Roberto Malinow - US grants

A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. It has been suggested that the cellular and molecular mechanisms responsible for LTP will elucidate several physiological phenomena including learning, memory, and developmental synapse specificity. An understanding of the mechanisms underlying LTP is important in deciphering the effect of experience on behavior and thus may give insight into the cellular basis of clinical psychiatric disorders. The cellular signalling responsible for generating LTP has been studied extensively. However, there is not yet a consensus in the scientific community regarding the final modification underlying potentiated transmission. In this project I have one specific aim: to determine the nature of synaptic changes responsible for the persistent enhancement of transmission during LTP. Answers to this central question should serve as the cornerstone in understanding the cellular and molecular mechanisms underlying LTP. Due to the recent application of whole-cell voltage-clamp recordings to hippocampal slices, a technique that allows much greater signal resolution, we are in a position to see, at a very basic mechanistic level, the intricate processes responsible for LTP. This powerful technique will be applied to answer two questions: 1) Does quantal size change with LTP? and 2) Is there a change in paired-pulse facilitation with LTP? I will use these two questions to test a specific hypothesis that could reconcile the different views currently in the literature regarding expression of LTP: for low levels of potentiation there is primarily a postsynaptic change; large levels of potentiation are due to presynaptic changes. It is hoped that a detailed knowledge of the changes during LTP will elucidate the sites of synaptic modulation in physiological conditions as well as point to critical mechanisms that may be perturbed in pathological states.

A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. It has been suggested that the cellular and molecular mechanisms responsible for LTP will elucidate several physiological phenomena including learning, memory, and developmental synapse specificity. An understanding of the mechanisms underlying LTP is important in deciphering the effect of experience on behavior and thus may give insight into the cellular basis of clinical psychiatric disorders. The cellular signalling responsible for generating LTP has been studied extensively. However, there is not yet a consensus in the scientific community regarding the final modification underlying potentiated transmission. In this project I have one specific aim: to determine the nature of synaptic changes responsible for the persistent enhancement of transmission during LTP. Answers to this central question should serve as the cornerstone in understanding the cellular and molecular mechanisms underlying LTP. Due to the recent application of whole-cell voltage-clamp recordings to hippocampal slices, a technique that allows much greater signal resolution, we are in a position to see, at a very basic mechanistic level, the intricate processes responsible for LTP. This powerful technique will be applied to answer two questions: 1) Does quantal size change with LTP? and 2) Is there a change in paired-pulse facilitation with LTP? I will use these two questions to test a specific hypothesis that could reconcile the different views currently in the literature regarding expression of LTP: for low levels of potentiation there is primarily a postsynaptic change; large levels of potentiation are due to presynaptic changes. It is hoped that a detailed knowledge of the changes during LTP will elucidate the sites of synaptic modulation in physiological conditions as well as point to critical mechanisms that may be perturbed in pathological states.

A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of intense synaptic activity. The cellular and molecular mechanisms underlying LTP may elucidate several physiological and pathological processes, including learning, memory, developmental synapse specificity, pain, neuronal death, epilepsy and dementia. The cellular signaling responsible for generating LTP has been studied extensively. A molecule thought to play an important role in LTP is the Ca++/calmodulin-dependent protein kinase II (CaMKII). In this project I will test the hypothesis that an increase in postsynaptic CaMKII activity is sufficient to produce LTP. The specific aim of this project is to test this hypothesis by answering the following: 1. Does acute expression of a constitutively active CaMKII enzyme in postsynaptic neurons mimic and occlude LTP? i.e. does it enhance transmission and use up the LTP process? 2. Does acute expression of the wild type CamKII enzyme in postsynaptic cells rescue LTP in slices prepared from mice in which CaMKII has been genetically "knocked out"? These questions will be addressed using a novel viral infection technique that allows acute expression of a gene of interest in targeted regions of hippocampal slices. Extensive data shows the efficacy of this technique and preliminary data indicates that expression of a constitutively active CaMKII in postsynaptic hippocampal slice neurons enhances synaptic transmission and prevents more LTP. The proposed experiments will combine this new method with modern electrophysiological assays of synaptic transmission. The broad long-term objectives of my laboratory are to delineate the biochemical and biophysical mechanisms underlying activity-dependent synaptic plasticity in the CNS. This project will serve as a foundation by identifying a central piece in the puzzle of LTP. More generally we will continue to develop a methodology applicable to a large range of signal transduction problems in the CNS. Furthermore, it is not unreasonable that such viral strategies will soon be used in gene therapy for CNS pathologies. It will be important to continue to develop these experimental systems, to test the effect of viral infection and recombinant expression on synaptic transmission and plasticity. Acute expression of recombinant products in the hippocampal slice offers many advantages since numerous physiological and pathological processes have been carefully studied in this preparation.

