Potassium channels in hippocampal neurones are absent in a transgenic but not in a chemical model of Alzheimer's disease.

We have investigated using single channel patch-clamp methods potassium channel prevalence in hippocampal neurones from two animal models of AD. Experiments have been carried out on transgenic mice (Tg2576) carrying the Swedish mutation (K670N/M671L) and rats receiving ventricular infusions of okadaic acid. In cell-attached patches from hippocampal neurones from the Tg2576 and control littermate mice there were three principal unitary conductance - 22 pS, 111 pS and 178 pS. The two channels of intermediate and large conductance were voltage-dependent, highly active in cell-attached patches, activity decreasing markedly on hyperpolarisation. The large conductance channel was sensitive to TEA, iberiotoxin, was activated in excised inside-out patches by Ca 2+(i) and is the type I maxi-K+ channel. Significantly, there was a reduction in the prevalence of a TEA-sensitive 113 pS channel in neurones from TG2576 mice with a corresponding increase in prevalence of the maxi-K+ channel. There was no difference in the characteristics of maxi-K+ between patches in neurones from the transgenic and littermate controls. In the rat model single channel analysis was performed on hippocampal neurons from three groups of animals i.e. non-operated, and these receiving an infusion of vehicle or vehicle with okadaic acid. Three principal unitary conductances of around 18 pS, 118 pS and 185 pS were also observed in cell-attached recordings from these three groups. The intermediate and high conductance channels were blocked by TEA or 4-AP or 140 mM RbCl. There were no statistically significant differences in the channel prevalence or channel density between the control and test groups.

Case Study Part 2: Probabilistic modelling of long-term effects of pesticides on individual breeding success in birds and mammals.

Long term exposure of skylarks to a fictitious insecticide and of wood mice to a fictitious fungicide were modelled probabilistically in a Monte Carlo simulation. Within the same simulation the consequences of exposure to pesticides on reproductive success were modelled using the toxicity-exposure-linking rules developed by R.S. Bennet et al. (2005) and the interspecies extrapolation factors suggested by R. Luttik et al. (2005). We built models to reflect a range of scenarios and as a result were able to show how exposure to pesticide might alter the number of individuals engaged in any given phase of the breeding cycle at any given time and predict the numbers of new adults at the season's end.

Case Study Part 1: How to calculate appropriate deterministic long-term toxicity to exposure ratios (TERs) for birds and mammals.

In the European Union, first-tier assessment of the long-term risk to birds and mammals from pesticides is based on calculation of a deterministic long-term toxicity/exposure ratio (TER(lt)). The ratio is developed from generic herbivores and insectivores and applied to all species. This paper describes two case studies that implement proposed improvements to the way long-term risk is assessed. These refined methods require calculation of a TER for each of five identified phases of reproduction (phase-specific TERs) and use of adjusted No Observed Effect Levels (NOELs) to incorporate variation in species sensitivity to pesticides. They also involve progressive refinement of the exposure estimate so that it applies to particular species, rather than generic indicators, and relates spraying date to onset of reproduction. The effect of using these new methods on the assessment of risk is described. Each refinement did not necessarily alter the calculated TER value in a way that was either predictable or consistent across both case studies. However, use of adjusted NOELs always reduced TERs, and relating spraying date to onset of reproduction increased most phase-specific TERs. The case studies suggested that the current first-tier TER(lt )assessment may underestimate risk in some circumstances and that phase-specific assessments can help identify appropriate risk-reduction measures. The way in which deterministic phase-specific assessments can currently be implemented to enhance first-tier assessment is outlined.

Increased anxiety and synaptic plasticity in estrogen receptor beta -deficient mice.

Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ERalpha or ERbeta in mice. Behavior consistent with increased anxiety was observed principally in ERbeta mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression in medial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERbeta-mediated estrogen signaling in the processing of emotional behavior.

Genes, models and Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that is claiming an increasing number of victims as the world population ages. The identification of gene mutations and polymorphisms that either cause AD or significantly increase the risk for developing it enabled the creation of a whole generation of realistic rodent models of the disease. Animals expressing mutated human amyloid precursor protein and presenilin 1 show dramatic parallels to AD, although none of the models appear to capture the full range of pathologies that characterize the human disease. Increased refinement of these models will enhance the already tantalizing possibility of treatment.

Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice.

We investigated synaptic communication and plasticity in hippocampal slices from mice overexpressing mutated 695-amino-acid human amyloid precursor protein (APP695SWE), which show behavioral and histopathological abnormalities simulating Alzheimer's disease. Although aged APP transgenic mice exhibit normal fast synaptic transmission and short term plasticity, they are severely impaired in in-vitro and in-vivo long-term potentiation (LTP) in both the CA1 and dentate gyrus regions of the hippocampus. The LTP deficit was correlated with impaired performance in a spatial working memory task in aged transgenics. These deficits are accompanied by minimal or no loss of presynaptic or postsynaptic elementary structural elements in the hippocampus, suggesting that impairments in functional synaptic plasticity may underlie some of the cognitive deficits in these mice and, possibly, in Alzheimer's patients.

Is the Ras-MAPK signalling pathway necessary for long-term memory formation?

