RESUMEN
Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.
The spinal cord contains circuits of nerve cells that control how the body moves. Within these networks are interneurons that project to motor neurons, which innervate different types of muscle to contract: flexors (such as the biceps), which bend, or 'flex', the body's joints, and extensors (such as the triceps), which lead to joint extension. These motor signals must be carefully coordinated to allow precise and stable control of the body's movements. Previous studies suggest that where interneurons are placed in the spinal cord depends on whether they activate the motor neurons responsible for flexion or extension. To test if these findings were reproducible, Ronzano, Skarlatou, Barriga, Bannatyne, Bhumbra et al. studied interneurons which flex and extend the ankle joint in mice. In collaboration with several laboratories, the team used a combination of techniques to trace how interneurons and motor neurons were connected in the mouse spinal cord. This revealed that regardless of the method used or the laboratory in which the experiments were performed, the distribution of interneurons associated with flexion and extension overlapped one another. This finding contradicts previously published results and suggests that interneurons in the spinal cord are not segregated based on their outputs. Instead, they may be positioned based on the signals they receive, similar to motor neurons. Understanding where interneurons in the spinal cord are placed will provide new insights on how movement is controlled and how it is impacted by injuries and disease. In the future, this knowledge could benefit work on how neural circuits in the spinal cord are formed and how they can be regenerated.
Asunto(s)
Interneuronas , Músculos , Médula Espinal , Animales , Ratones , Interneuronas/fisiología , Neuronas Motoras/fisiología , Rabia , Médula Espinal/fisiologíaRESUMEN
The spinal cord can appropriately generate diverse movements, even without brain input and movement-related sensory feedback, using a combination of multifunctional and behaviorally specialized interneurons. The adult turtle spinal cord can generate motor patterns underlying forward swimming, three forms of scratching, and limb withdrawal (flexion reflex). We previously described turtle spinal interneurons activated during both scratching and swimming (multifunctional interneurons), interneurons activated during scratching but not swimming (scratch-specialized interneurons), and interneurons activated during flexion reflex but not scratching or swimming (flexion reflex-selective interneurons). How multifunctional and behaviorally specialized turtle spinal interneurons affect downstream neurons was unknown. Here, we recorded intracellularly from spinal interneurons activated during these motor patterns in turtles of both sexes in vivo and filled each with dyes. We labeled motoneurons using choline acetyltransferase antibodies or earlier intraperitoneal FluoroGold injection and used immunocytochemistry of interneuron axon terminals to identify their neurotransmitter(s) and putative synaptic contacts with motoneurons. We found that multifunctional interneurons are heterogeneous with respect to neurotransmitter, with some glutamatergic and others GABAergic or glycinergic, and can directly contact motoneurons. Also, scratch-specialized interneurons are heterogeneous with respect to neurotransmitter and some directly contact motoneurons. Thus, scratch-specialized interneurons might directly excite motoneurons that are more strongly activated during scratching than forward swimming, such as hip-flexor motoneurons. Finally, and surprisingly, we found that some motoneurons are behaviorally specialized, for scratching or flexion reflex. Thus, either some limb muscles are only used for a subset of limb behaviors or some limb motoneurons are only recruited during certain limb behaviors.SIGNIFICANCE STATEMENT Both multifunctional and behaviorally specialized spinal cord interneurons have been described in turtles, but their outputs are unknown. We studied responses of multifunctional interneurons (activated during swimming and scratching) and scratch-specialized interneurons, filled each with dyes, and used immunocytochemistry to determine their neurotransmitters and contacts with motoneurons. We found that both multifunctional and scratch-specialized interneurons are heterogeneous with respect to neurotransmitter, with some excitatory and others inhibitory. We found that some multifunctional and some scratch-specialized interneurons directly contact motoneurons. Scratch-specialized interneurons may excite motoneurons that are more strongly activated during scratching than swimming, such as hip-flexor motoneurons, or inhibit their antagonists, hip-extensor motoneurons. Surprisingly, we also found that some motoneurons are behaviorally specialized, for scratching or for flexion reflex.
