Modulating the Excitability of Olfactory Output Neurons Affects Whole-Body Metabolism.

Metabolic state can alter olfactory sensitivity, but it is unknown whether the activity of the olfactory bulb (OB) may fine tune metabolic homeostasis. Our objective was to use CRISPR gene editing in male and female mice to enhance the excitability of mitral/tufted projection neurons (M/TCs) of the OB to test for improved metabolic health. Ex vivo slice recordings of MCs in CRISPR mice confirmed increased excitability due the targeted loss of Kv1.3 channels, which resulted in a less negative resting membrane potential (RMP), enhanced action potential (AP) firing, and insensitivity to the selective channel blocker margatoxin (MgTx). CRISPR mice exhibited enhanced odor discrimination using a habituation/dishabituation paradigm. CRISPR mice were challenged for 25 weeks with a moderately high-fat (MHF) diet, and compared with littermate controls, male mice were resistance to diet-induced obesity (DIO). Female mice did not exhibit DIO. CRISPR male mice gained less body weight, accumulated less white adipose tissue, cleared a glucose challenge more quickly, and had less serum leptin and liver triglycerides. CRISPR male mice consumed equivalent calories as control littermates, and had unaltered energy expenditure (EE) and locomotor activity, but used more fats for metabolic substrate over that of carbohydrates. Counter to CRISPR-engineered mice, by using chemogenetics to decrease M/TC excitability in male mice, activation of inhibitory designer receptors exclusively activated by designer drugs (DREADDs) caused a decrease in odor discrimination, and resulted in a metabolic profile that was obesogenic, mice had reduced EE and oxygen consumption (VO2). We conclude that the activity of M/TC projection neurons canonically carries olfactory information and simultaneously can regulate whole-body metabolism.SIGNIFICANCE STATEMENT The olfactory system drives food choice, and olfactory sensitivity is strongly correlated to hunger and fullness. Olfactory function thereby influences nutritional balance and obesity outcomes. Obesity has become a health and financial crisis in America, shortening life expectancy and increasing the severity of associated illnesses. It is expected that 51% of Americans will be obese by the year 2030. Using CRISPR gene editing and chemogenetic approaches, we discovered that changing the excitability of output neurons in the olfactory bulb (OB) affects metabolism and body weight stabilization in mice. Our results suggest that long-term therapeutic targeting of OB activity to higher processing centers may be a future clinical treatment of obesity or type II Diabetes.

Consumption of dietary fat causes loss of olfactory sensory neurons and associated circuitry that is not mitigated by voluntary exercise in mice.

Excess nutrition causes loss of olfactory sensory neurons (OSNs) and reduces odour discrimination and odour perception in mice. To separate diet-induced obesity from the consumption of dietary fat, we designed pair-feeding experiments whereby mice were maintained on isocaloric diets for 5 months, which prevented increased fat storage. To test our hypothesis that adiposity was not a prerequisite for loss of OSNs and bulbar projections, we used male and female mice with an odorant receptor-linked genetic reporter (M72tauLacZ; Olfr160) to visualize neural circuitry changes resulting from elevated fat in the diet. Simultaneously we monitored glucose clearance (diagnostic for prediabetes), body fat deposition, ingestive behaviours, select inflammatory markers and energy metabolism. Axonal projections to defined olfactory glomeruli were visualized in whole-mount brains, and the number of OSNs was manually counted across whole olfactory epithelia. After being pair fed a moderately high-fat (MHF) diet, mice of both sexes had body weight, adipose deposits, energy expenditure, respiratory exchange ratios and locomotor activity that were unchanged from control-fed mice. Despite this, they were still found to lose OSNs and associated bulbar projections. Even with unchanged adipocyte storage, pair-fed animals had an elevation in TNF cytokines and an intermediate ability for glucose clearance. Albeit improving health metrics, access to voluntary running while consuming an ad libitum fatty diet still precipitated a loss of OSNs and associated axonal projections for male mice. Our results support that long-term macronutrient imbalance can drive anatomical loss in the olfactory system regardless of total energy expenditure. KEY POINTS: Obesity can disrupt the structure and function of organ systems, including the olfactory system that is important for food selection and satiety. We designed dietary treatments in mice such that mice received fat, but the total calories provided were the same as in control diets so that they would not gain weight or increase adipose tissue. Mice that were not obese but consumed isocaloric fatty diets still lost olfactory neuronal circuits, had fewer numbers of olfactory neurons, had an elevation in inflammatory signals and had an intermediate ability to clear glucose (prediabetes). Mice were allowed access to running wheels while consuming fatty diets, yet still lost olfactory structures. We conclude that a long-term imbalance in nutrition that favours fat in the diet disrupts the olfactory system of mice in the absence of obesity.

