Functional brain connectivity based on resting-state functional magnetic resonance imaging (fMRI) has been shown to be correlated with human personality and behavior. In this study, we sought to know whether capabilities and traits in dogs can be predicted from their resting-state connectivity, as in humans. We trained awake dogs to keep their head still inside a 3T MRI scanner while resting-state fMRI data was acquired. Canine behavior was characterized by an integrated behavioral score capturing their hunting, retrieving, and environmental soundness. Functional scans and behavioral measures were acquired at three different time points across detector dog training. The first time point (TP1) was prior to the dogs entering formal working detector dog training. The second time point (TP2) was soon after formal detector dog training. The third time point (TP3) was three months’ post detector dog training while the dogs were engaged in a program of maintenance training for detection work. We hypothesized that the correlation between resting-state FC in the dog brain and behavior measures would significantly change during their detection training process (from TP1 to TP2) and would maintain for the subsequent several months of detection work (from TP2 to TP3). To further study the resting-state FC features that can predict the success of training, dogs at TP1 were divided into a successful group and a non-successful group. We observed a core brain network which showed relatively stable (with respect to time) patterns of interaction that were significantly stronger in successful detector dogs compared to failures and whose connectivity strength at the first time point predicted whether a given dog was eventually successful in becoming a detector dog. A second ontologically based flexible peripheral network was observed whose changes in connectivity strength with detection training tracked corresponding changes in behavior over the training program. Comparing dog and human brains, the functional connectivity between the brain stem and the frontal cortex in dogs corresponded to that between the locus coeruleus and left middle frontal gyrus in humans, suggestive of a shared mechanism for learning and retrieval of odors. Overall, the findings point toward the influence of phylogeny and ontogeny in dogs producing two dissociable functional neural networks.

ABSTRACTThe colonial naked mole rat Heterocephalus glaber is a subterranean, eusocial rodent. The H. glaber vomeronasal organ neuroepithelium (VNE) displays little postnatal growth. However, the VNE remains neuronal in contrast to some mammals that possess nonfunctional vomeronasal organ remnants, for example, catarrhine primates and some bats. Here, we describe the vomeronasal organ (VNO) microanatomy in the naked mole rat and we make preliminary observations to determine if H. glaber shares its minimal postnatal VNE growth with other African mole rats. We also determine the immunoreactivity to the mitotic marker Ki67, growth‐associated protein 43 (GAP43), and olfactory marker protein (OMP) in six adult and three subadult H. glaber individuals. VNE volume measurements on a small sample of Cryptomys hottentotus and Fukomys damarensis indicate that the VNE of those African mole rat species are also likely to be growth‐deficient. Ki67(+) cells show that the sensory epithelium is mitotically active. GAP43 labelling indicates neurogenesis and OMP(+) cells are present though less numerous compared to GAP43(+) cells. In this respect, the VNO of H. glaber does not appear vestigial. The African mole rat VNE may be unusually variable, perhaps reflecting reduced selection pressure on the vomeronasal system. If so, African mole rats may provide a useful genetic model for understanding the morphological variability observed in the mammalian VNO. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 303:318–329, 2020. © 2019 American Association for Anatomy

The thermodynamic properties of olfactory sensory receptors were characterized by the measurement of electrical responses to odorants in isolated olfactory epithelia. Using the electroolfactogram (EOG), electrical activity was recorded within a temperature range of 16°C–35°C with epithelia obtained from animals being kept at ambient environmental conditions. The amplitude, the area under the curve, and the voltage kinetics of the electroolfactogram recordings were measured at different temperatures. The apparent values of the activation energy and the change of enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) of the odorant-receptor interaction were estimated using both Arrhenius and Eyring equations. The results reveal a broad variation of enthalpy (ΔH) and entropy contribution (TΔS) for odorant interaction with different sets of olfactory receptors. The observed enthalpy change was always followed by a corresponding change in the entropy contribution (TΔS), maintaining constant free energy (ΔG): ΔG=ΔH-TΔS and obeying the enthalpy-entropy compensation. Notably, in all experiments, the changes in enthalpy or entropy do not correlate with the change of free energy. Due to the fact that the values of ΔH and TΔS represent a summation of all chemical interactions between odorant and olfactory sensory neuron, it is difficult to expect the binding properties of odorants to serve as a foundation of molecular recognition by olfactory receptors. For this reason, our thermodynamic results do not support the shape mechanism of olfaction.

Canine β‐defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a β‐defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient‐rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient‐rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus . As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin‐4 receptor, acting as a weak allosteric agonist.

