RESUMEN
After spinal cord transection (SCT) the interaction between motoneurons (MNs) and muscle is impaired, due to reorganization of the spinal network after a loss of supraspinal inputs. Rats subjected to SCT, treated with intraspinal injection of a AAV-BDNF (brain-derived neurotrophic factor) construct, partially regained the ability to walk. The central effects of this treatment have been identified, but its impact at the neuromuscular junction (NMJ) has not been characterized. Here, we compared the ability of NMJ pre- and postsynaptic machinery in the ankle extensor (Sol) and flexor (TA) muscles to respond to intraspinal AAV-BDNF after SCT. The gene expression of cholinergic molecules (VAChT, ChAT, AChE, nAChR, mAChR) was investigated in tracer-identified, microdissected MN perikarya, and in muscle fibers with the use of qPCR. In the NMJs, a distribution of VAChT, nAChR and Schwann cells was studied by immunofluorescence, and of synaptic vesicles and membrane active zones by electron microscopy. We showed partial protection of the Sol NMJs from disintegration, and upregulation of the VAChT and AChE transcripts in the Sol, but not the TA MNs after spinal enrichment with BDNF. We propose that the observed discrepancy in response to BDNF treatment is an effect of difference in the TrkB expression setting BDNF responsiveness, and of BDNF demands in Sol and TA muscles.
RESUMEN
Spasticity impacts the quality of life of patients suffering spinal cord injury and impedes the recovery of locomotion. At the cellular level, spasticity is considered to be primarily caused by the hyperexcitability of spinal α-motoneurons (MNs) within the spinal stretch reflex circuit. Here, we hypothesized that after a complete spinal cord transection in rats, fast adaptive molecular responses of lumbar MNs develop in return for the loss of inputs. We assumed that early loss of glutamatergic afferents changes the expression of glutamatergic AMPA and NMDA receptor subunits, which may be the forerunners of the developing spasticity of hindlimb muscles. To better understand its molecular underpinnings, concomitant expression of GABA and Glycinergic receptors and serotoninergic and noradrenergic receptors, which regulate the persistent inward currents crucial for sustained discharges in MNs, were examined together with voltage-gated ion channels and cation-chloride cotransporters. Using quantitative real-time PCR, we showed in the tracer-identified MNs innervating extensor and flexor muscles of the ankle joint multiple increases in transcripts coding for AMPAR and 5-HTR subunits, along with a profound decrease in GABAAR, GlyR subunits, and KCC2. Our study demonstrated that both MNs groups similarly adapt to a more excitable state, which may increase the occurrence of extensor and flexor muscle spasms.
Asunto(s)
Traumatismos de la Médula Espinal , Simportadores , Animales , Cloruros/metabolismo , Neuronas Motoras/metabolismo , Espasticidad Muscular/metabolismo , Fenotipo , Calidad de Vida , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Simportadores/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2-5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.
Asunto(s)
Ácido Hialurónico , Terminales Presinápticos , Animales , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Matriz Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Masculino , Neuronas Motoras/metabolismo , Lectinas de Plantas , Terminales Presinápticos/metabolismo , Ratas , Receptores N-Acetilglucosamina/metabolismoRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are important cellular mechanisms underlying learning and memory processes. N-Methyl-D-aspartate receptor (NMDAR)-dependent LTP and LTD play especially crucial roles in these functions, and their expression depends on changes in the number and single channel conductance of the major ionotropic glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) located on the postsynaptic membrane. Structural changes in dendritic spines comprise the morphological platform and support for molecular changes in the execution of synaptic plasticity and memory storage. At the molecular level, spine morphology is directly determined by actin cytoskeleton organization within the spine and indirectly stabilized and consolidated by scaffold proteins at the spine head. Palmitoylation, as a uniquely reversible lipid modification with the ability to regulate protein membrane localization and trafficking, plays significant roles in the structural and functional regulation of LTP and LTD. Altered structural plasticity of dendritic spines is also considered a hallmark of neurodevelopmental disorders, while genetic evidence strongly links abnormal brain function to impaired palmitoylation. Numerous studies have indicated that palmitoylation contributes to morphological spine modifications. In this review, we have gathered data showing that the regulatory proteins that modulate the actin network and scaffold proteins related to AMPAR-mediated neurotransmission also undergo palmitoylation and play roles in modifying spine architecture during structural plasticity.
