Separating the wheat from the chaff: systematic identification of functionally relevant noncoding variants in ADHD.

Attention deficit hyperactivity disorder (ADHD) is a highly heritable psychiatric condition with negative lifetime outcomes. Uncovering its genetic architecture should yield important insights into the neurobiology of ADHD and assist development of novel treatment strategies. Twenty years of candidate gene investigations and more recently genome-wide association studies have identified an array of potential association signals. In this context, separating the likely true from false associations ('the wheat' from 'the chaff') will be crucial for uncovering the functional biology of ADHD. Here, we defined a set of 2070 DNA variants that showed evidence of association with ADHD (or were in linkage disequilibrium). More than 97% of these variants were noncoding, and were prioritised for further exploration using two tools-genome-wide annotation of variants (GWAVA) and Combined Annotation-Dependent Depletion (CADD)-that were recently developed to rank variants based upon their likely pathogenicity. Capitalising on recent efforts such as the Encyclopaedia of DNA Elements and US National Institutes of Health Roadmap Epigenomics Projects to improve understanding of the noncoding genome, we subsequently identified 65 variants to which we assigned functional annotations, based upon their likely impact on alternative splicing, transcription factor binding and translational regulation. We propose that these 65 variants, which possess not only a high likelihood of pathogenicity but also readily testable functional hypotheses, represent a tractable shortlist for future experimental validation in ADHD. Taken together, this study brings into sharp focus the likely relevance of noncoding variants for the genetic risk associated with ADHD, and more broadly suggests a bioinformatics approach that should be relevant to other psychiatric disorders.

Integrated care pathways in the private sector.

ICPs are evidence- and outcome-based. The variances they expose can be analysed along with audit data. ICPs provide a useful audit tool, provided they are acted upon.

Prevention of phantom pain after major lower limb amputation by epidural infusion of diamorphine, clonidine and bupivacaine.

Phantom limb pain may appear in up to 85% of patients after amputation. There is no effective treatment. Perioperative epidural infusion of morphine and bupivacaine, alone or in combination, is effective in preventing phantom limb pain in patients with pre-existing limb pain. Serious side-effects, however, make them difficult to manage on a general ward. Clonidine has been shown to be an effective postoperative analgesia when applied epidurally. To mitigate the potentially serious side-effects of all these drugs, we have studied their combined efficiency in preventing phantom limb pain in a prospective controlled study of 24 patients undergoing lower limb amputation. In the study group (n = 13), an epidural infusion containing bupivacaine 75 mg, clonidine 150 micrograms and diamorphine 5 mg in 60 ml normal saline was given at 1-4 ml/h 24-48 h preoperatively and maintained for at least 3 days postoperatively. The control group (n = 11) received on-demand opioid analgesia. Pain was assessed by visual analogue scale at 7 days, 6 months and 1 year. At 1 year follow-up, one patient in the study group and eight patients in the control group had phantom pain (P < 0.002) and two patients in the study group versus eight patients in the control group had phantom limb sensation (P < 0.05). There was no significant improvement in stump pain. We conclude that perioperative epidural infusion of diamorphine, clonidine and bupivacaine is safe and effective in reducing the incidence of phantom pain after amputation.

Diaphragmatic fatigue in the rat.

We studied fatigue of rat diaphragm in response to repetitive brief and prolonged electrical stimulation of the phrenic nerve, at 0.2, 1-100 Hz. Low and high frequency of stimulation produced twitch and tetanic contractions in the rat diaphragm. A mean maximum twitch tension of 1.4 +/- 0.1 g was produced at 1 Hz, and a mean maximum tetanic tension of 5.6 +/- 0.3 g was obtained at 100 Hz (means +/- S.E., n = 8). Twitch and tetanic fatigue was produced at all frequencies of stimulations, but with different time scale, or duration, and with different number of stimuli delivered to the muscle. At low rates of stimulation, e.g. 10 Hz, fewer stimuli were needed to fatigue the muscle (3000 in 5 min), whereas at high rates of stimulation, e.g. 50 Hz, more stimuli were needed to fatigue the muscle (6600 in 2.2 min). The amplitude of the tetanic tensions elicited at 10 and 50 Hz, at the end of 5 or 2 min fatiguing stimulation, was 39 +/- 2.7% and 80 +/- 3.1% of their respective control tensions (2.8 +/- 0 2 g and 5.3 +/- 0.5 g, n = 8, P 0.001). It was concluded that fatigue in the rat diaphragm depended on the frequency and duration of stimulation as well as on the number of stimuli delivered to the muscle. Various mechanisms of muscle fatigue are described in the discussion to explain the observations made in the present investigation.

