Gregory Jacobson

Excitatory effects of gap junction blockers on cerebral cortex seizure-like activity in rats and mice

Purpose:  The role of gap junctions in seizures is an area of intense research. Many groups have reported anticonvulsant effects of gap junction blockade, strengthening the case for a role for gap junctions in ictogenesis. The cerebral cortex is underrepresented in this body of research. We have investigated the effect of gap junction blockade on seizure‐like activity in rat and mouse cerebral cortex slices.

Connexin36 knockout mice display increased sensitivity to pentylenetetrazol-induced seizure-like behaviors

OBJECTIVE Large-scale synchronous firing of neurons during seizures is modulated by electrotonic coupling between neurons via gap junctions. To explore roles for connexin36 (Cx36) gap junctions in seizures, we examined the seizure threshold of connexin36 knockout (Cx36KO) mice using a pentylenetetrazol (PTZ) model. METHODS Mice (2-3months old) with Cx36 wildtype (WT) or Cx36KO genotype were treated with vehicle or 10-40mg/kg of the convulsant PTZ by intraperitoneal injection. Seizure and seizure-like behaviors were scored by examination of video collected for 20min. Quantitative real-time PCR (QPCR) was performed to measure potential compensatory neuronal connexin (Cx30.2, Cx37, Cx43 and Cx45), pannexin (PANX1 and PANX2) and gamma-aminobutyric acid type A (GABA(A)) receptor α1 subunit gene expression. RESULTS Cx36KO animals exhibited considerably more severe seizures; 40mg/kg of PTZ caused severe generalized (≥grade III) seizures in 78% of KO, but just 5% of WT mice. A lower dose of PTZ (20mg/kg) induced grade II seizure-like behaviors in 40% KO vs. 0% of WT animals. There was no significant difference in either connexin, pannexin or GABA(A) α1 gene expression between WT and KO animals. CONCLUSION Increased sensitivity of Cx36KO animals to PTZ-induced seizure suggests that Cx36 gap junctional communication functions as a physiological anti-convulsant mechanism, and identifies the Cx36 gap junction as a potential therapeutic target in epilepsy.

GABAergic compensation in connexin36 knock-out mice evident during low-magnesium seizure-like event activity

Gap junctions within the cerebral cortex may facilitate cortical seizure formation by their ability to synchronize electrical activity. To investigate this, one option is to compare wild-type (WT) animals with those lacking the gene for connexin36 (Cx36 KO); the protein that forms neuronal gap junctions between cortical inhibitory cells. However, genetically modified knock-out animals may exhibit compensatory effects; with the risk that observed differences between WT and Cx36 KO animals could be erroneously attributed to Cx36 gap junction effects. In this study we investigated the effect of GABA(A)-receptor modulation (augmentation with 16μM etomidate and blockade with 100μM picrotoxin) on low-magnesium seizure-like events (SLEs) in mouse cortical slices. In WT slices, picrotoxin enhanced both the amplitude (49% increase, p=0.0006) and frequency (37% increase, p=0.005) of SLEs; etomidate also enhanced SLE amplitude (18% increase, p=0.003) but reduced event frequency (25% decrease, p<0.0001). In Cx36 KO slices, the frequency effects of etomidate and picrotoxin were preserved, but the amplitude responses were abolished. Pre-treatment with the gap junction blocker mefloquin in WT slices did not significantly alter the drug responses, indicating that the reduction in amplitude seen in the Cx36 KO mice was not primarily mediated by their lack of interneuronal gap junctions, but was rather due to pre-existing compensatory changes in these animals. Conclusions from studies comparing seizure characteristics between WT and Cx36 KO mice must be viewed with a degree of caution because of the possible confounding effect of compensatory neurophysiological changes in the genetically modified animals.

Role of Cx36 gap junction modulation in general anaesthetic anticonvulsant action.

Many GABAergic anaesthetics reduce gap junction coupling but it is currently unknown whether this effect contributes to anaesthetic anticonvulsant action. In this study we examined the possible role of connexin36 gap junctions in the anticonvulsant action of isoflurane and compared this to etomidate, an anaesthetic known for having proconvulsant effects. We compared the effect of anaesthetic concentrations of isoflurane (1 MAC) and etomidate (16 microM) on low-magnesium-induced interictal-like activity in isolated neocortical slices. The effect of connexin36 gap junction blockade was explored by comparing effects in slices from wild-type mice and from a transgenic mouse strain lacking the gene for connexin36. In slices from wild-type mice, both isoflurane (1 MAC) and etomidate (16 microM) reduced interictal-like event frequency; mean(S.D.) reduction of 44(13)% (P<0.0001) and 25(24)% (P<0.0001), respectively. The reduction in event frequency was greater for isoflurane (P<0.005). Isoflurane had no effect on the amplitude of interictal-like events, but event amplitude was enhanced by etomidate (18(28)% increase, P<0.005). The capacity for isoflurane to reduce event frequency was significantly reduced, but not eliminated in slices from connexin36 knock-out mice (33(15)% reduction, P<0.05 for the difference with wild-type), while that of etomidate remained unchanged (23(39)% reduction). The etomidate-mediated increase in event amplitude was eliminated in connexin36 knock-out slices. The results from this study support the hypothesis that the anticonvulsant effect of isoflurane is in part mediated by gap junction blockade. The role of gap junction modulation by etomidate is more complicated and may be important in the mechanism of action of etomidates proconvulsant effects.

