Alfredo Fontanini - US grants

[unreadable] DESCRIPTION (provided by applicant): The brain processes sensory stimuli differently depending on whether an animal is awake or anesthetized. In anesthetized animals no internal cognitive activity influences the brain, and so stimuli are passively received and coded solely for their physical properties. In awake subjects, on the other hand, responses to stimuli are heavily modulated by ongoing mental processes (i.e. attention, expectation). The same stimulus, for instance, is interpreted differently depending on what the animal expects: humans - and their brains - led to expect a mild taste, for instance, can be "fooled" into thinking that a bitter taste is milder than it actually is. The present proposal is designed to study the neural underpinnings of such phenomena: in particular, the experiments outlined here will address the extent to which expectation influences background activity of the brain and responses to sensory stimuli. The intrinsic rewarding/aversive nature of gustatory stimuli and the importance of food selection for animal survival make taste an ideal sensory system for directly studying the effects of expectation at both the neural and behavioral level. The experiments proposed will be conducted in awake rats; ensembles of single neurons will be recorded with multiple electrodes implanted in areas known to be involved in taste and expectation (GC, orbitofrontal cortex [OFC], amygdala [AM]). Two different aspects of expectation will be investigated: generic expectation of an unknown stimulus (a process somewhat analogous to attention) and specific expectation of a known stimulus. For the first, rats will be trained to collect a taste by pressing a lever in response to a tone. In this paradigm, the tone will not provide any information regarding the identity of the taste-it will simply trigger an attentional shift related to the expectation of an unknown stimulus. Expectation of a specific stimulus, on the other hand, will be addressed in another subset of experiments involving the use of two tones, each specifically associated with a different taste. In these experiments, the auditory cues will provide information about the specific taste anticipated. Spontaneous activity, responses to anticipatory cues, and responses to unexpected and expected tastes will be recorded to parse the effects of generic and specific expectation on taste. Finally, electrodes will be implanted also in OFC and AM - two areas known to be involved in expectation - and the relationship between these two areas and GC will be studied in the experimental conditions described above. Understanding the degree to which internal states, such as expectation, shape sensory responses is a central question in the field of sensory neuroscience. The comprehension of how the brain responds to the expectation of tastes will shed light on the complexities of food selection choices of all mammals - including humans - and will provide important information which could potentially be applied to the study of obesity and eating disorders. This proposal is designed to understand the neural underpinnings of expectation of taste stimuli. Expectation plays an important role in the perception of food and in its evaluation (Deliza and MacFie 1996; Hurling and Shepherd 2003); as such, this subject has important implications on issues relevant to public health, such as food liking and selection choices. Additionally, understanding the neural basis of expectation is relevant for the study of the neural mechanisms of placebo (Nitschke et al. 2006a; Nitschke et al. 2006b; Sarinopoulos et al. 2006) - a growing field of interest in neuroscience (Petrovic et al. 2005; Ploghaus et al. 1999). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The brain processes sensory stimuli differently depending on whether an animal is awake or anesthetized. In anesthetized animals no internal cognitive activity influences the brain, and so stimuli are passively received and coded solely for their physical properties. In awake subjects, on the other hand, responses to stimuli are heavily modulated by ongoing mental processes (i.e. attention, expectation). The same stimulus, for instance, is interpreted differently depending on what the animal expects: humans - and their brains - led to expect a mild taste, for instance, can be "fooled" into thinking that a bitter taste is milder than it actually is. The present proposal is designed to study the neural and systems underpinnings of such phenomena: in particular, the experiments outlined here will address the extent to which expectation influences background activity of the brain and responses to sensory stimuli. The intrinsic rewarding/aversive nature of gustatory stimuli and the importance of food selection for animal survival make taste an ideal sensory system for directly studying the effects of expectation at both the neural and behavioral level. The experiments proposed will be conducted in awake rats;ensembles of single neurons will be recorded with multiple electrodes implanted in connected areas known to be involved in taste and expectation (GC, orbitofrontal cortex [OFC], basolateral amygdala [BLA]). Two different aspects of expectation will be investigated: generic expectation of an unknown stimulus (a process somewhat analogous to attention) and specific expectation of a known stimulus. For the