Asunto(s)
Lipoilación , Plasticidad Neuronal/fisiología , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Modelos Neurológicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury (ONI) or laser-induced ocular hypertension (OHT). In models of glaucoma, BDNF therapy can delay or halt RGCs loss, but this protection is time-limited. The decreased efficacy of BDNF supplementation has been in part attributed to BDNF TrkB receptor downregulation. However, whether BDNF overexpression causes TrkB downregulation, impairing long-term BDNF signaling in the retina, has not been conclusively proven. After ONI or OHT, when increased retinal BDNF was detected, a concomitant increase, no change or a decrease in TrkB was reported. We examined quantitatively the retinal concentrations of the TrkB protein in relation to BDNF, in a course of adeno-associated viral vector gene therapy (AAV2-BDNF), using a microbead trabecular occlusion model of glaucoma. We show that unilateral glaucoma, with intraocular pressure ( IOP) increased for five weeks, leads to a bilateral decrease of BDNF in the retina at six weeks, accompanied by up to four-fold TrkB upregulation, while a moderate BDNF overexpression in a glaucomatous eye triggers changes that restore normal TrkB concentrations, driving signaling towards long-term RGCs neuroprotection. We conclude that for glaucoma therapy, the careful selection of the appropriate BDNF concentration is the main factor securing the long-term responsiveness of RGCs and the maintenance of normal TrkB levels.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Vectores Genéticos/administración & dosificación , Glaucoma/terapia , Receptor trkB/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glaucoma/genética , Glaucoma/metabolismo , Humanos , Inyecciones Intravítreas , Masculino , RatasRESUMEN
L1 cell adhesion molecule (L1CAM) supports spinal cord cellular milieu after contusion and compression lesions, contributing to neuroprotection, promoting axonal outgrowth, and reducing outgrowth-inhibitory molecules in lesion proximity. We extended investigations into L1CAM molecular targets and explored long-distance effects of L1CAM rostral and caudal to complete spinal cord transection (SCT) in adult rats. L1CAM overexpression in neurons and glia after Th10/Th11 SCT was achieved using adeno-associated viral vector serotype 5 (AAV5) injected into an L1-lumbar segment immediately after transection. At 5 weeks, a L1CAM mRNA profound decrease detected rostral and caudal to the transection site was alleviated by AAV5-L1CAM treatment, with increased endogenous L1CAM rostral to the SCT. Transected corticospinal tract fibers showed attenuated retraction after treatment, accompanied by a multi-segmental increase of lesion-reduced expression of adenylate cyclase 1 (Adcy1), synaptophysin, growth-associated protein 43, and myelin basic protein genes caudal to transection, and Adcy1 rostral to transection. In parallel, chondroitin sulfate proteoglycan phosphacan elevated after SCT was downregulated after treatment. Low-molecular L1CAM isoforms generated after spinalization indicated the involvement of sheddases in L1CAM processing and long-distance effects. A disintegrin and metalloproteinase (ADAM)10 sheddase immunoreactivity, stronger in AAV5-L1CAM than AAV5- enhanced green fluorescent protein (EGFP)-transduced motoneurons indicated local ADAM10 upregulation by L1CAM. The results suggest that increased L1CAM availability and penetration of diffusible L1CAM fragments post-lesion induce both local and long-distance neuronal and glial responses toward better neuronal maintenance, neurite growth, and myelination. Despite the fact that intervention promoted beneficial molecular changes, kinematic analysis of hindlimb movements showed minor improvement, indicating that spinalized rats require longer L1CAM treatment to regain locomotor functions.
