Pablo Castillo - US grants

? DESCRIPTION (provided by applicant): Short and long-term activity-dependent changes in synaptic efficacy are essential to brain function. Experimental evidence indicates that activity-induced long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission are cellular correlates to learning and memory and experience-dependent refinement of neural connections. The molecular mechanisms underlying synaptic plasticity are diverse and can be characterized as presynaptic or postsynaptic, depending on whether neurotransmitter release, or a target neuron's sensitivity to the released transmitter, is modified. Presynaptic LTP and LTD have now been observed across many brain regions, including the hippocampus, both at excitatory and inhibitory synapses, and growing evidence indicates that presynaptic LTP/LTD may underlie important forms of learning. However, the synaptic learning rules for these forms of plasticity are poorly characterized, and our understanding of presynaptic mechanisms lags far behind the postsynaptic side. Presynaptic plasticity can originate entirely in the presynaptic terminal or it may require retrograde signaling from postsynaptic to presynaptic compartments. In the last decade, retrograde signaling emerged as a widely expressed mechanism by which postsynaptic neurons can control their own inputs and, by this means, regulate neural circuits over short and long-time scales. The best characterized retrograde signaling system is the endocannabinoid (eCB) system, and while much has been learned from this system, important knowledge gaps remain for other retrograde messengers including the type of activity required for mobilization, the mechanisms of postsynaptic release and presynaptic action, and ultimately, the precise physiological role of retrograde signaling at a synapse in a given neural circuit. In this research proposal, we will address these outstanding questions by focusing on three distinct hippocampal synapses using state-of-the-art electrophysiology, molecular pharmacology, optogenetics, and live imaging in acute brain slices. Specifically, we will test the hypothesis that presynaptic protein synthesis is necessary for eCB-mediated LTD at inhibitory synapses. In addition, we will determine the mechanism and functional consequence of a novel form of presynaptic LTP at a key, but remarkably understudied, excitatory synapse in dentate gyrus. Finally, we will test the hypothesis that retrograde signaling negatively regulates a powerful detonator synapse. Knowledge derived from these investigations will provide new mechanistic insights on retrograde signaling at central synapses and may also uncover novel roles for presynaptic plasticity in the hippocampal network. A better understanding of presynaptic plasticity represents a significant step forward in the development of strategies to restore synaptic function in diseased brain states, such as autism, neurodegenerative diseases (e.g. Alzheimer's disease, Huntington's disease), schizophrenia, epilepsy and addictive behaviors.

DESCRIPTION (provided by applicant): Unique ion channels activated by both glutamate and voltage, NMDA receptors (NMDARs) play crucial roles in synapse formation, synaptic plasticity, learning, and memory. NMDARs have been associated with either the pathogenesis or the damage caused by several neurological disorders including schizophrenia, epilepsy, Parkinson's disease, drug addiction, and ischemia/stroke. The modulation of NMDARs by intracellular signaling pathways is an active area of investigation. Growing evidence now suggests that activity can dynamically regulate NMDARs, though much remains unexplored. We are currently aware of activity-dependent NMDAR regulation at only a few synapses, although its underlying molecular mechanisms and functional consequences remain unknown. We propose experiments to identify the mechanisms and specific functional contributions of activity-dependent NMDAR modulation, beginning with a novel form recently discovered in our laboratory. Recently we found that brief tetanic activity can induce long-term potentiation of NMDAR-mediated transmission at the hippocampal mossy fiber-CA3 pyramidal cell synapse (NMDAR-mfLTP). Preliminary data suggests that NMDAR-mfLTP is induced and expressed postsynaptically in a Ca2+-dependent process and is restricted to NMDARs. We propose to investigate the molecular mechanisms and functional consequences of NMDAR-mfLTP using electrophysiological recording and functional analysis, pharmacological manipulation, and Ca2+ imaging in acute hippocampal slices. In studies of the induction mechanism, we will use inhibitors and activators of signal transduction pathways including those known to regulate NMDARs in expression systems and cultured neurons. We will investigate whether this potentiation is expressed as an increase in NMDAR number and/or function. In terms of function consequences, we will determine whether NMDAR-mfLTP is associated with long-term enhancement of Ca2+ signaling at mf-CA3 synapses, and we will test the hypothesis that NMDAR-mfLTP will substantially modify the input/output function of this synapse. In addition, we will test whether NMDAR-mfLTP, once established, might modify the subsequent modifiability of excitatory and inhibitory CA3 synapses, a phenomenon known as metaplasticity. Finally, we will look for other forms of NMDAR plasticity at this synapse, including long-term depression, de-potentiation, and de-depression. Dynamic long-term modification of NMDARs may have important consequences for both normal and pathological physiology. For this reason, understanding the role of these forms of plasticity at the cellular and network level is critical to a more realistic representation of brain function, and may contribute to the development of therapeutic strategies to reverse or prevent NMDAR-mediated dysregulation or damage. PUBLIC HEALTH RELEVANCE: NMDA receptors are a subtype of receptors in the brain that participate in excitatory neurotransmission and are crucial for synapse formation, synaptic plasticity and learning and memory. Dynamic long-term modification of NMDA receptors by neuronal activity may have important consequences for both normal and pathological physiology with potential involvement in ischemia/stroke, epilepsy, schizophrenia, drug addiction, chronic pain, and Parkinson's disease. Understanding how these receptors are regulated is critical to a more realistic representation of brain function, and may contribute to the development of therapeutic strategies to reverse or prevent NMDAR-mediated dysregulation or brain damage.

