Urocortin-1 and -2 double-deficient mice show robust anxiolytic phenotype and modified serotonergic activity in anxiety circuits.

The urocortin (Ucn) family of neuropeptides is suggested to be involved in homeostatic coping mechanisms of the central stress response through the activation of corticotropin-releasing factor receptor type 2 (CRFR2). The neuropeptides, Ucn1 and Ucn2, serve as endogenous ligands for the CRFR2, which is highly expressed by the dorsal raphe serotonergic neurons and is suggested to be involved in regulating major component of the central stress response. Here, we describe genetically modified mice in which both Ucn1 and Ucn2 are developmentally deleted. The double knockout mice showed a robust anxiolytic phenotype and altered hypothalamic-pituitary-adrenal axis activity compared with wild-type mice. The significant reduction in anxiety-like behavior observed in these mice was further enhanced after exposure to acute stress, and was correlated with the levels of serotonin and 5-hydroxyindoleacetic acid measured in brain regions associated with anxiety circuits. Thus, we propose that the Ucn/CRFR2 serotonergic system has an important role in regulating homeostatic equilibrium under challenge conditions.

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex.

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to 11 days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.

Identification of an immune-responsive mesolimbocortical serotonergic system: potential role in regulation of emotional behavior.

Peripheral immune activation can have profound physiological and behavioral effects including induction of fever and sickness behavior. One mechanism through which immune activation or immunomodulation may affect physiology and behavior is via actions on brainstem neuromodulatory systems, such as serotonergic systems. We have found that peripheral immune activation with antigens derived from the nonpathogenic, saprophytic bacterium, Mycobacterium vaccae, activated a specific subset of serotonergic neurons in the interfascicular part of the dorsal raphe nucleus (DRI) of mice, as measured by quantification of c-Fos expression following intratracheal (12 h) or s.c. (6 h) administration of heat-killed, ultrasonically disrupted M. vaccae, or heat-killed, intact M. vaccae, respectively. These effects were apparent after immune activation by M. vaccae or its components but not by ovalbumin, which induces a qualitatively different immune response. The effects of immune activation were associated with increases in serotonin metabolism within the ventromedial prefrontal cortex, consistent with an effect of immune activation on mesolimbocortical serotonergic systems. The effects of M. vaccae administration on serotonergic systems were temporally associated with reductions in immobility in the forced swim test, consistent with the hypothesis that the stimulation of mesolimbocortical serotonergic systems by peripheral immune activation alters stress-related emotional behavior. These findings suggest that the immune-responsive subpopulation of serotonergic neurons in the DRI is likely to play an important role in the neural mechanisms underlying regulation of the physiological and pathophysiological responses to both acute and chronic immune activation, including regulation of mood during health and disease states. Together with previous studies, these findings also raise the possibility that immune stimulation activates a functionally and anatomically distinct subset of serotonergic neurons, different from the subset of serotonergic neurons activated by anxiogenic stimuli or uncontrollable stressors. Consequently, selective activation of specific subsets of serotonergic neurons may have distinct behavioral outcomes.

Investigation of a central nucleus of the amygdala/dorsal raphe nucleus serotonergic circuit implicated in fear-potentiated startle.

Serotonergic systems are thought to play an important role in control of motor activity and emotional states. We used a fear-potentiated startle paradigm to investigate the effects of a motor-eliciting stimulus in the presence or absence of induction of an acute fear state on serotonergic neurons in the dorsal raphe nucleus (DR) and cells in subdivisions of the central amygdaloid nucleus (CE), a structure that plays an important role in fear responses, using induction of the protein product of the immediate-early gene, c-Fos. In Experiment 1 we investigated the effects of fear conditioning training, by training rats to associate a light cue (conditioned stimulus, CS; 1000 lx, 2 s) with foot shock (0.5 s, 0.5 mA) in a single session. In Experiment 2 rats were given two training sessions identical to Experiment 1 on days 1 and 2, then tested in one of four conditions on day 3: (1) placement in the training context without exposure to either the CS or acoustic startle (AS), (2) exposure to 10 trials of the 2 s CS, (3) exposure to 40 110 dB AS trials, or (4) exposure to 40 110 dB AS trials with 10 of the trials preceded by and co-terminating with the CS. All treatments were conducted during a 20 min session. Fear conditioning training, by itself, increased c-Fos expression in multiple subdivisions of the CE and throughout the DR. In contrast, fear-potentiated startle selectively increased c-Fos expression in the medial subdivision of the CE and in serotonergic neurons in the dorsal part of the dorsal raphe nucleus (DRD). These data are consistent with previous studies demonstrating that fear-related stimuli selectively activate DRD serotonergic neurons. Further studies of this mesolimbocortical serotonergic system could have important implications for understanding mechanisms underlying vulnerability to stress-related psychiatric disorders, including anxiety and affective disorders.

The impact of meconium ileus on the clinical course of children with cystic fibrosis.