Asunto(s)
Regulación hacia Abajo/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Plasticidad Neuronal/fisiología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Regulación hacia Arriba/fisiología , Animales , Expresión Génica , Masculino , Molécula L1 de Adhesión de Célula Nerviosa/genética , Ratas , Ratas Wistar , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Traumatismos de la Médula Espinal/genética , Vértebras Torácicas/lesionesRESUMEN
Alpha-motoneurons (MNs) innervating ankle extensor muscles show reduced peripheral inputs from Ia proprioceptive afferents and cholinergic afferents after chronic spinalization (SCT). That phenomenon is not observed on ankle flexor MNs, indicating a smaller vulnerability of the latter MNs circuit to SCT. Locomotor training of spinal rats which partially restored those inputs to extensor MNs tended to hyper innervate flexor MNs, disclosing a need for selective approaches. In rats with intact spinal cord 7-days of low-threshold proprioceptive stimulation of the tibial nerve enriched glutamatergic Ia and cholinergic innervation of lateral gastrocnemius (LG) MNs, suggesting usefulness of selective stimulation for restoration of inputs to extensor MNs after SCT. Accordingly, to examine its effectiveness after SCT, tibial nerves and soleus muscles were implanted bilaterally, and for MN identification fluorescence tracers to LG and tibialis anterior (TA) muscles were injected two weeks prior to spinalization. Stimulation of tibial nerve, controlled by H-reflex recorded in the soleus muscle, started on the third post-SCT day and continued for 7 days. Nine days post-SCT the number and volume of glutamatergic Ia and of cholinergic C-boutons on LG MNs was decreased, but stimulation affected neither of them. Postsynaptically, a threefold decrease of NMDAR NR1 subunit and thirtyfold decrease of M2 muscarinic receptor transcripts caused by SCT were not counteracted by stimulation, whereas a threefold decrease of AMPAR GluR2 subunit tended to deepen after stimulation. We conclude that LG MNs, supported with proprioceptive stimuli after SCT, do not transcribe the perceived cues into compensatory response at the transcriptional level in the early post-SCT period.
Asunto(s)
Tobillo/fisiopatología , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Traumatismos de la Médula Espinal/fisiopatología , Nervio Tibial/fisiopatología , Animales , Modelos Animales de Enfermedad , Estimulación Eléctrica/instrumentación , Electrodos Implantados , Reflejo H/fisiología , Humanos , Masculino , Músculo Esquelético/fisiopatología , Terminales Presinápticos/fisiología , Propiocepción/fisiología , Ratas , Receptor Muscarínico M2/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/fisiología , Médula Espinal/cirugíaRESUMEN
Complete thoracic spinal cord transection (SCT) impairs excitatory cholinergic inputs to ankle extensor (soleus; Sol) but not to flexor (tibialis anterior; TA) α-motoneurons (MNs) modifiable by locomotor training applied post-transection. The purpose of this study was to investigate whether Sol and TA MNs adapt to changes in cholinergic environment by differential regulation of their muscarinic receptors M2 (M2R). We examined Chrm2 (M2R gene) transcript level, high-affinity 3-quinuclidinyl benzilate-3 H ([3 H]QNB) ligand binding, distribution and density of M2R immunolabeling in lumbar (L) segments in intact and SCT rats, with or without inclusion of 5-week treadmill locomotor training. We show that at the second week after SCT the levels of Chrm2 transcript are reduced in the L3-6 segments, with [3 H]QNB binding decreased selectively in the L5-6 segments, where ankle extensor MNs are predominantly located. At 5 weeks after SCT, [3 H]QNB binding differences between the L3-4 and L5-6 segments are maintained, accompanied by higher density of M2R immunolabeling in the plasma membrane and cytoplasm of TA than Sol MNs and by enriched synaptic versus extrasynaptic M2R pools (52% TA vs. 25% Sol MNs). Training normalized M2R in TA MNs, improved locomotion, and reduced frequency of clonic episodes. Our findings indicate higher sensitivity of TA than Sol MNs to cholinergic signaling after SCT, which might shorten flexor twitches duration and contribute to generation of clonic movements. Synaptic enrichment in M2R density may reflect a compensatory mechanism activated in TA and Sol MNs to different extent in response to reduced strength of cholinergic signaling to each MN pool. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.
