John G. Mielke

Cellular distribution of the nicotinic acetylcholine receptor α7 subunit in rat hippocampus

The hippocampus is a region of the mammalian brain that has been extensively studied due to its role in many forms of memory. To better understand hippocampal function, significant attention has focused upon the cellular distribution of ligand-gated ion channels. Despite strong cholinergic innervation from the basal forebrain and a dense expression of nicotinic acetylchoine receptors (nAChRs), the cellular distribution of subunits forming these receptors has received little attention. We used organotypic hippocampal slice cultures (OHSCs) to study native alpha7 subunits, which, unlike other nAChR subunits, form a homomeric receptor. Cell-surface biotinylation, cross-linking of surface proteins, and sub-cellular fractionation all revealed a very limited presence of the subunit at the plasma membrane. In contrast, subunits of other receptors displayed significant surface expression. Notably, subunits in adult hippocampal tissue were distributed in a fashion similar to that observed in OHSCs. To monitor alpha7 subunits contained in functional nAChRs, a colourimetric assay using alpha-bungarotoxin (a specific alpha7 nAChR antagonist) was developed, and revealed a majority of binding at the cell surface. To change alpha7 subunit distribution, OHSCs were treated with compounds known to affect other ionotropic receptors-insulin, genistein, and elevated external K(+); however, neither subunit surface expression nor antagonist binding was affected. Our data reveal that hippocampal neurons possess a large internal population of alpha7 subunits under basal conditions, which persists during stimuli affecting tyrosine phosphorylation or neuronal activity. The nature of the internal pool of alpha7 subunits remains to be determined, but should have important implications for hippocampal activity.

Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.

Insulin, synaptic function, and opportunities for neuroprotection.

A steadily growing number of studies have begun to establish that the brain and insulin, while traditionally viewed as separate, do indeed have a relationship. The uptake of pancreatic insulin, along with neuronal biosynthesis, provides neural tissue with the hormone. As well, insulin acts upon a neuronal receptor that, although a close reflection of its peripheral counterpart, is characterized by unique structural and functional properties. One distinction is that the neural variant plays only a limited part in neuronal glucose transport. However, a number of other roles for neural insulin are gradually emerging; most significant among these is the modulation of ligand-gated ion channel (LGIC) trafficking. Notably, insulin has been shown to affect the tone of synaptic transmission by regulating cell-surface expression of inhibitory and excitatory receptors. The manner in which insulin regulates receptor movement may provide a cellular mechanism for insulin-mediated neuroprotection in the absence of hypoglycemia and stimulate the exploration of new therapeutic opportunities.

5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.

Selective vulnerability of hippocampal sub-fields to oxygen-glucose deprivation is a function of animal age.

For more than a century, the hippocampal sub-fields have been recognized as being differentially vulnerable to injury. While the cause remains unknown, the explanations generally considered have involved either vascular differences, or innate variability among cells. To examine the latter possibility, we prepared acute hippocampal slices from Sprague-Dawley rats, applied a brief period of oxygen-glucose deprivation (OGD; an in vitro model of ischemia), and assessed the viability of dissected sub-fields (CA1, CA3, dentate gyrus) by measuring mitochondrial 2,3,5-triphenyltetrazolium chloride (TTC) metabolism. In slices from young animals (15 weeks of age), post-OGD TTC metabolism was significantly reduced in the CA sub-fields relative to the dentate gyrus. Since previous studies found increasing age may worsen ischemic injury, we completed the same experiment using tissue from animals at 52 weeks of age, and found no differences in TTC metabolism across sub-fields. Given the established role of glutamate receptors in ischemic cell death, we examined two key subunit proteins (GluN1, found in all NMDA receptors, and GluA2, found in most AMPA receptors) across sub-fields and age to determine whether their expression complemented our viability data. We found that, relative to the CA1, the DG displayed greater GluN1 expression and lower GluA2 expression in both young and old animals. Our results confirm that regional vulnerability can be shown in a slice model, that the property is not intransigent, and that these features are likely not attributable to the expression pattern of key glutamate receptor subunits, but another molecular variable that changes over the lifespan.

