Myelination of peripheral nerves is controlled by PI4KB through regulation of Schwann cell Golgi function.

Better understanding myelination of peripheral nerves would benefit patients affected by peripheral neuropathies, including Charcot-Marie-Tooth disease. Little is known about the role the Golgi compartment plays in Schwann cell (SC) functions. Here, we studied the role of Golgi in myelination of peripheral nerves in mice through SC-specific genetic inactivation of phosphatidylinositol 4-kinase beta (PI4KB), a Golgi-associated lipid kinase. Sciatic nerves of such mice showed thinner myelin of large diameter axons and gross aberrations in myelin organization affecting the nodes of Ranvier, the Schmidt-Lanterman incisures, and Cajal bands. Nonmyelinating SCs showed a striking inability to engulf small diameter nerve fibers. SCs of mutant mice showed a distorted Golgi morphology and disappearance of OSBP at the cis-Golgi compartment, together with a complete loss of GOLPH3 from the entire Golgi. Accordingly, the cholesterol and sphingomyelin contents of sciatic nerves were greatly reduced and so was the number of caveolae observed in SCs. Although the conduction velocity of sciatic nerves of mutant mice showed an 80% decrease, the mice displayed only subtle impairment in their motor functions. Our analysis revealed that Golgi functions supported by PI4KB are critically important for proper myelination through control of lipid metabolism, protein glycosylation, and organization of microvilli in the nodes of Ranvier of peripheral nerves.

Optimality of sparse olfactory representations is not affected by network plasticity.

The neural representation of a stimulus is repeatedly transformed as it moves from the sensory periphery to deeper layers of the nervous system. Sparsening transformations are thought to increase the separation between similar representations, encode stimuli with great specificity, maximize storage capacity of associative memories, and provide an energy efficient instantiation of information in neural circuits. In the insect olfactory system, odors are initially represented in the periphery as a combinatorial code with relatively simple temporal dynamics. Subsequently, in the antennal lobe this representation is transformed into a dense and complex spatiotemporal activity pattern. Next, in the mushroom body Kenyon cells (KCs), the representation is dramatically sparsened. Finally, in mushroom body output neurons (MBONs), the representation takes on a new dense spatiotemporal format. Here, we develop a computational model to simulate this chain of olfactory processing from the receptor neurons to MBONs. We demonstrate that representations of similar odorants are maximally separated, measured by the distance between the corresponding MBON activity vectors, when KC responses are sparse. Sparseness is maintained across variations in odor concentration by adjusting the feedback inhibition that KCs receive from an inhibitory neuron, the Giant GABAergic neuron. Different odor concentrations require different strength and timing of feedback inhibition for optimal processing. Importantly, as observed in vivo, the KC-MBON synapse is highly plastic, and, therefore, changes in synaptic strength after learning can change the balance of excitation and inhibition, potentially leading to changes in the distance between MBON activity vectors of two odorants for the same level of KC population sparseness. Thus, what is an optimal degree of sparseness before odor learning, could be rendered sub-optimal post learning. Here, we show, however, that synaptic weight changes caused by spike timing dependent plasticity increase the distance between the odor representations from the perspective of MBONs. A level of sparseness that was optimal before learning remains optimal post-learning.

Trade-off between information format and capacity in the olfactory system.

As information about the sensory environment passes between layers within the nervous system, the format of the information often changes. To examine how information format affects the capacity of neurons to represent stimuli, we measured the rate of information transmission in olfactory neurons in intact, awake locusts (Schistocerca americana) while pharmacologically manipulating patterns of correlated neuronal activity. Blocking the periodic inhibition underlying odor-elicited neural oscillatory synchronization increased information transmission rates. This suggests oscillatory synchrony, which serves other information processing roles, comes at a cost to the speed with which neurons can transmit information. Our results provide an example of a trade-off between benefits and costs in neural information processing.

Spatiotemporal Coding of Individual Chemicals by the Gustatory System.

Four of the five major sensory systems (vision, olfaction, somatosensation, and audition) are thought to use different but partially overlapping sets of neurons to form unique representations of vast numbers of stimuli. The only exception is gustation, which is thought to represent only small numbers of basic taste categories. However, using new methods for delivering tastant chemicals and making electrophysiological recordings from the tractable gustatory system of the moth Manduca sexta, we found chemical-specific information is as follows: (1) initially encoded in the population of gustatory receptor neurons as broadly distributed spatiotemporal patterns of activity; (2) dramatically integrated and temporally transformed as it propagates to monosynaptically connected second-order neurons; and (3) observed in tastant-specific behavior. Our results are consistent with an emerging view of the gustatory system: rather than constructing basic taste categories, it uses a spatiotemporal population code to generate unique neural representations of individual tastant chemicals. SIGNIFICANCE STATEMENT: Our results provide a new view of taste processing. Using a new, relatively simple model system and a new set of techniques to deliver taste stimuli and to examine gustatory receptor neurons and their immediate followers, we found no evidence for labeled line connectivity, or basic taste categories such as sweet, salty, bitter, and sour. Rather, individual tastant chemicals are represented as patterns of spiking activity distributed across populations of receptor neurons. These representations are transformed substantially as multiple types of receptor neurons converge upon follower neurons, leading to a combinatorial coding format that uniquely, rapidly, and efficiently represents individual taste chemicals. Finally, we found that the information content of these neurons can drive tastant-specific behavior.