Asunto(s)
Locomoción , Neuronas Motoras/metabolismo , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/métodos , Receptor Muscarínico M2/biosíntesis , Receptor Muscarínico M2/genética , Traumatismos de la Médula Espinal/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citoplasma/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Miembro Posterior/inervación , Masculino , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/rehabilitaciónRESUMEN
Neurotrophins (NT) were "brought to life" by Rita Levi-Montalcini and Stanley Cohen, thanks to their discovery of the nerve growth factor (NGF) isolated from the malignant tumor. A hint got from the cancer studies on the presence of NTs and other molecules like metalloproteases, which control neuronal remodeling and extracellular matrix in tumors, was the stimulus to investigate the role of these molecules in physiology and post-lesion plasticity of the nervous system. NTs have long been identified as drivers of neurogenesis during development and regeneration of the nervous system. Thousands of investigations spanned from early sixties till now provided strong evidence that this small family of molecules is indispensable for central and peripheral nervous system maintenance throughout the whole life of different animal species. In mammals NTs regulate sensation, movement, behavior, and cognition. A problem, which accompanies the majority of experimental therapies using NTs following injury of the nervous system, stems from unspecific and uncontrolled stimulation of the whole circuitry of preserved neurons. Among the already identified causes of clinical trial failure with the use of NT therapy in the treatment of neurodegenerative diseases are: (1) disregarding neuronal preferences to selected NTs; (2) poor knowledge on NT pharmacokinetics (3) uncontrolled spread of NTs when administered systemically, which could have unpredictable effects on other than intended neuronal populations (4) limitations of tissue penetration of some NTs (5) disturbances of the balance between signal transmission through their Trk and LNGTR receptors. I present our contribution to the field and efforts to control spatially and temporally postinjury NT signaling.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/uso terapéutico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Animales , Factores de Crecimiento Nervioso/farmacología , Enfermedades Neurodegenerativas/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Transducción de Señal/efectos de los fármacosRESUMEN
The effects of stimulation of low-threshold proprioceptive afferents in the tibial nerve on two types of excitatory inputs to α-motoneurons were tested. The first input is formed by glutamatergic Ia sensory afferents contacting monosynaptically α-motoneurons. The second one is the cholinergic input originating from V0c-interneurons, located in lamina X of the spinal cord, modulating activity of α-motoneurons via C-terminals. Our aim was to clarify whether enhancement of signaling to ankle extensor α-motoneurons, via direct electrical stimulation addressed predominantly to low-threshold proprioceptive fibers in the tibial nerve of awake rats, will affect Ia glutamatergic and cholinergic innervation of α-motoneurons of lateral gastrocnemius (LG). LG motoneurons were identified with True Blue tracer injected intramuscularly. Tibial nerve was stimulated for 7 days with continuous bursts of three pulses applied in four 20 min sessions daily. The Hoffmann reflex and motor responses recorded from the soleus muscle, LG synergist, allowed controlling stimulation. Ia terminals and C-terminals abutting on LG-labeled α-motoneurons were detected by immunofluorescence (IF) using input-specific anti- VGLUT1 and anti-VAChT antibodies, respectively. Quantitative analysis of confocal images revealed that the number of VGLUT1 IF and VAChT IF terminals contacting the soma of LG α-motoneurons increased after stimulation by 35% and by 26%, respectively, comparing to the sham-stimulated side. The aggregate volume of VGLUT1 IF and VAChT IF terminals increased by 35% and by 30%, respectively. Labeling intensity of boutons was also increased, suggesting an increase of signaling to LG α-motoneurons after stimulation. To conclude, one week of continuous burst stimulation of proprioceptive input to LG α-motoneurons is effective in enrichment of their direct glutamatergic but also indirect cholinergic inputs. The effectiveness of such and longer stimulation in models of injury is a prerequisite to propose it as a therapeutic method to improve inputs to selected group of α-motoneurons after damage.
