Synaptic plasticity in neuronal circuits regulating energy balance

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1 F o c u s o n n e u r a l c o n t r o l o f f e e d i n g Synaptic plasticity in neuronal circuits regulating energy balance Lori M Zeltser 1, Randy J Seeley 2 & Matthias H Tschöp 3 Maintaining energy balance is of paramount importance for metabolic health and survival. It is achieved through the coordinated regulation of neuronal circuits that control a wide range of physiological processes affecting energy intake and expenditure, such as feeding, metabolic rate, locomotor activity, arousal, growth and reproduction. Neuronal populations distributed throughout the CNS but highly enriched in the mediobasal hypothalamus, sense hormonal, nutrient and neuronal signals of systemic energy status and relay this information to secondary neurons that integrate the information and regulate distinct physiological parameters in a manner that promotes energy homeostasis. To achieve this, it is critical that neuronal circuits provide information about short-term changes in nutrient availability in the larger context of long-term energy status. For example, the same signals lead to different cellular and physiological responses if delivered under fasted versus fed conditions. Thus, there is a clear need to have mechanisms that rapidly and reversibly adjust responsiveness of hypothalamic circuits to acute changes in nutrient availability. Synaptic plasticity, a change in synaptic strength in response to stimuli, is an essential feature of neuronal circuits that need to adapt in an experience-dependent manner 1. The ability to alter synaptic strength in response to different degrees of activation endows circuits with the capacity to maintain responsiveness across a broad range of stimuli. This attribute would promote an organism s survival under different conditions of nutrient availability. Extrapolating from what is known about synaptic plasticity in other CNS circuits, it is possible to hypothesize several roles for synaptic plasticity in circuits regulating energy balance. First, changes in synapse number, probability of presynaptic transmitter release and/or postsynaptic responses could establish thresholds of activation in key neuronal populations 2. This type of plasticity acts to match the dynamic range of circuit responsiveness to the relevant physiological range of stimuli in the environment and serves as a gate to control the output of neuronal circuits 2,3. It would be beneficial, from an evolutionary standpoint, for animals in a nutrient-poor environment or those with intermittent access to food to respond more vigorously to food than animals with easy or abundant access to food. Second, as most animals eat in discrete periods of the day, the ability to modulate synaptic strength in response to frequent changes in short-term signals of nutrient availability would help to coordinate physiological responses in a manner appropriate to the prandial state. For example, the drive to eat should be greater in animals that have not recently eaten than in those that have just finished a meal. The field of synaptic plasticity in circuits regulating energy balance is still in its infancy. As such, initial research efforts in this area have been focused on demonstrating that 1 Naomi Berrie Diabetes Center, Division of Molecular Genetics, and Department of Pathology and Cell Biology, Columbia University, New York, New York, USA. 2 Metabolic Diseases Institute, University of Cincinnati, Cincinnati, Ohio, USA. 3 Institute for Diabetes and Obesity, Helmholtz Zentrum München and Division of Metabolic Diseases, Department of Medicine, Technische Universität München, Munich, Germany. Correspondence should be addressed to M.H.T. (tschoep@helmholtz-muenchen.de). Published online 25 September 2012; doi: /nn.3219 synaptic architecture changes in response to a variety of hormonal and nutrient signals 4 6. As there has been substantial progress in characterizing some of the mechanisms underlying synaptic plasticity in the hypothalamus, it is a good time to assess the current state of the field and to reflect on directions for future research. The important question for this Review is whether such mechanisms of synaptic plasticity are crucial to the accurate matching of energy intake to energy expenditure exhibited by most adult mammals, as has been proposed 4 6. The corollary to this overarching hypothesis is that the failure of these circuits to adapt appropriately to a changing environment that includes caloric surpluses and calorically dense and highfat diets degrades the ability of these key hypothalamic circuits to match eating behavior to the rate of caloric utilization, resulting in obesity, and to match appropriate fuel availability to fuel needs, resulting in type 2 diabetes 5. These are pressing