John F. Guzowski

Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles

We used fluorescent in-situ hybridization and confocal microscopy to monitor the subcellular distribution of the immediate-early gene Arc. Arc RNA appeared in discrete intranuclear foci within minutes of neuronal activation and subsequently disappeared from the nucleus and accumulated in the cytoplasm by 30 minutes. The time course of nuclear versus cytoplasmic Arc RNA accumulation was distinct, and could therefore be used to infer the activity history of individual neurons at two times. Following sequential exposure of rats to two different environments or to the same environment twice, the proportion of CA1 neurons with cytoplasmic, nuclear or overlapping Arc expression profiles matched predictions derived from ensemble neurophysiological recordings of hippocampal neuronal ensembles. Arc gene induction is thus specifically linked to neural encoding processes.

The Immediate Early Gene Arc/Arg3.1: Regulation, Mechanisms, and Function

In a manner unique among activity-regulated immediate early genes (IEGs), mRNA encoded by Arc (also known as Arg3.1) undergoes rapid transport to dendrites and local synaptic translation. Despite this intrinsic appeal, relatively little is known about the neuronal and behavioral functions of Arc or its molecular mechanisms of action. Here, we attempt to distill recent advances on Arc spanning its transcriptional and translational regulation, the functions of the Arc protein in multiple forms of neuronal plasticity [long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity], and its broader role in neural networks of behaving animals. Worley and colleagues have shown that Arc interacts with endophilin and dynamin, creating a postsynaptic trafficking endosome that selectively modifies the expression of AMPA-type glutamate receptors at the excitatory synapses. Both LTD and homeostatic plasticity in the hippocampus are critically dependent on Arc-mediated endocytosis of AMPA receptors. LTD evoked by activation of metabotropic glutamate receptors depends on rapid Arc translation controlled by elongation factor 2. Bramham and colleagues have shown that sustained translation of newly induced Arc mRNA is necessary for cofilin phosphorylation and stable expansion of the F-actin cytoskeleton underlying LTP consolidation in the dentate gyrus of live rats. In addition to regulating F-actin, Arc synthesis maintains the activity of key translation factors during LTP consolidation. This process of Arc-dependent consolidation is activated by the secretory neurotrophin, BDNF. Moore and colleagues have shown that Arc mRNA is a natural target for nonsense-mediated mRNA decay (NMD) by virtue of its two conserved 3′-UTR introns. NMD and other related translation-dependent mRNA decay mechanisms may serve as critical brakes on protein expression that contribute to the fine spatial-temporal control of Arc synthesis. In studies in behaving rats, Guzowski and colleagues have shown that location-specific firing of CA3 and CA1 hippocampal neurons in the presence of theta rhythm provides the necessary stimuli for activation of Arc transcription. The impact of Arc transcription in memory processes may depend on the specific context of coexpressed IEGs, in addition to posttranscriptional regulation of Arc by neuromodulatory inputs from the amygdala and other brain regions. In sum, Arc is emerging as a versatile, finely tuned system capable of coupling changes in neuronal activity patterns to diverse forms of synaptic plasticity, thereby optimizing information storage in active networks.

Differences in Hippocampal Neuronal Population Responses to Modifications of an Environmental Context: Evidence for Distinct, Yet Complementary, Functions of CA3 and CA1 Ensembles

