Steven A. Siegelbaum - US grants

Slow post-synaptic transmitter actions are important as a means of achieving long lasting changes in the electrical activity of nerve and muscle cells and are of interest as they often involve novel ionic and molecular mechanisms. Here I propose to study, in central neurons of the marine snail Aplysia, two slow transmitter actions that have been shown to mediate presynaptic facilitation and presynaptic inhibition, two forms of plastic changes in synaptic effectiveness: 1. Serotonin (5-HT) and stimulation of a putative serotonergic interneuron elicit a slow excitatory post-synaptic potential and facilitate transmitter release from a cluster of mechanoreceptor sensory neurons by a decrease in a specific membrane K+ conductance via cAMP-dependent phosphorylation. This increase in synaptic efficacy is thought to underlie behavioral sensitization, a simple form of learning. 2. Histamine and the stimulation of a putative histaminergic neuron produce a slow inhibitory post-synaptic potential and inhibit transmitter release from the cholinergic neuron L10 due to a decrease in the inward membrane calcium current. In this study, the recently developed patch clamp technique will be applied to investigate these transmitter actions at the level of single ion channel function. The research should provide insight into a number of general and specific questions, including: 1. What are the characteristics of transmitter action on single channel currents. 2. What are the biophysical properties of the individual potassium and calcium ion channels that underlie the action of serotonin and histamine, respectively. 3. What is the role of cAMP-dependent phosphorylation in channel modulation and what is the molecular mechanism of this modulation. An understanding of the basic mechanisms of a particular cAMP mediated transmitter action is likely to provide insight into such transmitter actions in general, including those occuring in the central nervous system of higher vertebrates.

The long term goal of this project is a molecular understanding of the mechanisms by which neurotransmitters modulate ion channels through second messenger cascades and thus regulate transmitter release from presynaptic terminals. Modulation of transmitter release is an important form of plasticity involved in learning and memory. Moreover, pathophysiological changes in modulatory transmitter actions are thought to underlie certain neurologic and psychiatric diseases. The specific goal of this study is to investigate the molecular bases for the antagonistic modulation by serotonin (5-HT) and the neuropeptide FMRFamide of the background conductance S-K channel in mechanoreceptor sensory neurons of Aplysia. In Aplysia, 5-HT causes presynaptic facilitation of transmitter release from sensory terminals whereas FMRFamide causes presynaptic inhibition. These transmitters also exert antagonistic actions at the single channel level: 5-HT closes S channels whereas FMRFamide increases S channel opening. The changes in S-K current are thought to alter Ca influx into the terminals and alter release indirectly. This action of 5-HT is mediated by cAMP-dependent protein kinase (cAMP-PK). The action of FMRFamide is mediated by the 12-lipoxygenase metabolite of arachidonic acid, 12-HPETE. FMRFamide also reopens S channels closed by 5-HT or cAMP and causes protein dephosphorylation. The specific goals of this project are to study the molecular mechanisms whereby 5-HT and FMRFamide modulate S channel function and address the following questions: Do the actions of cAMP and/or 12-HPETE depend on the down- or up-modulation of phosphatase activity? Is 12-HPETE the final active metabolite or are downstream metabolites required? Where in the membrane is the 12-HPETE receptor located? Might 12-HPETE act as a first messenger to alter the activity of neighboring cells? The above questions will be addressed using a combined biochemical and electrophysiological approach. Single S channel currents will be recorded in cell-free patches and whole cell S currents recorded under voltage clamp. Purified kinases, phosphatases, and various inhibitors will be applied to patches or injected into sensory neurons to determine how they alter S channel function or interact with the modulatory actions of cAMP-PK and arachidonate metabolites. Parallel biochemical assays of phosphatase activity and modulation of this activity in homogenates of sensory neuron clusters will also be performed.