Asunto(s)
Tobillo/inervación , Fibras Colinérgicas/fisiología , Estimulación Eléctrica , Glutamatos/metabolismo , Neuronas Motoras/fisiología , Unión Neuromuscular/fisiología , Propiocepción , Animales , Transporte Biológico , Interneuronas/fisiología , Masculino , Plasticidad Neuronal/fisiología , Ratas , Umbral Sensorial , Médula Espinal/fisiología , Nervio Tibial/patología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Glaucoma is a chronic optic neuropathy characterized by progressive damage to the optic nerve, death of retinal ganglion cells and ultimately visual field loss. It is one of the leading causes of irreversible loss of vision worldwide. The most important trigger of glaucomatous damage is elevated eye pressure, and the current standard approach in glaucoma therapy is reduction of intraocular pressure (IOP). However, despite the use of effective medications or surgical treatment leading to lowering of IOP, progression of glaucomatous changes and loss of vision among patients with glaucoma is common. Therefore, it is critical to prevent vision loss through additional treatment. To implement such treatment(s), it is imperative to identify pathophysiological changes in glaucoma and develop therapeutic methods taking into account neuroprotection. Currently, there is no method of neuroprotection with long-term proven effectiveness in the treatment of glaucoma. Among the most promising molecules shown to protect the retina and optic nerve are neurotrophic factors. Thus, the current focus is on the development of safe and non-invasive methods for the long-term elevation of the intraocular level of neurotrophins through advanced gene therapy and topical eye treatment and on the search for selective agonists of neurotrophin receptors affording more efficient neuroprotection.
Asunto(s)
Glaucoma/terapia , Animales , Glaucoma/fisiopatología , HumanosRESUMEN
Beneficial effects of locomotor training on the functional recovery after complete transection of the spinal cord indicate that in chronic spinal animals spontaneous recovery processes are enhanced and shaped by the training. The mechanisms of that use-dependent improvement are still not fully understood. This review tackles three aspects of this issue: (1) neurochemical attributes of functional improvement showing that concentrations of excitatory and inhibitory amino acids in the lumbar spinal segments, which were changed after transection, normalize after the training, or even raise beyond normal. As it does not translate to functional equilibrium between excitatory and inhibitory neurotransmission and may lead to hyperexcitability, the postsynaptic mechanisms which might be responsible for the hyperexcitability are discussed, including (i) dysfunction of K(+)-Cl(-) cotransporter KCC2, which controls the strength and robustness of inhibition, and (ii) altered function of 5-HT2 receptors, which may be targeted to restore KCC2 activity and intrinsic inhibition; (2) morphological changes of lumbar motoneurons and their inputs related to functional improvement of spinal animals, pointing to use-dependent diminution/reversal of the atrophy of the dendritic tree of the hindlimb motoneurons and of their synaptic impoverishment, which in paraplegic animals differs depending on the degree of disuse of the muscles; (3) the role of neurotrophins in motor improvement of spinal animals showing, that increases in neurotrophins due to training or due to efficient viral vector-based transgene expression, that might be responsible for the enrichment of the dendritic tree, elongation of processes and influence neurotransmitter systems in the areas subjected to plastic modifications after injury, correlate with improvement of locomotor functions.
Asunto(s)
Terapia por Ejercicio/métodos , Factores de Crecimiento Nervioso/uso terapéutico , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/rehabilitación , Animales , Humanos , Factores de Crecimiento Nervioso/metabolismo , Recuperación de la Función/fisiologíaRESUMEN
Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Vértebras Lumbares/fisiopatología , Actividad Motora , Recuperación de la Función , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Animales , Dependovirus/metabolismo , Vectores Genéticos/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Glicina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Vértebras Lumbares/enzimología , Vértebras Lumbares/patología , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rango del Movimiento Articular , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/metabolismo , Simportadores/metabolismo , Vértebras Torácicas/patología , Vértebras Torácicas/fisiopatología , Transducción Genética , Proteínas de Transporte Vesicular de Glutamato/genética , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