Assessment of neuromuscular blockade produced by atracurium in the rat diaphragm preparation. Measurements of tetanic fade, depression and recovery profile.

The effect of atracurium, a relatively new muscle relaxant, on neuromuscular transmission, in the rat diaphragm preparation, was studied, by analysing the characteristic features of tetanic fade and recovery pattern following a blocking concentration of atracurium (10 microns). Tetanic fade (TF) and peak tetanic tension (Tp) and its depression by atracurium, were analysed and the results were interpreted in terms of atracurium action at the neuromuscular junction. Atracurium reduced the sustained tetanic tension, elicited at 50 Hz for 0.5 s duration, and produced a marked tetanic fade in 38 s. Atracurium also reduced the peak tetanic tension by 40%, of the control value, in 38 s. Maximum tetanic tension was 5.7 g tension, and the time taken to completely block the tetanus was 4.75 +/- 0.15 min (means +/- SE, n = 8). Recovery from atracurium-induced blockade occurred in 30s (tetanic fade) and in 3-4 min (peak tetanic tension). It was concluded that atracurium produces a profound tetanic fade, at a time when the peak tetanic tension is reduced by only 40%. The data presented indicate that atracurium has a rapid onset of blockade, intermediate duration and a quick recovery profile at the rat neuromuscular junction.

Tetanic fade following atracurium-induced neuromuscular blockade in the rat diaphragm preparation.

1. The effect of atracurium on neuromuscular transmission was studied in the rat diaphragm preparation, by analysing the characteristics of tetanic fade and its recovery profile after using a blocking concentration of atracurium (10 mumol.litre-1). 2. Tetanic fade (TF), peak tetanic tension (Tp), and its depression, and end tetanic tension (Te), sustained tension, were analysed and compared to their respective control values before administration of atracurium. 3. The results showed that atracurium reduced the tetanic tension, i.e., the peak and end tetanic tensions, elicited at 50 Hz for 0.5 s duration, and produced a marked tetanic fade, which was developed fully in about 38 s. On the other hand, the peak tetanic tension (Tp) was only reduced by 40% (at 38 s) of its control value (5.7 g tension). The time taken to completely block Tp was about 5 min. 4. After washing out atracurium, recovery of the peak tetanic tension occurred within 3-4 min., while tetanic fade was reversed within 30 s. 5. It was concluded that atracurium produces a profound tetanic fade, at a time when the peak tetanic tension is only depressed by about 40% of the control value. The results indicated that atracurium had a powerful neuromuscular blocking action at the rat diaphragm preparation.

Effects of atropine and glycopyrrolate on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation.

1. The effects of atropine and glycopyrrolate on neuromuscular transmission and on muscle contraction, were studied, in the rat diaphragm preparation, by analyzing their effects on the indirectly (and directly)-elicited twitch (0.2 Hz), tetanic (50 Hz for 20 sec duration), post-tetanic twitch responses (at 5 sec after the tetanus), and on the phenomenon of post-tetanic twitch potentiation (PTP), which is thought to be of a presynaptic origin, i.e. due to increased transmitter release. 2. Atropine (0.001-10 microM) increased the indirectly-elicited twitch tension by 22 +/- 2.1% (control 0.9 +/- 0.1 g, P less than 0.02), the tetanus by 15 +/- 1.1% (control 3.9 +/- 0.7 g, P less than 0.05), the post-tetanic twitch response by 33 +/- 3.1% (control 1.2 +/- 0.1 g, P less than 0.01) and the PTP value by 36 +/- 1.9% (control 33 +/- 2.3%, P less than 0.01, means +/- SEM = 6). 3. Atropine (0.001-10 microM) had little effect on the directly-elicited twitch tension, but in high concentrations (e.g. 20 microM), it blocked the twitch tension. 4. In contrast, glycopyrrolate (0.1-100 microM) had little effect on the twitch tension (direct or indirect), but it significantly reduced the tetanus (by 38 +/- 3.5%, P less than 0.01), the post-tetanic twitch response (by 17 +/- 1.2%, P less than 0.05) and the PTP values (by 24 +/- 3.1% P less than 0.02). 5. In the presence of hemicholinium (1.3 microM) the responses to atropine and glycopyrrolate were altered (decreased), indicating a possible action on presynaptic mechanism of transmission. 6. It is concluded that atropine and glycopyrrolate produce different (opposite) effects at the rat neuromuscular junction, atropine enhances whereas glycopyrrolate depresses neuromuscular transmission. The effects of these two antimuscarinic drugs may be exerted at the presynaptic nerve terminals, i.e. on presynaptic muscarinic receptors, which are involved in the feedback mechanism of transmitter release.

