Marcus Rattray

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels.

Glutamate‐induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT‐1 in transgenic mice carrying a mutated (G93A) human copper–zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT‐1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14‐ and 18‐week‐old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT‐1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT‐1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age‐matched controls. These findings indicate that the loss of GLT‐1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT‐1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Rapid increase of NGF, BDNF and NT-3 mRNAs in inflamed bladder.

NERVE growth factor (NGF) is a mediator of hyperalgesia and has been previously associated with sensory and reflex changes after inflammation of the urinary bladder. A sensitive assay was developed to examine neurotrophin gene expression after bladder inflammation by turpentine, which causes a short-lived inflammatory response. Two hours, but not 6 or after induction of inflammation, there were significant increases in levels of NGF, brain-derived neurotrophic factor and neurotrophin-3 mRNAs. NGF immunoreactivity was elevated with a similar time course to its mRNA. Our results suggest that during bladder inflammation, endogenous NGF is rapidly up-regulated and released to mediating sensory and reflex changes. Brain-derived neurotrophic factor and neurotrophin-3 may also have a role in the inflammatory response.

Acute nicotine decreases, and chronic nicotine increases the expression of brain-derived neurotrophic factor mRNA in rat hippocampus

Acute nicotine administration (0.5 mg/kg i.p.) significantly decreased BDNF mRNA levels in dentate gyrus, CA3 and CA1 subfields of the rat hippocampus 2 h and 24 h after administration. However, with 7 days nicotine treatment, tolerance developed to the inhibitory effect of nicotine on BDNF mRNA expression and there was a significant increase in BDNF expression 2 h after the final injection in the CA1 region. These data suggests that changes in expression of hippocampal BDNF may be involved in the behavioural effects of nicotine observed after acute and chronic treatment.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan‐3‐ol, (‐)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP‐response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (‐)Epicatechin at 100–300 nmol/L stimulated a rapid, extracellular signal‐regulated kinase (ERK)‐ and PI3K‐dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 μmol/L) (‐)epicatechin was inhibitory. (‐)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell‐shaped concentration‐response characteristics. The human metabolite 3′‐O‐methyl‐(‐)epicatechin was as effective as (‐)epicatechin at stimulating ERK phosphorylation, but (‐)epicatechin glucuronide was inactive. (‐)Epicatechin and 3′‐O‐methyl‐(‐)epicatechin treatments (100 nmol/L) increased CRE‐luciferase activity in cortical neurons in a partially ERK‐dependent manner, suggesting the potential to increase CREB‐mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (‐)epicatechin and this translated into an increase in GluR2 protein. Thus, (‐)epicatechin has the potential to increase CREB‐regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

Hippocampal neurotrophin and trk receptor mRNA levels are altered by local administration of nicotine, carbachol and pilocarpine

Cholinergic receptor agonists nicotine (nicotinic), carbachol (nicotinic/muscarinic) and pilocarpine (muscarinic) were administered into the hippocampus and mRNA levels of neurotrophins and their receptors determined using in situ hybridisation. Drug doses were carefully chosen to avoid the potentially confounding effects of seizure and cell death. Nicotine caused a long-lasting increase in nerve growth factor (NGF) mRNA in all subfields of the hippocampus. The increase was evident from 24 h up to 72 h after drug administration. This increase was dependent on excitatory amino acid neurotransmission as it was blocked by administration of an AMPA or NMDA receptor antagonist. In contrast, carbachol and pilocarpine produced a transient increase in NGF mRNA levels present 4-8 h after drug administration. Pilocarpine caused a transient increase in hippocampal brain-derived neurotrophic factor (BDNF) levels, with carbachol and nicotine showing the same trend. Nicotine and carbachol caused transient decreases in NT-3 mRNA levels in dentate gyrus and CA2 with pilocarpine showing a similar trend. Increases in mRNA encoding full-length trkB were seen 8 h after nicotine, with nicotine also causing elevations in a mRNA encoding a truncated isoform (trkB.T2). TrkC mRNA was not altered by any of the conditions used. The study suggests that muscarinic and nicotinic receptor activation in the hippocampus causes transient changes in all of the neurotrophins, but that NGF levels are selectively up-regulated by nicotinic receptor stimulation. The reciprocal interaction between NGF and ascending cholinergic systems may be a component of the cognitive enhancing effects of nicotine.

