Distinct forms of Gq-receptor-dependent plasticity of excitatory transmission in the BNST are differentially affected by stress

Long-term depression (LTD) is an important synaptic mechanism for limiting excitatory influence over circuits subserving cognitive and emotional behavior. A major means of LTD induction is through the recruitment of signaling via G_q-linked receptors activated by norepinephrine (NE), acetylcholine, and glutamate. Receptors from these transmitter families have been proposed to converge on a common postsynaptic LTD maintenance mechanism, such that hetero- and homosynaptic induction produce similar alterations in glutamate synapse efficacy. We report that in the dorsolateral and ventrolateral bed nucleus of the stria terminalis (BNST), recruitment of G_q-linked receptors by glutamate or NE initiates mechanistically distinct forms of postsynaptically maintained LTD and these LTDs are differentially regulated by stress exposure. In particular, we show that although both mGluR5- and α₁-adrenergic receptor (AR)-dependent LTDs involve postsynaptic endocytosis, the α₁-AR-initiated LTD exclusively involves modulation of signaling through calcium-permeable AMPA receptors. Further, α₁-AR- but not mGluR5- dependent LTD is disrupted by restraint stress. α₁-AR LTD is also impaired in mice chronically exposed to ethanol. These data thus suggest that in the BNST, NE- and glutamate-activated G_q-linked signaling pathways differentially tune glutamate synapse efficacy in response to stress.     

Feed-forward inhibition as a buffer of the neuronal input-output relation

Neuronal processing depends on the input-output (I/O) relation between the frequency of synaptic stimulation and the resultant axonal firing rate. The all-or-none properties of spike generation and active membrane mechanisms can make the neuronal I/O relation very steep. The ensuing nearly bimodal behavior may severely limit information coding, as minimal input fluctuations within the expected natural variability could cause neuronal output to jump between quiescence and maximum firing rate. Here, using biophysically and anatomically realistic computational models of individual neurons, we demonstrate that feed-forward inhibition, a ubiquitous mechanism in which inhibitory interneurons and their target cells are activated by the same excitatory input, can change a steeply sigmoid I/O curve into a double-sigmoid typical of buffer systems. The addition of an intermediate plateau stabilizes the spiking response over a broad dynamic range of input frequency, ensuring robust integration of noisy synaptic signals. Both the buffered firing rate and its input firing range can be independently and extensively modulated by biologically plausible changes in the weight and number of excitatory synapses on the feed-forward interneuron. By providing a soft switch between essentially digital and analog rate-code, this continuous control of the circuit I/O could dramatically increase the computational power of neuronal integration.     

The relationship between birth timing,circuit wiring,and physiological response properties of cerebellar granule cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca²⁺ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Cerebellar granule cells (GrCs) comprise the majority of neurons in the mammalian brain (1, 2). Each GrC receives only four excitatory inputs from mossy fibers, which originate in a variety of brainstem nuclei and the spinal cord, and the vast number of GrCs permits diverse combinations of mossy fiber inputs. Classical theories of cerebellar function have therefore proposed that GrCs integrate diverse, multimodal mossy fiber inputs and thus collectively expand coding space in the cerebellum (3–5). Until recently, studies have focused on the role of GrCs in implementing sparse coding of sensorimotor variables and stimuli (6–9). However, recent physiological studies of GrCs in awake, behaving animals highlight GrC encoding of cognitive signals in addition to sensorimotor signals (10–13). GrCs have also been recently shown to encode denser representations than expected by classical theory (10–12, 14–18), including a lack of dimensionality expansion under certain conditions (18).Despite the vast number of GrCs, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region or lobule have not been described (19–22), although variation in gene expression across different regions has been reported (22, 23). The only known axis along which spatially intermingled GrCs can be distinguished from each other is the depth of the molecular layer to which their parallel fiber axons (PFs) project, which is dictated by GrC lineage and birth timing (24, 25). Birth timing predicts the wiring and functional properties of diverse neuron types in many neural systems (26), including the neocortex (27, 28), other forebrain regions (29, 30), olfactory bulb (31–33), and ventral spinal cord (34, 35). Furthermore, classic studies utilizing γ-irradiation at different times during rat postnatal development to ablate different cerebellar GrC and interneuron populations suggested that GrCs born at different times could contribute differentially to motor vs. action coordination (36). These observations also led to an as-of-yet untested hypothesis that mossy fibers arriving at different times during development could connect with different GrC populations. Could GrC birth timing be an organizing principle for information processing in the cerebellum?Recent evidence and modeling point to the possibility of spatial clusters of coactivated PFs (15, 37), suggesting that GrCs born around the same time may disproportionally receive coactive mossy fiber inputs. However, another study using different methods and stimuli did not find differences in the physiological responses of early- and late-born GrCs to various sensorimotor stimuli (38). Here, we address the role of birth timing in GrC wiring and function. We developed strategies to gain genetic access to early- and late-born GrCs, as well as control GrCs not restricted by birth timing. We report the first monosynaptic input tracing to GrCs, finding differential mossy fiber inputs to GrCs in vermis lobule 6 and simplex, as well as different patterns of input to early- and late-born GrCs in vermis lobule 6. Finally, we performed in vivo multidepth two-photon Ca²⁺ imaging of PFs of early- and late-born GrCs during an operant task and presentation of a panel of sensory, appetitive, and aversive stimuli. We found modest differences in the proportions of early- and late-born GrCs encoding of a subset of movement and reward parameters. Together, these results reveal a contribution of GrC birth timing to their input wiring and diverse encoding properties.     

