Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia.

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

Semliki Forest virus-mediated gene therapy of the RG2 rat glioma.

AIMS: Glioblastoma multiforme is the most common and most malignant adult brain tumour. Despite numerous advances in cancer therapy there has been little change in the prognosis of glioblastoma multiforme, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic RG2 rat glioma model. METHODS: Glioma-bearing rats were treated with IL-12-encoding SFV VLPs via an implanted cannula. Animals were treated with 5 × 107 (low-dose) or 5 × 108 (high-dose) VLPs per treatment and the effect on glioma growth and survival was assessed. RESULTS: Low-dose treatment produced a 70% reduction in tumour volume, associated with a significant extension (20.45%) in survival that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumour volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the central nervous system (CNS) demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus or high-level IL-12 expression. Treatment-related death was pronounced in high dose-treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site. CONCLUSION: The efficacy of an IL-12 gene therapy approach for the treatment of the RG2 glioma model has been demonstrated in addition to the oncolytic capacity of the VLP vector system. Despite this, the broad tropism of the SFV-based expression vector may limit use as a CNS gene therapy vector unless this inherent limitation can be overcome.

COX-2, but not COX-1, activity is necessary for the induction of perforant path long-term potentiation and spatial learning in vivo.

The objectives of this research were to investigate the role played by the enzyme cyclooxygenase (COX) in learning and memory, synaptic plasticity and synaptic transmission in the rat brain in vivo. Male Wistar rats were treated with isoform-selective inhibitors for COX-1 and COX-2, either chronically and tested in the watermaze or acutely before electrophysiological recordings were made. We found a significant impairment in acquisition of the watermaze with inhibition of COX-2. Furthermore, we found COX-2 but not COX-1 inhibition significantly blocked long-term potentiation (LTP) induction but had no effect on already established LTP. Moreover, exogenous replacement of the main metabolite of COX-2 activity, PGE(2), was sufficient to restore LTP induction and for normal downstream signalling to ensue, namely extracellular signalling-regulated kinase (ERK)-phosphorylation and c-FOS expression. We conclude that endogenous basal levels of PGE(2) resulting from COX-2 but not COX-1 activity are necessary for synaptic plasticity and memory acquisition.

Assessment of behavioural markers of autonoetic consciousness during episodic autobiographical memory retrieval: a preliminary analysis.

There is ongoing theoretical debate regarding episodic memory and how it can be accurately measured, in particular if the focus should be content-based recall of episodic details or something more experiential involving the subjective capacity to mentally travel back in time and "re-live" aspects of the original event. The autonoetic subscale of the Episodic Autobiographical Memory Interview (EAMI) is presented here as a new test instrument that attempts to redress theoretical and methodological shortcomings in autobiographical memory assessment. The EAMI merges a phenomenological detail-based approach with an assessment of autonoetic consciousness, departing considerably from traditional Remember/Know paradigms used within this field. We present findings from an initial pilot study investigating the potential markers of autonoetic consciousness that may accompany episodic retrieval. Key behavioural indices of autonoetic consciousness, notably those of viewer perspective, visual imagery, and emotional re-experiencing, emerged as being inextricably bound with the level of phenomenological detail recalled and the overall re-living judgment. The autonoetic subscale of the EAMI permits conceptually refined assessment of episodic personal memories and the accompanying subjective experience of mental re-living, characteristic of episodic memory.

Heterogeneous spatial representation by different subpopulations of neurons in the subiculum.

