Lynn E. Dobrunz - US grants

Postnatal development produces profound changes in synaptic transmission in the brain. Many developmental studies have focused on changes in the connections between neurons through consolidation or elimination of synaptic connection. Much less is known, however, about the developmental modulation of the strength of synapses, which is also likely to play a critical role in normal neonatal development. This modulation could occur pre-synaptically, or both While several studies have looked at developmental changes in postsynaptic properties, little attention has been paid to developmental changes in presynaptic function. This is an important area since considerable evidence suggests that presynaptic mechanisms are involved in the molecular implementation and maintenance of several types of synaptic plasticity implicated as substrates for learning and memory that are subject to impairment in various forms of mental retardation. Thus, the goal of this proposal is to examine the role of changes in presynaptic function at cerebral cortical excitatory synapses, that utilize the neurotransmitter glutamate, during neonatal development. A unique aspect of this approach is the application of behaviorally salient, natural temporal patterns of activation for testing the dynamic features of neonatal synapses. This proposal describes a series of experiments to determine the developmental regulation of presynaptic function in neonatal hippocampus. Using electrophysiological and pharmacological techniques, developmental changes in synaptic release probability, short- term plasticity, and functional connectivity will be assessed during the first five postnatal weeks. Understanding the normal development of presynaptic properties is a necessary first step to investigating the effects of pre- and postnatal environmental factors, such as maternal malnutrition cause cognitive deficits in short-term memory. Since short-term plasticity is likely to be a cellular substrate for short-term memory, understanding the normal developmental properties of short-term plasticity is a key prerequisite to determining how short-term plasticity and short-term memory are impaired in these different syndromes.

Inhibitory synaptic transmission provided by inhibitory interneurons is essential both for the functioning of neuronal circuits and for normal brain development. The balance between inhibitory and excitatory synaptic transmission is critical for the proper wiring of brain circuits during early postnatal development. Alterations in the balance between inhibition and excitation have been found in many neurodevelopmental and neuropsychiatric disorders, including autism, Down Syndrome, Fragile X Syndrome, and schizophrenia. The balance between excitation and inhibition is not static, but is dynamically changed during different patterns of stimulation by short-term plasticity. The strengths of both excitation and inhibition are modulated by short-term plasticity, and differences in short-term plasticity between these two components causes the balance of excitation to inhibition to be frequency-dependent. In hippocampus, GABAergic interneurons provide powerful inhibition to the excitatory pyramidal cells that is vital to prevent epilepsy and excitotoxicity. In addition, hippocampal interneurons can synchronize the firing of pyramidal cells and drive population oscillations that are essential for learning and memory. Hippocampal interneurons are highly diverse in their anatomical, neurochemical, and physiological properties. Knowledge of the physiological properties and functional roles of these different interneuron subtypes, although required for understanding the relationship between hippocampal circuit function and behavior in both normal and disease states, is still limited. In the CA1 region of hippocampus, feedforward inhibition is provided through two pathways, the Schaffer collateral (SC) pathway onto interneurons in stratum radiatum (SR), and the temporoammonic (TA) pathway onto interneurons in stratum lacunosum-moleculare (SLM). However, the little is known about the frequency-dependence of feed-forward inhibition and how it affects the balance of excitation to inhibition. In this proposal we will test the mechanisms, functional effects, and consequences for circuit function of short-term plasticity of feed-forward inhibition onto CA1 pyramidal cells from both the SC and TA pathways. Together these data will provide information that is essential to understanding how the balance of excitation and inhibition is regulated, how short-term plasticity helps gate the flow of information through hippocampus, and the normal functional roles of different interneuron subtypes. This information may provide potential therapeutic targets for selectively modifying inhibition in neurological disease, as well as providing a foundation for future studies investigating the role of short-term plasticity of feed-forward inhibition in neuropsychiatric disorders and neurodevelopmental disorders that cause mental retardation.