Tetanic fade during neuromuscular blockade produced by atracurium in the rat diaphragm preparation.

In the present investigation, we studied and measured the phenomenon of tetanic fade and peak tetanic tension depression in the rat diaphragm preparation in the presence of a blocking concentration of atracurium (e.g., 10 mumol.l-1). Atracurium (10 mumol.l-1) produced a pronounced tetanic fade (i.e., 47-69% reduction of total sustained tetanic tension) at a time (15 s) when it reduced the peak tetanic tension by only 25%. The time course for total tetanic fade was 30-35 s, whereas the time taken for complete peak tetanic tension depression was 3-3.5 min, suggesting that the two effects were produced via different mechanisms, involving presynaptic and postsynaptic mechanism. It was concluded that atracurium produces a profound tetanic fade, with respect to its effect on twitch or tetanic tension, suggesting that the drug is a potent neuromuscular blocker, with rapid onset of blockade.

Glycopyrrolate intensifies neuromuscular blockade produced by atracurium in the rat diaphragm preparation.

The effect of glycopyrrolate (Glycopyrronium, a muscarinic antagonist) (10 mumol.litre-1) and neostigmine (1 mumol.litre-1) on atracurium (0.1-100 mumol.litre-1) - induced neuromuscular blockade was studied in the rat isolated phrenic nerve-diaphragm preparation, to see if glycopyrrolate intensified the neuromuscular blockade produced by atracurium in this preparation. Atracurium had a rapid onset of blockade, reaching a complete block in 30-40 s. Glycopyrrolate had no significant effect on indirectly-elicited twitch (0.2 Hz) tension, whereas it significantly increased atracurium-induced depression of twitch tension and shortened the time needed to a complete block by 10 s. Combinations of glycopyrrolate+ neostigmine, only slightly reversed atracurium-induced blockade, if compared to the reversal by neostigmine alone. The mean concentrations to produce 50% depression of twitch tension were: 1.6 +/- 0.1 (atracurium), 0.3 +/- 0.1 (atracurium +glycopyrrolate), 4.8 +/- 0.2 (atracurium +neostigmine) and 2.7 +/- 0.1 mumol.litre-1 (atracurium +glycopyrrolate +neostigmine) (means +/- SEM, n = 6, P less than 0.001, with respect to control value of atracurium alone). It was concluded that glycopyrrolate enhanced atracurium-induced neuromuscular blockade in the rat diaphragm preparation, and that this effect should be noted when dosing glycopyrrolate in man.

Atropine sulphate enhances neuromuscular transmission in the rat.

The effect of atropine (0.001-10 mumol.l-1) on neuromuscular transmission in the rat hemidiaphragm preparation was investigated by analysing its effects on directly and indirectly-elicited twitch, tetanic, post-tetanic twitch responses and on the phenomenon of post-tetanic twitch potentiation. The effect of atropine on contractions produced by endogenous acetylcholine (ACh) or exogenous ACh (added directly into organ bath containing muscle) was studied in rat ileum. The results showed that atropine in low concentrations (1 mumol.l-1 or less), enhanced the indirectly-elicited twitch, tetanic and post-tetanic twitch responses in the rat diaphragm preparation. The mean EC50 value of atropine-induced increase in twitch tension was 0.08 +/- 0.01 mumol.l-1 (mean +/- s.e. mean, n = 6). Atropine had little effect on directly-elicited twitch tension, but in high concentrations (10 mumol.l-1 or more), it reduced the directly, and indirectly-elicited twitch contractions and produced a neuromuscular block in the rat diaphragm preparation. Atropine increased the contraction produced, in rat ileum, by endogenous ACh, i.e. ACh released from the phrenic nerve stimulated at 50 Hz for 20 s duration (control contraction: 1.3 +/- 0.1 g, contraction in atropine: 1.7 +/- 0.2 g). In contrast, atropine significantly reduced the contraction produced by exogenous ACh in the same preparation (control contraction: 3.0 +/- 0.5 g, atropine: 2.0 +/- 0.1 g), suggesting that a different mechanism may be involved in the latter effect of atropine. It was concluded that atropine, in low concentration, enhanced neuromuscular transmission, possibly via a presynaptic mechanism. In high concentration, atropine may reduce and then block transmission, possibly via pre- and postsynaptic mechanisms.

