Laszlo Zaborszky - US grants

DESCRIPTION (provided by applicant): The broad objective of this research program is to understand how basal forebrain (BF) cholinergic (BFC) and non-cholinergic neurons are organized to modulate specific cortical regions. Despite its involvement in cortical activation, attention, and memory, the functional details of the BF are not well understood due to the anatomical complexity of the region. Patients with Alzheimer's disease and related dementias have a significant decrease of acetylcholine in the cortex and show pathological changes in cholinergic neurons in the BF. Thus, a complete understanding of its functional organization is warranted. The central hypothesis of this application is that cholinergic neurons constitute local ensembles in the BF ('cell clusters') that via local collaterals and/or common inputs with their projections to cortical areas provide the neural basis of a distributed functional network to selectively modulate cognitive processes. We will test this hypothesis in 4 interrelated Specific Aims using traditional and monosynaptic viral tracing, computational analysis of large-scale networks, in vitro patch-clamp recording of BF neurons, and high-resolution monitoring of cortical network activity in freely-moving rats with optogenetic stimulation of defined BF cholinergic neurons. In Aim 1 we will build up a relatively complete database with 200 ?m resolution of mapped BF cholinergic and non-cholinergic neurons using conventional retrograde tracing techniques. Cholinergic clusters will be defined in the resulting 'database' and in the cluster volume significant association of projection cell populations will be determined. In Aim 2 we will validate of the functional significance of the specific organization of the BFC system in wake-behaving rats. With newly developed multi-array silicon probes implanted into two specific cortical areas and light-assisted perturbation of various cholinergic cell groups in ChAT-Cre rats, in which cholinergic cells were transfected to express channelrhodopsin (ChR2), we will determine the emerging cholinergic ensembles in the BF and their effect on the functional connectivity of various large-scale cortical networks during various brain states. In Aim 3 we will define the input to cholinergic neurons in various subdivisions of the BF using commercially available Cre-dependent AAV helper viruses (AAV-EF1a-FLEX- TVAmCherry and AAV-CA-FLEX-RG) and a replication deficient rabies vector (RV: EnvA G-deleted Rabies- eGFP). In Aim 4 we will determine, using in vitro patch clamp recording and retrograde tracing in ChAT-cre X ChAT-eGFP crossbred mice, how does the system of early (EF) and late firing (LF) cholinergic neurons and local cholinergic axon arborizations fit into the global organization of the BFC system. The in vivo large-scale, high-density recording design that is built on a realistic forebran model will lead to substantially improved animal models for addressing function in behavioral studies. Concomitantly, it will facilitate the understanding of the aberrant processing in basalo-cortical networks and may help the development of new treatment strategies to ameliorate the cognitive symptoms in Alzheimer's and related disorders.

A grant has been awarded to Rutgers University under the direction of Dr. Esther Nimchinsky to acquire a two-photon laser scanning microscopy (2PLSM) suite, consisting of two custom-designed microscopes operating off a single laser source. The research projects described below represent some of the first studies in what will undoubtedly be the next phase of synaptic physiology research. They go to the heart of the question of how individual synapses interact with their immediate microenvironment, and how neurons are able to receive so many diverse inputs, respond individually to each, and maintain precisely an appropriate level of functioning for the constantly changing demands of the outside world.

The understanding of how neurons communicate with one another has come largely from studies where large numbers of synapses are sampled at the same time, and inferences are drawn regarding their individual behavior from the population averages. While this approach has yielded a great deal of knowledge, there is no escaping the fact that synapses are individual structures. In fact, one of their fascinating properties is that they can be modified separately-with over 10,000 synapses on each neuron, this is an ability that permits an exquisite degree of fine-tuning. However, their extremely small size makes them very difficult to study. In recent years there have been several important technological advances that greatly improve the ability to study individual synapses and their modulation. 2PLSM is an advanced imaging technique that was developed to permit imaging of structures deep in live tissue in vitro and in vivo for extended periods. It thus permits very high-resolution studies at the level of individual synapses in intact tissue, as well as time-lapse studies, which are critical for the uncovering of time-dependent processes. At the same time genetically encoded fluorophores have been characterized and improved, permitting the labeling of living cells with relatively low toxicity. Dyes sensitive to changes in intracellular calcium have also improved dramatically, and these allow the study of functional aspects of neuronal behavior. The system proposed here would be flexible enough to take full advantage of all these innovations. Using 2PLSM and new fluorescent dyes, individual synapses can, for the first time, be studied optically in intact tissue. Specifically, all these techniques will be combined to study the interactions of astrocytes, the major non-neuronal cell type in the brain, with synapses; the ways in which neurons balance the strengths of their synapses across their branches; and the roles of the neurotransmitters dopamine and serotonin in synaptic function, and their mechanisms of action.

This 2PLSM suite will greatly benefit projects that have a broad relevance in neuroscience, and which will be publicized by publication in major journals and presentation at national and international meetings such as that of the Society for Neuroscience. The acquisition of this flexible system will further the teaching mission of the university. Students and postdoctoral fellows in the participating labs and beyond will learn not only how neurons look and how synapses function, but will also acquire hands-on experience in the fundamentals of optics and microscopy, and learn how to optimize experimental conditions and the instruments themselves to make the most of their preparations. In addition, the faculty themselves will learn to use and exploit this important new technology, and perhaps also further to advance it. Furthermore, the acquisition of this microscopy system at Rutgers University-Newark, a campus where underrepresented minorities comprise a very sizeable proportion (40%) of the student body, will put state-of-the-art technology and a cutting-edge approach within reach of a large number of motivated students who would otherwise be very unlikely to have access to them. Finally, the presence at the campus of these microscopes would enhance the strengths of the CMBN in the field of neuronal plasticity, and help to attract faculty and students that are interested in this rapidly expanding field.