Microtubules are not required to generate a nascent axon in embryonic spinal neurons in vivo.

Our understanding of the cell behaviours and cytoskeletal requirements of axon formation is largely derived from in vitro models but how these relate to axon formation in vivo is not clear. In vitro, neurons progress through a well-defined multineurite stage to form an axon and both actin and microtubules cooperate to drive the first steps in neurite and axon morphogenesis. However, these steps are not recapitulated in vivo, and it is not clear whether the underlying cell biological mechanisms may differ also. Here, we investigate the mechanisms that regulate axon formation in embryonic zebrafish spinal neurons in vivo. We find microtubule organising centres are located distant from the site of axon initiation, and microtubule plus-ends are not enriched in the axon during axon initiation. Focal F-actin accumulation precedes axon formation, and we find that nocodazole-treated neurons with no detectable microtubules are still able to form nascent axonal protrusions that are approximately 10-µm long, dilated and relatively long-lived. We suggest spinal axon formation in vivo is fundamentally different from axon formation in in vitro models.

Evolution of central neural circuits: state of the art and perspectives.

The wide variety of animal behaviours that can be observed today arose through the evolution of their underlying neural circuits. Advances in understanding the mechanisms through which neural circuits change over evolutionary timescales have lagged behind our knowledge of circuit function and development. This is particularly true for central neural circuits, which are experimentally less accessible than peripheral circuit elements. However, recent technological developments - including cross-species genetic modifications, connectomics and transcriptomics - have facilitated comparative neuroscience studies with a mechanistic outlook. These advances enable knowledge from two classically separate disciplines - neuroscience and evolutionary biology - to merge, accelerating our understanding of the principles of neural circuit evolution. Here we synthesize progress on this topic, focusing on three aspects of neural circuits that change over evolutionary time: synaptic connectivity, neuromodulation and neurons. By drawing examples from a wide variety of animal phyla, we reveal emerging principles of neural circuit evolution.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the enhancer of microexons (eMIC) domain in Srrm234. In Drosophila, this processing is dependent on regulation by Elav/Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. We found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient posttranscriptional programs can evolve independently in different phyla to affect distinct cellular modules while maintaining cell-type specificity.

Extensive and diverse patterns of cell death sculpt neural networks in insects.

Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death (PCD) appears to play a key role. We show that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Mimicking an evolutionary role for increasing cell numbers, we artificially block in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of 'undead' neurons with complex arborisations and distinct neurotransmitter identities. Activation of these 'undead' neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Focusing on two dipterans, which have lost flight during evolution, we reveal that reductions in populations of flight interneurons are likely caused by increased cell death during development. Our findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations.

Just like a sculptor chips away at a block of granite to make a statue, the nervous system reaches its mature state by eliminating neurons during development through a process known as programmed cell death. In vertebrates, this mechanism often involves newly born neurons shrivelling away and dying if they fail to connect with others during development. Most studies in insects have focused on the death of neurons that occurs at metamorphosis, during the transition between larva to adult, when cells which are no longer needed in the new life stage are eliminated. Pop et al. harnessed a newly designed genetic probe to point out that, in fruit flies, programmed cell death of neurons at metamorphosis is not the main mechanism through which cells die. Rather, the majority of cell death takes place as soon as neurons are born throughout all larval stages, when most of the adult nervous system is built. To gain further insight into the role of this 'early' cell death, the neurons were stopped from dying, showing that these cells were able to reach maturity and function. Together, these results suggest that early cell death may be a mechanism fine-tuned by evolution to shape the many and varied nervous systems of insects. To explore this, Pop et al. looked for hints of early cell death in relatives of fruit flies that are unable to fly: the swift lousefly and the bee lousefly. This analysis showed that early cell death is likely to occur in these two insects, but it follows different patterns than in the fruit fly, potentially targeting the neurons that would have controlled flight in these flies' ancestors. Brains are the product of evolution: learning how neurons change their connections and adapt could help us understand how the brain works in health and disease. This knowledge may also be relevant to work on artificial intelligence, a discipline that often bases the building blocks and connections in artificial 'brains' on how neurons communicate with one another.

Neurexin and Neuroligin-based adhesion complexes drive axonal arborisation growth independent of synaptic activity.

Building arborisations of the right size and shape is fundamental for neural network function. Live imaging in vertebrate brains strongly suggests that nascent synapses are critical for branch growth during development. The molecular mechanisms underlying this are largely unknown. Here we present a novel system in Drosophila for studying the development of complex arborisations live, in vivo during metamorphosis. In growing arborisations we see branch dynamics and localisations of presynaptic proteins very similar to the 'synaptotropic growth' described in fish/frogs. These accumulations of presynaptic proteins do not appear to be presynaptic release sites and are not paired with neurotransmitter receptors. Knockdowns of either evoked or spontaneous neurotransmission do not impact arbor growth. Instead, we find that axonal branch growth is regulated by dynamic, focal localisations of Neurexin and Neuroligin. These adhesion complexes provide stability for filopodia by a 'stick-and-grow' based mechanism wholly independent of synaptic activity.

Local Signals in Mouse Horizontal Cell Dendrites.

The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically coupled network, they have been implicated in global signal processing, such as large-scale contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptors. To test this possibility physiologically, we used two-photon microscopy to record light stimulus-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain isolated within horizontal cell dendritic tips or whether they spread across the dendritic arbor. Consistent with the mouse's opsin expression gradient, we found that the Ca2+ signals recorded from dendrites of dorsal horizontal cells were dominated by M-opsin and those of ventral horizontal cells by S-opsin activation. The signals measured in neighboring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our experimental data and results from biophysically realistic modeling support the idea that horizontal cells can process cone input locally, extending the classical view of horizontal cell function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.

Secondary hyperpigmentation during interferon alfa treatment for chronic hepatitis C virus infection.

IMPORTANCE: Interferon alfa remains the central treatment for chronic hepatitis C virus (HCV) infection. Cases of cutaneous and mucous hyperpigmentations during interferon alfa treatment have been reported, but they are considered rare adverse effects. OBJECTIVE: To study the clinical presentation and frequency of hyperpigmentation in patients receiving interferon alfa treatment for chronic HCV infection. DESIGN: Prospective, descriptive clinical trial. SETTING: Monocentric study performed in the Departments of Hepatology and Dermatology of the University Hospital of Nice, Nice, France. PARTICIPANTS: Consecutive patients treated with pegylated interferon alfa-2b and ribavirin for chronic HCV infection. MAIN OUTCOME MEASURES: Demographic data and medical history were noted. A systematic clinical and dermoscopic examination of skin, nails, and mucous membranes was performed, and skin biopsies were performed if needed. RESULTS: Of 77 patients who were included, 16 (21%) presented with hyperpigmentation. Hyperpigmentation of the oral mucous membrane, acquired longitudinal melononychia, and hyperpigmentation of the face were each observed in 7 patients (9%). All patients with hyperpigmentation of the skin had skin type III or IV and worked outside without sun protection. The intensity of pigmentation was reported to decrease progressively when interferon treatment was discontinued. Most patients with hyperpigmentation of the oral mucosa also had melanonychia. However, patients with hyperpigmentation of the skin did not have mucosal or nail involvement, suggesting 2 distinct mechanisms. CONCLUSIONS AND RELEVANCE: Secondary hyperpigmentation during interferon alfa treatment occurs as an adverse event in 21% of patients, especially in those with dark skin types who have unprotected sun exposure. Physicians should be aware of the adverse effects of interferon treatment and advise patients in the use of sun protection, especially patients with darker skin types.