The present study was designed to compare the clinical course of children diagnosed with cystic fibrosis (CF) in infancy due to the presence of meconium ileus (MI) with children diagnosed by way of a newborn screening programme (non-MI). A matched case-control study design was used. Matching was performed on the basis of sex and date of birth. All children born in New South Wales, Australia after 1980 and who had attended the CF clinic at The Children's Hospital at Westmead since diagnosis were included as possible cases or controls. Parameters pertaining to the clinical course were compared in 39 matched pairs. MI children had a significantly worse pulmonary status. The forced expiratory volume in one second was 16.3 +/- 5.2% higher (p<0.001, n=21 pairs) and the forced vital capacity value 10.5 +/- 4.7%, higher (p<0.05, n=21 pairs) in non-MI children. The difference between the pairs (18.6 +/- 4.4 MI and 20.5 +/- 3.4 non-MI) in the Shwachman chest radiograph score was statistically significant (p<0.05, n=39 pairs). There were no significant differences in any other assessed parameters, such as height, weight, the presence of liver function abnormalities, the frequency of hospitalization or airway microbial colonization. Meconium ileus may be an early indication of a more severe phenotype of cystic fibrosis. This was suggested by the significantly lower pulmonary function found in children with a history of meconium ileus compared to age- and sex-matched children who did not have meconium ileus.

The QRST deflection area of electrograms during global alterations of ventricular repolarization.

Changes of refractory period and QRST deflection area in cardiac electrograms due to localized myocardial warming and altered cycle length were determined. Localized myocardial warming consistently resulted in increased QRST deflection area which was highly correlated with reduction of refractory period. Similar reductions of refractory period by decreased cycle length were associated with insignificant changes of small QRST areas and significant reductions of larger QRST areas. The different effect of local thermal alteration of repolarization and global alteration due to cycle length changes is experimental evidence that QRST deflection area depends on differences in duration of ventricular repolarization properties. The finding of decreased deflection area with decreased cycle length is also evidence of decreased disparity of repolarization properties of normal myocardium at rapid heart rates.

Ventricular recovery properties and QRST deflection area in cardiac electrograms.

We measured QRST deflection areas in cardiac surface electrograms of dogs and determined their relation to changes of refractory period induced by localized myocardial warming. Refractory period changes and QRST deflection areas were highly correlated with an average correlation of 0.95 over multiple activation sequences. For a single activation sequence, refractory period change was also highly correlated with ST-T deflection area, but over multiple activation sequences this correlation was reduced and the pooled variance of ST-T deflection areas was over 18 times greater than that of QRST area. No relation between QRST deflection area and the absolute value of refractory period over various cardiac sites was evident. Findings suggest that a particular set of ventricular recovery properties cannot be defined on the basis of QRST deflection areas, but that changes of recovery properties can be recognized on that basis.

Clinically practical lead systems for improved electrocardiography: comparison with precordial grids and conventional lead systems.

The use of limited leads for estimating total body surface potential distributions was investigated as a practical solution to the problem associated with extensive electrocardiographic sampling used in surface potential mapping. Two practical, limited lead sets of 32 leads each were derived and contrasted to a set of 30 precordial leads similar to those used in ST-segment and QRS mapping for estimating infarct size, and to a set of nine leads simulating those used in conventional 12-lead examinations. The two arrays, one of which excluded posterior sites for use in recumbent patients, showed little difference in ability to estimate 192 lead measured maps (average rms voltage error of 35 muV and average correlation coefficient of 0.97). The 30- and 9-lead arrays consistently showed twice the voltage (72 muV) and poorer pattern estimation (average correlation coefficient of 0.91) than either of the 32 lead arrays. These findings indicate the need for 20-35 properly located electrodes for accurate total body surface potential estimation. They also show that there is no difference in the abilities of a 30-lead precordial array and conventional leads to estimate maps.

Redundancy reduction for improved display and analysis of body surface potential maps. I. Spatial compression.

The Karhunen-Loeve technique of random process representation was investigated as a method of quantitatively characterizing body surface potential maps. One hundred ninety-two lead body surface potential maps from 124 normal subjects and 97 patients with independently documented heart disease were used in the study. Each map frame in QRS and ST-T of 34 maps in a test set was represented as a linear sum of orthonormal distributions derived from the covariance matrix estimated from all QRS frames in the 221 training maps. A 16:1 reduction in spatial data of the test set was achieved with rms errors of 45 and 21 microV in QRS and ST-T, respectively. Results suggest that 12 independent waveforms, derived from the 192 measured ECGs, may be used in place of those 192 ECGs. In addition to providing a convenient and familiar method of display for map data, the technique puts the data in an appropriate form for quantitative statistical analysis.

Redundancy reduction for improved display and analysis of body surface potential maps. II. Temporal compression.

This paper describes use of the Karhunen-Loeve expansion to identify and reduce temporal redundancy in electrocardiographic body surface potential maps (192 body surface leads recorded simultaneously at 1 kHz/channel for approximately 600 msec). Temporal data compression of about 20 to 1 was obtained with accurate representation of the original data. Use of separate sets of orthonormal basis functions for QRS and ST-T provided a more accurate representation than the basis derived from QRST. Combined with the spatial compression described in the preceding paper, overall map data compression of about 320 to 1 was obtained without significant loss of accuracy of representation or map appearance. With both spatial and temporal compression the 100,000 numbers which typically comprise a single cardiac complex were accurately represented by 216 coefficients. Using basis functions derived from a single cardiac complex were accurately represented by 216 coefficients. Using basis functions derived from a training set of 221 maps, the estimated average rms error of representation was 60 microV during the ST-T. For 34 test maps which were not part of the training set, measured average errors were 64 microV during the QRS and 23 microV during the ST-T. This technique provides a basis for quantification of the diagnostic content of maps and automated classification of maps.