Petter Laake

Presynaptic Ca2+-Activated K+ Channels in Glutamatergic Hippocampal Terminals and Their Role in Spike Repolarization and Regulation of Transmitter Release

Large-conductance Ca2+-activated K+ channels (BK, also called Maxi-K orSlo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca2+ influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40–100 μm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction

In cardiac myocytes, initiation of excitation–contraction coupling is highly localized near the T‐tubule network. Myocytes with a dense T‐tubule network exhibit rapid and homogeneous sarcoplasmic reticulum (SR) Ca2+ release throughout the cell. We examined whether progressive changes in T‐tubule organization and Ca2+ release synchrony occur in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by ligation of the left coronary artery, and CHF was diagnosed by echocardiography (left atrial diameter >2.0 mm). CHF mice were killed at 1 or 3 weeks following MI (1‐week CHF, 3‐week CHF) and cardiomyocytes were isolated from viable regions of the septum, excluding the MI border zone. Septal myocytes from SHAM‐operated mice served as controls. T‐tubules were visualized by confocal microscopy in cells stained with di‐8‐ANEPPS. SHAM cells exhibited a regular striated T‐tubule pattern. However, 1‐week CHF cells showed slightly disorganized T‐tubule structure, and more profound disorganization occurred in 3‐week CHF with irregular gaps between adjacent T‐tubules. Line‐scan images of Ca2+ transients (fluo‐4 AM, 1 Hz) showed that regions of delayed Ca2+ release occurred at these gaps. Three‐week CHF cells exhibited an increased number of delayed release regions, and increased overall dyssynchrony of Ca2+ release. A common pattern of Ca2+ release in 3‐week CHF was maintained between consecutive transients, and was not altered by forskolin application. Thus, progressive T‐tubule disorganization during CHF promotes dyssynchrony of SR Ca2+ release which may contribute to the slowing of SR Ca2+ release in this condition.

DIETARY FAT INTAKE AND RISK OF PROSTATE CANCER: A PROSPECTIVE STUDY OF 25,708 NORWEGIAN MEN

The relationship between incidence of prostate cancer and intake of dietary fat and foods rich in fat was studied in 25,708 men aged 16–56 years attending a Norwegian health screening in 1977–1983. Linkage to the Cancer Registry of Norway and the Central Bureau of Statistics of Norway ensured a complete follow‐up until December 31, 1992. Diet was recorded on a semi‐quantitative food‐frequency questionnaire at the time of screening, and 72 cases of prostate cancer were identified during follow‐up. At the end of follow‐up, mean age of the total study sample was 56 years (range 19–68), while mean age at diagnosis of prostate cancer was 60 years (range 47–67). No association was found between energy‐adjusted intake of total fat, saturated fat, mono‐unsaturated fat or poly‐unsaturated fat and the incidence of prostate cancer. Significant positive associations were found for body mass index (BMI) and consumption of hamburgers/meatballs, while no association was found with consumption of frankfurters/sausages and a significant negative association with the weekly number of main meals with meat. A significantly increased risk of prostate cancer was associated with skim milk as compared to whole milk. Milk preference (skim vs. whole) was associated significantly positively with BMI. Our study of a relatively young cohort does not confirm previous case‐control and cohort studies suggesting that dietary fat, especially from animal sources, is associated positively with risk of prostate cancer. Int. J. Cancer 73:634–638, 1997.

Recommended tests for association in 2×2 tables

The asymptotic Pearsons chi-squared test and Fishers exact test have long been the most used for testing association in 2x2 tables. Unconditional tests preserve the significance level and generally are more powerful than Fishers exact test for moderate to small samples, but previously were disadvantaged by being computationally demanding. This disadvantage is now moot, as software to facilitate unconditional tests has been available for years. Moreover, Fishers exact test with mid-p adjustment gives about the same results as an unconditional test. Consequently, several better tests are available, and the choice of a test should depend only on its merits for the application involved. Unconditional tests and the mid-p approach ought to be used more than they now are. The traditional Fishers exact test should practically never be used.

Diet and risk of cutaneous malignant melanoma: A prospective study of 50,757 Norwegian men and women

The relationship between dietary habits and subsequent risk of cutaneous malignant melanoma (CMM) was studied in 25,708 men and 25,049 women aged 16‐56 years attending a Norwegian health screening in 1977‐1983. Linkage to the Cancer Registry of Norway and the Central Bureau of Statistics of Norway ensured a complete follow‐up until December 31, 1992. Diet was recorded through a semi‐quantitative food‐frequency questionnaire at the time of screening, and 108 cases of CMM were identified during follow‐up. Use of cod liver oil supplementation and intake of polyunsaturated fat were associated with significant increased risk and drinking coffee with significant decreased risk of CMM in women. Adjusting for height, body mass index, body surface area, education, smoking or occupational or recreational physical activity did not change the results. No significant association was found between the incidence of CMM and any of the dietary factors in men. Important aspects are residual confounding by sun exposure and social class, as well as concern with multiple comparisons. Int. J. Cancer 71:600‐604, 1997.

Regression among factor scores

Structural equation models with latent variables are sometimes estimated using an intuitive three-step approach, here denoted factor score regression. Consider a structural equation model composed of an explanatory latent variable and a response latent variable related by a structural parameter of scientific interest. In this simple example estimation of the structural parameter proceeds as follows: First, common factor models areseparately estimated for each latent variable. Second, factor scores areseparately assigned to each latent variable, based on the estimates. Third, ordinary linear regression analysis is performed among the factor scores producing an estimate for the structural parameter. We investigate the asymptotic and finite sample performance of different factor score regression methods for structural equation models with latent variables. It is demonstrated that the conventional approach to factor score regression performs very badly. Revised factor score regression, using Regression factor scores for the explanatory latent variables and Bartlett scores for the response latent variables, produces consistent estimators for all parameters.

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with δ-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses

Abstract. Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber–Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the δ2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood–brain water uptake and confers barrier function on perivascular astrocyte endfeet

Tissue- and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood–brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood–brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement.

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.

Childhood and adult milk consumption and risk of premenopausal breast cancer in a cohort of 48,844 women—the Norwegian women and cancer study

Analyses of dairy consumption and breast cancer incidence have yielded conflicting results. In this prospective cohort study of 48,844 premenopausal Norwegian women, we examined the relationship between childhood and adult milk consumption and breast cancer incidence. During a mean follow‐up time of 6.2 years, 317 incident cases of breast cancer were diagnosed. Information on childhood and adult milk consumption was obtained from frequency questions mailed to the participants in 1991–92. Milk consumption as a child was negatively associated with subsequent breast cancer among the youngest women (34–39 years) (p for trend = 0.001), but not among older ones (40–49 years). Adult milk consumption tended to be negatively related to breast cancer incidence (p for trend = 0.12) after adjustment for age, reproductive and hormonal factors, body mass index, education, physical activity, and alcohol consumption. Women drinking more than 3 glasses of milk per day had an incidence rate ratio of breast cancer of 0.56 (95% confidence interval 0.31–1.01) compared with women not drinking milk. Analyses according to type of milk consumed and milk fat consumption did not reveal any clear associations. A combination of childhood and adult milk consumption produced a clear negative trend in breast cancer incidence rate ratios with increasing milk consumption (p = 0.03).