The relationship between frequency of ventilator circuit changes and infectious hazard.

The relationship between frequency of ventilator circuit changes and risk of ventilator-associated pneumonia was studied using 2 independent approaches. The first was an in-use aerosol contamination study with patients on 8-hour, 16-hour, or 24-hour ventilator changing schedules. The second approach was a study comparing the incidence of pneumonia in patients on ventilators for 2 one-year periods when the ventilator circuit changing time differed. In-use aerosol sampling of 513 ventilator treatment periods showed bacterial contamination greater than 100 organisms per aerosol in 1.8 per cent of 8-hour cycles, in 2.5 per cent of 16-hour cycles, and in 5.4 per cent of 24-hour cycles. These differences were not significant. The incidence of ventilator-associated pneumonia was the same for one-year periods when ventilator circuits were changed either every 8 or every 24 hours. It was concluded that changing ventilator circuits every 24 hours provides adequate protection from ventilator-associated pneumonia.     

Vasculin,a novel vascular protein differentially expressed in human atherogenesis

Recent suppressive subtractive hybridization analysis on human atherosclerotic plaque-derived RNA revealed genes upregulated in plaques with a thrombus versus stable plaques. Clone SSH6, containing part of a putative open reading frame of an unknown protein, was further investigated. Full-length cDNA, coding for a 473-amino acid (aa) protein, was identified in a vascular smooth muscle cell (SMC) cDNA library. Bioinformatics suggested the presence of multiple SSH6 variants due to alternative splicing of exon 3. Multiple-tissue Northern blot analysis demonstrated a differential expression pattern of these variants, as a ubiquitously expressed SSH6 mRNA missing exon 3, was detected apart from a putative vascular SMC-specific form containing exon 3. Western blot analysis indicated a ubiquitous 35-kDa protein (SSH6-beta), in addition to a 45-kDa protein (vasculin), detected in the vascular wall and in plasma. Analysis of arteries displaying various stages of atherosclerosis indicated that the vasculin/SSH6-beta ratio increases throughout atherogenesis. Immunohistochemical analysis demonstrated cytoplasmic expression of SSH6 gene products in macrophages, endothelial cells, and SMCs. In summary, we identified a novel mRNA/protein, vasculin, in the arterial wall and plasma. The regulated expression of vasculin in plaques suggests a role in atherogenesis. Moreover, its presence in plasma opens perspectives for vasculin as a marker for atherosclerosis.     

