We compared performance of recently developed silicon photomultipliers (SiPMs) to GaAsP photomultiplier tubes (PMTs) for two-photon imaging of neural activity. Despite higher dark counts, SiPMs match or exceed the signal-to-noise ratio of PMTs at photon rates encountered in typical calcium imaging experiments due to their low pulse height variability. At higher photon rates encountered during high-speed voltage imaging, SiPMs substantially outperform PMTs.

Recent advances in optogenetic techniques have generated new tools for controlling neuronal activity, with a wide range of neuroscience applications. The most commonly used approach has been the optical activation of the light-gated ion channel channelrhodopsin-2 (ChR2). However, targeted single-cell-level optogenetic activation with temporal precessions comparable to the spike timing remained challenging. Here we report fast (< or = 1 ms), selective, and targeted control of neuronal activity with single-cell resolution in hippocampal slices. Using temporally focused laser pulses (TEFO) for which the axial beam profile can be controlled independently of its lateral distribution, large numbers of channels on individual neurons can be excited simultaneously, leading to strong (up to 15 mV) and fast (< or = 1 ms) depolarizations. Furthermore, we demonstrated selective activation of cellular compartments, such as dendrites and large presynaptic terminals, at depths up to 150 microm. The demonstrated spatiotemporal resolution and the selectivity provided by TEFO allow manipulation of neuronal activity, with a large number of applications in studies of neuronal microcircuit function in vitro and in vivo.

External cues, including touch, enable walking animals to flexibly maneuver around obstacles and extricate themselves from dead-ends (for reviews, see [1-3]). In a screen for neurons that enable Drosophila melanogaster to retreat when it encounters a dead-end, we identified a pair of ascending neurons, the TwoLumps Ascending (TLA) neurons. Silencing TLA activity impairs backward locomotion, whereas optogenetic activation triggers backward walking. TLA-induced reversal is mediated in part by the Moonwalker Descending Neurons (MDNs) [4], which receive excitatory input from the TLAs. Silencing the TLAs decreases the extent to which freely walking flies back up upon encountering a physical barrier in the dark, and TLAs show calcium responses to optogenetic activation of neurons expressing the mechanosensory channel NOMPC. We infer that TLAs convey feedforward mechanosensory stimuli to transiently activate MDNs in response to anterior body touch.

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the role of the other set of T4P genes is that they could comprise a system analogous to the type II secretion systems (TTSS) found in Gram-negative bacteria, which is used to export folded proteins from the periplasm through the outer membrane to the extracellular environment. Gram-positive bacteria have a similar secretion barrier in the thick peptidoglycan (PG) layer, which blocks secretion of folded proteins >25 kD. To determine if the T4P-associated genes comprise a Gram-positive TTSS, the secretome of mutants lacking type IV pilins were examined and a single protein, a von Willebrand A domain containing protein BsaC (CPE0517) was identified as being dependent on PilA3 for secretion. BsaC is in an operon with a signal peptidase and two putative biofilm matrix proteins with homology to Bacillus subtilis TasA. One of these proteins, BsaA, was shown by another group to produce high mol wt oligomers. We analyzed BsaA monomer interactions with de novo modeling, which projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to loss of oligomerization, supporting the predicted bond formation process. Phylogenetic analysis showed the BsaA family of proteins are widespread among bacteria and archaea but only a subset are predicted to form isopeptide bonds.

To understand the neural basis of behavior, it is essential to sensitively and accurately measure neural activity at single neuron and single spike resolution. Extracellular electrophysiology delivers this, but it has biases in the neurons it detects and it imperfectly resolves their action potentials. To minimize these limitations, we developed a silicon probe with much smaller and denser recording sites than previous designs, called Neuropixels Ultra (NP Ultra). This device samples neuronal activity at ultra-high spatial density ( 10 times higher than previous probes) with low noise levels, while trading off recording span. NP Ultra is effectively an implantable voltage-sensing camera that captures a planar image of a neuron’s electrical field. We use a spike sorting algorithm optimized for these probes to demonstrate that the yield of visually-responsive neurons in recordings from mouse visual cortex improves up to 3-fold. We show that NP Ultra can record from small neuronal structures including axons and dendrites. Recordings across multiple brain regions and four species revealed a subset of extracellular action potentials with unexpectedly small spatial spread and axon-like features. We share a large-scale dataset of these brain-wide recordings in mice as a resource for studies of neuronal biophysics. Finally, using ground-truth identification of three major inhibitory cortical cell types, we found that these cell types were discriminable with approximately 75% success, a significant improvement over lower-resolution recordings. NP Ultra improves spike sorting performance, detection of subcellular compartments, and cell type classification to enable more powerful dissection of neural circuit activity during behavior.

