Examining Form and Function of Dendritic Spines

The majority of fast excitatory synaptic transmission in the central nervous system takes place at protrusions along dendrites called spines. Dendritic spines are highly heterogeneous, both morphologically and functionally. Not surprisingly, there has been much speculation and debate on the relationship between spine structure and function. The advent of multi-photon laser-scanning microscopy has greatly improved our ability to investigate the dynamic interplay between spine form and function. Regulated structural changes occur at spines undergoing plasticity, offering a mechanism to account for the well-described correlation between spine size and synapse strength. In turn, spine structure can influence the degree of biochemical and perhaps electrical compartmentalization at individual synapses. Here, we review the relationship between dendritic spine morphology, features of spine compartmentalization and synaptic plasticity. We highlight emerging molecular mechanisms that link structural and functional changes in spines during plasticity, and also consider circumstances that underscore some divergence from a tight structure-function coupling. Because of the intricate influence of spine structure on biochemical and electrical signalling, activity-dependent changes in spine morphology alone may thus contribute to the metaplastic potential of synapses. This possibility asserts a role for structural dynamics in neuronal information storage and aligns well with current computational models

Structure-function analysis on the level of individual synapses

Excitatory synapses in the mammalian brain are made on small protrusions of the postsynaptic cell called dendritic spines. Dendritic spines are highly variable in their morphology and in their microanatomy (e.g. presence of subsynaptic organelles). It is unclear whether and how variability in spine morphological and anatomical properties translates into differences in synaptic function. Using two photon imaging, we analyzed how spine properties can affect synaptic signals and the potential for synaptic plasticity at single identified spine synapses. We show that synaptic signals can be tightly regulated on the level of individual synapses and that differences in spine morphology and microanatomy regulate synaptic function. We also provide evidence for the existence of functionally distinct populations of synapses in regard to their potential for synaptic plasticity. The present thesis is subdivided into three main sections. The first section is dedicated to the analysis of the function of specialized subsynaptic organelles in regulating synaptic plasticity. In the second section we studied the impact of spine morphology on synaptic signals and in the third section we examined whether critical proteins can be tagged to individual synapses in response to plasticity inducing stimuli. In pyramidal cells, only a subset of dendritic spines contains endoplasmic reticulum (ER). Spine ER often forms a ‘spine apparatus’, a specialized organelle with unknown function. It is unclear whether these specialized subsynaptic structures can affect the function of the synapse on the spine head. The possible involvement of spine ER in shaping spine calcium transients, a key trigger for synaptic plasticity, raises the possibility that spine ER could modulate the potential of a given synapse to undergo activity dependent modifications. Using a genetic approach to label the ER in living neurons, we find that the ER preferentially localizes to spines containing strong synapses. We demonstrate that spine ER represents a specialized calcium signaling machinery required for the induction of metabotropic glutamate receptor dependent long term depression at individual synapses. We demonstrate that different subsets of synapses exist in regard to their potential to undergo specific forms of plasticity. Spine ER represents the anatomical correlate for a mechanism by which strong synapses can be retuned in an activity dependent manner. Dendritic spines are separated from their parent dendrite by a thin spine neck. The spine neck slows down diffusion of molecules from the spine head to the parent dendrite, allowing spine-specific action of second messengers and activated enzymes. The resistance of the spine neck is crucial in determining whether spines can also be considered electrical compartments. Only a high enough spine neck resistance leads to electrical compartmentalization and activation of voltage gated channels in the spine in response to synaptic stimulation. We show that spine neck resistance can change in an activity dependent manner. Using single spine calcium imaging as a reporter of NMDA receptor activation and spine head depolarization, we show that spines can indeed act as electrical compartments. Using pharmacological experiments and modeling, we demonstrate that different voltage dependent channels cooperatively participate in shaping spine head depolarization and spine calcium transients. We also show that in vivo the spine neck resistance is higher compared to the situation in acutely sliced brain tissue, demonstrating that in the living animal a higher fraction of spines can be considered electrical compartments compared to the in vitro situation. We provide strong evidence that the spine neck can profoundly affect synaptic calcium signals. Biochemical and electrical compartmentalization is dynamically regulated in an activity dependent way. Spine calcium signals can activate key signaling cascades responsible for the induction of synaptic plasticity. Long term potentiation (LTP) has been shown to require the activity of CaMKII, a serine/ threonine kinase. A chemical protocol leading to LTP has been shown to induce translocation of CaMKII to dendritic spines. It is however unclear whether this molecule acts at single synapses or whether it can spread and modulate neighboring synapses in response to more physiological protocols. Using a new optical approach to induce LTP at single visualized synapses, we show that LTP induction is accompanied by a long-lasting increase of CaMKII at the stimulated synapse. This increase was specific to the stimulated spine and did not spread to neighboring spines. We provide evidence that CaMKII acts locally, on the micrometer scale, to regulate plasticity. We show that the concentration of proteins involved in regulating synaptic plasticity can be tightly regulated at the level of single synapses

Diffusion laws in dendritic spines

Dendritic spines are small protrusions on a neuronal dendrite that are the main locus of excitatory synaptic connections. Although their geometry is variable over time and along the dendrite, they typically consist of a relatively large head connected to the dendritic shaft by a narrow cylindrical neck. The surface of the head is connected smoothly by a funnel or non-smoothly to the narrow neck, whose end absorbs the particles at the dendrite. We demonstrate here how the geometry of the neuronal spine can control diffusion and ultimately synaptic processes. We show that the mean residence time of a Brownian particle, such as an ion or molecule inside the spine, and of a receptor on its membrane, prior to absorption at the dendritic shaft depends strongly on the curvature of the connection of the spine head to the neck and on the neck's length. The analytical results solve the narrow escape problem for domains with long narrow necks