A Multifunctional Contrast Agent for 19F-Based Magnetic Resonance Imaging.

Magnetic resonance imaging, MRI, relying on 19F nuclei has attracted much attention, because the isotopes exhibit a high gyromagnetic ratio (comparable to that of protons) and have 100% natural abundance. Furthermore, due to the very low traces of intrinsic fluorine in biological tissues, fluorine labeling allows easy visualization in vivo using 19F-based MRI. However, one of the drawbacks of the available fluorine tracers is their very limited solubility in water. Here, we detail the design and preparation of a set of water-compatible fluorine-rich polymers as contrast agents that can enhance the effectiveness of 19F-based MRI. The agents are synthesized using the nucleophilic addition reaction between poly(isobutylene-alt-maleic anhydride) copolymer and a mixture of amine-appended fluorine groups and polyethylene glycol (PEG) blocks. This allows control over the polymer architecture and stoichiometry, resulting in good affinity to water solutions. We further investigate the effects of introducing additional segmental mobility to the fluorine moieties in the polymer, by inserting a PEG linker between the moieties and the polymer backbone. We find that controlling the polymer stoichiometry and introducing additional segmental mobility enhance the NMR signals and narrow the peak profile. In particular, we assess the impact of the PEG linker on T2* and T1 relaxation times, using a series of gradient-recalled echo images with varying echo times, TE, or recovery time, TR, respectively. We find that for equivalent concentrations, the PEG linker greatly increases T2*, while maintaining high T1 values, as compared to polymers without this linker. Phantom images collected from these compounds show bright signals over a background with high intensities.

Olfactory bulb-targeted quantum dot (QD) bioconjugate and Kv1.3 blocking peptide improve metabolic health in obese male mice.

The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the individual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage-dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon-like peptide-1. Because gene-targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal-histidine coordination to self-assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch-clamp electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet-induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini-pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses.

Loss of odor-induced c-Fos expression of juxtaglomerular activity following maintenance of mice on fatty diets.

Diet-induced obesity (DIO) decreases the number of OMP+ olfactory sensory neurons (OSN) in the olfactory epithelium by 25% and reduces correlate axonal projections to the olfactory bulb (OB). Whether surviving OSNs have equivalent odor responsivity is largely unknown. Herein, we utilized c-fos immediate-early gene expression to map neuronal activity and determine whether mice weaned to control (CF), moderately-high fat (MHF), or high-fat (HF) diet for a period of 6 months had changes in odor activation. Diet-challenged M72-IRES-tau-GFP mice were exposed to either a preferred M72 (Olfr160) ligand, isopropyl tiglate, or clean air in a custom-made Bell-jar infusion chamber using an alternating odor exposure pattern generated by a picosprizer™. Mice maintained on fatty diets weighed significantly more and cleared glucose less efficiently as determined by an intraperitoneal glucose tolerance test (IPGTT). The number of juxtaglomerular cells (JGs) decreased following maintenance of the mice on the MHF diet for cells surrounding the medial but not lateral M72 glomerulus within a 4 cell-column distance. The percentage of c-fos + JGs surrounding the lateral M72 glomerulus decreased in fat-challenged mice whereas those surrounding the medial glomerulus were not affected by diet. Altogether, these results show an asymmetry in the responsiveness of the 'mirror image' glomerular map for the M72 receptor that shows greater sensitivity of the lateral vs. medial glomerulus upon exposure to fatty diet.