Genes encoding multiple forms of phospholipase A2 are expressed in rat brain

The polymerase chain reaction was utilized to determine which of six cloned phospholipase A2 (PLA2) messenger RNAs (mRNAs), encoding four distinct low molecular weight forms of secretory PLA2 (PLA2-IB, PLA2-IIA, PLA2-IIC and PLA2-V), a calcium-dependent high molecular weight PLA2 (PLA2-IV, cPLA2) and a calcium-independent high molecular weight PLA2 (PLA2-VI, iPLA2), were expressed in different regions of rat brain and in a number of peripheral tissues. Pancreatic PLA2-IB mRNA was not expressed in the brain. PLA2-IIA, PLA2-IV and PLA2-VI mRNAs appeared to be ubiquitously expressed in brain, with relatively similar levels detected in all regions. PLA2-IIC mRNA was expressed in all brain regions but not in any of the peripheral tissues studied. PLA2-V mRNA was found at low levels in most areas of the brain, but at very high levels in the hippocampus. These results indicate that mammalian brain has the potential to express multiple isoforms of PLA2 which could be important given the potential role of these enzyme activities in ischaemic damage and in the regulation of synaptic plasticity.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood–brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse‐transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up‐regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood–brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood–brain barrier endothelium. Reverse‐transcriptase–PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non‐brain endothelia, is suppressed in the specialized endothelium of the blood–brain barrier.

The ‘glial’ glutamate transporter, EAAT2 (Glt‐1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse‐transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt‐1) in both synaptosomes and GPVs. Uptake of [3H]D‐aspartate or [3H]L‐glutamate into these preparations revealed sodium‐dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine‐o‐sulfate, l‐trans‐2,4‐pyrrolidine dicarboxylate (PDC) (+/–)‐threo‐3‐methylglutamate and (2S,4R )‐4‐methylglutamate. The IC50 values found for these compounds suggested functional expression of the ‘glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 µm dihydrokainate, failed to unmask any functional non‐EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Dietary levels of pure flavonoids improve spatial memory performance and increase hippocampal brain-derived neurotrophic factor.

Evidence suggests that flavonoid-rich foods are capable of inducing improvements in memory and cognition in animals and humans. However, there is a lack of clarity concerning whether flavonoids are the causal agents in inducing such behavioral responses. Here we show that supplementation with pure anthocyanins or pure flavanols for 6 weeks, at levels similar to that found in blueberry (2% w/w), results in an enhancement of spatial memory in 18 month old rats. Pure flavanols and pure anthocyanins were observed to induce significant improvements in spatial working memory (p = 0.002 and p = 0.006 respectively), to a similar extent to that following blueberry supplementation (p = 0.002). These behavioral changes were paralleled by increases in hippocampal brain-derived neurotrophic factor (R = 0.46, p<0.01), suggesting a common mechanism for the enhancement of memory. However, unlike protein levels of BDNF, the regional enhancement of BDNF mRNA expression in the hippocampus appeared to be predominantly enhanced by anthocyanins. Our data support the claim that flavonoids are likely causal agents in mediating the cognitive effects of flavonoid-rich foods.

Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes

1 5‐Hydroxytryptamine (5‐HT) elicited a dose‐dependent stimulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 ± 0.08 (mean ± s.e.mean). 2 In order to characterize the 5‐HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5‐carboxamidotryptamine (5‐CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5‐CT (pEC50 = 7.81 ±+ 0.09)>5‐methoxytryptamine (5‐MeOT) (pEC50 = 6.86 ±0.36) > 5‐HT (pEC50 = 6.68± 0.08). The following compounds, at concentrations up to 10 μm, did not affect basal cyclic AMP levels 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was meth‐iothepin (p Ki = 7.98 ±0.25)>mesulergine (p Ki = 7.58 ± 0.18)>ritanserin (p Ki = 7.20 ±0.24) >clozapine (p Ki = 7.03±0.19)>mianserin (p Ki= 6.41 ±0.19). The following compounds, at concentrations up to 10 μM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5‐HT7 receptor subtype‐mediated effects. 3 The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 ± 75% and 572 ± 63% of basal levels for 5‐CT and 5‐HT, respectively) compared to brainstem (274 ± 51% and 318 ± 46%, respectively) and colliculus astrocytes (244 ± 15% and 301±24%, respectively). No significant differences in pEC50 (for either 5‐HT or 5‐CT) values were observed. 4 Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers specific for the 5‐HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5‐HT6 receptor also amplified a cDNA fragment from the same samples. 5 From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5‐HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally‐dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5‐HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5‐HT6 and 5‐HT7 receptor messenger RNA.