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1.
Neuroscience ; 262: 129-42, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24412234

RESUMO

GABA is a predominant inhibitory neurotransmitter in the CNS. Released GABA is removed from the synaptic cleft by two GABA transporters (GATs), GAT-1 and GAT-3, and their dysfunction affects brain functions. The present study aimed to reveal the ontogeny of the GABA-removal system by examining the immunohistochemical localization of GAT-1 and GAT-3 in the embryonic and postnatal mouse cervical spinal cord. In the dorsal horn, GAT-1 was localized within the presynapses of inhibitory axons after embryonic day 15 (E15), a little prior to GABAergic synapse formation. The GAT-1-positive dots increased in density until postnatal day 21 (P21). By contrast, in the ventral horn, GAT-1-positive dots were sparse during development, although many transient GABAergic synapses were formed before birth. GAT-3 was first localized within the radial processes of radial glia in the ventral part on E12 and the dorsal part on E15. The initial localization of the GAT-3 was almost concomitant with the dispersal of GABAergic neurons. GAT-3 continued to be localized within the processes of astrocytes, and increased in expression until P21. These results suggested the following: (1) before synapse formation, GABA may be transported into the processes of radial glia or immature astrocytes by GAT-3. (2) At the transient GABAergic synapses in the ventral horn, GABA may not be reuptaken into the presynapses. (3) In the dorsal horn, GABA may start to be reuptaken by GAT-1 a little prior to synapse formation. (4) After synapse formation, GAT-3 may continue to remove GABA from immature and mature synaptic clefts into the processes of astrocytes. (5) Development of the GABA-removal system may be completed by P21.


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Células do Corno Anterior/embriologia , Células do Corno Anterior/crescimento & desenvolvimento , Células do Corno Anterior/metabolismo , Astrócitos/metabolismo , Axônios/metabolismo , Vértebras Cervicais , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Nestina/metabolismo , Células do Corno Posterior/embriologia , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/metabolismo , Medula Espinal/embriologia , Sinapses/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
2.
Neuron ; 80(6): 1392-406, 2013 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-24360543

RESUMO

In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Células do Corno Posterior/metabolismo , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismo , Animais , Axônios/metabolismo , Rastreamento de Células , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Interneurônios/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Receptor EphA4/metabolismo , Receptores de Superfície Celular , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Tálamo/citologia , Tálamo/crescimento & desenvolvimento , Tálamo/metabolismo
3.
Neuron ; 80(6): 1407-20, 2013 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-24360544

RESUMO

The spinal cord contains many descending and ascending longitudinal tracts whose development appears to be controlled by distinct guidance systems. We identified a population of dorsal spinal neurons marked by coexpression of the transcription factor Zic2 and the guidance receptor EphA4. Zic2+;EphA4+ neurons are surrounded by mechanosensory terminals, suggesting innervation by mechanoreceptor afferents. Their axons form an ipsilateral ascending pathway that develops during embryogenesis and projects within the ventral aspect of the dorsal funiculus, the same location as the descending corticospinal tract (CST), which develops postnatally. Interestingly, the same guidance mechanism, namely, ephrinB3-induced EphA4 forward signaling, is required for the guidance of both ascending and descending axon tracts. Our analysis of conditional EphA4 mutant mice also revealed that the development of the dorsal funiculus occurs independently of EphA4 expression in descending CST axons and is linked to the distribution of Zic2+;EphA4+ spinal neurons and the formation of the ascending pathway.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Efrina-B3/metabolismo , Células do Corno Posterior/metabolismo , Receptor EphA4/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Animais , Axônios/metabolismo , Rastreamento de Células , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Interneurônios/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Receptor EphA4/genética , Medula Espinal/citologia , Fatores de Transcrição/metabolismo
4.
J Physiol ; 588(Pt 14): 2571-87, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20498232