Differential serotonergic modulation across the main and accessory olfactory bulbs.

KEY POINTS: There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current-clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5-HT2A receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5-HT2 receptors on GABAergic interneurons and direct inhibition via the 5-HT1 receptors on MCs. Differential 5-HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression. ABSTRACT: Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5-HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15-30 day mice. In the MOB, 5-HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)- first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5-HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5-HT2A receptors through a direct activation. In comparison, 5-HT-evoked inhibitory responses in the AOB arose due to a polysynaptic, slow-onset inhibition attributed to 5-HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5-HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.

Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.

Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo.

The incretin hormone glucagon-like peptide 1 increases mitral cell excitability by decreasing conductance of a voltage-dependent potassium channel.

KEY POINTS: The gut hormone called glucagon-like peptide 1 (GLP-1) is a strong moderator of energy homeostasis and communication between the peripheral organs and the brain. GLP-1 signalling occurs in the brain; using a newly developed genetic reporter line of mice, we have discovered GLP-synthesizing cells in the olfactory bulb. GLP-1 increases the firing frequency of neurons (mitral cells) that encode olfactory information by decreasing activity of voltage-dependent K channels (Kv1.3). Modifying GLP-1 levels, either therapeutically or following the ingestion of food, could alter the excitability of neurons in the olfactory bulb in a nutrition or energy state-dependent manner to influence olfactory detection or metabolic sensing. The results of the present study uncover a new function for an olfactory bulb neuron (deep short axon cells, Cajal cells) that could be capable of modifying mitral cell activity through the release of GLP-1. This might be of relevance for the action of GLP-1 mimetics now widely used in the treatment of diabetes. ABSTRACT: The olfactory system is intricately linked with the endocrine system where it may serve as a detector of the internal metabolic state or energy homeostasis in addition to its classical function as a sensor of external olfactory information. The recent development of transgenic mGLU-yellow fluorescent protein mice that express a genetic reporter under the control of the preproglucagon reporter suggested the presence of the gut hormone, glucagon-like peptide (GLP-1), in deep short axon cells (Cajal cells) of the olfactory bulb and its neuromodulatory effect on mitral cell (MC) first-order neurons. A MC target for the peptide was determined using GLP-1 receptor binding assays, immunocytochemistry for the receptor and injection of fluorescence-labelled GLP-1 analogue exendin-4. Using patch clamp recording of olfactory bulb slices in the whole-cell configuration, we report that GLP-1 and its stable analogue exendin-4 increase the action potential firing frequency of MCs by decreasing the interburst interval rather than modifying the action potential shape, train length or interspike interval. GLP-1 decreases Kv1.3 channel contribution to outward currents in voltage clamp recordings as determined by pharmacological blockade of Kv1.3 or utilizing mice with Kv1.3 gene-targeted deletion as a negative control. Because fluctuations in GLP-1 concentrations monitored by the olfactory bulb can modify the firing frequency of MCs, olfactory coding could change depending upon nutritional or physiological state. As a regulator of neuronal activity, GLP-1 or its analogue may comprise a new metabolic factor with a potential therapeutic target in the olfactory bulb (i.e. via intranasal delivery) for controlling an imbalance in energy homeostasis.

Ubiquitin ligase Nedd4-2 modulates Kv1.3 current amplitude and ion channel protein targeting.

Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493-498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density.

Maternal ingestion of cannabidiol (CBD) in mice leads to sex-dependent changes in memory, anxiety, and metabolism in the adult offspring, and causes a decrease in survival to weaning age.

