Transient receptor potential (TRP) channels are a class of six-transmembrane (6-TM) cation-permeable channels that mediate flux of calcium and sodium into cells, leading to depolarization as well as activation of calcium-mediated second-messenger signaling pathways. The TRP channel family is large and diverse in terms of tissue expression, mechanism, and function; therefore, sub-classification is primarily through amino acid homology. A general role has emerged for TRP channels, though, in the processing of sensory stimuli at both the cellular and organismal level. The goal of this study was to perform mutagenesis screens of mammalian TRP channels to reveal key structural determinants of channel activity (such as gating, permeation, and selectivity). We screened for gain-of-function alleles of TRP channels by their ability to rescue growth deficiency of a strain of the yeast Saccharomyces cerevisiae caused by lack of ion efflux. Channels were further characterized through electrophysiological analysis of their activity when heterologously expressed in Xenopus laevis oocytes. Of the subset of mammalian TRP channels tested, only wild type TRPV4 rescued the ability of the yeast strain trk1Δ trk2Δ to grow on low potassium media. The TRPV4 channel is important in thermosensitive, osmosensitive, and mechanosensitive processes; recently, mutations of TRPV4 have been linked to human skeletal and neurodegenerative disorders. We obtained a loss-of-function variant of TRPV4 containing the substitutions K70E (N-terminal tail) and M605T (intracellular linker between transmembrane helices S4 and S5) that failed to rescue low potassium growth of trk1Δ trk2Δ. Therefore, we screened for compensatory mutations that would restore the ability of the V4-K70E/M605T channel to rescue the yeast growth phenotype. Five gain-of-function clones were isolated, containing a total of seven mutations: three substitutions in the N-terminal tail (R151W, P152S, L154F), one substitution in the pore-lining S5 transmembrane helix (M625I), one substitution in the C-terminal tail (H787Y), and two truncations of the C-terminal tail (N789Δ and Q790Δ). Each of these mutations was assayed, in both the variant V4-K70E/M605T and the wild type TRPV4 background, for effect on rescue of trk1Δ trk2Δ yeast low-potassium growth, as well as degree of salt sensitivity conferred on wild type yeast. We also performed two-electrode voltage-clamp (TEVC) recordings of the mutant channels expressed in Xenopus oocytes, obtaining preliminary data on the ability of the mutations to restore a calcium-activated sodium current to V4-K70E/M605T that was present in wild type TRPV4. Given the known importance of the S5 helix in gating, the mutation M625I most likely has an effect on gating of the intracellular pore. This mutation showed strong rescue of low potassium growth and salt sensitivity in yeast, and preliminary data showed strong rescue of calcium-activated current in oocytes. An autoinhibitory channel structure is formed by binding of the C-terminal calmodulin-binding domain to a portion of the N-terminus, which is disrupted by the binding of calcium-calmodulin to the C-terminal domain. The point mutations we isolated in the N- and C-termini lie just outside these respective regions, leading us to believe that the gain-of-function phenotype could be due to disruption of this autoinhibitory structure. Although the C-terminal truncations were isolated with a gain-of-function phenotype in V4-K70E/M605T (rescue of low-potassium yeast growth), introduction of the truncations into wild type TRPV4 led to a loss-of-function phenotype: truncated channels no longer induced yeast salt sensitivity and exhibited no calcium-activated current in oocytes. This phenotype could be due to the loss of the calmodulin-binding domain, suggesting that the potentiation of channel activity by calcium involves mechanisms other than simply the disruption of the autoinhibitory domain. However, it is also possible that the phenotype is merely the result of reduced membrane expression: some studies indicate that truncation of the C-terminal tail leads to ER retention of the protein. Taken together, the results of our three assays provide insight into the mechanisms of TRP channel function. Combined with what is already known about these channel regions, we are able to draw conclusions as to the potential contribution of these residues on channel activity and add to the body of evidence regarding mechanisms of function of the TRPV4 channel.