RESUMO

Inhibition mediated by glycine and GABA in the spinal cord dorsal horn is essential for controlling sensitivity to painful stimuli. Loss of inhibition results in hyperalgesia, a sensitized response to a painful stimulus, and allodynia, a pain-like response to an innocuous stimulus like touch. The latter is due, in part, to disinhibition of an excitatory polysynaptic pathway linking low threshold touch input to pain projection neurons. This critical impact of disinhibition raises the issue of what regulates the activity of inhibitory interneurons in the dorsal horn under non-pathological conditions. We have found that inhibitory neurons throughout lamina I-III, identified by the GAD67 promoter-driven EGFP, are tonically inhibited by glycine or GABA in a regionally distinct way that is mirrored by their inhibitory synaptic input. This tonic inhibition strongly modifies action potential firing properties. Surprisingly, we found that inhibitory neurons at the lamina II/III border are under tonic glycinergic control and receive synapses that are predominantly glycinergic. Futhermore, this tonic glycinergic inhibition remains strong as the mice mature postnatally. Interestingly, GlyT1, the glial glycine transporter, regulates the strength of tonic glycinergic inhibition of these glycine-dominant neurons. The more dorsal lamina I and IIo inhibitory neurons are mainly under control by tonic GABA action and receive synapses that are predominantly GABAergic. Our work supports the hypothesis that tonic glycine inhibition controls the inhibitory circuitry deep in lamina II that is likely to be responsible for separating low threshold input from high threshold output neurons of lamina I.


Assuntos
Glicina/fisiologia , Interneurônios/fisiologia , Inibição Neural/fisiologia , Células do Corno Posterior/fisiologia , Ácido gama-Aminobutírico/fisiologia , Potenciais de Ação/fisiologia , Animais , Proteínas da Membrana Plasmática de Transporte de Glicina/fisiologia , Camundongos , Camundongos Transgênicos , Células do Corno Posterior/crescimento & desenvolvimento
5.
J Neurosci ; 29(46): 14415-22, 2009 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-19923276

RESUMO

Partial peripheral nerve injury in adult rats results in neuropathic pain-like hypersensitivity, while that in neonatal rats does not, a phenomenon also observed in humans. We therefore compared gene expression profiles in the dorsal horn of adult and neonatal rats in response to the spared nerve injury (SNI) model of peripheral neuropathic pain. The 148 differentially regulated genes in adult, but not young, rat spinal cords indicate a greater microglial and T-cell response in adult than in young animals. T-cells show a large infiltration in the adult dorsal horn but not in the neonate after SNI. T-cell-deficient Rag1-null adult mice develop less neuropathic mechanical allodynia than controls, and central expression of cytokines involved in T-cell signaling exhibits large relative differences between young and adult animals after SNI. One such cytokine, interferon-gamma (IFNgamma), is upregulated in the dorsal horn after nerve injury in the adult but not neonate, and we show that IFNgamma signaling is required for full expression of adult neuropathic hypersensitivity. These data reveal that T-cell infiltration and activation in the dorsal horn of the spinal cord following peripheral nerve injury contribute to the evolution of neuropathic pain-like hypersensitivity. The neuroimmune interaction following peripheral nerve injury has therefore a substantial adaptive immune component, which is absent or suppressed in the young CNS.


Assuntos
Movimento Celular/imunologia , Hipersensibilidade/patologia , Neuralgia/imunologia , Dor/imunologia , Células do Corno Posterior/imunologia , Transdução de Sinais , Medula Espinal/imunologia , Linfócitos T/patologia , Fatores Etários , Animais , Hipersensibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Neuralgia/patologia , Dor/patologia , Medição da Dor/métodos , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/imunologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/patologia , Linfócitos T/imunologia
6.
J Neurophysiol ; 102(4): 2208-19, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19675290

RESUMO

Tissue injury in early life can produce distinctive effects on pain processing, but little is known about the underlying neural mechanisms. Neonatal inflammation modulates excitatory synapses in spinal nociceptive circuits, but it is unclear whether this results directly from altered afferent input. Here we investigate excitatory and inhibitory synaptic transmission in the rat superficial dorsal horn following neonatal hindlimb surgical incision using in vitro patch-clamp recordings and test the effect of blocking peripheral nerve activity on the injury-evoked changes. Surgical incision through the skin and muscle of the hindlimb at postnatal day 3 (P3) or P10 selectively increased the frequency, but not amplitude, of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) recorded 2-3 days after injury, without altering miniature inhibitory postsynaptic current frequency or amplitude at this time point. Meanwhile, incision at P17 failed to affect excitatory or inhibitory synaptic function at 2-3 days postinjury. The elevated mEPSC frequency was accompanied by increased inward rectification of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents, but no change in AMPAR/N-methyl-D-aspartate receptor ratios, and was followed by a persistent reduction in mEPSC frequency by 9-10 days postinjury. Prolonged blockade of primary afferent input from the time of injury was achieved by administration of bupivacaine hydroxide or tetrodotoxin to the sciatic nerve at P3. The increase in mEPSC frequency evoked by P3 incision was prevented by blocking sciatic nerve activity. These results demonstrate that increased afferent input associated with peripheral tissue injury selectively modulates excitatory synaptic drive onto developing spinal sensory neurons and that the enhanced glutamatergic signaling in the dorsal horn following neonatal surgical incision is activity dependent.


