Cell type-specific and frequency-dependent centrifugal modulation in olfactory bulb output neurons in vivo.

Mitral/tufted cells (M/TCs) form complex local circuits with interneurons in the olfactory bulb and are powerfully inhibited by these interneurons. The horizontal limb of the diagonal band of Broca (HDB), the only GABAergic/inhibitory source of centrifugal circuit with the olfactory bulb, is known to target olfactory bulb interneurons, and we have shown targeting also to olfactory bulb glutamatergic neurons in vitro. However, the net efficacy of these circuits under different patterns of activation in vivo and the relative balance between the various targeted intact local and centrifugal circuits was the focus of this study. Here channelrhodopsin-2 (ChR2) was expressed in HDB GABAergic neurons to investigate the short-term plasticity of HDB-activated disinhibitory rebound excitation of M/TCs. Optical activation of HDB interneurons increased spontaneous M/TC firing without odor presentation and increased odor-evoked M/TC firing. HDB activation induced disinhibitory rebound excitation (burst or cluster of spiking) in all classes of M/TCs. This excitation was frequency dependent, with short-term facilitation only at higher HDB stimulation frequency (5 Hz and above). However, frequency-dependent HDB regulation was more potent in the deeper layer M/TCs compared with more superficial layer M/TCs. In all neural circuits the balance between inhibition and excitation in local and centrifugal circuits plays a critical functional role, and this patterned input-dependent regulation of inhibitory centrifugal inputs to the olfactory bulb may help maintain the precise balance across the populations of output neurons in different environmental odors, putatively to sharpen the enhancement of tuning specificity of individual or classes of M/TCs to odors.NEW & NOTEWORTHY Neuronal local circuits in the olfactory bulb are modulated by centrifugal long circuits. In vivo study here shows that inhibitory horizontal limb of the diagonal band of Broca (HDB) modulates all five types of mitral/tufted cells (M/TCs), by direct inhibitory circuits HDB → M/TCs and indirect disinhibitory long circuits HDB → interneurons → M/TCs. The HDB net effect exerts excitation in all types of M/TCs but more powerful in deeper layer output neurons as HDB activation frequency increases, which may sharpen the tuning specificity of classes of M/TCs to odors during sensory processing.

Generative Adversarial Networks in Digital Histopathology: Current Applications, Limitations, Ethical Considerations, and Future Directions.

Generative adversarial networks (GANs) have gained significant attention in the field of image synthesis, particularly in computer vision. GANs consist of a generative model and a discriminative model trained in an adversarial setting to generate realistic and novel data. In the context of image synthesis, the generator produces synthetic images, whereas the discriminator determines their authenticity by comparing them with real examples. Through iterative training, the generator allows the creation of images that are indistinguishable from real ones, leading to high-quality image generation. Considering their success in computer vision, GANs hold great potential for medical diagnostic applications. In the medical field, GANs can generate images of rare diseases, aid in learning, and be used as visualization tools. GANs can leverage unlabeled medical images, which are large in size, numerous in quantity, and challenging to annotate manually. GANs have demonstrated remarkable capabilities in image synthesis and have the potential to significantly impact digital histopathology. This review article focuses on the emerging use of GANs in digital histopathology, examining their applications and potential challenges. Histopathology plays a crucial role in disease diagnosis, and GANs can contribute by generating realistic microscopic images. However, ethical considerations arise because of the reliance on synthetic or pseudogenerated images. Therefore, the manuscript also explores the current limitations and highlights the ethical considerations associated with the use of this technology. In conclusion, digital histopathology has seen an emerging use of GANs for image enhancement, such as color (stain) normalization, virtual staining, and ink/marker removal. GANs offer significant potential in transforming digital pathology when applied to specific and narrow tasks (preprocessing enhancements). Evaluating data quality, addressing biases, protecting privacy, ensuring accountability and transparency, and developing regulation are imperative to ensure the ethical application of GANs.

Social odor choice buffers drug craving.

