Thomas Gerald Mast

Awake Intranasal Insulin Delivery Modifies Protein Complexes and Alters Memory, Anxiety, and Olfactory Behaviors

The role of insulin pathways in olfaction is of significant interest with the widespread pathology of diabetes mellitus and its associated metabolic and neuronal comorbidities. The insulin receptor (IR) kinase is expressed at high levels in the olfactory bulb, in which it suppresses a dominant Shaker ion channel (Kv1.3) via tyrosine phosphorylation of critical N- and C-terminal residues. We optimized a 7 d intranasal insulin delivery (IND) in awake mice to ascertain the biochemical and behavioral effects of insulin to this brain region, given that nasal sprays for insulin have been marketed notwithstanding our knowledge of the role of Kv1.3 in olfaction, metabolism, and axon targeting. IND evoked robust phosphorylation of Kv1.3, as well as increased channel protein–protein interactions with IR and postsynaptic density 95. IND-treated mice had an increased short- and long-term object memory recognition, increased anxiolytic behavior, and an increased odor discrimination using an odor habituation protocol but only moderate change in odor threshold using a two-choice paradigm. Unlike Kv1.3 gene-targeted deletion that alters metabolism, adiposity, and axonal targeting to defined olfactory glomeruli, suppression of Kv1.3 via IND had no effect on body weight nor the size and number of M72 glomeruli or the route of its sensory axon projections. There was no evidence of altered expression of sensory neurons in the epithelium. In mice made prediabetic via diet-induced obesity, IND was no longer effective in increasing long-term object memory recognition nor increasing anxiolytic behavior, suggesting state dependency or a degree of insulin resistance related to these behaviors.

Impact of experience-dependent and -independent factors on gene expression in songbird brain

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical “constitutive plasticity” (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as “ribosome” (expressed more highly in juvenile brain) and “dopamine metabolic process” (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

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 Golf 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. J. Comp. Neurol. 506:161–179, 2008.

The TRPC2 Channel Forms Protein-Protein interactions with Homer and RTP in the Rat Vomeronasal Organ

BackgroundThe signal transduction cascade operational in the vomeronasal organ (VNO) of the olfactory system detects odorants important for prey localization, mating, and social recognition. While the protein machinery transducing these external cues has been individually well characterized, little attention has been paid to the role of protein-protein interactions among these molecules. Development of an in vitro expression system for the transient receptor potential 2 channel (TRPC2), which establishes the first electrical signal in the pheromone transduction pathway, led to the discovery of two protein partners that couple with the channel in the native VNO.ResultsHomer family proteins were expressed in both male and female adult VNO, particularly Homer 1b/c and Homer 3. In addition to this family of scaffolding proteins, the chaperones receptor transporting protein 1 (RTP1) and receptor expression enhancing protein 1 (REEP1) were also expressed. RTP1 was localized broadly across the VNO sensory epithelium, goblet cells, and the soft palate. Both Homer and RTP1 formed protein-protein interactions with TRPC2 in native reciprocal pull-down assays and RTP1 increased surface expression of TRPC2 in in vitro assays. The RTP1-dependent TRPC2 surface expression was paralleled with an increase in ATP-stimulated whole-cell current in an in vitro patch-clamp electrophysiological assay.ConclusionsTRPC2 expression and channel activity is regulated by chaperone- and scaffolding-associated proteins, which could modulate the transduction of chemosignals. The developed in vitro expression system, as described here, will be advantageous for detailed investigations into TRPC2 channel activity and cell signalling, for a channel protein that was traditionally difficult to physiologically assess.

Mature and Precursor Brain-Derived Neurotrophic Factor Have Individual Roles in the Mouse Olfactory Bulb

Background Sensory deprivation induces dramatic morphological and neurochemical changes in the olfactory bulb (OB) that are largely restricted to glomerular and granule layer interneurons. Mitral cells, pyramidal-like neurons, are resistant to sensory-deprivation-induced changes and are associated with the precursor to brain-derived neurotrophic factor (proBDNF); here, we investigate its unknown function in the adult mouse OB. Principal Findings As determined using brain-slice electrophysiology in a whole-cell configuration, brain-derived neurotrophic factor (BDNF), but not proBDNF, increased mitral cell excitability. BDNF increased mitral cell action potential firing frequency and decreased interspike interval in response to current injection. In a separate set of experiments, intranasal delivery of neurotrophic factors to awake, adult mice was performed to induce sustained interneuron neurochemical changes. ProBDNF, but not BDNF, increased activated-caspase 3 and reduced tyrosine hydroxylase immunoreactivity in OB glomerular interneurons. In a parallel set of experiments, short-term sensory deprivation produced by unilateral naris occlusion generated an identical phenotype. Conclusions Our results indicate that only mature BDNF increases mitral cell excitability whereas proBDNF remains ineffective. Our demonstration that proBDNF activates an apoptotic marker in vivo is the first for any proneurotrophin and establishes a role for proBDNF in a model of neuronal plasticity.