Assuntos
Ácido Glutâmico/metabolismo , Músculo Esquelético/lesões , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/fisiopatologia , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Bupivacaína/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Bloqueio Nervoso , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Nervo Isquiático/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Tetrodotoxina/farmacologia , Fatores de Tempo
7.
J Neurochem ; 110(5): 1695-706, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19619140

RESUMO

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca(2+) concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.


Assuntos
AMP Cíclico/fisiologia , Glicina/fisiologia , Potenciação de Longa Duração/fisiologia , Células do Corno Posterior/crescimento & desenvolvimento , Substância Gelatinosa/citologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Colforsina/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Glicina/fisiologia , Substância Gelatinosa/efeitos dos fármacos , Substância Gelatinosa/crescimento & desenvolvimento , Transmissão Sináptica/efeitos dos fármacos
8.
J Comp Neurol ; 508(4): 592-604, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18383051

RESUMO

In this study, we investigated postnatal changes in Rexed's laminae and distribution of nociceptive afferents in the dorsal horn of the rat lumbar spinal cord at postnatal days 0, 5, 10, 15, 20, and 60. Transverse sections of the L4-L5 segments were processed for triple labeling with isolectin B4 (IB4)-binding as a marker of nonpeptidergic C-fibers, calcitonin gene-related peptide (CGRP) immunoreactivity to label peptidergic nociceptive afferents, and a fluorescent Nissl stain to visualize cells and lamination at different stages of postnatal development. The Nissl staining revealed that the thickness of lamina I (LI) and outer lamina II remained mostly unchanged from birth until adulthood. CGRP afferents terminated mostly in LI and the outer two-thirds of lamina II, whereas the termination area of fibers binding IB4 was centered on the middle one-third of lamina II at all ages studied. In absolute values, the overall width of the bands of intense CGRP and IB4 labeling increased with age but decreased as a percentage of the overall thickness of the dorsal horn with maturation. The overlap of CGRP termination area with that of IB4 afferents increased with age. The consequences of these findings are twofold. First, the size of the different laminae does not grow evenly across the dorsal horn. Second, CGRP and IB4 labeling cannot be considered per se to be reliable markers of lamination during development. These findings have implications for comparing data obtained in immature and mature tissues with respect to localization of structures in the dorsal horn.


Assuntos
Neurônios Aferentes/fisiologia , Nociceptores/crescimento & desenvolvimento , Células do Corno Posterior/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Biomarcadores/química , Masculino , Fibras Nervosas Amielínicas/química , Fibras Nervosas Amielínicas/fisiologia , Neurônios Aferentes/química , Nociceptores/química , Células do Corno Posterior/química , Ratos , Ratos Sprague-Dawley
9.
IEEE Trans Biomed Eng ; 55(4): 1447-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18390337

RESUMO

xonal outgrowth on smooth and porous silicon surfaces was studied in organ culture. The pore size of the silicon substrata varied between 100 and 1500 nm. We found that axons preferred to grow and elongate on porous silicon surfaces only when pores of (150-500 nm) are available.


Assuntos
Axônios/fisiologia , Axônios/ultraestrutura , Materiais Biocompatíveis/química , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Silício/química , Animais , Crescimento Celular , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Teste de Materiais , Camundongos , Porosidade
10.
Dev Biol ; 314(1): 23-39, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18155191

