Principal neuron spiking: neither necessary nor sufficient for cerebral blood flow in rat cerebellum

Neuronal activity, cerebral blood flow, and metabolic responses are all strongly coupled, although the mechanisms behind the coupling remain unclear. One of the key questions is whether or not increases in spiking activity in the stimulated neurons are sufficient to drive the activity-dependent rises in cerebral blood flow (CBF) that form the basis of the signals used in functional neuroimaging such as the blood oxygen level-dependent (BOLD) signal. To this end the present study examined the effect of enhanced spike activity per se on CBF in rat cerebellar cortex under conditions of disinhibition, achieved by blocking GABA_A receptors using either bicuculline or picrotoxin. Purkinje cell spiking activity and local field potentials were recorded by glass microelectrodes, and laser Doppler flowmetry was used to monitor CBF. Disinhibition increased Purkinje cell spiking rate to 200–300% of control without incurring any increase in basal CBF. This demonstrates that increased spike activity per se is not sufficient to affect basal CBF. The neurovascular coupling between excitatory synaptic activity and CBF responses evoked by inferior olive (climbing fibre) stimulation was preserved during disinhibition. Thus, the unchanged basal CBF in the presence of the dramatic rise in Purkinje cell spiking rate was not explained by impaired synaptic activity–CBF coupling. On the basis of our previous and the present studies, we conclude that increased spiking activity of principal neurons is neither sufficient nor necessary to elicit CBF responses and in turn BOLD signals, and that activation-dependent vascular signals reflect excitatory synaptic activity.     

Neuromuscular defects in a Drosophila survival motor neuron gene mutant

Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. We have isolated a Drosophila smn mutant. The fly alleles contain point mutations in smn similar to those found in SMA patients. We find that zygotic smn mutant animals show abnormal motor behavior and that smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects.     

A comparison of micropuncture and lithium clearance methods in the assessment of renal tubular function in rats with diabetes insipidus

The lithium clearance technique has been proposed as a non-invasive method whereby fluid delivery from the pars recta and pars convoluta of proximal tubules can be measured as C_Li and C_IN [0.78 C_Li/C_IN+0.22], respectively [12], C_Li being the clearance of lithium and C_IN that of inulin. In the present study, fluid delivery from proximal tubules was estimated simultaneously by micropuncture and lithium clearance techniques in anaesthetized Brattleboro rats with diabetes insipidus, under control conditions and following chronic treatment with hydrochlorothiazide. Absolute deliveries from the proximal convoluted tubules as determined by the micropuncture and lithium clearance methods were 437 and 427 μl/min, respectively, in untreated animals and 348 and 355 μl/min, respectively, in thiazide-treated animals. The individual results obtained by the two methods showed a high degree of correlation (r=0.85,P<0.001). In untreated Brattleboro rats, proximal fluid delivery as estimated by both the micropuncture and lithium clearance techniques showed significant (P<0.001) correlations with urine flow rate. These results provide further evidence for the acceptance of lithium clearance as a valid estimate of proximal tubular fluid delivery.     

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides.

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)-DR1 and HLA-DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the beta chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306-318 (IC50). Among the high-affinity peptides (IC50 < or = 1 microM), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty-two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1-specific or DR103-specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR-binding peptides are able to outcompete specific T-cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T-cell response and mutation at position 71 abolishes completely the T-cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T-cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele-specific peptides and also influence the conformation of promiscuous peptides.     

A case of broken bones and systems: The threat of irresponsible testimony

A 2‐month‐old healthy baby presented to the emergency room with an arm that was not moving and was found to have multiple and extensive fractures of her long bones. An extensive medical work‐up was done, and the hospital's multidisciplinary child abuse team was consulted, including child protection, genetics, radiology, and general pediatrics. It was determined that the history, clinical findings, radiographic findings, and laboratory findings were consistent with child abuse. Child protection services removed the child from the home, and for the next 10 months, the infant was well, and did not sustain a single new fracture. At a civil proceeding to determine the infant's custody, an expert witness for the defense concluded that the child had hypermobile Ehlers‐Danlos syndrome and low vitamin D. He stated that because of these conditions, the baby was vulnerable to fractures with routine handling. This is a personal story of a clinical geneticist who explored fracture fact versus fracture fiction and learned about the difference between responsible and irresponsible testimony. This story gives insight into how physicians can prepare to transition from the clinic to the courtroom. It is also a story about how medical experts must and should remain unbiased, evidence‐based, and committed to accuracy and truth.     

Glucose-6-phosphate dehydrogenase activity in monolayer cultures of thyroid epithelial cells: TSH and inhibition of nitrogen oxide synthase affect the enzyme activity and the oxygen sensitivity of the histochemical assay

The objective was to investigate glucose-6-phosphate dehydrogenase (G6PD) activity in monolayer cultures of thyroid epithelial cells and to examine whether inhibition of nitric oxide synthase affects activity of G6PD or oxygen sensitivity of the assay. Primary cultures without TSH addition prior to experiments demonstrated a TSH-dependent increase in G6PD activity. G6PD activity was higher in F12 medium than in a serum-free physiological medium. Secondary cultures grown in F12 medium demonstrated a diminished activity of G6PD and a lack of response to TSH. In the serum-free physiological medium, G6PD activity was comparable to that found in primary cultures and a response to high concentrations of TSH was maintained. In primary cultures grown in F12 medium devoid of TSH, G6PD activity decreased dose-dependently when nitric oxide synthase activity was inhibited. The oxygen sensitivity of the assay was comparable to that reported previously in malignant cells and correlated with the activity of G6PD in primary cultures. We suggest that thyroid epithelial cells may be an appropriate system to investigate oxygen sensitivity of the G6PD assay as the cells demonstrate a reduced oxygen sensitivity which can be influenced by culture conditions.     

Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity

The activity of the homeobox gene Prox1 is necessary and sufficient for venous blood endothelial cells (BECs) to acquire a lymphatic endothelial cell (LEC) fate. We determined that the differentiated LEC phenotype is a plastic, reprogrammable condition that depends on constant Prox1 activity for its maintenance. We show that conditional down-regulation of Prox1 during embryonic, postnatal, or adult stages is sufficient to reprogram LECs into BECs. Consequently, the identity of the mutant lymphatic vessels is also partially reprogrammed as they acquire some features typical of the blood vasculature. siRNA-mediated down-regulation of Prox1 in LECs in culture demonstrates that reprogramming of LECs into BECs is a Prox1-dependent, cell-autonomous process. We propose that Prox1 acts as a binary switch that suppresses BEC identity and promotes and maintains LEC identity; switching off Prox1 activity is sufficient to initiate a reprogramming cascade leading to the dedifferentiation of LECs into BECs. Therefore, LECs are one of the few differentiated cell types that require constant expression of a certain gene to maintain their phenotypic identity.     

Digital peer‐to‐peer information seeking and sharing: Opportunities for education and collaboration in newborn screening

Parents use the internet to connect with their peers and access information about a multitude of health topics, including newborn screening (NBS). As the NBS system evolves, education about NBS must be evaluated and updated to remain accessible and beneficial to parents. In this article, we aim to describe parents' current NBS educational needs and highlight areas to improve newborn screening education by detailing an analysis of NBS posts on an online parenting discussion platform. We analyzed a total of 317 discussion posts on BabyCenter®, finding that parents had questions about and desired support around many aspects of NBS including processes, results, and follow‐up. As a result of this analysis, three recommendations to improve NBS education were developed. Through collaboration and by leveraging technology, we can provide parents with accessible, timely, and desired NBS informational and social support.