first, rats will be trained to collect tastes by pressing a lever in response to a tone. In this paradigm, the tone will not provide any information regarding the identity of the taste-it will simply trigger an attentional shift related to the expectation of an unknown stimulus. Expectation of a specific stimulus, on the other hand, will be addressed in another subset of experiments involving the use of two tones, each specifically associated with the availability of a different taste at a press of a lever. In these experiments, the auditory cues will provide information about the specific taste anticipated. Two behavioral tasks will be used to study different aspects of specific expectation: the first task, a go/no-go in which one tone cues the availability of a palatable taste (sucrose or NaCl) and the other a punishment (quinine) at a press of a lever, will reveal the effects of expectation of particular tastes having opposite affective value. The second task, a two tone, two levers task in which each tone is associated with a taste available at a specific lever, relies on tastes with similar palatability. This task will specifically address the effects of expectation for stimuli with similar affective value but different chemical identity. Spontaneous activity, responses to anticipatory cues, and responses to unexpected and expected tastes will be recorded in GC, OFC and BLA to parse the effects of generic and specific expectations on taste processing. Finally, intracranial cannulae will be implanted in OFC and BLA and the contribution of these two areas in modulating GC activity will be studied for the experimental conditions described above. Understanding the degree to which internal states, such as expectation, shape sensory responses is a central question in the field of sensory neuroscience. The comprehension of how the brain responds to the expectation of tastes will shed light on the complexities of food selection choices of all mammals - including humans - and will provide important information which could potentially be applied to the study of obesity and eating disorders. PUBLIC HEALTH RELEVANCE: This proposal is designed to understand the neural underpinnings of expectation of incipient taste stimuli. Expectation plays an important role in the perception of food and in its evaluation (Deliza and MacFie 1996;Hurling and Shepherd 2003;Small 2008);as such, this subject has important implications on issues relevant to public health, such as food liking and selection choices. Additionally, understanding the neural basis of expectation is relevant for the study of the neural mechanisms of placebo (Nitschke et al. 2006a;Nitschke et al. 2006b;Sarinopoulos et al. 2006) - a growing field of interest in neuroscience (Petrovic et al. 2005;Ploghaus et al. 1999).

DESCRIPTION (provided by applicant): Neurons in the gustatory cortex (GC) respond to sensory stimuli with time-varying modulations of their firing rates. Electrophysiological recordings from anesthetized and awake animals have shown that firing activity can change over few seconds following the onset of stimulus presentation (Grossman et al., 2008; Gutierrez et al., 2010; Jones et al., 2007; Stapleton et al., 2006; Yamamoto et al., 1984b; Yokota et al., 2011). These dynamics are a critical feature of gustatory responses and are believed to mediate the processing of different aspects of gustatory information (Fontanini and Katz, 2006, 2009; Gutierrez et al., 2010; Katz et al., 2002a). While a great deal of work has focused on understanding the functional significance of these patterns, little is known about their genesis. I is not known, for instance, why some neurons display rapid taste responses, some are activated at much longer latencies (i.e. hundreds of milliseconds) and some others do not respond at all. Pharmacological experiments relying on local infusions of the GABA blocker bicuculline point to inhibition as a key player in shaping responses (Ogawa et al., 1998). However, the lack of synaptic resolution of extracellular techniques has limited our understanding of how interactions between inhibition and excitation could influence the time course of responses in different neurons. The experiments in this proposal are designed to test the general hypothesis that time-varying spiking responses are determined by specific combinations of excitation and inhibition. The use of in vivo intracellular techniques will allow to resolve synaptic potentials ad dissect the balance between excitation and inhibition in anesthetized rats (Haider et al., 2006; Stone et al., 2011; Wilent and Contreras, 2005). Intracellular injection of biocytin, followed by histological reconstructions, will be used to identify the cell type and the location of the neuron recorded. The ability to identify the recorded neurons will be instrumental in understanding whether cells in the different layers and divisions (granular, dysgranular and agranular) of GC show specific patterns of synaptic interactions. The analysis of synaptic responses to thalamic, amygdalar and gustatory stimulation will allow us to determine how specific elements in the circuit integrate bottom-up sensory inputs with top-down modulations This framework represents an entirely novel approach to the study of GC in intact animals and promises to provide the first integrative view of the synaptic bases of gustatory cortical processing.