RESUMO

The transcription cofactor Pbx3 is critical for the function of hindbrain circuits controlling respiration in mammals, but the perinatal lethality caused by constitutively null mutations has hampered investigation of other roles it may play in neural development and function. Here we report that the conditional loss of Pbx3 function in most tissues caudal to the hindbrain resulted in progressive deficits of posture, locomotion, and sensation that became apparent during adolescence. In adult mutants, the size of the dorsal horn of the spinal cord and the numbers of calbindin-, PKC-gamma, and calretinin-expressing neurons in laminae I-III were markedly reduced, but the ventral cord and peripheral nervous system appeared normal. In the embryonic dorsal horn, Pbx3 expression was restricted to a subset of glutamatergic neurons, but its absence did not affect the initial balance of excitatory and inhibitory interneuron phenotypes. By embryonic day 15 a subset of Meis(+) glutamatergic neurons assumed abnormally superficial positions and the number of calbindin(+) neurons was increased three-fold in the mutants. Loss of Pbx3 function thus leads to the incorrect specification of some glutamatergic neurons in the dorsal horn and alters the integration of peripheral sensation into the spinal circuitry regulating locomotion.


Assuntos
Proteínas de Homeodomínio/fisiologia , Atividade Motora/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Rombencéfalo/crescimento & desenvolvimento , Medula Espinal/crescimento & desenvolvimento , Animais , Calbindina 2 , Calbindinas , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Mutantes , Mutação , Células do Corno Posterior/embriologia , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/fisiologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/genética , Rombencéfalo/embriologia , Rombencéfalo/fisiologia , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteína G de Ligação ao Cálcio S100/fisiologia , Medula Espinal/embriologia , Medula Espinal/fisiologia
11.
J Comp Neurol ; 505(5): 547-61, 2007 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-17924532

RESUMO

Despite intensive study, our understanding of the neuronal structures responsible for transducing the broad spectrum of environmental energies that impinge upon the skin has rested on inference and conjecture. This major shortcoming motivated the development of ex vivo somatosensory system preparations in neonatal mice in the hope that their small size might allow the peripheral terminals of physiologically identified sensory neurons to be labeled intracellularly for direct study. The present report describes the first such study of the peripheral terminals of four slowly adapting type I low-threshold mechanoreceptors (SAIs) that innervated the back skin of neonatal mice. In addition, this report includes information on the central anatomy of the same SAI afferents that were identified peripherally with both physiological and anatomical means, providing an essentially complete view of the central and peripheral morphology of individual SAI afferents in situ. Our findings reveal that SAIs in neonates are strikingly adult-like in all major respects. Afferents were exquisitely sensitive to mechanical stimuli and exhibited a distinctly irregular, slowly adapting discharge to stimulation of 1-4 punctate receptive fields in the skin. Their central collaterals formed transversely oriented and largely nonoverlapping arborizations limited to regions of the dorsal horn corresponding to laminae III-V. Their peripheral arborizations were restricted entirely within miniaturized touch domes, where they gave rise to expanded disc-like endings in close apposition to putative Merkel cells in basal epidermis. These findings therefore provide the first direct confirmation of the functional morphology of this physiologically unique afferent class.


Assuntos
Adaptação Fisiológica/fisiologia , Mecanorreceptores/citologia , Células de Merkel/citologia , Camundongos/anatomia & histologia , Pele/inervação , Animais , Animais Recém-Nascidos , Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/fisiologia , Mecanorreceptores/crescimento & desenvolvimento , Mecanorreceptores/fisiologia , Células de Merkel/fisiologia , Camundongos/fisiologia , Terminações Nervosas/citologia , Terminações Nervosas/crescimento & desenvolvimento , Terminações Nervosas/fisiologia , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/fisiologia , Pele/crescimento & desenvolvimento , Tato/fisiologia
12.
Am J Physiol Heart Circ Physiol ; 293(1): H266-73, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17337595

RESUMO

Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor alpha3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 +/- 11.9 mm, and that in the absence of FA was 26.3 +/- 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.


Assuntos
Axônios/fisiologia , Vasos Sanguíneos/inervação , Vasos Sanguíneos/metabolismo , Proteínas do Tecido Nervoso/administração & dosagem , Células do Corno Posterior/crescimento & desenvolvimento , Sistema Nervoso Simpático/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Modelos Biológicos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/efeitos dos fármacos
13.
Brain Behav Immun ; 21(5): 617-23, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17158026