Choline-mediated depression of hippocampal synaptic transmission

Abstract Choline is a micronutrient essential for the structural integrity of cellular membranes, and its presence at synapses follows either depolarization-induced pre-synaptic release or degradation of acetylcholine. Previous studies using whole-cell recording have shown that choline can modulate inhibitory input to hippocampal pyramidal neurons by acting upon nicotinic acetylcholine receptors (nAChRs) found on interneurons. However, little is known about how choline affects neuronal activity at the population level; therefore, we used extracellular recordings to assess its influence upon synaptic transmission in acutely prepared hippocampal slices. Choline caused a reversible depression of evoked field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependant manner (10, 500, and 1000 µM). When applied after the induction of long-term potentiation, choline-mediated depression (CMD) was still observed, and potentiation returned on wash-out. Complete blockade of CMD could not be achieved with antagonists for the α7 nAChR, to which choline is a full agonist, but was possible with a general nAChR antagonist. The ability of choline to increase paired-pulse facilitation, and the inability of applied gamma-aminobutyric acid (GABA) to mediate further depression of fEPSPs, suggests that the principal mechanism of cholines action was on the facilitation of neurotransmitter release. Our study provides evidence that choline can depress population-level activity, quite likely by facilitating the release of GABA from interneurons, and may thereby influence hippocampal function.

Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia

Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

Does Resuscitation Training Reduce Neonatal Deaths in Low-Resource Communities? A Systematic Review of the Literature.

Every year, nearly 1 million babies succumb to birth asphyxia (BA) within the Asia-Pacific region. The present study sought to determine whether educational interventions containing some element of resuscitation training would decrease the relative risk (RR) of neonatal mortality attributable to BA in low-resource communities. We systematically reviewed 3 electronic databases and identified 14 relevant reports. For community deliveries, providing traditional birth attendants (TBAs) with neonatal resuscitation training modestly reduced the RR in 3 of 4 studies. For institutional deliveries, training a range of clinical staff clearly reduced the RR within 2 of 8 studies. When resuscitation-specific training was directed to community and institutional health care workers, a slight benefit was observed in 1 of 2 studies. Specific training in neonatal resuscitation appears most effective when provided to TBAs (specifically, those presented with ongoing opportunities to review and update their skills), but this particular intervention alone may not appreciably reduce mortality.

Susceptibility to oxygen-glucose deprivation is reduced in acute hippocampal slices from euthermic Syrian golden hamsters relative to slices from Sprague-Dawley rats

Hibernation in mammals is characterised by a marked decrease in body temperature and a dramatic suppression of metabolism. In addition, despite experiencing a reduced cardiac output that would normally cause profound cerebral ischaemia, hibernating animals display robust neuroprotection. However, whether the reduced susceptibility to neural injury displayed by hibernators is attributable to an innate factor, or to the physiologic changes that accompany hibernation, remains uncertain. To help clarify the nature of the ischaemic tolerance displayed by hibernators, the current study examined hippocampal slices from rodents not capable of hibernation (rat) and rodents that could undergo hibernation (hamsters), but were active immediately prior to slice preparation. Slices from each species were subjected to oxygen-glucose deprivation (OGD; a commonly used in vitro model of ischaemia), and their viability examined after a recovery period. Although OGD reduced plasma membrane integrity in each species, rat-derived slices displayed a nearly threefold greater degree of effect. In addition, only slices harvested from rats showed reductions in synaptic mitochondrial function. While the improved ischaemic tolerance displayed by euthermic hamster brain slices maintained at a physiological temperature suggests an intrinsic, protection-related variable, the synaptic level of the GluN1 subunit (which is required to form functional NMDA receptors) was not found to be different between the two species. Further work is needed to improve understanding of the molecular mechanisms underlying the intrinsic injury tolerance of hibernator brain, which should help provide inspiration for new approaches to neuroprotection.