Glycoprofiling with micro-arrays of glycoconjugates and lectins.

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.

alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive loss of substantia nigra dopaminergic neurons and the presence of cytoplasmic inclusions named Lewy bodies. Two missense mutations of the alpha-synuclein (alpha-syn; A30P and A53T) have been described in several families with an autosomal dominant form of PD. alpha-Syn also constitutes one of the main components of Lewy bodies in sporadic cases of PD. To develop an animal model of PD, lentiviral vectors expressing different human or rat forms of alpha-syn were injected into the substantia nigra of rats. In contrast to transgenic mice models, a selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum was observed in animals expressing either wild-type or mutant forms of human alpha-syn. This neuronal degeneration correlates with the appearance of abundant alpha-syn-positive inclusions and extensive neuritic pathology detected with both alpha-syn and silver staining. Lentiviral-mediated expression of wild-type or mutated forms of human alpha-syn recapitulates the essential neuropathological features of PD. Rat alpha-syn similarly leads to protein aggregation but without cell loss, suggesting that inclusions are not the primary cause of cell degeneration in PD. Viral-mediated genetic models may contribute to elucidate the mechanism of alpha-syn-induced cell death and allow the screening of candidate therapeutic molecules.

Recombinant proteins for neurodegenerative diseases: the delivery issue.

Tackling neurodegenerative diseases represents a formidable challenge for our ageing society. Recently, major achievements have been made in understanding the molecular mechanisms responsible for such diseases, and, simultaneously, numerous proteins such as neurotrophic factors, anti-apoptotic or anti-oxidant have been identified as potential therapeutic agents. Although many neurotrophic factors have been tested on individuals suffering from various neurodegenerative disorders, to date none has shown efficacy. Inadequate protein delivery is believed to be part of the problem. Recent improvements in pump technology, as well as in cell and gene therapy, are providing innovative ways to allow localized, regulatable delivery of proteins in brain parenchyma, opening new avenues for clinical trials in the not so distant future.

Primary adult human astrocytes as an ex vivo vehicle for beta-glucuronidase delivery in the brain.

Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.

Gene transfer techniques for the delivery of GDNF in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor disturbances caused by an alteration of the dopaminergic nigrostriatal system. Current symptomatic treatments for PD include dopaminergic drug administration, deep brain stimulation, ablative surgery and fetal cell transplantation. Though these approaches have significant beneficial effects, they are hampered by limiting side-effects, but more importantly they do not change the disease progression. Alternative restorative and neuroprotective strategies have therefore to be considered. Neuroprotective effects of neurotrophic factors, anti-apoptotic and antioxidant molecules are currently being investigated for this purpose. Among neurotrophic molecules, the potential of the glial cell line-derived neurotrophic factor (GDNF) to protect the nigral dopaminergic neurons and/or rescue striatal dopamine levels has been extensively documented. For GDNF to become a clinical reality, appropriate delivery techniques will have to be developed. This chapter focuses on the potential of encapsulated cells and viral vectors to locally release neurotrophic factors in experimental models of PD.

Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector.

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.

Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF.

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson's disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson's disease.

Effects of spinal cord X-irradiation on the recovery of paraplegic rats.

Axonal regrowth is limited in the adult CNS, especially in the spinal cord, one of the major sites of traumatic lesions. Pathophysiological changes occurring after spinal cord injury include complex acute, subacute, and late processes. In this study, we assessed whether X-irradiation interferes with the acute/subacute phases, thereby improving the functional recovery of paraplegic animals. Two days after acute compression of adult rat spinal cords, various doses (0, 2, 5, 10, 20 Gy) of X-rays were administered as one single dose to the compression site. The animals were functionally evaluated over the course of 1 month after injury, using the Tarlov scale and the Rivlin and Tator scale. We also designed a "physiological" scale, including an assessment of urinary function and infection, appropriate for the evaluation of spinal-cord-lesioned animals. Behavioral analysis suggested that the high doses, 20 Gy and, to a lesser extent, 5 and 10 Gy, were toxic, as shown by morbidity rate and "physiological" score. The 2-Gy group showed better motor performances than the lesioned nonirradiated (LNI) animals and the 5- and 20-Gy groups. Motor performance in the 5-, 10-, and 20-Gy groups was poorer than that seen in the LNI group. Gliosis was reduced in the 2-Gy group compared to LNI animals, and there was high levels of gliosis in the highly (>/=5 Gy) irradiated animals. There was a 23% less lesion-induced syringomyelia in the 2-Gy group than in the other groups (LNI and 5-20 Gy). Thus, low doses of X-rays may interfere with the formation of syringomyelia and glial scar, thereby facilitating the recovery of paraplegic animals. These findings suggest that low-dose irradiation of the lesion site, in association with other therapies, is a potentially promising treatment for improving recovery after spinal cord injury.

