The CB1 cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway.

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. In particular, the CB1 receptor is highly expressed in the basal ganglia, mostly on terminals of medium-sized spiny neurons, where it plays a key neuromodulatory function. The CB1 receptor also confers neuroprotection in various experimental models of striatal damage. However, the assessment of the physiological relevance and therapeutic potential of the CB1 receptor in basal ganglia-related diseases is hampered, at least in part, by the lack of knowledge of the precise mechanism of CB1 receptor neuroprotective activity. Here, by using an array of pharmacological, genetic and pharmacogenetic (designer receptor exclusively activated by designer drug) approaches, we show that (1) CB1 receptor engagement protects striatal cells from excitotoxic death via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin complex 1 pathway, which, in turn, (2) induces brain-derived neurotrophic factor (BDNF) expression through the selective activation of BDNF gene promoter IV, an effect that is mediated by multiple transcription factors. To assess the possible functional impact of the CB1/BDNF axis in a neurodegenerative-disease context in vivo, we conducted experiments in the R6/2 mouse, a well-established model of Huntington's disease, in which the CB1 receptor and BDNF are known to be severely downregulated in the dorsolateral striatum. Adeno-associated viral vector-enforced re-expression of the CB1 receptor in the dorsolateral striatum of R6/2 mice allowed the re-expression of BDNF and the concerted rescue of the neuropathological deficits in these animals. Collectively, these findings unravel a molecular link between CB1 receptor activation and BDNF expression, and support the relevance of the CB1/BDNF axis in promoting striatal neuron survival.

Neurotrophins: transcription and translation.

Neurotrophins are powerful molecules. Small quantities of these secreted proteins exert robust effects on neuronal survival, synapse stabilization, and synaptic function. Key functions of the neurotrophins rely on these proteins being expressed at the right time and in the right place. This is especially true for BDNF, stimulus-inducible expression of which serves as an essential step in the transduction of a broad variety of extracellular stimuli into neuronal plasticity of physiologically relevant brain regions. Here we review the transcriptional and translational mechanisms that control neurotrophin expression with a particular focus on the activity-dependent regulation of BDNF.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease.

Huntingtin is a 350-kilodalton protein of unknown function that is mutated in Huntington's disease (HD), a neurodegenerative disorder. The mutant protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Here we demonstrate that wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. We show that this beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD.

Neuron-specific Bcl-2 homology 3 domain-only splice variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells.

We have identified and characterized N-Bak, a neuron-specific isoform of the pro-apoptotic Bcl-2 family member Bak. N-Bak is generated by neuron-specific splicing of a novel 20-base pair exon, which changes the previously described Bak, containing Bcl-2 homology (BH) domains BH1, BH2, and BH3, into a shorter BH3-only protein. As demonstrated by reverse transcription-polymerase chain reaction and RNase protection assay, N-Bak transcripts are expressed only in central and peripheral neurons, but not in other cells, whereas the previously described Bak is expressed ubiquitously, but not in neurons. Neonatal sympathetic neurons microinjected with N-Bak resisted apoptotic death caused by nerve growth factor (NGF) removal, whereas microinjected Bak accelerated NGF deprivation-induced death. Overexpressed Bak killed sympathetic neurons in the presence of NGF, whereas N-Bak did not. N-Bak was, however, still death-promoting when overexpressed in non-neuronal cells. Thus, N-Bak is an anti-apoptotic BH3-only protein, but only in the appropriate cellular environment. This is the first example of a neuron-specific Bcl-2 family member.

Human glial cell line-derived neurotrophic factor receptor alpha 4 is the receptor for persephin and is predominantly expressed in normal and malignant thyroid medullary cells.

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.

Expression and alternative splicing of mouse Gfra4 suggest roles in endocrine cell development.

Members of the GDNF protein family signal through receptors consisting of a GPI-linked GFRalpha subunit and the transmembrane tyrosine kinase Ret. Here we characterize the mouse Gfra4 and show that it undergoes developmentally regulated alternative splicing in several tissues. The mammalian GFRalpha4 receptor lacks the first Cys-rich domain characteristic of other GFRalpha receptors. Gfra4 is expressed in many tissues, including nervous system, in which intron retention leads to a putative intracellular or secreted GFRalpha4 protein. Efficient splicing occurs only in thyroid, parathyroid, and pituitary and less in adrenal glands. A splice form that leads to a GPI-linked GFRalpha4 receptor is expressed in juvenile thyroid and parathyroid glands. In newborn and mature thyroid as well as in parathyroid and pituitary glands major transcripts encode for a putative transmembrane isoform of GFRalpha4. Significant loss of thyroid C cells in Ret-deficient mice suggests that C cells and cells in adrenal medulla, which also express Ret, may require signaling via the GFRalpha4-Ret receptor. Finally, in human, GFRalpha4 expression may restrict the inherited cancer syndrome multiple endocrine neoplasia type 2, associated with mutations in RET, to these cells.

