Systematic constellations of neurons are activated in lateral amygdala following Pavlovian fear conditioning

How memory is organized within neural networks is a fundamental question in neuroscience. We used Pavlovian fear conditioning to study the discrete organization patterns of neurons activated in an associative memory paradigm. In Pavlovian fear conditioning a neutral stimulus, such as an auditory tone, is temporally paired with an aversive unconditioned stimulus (US), such as a foot shock...

Afferent specific regulation of cortical and subcortical synaptic input to the lateral amygdala by norepinephrine

The lateral amygdala (LA) has been extensively implicated in the neurobiology of conditioned fear paradigms. Norepinepherine (NE), especially its beta receptors, has been implicated in consolidation, reconsolidation and extinction of fear memories, and has been proposed as a potential treatment for PTSD (Berlau and McGaugh, NLM, 2006; Debiec and LeDoux, N, 2005)...

A recurrent network in the lateral amygdala: A mechanism for temporal coincidence detection

The dorsal lateral amygdala (LAd) is a vital nucleus for the formation of associations between aversive unconditioned stimuli (US) and neutral stimuli, such as auditory tones, which can become conditioned (CS) to the US through temporal pairing. Important aspects of CS-US associations are believed to occur within the LAd, however relatively little is known about the temporal behavior of local LAd networks. Information about the CS and US enters the LA via a rapid and direct thalamic input and a longer latency cortical path...

GABAA and NMDA receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS), is paired with an aversive unconditioned stimulus (US). The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principal neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs...

Polysynaptic potentials within the lateral amygdala networks as indicators of reverberatory activity

Synaptic plasticity in the lateral amygdala (LA) may underlie auditory fear conditioning. Hebb postulated that sustained activity in reverberating cellular ensembles can facilitate temporal coincidence detection. Our anatomical data show that LA neurons have extensive local axon collaterals that are topographically organized and that could provide the anatomical basis for reverberatory activity...

Amygdala-dependent learning increases the number of spinophilin-immunoreactive dendritic spines in the lateral amygdala

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons in the lateral nucleus of the amygdala (LA). One proposed form of cellular plasticity involves structural changes in the number and morphology of dendritic spines...

Differences in intrinsic properties of dorsally and ventrally located lateral amygdala neurons of the rat

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons of the lateral nucleus of the amygdala (LA). While the LA has been implicated as a key brain structure for fear learning, how its network of cellular components performs these operations is not yet known...

GABAa receptors contribute to synaptic integration in lateral amygdala neurons

In classical fear conditioning a neutral conditioned stimulus (CS) such as a tone, is paired with an aversive unconditioned stimulus (US) such as a shock. The CS thereby acquires the capacity to elicit a fear response. This type of associative learning is thought to require co-activation of principle neurons in the lateral nucleus of the amygdala (LA) by two sets of synaptic inputs, a weak CS and a strong US...

Propagating excitatory potentials within the fear conditioning circuit of the lateral amygdala

In the Hebbian postulate, transiently reverberating cellular ensembles can sustain activity to facilitate temporal coincidence detection. Auditory fear conditioning is believed to be formed in the lateral amygdala (LA), by way of plasticity at auditory input synapses on principal neurons. To evaluate the contribution of LA cellular ensembles in the formation of conditioned fear memories, we investigated the LA micro-circuitry by electrophysiological and anatomical approaches. Polysynaptic field potentials evoked in the LA by stimulation of auditory thalamus(MGm/PIN) or auditory cortical (TE3) afferents were analyzed in vitro and in vivo. In vivo, two potentials were identified following stimulation of either pathway. In vitro, these multiple potentials were revealed by adding 75uM Picrotoxin or 30uM Bicuculine, with the first potential peaking at 15-20 ms, followed by two additional potentials at 20 – 25 and 30 – 35 ms, respectively. These data show single stimulation events can result in multiple synchronized excitatory events within the lateral amygdala. In order to determine underlying mechanisms of auditory signal propagation, LA principal neuron axon collateral trajectory patterns and morphology were analyzed. Neurons were found to have local axon collaterals that are topographically organized. Each axon collateral within the LA totaled 14.1 ± 2.73mm, had 29.8 ± 9.1 branch points and 1870.8 ± 1035 boutons (n=9). Electrophysiological and anatomical data show that a network of extensive axon collaterals within the LA may facilitate preservation of auditory afferent signals.