DESCRIPTION (provided by applicant): The gustatory cortex (GC) receives modulatory inputs from multiple regions of the limbic system. Lateral hypothalamus, medial prefrontal cortex, mediodorsal thalamus and basolateral amygdala (BLA) are all known to send projections to GC (Saper 1982; Allen, Saper et al. 1991; Maffei, Haley et al. 2012). Among these inputs, those from the BLA are the ones whose functional significance has been studied the most. Pharmacological and electrophysiological experiments in alert animals have suggested that inputs from BLA are necessary for GC to process information pertaining to the hedonic value of gustatory stimuli (Piette, Baez- Santiago et al. 2012) and the anticipatory value of taste-predictive cues (Samuelsen, Gardner et al. 2012). In addition, plastic changes of the BLA-GC connection have been associated with taste aversion learning (Guzman-Ramos and Bermudez-Rattoni 2012). Despite the abundance of studies investigating the functional role of the BLA-GC connection (Jones, French et al. 1999; Grossman, Fontanini et al. 2008; Guzman-Ramos and Bermudez-Rattoni 2012; Piette, Baez- Santiago et al. 2012; Parkes and Balleine 2013), very little is known on the synaptic organization and plasticity of these inputs. Evidence from intracellular and extracellular recordings in vivo suggests that BLA-GC inputs might exert complex excitatory as well as inhibitory actions (Yamamoto, Azuma et al. 1984; Hanamori 2009; Stone, Maffei et al. 2011), yet no information is available on the synaptic mechanisms underlying these effects. Furthermore, while analysis of BLA evoked potentials in GC provides evidence for learning-related plasticity at this connection (Escobar, Chao et al. 1998; Jones, French et al. 1999; Escobar and Bermudez-Rattoni 2000; Rodriguez-Duran, Castillo et al. 2011), the mechanisms, rules and postsynaptic targets of this plasticity are unknown. Until recently it has been impossible to selectively activate BLA afferents in vitro to finely dissect th GC circuits recruited by amygdalar inputs. The availability of optogenetic tools has however changed the situation (Zhang, Gradinaru et al. 2010; Stuber, Sparta et al. 2011; Yizhar, Fenno et al. 2011; Britt and Bonci 2013; Wang, Kloc et al. 2013), finally allowing us the fundamental questions pertaining to the synaptic organization of amygdalar afferents to GC to be addressed. The experiments proposed here rely on these novel techniques, combined with in vitro whole cell patch clamp recordings, to directly measure the properties of amygdalar synapses onto pyramidal cells and inhibitory interneurons in GC. This methodological approach will be complemented with pharmacological and behavioral manipulations to test the following hypotheses: 1) BLA afferents make direct functional synapses onto different cell types within GC local circuits; 2) Synaptic inputs onto pyramidal neurons and inhibitory interneurons show activity-dependent plasticity; 3) The strength of BLA- GC synapses is affected by hedonic learning. Altogether these experiments will allow us to investigate the synaptic organization of BLA-GC inputs, their plasticity and the changes associated with aversion learning. This framework represents an entirely novel approach to the study of GC inputs in brain slices and promises to provide the first circuit-level description of amygdalar synapses in the gustatory cortex.

? DESCRIPTION (provided by applicant): How is gustatory information represented in the cortex? The answer to this question has been one of the mostly intensely debated topics in the field of gustatory neuroscience1-3. Two theories have been proposed and discussed over the past decades. According to the labeled-line theory the physiochemical identity of a gustatory stimulus is encoded by specific subsets of neurons, each of which is narrowly tuned to a single taste quality (i.e. salty, sweet, sour, bitter or umami)4-7. An alternative theory, the across-neuron pattern theory, postulates that taste coding relies on the combined activity of large ensembles of cortical neurons8. According to this theory, neurons do not need to be selective for specific taste qualities, instead each neuron can densely represent information by encoding multiple qualities3,9. Both theories are supported by experimental evidence. While a recent 2-photon calcium imaging study suggested the exclusive presence of narrowly tuned neurons in GC of anesthetized mice7; years of electrophysiological recordings in anesthetized and alert rodents demonstrate the presence of both narrowly tuned and densely coding neurons in GC10-13. Despite evidence that neurons using different coding strategies exist in GC, the debate is still polarized and no unifying view of taste coding in the cortex has emerged. The overarching goal of this proposal is to test the hypothesis that narrowly tuned and densely coding neurons reflect different stages of cortical processing. Specifically, we propose that the coding scheme varies depending on the cortical layers, with superficial layers (i.e., layers 2/3) featuring narroly tuned neurons and deep layers (i.e., layers 4 and 5) containing densely coding neurons. In addition, the experiments in this proposal aim at providing a mechanistic explanation for these differences, linking coding properties with laminar-dependent variations in inhibitory drive. The experiments will rely on a combination of sophisticated electrophysiological approaches to directly relate coding properties, balance between excitation and inhibition, and properties of local microcircuits. The framework of this grant is firmly grounded in the literature on cortical coding of sensory information14-17, and supported by our preliminary results, showing layer-specific differences in taste response properties and local connectivity. This research will help the field go beyond a dated debate and will move the discussion on taste coding toward a more circuit-oriented perspective. If successful this research will provide, for the first time, a unifid view of taste coding in cortical circuits of alert rodents.