The importance of neurotrophin 3 (NT-3) for motor control prompted us to ask the question whether direct electrical stimulation of low-threshold muscle afferents, strengthening the proprioceptive signaling, could effectively increase the endogenous pool of this neurotrophin and its receptor TrkC in the Hoffmann-reflex (H-reflex) circuitry. The effects were compared with those of brain-derived neurotrophic factor (BDNF) and its TrkB receptor. Continuous bursts of stimuli were delivered unilaterally for seven days, 80 min daily, by means of a cuff-electrode implanted over the tibial nerve in awake rats. The H-reflex was recorded in the soleus muscle to control the strength of stimulation. Stimulation aimed at activation of Ia fibers produced a strong increase of NT-3 protein, measured with ELISA, in the lumbar L3-6 segments of the spinal cord and in the soleus muscle. This stimulation exerted much weaker effect on BDNF protein level which slightly increased only in L3-6 segments of the spinal cord. Increased protein level of NT-3 and BDNF corresponded to the changes of NT-3 mRNA and BDNF mRNA expression in L3-6 segments but not in the soleus muscle. We disclosed tissue-specificity of TrkC mRNA and TrkB mRNA responses. In the spinal cord TrkC and TrkB transcripts tended to decrease, whereas in the soleus muscle TrkB mRNA decreased and TrkC mRNA expression strongly increased, suggesting that stimulation of Ia fibers leads to sensitization of the soleus muscle to NT-3 signaling. The possibility of increasing NT-3/TrkC signaling in the neuromuscular system, with minor effects on BDNF/TrkB signaling, by means of low-threshold electrical stimulation of peripheral nerves, which in humans might be applied in non-invasive way, offers an attractive therapeutic tool.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Reflejo H/fisiología , Neuronas Motoras/metabolismo , Neurotrofina 3/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Electrofisiología , Reflejo H/genética , Masculino , Neurotrofina 3/genética , Ratas , Ratas Wistar , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cholinergic input modulates excitability of motoneurons and plays an important role in the control of locomotion in both intact and spinalized animals. However, spinal cord transection in adult rats affects cholinergic innervation of only some hindlimb motoneurons, suggesting that specificity of this response is related to functional differences between motoneurons. Our aim was therefore to compare cholinergic input to motoneurons innervating the soleus (Sol) and tibialis anterior (TA) motoneurons following spinal cord transection at a low-thoracic level. The second aim was to investigate whether deficits in cholinergic input to these motoneurons could be modified by locomotor training. The Sol and TA motoneurons were identified by retrograde labelling with fluorescent dyes injected intramuscularly. Cholinergic terminals were detected using anti-vesicular acetylcholine transporter (VAChT) antibody. Overall innervation of motoneurons was evaluated with anti-synaptophysin antibody. After spinalization we found a decrease in the number of VAChT-positive boutons apposing perikarya of the Sol (to 49%) but not TA motoneurons. Locomotor training, resulting in moderate functional improvement, partly reduced the deficit in cholinergic innervation of Sol motoneurons by increasing the number of VAChT-positive boutons. However, the optical density of VAChT-positive boutons terminating on various motoneurons, which decreased after spinalization, continued to decrease despite the training, suggesting an impairment of acetylcholine availability in the terminals. Different effects of spinal cord transection on cholinergic innervation of motoneurons controlling the ankle extensor and flexor muscles point to different functional states of these muscles in paraplegia as a possible source of activity-dependent signaling regulating cholinergic input to the motoneurons.
Asunto(s)
Neuronas Colinérgicas/fisiología , Locomoción/fisiología , Neuronas Motoras/fisiología , Médula Espinal/fisiología , Acetilcolina/fisiología , Animales , Masculino , Músculo Esquelético/inervación , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Médula Espinal/cirugía , Tarso Animal/inervación , Proteínas de Transporte Vesicular de Acetilcolina/fisiologíaRESUMEN
BACKGROUND: It has been postulated that exercise-induced activation of brain-derived neurotrophic factor (BDNF) may account for improvement of stepping ability in animals after complete spinal cord transection. As we have shown previously, treadmill locomotor exercise leads to up-regulation of BDNF protein and mRNA in the entire neuronal network of intact spinal cord. The questions arise: (i) how the treadmill locomotor training, supplemented with tail stimulation, affects the expression of molecular correlates of synaptic plasticity in spinal rats, and (ii) if a response is related to BDNF protein level and distribution. We investigated the effect of training in rats spinalized at low thoracic segments on the level and distribution of BDNF immunoreactivity (IR) in ventral quadrants of the lumbar segments, in conjunction with markers of presynaptic terminals, synaptophysin and synaptic zinc. RESULTS: Training improved hindlimb stepping in spinal animals evaluated with modified Basso-Beattie-Bresnahan scale. Grades of spinal trained animals ranged between 5 and 11, whereas those of spinal were between 2 and 4. Functional improvement was associated with changes in presynaptic markers and BDNF distribution. Six weeks after transection, synaptophysin IR was reduced by 18% around the large neurons of lamina IX and training elevated its expression by over 30%. The