Neurobiology

Bernardo Sabatini, M.D, Ph.D

Associate Professor of Neurobiology

The modification of synaptic connections between neurons is thought to underlie our ability to form memories and acquire new behaviors. The majority of excitatory synapses in the brain are formed onto specialized cellular structures known as dendritic spines that consist of a bulbous spine head that is separated from the remainder of the dendrite by a thin neck. It has long been speculated the function of spines is to provide the cell with a compartmentalized space in which the machinery necessary to modify and read the activity of a particular synapse can operate without interference from neighboring synapses. However, until recent technical advances, the small size of spines (usually less than 0.1 femtoliters in volume) had prevented the direct examination of their role in spatially restricting biochemical signaling. Advances in optical techniques and in particular the two-photon microscope have made possible imaging of spines located deep within scattering tissue, such as in a brain slice or within an intact animal. It is now known that spines are dynamic structures that grow, structurally reorganize, and sometimes disappear within tens of minutes and spine motility has been correlated with the ability of animals of reorganize their cerebral cortex in response to sensory deprivation. However, many questions concerning the function and biogenesis of spines remain unanswered. In particular, it is not known to what degree spines are functionally specialized and segregated compartments. Nor have the intracellular mechanisms by which neurons regulate the formation and maintenance of spines been elucidated.

The goal of our laboratory is to study biochemical signaling within spines and boutons in order to understand the pathways that trigger the formation of new synapses and the regulation of existing ones. The principal challenge in these studies is that much of the relevant biochemical signaling occurs in very small subcellular compartments such as dendritic spines and axonal boutons. We are combining molecular biology, electrophysiology, and microscopy to overcome this obstacle. Principal among these is the use of 2-photon laser scanning microscopy (2PLSM), which is ideally suited for measuring fluorescent signals from individual boutons or spines located within brain slices or in vivo.

Using combined 2PLSM and 2-photon laser photoactivation (2PLP) of caged glutamate we are examining the degree to which spines and dendrites operate as independent electrical and calcium signaling compartments.  These studies include exploring how a neuron responds to a single active input and to groups of active synapses.  By stimulating synapses with 2PLP of caged glutamate, we can exactly determine the spatial layout and temporal ordering of active synapse and thus explore how these parameters regulate neuronal responses. Furthermore, using photoactivatable intracellular fluorophore we are examining the mechanism and regulation of biochemical isolation of synapses in spiny and aspiny neurons.

Similar optical approaches can also be used to explore other facets of cell biological function within dendrites.  For example, we are investigating the mechanisms that reciprocally couple synaptic activity and regulated protein translation. Ample evidence now exists that synaptic stimuli can influence the translation of dendritic mRNAs and that the newly synthesized proteins in turn regulate synaptic activity. We have developed fluorescent reporters of protein translation and have begun to explore the synaptic stimuli and signaling cascades that activate protein translation in dendrites.   Similarly, using fluorescence reporters of membrane lipid composition, we are asking if phosphoinositide distribution in spines in dendrites is inhomogeneous and regulated by synaptic activity.