Segmental nerve loss presents a challenge to the reconstructive surgeon. Collagen nerve conduits are one strategy for reconstruction of segmental defects, but no conduit-based reconstructive strategy has been as successful as autograft reconstruction. We hypothesized that collagen nerve conduits used to bridge segmental sciatic nerve defects may be enhanced by grafting with vomeronasal organ (VNO), owing to this tissue’s capacity for regeneration. Fourteen rats underwent resection of a 1.0 cm segment of sciatic nerve. Seven rats underwent reconstruction of the defect using a commercially available collagen nerve conduit. Seven rats underwent reconstruction using conduit filled with fresh VNO allograft. An additional seven rats underwent transection and direct epineural repair. Fourteen weeks postoperatively, all animals underwent calculation of sciatic functional index (SFI) via walking track analysis. After sacrifice, tissues were processed for histomorphometric analysis, including axon quantification and axon density. All reconstructed nerves were in continuity at sacrifice. At 14 weeks, the mean SFI was significantly higher in the vomeronasal organ-enhanced (VNOE) group and epineural repair (ER) group than the conduit-only (CO) group. `SFI was equivalent between VNOE and ER groups. Axon density was greater in the VNOE and ER groups than the CO group. Axon density was equivalent between VNOE and ER groups. In conclusion, this rat sciatic nerve segmental defect model achieved greater functional recovery and axon density using collagen nerve conduits filled with a pluripotent neuroepithelium relative to reconstruction with an empty conduit. These results suggest a promising strategy for repair of segmental peripheral nerve defects.

The olfactory sensory neural system contains the highest levels of zinc found throughout the central nervous system. Zinc is one of many metals known to play a role in multiple facets of biology. Metal nanoparticles such as copper, gold, and zinc have been isolated from human and animal blood. The blood isolated zinc nanoparticles (ZnNPs) were unique in their ability to enhance the electrophysiological olfactory sensory neuron response to odorant by about threefold. Characterization of the physiochemical properties of ZnNPs determined that small (1.2±0.3 nm), non‐oxidized, primarily elemental, zinc nanoparticles were capable of olfaction enhancement. At low concentrations, ZnNPs added to odorant display a dose‐dependent, specific, and reversible effect. In conjunction with the enhancement of olfactory sensory neurons in the isolated rodent olfactory epithelium, cognitive processing of the sensory amplification is demonstrated by a significant increase of canine brain excitation in response to odorants resulted from the addition of ZnNPs. The presence of ZnNPs at the olfactory mucosa level has not been demonstrated. To evaluate the potential biological role ZnNPs have in the initial events of olfaction, the presence of ZnNPs in the olfactory and respiratory epithelia was examined by transmission electron microscopy (TEM). Detailed structures of nanoparticles were characterized by selected area diffraction. The physiological effects of nanoparticles were tested ex vivo by electroolfactogram (EOG). Zinc was identified in both the respiratory and olfactory filtrate samples. TEM demonstrated an organization of high homogeneity, crystalline structure, and within nanoparticle size range. The EOG responses to the epithelia filtrates mixed with odorant evoked higher olfactory responses compared to the response induced by odorant alone. These effects are similar to those produced by the engineered ZnNPs mixed with odorant. Calculation of the concentration of endogenous nanoparticles was performed using the relative EOG peaks as a function of engineered zinc concentration for a calibration curve. The calculated concentrations of ZnNPs in the olfactory epithelium and respiratory epithelium filtrates are 5.3×10−3 ± 5×10−4 nM and 2.7×10−3 ± 5×10−4 nM, respectively. The concentrations of ZnNPs in the olfactory epithelium and respiratory epithelia are 10.3 ± 1.0 nM and 7.9 ± 1.5 nM, respectively. The presence of ZnNPs within the olfactory and respiratory epithelia may have physiological significance. To determine the role of ZnNPs in olfaction, many questions still have to be answered. However, the presence of olfaction enhancing ZnNPs within the olfactory epithelium at the initial site of olfactory signaling suggests a physiological role in the initial events of olfaction at the receptor level. The confirmation of ZnNPs presence by TEM in combination with the electrophysiological enhancement of EOG provide support for this hypothesis.Support or Funding InformationSupported by grant from the National Institute of Standards and Technology: 70NANB14H324This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prior functional Magnetic Resonance Imaging (fMRI) studies have indicated increased neural activation when zinc nanoparticles are added to odorants in canines. Here we demonstrate that zinc nanoparticles up-regulate directional brain connectivity in parts of the canine olfactory network. This provides an explanation for previously reported enhancement in the odor detection capability of the dogs in the presence of zinc nanoparticles. In this study, we obtained fMRI data from awake and unrestrained dogs while they were being exposed to odorants with and without zinc nanoparticles, zinc nanoparticles suspended in water vapor, as well as just water vapor alone. We obtained directional connectivity between the brain regions of the olfactory network that were significantly stronger for the condition of odorant + zinc nanoparticles compared to just odorants, water vapor + zinc nanoparticles and water vapor alone. We observed significant strengthening of the paths of the canine olfactory network in the presence of zinc nanoparticles. This result indicates that zinc nanoparticles could potentially be used to increase canine detection capabilities in the environments of very low concentrations of the odorants. which would have otherwise been undetected.

