Decoding the rules of recruitment of excitatory interneurons in the adult zebrafish locomotor network.

Neural networks in the spinal cord transform signals from the brain into coordinated locomotor movements. An optimal adjustment of the speed of locomotion entails a precise order of recruitment of interneurons underlying excitation within these networks. However, the mechanisms encoding the recruitment threshold of excitatory interneurons have remained unclear. Here we show, using a juvenile/adult zebrafish preparation, that excitatory V2a interneurons are incrementally recruited with increased swimming frequency. The order of recruitment is not imprinted by the topography or the input resistance of the V2a interneurons. Rather, it is determined by scaling the effect of excitatory synaptic currents by the input resistance. We also show that the locomotor networks are composed of multiple microcircuits encompassing subsets of V2a interneurons and motoneurons that are recruited in a continuum with increased swimming speeds. Thus, our results provide insights into the organization and mechanisms determining the recruitment of spinal microcircuits to ensure optimal execution of locomotor movements.

Initiation of locomotion in adult zebrafish.

Motor behavior is generated by specific neural circuits. Those producing locomotion are located in the spinal cord, and their activation depends on descending inputs from the brain or on sensory inputs. In this study, we have used an in vitro brainstem-spinal cord preparation from adult zebrafish to localize a region where stimulation of descending inputs can induce sustained locomotor activity. We show that a brief stimulation of descending inputs at the junction between the brainstem and spinal cord induces long-lasting swimming activity. The swimming frequencies induced are remarkably similar to those observed in freely moving adult fish, arguing that the induced locomotor episode is highly physiological. The motor pattern is mediated by activation of ionotropic glutamate and glycine receptors in the spinal cord and is not the result of synaptic interactions between neurons at the site of the stimulation in the brainstem. We also compared the activity of motoneurons during locomotor activity induced by electrical stimulation of descending inputs and by exogenously applied NMDA. Prolonged NMDA application changes the shape of the synaptic drive and action potentials in motoneurons. When escape activity occurs, the swimming activity in the intact zebrafish was interrupted and some of the motoneurons involved became inhibited in vitro. Thus, the descending inputs seem to act as a switch to turn on the activity of the spinal locomotor network in the caudal spinal cord. We propose that recurrent synaptic activity within the spinal locomotor circuits can transform a brief input into a well coordinated and long-lasting swimming pattern.

Principles governing recruitment of motoneurons during swimming in zebrafish.

Locomotor movements are coordinated by a network of neurons that produces sequential muscle activation. Different motoneurons need to be recruited in an orderly manner to generate movement with appropriate speed and force. However, the mechanisms governing recruitment order have not been fully clarified. Using an in vitro juvenile/adult zebrafish brainstem-spinal cord preparation, we found that motoneurons were organized into four pools with specific topographic locations and were incrementally recruited to produce swimming at different frequencies. The threshold of recruitment was not dictated by the input resistance of motoneurons, but was instead set by a combination of specific biophysical properties and the strength of the synaptic currents. Our results provide insights into the cellular and synaptic computations governing recruitment of motoneurons during locomotion.

Nitric oxide potentiation of locomotor activity in the spinal cord of the lamprey.

To understand the intrinsic operation of spinal networks generating locomotion, we need to not only characterize the constituent neurons and their connectivity, but also determine the role of intrinsic modulation in shaping the final motor output. We have focused on the effects of nitric oxide (NO) on the locomotor frequency and the underlying synaptic mechanisms in the lamprey spinal cord. To identify the source of NO, we used NADPH-diaphorase histochemistry and nNOS immunocytochemistry. Gray matter and sensory neurons were positively labeled using both methods. Preparations preincubated with NO synthase inhibitors displayed slower locomotor frequency that increased upon washout of the inhibitors, suggesting that NO is an endogenous neuromodulator in the spinal cord. Application of NO donors increased the locomotor frequency that was blocked by an NO scavenger and partially reduced by an inhibitor of sGC. To analyze the synaptic modulation underlying the NO-induced increase of the locomotor frequency we performed intracellular recordings from motoneurons and interneurons. The NO-induced increase in locomotor frequency was associated with a decrease in the midcycle inhibition and an increase in on-cycle excitation. To determine the site of action of NO, we examined the effect of NO donors on miniature PSCs. NO increased both the frequency and amplitude of mEPSCs while it only decreased the frequency of mIPSCs, suggesting the increased excitation is mediated by both presynaptic and postsynaptic mechanisms, while the decrease in inhibition involves only presynaptic mechanisms. Our results demonstrate a significant role of NO in adult vertebrate motor control which, via modulation of both excitatory and inhibitory transmission, increases the locomotor burst frequency.