The subiculum is a pivotal structure located in the hippocampal formation that receives inputs from grid and place cells and that mediates the output from the hippocampus to cortical and sub-cortical areas. Previous studies have demonstrated the existence of boundary vector cells (BVC) in the subiculum, as well as exceptional stability during recordings conducted in the dark, suggesting that the subiculum is involved in the coding of allocentric cues and also in path integration. In order to better understand the role of the subiculum in spatial processing and the coding of external cues, we recorded subicular units in freely moving rats while performing two experiments: the "size experiment" in which we modified the arena size, and the "barrier experiment" in which we inserted new barriers in a familiar open field thus dividing the enclosure into four comparable sub-chambers. We hypothesized that if physical boundaries were deterministic of the firing of subicular units a strong spatial replication pattern would be found in most spatially modulated units. In contrast, our results demonstrate heterogeneous space coding by different cell types: place cells, barrier-related units and BVC. We also found units characterized by narrow spike waveforms, most likely belonging to axonal recordings, that showed grid-like patterns. Our data indicate that the subiculum codes space in a flexible manner, and that it is involved in the processing of allocentric information, external cues and path integration, thus broadly supporting spatial navigation.

The subiculum: a review of form, physiology and function.

We review the neuroanatomical, neurophysiological and functional properties of the mammalian subiculum in this paper. The subiculum is a pivotal structure positioned between the hippocampus proper and entorhinal and other cortices, as well as a range of subcortical structures. It is an under-investigated region that plays a key role in the mediation of hippocampal-cortical interaction. We argue that on neuroanatomical, physiological and functional grounds, the subiculum is properly part of the hippocampal formation, given its pivotal role in the hippocampal circuit. We suggest that the term "subicular complex" embraces a heterogenous range of distinct structures and this phrase does not connote a functionally or anatomically meaningful grouping of structures. The subiculum has a range of electrophysiological and functional properties which are quite distinct from its input areas; given the widespread set of cortical and subcortical areas with which it interacts, it is able to influence activity in quite disparate brain regions. The rules which govern the plasticity of synaptic transmission are not well-specified; it shares some properties in common with the hippocampus proper, but behaves quite differently in other respects. Equally, its functional properties are not well-understood, it plays an important but ill-defined role both in spatial navigation and in mnemonic processing. The important challenges for the future revolve around the theoretical specification of its unique contribution to hippocampal formation processing on the one hand, and the experimental investigation of the many open questions (anatomical, physiological, pharmacological, functional) regarding its properties, on the other.

Exercise, but not environmental enrichment, improves learning after kainic acid-induced hippocampal neurodegeneration in association with an increase in brain-derived neurotrophic factor.

Previous studies have suggested that exercise in a running wheel can be neuroprotective, perhaps due to, among others, gene-expression changes after exercise, increases in trophic proteins and/or enhanced cardiovascular responsivity. Here we ask whether physical exercise or environmental enrichment provide protection after brain damage, especially in terms of recovery of cognitive function. To evaluate the neuroprotective effect of these conditions, we used the kainic acid (KA) model of neuronal injury. Systemically-administered KA induces excitotoxicity by overstimulation of glutamate receptors, resulting in neuronal death by necrosis and apoptosis. Our results show that exercise, but not enriched environment, prior to KA-induced brain damage, improved behavioural performance in both Morris watermaze and object exploration tasks. However, prior exercise did not decrease to control levels the hyperactivity normally seen in KA-treated animals, as measured by ambulation in the open field. Furthermore, both exercise and enriched environment did not protect against neuron loss in CA1, CA2 and CA3 areas of the hippocampus, despite a substantial increase in brain-derived neutrophic factor (BDNF) levels in dentate gyrus of the exercise and KA-treated animals.

Combining exercise and cyclooxygenase-2 inhibition does not ameliorate learning deficits after brain insult, despite an increase in BDNF levels.

Neurodegeneration can produce behavioral impairments. Previously, we have found that inhibition of cyclooxygenase-2 activity or physical activity was neuroprotective during kainic-acid-induced neural loss. Here, we investigated the combined effect of exercise pre-insult and cyclooxygenase inhibitor treatment post-kainate-induced brain damage. However, in spite of an increase in BDNF levels, the combination did not improve behavioral performance in Morris watermaze and object exploration tasks.

Metabotropic glutamate receptor activation and blockade: their role in long-term potentiation, learning and neurotoxicity.