RESUMO

Neuropathic pain, arising from nerve injury or secondary to other diseases, occurs in young children as well as adults but little is known about its postnatal development. Neonatal rat pups do not display mechanical allodynia following nerve injury and young rats recover faster from spinal nerve damage. Since both spinal microglia and astrocytes are strongly implicated in the maintenance of persistent pain, we hypothesized that the magnitude and time course of spinal cord glial activation following nerve injury change throughout postnatal development. To test this, we have compared the time course and intensity of the microglial and astrocytic response in the spinal cord dorsal horn at various times following spared nerve injury in postnatal day 3, 10, 21 and adult rats. The levels of the microglial markers OX-42 and IBA-1 and of the astrocytic marker GFAP were analysed using immunohistochemistry and Western blots. We show that in the adult SNI evokes clear dorsal horn microglial activation at 5 days and astrocytic activation at 7 days post surgery. In contrast, SNI in young animals evokes a weak microglial response but a robust astrocytic response with an early onset at day 1 that is not observed in adults, followed by a second activation at day 7. These results highlight the differential development of the glial response to nerve injury which may explain the lack of neuropathic allodynia in young animals.


Assuntos
Envelhecimento/fisiologia , Astrócitos/metabolismo , Microglia/metabolismo , Nervo Isquiático/lesões , Neuropatia Ciática/imunologia , Medula Espinal/citologia , Envelhecimento/imunologia , Análise de Variância , Animais , Astrócitos/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Masculino , Proteínas dos Microfilamentos , Microglia/imunologia , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/imunologia , Células do Corno Posterior/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/imunologia , Nervo Isquiático/metabolismo , Neuropatia Ciática/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/imunologia , Medula Espinal/metabolismo , Fatores de Tempo
14.
Mol Cell Neurosci ; 33(1): 88-95, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16875841

RESUMO

Superficial dorsal horn neurones undergo marked structural and functional activity-dependent development during the early postnatal period, but little is known about the molecular mechanisms underlying these changes. Calcium signalling, through activation and autophosphorylation of CaMKII, has been shown to play a major role in the maturation of neuronal morphology and connectivity in the cortex. Here, we show that the normal structural and functional development of superficial dorsal horn neurones requires CaMKII autophosphorylation at the Thr286 residue. The dendritic branching of neurones from mice containing a point mutation at this site (T286A) was significantly increased compared with wild-type littermates. This was accompanied by significant increases in receptive field size, recorded from intact preparations. Whole-cell patch clamp recordings of superficial dorsal horn slices revealed a selective deficit in low-threshold A fibre-evoked synaptic input. These results show that CaMKII autophosphorylation is required for the normal development of spinal sensory circuits.


Assuntos
Vias Aferentes/anatomia & histologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Dendritos/ultraestrutura , Células do Corno Posterior , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Dendritos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Mutação Puntual , Células do Corno Posterior/citologia , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/patologia
15.
J Physiol ; 573(Pt 3): 753-63, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16613873

RESUMO

ATP mediates somatosensory transmission in the spinal cord through the activation of P2X receptors. Nonetheless, the functional significance of postsynaptic P2X receptors in spinal deep dorsal horn neurones is still not yet well understood. Using the whole-cell patch-clamp technique, we investigated whether the activation of postsynaptic P2X receptors can modulate the synaptic transmission in lamina V neurones of postnatal day (P) 9-12 spinal cord slices. At a holding potential of -70 mV, ATPgammaS (100 microm), a nonhydrolysable ATP analogue, generated an inward current, which was resistant to tetrodotoxin (1 microm) in 61% of the lamina V neurones. The ATPgammaS-induced inward current was accompanied by a significant increase in the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the majority of lamina V neurones. The ATPgammaS-induced inward current was not reproduced by P2Y receptor agonists, UTP (100 microm), UDP (100 microm), and 2-methylthio ADP (100 microm), and it was also not affected by the addition of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) into the pipette solution, thus suggesting that ionotropic P2X receptors were activated by ATPgammaS instead of metabotropic P2Y receptors. On the other hand, alpha,beta-methylene ATP (100 microm) did not change any membrane current, but instead increased the mEPSC frequency in the majority of lamina V neurones. The ATPgammaS-induced inward current was suppressed by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10 microm), but not by trinitrophenyl-ATP (TNP-ATP) (1 microm). Furthermore, we found that ATPgammaS (100 microm) produced a clear inward current which was observed in all lamina V neurones over P16 spinal cord slices, in contrast to P9-12. These results indicate that distinct subtypes of P2X receptors were functionally expressed at the post- and presynaptic sites in lamina V neurones, both of which may contribute to the hyperexcitability of lamina V in a different manner. In addition, the data relating to the developmental increase in the functional P2X receptors suggest that purinergic signalling may thus be more common in somatosensory transmission with maturation.