RATIONALE: The consequences of perinatal cannabidiol (CBD) exposure are severely understudied, but are important, given its widespread use and believed safety as a natural supplement. OBJECTIVE: The objective of this study was to test the health, metabolic, and behavioral consequences of perinatal CBD exposure on dams and their offspring raised to adult. METHODS: Primiparous female C57BL/6 J mice were orally administered 100 mg/kg CBD in strawberry jam to expose offspring during gestation, lactation, or both using a cross-fostering design. Adult offspring were metabolically profiled using indirect calorimetry and intraperitoneal glucose tolerance testing. Adults were behaviorally phenotyped, video recorded, and mouse position tracked using DeepLabCut. RESULTS: CBD was detected in maternal plasma using LC-MS 10-min post consumption (34.2 ±â€¯1.7 ng/ul) and peaked within 30 min (371.0 ±â€¯34.0 ng/ul). Fetal exposure to CBD significantly decreased survival of the pups, and decreased male postnatal development, but did not alter litter size, maternal body weight or pup birth weight. We observed many sex-dependent effects of perinatal CBD exposure. Exposure to CBD during gestation and lactation increased meal size, caloric intake, and respiratory exchange ratio for adult male offspring, while exposure during lactation decreased fasting glucose, but had no effect on clearance. Adult female offspring exposed to CBD during lactation showed increased drink size. Perinatal CBD exposure increased obsessive compulsive- and decreased anxiety-like behaviors (marble burying, light-dark box, elevated-plus maze) in female mice, decreased long-term object memory in male mice, and had no effect on attention tasks for either sex. CONCLUSIONS: We conclude that orally-administered CBD during pregnancy affects behavior and metabolism in a sex-dependent manner, and mice are differentially sensitive to exposure during gestation vs. lactation, or both. Because long-term changes are observed following perinatal exposure to the drug, and exposure significantly decreases survival to weaning, more research during development is warranted.

Glucose sensitivity of mouse olfactory bulb neurons is conveyed by a voltage-gated potassium channel.

The olfactory bulb has recently been proposed to serve as a metabolic sensor of internal chemistry, particularly that modified by metabolism. Because the voltage-dependent potassium channel Kv1.3 regulates a large proportion of the outward current in olfactory bulb neurons and gene-targeted deletion of the protein produces a phenotype of resistance to diet-induced obesity in mice, we hypothesized that this channel may play a role in translating energy availability into a metabolic signal. Here we explored the ability of extracellular glucose concentration to modify evoked excitability of the mitral neurons that principally regulate olfactory coding and processing of olfactory information. Using voltage-clamp electrophysiology of heterologously expressed Kv1.3 channels in HEK 293 cells, we found that Kv1.3 macroscopic currents responded to metabolically active (d-) rather than inactive (l-) glucose with a response profile that followed a bell-shaped curve. Olfactory bulb slices stimulated with varying glucose concentrations showed glucose-dependent mitral cell excitability as evaluated by current-clamp electrophysiology. While glucose could be either excitatory or inhibitory, the majority of the sampled neurons displayed a decreased firing frequency in response to elevated glucose concentration that was linked to increased latency to first spike and decreased action potential cluster length. Unlike modulation attributed to phosphorylation, glucose modulation of mitral cells was rapid, less than one minute, and was reversible within the time course of a patch recording. Moreover, we report that modulation targets properties of spike firing rather than action potential shape, involves synaptic activity of glutamate or GABA signalling circuits, and is dependent upon Kv1.3 expression. Given the rising incidence of metabolic disorders attributed to weight gain, changes in neuronal excitability in brain regions regulating sensory perception of food are of consequence.

An anatomically distinct subpopulation of orexin neurons project from the lateral hypothalamus to the olfactory bulb.

