Global transgenic upregulation of KCC2 confers enhanced diazepam efficacy in treating sustained seizures.

Reduced anticonvulsant efficacy of benzodiazepines is a problem in the treatment of status epilepticus, with up to 50% of patients failing to respond to their first dose. KCC2 is a neuronal K+ -Cl- co-transporter that helps set and maintain intracellular Cl- concentrations. KCC2 functional downregulation is a potential contributor to benzodiazepine resistance. We tested this idea using male and female doxycycline-inducible, conditional transgenic mice to increase the functional expression of KCC2 in pyramidal neurons. We administered mice with two doses of the chemoconvulsant kainic acid (5 mg/kg, i.p.) 60 min apart and quantified the resultant seizures with electroencephalography (EEG) recordings. Overexpression of KCC2 prior to the chemoconvulsant challenge did not affect seizure latency or other measures of seizure severity, but it did increase diazepam's efficacy in stopping EEG seizures. Spike rate, time in seizure, and EEG spectral power following diazepam (5 mg/kg, i.p) were all significantly lower in KCC2 overexpression mice as compared to control mice. Our results indicate that, in the context of benzodiazepine resistance during sustained seizures, addressing impaired Cl- homeostasis alone appreciably improves the efficacy of γ-aminobutyric acid (GABA)ergic inhibition. We therefore suggest the simultaneous targeting of KCC2 and GABAA receptors as a pathway for improving current anticonvulsant therapeutic strategies.

The effect of route of administration on the enantioselective pharmacokinetics of ketamine and norketamine in rats.

BACKGROUND: Ketamine has been shown to produce a rapid and potent antidepressant response in patients with treatment-resistant depression. Currently ketamine is most commonly administered as a 40-minute intravenous infusion, though it is unknown whether this is the optimal route of administration. AIMS: To determine the plasma concentration time course of the R- and S-enantiomers of ketamine and norketamine following administration of ketamine by four different routes of administration. METHODS: Plasma from conscious non-anaesthetised rats was collected following administration of ketamine by either subcutaneous (SC), intramuscular (IM), intravenous infusion (IVI) or intravenous bolus (IVB) routes of administration. Concentrations of the enantiomers of ketamine and norketamine were determined by LC/MS. RESULTS: Administration by the SC, IM and IVI routes produced an overall similar drug exposure. In contrast, administration by the IVB route produced approximately 15-fold higher peak plasma concentrations for the enantiomers of ketamine and an approximately four-fold lower AUC for the enantiomers of norketamine. CONCLUSIONS: Route of administration can significantly influence ketamine and norketamine exposures. These differences may influence safety and tolerability, and potentially drug efficacy in humans. This knowledge adds to current research into the optimisation of the use of ketamine for the treatment of depression.

Acute low-dose ketamine produces a rapid and robust increase in plasma BDNF without altering brain BDNF concentrations.

Peripheral BDNF changes after ketamine administration have been proposed as a biomarker for brain BDNF changes. However, published data are conflicting and come from studies in paired animal groups. This study determined the time course of plasma BDNF concentrations following the administration of a single 10 mg/kg dose of ketamine by different routes of administration in rats. Brain BDNF concentrations in prefrontal cortex, hippocampus and cortex were measured in the same animals. Ketamine administration resulted in a rapid and robust increase in plasma BDNF concentrations that were sustained for 240 min. In contrast, there were no changes in brain BDNF concentrations in prefrontal cortex, hippocampus or cortex and there were no correlations between peripheral and central BDNF concentrations. These data suggest that peripheral BDNF is unlikely to be a useful biomarker of acute central BDNF changes following ketamine.

Validation of optical voltage reporting by the genetically encoded voltage indicator VSFP-Butterfly from cortical layer 2/3 pyramidal neurons in mouse brain slices.