Assuntos
Células do Corno Posterior/metabolismo , Receptores Purinérgicos P2/metabolismo , Medula Espinal/metabolismo , Transmissão Sináptica , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores , Técnicas In Vitro , Técnicas de Patch-Clamp , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/crescimento & desenvolvimento , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Agonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y2 , Medula Espinal/efeitos dos fármacos , Medula Espinal/crescimento & desenvolvimento , Uridina Trifosfato/farmacologia
16.
J Comp Neurol ; 495(1): 37-52, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16432898

RESUMO

The development of the primary sensory innervation of the superficial dorsal horn (SDH) was studied in postnatal opossums Monodelphis domestica by using DiI labelling of primary afferents and with GSA-IB(4) lectin binding and calcitonin gene-related peptide (CGRP) immunoreactivity to label primary afferent subpopulations. We also compared the timing of SDH innervation in the cervical and lumbar regions of the spinal cord. The first primary afferent projections to SDH emerge from the most lateral part of the dorsal root entry zone at postnatal day 5 and project around the lateral edge of the SDH toward lamina V. Innervation of the SDH occurs slowly over the second and third postnatal weeks, with the most dorsal aspect becoming populated by mediolaterally oriented varicose fibers before the rest of the dorsoventral thickness of the SDH becomes innervated by fine branching varicose fibers. Labelling with GSA-IB(4) lectin also labelled fibers at the lateral edge of the dorsal horn and SDH at P5, indicating that the GSA-IB(4) is expressed on SDH/lamina V primary afferents at the time when they are making their projections into the spinal cord. In contrast, CGRP-immunoreactive afferents were not evident until postnatal day 7, when a few short projections into the lateral dorsal horn were observed. These afferents then followed a pattern similar to the development of GSA-IB(4) projects but with a latency of several days. The adult pattern of labelling by GSA-IB(4) is achieved by about postnatal day 20, whereas the adult pattern of CGRP labelling was not seen until postnatal day 30. Electron microscopy revealed a few immature synapses in the region of the developing SDH at postnatal day 10, and processes considered to be precursors of glomerular synapses (and thus of primary afferent origin) were first seen at postnatal day 16 and adopted their definitive appearance between postnatal days 28 and 55. Although structural and functional development of forelimbs of neonatal Monodelphis is more advanced than the hindlimbs, we found little evidence of a significant delay in the invasion of the spinal cord by primary afferents in cervical and lumbar regions. These observations, together with the broadly similar maturational appearance of histological sections of rostral and caudal spinal cord, suggest that, unlike the limbs they innervate, the spinal regions do not exhibit a large rostrocaudal gradient in their maturation.


Assuntos
Vias Aferentes/crescimento & desenvolvimento , Monodelphis/crescimento & desenvolvimento , Neurônios Aferentes/metabolismo , Células do Corno Posterior/crescimento & desenvolvimento , Raízes Nervosas Espinhais/crescimento & desenvolvimento , Vias Aferentes/ultraestrutura , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Carbocianinas , Diferenciação Celular/fisiologia , Vértebras Cervicais , Extremidades/crescimento & desenvolvimento , Extremidades/inervação , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/ultraestrutura , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Imuno-Histoquímica , Vértebras Lombares , Microscopia Eletrônica de Transmissão , Monodelphis/anatomia & histologia , Neurônios Aferentes/ultraestrutura , Lectinas de Plantas , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Raízes Nervosas Espinhais/ultraestrutura
17.
J Neurosci ; 25(42): 9613-23, 2005 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-16237166

RESUMO

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.


Assuntos
Canais de Cloreto/fisiologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/crescimento & desenvolvimento , Receptores de GABA-A/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Animais Recém-Nascidos , Ânions , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Agonistas de Receptores de GABA-A , Células do Corno Posterior/fisiologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos
18.
Neuroreport ; 16(12): 1325-8, 2005 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-16056133

RESUMO

Neonatal superficial dorsal horn neurons exhibit distinct firing properties in response to nociceptive and tactile inputs, but it is not known whether the intrinsic membrane excitability of these neurons changes during the early postnatal period. We have investigated the evoked firing properties of dorsal horn cells in rat spinal cord slices at different postnatal ages (P3, P10 and P21) and found no significant differences in mean firing frequency, spike frequency adaptation, regularity of action potential discharge or rheobase current levels between age groups. These results demonstrate that the intrinsic excitability of superficial dorsal horn neurons remains stable during early postnatal development and suggest that alterations in the synaptic inputs to these cells explain the changes in response to peripheral stimulation.