This REU site in neuroscience at the University of Alabama at Birmingham (UAB) will provide motivated undergraduates with the opportunity to experience exciting independent research under the guidance of a faculty mentor. The REU site is designed to increase student interest in graduate education leading to careers in research and to enhance student's written and oral communication skills. Under the supervision of a faculty mentor, students have the opportunity to design their own projects and learn basic skills necessary to conduct a research project. Students receive training in the research methods applicable to their research plan, analyze their data, attend lab meetings and journal clubs, and create written and oral presentations of their results at a research forum at the end of the summer program and at weekly journal clubs. Projects in developmental, molecular, cellular, and integrative/behavioral/cognitive neuroscience are available. Students are paired with faculty mentors and participate in weekly faculty-student research discussions and lunches, and cultural and social events. Twelve students will be selected for the ten-week research experience each summer. Under-represented minority students and undergraduates from institutions at which research opportunities are limited are encouraged to apply. Application and other information on the REU program are available at: http://www.neurobiology.uab.edu/REU.htm or by contacting Ms. Cindy Urthaler, 205-934-1550 (voice); Email: urthaler@nrc.uab.edu

[unreadable] DESCRIPTION (provided by applicant): Activity dependent modulation of neurotransmitter release, called short-term plasticity, has been implicated in several forms of behavior and learning and memory. Although it is mediated by presynaptic mechanisms, short-term plasticity has been shown at many synapses to be target-cell specific. Thus synapses made by axons from the same cell type onto target neurons of different types can exhibit markedly different properties of short-term plasticity. This implies the existence of a retrograde signal from the postsynaptic neuron to the presynaptic cell which alters the structure and/or function of the presynaptic terminal depending on the identity of the postsynaptic target neuron. While it has been known for over 30 years that presynaptic function is target cell specific, the identity of this retrograde messenger(s) is still not known. The long term goal of this project is to determine the postsynaptic molecules that are responsible for target-cell specific short-term plasticity. The synaptic cell adhesion molecules neuroligin 1 and SynCAM have recently been shown to induce presynaptic differentiation in neurons when expressed in non-neuronal cells in co-culture. This shows that neuroligin 1 and SynCAM are sufficient to trigger presynaptic differentiation, even in the absence of other normal components of the postsynaptic terminal. While neuroligin 1 and SynCAM have each been shown to be sufficient to induce formation of presynaptic terminals in neurons (referred to here as hemisynapses) that are functional at a basic level, it is not known whether they are sufficient to enable presynaptic terminals of hemisynapses to have the same complex presynaptic properties that neuron-neuron synapses possess. In particular, has not previously been shown whether they are capable of evoked neurotransmitter release, and if so, whether they have a similar release probability and mechanisms of short-term plasticity as neuron-neuron synapses. In this proposal, we will develop the hemisynapse preparation and measure evoked neurotransmitter release and short-term plasticity, in order to test the hypothesis that the cell adhesion molecules that trigger synapse formation are also involved in the retrograde signaling that modulates the functional properties of presynaptic terminals, yet are not sufficient to induce formation of presynaptic terminals with all of the same properties of neuron- neuron synapses. This system will provide a simple but powerful assay for a large range of future experiments investigating the influences of specific cell adhesion molecules, extracellular matrix proteins, neurotrophins, secreted factors, and postsynaptic density components on the formation and function of presynaptic terminals. These experiments address fundamental questions of presynaptic function, synaptic specialization and development that are important to our understanding of circuits in the brain, and which will have implications for learning and memory as well as neurodegenerative diseases and developmental disorders that cause mental retardation. These experiments address fundamental questions of presynaptic function, synaptic specialization and development that are important to our understanding of circuits in the brain, and which will have implications for learning and memory as well as neurodegenerative diseases and developmental disorders that cause mental retardation. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The entorhinal cortex and hippocampus are both critical for learning and memory. The entorhinal cortex has been implicated in several neurological diseases, including developmental disorders that cause mental retardation. One major input pathway from entorhinal cortex to hippocampus is the temporoammonic (TA) pathway to the CA1 region of hippocampus. The TA pathway provides specific sensory information to CA1, and it has been shown to be involved in memory consolidation. Because of the importance of the TA pathway to learning and memory, alterations in the functional properties of TA synapses could contribute to cognitive impairment in developmental disorders. Such changes might not be apparent at the morphological level, but would require electrophysiology to be detected. The investigators' long term objective is to use electrophysiology to look for changes in the function of synapses and circuits in animal models of developmental disorders. To lay the foundation for that research they will first investigating the changes in synaptic function that occur during normal development. They have previously shown changes during early postnatal development in presynaptic function of synapses in the other major input pathway to CA1, the Schaffer collateral (SC) pathway. In order to understand the impact of these developmental changes for hippocampal function, the investigators also need to know the properties of TA synapses and how they are modulated during development. Relatively little is known about the presynaptic function of TA synapses and how they compare to SC synapses, and nothing is known about whether the properties of TA synapses are also developmentally regulated. Because both TA and SC synapses influence the firing of CA1 neurons, the main output neurons of hippocampus, their dynamic properties are crucial determinants of normal hippocampal function and development of hippocampal circuitry. In this small grant application, the investigators will use hippocampal brain slices from rats as a model system, and use electrophysiology to measure the presynaptic properties of TA synapses in slices from juveniles versus young adults. They will test the hypothesis that developmental modulation of presynaptic properties is fundamentally different between TA synapses and SC synapses, and that short-term plasticity is different between TA synapses and SC synapses in both juveniles and young adults. Understanding the developmental changes in the different input pathways that influence CA1 cell firing will be important for understanding how the function of the hippocampal circuit changes during normal neonatal development. Together, this research provides the foundation for future studies on the functional properties and developmental modulation of hippocampal synapses and circuits that could contribute to cognitive impairment in animal models of developmental disorders that cause mental retardation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Because of the enormous impact of neuropsychiatric disorders on human health, and the scarcity of effective treatments, it is essential to advance the understanding of the synaptic and circuit mechanisms underlying neuropsychiatric disorders such as schizophrenia. Alteration in the balance of inhibition and excitation (I/E) is emerging as a fundamental unifying principle underlying a wide variety of complex brain disorders, including neuropsychiatric and neurodevelopmental disorders such as schizophrenia, bipolar disorder, autism, Down Syndrome, Rett Syndrome and Fragile X. I/E imbalance is often caused by alterations in GABAergic interneurons, particularly interneurons containing the calcium binding protein parvalbumin (PV). Transcriptional dysregulation in PV interneurons and GABAergic dysfunction are consistent finding in postmortem tissue of SZ patients. The effects of these changes on synaptic and circuit function are not well understood. In this proposal we will investigate alterations in the dynamic I/E balance in an animal model of inhibitory dysfunction caused by genetic deletion of PGC-1¿. PGC-1¿ (peroxisome proliferator activated receptor ? coactivator 1¿) is a transcriptional co-activator in interneurons that regulates transcription of P. Genetic deletion of PGC-1¿ in mice results in decreased expression of PV in interneurons and alterations in GABAergic inhibition. PGC-1¿ is therefore a potential mediator of the decreased PV seen in SZ. In addition, the gene for PGC-1¿ is associated with SZ and bipolar disorder. PGC-1¿-/- mice provide a way to investigate the multi-factorial effects on synaptic and circuit function of interneuron dysfunction cause by transcriptional dysregulation in interneurons. We will determine the mechanisms underlying the inhibitory dysfunction in PGC-1¿ deficient mice, as well as the overall effects on the dynamics of the I/E balance and on hippocampal circuit function. In addition, we will utilize pharmacological and optogenetic manipulation of the I/E balance as a means to restore the I/E balance in PGC-1¿ deficient mice, and determine the resulting effects on circuit function. The proposed studies will greatly advance our understanding of the effects of inhibitory dysfunction due to transcripitional dysregulation on the dynamics of I/E balance in hippocampus. This will provide insights into new strategies or therapeutic targets for correcting I/E imbalances, with implications for treatment of SZ and a wide range of other complex brain disorders involving I/E imbalance and circuit dysfunction.