concerns given the rising rates of both of these disorders. How we view these epidemics and what we do about them are critically informed by answering these questions. Dynamic hypothalamic responses suggest synaptic plasticity The first demonstration of synaptic plasticity in energy balance circuits by Horvath and colleagues 7 arose from analyses of two neuronal populations in the arcuate nucleus of the hypothalamus (): orexigenic neurons expressing neuropeptide Y (NPY) and Agoutirelated peptide (AgRP) and anorexigenic neurons expressing proopiomelanocortin () 8,9. Major targets of both populations of neurons are downstream neurons that express the melanocortin receptor 4 () Although products such as alphamelanocyte stimulating hormone (α-msh) increase signaling, AgRP reduces signaling 11, It is clear that reduced signaling results in rapid obesity in both humans and animals Thus, the balance between the firing rate of and neurons is a major regulator of feeding behavior and body weight. These neurons were ideal candidates for examining the possibility that synaptic plasticity contributes to their activity, as the neurons 1336 VOLUME 15 NUMBER 10 OCTOBER 2012 nature neuroscience

2 sense a wide range of nutrient and hormonal signals (including leptin, ghrelin, insulin, glucose, amino acids and free fatty acids) and their responses to many of these signals change dramatically under fed versus fasted conditions 9,19. For example, neuronal activity and Pomc mrna expression and peptide release increase following feeding and decrease following fasting, whereas neurons show the opposite pattern of changes in activity, expression and secretion These observations have been replicated using a wide variety of experimental approaches, including quantification of Npy, Agrp and Pomc transcripts, immunohistological detection of c-fos expression, and recording of electrophysiological activity from these specific neuronal subpopulations. For example, Takahashi and Cone found that fasting in mice increases the firing rate of AgRP neurons 23. A key point is that a wide range of signals that are altered by feeding versus fasting also have opposite effects on these two populations of neurons 22,24. Furthermore, in acute hypothalamic slices, the fastinginduced increase in neuronal activity is inhibited by leptin and stimulated by ghrelin, whereas subpopulations of neurons are stimulated by leptin 21,23, Electrophysiological recordings in ex vivo preparations provided the first mechanistic insights into regulation of neurons by prandial state and by hormonal inputs 21,26. neurons are inhibited in the fasting condition and are activated following re-feeding 28. In addition, immunohistochemistry and electrophysiology in brain slices support the idea that a subset (30 40%) of neurons are leptin sensing; these neurons depolarize in response to bath application of leptin 21,24. In addition to direct modulation of neuron activity by prandial state or hormonal signals there is also evidence for indirect regulation via changes to presynaptic inhibitory inputs under these conditions. Inhibitory synapses on soma containing NPY and AgRP have been described 21,29. Stimulation of neurons by fasting or ghrelin administration increased inhibitory currents in neurons in slice preparations 21,26, but efforts to search for -mediated regulation of neurons were not successful 30. From these findings, it was hypothesized that leptin could modulate the balance of anabolic and catabolic melanocortin signals through direct effects via the leptin receptor on neuron and neuron firing rates and indirect effects through its actions to suppress GABA release from neurons 21,26,29. Another notable feature of neuronal populations is that the magnitude of the response to many important hormonal signals varies between fed and fasted conditions. If firing thresholds were hard-wired, the degree of neuronal activation would be determined exclusively by the strength of the stimulus, in this case, the concentration of hormone. There is evidence supporting the idea that cellular and physiological responses to leptin and ghrelin are different under fasted versus fed conditions Change in the magnitude of the response evoked by a fixed stimulus under different environmental conditions can be regulated at several different levels, including synapse strength, synapse number and intrinsic neuronal excitability. However, it is also possible that differences in responsiveness reflect floor and ceiling effects and are not a result of synaptic plasticity per se. Although there is evidence that feeding state and hormonal signals influence circuit responsiveness via effects on both synaptic plasticity and intrinsic membrane properties, this Review will focus on mechanisms of hypothalamic synaptic plasticity. First reports of synaptic plasticity in melanocortin circuits The