Biomedical subjects

John F Guzowski

Publications and source records attributed to John F Guzowski.

13 recordsLinked to original sources

PubMed2006

Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

Alleles↗

PubMed2005

Memory-influencing intra-basolateral amygdala drug infusions modulate expression of Arc protein in the hippocampus.

Activation of beta-adrenoceptors in the basolateral complex of the amygdala (BLA) modulates memory storage processes and long-term potentiation in downstream targets of BLA efferents, including the hippocampus. Here, we show that this activation also increases hippocampal levels of activity-regulated cytoskeletal protein (Arc), an immediate-early gene (also termed Arg 3.1) implicated in hippocampal synaptic plasticity and memory consolidation processes. Infusions of the beta-adrenoreceptor agonist, clenbuterol, into the BLA immediately after training on an inhibitory avoidance task enhanced memory tested 48 h later. The same dose of clenbuterol significantly increased Arc protein levels in the dorsal hippocampus. Additionally, posttraining intra-BLA infusions of a memory-impairing dose of lidocaine significantly reduced Arc protein levels in the dorsal hippocampus. Increases in Arc protein levels were not accompanied by increases in Arc mRNA, suggesting that amygdala modulation of Arc protein and synaptic plasticity in efferent brain regions occurs at a posttranscriptional level. Finally, infusions of Arc antisense oligodeoxynucleotides into the dorsal hippocampus impaired performance of an inhibitory avoidance task, indicating that the changes in Arc protein expression are related to the observed changes in memory performance.

Adrenergic beta-Agonists↗

PubMed2005

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

BACKGROUND: 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. METHODS: A recursive tree-based algorithm that can consider multiple object fragments simultaneously is described. Starting with oversegmented data, it searches efficiently for the optimal merging pattern guided by a quantitative scoring criterion based on object modeling. Computation is bounded by limiting the depth of the merging tree. RESULTS: The proposed method was found to perform consistently better, achieving merging accuracy in the range of 92% to 100% compared with our previous algorithm, which varied in the range of 75% to 97%, even with a modest merging tree depth of 3. The overall average accuracy improved from 90% to 96%, with roughly the same computational cost for a set of representative images drawn from the CA1, CA3, and parietal cortex regions of the rat hippocampus. CONCLUSION: Hierarchical tree model-based algorithms significantly improve the accuracy of automated nuclear segmentation without sacrificing speed.

Algorithms↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

3D-catFISH: a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) of neural activity-regulated, immediate-early gene (IEG) expression provides a method of functional brain imaging with cellular resolution. This enables the identification, in one brain, of which specific principal neurons were active during each of two distinct behavioral epochs. The unprecedented potential of this differential method for large-scale analysis of functional neural circuits is limited, however, by the time-intensive nature of manual image analysis. A comprehensive software tool for processing three-dimensional, multi-spectral confocal image stacks is described which supports the automation of this analysis. Nuclei counterstained with conventional DNA dyes and FISH signals indicating the sub-cellular distribution of specific, IEG RNA species are imaged using different spectral channels. The DNA channel data are segmented into individual nuclei by a three-dimensional multi-step algorithm that corrects for depth-dependent attenuation, non-isotropic voxels, and imaging noise. Intra-nuclear and cytoplasmic FISH signals are associated spatially with the nuclear segmentation results to generate a detailed tabular/database and graphic representation. Here we present a comprehensive validation of data generated by the automated software against manual quantification by human experts on hippocampal and parietal cortical regions (96.5% concordance with multi-expert consensus). The high degree of reliability and accuracy suggests that the software will generalize well to multiple brain areas and eventually to large-scale brain analysis.

Algorithms↗

PubMed2004

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 approximately 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 (approximately 18%) than in CA1 (approximately 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.

Animals↗

PubMed2004

Amyloid suppresses induction of genes critical for memory consolidation in APP + PS1 transgenic mice.

Mice transgenic for mutated forms of the amyloid precursor protein (APP) plus presenilin-1 (PS1) genes (APP + PS1 mice) gradually develop memory deficits which correlate with the extent of amyloid deposition. The expression of several immediate-early genes (IEGs: Arc, Nur77 and Zif268) and several other plasticity-related genes (GluR1, CaMKIIalpha and Na-K- ATPase alphaIII) critical for learning and memory was normal in young APP + PS1 mice preceding amyloid deposition, but declined as mice grew older and amyloid deposits accumulated. Gene repression was less in APP + PS1 mouse brain regions that contain less Abeta and in APP mice compared with APP + PS1 mice, further linking the extent of amyloid deposition and the extent of gene repression. Critically, we demonstrated that amyloid deposition led specifically to impaired induction of the IEGs with no effects on basal expression using exposure to a novel environment 30 min prior to being killed to induce IEGs. These data imply that Abeta deposition can selectively reduce expression of multiple genes linked to synaptic plasticity, and provide a molecular basis for memory deficiencies found in transgenic APP mice and, most likely, in early stage Alzheimer's disease (AD). Presumably, pharmacological agents blocking the Abeta-related inhibition of gene expression will have benefit in AD.

Amyloid↗

PubMed2003

A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks.