Toward autologous ex vivo gene therapy for the central nervous system with human adult astrocytes.

The combination of gene transfer techniques and cell transplantation is a promising approach to deliver therapeutic molecules into the CNS. To optimize gene transfer systems, several neural and nonneural cell types are currently under investigation. Among these cells, astrocytes are particularly well suited because of their CNS origin, their efficient secretory mechanisms, and their role as neuronal support. Most importantly, the use of human adult astrocytes as cellular vehicles for ex vivo gene transfer may open the way to autologous transplantation, thus obviating immunological rejection and the side effects of immunosuppressors. In the present study, we report the ability of these cells to be expanded and genetically modified in vitro. Astrocytes derived from human adult cerebral cortex were grown and maintained in vitro as pure primary cultures for at least 10 months. In addition, cells were efficiently transduced by an adenoviral vector encoding human tyrosine hydroxylase (hTH) under the negative control of the tetracycline-based regulatory system (tet-off). The infected cells synthesized large amounts of active hTH and released L-dopa. In addition, doxycycline, a potent analog of tetracycline, efficiently regulated transgene expression. This work is a first step toward the development of therapeutic strategies based on the use of genetically engineered human adult astrocytes for autologous transplantation in human neurodegenerative diseases and CNS trauma.

Expression of L1 and PSA during sprouting and regeneration in the adult hippocampal formation.

The objective of the present study was to evaluate the expression of polysialic acid (PSA) and the cell adhesion molecule L1 during axonal regeneration and sprouting after injury to the adult rat brain. All animals received a complete lesion of the fimbria-fornix (FF). Grafts of nerve growth factor (NGF)- or beta-galactosidase (betaGal)-producing fibroblasts were placed in the FF lesion cavity and induced septohippocampal cholinergic regeneration or sympathetic tyrosine hydroxylase (TH)-positive sprouting, respectively. Cholinergic regeneration was evaluated from 2 to 8 weeks following grafting of NGF-producing fibroblasts in the FF lesion cavity. In the graft area, choline acetyltransferase (ChAT)-positive fibers expressed L1 and PSA. Once cholinergic axons reached the hippocampal formation (HF), they no longer expressed L1 or PSA. Eight weeks after a lesion of the FF and transplantation of betaGal-producing fibroblasts, TH-positive fibers sprouted in the denervated HF and expressed L1 but not PSA. At the zone of reactive gliosis, PSA but not L1 expression was increased following a lesion of the FF and transplantation of NGF- or betaGal-producing fibroblasts. In animals that received a graft of NGF-producing fibroblasts in the FF lesion cavity, numerous ChAT-positive axons were observed along these areas rich in PSA and reactive astrocytes. Taken together, these results suggest that the expression of PSA and L1 is upregulated on regenerating cholinergic axons during axonal elongation and downregulated upon target innervation. In contrast, TH-positive fibers that sprout in the denervated HF express and maintain their expression of L1. Finally, the expression of PSA in the area of reactive gliosis may contribute to a permissive environment for axonal regrowth.

Reactive astrocytes: cellular and molecular cues to biological function.

For several decades, the reactive gliosis that occurs after an injury to the CNS has been considered one of the major impediments to axonal regeneration. Nevertheless, recent studies have suggested that in certain conditions, reactive astrocytes may provide a permissive substratum to support axonal regrowth. The important criteria, allowing for the distinction between permissive and non-permissive gliosis, are the ultrastructural 3D organization of the scar and more importantly the recognition molecules expressed by reactive astrocytes. Reactive astrocytes express surface molecules and produce various neurotrophic factors and cytokines. The latter in turn might modulate the production of recognition molecules by reactive astrocytes, allowing them to support post-lesional axonal regrowth. Although numerous recent articles have focused on cytokines and cell adhesion molecules, scant attention has been paid to reactive astrocytes. Reactive astrocytes should be considered a key element, like neurons, of a dynamic environment, thus forming with neurons a functional unit involved in homeostasis, plasticity and neurotransmission. Attempts are in progress to identify molecular markers for reactive astrocytes.

Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.

Immunocytochemical characterization of a new marker of fibrous and reactive astrocytes.

A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin.

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine.

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.

Gene therapy in the central nervous system: direct versus indirect gene delivery.

Over the last decade, the combination of molecular biology and cell transplantation techniques has given rise to a powerful method for gene therapy. The implantation of genetically modified cultured cells has been extensively used in the central nervous system (CNS) in various experimental models of neurologic disorders. More recently, viral and chemical methods have been developed to further efforts to shuttle transgenes into the relatively inaccessible brain. Adenoviral and liposomal synthetic vectors carry transgenes into neural tissue in situ and are beginning to show promise as new methods for CNS therapy.

Regeneration in the adult mammalian CNS: guided by development.

In the past year, the roles and mechanisms of molecules involved in cell survival (glial-derived neurotrophic growth factor), growth cone guidance (netrins and semaphorins), axonal outgrowth and sorting (neural cadherin, polysialylated neural cell adhesion molecules, and L1), and neuronal connectivity (cell adhesion molecules, dystroglycan, and agrin) have been described during development and, to a limited extent, in the mature CNS. Evidence is now emerging that some developmental events, such as the expression of polysialylated neural cell adhesion molecule and L1, are recapitulated during adult CNS regeneration. These results suggest new avenues to address more accurately the challenges of axonal regrowth in the adult mammalian CNS.

B-50 (GAP-43) immunoreactivity is rarely detected within intact catecholaminergic and serotonergic axons innervating the brain and spinal cord of the adult rat, but is associated with these axons following lesion.

The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B-50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts.

Encephalization and brain organization of the fishes of the family Osteoglossidae.

Despite their primitive place in the classification of fishes, Osteoglossids have a high level of encephalization, which can be analyzed by studying their brain organization. The quantitative analysis of their main brain parts shows small relative volumes of the olfactory bulbs, normal importance of the optic tectum (linked to vision) and of the cerebellum (linked to motor abilities). The medulla oblongata shows very large gustatory lobes in Heterotis, due to the special microphagous mode of feeding of this species. The cerebral hemispheres are larger in Arapaima than in other osteoglossids, but the lack of data on the comparative behaviour of these fishes does not allow us to give a reliable explantation of this peculiarity.

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

Light and electron microscopic studies of the effects of p-chloroamphetamine on the monoaminergic innervation of the rat spinal cord.

A previous report has shown that small diameter serotoninergic (5-HT) axons innervating the forebrain are selectively eliminated by treatment with an amphetamine derivative, (+/-)p-chloroamphetamine (PCA; Mamounas et al., [1991] J. Comp. Neurol. 314:558-586). It is well known that the spinal cord is the target of numerous monoaminergic fibers of different types. We have previously shown that the dorsal and ventral horns and the intermediolateral cell column are innervated by numerous serotonin-, noradrenaline- and dopamine-containing axons, including both thin axons with small varicosities and beaded axons with large varicosities. In all these regions, the large majority of fine indolaminergic fibers do not establish synaptic contacts, contrasting with the beaded axons which mostly exhibit synapses. The present work was conducted to study the effect of PCA on the monoaminergic innervation of the adult rat spinal cord. Animals received two subcutaneous doses of PCA 24 hours apart and were perfused 3 weeks later. Immunocytochemistry was performed to detect 5-HT, noradrenergic and dopaminergic fibers by using light and electron microscopy. Light microscopy revealed that PCA treatment caused a marked and selective elimination of the fine 5-HT-immunoreactive fibers, mainly found in the dorsal horn, but spared all other larger axons. This selective effect on the dorsal horn innervating thin 5-HT fibers was confirmed with the electron microscope by calculating the synaptic incidence(s) of monoaminergic innervation. These results suggest that fine and beaded 5-HT axons correspond to two anatomically, biochemically and pharmacologically different types of fibers, which could arise from two subpopulations of brainstem neurons. In addition, this drug could be used to provide an experimental animal, devoid of 5-HT nonsynaptic fibers, thereby facilitating a study on the role of dorsal horn nonsynaptic system in pain modulation.