Understanding how behavior emerges from brain electrical activity is one of the ultimate goals of neuroscience. To achieve this goal we require methods for large-scale recording of the electrical activity of specific neuronal circuits. A very promising approach is to use optical reporting of membrane voltage transients, particularly if the voltage reporter is genetically targeted to specific neuronal populations. Targeting in this way allows population signals to be recorded and interpreted without blindness to neuronal diversity. Here, we evaluated the voltage-sensitive fluorescent protein, VSFP Butterfly 2.1, a genetically encoded voltage indicator (GEVI), for monitoring electrical activity of layer 2/3 cortical pyramidal neurons in mouse brain slices. Standard widefield fluorescence and two-photon imaging revealed robust, high signal-to-noise ratio read-outs of membrane voltage transients that are predominantly synaptic in nature and can be resolved as discrete areas of synaptically connected layer 2/3 neurons. We find that targeted expression of this GEVI in the cortex provides a flexible and promising tool for the analysis of L2/3 cortical network function.

Cardiac electrographic and morphological changes following status epilepticus: effect of clonidine.

PURPOSE: Status epilepticus has been increasingly associated with cardiac injury in both clinical and animal studies. Our group has previously shown that excitotoxic seizure induction results in the formation of ischaemic myocardial infarcts and loss of cardiac haemodynamic function. We hypothesised that attenuation of cardiac sympathetic/parasympathetic balance with a central presynaptic α2 agonist, clonidine, can reduce the development of interictal ECG and ventricular morphological changes resulting from kainic acid (KA; 10mg/kg) induced status epilepticus in a conscious rat model. METHODS: Using simultaneous ECG and electrocorticogram (ECoG) radiotelemetry, animals were randomised into saline controls, saline-pretreated KA and clonidine (100 µg/kg, b.i.d.)-pretreated KA groups. Baseline ECG, ECoG and behavioural score recordings were acquired in conscious animals for 2h post-KA administration. RESULTS: Bradycardia and low level seizure activity occurred immediately following KA administration. As seizure activity (ECoG spiking and high level seizure behavioural scoring) progressively increased, tachycardia developed. Both QTc prolongation and T wave amplitude were transiently but significantly increased. Clonidine treatment attenuated seizure activity, increased the latency to onset of seizure behaviour and reduced seizure-induced changes in heart rate, QTc interval, and T wave amplitude. Histological examination of the ventricular myocardium revealed hypercontraction band necrosis, inflammatory cell infiltration, and oedema at 48 h post-KA. In contrast, clonidine-treatment in seizure animals preserved tissue integrity and structure. CONCLUSION: These results demonstrate that KA-induced seizures are associated with altered ECG activity and cardiac structural pathology. We suggest that pharmacological modulation of sympathetic/parasympathetic activity in status epilepticus provides a promising therapeutic approach to reduce seizure-induced cardiomyopathy.

Pharmacological Preconditioning with GYKI 52466: A Prophylactic Approach to Neuroprotection.

Some toxins and drugs can trigger lasting neuroprotective mechanisms that enable neurons to resist a subsequent severe insult. This "pharmacological preconditioning" has far-reaching implications for conditions in which blood flow to the brain is interrupted. We have previously shown that in vitro preconditioning with the AMPA receptor antagonist GYKI 52466 induces tolerance to kainic acid (KA) toxicity in hippocampus. This effect persists well after washout of the drug and may be mediated via inverse agonism of G-protein coupled receptors (GPCRs). Given the amplifying nature of metabotropic modulation, we hypothesized that GYKI 52466 may be effective in reducing seizure severity at doses well below those normally associated with adverse side effects. Here we report that pharmacological preconditioning with low-dose GYKI imparts a significant protection against KA-induced seizures in vivo. GYKI (3 mg/kg, s.c.), 90-180 min prior to high-dose KA, markedly reduced seizure scores, virtually abolished all level 3 and level 4 seizures, and completely suppressed KA-induced hippocampal c-FOS expression. In addition, preconditioned animals exhibited significant reductions in high frequency/high amplitude spiking and ECoG power in the delta, theta, alpha, and beta bands during KA. Adverse behaviors often associated with higher doses of GYKI were not evident during preconditioning. The fact that GYKI is effective at doses well-below, and at pre-administration intervals well-beyond previous studies, suggests that a classical blockade of ionotropic AMPA receptors does not underlie anticonvulsant effects. Low-dose GYKI preconditioning may represent a novel, prophylactic strategy for neuroprotection in a field almost completely devoid of effective pharmaceuticals.