Monica K. Chawla

Hippocampal granule cells opt for early retirement.

Increased excitability and plasticity of adult‐generated hippocampal granule cells during a critical period suggests that they may “orthogonalize” memories according to time. One version of this “temporal tag” hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90–95%, are effectively “retired.” If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard “orthogonalizer” theory of the role of the dentate gyrus in memory.

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.

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.

Neuronal ensembles in amygdala, hippocampus, and prefrontal cortex track differential components of contextual fear.

Although the circuit mediating contextual fear conditioning has been extensively described, the precise contribution that specific anatomical nodes make to behavior has not been fully elucidated. To clarify the roles of the dorsal hippocampus (DH), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) in contextual fear conditioning, activity within these regions was mapped using cellular compartment analysis of temporal activity using fluorescence in situ hybridization (catFISH) for Arc mRNA. Rats were delay-fear conditioned or immediately shocked (controls) and thereafter reexposed to the shocked context to test for fear memory recall. Subsequent catFISH analyses revealed that in the DH, cells were preferentially reactivated during the context test, regardless of whether animals had been fear conditioned or immediately shocked, suggesting that the DH encodes spatial information specifically, rather then the emotional valence of an environment. In direct contrast, neuronal ensembles in the BLA were only reactivated at test if animals had been fear conditioned, suggesting that the amygdala specifically tracks the emotional properties of a context. Interestingly, Arc expression in the mPFC was consistent with both amygdala- and hippocampus-like patterns, supporting a role for the mPFC in both fear and contextual processing. Collectively, these data provide crucial insight into the region-specific behavior of neuronal ensembles during contextual fear conditioning and demonstrate a dissociable role for the hippocampus and amygdala in processing the contextual and emotional properties of a fear memory.

Differential Encoding of Behavior and Spatial Context in Deep and Superficial Layers of the Neocortex

Rodent hippocampal activity is correlated with spatial and behavioral context, but how context affects coding in association neocortex is not well understood. The cellular distribution of the neural activity-regulated immediate-early gene Arc was used to monitor the activity history of cells in CA1, and in deep and superficial layers of posterior parietal and gustatory cortices (which encode movement and taste, respectively), during two behavioral epochs in which spatial and behavioral context were independently manipulated while gustatory input was held constant. Under conditions in which the hippocampus strongly differentiated behavioral and spatial contexts, deep parietal and gustatory layers did not discriminate between spatial contexts, whereas superficial layers in both neocortical regions discriminated well. Deep parietal cells discriminated behavioral context, whereas deep gustatory cortex neurons encoded the two conditions identically. Increased context sensitivity of superficial neocortical layers, which receive more hippocampal outflow, may reflect a general principle of neocortical organization for memory retrieval.

Localization of neurons expressing substance P and neurokinin B gene transcripts in the human hypothalamus and basal forebrain

In situ hybridization histochemistry was used to map the distribution of neurons expressing the substance P (SP) or neurokinin B (NKB) genes in the human hypothalamus and basal forebrain. Hypothalami from five adult males were frozen in isopentane at −30°C and serially sectioned at 20 μm thickness. Every 20th section was hybridized with [35S]‐labeled, 48‐base synthetic cDNA probes that were complementary to either SP or NKB mRNAs. Slides were dipped into nuclear emulsion for visualization of mRNAs at the single‐cell level. The location of labeled neurons (greater than ×5 background) was mapped by using an image‐combining computer microscope system. A distinct and complementary distribution pattern of SP and NKB neurons was observed in the human hypothalamus and basal forebrain. NKB was the predominant tachykinin in the rostral hypothalamus, whereas SP mRNA predominated in the posterior hypothalamus. Numerous NKB neurons were identified in the magnocellular basal forebrain, the bed nucleus of stria terminalis, and the anterior hypothalamic area. Scattered NKB neurons were present in the infundibular and paraventricular nuclei, paraolfactory gyrus, posterior hypothalamic area, lateral division of the medial mammillary nucleus, and amygdala. Numerous neurons expressing SP mRNAs were identified in the premammillary, supramammillary, and medial mammillary nuclei; the posterior hypothalamic area; and the corpus striatum. Scattered SP neurons were also observed in the preoptic area; the infundibular, intermediate, dorsomedial, and ventromedial nuclei; the infundibular stalk; the amygdala; the bed nucleus of stria terminalis; and the paraolfactory gyrus. These studies provide the first description of the location of neurons that express tachykinin gene transcripts in the human hypothalamus. J. Comp. Neurol. 384:429–442, 1997.

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.

Advanced imaging of multiple mRNAs in brain tissue using a custom hyperspectral imager and multivariate curve resolution.

Simultaneous imaging of multiple cellular components is of tremendous importance in the study of complex biological systems, but the inability to use probes with similar emission spectra and the time consuming nature of collecting images on a confocal microscope are prohibitive. Hyperspectral imaging technology, originally developed for remote sensing applications, has been adapted to measure multiple genes in complex biological tissues. A spectral imaging microscope was used to acquire overlapping fluorescence emissions from specific mRNAs in brain tissue by scanning the samples using a single fluorescence excitation wavelength. The underlying component spectra obtained from the samples are then separated into their respective spectral signatures using multivariate analyses, enabling the simultaneous quantitative measurement of multiple genes either at regional or cellular levels.

Hippocampal granule cells in normal aging: insights from electrophysiological and functional imaging experiments

Normal aging, in the absence of neurodegenerative disease, can provide important insights into the mechanisms by which the brain can maintain cognitive abilities across the lifespan. Hippocampal-dependent memory processes can become vulnerable as age advances. The focus of this chapter is the contribution of hippocampal granule cells to cognitive impairments that are observed during aging. A number of alterations in structure, function, and gene expression have been observed in aged granule cells, any of which may lead to adaptive, compensatory or detrimental consequences to hippocampal function. As the average life span of humans continues to increase, those who reach 100 years or beyond is more common. Individuals that have aged successfully, and exhibit high levels of cognitive ability can provide useful clues into the enormous potential possessed by the mammalian brain.

STUDIES IN PHLEBITIS : DETECTION AND QUANTITATION USING A THERMOGRAPHIC CAMERA

A new method for the detection of acute phlebitis in superficial veins is investigated. A thermographic camera is utilized for the quantitation of temperature changes in a rabbit ear model. A control group receiving no injection is compared against each of five treatment groups receiving these commercially available parenterals: amiodarone hydrochloride, phenytoin sodium, mechlorethamine hydrochloride, cephalothin sodium, and diazepam. The vehicles of the above-mentioned drugs as well as several commonly used organic cosolvents are also investigated. Local tissue responses to the parenteral challenges are measured and a good correlation between the visual and the thermographic data was seen.