DESCRIPTION (provided by applicant): Excitatory synaptic transmission in the central nervous system is largely mediated by the AMPA and NMDA subtypes of ionotropic glutamate receptors (AMPAR and NMDAR, respectively). Because AMPARs mediate the bulk of glutamatergic synaptic transmission, excitatory efficacy is commonly associated with the magnitude of AMPAR-mediated synaptic responses. While postsynaptic NMDARs are well-known for gating several forms of activity-dependent plasticity (e.g. long-term potentiation and long-term depression) of AMPAR-mediated transmission, NMDARs can also contribute to information transfer at synapses and to neuronal excitability. In addition, certain synapses localize NMDARs to the presynaptic compartment where their activation by synaptically-released glutamate can regulate neurotransmitter release. Moreover, an expanding body of evidence indicates that NMDARs themselves are also dynamically regulated and subject to activity-dependent long-term plasticity. However, many of the mechanisms underlying NMDAR plasticity are poorly understood. Thus far, most studies addressing NMDAR regulation have been performed in expression systems and cultured neurons. As a result, the extent to which similar mechanisms apply to the in vivo situation remains largely unknown. Furthermore, scant knowledge exists on the mechanisms of induction and expression of NMDAR plasticity. In this proposal, we will attempt to fill this knowledge gap by analyzing two key hippocampal synapses that express robust NMDAR plasticity. The overarching hypothesis is that common mechanisms underlie dynamic regulation of NMDARs across synapses. Using a combination of complementary experimental approaches, such as electrophysiology in acute hippocampal slices, optogenetics, immunoelectron microscopy, calcium imaging, in vivo knockdown strategies and transgenic mice, we will investigate the role of specific signaling pathways and receptor subunits in NMDAR plasticity. In addition, we will determine whether presynaptic NMDARs are regulators of short-term and long-term synaptic plasticity, and whether NMDAR plasticity is developmentally regulated. Dysregulation of NMDARs has been implicated in a wide range of neuropsychiatric disorders, such as schizophrenia, epilepsy, chronic pain, addiction to drugs, Alzheimer's disease, and Huntington's disease. Understanding the molecular mechanisms underlying NMDAR plasticity could help elucidate the precise contribution of these receptors to normal brain function, and also provide significant insights in developing novel strategies for restoring receptor function in specific disease states.