Cyclic nucleotide-gated (CNG) ion channels play important roles in vision and olfaction and have recently been identified in neurons and cardiac muscle. These channels are likely to be involved in disease, including retinal degeneration, and are potential targets for therapeutic drugs. The long-term goals of this project are to understand the molecular mechanism of channel of channel activation gating. This proposal investigates how cyclic nucleotide binding leads to the opening of the CNG channels, building upon the previous identification of two domains important for ligand gating: the C-helix of the carboxyl terminus ligand-binding domain and an amino terminus gating domain (N-S2 domain). Activation can be described by the Monod-Wyman-Changeyx (MWC) model in which the N-S2 domain participates in the concerted allosteric transition which mediates channel opening. The C-helix is important for enhanced binding of ligand to the open channel, thus stabilizing the open state. This hypothesis will be tested by investigating three specific questions: 1. What is the functional stoichiometry of ligan-gating? How many ligands must bind to activate the channel? Does the MWC model adequately describe gating? The properties of channels will be investigated in which the binding to zero to three of the channel's four subunits has been inactivated using point mutations of deletions in the binding site. These experiments will provide information as to whether ligand binding is cooperative or independent. They will revieal how much free energy for activation each binding site contributes. Finally they may suggest alternative schemes for channel activation gating other than the MWC model. 2. How does the C helix participate in activation gating? Does the C helix act to selectively stabilize cyclic mucleotide binding to the open channel? Does this stabilization require the formation of intrasubunit bonds? 3. What is the role of the N-S2 domain in subunit assembly? Does the allosteric gating transition involve a change in subunit-subunit interactions? This hypothesis is based on the fact that regions homologous to the N-S2 domain mediate subunit assembly in voltage-gated K channels. It is our hypothesis that the N-S2 domain contributes to subunit assembly of CNG channels. These experiments will thus provide a powerful means of exploring both the basic mechanism of CNG channel gating as well as a test of the role of two domains of the channel in activation gating. Such information will be important for understanding how the structure of this family of channels underlies the unique physiological roles of different types of CNG channels in sensory information processing.

[unreadable] DESCRIPTION (provided by applicant): Two related classes of ion channels are directly regulated by cyclic nucleotide (CN) binding. The cyclic nucleotide-gated (CNG) channels are important for visual and olfactory signaling; inherited mutations in CNG channels underlie forms of retinal degeneration and color blindness. The hyperpolarization-activated, Cnmodulated (HCN) channels contribute to spontaneous firing in the brain and heart. Misregulation of HCN channels has been implicated in epilepsy, neuropathic pain, and cardiac disease. Thus, a deeper knowledge of how these channels are regulated by CNs, the focus of this proposal, is important for understanding their roles in normal neuronal function and disease. An X-ray crystal structure of a soluble C-terminal region of HCN2, including the CN-binding domain, has shown that four C-terminal regions assemble into a symmetric gating ring. However, structural and functional studies on CNG channels and functional studies of HCN2 channels indicate that subunits associate and gate as a dimer-of-dimers. The proposed studies will use several strategies to determine the nature and importance of subunit interactions in HCN and CNG gating, an unresolved issue since the studies of Hodgkin and Huxley. Mutagenesis experiments will further define the importance of specific C-terminal regions. The role of subunit interactions will be tested with tandem HCN2 tetramers in which such regions are deleted from 1,2,3 or 4 subunits. The physical proximity of neighboring HCN2 CNBDs and changes in proximity during gating will be assessed by disulfide bond formation between substituted cysteines in neighboring CNBDs. Polymer-linked CN dimers will also probe the distance between CNBDs during gating. Finally, the importance of subunit interactions in the transmembrane region will be assessed in voltage-gating of CNG channels. These studies should provide novel insights into the mechanisms of CN gating of two important classes of channels and aid in the design of therapeutic compounds to treat neurologic and cardiac diseases in which these channels participate. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Disorders in the function of the hippocampus, a brain region important for declarative learning and memory, have been implicated in a variety of psychiatric and neurological disorders, including Alzheimer's disease, schizophrenia, depression and epilepsy. Long-term synaptic plasticity in hippocampal circuits has been widely implicated in both learning and memory and as an underlying cause of disease. However, comparatively little is known about how the integrative membrane properties of hippocampal neurons contribute to spatial information processing or disease processes. This proposal focuses on the contribution to spatial learning and memory of the HCN1 hyperpolarization-activated channel, which is highly expressed in the dendrites of hippocampal CA1 pyramidal neurons where it regulates synaptic integration. HCN1 is of further interest as its level of expression is dynamically regulated by neural activity. Indeed, downregulation of HCN1 during seizures has been proposed to contribute to development of epilepsy. The role of HCN1 in brain function was previously studied in a line of mice in which HCN1 was deleted selectively in the forebrain. Surprisingly, these mice showed an enhancement in spatial learning and memory. This behavioral effect was associated with an enhancement in synaptic transmission and long-term potentiation (LTP) at the direct cortical, temperoammonic (TA) inputs to CA1 neurons, which terminate on the distal CA1 dendrites where HCN1 expression is normally greatest. These results indicate that HCN1 provides an inhibitory constraint on both synaptic plasticity at the TA synapses and on spatial learning and memory. However, the mechanism by which HCN1 constrains these processes is not known. Moreover, relatively little is known about synaptic plasticity or its behavioral significance at the TA synapses, compared to the wealth of information on synaptic plasticity at the major Schaffer collateral inputs to CA1 neurons. This proposal represents a multidisciplinary study, combining in vivo recordings of CA1 neuronal activity with hippocampal slice recordings of TA synaptic plasticity and two-photon imaging of calcium in distal CA1 dendrites, to address the following questions: How does HCN1 deletion enhance LTP at TA synapses? Does it alter the firing in the distal dendrites of calcium spikes - events that have been implicated in LTP? Is TA LTP associated with dynamic changes in HCN1 expression in the distal dendrites and does this alter synaptic transmission? Does HCN1 constrain spatial learning and memory by affecting the in vivo encoding of spatial information in CA1 neurons? By focusing on the role of a specific ion channel that is enriched in a specific region of CA1 neuron dendrites, this study will help elucidate the role of HCN1 and the defined synaptic element that it regulates in the encoding of spatial memory. Such studies may validate the HCN1 channel as a potential target for novel therapies for epilepsy, age-related memory loss, major depression, schizophrenia and related diseases of hippocampal function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The HCN1 hyperpolarization-activated cyclic nucleotide regulated cation channel regulates the electrical activity of several types of neurons in the brain and spinal cord. Whereas forebrain-restricted HCN1 knockout mice show an improvement in hippocampal-dependent spatial learning and memory, the mice also have an enhanced susceptibility to seizures. In both humans and wild-type mice an initial precipitating seizure alters HCN1 expression, which is thought to contribute to development of epilepsy. One striking feature of HCN1 is that the channel plays distinct physiological roles in different neuron as a result of its differential targeting to distinct neuronal compartments. Whereas HCN1 is targeted to distal dendrites in hippocampal CA1 pyramidal neurons, the channel is targeted to presynaptic terminals in inhibitory basket cells. Moreover the pattern of channel expression is dynamic. Following a seizure, HCN1 becomes mislocalized in CA1 neurons, appearing in the soma instead of dendrites. How can a single macromolecule be differentially targeted to distinct locales based on neural identity? What signaling mechanisms might be important in regulating channel location? HCN1 channel expression and function are powerfully regulated by a brain-specific auxiliary subunit of HCN1 termed TRIP8b. The brain contains at least ten different TRIP8b splice variants that differ in their cellular localization and effects on channel surface expression. Knockdown of all TRIP8b isoforms greatly reduces expression and prevents targeting of HCN1 to CA1 distal dendrites. In contrast a TRIP8b hypomorph mouse that lacks all but two TRIP8b isoforms normally expressed in brain shows normal levels of HCN1 expression and dendritic targeting in CA1. What is the role of TRIP8b in targeting HCN1 to its distinct subcellular locales in different neurons? Do different TRIP8b isoforms differentially target HCN1 in different neurons? What signals define the identity of neural compartments, such as the distal dendrites? Might such signals regulate HCN1 trafficking through phosphorylation? Answers to such questions will provide insight into the mechanisms by which neurons regulate their electrical signaling properties and how such mechanisms might provide novel disease substrates.

