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.
Confocal microscopy; electron microscopy; image analysis; stereology; PSD-95; postsynaptic densities
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.
microinjection; dendritic spines; confocal microscopy; morphometrics; image analysis
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.
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.
Prefrontal cortex; estrogen; aging; primate; cognition; hormone replacement therapy
The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.
The surprising discovery in 1990 that estrogen modulates hippocampal structural plasticity launched a whole new field of scientific inquiry. Over the past two decades, estrogen-induced spinogenesis has been described in several brain areas involved in cognition, in a number of species, in both sexes, and on multiple time-scales. Exploration into the interaction between estrogen and aging has illuminated some of the hormone’s neuroprotective effects, most notably on age-related cognitive decline in non-human primates. While there is still much to be learned about the mechanisms by which estrogen exerts its actions, key components of the signal transduction pathways are beginning to be elucidated and non-genomic actions via membrane bound estrogen receptors are of particular interest. The goal of this line of investigation is the eventual development of new therapeutics that can prevent or reverse age-related cognitive impairment.
estrogen; aging; hippocampus; prefrontal cortex; rat; monkey; dendritic spines; cognition
Addictive drugs cause persistent restructuring of several neuronal cell types in the brain’s limbic regions thought to be responsible for long-term behavioral plasticity driving addiction. Although these structural changes are well documented in nucleus accumbens medium spiny neurons, little is known regarding the underlying molecular mechanisms. Additionally, it remains unclear whether structural plasticity and its synaptic concomitants drive addictive behaviors, or whether they reflect homeostatic compensations to the drug not related to addiction per se. Here, we discuss recent paradoxical data, which either support or oppose the hypothesis that drug-induced changes in dendritic spines drive addictive behavior. We define areas where future investigation can provide a more detailed picture of drug-induced synaptic reorganization, including ultrastructural, electrophysiological, and behavioral studies.
dendritic spines; drug addiction; relapse; mesolimbic dopamine system; long-term potentiation (LTP); long-term depression (LTD); medium spiny neuron (MSN); α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA); N-methyl D-aspartate (NMDA); ΔFosB; cyclic AMP response element binding protein (CREB); nuclear factor kappaB (NFκB); and myocyte-enhancing factor 2 (MEF-2)
Despite abundant expression of DNA methyltransferases (Dnmt’s) in brain, the regulation and behavioral role of DNA methylation remain poorly understood. We find that Dnmt3a expression is regulated in mouse nucleus accumbens (NAc) by chronic cocaine and chronic social defeat stress. Moreover, NAc specific manipulations that block DNA methylation potentiate cocaine reward and exert antidepressant-like effects, whereas NAc specific Dnmt3a overexpression attenuates cocaine reward and is pro-depressant. On a cellular level, we show that chronic cocaine selectively increases thin dendritic spines on NAc neurons and that DNA methylation is both necessary and sufficient to mediate these effects. These data establish the importance of Dnmt3a in the NAc in regulating cellular and behavioral plasticity to emotional stimuli.
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.
Synaptogenesis; protein synthesis; vesicle release; CAM kinase; PKA; AKAP