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1.
medRxiv ; 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38883729

RESUMO

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by a varying degree of severity that correlates with the reduction of SMN protein levels. Motor neuron degeneration and skeletal muscle atrophy are hallmarks of SMA, but it is unknown whether other mechanisms contribute to the spectrum of clinical phenotypes. Here, through a combination of physiological and morphological studies in mouse models and SMA patients, we identify dysfunction and loss of proprioceptive sensory synapses as key signatures of SMA pathology. We demonstrate that SMA patients exhibit impaired proprioception, and their proprioceptive sensory synapses are dysfunctional as measured by the neurophysiological test of the Hoffmann reflex (H-reflex). We further show that loss of excitatory afferent synapses and altered potassium channel expression in SMA motor neurons are conserved pathogenic events found in both severely affected patients and mouse models. Lastly, we report that improved motor function and fatigability in ambulatory SMA patients and mouse models treated with SMN-inducing drugs correlate with increased function of sensory-motor circuits that can be accurately captured by the H-reflex assay. Thus, sensory synaptic dysfunction is a clinically relevant event in SMA, and the H-reflex is a suitable assay to monitor disease progression and treatment efficacy of motor circuit pathology.

2.
J Neurophysiol ; 97(4): 2875-86, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17287435

RESUMO

The effects of opioids on sacrocaudal afferent (SCA) pathways and the pattern-generating circuitry of the thoracolumbar and sacrocaudal segments of the spinal cord were studied in isolated spinal cord and brain stem-spinal cord preparations of the neonatal rat. The locomotor and tail moving rhythm produced by activation of nociceptive and nonnociceptive sacrocaudal afferents was completely blocked by specific application of the mu-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate salt (DAMGO) to the sacrocaudal but not the thoracolumbar segments of the spinal cord. The rhythmic activity could be restored after addition of the opioid receptor antagonist naloxone to the experimental chamber. The opioid block of the SCA-induced rhythm is not due to impaired rhythmogenic capacity of the spinal cord because a robust rhythmic activity could be initiated in the thoracolumbar and sacrocaudal segments in the presence of DAMGO, either by stimulation of the ventromedial medulla or by bath application of N-methyl-d-aspartate/serotonin. We suggest that the opioid block of the SCA-induced rhythm involves suppression of synaptic transmission through sacrocaudal interneurons interposed between SCA and the pattern-generating circuitry. The expression of mu opioid receptors in several groups of dorsal, intermediate and ventral horn interneurons in the sacrocaudal segments of the cord, documented in this study, provides an anatomical basis for this suggestion.


Assuntos
Vias Aferentes/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Animais Recém-Nascidos/fisiologia , Tronco Encefálico/fisiologia , Instinto , Medula Espinal/fisiologia , Animais , Tronco Encefálico/efeitos dos fármacos , Interpretação Estatística de Dados , Estimulação Elétrica , Eletrofisiologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Imuno-Histoquímica , Locomoção/fisiologia , Microscopia Confocal , Movimento/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Ratos , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Receptores Opioides mu/fisiologia , Medula Espinal/efeitos dos fármacos , Cauda/inervação , Cauda/fisiologia
3.
Neuroscience ; 108(3): 493-506, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11738262

RESUMO

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.


Assuntos
Neuritos/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Receptores de Glicina/fisiologia , Medula Espinal/fisiologia , Animais , Cálcio/metabolismo , Embrião de Mamíferos , Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Glicina/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Sinapses/fisiologia , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismo
4.
Neuroreport ; 11(13): 3007-10, 2000 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-11006984

RESUMO

The effect of glycine receptor activation on neurite outgrowth and survival was studied in 5 DIV (days in vitro) spinal neurons. These neurons were depolarized by spontaneous synaptic activity and by glycine, but not by glutamate. These responses were accompanied by increases in intracellular calcium concentration measured with Indo-1 and Fluo-3. Glycine (100 microM, 48 h) increased (46 +/- 6%) the number of primary neurites and total neuritic length. This effect was mediated by synaptic activity and calcium influx because TTX (1 microM) and nimodipine (4 microM) blocked the stimulatory effect of glycine. Neuronal survival, on the other hand, was not affected. This study shows for the first time the modulatory effect of glycine receptors on spinal neuron development.


