A cortico-collicular circuit for orienting to shelter during escape.

When faced with predatory threats, escape towards shelter is an adaptive action that offers long-term protection against the attacker. Animals rely on knowledge of safe locations in the environment to instinctively execute rapid shelter-directed escape actions1,2. Although previous work has identified neural mechanisms of escape initiation3,4, it is not known how the escape circuit incorporates spatial information to execute rapid flights along the most efficient route to shelter. Here we show that the mouse retrosplenial cortex (RSP) and superior colliculus (SC) form a circuit that encodes the shelter-direction vector and is specifically required for accurately orienting to shelter during escape. Shelter direction is encoded in RSP and SC neurons in egocentric coordinates and SC shelter-direction tuning depends on RSP activity. Inactivation of the RSP-SC pathway disrupts the orientation to shelter and causes escapes away from the optimal shelter-directed route, but does not lead to generic deficits in orientation or spatial navigation. We find that the RSP and SC are monosynaptically connected and form a feedforward lateral inhibition microcircuit that strongly drives the inhibitory collicular network because of higher RSP input convergence and synaptic integration efficiency in inhibitory SC neurons. This results in broad shelter-direction tuning in inhibitory SC neurons and sharply tuned excitatory SC neurons. These findings are recapitulated by a biologically constrained spiking network model in which RSP input to the local SC recurrent ring architecture generates a circular shelter-direction map. We propose that this RSP-SC circuit might be specialized for generating collicular representations of memorized spatial goals that are readily accessible to the motor system during escape, or more broadly, during navigation when the goal must be reached as fast as possible.

Preparation of acute midbrain slices containing the superior colliculus and periaqueductal Gray for patch-clamp recordings.

This protocol is a practical guide for preparing acute coronal slices from the midbrain of young adult mice for electrophysiology experiments. It describes two different sets of solutions with their respective incubation strategies and two alternative procedures for brain extraction: decapitation under terminal isoflurane anaesthesia and intracardial perfusion with artificial cerebrospinal fluid under terminal isoflurane anaesthesia. Slices can be prepared from wild-type mice as well as from mice that have been genetically modified or transfected with viral constructs to label subsets of cells. The preparation can be used to investigate the electrophysiological properties of midbrain neurons in combination with pharmacology, opto- and chemogenetic manipulations, and calcium imaging; which can be followed by morphological reconstruction, immunohistochemistry, or single-cell transcriptomics. The protocol also provides a detailed list of materials and reagents including the design for a low-cost and easy to assemble 3D printed slice recovery chamber, general advice for troubleshooting common issues leading to suboptimal slice quality, and some suggestions to ensure good maintenance of a patch-clamp rig.

Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling.

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Nerve cells communicate using tiny electrical impulses called action potentials. Special proteins termed ion channels produce these electric signals by allowing specific charged particles, or ions, to pass in or out of cells across its membrane. When a nerve cell 'fires' an action potential, specific ion channels briefly open to let in a surge of positively charged ions which electrify the cell. Action potentials begin in the same place in each nerve cell, at an area called the axon initial segment. The large number of sodium channels at this site kick-start the influx of positively charged sodium ions ensuring that every action potential starts from the same place. Previous research has shown that, when action potentials begin, the concentration of calcium ions at the axon initial segment also increases, but it was not clear which ion channels were responsible for this entry of calcium. Channels that are selective for calcium ions are the prime candidates for this process. However, research in squid nerve cells gave rise to an unexpected idea by suggesting that sodium channels may not exclusively let in sodium but also allow some calcium ions to pass through. Hanemaaijer, Popovic et al. therefore wanted to test the routes that calcium ions take and see whether the sodium channels in mammalian nerve cells are also permeable to calcium. Experiments using fluorescent dyes to track the concentration of calcium in rat and human nerve cells showed that calcium ions accumulated at the axon initial segment when action potentials fired. Most of this increase in calcium could be stopped by treating the neurons with a toxin that prevents sodium channels from opening. Electrical manipulations of the cells revealed that, in this context, the calcium ions were effectively behaving like sodium ions. Human kidney cells were then engineered to produce the sodium channel protein. This confirmed that calcium and sodium ions were indeed both passing through the same channel. These results shed new light on the relationship between calcium ions and sodium channels within the mammalian nervous system and that this interplay occurs at the axon initial segment of the cell. Genetic mutations that 'nudge' sodium channels towards favoring calcium entry are also found in patients with autism spectrum disorders, and so this new finding may contribute to our understanding of these conditions.

The Neuropeptide Galanin Is Required for Homeostatic Rebound Sleep following Increased Neuronal Activity.

Sleep pressure increases during wake and dissipates during sleep, but the molecules and neurons that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole-brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin (Galn)-expressing neurons as selectively active during rebound sleep, and the relative induction of galn transcripts was predictive of total rebound sleep time. Galn is required for sleep homeostasis, as galn mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galn plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.