Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Animal Poultry Sciences
Non Technical Summary
Summary and Justification:Problem Statement: The growing human population has increased the demand for high-quality protein sources, including meat from broiler chickens. Suppliers have responded to this demand by significantly increasing broiler chicken production through intense genetic selection for growth-related traits. This process has broadened our understanding of food intake and metabolic energy expenditure in chickens. Most research on this topic has focused exclusively on the role of hypothalamic neurons in the brain, establishing that food intake is controlled by tight coordination between multiple populations of neural brain cells. Astrocytes are non-neuronal cells that form intimate structural connections with neurons and astrocyte function is vital for neuronal activity. Although astrocytes are the most abundant cells in the central nervous system (CNS), they have often been relegated to a less than prominent role in the control of complex brain functions. Increasing evidence now supports a role for astrocytes in normal health of animals and humans, and its dysfunction is a trigger for numerous diseases. Importantly, astrocytes have been recently implicated in the process of feeding behaviors in both animals and humans; however, there is a substantive gap in our knowledge concerning astrocyte contribution to food intake in chickens.This proposal evaluates astrocyte function in physiological processes that regulate dietary fat intake in chickens. We focus on dietary fat, because it is one of the major macronutrients in the diet and dietary fat quantity and composition is known to affect feeding behavior and growth performance. Findings from this dietary fat study will deepen our understanding of fat production and raise the possibility of identifying novel mechanisms that regulate fat in chickens.Relevance to advancing Virginia/Region and the US: The broiler chicken industry is the number one agricultural commodity for the state of Virginia with an estimated $733 million economic impact for the state (USDA). The U.S. has one of the largest broiler chicken industries in the world and is the second largest exporter of poultry meat (1). Americans consume more chicken than any other country in the world, more than 92 pounds per capita (USDA). This proposal aims to decipher the contribution of astrocytes in molecular mechanisms involved in food intake in broilers. Results from this proposal will provide insight on mechanisms to modulate feeding behavior and fat composition in the broiler industry.Approach: The proposed study examines the role of astrocytes in hypothalamic neuronal circuits involved in dietary fat intake. To address this, we propose to study the influence of regular starter diets (RD) and high-fat diets (HFD) on astrocyte: 1) gene expression, 2) function, 3) interaction with key neurons that play a vital role in appetite regulation, and 4) response to hypothalamic neuropeptides.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Major Goals and Objectives:The overall goal of this project is to discern the contribution of astrocyte function to the relationship between dietary quantity and feeding behavior in broiler chickens. Objective 1:Hypothesis: I hypothesize that chicken hypothalamic astrocytes will be reactive to HFD feeding.The objective is to determine if astrocyte expression is altered by a HFD. Experiments in this aim will determine changes in the expression (mRNA, protein, cellular) of astrocytes in response to a HFD. It will also determine changes in the interactions between astrocytes and neurons.Aim 1.1: Show changes in the expression of astrocytes in response to HFD. Aim 1.2 Demonstrate that astrocytic interactions with NYP/AgRP and POMC neurons are compromised by a HFD.Objective 2Hypothesis 2: I hypothesize that astrocyte K+ and Glu uptake is altered in the presence of a HDF.The experiments in this aim will determine if HFD feeding in chickens cause changes in key astrocyte functions.Aim 2.1: Show that K+ uptake in astrocytes is affected by a HFD. Aim 2.2: Show that astrocytic Glu uptake is altered in response to a HFD.Objective 3:Hypothesis 3: I hypothesize that α-MSH administration alters the astrocyte response to affect food intake.Determine the astrocyte response to α-MSH in response to feeding the RD and HFD. If astrocytes are important in the neuronal circuitry involved in food intake, they should respond to hypothalamic neuropeptides. These experiments will mechanistically link astrocyte function to changes in α-MSH, which affects food intake.Aim 3.1: Determine if α-MSH administration affects astrocyte expression in chickens.Aim 3.2: Determine the effect of α-MSH on the astrocyte response to HFD and RD. Aim 3.3: Show that key astrocytic functions are differentially affected by exogenous application of α-MSH in HFD and RD.