Olfactory sensory deprivation increases the number of proBDNF-immunoreactive mitral cells in the olfactory bulb of mice

In the olfactory bulb, apoptotic cell-death induced by sensory deprivation is restricted to interneurons in the glomerular and granule cell layers, and to a lesser extent in the external plexiform layer, whereas mitral cells do not typically undergo apoptosis. With the goal to understand whether brain-derived neurotrophic factor (BDNF) mediates mitral cell survival, we performed unilateral naris occlusion on mice at postnatal day one (P1) and examined the subsequent BDNF-immunoreactive (BDNF-ir) profile of the olfactory bulb at P20, P30, and P40. Ipsilateral to the naris occlusion, there was a significant increase in the number of BDNF-ir mitral cells per unit area that was independent of the duration of the sensory deprivation induced by occlusion. The number of BDNF-ir juxtaglomerular cells per unit area, however, was clearly diminished. Western blot analysis revealed the presence of primarily proBDNF in the olfactory bulb. These data provide evidence for a neurotrophic role of proBDNF in the olfactory system of mice and suggest that proBDNF may act to protect mitral cells from the effects of apoptotic changes induced by odor sensory deprivation.

Statistical Analysis and Decoding of Neural Activity in the Rodent Geniculate Ganglion Using a Metric-Based Inference System

We analyzed the spike discharge patterns of two types of neurons in the rodent peripheral gustatory system, Na specialists (NS) and acid generalists (AG) to lingual stimulation with NaCl, acetic acid, and mixtures of the two stimuli. Previous computational investigations found that both spike rate and spike timing contribute to taste quality coding. These studies used commonly accepted computational methods, but they do not provide a consistent statistical evaluation of spike trains. In this paper, we adopted a new computational framework that treated each spike train as an individual data point for computing summary statistics such as mean and variance in the spike train space. We found that these statistical summaries properly characterized the firing patterns (e. g. template and variability) and quantified the differences between NS and AG neurons. The same framework was also used to assess the discrimination performance of NS and AG neurons and to remove spontaneous background activity or “noise” from the spike train responses. The results indicated that the new metric system provided the desired decoding performance and noise-removal improved stimulus classification accuracy, especially of neurons with high spontaneous rates. In summary, this new method naturally conducts statistical analysis and neural decoding under one consistent framework, and the results demonstrated that individual peripheral-gustatory neurons generate a unique and reliable firing pattern during sensory stimulation and that this pattern can be reliably decoded.

Thirst Increases Chorda Tympani Responses to Sodium Chloride

In nature, water is present as a low-salt solution, thus we hypothesized that thirst would increase taste responses to low-salt solutions. We investigated the effect of thirst on the 2 different salt detection mechanisms present in the rat chorda tympani (CT) nerve. The first mechanism is dependent upon the epithelial sodium channel (ENaC), is blocked by benzamil, and is specific to the cation sodium. The second mechanism, while undefined, is independent of ENaC, and detects multiple cations. We expected thirst to increase benzamil-sensitive sodium responses due to mechanistically increasing the benzamil-sensitive ENaC. We recorded CT whole-nerve electrophysiological responses to lingual application of NaCl, KCl (30, 75, 150, 300, 500, and 600 mM), and imitation rainwater in both control and 24-h water-restricted male rats. NaCl solutions were presented in artificial saliva before and after lingual application of 5µM benzamil. Water restriction significantly increased the integrated CT responses to NaCl but not to KCl or imitation rainwater. Consistent with our hypothesis, only the benzamil-sensitive, and not the benzamil-insensitive, CT sodium response significantly increased. Additionally, CT responses to salt were recorded following induction of either osmotic or volemic thirst. Both thirsts significantly enhanced the integrated CT responses to NaCl and KCl, but not imitation rainwater. Interestingly, osmotic and volemic thirsts increased CT responses by increasing both the benzamil-sensitive and benzamil-insensitive CT sodium responses. We propose that thirst increases the sensitivity of the CT nerve to sodium.

Abstract 1957: Loss of TGFβ1 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Introduction: The TGFB pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Tgfb1 −/− Rag2 −/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 on colonic bacterial composition we used GEM models, Tgfb1 −/− Rag2 −/− and Tgfb1 +/+ Rag2 −/− . The cecal microflora of 10 Tgfb1 −/− Rag2 −/− and 10 Tgfb1 +/+ Rag2 −/− mice were isolated and serially diluted onto brucella (BRU), bacteroides bile esculin (BBE), and laked kanomycin-vancomycin (LKV) media under anaerobic conditions. Colony forming units (CFUs) were enumerated. Individual colony types were then streaked onto trypitcase soy agar with 5% sheep blood and grown anaerobically and aerobically. The bacteria were then gram-stained and biotyped utilizing a Dade Berhing MicroScan instrument. To further examine the bacterial composition we designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on fecal DNA. Results: The Dade Berhing Instrument showed that Tgfb1 −/− Rag2 −/− mice had a 4 fold increase in bacterial load and a 28 fold increase in Bacteroides species when compared with Tgfb1 +/+ Rag2 −/− mice. The qRT-PCR results showed an increase in Bacteroides fragilis and Bacteroides distasonis and a significant decrease in Bacteroides thetaiotaomicron in the Tgfb1 −/− Rag2 −/− mice. These data suggest that loss of TGFβ1 alters the colonic microflora. Previous studies illustrated the importance of bacterial nutrient sources on bacterial composition. To examine if TGFβ1 is altering nutrient availability in the colon a previous micro-array was analyzed for candidate genes associated with glycoprotein metabolism. This showed changes in fucose metabolizing enzymes with the loss of TGFβ1. Conclusion: These findings suggest that TGFβ1 plays a role in bacterial load maintenance, possibly by altering available nutrient sources and when disrupted, can cause abnormalities in pathobionts (commensal bacterial with pathogenic potential) which could then lead to increased inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1957.