A model-based hierarchical Bayesian approach to Sholl analysis.

MOTIVATION: Due to the link between microglial morphology and function, morphological changes in microglia are frequently used to identify pathological immune responses in the central nervous system. In the absence of pathology, microglia are responsible for maintaining homeostasis, and their morphology can be indicative of how the healthy brain behaves in the presence of external stimuli and genetic differences. Despite recent interest in high throughput methods for morphological analysis, Sholl analysis is still widely used for quantifying microglia morphology via imaging data. Often, the raw data are naturally hierarchical, minimally including many cells per image and many images per animal. However, existing methods for performing downstream inference on Sholl data rely on truncating this hierarchy so rudimentary statistical testing procedures can be used. RESULTS: To fill this longstanding gap, we introduce a parametric hierarchical Bayesian model-based approach for analyzing Sholl data, so that inference can be performed without aggressive reduction of otherwise very rich data. We apply our model to real data and perform simulation studies comparing the proposed method with a popular alternative. AVAILABILITY AND IMPLEMENTATION: Software to reproduce the results presented in this article is available at: https://github.com/vonkaenelerik/hierarchical_sholl. An R package implementing the proposed models is available at: https://github.com/vonkaenelerik/ShollBayes.

A Model-Based Hierarchical Bayesian Approach to Sholl Analysis.

Due to the link between microglial morphology and function, morphological changes in microglia are frequently used to identify pathological immune responses in the central nervous system. In the absence of pathology, microglia are responsible for maintaining homeostasis, and their morphology can be indicative of how the healthy brain behaves in the presence of external stimuli and genetic differences. Despite recent interest in high throughput methods for morphological analysis, Sholl analysis is still the gold standard for quantifying microglia morphology via imaging data. Often, the raw data are naturally hierarchical, minimally including many cells per image and many images per animal. However, existing methods for performing downstream inference on Sholl data rely on truncating this hierarchy so rudimentary statistical testing procedures can be used. To fill this longstanding gap, we introduce a fully parametric model-based approach for analyzing Sholl data. We generalize our model to a hierarchical Bayesian framework so that inference can be performed without aggressive reduction of otherwise very rich data. We apply our model to three real data examples and perform simulation studies comparing the proposed method with a popular alternative.

Loss of P2Y12 Has Behavioral Effects in the Adult Mouse.

While microglia have been established as critical mediators of synaptic plasticity, the molecular signals underlying this process are still being uncovered. Increasing evidence suggests that microglia utilize these signals in a temporally and regionally heterogeneous manner. Subsequently, it is necessary to understand the conditions under which different molecular signals are employed by microglia to mediate the physiological process of synaptic remodeling in development and adulthood. While the microglial purinergic receptor P2Y12 is required for ocular dominance plasticity, an adolescent form of experience-dependent plasticity, it remains unknown whether P2Y12 functions in other forms of plasticity at different developmental time points or in different brain regions. Using a combination of ex vivo characterization and behavioral testing, we examined how the loss of P2Y12 affects developmental processes and behavioral performance in adulthood in mice. We found P2Y12 was not required for an early form of plasticity in the developing visual thalamus and did not affect microglial migration into barrels in the developing somatosensory cortex. In adult mice, however, the loss of P2Y12 resulted in alterations in recognition and social memory, as well as anxiety-like behaviors, suggesting that while P2Y12 is not a universal regulator of synaptic plasticity, the loss of P2Y12 is sufficient to cause functional defects.

Microglia and astrocytes show limited, acute alterations in morphology and protein expression following a single developmental alcohol exposure.