The olfactory epithelium (OE) possesses the rare capacity among neuronal tissues to regenerate throughout life. As a result, progenitor cells continuously proliferate and differentiate into olfactory sensory neurons (OSNs) that project their axons to the olfactory bulb (OB) where they establish connections to the central nervous system. The olfactory epithelium is therefore an attractive model for the study of axonal growth and synapse formation. The present set of studies attempts to provide insights into synapse formation and axonal development of olfactory sensory neurons. First, I sought to understand the regulation of expression of pre-synaptic molecules in the olfactory epithelium. I established by in situ hybridization that as OSNs mature, they express sequentially groups of pre-synaptic genes. Genes encoding for proteins that play a structural role at the active zone showed an early onset of expression, whereas genes encoding for proteins associated with synaptic vesicles showed a later onset of expression. In particular, the signature molecule for glutamatergic neurons VGLUT2 shows the latest onset of expression. The sequential onset of expression suggests the existence of discrete steps in pre-synaptic development. In addition, contact with the targets in the olfactory bulb is not controlling pre-synaptic protein gene expression, suggesting that olfactory sensory neurons follow an intrinsic program of development. Second, in order to visualize simultaneously OSN axonal arborizations and their pre-synaptic specializations in vivo, I developed a method based on post-natal electroporation of the mouse nasal cavity. This technique allowed me to perform a temporal study where I followed the elaboration of axons and synapses in olfactory sensory neurons at different post-natal ages. The results show that olfactory sensory axons develop with exuberant growth and synapse formation. Exuberant branches and synapses are eliminated to achieve the mature pattern of connectivity in a process likely to be regulated by neural activity. Finally I investigated the consequences of suppressing neural activity in olfactory sensory neuron axonal morphology and synaptic composition. To this end I utilized two anosmic mouse models: cyclic nucleotide gated (CNG) channel and adenylyl cyclase 3 (AC3) knock-out mice. I observed that in the CNG knock-out mice, where OSNs do not generate action potentials after odor stimulation, the morphology of terminal arborizations and the synaptic composition were indistinguishable from wild-type littermates. In sharp contrast, AC3 knock-out mice, where there is no induction of cAMP production after odor stimulation, both the morphology and synaptic composition of OSN axons are severely altered. These results provide evidence that, unlike odor-induced membrane depolarization, odor-induced cAMP signaling events are critical for axonal growth and synapse formation in OSNs.

Serotonin, arising from neurons of the raphe nuclei, is intimately involved in the treatment of depression and anxiety disorders. Serotonin selective reuptake inhibitors (SSRIs) are frontline therapy for both of these conditions, and have well-described behavioral effects in animal models of emotional behavior. Yet, administration of SSRIs during postnatal development produces an opposing phenotype, including increased anxiety- and depression-like and reduced social behaviors. Because the serotonin transporter, the target of these drugs, is expressed primarily in serotonergic neurons of the raphe, I sought to identify biological mechanisms for behavioral effects of both adult and postnatal SSRI treatment in this cell population. Initially, we compared the transcriptome of serotonergic neurons to whole brain homogenate, in order to both validate the experimental methods, and to provide a descriptive analysis of gene expression in this neuron type. Many transcripts were enriched in raphé samples, including both known markers of serotonergic neurons, and novel genes, most of which could be confirmed from by in situ hybridization data from the Allen Brain Atlas. In postnatal fluoxetine treated mice, we report alterations at the anatomical, electrophysiological, and transcriptional levels. Serotonergic innervation of the prefrontal cortex and hippocampus was reduced by fluoxetine treatment, consistent with the neurodevelopmental role serotonin plays. The firing rate of serotonergic neurons was also reduced due to an increase in inhibitory transmission. Additionally, the transcriptome of serotonergic neurons was altered by postnatal SSRI: a preponderance of downregulated transcripts was observed, particularly among genes involved in mitochondrial and ribosomal function. These findings combine to suggest a hypotrophic serotonergic system is produced by postnatal SSRI treatment. Studying the effect of adult treatment with SSRIs, we report a normalization of elevated serotonin 1A receptors in depressed, medication-naive patients. However, we did not detect a relationship with clinical response, raising the possibility that serotonin 1A downregulation is an epiphenomenon of SSRI treatment. In adult fluoxetine treated mice, gene expression profiling identified a number of differentially regulated transcripts. We further used postnatal fluoxetine treatment as a model of treatment resistance, investigating the transcriptional actions of adult fluoxetine treatment in postnatal fluoxetine- vs. saline-treated mice, in order to identify transcripts that track with behavior. We found upregulation of a number of promising candidate genes, including vesicular glutamate transporter 3, histamine receptor 2, and the neuropeptide processing enzyme Ece2.