Neuropeptide Y (NPY) has robust anxiolytic (anti-anxiety) properties and is thought to be a stress resilience factor. Clinical and pre-clinical studies have shown that NPY signaling regulates stress-dependent behavior in both humans and rodents. Our knowledge of the mechanisms by which NPY affects synaptic and circuit function to alter behavior is incomplete. NPY has been implicated in a wide variety of anxiety disorders, including post- traumatic stress disorder (PTSD). Low levels of NPY have been measured in patients with PTSD and in animals exposed to traumatic stress. Modulation of NPY has been proposed as a potential therapy for PTSD and other anxiety disorders. The hippocampus has been implicated in anxiety disorders including PTSD, which can be considered maladaptive forms of learning. NPY and its receptors are found at high levels in hippocampus, and direct injections of NPY into hippocampus attenuate avoidance behavior in rodents. Importantly, the levels of NPY in hippocampus are reduced in rodents exposed to traumatic stress. The mechanism underlying this decrease is not known. However, the reduction in NPY expression by traumatic stress is important, because injection of NPY into hippocampus alleviates behavioral symptoms. In the previous funding period, we showed that traumatic stress causes the loss of NPY release in the temporammonic pathway in the CA1 region of hippocampus, a pathway that is important for memory consolidation and fear learning. In this funding period, we will determine the effects of NPY release in the TA pathway on hippocampal circuit function and behavior, investigate the mechanisms of decreased NPY release caused by traumatic stress, and test whether enhancing release of NPY in the TA pathway can rescue the behavioral effects of traumatic stress. These studies could lead to new therapeutic strategies to alleviate anxiety symptoms in PTSD patients.

The regulation of intracellular pH (pHi) and extracellular pH (pHo) of brain is critical for optimizing neuronal excitability. Na-Coupled Bicarbonate Transporters (NCBTs) ?particularly the astrocytic electrogenic Na/bicarbonate cotransporter NBCe1 that couples changes in pH with neuronal activity? are key regulators of brain pH. Despite the clear importance of pH regulation and the abundance of NBCe1 in brain, the role of NBCe1 and associated pH changes in modulating synaptic transmission and synaptic plasticity has not be identified. The current objective is to use molecular and pharmacological tools with electrophysiological approaches in brain-slice studies to investigate the role of NBCe1 and associated pH changes in modulating hippocampal synaptic function. Aim 1 is to address the hypothesis that NBCe1 dampens basal, low-frequency synaptic transmission. A combination of extracellular and whole-cell recordings in CA1 of acute hippocampal slices from wild-type and NBCe1 knockout (KO) mice will be used to investigate the role of NBCe1 in regulating synaptic transmission and spiking in hippocampal CA1 in response to low-frequency (0.1 Hz) extracellular stimulation. The effects of NBCe1 inhibitors (S0859 and a function blocking L3 antibody), a function stimulating L4 antibody, and viral restoration of NBCe1 into astrocytes in NBCe1 KO mice will be determined. pH-sensitive microelectrodes and dyes will be used to examine associated changes in pHo, as well as the pH of astrocytes and presynaptic terminals to test the hypothesis that inhibiting NBCe1 stimulates basal synaptic transmission by enhancing the extracellular alkaline shift (established conventional model). Aim 2 is to address the hypothesis that NBCe1 enhances high-frequency synaptic transmission and long-term plasticity. The molecular and pharmacological tools and approaches described for Aim 1 will be used to examine excitatory synaptic responses, but in response to high-frequency (50 Hz) extracellular stimulation. pH measurements will be made in the extracellular space, astrocytes, and presynaptic nerve terminals to test the hypothesis that NBCe1 stimulation of high-frequency synaptic transmission and long-term potentiation (LTP) involves dampening of the activity-evoked presynaptic pHi decrease (new mechanism). Underlying mechanisms such as pre- vs postsynaptic responses and the role of specific receptors will be evaluated for both Aims. Results will reveal that NBCe1 is a physiologically important acid-base transporter that modulates synaptic transmission and LTP through changes in pH that are dependent on frequency stimulation. The results will contribute to our understanding of NBCe1 and associated pH changes in neuronal activity, seizures, ischemia, and hypoxia.