Assuntos
Potenciais de Ação/fisiologia , Envelhecimento/fisiologia , Células do Corno Posterior/fisiologia , Medula Espinal/crescimento & desenvolvimento , Potenciais de Ação/efeitos da radiação , Análise de Variância , Animais , Animais Recém-Nascidos , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Técnicas In Vitro , Técnicas de Patch-Clamp/métodos , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia
19.
Biomaterials ; 26(1): 47-61, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15193880

RESUMO

Study of axonal growth and ligand-receptor interactions requires specificity and careful characterization of the biomaterial substrates to which the neurons bind. It would be impossible to predict the effects of important variables such as composition, surface density, spatial distribution, and conformation of the ligands on axonal growth of a neuron without highly specific surface characterization. Here, we compare two methods of surface modification (hereafter referred to as "Heterobifunctional Crosslinker" and "Pluronics" methods) used for immobilization of fibronectin (FN) and FN-derived, RGD-containing peptides to the substrates. We also characterized their performance in neurite outgrowth experiments. Various surface analytical techniques such as contact angle measurement, XPS, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used for the analysis of the substrates at each step of the two different chemistries involved. FN-patterned surfaces were created by micro-contact printing methods and confirmed by imaging TOF-SIMS, and AFM techniques. After immobilization of FN and/or FN-derived RGD-containing peptide, including the formation of micron-scale patterns of FN, the modified surfaces were plated with neurons from postnatal rat dorsal root ganglia (DRG) and incubated in serum-free medium. Both the peptide- and/or protein-modified substrates supported significantly greater neurite outgrowth than controls, and outgrowth on both substrate chemistries was inhibited by the addition of soluble RGD peptide. Patterned FN surfaces were successful in spatially controlling the neuron attachment and outgrowth.


Assuntos
Técnicas de Cultura de Células/métodos , Materiais Revestidos Biocompatíveis/síntese química , Cristalização/métodos , Fibronectinas/química , Neuritos/fisiologia , Neuritos/ultraestrutura , Oligopeptídeos/química , Adsorção , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/fisiologia , Células Imobilizadas/ultraestrutura , Reagentes de Ligações Cruzadas/química , Fibronectinas/farmacologia , Teste de Materiais , Neuritos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/crescimento & desenvolvimento , Células do Corno Posterior/ultraestrutura , Ligação Proteica , Ratos , Engenharia Tecidual
20.
J Comp Neurol ; 467(3): 403-17, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14608602

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) is the prototypical member of a growth factor family that signals via the cognate receptors ret and GDNF-receptor alpha-1. The latter receptors are expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. Although effects of GDNF on neuronal survival in the brain have previously been reported, GDNF effects on injured axons of the adult spinal cord have not been investigated. Using an ex vivo gene delivery approach that provides both trophic support and a cellular substrate for axonal growth, we implanted primary fibroblasts genetically modified to secrete GDNF into complete and partial mid-thoracic spinal cord transection sites. Compared to recipients of control grafts expressing a reporter gene, GDNF-expressing grafts promoted significant regeneration of several spinal systems, including dorsal column sensory, regionally projecting propriospinal, and local motor axons. Local GDNF expression also induced Schwann cell migration to the lesion site, leading to remyelination of regenerating axons. Thus, GDNF exerts tropic effects on adult spinal axons and Schwann cells that contribute to axon growth after injury.


Assuntos
Fibroblastos/transplante , Neurônios Motores/fisiologia , Bainha de Mielina/fisiologia , Fatores de Crescimento Neural/administração & dosagem , Neurônios Aferentes/fisiologia , Células do Corno Posterior/crescimento & desenvolvimento , Traumatismos da Medula Espinal/terapia , Animais , Axônios/fisiologia , Feminino , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Bainha de Mielina/genética , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/fisiologia , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Células do Corno Posterior/fisiologia , Ratos , Ratos Endogâmicos F344 , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologia
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