Asunto(s)
Conducta Animal/fisiología , Interneuronas/fisiología , Actividad Motora/fisiología , Neuronas Motoras/fisiología , Médula Espinal/fisiología , Potenciales de Acción/fisiología , Animales , Femenino , Masculino , Reflejo/fisiología , Natación/fisiología , TortugasRESUMEN
Although Renshaw cells (RCs) were discovered over half a century ago, their precise role in recurrent inhibition and ability to modulate motoneuron excitability have yet to be established. Indirect measurements of recurrent inhibition have suggested only a weak modulatory effect but are limited by the lack of observed motoneuron responses to inputs from single RCs. Here we present dual recordings between connected RC-motoneuron pairs, performed on mouse spinal cord. Motoneuron responses demonstrated that Renshaw synapses elicit large inhibitory conductances and show short-term potentiation. Anatomical reconstruction, combined with a novel method of quantal analysis, showed that the strong inhibitory input from RCs results from the large number of synaptic contacts that they make onto individual motoneurons. We used the NEURON simulation environment to construct realistic electrotonic models, which showed that inhibitory conductances from Renshaw inputs exert considerable shunting effects in motoneurons and reduce the frequency of spikes generated by excitatory inputs. This was confirmed experimentally by showing that excitation of a single RC or selective activation of the recurrent inhibitory pathway to generate equivalent inhibitory conductances both suppress motoneuron firing. We conclude that recurrent inhibition is remarkably effective, in that a single action potential from one RC is sufficient to silence a motoneuron. Although our results may differ from previous indirect observations, they underline a need for a reevaluation of the role that RCs perform in one of the first neuronal circuits to be discovered.
Asunto(s)
Interneuronas/fisiología , Neuronas Motoras/fisiología , Inhibición Neural/fisiología , Potenciales de Acción/fisiología , Animales , Femenino , Interneuronas/citología , Masculino , Ratones , Modelos Neurológicos , Médula Espinal/citología , Médula Espinal/fisiologíaRESUMEN
The cerebellum receives information from the hindlimbs through several populations of spinocerebellar tract neurons. Although the role of these neurons has been established in electrophysiological experiments, the relative contribution of afferent fibres and central neurons to their excitatory input has only been estimated approximately so far. Taking advantage of differences in the immunohistochemistry of glutamatergic terminals of peripheral afferents and of central neurons (with vesicular glutamate transporters VGLUT1 or VGLUT2, respectively), we compared sources of excitatory input to four populations of spinocerebellar neurons in the thoraco-lumbar spinal cord: dorsal spinocerebellar tract neurons located in Clarke's column (ccDSCT) and in the dorsal horn (dhDSCT) and ventral spinocerebellar tract (VSCT) neurons including spinal border (SB) neurons. This was done on 22 electrophysiologically identified intracellularly labelled neurons in cats and on 80 neurons labelled by retrograde transport of cholera toxin b subunit injected into the cerebellum of rats. In both species distribution of antibodies against VGLUT1 and VGLUT2 on SB neurons (which have dominating inhibitory input from limb muscles), revealed very few VGLUT1 contacts and remarkably high numbers of VGLUT2 contacts. In VSCT neurons with excitatory afferent input, the number of VGLUT1 contacts was relatively high although VGLUT2 contacts likewise dominated, while the proportions of VGLUT1 and VGLUT2 immunoreactive terminals were the reverse on the two populations of DSCT neurons. These findings provide morphological evidence that SB neurons principally receive excitatory inputs from central neurons and provide the cerebellum with information regarding central neuronal activity.
