5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders

Enhanced axonal response of mitochondria to demyelination offers neuroprotection:implications for multiple sclerosis

Axonal loss is the key pathological substrate of neurological disability in demyelinating disorders, including multiple sclerosis (MS). However, the consequences of demyelination on neuronal and axonal biology are poorly understood. The abundance of mitochondria in demyelinated axons in MS raises the possibility that increased mitochondrial content serves as a compensatory response to demyelination. Here, we show that upon demyelination mitochondria move from the neuronal cell body to the demyelinated axon, increasing axonal mitochondrial content, which we term the axonal response of mitochondria to demyelination (ARMD). However, following demyelination axons degenerate before the homeostatic ARMD reaches its peak. Enhancement of ARMD, by targeting mitochondrial biogenesis and mitochondrial transport from the cell body to axon, protects acutely demyelinated axons from degeneration. To determine the relevance of ARMD to disease state, we examined MS autopsy tissue and found a positive correlation between mitochondrial content in demyelinated dorsal column axons and cytochromecoxidase (complex IV) deficiency in dorsal root ganglia (DRG) neuronal cell bodies. We experimentally demyelinated DRG neuron-specific complex IV deficient mice, as established disease models do not recapitulate complex IV deficiency in neurons,and found that these mice are able to demonstrate ARMD, despite the mitochondrial perturbation.Enhancement of mitochondrial dynamics in complex IV deficient neurons protects the axon upon demyelination. Consequently, increased mobilisation of mitochondria from the neuronal cell body to the axon is a novel neuroprotective strategy for the vulnerable, acutely demyelinated axon. We propose that promoting ARMD is likely to be a crucial preceding step for implementing potential regenerative strategies for demyelinating disorders.</p

Assessment of MRI scanner performance for preclinical functional studies

Functional Magnetic Resonance Imaging (fMRI) based studies are rapidly expanding in the field of preclinical research. The majority of these studies use Echo Planar Imaging (EPI) to measure Blood Oxygenation Level Dependent (BOLD) signal contrasts in the brain. In such studies the magnitude and statistical significances of these contrasts are then related to brain function and cognition. It is assumed that any observed signal contrast is ultimately due to differences in biological state and that scanner performance is stable and repeatable between subjects and studies. However, due to confounding issues introduced by in vivo subjects, little work has been undertaken to test this basic assumption. As the BOLD signal contrasts generated in such experiments are often very low, even small changes in scanner performance may dominate the BOLD contrast, distorting any biological conclusions drawn. A series of fMRI phantoms were produced to measure scanner performance independent of biological subjects. These phantoms produce specified signal contrast levels on demand during an fMRI scan by means of current-induced magnetic field gradients. These were used to generate data sets that emulated the BOLD signal contrast of in vivo imaging. Two studies examining scanner performance were then conducted on high-field preclinical MRI scanners. Firstly, in a longitudinal study on a single scanner, measurements were taken over a number of days across a week long period and then every two months over a year long period. Secondly, the behaviour of four preclinical scanners (three at 7T, one at 9.4T) was comparatively assessed. Measurements of several imaging parameters including contrast generated and functional contrast to noise ratio (fCNR) were obtained in both studies. If the scanners involved are truly comparable then they should generate similar measurement values. Across both studies parameter measurements showed significant differences for identical contrast settings on the phantom. Although signal contrast itself proved very comparable across the studies fCNR proved to be highly variable. As well as these measurements of longer tem behaviour proving variable, short and mid-term signal stability displayed a wide range of variability. Variations in the level and quality of both signal and noise were observed. Modelling of signal changes based on fundamental physical principles was also performed for comparison. The impact of these behaviours and variations on in vivo studies could result in skewed biological conclusions at any single site, with some sites exhibiting greater problems than others. The multisite results suggest potential difficulties when comparing biological conclusions between sites, even when using identical imaging parameters. In summary, these results suggest that a cautious approach should be taken with the conclusions of both fMRI and associated resting state connectivity studies that use EPI as their acquisition sequence. Improvements to both the experimental design of studies and regular quality monitoring of scanners should be undertaken to minimise these effects. Clinical MRI scanners should also be assessed for similar aberrations in behaviour

Training and scanning procedure.

<p>Animals were acclimatised to the scanning procedure using a mock scanner, conditioned and then tested during retrieval in the scanner 24 h later.</p

Effects of early life stress on brain activation.

<p>Within and between group analysis comparing BOLD response to first CS presentation over baseline in rats previously exposed to early life stress (ELS) compared to unstressed controls (CON). KE  =  cluster extent in voxels. T  =  peak voxel t-statistic. Clusters reaching <i>P</i>_corrected <0.05 are shown. R =  right, L =  left, RSG =  retrospenial granular cortex. CON, n =  14; ELS, n = 8.</p

Brain activation maps in response to a fear conditioned stimulus.

<p>(a) Activation in PG in response to CS presentation is observed in right lateral amygdala (LA) and hypothalamus (Hyp). (b) Comparison of response between groups (PG:UG) confirms LA, Hyp and granular insular cortex (GI) activation in PG in response to the CS presentation. Extinction modeling of (c) the response across CS presentations within the PG or (d) between PG and UG groups reveals right LA, Hyp, GI and somatosensory cortex activation (SSC). PG, n = 14; UG, n = 10.</p

Heart rate levels obtained during preliminary testing using a mock scanner.

<p>Data Mean ±SEM, n = 6 rats. Previous telemetry evidence suggests a resting heart rate of around 300–380 bpm in adult rats.</p

Consequence of early life stress on fear associated brain activation.

<p>Greater activation in response to CS presentation is observed in right and left lateral amygdalae (LA), hypothalamus (Hyp) and optic grey (optic) when ELS is compared to CON. (a) Coronal, (b) axial and (c) sagittal sections of rat brain. Control, n = 14; ELS, n = 8.</p

Brain activation during first CS presentation versus baseline.

<p>Significant regional brain activation (BOLD responses) to the first CS presentation compared to baseline. PG  =  paired group, UG  =  unpaired group. KE  =  cluster extent in voxels. T  =  peak voxel t-statistic. Clusters reaching <i>P</i>_corrected <0.05 are shown.*  =  significant (<i>P</i>_corrected <0.05) within lateral amygdala ROI. R =  right, L =  left, PVN =  paraventricular nucleus. PG, n =  14; UG, n = 10.</p