Social interactions are rewarding and protective against substance use disorders, but it is unclear which specific aspect of the complex sensory social experience drives these effects. Here, we investigated the role of olfactory sensory experience on social interaction, social preference over cocaine, and cocaine craving in rats. First, we conducted bulbectomy on both male and female rats to evaluate the necessity of olfactory system experience on the acquisition and maintenance of volitional social interaction. Next, we assessed the effect of bulbectomy on rats given a choice between social interaction and cocaine. Finally, we evaluated the influence of olfactory sensory experience by training rats on volitional partner-associated odors, assessing their preference for partner odors over cocaine to achieve voluntary abstinence and assessing its effect on the incubation of cocaine craving. Bulbectomy impaired operant social interaction without affecting food and cocaine self-administration. Rats with intact olfactory systems preferred social interaction over cocaine, while rats with impaired olfactory sense showed a preference for cocaine. Providing access to a partner odor in a choice procedure led to cocaine abstinence, preventing incubation of cocaine craving, in contrast to forced abstinence or non-contingent exposure to cocaine and partner odors. Our data suggests the olfactory sensory experience is necessary and sufficient for volitional social reward. Furthermore, the active preference for partner odors over cocaine buffers drug craving. Based on these findings, translational research should explore the use of social sensory-based treatments utilizing odor-focused foundations for individuals with substance use disorders.

Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses.

BACKGROUND: Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. METHODS: We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. RESULTS: Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. CONCLUSIONS: These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79-97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board.

Frequency-dependent centrifugal modulation of the activity of different classes of mitral and tufted cells in olfactory bulb.

Mitral/tufted cells (M/TCs), the principal output neuron classes form complex circuits with bulbar neurons and long-range centrifugal circuits with higher processing areas such as the horizontal limb of the diagonal band of Broca (HDB). The precise excitability of output neurons is sculpted by local inhibitory circuits. Here, light-gated cation channel channelrhodopsin-2 (ChR2) was expressed in HDB GABAergic neurons to investigate the short-term plasticity of evoked postsynaptic currents/potentials of HDB input to all classes of M/TCs and effects on firing in the acute slice preparation. Activation of the HDB directly inhibited all classes of output neurons exhibiting frequency-dependent short-term depression of evoked inhibitory postsynaptic current (eIPSC)/potential (eIPSP), resulting in decreased inhibition of responses to olfactory nerve input as a function of input frequency. In contrast, activation of an indirect circuit of HDB→interneurons→M/TCs induced frequency-dependent disinhibition, resulting in short-term facilitation of evoked excitatory postsynaptic current (eEPSC) eliciting a burst or cluster of spiking in M/TCs. The facilitatory effects of elevated HDB input frequency were strongest on deeper output neurons (deep tufted and mitral cells) and negligible on peripheral output neurons (external and superficial tufted cells). Taken together, GABAergic HDB activation generates frequency-dependent regulation that differentially affects the excitability and responses across the five classes of M/TCs. This regulation may help maintain the precise balance between inhibition and excitation of neuronal circuits across the populations of output neurons in the face of changes in an animal sniffing rate, putatively to enhance and sharpen the tuning specificity of individual or classes of M/TCs to odors.NEW & NOTEWORTHY Neuronal circuits in the olfactory bulb closely modulate olfactory bulb output activity. Activation of GABAergic circuits from the HDB to the olfactory bulb has both direct and indirect action differentially across the five classes of M/TC bulbar output neurons. The net effect enhances the excitability of deeper output neurons as HDB frequency increases, altering the relative inhibition-excitation balance of output circuits. We hypothesize that this sharpens the tuning specificity of classes of M/TCs to odors during sensory processing.

Fluorescence Lifetime Imaging of Human Sub-RPE Calcification In Vitro Following Chlortetracycline Infusion.

