exon 1b, 1d and 1e containing transcripts are mediated by putative calcium-responsive regions flanking all three of these exons.

Lumbar transplants of immortalized serotonergic neurons alleviate chronic neuropathic pain

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to non-permissive temperature. Brain-derived neurotrophic factor (BDNF) induced the serotonergic phenotype and increased the survival of RN46A cells in vitro. After transfection of the rat BDNF gene into RN46A cells, an autocrine BDNF-secreting cell line, 46A-B14, was isolated and transplanted into the rat CNS. Transplanted 46A-B14 cells had increased survival and enhanced serotonin (5HT) synthesis compared to 46A-V1 cells, RN46A cells transfected with vector-alone. When 46A-B14 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a chronic constriction injury (CCI) of the sciatic nerve, they survived longer than 6 weeks on the pia mater. Furthermore, the tactile and cold allodynia and thermal hyperalgesia induced by CCI was significantly reduced during a 4–6- week period. The maximal effect occurred 1 week after transplantation. 46A-V1 cells, transplanted after CCI, did not survive beyond 2–3 weeks and had no effect on the allodynia and hyperalgesia induced by CCI. Acute intrathecal injection of the 5HT receptor antagonist methysergide decreased the antinociceptive effects of the 46A-B14 cells to pre-transplant levels. These data suggest that a chronically applied, low local dose of serotonin near the dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters such as serotonin, in a model of chronic pain offers a novel approach to pain management.

Distribution of Brain-Derived Neurotrophic Factor (BDNF) Protein and mRNA in the Normal Adult Rat CNS: Evidence for Anterograde Axonal Transport

A sensitive immunohistochemical technique was used, along with highly specific affinity-purified antibodies to brain-derived neurotrophic factor (BDNF), to generate a detailed mapping of BDNF immunoreactivity (BDNF-ir) throughout the adult rat CNS. A parallel analysis of sites of BDNF synthesis was performed with in situ hybridization techniques using a cRNA probe to the exon encoding mature rat BDNF protein. These combined data revealed (1) groups of cell bodies containing diffuse BDNF-ir throughout the CNS that were strongly correlated with fields of cells containing BDNF mRNA; (2) varying degrees of BDNF-ir outside of cell bodies, in what appeared to be fibers and/or terminals; and (3) many regions containing extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord). The latter observation suggested that in these regions BDNF was derived from anterograde axonal transport by afferent systems. In the two cases in which this hypothesis was tested by the elimination of select afferents, BDNF immunostaining was completely eliminated. These data, along with the observation that BDNF-ir was rarely found within dendrites or fibers en passage, suggest that BDNF protein produced in adult CNS neurons is polarized primarily along axonal processes and is preferentially stored in terminals within the innervation target.

Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons: induction of NPY production in aggregate cultures derived from rat but not from human fetal brains

Previous studies established that brain-derived neurotrophic factor (BDNF) induces neuropeptide Y (NPY) production and accumulation of NPY-mRNA in cultures of rat fetal brain tissues. In this study, we addressed the question: Are cultured human NPY neurons regulated by BDNF and/or by another member of the neurotrophin (NT) family of growth factors? Using aggregate cultures derived from human fetal cortical hemispheres, we assessed the effect of BDNF on NPY production varying the following experimental conditions: fetal and culture age; medium composition (with and without serum), dose and duration of exposure to BDNF, and neurotrophin species tested (BDNF, NT-4/5, NT-3 or NGF). Under none of these conditions did BDNF, NT-4/5, NT-3 or NGF induce an increase in NPY production. This was in contrast to forskolin + phorbol 12 myristate 13-acetate (PMA) which were highly effective in inducing NPY production, verifying that expression of NPY is a regulated process in these cultures. None of these neurotrophins enhanced the response to forskolin + PMA. By comparison, using aggregate cultures derived from rat fetal cortices, BDNF and NT-4/5 were equipotent in inducing NPY production but NT-3 and NGF were essentially ineffective. Moreover, the effects of BDNF or NT-4/5 and forskolin + PMA on NPY production were additive, indicating the involvement of distinct intracellular signalling pathways. Western blot analyses of human- and rat-derived aggregates indicated the presence of full-length Trk receptors which are tyrosine-phosphorylated in response to either BDNF, NT-4/5 or NT-3. Primary cultures of astrocytes (rat as well as human) were devoid of a functional TrkB receptor, strongly suggesting a neuronal expression of TrkB in the aggregates. Thus, a functional TrkB receptor is expressed by both the human and rat aggregates, but only the rat aggregates responded to BDNF or NT-4/5. These results are consistent with a difference in a post TrkB-receptor event(s) mediating BDNF action in the cultured human and rat fetal NPY neurons.

Autocrine BDNF Secretion Enhances the Survival and Serotonergic Differentiation of Raphe Neuronal Precursor Cells Grafted into the Adult Rat CNS

RN46A cells are a temperature-sensitive neuronal cell line derived from the E13 rat raphe nucleus. RN46A cells grafted into the adult rat hippocampus and cerebral cortex do not survive beyond 2 weeks. Brain-derived neurotrophic factor (BDNF) regulates thein vitrosurvival and serotonergic phenotype of RN46A cells, and we hypothesized that expression of BDNF in RN46A cells would potentiate their survival and serotonin (5HT) expressionin vivo.The gene encoding rat BDNF was transfected into RN46A cells and the clonal 46A-B14 cell line isolated. 46A-B14 cells synthesize and secrete biologically active BDNFin vitroand synthesize 5HT following partial membrane depolarization. Two weeks following 46A-B14 cell transplantation into the adult rat cortex and hippocampus, there is a threefold increase in survival of 46A-B14 cells compared to RN46A cells transfected with the vector alone. The grafted 46A-B14 cells immunohistochemically stain for BDNF and 5HT, while RN46A cells transfected with vector only are negative for both BDNF and 5HT. In addition, 46A-B14 cells attain more morphologically complex phenotypes, indicating enhanced neuronal differentiation. Autocrine secretion of BDNF by RN46A cells thus potentiates survival and can be used to deliver both BDNF and 5HTin vivo.