Olfactory investigation in the home cage.

We have developed a behavioral paradigm to study volitional olfactory investigation in mice over several months. We placed odor ports in the wall of a standard cage that administer a neutral odorant stimulus when a mouse pokes its nose inside. Even though animals were fed and watered ad libitum, and sampling from the port elicited no outcome other than the delivery of an odor, mice readily sampled these stimuli hundreds of times per day. This self-paced olfactory investigation persisted for weeks with only modest habituation following the first day of exposure to a given set of odorants. If an unexpected odorant stimulus was administered at the port, the sampling rate increased transiently (in the first 20 min) by an order of magnitude and remained higher than baseline throughout the subsequent day, indicating learned implicit knowledge. Thus, this system may be used to study naturalistic olfactory learning over extended time scales outside of conventional task structures.

The representational geometry of emotional states in basolateral amygdala.

Sensory stimuli associated with aversive outcomes can cause multiple behavioral responses related to an animal's evolving emotional state. We employed chemogenetic inactivation and two-photon imaging to reveal how the basolateral amygdala (BLA) mediates these state changes. Mice were presented stimuli in a virtual burrow, causing two responses reflecting fear and flight to safety: tremble and ingress into the burrow. Inactivation eliminated differential tremble and ingress to aversive and neutral stimuli without eliminating responses themselves. Multiple variables, including stimulus valence and identity, and being in the tremble or ingressed state, typically modulated each neuron's activity (mixed-selectivity). BLA neural ensembles represented these variables even after neurons with apparent specialized selectivity were eliminated from analyses. Thus, implementing different readouts of BLA ensembles comprised of mixed-selectivity neurons can identify distinct emotional states defined by responses, like tremble for fear and ingress for safety. This mechanism relies on BLA's representational geometry, not its circuit specialization.

Representational drift in primary olfactory cortex.

Perceptual constancy requires the brain to maintain a stable representation of sensory input. In the olfactory system, activity in primary olfactory cortex (piriform cortex) is thought to determine odour identity1-5. Here we present the results of electrophysiological recordings of single units maintained over weeks to examine the stability of odour-evoked responses in mouse piriform cortex. Although activity in piriform cortex could be used to discriminate between odorants at any moment in time, odour-evoked responses drifted over periods of days to weeks. The performance of a linear classifier trained on the first recording day approached chance levels after 32 days. Fear conditioning did not stabilize odour-evoked responses. Daily exposure to the same odorant slowed the rate of drift, but when exposure was halted the rate increased again. This demonstration of continuous drift poses the question of the role of piriform cortex in odour perception. This instability might reflect the unstructured connectivity of piriform cortex6-12, and may be a property of other unstructured cortices.

A virtual burrow assay for head-fixed mice measures habituation, discrimination, exploration and avoidance without training.

We have designed an assay that measures approach and avoidance behaviors in head-fixed mice at millisecond timescale, is compatible with standard electrophysiological and optical methods for measuring neuronal activity, and requires no training. The Virtual Burrow Assay simulates a scenario in which a mouse, poised at the threshold of its burrow, evaluates whether to exit the enclosure or to retreat inside. The assay provides a sensitive readout of habituation, discrimination and exploration, as well as avoidance of both conditioned and innately aversive cues.

Comparative strength and dendritic organization of thalamocortical and corticocortical synapses onto excitatory layer 4 neurons.

Thalamus is a potent driver of cortical activity even though cortical synapses onto excitatory layer 4 neurons outnumber thalamic synapses 10 to 1. Previous in vitro studies have proposed that thalamocortical (TC) synapses are stronger than corticocortical (CC) synapses. Here, we investigated possible anatomical and physiological differences between these inputs in the rat in vivo. We developed a high-throughput light microscopy method, validated by electron microscopy, to completely map the locations of synapses across an entire dendritic tree. This demonstrated that TC synapses are slightly more proximal to the soma than CC synapses, but detailed compartmental modeling predicted that dendritic filtering does not appreciably favor one synaptic class over another. Measurements of synaptic strength in intact animals confirmed that both TC and CC synapses are weak and approximately equivalent. We conclude that thalamic effectiveness does not rely on enhanced TC strength, but rather on coincident activation of converging inputs.

Functional architecture of the inner pore of a voltage-gated Ca2+ channel.

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.