A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. It has been suggested that the cellular and molecular mechanisms responsible for LTP will elucidate several physiological phenomena including cognitive functions such as learning a memory. Understanding the mechanisms underlying LTP is important in deciphering the effects of experience on behavior and thus will likely give insight into the cellular basis of psychiatric diseases. The cellular signaling responsible for generating LTP in CA1 hippocampus has been studied extensively with no general consensus on the nature of the persistent modifications. This project will focus on testing a recent model proposed for LTP: synapses that have only functional NMDA receptors (and are silent at hyperpolarized potentials) acquire functional AMPA receptors during LTP. This possible mechanism of potentiation is import because it can incorporate virtually all results that have previously led to disparate views regarding the locus of modification during LTP in CA1 hippocampus. This model will be tested using electrophysiological and analytical techniques previously developed. In addition, rapid photolysis of caged glutamate will be used to mimic synaptic transmitter release. With these techniques four specific aims will be addressed. 1. Determine the prevalence of postsynaptically silent synapses in different nueronal types and at different points in development. 2. Determine if the changes observed during LTP are different at different points in development. 3. Determine if there is an increased sensitivity to photolytically delivered transmitter during LTP, 4. Determine the biophysical basis for postsynatically silent synapses. It is hoped that a detailed knowledge of the changes during LTP will elucidate the sites of synaptic modulation in physiological conditions as well as point to critical mechanisms that may be perturbed in pathologicalstates.

A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. The cellular and molecular mechanisms responsible for LTP will likely elucidate physiological and pathological phenomena including learning, memory, developmental synapse specificity, neuronal death, and dementia. The cellular signaling responsible for generating LTP has been studied extensively. There is now compelling evidence that there is delivery of AMPA-type glutamate receptors to synapses during LTP. Here we will examine the cellular and molecular mechanisms of AMPA receptor delivery to synapses. We will characterize constitutive and regulated synaptic delivery. A specific model is proposed and tested. A regulated pathway effects transient delivery of receptors. The increased levels of receptors at synapses are maintained by constitutive one-for-one exchange between intracellular and synaptic pools. These issues will be examined with several complementing methodologies including electrophysiology, two-photon imaging of GFP-tagged receptors, molecular biology and transgenic technology. These studies will use rodent hippocampal slices (acute and organotypic). In this grant period we plan to: 1. measure delivery of recombinant AMPA receptors to synapses 2. determine which AMPA receptor subunits and domains control constitutive and regulated synaptic delivery 3. generate transgenic mice with dominant negative AMPA-receptor mutations that perturb regulated or constitutive delivery

DESCRIPTION (provided by applicant): A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. It has been suggested that the cellular and molecular mechanisms responsible for LTP will elucidate physiological and pathological phenomena including learning, memory, developmental synapse specificity, pain, neuronal death, epilepsy and dementia. A number of recent studies indicate that regulated synaptic delivery of AMPA receptors (-Rs) is a critical aspect of LTP. Here we will examine molecular mechanisms controlling the regulated synaptic delivery of AMPA-Rs The central hypothesis to be tested is that protein kinases drive AMPA-Rs to synapses by relieving their extrasynaptic retention. We will examine the role of two protein kinases, PKA and CaMKII, in controlling retention interactions that keep AMPA-Rs extrasynaptically. This will be examined early in development, when GluR4 controls synaptic delivery, and later in life, when synaptic delivery is controlled by GluR1. Several complementing methodologies will be used, including molecular biology, electrophysiology, two-photon imaging of GFP-tagged receptors, and immunohistochemistry with light and electron microscopy. These studies will use organotypic rat hippocampal slices. The specific aims are to determine the mechanism by which: SA1: PKA controls synaptic delivery of GluR4-eontaining-AMPA-R early in postnatal development. SA2: PKA controls synaptic delivery of GluR1-containing-AMPA-R driven by CaMKII later in life. SA3: PKA controls generation of LTP later in life. SA4: extrasynaptic retention of AMPA-Rs is regulated by protein-protein interactions.