Neuron-specific splicing of zinc finger transcription factor REST/NRSF/XBR is frequent in neuroblastomas and conserved in human, mouse and rat.

Neuron-restrictive silencer factor (NRSF), also known as repressor element RE1 binding transcription factor (REST) or repressor binding to the X2 box (XBR) (REST/NRSF/XBR), is a zinc finger transcription factor that during early embryogenesis is required to repress a subset of neuron-specific genes in non-neural tissues and undifferentiated neural precursors. We have previously shown that splicing within the coding region of rat REST/NRSF/XBR (rREST) generates several different transcripts all of which are expressed in the adult nervous system. rREST transcripts with short neuron-specific exons (exon N) have in-frame stop codons and encode truncated proteins which have an N-terminal repressor domain and weakened DNA binding activity. The aim of this study was to analyze the regulatory mechanisms underlying REST/NRSF/XBR activity in human and mouse as compared to rat. We show that the structure of REST/NRSF/XBR gene and its regulation by neuron-specific splicing is conserved in human, mouse and rat. Expression levels of REST/NRSF/XBR transcripts with the insertion of exon N are increased during the neuronal differentiation of mouse teratocarcinoma PCC7 and rat pheocromocytoma PC12 cells and are high in several human and mouse neuroblastoma cells as compared to the relatively low levels in the developing and adult nervous system. The exclusive expression of the neuronal forms of REST/NRSF/XBR mRNAs in mouse neuroblastoma Neuro-2A cells is not caused by rearrangement of the REST/NRSF/XBR gene nor by mutations in the sequence of the splice sites flanking exon N. These data suggest that changes in REST/NRSF/XBR splicing pattern may result from altered levels of splicing factors reflecting the formation and/or progression of neuroblastoma tumors.

Brain-derived neurotrophic factor expression in vivo is 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 vivo role 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.

Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons.

Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, pan-neurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors. In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries.

Identification of a signaling pathway involved in calcium regulation of BDNF expression.

A signaling pathway by which calcium influx regulates the expression of the major activity-dependent transcript of BDNF in cortical neurons has been elucidated. Deletion and mutational analysis of the promoter upstream of exon III reveals that transactivation of the BDNF gene involves two elements 5' to the mRNA start site. The first element, located between 72 and 47 bp upstream of the mRNA start site, is a novel calcium response element and is required for calcium-dependent BDNF expression in both embryonic and postnatal cortical neurons. The second element, located between 40 and 30 bp upstream of the mRNA start site, matches the consensus sequence of a cAMP response element (CRE) and is required for transactivation of the promoter in postnatal but not embryonic neurons. The CRE-dependent component of the response appears to be mediated by CREB since it is part of the complex that binds to this CRE, and since dominant negative mutants of CREB attenuate transactivation of the promoter. A constitutively active mutant of CaM kinase IV, but not of CaM kinase II, leads to activation of the promoter in the absence of extracellular stimuli, and partially occludes calcium-dependent transactivation. The effects of CaM kinase IV on the promoter require an intact CRE. These mechanisms, which implicate CaM kinase IV and CREB in the control of BDNF expression, are likely to be centrally involved in activity-dependent plasticity during development.

Neuronal expression of zinc finger transcription factor REST/NRSF/XBR gene.

The identification of a common cis-acting silencer element, a neuron-restrictive silencer element (NRSE), in multiple neuron-specific genes, together with the finding that zinc finger transcription factor REST/NRSF/XBR could confer NRSE-mediated silencing in non-neuronal cells, suggested that REST/NRSF/XBR is a master negative regulator of neurogenesis. Here we show that, although REST/NRSF/XBR expression decreases during neuronal development, it proceeds in the adult nervous system. In situ hybridization analysis revealed neuronal expression of rat REST/NRSF/XBR mRNA in adult brain, with the highest levels in the neurons of hippocampus, pons/medulla, and midbrain. The glutamate analog kainic acid increased REST/NRSF/XBR mRNA levels in various hippocampal and cortical neurons in vivo, suggesting that REST/NRSF/XBR has a role in neuronal activity-implied processes. Several alternatively spliced REST/NRSF/XBR mRNAs encoding proteins with nine, five, or four zinc finger motifs are transcribed from REST/NRSF/XBR gene. Two of these transcripts are generated by neuron-specific splicing of a 28-bp-long exon. Rat REST/NRSF/XBR protein isoforms differ in their DNA binding specificities; however, all mediate repression in transient expression assays. Our data suggest that REST/NRSF/XBR is a negative regulator rather than a transcriptional silencer of neuronal gene expression and counteracts with positive regulators to modulate target gene expression quantitatively in different cell types, including neurons.

