Mats Julius Stensrud

VGLUT1 is localized in astrocytic processes in several brain regions

During the last years, the concept of gliotransmission has been established. Glutamate has been shown to be released from astrocytes by different mechanisms, e.g., in an exocytotic manner. The authors have previously shown that astrocytes in the dentate‐molecular layers express vesicular glutamate transporters on synaptic‐like microvesicles (SLMVs). By confocal microscopy, the authors, in this study, show that vesicles by a family of glutamate transporters 1 (VGLUT1) labeling was clearly present within astrocytic processes (diameter > 1 μm) in several brain regions; the dentate‐molecular layers, the stratum radiatum of CA1 hippocampus, the frontal cortex, and the striatum. At the electron microscopic level, immunogold cytochemistry showed the presence of VGLUT1 gold particles over SLMVs in delicate astrocytic processes (cross‐sectional diameter < 500 nm) in all the above‐mentioned brain regions. When measuring the distance from the membrane of SLMVs in astrocytes to the closest VGLUT1 gold particle, it turned out that most gold particles (above 95 %) were located within 25 nm from the membrane, strongly suggesting that VGLUT1 is present in SLMVs in the astrocytes. Finally, electron microscopic immunocytochemistry shows that VGLUT1 labeling was concentrated in astrocytic processes from wild type, and not in VGLUT1 knock out hippocampus. The authors have concluded that astrocytes not only in the dentate‐molecular layers but also in stratum radiatum of CA1 hippocampus, frontal cortex, and the striatum possess SLMVs carrying VGLUT1, suggesting that astrocytes in all these brain regions are capable of vesicular release of glutamate.

A distinct set of synaptic-like microvesicles in atroglial cells contain VGLUT3.

There is increasing evidence for vesicular release of glutamate from astrocytes. We have previously demonstrated existence of VGLUT1 on astrocytic synaptic‐like microvesicles (SMLVs) in several brain regions indicating a role in astroglial glutamate release. As VGLUT3 is prominently expressed in non‐neuronal cells, this prompted us to investigate whether VGLUT3 is also involved in astroglial release of glutamate. Confocal microscopic investigations revealed that astrocytes in the hippocampus and the frontal cortex, as well as Bergmann glia in the cerebellum were labeled for VGLUT3. Immunogold cytochemistry showed that VGLUT3 gold particles were located over SMLVs in perisynaptic astrocytic and Bergmann glial processes. The specificity of the VGLUT3 immunoreactivity was demonstrated by abolished VGLUT3 labeling in astroglia in VGLUT3 knock‐out mice. Double immunogold labeling showed that astrocytic processes contained labeling for VGLUT3 and VGLUT1, but the antibodies labeled separate subpopulations of vesicles in the processes. The ratio of gold particle densities between glial processes and nerve terminals were higher for VGLUT3 than for VGLUT1, suggesting that VGLUT3 is particularly abundant in astrocytic processes. Thus, our data show that VGLUT3 localizes to a distinct set of SMLVs in perisynaptic astroglial processes and suggest that VGLUT3 is important for glutamate release from astrocytes.

Vesicular glutamate transporter‐3 in the rodent brain: Vesicular colocalization with vesicular γ‐aminobutyric acid transporter

Vesicular glutamate transporters (VGLUT1–3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ‐aminobutyric acid (GABA)‐ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT‐positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light‐microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals. J. Comp. Neurol. 521: 3042–3056, 2013.

Exploring Selection Bias by Causal Frailty Models: The Magnitude Matters

Counter-intuitive associations appear frequently in epidemiology, and these results are often debated. In particular, several scenarios are characterized by a general risk factor that appears protective in particular subpopulations, for example, individuals suffering from a specific disease. However, the associations are not necessarily representing causal effects. Selection bias due to conditioning on a collider may often be involved, and causal graphs are widely used to highlight such biases. These graphs, however, are qualitative, and they do not provide information on the real life relevance of a spurious association. Quantitative estimates of such associations can be obtained from simple statistical models. In this study, we present several paradoxical associations that occur in epidemiology, and we explore these associations in a causal, frailty framework. By using frailty models, we are able to put numbers on spurious effects that often are neglected in epidemiology. We discuss several counter-intuitive findings that have been reported in real life analyses, and we present calculations that may expand the understanding of these associations. In particular, we derive novel expressions to explain the magnitude of bias in index-event studies.

Radiologic evaluation of lumps in the male breast

Background Gynecomastia has a typical appearance on mammography, and occurs frequently in men. However, imaging is often performed on men with breast lumps to exclude breast cancer, which only comprises 1% of male breast masses. Purpose To assess whether ultrasound and fine needle aspiration cytology (FNAC) are necessary investigations when mammograms show classical gynecomastia. Material and Methods We have retrospectively collected data on male patients referred for mammography during the period 2011–2013 (a total of 539 patients). All radiological images were re-read, and descriptions of ultrasound images were reviewed. Clinical information supplied with the original referrals was assessed, along with pathology and cytology reports. Results Among the 539 male patients who underwent mammography, 483 were also examined with ultrasound, and 335 were further evaluated with FNAC. Mammograms showed gynecomastia in 350 patients, and among these subjects ultrasound was performed in 340 (97%), FNAC in 261 (75%), and core biopsies in four (1%) patients. The diagnosis gynecomastia was unchanged in all patients who underwent FNAC or biopsy. Malignant tumors were found in eight patients, six of which were invasive ductal carcinomas. Conclusion In patients with a classical appearance of gynecomastia on mammography, supplemental ultrasound, FNAC, or biopsy is superfluous and contributes to unnecessary costs.