PROJECT SUMMARY This application is to renew funding for the Summer Program for Under-Represented Students (SPURS). Columbia University's SPURS Program provides an intense undergraduate research experience on the campus of Columbia's College of Physicians & Surgeons (P&S) for talented students from backgrounds that are under-represented in biomedical research. SPURS participants are accepted primarily from the City University of New York (CUNY) senior colleges, including Hunter, Brooklyn, Queens, and City Colleges, though in the last year we have extended our outreach and have accepted applications nationwide. This includes students from Columbia University, Barnard College, New York University, Harvard University, University of California ? Berkeley, and Amherst College. SPURS provides extensive training in basic science research, and enhances the likelihood that the students will achieve a career in science by pursuing an advanced degree (M.D. and/or Ph.D.). To expand opportunities for under- represented minority undergraduate students to participate in high quality, focused and sustained research experiences in the neurosciences, Dr. Steven Siegelbaum (P.I.), Chair of the Department of Neuroscience at P&S, and Dr. Andrew Marks, Department Chair of Physiology and Cellular Biophysics and founder of Columbia University's SPURS program have joined to expand the SPURS program. Over the last funding period, applications have soared and in the upcoming period, we propose the expansion of the program to include even more highly qualified minority student participants. Students selected for support through NIH's R25 Summer Research Experience Programs solicitation (PAR-15-184) perform hands-on research for nine summer weeks under the mentorship of NINDS-supported Columbia University neuroscientists (currently 89 Columbia University researchers receive NINDS support). In addition to specific training in neuroscience, the students have received in-depth training in research methodology including: (a) the design and analysis of experiments; (b) critical reading of scientific literature through journal clubs and discussions of ethics in science; (c) the presentation of scientific results at laboratory meetings; (d) presentation of their research at poster sessions; (e) an oral presentation of their research to an audience of scientists; and (f) career counseling. Finally, the research training will be provided in a uniquely enriching setting that includes weekly meetings with under-represented minority role models in biomedical research. The SPURS program addresses the critical need to increase the pipeline of highly qualified minority trainees into neuroscience.      

DESCRIPTION (provided by applicant): This project examines the neural circuitry of the CA2 region of the hippocampus and its role in hippocampal-dependent learning and behavior. Although the hippocampus has been one of the most intensively studied brain areas, based on its importance for declarative memory, relatively little is known about the CA2 region since its initial description by Lorente de N? in 1934. In contrast there is a wealth of information about th functional properties and synaptic connections of the other major regions of hippocampus including dentate gyrus, CA3 and CA1. The lack of attention paid to CA2 has been largely due to experimental and technical difficulties in studying this relatively small region that occupies a transitional zone between CA3 and CA1. This situation has impeded our understanding of how hippocampus encodes memories and how alterations in hippocampal function contribute to psychiatric and neurological disorders as CA2 has been implicated schizophrenia and bipolar disorder, as well as in epilepsy. Moreover, CA2 pyramidal neurons exhibit some of the highest levels of expression in the brain of the vasopressin 1b receptor, which has been implicated in both normal social behavior and autism. Over the past several years it has become increasingly clear that CA2 does indeed form a separate region with its own molecular identity and distinct electrophysiological properties (as shown, in part, by recent data from our laboratory). These molecular studies have enabled us to generate a mouse line that expresses Cre recombinase in CA2 pyramidal neurons, thereby allowing us to selectively label and manipulate CA2 excitatory output. Our initial experiments have used this mouse to identify some of the major inputs and outputs of the CA2 pyramidal neurons. Moreover by expressing tetanus toxin selectively in CA2 we have been able to inactivate its synaptic output and explore the behavioral consequences of CA2 silencing. Surprisingly, we find that inactivation of CA2 has little effect on a number of mouse behaviors, with no significant change in hippocampal-dependent spatial memory (Morris water maze), contextual fear conditioning, or novel object recognition. In stark contrast, silencing of CA2 results in a profound loss of social memory, the ability of a mouse to recognize a previously encountered mouse. Here we propose to employ this mouse line to examine in more detail both the anatomical and functional synaptic connectivity of CA2 pyramidal neurons and to explore more deeply the role of CA2 in various social and non-social forms of hippocampal-dependent learning and memory. Given the changes in social behavior associated with various neurological and psychiatric disorders, some of which have been linked to CA2, our experiments offer potential insights into both basic mechanisms of memory storage and the neural bases of altered cognitive processing important for social interactions.