Metabotropic glutamate receptors represent a fairly recent addition to the family of glutamate receptors. These receptors have the distinguishing feature of being coupled to G-proteins rather than ion channels and they appear to have a variety of functional characteristics. These receptors play a vital role, for example, in the induction and maintenance of long-term potentiation, the most popular current model of the biological correlates of learning and memory. Blockade of metabotropic glutamate receptors prevents long-term potentiation induction and learning in a variety of tasks in different species. Chronic metabotropic glutamate receptor activation is also associated with neurodegeneration and selective neuronal loss when agonists of these receptors are injected in high concentrations directly into the brain. Metabotropic glutamate receptors also play a role in the normal development of the nervous system and these sites within the central nervous system offer possible routes for drug therapies; selective receptor antagonists, for example, may prove to have the very desirable feature of endowing neuroprotection during ischaemic episodes whilst allowing normal excitatory neurotransmission to occur.

Analysis of expressed N-ras mutations in human melanoma short-term cell lines with allele specific restriction analysis induced by the polymerase chain reaction.

Mutations in ras genes have been found in the DNA of numerous cancer types including melanomas, but the expression of these mutations in melanomas has not yet been addressed. We have used the polymerase chain reaction (PCR) and allele-specific restriction analysis (ASRA) to determine the frequency of expressed N-ras mutations on 25 short-term melanoma tissue culture samples. N-ras cDNA generated using reverse transcriptase from whole cells was used as the PCR template. 14 secondary melanoma cultures that varied in differentiation patterns were analysed. Only 2 were found to express N-ras mutations; in both, the mutation was localised to one of the first two positions of the 61st codon of N-ras. These tumour lines, KMI-M8412a and KMI-M8412b, were established from separate tumour deposits in the same patient. Codons 12 and 13 were found to be free of mutations in all of the lines studied. 8 primary melanomas and 3 unclassified skin lesions were also analysed and found free of N-ras mutations. These results suggest that N-ras may not play such an important role in melanoma tumorigenesis as is speculated by others.

Metabotropic glutamate receptor-induced homosynaptic long-term depression and depotentiation in the dentate gyrus of the rat hippocampus in vitro.

We have investigated the role of metabotropic glutamate receptors (mGluR) in the induction of homosynaptic long-term depression (LTD) and depotentiation (DP) in the dentate gyrus of the adult rat. Perfusion of the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) for a prolonged period (20 min) induced long-term depression (LTD) of field excitatory postsynaptic field potentials (epsps) from the baseline level and also depotentiation (DP) from the long-term potentiated level. Both the ACPD-and the low frequency stimulation (LFS)-induced LTD and DP were inhibited in the presence of the mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), demonstrating the necessity for the activation of metabotropic glutamate receptors in the induction of LTD/DP. The LFS and ACPD-induced LTD were independent of the activation of N-methyl-D-aspartate (NMDA) receptors, as they were not blocked by the NMDA receptor antagonist D-2-amino-5-phophonopentanoate (AP5).

Preparation, DNA binding, and in vitro cytotoxicity of a pair of enantiomeric platinum(II) complexes, [(R)- and (S)-3-aminohexahydroazepine]dichloroplatinum(II). Crystal structure of the S enantiomer.

A pair of enantiomeric Pt(II) complexes, [Pt(R-ahaz)Cl2] and [Pt(S-ahaz)Cl2] (ahaz = 3-aminohexahydroazepine), has been investigated for their ability to bind enantioselectively to DNA. Improved synthetic procedures were developed for preparing both the ligands and the Pt complexes. The structure of the complex of the S enantiomer was determined by X-ray crystallographic methods. Crystals of [Pt(S-ahaz)Cl2] are orthorhombic, space group P2(1)2(1)2(1), with a = 6.917(1) A, b = 11.167(1) A, c = 12.373(2) A, Z = 4, and the structure was refined to R = 0.023 (1505F). Molecular modeling techniques were used to investigate the role of steric interactions between the ligand and DNA in influencing the bifunctional binding of the two enantiomers, and it was found that the S enantiomer should bind more readily. The binding of the S enantiomer, to calf thymus DNA, was indeed found to be slightly greater than that for the R enantiomer though slightly less than that for cis-DDP. Assays of the proportion of monofunctional adducts showed that a substantially greater proportion of monofunctional adducts remained for the R enantiomer and cisplatin than for the S enantiomer. Each of the enantiomers was subjected to in vitro cytotoxicity assays using cultures of human bladder (BL13/0), lung and resistant lung (PC9 and PC9cisR), and prostate (DU145) cancer cells. The R enantiomer was found to be slightly more cytotoxic in the bladder cell line and may be less cytotoxic in the lung cell line but there were no significant differences in the resistant cell line nor in the prostate cell line. The two enantiomers were taken up equally by the bladder cancer cells.