Understanding how the hippocampus processes information critical for establishing spatial and declarative memories will benefit greatly from determining not only what kind of information the hippocampus registers, but also how this information is processed across the different hippocampal subfields. We addressed this question using a novel immediate-early gene-based brain-imaging method (Arc/H1a catFISH) that allows comparisons of neuronal ensembles activated by two experiences separated by ∼30 min. Rats exposed to the same environment twice activated CA3 and CA1 ensembles with a similarly high degree of overlap. Changing the identity or configuration of local cues, or changing distal cues, activated CA3 and CA1 ensembles with reduced overlap. Yet, the overlap was greater in CA3 than in CA1. In contrast, rats exposed to two completely different environments activated CA3 and CA1 ensembles with low overlap, and this overlap was even lower in CA3 compared with CA1. Thus, CA3 has a discontinuous, whereas CA1 has a graded, population response to alterations of an environment. Additionally, as indicated by the percentage of active neurons, the context representation was more sparse in CA3 (∼18%) than in CA1 (∼35%). Finally, CA3 and CA1 activity levels were not correlated within a session, arguing against a simple coactivation of these regions. Instead, the within-rat ratio of CA3/CA1 cell activity was correlated across sessions, suggesting that the balance of CA3/CA1 activity is individual specific. Taken together, these findings suggest that CA3 and CA1 neuronal ensembles perform distinct, yet complementary, functions in the processing of spatial and contextual information.

A Hybrid 3D Watershed Algorithm Incorporating Gradient Cues and Object Models for Automatic Segmentation of Nuclei in Confocal Image Stacks

Automated segmentation of fluorescently‐labeled cell nuclei in 3D confocal microscope images is essential to many studies involving morphological and functional analysis. A common source of segmentation error is tight clustering of nuclei. There is a compelling need to minimize these errors for constructing highly automated scoring systems.

Mapping behaviorally relevant neural circuits with immediate-early gene expression

Immediate-early genes have gained widespread popularity as activity markers for mapping neuronal circuits involved in specific behaviors in many different species. In situ immediate early gene detection methods provide cellular level resolution, a major benefit for mapping neuronal networks. Recent advances using fluorescence in situ hybridization also afford temporal resolution, enabling within-animal activity maps for two distinct behaviors. Moreover, use of transgenic mice with fluorescent reporter proteins driven by immediate early gene promoters is enabling repeated measurements, over long time scales, of cortical activity within the same animal. These methodological innovations, coupled with recent advances in fluorescence imaging and probe development, will enable large scale mapping of behaviorally relevant circuits with temporal and three-dimensional spatial resolution in experimental animals.

Ensemble Dynamics of Hippocampal Regions CA3 and CA1

Computational models based on hippocampal connectivity have proposed that CA3 is uniquely positioned as an autoassociative memory network, capable of performing the competing functions of pattern completion and pattern separation. Recently, three independent studies, two using parallel neurophysiological recording methods and one using immediate-early gene imaging, have examined the responses of CA3 and CA1 ensembles to alterations of environmental context in rats. The results provide converging evidence that CA3 is capable of performing nonlinear transformations of sensory input patterns, whereas CA1 may represent changes in input in a more linear fashion.

Spatial exploration induces ARC, a plasticity-related immediate-early gene, only in calcium/calmodulin-dependent protein kinase II-positive principal excitatory and inhibitory neurons of the rat forebrain.

Active behavior, such as exploring a novel environment, induces the expression of the immediate‐early gene Arc (activity‐regulated cytoskeletal associated protein, or Arg 3.1) in many brain regions, including the hippocampus, neocortex, and striatum. Arc messenger ribonucleic acid and protein are localized in activated dendrites, and Arc protein is required for the maintenance of long‐term potentiation and memory consolidation. Although previous evidence suggests that Arc is expressed in neurons, there is no direct demonstration that only neurons can express Arc. Furthermore, there is no characterization of the main neuronal types that express Arc. The data reported here show that behavior‐ or seizure‐induced Arc expression in the hippocampus, primary somatosensory cortex, and dorsal striatum of rats colocalizes only with neuronal (NeuN‐positive) and not with glial (GFAP‐positive) cells. Furthermore, Arc was found exclusively in non‐GABAergic α‐CaMKII‐positive hippocampal and neocortical neurons of rats that had explored a novel environment. Some GAD65/67‐positive neurons in these regions were observed to express Arc, but only after a very strong stimulus (electroconvulsive seizure). In the dorsal striatum, spatial exploration induced Arc only in GABAergic and α‐CaMKII‐positive neurons. Combined, these results show that although a very strong stimulus (seizure) can induce Arc in a variety of neurons, behavior induces Arc in the CaMKII‐positive principal neurons of the hippocampus, neocortex, and dorsal striatum. These results, coupled with recent in vitro findings of interactions between Arc and CaMKII, are consistent with the hypothesis that Arc and CaMKII act as plasticity partners to promote functional and/or structural synaptic modifications that accompany learning. J. Comp. Neurol. 498:317–329, 2006.