Olfactory cues play a key role in natural behaviors such as finding food, finding mates, and avoiding predators. In principle, the ability of the olfactory system to carry out these perceptual functions would be facilitated by signaling related to an organism's physiological state. One candidate pathway includes a direct projection from the hypothalamus to the main olfactory bulb, the first stage of olfactory sensory processing. The pathway from the hypothalamus to the main olfactory bulb is thought to include neurons that express the neuropeptide orexin, although the proportion that is orexinergic remains unknown. A current model proposes that the orexin population is heterogeneous, yet it remains unknown whether the proportion that innervates the main olfactory bulb reflects a distinct subpopulation of the orexin population. Herein, we carried out combined retrograde tract tracing with immunohistochemistry for orexin-A in the mouse to define the proportion of hypothalamic input to the main olfactory bulb that is orexinergic and to determine what fraction of the orexin-A population innervates the bulb. The numbers and spatial positions of all retrogradely labeled neurons and all the orexin-A-expressing neurons were quantified in sequential sections through the hypothalamus. Retrogradely labeled neurons were found in the ipsilateral hypothalamus, of which 22% expressed orexin-A. The retrogradely labeled neurons that did and did not express orexin-A could be anatomically distinguished based on their spatial position and cell body area. Remarkably, only 7% of all the orexin-A neurons were retrogradely labeled, suggesting that only a small fraction of the orexin-A population directly innervate the main olfactory bulb. These neurons spatially overlapped with the orexin-A neurons that did not innervate the bulb, although the two cell populations were differentiated based on cell body area. Overall, these results support a model in which olfactory sensory processing is influenced by orexinergic feedback at the first synapse in the olfactory processing pathway.

Odor Discrimination by Lipid Membranes.

Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general.

Single cannabidiol administration affects anxiety-, obsessive compulsive-, object memory-, and attention-like behaviors in mice in a sex and concentration dependent manner.

RATIONALE: The behavioral effects of cannabidiol (CBD) are understudied, but are important, given its therapeutic potential and widespread use as a natural supplement. OBJECTIVE: The objective of this study was to test whether a single injection of CBD affected anxiety-like or attention-like behavior, or memory in wildtype mice or mice with reported trait anxiety due to a targeted gene-deletion in a voltage-dependent potassium channel, Kv1.3. METHODS: Wildtype C57BL/6 J and Kv1.3-/- mice of both sexes were reared to adulthood and then administered an intraperitoneal injection of 10 or 20 mg/kg CBD. Mice were behaviorally-phenotyped using the marble-burying test, the light-dark box (LDB), short (1 h) and long-term (24 h) object memory test, the elevated-plus maze (EPM), and the object-based attention task in order to assess obsessive compulsive-, anxiety-, and attention-like behaviors, and memory. RESULTS: We discovered that acute CBD treatment reduced marble burying in male, but not female mice. CBD was effective in lessening anxiety-like behaviors determined by the LDB test in both male and female wildtype mice, whereby the effective dose required to observe the effect in females was less. In Kv1.3-/- mice, CBD increased anxiety-like behaviors in the LDB in both sexes at the higher concentration of CBD and it similarly increased anxiety-like behavior in females in the EPM at the lower concentration of CBD. Long-term object memory was reduced in male wildtype mice at the lower concentration of CBD. Finally, ADHD- or attention-like behaviors were not altered by CBD in wildtype mice, but in Kv1.3-/- mice, females were observed to have a loss in attention while males demonstrated improved attention. CONCLUSIONS: We conclude that administration of a single dose of CBD has immediate effects on mouse behavior that is dose, sex, and anxiety-state dependent - and that these behavioral outcomes are important to examine in parallel human trials.

Olfaction under metabolic influences.