Plasticity in the projection from the anterior thalamic nuclei to the anterior cingulate cortex in the rat in vivo: paired-pulse facilitation, long-term potentiation and short-term depression.

Several neurophysiological and computational theories of the rodent navigational system suggest that the differing cortices of the frontal lobe and thalamus share information and therefore undergo changes in synaptic strength. We examine here for the first time three forms of synaptic plasticity in the projection from the anterior thalamic nuclei to the anterior cingulate cortex: we demonstrate that this projection is capable of expressing paired-pulse facilitation, long-term potentiation, and short-term depression. Furthermore, input/output curves show that field excitatory post-synaptic potential amplitude increased at all stimulus intensities following high-frequency stimulation. These findings add important information to our understanding of synaptic plasticity in this important pathway, which has been widely hypothesized to play important roles in memory and spatial representation in the rodent.

Post-treatment, but not pre-treatment, with the selective cyclooxygenase-2 inhibitor celecoxib markedly enhances functional recovery from kainic acid-induced neurodegeneration.

We have investigated the role of inflammation in the excitotoxicity induced by overstimulation of glutamate receptors using kainic acid, an important tool for studying functions related to excitatory amino acid transmission and for producing neuronal death, especially in areas CA1 and CA3 of the hippocampus. We hypothesised that by inhibiting one of the major components of the neuroinflammation response, after kainic acid injection, that there would be less inflammation and therefore a reduction in cell loss, an enhancement of cognitive function (using spatial learning and object exploration tasks) or both. We examined brain-derived neurotrophic factor levels, expecting that there would be a correlation between its level and subsequent recovery. Our results confirmed our hypothesis: the kainic acid injected-rats treated with celecoxib (after kainic injection) performed better in the spatial and non-spatial tasks than the kainic acid-treated group. However, there was not any improvement if celecoxib was given before kainic acid treatment, underlining also the importance of the production of prostaglandin at the beginning of inflammation.

Dantrolene inhibits long-term depression and depotentiation of synaptic transmission in the rat dentate gyrus.

The involvement of Ca release from intracellular stores in the induction of long-term depression and depotentiation of excitatory synaptic transmission was investigated in the rat dentate gyrus using dantrolene, an agent known to block Ca release via the ryanodine receptor. In control slices, low-frequency stimulation (1 Hz for 15 min) induced robust long-term depression of baseline field excitatory postsynaptic potentials and depotentiation of previously established long-term potentiation. Dantrolene (50 microM) was found to block completely both long-term depression of baseline responses and depotentiation. Moreover, long-term potentiation induced by high-frequency stimulation was enhanced in the presence of dantrolene.

The effects of the bacterial endotoxin lipopolysaccharide on synaptic transmission and plasticity in the CA1-subiculum pathway in vivo.

