In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

The distribution of glycinergic cells in the brain of nonmammalian vertebrates is still unknown. Lampreys are the most primitive extant vertebrates, and they may provide important data on the phylogeny of this system. Here, we studied for the first time the distribution of glycine immunoreactivity in the sea lamprey brain and compared it with gamma-aminobutyric acid (GABA)-ergic populations. Most glycine-immunoreactive neurons were found at midbrain and hindbrain levels, and most of these cells did not exhibit GABA immunoreactivity. We describe glycine-immunoreactive cell populations in the olfactory bulbs, the preoptic nucleus, and the thalamus of the sea lamprey, which is in striking contrast to their lack in the mammalian forebrain. We also observed glycine-immunoreactive populations in the optic tectum, the torus semicircularis and the midbrain tegmentum, the isthmus, the octavolateral area, the dorsal column nucleus, the abducens nucleus, the trigeminal motor nucleus, the facial motor nucleus, and the rhombencephalic reticular formation. In these populations, colocalization with GABA was observed in only some cells of the tegmental M5 nucleus, ventral isthmus, medial octavolateral nucleus, dorsal column nucleus, and lateral reticular region. The present results allow us to conclude that the distribution of glycine-immunoreactive cells changed notably from lamprey to mammals, with a decrease in glycinergic populations in the forebrain and a specialization of brainstem cell groups. Although knowledge of the glycinergic populations in lampreys is important for understanding the early evolution of this system, there is a notable gap of information regarding its organization in brains of other nonmammalian vertebrates.

Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium-sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract-tracing methods or γ-aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium-sized cells of some reticulospinal-projecting groups were often glutamate-immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal-projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non-glutamatergic cells or neurons that co-release both GABA and glutamate.