We have shown that all sub-retinal pigment epithelial (sub-RPE) deposits examined contain calcium phosphate minerals: hydroxyapatite (HAP), whitlockite (Wht), or both. These typically take the form of ca. 1 µm diameter spherules or >10 µm nodules and appear to be involved in the development and progression of age-related macular degeneration (AMD). Thus, these minerals may serve as useful biomarkers the for early detection and monitoring of sub-RPE changes in AMD. We demonstrated that HAP deposits could be imaged in vitro by fluorescence lifetime imaging microscopy (FLIM) in flat-mounted retinas using legacy tetracycline antibiotics as selective sensors for HAP. As the contrast on a FLIM image is based on the difference in fluorescence lifetime and not intensity of the tetracycline-stained HAP, distinguishing tissue autofluorescence from the background is significantly improved. The focus of the present pilot study was to assess whether vascular perfusion of the well tolerated and characterized chlortetracycline (widely used as an orally bioavailable antibiotic) can fluorescently label retinal HAP using human cadavers. We found that the tetracycline delivered through the peripheral circulation can indeed selectively label sub-RPE deposits opening the possibility for its use for ophthalmic monitoring of a range of diseases in which deposit formation is reported, such as AMD and Alzheimer disease (AD).

OpNotes and Clinical Exercises: Activities to Enhance the Clinical Context of the Preclerkship Anatomy Dissection Laboratory.

PROBLEM: Despite numerous pedagogical approaches and technologies now available for medical gross anatomy, students can find it difficult to translate what occurs in a dissection laboratory into the context of clinical practice. APPROACH: Using complementary and collaborative approaches at 2 different medical schools, Virginia Commonwealth University (VCU) and University of Maryland (UM), we designed and implemented a series of clinical activities in the preclerkship medical gross anatomy laboratory that directly link dissected structures to clinical procedures. These activities specifically direct students to perform simulated clinically related procedures on anatomic donors during laboratory dissection sessions. The activities are called OpNotes at VCU and Clinical Exercises at UM. Each activity in the VCU OpNotes requires about 15 minutes of group activity at the end of a scheduled laboratory and involves faculty to grade the student responses submitted via a web-based-assessment form. Each exercise in UM Clinical Exercises also requires about 15 minutes of group activity during the schedule laboratory but does not involve faculty to complete grading. OUTCOMES: Cumulatively, the activities in OpNotes and Clinical Exercises both brought clinical context directly to anatomical dissections. These activities began in 2012 at UM and 2020 at VCU, allowing a multiyear and multi-institute development and testing of this innovative approach. Student participation was high, and perception of its effectiveness was almost uniformly positive. NEXT STEPS: Future iterations of the program will work to assess the efficacy of the program as well as to streamline the scoring and delivery of the formative components. Collectively, we propose that the concept of executing clinic-like procedures on donors in anatomy courses is an effective means of enhancing learning in the anatomy laboratory while concurrently underscoring the relevance of basic anatomy to future clinical practice.

Bulbar projecting subcortical GABAergic neurons send collateral branches extensively and selectively to primary olfactory cortical regions.

Circuit operations of the olfactory bulb are modulated by higher order projections from multiple regions, many of which are themselves targets of bulbar output. Multiple glutamatergic regions project to the olfactory bulb, including the anterior olfactory nucleus (AON), prefrontal cortex (PFC), piriform cortex (PC), entorhinal cortex (EC), and tenia tecta (TT). In contrast, only one region provides GABAergic projections to the bulb. These GABA neurons are located in the horizontal limb of the diagonal band of Broca extending posteriorly through the magnocellular preoptic nucleus to the nucleus of the lateral olfactory bulb. However, it was unclear whether bulbar projecting GABAergic neurons collaterallize projecting to other brain regions. To address this, we mapped collateral projections from bulbar projecting GABAergic neurons using intersectional strategies of viral and traditional tract tracers. This approach revealed bulbar projecting GABAergic neurons show remarkable specificity targeting other primary olfactory cortical regions exhibiting abundant collateral projections into the accessory olfactory bulb, AON, PFC, PC, and TT. The only "nonolfactory" region receiving collateral projections was sparse connectivity to the medial prefrontal orbital cortex. This suggests that basal forebrain inhibitory feedback also modulates glutamatergic feedback areas that are themselves prominent bulbar projection regions. Thus, inhibitory feedback may be simultaneously modulating both synaptic processing of olfactory information in the bulb and associational processing of olfactory information from primary olfactory cortex. We hypothesize that these olfactory GABAergic feedback neurons are a regulator of the entire olfactory system.