? DESCRIPTION (provided by applicant): One of the outstanding problems in temporal lobe epilepsy is to understand the mechanisms contributing to treatment-resistant seizures that involve the hippocampus, and then to design mechanism-based novel therapeutic approaches. Such treatments are important both to control intractable seizures and to ameliorate the hippocampal-dependent memory deficits associated with epilepsy. This project will challenge current dogma that focuses on the role of neurons in the three major regions of the hippocampus: dentate gyrus, CA3 and CA1. Instead, this proposal focuses on area CA2, a relatively small region of hippocampus that has received little attention but is known to survive relatively intact in epileptic patients and may serve as a hyperexcitable seizure focus. Experimental tools developed in the laboratories of the two Principal Investigators now enable the direct investigation of the importance of CA2 in mouse models of epilepsy by employing a mouse line that selectively expresses the bacterial enzyme Cre recombinase only in CA2 neurons. Viral vectors that express tetanus toxin (or other proteins that can silence neural activity) in the presence of Cre will be injected into the hippocampus of these mice to turn off CA2 activity. In this manner it will be possible to test whether CA2 controls the pharmacological induction of seizures in the healthy brain and/or spontaneous seizures in the diseased, epileptic brain. The methods alone will advance the field because they are novel and provide more specificity and control than has been previously possible in epilepsy research. Moreover, by evaluating the role of CA2 in epilepsy, this project will result in the potential validation of sevral novel drug targets highly enriched in CA2 neurons.

DESCRIPTION (provided by applicant): The consequences of alcohol abuse on the American public are profound, both in terms of individual well-being and impact on the family structure, as well as the enormous cost to society in terms of lost productivity and associated health care expenses. Despite increasing efforts, our understanding of the neurobiological mechanisms that underlie the effects of alcohol and the development of alcohol use disorders (AUD) remains incomplete. Epidemiological research has pointed to adolescence as a critical period in the development of alcohol disorders. The prefrontal cortex (PFC) is a brain region that is not yet mature at the onset of human adolescence and continues to develop during this period, during which some individuals may be highly susceptible to the effects of alcohol. The PFC mediates control over goal-directed behaviors and dysfunction of the PFC is thought to underlie compulsive drug-taking and relapse in substance abusers. Binge drinking is highly prevalent in adolescents, and episodes of high alcohol intake have been associated with decreased PFC activity and function (hypofrontality). Imaging studies suggest that hypofrontality persists in chronic alcohol abusers, and may therefore be a contributory factor in the development of AUDs and behavioral pathologies in adulthood. The underlying mechanisms of this PFC hypoactivity are unknown, and the development of robust animal models would therefore be useful in investigating the underlying changes in neuronal excitability. A better understanding of these changes would enable possible molecular and therapeutic interventions in order to prevent the development of alcoholism. One plausible mechanism for hypofrontality involves the depression of persistent activity, a mode of firing that can be observed in recordings from pyramidal neurons in the PFC of rodents. This type of activity is seen at more depolarized membrane potentials and is associated with performance in working memory tasks, and is dependent on the Ih current, which is mediated by a family of hyperpolarization-activated and cyclic nucleotide modulated (HCN) channels. We propose that chronic changes in persistent firing might result from prolonged alcohol exposure. To date there have been few detailed studies of excitability in the PFC after drinking and none in adolescent rodents. In three separate but integrated aims, we plan to test the overall hypothesis that the HCN1 channel that contributes to the Ih current in PFC PNs is important for the regulation of alcohol drinking, and specifically that (a) binge drinking of alcohol during adolescence inhibits persistent firing and excitability in the PFC via reduction of Ih and (b) reduction in HCN1 channel activity in layer 5 of PFC can mimic the effects of alcohol consumption during adolescence, while (c) activation or over-expression of HCN1 channels can restore persistent activity and normal levels of excitability in PFC of binge drinking adolescent animals.