level of synaptic zinc staining in the ventral horn was unaltered, whereas in ventral funiculi it was decreased by 26% postlesion and tended to normalize after the training. Overall BDNF IR levels in the ventral horn, which were higher by 22% postlesion, were unchanged after the training. However, training modified distribution of BDNF in the processes with its predominance in the longer and thicker ones. It also caused selective up-regulation of BDNF in two classes of cells (soma ranging between 100-400 microm2 and over 1000 microm2) of the ventrolateral and laterodorsal motor nuclei. CONCLUSION: Our results show that it is not BDNF deficit that determines lack of functional improvement in spinal animals. They indicate selectivity of up-regulation of BDNF in distinct subpopulations of cells in the motor nuclei which leads to changes of innervation targeting motoneurons, tuned up by locomotor activity as indicated by a region-specific increase of presynaptic markers.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Terapia por Ejercicio , Condicionamiento Físico Animal/métodos , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/rehabilitación , Médula Espinal/metabolismo , Sinapsis/metabolismo , Animales , Biomarcadores/metabolismo , Prueba de Esfuerzo , Técnica del Anticuerpo Fluorescente/métodos , Región Lumbosacra , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Movimiento , Terminales Presinápticos/metabolismo , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/patología , Sinaptofisina/metabolismo , Distribución Tisular , Zinc/metabolismoRESUMEN
Metabotropic glutamate (mGlu) receptors, which are present on neurons and glial cells, have been shown to play a role in neuropathic pain. The present study sought to investigate how the glial inhibitors minocycline and pentoxifylline alter the effect that chronic constriction injury (CCI) has on the expression of mGlu receptors and on their associated ligands. RT-PCR analysis revealed that seven days after CCI, the mRNA levels of glial markers C1q and GFAP, as well as those of mGlu5 and mGlu3, but not mGlu7, were elevated in the lumbar spinal cord - ipsilateral to the injury. The protein levels of the microglial marker OX42, the astroglial marker GFAP, and mGlu5 receptor protein were increased, whereas the levels of mGlu2/3 and mGlu7 receptor proteins were reduced. Preemptive and repeated intraperitoneal (i.p.) administration (16 and 1h before nerve injury and then twice daily for seven days) of minocycline (30mg/kg) and pentoxifylline (20mg/kg) prevented the injury-induced changes in the levels of mGlu3 and mGlu5 receptor mRNAs and the injury-induced changes in the protein levels of all the receptors. Repeated administration of minocycline and pentoxifylline significantly attenuated CCI-induced allodynia (von Frey test) and hyperalgesia (cold plate test) measured on day seven after injury and potentiated the antiallodynic and antihyperalgesic effects of single i.p. and intrathecal (i.t.) injections of mGlu receptor ligands: MPEP, LY379268 or AMN082. We conclude that attenuation of injury-induced glial activation can reduce glutamatergic activity, thereby contributing to regulation of pain sensation.
Asunto(s)
Complemento C1q/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Ciática/tratamiento farmacológico , Ciática/metabolismo , Aminoácidos/farmacología , Análisis de Varianza , Animales , Compuestos de Bencidrilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Complemento C1q/antagonistas & inhibidores , Complemento C1q/genética , Modelos Animales de Enfermedad , Esquema de Medicación , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/antagonistas & inhibidores , Proteína Ácida Fibrilar de la Glía/genética , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Masculino , Ratones , Minociclina/farmacología , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Pentoxifilina/farmacología , Piridinas/farmacología , ARN Mensajero/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Ciática/complicaciones , Ciática/etiología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
The article summarizes the most meaningful studies which have provided evidence that protein synthesis in neurons can occur not only in cell perikarya but also locally in dendrites. The presence of the complete machinery required to synthesize cytoplasmic and integral membrane proteins in dendrites, identification of binding proteins known to mediate mRNA trafficking in dendrites and the ability to trigger "on-site" translation make it possible for the synthesis of particular proteins to be regulated by synaptic signals. Until now over 100 different mRNAs coding the proteins involved in neurotransmission and modulation of synaptic activity have been identified in dendrites. Local protein synthesis is postulated to provide the basic mechanism of fast changes in the strength of neuronal connections and to be an important factor in the molecular background of synaptic plasticity, giving rise to enduring changes in synaptic function, which in turn play a role in local homeostatic responses. Local protein synthesis points to some autonomy of dendrites which makes them "the brains of the neurons" (Jim Eberwine; from the interview with J. Eberwine - Barinaga 2000).