Asunto(s)
Movimiento/fisiología , Neuronas/fisiología , Médula Espinal/fisiología , Tractos Espinocerebelares/fisiología , Animales , Gatos , Estimulación Eléctrica , Miembro Posterior/inervación , Miembro Posterior/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Tractos Espinocerebelares/citología , Proteína 1 de Transporte Vesicular de Glutamato/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/fisiologíaRESUMEN
The intermediate zone of the spinal grey matter contains premotor interneurons mediating reflex actions of group I and II muscle afferents. However, limited information is available on how activity of inhibitory versus excitatory interneurons in this population are modulated and how they contribute to motor networks. There were three aims of this study: (1) to characterize excitatory axonal contacts on interneurons; (2) to determine if contact patterns on excitatory and inhibitory interneurons are different; (3) to determine if there are differences in presynaptic inhibitory control of excitatory and inhibitory interneurons. We used intracellular labelling of electrophysiologically identified cells along with immunochemistry to characterise contacts formed by axons that contain vesicular glutamate transporters (VGLUT1 and VGLUT2) and contacts formed by VGLUT1 terminals which in turn were contacted by GABAergic terminals on cells that were characterised according to their transmitter phenotype. All 17 cells investigated were associated with numerous VGLUT1 contacts originating from primary afferents, and similar contact densities were found on excitatory and inhibitory cells, but VGLUT2-immunoreactive terminals originating from intraspinal neurons were less frequent, or were practically absent, especially on excitatory cells. Similar numbers of VGLUT1 contacts with associated GABAergic terminals were found on excitatory and inhibitory cells indicating a similar extent of presynaptic GABAergic control. However, scarce VGLUT2 terminals on intermediate zone excitatory premotor interneurons with input from muscle afferents suggest that they are not significantly excited by other spinal neurons but are under direct excitatory control of supraspinal neurons and, principally inhibitory, control of spinal neurons.
Asunto(s)
Interneuronas/fisiología , Músculo Esquelético/inervación , Neuronas Aferentes/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Animales , Gatos , Comunicación Celular/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Modelos Animales , Proteína 1 de Transporte Vesicular de Glutamato/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/fisiologíaRESUMEN
Dorsal horn interneurons with input from group II muscle spindle afferents are components of networks involved in motor control. Thirteen dorsal horn interneurons with monosynaptic group II input were characterized electrophysiologically and labeled intracellularly with Neurobiotin. Their axonal projections were traced, and neurotransmitter content was established by using immunocytochemistry. Two subpopulations were identified: five interneurons had axons that contained vesicular glutamate transporter 2 and hence were glutamatergic and excitatory. Terminals of the remaining eight interneurons were immunoreactive for the glycine transporter 2 or were apposed to gephyrin but did not contain the GABA-synthesizing enzyme glutamic acid decarboxylase and were therefore glycinergic and inhibitory. Excitatory cells were located mainly in the central region of lamina IV and had relatively small somata and restricted dendritic trees. In contrast, inhibitory interneurons were located more ventrally, in lamina V and had relatively larger somata and more extensive dendritic trees. Axonal projections of the two subpopulations differed considerably. Excitatory interneurons predominantly projected ipsilaterally, whereas most inhibitory interneurons projected both ipsilaterally and contralaterally. Three inhibitory axons formed contacts with large cholinergic cells in motor nuclei, thus revealing a novel direct coupling between inhibitory dorsal horn interneurons and motoneurons. The organization of the excitatory interneurons is consistent with current knowledge of reflex pathways to motoneurons, but the existence and connections of the inhibitory subpopulation could not be predicted from previous data. Our results indicate that these latter interneurons exercise widespread inhibitory control over a variety of cell types located on both sides of the spinal cord.