? DESCRIPTION (provided by applicant): This project examines the inhibitory neural circuitry of the CA2 region of the hippocampus and its role in neuropsychiatric disorders, focusing on neurodevelopmental disease including schizophrenia (SCZ) and autism spectrum disorder (ASD). The motivation for this proposal comes from two independent findings. First, we have recently found that CA2 is crucial for social memory, the ability of an animal to recognize a conspecific. Given the altered social behaviors characteristic of SCZ and ASD, we postulated that pathological changes in CA2 function might be contributing factors. Indeed, two independent studies on the brains of individuals with SCZ and bipolar disorder have found a significant loss in the number of parvalbumin positive (PV+) inhibitory neurons in the CA2 region of the hippocampus, but not in neighboring hippocampal regions (CA1 and CA3). To examine the possible role of CA2 in SCZ, we examined a mouse model (Df(16)A+/- mice) of the human 22q11.2 deletion syndrome, a neurodevelopmental disorder that provides one of the strongest known genetic links with SCZ. Individuals harboring this deletion also display a number of autistic-like changes in social behaviors. The Df(16)A+/- mice were shown to have a number of cognitive changes associated with SCZ, including altered prepulse inhibition and contextual and working memory. Our preliminary results demonstrate that these mice also have a profound deficit in social memory. Remarkably, we find that the Df(16)A+/- mice show a specific reduction in PV+ inhibitory neurons in CA2, but no change in CA1 or CA3, identical to the results in human neuropsychiatric disorders. Moreover, the loss of inhibition in mice is first seen in late adolescence to early adulthood, similar to the developmental onset of SCZ. Here we will expand upon these initial findings by examining the importance of CA2 in the behavioral phenotypes of the Df(16)A+/- mice. We will first explore in more detail the function of CA2 inhibitory neurons that are targeted by the deletion mutation. We will also probe more deeply the behavioral changes in the Df(16)A+/- mice and ask whether we can rescue the behavioral changes by either silencing or activating CA2 in these animals. Thus the experiments we propose will both provide new insight into the neural circuitry and function of inhibition in the underexplored CA2 region of the hippocampus and will help determine the potential role of altered CA2 function in a mouse model of SCZ. These experiments offer the possibility of identifying new targets for treating disease, particularly as CA2 PNs display a unique pattern of gene expression not seen in other hippocampal areas. Given the changes in social behavior associated with other neuropsychiatric disorders, including ASD, our experiments may also provide general insights into basic brain mechanisms contributing to a variety of disorders of social behavior.