Serotonergic modulation of locomotion in zebrafish: endogenous release and synaptic mechanisms.

Serotonin (5-HT) plays an important role in shaping the activity of the spinal networks underlying locomotion in many vertebrate preparations. At larval stages in zebrafish, 5-HT does not change the frequency of spontaneous swimming; and it only decreases the quiescent period between consecutive swimming episodes. However, it is not known whether 5-HT exerts similar actions on the locomotor network at later developmental stages. For this, the effect of 5-HT on the fictive locomotor pattern of juvenile and adult zebrafish was analyzed. Bath-application of 5-HT (1-20 mum) reduced the frequency of the NMDA-induced locomotor rhythm. Blocking removal from the synaptic cleft with the reuptake inhibitor citalopram had similar effects, suggesting that endogenous serotonin is modulating the locomotor pattern. One target for this modulation was the mid-cycle inhibition during locomotion because the IPSPs recorded in spinal neurons during the hyperpolarized phase were increased both in amplitude and occurrence by 5-HT. Similar results were obtained for IPSCs recorded in spinal neurons clamped at the reversal potential of excitatory currents (0 mV). 5-HT also slows down the rising phase of the excitatory drive recorded in spinal cord neurons when glycinergic inhibition is blocked. These results suggest that the decrease in the locomotor burst frequency induced by 5-HT is mediated by a potentiation of mid-cycle inhibition combined with a delayed onset of the subsequent depolarization.

Endocannabinoid signaling in the spinal locomotor circuitry.

To understand how the spinal central pattern generators produce locomotor movements, it is necessary to characterize the network's connectivity, the intrinsic properties of the constituent neurons and the modulatory mechanisms. Modulation operating within spinal locomotor networks is required for the generation of the final motor output. In this review, we have summarized how endocannabinoids released by locomotor network neurons contribute to setting the baseline locomotor frequency. They are synthesized on demand as a result of activation of mGluR1 and act as retrograde messengers to depress inhibitory synaptic transmission. We also discuss how endogenous activation of mGluR1 contributes to the normal operation of the spinal locomotor network and the underlying cellular and synaptic mechanisms.

Locomotor pattern in the adult zebrafish spinal cord in vitro.

The zebrafish is an attractive model system for studying the function of the spinal locomotor network by combining electrophysiological, imaging, and genetic approaches. Thus far, most studies have been focusing on embryonic and larval stages. In this study we have developed an in vitro preparation of the isolated spinal cord from adult zebrafish in which locomotor activity can be induced while the activity of single neurons can be monitored using whole cell recording techniques. Application of NMDA elicited rhythmic locomotor activity that was monitored by recording from muscles or ventral roots in semi-intact or isolated spinal cord preparations, respectively. This rhythmic activity displayed a left-right alternation and a rostrocaudal delay. Blockade of glycinergic synaptic transmission by strychnine switched the alternating activity into synchronous bursting in the left and right sides as well as along the rostrocaudal axis. Whole cell recordings from motoneurons showed that they receive phasic synaptic inputs that were correlated with the locomotor activity recorded in ventral roots. This newly developed in vitro preparation of the adult zebrafish spinal cord will allow examination of the organization of the spinal locomotor network in an adult system to complement studies in zebrafish larvae and new born rodents.