Asunto(s)
Ácido Glutámico/fisiología , Glicina/fisiología , Interneuronas/fisiología , Músculo Esquelético/inervación , Células del Asta Posterior/fisiología , Médula Espinal/anatomía & histología , Vías Aferentes/fisiología , Animales , Axones/química , Axones/ultraestructura , Biotina/análogos & derivados , Biotina/análisis , Proteínas Portadoras/análisis , Gatos , Colorantes Fluorescentes/análisis , Glutamato Descarboxilasa/análisis , Proteínas de Transporte de Glicina en la Membrana Plasmática/análisis , Interneuronas/química , Interneuronas/clasificación , Proteínas de la Membrana/análisis , Neuronas Motoras/fisiología , Terminaciones Nerviosas/química , Terminaciones Nerviosas/ultraestructura , Proteínas del Tejido Nervioso/análisis , Células del Asta Posterior/química , Médula Espinal/citología , Médula Espinal/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/análisisRESUMEN
Modulatory actions of a metabotropic 5-HT1A&7 membrane receptor agonist and antagonist [(+/-)-8-hydroxy-2-(di-n-propylamino)-tetralin; N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide] and an ionotropic 5-HT3 membrane receptor agonist and antagonist [2-methyl-serotonin (2-Me 5-HT); N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride] were investigated on dorsal horn interneurons mediating reflex actions of group II muscle afferents. All drugs were applied ionophoretically in deeply anesthetized cats. Effects of agonists were tested on extracellularly recorded responses of individual interneurons evoked by electrical stimulation of group II afferents in a muscle nerve. Effects of antagonists were tested against the depression of these responses after stimulation of raphe nuclei. The results show that both 5-HT1A&7 and 5-HT3 membrane receptors are involved in counteracting the activation of dorsal horn interneurons by group II afferents. Because only quantitative differences were found within the sample of the tested neurons, these results suggest that modulatory actions of 5-HT on excitatory and inhibitory interneurons might be similar. The relationship between 5-HT axons and axons immunoreactive for the 5-HT3A receptor subunit, which contact dorsal horn interneurons, was analyzed using immunofluorescence and confocal microscopy. Contacts from both types of axons were found on all interneurons, but their distribution and density varied, and there was no obvious relationship between them. In two of six interneurons, 5-HT3A-immunoreactive axons formed ring-like arrangements around the cell bodies. In previous studies, axons possessing 5-HT3 receptors were found to be excitatory, and as 2-Me 5-HT depressed transmission to dorsal horn interneurons, the results indicate that 5-HT operates at 5-HT3 receptors presynaptic to these neurons to depress excitatory transmission.
Asunto(s)
Interneuronas/fisiología , Células del Asta Posterior/fisiología , Receptores de Serotonina/fisiología , Serotonina/análogos & derivados , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Bicuculina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Gatos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Inmunohistoquímica , Interneuronas/efectos de los fármacos , Iontoforesis , Músculos/inervación , Oxazinas/farmacología , Piperazinas/farmacología , Células del Asta Posterior/efectos de los fármacos , Piridinas/farmacología , Núcleos del Rafe/fisiología , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Receptor de Serotonina 5-HT1A/fisiología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina 5-HT3/efectos de los fármacos , Receptores de Serotonina 5-HT3/fisiología , Serotonina/farmacologíaRESUMEN
Modulatory actions of monoamines were investigated on spinal commissural interneurons which coordinate left-right hindlimb muscle activity through direct projections to the contralateral motor nuclei. Commissural interneurons located in Rexed lamina VIII, with identified projections to the contralateral gastrocnemius-soleus motor nuclei, were investigated in deeply anaesthetized cats. Most interneurons had dominant input from either the reticular formation or from group II muscle afferents; a small proportion of neurons had input from both. Actions of ionophoretically applied serotonin and noradrenaline were examined on extracellularly recorded spikes evoked monosynaptically by group II muscle afferents or reticulospinal tract fibres. Activation by reticulospinal fibres was facilitated by both serotonin and noradrenaline. Activation by group II afferents was also facilitated by serotonin but was strongly depressed by noradrenaline. To investigate the possible morphological substrates of this differential modulation, seven representative commissural interneurons were labelled intracellularly with tetramethylrhodamine-dextran and neurobiotin. Contacts from noradrenergic and serotoninergic fibres were revealed by immunohistochemistry and analysed with confocal microscopy. There were no major differences in the numbers and distributions of contacts among the interneurons studied. The findings suggest that differences in modulatory actions of monoamines, and subsequent changes in the recruitment of subpopulations of commissural interneurons in various behavioural situations, depend on intrinsic interneuron properties rather than on the patterns of innervation by monoaminergic fibres. The different actions of noradrenaline on different populations of interneurons might permit reconfiguration of the actions of the commissural neurons according to behavioural context.