Background: Docetaxel (DOC), or Taxotere, is an anthracycline antibiotic used to treat multiple types of cancer. It is a first-line chemotherapy treatment for patients with metastasized, hormone-resistant prostate cancer (PCa) or for patients with high-risk, localized PCa that could benefit from early chemotherapy treatment. Previously, we showed that stearidonic acid (SDA), an omega-3 fatty acid, enhances the cytotoxicity of doxorubicin (DOX) in human PCa cells. This observation suggests that PCa therapies using SDA and chemotherapeutic drugs in combination offer attractive possibilities for developing treatments that ameliorate toxic side effects of some commonly used chemotherapy drugs. Objectives: We used androgen-resistant PC3 and DU 145 cells derived from human prostate cancer to quantify the effects of combined SDA and DOC on proliferation/viability and on the production of pro-apoptotic caspases 9 and 3. We also compared the effects of SDA with those of BAY, a pharmacological inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), in androgen-sensitive LNCaP cells. Finally, we qualitatively and quantitatively assessed the drug combination on androgen receptor (AR) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in LNCaP and PC3 cells, respectively. Methods: The half maximal inhibitory concentration (IC50) and combination indices of SDA and DOC in PC3 and DU 145 cells were determined using the MTT cell viability assay. To quantify the effects of SDA and BAY on NF-ĸB activity, we used luciferase reporter assays in LNCaP cells that were stably transduced with lentiviral vectors carrying NF-ĸB response element sequence upstream of the luciferase gene sequence. AR and PPARγ expression were assessed by western blotting and immunocytochemistry. We considered caspase 9 and 3 cleavage to be apoptosis markers and determined the drug combination effect on the extent of that cleavage by western blot analysis. Results: The cytotoxic effects of DOC were synergistically enhanced by SDA when the two were added to DU145 and PC3 cell cultures. Combination index (CI) analyses based on the Chou-Talalay method and mass action law showed synergistic interaction with CI <1. SDA suppressed TNFα-induced NF-κB activity similarly to BAY. The SDA/DOC combination down regulated testosterone (T)-induced AR and troglitazone-induced PPARγ protein expression when compared to using the drugs singly. Similarly, the SDA/DOC combination induced caspase 9 and 3 production and cleavage suggesting apoptosis induction. Like our DOX studies, this work provides proof-of-concept for using SDA and DOC in combination to reduce the dose, and therefore the toxicity, of DOC and possibly increasing the survival benefit in DOC clinical translation studies.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB.

Olfactory responses are intensely enhanced with the addition of endogenous and engineered primarily-elemental small zinc nanoparticles (NPs). With aging, oxidation of these Zn nanoparticles eliminated the observed enhancement. The design of a polyethylene glycol coating to meet storage requirements of engineered zinc nanoparticles is evaluated to achieve maximal olfactory benefit. The zinc nanoparticles were covered with 1000 g/mol or 400 g/mol molecular weight polyethylene glycol (PEG). Non-PEGylated and PEGylated zinc nanoparticles were tested by electroolfactogram with isolated rat olfactory epithelium and odorant responses evoked by the mixture of eugenol, ethyl butyrate and (±) carvone after storage at 278 K (5 oC), 303 K (30 oC) and 323 K (50 oC). The particles were analyzed by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and laser Doppler velocimetry. Our data indicate that stored ZnPEG400 nanoparticles maintain physiologically-consistent olfactory enhancement for over 300 days. These engineered Nanoparticles support future applications in olfactory research, sensitive detection, and medicine.

Electrical responses of olfactory sensory neurons to odorants were examined in the presence of zinc nanoparticles of various sizes and degrees of oxidation. The zinc nanoparticles were prepared by the underwater electrical discharge method and analyzed by atomic force microscopy and X-ray photoelectron spectroscopy. Small (1.2 ± 0.3 nm) zinc nanoparticles significantly enhanced electrical responses of olfactory neurons to odorants. After oxidation, however, these small zinc nanoparticles were no longer capable of enhancing olfactory responses. Larger zinc oxide nanoparticles (15 nm and 70 nm) also did not modulate responses to odorants. Neither zinc nor zinc oxide nanoparticles produced olfactory responses when added without odorants. The enhancement of odorant responses by small zinc nanoparticles was explained by the creation of olfactory receptor dimers initiated by small zinc nanoparticles. The results of this work will clarify the mechanisms for the initial events in olfaction, as well as to provide new ways to alleviate anosmia related to the loss of olfactory receptors.

