No abstract

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.

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 degrees C), when the cells were proliferating, and during differentiation at non-permissive temperature (39 degrees 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.

Caenorhabditise/egans possesses two classes of inhibitory locomotory neurons, the DD and VD motoneurons (mns), and they form complementary components of a cross-inhibitory neuronal network innervating dorsal and ventral body muscles. The DD and VD mns (collectively called the D mns) share a number of morphological and neurochemical features, and mutations in a number of different genes disrupt both cell types in identical ways; however, the DD and VD mns have different lineal origins and different synaptic patterns. Given the number of phenotypic features shared by the D mns, it was of interest to determine what is responsible for the synaptic patterns that distinguishthem. An analysis of the locomotor-y defect along with a genetic epistasis test suggested that uric-55 mutations alter the function of the VD but not the DD mns. Correlated with the defective locomotory behavior of uric-55 mutants was an alteration in the distribution of varicosities, structures associated with presynaptic elements, on the VD mns. The pattern of varicosities of the uric-55 VD mns resembled that of the wild-type DD mns. Moreover, the selective removal of the DD mns revealed that uric-55 VD mns had adopted a functional role appropriate for the DD mns. Thus, uric-55 appears to be involved in producing the synaptic patterns that distinguish the two D mn classes from one another; when the gene is mutated the VD and DD mns become structurally similar and functionally equivalent.[Key words: uric-55, synaptic specificity, Caenorhabditis elegans, motoneuron, locomotory behavior, GABAergic neurons]The selection of specific synaptic targets by a neuron represents the completion of a defined sequence of steps in which individual neurons are assembled into a functional neuronal circuit. First, presumptive neurons are generated and positioned, second, the undifferentiated neurons extend processes to appropriate target regions; and finally, synapses are formed. Each step is dependent upon the preceding step and each is influenced to varying degrees by cell intrinsic and extrinsic events (Goodman Received Feb. 28, 1994; revised May 31, 1994; accepted July 15, 1994. This and Schatz, 1993). The interdependence between the steps and the inability to distinguish between intrinsic and extrinsic influences contribute to the difficulty in characterizing mechanisms involved in synapse specification. Animals amenable to genetic manipulations offer the potential to circumvent early steps in the sequence and identify directly genes whose products are involved in synapse specification. The nematode Caenorhabditis elegans is well suited for sophisticated genetic manipulations (Brenner, 1974;Herman, 1986). In addition, a stereotyped pattern of cell divisions produces a nervous system with exactly 302 individually identifiable neurons (Sulston and Horvitz, 1977;Sulston et al., 1983). The associated synaptic patterns that interconnect the neurons and their targets have been determined through reconstructions of serially sectioned animals. These synaptic relat...

Spinal cord injury (SCI) in mammals results in functional deficits that are mostly permanent due in part to the inability of severed axons to regenerate. Several types of growth-inhibitory molecules expressed at the injury site contribute to this regeneration failure. The responses of axons to these inhibitors vary greatly within and between organisms, reflecting axons' characteristic intrinsic propensity for regeneration. In the zebrafish (Danio rerio) many but not all axons exhibit successful regeneration after SCI. This review presents and compares the intrinsic and extrinsic determinants of axonal regeneration in the injured spinal cord in mammals and zebrafish. A better understanding of the molecules and molecular pathways underlying the remarkable individualism among neurons in mature zebrafish may support the development of therapies for SCI and their translation to the clinic.