Classification of odorants across layers in locust olfactory pathway.

Olfactory processing takes place across multiple layers of neurons from the transduction of odorants in the periphery, to odor quality processing, learning, and decision making in higher olfactory structures. In insects, projection neurons (PNs) in the antennal lobe send odor information to the Kenyon cells (KCs) of the mushroom bodies and lateral horn neurons (LHNs). To examine the odor information content in different structures of the insect brain, antennal lobe, mushroom bodies and lateral horn, we designed a model of the olfactory network based on electrophysiological recordings made in vivo in the locust. We found that populations of all types (PNs, LHNs, and KCs) had lower odor classification error rates than individual cells of any given type. This improvement was quantitatively different from that observed using uniform populations of identical neurons compared with spatially structured population of neurons tuned to different odor features. This result, therefore, reflects an emergent network property. Odor classification improved with increasing stimulus duration: for similar odorants, KC and LHN ensembles reached optimal discrimination within the first 300-500 ms of the odor response. Performance improvement with time was much greater for a population of cells than for individual neurons. We conclude that, for PNs, LHNs, and KCs, ensemble responses are always much more informative than single-cell responses, despite the accumulation of noise along with odor information.

Feed-Forward versus Feedback Inhibition in a Basic Olfactory Circuit.

Inhibitory interneurons play critical roles in shaping the firing patterns of principal neurons in many brain systems. Despite difference in the anatomy or functions of neuronal circuits containing inhibition, two basic motifs repeatedly emerge: feed-forward and feedback. In the locust, it was proposed that a subset of lateral horn interneurons (LHNs), provide feed-forward inhibition onto Kenyon cells (KCs) to maintain their sparse firing--a property critical for olfactory learning and memory. But recently it was established that a single inhibitory cell, the giant GABAergic neuron (GGN), is the main and perhaps sole source of inhibition in the mushroom body, and that inhibition from this cell is mediated by a feedback (FB) loop including KCs and the GGN. To clarify basic differences in the effects of feedback vs. feed-forward inhibition in circuit dynamics we here use a model of the locust olfactory system. We found both inhibitory motifs were able to maintain sparse KCs responses and provide optimal odor discrimination. However, we further found that only FB inhibition could create a phase response consistent with data recorded in vivo. These findings describe general rules for feed-forward versus feedback inhibition and suggest GGN is potentially capable of providing the primary source of inhibition to the KCs. A better understanding of how inhibitory motifs impact post-synaptic neuronal activity could be used to reveal unknown inhibitory structures within biological networks.

A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain.

Although the gustatory system provides animals with sensory cues important for food choice and other critical behaviors, little is known about neural circuitry immediately following gustatory sensory neurons (GSNs). Here, we identify and characterize a bilateral pair of gustatory second-order neurons (G2Ns) in Drosophila. Previous studies identified GSNs that relay taste information to distinct subregions of the primary gustatory center (PGC) in the gnathal ganglia (GNG). To identify candidate G2Ns, we screened ∼5,000 GAL4 driver strains for lines that label neural fibers innervating the PGC. We then combined GRASP (GFP reconstitution across synaptic partners) with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate G2Ns and the axonal terminals of Gr5a-expressing GSNs, which are known to respond to sucrose. Results of the GRASP analysis, followed by a single-cell analysis by FLP-out recombination, revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, we expressed fluorescence-tagged acetylcholine receptor subunit (Dα7) and active-zone marker (Brp) in the G2Ns. We found that G2N input sites overlaid GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. GRASP analysis and further tests with the Syb-GRASP method suggested that the identified G2Ns receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the GNG, and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). Our findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.

Olfactory system structure and function in newly hatched and adult locusts.

