Studies of nicotinic acetylcholine receptors containing α4 and α6 subunits in nicotine-induced synaptic plasticity in brain reward areas

Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine, the primary psychoactive compound in tobacco smoke, activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine. Nicotine causes cellular changes in VTA DA neurons, including the enhancement of AMPA receptor (AMPAR) function. This enhancement sensitizes the VTA to excitatory input and promotes drug seeking in animal models. However, which nAChR subtype(s) are responsible for initiating these cellular changes is poorly understood. nAChRs containing the α6 subunit (α6* nAChRs) are highly and selectively expressed in DA neurons in the VTA. Therefore, we hypothesized that activation of α6* nAChRs is sufficient to enhance AMPAR function on the surface of VTA DA neurons. To test this, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9S mice). We found that low concentrations of nicotine could act selectively through α6* nAChRs to enhance the function of AMPARs on the surface of VTA DA neurons. Through pretreatment with pharmacological inhibitors, we found that NMDA receptors, as well as Ca2+/calmodulin dependent protein kinase II, are also required for this effect. We subsequently expanded these studies to include alcohol because of the high rate of tobacco and alcohol co-abuse. Just as with nicotine, we found that low concentrations of ethanol were sufficient to enhance AMPAR function on VTA DA neurons of α6L9S mice. Because ethanol and nicotine both modulate AMPAR function in a manner involving α6* nAChRs, we tested the hypothesis that low concentrations of ethanol and nicotine combine to modulate AMPAR function. Remarkably, co-incubation of α6L9S brain slices in concentrations of ethanol and nicotine that are sub-threshold when incubated alone resulted in robust enhancement of AMPAR function. Within the VTA, α6 nAChR subunits form nAChRs with and without the α4 nAChR subunit. Therefore, we studied the contribution of α4 nAChR subunits to nicotine-elicited changes in VTA synaptic plasticity. To address this, we removed α4 nAChR subunits from the VTA of adult mice by injecting viral vectors directing expression of Cre recombinase into the VTA of mice with loxP sites flanking the α4 subunit gene. We found that nicotine no longer increases AMPAR function when α4 nAChR subunits are removed from the VTA, indicating a role of nAChRs that contain both α4 and α6 nAChR subunits in VTA synaptic plasticity. Interestingly, we also saw that removing α4 subunits from the VTA of adult mice increases the excitability of VTA DA neurons. We hypothesized that removal of α4* nAChRs from GABAergic neurons in the VTA results in less tonic inhibition of VTA DA neurons. To test this we measured spontaneous inhibitory postsynaptic currents (IPSCs) on VTA DA neurons. Indeed, we saw that the instantaneous frequency of IPSCs was significantly reduced when α4 nAChR subunits are removed from the VTA. Overall, these studies highlight the importance of α4α6* nAChRs in the initiation of cellular changes that play a role in addiction to nicotine, suggesting α4α6* nAChRs may be a promising target for future smoking cessation pharmacotherapies

Use of an Episodic Food Intake Monitoring System to Evaluate Feeding Behavior in Mice

poster abstractThe measurement of food consumption in laboratory animals is critical to studies in metabolism and obesity. Unfortunately, feeding behavior is very sensitive to the environment. Many factors such as the change of cages, diet, and human interactions can introduce undesired experimental variation. Here we describe our experiences with a commercially available episodic food intake monitoring system, the BioDAQ Monitor. This system is designed to quantitatively record feeding behavior in mice. It continuously monitors the weight of the food and uses this information to determine bout length and size. Bouts that occur soon after one another can then be defined as meals. When an animal jostles the food hopper while eating, the weight of the hopper fluctuates and eating is considered to be in progress. Once the hopper weight has been stable for a specified time, that period of feeding is considered to be concluded. The system also has the capability to assess either food or liquid choice paradigms and to directly measure the administration of orally available drugs in either the feed or the water. In addition to these functions, the system uses an environment monitor to record temperature, humidity and lighting of the room every five minutes. Here we present data showing measurements taken in hyperphagic mutant mice, altered feeding paradigms, and under different drug and protein hormone treatments. Future studies using this system will continue to focus on the hyperphagia associated obesity phenotype observed in mice upon conditional disruption of primary cilia

