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.

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.

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.

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).

Hyperhomocysteinemia-Induced Oxidative Stress Exacerbates Cortical Traumatic Brain Injury Outcomes in Rats.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among military service members and civilians in the United States. Despite significant advances in the understanding of TBI pathophysiology, several clinical reports indicate that multiple genetic and epigenetic factors can influence outcome. Homocysteine (HCY) is a non-proteinogenic amino acid, the catabolism of which can be dysregulated by stress, lifestyle, aging, or genetic abnormalities leading to hyperhomocysteinemia (HHCY). HHCY is a neurotoxic condition and a risk factor for multiple neurological and cardiovascular disorders that occurs when HCY levels is clinically > 15 µM. Although the deleterious impact of HHCY has been studied in human and animal models of neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease, it has not been addressed in TBI models. This study tested the hypothesis that HHCY has detrimental effects on TBI pathophysiology. Moderate HHCY was induced in adult male Sprague Dawley rats via daily administration of methionine followed by impact-induced traumatic brain injury. In this model, HHCY increased oxidative stress, upregulated expression of proteins that promote blood coagulation, exacerbated TBI-associated blood-brain barrier dysfunction and promoted the infiltration of inflammatory cells into the cortex. We also observed an increase of brain injury-induced lesion size and aggravated anxiety-like behavior. These findings show that moderate HHCY exacerbates TBI outcomes and suggest that HCY catabolic dysregulation may be a significant biological variable that could contribute to TBI pathophysiology heterogeneity.

Enhanced Training Benefits of Video Recording Surgery With Automated Hand Motion Analysis.

BACKGROUND: Hand motion analysis by video recording during surgery has potential for evaluation of surgical performance. The aim was to identify how technical skill during open surgery can be measured unobtrusively by video recording during a surgical procedure. We hypothesized that procedural-step timing, hand movements, instrument use and Shannon entropy differ with expertise and training and are concordant with a performance-based validated individual procedure score. METHODS: Surgeon and non-surgeon participants with varying training and levels of expertise were video recorded performing axillary artery exposure and control (AA) on un-preserved cadavers. Color-coded gloves permitted motion-tracking and automated extraction of entropy data from recordings. Timing and instrument-use metrics were obtained through observational video reviews. Shannon entropy measured speed, acceleration and direction by computer-vision algorithms. Findings were compared with individual procedure score for AA performance RESULTS: Experts had lowest entropy values, idle time, active time and shorter time to divide pectoralis minor, using fewer instruments. Residents improved with training, without reaching expert levels, and showed deterioration 12-18 months later. Individual procedure scores mirrored these results. Non-surgeons differed substantially. CONCLUSIONS: Hand motion entropy and timing metrics discriminate levels of surgical skill and training, and these findings are congruent with individual procedure score evaluations. These measures can be collected using consumer-level cameras and analyzed automatically with free software. Hand motion with video timing data may have widespread application to evaluate resident performance and can contribute to the range of evaluation and testing modalities available to educators, training course designers and surgical quality assurance programs.

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.

Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations.

Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.

Innately activated TLR4 signal in the nucleus accumbens is sustained by CRF amplification loop and regulates impulsivity.

Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABAA receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.

The Interglomerular Circuit Potently Inhibits Olfactory Bulb Output Neurons by Both Direct and Indirect Pathways.

UNLABELLED: Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combined in vitro and in vivo electrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activation in vivo generates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition. SIGNIFICANCE STATEMENT: Odor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination of in vitro and in vivo optogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action.

Subsecond Regulation of Synaptically Released Dopamine by COMT in the Olfactory Bulb.