PROJECT SUMMARY The gustatory system processes tastants according to specific sensory and hedonic categories. In naïve animals, gustatory stimuli are represented for the taste quality they evoke ? i.e., sweet, salty, umami, bitter, sour ? and for their hedonic value (palatable or aversive). However, gustatory circuits are highly plastic and representations can be modified by learning. The best example of gustatory plasticity comes from studies on conditioned taste aversion (CTA) - a learning paradigm in which an innately palatable tastant becomes aversive once paired with gastric illness. Electrophysiological recordings and imaging experiments demonstrate that CTA leads to a persistent remapping of cortical representations of taste. While these studies have been instrumental in demonstrating that the gustatory cortex (GC) can reshape how it represents taste, they do not clarify whether persistent remapping is specific to aversive learning and to the hedonic dimension, or if it can occur also for other forms of taste experience. The experiments in this grant will address this unanswered question by investigating whether reward-driven, taste-action associations can modify and remap sensory representations. To this purpose, we modified a classic two-alternative choice procedure. In this task, mice sample one out of four tastants (two sweets: sucrose [S1] and maltose [S2]; two bitters: quinine [B1] and cycloheximide [B2]) from a central licking spout and respond by licking one of two lateral spouts. In the main version of this task, mice will produce the same response to incongruent pairs of tastants (i.e., sucrose or quinine -> lick lateral spout 1 vs maltose or cycloheximide -> lick lateral spout 2). This version of the paradigm requires mice to ignore innate taste similarities and produce i) similar responses for tastants with different qualities and opposite hedonics; ii) different responses for tastants with similar qualities and hedonics. A number of additional behavioral paradigms will be used as controls. The experiments in this grant will test the overarching hypothesis that GC allows mice to form taste-action associations and that this behavior is associated with task-related activity and plasticity of taste-evoked responses. The proposed research focuses on the following aims: Aim #1 will develop a well-controlled behavioral paradigm for training mice to form new taste-action associations. In addition, the experiments will rely on chemogenetic and optogenetic inactivation of GC to determine its involvement in the performance of a taste- action association task. Aim #2 will use electrophysiological methods to unveil plastic changes of single unit spiking activity in GC of alert mice learning and performing a taste-action association task. Waveform analyses will allow us to separate putative excitatory and inhibitory neurons and to follow their activity across days. Finally, Aim #3 will rely on 2-photon calcium imaging in Gad2-T2a-NLS-mCherry mice to track how associative learning changes the spatial patterns of activity in large ensembles of excitatory and GABAergic neurons across days. These experiments will allow us to investigate the possible role of inhibition in GC plasticity. Furthermore, comparing patterns of activity in alert and anesthetized mice will inform us on the state-dependency of remapping. Altogether, the proposed experiments will open a new alley of research on the function of GC and establish an experimental pipeline to investigate reward-based taste learning and GC plasticity. If successful, these studies will demonstrate that GC representation of taste accounts for taste-action associations. Proving this principle will have important consequences for our understanding of the role of GC in ingestive behaviors and decisions.

PROJECT SUMMARY The gustatory cortex (GC) has been the subject of great attention over the past years. While its role in processing taste is well established, several issues regarding the strategy used by GC for coding gustatory information remain unsolved. The recent adoption of 2-photon calcium imaging to map the surface of GC in anesthetized mice has highlighted the possibility that gustatory information may be encoded by spatial patterns of single neuron activity. An influential proposal suggested a ?gustotopic? organization of taste coding, featuring spatially localized ?hot spots? formed by single clusters of neurons responding exclusively to a single taste quality. More recent imaging data complemented, and partially challenged this hypothesis, by showing spatially distributed representations in portions of GC between hotspots. Alas, both the studies published so far were performed in anesthetized animals. The impervious anatomical location of GC has hindered the application of 2- photon calcium imaging to study the topographical organization GC in alert mice. As a result, the nature of spatial coding in alert animals remains unknown. The research proposed in this grant will establish the experimental protocols for imaging large ensembles of neurons from the surface of GC in alert mice. The proposed research focuses on the following aims: Aim #1 will establish the methods for 2-photon calcium imaging using microprisms implanted on the surface of GC in mice licking for different tastants; Aim #2 will perfect the ability to monitor activity in the same ensemble for multiple days; Aim #3 will combine calcium imaging with behavioral training to monitor neural activity in mice engaged in taste discriminations. In addition to achieving technical milestones, the experiments in each aim will also address important scientific questions. Specifically, Aim #1 will investigate spatial patterns of activity on the surface of GC, addressing how wakefulness affects gustotopy and introduces temporal dynamics. Aim #2 will investigate the balance between plasticity and stability in GC representations, addressing how familiarization and repeated exposure affect taste evoked activity. Finally, Aim #3 will unveil the relationship between spatio-temporal patterns of activity and perception, by imaging GC in mice learning and performing simple and difficult discriminations in a taste-based, two alternative forced choice task. Altogether, the proposed experiments will answer fundamental questions in the field of gustation and open an entirely new research pipeline for future studies. Future studies made possible by this approach will investigate GC responses to multiple stimuli (multiple tastants, odors, flavors and cross-modal cues), unveil whether anticipatory cues can recruit taste representations and investigate coding in different cell types.