Schizophrenia is highly heritable yet very few genetic risk variants have been unequivocally linked to the disease. Disrupted in Schizophrenia 1 (DISC1), was first discovered in a family with a balanced translocation t (1; 11) (q42; q14) and a history of psychiatric disease that segregates with the translocation. We created the Disc1Tm1Kara mice, an etiologically valid genetic mouse model which mimics the effect of the human translocation. Disc1 is highly expressed in the dentate gyrus of the hippocampus of adult mice, a region that is important for learning and memory. Disc1Tm1Kara mutant mice have specific deficits in spatial working memory, a process that requires the interconnectivity of the hippocampus and prefrontal cortex. Electrophysiological recordings in the dentate gyrus region find deficits in short term plasticity and decreases in the excitability of mature granule cells. Moreover, the dentate gyrus is one of two regions in the brain where adult neurogenesis occurs and this process is believed to be important for encoding new memories. Disc1Tm1Kara mutant mice have a 20% reduction in neurogenesis and alterations in the architecture of the dendrites and mossy fiber axonal projections of the granule cells in the dentate gyrus of both early postnatal and adult mice. Biochemical evidence suggests a link between Disc1 and the phosphodiesterase, PDE4B, which is important for the degradation of cAMP. Disc1Tm1Kara mutant mice also have decreases in levels of many isoforms of PDE4 as well as increases in cAMP and its downstream target pCREB. Finally, Nrp1 and Sema3A, guidance cues that are regulated by cAMP, are altered in the Disc1Tm1Kara mutant mice. Overall, this mouse is a valuable model of a known genetic variant of schizophrenia susceptibility and adds to our understanding of the pathology of the disease. Abnormalities of the structure, organization, and synaptic plasticity of granule cells of the dentate gyrus are likely caused by the changes in intracellular signaling and contribute to the alterations in connectivity within the trisynaptic circuit and the behavioral deficits of the Disc1Tm1Kara mutant mouse.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Although ALS has been extensively studied in post-mortem patient samples and animal models, there are currently no very effective treatments and there is no cure. One reason for the lack of treatment options in ALS may stem from the inaccessibility of living human motor neurons for use in disease research and subsequent therapeutic target validation. Recent developments in the field of stem cell biology can potentially provide access to living human motor neurons from individual ALS patients. It is now possible to derive induced pluripotent stem cells (iPS cells) from the somatic tissues of ALS patients and then to differentiate these iPS cells into motor neurons with the precise genetic makeup of the donor patient (iPS-MNs). Before iPS-MNs can be put to productive use, however, the iPS system as a whole must be validated as a reliable source of motor neurons with characteristics that closely resemble their endogenous or hES-derived counterparts. This thesis will first address a series of issues relating to the validation of iPS cells as a reliable source of motor neurons a then move on to expression profiling studies aimed at identifying a transcriptional signature of ALS in iPS-MNs. I will first describe a collaborative study aimed at determining whether or not iPS cells are as useful as ES cells for the production of motor neurons. By comparing motor neuron differentiation efficiency across a panel of 6 ES lines and 16 iPS lines, we demonstrated that iPS cells are equally capable of producing electrophysiologically active motor neurons as ES cells. Moreover, both ALS and control iPS lines produce motor neurons with equal efficiency, suggesting that iPS cells will be useful in the production of ALS iPS-MNs for disease research. In addition, our results identify some of the variables that contribute to differentiation efficiency, including donor identity and individual iPS/ES line identity. The following section will serve to provide a deeper molecular and electrophysiological understanding of human stem cell-derived motor neurons. I first generated expression profiles from purified hES-MNs to identify potential motor neuron-specific surface markers as well as maturational changes occurring in motor neurons in vitro. Using calcium imaging techniques, I then demonstrated that iPS-MNs behave functionally similarly to ES-MNs and described culture-wide rhythmic depolarizations that are likely influencing multiple properties of iPS-MNs. After characterizing the iPS-MN culture system, I made a first attempt at defining the transcriptional phenotypes of ALS in iPS-MNs. This work relied on the use of a motor neuron-specific lentiviral reporter that I developed to isolate and transcriptionally profile iPS-MNs from two control iPS lines and four ALS iPS lines. I show evidence of significant transcriptional differences between motor neurons isolated from ALS lines and those from control patients. These differences may in the future help to define ALS-specific phenotypes. Lastly, I conducted a meta-analysis comparing transcriptional changes in ALS iPS-MNs to those in existing models of ALS and identified some common stress-related features of ALS in iPS-MNs. In order to form new hypotheses about what sorts of individual patient-specific phenotypes may be present in iPS-MNs, I will then utilize published expression profiles from post-mortem ALS patient motor neurons to identify a previously-overlooked class of genes that exhibit expression levels highly correlated with individual age at ALS onset. This group of 43 onset-correlated genes contains many members with known or hypothesized relationships to neurodegenerative disease. I discuss how onset-correlated genes may function as disease-modifiers or biomarkers and design experiments to investigate these possibilities. Taken together, the work in this thesis will lay the foundations for developing a human iPS-based model of ALS and point toward numerous avenues of future investigation.