To study the neural basis of behavior, we require methods to sensitively and accurately measure neural activity at single neuron and single spike resolution. Extracellular electrophysiology is a principal method for achieving this, but it has biases in the neurons it detects and it imperfectly resolves their action potentials. To overcome these limitations, we developed a silicon probe with significantly smaller and denser recording sites than previous designs, called Neuropixels Ultra (NP Ultra). This device measures neuronal activity at ultra-high densities (>1300 sites per mm, 10 times higher than previous probes), with 6 µm center-to-center spacing and low noise. This device effectively comprises an implantable voltage-sensing camera that captures a planar image of a neuron's electrical field. We introduce a new spike sorting algorithm optimized for these probes and use it to find that the yield of visually-responsive neurons in recordings from mouse visual cortex improves ∼3-fold. Recordings across multiple brain regions and four species revealed a subset of unexpectedly small extracellular action potentials not previously reported. Further experiments determined that, in visual cortex, these do not correspond to major subclasses of interneurons and instead likely reflect recordings from axons. Finally, using ground-truth identification of cortical inhibitory cell types with optotagging, we found that cell type was discriminable with approximately 75% success among three types, a significant improvement over lower-resolution recordings. NP Ultra improves spike sorting performance, sampling bias, and cell type classification.

Nervous systems combine lower-level sensory signals to detect higher-order stimulus features critical to survival, such as the visual looming motion created by an imminent collision or approaching predator. Looming-sensitive neurons have been identified in diverse animal species. Different large-scale visual features such as looming often share local cues, which means loom-detecting neurons face the challenge of rejecting confounding stimuli. Here we report the discovery of an ultra-selective looming detecting neuron, lobula plate/lobula columnar, type II (LPLC2) in Drosophila, and show how its selectivity is established by radial motion opponency. In the fly visual system, directionally selective small-field neurons called T4 and T5 form a spatial map in the lobula plate, where they each terminate in one of four retinotopic layers, such that each layer responds to motion in a different cardinal direction. Single-cell anatomical analysis reveals that each arm of the LPLC2 cross-shaped primary dendrites ramifies in one of these layers and extends along that layer's preferred motion direction. In vivo calcium imaging demonstrates that, as their shape predicts, individual LPLC2 neurons respond strongly to outward motion emanating from the centre of the neuron's receptive field. Each dendritic arm also receives local inhibitory inputs directionally selective for inward motion opposing the excitation. This radial motion opponency generates a balance of excitation and inhibition that makes LPLC2 non-responsive to related patterns of motion such as contraction, wide-field rotation or luminance change. As a population, LPLC2 neurons densely cover visual space and terminate onto the giant fibre descending neurons, which drive the jump muscle motor neuron to trigger an escape take off. Our findings provide a mechanistic description of the selective feature detection that flies use to discern and escape looming threats.

BACKGROUND: In holometabolous insects such as Drosophila melanogaster, neuroblasts produce an initial population of diverse neurons during embryogenesis and a much larger set of adult-specific neurons during larval life. In the ventral CNS, many of these secondary neuronal lineages differ significantly from one body segment to another, suggesting a role for anteroposterior patterning genes. RESULTS: Here we systematically characterize the expression pattern and function of the Hox gene Ultrabithorax (Ubx) in all 25 postembryonic lineages. We find that Ubx is expressed in a segment-, lineage-, and hemilineage-specific manner in the thoracic and anterior abdominal segments. When Ubx is removed from neuroblasts via mitotic recombination, neurons in these segments exhibit the morphologies and survival patterns of their anterior thoracic counterparts. Conversely, when Ubx is ectopically expressed in anterior thoracic segments, neurons exhibit complementary posterior transformation phenotypes. CONCLUSION: Our findings demonstrate that Ubx plays a critical role in conferring segment-appropriate morphology and survival on individual neurons in the adult-specific ventral CNS. Moreover, while always conferring spatial identity in some sense, Ubx has been co-opted during evolution for distinct and even opposite functions in different neuronal hemilineages.

The need for optical sectioning in bio-imaging has amongst others led to the development of the two-photon scanning microscopy. However, this comes with some intrinsic fundamental limitations in the temporal domain as the focused spot has to be scanned mechanically in the sample plane. Hence for a large number of biological applications where imaging speed is a limiting factor, it would be significantly advantageous to generate widefield excitations with an optical sectioning comparable to the two-photon scanning microscopy. Recently by using the technique of temporal focusing it was shown that high axial resolution widefield excitation can be generated in picosecond time scales without any mechanical moving parts. However the achievable axial resolution is still well above that of a two-photon scanning microscope. Here we demonstrate a new ultrafast widefield two-photon imaging technique termed Multifocal Temporal Focusing (MUTEF) which relies on the generation of a set of diffraction limited beams produced by an Echelle grating that scan across a second tilted diffraction grating in picosecond time scale, generating a widefield excitation area with an axial resolution comparable to a two-photon scanning microscope. Using this method we have shown widefield two-photon imaging on fixed biological samples with an axial sectioning with a FWHM of  0.85 μm.

Chemogenetics enables non-invasive chemical control over cell populations in behaving animals. However, existing small molecule agonists show insufficient potency or selectivity. There is also need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the anti-smoking drug varenicline. We then synthesized novel sub-nanomolar potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.