PROJECT ABSTRACT A key challenge in temporal lobe epilepsy (TLE) is to determine the neural mechanisms contributing to seizures because current drugs fail to treat all seizures and usually come with debilitating side effects. Moreover, current drugs do not treat alterations in social behavior often manifest by individuals with epilepsy, including increased social aggression. This project will challenge current dogma as to the pathophysiological bases of how the hippocampus contributes to seizures in TLE, which has focused on three major regions of the hippocampus: dentate gyrus, CA3 and CA1. Instead, we examine area CA2, a relatively small region of hippocampus that has received little attention but is known to survive relatively intact in TLE patients and rodent models, and may serve as a seizure focus or facilitate seizure propagation. Experimental tools developed in the laboratories of the two Principal Investigators now enable the direct investigation of the importance of CA2 in mouse TLE models by employing a mouse line that expresses Cre recombinase relatively selectively in CA2 principal neurons. Cre-dependent viral vectors will be used to express genetically encoded tools in CA2 principal neurons to examine both alterations in CA2 circuitry in TLE and the effects of CA2 acute or chronic silencing on seizures. Thus, we will determine whether CA2 controls the pharmacological induction of acute seizures in the healthy brain and/or chronic seizures in the epileptic brain. We will also determine the importance of CA2 in reported deficits in social cognition and social aggression in mouse models of acquired TLE, as we find that CA2 is required for social recognition memory and is implicated in social aggression. As the social hormone arginine vasopressin promotes social memory and social aggression by enhancing CA2 input and output, and has been shown to regulate seizures in animals, we will examine the role of CA2 regulation by this hormone on social behavioral alterations in epileptic mice. By evaluating the role of CA2 in epilepsy, this project offers the promise of providing both basic mechanistic insight into seizures and social behavioral comorbidity, and may validate novel drug targets highly enriched in CA2 neurons.

Heightened levels of social aggression, a motivated behavior, are often associated with neuropsychiatric disease. Although our understanding of the neural mechanisms regulating aggression is incomplete, both lateral septum (LS) and ventral hypothalamus are known to be important. Even though LS receives one of its strongest inputs from hippocampus, a region important for declarative memory, little is known about how hippocampus regulates aggression. Moreover, as hippocampus is implicated in several neuropsychiatric disorders associated with altered social behavior and aggression, a basic understanding of how hippocampus and its circuitry regulate aggression will likely yield important new insights into disease mechanisms. Here we focus on the role of the hippocampal CA2 region in social aggression. Relatively little is known about CA2, largely because of technical problems that limit its study with conventional lesioning approaches. We therefore developed a Cre mouse line that enables us to label and manipulate the activity of CA2 pyramidal neurons. Using a genetic silencing approach, we found that CA2 was critical during non-aggressive social exploration for the formation of social memory, the ability of an animal to recognize and remember another mouse (conspecific), but CA2 was not needed for other forms of hippocampal memory. Our recent results now show that CA2 also promotes social aggression, through an excitatory projection to LS that disinhibits a subnucleus in ventral medial hypothalamus important for aggression. Moreover, we find that the social neuropeptide arginine vasopressin promotes aggression by enhancing the CA2 to LS synapse. Here we ask: How does a single brain region, CA2, participate in social memory storage during non- aggressive social exploration and promote social aggression? Is there a single population of CA2 neurons that is activated during both social exploration and social aggression? Or are there specialist neurons for each behavior? Does CA2 actively encode distinct representations of social exploration and social aggression, or does CA2 encode a single social salience signal that does not in itself encode aggression but that is gated by the internal state of an animal to promote aggression through CA2 inputs to LS? We will test the hypothesis that vasopressin release in LS acts as such a permissive gate. As vasopressin also enhances social memory by acting within CA2, we will ask: How can a single neuromodulator produce two such distinct actions? Do distinct sources of vasopressin input to CA2 and LS promote, respectively, social memory and aggression? We will address these questions by characterizing CA2 circuits and neural activity during aggressive and non-aggressive social interactions using: 1. Activity-dependent genetic marking of active CA2 ensembles; 2. Electrophysiological characterization of CA2?LS circuits and their regulation by vasopressin in ex vivo brain slices; 3. Behavioral control of aggression using chemogenetics and optogenetics; and 4. In vivo optical and electrophysiological recordings of CA2 activity during non-aggressive and aggressive social interactions.