The surprising implications of familial association in disease risk

BackgroundA wide range of diseases show some degree of clustering in families; family history is therefore an important aspect for clinicians when making risk predictions. Familial aggregation is often quantified in terms of a familial relative risk (FRR), and although at first glance this measure may seem simple and intuitive as an average risk prediction, its implications are not straightforward.MethodsWe use two statistical models for the distribution of disease risk in a population: a dichotomous risk model that gives an intuitive understanding of the implication of a given FRR, and a continuous risk model that facilitates a more detailed computation of the inequalities in disease risk. Published estimates of FRRs are used to produce Lorenz curves and Gini indices that quantifies the inequalities in risk for a range of diseases.ResultsWe demonstrate that even a moderate familial association in disease risk implies a very large difference in risk between individuals in the population. We give examples of diseases for which this is likely to be true, and we further demonstrate the relationship between the point estimates of FRRs and the distribution of risk in the population.ConclusionsThe variation in risk for several severe diseases may be larger than the variation in income in many countries. The implications of familial risk estimates should be recognized by epidemiologists and clinicians.

Can chance cause cancer? A causal consideration

The role of randomness, environment and genetics in cancer development is debated. We approach the discussion by using the potential outcomes framework for causal inference. By briefly considering the underlying assumptions, we suggest that the antagonising views arise due to estimation of substantially different causal effects. These effects may be hard to interpret, and the results cannot be immediately compared. Indeed, it is not clear whether it is possible to define a causal effect of chance at all.

Immunogold characteristics of VGLUT3‐positive GABAergic nerve terminals suggest corelease of glutamate

There is compelling evidence that glutamate can act as a cotransmitter in the mammalian brain. Interestingly, the third vesicular glutamate transporter (VGLUT3) is primarily found in neurons that were anticipated to be nonglutamatergic. Whereas the function of VGLUT3 in acetylcholinergic and serotoninergic neurons has been elucidated, the role of VGLUT3 in neurons releasing gamma‐aminobutyric acid (GABA) is not settled. We have previously shown that VGLUT3 is found together with the vesicular GABA transporter (VIAAT) on synaptic vesicle membranes in the hippocampus. Now we provide novel electron microscopic data from the rat hippocampus suggesting that glutamate is enriched in inhibitory nerve terminals containing VGLUT3 compared to those lacking VGLUT3. The opposite was found for GABA; VGLUT3‐positive inhibitory terminals contained lower density of GABA labeling compared to VGLUT3‐negative inhibitory terminals. In addition, semiquantitative confocal immunofluorescence showed that N‐methyl‐D‐aspartate (NMDA)‐receptor labeling was present more frequently in VGLUT3‐positive/VIAAT‐positive synapses versus in VGLUT3‐negative/VIAAT‐positive synapses. Electron microscopic immunogold data further suggest that NMDA receptors are enriched in VGLUT3 containing inhibitory terminals. Our data reveal new chemical characteristics of a subset of GABAergic interneurons in the hippocampus. The analyses suggest that glutamate is coreleased with GABA from hippocampal basket cell‐synapses to act on NMDA receptors. J. Comp. Neurol. 523:2698–2713, 2015.

Inequality in genetic cancer risk suggests bad genes rather than bad luck

Heritability is often estimated by decomposing the variance of a trait into genetic and other factors. Interpreting such variance decompositions, however, is not straightforward. In particular, there is an ongoing debate on the importance of genetic factors in cancer development, even though heritability estimates exist. Here we show that heritability estimates contain information on the distribution of absolute risk due to genetic differences. The approach relies on the assumptions underlying the conventional heritability of liability model. We also suggest a model unrelated to heritability estimates. By applying these strategies, we describe the distribution of absolute genetic risk for 15 common cancers. We highlight the considerable inequality in genetic risk of cancer using different metrics, e.g., the Gini Index and quantile ratios which are frequently used in economics. For all these cancers, the estimated inequality in genetic risk is larger than the inequality in income in the USA.Cancer heritability estimates can be obtained via decomposing trait variance into genetic and other factors. Here, the authors obtain the distribution of absolute genetic risk for 15 common cancers, and they use a number of metrics to show that the genetic risk varies considerably across individuals.

Can Collider Bias Explain Paradoxical Associations

To the Editor:Limiting the study population to diseased subjects may influence the effect estimates,1–4 because collider bias is introduced. Sperrin et al.5 recently suggested that “the bias is small relative to the causal relationships between the variables.” Furthermore, they stated that collider