Asunto(s)
Potenciales de Acción/fisiología , Interneuronas/fisiología , Norepinefrina/fisiología , Serotonina/fisiología , Médula Espinal/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Monoaminas Biogénicas/farmacología , Monoaminas Biogénicas/fisiología , Gatos , Femenino , Interneuronas/efectos de los fármacos , Masculino , Norepinefrina/farmacología , Serotonina/farmacología , Médula Espinal/efectos de los fármacosRESUMEN
Axonal projections and neurotransmitters used by commissural interneurons mediating crossed actions of reticulospinal neurons were investigated in adult cats. Eighteen interneurons, located in or close to lamina VIII in midlumbar segments, that were monosynaptically excited by reticulospinal tract fibres and projected to contralateral motor nuclei were labelled by intracellular injection of tetramethylrhodamine-dextran and Neurobiotin. The nine most completely labelled interneurons were analysed with combined confocal and light microscopy. None of the stem axons gave off ipsilateral axon collaterals. Seven cells had axon collaterals that arborized in the contralateral grey matter in the ventral horn of the same segments. Transmitters were identified by using antibodies raised against vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and the glycine transporter 2. The axons of two cells were immunoreactive for the glycine transporter 2 and hence were glycinergic. Three cells were immunoreactive for the vesicular glutamate transporter 2 and hence were glutamatergic. None of the axons displayed immunoreactivity for glutamic acid decarboxylase. Electron microscopy of two cells revealed direct synaptic connections with motoneurons and other neurons. Axonal swellings of one neuron formed synapses with profiles in motor nuclei whereas those of the other formed synapses with other structures, including cell bodies in lamina VII. The results show that this population of commissural interneurons includes both excitatory and inhibitory cells that may excite or inhibit contralateral motoneurons directly. They may also influence the activity of motoneurons indirectly by acting through interneurons located outside motor nuclei in the contralateral grey matter but are unlikely to have direct actions on interneurons in the ipsilateral grey matter.
Asunto(s)
Biotina/análogos & derivados , Interneuronas/fisiología , Proteínas de Transporte de Membrana , Red Nerviosa/fisiología , Inhibición Neural/fisiología , Formación Reticular/fisiología , Médula Espinal/fisiología , Proteínas de Transporte Vesicular , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animales , Axones/fisiología , Axones/ultraestructura , Biotina/metabolismo , Proteínas Portadoras/metabolismo , Gatos , Estimulación Eléctrica , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Lateralidad Funcional , Glutamato Descarboxilasa/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática , Inmunohistoquímica , Interneuronas/ultraestructura , Microscopía Confocal , Microscopía Electrónica , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Red Nerviosa/anatomía & histología , Tiempo de Reacción , Formación Reticular/citología , Rodaminas/metabolismo , Médula Espinal/citología , Sinapsis/ultraestructura , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de GlutamatoRESUMEN
The purpose of the present study was to characterize the transmitter content of structures in presynaptic apposition to the central terminals of cutaneous afferent fibers in the dorsal horn of the spinal cord. Axons in the Aalphabeta conduction velocity range were identified in adult cats, stained intra-axonally with horseradish peroxidase, and prepared for combined light and electron microscopy. In total, we labeled two slowly adapting (Type 1) axons, two hair-follicle afferents, and one rapidly adapting (Krause) afferent. Ninety-nine labeled boutons were examined through complete series of serial sections. Approximately 80% of boutons originating from rapidly adapting and hair-follicle afferents were postsynaptic to other axons, but only 50% of boutons from slowly adapting axons were associated with this type of arrangement. Postembedding immunogold reactions revealed that between 80% (for slowly adapting axons) and 100% (for rapidly adapting axons) of boutons presynaptic to primary afferents were immunoreactive for gamma-aminobutyric acid (GABA). The vast majority of these terminals (in excess of 80%) were also enriched with glycine. Therefore, presynaptic inhibition of these three functional classes of Aalphabeta cutaneous primary afferents is mediated principally by the subgroup of GABAergic interneuron that also contains glycine.