Project Summary The goal of this project is to understand how the gating of HCN channels is regulated by domains located in the cytoplasmic, intracellular portion of the protein. HCN channels regulate pacemaking in the heart, and neuronal excitability in the brain. They have the general structure of voltage-gated K+ channels, but are activated upon membrane hyperpolarization and conduct an inward, depolarizing current. Importantly, their gating is modulated by the direct binding of cAMP to an intracellular cyclic-nucleotide binding domain (CNBD) located in the C-terminal portion of the protein. Recent structural data have also revealed the presence of a second, highly structured domain in the intracellular N-terminal portion of the protein, immediately preceding the first transmembrane domain (HCN domain). In this study, we will use a combination of structural and functional approaches to determine the potential role of the HCN domain in the modulation of channel gating, and the nature of the interactions between the C-terminal CNBD and N-terminal HCN domain. As different HCN isoforms (HCN1-4) exhibit markedly distinct biophysical properties, we will exploit these differences to identify key contacts and residues that may contribute to modulate the gating of HCN channels. In Aim 1, we will use cryo-electron microscopy (cryoEM) to determine the structure of the HCN4 isoform, in the presence and absence of cAMP, and compare its features to the available cryoEM structure of HCN1. In Aim 2, we will resolve the structure of HCN4 in the presence of ligands that interfere with the cAMP-mediated facilitation of channel opening (auxiliary subunit TRIP8b, biphenylurea compound BPU), and use the structural data acquired through Aims 1 and 2 to inform functional experiments designed to test and interpret any inferred model of structure/function relation. Finally, in Aim 3, we will use a similar combination of cryoEM and functional studies to study select HCN1 and HCN4 mutations found in patients with genetic epilepsy and cardiac arrhythmias.

Project Summary Recent clinical findings implicate de novo mutations in the gene encoding the hyperpolarization-activated HCN1 cation channel in severe forms of childhood epilepsy. At the same time genome-wide association studies demonstrate a strong link of the HCN1 locus with schizophrenia. Here we aim to provide a detailed characterization of the role of HCN1 in normal neural function, and to determine how disease-causing HCN1 mutations perturb neural activity to generate disordered brain function. HCN1 channels are unusual in that they are activated by membrane hyperpolarization, yet conduct an inward depolarizing Na+/K+ current, and show a contrasting pattern of subcellular localization in the distinct classes of neurons in which they are expressed. Thus, the channel is strongly expressed in hippocampal CA1 and neocortical layer 5 pyramidal neurons, where it is targeted to the apical dendrites in a striking gradient of increasing density with increasing distance from the soma. HCN1 is also strongly expressed in parvalbumin-positive inhibitory neurons (PV INs), where, in contrast to pyramidal neurons, it is targeted to PV IN axons and presynaptic terminals. Studies of mice with a general or forebrain-restricted genetic deletion of HCN1 have revealed the important role of this channel as a negative constraint of hippocampal pyramidal neuron dendritic integration and long-term synaptic plasticity, and of hippocampal-dependent spatial memory. Loss of HCN1 decreases the precision of pyramidal neuron place cell spatial coding while increasing the stability of spatial representations. In contrast to the well-studied role of HCN1 in pyramidal neuron function, relatively little is known about the role of HCN1 in inhibitory neurons. This lack of information prevents a full appreciation as to how HCN1 contributes to both normal brain function and disease, given the importance of inhibitory neurons, and PV INs in particular, in these processes. In addition, because HCN1 was deleted from both excitatory and inhibitory neurons in the HCN1 knockout mice examined to date, the extent to which the reported alterations in learning and memory and in vivo firing properties reflect the role of HCN1 in excitatory versus inhibitory neurons is unclear. In our application we propose to examine in detail how HCN1 contributes to PV IN function at the cellular, in vivo network, and behavioral levels. We will thus explore: the role of wild-type HCN1 in regulating PV IN intrinsic excitability and presynaptic function (Aim 1); how PV IN function is perturbed by epilepsy-associated HCN1 mutations (Aim 3a); how selective deletion of wild-type HCN1 from PV INs alters the in vivo coding of spatial information, as well as spatial and non-spatial memory behavior (Aim 2); and the paradoxical effects of certain anti-epileptic drugs to increase seizures in mice harboring epilepsy-associated HCN1 mutations (Aim 3b). Our goal in these studies is to both provide basic information about how a given channel expressed in a specific class of neurons contributes to brain function, and to provide new insights into disease mechanisms that may suggest new therapeutic approaches.