Throughout the developing spinal cord, Olig2+ progenitors in the motor neuron progenitor domain give rise to an impressive array of motor neurons, oligodendrocytes and astrocytes. Motor neurons are further diversified into motor columns and pools based on cell body settling position, general axonal trajectories, and the individual muscles they innervate. Elegant studies have demonstrated that motor neuron columnar and pool diversity, along the rostral-caudal axis of spinal cord, is programmed by extrinsic signals that confer a combinatorial Hox code at each rostral-caudal coordinate. However, we are only beginning to understand the signals that control motor neuron diversification and neuronal versus glial competency within a given rostral-caudal segment level of spinal cord. As a key mediator of cell-to-cell communication, the Notch signaling pathway has been implicated as a primary player in the generation of intra-domain cellular diversity throughout development. Despite this, the role of Notch signaling in contributing to neural diversity within the motor neuron progenitor domain has remained elusive. The major hurdle to studying the role of Notch in the motor neuron progenitor domain has been the inability to specifically manipulate Notch signaling in motor neuron progenitors. In this dissertation, I use embryonic stem cell (ESC) to motor neuron differentiation technology to demonstrate that Notch signaling has a dual role during motor neuron differentiation. In Chapter 2, I demonstrate that Notch signaling is required for inhibiting motor neuron differentiation and maintaining a subset of progenitors for oligodendrocyte genesis via lateral inhibition. Activation or inactivation of Notch signaling during ESC to motor neuron differentiation is capable of disrupting lateral inhibition and generating homogenous cultures of either glial precursors or motor neurons. Interestingly, induction of Notch signaling during differentiation is sufficient to upregulate glial markers Sox9 and Sox10, suggesting that Notch also plays an instructive role in specifying glial cell fate. In Chapter 3, I show that Notch signaling regulates motor neuron columnar identity. Specifically, I demonstrate that Notch signaling is required for selection of medial motor column (MMC) identity and that inhibition of Notch signaling during motor neuron differentiation leads to rostral-caudal appropriate conversion of MMC identity into hypaxial motor column (HMC) identity in cervical conditions or lateral motor column (LMC) identity in brachial conditions. I further identify the transition from progenitor to postmitotic motor neuron as the critical period where Notch activity is necessary to select motor neuron columnar identity. Previous studies have proposed that an Olig2/Ngn2 competition model controls motor neuron differentiation. In Chapter 5, I show that contrary to this hypothesis, Olig2 does not inhibit motor neuron differentiation and that Olig2 and Ngn2 largely bind and regulate different sets of genes during motor neuron differentiation. Comparing genome-wide binding and gene expression data after Ngn2 induction, I identify the early gene expression program directly downstream of Ngn2 that drives motor neuron differentiation.