Differential regulation of BDNF and NT-3 mRNA levels in primary cultures of rat cerebellar neurons.

Reciprocal developmental patterns of expression for BDNF and NT-3 have been observed in several neuronal types, including cerebellar granule neurons: NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation. In the present study we analysed cultured cerebellar granule neurons prepared from postnatal rat cerebellum, a model system widely used for studies on the maturation and survival of these neurons. We show that chronic depolarization, induced by 25 mM K+ in the culture medium, is able to sustain a persistent increase of BDNF expression in cerebellar granule neurons. It has been suggested that chronic depolarization in vitro mimics the effect of the earliest afferent inputs received by granule cells in vivo: on this basis we suggest that the beginning of neuronal activity in differentiated granule neurons may represent one of the signals that trigger the developmental increase in BDNF expression. Interestingly, we observed that up-regulation of BDNF expression in vitro is accompanied by a dramatic decrease of NT-3 expression: a differential regulation that is highly reminiscent of the reciprocal developmental patterns of expression observed in vivo for BDNF and NT-3. Another point raised by the present results is the possible role of BDNF, acting in an autocrine or paracrine manner, in the trophic effect of high potassium concentration. Indeed, repeated additions of BDNF to the culture medium have a trophic effect on cerebellar granule neurons but reproduce only partially the survival effect observed with 25 mM K+ conditions, suggesting that the increased expression of BDNF is not the only mechanism responsible for the trophic effects of high potassium. In conclusion we show the existence of a reciprocal regulation of BDNF and NT-3 expression in cultured cerebellar granule neurons and we propose that this culture system could represent an in vitro model for the study of the molecular mechanisms underlying the developmental regulation of these neurotrophins in cerebellum.

Structural and functional characterization of the rat neurotrophin-4 gene.

Neurotrophin-4 (NT-4) is a member of the neurotrophin family of growth factors. To study the molecular mechanisms that govern NT-4 expression, we have cloned and characterized the rat genome region encoding NT-4. The rat NT-4 gene consists of three exons: two 5'-flanking noncoding exons and a coding exon. NT-4 mRNA transcription is controlled by two promoters flanking the noncoding exons. Alternative splicing of the second intron results in a NT-4 mRNA with a different open reading frame, encoding a shorter protein lacking pre-NT-4 sequence. A rat NT-4 gene fragment, containing all exons and introns in addition to 1.4 kb of the upstream genomic sequence, has been introduced into mice. This transgene enables partial recapitulation of the expression pattern of NT-4 mRNA and confers activity-dependent expression of the NT-4 mRNA in muscle.

Change in neurotrophins and their receptor mRNAs in the rat forebrain after status epilepticus induced by pilocarpine.

We studied the effects of status epilepticus (SE) induced by lithium chloride/pilocarpine treatment on gene expression of neurotrophins of the nerve growth factor (NGF) family and of their high-affinity receptors of the tyrosine protein kinase (trk) family in the forebrain. Using in situ hybridization (ISH), we demonstrated an early (3 h after treatment) increase in brain-derived neurotrophic factor (BDNF) and trkB mRNA expression in the dentate gyrus, amygdala, and piriform cortex, as well as widespread increases in the cerebral cortex. NGF mRNA, but not the mRNA of its receptor trkA, was increased in the dentate gyrus. In contrast, 12 h after treatment, neurotrophin-3 (NT-3) decreased, and its receptor trkC mRNA increased. There was no change in NT-4 mRNA levels. All changes were blocked by pretreatment with scopolamine, a muscarinic antagonist. The noncompetitive N-methyl-D-aspartate (NMDA) antagonist ketamine blocked NGF, BDNF, and trkB mRNA increases in the hippocampus and cerebral cortex, but not in the amygdala and piriform cortex. In contrast, ketamine did not affect NT-3 and trkC changes. These results provide a complete description of changes in mRNA levels of neurotrophins and their receptors in the forebrain after SE and supply additional data supporting the view that neurotrophin gene expression is related to abnormal neuronal activity.