The dentate gyrus (DG) of the hippocampus plays a key role in memory formation by transforming patterns of cortical inputs into new patterns of output to the CA3 area. Although the cellular and synaptic basis of this important transformation remain poorly understood, two excitatory cell types in the DG, granule cells (GC) and hilar mossy cells (MC), play a major role. MCs mediate an intrinsic, hetero-associative (GC-MC-GC) excitatory loop, receiving powerful input from a relatively small number of GCs, and providing highly distributed excitatory output to a large number of GCs. MCs project their associational and commissural axons to the ipsi- and contralateral inner molecular layer of the DG, where they synapse onto proximal dendrites of GCs. Moreover, MCs also project their axons along the septotemporal axis of the hippocampus, thereby connecting functionally diverse areas of this structure. By projecting to most areas of the DG along the septotemporal axis, MCs could provide important contextual content to the information arising from the cortex. In order to understand how information is processed in the DG and how dysregulation of this circuit may contribute to disease, a better knowledge of the hetero-associative GC-MC-GC circuit and its dynamic properties is required. We have recently reported that MC-GC synapses undergo a novel presynaptic, NMDA-receptor independent form of long-term potentiation (LTP) that requires postsynaptic brain-derived neurotrophic factor (BDNF)/TrkB and presynaptic cyclic AMP(cAMP)/PKA signaling. We hypothesize that this novel form of plasticity enhances GC output at the associative MC-GC recurrent circuit, and may contribute to DG-dependent forms of learning and brain disease, such as epilepsy. A large number of questions regarding this circuit remain unanswered. Preliminary data indicates that MC-GC LTP is induced in vivo by experience and epileptic activity, is critically regulated by endogenous systems (e.g. endocannabinoid and adenosine signaling), and it can be accompanied by LTP of inhibitory transmission. Here, using a combination of experimental approaches both in vitro and in vivo, we aim to (1) characterize the synaptic learning rules of MC plasticity, (2) identify the molecular mechanism underlying MC-GC LTP, (3) determine the properties and mechanism underlying inhibitory LTP, and (4) determine the functional relevance of MC plasticity in vivo. By identifying the main properties and mechanisms of activity-dependent plasticity in a crucial recurrent circuit in the DG, our proposed studies may not only improve our understanding of the precise role of this circuit in DG information processing and memory encoding, but also assess how dysregulation of this circuit may contribute to brain disease, including epilepsy, anxiety, schizophrenia and depression.

ABSTRACT Microglia are brain macrophages derived from yolk-sac progenitors, with classically assigned roles that center on immune surveillance and response to an injury or a disease state. Recent views, however, have moved microglia into the landscape of normal brain function, performing activities such as sculpting and refining of neuronal circuitry in the absence of external stimuli or disease. Thus, understanding the contribution of microglia- neuron interactions in mental health is an area of active interest. Ionized Ca2+-binding adapter molecule 1 (Iba1 a.k.a. AIF1) is a highly conserved protein expressed in microglia. Iba1 has been used widely as a marker of microglia, but its contributions to microglial and brain functions remain largely unknown. In preliminary studies of mice globally deficient for Iba1 function, we have found that this protein is essential for microglial activity, evidenced by reductions in microglial branching and alterations in the total brain expression of several microglial- enriched proteins that have roles in synaptic function and behavior. Furthermore, loss of Iba1 reduces developmental excitatory synaptic strength and synapse numbers but enhances excitation-inhibition ratio involving the pyramidal neurons of CA1 hippocampus. These developmental deficits correlate with a deficit in social interaction in adult Iba1-deficient mice. We hypothesize that microglial Iba1 has important synaptic remodeling functions during postnatal development, which in turn shapes adult behavior. Since, Iba1 is also expressed in CNS-associated macrophages, circulating monocytes and peripheral tissue macrophages, it is unclear whether Iba1 function specifically in microglia contributed to the aforementioned effects in synaptic physiology, gene expression and behavior. To test these ideas, we will take advantage of our unique conditional Iba1 loss-of-function model to manipulate microglial activity. Our two aims address in turn the neurophysiological and behavioral consequences of Iba1 loss from microglial cells during a critical developmental window, and to understand the contributions of Iba1 in the modulation of microglial and synaptic gene expression that underlie an altered synaptic remodeling and behavior. The proposed work will highlight the importance of synaptic developmental functions regulated by a microglia-intrinsic protein and further attempt to showcase the impact of such regulation on behavior. Knowledge gained from this study will advance our understanding of how microglia contribute to the refinement of neuronal circuitry in a developing brain, and thus may provide a rationale for therapeutic targeting of microglia, to advance treatment of neurodevelopmental and neuropsychiatric disorders.