BACKGROUND: 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. METHODS: A combination of two approaches is presented. First, an improved distance transform combining intensity gradients and geometric distance is used for the watershed step. Second, an explicit mathematical model for the anatomic characteristics of cell nuclei such as size and shape measures is incorporated. This model is constructed automatically from the data. Deliberate initial over-segmentation of the image data is performed, followed by statistical model-based merging. A confidence score is computed for each detected nucleus, measuring how well the nucleus fits the model. This is used in combination with the intensity gradient to control the merge decisions. RESULTS: Experimental validation on a set of rodent brain cell images showed 97% concordance with the human observer and significant improvement over prior methods. CONCLUSIONS: Combining a gradient-weighted distance transform with a richer morphometric model significantly improves the accuracy of automated segmentation and FISH analysis.

Algorithms↗

PubMed2002

Experience-dependent coincident expression of the effector immediate-early genes arc and Homer 1a in hippocampal and neocortical neuronal networks.

The transcription of the immediate-early genes Arc and Homer 1a (H1a) is dynamically regulated in response to synaptic activity; their protein products function at the postsynaptic sites of excitatory synapses. Previous studies demonstrate a role for Arc in the maintenance of long-term potentiation and in memory consolidation processes and indicate a role for H1a in modifying glutamatergic signaling pathways. Using double-label fluorescence in situ hybridization, we demonstrate that Arc and H1a RNA expression is induced strongly in the same neurons of rat hippocampus and neocortex after exploration of a novel environment. These findings support the view that novel experience activates a cell-specific genomic program and that Arc and H1a may function in concert in the structural and functional modifications of dendrites that lead to long-term changes in synaptic efficacy.

3' Untranslated Regions↗

PubMed2002

Synaptic activity-induced conversion of intronic to exonic sequence in Homer 1 immediate early gene expression.

Three Homer genes regulate the activity of metabotropic glutamate receptors mGluR1a and mGluR5 and their coupling to releasable intracellular Ca2+ pools and ion channels. Only the Homer 1 gene evolved bimodal expression of constitutive (Homer 1b and c) and immediate early gene (IEG) products (Homer 1a and Ania 3). The IEG forms compete functionally with the constitutive Homer proteins. The complex expression of the Homer 1 gene, unique for IEGs, focused our attention on the gene organization. In contrast to most IEGs, which have genes that are <5 kb, the Homer 1 gene was found to span approximately 100 kb. The constitutive Homer 1b/c forms are encoded by exons 1-10, whereas the IEG forms are encoded by exons 1-5 and parts of intron 5. RNase protection demonstrated a >10-fold activity-dependent increase in mRNA levels exclusively for the IEG forms. Moreover, fluorescent in situ hybridization documented that new primary Homer 1 transcripts are induced in neuronal nuclei within a few minutes after seizure, typical of IEGs, and that Homer 1b-specific exons are excluded from the activity-induced transcripts. Thus, at the resting state of the neurons, the entire gene is constitutively transcribed at low levels to yield Homer 1b/c transcripts. Neuronal activity sharply increases the rate of transcription initiation, with most transcripts now ending within the central intron. These coordinate transcriptional events rapidly convert a constitutive gene to an IEG and regulate the expression of functionally different Homer 1 proteins.

Alternative Splicing↗

PubMed2002

Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches.

In the 14 years since it was discovered that specific genes could be dynamically regulated in the brain by neural activity, there has been a substantial research focus attempting to understand the role immediate-early genes (IEGs) play in various brain functions. This article examines the involvement of IEGs in hippocampal synaptic plasticity and in memory consolidation processes performed by the hippocampus. Studies employing conventional IEG detection methodologies and a novel gene-imaging approach that provides temporal and cellular resolution (cellular compartment analysis of emporal activity by fluorescence in situ hybridization or catFISH) provide evidence supporting the assertion that IEG expression reflects the integration of information processed by hippocampal neurons. However, IEG expression is not merely correlated with neural activity, but also plays a pivotal role in stabilizing recent changes in synaptic efficacy. As such, localized disruption of IEGs Arc or c-fos by intrahippocampal administration of antisense oligonucleotides or germline disruption of the IEGs c-fos, tissue plasminogen activator, or zif268 impairs consolidation of long-term memory formation, without affecting learning or short-term memory. Further investigation into the expression and function of IEGs using catFISH and antisense approaches will likely increase understanding of the molecular and cellular bases of information processing involving the hippocampus.

Animals↗

PubMedDate not supplied

Mapping neuronal activation and the influence of adrenergic signaling during contextual memory retrieval.

We recently described a critical role for adrenergic signaling in the hippocampus during contextual and spatial memory retrieval. To determine which neurons are activated by contextual memory retrieval and its sequelae in the presence and absence of adrenergic signaling, transcriptional imaging for the immediate-early gene Arc was used in control and mutant mice lacking norepinephrine and epinephrine. This imaging approach permits the identification of neuronal genomic activation specific to one of two behavioral epochs in the same animal. Analysis revealed several brain regions that were more greatly activated by re-exposure to a salient versus neutral context 1 d after training in control mice (e.g., hippocampal CA3 and CA1, the amygdala, the dorsolateral caudate/putamen, the primary motor cortex, and parts of the rhinal cortices). In mice lacking norepinephrine and epinephrine, many of these regions exhibited significantly reduced activation (e.g., hippocampal CA1), while other regions did not (e.g., hippocampal CA3). In consideration with previous results, the current findings suggest a hypothesis in which adrenergic signaling may be critical for the transfer of retrieved contextual information from CA3 to CA1, where it would be compared to online sensory information coming directly from the cortex.

Animals↗