Understanding Cilia Function on POMC Neurons in Appetite and Satiety

poster abstractOver one-third of adults in the United States are obese. Obese individuals are at an increased risk for cardiovascular diseases, type 2 diabetes, cancer, and other health conditions, resulting in premature death. Interestingly, cilia have been linked to controlling satiety in both mice and humans, and individuals with dysfunctional cilia are often obese. Cilia are cellular appendages composed of microtubules and can be motile or immotile. Primary (immotile) cilia function as sensors for important signaling pathways. The loss of cilia, specifically from hypothalamic proopiomelanocortin (POMC) expressing cells, disrupts satiety, leading to overeating and obesity. While it is known that cilia loss in POMC cells in the hypothalamus causes obesity, the age or developmental stage at which cilia loss is important for this phenotype remains unclear. The aim of this research is to determine the time point critical for proper cilia function on POMC neurons to maintain normal feeding behaviors. To do this, we utilize an inducible POMC-CreER mouse model. This model allows us to disrupt cilia formation and maintenance at specific stages of life. We take a multifaceted approach to analyze the impact of cilia loss by measuring longterm body weight and feeding behavior in adult mice, studying changes in embryonic development, as well as analyzing physiological changes in cultured primary neurons. These studies will contribute to a better understanding of the role of cilia in satiety signaling which will help lead to the development of effective treatments for weight related diseases

Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures

Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address in vivo. Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity

Differential Expression and Function of Nicotinic Acetylcholine Receptors in Subdivisions of Medial Habenula

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and β4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, β2, β3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 μm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV

The Hedgehog Signaling Pathway is Expressed in the Adult Mouse Hypothalamus and Modulated by Fasting

The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies

A CreER Mouse to Study Melanin Concentrating Hormone Signaling in the Developing Brain

The neuropeptide, melanin concentrating hormone (MCH), and its G protein‐coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1‐Cre) exists, there is a need for an inducible Mchr1‐Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1‐Cre expression pattern are recapitulated by the Mchr1‐CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1‐CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1‐CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences

α6* Nicotinic Acetylcholine Receptor Expression and Function in a Visual Salience Circuit

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway

An N‐terminal fusion allele to study melanin concentrating hormone receptor 1

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs

Recognition of dance-like actions: memory for static posture or dynamic movement?

Dance-like actions are complex visual stimuli involving multiple changes in body posture across time and space. Visual perception research has demonstrated a difference between the processing of dynamic body movement and the processing of static body posture. Yet, it is unclear whether this processing dissociation continues during the retention of body movement and body form in visual working memory (VWM). When observing a dance-like action, it is likely that static snapshot images of body posture will be retained alongside dynamic images of the complete motion. Therefore, we hypothesized that, as in perception, posture and movement would differ in VWM. Additionally, if body posture and body movement are separable in VWM, as form- and motion-based items, respectively, then differential interference from intervening form and motion tasks should occur during recognition. In two experiments, we examined these hypotheses. In Experiment 1, the recognition of postures and movements was tested in conditions in which the formats of the study and test stimuli matched (movement-study to movement-test, posture-study to posture-test) or mismatched (movement-study to posture-test, posture-study to movement-test). In Experiment 2, the recognition of postures and movements was compared after intervening form and motion tasks. These results indicated that (1) the recognition of body movement based only on posture is possible, but it is significantly poorer than recognition based on the entire movement stimulus, and (2) form-based interference does not impair memory for movements, although motion-based interference does. We concluded that, whereas static posture information is encoded during the observation of dance-like actions, body movement and body posture differ in VWM