Temperature is one of the most challenging stressors, affecting human and animal health. Extreme heat stress can damage the olfactory sensory neurons. In this work, we examined the effects of elevated temperature on the kinetic properties of rat olfactory sensory receptors in vitro by the recording of electroolfactogram (EOG) of the olfactory epithelium (OE) removed from the animals before and after the heat stress at 45 ºC for 25 minutes. EOG were evoked by an odorant mixture of ethyl butyrate, eugenol, and (+) and (−) carvone. The biochemical properties of the animal blood related to heat stress were characterized. Exposure of rats to high temperature resulted in a significant increase of body temperature. The mean body temperatures of rats before and immediately after heat stress were 36.7±0.07 ºC and 40.3 ±0.17 ºC, respectively. The kinetic properties of electrical responses to odorant were significantly changed in animals subjected to heat stress. The mean half‐rise (tr)and –decay times (td) of control OE were 328±11 and 660±20 ms, respectively. The rise and decay of EOG peak amplitude after heat stress were much faster, being reduced by 175 %, and 72 %, respectively. Heat stress did not result in a change of IL‐1β; IL‐6, TNF‐α, INF‐γ cytokines level. However, a significant increase of the IL‐10 level was found in rats exposed to heat. The level of lipopolysaccharides (LPS) was significantly increased in the serum of heat‐stressed animals compared to control animals. The concentration of free vesicles in animal blood increased after exposure to heat stress from (1.4±0.2)×106 to (3.8±0.3)×106 vesicles μl−1, indicating a faster conversion of erythrocytes into echinocytes. The kinetic properties found in this work are consistent with the kinetics of cultured mouse olfactory neurons 1. The effect of elevated temperature on the kinetic properties of olfactory receptors is also consistent with those seen in other neurons2–4. Detrimental effects of elevated temperatures on olfaction are shown in other animals5. The change in kinetic properties of olfactory receptors as a result of elevated temperature is consistent with those in other neurons2–4. Cytokines were found to cause neurodegeneration of the olfactory bulb6. We conclude that heat stress causes a strong irreversible modulation of olfactory responses.Support or Funding InformationSupported by grant from the National Institute of Standards and Technology: 70NANB14H324

Diffusion tensor imaging (DTI) provides us an insight into the micro-architecture of white-matter tracts in the brain. This method has proved promising in understanding and investigating the neuronal tracts and structural connectivity between the brain regions in primates as well as rodents. The close evolutionary relationship between canines and humans may have spawned a unique bond in regard to social cognition rendering them useful as an animal model in translational research. In this study, we acquired diffusion data from anaesthetized dogs and created a DTI-based atlas for a canine model which could be used to investigate various white matter diseases. We illustrate the application of this atlas by calculating DTI tractography based structural connectivity between the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) regions of the default mode network (DMN) in dogs. White matter connectivity was investigated to provide structural basis for the functional dissociation observed between the anterior and posterior parts of DMN. A comparison of the integrity of long range structural connections (such as in the DMN) between dogs and humans is likely to provide us with new perspectives on the neural basis of the evolution of cognitive functions.

Aim: Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats. Methods: Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca 2+ transient. Results: A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca 2+ transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 μg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats. Conclusion: These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure.

Using noninvasive in vivo functional magnetic resonance imaging (fMRI), we demonstrate that the enhancement of odorant response of olfactory receptor neurons by zinc nanoparticles leads to increase in activity in olfaction-related and higher order areas of the dog brain. To study conscious dogs, we employed behavioral training and optical motion tracking for reducing head motion artifacts. We obtained brain activation maps from dogs in both anesthetized state and fully conscious and unrestrained state. The enhancement effect of zinc nanoparticles was higher in conscious dogs with more activation in higher order areas as compared with anesthetized dogs. In conscious dogs, voxels in the olfactory bulb and hippocampus showed higher activity to odorants mixed with zinc nanoparticles as compared with pure odorants, odorants mixed with gold nanoparticles as well as zinc nanoparticles alone. These regions have been implicated in odor intensity processing in other species including humans. If the enhancement effect of zinc nanoparticles observed in vivo are confirmed by future behavioral studies, zinc nanoparticles may provide a way for enhancing the olfactory sensitivity of canines for detection of target substances such as explosives and contraband substances at very low concentrations, which would otherwise go undetected.