Association of Innate Immune Single-Nucleotide Polymorphisms with the Electroencephalogram During Desflurane General Anaesthesia

The electroencephalogram (EEG) records the electrical activity of the brain and enables effects of anaesthetic drugs on brain functioning to be monitored. Identification of genes contributing to EEG variability during anaesthesia is important to the clinical application of anaesthesia monitoring and may provide an avenue to identify molecular mechanisms underlying the generation and regulation of brain oscillations. Central immune signalling can impact neuronal activity in the brain and accumulating evidence suggests an important role for cytokines as neuronal modulators. We tested 21 single-nucleotide polymorphisms (SNPs) in immune-related genes for associations with three anaesthesia-induced EEG patterns; spindle amplitude, delta power and alpha power, during general anaesthesia with desflurane in 111 patients undergoing general, gynaecological or orthopaedic surgery. Wide inter-patient variability was observed for all EEG variables. MYD88 rs6853 (p = 6.7 × 10−4) and IL-1β rs1143627 in conjunction with rs6853 (p = 1.5 × 10−3) were associated with spindle amplitude, and IL-10 rs1800896 was associated with delta power (p = 1.3 × 10−2) suggesting involvement of cytokine signalling in modulation of EEG patterns during desflurane anaesthesia. BDNF rs6265 was associated with alpha power (p = 3.9 × 10−3), suggesting differences in neuronal plasticity might also influence EEG patterns during desflurane anaesthesia. This is the first study we are aware of that has investigated genetic polymorphisms that may influence the EEG during general anaesthesia.

The role of connexin36 gap junctions in modulating the hypnotic effects of isoflurane and propofol in mice

Gap junction blockade is a possible mechanism by which general anaesthetic drugs cause unconsciousness. We measured the sensitivity of connexin36 knockout mice to the hypnotic effects of isoflurane and propofol. The experimental endpoint was recovery of the righting reflex of the anaesthetised animals during 0.2% step‐reductions in isoflurane concentration, or following intraperitoneal injection of propofol (100 mg.kg−1). Connexin36 knockout animals were more sensitive to the hypnotic effects of isoflurane than ‘normal’ wild‐type animals. The half maximal effective concentration (EC50) for recovery of righting reflex was 0.37% for connexin36 knockout vs 0.49% for wild‐type animals (p < 0.001). For propofol, connnexin36 knockout animals showed more rapid loss of righting reflex than wild‐type animals (mean (SD) 2.8 (0.13) vs 3.8 (0.27) min); and young (< 60 days) connexin36 knockout animals remained anaesthetised for longer than young wild‐type mice (47.2 (2.9) vs 30.5 (1.7) min; p < 0.00001). These findings suggest that the hypnotic effects of anaesthetic drugs may be moderately enhanced by gap junction blockade.

Antimicrobial peptides within the Yellowtail Kingfish (Seriola lalandi)

ABSTRACT A number of Seriola species are currently farmed or being investigated as future aquaculture species in countries around the world. However they face a number of issues and limitations which will need to be overcome to ensure future stability and growth, one of which are disease outbreaks. Despite this, very little has been done to understand the immune system of Seriola species and very few immune genes have been characterised. Antimicrobial peptides (AMP) are naturally occurring low molecular weight polypeptides that play a major role in an organisms immune system and act effectively as a first line of defence. This investigation isolates the full length cDNA sequences of two AMPs, piscidin and hepcidin from the yellowtail kingfish (Seriola lalandi). The full‐length cDNA of the piscidin gene encodes a 65 amino acid prepropeptide, containing a 25‐residue peptide, predicted to form an amphipathic helix‐loop‐helix structure. Phylogenetic analysis using fish piscidin sequences, showed that this AMP is only found in bony fish within the Acanthomorpha clade and that a possible three groups within the piscidin family exists, with S. lalandi belonging to a particular group. The full‐length cDNA of the hepcidin gene encodes a 90 amino acid preprohepcidin, which contains a typical RX(R/K)R motif for cleavage of the mature peptide which comprises of eight conserved cysteine residues. Phylogenetic analysis of known vertebrate hepcidin antimicrobial peptide (HAMP) sequences, shows sequences from the Neoteleostei clade of bony fish form two very separate groups, HAMP1 and HAMP2, with the S. lalandi hepcidin gene grouped with the HAMP1 sequences. HAMP2 sequences are found to have multiple copies within fish and genome analysis showed very clearly that these two groups of genes are located on separate regions on the genome, with the multiple HAMP2 copies formed from tandem gene duplications. Lastly, using qPCR the expression of the S. lalandi piscidin gene within healthy fish was highest within, spleen and gills and lowest in liver, whereas hepcidin was highest in the liver with little or no expression in the spleen and gills. HighlightsTwo antimicrobial peptides have been cloned within S. lalandi.Fish piscidin genes can be classified into three distinct groups, with S. lalandi piscidin belonging to one of them.Multiple hepcidin genes in Neoteleostei fish form two clear groups, with S. lalandi hepcidin belonging to one of these groups.Healthy S. lalandi have highest expression of piscidin within spleen and gills, whilst hepcidin is highest in the liver.

Omics and cytokine discovery in fish : Presenting the Yellowtail kingfish (Seriola lalandi) as a case study

ABSTRACT A continued programme of research is essential to overcome production bottlenecks in any aquacultured fish species. Since the introduction of genetic and molecular techniques, the quality of immune research undertaken in fish has greatly improved. Thousands of species specific cytokine genes have been discovered, which can be used to conduct more sensitive studies to understand how fish physiology is affected by aquaculture environments or disease. Newly available transcriptomic technologies, make it increasingly easier to study the immunogenetics of farmed species for which little data exists. This paper reviews how the application of transcriptomic procedures such as RNA Sequencing (RNA‐Seq) can advance fish research. As a case study, we present some preliminary findings using RNA‐Seq to identify cytokine related genes in Seriola lalandi. These will allow in‐depth investigations to understand the immune responses of these fish in response to environmental change or disease and help in the development of therapeutic approaches. HIGHLIGHTSSequence genomes and transcriptomes has accelerated cytokine discovery within fish.RNA‐Seq on selected tissues of S. lalandi identified a large number of cytokine and receptor genes.Cytokine and receptor genes discovered using transcriptomic approaches will allow the effective monitoring of fish immunity.

Randomized controlled trial of the effect of depth of anaesthesia on postoperative pain

BACKGROUND Our hypothesis was that deep anaesthesia, as estimated by a low target bispectral index (BIS) of 30-40, would result in less postoperative pain than that achieved at a conventional depth of anaesthesia. METHODS We undertook a randomized double-blind controlled study at two tertiary teaching hospitals in New Zealand (2010-1) recruiting 135 adult patients ASA I-II presenting for non-emergent surgery under general anaesthesia requiring tracheal intubation. Anaesthesia was maintained with desflurane and a multimodal analgesia regimen comprising fentanyl infusion, i.v. paracetamol, and parecoxib. Patients were randomly assigned to either a low BIS (30-40) group or a high BIS (45-60) group. Desflurane concentrations were titrated to achieve these targets. Postoperative pain was assessed by: the pain on awakening (0-10, verbal rating scale, VRS(awake)) in the post-anaesthetic care unit; pain on activity at 20-24 h after operation (VRS(d1A)); and the rate of morphine patient-controlled analgesia (PCA) usage over the first 24 h. RESULTS There was no statistically significant difference between the two groups for any of the pain scores. The median [inter-quartile range (IQR)] VRS(awake) was 4.0 (0-8) for the low and 4.0 (0-8) for the high BIS groups (P=0.56). The median (IQR) VRS(d1A) was 3.0 (1-5) for the low and 3.0 (1.5-4.5) for the high BIS groups (P=0.83). The median PCA morphine consumption in the low BIS group was 0.61 mg h(-1) (0.04-1.5) vs 0.43 mg h(-1) (0-1.59) in the high BIS group (P=0.98). CONCLUSIONS We conclude that there is no clinically useful analgesic effect of a deep anaesthesia regimen.

Sleep and stress hormone responses to training and competition in elite female athletes

ABSTRACT Stress hormone and sleep differences in a competition versus training setting are yet to be evaluated in elite female team-sport athletes. The aim of the current study was to evaluate salivary cortisol and perceptual stress markers during competition and training and to determine the subsequent effects on sleep indices in elite female athletes. Ten elite female netball athletes (mean ± SD; age: 23 ± 6 years) had their sleep monitored on three occasions; following one netball competition match (MATCH), one netball match simulation session (TRAIN), and one rest day (CONTROL). Perceived stress values and salivary cortisol were collected immediately pre- (17:15 pm) and post-session (19:30 pm), and at 22:00 pm. Sleep monitoring was performed using wrist actigraphy assessing total time in bed, total sleep time (TST), efficiency (SE%), latency, sleep onset time and wake time. Cortisol levels were significantly higher (p < .01) immediately post MATCH compared with TRAIN and CONTROL (mean ± SD; 0.700 ± 0.165, 0.178 ± 0.127 and 0.157 ± 0.178 μg/dL, respectively) and at 22:00 pm (0.155 ± 0.062, 0.077 ± 0.063, and 0.089 ± 0.083 μg/dL, respectively). There was a significant reduction in TST (−118 ± 112 min, p < .01) and SE (−7.7 ± 8.5%, p < .05) following MATCH vs. TRAIN. Salivary cortisol levels were significantly higher, and sleep quantity and quality were significantly reduced, following competition when compared to training and rest days.