UNLABELLED: The efficacy of neurotransmission depends on multiple factors, including presynaptic vesicular release of transmitter, postsynaptic receptor populations and clearance/inactivation of the transmitter. In the olfactory bulb (OB), short axon cells (SACs) form an interglomerular circuit that uses GABA and dopamine (DA) as cotransmitters. Selective optical activation of SACs causes GABA and DA co-release, resulting in a fast, postsynaptic GABA inhibitory response and a slower G-protein-coupled DA rebound excitation. In most systems, vesicular release of DA is cleared by the dopamine transporter (DAT). However, in the OB, high levels of specific DA metabolites suggest that enzymatic catalysis by catechol-O-methyl-transferase (COMT) predominates over DAT re-uptake. To assess this possibility we measured the amount of the DA breakdown enzyme, COMT, present in the OB. Compared with the striatum, the brain structure richest in DA terminals, the OB contains 50% more COMT per unit of tissue. Furthermore, the OB has dramatically less DAT compared with striatum, supporting the idea that COMT enzymatic breakdown, rather than DAT recycling, is the predominant mechanism for DA clearance. To functionally assess COMT inactivation of vesicular release of DA we used fast-scan cyclic voltammetry and pharmacological blockade of COMT. In mice expressing ChR2 in tyrosine hydroxylase-containing neurons, optical activation of SACs evoked robust DA release in the glomerular layer. The COMT inhibitor, tolcapone, increased the DA signal ∼2-fold, whereas the DAT inhibitor GBR12909 had no effect. Together, these data indicate that the OB preferentially employs COMT enzymatic inactivation of vesicular release of DA. SIGNIFICANCE STATEMENT: In the olfactory bulb (OB), odors are encoded by glomerular activation patterns. Dopaminergic short axon neurons (SACs) form an extensive network of lateral connections that mediate cross talk among glomeruli, releasing GABA and DA onto sensory nerve terminals and postsynaptic neurons. DA neurons are ∼10-fold more numerous in OB than in ventral tegmental areas that innervate the striatum. We show that OB has abundant expression of the DA catalytic enzyme catechol-O-methyl-transferase (COMT), but negligible expression of the dopamine transporter. Using optogenetics and fast-scan cyclic voltammetry, we show that inhibition of COMT increases DA signals ∼2-fold. Thus, in contrast to the striatum, which has the brain's highest proportion of DAergic synapses, the DA catalytic pathway involving COMT predominates over re-uptake in OB.

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.

Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output.

Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeruli, the initial site of synaptic integration in the olfactory system. Both nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs) are expressed in glomeruli. The activation of nAChRs directly excites both mitral/tufted cells (MTCs) and external tufted cells (ETCs), the two major excitatory neurons that transmit glomerular output. The functional roles of mAChRs in glomerular circuits are unknown. We show that the restricted glomerular application of ACh causes rapid, brief nAChR-mediated excitation of both MTCs and ETCs in the mouse olfactory bulb. This excitation is followed by mAChR-mediated inhibition, which is blocked by GABAA receptor antagonists, indicating the engagement of periglomerular cells (PGCs) and/or short axon cells (SACs), the two major glomerular inhibitory neurons. Indeed, selective activation of glomerular mAChRs, with ionotropic GluRs and nAChRs blocked, increased IPSCs in MTCs and ETCs, indicating that mAChRs recruit glomerular inhibitory circuits. Selective activation of glomerular mAChRs in the presence of tetrodotoxin increased IPSCs in all glomerular neurons, indicating action potential-independent enhancement of GABA release from PGC and/or SAC dendrodendritic synapses. mAChR-mediated enhancement of GABA release also presynaptically suppressed the first synapse of the olfactory system via GABAB receptors on sensory terminals. Together, these results indicate that cholinergic modulation of glomerular circuits is biphasic, involving an initial excitation of MTC/ETCs mediated by nAChRs followed by inhibition mediated directly by mAChRs on PGCs/SACs. This may phasically enhance the sensitivity of glomerular outputs to odorants, an action that is consistent with recent in vivo findings.

Serotonin increases synaptic activity in olfactory bulb glomeruli.