Lipopolysaccharide is derived from the cell wall of gram-negative bacteria and is a potent endotoxin which causes the release of cytokines in the CNS. We examined the effect of lipopolysaccharide on synaptic transmission and synaptic plasticity in the hippocampal area CA1-subicular pathway in vivo. We found that lipopolysaccharide did not affect baseline synaptic transmission in this pathway; it did, however, reduce the magnitude of paired-pulse facilitation, a form of short-term plasticity thought to be primarily presynaptic in origin. We then examined the interaction between lipopolysaccharide and two common models for the biological basis of memory: high-frequency stimulation induced long-term potentiation and low-frequency stimulation induced long-term depression of synaptic transmission. We found that lipopolysaccharide blocked long-term potentiation following high-frequency stimulation and also induced potentiation of synaptic transmission after low-frequency stimulation. Lipolysaccharide blocked paired-pulse facilitation selectively at short rather than longer interstimulus intervals. Thus, lipopolysaccharide has different effects on synaptic transmission in this pathway depending on the frequency and length of stimulation. These results provide new insights into the action of lipopolysaccharide on various forms of plasticity in the hippocampus, an area known to play a vital role in learning and memory.

Medial prefrontal cortex lesions cause deficits in a variable-goal location task but not in object exploration.

The role of the medial prefrontal cortex (PFC) in goal-directed behavior was examined in rats with aspiration lesions. In Experiment 1, PFC lesions resulted in an impaired ability to relearn the location of a behaviorally defined goal arm of a plus-maze after it was moved from an initially fixed position. Lesioned rats also exhibited a significantly greater degree of perseveration compared with control animals. Experiment 2 was an object exploration task in which rats had to respond to a change in the layout of the environment. PFC-lesioned rats performed identically to controls, therefore demonstrating that the deficits observed in Experiment 1 did not result from a deficit in the ability to explore the environment. The results are discussed in terms of several competing, but not mutually exclusive explanations of the role of the PFC in navigation and spatial representation.

Long-term potentiation and spatial learning are associated with increased phosphorylation of TrkB and extracellular signal-regulated kinase (ERK) in the dentate gyrus: evidence for a role for brain-derived neurotrophic factor.

In this study, the authors investigate changes in the presynaptic terminal of the dentate gyrus that accompany 2 types of hippocampal-dependent plasticity: spatial learning and long-term potentiation (LTP). Parallel changes occurred in the dentate gyrus of rats that had undergone training in the Morris water maze and had sustained LTP. In both cases, KCl-induced brain-derived neurotrophic factor release was increased, and this was accompanied by increased phosphorylation of TrkB and the mitogen-activated protein kinase, ERK. Glutamate release was also enhanced, and the data suggest that this may be a consequence of increased activation of TrkB and ERK. Because the data indicate that similar cellular modifications are shared by these 2 forms of plasticity, they provide circumstantial evidence that LTP satisfies some of the requirements of a memory-inducing cellular substrate.

Interaction between paired-pulse facilitation and long-term potentiation in the projection from hippocampal area CA1 to the subiculum.

Studies of the interaction between long-term potentiation (LTP) and paired-pulse facilitation (PPF) may throw light on the role of presynaptic factors in LTP. We examine here, for the first time, the nature of PPF in the CA1-subiculum projection. PPF peaks at a 50 ms interstimulus interval (ISI) and is evident at ISIs from 10 to 500 ms. There is no PPF effect at a 1000 ms ISI. PPF decreases in magnitude post-LTP induction across the middle range of ISI values tested (30, 50 and 100 ms). There is a positive correlation between initial PPF values and LTP; this correlation increases as the ISI increases. Initial values and the change in PPF post-LTP are also negatively correlated.

The projection from hippocampal area CA1 to the subiculum sustains long-term potentiation.

Long-term potentiation (LTP) is a popular model of the synaptic plasticity which may be engaged by the biological processes underlying learning and memory. Most available studies of LTP have concentrated on the analysis of LTP occurring in 'early' components of the hippocampal circuit (for example, dentate gyrus and area CA1). We examine here, for the first time, LTP as it occurs in the massive, unidirectional projection from CA1 to the subiculum in vivo. We show that this projection sustains high-frequency stimulus-induced LTP (10 trains of 20 stimuli at 200 Hz; intertrain interval 2 s; LTP 181 +/- 9% at 30 min post-LTP induction). In addition, input-output (I/O) curves show a leftward shift for all stimulation values.