Circulation of the cerebrospinal fluid (CSF) contributes to body axis formation and brain development. Here, we investigated the unexplained origins of the CSF flow bidirectionality in the central canal of the spinal cord of 30 hpf zebrafish embryos and its impact on development. Experiments combined with modeling and simulations demonstrate that the CSF flow is generated locally by caudally-polarized motile cilia along the ventral wall of the central canal. The closed geometry of the canal imposes the average flow rate to be null, explaining the reported bidirectionality. We also demonstrate that at this early stage, motile cilia ensure the proper formation of the central canal. Furthermore, we demonstrate that the bidirectional flow accelerates the transport of particles in the CSF via a coupled convective-diffusive transport process. Our study demonstrates that cilia activity combined with muscle contractions sustain the long-range transport of extracellular lipidic particles, enabling embryonic growth.

In contextual conditioning, a complex pattern of information is processed to associate the characteristics of a particular place with incentive or aversive reinforcements. This type of learning has been widely studied in mammals, but studies of other taxa are scarce. The context-signal memory (CSM) paradigm of the crab Chasmagnathus has been extensively used as a model of learning and memory. Although initially interpreted as habituation, some characteristics of contextual conditioning have been described. However, no anticipatory response has been detected for animals exposed to the training context. Thus, CSM could be interpreted either as an associative habituation or as contextual conditioning that occurs without a context-evoked anticipatory response. Here, we describe a training protocol developed for contextual Pavlovian conditioning (CPC). For each training trial, the context (conditioned stimulus, CS) was discretely presented and finished together with the unconditioned stimulus (US). In agreement with the CSM paradigm, a robust freezing response was acquired during the 15 training trials, and clear retention was found when tested with the US presentation after short (2 and 4 h) and long (1-4 days) delays. This CPC memory showed forward but not simultaneous presentation conditioning and was context specific and protein synthesis dependent. Additionally, a weak CPC memory was enhanced during consolidation. One day after training, CPC was extinguished by repeated CS presentation, while one presentation induced a memory labilisation-reconsolidation process. Finally, we found an anticipatory conditioned response (CR) during the CS presentation for both short-term (4 h) and long-term memory (24 h). These findings support the conditioning nature of the new paradigm.

N-Methyl-D-aspartate receptors (NMDARs) are involved in learning and memory processes in vertebrates and invertebrates. In Neohelice granulata, NMDARs are involved in the storage of associative memories (see references in text). The aim of this work was to characterize this type of glutamate receptor in Neohelice and to describe its distribution in the central nervous system (CNS). As a first step, a detailed study of the CNS of N. granulata was performed at the neuropil level, with special focus on one of the main structures involved in this type of memory, the supraesophageal ganglion, called central brain. The characterization of the NMDAR was achieved by identifying the essential subunit of these receptors, the NR1-like subunit. The NR1-like signals were found via western blot and immunohistochemistry techniques in each of the major ganglia: the eyestalk ganglia, the central brain, and the thoracic ganglion. Western blots yielded two bands for the crab NR1-like subunit, at ∼88 and ∼84 kDa. This subunit is present in all the major ganglia, and shows a strong localization in synaptosomal membranes. NMDARs are distributed throughout the majority of each ganglion but show prominent signal intensity in some distinguishable neuropils and neurons. This is the first general description of the N. granulata nervous system as a whole and the first study of NMDARs in the CNS of decapods. The preferential localization of the receptor in some neuropils and neurons indicates the presence of possible new targets for memory processing and storage.

Highlights d Rostralmost CSF-cNs form inhibitory synapses onto occipital motor neurons d Ventral CSF-cNs inhibit reticulospinal neurons driving fast and powerful locomotion d Dorsal CSF-cNs inhibit reticulospinal neurons eliciting slow and refined locomotion d Rostral CSF-cN inhibitory sensory feedback enhances speed, power, and posture

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Locomotion in vertebrates relies on high brain centers converging onto the mesencephalic locomotor region (MLR). How the MLR recruits brainstem reticulospinal neurons (RSNs) to initiate locomotion is incompletely understood due to the challenge of recording these cells in vivo. To tackle this question, we leveraged the transparency and genetic accessibility of larval zebrafish. In this model organism, we uncovered the locus of the MLR as a small region dorsal to the locus coeruleus containing glutamatergic and cholinergic neurons. MLR stimulations reliably elicited forward bouts of controlled duration and speed. We find that the MLR elicits forward locomotion by recruiting V2a RSNs in the pontine and retropontine regions, and gradually in the medulla. Remarkably, recruited V2a RSNs in the medulla act as maintain cells encoding speed of forward locomotion. Altogether, our study reveals that the MLR recruits genetically-identified reticulospinal neurons in the medulla to control the kinematics of exploration.

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