Validation of Muscle Fiber Architecture of the Human Tongue Revealed by Diffusion Magnetic Resonance Imaging With Histology Verification.

PURPOSE: The goal of this study is to validate the muscle architecture derived from both ex vivo and in vivo diffusion-weighted magnetic resonance imaging (dMRI) of the human tongue with histology of an ex vivo tongue. METHOD: dMRI was acquired with a 200-direction high angular resolution diffusion imaging (HARDI) diffusion scheme for both a postmortem head (imaged within 48 hr after death) and a healthy volunteer. After MRI, the postmortem head was fixed and the tongue excised for hematoxylin and eosin (H&E) staining and histology imaging. Structure tensor images were generated from the stained images to better demonstrate muscle fiber orientations. The tongue muscle fiber orientations, estimated from dMRI, were visualized using the tractogram, a novel representation of crossing fiber orientations, and compared against the histology images of the ex vivo tongue. RESULTS: Muscle fibers identified in the tractograms showed good correspondence with those appearing in the histology images. We further demonstrated tongue muscle architecture in in vivo tractograms for the entire tongue. CONCLUSION: The study demonstrates that dMRI can accurately reveal the complex muscle architecture of the human tongue and may potentially benefit planning and evaluation of oral surgery and research on speech and swallowing.

Quantitative analysis of intermuscular septa in the leg: implications for trauma surgery.

BACKGROUND: Compartment syndrome is the excess swelling within an inelastic compartment leading to excessive compartment pressure. Lower limb trauma has a high risk of compartment syndrome, which is typically mitigated using a two-incision fasciotomy. Our previous findings showed surgeons sometimes perform incomplete fasciotomies due to misidentifying the septum between the lateral and superficial posterior compartments as the septum between the anterior and lateral compartments. We conjectured this may be due to variability in the septal position between individuals leading to misinterpretation of the septal identity. METHODS: A retrospective analysis was performed using CT angiograms to analyze septal position between the anterior and lateral compartments of the leg of 100 patients randomly selected from the University of Maryland Shock Trauma Center database. RESULTS: Analysis of septal position showed that (1) as the septum progresses distally down the leg, the relative septum position shifts anteriorly; and that (2) there was considerable variability in the intermuscular septum position between individuals even when accounting for the anterior to posterior progression of septal position. DISCUSSION: This variability could lead to erroneous septal identification in individuals with a very anteriorly located septum during a leg fasciotomy with the classic initial incision being insufficiently anterior. We propose making the lateral initial incision 'two finger breadths posterior the tibia' rather than the traditional 'one finger breadth anterior' to the fibula. This moves the initial incision slightly anteriorly, uses the more readily palpable tibia, and makes the medial and lateral incisions symmetrical at 'two finger breadths' from the tibia, simplifying the procedure. LEVEL OF EVIDENCE: Level 3.

Short-Term Plasticity in Cortical GABAergic Synapses on Olfactory Bulb Granule Cells Is Modulated by Endocannabinoids.