earliest evidence of plasticity in hypothalamic circuits regulating energy balance came from electron microscopic and electrophysiological analyses that quantified excitatory and inhibitory synapses as well as postsynaptic currents on fluorescently tagged and neurons 7. In their pioneering study on changes in synaptic organization in ob/ob (leptin deficient) mice, the Horvath and Friedman laboratories found that leptin induced more excitatory inputs on neurons and fewer on neurons; inhibitory synapses were affected in the opposite direction, albeit to a lesser degree 7. This alteration in neuronal inputs would serve to increase activity of neurons while decreasing activity of neurons, a response that is conducive to leptin increasing signaling and reducing food intake. Moreover, this powerful change in synapses occurs within 6 h of leptin treatment. Other important factors sensed by these neurons also regulate the composition of synaptic inputs in a similar fashion. These include ghrelin 7,26, estradiol 34 and corticosterone 35, which affects synaptology in a manner consistent with their effects on food intake and energy balance. If synaptological analyses accurately reflect the relative contribution of changes in inhibitory and excitatory inputs to modulate neuronal firing rate in response to hormonal signals, the data would support a mechanism in which leptin acts primarily via direct changes in excitatory inputs onto both and neurons, as well as indirect effects resulting from suppression of inhibitory neurons onto neurons 6,7. Taken together, these studies provide evidence that the number of synaptic connections onto and neurons can change dramatically in the time frame that is necessary to coordinate between changes in available fuel caused by fasting and long-term signals of energy balance 4 6. One issue that is not addressed by these studies, however, is whether such synaptic plasticity is recapitulated with the fluctuation of fuels that comes after meals, as each of these studies restores a signal that is nearly absent and examines its potent effects, rather than the more graded responses that happen over the course of normal physiology. Moreover, as synaptic reorganization has not been examined in a time frame consistent with the acute action of meal initiation and termination signals, it is less clear that this mechanism of plasticity contributes to short-term regulation of food intake (Fig. 1). Synaptic plasticity in melanocortin neurons: recent insights Over the past few years, scientists have begun to use combinations of genetic and pharmacological tools to manipulate discrete components of the circuits regulating energy balance in conjunction with the assessments of both metabolic and electrophysiological endpoints. These approaches are yielding insights into how and NPY- AgRP neurons are regulated by pharmacological administration of leptin, as well as by physiological changes in feeding state. It should be noted that studies using transgenic GFP reporters to characterize electrophysiological properties or synaptic inputs of or NPY- AgRP neurons often produced discordant results, particularly in the case of neurons. For example, some groups have found that the vast majority of neurons are depolarized by leptin 21,36, whereas other groups have reported that leptin signaling or its effects on neuronal activity are only observed in a subpopulation (30 40%) of neurons 24, In addition, there is disagreement in the literature over whether or not a subset of neurons expresses the inhibitory neurotransmitter GABA 28,40. These discrepancies raise the possibility that variations in culture and recording conditions can substantially affect results. Moreover, there is a growing body of evidence to support the idea that there are functionally distinct populations of and populations that exhibit distinct responses to energy-related signals and project to different downstream targets 21,38,39,41 44 ; heterogeneity in these populations would confound analyses of a generic or NPY neuron. nature neuroscience VOLUME 15 NUMBER 10 OCTOBER

3 Plasticity in neurons Leptin rapidly depolarizes neurons through intrinsic effects on channel conductance and by decreasing inhibitory synaptic inputs 21. Fasting is associated with a reduction in excitatory currents in neurons 45,46, and leptin or estrogen treatment increases the number of excitatory synapses onto neurons in ob/ob mice 7,34. However, as marked reductions in the number of excitatory synapses onto neurons in ob/ob mice are not translated into substantial decreases in excitatory currents 7, it raises the question of whether leptin-induced changes in synaptic architecture are physiologically relevant. Moreover, disrupting leptin signals in glutamatergic neurons does not change excitatory currents in neurons 28. Together, these observations raise the