A promising and widely studied example of vertebrate synaptic plasticity is long-term[unreadable] potentiation (LTP), the persistent synaptic enhancement seen following a brief period of[unreadable] coincident pre- and postsynaptic activity. The cellular and molecular mechanisms[unreadable] responsible for LTP will likely elucidate physiological and pathological phenomena[unreadable] including learning, memory, developmental synapse specificity, neuronal death, and[unreadable] dementia. The cellular signaling responsible for generating LTP has been studied[unreadable] extensively. There is now compelling evidence that there is delivery of AMPA-type[unreadable] glutamate receptors to synapses during LTP. Here we will examine the cellular and[unreadable] molecular mechanisms of AMPA receptor delivery to synapses. We will characterize[unreadable] constitutive and regulated synaptic delivery. A specific model is proposed and tested. A[unreadable] regulated pathway effects transient delivery of receptors. The increased levels of[unreadable] receptors at synapses are maintained by constitutive one-for-one exchange between[unreadable] intracellular and synaptic pools. These issues will be examined with several[unreadable] complementing methodologies including electrophysiology, two-photon imaging of GFP-tagged[unreadable] receptors, molecular biology and transgenic technology. These studies will use rodent[unreadable] hippocampal slices (acute and organotypic). In this grant period we plan to: 1. measure[unreadable] delivery of recombinant AMPA receptors to synapses 2. determine which AMPA receptor[unreadable] subunits and domains control constitutive and regulated synaptic delivery 3. generate[unreadable] transgenic mice with dominant negative AMPA-receptor mutations that perturb regulated or[unreadable] constitutive delivery.

DESCRIPTION (provided by applicant): Long-term potentiation and depression (LTP and LTD) are promising and widely studied examples of vertebrate synaptic plasticity. In LTP and LTD there is a persistent synaptic enhancement or decrement, respectively, seen following brief conditioning periods of synaptic activity. In both these forms of plasticity, which are leading models of memory, the trafficking of AMPA receptors (-Rs) at synapses plays a key role. The general aim of this grant has been to examine the subcellular signaling controlling AMPA-R trafficking. Recently, we have found that beta amyloid (A?), a peptide strongly implicated as a causative agent in Alzheimer's disease, has pronounced effects on AMPA-R trafficking. In this grant period, we will examine how A? can control the trafficking of synaptic AMPA receptors. Our recent studies show that A? recruits signaling used in LTD to remove synaptic AMPA receptors. Furthermore, loss of synaptic AMPA receptors leads to loss of dendritic spines and NMDA receptors;that is, loss of the synapse. Here we will determine the mechanisms by which A? leads to these events. Several complementing methodologies will be used, including molecular biology, electrophysiology, two-photon laser scanning microscopy, and electron microscopy. These studies will use organotypic rat hippocampal slices, dissociated cultured neurons and transgenic mice. The results of these studies will elucidate the mechanisms underlying Alzheimer's disease as well as provide potentially efficacious treatment strategies. The specific aims are to determine: SA1: If A? allosterically up-modulates NMDA-R function;SA2: How A? interacts with synaptic plasticity;SA3: How A? leads to removal of synaptic AMPA-Rs;SA4: How A? leads to loss of synapses. There is growing evidence that one of the first targets of dysfunction in Alzheimer's disease is the synapse. We will examine the mechanisms by which beta amyloid, a molecule strongly implicated in the etiology of the disease, leads to synaptic dysfunction. By elucidating these mechanisms we will identify potentially therapeutic targets in the treatment of Alzheimer's disease.

DESCRIPTION (provided by applicant): A promising and widely studied example of vertebrate synaptic plasticity is long-term potentiation (LTP), the persistent synaptic enhancement seen following a brief period of coincident pre- and postsynaptic activity. The cellular and molecular mechanisms responsible for LTP are thought to participate in physiological and pathological processes including learning, memory, developmental synapse specificity, pain, neuronal death, and dementia. For many years the locus undergoing changes during LTP (pre- and/or postsynaptic) was debated. Identification of the post-synapse as a site of modification has led to considerable advancement in the field. Evidence accrued over the past ten years indicates that delivery of AMPA-type glutamate receptors to synapses plays a critical role during LTP. However, the mechanisms controlling synaptic incorporation of AMPA receptors are not clear. In particular, the path by which AMPA receptors reach synapses during LTP, lateral diffusion and/or exocytosis, is hotly contested. Since these paths employ such mechanistically distinct processes, knowing each of their roles will shed light on the underlying molecular machinery operating during LTP. We have developed molecular, optical and electrophysiological methods in rodent brain slices to elucidate the mechanisms controlling AMPA receptor synaptic incorporation during LTP and experience-driven plasticity. In this grant period we plan to: 1. Measure optically synaptic incorporation of recombinant AMPA receptors. 2. Determine the role played by AMPA receptor exocytosis in LTP. 3. Determine the role played by AMPA receptor lateral diffusion in LTP. 4. Determine the pattern of synaptic potentiation in single neurons following experience-driven plasticity. PUBLIC HEALTH RELEVANCE: Synapses, the sites of communication between nerve cells, are modified during learning and memory. How this modification takes place, at the molecular level, will help scientists understand the biological basis of learning and memory, as well as what goes wrong during diseases such as Alzheimer's disease.