Sleep transforms the cerebral trace of declarative memories

After encoding, memory traces are initially fragile and have to be reinforced to become permanent. The initial steps of this process occur at a cellular level within minutes or hours. Besides this rapid synaptic consolidation, systems consolidation occurs within a time frame of days to years. For declarative memory, the latter is presumed to rely on an interaction between different brain regions, in particular the hippocampus and the medial prefrontal cortex (mPFC). Specifically, sleep has been proposed to provide a setting that supports such systems consolidation processes, leading to a transfer and perhaps transformation of memories. Using functional MRI, we show that postlearning sleep enhances hippocampal responses during recall of word pairs 48 h after learning, indicating intrahippocampal memory processing during sleep. At the same time, sleep induces a memory-related functional connectivity between the hippocampus and the mPFC. Six months after learning, memories activated the mPFC more strongly when they were encoded before sleep, showing that sleep leads to long-lasting changes in the representation of memories on a systems level.     

Low osmolarity transforms ventricular fibrillation from complex to highly organized, with a dominant high-frequency source

BACKGROUND: An osmotic challenge activates volume-regulated chloride currents (I(Cl,vol)), resulting in depolarization of the resting membrane potential and shortening of action potential duration (APD). I(Cl,vol) is activated in ischemia/reperfusion, but the effects of osmotic challenges and I(Cl,vol) on ventricular fibrillation (VF) are unknown. OBJECTIVES: The purpose of this study was to investigate the influence of hypo-osmotic and hypotonic stress and I(Cl,vol) activation on VF dynamics. METHODS: Guinea pig hearts were isolated, stained with di-4 ANEPPS to optically map action potentials (APs) from epicardium using a photodiode array, and perfused with iso-osmotic (low NaCl Ringer plus 45 mM mannitol) or hypo-osmotic (low NaCl Ringer) solution. RESULTS: Hypo-osmotic solution shortened APDs (143 +/- 5 ms --> 115 +/- 10 ms) and increased APD gradients between right and left ventricles (21 +/- 7 ms --> 41 +/- 10 ms, n = 4). In VF induced by burst stimulation, switching to hypo-osmotic solution increased VF frequencies (15.3 +/- 1.2 Hz to 28.9 +/- 3.6 Hz, n = 11), transforming complex fast Fourier transformation spectra to a single dominant high frequency on the left but not the right ventricle. Perfusion with the I(Cl,vol) blocker indanyloxyacetic acid-94 (10 muM) reversed organized VF to complex VF with lower (13.5 +/- 3.7 Hz in left ventricle) frequencies (n = 8), indicating that I(Cl,vol) underlies the changes in VF dynamics. Consistent with this interpretation, the levels of ClC-3 channel protein were 27% greater on left than right ventricles (n = 10), and computer simulations showed that insertion of I(Cl,vol) transformed complex VF to a stable spiral. CONCLUSION: Activation of I(Cl,vol) by decreasing osmolarity (45 mOsm) has a major impact on VF dynamics by transforming random multiple wavelets to a highly organized VF with a single dominant frequency.     

Development of a new analytical method for the electrocardiogram using short-time first Fourier transforms

To detect the minute electric potential inside the QRS complex, a new frequency domain method was designed using short-time First Fourier Transforms (SFFT) and high-frequency sampling (oversampling). SFFT improved the frequency resolution by oversampling that was applied to this analysis. The electric potential data of 15,000 points received weighted, running average processing and was subtracted from the original waveform to reduce the low-frequency component. The data in a segment of 160 ms, including QRS, was processed by frequency analysis with the SFFT computation routine. The ECG of healthy individuals was analyzed by this method and its usefulness evaluated. The processing waves of the X-axis, Y-axis, and Z-axis of a representative normal subject were formed into 3 groups of peak electric potential. SFFT enabled the detection of the structure inside the QRS complex without signal averaging, and is considered capable of evaluating the process of excitement inside the QRS complex in the various heart diseases.     

BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H+ antiport activity.