An important question in neuroscience is how sensory systems change as animals grow and interact with the environment. Exploring sensory systems in animals as they develop can reveal how networks of neurons process information as the neurons themselves grow and the needs of the animal change. Here we compared the structure and function of peripheral parts of the olfactory pathway in newly hatched and adult locusts. We found that populations of olfactory sensory neurons (OSNs) in hatchlings and adults responded with similar tunings to a panel of odors. The morphologies of local neurons (LNs) and projection neurons (PNs) in the antennal lobes (ALs) were very similar in both age groups, though they were smaller in hatchlings, they were proportional to overall brain size. The odor evoked responses of LNs and PNs were also very similar in both age groups, characterized by complex patterns of activity including oscillatory synchronization. Notably, in hatchlings, spontaneous and odor-evoked firing rates of PNs were lower, and LFP oscillations were lower in frequency, than in the adult. Hatchlings have smaller antennae with fewer OSNs; removing antennal segments from adults also reduced LFP oscillation frequency. Thus, consistent with earlier computational models, the developmental increase in frequency is due to increasing intensity of input to the oscillation circuitry. Overall, our results show that locusts hatch with a fully formed olfactory system that structurally and functionally matches that of the adult, despite its small size and lack of prior experience with olfactory stimuli.

Functional analysis of a higher olfactory center, the lateral horn.

The lateral horn (LH) of the insect brain is thought to play several important roles in olfaction, including maintaining the sparseness of responses to odors by means of feedforward inhibition, and encoding preferences for innately meaningful odors. Yet relatively little is known of the structure and function of LH neurons (LHNs), making it difficult to evaluate these ideas. Here we surveyed >250 LHNs in locusts using intracellular recordings to characterize their responses to sensory stimuli, dye-fills to characterize their morphologies, and immunostaining to characterize their neurotransmitters. We found a great diversity of LHNs, suggesting this area may play multiple roles. Yet, surprisingly, we found no evidence to support a role for these neurons in the feedforward inhibition proposed to mediate olfactory response sparsening; instead, it appears that another mechanism, feedback inhibition from the giant GABAergic neuron, serves this function. Further, all LHNs we observed responded to all odors we tested, making it unlikely these LHNs serve as labeled lines mediating specific behavioral responses to specific odors. Our results rather point to three other possible roles of LHNs: extracting general stimulus features such as odor intensity; mediating bilateral integration of sensory information; and integrating multimodal sensory stimuli.

Spontaneous olfactory receptor neuron activity determines follower cell response properties.

Noisy or spontaneous activity is common in neural systems and poses a challenge to detecting and discriminating signals. Here we use the locust to answer fundamental questions about noise in the olfactory system: Where does spontaneous activity originate? How is this activity propagated or reduced throughout multiple stages of neural processing? What mechanisms favor the detection of signals despite the presence of spontaneous activity? We found that spontaneous activity long observed in the secondary projection neurons (PNs) originates almost entirely from the primary olfactory receptor neurons (ORNs) rather than from spontaneous circuit interactions in the antennal lobe, and that spontaneous activity in ORNs tonically depolarizes the resting membrane potentials of their target PNs and local neurons (LNs) and indirectly tonically depolarizes tertiary Kenyon cells (KCs). However, because these neurons have different response thresholds, in the absence of odor stimulation, ORNs and PNs display a high spontaneous firing rate but KCs are nearly silent. Finally, we used a simulation of the olfactory network to show that discrimination of signal and noise in the KCs is best when threshold levels are set so that baseline activity in PNs persists. Our results show how the olfactory system benefits from making a signal detection decision after a point of maximal information convergence, e.g., after KCs pool inputs from many PNs.

Excitatory local interneurons enhance tuning of sensory information.

Neurons in the insect antennal lobe represent odors as spatiotemporal patterns of activity that unfold over multiple time scales. As these patterns unspool they decrease the overlap between odor representations and thereby increase the ability of the olfactory system to discriminate odors. Using a realistic model of the insect antennal lobe we examined two competing components of this process -lateral excitation from local excitatory interneurons, and slow inhibition from local inhibitory interneurons. We found that lateral excitation amplified differences between representations of similar odors by recruiting projection neurons that did not receive direct input from olfactory receptors. However, this increased sensitivity also amplified noisy variations in input and compromised the ability of the system to respond reliably to multiple presentations of the same odor. Slow inhibition curtailed the spread of projection neuron activity and increased response reliability. These competing influences must be finely balanced in order to decorrelate odor representations.

Innate attraction and aversion to odors in locusts.

Many animals display innate preferences for some odors, but the physiological mechanisms underlying these preferences are poorly understood. Here, with behavioral tests, we establish a model system well suited to investigating olfactory mechanisms, the locust Schistocerca americana. We conducted open field tests in an arena designed to provide only olfactory cues to guide navigation choices. We found that newly hatched locusts navigated toward, and spent more time near, the odor of wheat grass than humidified air. In similar tests, we found that hatchlings avoided moderate concentrations of major individual components of the food blend odor, 1-hexanol (1% v/v) and hexanal (0.9% v/v) diluted in mineral oil relative to control presentations of unscented mineral oil. Hatchlings were neither attracted nor repelled by a lower concentration (0.1% v/v) of 1-hexanol but were moderately attracted to a low concentration (0.225% v/v) of hexanal. We quantified the behavior of the animals by tracking their positions with the Argos software toolkit. Our results establish that hatchlings have a strong, innate preference for food odor blend, but the valence of the blend's individual components may be different and may change depending on the concentration. Our results provide a useful entry point for an analysis of physiological mechanisms underlying innate sensory preferences.

