Biochemical mechanisms of action of Hypericum LI 160 in glial and neuronal cells: inhibition of neurotransmitter uptake and stimulation of extracellular signal regulated protein kinase.

We have investigated biochemical mechanisms that may underlie the antidepressant effects of Hypericum LI 160. We found that LI 160 inhibits uptake of serotonin and norepinephrine in cultures of rat cortical astrocytes. Observed differences in the kinetic parameters Km and Vmax as well as in the recovery of uptake after removal of Hypericum indicates that LI 160 does not affect serotonin and norepinephrine transport in the same manner. This suggests that multiple components of the extract can mediate inhibition of these neurotransmitter transporters. Hypericum LI 160 also inhibits serotonin uptake in neuronal cultures from serotonergic-rich raphe nuclei; concentration-response studies indicate LI 160 is 25 times more potent in terms of inhibition of serotonin uptake in neurons than in astrocytes. In addition, Hypericum LI 160 inhibits norepinephrine uptake in SK-N-SH cells, a human neuroblastoma cell line enriched in norepinephrine transporters. A chemical constituent of LI 160, hyperforin, is about 10 to 20 times more potent than LI 160 in inhibiting neurotransmitter uptake in astrocytes and neuronal cells; this finding is consistent with the observation that hyperforin comprises 5% of LI 160 extracts. As several weeks are needed to achieve a clinical response with antidepressants, we have also investigated whether Hypericum LI 160 affects biochemical mechanisms that underlie long-term changes such as gene expression. We found that LI 160 stimulates a sustained activation of extracellular signal regulated protein kinase (ERK), a key component of a signal transduction pathway involved in gene expression. Taken together, our findings suggest that Hypericum LI 160 can affect rapidly-acting as well as slower-developing, long-term biochemical mechanisms related to depressive disorders.

Embryonic brain precursors transplanted into kainate lesioned rat spinal cord.

Embryonic day 14 rat cerebral cortex-derived precursors were expanded with FGF2 and labeled with BrdU prior to being transplanted into the kainic acid-lesioned adult rat spinal cord. While these precursors give rise to cells with neuronal, astrocytic and oligodendroglial phenotypes vitro, they remained largely undifferentiated up to 12 weeks in vivo. Numerous BrdU-labeled cells were found in injured gray matter, and also lining the dilated central canal that sometimes accompanies these lesions. BrdU-labeled cells never co-expressed Map2ab, rarely co-expressed GFAP but often co-expressed nestin, even after 12 weeks in vivo. These observations suggest that the environment of the kainic acid-injured spinal cord is not hostile to transplanted embryonic cerebral cortex-derived precursors, but also is not conducive to their neuronal differentation.

Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage.

Proliferating populations of undifferentiated neural stem cells were isolated from the embryonic day 14 rat cerebral cortex or the adult rat subventricular zone. These cells were pluripotent through multiple passages, retaining the ability to differentiate in vitro into neurons, astrocytes, and oligodendrocytes. Two weeks to 2 months after engraftment of undifferentiated, BrdU-labeled stem cells into the normal adult spinal cord, large numbers of surviving cells were seen. The majority of the cells differentiated with astrocytic phenotype, although some oligodendrocytes and undifferentiated, nestin-positive cells were detected; NeuN-positive neurons were not seen. Labeled cells were also engrafted into the contused adult rat spinal cord (moderate NYU Impactor injury), either into the lesion cavity or into the white or gray matter both rostral and caudal to the injury epicenter. Up to 2 months postgrafting, the majority of cells either differentiated into GFAP-positive astrocytes or remained nestin positive. No BrdU-positive neurons or oligodendrocytes were observed. These results show robust survival of engrafted stem cells, but a differentiated phenotype restricted to glial lineages. We suggest that in vitro induction prior to transplantation will be necessary for these cells to differentiate into neurons or large numbers of oligodendrocytes.

Immortalizing central nervous system cells.

This unit presents methods for isolating clonal, neural-derived cell lines. One approach for isolating such neural cell lines involves a replication-deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.

Developmental regulation of tryptophan hydroxylase messenger RNA expression and enzyme activity in the raphe and its target fields.

Tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin and during development, brain serotonin levels and tryptophan hydroxylase activities increase. Increased tryptophan hydroxylase activity could result from alterations in tryptophan hydroxylase messenger RNA levels, translation, and/or post-translational regulation. Tryptophan hydroxylase messenger RNA levels in the dorsal raphe nucleus increased 35-fold between embryonic day 18 and postnatal day 22, measured by quantitative in situ hybridization, then decreased by 40% between postnatal days 22 and 61. These changes correlated with tryptophan hydroxylase enzyme activities in the raphe nuclei as expected, but not in cortical or hippocampal targets. Tryptophan hydroxylase messenger RNA expression in the nucleus raphe obscuris increased 2.5-fold between postnatal days 8 and 22 but did not correlate with enzyme activity in the spinal cord. Using an in vitro model of serotonergic raphe neuron differentiation, serotonergic differentiation was associated with an increase in both tryptophan hydroxylase promoter activity and protein expression. In vivo, tryptophan hydroxylase messenger RNA levels per single cell and per brain section were correlated during development up to postnatal day 22, but not beyond for both the dorsal raphe nucleus and nucleus raphe obscuris. Between postnatal days 22 and 61 single cell levels of tryptophan hydroxylase messenger RNA in the dorsal raphe nucleus did not change yet the levels per brain section significantly decreased by 40%. During the same period in the nucleus raphe obscuris, tryptophan hydroxylase messenger RNA levels per single cell signifcantly increased by 30% yet levels per brain section did not change. Comparison of tryptophan hydroxylase messenger RNA levels per cell and per brain section indicated a serotonergic loss between postnatal days 22 and 61 in both the dorsal raphe nucleus and nucleus raphe obscuris and may reflect either a loss of neurotransmitter phenotype or cell death. This study is the first to characterize the expression of brain tryptophan hydroxylase messenger RNA during rat development. In addition, this study is the first to report the activity of tryptophan hydroxylase in the spinal cord and hippocampus in the embryonic and neonatal rat. Together, the data provide a better understanding of the intricate relationship between patterns of tryptophan hydroxylase messenger RNA expression and enzyme activity.

Human cytomegalovirus causes productive infection and neuronal injury in differentiating fetal human central nervous system neuroepithelial precursor cells.

OBJECTIVES: To study the effect of cell differentiation on the vulnerability of human neural cell types to human cytomegalovirus (HCMV) infection. STUDY DESIGN/METHODS: Primary cultures of human fetal neuroepithelial stem cells and differentiating neuroepithelial precursor cells were infected with HCMV strain AD169. Infectious virus production, apoptosis, and viral-associated cytopathic effects then were examined over a 5-day period. RESULTS: HCMV established productive infection in these cells, generating 10-fold amplification of infectious virus. There was no significant difference in the percentage of apoptotic cells in HCMV-infected versus mock-infected cultures. HCMV antigen and specific cytopathic effects were observed in differentiating astrocytes and neurons, although HCMV antigen was 2-fold more frequent among postmitotic neurons. CONCLUSIONS: Neuroepithelial precursor cells and differentiating astrocytes and neurons are permissive to cytopathic HCMV infection, suggesting that the fetal human central nervous system is vulnerable to HCMV-induced neuronal injury at its earliest stages of development.

Divergent regulation of GAP-43 expression and CNS neurite outgrowth by cyclic AMP.

Robust process outgrowth and high expression of the growth-associated protein GAP-43 seem to be intrinsic features of neurons, but both are down-regulated after axonal contact of target cells. We report that chronic exposure of the serotonergic CNS cell line RN46A to cyclic AMP analogs, forskolin, or cholera toxin represses GAP-43 expression in a dose dependent manner. Thus, cAMP could mediate a GAP-43 repressive signal that is initiated extracellularly. Activation of the cyclic AMP pathway by these same reagents, however, enhances the rate that RN46A cells extend neurites. This stimulation of neurite growth can occur during inhibition of new transcription, and in the absence of high levels of GAP-43. These findings demonstrate that a GAP-43-repressing intracellular signaling pathway exists, that repression of GAP-43 expression by cAMP is not directly coupled to inhibition of neurite growth, and that acceleration of growth cone advancement by cAMP is not dependent on the presence of GAP-43.

Neuronal replacement strategies for spinal cord injury.