Fetal alcohol spectrum disorders (FASD) are the most common cause of nonheritable, preventable mental disability and are characterized by cognitive, behavioral, and physical impairments. FASD occurs in almost 5% of births in the United States, but despite this prevalence there is no known cure, largely because the biological mechanisms that translate alcohol exposure to neuropathology are not well understood. While the effects of early ethanol exposure on neuronal survival and circuitry have received more attention, glia, the cells most closely tied to initiating and propagating inflammatory events, could be an important target for alcohol in the developing brain. Inflammation is known to alter developmental trajectories, but it has recently been shown that even small changes in both astrocytes and microglia in the absence of full-blown inflammatory signaling can alter brain function long-term. Here, we studied the acute response of astrocytes and microglia to a single exposure to ethanol in development across sexes in a mouse model of human third trimester exposure, in order to understand how these cells may transition from their normal developmental path to a different program that leads to FASD neuropathology. We found that although a single ethanol exposure delivered subcutaneously on postnatal day 4 did not cause large changes in microglial morphology or the expression of AldH1L1 and GFAP in the cortex and hippocampus, subtle effects were observed. These findings suggest that even a single, early ethanol exposure can induce mild acute alterations in glia that could contribute to developmental deficits.

The microglial fractalkine receptor is not required for activity-dependent plasticity in the mouse visual system.

Microglia have recently been implicated as key regulators of activity-dependent plasticity, where they contribute to the removal of inappropriate or excess synapses. However, the molecular mechanisms that mediate this microglial function are still not well understood. Although multiple studies have implicated fractalkine signaling as a mediator of microglia-neuron communications during synaptic plasticity, it is unclear whether this is a universal signaling mechanism or whether its role is limited to specific brain regions and stages of the lifespan. Here, we examined whether fractalkine signaling mediates microglial contributions to activity-dependent plasticity in the developing and adolescent visual system. Using genetic ablation of fractalkine's cognate receptor, CX3 CR1, and both ex vivo characterization and in vivo imaging in mice, we examined whether fractalkine signaling is required for microglial dynamics and modulation of synapses, as well as activity-dependent plasticity in the visual system. We did not find a role for fractalkine signaling in mediating microglial properties during visual plasticity. Ablation of CX3 CR1 had no effect on microglial density, distribution, morphology, or motility, in either adolescent or young adult mice across brain regions that include the visual cortex. Ablation of CX3 CR1 also had no effect on baseline synaptic turnover or contact dynamics between microglia and neurons. Finally, we found that fractalkine signaling is not required for either early or late forms of activity-dependent visual system plasticity. These findings suggest that fractalkine is not a universal regulator of synaptic plasticity, but rather has heterogeneous roles in specific brain regions and life stages.

Microglial interactions with synapses are modulated by visual experience.

Microglia are the immune cells of the brain. In the absence of pathological insult, their highly motile processes continually survey the brain parenchyma and transiently contact synaptic elements. Aside from monitoring, their physiological roles at synapses are not known. To gain insight into possible roles of microglia in the modification of synaptic structures, we used immunocytochemical electron microscopy, serial section electron microscopy with three-dimensional reconstructions, and two-photon in vivo imaging to characterize microglial interactions with synapses during normal and altered sensory experience, in the visual cortex of juvenile mice. During normal visual experience, most microglial processes displayed direct apposition with multiple synapse-associated elements, including synaptic clefts. Microglial processes were also distinctively surrounded by pockets of extracellular space. In terms of dynamics, microglial processes localized to the vicinity of small and transiently growing dendritic spines, which were typically lost over 2 d. When experience was manipulated through light deprivation and reexposure, microglial processes changed their morphology, showed altered distributions of extracellular space, displayed phagocytic structures, apposed synaptic clefts more frequently, and enveloped synapse-associated elements more extensively. While light deprivation induced microglia to become less motile and changed their preference of localization to the vicinity of a subset of larger dendritic spines that persistently shrank, light reexposure reversed these behaviors. Taken together, these findings reveal different modalities of microglial interactions with synapses that are subtly altered by sensory experience. These findings suggest that microglia may actively contribute to the experience-dependent modification or elimination of a specific subset of synapses in the healthy brain.

Intracranial injection of adeno-associated viral vectors.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Rapid, long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.

Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.

Long-term use of clonidine in a critically-ill infant.

We report the use of clonidine in an infant as an adjunct to sedation and analgesia for 4.5 months in the critical care setting. Advantages, potential side effects, and dosing for multiple modes of delivery are discussed.