Imaging synaptic plasticity

Over the past decade, the use and development of optical imaging techniques has advanced our understanding of synaptic plasticity by offering the spatial and temporal resolution necessary to examine long-term changes at individual synapses. Here, we review the use of these techniques in recent studies of synaptic plasticity and, in particular, long-term potentiation in the hippocampus

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Dendritic spines compartmentalize synaptically-evoked biochemical signals. The authors show that electrical compartmentalization provided by a spine endows the associated synapse with additional modes of calcium signaling by shaping the kinetics of synaptic calcium currents

The Role of SRGAP2 in Modulating Synaptic Dynamics in Adult Sensory Cortex

Human brain evolution granted us cognitive and behavioral capabilities that are unique amongst animals. SRGAP2 is a gene that was specifically duplicated in the human lineage and plays roles in the regulation of cortical development and synapse dynamics. As paralogs of one of the few known genes that regulates excitatory and inhibitory synapses concurrently, the duplications of SRGAP2 were well-positioned during human evolution to gain novel functions leading to the cognitive and behavioral phenotypes exhibited in humans. SRGAP2C, a human-specific paralog of the ancestral SRGAP2 gene, inhibits every known function of SRGAP2 and induces a phenotype similar to SRGAP2 knockdown. This induces neoteny in the maturation of synapses in mice, allowing us to study a putatively “human-like” phenotype in the mouse brain. While studies have been conducted on the effects of SRGAP2 manipulation in juvenile and young adult mice, its effects on older mice has yet to be determined. In this dissertation, we perform longitudinal imaging experiments to determine the effects of SRGAP2 manipulation in the cortex of adult mice. In Chapter 3, we first examine the effects of SRGAP2 knockdown on the spine dynamics on apical dendrites of layer 5 pyramidal cells in the barrel cortex of adult mice, determining how it regulates spine density, turnover, and survival at baseline and in response to sensory deprivation. In Chapter 4, we study how SRGAP2 knockdown affects the clustered formations of new dendritic spines on the apical dendrites of layer 5 pyramidal cells in the barrel cortex of adult mice. Together, these results represent the first demonstration of SRGAP2 regulating on synapse dynamics in vivo and show that SRGAP2 knockdown can be used to model human brain evolution in adult mice

Prelimbic Cortical Synaptic and Structural Plasticity Following Cocaine Self-administration and Abstinence in Rats: Role of Glutamatergic Pathway Specificity

The primary goal of this dissertation is to further examine the role of the prelimbic (PrL) subdivision of the rodent medial prefrontal cortex in relapse to cocaine seeking following abstinence, and to extend our understanding of pathway-specific adaptations in the PrL cortex projection to the nucleus accumbens (NAc) core that drives relapse. Previous findings indicate that the PrL cortex shows a biphasic response to abstinence from cocaine exposure. Specifically, early withdrawal (two hours after the final self-administration session) results in dephosphorylation of glutamate NMDA receptors and glutamate signaling regulators including extracellular signal-regulated kinase and the downstream transcription factor cAMP response-element binding protein (CREB). One week of abstinence enhances p-CREB and AMPA receptor subunit GluA1 phosphorylation in the PrL cortex, and Synapsin I in the NAc core. Interventions that act to normalize glutamate transmission in the PrL cortex during early withdrawal provide an enduring suppression of drug-seeking by normalizing activity in the PrL-NAc core pathway. Using a combination of biochemical and behavioral pharmacology techniques, we have found that the cocaine-induced activation of STriatal-Enriched protein tyrosine Phosphatase in the PrL cortex during early withdrawal plays a role in subsequent cocaine seeking by dephosphorylating extra-cellular signal-regulated kinase. We also show that chemogenetic-mediated activation of the PrL cortex, or PrL-NAc core neurons, immediately after self-administration transiently reduces drug seeking which is not sustained. Finally, using an array of immunohistochemistry, pathway-specific viral vectors, and high-resolution confocal microscopy techniques, we provide evidence that PrL-NAc core neurons show reduced immunoreactivity of the activity markers Fos and p-CREB, reduced dendritic spine head diameter, and reduced GluA1/2 expression in subsets of dendritic spines during early withdrawal. The opposite effect was found after one week of abstinence. At this timepoint, PrL-NAc core neurons showed heightened nuclear p-CREB, spine head diameter, and GluA1/2 expression in dendritic spines. These findings suggest that the PrL cortex, and specifically PrL-NAc core neurons, undergoes an abstinence duration-dependent transformation in glutamate transmission which may be regulated by the activation of STEP during early withdrawal

Rapid Redistribution of Synaptic PSD-95 in the Neocortex In Vivo

Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10–21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times τ (r) ~ 22–63 min from P10–P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (τ (r) ~ 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size

Membrane Trafficking in Neuronal Development: Ins and Outs of Neural Connectivity

During development, neurons progress through rapid yet stereotypical shape changes to achieve proper neuronal connectivity. This morphological progression requires carefully orchestrated plasma membrane expansion, insertion of membrane components including receptors for extracellular cues into the plasma membrane and removal and trafficking of membrane materials and proteins to specific locations. This review outlines the cellular machinery of membrane trafficking that play an integral role in neuronal cell shape change and function from initial neurite formation to pathway navigation and synaptogenesis