Renal hypertrophy is characterized by an increase in cell size and protein content with minimal hyperplasia. The mechanisms of control of this pattern of cell growth have not been determined. The present studies examined whether the growth inhibitor elaborated by BSC-1 kidney epithelial cells (GI), which has nearly identical biological properties to transforming growth factor beta (TGF-beta), could transform a mitogenic stimulus into a hypertrophic stimulus for rabbit renal proximal tubular cells in primary culture. Insulin (10 micrograms/ml) plus hydrocortisone (50 nM) increased the amount of protein per cell, cell volume, and [3H]thymidine incorporation at 24 and 48 hr in these cells. GI/TGF-beta (10 units/ml) led to a minimal stimulation of [3H]thymidine incorporation. When added together with insulin plus hydrocortisone, GI/TGF-beta inhibited the stimulatory effect of these mitogens on [3H]thymidine incorporation but did not block the increase in protein per cell and cell volume--i.e., the cells underwent hypertrophy. The fact that this pattern persisted for 48 hr indicated that GI/TGF-beta exerted a prolonged inhibitory effect on mitogenic-stimulated DNA synthesis rather than delaying its onset. Amiloride-sensitive Na+ uptake (indicative of Na+/H+ antiport activity) correlated with protein per cell and cell volume rather than with DNA synthesis. P60 gel chromatographic fractionation of conditioned medium harvested from proximal tubular cells yielded a fraction that inhibited [3H]thymidine incorporation in BSC-1 cells and CCL 64 cells; the relative inhibitory activity on these cell lines and the chromatographic behavior were similar to those observed with GI/TGF-beta. These studies indicate that the control of cell size may be regulated by autocrine mechanisms mediated by the elaboration of growth inhibitory factors that alter the pattern of the growth response to mitogens.     

Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die. Here we report the complete genome of B. pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them. The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins. A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome. Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B. mallei. We propose that variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.     

The relationship between ST segment deviation projected to the front of the chest during exercise and simultaneous Holter monitoring

Simultaneous two-channel Holter monitoring, with a direct recordingsystem, and maximal exercise testing with a 12-lead precordialelectrocardiographic mapping system were performed in 50 patientswith chest pain (41 with documented coronary artery disease,9 without). The exploring Holter leads were placed to correspondto CM5 and an aVF-like lead. In 36 patients, ST segment changeswere found with both Holter and the 12-lead precordial electrocardiogram,while in 12 patients no ischaemic changes were detected by eithermethod. Thus the results of the two methods concurred in 48of 50 patient (96%). The magnitude of the ischaemic change wassimilar in 24 of 36 patients (67%), while the Holter systemunderestimated the ischaemic change by 0.5–2.0 mm in 12patients. When the maximal ST segment deviation in V5 was comparedwith CM5, the deviations with both systems were identical inall but one patient in whom a difference of 0.5 mm was found.The use of a Holter lead resembling aVF identified maximal STsegment change on only one occasion, and in only four patientswas an ST segment change of 1 mm noted. In conclusion, ambulatory monitoring utilizing only CM5 seemsto detect most episodes with ST segment changes, but the useof a 12-lead precordial mapping system during exercise testingmay expand the possibility of defining the optimal sites forthe exploring Holter leads to detect maximal ST segment change.     

Calcium, dexamethasone, and the antiglucocorticoid RU-486 differentially regulate neuropeptide synthesis in a rat C cell line

The differential regulation of neurotensin (NT), calcitonin (CT), and CT gene-related peptide (CGRP) production was studied in the clonal, rat C cell-derived, 44-2C cell line. Two experimental paradigms were used: cells were incubated with maximally effective concentrations of calcium (4.0 mM); alternatively, cells were treated with the synthetic glucocorticoid, dexamethasone (DEX). The specificity of the DEX-mediated response was assessed by using the synthetic antiglucocorticoid, RU-486. Calcium was not mitogenic in 44-2C cells and did not affect cell growth. Calcium increased the secretion and cellular accumulation of NT. In contrast, calcium treatment decreased CT content and release while it diminished the levels of CT- and CGRP-specific messenger RNA (mRNA) levels. DEX (10(-8) M) inhibited cell proliferation. NT content and secretion increased after DEX treatment, and this was potentiated by the addition of calcium. DEX-treated cells showed diminished CT content and secretion. The levels of CT- and CGRP-specific mRNA were significantly reduced in DEX-treated cultures. RU-486 antagonized the action of DEX and blocked DEX-inhibited cell proliferation. Inhibition of CT secretion by DEX was blocked by RU-486; CT- and CGRP-specific mRNA levels were increased in response to treatment with equimolar or 100-fold excess concentrations of RU-486. We conclude that NT secretion as well as CT/CGRP expression and release can be differentially regulated in the 44-2C cell line.