Expression of mRNAs for neurotrophins and their receptors in the rat choroid plexus and dura mater.

The presence of the neurotrophin, nerve growth factor, brain derived neurotrophic factor, neurotrophin-3 and neurotrophin-4 (NGF, BDNF, NT-3 and NT-4) and their receptors of the tyrosine kinase family (trkA, trkB and trkC) have been investigated in the choroid plexus and dura mater of the adult rat by ribonuclease protection assay. The choroid plexus contained high levels of mRNAs for NGF and NT-4, and low levels of NT-3 and BDNF mRNA; and high levels of trkB mRNA, and undetectable levels of trkA and trkC mRNA. In the dura mater there were high levels of NT-3 and NGF, and low levels of BDNF and NT-4 mRNAs; and high levels of trkC mRNA, and relatively high amount of trkB mRNA, while levels of trkA mRNA was undetectable. The present analysis revealed a different distribution of neurotrophins and their related receptors in the choroid plexus and dura mater.

Neurotoxic injury in rat hippocampus differentially affects multiple trkB and trkC transcripts.

In the present work we determined, by Northern blotting, ribonuclease assay and in situ hybridization, the level of multiple trkB and trkC transcripts at different times after ibotenic acid-induced neuronal injury in the rat hippocampus. All the transcripts (7.0-7.5, 2.4 and 1.8 kb) encoding the truncated TrkB receptor are coordinately up-regulated following neurotoxic injury, with a time-course similar to that observed for the glial fibrillary acidic protein mRNA, a molecular marker of reactive astrocytes. The highest level of induction was observed for the 2.4 kb mRNA level. The 1.8 kb mRNA, whose relative level is higher in astroglial cultures compared to normal brain tissue, is detectable only in the gliotic hippocampus. The 9 kb trkB mRNA, which encodes the full-length TrkB receptor, rapidly decreases with a time-course similar to that previously observed for other neuronal markers. In situ hybridization studies show that the increased mRNA level per cell is a major determinant in the up-regulation of truncated trkB expression. A decrease of truncated and full-length trkC mRNA was observed in the neuron-depleted astroglia-enriched hippocampus, suggesting that this mRNA is mainly localized in the neuronal layers and that no induction of its expression occurs in reactive astrocytes.

Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF-beta), originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF beta in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 x 10(-9) M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system.

Up-regulation of trkB mRNA expression in the rat striatum after seizures.

The present study investigates the expression of a tyrosine kinase receptor (trkB), its specific ligands brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) mRNAs in the striatum after seizures. The result showed an increase of trkB mRNA expression, both with and without tyrosine kinase domain, in the caudate-putamen and nucleus accumbens, but not in the globus pallidus. This increase peaked 3 h after treatment, and returned to normal levels by 12 h. The BDNF and NT-4 mRNAs showed no change at any time. In conclusion, the widespread and massive trkB mRNA induction after abnormal neuronal activity suggests local trophic support for this receptor, and a potential role in basal ganglia diseases involving non-dopaminergic components.

Seizures increase trkC mRNA expression in the dentate gyrus of rat hippocampus. Role of glutamate receptor activation.

In this study we have shown, by in situ hybridization and RNase protection assay, a significant trkC mRNA increase confined to the dentate gyrus of hippocampus, both after seizures induced by intracerebroventricular injection of kainic acid and bicuculline. Moreover, after bicuculline treatment we observed an earlier increase of trkC mRNA level, which peaked after 3 h and returned back to normal levels by 12 h. In contrast, the kainic acid treatment produced a delayed increase of trkC mRNA, which initiated after 6 h, peaked at 12 h, and returned to normal levels at 24 h. This increase, which involves also trkC mRNA receptor with tyrosine kinase activity, was mediated by non-NMDA receptors and counteracted by GABA potentiating agent diazepam. Using embryonic neuronal cultures from cerebral hemispheres, including hippocampus, we found that glutamate receptor agonists, including glutamate, kainate, NMDA, and t-ACPD, increase trkC mRNA levels with the following rank order of efficacy: NMDA > t-ACPD > kainic acid > glutamate. In conclusion, our data show that trkC mRNA expression in granule cells of the hippocampus dentate gyrus is increased during seizure activity and that it is mediated by non-NMDA receptors.