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1.  Right Brain: Past medical story 
Neurology  2012;79(12):e98-e100.
PMCID: PMC3440451  PMID: 22987749
2.  Vamping: Stereology-based automated quantification of fluorescent puncta size and density 
Journal of Neuroscience Methods  2012;209(1):97-105.
The size of dendritic spines and postsynaptic densities (PSDs) is well-known to be correlated with molecular and functional characteristics of the synapse. Thus, the development of microscopy methods that allow high throughput quantification and measurement of PSDs is a contemporary need in the field of neurobiology. While the gold standard for measurement of sub-micrometer structures remains electron microscopy (EM), this method is exceedingly laborious and therefore not always feasible. Immunohistochemistry (IHC) is a much faster technique for identifying biological structures such as PSDs, but the fluorescent images resulting from it have traditionally been harder to interpret and quantify. Here, we report on two new image analysis tools that result in accurate size and density measurements of fluorescent puncta. Anti-PSD-95 staining was used to target synapses. The new technique of vamping, using Volume Assisted Measurement of Puncta in 2 and 3 Dimensions (VAMP2D and VAMP3D) respectively, is based on stereological principles. The fully automated image analysis tool was tested on the same subjects for whom we had previously obtained EM measurements of PSD size and/or density. Based on highly consistent results between data obtained by each of these methods, vamping offers an expedient alternative to EM that can nonetheless deliver a high level of accuracy in measuring sub-cellular structures.
PMCID: PMC3402578  PMID: 22683698
Confocal microscopy; electron microscopy; image analysis; stereology; PSD-95; postsynaptic densities
3.  High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy 
Nature protocols  2011;6(9):1391-1411.
Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought.
PMCID: PMC3566769  PMID: 21886104
microinjection; dendritic spines; confocal microscopy; morphometrics; image analysis
4.  Subregional, dendritic compartment, and spine subtype specificity in cocaine-regulation of dendritic spines in the nucleus accumbens 
The Journal of Neuroscience  2012;32(20):6957-6966.
Numerous studies have found that chronic cocaine increases dendritic spine density of medium spiny neurons in the nucleus accumbens (NAc). Here, we employed single cell microinjections and advanced 3D imaging and analysis techniques to extend these findings in several important ways: by assessing cocaine regulation of dendritic spines in the core versus shell subregions of NAc in the mouse, over a broad time course (4 hours, 24 hours, or 28 days) of withdrawal from chronic cocaine, and with a particular focus on proximal versus distal dendrites. Our data demonstrate subregion-specific, and in some cases opposite, regulation of spines by cocaine on proximal but not distal dendrites. Notably, all observed density changes were attributable to selective regulation of thin spines. At 4 hours post-injection, the proximal spine density is unchanged in the core but significantly increased in the shell. At 24 hours, the density of proximal dendritic spines is reduced in the core but increased in the shell. Such down-regulation of thin spines in the core persists through 28 days of withdrawal, while the spine density in the shell returns to baseline levels. Consistent with previous results, dendritic tips exhibited up-regulation of dendritic spines after 24 hours of withdrawal, an effect localized to the shell. The divergence in regulation of proximal spine density in NAc core versus shell by cocaine correlates with recently reported electrophysiological data from a similar drug administration regimen and might represent a key mediator of changes in the reward circuit that drive aspects of addiction.
PMCID: PMC3360066  PMID: 22593064
5.  Interactive Effects of Age and Estrogen on Cortical Neurons: Implications for Cognitive Aging 
Neuroscience  2011;191:148-158.
In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: 1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and 2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: 1) synaptic ER-α is present in axospinous synapses in area 46; 2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and 3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women.
PMCID: PMC3166405  PMID: 21664255
Prefrontal cortex; estrogen; aging; primate; cognition; hormone replacement therapy
6.  Requirement for Protein Synthesis at Developing Synapses 
Activity and protein synthesis act cooperatively to generate persistent changes in synaptic responses. This forms the basis for enduring memory in adults. Activity also shapes neural circuits developmentally, but whether protein synthesis plays a congruent function in this process is poorly understood. Here, we show that brief periods of global or local protein synthesis inhibition decrease the synaptic vesicles available for fusion and increase synapse elimination. CaMKII is a critical target; its levels are controlled by rapid turnover, and blocking its activity or knocking it down recapitulates the effects of protein synthesis inhibition. Mature presynaptic terminals show decreased sensitivity to protein synthesis inhibition, and resistance coincides with a developmental switch in regulation from CaMKII to PKA. These findings demonstrate a novel mechanism regulating presynaptic activity and synapse elimination during development, and suggest that protein translation acts coordinately with activity to selectively stabilize appropriate synaptic interactions.
PMCID: PMC2771567  PMID: 19657031
Synaptogenesis; protein synthesis; vesicle release; CAM kinase; PKA; AKAP

Results 1-6 (6)