Over the last two decades technological developments in multi-electrode arrays and fluorescence microscopy have made it possible to simultaneously record from hundreds to thousands of neurons. Developing methods for analyzing these data in order to learn how networks of neurons respond to external stimuli and process information is an outstanding challenge for neuroscience. In this dissertation, I address the challenge of developing and testing models that are both flexible and computationally tractable when used with high dimensional data. In chapter 2 I will discuss an approximation to the generalized linear model (GLM) log-likelihood that I developed in collaboration with my thesis advisor. This approximation is designed to ease the computational burden of evaluating GLMs. I will show that our method reduces the computational cost of evaluating the GLM log-likelihood by a factor proportional to the number of parameters in the model times the number of observations. Therefore it is most beneficial in typical neuroscience applications where the number of parameters is large. I then detail a variety of applications where our method can be of use, including Maximum Likelihood estimation of GLM parameters, marginal likelihood calculations for model selection and Markov chain Monte Carlo methods for sampling from posterior parameter distributions. I go on to show that our model does not necessarily sacrifice accuracy for speed. Using both analytic calculations and multi-unit, primate retinal responses, I show that parameter estimates and predictions using our model can have the same accuracy as that of generalized linear models. In chapter 3 I study the neural decoding problem of predicting stimuli from neuronal responses. The focus is on reconstructing zebra finch song spectrograms, which are high-dimensional, by combining the spike trains of zebra finch auditory midbrain neurons with information about the correlations present in all zebra finch song. I use a GLM to model neuronal responses and a series of prior distributions, each carrying different amounts of statistical information about zebra finch song. For song reconstruction I make use of recent connections made between the applied mathematics literature on solving linear systems of equations involving matrices with special structure and neural decoding. This allowed me to calculate \textit{maximum a posteriori} (MAP) estimates of song spectrograms in a time that only grows linearly, and is therefore quite tractable, with the number of time-bins in the song spectrogram. This speed was beneficial for answering questions which required the reconstruction of a variety of song spectrograms each corresponding to different priors made on the distribution of zebra finch song. My collaborators and I found that spike trains from a population of MLd neurons combined with an uncorrelated Gaussian prior can estimate the amplitude envelope of song spectrograms. The same set of responses can be combined with Gaussian priors that have correlations matched to those found across multiple zebra finch songs to yield song spectrograms similar to those presented to the animal. The fidelity of spectrogram reconstructions from MLd responses relies more heavily on prior knowledge of spectral correlations than temporal correlations. However the best reconstructions combine MLd responses with both spectral and temporal correlations.

The question of how one's emotional state affects one's ability to perform cognitive tasks has long captivated scientists. In the work described in this thesis, a visual search task was employed as a proxy for cognition while images of emotional facial expression served to influence emotional experience. Previous models of the interaction between emotion, cognition, and visual perception have focused on the negative impact of emotion on cognition and behavioral performance. The goal of the experiment described in this thesis was to investigate whether exposure to an emotional stimulus can have positive or negative effects on a subsequent visual search task. Specifically, the study was aimed at exploring the neural correlates of behavioral effects, BOLD effects, and functional connectivity between the seed regions amygdala, V1, and V2 with networks in the brain corresponding to cognition, particularly visual attention. Nineteen subjects performed the search task during fMRI, while their eye movements, pupillometric data and manual responses were measured. Furthermore, the subjects completed several emotional rating scales to assess their individual levels of anxiety and hedonic capacity. Subjects performed more accurately on the visual search task in trials preceded by fearful or happy face stimuli as compared with a neutral one. Functional connectivity measures based on psychophysiological interaction and the contrast of the fearful and neutral conditions revealed a widespread pattern of enhanced functional connections between the amygdala seed and areas located in early and higher order extrastriate cortex including inferotemporal gyrus and fusiform gyrus. In addition, higher connectivity with the medial dorsal nucleus of the thalamus was observed. Also for the Fearful → Neutral contrast, V1 had higher functional connectivity with medial prefrontal cortex, superior frontal gyrus, posterior cingulate, and the pulvinar. Furthermore, the exposure to a happy stimulus relative to a neutral one resulted in increased connectivity to the inferior parietal lobule and precuneus, both of which are involved in the frontoparietal network. Comparison of fearful and happy functional connectivity patterns revealed higher V1 and V2 connectivity with medial frontal gyrus and anterior cingulate during the fearful condition, a difference which was also correlated with subject trait anxiety. Taken together, the results indicate that exposure to emotional stimuli can have enhancing effects on visual search performance which are related to changes in the functional relationships between brain regions including the amygdala (emotion processing), inferior parietal lobule and precuneus (cognition), and striate/extrastriate cortex (visual).

Two cellular reprogramming technologies have emerged that demonstrate that cell-fate can be converted by ectopic expression of defined transcription factors: induced pluripotent stem (iPS) cell technology and induced neuronal (iN) cell technology. These recent advances in cell reprogramming strategies have great potential utility for patient-specific disease modeling and for applications in regenerative medicine. Current models of neurodegenerative diseases are limited in their representation of disease phenotypes and there is an essential need for human cellular models of neurodegenerative disorders. Induced pluripotent stem (iPS) cell technology offers a two-step approach to disease modeling, in which patient somatic cells are first reprogrammed to a pluripotent state and subsequently differentiated in neurons. In contrast, induced neuronal (iN) cell technology allows for the direct conversion of somatic cells to neurons. Here I demonstrate the modeling of Alzheimer's disease (AD) using both iPS and iN cellular reprogramming technologies. These bioengineered human cell-based models of AD provide unique and invaluable tools for elucidating the mechanism of AD pathogenesis.