Olfactory bulb and higher processing areas are synaptically interconnected, providing rapid regulation of olfactory bulb circuit dynamics and sensory processing. Short-term plasticity changes at any of these synapses could modulate sensory processing and potentially short-term sensory memory. A key olfactory bulb circuit for mediating cortical feedback modulation is granule cells, which are targeted by multiple cortical regions including both glutamatergic excitatory inputs and GABAergic inhibitory inputs. There is robust endocannabinoid modulation of excitatory inputs to granule cells and here we explored whether there was also endocannabinoid modulation of the inhibitory cortical inputs to granule cells. We expressed light-gated cation channel channelrhodopsin-2 (ChR2) in GABAergic neurons in the horizontal limb of the diagonal band of Broca (HDB) and their projections to granule cells in olfactory bulb. Selective optical activation of ChR2 positive axons/terminals generated strong, frequency-dependent short-term depression of GABA A -mediated-IPSC in granule cells. As cannabinoid type 1 (CB1) receptor is heavily expressed in olfactory bulb granule cell layer (GCL) and there is endogenous endocannabinoid release in GCL, we investigated whether activation of CB1 receptor modulated the HDB IPSC and short-term depression at the HDB→granule cell synapse. Activation of the CB1 receptor by the exogenous agonist Win 55,212-2 significantly decreased the peak amplitude of individual IPSC and decreased short-term depression, while blockade of the CB1 receptor by AM 251 slightly increased individual IPSCs and increased short-term depression. Thus, we conclude that there is tonic endocannabinoid activation of the GABAergic projections of the HDB to granule cells, similar to the modulation observed with glutamatergic projections to granule cells. Modulation of inhibitory synaptic currents and frequency-dependent short-term depression could regulate the precise balance of cortical feedback excitation and inhibition of granule cells leading to changes in granule cell mediated inhibition of olfactory bulb output to higher processing areas.

Imaging hydroxyapatite in sub-retinal pigment epithelial deposits by fluorescence lifetime imaging microscopy with tetracycline staining.

SIGNIFICANCE: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of age-related macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits. However, HAP-bound tetracycline fluorescence excitation and emission spectra overlap with the well-known autofluorescence of outer retinal tissues, making them difficult to resolve. AIM: In this initial study, we sought to determine if the HAP-bound tetracyclines also exhibit enhanced fluorescence lifetimes, providing a useful difference in lifetime compared with the short lifetimes observed in vivo in the human retina by the pioneering work of Schweitzer, Zinkernagel, Hammer, and their colleagues, and thus a large enough effect size to resolve the HAP from background by fluorescence lifetime imaging. APPROACH: We stained authentic HAP with tetracyclines and measured the lifetime(s) by phase fluorometry, and stained aged, fixed human cadaver retinas with drusen with selected tetracyclines and imaged them by fluorescence lifetime imaging microscopy (FLIM). RESULTS: We found that chlortetracycline and doxycycline exhibited substantial increase in fluorescence lifetime compared to the free antibiotics and the retinal background, and the drusen were easily resolvable from the retinal background in these specimens by FLIM. CONCLUSIONS: These findings suggest that FLIM imaging of tetracycline (and potentially other molecules) binding to HAP could become a diagnostic tool for the development and progression of AMD.

Short-term plasticity in glomerular inhibitory circuits shapes olfactory bulb output.

