GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

Actions expressed prematurely without regard for their consequences are considered impulsive. Such behaviour is governed by a network of brain regions including the prefrontal cortex (PFC) and nucleus accumbens (NAcb) and is prevalent in disorders including attention deficit hyperactivity disorder (ADHD) and drug addiction. However, little is known of the relationship between neural activity in these regions and specific forms of impulsive behaviour. In the present study we investigated local field potential (LFP) oscillations in distinct sub-regions of the PFC and NAcb on a 5-choice serial reaction time task (5-CSRTT), which measures sustained, spatially-divided visual attention and action restraint. The main findings show that power in gamma frequency (50-60 Hz) LFP oscillations transiently increases in the PFC and NAcb during both the anticipation of a cue signalling the spatial location of a nose-poke response and again following correct responses. Gamma oscillations were coupled to low-frequency delta oscillations in both regions; this coupling strengthened specifically when an error response was made. Theta (7-9 Hz) LFP power in the PFC and NAcb increased during the waiting period and was also related to response outcome. Additionally, both gamma and theta power were significantly affected by upcoming premature responses as rats waited for the visual cue to respond. In a subgroup of rats showing persistently high levels of impulsivity we found that impulsivity was associated with increased error signals following a nose-poke response, as well as reduced signals of previous trial outcome during the waiting period. Collectively, these in-vivo neurophysiological findings further implicate the PFC and NAcb in anticipatory impulsive responses and provide evidence that abnormalities in the encoding of rewarding outcomes may underlie trait-like impulsive behaviour.

Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management.

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

It is known that sensory deprivation, including postnatal whisker trimming, can lead to severe deficits in the firing rate properties of cortical neurons. Recent results indicate that development of synchronous discharge among cortical neurons is also activity influenced, and that correlated discharge is significantly impaired following loss of bilateral sensory input in rats. Here we investigate whether unilateral whisker trimming (unilateral deprivation or UD) after birth interferes in the same way with the development of synchronous discharge in cortex. We measured the coincidence of spikes among pairs of neurons recorded under urethane anesthesia in one whisker barrel field deprived by trimming all contralateral whiskers for 60 days after birth (UD), and in untrimmed controls (CON). In the septal columns around barrels, UD significantly increased the coincident discharge among cortical neurons compared with CON, most notably in layers II/III. In contrast, synchronous discharge was normal between layer IV UD barrel neurons: i.e., not different from CON. Thus, while bilateral whisker deprivation (BD) produced a global deficit in the development of synchrony in layer IV, UD did not block the development of synchrony between neurons in layer IV barrels and increased synchrony within septal circuits. We conclude that changes in synchronous discharge after UD are unexpectedly different from those recorded after BD, and we speculate that this effect may be due to the driven activity from active commissural inputs arising from the contralateral hemisphere that received normal activity levels during postnatal development.

Behavioral choices that ignore prior experience promote exploration and unpredictability but are seemingly at odds with the brain's tendency to use experience to optimize behavioral choice. Indeed, when faced with virtual competitors, primates resort to strategic counterprediction rather than to stochastic choice. Here, we show that rats also use history- and model-based strategies when faced with similar competitors but can switch to a "stochastic" mode when challenged with a competitor that they cannot defeat by counterprediction. In this mode, outcomes associated with an animal's actions are ignored, and normal engagement of anterior cingulate cortex (ACC) is suppressed. Using circuit perturbations in transgenic rats, we demonstrate that switching between strategic and stochastic behavioral modes is controlled by locus coeruleus input into ACC. Our findings suggest that, under conditions of uncertainty about environmental rules, changes in noradrenergic input alter ACC output and prevent erroneous beliefs from guiding decisions, thus enabling behavioral variation.

In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell.

The superficial superior colliculus (sSC) occupies a critical node in the mammalian visual system; it is one of two major retinorecipient areas, receives visual cortical input, and innervates visual thalamocortical circuits. Nonetheless, the contribution of sSC neurons to downstream neural activity and visually guided behavior is unknown and frequently neglected. Here we identified the visual stimuli to which specific classes of sSC neurons respond, the downstream regions they target, and transgenic mice enabling class-specific manipulations. One class responds to small, slowly moving stimuli and projects exclusively to lateral posterior thalamus; another, comprising GABAergic neurons, responds to the sudden appearance or rapid movement of large stimuli and projects to multiple areas, including the lateral geniculate nucleus. A third class exhibits direction-selective responses and targets deeper SC layers. Together, our results show how specific sSC neurons represent and distribute diverse information and enable direct tests of their functional role.

Mitochondria are complex organelles at the center of cellular metabolism, apoptosis, and signaling. They continue to be the subject of intense basic investigation to understand their composition and function, but they have also captivated the attention of clinical researchers because of the growing knowledge of the (sometimes unexpected) roles of mitochondria in human diseases and aging. A full understanding of these intriguing organelles often requires their purification from cells or tissues under specific physiological or pathological conditions. Here we provide some introductory considerations for those interested in purifying mitochondria for subsequent downstream biophysical, structural, and functional analysis.