Serotoninergic fibers densely innervate olfactory bulb glomeruli, the first sites of synaptic integration in the olfactory system. Acting through 5HT2A receptors, serotonin (5HT) directly excites external tufted cells (ETCs), key excitatory glomerular neurons, and depolarizes some mitral cells (MCs), the olfactory bulb's main output neurons. We further investigated 5HT action on MCs and determined its effects on the two major classes of glomerular interneurons: GABAergic/dopaminergic short axon cells (SACs) and GABAergic periglomerular cells (PGCs). In SACs, 5HT evoked a depolarizing current mediated by 5HT2C receptors but did not significantly impact spike rate. 5HT had no measurable direct effect in PGCs. Serotonin increased spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in PGCs and SACs. Increased sEPSCs were mediated by 5HT2A receptors, suggesting that they are primarily due to enhanced excitatory drive from ETCs. Increased sIPSCs resulted from elevated excitatory drive onto GABAergic interneurons and augmented GABA release from SACs. Serotonin-mediated GABA release from SACs was action potential independent and significantly increased miniature IPSC frequency in glomerular neurons. When focally applied to a glomerulus, 5HT increased MC spontaneous firing greater than twofold but did not increase olfactory nerve-evoked responses. Taken together, 5HT modulates glomerular network activity in several ways: 1) it increases ETC-mediated feed-forward excitation onto MCs, SACs, and PGCs; 2) it increases inhibition of glomerular interneurons; 3) it directly triggers action potential-independent GABA release from SACs; and 4) these network actions increase spontaneous MC firing without enhancing responses to suprathreshold sensory input. This may enhance MC sensitivity while maintaining dynamic range.

Cholinergic inputs from Basal forebrain add an excitatory bias to odor coding in the olfactory bulb.

Cholinergic modulation of central circuits is associated with active sensation, attention, and learning, yet the neural circuits and temporal dynamics underlying cholinergic effects on sensory processing remain unclear. Understanding the effects of cholinergic modulation on particular circuits is complicated by the widespread projections of cholinergic neurons to telencephalic structures that themselves are highly interconnected. Here we examined how cholinergic projections from basal forebrain to the olfactory bulb (OB) modulate output from the first stage of sensory processing in the mouse olfactory system. By optogenetically activating their axons directly in the OB, we found that cholinergic projections from basal forebrain regulate OB output by increasing the spike output of presumptive mitral/tufted cells. Cholinergic stimulation increased mitral/tufted cell spiking in the absence of inhalation-driven sensory input and further increased spiking responses to inhalation of odorless air and to odorants. This modulation was rapid and transient, was dependent on local cholinergic signaling in the OB, and differed from modulation by optogenetic activation of cholinergic neurons in basal forebrain, which led to a mixture of mitral/tufted cell excitation and suppression. Finally, bulbar cholinergic enhancement of mitral/tufted cell odorant responses was robust and occurred independent of the strength or even polarity of the odorant-evoked response, indicating that cholinergic modulation adds an excitatory bias to mitral/tufted cells as opposed to increasing response gain or sharpening response spectra. These results are consistent with a role for the basal forebrain cholinergic system in dynamically regulating the sensitivity to or salience of odors during active sensing of the olfactory environment.

Olfactory bulb short axon cell release of GABA and dopamine produces a temporally biphasic inhibition-excitation response in external tufted cells.

Evidence for coexpression of two or more classic neurotransmitters in neurons has increased, but less is known about cotransmission. Ventral tegmental area (VTA) neurons corelease dopamine (DA), the excitatory transmitter glutamate, and the inhibitory transmitter GABA onto target cells in the striatum. Olfactory bulb (OB) short axon cells (SACs) form interglomerular connections and coexpress markers for DA and GABA. Using an optogenetic approach, we provide evidence that mouse OB SACs release both GABA and DA onto external tufted cells (ETCs) in other glomeruli. Optical activation of channelrhodopsin specifically expressed in DAergic SACs produced a GABA(A) receptor-mediated monosynaptic inhibitory response, followed by DA-D(1)-like receptor-mediated excitatory response in ETCs. The GABA(A) receptor-mediated hyperpolarization activates I(h) current in ETCs; synaptically released DA increases I(h), which enhances postinhibitory rebound spiking. Thus, the opposing actions of synaptically released GABA and DA are functionally integrated by I(h) to generate an inhibition-to-excitation "switch" in ETCs. Consistent with the established role of I(h) in ETC burst firing, we show that endogenous DA release increases ETC spontaneous bursting frequency. ETCs transmit sensory signals to mitral/tufted output neurons and drive intraglomerular inhibition to shape glomerulus output to downstream olfactory networks. GABA and DA cotransmission from SACs to ETCs may play a key role in regulating output coding across the glomerular array.

Human airway epithelia express catalytically active NEU3 sialidase.

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

Binge alcohol drinking is associated with GABAA alpha2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala.

Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABA(A) α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABA(A) receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABA(A) α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABA(A) α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.

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.