Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post-fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally-extending neurites resembling dendrites and a caudally-extending neurite resembling an axon, which terminated with an enlarged growth cone-like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally-produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin-2 each produced similar effects to 5HT, indicating that spinally-intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018.

The spinal cord (SC) contains neural networks that are capable of producing organized locomotor activity autonomously from the brain. Locomotor activity can be induced in spinally transected (spinalized) animals by adding a source of tonic excitation to activate spinal networks. This is commonly accomplished by activating N-methyl-d-aspartate (NMDA) glutamate receptors through bath application of NMDA. More recently, optogenetic approaches have enabled both activation and inactivation of neuronal cell populations to control the activity of locomotor networks. Larval zebrafish are exceptionally amenable to optogenetic techniques due to their transparency, which permits noninvasive light delivery. In this study, we induced locomotor activity in spinalized transgenic zebrafish larvae that expressed channelrhodopsin-2 in all subtypes of spinal vesicular glutamate transporter 2a ( vglut2a)-expressing neurons by applying 10 s of constant blue light to the preparations. The resultant locomotor activity possessed all of the characteristics of swimming: bilateral alternation, rostrocaudal progression, and organization into discrete swimming episodes. Spatially restricted light application revealed that illumination of the rostral SC produced more robust activity than illumination of the caudal SC. Moreover, illumination of only three body segments was sufficient to produce fictive swimming. Intriguingly, organized swimming activity persisted during NMDA receptor antagonism but was disrupted by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonism. Hence, AMPA receptor signaling is required for episodically-organized swimming, whereas NMDA receptor signaling is not necessary. NEW & NOTEWORTHY Spinal locomotor networks have the intrinsic capacity to transform unpatterned excitatory input into patterned output. Conventionally, spinally mediated fictive locomotor activity is experimentally elicited by N-methyl-d-aspartate (NMDA) application to bias the network toward activation. We present a novel experimental paradigm that permits spatially and temporally controllable activation of spinal vesicular glutamate transporter 2a-expressing neurons in larval zebrafish, eliciting patterned locomotor activity that is not dependent on NMDA receptor signaling.

Key pointsr Cortico-hippocampal feed-forward circuits formed by the temporoammonic (TA) pathway exhibit a marked increase in excitation/inhibition ratio and abnormal spike modulation functions in Fmr1 knock-out (KO) mice.r Inhibitory, but not excitatory, synapse dysfunction underlies cortico-hippocampal feed-forward circuit abnormalities in Fmr1 KO mice.r GABA release is reduced in TA-associated inhibitory synapses of Fmr1 KO mice in a GABA B receptor-dependent manner.r Inhibitory synapse and feed-forward circuit defects are mediated predominately by presynaptic GABA B receptor signalling in the TA pathway of Fmr1 KO mice.r GABA B receptor-mediated inhibitory synapse defects are circuit-specific and are not observed in the Schaffer collateral pathway-associated inhibitory synapses in stratum radiatum.Abstract Circuit hyperexcitability has been implicated in neuropathology of fragile X syndrome, the most common inheritable cause of intellectual disability. Yet, how canonical unitary circuits are affected in this disorder remains poorly understood. Here, we examined this question in the context of the canonical feed-forward inhibitory circuit formed by the temporoammonic (TA) branch of the perforant path, the major cortical input to the hippocampus. TA feed-forward circuits exhibited a marked increase in excitation/inhibition ratio and major functional defects in spike modulation tasks in Fmr1 knock-out (KO) mice, a fragile X mouse model. Changes in feed-forward circuits were caused specifically by inhibitory, but not excitatory, synapse defects. TA-associated inhibitory synapses exhibited increase in paired-pulse ratio and in the coefficient of variation of IPSPs, consistent with decreased GABA release probability. TA-associated inhibitory synaptic transmission in Fmr1 KO mice was also more sensitive to inhibition of GABA B receptors, suggesting an increase in presynaptic GABA B receptor (GABA B R) signalling. Indeed, the differences in inhibitory synaptic transmission between Fmr1 KO and wild-type (WT) mice were eliminated by a GABA B R antagonist. Inhibition of GABA B Rs or selective activation of presynaptic GABA B Rs also abolished the differences in the TA feed-forward circuit properties between Fmr1 KO and WT mice. These GABA B R-mediated defects were circuit-specific and were not observed in the Schaffer collateral pathway-associated inhibitory synapses. Our results suggest that the inhibitory synapse dysfunction in the cortico-hippocampal pathway of Fmr1 KO mice causes hyperexcitability and feed-forward circuit defects, which are mediated in part by a presynaptic GABA B R-dependent reduction in GABA release.

Feed-forward inhibitory (FFI) circuits are important for many information-processing functions. FFI circuit operations critically depend on the balance and timing between the excitatory and inhibitory components, which undergo rapid dynamic changes during neural activity due to short-term plasticity (STP) of both components. How dynamic changes in excitation/inhibition (E/I) balance during spike trains influence FFI circuit operations remains poorly understood. In the current study we examined the role of STP in the FFI circuit functions in the mouse hippocampus. Using a coincidence detection paradigm with simultaneous activation of two Schaffer collateral inputs, we found that the spiking probability in the target CA1 neuron was increased while spike precision concomitantly decreased during high-frequency bursts compared with a single spike. Blocking inhibitory synaptic transmission revealed that dynamics of inhibition predominately modulates the spike precision but not the changes in spiking probability, whereas the latter is modulated by the dynamics of excitation. Further analyses combining whole cell recordings and simulations of the FFI circuit suggested that dynamics of the inhibitory circuit component may influence spiking behavior during bursts by broadening the width of excitatory postsynaptic responses and that the strength of this modulation depends on the basal E/I ratio. We verified these predictions using a mouse model of fragile X syndrome, which has an elevated E/I ratio, and found a strongly reduced modulation of postsynaptic response width during bursts. Our results suggest that changes in the dynamics of excitatory and inhibitory circuit components due to STP play important yet distinct roles in modulating the properties of FFI circuits.

N-methyld-aspartate (NMDA) application has conventionally been used to activate spinal networks to induce locomotion in spinalized animals. We recently described an alternative approach in which application of continuous blue light activates channelrhodopsin-2 in vesicular glutamate transporter 2a (vglut2a)-expressing spinal neurons to produce organized, rhythmic locomotor activity in spinally-transected larval zebrafish. This technique arguably enhances research validity, because endogenous glutamate is released into existing synapses instead of activating only a subset of glutamatergic (NMDA) receptors with an exogenous compound. Here, we explored the viability of this approach in the context of using it for longer-term experiments. Fictive swimming was induced through repetitive application of 10-s blue light stimuli to spinalized preparations for up to 60 min at intervals of 1, 3, or 15 min. Locomotor activity was maintained throughout the experimental timecourse, demonstrating the robustness of the system. Although locomotor bursts remained organized into episodes of activity, the number of bursts elicited during each successive stimulus decreased. This was in contrast to NMDA bath application, in which bursts became less episodically organized while the overall number of bursts remained unchanged. The efficacy of the repetitive optogenetic stimulation paradigm was demonstrated through application of exogenous dopamine, which reversibly decreased the number of bursts produced per stimulus compared with untreated preparations. Finally, increasing the stimulus interval to 15 min lessened, but did not eliminate locomotor fatigue from repetitive activation. Altogether, we established repetitive optogenetic stimulation of vglut2a-expressing neurons as a viable alternative to NMDA application for activation of the zebrafish spinal locomotor network.