Quantification of the total neuronal structure of the fear conditioning circuit of the lateral amygdala of the rat

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons in the lateral nucleus of the amygdala (LA). In order to begin to understand how fear memories are stored and processed by synaptic changes in the LA, we have quantified both the entire neural number and the sub-cellular structure of LA principal neurons.We first used stereological cell counting methods on Gimsa or GABA immunostained rat brain. We identified 60,322+/-1408 neurons in the LA unilaterally (n=7). Of these 16,917+/-471 were GABA positive. The intercalated nuclei were excluded from the counts and thus GABA cells are believed to represent GABAergic interneurons. The sub-nuclei of the LA were also independently counted. We then quantified the morphometric properties of in vitro electrophysiologically identified principal neurons of the LA, corrected for shrinkage in xyz planes. The total dendritic length was 9.97+/-2.57mm, with 21+/-4 nodes (n=6). Dendritic spine density was 0.19+/-0.03 spines/um (n=6). Intra-LA axon collaterals had a bouton density of 0.1+/-0.02 boutons/um (n=5). These data begin to reveal the finite cellular and sub-cellular processing capacity of the lateral amygdala, and should facilitate efforts to understand mechanisms of plasticity in LA.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Quantitative genetic modeling of lateral ventricular shape and volume using multi-atlas fluid image alignment in twins

Despite substantial progress in measuring the 3D profile of anatomical variations in the human brain, their genetic and environmental causes remain enigmatic. We developed an automated system to identify and map genetic and environmental effects on brain structure in large brain MRI databases . We applied our multi-template segmentation approach ("Multi-Atlas Fluid Image Alignment") to fluidly propagate hand-labeled parameterized surface meshes into 116 scans of twins (60 identical, 56 fraternal), labeling the lateral ventricles. Mesh surfaces were averaged within subjects to minimize segmentation error. We fitted quantitative genetic models at each of 30,000 surface points to measure the proportion of shape variance attributable to (1) genetic differences among subjects, (2) environmental influences unique to each individual, and (3) shared environmental effects. Surface-based statistical maps revealed 3D heritability patterns, and their significance, with and without adjustments for global brain scale. These maps visualized detailed profiles of environmental versus genetic influences on the brain, extending genetic models to spatially detailed, automatically computed, 3D maps.

Automated 3D mapping & shape analysis of the lateral ventricles via fluid registration of multiple surface-based atlases

We developed and validated a new method to create automated 3D parametric surface models of the lateral ventricles, designed for monitoring degenerative disease effects in clinical neuroscience studies and drug trials. First we used a set of parameterized surfaces to represent the ventricles in a manually labeled set of 9 subjects' MRIs (atlases). We fluidly registered each of these atlases and mesh models to a set of MRIs from 12 Alzheimer's disease (AD) patients and 14 matched healthy elderly subjects, and we averaged the resulting meshes for each of these images. Validation experiments on expert segmentations showed that (1) the Hausdorff labeling error rapidly decreased, and (2) the power to detect disease-related alterations monotonically improved as the number of atlases, N, was increased from 1 to 9. We then combined the segmentations with a radial mapping approach to localize ventricular shape differences in patients. In surface-based statistical maps, we detected more widespread and intense anatomical deficits as we increased the number of atlases, and we formulated a statistical stopping criterion to determine the optimal value of N. Anterior horn anomalies in Alzheimer's patients were only detected with the multi-atlas segmentation, which clearly outperformed the standard single-atlas approach.

A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

Incorporation of human recombinant tropoelastin into silk fibroin membranes with the view to repairing Bruch’s membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.