Functional recovery following central nervous system (CNS) trauma or neurodegenerative disease is likely to require the transplantation of exogenous neurons. Given the logistical constraints of the potential widespread use of fetal human CNS tissues for therapeutic treatment, alternative sources of exogenous neurons for grafting will likely be necessary. Described here are studies examining the use of an immortalized, CNS-derived, neuronal precursor cell line, RN33B, as such a source. Results demonstrate that RN33B cells show remarkable plasticity to respond to local microenvironmental cues to differentiate in a direction that is morphologically indistinguishable from endogenous neurons at the site of transplantation. Concomitantly, the adult CNS retains the capacity to direct very specific differentiation of those engrafted precursor cells. However, the type and extent of site-specific appropriate differentiation is influenced by the type and extent of the lesion at the graft site. Attempts at immortalizing human CNS cells wtih similar approaches led to significant chromosomal aberrations, obviating such a strategy for therapeutic treatment. Thus, we utilized mitogen expansion of fetal human spinal cord cells as a means to generate populations of undifferentiated human neural precursor cells. Cells were expanded in the presence of epidermal growth factor and fibroblast growth factor 2, were readily passaged, and retained a pluriopotential to differentiate into neurons and astrocytes through at least 4 passages, after which the proliferating precursors became restricted to the astrocytic lineage. Further delineation of the variable needed to maintain these cells as undifferentiated, pluripotent precursor cells should ultimately enable examination of the ability of these cells to restore function in the damaged CNS.

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.

Protein kinase C and cAMP-dependent protein kinase regulate the neuronal differentiation of immortalized raphe neurons.

These studies examined the extent to which protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) regulate the neuronal differentiation of the raphe-derived neuronal cell line, RN33B. A differentiation-specific 2.25-fold increase in soluble PKA activity was observed. Neither membrane-associated-PKA, -PKC, or soluble PKC activities changed concomitant with differentiation. The PKC activity was derived from PKC alpha, gamma, epsilon, and theta isoenzymes. Activation of PKC inhibited the immunocytochemical expression of low and medium molecular weight neurofilament proteins, an effect due at least in part to decreased steady-state levels of protein. PKC activation also decreased glutamate immunoreactivity and increased cell number, protein synthesis, and bromodeoxyuridine uptake by 2.4-fold, 25%, and 32%, respectively. Coupled with the decrease in mature neuronal antigen expression, these data suggest that PKC activation inhibits neuronal differentiation by inducing proliferation. Inhibition of PKC markedly upregulated glutamate immunoreactivity. PKA activation potentiated the glutamatergic phenotype of RN33B cells, but inhibition of PKA was without effect on the expression of all neuronal antigens examined. Thus, both PKC and PKA regulate the differentiation of RN33B cells, although neither is absolutely necessary for expression of the differentiated neuronal phenotype. These results suggest the existence of parallel pathways regulating raphe neuronal differentiation.

Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord.

In the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2), neuroepithelial precursor cells from dissociated fetal human spinal cord are mitotically active and form free-floating spheres of undifferentiated cells. Proliferating cells were obtained in approximately 40% of preparations with each mitogen, were immunoreactive for the intermediate filament nestin, and did not express neuronal- or glial-specific markers. Early passage neuroepithelial precursor cells were pluripotent and differentiated into neurons expressing MAP2a,b, NF-M, and TuJ1, and GFAP-positive astrocytes; however, oligodendrocytes were never seen. As the cells were passaged from P0 to P4, the percentage of differentiating neurons significantly decreased and the prevalence of astrocytes significantly increased. While the majority of cell populations from individual preparations stopped proliferating between 3 and 6 passages, two expanding cell lines have been successfully expanded in EGF and FGF2 for over 25 passages and have been maintained in culture for over one year. These cells express nestin and not other cell-specific lineage markers. When differentiated, these neuroepithelial cell lines differentiate only into astrocytes, showing no expression of any neuronal marker. These data suggest that continued passage under these conditions preferentially selects for spinal cord neural precursors that are restricted to the astrocytic lineage. Despite the lineage restriction of later passage cell populations, these results provide a rationale for future investigation into the lineage potential of these cells in vivo following transplantation into the adult CNS, potentially as a therapeutic approach for traumatic injury and neurodegenerative disease.

Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

Ret-dependent and -independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to signal through a multicomponent receptor complex consisting of the Ret receptor tyrosine kinase and a member of the GFRalpha family of glycosylphosphatidylinositol-anchored receptors. In the current model of GDNF signaling, Ret delivers the intracellular signal but cannot bind ligand on its own, while GFRalphas bind ligand but are thought not to signal in the absence of Ret. We have compared signaling pathways activated by GDNF in two neuronal cell lines expressing different complements of GDNF receptors. In a motorneuron-derived cell line expressing Ret and GFRalphas, GDNF stimulated sustained activation of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways, cAMP response element-binding protein phosphorylation, and increased c-fos expression. Unexpectedly, GDNF also promoted biochemical and biological responses in a line of conditionally immortalized neuronal precursors that express high levels of GFRalphas but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFRalpha1-associated Src-like kinase activity in detergent-insoluble membrane compartments, rapid phosphorylation of cAMP response element-binding protein, up-regulation of c-fos mRNA, and cell survival. Together, these results offer new insights into the dynamics of GDNF signaling in neuronal cells, and indicate the existence of novel signaling mechanisms directly or indirectly mediated by GFRalpha receptors acting in a cell-autonomous manner independently of Ret.

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic "minipumps" to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39 degrees C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These 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 hind paw. When 33G10.17 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. 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 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2-3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably 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 inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.

Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells.

To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized. The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A, three cis-acting, cell specific, activating elements and a silencer were located. One of the activators and the silencer are located in the repeat region and one activator is positioned in the novel fragment. A fourth activating element was found to be active in both RN46A cells and in a non-neuronal serotonergic cell line, JAR. A 3.5 kb fragment from intron 1 was cloned and found to possess cell specific activity in JAR cells indicating the presence of an alternative promoter in intron 1.

Induction of Eph B3 after spinal cord injury.

Spinal cord injury (SCI) in adult rats initiates a cascade of events producing a nonpermissive environment for axonal regeneration. This nonfavorable environment could be due to the expression of repulsive factors. The Eph receptor protein tyrosine kinases and their respective ligands (ephrins) are families of molecules that play a major role in axonal pathfinding and target recognition during central nervous system (CNS) development. Their mechanism of action is mediated by repellent forces between receptor and ligand. The possible role that these molecules play after CNS trauma is unknown. We hypothesized that an increase in the expression of Eph proteins and/or ephrins may be one of the molecular cues that restrict axonal regeneration after SCI. Rats received a contusive SCI at T10 and in situ hybridization studies 7 days posttrauma demonstrated: (i) a marked up-regulation of Eph B3 mRNA in cells located in the white matter at the lesion epicenter, but not rostral or caudal to the injury site, and (ii) an increase in Eph B3 mRNA in neurons in the ventral horn and intermediate zone of the gray matter, rostral and caudal to the lesion. Immunohistochemical analyses localizing Eph B3 protein were consistent with the mRNA results. Colocalization studies performed in injured animals demonstrated increased Eph B3 expression in white matter astrocytes and motor neurons of the gray matter. These results suggest that Eph B3 may contribute to the unfavorable environment for axonal regeneration after SCI.

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord.

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 (Xu et al., 1995b). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 106 cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DbetaH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.

BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain.

We have previously demonstrated an augmentation of serotonergic activity within various brain areas following infusion of brain-derived neurotrophic factor (BDNF) into the midbrain near the periaqueductal gray and dorsal and median raphe nuclei (PAG/DR). However, the mechanism of this BDNF-induced modulatory effect on serotonergic systems was unclear. The aim of the present work was to study the regulation of tryptophan hydroxylase (TPH) mRNA levels after chronic BDNF administration in vivo. TPH mRNA levels were measured using a quantitative competitive reverse transcription polymerase chain reaction (RT-PCR) assay. A significant increase in the expression of TPH mRNA (13-fold) was found within the PAG/DR as early as 24 hr after onset of BDNF infusion and was sustained throughout the duration of infusion (11 days). This was accompanied by increased serotonin (5-hydroxytryptamine, 5-HT) levels and decreased nociceptive responsiveness assessed by tail-flick latency. BDNF induction of TPH mRNA levels was also observed in a serotonergic cell line derived from raphe neurons, indicating that BDNF can directly regulate TPH mRNA levels. These results suggest that BDNF augments 5-HT synthesis in vivo by directly enhancing steady-state TPH mRNA levels, and subsequently leading to marked behavioral alterations.

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.