possibility that fasting-induced changes in excitatory inputs to neurons are mediated by signals other than leptin. As neurons lack dendritic spines 47, a major site of synaptic plasticity for excitatory inputs 48, efforts to investigate excitatory synaptic plasticity should consider mechanisms that influence the probability of transmitter release from presynaptic neurons and postsynaptic processes that influence cellular responses to excitatory signals. Data from wild-type and ob/ob mice support the idea that leptin suppresses inhibitory currents in neurons 7,21. The effects of leptin on inhibitory currents are likely mediated presynaptically, as genetic disruption of leptin signaling in neurons does not affect inhibitory currents in neurons at baseline, nor does it impair leptin s well-established ability to suppress fasting-mediated increases in inhibitory currents in these neurons 28. In contrast, genetic strategies to disrupt leptin signaling in all GABAergic neurons leads to hyperpolarization of neurons at baseline conditions and a failure of leptin to suppress fasting-induced inhibitory currents 28. These observations support the idea that leptin signaling in presynaptic neurons reduces GABA release onto neurons, thereby increasing responsiveness of neurons to other excitatory signals, and implicate leptin as a signal that gates responsiveness of the circuit to the fed or fasted states. The fact that these mice are severely obese underscores the assertion that the global modulation of inhibitory tone has a major role in leptin-mediated body-weight regulation 28. It is important to understand how leptin functions to modulate GABA release, whether it is a result of the activation of an intracellular signaling cascade that ultimately modulates synaptic function or whether it acts directly on the synapse. The only CNS sites at which leptin receptor is expressed in GABAergic neurons are in the, dorsomedial nucleus of the hypothalamus (DMH) and lateral hypothalamus 28. The assertion that GABAergic neurons in the are the primary source of inhibitory leptin-mediated inputs onto neurons is called into question by the observation that disrupting leptin signaling in these neurons does not impair leptin s ability to reduce fastingmediated increases in inhibitory currents in neurons 28. Although these findings support the idea that inhibitory inputs from neurons are not necessary for this effect of leptin, as these circuits have an extraordinary capacity to compensate for the loss of signals from neurons 49, they do not disprove previous reports that neurons do provide inhibitory inputs to neurons 21,30,50. In fact, activation of neurons directly or by the appetite-stimulating hormone ghrelin has been shown to inhibit neuronal activity The role of ghrelin in synaptic plasticity on neurons is discussed below. Evidence supporting the existence of leptin-mediated inhibitory synaptic plasticity on neurons is compelling; however, this mechanism of plasticity is neither necessary nor sufficient to maintain normal body weight. Disruption of GABA release from leptin receptor expressing neurons causes a much less dramatic effect on body weight 53 than impairments in leptin signaling in GABAergic neurons 28. These findings support the idea that GABA transmission is not required for proper circuit function and that relevant populations of leptin-sensing GABAergic neurons can regulate hypothalamic circuits through the release of other neuromodulators. On the other hand, preservation of leptin-mediated suppression of inhibitory currents in some neurons is not sufficient to maintain body weight in mice with deficits in leptin-mediated phospho-stat3 signaling 54. These observations highlight the need to consider that, although neuronal activity is often the primary endpoint assessed, there are many other neurons in the and VMH that likely contribute to leptin s effects on body weight regulation. As discussed above, leptin is not the only regulator of synaptic input organization of neurons. Other circulating hormones, such as ghrelin, estradiol and corticosterone, have all been found to affect the connectivity and activity of neurons, consistent with these hormones respective roles in metabolism regulation 7,34,35,51. Plasticity in neurons It is well established that neurons are activated both by fasting and by ghrelin, as measured by increased numbers of cells that are positive for c-fos and by electrophysiological recordings of action potentials 23,26,51. Early electrophysiological studies reported that fastingand orexigen-induced changes in the frequency of firing activity do not require synaptic inputs 23,27 but are modulated by potassium channel conductance or other effects on intrinsic membrane potential, consistent with a direct effect on neurons. Recent work also showed that the AMPA receptor (AMPAR) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) prevents ghrelin-induced AgRP neuron activation in brain slices 46. Recent studies using genetic approaches provide evidence consistent with a physiological role for fasting- and hormone-mediated modulation of excitatory synaptic inputs onto neurons. neurons have many dendritic spines 47, which serve as the primary site of synaptic plasticity of excitatory inputs 48. The number of dendritic spines is increased by fasting and gradually decreases to baselines levels after several days of feeding with the same temporal profile as the restoration of baseline levels of food intake 25,46,47. Consistent with previous observations that ghrelin signaling activates neurons 26, it is also reported to be necessary and sufficient to modulate fasting-induced increases in excitatory currents in neurons 46. As enhanced excitatory inputs would potentiate orexigenic actions of neurons under fasted conditions, this type of plasticity would promote adaptive responses to alterations in nutrient availability that restore the levels of stored fuel and energy balance. Notably, promotion of spine synapse formation by the hunger hormone ghrelin was first identified in the dentate gyrus and CA1 region of the hippocampal formation 55, suggesting that hunger state triggered alteration in spine density is characteristic of specific subpopulations of neurons that include principal cells of the hippocampal formation and arcuate nucleus neurons. Activation of postsynaptic NMDA receptors (NMDARs) has a well-established role in mediating plasticity in excitatory synapses 48. Using a genetic approach, the Lowell group found that loss of NMDARs in neurons leads to a reduction in fasting-induced c-fos, Npy and Agrp mrna expression, dendritic spine number and firing rate 47. The fact that modulation of synaptic organization onto neurons is correlated with a decrease in neuronal activity, as well as a reduction in food intake and 1338 VOLUME 15 NUMBER 10 OCTOBER 2012 nature neuroscience

4 a Previous model b Previous model c Updated model d Updated model Fasting Feeding Fasting Feeding GABA, AgRP, NPY GABA, AgRP, NPY AMPK UCP2 Sirt1 Glutamate Ghrelin Leptin Excitatory synaptic inputs Ghrelin Leptin Inhibitory synaptic inputs Unidentified leptinsensing cells GABA AMPK Unidentified ghrelinsensing cells Unidentified inhibitory neuromodulators AMPK β-endorphin Leptin receptor Ghrelin receptor AMPAR NMDAR Opioid receptor Figure 1 Changes in the balance between the firing rates of and neurons in response to the nutrient and hormonal environment are thought to be important for regulating feeding behavior and body weight. (a,b) Initial models of the role of synaptic plasticity in feeding circuits were largely based on electron microscopic and electrophysiological analyses of the consequences of restoring a signal that is nearly absent (that is, leptin in ob/ob mice) on excitatory and inhibitory synaptic organization onto fluorescently tagged and neurons. In the fasted state, which is associated with high ghrelin and low leptin levels, excitatory inputs to neurons are increased, whereas neuronal activity is reduced through a decrease in excitatory transmission, as well as through inhibitory inputs from neurons (a). Conversely, in the fed state, the balance of excitatory versus inhibitory synaptic inputs is reversed, increasing the anorexigenic melanocortin tone (b). (c,d) The application of genetic and pharmacological tools to manipulate discrete circuit components has yielded insights into the role of synaptic plasticity in energy balance circuits. Ghrelin signaling is reported to be necessary and sufficient to mediate fasting-induced increases in glutamatergic transmission onto dendrites through an AMPK-dependent mechanism (c). There are leptin-sensing GABAergic neurons that supply inhibitory inputs to neurons in addition to neurons in the fasted state. In the fed state, leptin-dependent increases in β-endorphin release from neurons provide negative feedback to suppress ghrelin-induced increases in excitatory transmission onto neurons (d). Leptin-dependent increases in β-endorphin release may stem from direct effects on neurons, as well indirect effects through reductions in leptin-sensitive inhibitory inputs. body weight, supports the idea that this postsynaptic mechanism contributes to normal body-weight regulation 47. Findings from experiments involving pharmacological (as opposed to genetic) manipulations of neuronal signaling in acute slices invoke a presynaptic rather than a postsynaptic mechanism for fasting-mediated activation of excitatory glutamatergic transmission onto neurons. For example, AMPK signaling is necessary for ghrelin-induced increases in neuronal activity 51 ; however, it is not clear whether it is required in AgRP neurons 46,56. In addition, common mechanisms of postsynaptic plasticity (such as changes in AMPAR/NMDAR synaptic current ratio and rectification of glutamatergic synaptic currents) in neurons do not appear to be influenced by fed and fasted states 46. Together with additional electrophysiological recording, these findings led the Sternson group to propose that presynaptic ghrelin-mediated activation of an AMPK-dependent positivefeedback loop promotes glutamatergic transmission onto neurons 46. Localizing the presynaptic glutamatergic ghrelin-sensing neuronal population and identifying genetic reagents that target these neurons would facilitate mechanistic studies and are critical for examining the consequences of direct perturbations of this form of plasticity on feeding behavior. Leptin inhibits neurons through rapid and reversible effects on excitatory synaptic inputs 25 and persistent effects on intrinsic neuronal membrane properties 23,25,27,57. There are some key methodological issues that should be considered at this point. Parsing the contribution of synaptic plasticity versus intrinsic changes to leptin s effects on neuronal activity has been complicated by inconsistencies in findings with ex vivo leptin treatment. Although bath application of leptin to acute slices from fed mice has been reported to decrease excitatory currents in neurons 25, it does not when slices are prepared from fasted mice 46. As leptin injection in vivo is capable of suppressing fasting-induced increases in excitatory inputs 46, these observations raise the possibility that critical leptin-sensing neurons are outside the plane of section of the brain slice. Although initial models of circuit function proposed that neurons inhibit neuronal activity 21,26, recent findings from the Sternson laboratory provide evidence that also provides negative feedback to neurons 46. The effects of bath application of ghrelin and/or the opioid antagonist naltrexone on brain slices from fed versus fasted mice are consistent with a model in which leptin-mediated increases in opioid tone provide negative feedback to suppress fasting- or ghrelin-induced increases in excitatory transmission but do not influence neuronal activity under fed conditions 46. As early efforts to identify examples of -mediated regulation of neurons were largely dependent on melanocortin-related rather than opioid-related reagents, it is understandable that this mechanism was not observed previously 21,30. Optogenetic neuronal stimulation in slices from fasted mice leads to a reduction in excitatory currents in neurons, which is reversed with naltrexone treatment, consistent with the idea that neurons can serve as a source of opioid signals to NPY- AgRP neurons 46. These findings raise the question of why neurons are not capable of inhibiting fasting-induced excitatory transmission following bath application of leptin 46. It is possible that leptin-mediated opioid release from neurons is mediated by neurons that lie outside the plane of the brain slice. Another explanation is that the physiologically relevant neuron lies outside the plane of the section. Fortunately, genetic reagents are available to test whether one or both of these explanations are valid. nature neuroscience VOLUME 15 NUMBER 10 OCTOBER

5 Investigating causality in synaptic plasticity Prandial state and associated hormonal and nutrient signals cause changes in synaptic inputs onto and neurons and changes to intrinsic membrane properties; however, the relative contributions of either mechanism to potent changes in energy balance remains unknown. This is partly a result of technical limitations associated with the primary techniques used to assess the effects of synaptic plasticity on circuit function: electrophysiological recordings from brain slices and genetically induced loss-of-function mutations. The major limitation of electrophysiological recordings from ex vivo brain preparations is that neurons in circuits regulating body weight are broadly distributed, and therefore many physiologically relevant synaptic partners are not contained in the same coronal brain slice. Development of tools to directly modulate synaptic terminals without the need for intact soma could overcome this obstacle 58. Another issue with slice studies is that the culture conditions are often very different from physiological conditions in which neurons normally function; variations in these conditions could contribute to some inconsistencies in data from different groups. Finally, the most substantial limitation of ex vivo approaches is that they are not able to simultaneously assess effects of synaptic events on physiological behaviors. Given these constraints, investigations of the role of synaptic plasticity in energy balance regulation should involve combinations of ex vivo electrophysiological recordings and parallel studies of synaptic structure. Going forward, the identification