Lentiviral delivery of a vesicular glutamate transporter 1 (VGLUT1)-targeting short hairpin RNA vector into the mouse hippocampus impairs cognition.

Glutamate is the principle excitatory neurotransmitter in the mammalian brain, and dysregulation of glutamatergic neurotransmission is implicated in the pathophysiology of several psychiatric and neurological diseases. This study utilized novel lentiviral short hairpin RNA (shRNA) vectors to target expression of the vesicular glutamate transporter 1 (VGLUT1) following injection into the dorsal hippocampus of adult mice, as partial reductions in VGLUT1 expression should attenuate glutamatergic signaling and similar reductions have been reported in schizophrenia. The VGLUT1-targeting vector attenuated tonic glutamate release in the dorsal hippocampus without affecting GABA, and selectively impaired novel object discrimination (NOD) and retention (but not acquisition) in the Morris water maze, without influencing contextual fear-motivated learning or causing any adverse locomotor or central immune effects.

Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms.

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days.

Detection, quantification, and microlocalisation of targets of pesticides using microchannel plate autoradiographic imagers.

Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited.

Differential susceptibility of astrocytic and neuronal function to 3-chloropropanediol in the rat inferior colliculus.

We have previously shown that systemic administration of S(+)3-chloropropanediol (3-CPD) produces a morphological loss of astrocytes in specific nuclei of the rodent brain that precedes loss of both neurones and endothelial tight junctions. Here, we have evaluated the differential susceptibility of neuronal and astrocytic function to 3-CPD, in order to see if this parallels the morphological selectivity. To do this, we have developed an in vivo method for monitoring astrocyte function over time by giving hourly 20-min bolus challenge exposures to ammonia via an implanted microdialysis probe and measuring the resulting transient increases in the extracellular glutamine : glutamate ratio.

Analytical approaches to investigate protein-pesticide adducts.

Organophosphorus pesticides primarily elicit toxicity via their common covalent adduction of acetylcholinesterase (AChE), but pesticide binding to additional sensitive secondary targets may also compromise health. We have utilised tritiated-diisopropylfluorophosphate ((3)H-DFP) binding to quantify the levels of active immune and brain tissue serine hydrolases, and visualise them using autoradiography after protein separation by one-dimensional and two-dimensional techniques. Preincubation of protein extracts with pesticide in vitro or dosing of rats with pesticide in vivo was followed by (3)H-DFP radiolabelling.

Proteomics reveal a concerted upregulation of methionine metabolic pathway enzymes, and downregulation of carbonic anhydrase-III, in betaine supplemented ethanol-fed rats.

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques.

Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides.

We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding.

Microvascular P-glycoprotein expression at the blood-brain barrier following focal astrocyte loss and at the fenestrated vasculature of the area postrema.

The multidrug transporter, P-glycoprotein, expressed at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but its relationship to astrocyte expression is unclear. We have studied P-glycoprotein expression in the inferior colliculus after a temporary loss of blood-brain barrier integrity following chemically induced astrocyte loss and at the fenestrated vascular endothelium of the area postrema. Male Fisher F344 rats given 3-chloropropanediol showed astrocyte loss from 12 to 24 h until the lesion was repopulated 8-28 days later.

Differential protein adduction by seven organophosphorus pesticides in both brain and thymus.

There is a need for mechanistic understanding of the lasting ill health reported in several studies of workers exposed to organophosphorus (OP) pesticide. Although the acute toxicity is largely explicable by acetylcholinesterase inhibition and the lasting effects of frank poisoning by direct excitotoxicity or indirect consequences of the cholinergic syndrome, effects at lower levels of exposure would not be predicted from these mechanisms. Similarly, reversible interactions with nicotinic and muscarinic receptors in adults would not predict continuing ill health.

Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death.

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential.

Antioxidants attenuate MK-801-induced cortical neurotoxicity in the rat.

Oxidative stress has been implicated in the pathogenesis of several neurodegenerative diseases and may result from excessive free radical production due to increased local metabolism. Non-competitive N-methyl-D-aspartate (NMDA) antagonists (MK-801 and phencyclidine) increase glucose metabolism in many brain areas and induce cytoplasmic vacuoles, heat shock protein and necrotic cell death in neurones of the rodent posterior cingulate and retrosplenial cortex. We have investigated the effect of several antioxidants with differing properties on MK-801-induced neuronal loss.

Basal forebrain cholinergic lesions reduce heat shock protein 72 response but not pathology induced by the NMDA antagonist MK-801 in the rat cingulate cortex.

Non-competitive N-methyl-D-aspartate (NMDA) antagonists, in addition to their neuroprotective potential, possess neurotoxic properties and induce seizures and psychosis. MK-801 induces cytoplasmic vacuoles and heat shock protein in pyramidal neurones in the rodent posterior cingulate and retrosplenial cortex. The mechanism of this neurotoxicity is unclear, involving many neurotransmitter systems.

Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme.

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain.

The effects of combined exposure to the pyrethroids deltamethrin and S-bioallethrin on hippocampal inhibition and skeletal muscle hyperexcitability in rats.

The default assumption that different pyrethroid insecticides, sharing a common mode of action, will show additivity of toxicity has not always been supported by in vitro measures, some of which have indicated antagonism. Our intention was to see whether the antagonism between pyrethroids of different classes seen in vitro could be reproduced in vivo. We therefore investigated the effects of single and combined exposures to two commonly used pyrethroids, deltamethrin (type II) and S-bioallethrin (type I) given intravenously to anaesthetised rats.

Fructose metabolism in the adult mouse optic nerve, a central white matter tract.

Our recent report that fructose supported the metabolism of some, but not all axons, in the adult mouse optic nerve prompted us to investigate in detail fructose metabolism in this tissue, a typical central white matter tract, as these data imply efficient fructose metabolism in the central nervous system (CNS). In artificial cerebrospinal fluid containing 10 mmol/L glucose or 20 mmol/L fructose, the stimulus-evoked compound action potential (CAP) recorded from the optic nerve consisted of three stable peaks. Replacing 10 mmol/L glucose with 10 mmol/L fructose, however, caused delayed loss of the 1st CAP peak (the 2nd and 3rd CAP peaks were unaffected).

A reassessment of the neurotoxicity of pyrethroid insecticides.

The pyrethroids are a widely used class of insecticides to which there is significant human exposure. They are however generally regarded as safe to man, and there have been few reports of human fatalities. Their acute toxicity is dominated by pharmacological actions upon the central nervous system (CNS), predominantly mediated by prolongation of the kinetics of voltage-gated sodium channels, although other mechanisms operate.

Fructose supports energy metabolism of some, but not all, axons in adult mouse optic nerve.

We used transmission electron microscopy (TEM) and electrophysiological techniques to characterize the morphology and stimulus-evoked compound action potential (CAP), respectively, of the adult mouse optic nerve (MON). Electrophysiological recordings demonstrated an identical CAP profile for each MON. An initial peak, smallest in area and presumably composed of the fastest-conducting axons displayed the lowest threshold for activation as expected for large axons.

Novel protein targets for organophosphorus pesticides in rat brain.

We report preliminary results from a proteomic search for rat brain protein targets adducted by organophosphorous pesticides. Azamethaphos, chlorfenvinphos, diazinon, malathion and chlorpyrifos oxons (in rat brain homogenates) or pirimiphos-methyl (after systemic treatment) were tested at levels producing no more than 30% inhibition of brain acetylcholinesterase. Loss of reactivity with tritiated diisopropylflurophosphate was taken as proof of adduction by the test organophosphate.

Reversible disruption of tight junction complexes in the rat blood-brain barrier, following transitory focal astrocyte loss.

Breakdown of the blood-brain barrier is a feature of acute and chronic neurodegenerative changes, yet the relationship between astrocytes and the mature barrier remains unclear. We studied this role of astrocytes in vivo using a gliotoxin and evaluated changes in three vascular tight junction markers. Male Fisher F344 rats given systemic 3-chloropropanediol showed astrocytic loss in the inferior colliculus from 12-24 h until the lesion was repopulated 8-28 days later.

Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact.

Blood-brain barrier (BBB) breakdown is a feature of cerebral ischaemia, multiple sclerosis, and other neurodegenerative diseases, yet the relationship between astrocytes and the BBB integrity remains unclear. We present a simple in vivo model in which primary astrocyte loss is followed by microvascular damage, using the metabolic toxin 3-chloropropanediol (S-alpha-chlorohydrin). This model is uncomplicated by trauma, ischaemia, or primary immune involvement, permitting the study of the role of astrocytes in vascular endothelium integrity, maintenance of the BBB, and neuronal function.

Structure-activity and interaction effects of 14 different pyrethroids on voltage-gated chloride ion channels.

We have proposed that since the type II pyrethroids deltamethrin and cypermethrin, but not the type I pyrethroid cismethrin act on chloride channels, this could contribute to the bimodal nature of pyrethroid poisoning syndromes. We now examine a wider range of pyrethroid structures on the activity of these calcium-independent voltage-gated maxi-chloride channels. Excised inside-out membrane patches from differentiated mouse neuroblastoma cells were used, and mean channel open probabilities calculated.

Microglial activation and increased synthesis of complement component C1q precedes blood-brain barrier dysfunction in rats.

A reliable way to visualise the state of microglial activation is to monitor the microglial gene expression profile. Microglia are the only CNS resident cells that synthesise C1q, the recognition sub-component of the classical complement pathway, in vivo. C1q biosynthesis in resting ramified microglia is often low, but it increases dramatically in activated microglia.