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Dendritas/fisiología , Neuronas/citología , Biosíntesis de Proteínas/fisiología , Animales , Modelos BiológicosRESUMEN
Brain infarct triggers neurodegeneration that often shades spontaneous plasticity, occurring in the areas related anatomically and functionally to the infarcted structures. Neurotrophins which promote neuronal survival and plasticity, may protect neurons and enhance remodeling of the remaining circuits, leading to restoration of function. In particular, the crucial role of brain-derived neurotrophic factor (BDNF) in cortical function is well documented. Since BDNF was implicated in the mechanism of postinfarct recovery, we investigated whether focal photothrombosis in the motor cortex of adult rats modifies cortical BDNF protein levels in a time- and region-dependent fashion. In parallel, we aimed to establish, which cortical cells respond with altered BDNF expression and whether these alterations are reflected by forelimb motor skill impairment and recovery, evaluated up to 1 month postinfarct. The distribution of BDNF protein was visualized immunohistochemically and BDNF tissue levels were evaluated with enzyme-linked immunosorbent assay (ELISA). Ipsilateral to the infarct, an increase in BDNF levels occurred both in injured and neighboring regions already 24 h after photothrombosis. This increase was sustained up to postlesion day 7 in the motor cortex and reduced at 28 days. No BDNF changes were detected in homotopic regions of the contralateral cortex. The time-course of enhanced neurotrophic expression was paralleled by bilateral deficits in skilled reaching, which was the only clear and measurable motor impairment observed in the study. We conclude that the spontaneous increase of BDNF is not sufficient to protect neurons from degeneration in the lesion proximity whereas plasticity reported in the adjacent regions may be attributable to enhanced BDNF-related stimuli, which do not counteract the impairment of skilled reaching but might be, at least in part, responsible for the absence of deficits in other functional/behavioral tests.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Infarto Cerebral/metabolismo , Actividad Motora/fisiología , Corteza Motora/metabolismo , Recuperación de la Función/fisiología , Animales , Infarto Cerebral/patología , Infarto Cerebral/fisiopatología , Modelos Animales de Enfermedad , Miembro Anterior , Masculino , Neuroglía/fisiología , Neuronas/fisiología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Previous evidence indicates that locomotor exercise is a powerful means of increasing brain-derived neurotrophic factor (BDNF) and its signal transduction receptor TrkB mRNA levels, immunolabeling intensity and number of BDNF- and TrkB-immunopositive cells in the spinal cord of adult rats but the contribution of specific cell types to changes resulting from long-term activity is unknown. As changes in BDNF protein distribution due to systemic stimuli may reflect either its in-situ synthesis or its translocation from other sources, we investigated where BDNF and TrkB mRNA are expressed in the spinal lumbar segments. We report on the cell types defined by size, BDNF mRNA levels and number of cells with TrkB transcripts in sedentary and exercised animals following 28 days of treadmill walking. In the majority of cells, exercise increased perikaryonal levels of BDNF mRNA but did not affect TrkB transcript levels. Bidirectional changes in a number of TrkB mRNA-expressing cells occurred in small groups of ventral horn neurons. An increase in BDNF transcripts was translated into changes in pro-BDNF and BDNF levels. A 7-day walking regimen increased BDNF protein levels similarly to 28-day treadmill walking. Our observations indicate that long- and short-term locomotor activity of moderate intensity produce stimuli sufficient to recruit a majority of spinal cells to increased BDNF synthesis, suggesting that continuous tuning of pro-BDNF and BDNF levels permits spinal networks to undergo trophic modulation not requiring changes in TrkB mRNA supply.