of immunohistochemical reagents that label relevant synapses in energy balance circuits 59 would facilitate analyses of synaptic elements on dendrites and axons and would also broaden the number of laboratories that are capable of examining synaptic structure, as this approach does not require electron microscopy. There are also several important caveats associated with the use of genetic ablations of transmitter or hormone receptors to study synaptic plasticity. First, as neurotransmitter receptors such as NMDAR are involved in synapse formation and maturation 60,61, deficits in circuit function could be a result of earlier developmental effects. Second, as circuits can compensate for loss of neurons in neonates 49, adaptations in the circuit could misrepresent the physiological contributions of certain neuronal connections. Finally, as transmitter receptors could influence neuronal activity by influencing membrane properties or postsynaptic conductance, changes cannot be definitively attributed to synaptic plasticity per se. Thus, the development of viral reagents that target specific pathways mediating a particular type of synaptic plasticity (as opposed to neurotransmitter receptors themselves) in adult animals would provide important insights into their contribution to body-weight regulation. Evidence of plasticity outside of the melanocortin system Efforts to characterize synaptic plasticity outside of melanocortin neurons in the have been hampered by neuroanatomical and technological constraints. Electrophysiological studies of synaptic plasticity are most productive when the relevant synaptic partners are contained in the slice preparation. In the case of the hypothalamus, many important connections cannot be captured in a slice and therefore cannot be measured. As neurons are extensively interconnected 62, this is one area that is amenable to electrophysiological analyses; another area is the lateral hypothalamus. Tools to overcome this limitation are being developed 52,58 and will certainly facilitate studies of synaptic plasticity in long-range projections that have been proposed to affect energy expenditure, such as those from the to the paraventricular nucleus (PVH). Extra- hypothalamic nuclei The ability to reliably record from synaptic partners is enhanced in brain regions with a laminated organization, such as the cortex, where distinct classes of neurons occupy characteristic topographical positions and exhibit stereotyped synaptic connections. As functionally distinct classes of hypothalamic neurons are interspersed, mice with fluorescently tagged populations of interest are needed to investigate synaptic plasticity at the electrophysiological level. As only a few of these lines reliably mark subsets of neurons in hypothalamic circuits regulating energy balance, progress has been limited. The first studies of synaptic plasticity outside of the were reported in orexin neurons of the lateral hypothalamus by the Horvath laboratory 63. Orexin (also known as hypocretin) neurons are critical for arousal 64 and have been synaptically tied to the melanocortin system 65 and feeding regulation 66. It was found that the synaptic inputs onto orexin neurons, revealed by both electron microscopy and analyses of miniature postsynaptic events, are rapidly induced by fasting and involve leptin action 63. This is the first, and to date only, metabolically triggered synaptic plasticity in the brain that was confirmed by in vivo, real-time analyses of synaptic changes in a zebrafish model 67. Electrophysiological studies in the DMH and lateral hypothalamus by the Role and Bains laboratories provided compelling evidence of synaptic plasticity through retrograde endocannabinoid (ecb) and nitrous oxide (N 2 O) signaling. Activation of neurons in the perifornical lateral hypothalamus induces an ecb-mediated suppression of inhibitory tone to lateral hypothalamus neurons, which is blocked by leptin 68. Retrograde ecb signaling is reported to suppress transmission from GABAergic synapses in the DMH, whereas retrograde N 2 O signaling has the opposite effect 69. N 2 O is preferentially released in response to short bursts of high-frequency stimulation in the DMH, leading to long-term potentiation, whereas longer stimulation induces negative feedback to the system through ecb induction. The balance between ecb and N 2 O determines whether inhibitory tone is reduced or enhanced. Notably, this polarity switch is not only observed with pharmacological manipulations of slices but is also recapitulated with food deprivation and re-feeding 69. Defining the identity of these neurons, as well as their contribution to the regulation of energy balance, is an important area for future research. Appetitive motivation and reward-related feeding There has been increasing interest in determining the relationship between