Olfactory receptor neurons generate multiple response motifs, increasing coding space dimensionality.

Odorants binding to olfactory receptor neurons (ORNs) trigger bursts of action potentials, providing the brain with its only experience of the olfactory environment. Our recordings made in vivo from locust ORNs showed that odor-elicited firing patterns comprise four distinct response motifs, each defined by a reliable temporal profile. Different odorants could elicit different response motifs from a given ORN, a property we term motif switching. Further, each motif undergoes its own form of sensory adaptation when activated by repeated plume-like odor pulses. A computational model constrained by our recordings revealed that organizing responses into multiple motifs provides substantial benefits for classifying odors and processing complex odor plumes: each motif contributes uniquely to encode the plume's composition and structure. Multiple motifs and motif switching further improve odor classification by expanding coding dimensionality. Our model demonstrated that these response features could provide benefits for olfactory navigation, including determining the distance to an odor source.

Peripheral and central olfactory tuning in a moth.

Animals can be innately attracted to certain odorants. Because these attractants are particularly salient, they might be expected to induce relatively strong responses throughout the olfactory pathway, helping animals detect the most relevant odors but limiting flexibility to respond to other odors. Alternatively, specific neural wiring might link innately preferred odors to appropriate behaviors without a need for intensity biases. How nonpheromonal attractants are processed by the general olfactory system remains largely unknown. In the moth Manduca sexta, we studied this with a set of innately preferred host plant odors and other, neutral odors. Electroantennogram recordings showed that, as a population, olfactory receptor neurons (ORNs) did not respond with greater intensity to host plant odors, and further local field potential recordings showed that no specific amplification of signals induced by host plant odors occurred between the first olfactory center and the second. Moreover, when odorants were mutually diluted to elicit equally intense output from the ORNs, moths were able to learn to associate all tested odorants equally well with food reward. Together, these results suggest that, although nonpheromonal host plant odors activate broadly distributed responses, they may be linked to attractive behaviors mainly through specific wiring in the brain.

Argos: A toolkit for tracking multiple animals in complex visual environments.

Automatically tracking the positions of multiple animals is often necessary for studying behaviours. This task involves multiple object tracking, a challenging problem in computer vision. Recent advances in machine learning applied to video analysis have been helpful for animal tracking. However, existing tools work well only in homogeneous environments with uniform illumination, features rarely found in natural settings. Moreover, available algorithms cannot effectively process discontinuities in animal motion such as sudden jumps, thus requiring laborious manual review.Here we present Argos, a software toolkit for tracking multiple animals in inhomogeneous environments. Argos includes tools for compressing videos based on animal movement, for generating training sets for a convolutional neural network (CNN) to detect animals, for tracking multiple animals in a video and for facilitating review and correction of the tracks manually, with simple graphical user interfaces.We demonstrate that Argos can help reduce the amount of video data to be stored and analysed, speed up analysis and allow analysing difficult and ambiguous conditions in a scene.Thus, Argos supports multiple approaches to animal tracking suited for varying recording conditions and available computational resources. Together, these tools allow the recording and tracking of movements of multiple markerless animals in inhomogeneous environments over many hours.

Insect neuroscience: Filling the knowledge gap on gap junctions.

Gap junctions, too small to spot in images used to create connectome maps, play outsized roles in shaping neural activity. A recent study reveals a surprising new gap junction function: they can stabilize a neuron's membrane potential against unwanted oscillations.

Identification and analysis of odorant receptors expressed in the two main olfactory organs, antennae and palps, of Schistocerca americana.

Locusts depend upon their sense of smell and provide useful models for understanding olfaction. Extending this understanding requires knowledge of the molecular and structural organization of the olfactory system. Odor sensing begins with olfactory receptor neurons (ORNs), which express odorant receptors (ORs). In insects, ORNs are housed, in varying numbers, in olfactory sensilla. Because the organization of ORs within sensilla affects their function, it is essential to identify the ORs they contain. Here, using RNA sequencing, we identified 179 putative ORs in the transcriptomes of the two main olfactory organs, antenna and palp, of the locust Schistocerca americana. Quantitative expression analysis showed most putative ORs (140) are expressed in antennae while only 31 are in the palps. Further, our analysis identified one OR detected only in the palps and seven ORs that are expressed differentially by sex. An in situ analysis of OR expression suggested ORs are organized in non-random combinations within antennal sensilla. A phylogenetic comparison of OR predicted protein sequences revealed homologous relationships among two other Acrididae species. Our results provide a foundation for understanding the organization of the first stage of the olfactory system in S. americana, a well-studied model for olfactory processing.