Summary The perception of taste is a complex process that includes the chemosensory detection of a stimulus as well as information regarding its hedonic value, whether a stimulus is pleasant or unpleasant. The gustatory cortex is thought to be a central component of the taste system, and is hypothesized to integrate both of chemosensory and affective dimensions of taste stimuli. However, to date, the neuronal and circuit mechanisms involved in this process remain unclear. In this proposal, we will fill a gap in the current understanding of circuit underpinning for taste by investigating the synaptic mechanisms underlying the integration of thalamocortical and amygdalocortical inputs to the primary gustatory cortex. The thalamocortical projection from the taste thalamus provides the gateway for chemosensory information to the gustatory cortex, while the basolateral nucleus of the amygdala has been identified as a major contributor of hedonic information regarding taste stimuli. Taking advantage of cutting-edge experimental approaches for circuit and synaptic analysis, and of a well-established learning paradigm, conditioned taste aversion, that preserves the chemosensory identity, but alters a stimulus affective dimension, we will determine how sensory and hedonic components of taste are integrated in the gustatory cortical circuit. The approach we propose will begin to bridge the gap between the extensive behavioral/ pharmacological and cellular/molecular work that has significantly advanced the field, but without information about underlying synaptic and circuit mechanisms remains difficult to reconcile in an overarching framework. In addition, our study has important implication for public health. Many neurological and psychiatric disorders are associated with altered sensory perception, or by anhedonia. Taste perception in human and rodents is characterized by sensory and affective dimensions, therefore it provides a unique model for understanding common mechanism of sensory processing, as well as for investigating the circuit underpinning for the integration of sensory stimuli with their hedonic value. Therefore, results from our current study have the potential of identifying novel targets for the development of therapeutic intervention.

PROJECT SUMMARY Cortical circuits generate dynamic patterns of activity. One of the great challenges of modern neuroscience is to determine the circuit architectures that generate such dynamics patterns, and understand their genesis and functional significance. Most research on brain dynamics focused on stable patterns of activity showing continuous transitions (e.g., oscillations). However, in recent years there has been an increased interest on transient dynamics, including the ones resulting from the sequential switching between metastable states. Extracellular recordings of cortical ensembles indicated that sequences of metastable states, characterized by correlated changes in activity can be detected across subpopulations of neurons. Metastable states have been associated with specific cognitive or sensory variables, suggesting an important role for brain function. Metastability was also observed in the absence of any behavior or stimulation ? suggesting that metastable states may be generated locally and may reflect intrinsic architectures of cortical circuits. Despite evidence for their functional significance, little is known about metastable dynamics in cortical circuits. Indeed, lack of a coordinated and systematic approach to study both temporal and spatial signatures of these patterns has limited progress in this area. This proposal aims at developing an integrated experimental-computational platform for detecting metastable dynamics in cortical ensembles, inferring the circuit organizational principles underlying them, and understanding how plasticity affects metastability. Our team is formed by six PIs with complementary expertise in the experimental and computational approaches necessary to successfully accomplish this program. We will focus on circuits in the superficial layers of the gustatory portion of the insular cortex, a well-established model for understanding metastability. Our long-term goal is to generalize our findings to the study of transient dynamics in other cortical areas and understand their relevance for sensory, motor and/or cognitive tasks. Successfully accomplishing the proposed research will allow us to identify universal principles of collective network dynamics underlying behavior and experience-dependent learning.