homeostatic and hedonic aspects of feeding in relation to metabolic disorders such as obesity. For example, studies have shown that ghrelin can modify midbrain dopamine function while promoting feeding The Horvath laboratory found that, during this action of ghrelin in the ventral tegmental area, synaptic input organization of dopamine neurons rapidly changes, as assessed by both electrophysiology and morphology 71. The effect of ghrelin in modifying VTA dopamine neuronal activity and output was found to involve changes in miniature postsynaptic events 71, a process that was recently recapitulated regarding ghrelin s action on AgRP neurons 46. A very recent development introduces yet another twist in the complexity of energy metabolism related synaptic plasticity with specific relevance to a relationship between the circuitry and the midbrain dopamine system 73. Here, the integrity of the neuronal circuitry via AgRP neuron efferents to the ventral tegmental area were reported to directly affect synaptic plasticity, most notably long-term potentiation propagation in midbrain dopamine neurons 73. A recent study from the Malenka group provides anatomical, electrophysiological and behavioral evidence to support a direct role for projections from neurons to D1 dopamine 1340 VOLUME 15 NUMBER 10 OCTOBER 2012 nature neuroscience

6 receptor expressing medium spiny neurons in the nucleus accumbens in mediating stress-induced anhedonia and weight loss 74. α-msh production and release in neurons 75 and Mc4r expression in the nucleus accumbens are induced by stress 74. In acute brain slices, both α-msh and chronic stress reduce excitatory postsynaptic currents specifically in D1 medium spiny neurons via changes in AMPAR subunit composition 74. Moreover, signaling in D1 neurons is required for the stress-induced modulation of D1 excitatory transmission, AMPAR stoichiometry and NMDAR-induced long-term depression, as well as effects on food intake 74. Together, these findings have implications for behaviors not limited to feeding and energy expenditure, and they suggest that hormonal action on hypothalamic synaptic plasticity may have long-range, indirect effects on synaptic plasticity in a broad array of brain regions. The key question: a cause or a consequence? Taken together, it is clear that synaptic plasticity occurs in a wide variety of neurons that contribute to the regulation of food intake and energy balance. Moreover, a number of crucial signals that make it possible to maintain energy homeostasis have also been shown to modulate synaptic organization and/or function. However, what remains to be determined is the degree to which synaptic plasticity contributes to either normal energy homeostasis or the dysregulation that underlies obesity or diabetes. For example, exposure to high-fat diets results in reduced ability of leptin to constrain further food intake, and this process is thought to contribute to the defense of higher body weights when animals are maintained on a high-fat diet Such leptin resistance has been hypothesized to be a defect of either leptin transport into the CNS or reduced leptin action on important leptin-responsive neurons 78. The degree to which leptin resistance is the result of impairments in leptin-mediated synaptic plasticity in key hypothalamic circuits is not yet clear. A recent study began to address questions of cause or consequence and found that the synaptic constellation on neurons is predictive of vulnerability to diet-induced obesity in rats and that synaptic organization is affected differentially by high-fat diet in vulnerable and resistant animals 79. On the other hand, they also reported that exposure to a high-fat diet is associated with changes in synaptic inputs onto both and AgRP neurons in directions that were consistent with elevated leptin levels 79 at a time when the electric activity of these neurons showed leptin resistance 80. Ultimately, however, additional tools that will allow for inducible, site-specific manipulation of key molecular pathways that underlie the regulation of synaptic plasticity in specific neuronal populations in conjunction with assessment of metabolic endpoints would provide a more direct way to test the role of alterations in the overall regulation of energy balance. The ability to do this is crucial. There are two competing hypotheses about how plasticity might contribute to metabolic disease. The first is that exposure to diets that induce weight gain and impair glucose regulation inhibit the normal plasticity responses that underlie accurate metabolic regulation. The second is that exposure to such diets induces its own alteration in synaptic strengths that serve to undermine the ability of these homeostatic circuits to protect the organism from external homeostatic challenges. 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