Recently published work and emerging research efforts have suggested that the olfactory system is intimately linked with the endocrine systems that regulate or modify energy balance. Although much attention has been focused on the parallels between taste transduction and neuroendocrine controls of digestion due to the novel discovery of taste receptors and molecular components shared by the tongue and gut, the equivalent body of knowledge that has accumulated for the olfactory system, has largely been overlooked. During regular cycles of food intake or disorders of endocrine function, olfaction is modulated in response to changing levels of various molecules, such as ghrelin, orexins, neuropeptide Y, insulin, leptin, and cholecystokinin. In view of the worldwide health concern regarding the rising incidence of diabetes, obesity, and related metabolic disorders, we present a comprehensive review that addresses the current knowledge of hormonal modulation of olfactory perception and how disruption of hormonal signaling in the olfactory system can affect energy homeostasis.

Modulation of Neural Microcircuits That Control Complex Dynamics in Olfactory Networks.

Neuromodulation influences neuronal processing, conferring neuronal circuits the flexibility to integrate sensory inputs with behavioral states and the ability to adapt to a continuously changing environment. In this original research report, we broadly discuss the basis of neuromodulation that is known to regulate intrinsic firing activity, synaptic communication, and voltage-dependent channels in the olfactory bulb. Because the olfactory system is positioned to integrate sensory inputs with information regarding the internal chemical and behavioral state of an animal, how olfactory information is modulated provides flexibility in coding and behavioral output. Herein we discuss how neuronal microcircuits control complex dynamics of the olfactory networks by homing in on a special class of local interneurons as an example. While receptors for neuromodulation and metabolic peptides are widely expressed in the olfactory circuitry, centrifugal serotonergic and cholinergic inputs modulate glomerular activity and are involved in odor investigation and odor-dependent learning. Little is known about how metabolic peptides and neuromodulators control specific neuronal subpopulations. There is a microcircuit between mitral cells and interneurons that is comprised of deep-short-axon cells in the granule cell layer. These local interneurons express pre-pro-glucagon (PPG) and regulate mitral cell activity, but it is unknown what initiates this type of regulation. Our study investigates the means by which PPG neurons could be recruited by classical neuromodulators and hormonal peptides. We found that two gut hormones, leptin and cholecystokinin, differentially modulate PPG neurons. Cholecystokinin reduces or increases spike frequency, suggesting a heterogeneous signaling pathway in different PPG neurons, while leptin does not affect PPG neuronal firing. Acetylcholine modulates PPG neurons by increasing the spike frequency and eliciting bursts of action potentials, while serotonin does not affect PPG neuron excitability. The mechanisms behind this diverse modulation are not known, however, these results clearly indicate a complex interplay of metabolic signaling molecules and neuromodulators that may fine-tune neuronal microcircuits.

Long-term obesogenic diet and targeted deletion of potassium channel Kv 1.3 have differing effects on voluntary exercise in mice.

Voluntary exercise is frequently employed as an intervention for obesity. The voltage-gated potassium channel Kv 1.3 is also receiving attention as a therapeutic target for obesity, in addition to potential therapeutic capabilities for neuroinflammatory diseases. To investigate the combinatorial effects of these two therapies, we have compared the metabolic status and voluntary exercise behavior of both wild-type mice and a transgenic line of mice that are genetic knockouts for Kv 1.3 when provided with a running wheel and maintained on diets of differing fat content and caloric density. We tracked the metabolic parameters and wheel running behavior while maintaining the mice on their assigned treatment for 6 months. Wild-type mice maintained on the fatty diet gain a significant amount of bodyweight and adipose tissue and display significantly impaired glucose tolerance, though all these effects were partially reduced with provision of a running wheel. Similar to previous studies, the Kv 1.3-null mice were resistant to obesity, increased adiposity, and impaired glucose tolerance. Both wild-type and Kv 1.3-null mice maintained on the fatty diet displayed increased wheel running activity compared to control-fed mice, which was caused primarily by a significant increase in the amount of time spent running as opposed to an increase in running velocity. Interestingly, the patterns of running behavior differed between wild-type and Kv 1.3-null mice. Kv 1.3-null mice spent significantly less time running during the light phase and displayed a decrease in running 1-2 h before the onset of the light phase, seemingly in anticipation of the dark-to-light phase transition. These studies indicate that voluntary exercise combats metabolic maladies and running behavior is modified by both consumption of an obesogenic diet and deletion of the Kv 1.3 channel.