Project Methods
Objective 1: Show that astrocytes become reactive in response to HFD.Aim 1.1 Experiment 1: GFAP mRNA expression: To demonstrate that the mRNA expression of astrocytes is altered in response to HFD, GFAP in the hypothalamic nuclei of chicks administered RD (n=10) and HFD (n=10) (Table) will be compared using qPCR. Each chick will be deeply anesthetized at 60 minutes post-injection, decapitated, brains perfused via the carotid artery with 2.5 mL of RNA stabilizing buffer (16.7 mM sodium citrate, 13.3 mM EDTA, and 3.5 M ammonium sulfate; pH=5.2), and 500 µm thick coronal sections collected in a cryostat that correspond to 7.8 (PVN and LH) and 5.6 (DMN and ARC) interaural (35). Nuclei samples will be collected using sterile disposable biopsy instruments (1 mm, Braintree Scientific Inc., Braintree, MA) and total RNA will be isolated using the Total RNA Purification Micro Kit and Rnase-Free DNase I kit (Norgen Biotek), following the manufacturer's instructions (36). First-strand cDNA will be synthesized and real-time PCR reactions performed, with all primer sequences published (34, 37). Data will be analyzed using analysis of variance (ANOVA) within each group at 5 and 7 days post-hatch.Immunohistochemistry: Chickens will be anesthetized and perfused with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.6) at 4°C. After decapitation, the brains will be removed and fixed by immersion in the same solution overnight at 4°C. The tissue will be dehydrated with a series of ethanol solutions and embedded in paraffin. Transverse and sagittal sections (10 μm thick) will be cut and processed for IHC against GFAP. The primary antibodies will be mouse monoclonal antibodies against GFAP (1:400, Sigma, St. Louis, MO, U.S.A.). Sections will be incubated with the primary antibodies overnight at 4°C. Rabbit anti-mouse IgG (1:100, Jackson, West Grove, PA, U.S.A.) will be used as a secondary antibody (for 60 min at room temperature), followed by mouse peroxidase-anti-peroxidase complex (1:100, Jackson) for 90 min at room temperature (38). The cellular expression of astrocytes in response to HFD will be determined by determining GFAP labelled cells in the hypothalamic nuclei of chickens administered RD (n=10) and HFD (n=10). Slices will be observed under a confocal laser microscope and quantified using unbiased stereology. Comparison of GFAP expression will be conducted as described above.Aim 1.1 Experiment 2: To demonstrate structural changes in astrocytic processes in response to HFD, GFAP stained astrocytes from chickens administered HFD and RD will be used to analyze the density, ramification and the length and number of astrocytic primary processes. For the analysis of astrocytic ramification, an adaptation of Sholl's concentric circles technique will be used (39,40). Primary process quantification will be performed by counting the processes extending directly from the soma in both the lateral and central quadrants of astrocytes in the same sections using the Image Pro Plus software. Comparisons will be conducted as described above using Graph Pad 4.0 software.Objective 1.2: Experiment 1: The ratio of GFAP to POMC and NPY mRNA levels will be compared between HFD and RD as described above. Similar methods will be employed, except specific sense and antisense primers for NPY, POMC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard) will be used.Objective 2: Determine if key astrocyte functions are altered by HFD. Aim 2.1: Experiment 1: To assess changes in astrocytes ability to remove extracellular K+, electrophysiological recordings will be conducted in astrocytes from HFD and RD animals. Hypothalamic brain slices from day 7 post-hatch chicks from both groups will be prepared as described previously (39). Chicks will be anesthetized and decapitated. The brain will be removed quickly and placed in ice-cold saline. Astrocytes will be identified based on cell morphology, lack of spontaneous synaptic activity and absence of action potentials. K+ will be puffed onto the astrocytes and the amplitude and response area will be compared between groups. In some instances, biocytin (Sigma-Aldrich, St. Louis, MO) will be added to the pipette solution (0.5 mg/mL) forpost-hocidentification and assessment of cell coupling. The amplitude and response area will be compared using two-tailedt-tests.Aim 2.2: Experiment 1: To show changes in astrocyte Glu uptake in chicks administered HFD, we will exogenously apply Glu onto astrocytes using a Picospritzer (Warner, Hamden, CT) as described previously (41). Glutamate-induced responses will be recorded in astrocytes using a cocktail that will allow us to isolate Glu transporter currents (42). The amplitude and response area will be compared and two-tailedt-tests will be performed as described in Experiment 1 of this aim.Aim 2.2: Experiment 2: To demonstrate changes in synaptically-evoked astrocyte Glu uptake in chicks administered HFD, Glu transporter currents will be recorded from astrocytes. Currents will be evoked with a bipolar stimulating electrode positioned within 150-200 µm of the recording pipette. Current pulses 10-100 µA in amplitude and 50-100 µs in duration will be used. The amplitude and response area will be compared and two-tailedt-tests will be performed as described in Experiment 1 of this aim.Objective 3: Determine astrocyte response to α-MSH in RD and HFD.Aim 3.1: Experiment 1: Use qPCR and IHC analysis of the mRNA and cellular expression of GFAP in response to α-MSH administration in chickens using methods described in Aim 1. At day 4, chicks will be assigned to treatments according to a randomized complete block design with body weight as the blocking factor. Treatments include: 1) control diet and vehicle (artificial cerebrospinal fluid) injection (no α-MSH), 2) control diet and 0.12 nmol α-MSH, 3) HF diet and vehicle, and 4) HF diet and 0.12 nmol α-MSH, = 20 chicks per group. The dose is based on a preliminary experiment that was conducted to determine the dose threshold response. The selected dose produces a significant decrease in food intake in the first hour post-injection. Chicks will be injected intracerebroventricularly (ICV), using a method (43) that does not induce stress (44) as previously described (45), and returned to their home cage with ad libitum access to feed and water. Chicks will be decapitated and brain slices will be made for electrophysiology recordings.Aim 3.2: Experiment 1: Using slices from α-MSH-treated and untreated chicks on RD and HFD, whole-cell patch-clamp recordings will be used to establish astrocyte response to α-MSH-treated and untreated chicks. The effect of α-MSH on Glu uptake will be determined using methods described in Aim 2. The magnitude of the current amplitude, response area and decay kinetics will be compared.Aim 3.2: Experiment 2: Repeat the experiment above but assess astrocytic K+ uptake.Aim 3.3: Experiment 1: Using slices from α-MSH-treated and untreated chicks on RD and HFD, the effect of local application of α-MSH will be discerned by puffing increasing concentrations of α-MSH (0.05, 1.0. 2.0 µM) onto astrocytes, as described in Aim 2. The effect of α-MSH on astrocyte Glu and K+ uptake will be compared among groups.