Hierarchical, model-based merging of multiple fragments for improved three-dimensional segmentation of nuclei

Automated segmentation of fluorescently labeled cell nuclei in three‐dimensional confocal images is essential for numerous studies, e.g., spatiotemporal fluorescence in situ hybridization quantification of immediate early gene transcription. High accuracy and automation levels are required in high‐throughput and large‐scale studies. Common sources of segmentation error include tight clustering and fragmentation of nuclei. Previous region‐based methods are limited because they perform merging of two nuclear fragments at a time. To achieve higher accuracy without sacrificing scale, more sophisticated yet computationally efficient algorithms are needed.

Rapid activation of plasticity-associated gene transcription in hippocampal neurons provides a mechanism for encoding of one-trial experience

The hippocampus is hypothesized to support rapid encoding of ongoing experience. A critical prerequisite for such function is the ability to readily recruit enduring synaptic plasticity in hippocampal neurons. Hippocampal long-term potentiation (LTP) and memory consolidation require expression of the immediate-early gene (IEG) Arc. To determine whether Arc transcription could be driven by limited and controlled behavioral experience, we used a rectangular track paradigm. In past electrophysiological studies, pyramidal neurons recorded from rats running in one direction on similar tracks typically exhibited a single firing field. Using fluorescence in situ hybridization, we show that the behavioral activity associated with a single lap around the track was sufficient to trigger Arc transcription in complete CA3 neuronal ensembles, as predicted given the role of CA3 in one-trial learning. In contrast, Arc transcription in CA1 ensembles was recruited incrementally, with maximal activation achieved after four laps a day for 4 consecutive days. To test whether Arc transcription is linked to learning and plasticity, or merely elicited by location-specific firing, we inactivated the medial septum, a treatment that compromises hippocampus-dependent learning and LTP but spares location-specific firing in CA1 neurons. Septal inactivation abolished track training-induced Arc transcription in CA1 and CA3 neurons, showing that Arc transcription requires plasticity-inducing stimuli. Accordingly, LTP induction activated Arc transcription in CA1 neurons in vivo. These findings demonstrate for the first time that a single brief experience, equivalent to a single crossing of a firing field, can trigger IEG expression required for long-term plasticity in the hippocampus.

A multi‐model approach to simultaneous segmentation and classification of heterogeneous populations of cell nuclei in 3D confocal microscope images

Automated segmentation and morphometry of fluorescently labeled cell nuclei in batches of 3D confocal stacks is essential for quantitative studies. Model‐based segmentation algorithms are attractive due to their robustness. Previous methods incorporated a single nuclear model. This is a limitation for tissues containing multiple cell types with different nuclear features. Improved segmentation for such tissues requires algorithms that permit multiple models to be used simultaneously. This requires a tight integration of classification and segmentation algorithms. Two or more nuclear models are constructed semiautomatically from user‐provided training examples. Starting with an initial over‐segmentation produced by a gradient‐weighted watershed algorithm, a hierarchical fragment merging tree rooted at each object is built. Linear discriminant analysis is used to classify each candidate using multiple object models. On the basis of the selected class, a Bayesian score is computed. Fragment merging decisions are made by comparing the score with that of other candidates, and the scores of constituent fragments of each candidate. The overall segmentation accuracy was 93.7% and classification accuracy was 93.5%, respectively, on a diverse collection of images drawn from five different regions of the rat brain. The multi‐model method was found to achieve high accuracy on nuclear segmentation and classification by correctly resolving ambiguities in clustered regions containing heterogeneous cell populations.