Short-term plasticity is a fundamental synaptic property thought to underlie memory and neural processing. The glomerular microcircuit comprises complex excitatory and inhibitory interactions and transmits olfactory nerve signals to the excitatory output neurons, mitral/tufted cells (M/TCs). The major glomerular inhibitory interneurons, short axon cells (SACs) and periglomerular cells (PGCs), both provide feedforward and feedback inhibition to M/TCs and have reciprocal inhibitory synapses between each other. Olfactory input is episodically driven by sniffing. We hypothesized that frequency-dependent short-term plasticity within these inhibitory circuits could influence signals sent to higher-order olfactory networks. To assess short-term plasticity in glomerular circuits and MC outputs, we virally delivered channelrhodopsin-2 (ChR2) in glutamic acid decarboxylase-65 promotor (GAD2-cre) or tyrosine hydroxylase promoter (TH-cre) mice and selectively activated one of these two populations while recording from cells of the other population or from MCs. Selective activation of TH-ChR2-expressing SACs inhibited all recorded GAD2-green fluorescent protein(GFP)-expressing presumptive PGC cells, and activation of GAD2-ChR2 cells inhibited TH-GFP-expressing SACs, indicating reciprocal inhibitory connections. SAC synaptic inhibition of GAD2-expressing cells was significantly facilitated at 5-10 Hz activation frequencies. In contrast, GAD2-ChR2 cell inhibition of TH-expressing cells was activation-frequency independent. Both SAC and PGC inhibition of MCs also exhibited short-term plasticity, pronounced in the 5-20 Hz range corresponding to investigative sniffing frequency ranges. In paired SAC and olfactory nerve electrical stimulations, the SAC to MC synapse was able to markedly suppress MC spiking. These data suggest that short-term plasticity across investigative sniffing ranges may differentially regulate intra- and interglomerular inhibitory circuits to dynamically shape glomerular output signals to downstream targets.NEW & NOTEWORTHY Short-term plasticity is a fundamental synaptic property that modulates synaptic strength based on preceding activity of the synapse. In rodent olfaction, sensory input arrives episodically driven by sniffing rates ranging from quiescent respiration (1-2 Hz) through to investigative sniffing (5-10 Hz). Here we show that glomerular inhibitory networks are exquisitely sensitive to input frequencies and exhibit plasticity proportional to investigative sniffing frequencies. This indicates that olfactory glomerular circuits are dynamically modulated by episodic sniffing input.

Reciprocal Inhibitory Glomerular Circuits Contribute to Excitation-Inhibition Balance in the Mouse Olfactory Bulb.

The major inhibitory interneurons in olfactory bulb (OB) glomeruli are periglomerular cells (PGCs) and short axon cells (SACs). PGCs and SACs provide feedforward inhibition to all classes of projection neurons, but inhibition between PGCs and SACs is not well understood. We crossed Cre and GFP transgenic mice and used virally-delivered optogenetic constructs to selectively activate either SACs or GAD65cre-ChR2-positive PGCs while recording from identified GAD65cre-ChR2-positive PGCs or SACs, respectively, to investigate inhibitory interactions between these two interneuron types. We show that GAD65cre-ChR2-positive PGCs robustly inhibit SACs and SACs strongly inhibit PGCs. SACs form the interglomerular circuit, which inhibits PGCs in distant glomeruli. Activation of GAD65cre-ChR2-positive PGCs monosynaptically inhibit mitral cells (MCs), which complements recent findings that SACs directly inhibit MCs. Thus, both classes of glomerular inhibitory neurons inhibit each other, as well as OB output neurons. We further show that olfactory nerve input to one glomerulus engages the interglomerular circuit and inhibits PGCs in distant glomeruli. Sensory activation of the interglomerular circuit directly inhibits output neurons in other glomeruli and by inhibiting intraglomerular PGCs, may potentially disinhibit output neurons in other glomeruli. The nature and context of odorant stimuli may determine whether inhibition or excitation prevails so that odors are represented in part by patterns of active and inactive glomeruli.

Short-Term Plasticity at Olfactory Cortex to Granule Cell Synapses Requires CaV2.1 Activation.

Output projections of the olfactory bulb (OB) to the olfactory cortex (OCX) and reciprocal feedback projections from OCX provide rapid regulation of OB circuit dynamics and odor processing. Short-term synaptic plasticity (STP), a feature of many synaptic connections in the brain, can modulate the strength of feedback based on preceding network activity. We used light-gated cation channel channelrhodopsin-2 (ChR2) to investigate plasticity of excitatory synaptic currents (EPSCs) evoked at the OCX to granule cell (GC) synapse in the OB. Selective activation of OCX glutamatergic axons/terminals in OB generates strong, frequency-dependent STP in GCs. This plasticity was critically dependent on activation of CaV2.1 channels. As acetylcholine (ACh) modulates CaV2.1 channels in other brain regions and as cholinergic projections from the basal forebrain heavily target the GC layer (GCL) in OB, we investigated whether ACh modulates STP at the OCX→GC synapse. ACh decreases OCX→GC evoked EPSCs, it had no effect on STP. Thus, ACh impact on cortical feedback is independent of CaV2.1-mediated STP. Modulation of OCX feedback to the bulb by modulatory transmitters, such as ACh, or by frequency-dependent STP could regulate the precise balance of excitation and inhibition of GCs. As GCs are a major inhibitory source for OB output neurons, plasticity at the cortical feedback synapse can differentially impact OB output to higher-order networks in situations where ACh inputs are activated or by active sniff sampling of odors.