Assuntos
Fatores de Crescimento Neural/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Receptores de Glicina/metabolismo , Medula Espinal/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Feto , Glicina/metabolismo , Glicina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Receptores de Glicina/efeitos dos fármacos , Medula Espinal/citologia
5.
Neuropathol Appl Neurobiol ; 26(3): 258-72, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10886684

RESUMO

The vulnerability of motoneurones to glutamate has been implicated in neurological disorders such as amyotrophic lateral sclerosis but it is not known whether specific receptor subtypes mediate this effect. In order to investigate this further, the expression of N-methyl-D-aspartate (NMDA) receptor subunits was studied during the first three post-natal weeks when motoneurones are differentially vulnerable to injury following neonatal nerve crush compared to the adult. Unilateral nerve crush was carried out at day 2 after birth (P2) which causes a decrease of 66% in motoneurone number by 14 days (P14). To study receptor expression in identified motoneurones, serial section analysis was carried out on retrogradely labelled common peroneal (CP) motoneurones by combined immunocytochemistry and in situ hybridization (ISH). mRNA levels were also quantified in homogenates from lumbar spinal cords in which the side ipsilateral to the crush was separated from the contralateral side. The NR1 subunit of the NMDA receptor was widely distributed in the spinal cord being expressed most strongly in motoneurone somata particularly during the neonatal period (P3-P7). The NR2 subunits were also expressed at higher levels in the somata and dendrites of neonatal motoneurones compared to older animals. NR2B mRNA was expressed at low to moderate levels throughout the studied period whereas NR2A mRNA levels were low until P21. Following unilateral nerve crush, an initial decrease in NR1 mRNA occurred at one day after nerve crush (P3) in labelled CP motoneurones ipsilateral to the crush which was followed by a significant increase in NR1 subunit expression at 5 days post-injury. This increase was bilateral although reaching greater significance ipsilateral to the crush compared with sham-operated animals. A significant increase in NR1 and NR2B mRNA post injury was also detected in spinal cord homogenates. In addition, the changes in levels of NR1 and NR2B mRNA were reflected by comparable bilateral changes at P7 in receptor protein determined by quantitative immunocytochemical analysis of NR1 and NR2 subunit expression in identified CP motoneurones indicating a co-ordinated regulation of receptor subunits in response to injury.


Assuntos
Neurônios Motores/metabolismo , Nervo Fibular/lesões , Receptores de N-Metil-D-Aspartato/genética , Medula Espinal/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Músculo Esquelético/inervação , Compressão Nervosa , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
6.
Neuroscience ; 64(1): 109-15, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7708198

RESUMO

Paralysis of the soleus muscle in newborn rats causes a large proportion of motoneurons to die by 10 weeks of age. However, all of these neurons are still present at three to four weeks of age. We have previously shown that although nerve injury at five days does not result in any motoneuron death, it does render these neurons susceptible to the toxic effects of the glutamate agonist N-methyl-D-aspartate. Using retrograde labelling of soleus motoneurons, in this study we show that an increased susceptibility to glutamate also plays a role in the eventual death of those motoneurons which survive for three weeks after interruption of neuromuscular transmission at birth but die by 10 weeks. Treatment with dizocilpine maleate an antagonist of the N-methyl-D-aspartate receptor increased the survival of motoneurons to alpha-bungarotoxin-treated soleus muscles. By 10 weeks of age the size of motoneurons to alpha-bungarotoxin-treated soleus muscles is smaller than that of controls, but after treatment with dizocilpine maleate the sizes of motoneurons to control and treated muscles are similar. Moreover, only 55 +/- 2.7% of motoneurons to the soleus muscle paralysed at birth with alpha-bungarotoxin survive for three weeks after a single injection of N-methyl-D-aspartate at 12 days of age. This motoneuron death is due to the application of N-methyl-D-aspartate since treatment with alpha-bungarotoxin alone causes no loss of neurons at this age.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Neurônios Motores/efeitos dos fármacos , Músculos/efeitos dos fármacos , N-Metilaspartato/farmacologia , Paralisia/classificação , Animais , Maleato de Dizocilpina/farmacologia , Humanos , Recém-Nascido , Neurônios Motores/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia
7.
Brain Res Dev Brain Res ; 81(2): 162-70, 1994 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-7813038