Comparative effects of atropine and oxotremorine on neuromuscular transmission at the rat hemidiaphragm preparation.

The effects of atropine (0.001-10 microM) and oxotremorine (0.51-51.3 microM) on neuromuscular transmission and muscle contraction were studied by analysing their effects on the twitch, tetanic and post-tetanic twitch responses and on the phenomenon of post-tetanic twitch potentiation (PTP) in a rat phrenic nerve-diaphragm preparation. In addition, the effects of atropine (0.1 microM) and oxotremorine (0.6 microM) were studied on the contractions produced in rat ileum by superfusate from repetitively stimulated phrenic nerve diaphragm preparation. The results showed that atropine had a dual action at the neuromuscular junction; at low concentrations (0.001-1.0 microM) enhanced, whereas at high concentration (greater than 1.0 microM) decreased the twitch, tetanic and post-tetanic twitch responses in the rat diaphragm preparation. Oxotremorine, on the other hand, always reduced these responses and also reduced the contractions produced by diaphragm perfusate and by exogenous acetylcholine (ACh) in the rat ileum. Since atropine enhanced the contractions, in rat diaphragm, to electrical stimulation, and those in rat ileum, to superfusate of stimulated diaphragm preparation, the results may suggest that atropine has a presynaptic action at the rat neuromuscular junction. The presence of more than one type of presynaptic muscarinic autoreceptors is discussed in relation to the results obtained.

Atropine enhances neuromuscular transmission in humans.

The effect of atropine (1-10 micrograms . kg-1) on neuromuscular transmission in humans was studied by analysing its effects on the amplitude of indirectly-elicited twitch (0.2 Hz) and tetanic (50 and 100 Hz for 1 s duration) contractions. Six patients, free from any neuromuscular disorders, undergoing orthopaedic surgery, were included in the present study. The patients received either no premedication or the oral benzodiazepine, temazepam, 30 mg 1-2 h pre-operatively. Anaesthesia was induced with propofol (1-2 mg . kg-1, i.v., over 20 s). The patients breathed no less than 30% oxygen in nitrous oxide, halothane (1%) or enflurane (1-2%). Incremental doses of fentanyl (50-100 micrograms) were given to provide additional analgesia. The ulnar nerve was stimulated, supramaximally, at the wrist, and control mechanical responses of the adductor pollicis muscle, to nerve stimulation at 50 and 100 Hz for 1 s duration, were recorded. Theses responses were repeated at 2, 5 and 10 min intervals, after injection of atropine (1-10 micrograms . kg-1). At the same time, heart rate and blood pressures (systolic and diastolic) were recorded. The results showed that atropine enhanced the tetanic contractions, elicited at 50 and 100 Hz for 1 s duration, by 27 +/- 1.2% (50 Hz and atropine, control 220 +/- 13 g tension), and by 43 +/- 7% (100 Hz and atropine, control 333 +/- 26 g tension) at the 5 min intervals (mean +/- S.E., n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of starvation on the planarian worm Polycelis tenuis Iijima.

Employing a combination of microscopical, biochemical and autoradiographic techniques, the primary effects of starvation on adult polycelis tenuis have been studied. Over a five week period of starvation there is on average a 32% decrease in the size of the organism. This decrease is contributed to by a reduction in mitosis and an increase in cell shrinkage autolysis and death. During starvation (following a sharp rise in RNA synthesis) there is a distinct sequence of events; four peaks of acid phosphatase activity can be resolved. The first is associated with the immediate response of the gastrodermis to feeding; the second (after 6 to 7 days) with increased autophagy and dedifferentiation in the gland cells and with muscle lysis of cells. The third peak (after 14 to 15 days) is contributed to largely by the lysis of cells in the gut and the fourth peak (after 25 to 26 days) is caused by an extensive lysis of the reproductive system. Fine structural changes involving increased intracellular vacuolation, autophagy, crinophagy, atrophy of muscle, increased intercellular space and loss of basement membrane matrix have been related to changes in enzyme pattern. Nerve cells appear unchanged throughout the first five weeks of starvation. Pigment and gland cells loose their characteristic granules, dedifferentiate and become morphologically similar to the undifferentiatied neoblasts. Dedifferentiation and the mechanisms involved in the survival of starvation are discussed.