Aeromedical evacuation-relevant hypobaria worsens axonal and neurologic injury in rats after underbody blast-induced hyperacceleration.

BACKGROUND: Occupants of military vehicles targeted by explosive devices often suffer from traumatic brain injury (TBI) and are typically transported by the aeromedical evacuation (AE) system to a military medical center within a few days. This study tested the hypothesis that exposure of rats to AE-relevant hypobaria worsens cerebral axonal injury and neurologic impairment caused by underbody blasts. METHODS: Anesthetized adult male rats were secured within cylinders attached to a metal plate, simulating the hull of an armored vehicle. An explosive located under the plate was detonated, resulting in a peak vertical acceleration force on the plate and occupant rats of 100G. Rats remained under normobaria or were exposed to hypobaria equal to 8,000 feet in an altitude chamber for 6 hours, starting at 6 hours to 6 days after blast. At 7 days, rats were tested for vestibulomotor function using the balance beam walking task and euthanized by perfusion. The brains were then analyzed for axonal fiber injury. RESULTS: The number of internal capsule silver-stained axonal fibers was greater in animals exposed to 100G blast than in shams. Animals exposed to hypobaria starting at 6 hours to 6 days after blast exhibited more silver-stained fibers than those not exposed to hypobaria. Rats exposed to 100% oxygen (O2) during hypobaria at 24 hours postblast displayed greater silver staining and more balance beam foot-faults, in comparison with rats exposed to hypobaria under 21% O2. CONCLUSION: Exposure of rats to blast-induced acceleration of 100G increases cerebral axonal injury, which is significantly exacerbated by exposure to hypobaria as early as 6 hours and as late as 6 days postblast. Rats exposed to underbody blasts and then to hypobaria under 100% O2 exhibit increased axonal damage and impaired motor function compared to those subjected to blast and hypobaria under 21% O2. These findings raise concern about the effects of AE-related hypobaria on TBI victims, the timing of AE after TBI, and whether these effects can be mitigated by supplemental oxygen.

Decreased Nucleus Accumbens Expression of Psychiatric Disorder Risk Gene Cacna1c Promotes Susceptibility to Social Stress.

Background: Polymorphisms in the CACNA1C gene are associated with human mood disorders. The rodent social defeat model of stress/mood-disorder susceptibility results in maladaptive consequences mediated by altered function of mesolimbic circuits. Methods: mRNA levels of Cacna1c in the nucleus accumbens of mice exposed to social defeat were assessed. Cacna1c was selectively deleted in the nucleus accumbens of floxed Cacna1c mice using viral Cre-recombinase to examine Cacna1c in social defeat susceptibility. Results: Reduced expression of Cacan1c in the nucleus accumbens is associated with increased susceptibility to social defeat stress, and a knockdown of Cacna1c in the nucleus accumbens significantly increases susceptibility measured by social interaction and female urine preference. Conclusions: Cacna1c reduction causally predisposes to the maladaptive outcomes of social stress. Normal Cacna1c function in the nucleus accumbens is crucial for resiliency to social stressors. Variations in expression of CACNA1C in the nucleus accumbens may mediate human risk for developing mood disorders and be a target for therapeutic intervention.

Using Intrinsic Flavoprotein and NAD(P)H Imaging to Map Functional Circuitry in the Main Olfactory Bulb.

Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB.