Jill E. Maddison

Brain γ-aminobutyric acid receptor binding is normal in rats with thioacetamide-induced hepatic encephalopathy despite elevated plasma γ-aminobutyric acid-like activity

Brain gamma-aminobutyric acid (GABA) receptor density, affinity, and function, and plasma GABA-like activity were determined in rats with acute hepatic encephalopathy induced by an intraperitoneal injection of thioacetamide. In addition, the effect of various stress factors on brain GABA binding was assessed. Plasma GABA-like activity was significantly increased in rats with thioacetamide-induced hepatic encephalopathy compared with rats injected with vehicle alone (1506 +/- 993 nM, n = 7 vs. 367 +/- 97 nM, n = 9, mean +/- SD; p less than 0.001). In contrast, there were no alterations in either brain GABA receptor binding or in GABA-enhanced benzodiazepine binding in rats with hepatic encephalopathy when compared with relevant controls. However, rats that had received intraperitoneal injections of thioacetamide or vehicle (0.15 M NaCl) had significantly more low-affinity GABA receptors than rats that had neither been injected nor handled before killing (8769 +/- 1101 vs. 2710 +/- 757 fmol/mg protein, mean +/- SEM, p less than 0.001). We concluded that stress factors appear to be important causes of altered brain GABA binding. Brain GABA receptor binding and function, however, are unaltered in rats with thioacetamide-induced hepatic encephalopathy despite elevated plasma GABA-like activity.

Alterations in Cortical [3H]Kainate and α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Abstract: Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L‐[3H]Glutamate, (+)‐[3H]‐5‐methyl‐10, 11 ‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate ([3H]MK‐801), [3H]kainate, and α‐[3H]‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L‐[3H]‐glutamate or [3H]MK‐801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low‐affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non‐N‐methyl‐D‐aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.

Influence of the oestrous cycle on L-glutamate and L-aspartate transport in rat brain synaptosomes

Oestrous cycle and sex differences in sodium-dependent transport of L-[3H]glutamate and L-[3H]aspartate were investigated employing well washed synaptosomes prepared from rat brain cortex. Transport was best analysed on the basis of two components, a high and low affinity transport site. Oestrous cycle and sex differences were observed for both substrates. The high affinity transporter displayed highest affinity for glutamate transport in synaptosomes from female rats during proestrous and oestrous. This differed significantly from glutamate transport during dioestrous and in male rats. High affinity aspartate transport displayed highest affinity during oestrous and differed significantly from transport during dioestrous. Maximal velocity of high affinity glutamate transport was higher in synaptosomes from females during dioestrous compared with oestrous and lower in synaptosomes from male rats when compared with female rats in dioestrous and metoestrous. The low affinity sodium-dependent glutamate transporter displayed a 10-fold higher affinity for glutamate during proestrous than during the other three phases of oestrous and in male rats. Exogenously applied oestradiol and progesterone to synaptosomes from male rats showed no effect on glutamate or aspartate transport. No acute effect of oestradiol or progesterone on glutamate currents in oocytes expressing EAAT1 or EAAT2 subtype of glutamate transporter was observed. These results suggest hormonal regulation of high and low affinity sodium-dependent excitatory amino acid transporters over the four day oestrous cycle in synaptosomes from rat cortex. This regulation is unlikely to be due to a direct effect of oestradiol or progesterone on glutamate transporters.

Subcellular localization of rat brain insulin binding sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, γ‐aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mi‐tochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r= 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.

Synaptosomal and brain slice cerebrocortical [3H]L-glutamate uptake in a rat model of chronic hepatic encephalopathy

Cerebrocortical [3H]L-glutamate uptake was examined using brain slices and synaptosomes obtained from rats with portal vein and bile duct ligation. In addition, the effect of in vitro addition of 5 mM ammonia on glutamate uptake parameters was determined. There was no significant difference in brain slice or synaptosomal glutamate uptake in rats with portal vein and bile duct ligation compared to control rats. In vitro addition of ammonia had no effect on uptake kinetics in either brain slices or synaptosomes. These results suggest that glutamate uptake kinetics are not perturbed in this animal model of chronic hepatic encephalopathy.

What is the veterinary professional identity? Preliminary findings from web-based continuing professional development in veterinary professionalism

Professionalism and professional skills are increasingly being incorporated into veterinary curricula; however, lack of clarity in defining veterinary professionalism presents a potential challenge for directing course outcomes that are of benefit to the veterinary professional. An online continuing education course in veterinary professionalism was designed to address a deficit in postgraduate support in this area; as part of this course, delegates of varying practice backgrounds participated in online discussions reflecting on the implications of professional skills for their clinical practice. The discussions surrounding the role of the veterinary professional and reflecting on strengths and weaknesses in professional skills were analysed using narrative methodology, which provided an understanding of the defining skills and attributes of the veterinary professional, from the perspectives of those involved (i.e. how vets understood their own career identity). The veterinary surgeon was understood to be an interprofessional team member, who makes clinical decisions in the face of competing stakeholder needs and works in a complex environment comprising multiple and diverse challenges (stress, high emotions, financial issues, work–life balance). It was identified that strategies for accepting fallibility, and those necessary for establishing reasonable expectations of professional behaviour and clinical ability, are poorly developed.

Veterinary e-CPD: a new model for providing online continuing professional development for the veterinary profession.

Continuing professional development (CPD) is widely recognized as an important element in effective lifelong learning for veterinary surgeons. Traditional methods of CPD do not suit all learners, as issues such as location, time, cost, and structure sometimes prevent individuals from completing the required number of CPD study hours per year. The rapid development of the Internet, and with it the increasing scope and sophistication of e-learning, provides new opportunities to address some of these constraints on the provision of CPD. This article describes one way in which e-learning has been deployed effectively to support veterinary surgeons in practice. Since 2003, a series of six-week e-CPD courses has been offered by the Royal Veterinary College (RVC) in an online format, with no face-to-face teaching component. Participants enrolled in courses from May 2006 to January 2007 were found to come from 23 different countries. Analysis of feedback forms indicates a general satisfaction with this new way of studying, with a significant majority of participants stating that they would wish to use this approach again in future. The feedback indicates that e-learning can offer an effective alternative to traditional face-to-face courses and that its popularity is likely to grow in future as veterinarians become increasing familiar with and confident about working online.

L-glutamate and γ-aminobutyric acid uptake in synaptosomes from the cerebral cortex of dogs with congenital chronic hepatic encephalopathy

High affinity [3H]γ-aminobutyric acid (GABA) and [3H]L-glutamate uptake were determined in synaptosomes prepared from the cerebral cortex of dogs with congenital hepatic encephalopathy and control dogs. The Km value for GABA uptake was increased by 35% but there was a concomitant 34% increase in Vmax suggesting that GABA uptake capacity was not changed in HE dogs. In contrast, mean Vmax for glutamate uptake in HE dogs was 85% greater than mean Vmax in control dogs; mean Km was increased by 25% in HE dogs. Therefore, overall synaptosomal high affinity glutamate uptake capacity was increased in HE dogs compared to controls.

Erythrocyte protoporphyrin concentrations in clinically normal cats and cats with lead toxicity

Erythrocyte protoporphyrin (EPP) and blood lead concentrations were determined in 91 clinically healthy cats living in the inner suburban area of Sydney, Australia. The mean EPP concentration was 223.4 +/- 186.1 micrograms litre-1 whole blood and the mean blood lead concentration 0.62 +/- 0.25 mumol litre-1. EPP concentrations were also monitored in three cats with confirmed lead toxicity--at the time of diagnosis and one week and one month after chelation therapy with calcium EDTA. EPP concentrations were elevated in two cats and within the normal range in the third cat at the time of diagnosis. EPP concentration were higher in two cats one week after treatment than at the time of diagnosis. One month after chelation therapy, EPP concentrations were normal in two cats but still substantially elevated in the third cat although its blood lead concentration had returned to normal and all clinical signs of lead toxicity had resolved. It was determined that the predominant form of protoporphyrin present in cats with lead toxicity was zinc protoporphyrin. The EPP assay may have limited value in the diagnosis of acute lead toxicity and in monitoring the success of chelation therapy in cats.

CNQX binding to non-NMDA glutamate receptors in canine cerebro-cortical crude synaptosomal membranes: Pharmacological characterization and comparison of binding parameters in dogs with congenital portosystemic encephalopathy and control dogs

The pharmacological profile and binding characteristics of the non-NMDA antagonist of glutamate receptors [3H]6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), were investigated in triton-washed crude synaptosomal membranes prepared from canine cerebral cortex. [3H]CNQX binding was inhibited by various glutamate agonists and antagonists, the rank order of potency being CNQX>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)=quisqualate=kainate>glutamate. Two binding sites for [3H]CNQX were apparent when non-specific binding (NSB) was defined with unlabelled CNQX. In contrast, when NSB was defined with saturating concentrations of unlabelled AMPA and kainate, only one binding site was identified which corresponded to the high affinity site identified when CNQX was used to define NSB. No physiologically relevant differences were found in binding parameters for [3H]CNQX membranes from dogs with congenital portosystemic encephalopathy (PSE) when compared with control dogs. The affinity constant (Ki of AMPA displacement of [3H]CNQX binding was not significantly different in PSE dogs compared with control dogs. These results suggest that the antagonist site on cortical non-NMDA receptors is not perturbed in dogs with congenital PSE.