? DESCRIPTION (provided by applicant): Depression is a common disease that causes significant morbidity and mortality in humans. There is currently no clarity regarding the underlying molecular, cellular or circuit mechanisms. Presently, therapeutic intervention is not well understood mechanistically and often unsuccessful. It is important to derive a mechanistic understand of depressive disorders so that effective treatment can be developed. Abnormalities in parts of the brain that participate in the reward system are thought to play important roles. The lateral habenula (LHb) is an important part of the reward circuit by providing `reward prediction error' signals: when an animal receives a reward that is less than expected (i.e. is disappointed) or anticipates punishment (i.e. expects something bad), the LHb is active, and this information is thought normally to be used to shape future behavior to maximize reward and avoid unpleasant events. An individual with overly active LHb would be expected to be easily or continually disappointed and generally expect bad outcomes. It is therefore not a surprise that a number of studies in humans and rodents indicate that excessive activity in the LHb contributes to major depression. This grant proposes to examine how information in the LHb is processed in normal rodents, and modified by manipulations related to depression.

DESCRIPTION (provided by applicant): Novel roles by glutamatergic receptors in the synaptic effects of beta amyloid Long-term potentiation and depression (LTP and LTD) are promising and widely studied examples of vertebrate synaptic plasticity in which there is a persistent synaptic enhancement or decrement, respectively, seen following brief conditioning periods of synaptic activity. In both these forms of plasticity, which are leading models of memory, NMDA receptors (-Rs) and AMPA receptors (-Rs) at synapses play key and distinct roles. The general aim of this grant has been to examine the subcellular signaling controlling LTP and LTD. Recently, we have found that beta amyloid (A?), a peptide strongly implicated as a causative agent in Alzheimer's disease, has pronounced effects on AMPA-R trafficking requiring a novel form of NMDA-R signaling. In this grant period, we will examine the different roles played by NMDA-Rs and AMPA-Rs and their associated proteins in the effects of A? on synapses. Our preliminary studies show that a non-ionic form of NMDA-R signaling as well as a specific subunit of AMPA-Rs are required for A? to modify excitatory synapses. Here we will examine these findings using several complementing methodologies including molecular biology, electrophysiology, and two-photon laser scanning microscopy. These studies will use heterologous cell lines, organotypic rat hippocampal slices and genetically modified mice. The results of these studies will elucidate the mechanisms underlying Alzheimer's disease as well as provide potentially efficacious treatment strategies. The specific aims are to determine: Specific Aim 1: The role played by NMDA-Rs and associated molecules in A?-induced synaptic depression Specific Aim 2: The role played by AMPA-Rs and associated molecules in A?-induced synaptic depression

DESCRIPTION (provided by applicant): A major goal of modern neuroscience is to identify how and where in the brain experience modifies synapses. Such knowledge would advance the understanding and treatment of major neurological and neuropsychiatric diseases, such as post-traumatic stress disorder, schizophrenia, dementia and substance abuse disorders. The general goal of this grant has been to understand the nature of synaptic changes triggered by brief conditioning periods of synaptic activity (such as long-term potentiation, LTP; and long-term depression, LTD) and determine their functional consequences. In this grant period, we will focus on the role that LTP and LTD play in associative memory. In particular, we will test the hypothesis that modification of synapses by LTD and LTP can turn off and subsequently turn back on a previously established associative memory. Such experiments will investigate the causal role between these well-studied cellular processes and memory.

? DESCRIPTION (provided by applicant): The goal of this application is to develop SYNPLA, a specific, selective and high-throughput method for marking experience-induced plasticity with single synapse resolution. SYNPLA exploits our understanding of the molecular mechanism of long-term potentiation, which is mediated by the insertion of a specific subtype of glutamate receptor at recently potentiated synapses. In Aim 1 we develop SYNPLA in cultured neurons. In Aims 2 and 3 we apply it to two brain circuits, cortico-amygdalar and cortico-striatal respectively which are known to undergo experience dependent plasticity. SYNPLA has the potential to emerge as a powerful tool for dissecting the circuit mechanisms underlying behavioral plasticity.