A unique olfactory bulb microcircuit driven by neurons expressing the precursor to glucagon-like peptide 1.

The presence of large numbers of local interneurons in the olfactory bulb has demonstrated an extensive local signaling process, yet the identification and purpose of olfactory microcircuits is poorly explored. Because the discrimination of odors in a complex environment is highly dependent on the tuning of information by local interneurons, we studied for the first time the role of preproglucagon (PPG) neurons in the granule cell layer of the olfactory bulb. Combining electrophysiological recordings and confocal microscopy, we discovered that the PPG neurons are a population of cells expressing the precursor of glucagon-like peptide 1 and are glutamatergic; able to modulate the firing pattern of the mitral cells (M/TCs). Optogenetic activation of PPG neurons resulted in a mixed excitation and inhibition that created a multiphasic response shaping the M/TCs firing pattern. This suggests that PPG neurons could drive neuromodulation of the olfactory output and change the synaptic map regulating olfactory coding.

Modulation of olfactory-driven behavior by metabolic signals: role of the piriform cortex.

Olfaction is one of the major sensory modalities that regulates food consumption and is in turn regulated by the feeding state. Given that the olfactory bulb has been shown to be a metabolic sensor, we explored whether the anterior piriform cortex (aPCtx)-a higher olfactory cortical processing area-had the same capacity. Using immunocytochemical approaches, we report the localization of Kv1.3 channel, glucose transporter type 4, and the insulin receptor in the lateral olfactory tract and Layers II and III of the aPCtx. In current-clamped superficial pyramidal (SP) cells, we report the presence of two populations of SP cells: glucose responsive and non-glucose responsive. Using varied glucose concentrations and a glycolysis inhibitor, we found that insulin modulation of the instantaneous and spike firing frequency are both glucose dependent and require glucose metabolism. Using a plethysmograph to record sniffing frequency, rats microinjected with insulin failed to discriminate ratiometric enantiomers; considered a difficult task. Microinjection of glucose prevented discrimination of odorants of different chain-lengths, whereas injection of margatoxin increased the rate of habituation to repeated odor stimulation and enhanced discrimination. These data suggest that metabolic signaling pathways that are present in the aPCtx are capable of neuronal modulation and changing complex olfactory behaviors in higher olfactory centers.

Deletion of voltage-gated channel affects glomerular refinement and odorant receptor expression in the mouse olfactory system.

Olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) gene send axonal projections to specific glomeruli, creating a stereotypic olfactory sensory map. Odorant receptor sequence, G-protein cAMP signaling, and axon guidance molecules have been shown to direct axons of OSNs toward central targets in the olfactory bulb (OB). Although the OR sequence may act as one determinant, our objective was to elucidate the extent by which voltage-dependent activity of postsynaptic projection neurons in the OB centrally influences peripheral development and target destination of OSNs. We bred OR-tagged transgenic mice to homozygosity with mice that had a gene-targeted deletion of the Shaker potassium ion channel (Kv1.3) to elucidate how activity modulates synaptic connections that formulate the sensory map. Here we report that the Kv1.3 ion channel, which is predominantly expressed in mitral cells and whose gene-targeted deletion causes a "super-smeller" phenotype, alters synaptic refinement of axonal projections from OSNs expressing P2, M72, and MOR28 ORs. Absence of Kv1.3 voltage-gated activity caused the formation of small, heterogeneous, and supernumerary glomeruli that failed to undergo neural pruning over development. These changes were accompanied by a significant decrease in the number of P2-, M72-, and MOR28-expressing OSNs, which contained an overexpression of OR protein and G-protein G(olf) in the cilia of the olfactory epithelium. These findings suggest that voltage-gated activity of projection neurons is essential to refine primary olfactory projections and that it regulates proper expression of the transduction machinery at the periphery.