RESUMO

In rats following nerve injury at birth a large proportion of motoneurones to the soleus muscle dies. Blocking of N-methyl-D-aspartate (NMDA) receptors by MK-801 (dizocilpine maleate) for 12 days after nerve injury at birth leads to rescue of a proportion of motoneurones destined to die. Retrograde labelling of soleus motoneurones shows that 6-8 weeks after crushing the sciatic nerve in one hindlimb, only 10.9 +/- 2.3% of the motoneurones have survived. In animals treated with an NMDA receptor blocker MK-801 (2 mg/kg i.p., from birth to 12 days old) 50.6 +/- 3.8% of soleus motoneurones survived. This neuroprotective effect of MK-801 was dose dependant, since after treatment with lower doses (0.5 mg/kg; 1 mg/kg) fewer motoneurones survived (13.7% and 34.5%, respectively). To assess the effect of treatment with MK-801 on survival of alpha-motoneurones only, the number of soleus motor units was established physiologically. After nerve injury alone only 4.2 +/- 1.2 of the 29-30 soleus motor units were present, while in animals treated with MK-801 (2 mg/kg) 14 +/- 1.5 motor units were identified. The neuroprotective effect of MK-801 was not confined to soleus motoneurones but was also apparent on motoneurones to the extensor digitorum longus (EDL). In untreated EDL muscles of the 40 motor units only 5.5 +/- 1.7 motor units survived neonatal nerve injury and this number increased to 18 +/- 2.6 after treatment with MK-801. The neuroprotective effect of MK-801 was apparent regardless of whether the nerve lesion was carried out close to or far from the soleus muscle.


Assuntos
Maleato de Dizocilpina/farmacologia , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/inervação , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Comportamento Animal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenvolvimento Muscular , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Ratos , Ratos Wistar
8.
Neuroscience ; 54(2): 283-5, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-8336825

RESUMO

Motoneurons to soleus muscle die if their axons are injured at birth. We tested the possibility that their death may be caused by toxic effects of excitatory transmitters such as glutamate. Animals that had their sciatic nerves crushed at birth were treated either with a blocker of the N-methyl-D-aspartate receptor, dizocilpine maleate (MK-801), or saline. The number of surviving soleus motoneurons and motor units was assessed 60-90 days later. Treatment of rats with MK-801 rescued a large proportion of injured motoneurons destined to die. Moreover, the weight loss of soleus muscles seen after nerve injury at birth was reduced in animals treated with MK-801. These results suggest that motoneurons axotomized at birth are unable to withstand the excitotoxic effects of glutamate and die. Blocking glutamate receptors in conditions where motoneuron loss occurs could be an effective way of rescuing them.


Assuntos
Morte Celular/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Neurônios Motores/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Nervo Isquiático/fisiologia , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Neurônios Motores/citologia , Compressão Nervosa , Ratos , Nervo Isquiático/citologia , Nervo Isquiático/efeitos dos fármacos
9.
Exp Physiol ; 77(5): 765-8, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1418959

RESUMO

We investigated whether flare crossed the mid-line in various positions in humans using laser Doppler flowmetry to confirm visual observations. On the back and forehead, flare crossed the mid-line. However, on the posterior part of the neck, flare did not cross the mid-line, an observation that may be related to the elongated shape of flare in this region.


Assuntos
Pele/irrigação sanguínea , Pele/lesões , Vasodilatação/fisiologia , Adulto , Axônios/fisiologia , Feminino , Temperatura Alta/efeitos adversos , Humanos , Fluxometria por Laser-Doppler , Masculino , Pessoa de Meia-Idade , Fibras Nervosas/fisiologia , Nociceptores/fisiologia , Pele/patologia
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