Drosophila exhibits a rich repertoire of simple and complex behaviors. In addition, the ability to allow genetic manipulations of specific neuronal populations makes the numerically simple fly brain an attractive model system to study the mechanisms that translate neural circuits to meaningful behavioral responses. Delineation of neural circuits requires development of approaches that trace functional synaptic connections. We have developed HA-Tango-trace, an activity-dependent trans-synaptic tracer to define neural circuits that convey information from the inner photoreceptors in the retina to the lobula complex in the Drosophila visual system. Elucidation of neural circuits and the mechanisms involved in translating the circuitry into a meaningful behavioral response with Tango-trace involves labeling of neurons in an activity-dependent manner based on the release of an endogenous neurotransmitter at a synapse. This strategy can be extended to any neural circuit in the brain with a known neurotransmitter in both flies and mice. In the visual system, specific features of the visual image like motion, color, form and shape are extracted and processed in neural pathways. This information is transmitted to the brain where it must be processed to translate stimulus features into appropriate behavioral output. Here we investigate how this information is represented in higher visual centers in flies. The stochastically distributed p/yR7s and p/y R8s in the retina project to the medulla and make precise connections with four unique connectors that relay information to the lobula complex. Thus, the p/yR7s and p/y R8s process spectral information in separate pathways and relay information to the lobula and lobula plate. The projections to the lobula plate afford the opportunity for inputs to the motion pathway. Moreover, our behavioral data show that R8s influence motion-evoked behavioral responses under bright light conditions. Gap junctions between the inner and outer photoreceptors could afford an explanation for the convergence of the two pathways. This by itself is sufficient for visual discrimination of objects during navigation or, alternatively, the postsynaptic partners of R7 and R8 may additionally provide inputs to the motion pathway. Thus, spectral and motion pathways may converge repetitively at each stage of the circuit and reorganize into pathways of behavioral significance. Furthermore, histaminergic neurons have been implicated in temperature preference and circadian rhythms. These behaviors are likely to result from neuromodulation of central brain circuits mediated by histamine. Tango assay can be used to study this other important aspect of neural circuits by measuring the intensity of signal before and after neuromodulation. This approach was successfully used to map neuromodulation of dopamine mediated sugar sensitivity in flies using dopamine tango-map. Hunger enhances behavioral sensitivity to sugar and this is mediated by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx in a DopEcR-dependent manner. Tango-map permits the detection of increases in endogenous neuromodulator release in vivo. In addition, histamine has been detected in mechanosensory neurons in Drosophila. Auditory systems are critical to the behavior of many insects. In Drosophila melanogaster, acoustic communication is essential for making decisions related to mate selection. The projections of the HA-Tango labeled neurons overlap with the proposed higher order auditory neurons in the protocerebral areas. Further characterization of these circuits with HA-Tango-trace will provide insights into the representation of mechanosensory and auditory information that drive diverse behaviors in Drosophila. Acetylcholine is a major neurotransmitter of the olfactory and gustatory systems in Drosophila. We have designed Ach-Tango to trace connections in the olfactory and gustatory systems in an activity-dependent manner. Characterization of circuits in higher brain areas may help us understand how odor and taste percepts are formed and how these sensory modalities are processed in the higher brain centers to generate diverse olfactory and gustatory behaviors. The studies described in this thesis provide approaches to analyze circuits and understand their functional implications. Tango-Trace is a genetically encoded trans-synaptic tracer designed to identify synaptic connections in an activity-dependent manner by chronic activation of the presynaptic neuron with a genetically targeted neuronal activator, dTrpA1 and the identification of postsynaptic partners by GFP or any other reporter of choice. Tango-Map is designed to detect volume transmission of a neuromodulator by measuring the signal intensity of the reporter before and after a neuromodulatory effect. Furthermore, deciphering the circuit mechanisms that translate into complex behaviors will provide an understanding of more complex processes in the brain like emotion, cognition and consciousness. Our understanding of the nervous system can benefit greatly from these tools.