Genetic and pharmacological experiments have recently implicated several protein kinase cascades in LTP and memory formation. The small GTPases of the Ras subfamily are activated by multiple extracellular stimuli and, via a complex array of downstream effectors, they control a variety of cellular events that culminate in gene transcription. In the well-characterized Aplysia gill-withdrawal reflex, activation of the Ras-dependent mitogen-activated protein kinase (MAPK) cascade is essential for the long-term, but not the short-term, facilitation process. In addition, in the rodent hippocampus, specific inhibition of the MAPK pathway significantly impairs the induction of LTP, which implicates this signalling cascade in hippocampal-dependent behaviour. Mice that lack the neuronal-specific Ras regulator, Ras-GRF (guanine-releasing factor), have severely impaired LTP in the amygdala and a corresponding deficit in long-term memory for aversive events. The results obtained from these different systems demonstrate the involvement of Ras-dependent signalling in neuronal plasticity and behaviour and raise a number of intriguing questions.

Long-term potentiation in vivo in layers II/III of rat barrel cortex.

Long-term potentiation was studied in vivo in the rat barrel cortex. It was found that LTP lasting several hours could be induced in layer II/III by tetanic stimuli applied in layer IV. The probability of inducing LTP at a given site was high (86%) provided that the electrodes were not displaced too far horizontally. LTP was not observed if the stimulating electrode was located on the far side of the neighbouring barrel-column from the recording electrode. The strongest LTP was induced by stimulating layer IV septal locations or the edge of the barrel and recording in the near half of the neighbouring barrel. However, examples were found of LTP from layer IV to II/III within the same barrel, within the same septum and from barrel to adjacent septum. The probability of inducing LTP on a particular occasion was greatly increased by iontophoresis of bicuculline at the recording site during the tetanus (from 20 to 55% judged by a change in peak amplitude). The average increase in the peak amplitude was 29 +/- 3.2% for protocol 1 (urethane anesthesia, monopolar stimulation) and 23 +/- 7% for protocol 2 (barbiturate anesthesia, bipolar stimulation). The probability of inducing LTP was greater if the first tetanus was accompanied by BMI application (67%) than for any subsequent attempts (39%). These results suggest it should be possible to study the effect of LTP on sensory processing in defined positions within the barrel field.

A role for the Ras signalling pathway in synaptic transmission and long-term memory.

Members of the Ras subfamily of small guanine-nucleotide-binding proteins are essential for controlling normal and malignant cell proliferation as well as cell differentiation. The neuronal-specific guanine-nucleotide-exchange factor, Ras-GRF/CDC25Mm, induces Ras signalling in response to Ca2+ influx and activation of G-protein-coupled receptors in vitro, suggesting that it plays a role in neurotransmission and plasticity in vivo. Here we report that mice lacking Ras-GRF are impaired in the process of memory consolidation, as revealed by emotional conditioning tasks that require the function of the amygdala; learning and short-term memory are intact. Electrophysiological measurements in the basolateral amygdala reveal that long-term plasticity is abnormal in mutant mice. In contrast, Ras-GRF mutants do not reveal major deficits in spatial learning tasks such as the Morris water maze, a test that requires hippocampal function. Consistent with apparently normal hippocampal functions, Ras-GRF mutants show normal NMDA (N-methyl-D-aspartate) receptor-dependent long-term potentiation in this structure. These results implicate Ras-GRF signalling via the Ras/MAP kinase pathway in synaptic events leading to formation of long-term memories.

Nitric oxide facilitates long-term potentiation, but not long-term depression.

Reports that nitric oxide synthase (NOS) inhibition prevents the induction of long-term potentiation (LTP) have been controversial. Recent evidence suggests that NO may help to regulate the threshold for LTP induction. We have tested this hypothesis by examining the effects of stimulus frequency and train duration on synaptic plasticity in the presence of either NO donors or NOS inhibitors. Two different NO donors facilitated LTP induction by stimuli that normally produced only short-term potentiation, whereas NOS inhibitors blocked LTP to stimuli that normally produce small LTP. NO donors facilitated LTP induction even when NMDA receptors were blocked, indicating that NO need not act via NMDA receptors. NO donors and NOS inhibitors were without effect on long-term depression (LTD), suggesting that they act on a distinct potentiating mechanism. Thus, NO could contribute to the establishment of plasticity under physiologically relevant conditions by selectively increasing the probability of LTP induction.

Impaired learning in mice with abnormal short-lived plasticity.

BACKGROUND: Many studies suggest that long term potentiation (LTP) has a role in learning and memory. In contrast, little is known about the function of short-lived plasticity (SLP). Modeling results suggested that SLP could be responsible for temporary memory storage, as in working memory, or that it may be involved in processing information regarding the timing of events. These models predict that abnormalities in SLP should lead to learning deficits. We tested this prediction in four lines of mutant mice with abnormal SLP, but apparently normal LTP-mice heterozygous for a alpha-calcium calmodulin kinase II mutation (alpha CaMKII +/-) have lower paired-pulse facilitation (PPF) and increased post-tetanic potentiation (PTP); mice lacking synapsin II (SyII-/-), and mice defective in both synapsin I and synapsin II (SyI/II-/-), show normal PPF but lower PTP; in contrast, mice just lacking synapsin I (SyI-/-) have increased PPF, but normal PTP. RESULTS: Our behavioral results demonstrate that alpha CaMKII +/-, SyII-/- and SyI/II-/- mutant mice, which have decreased PPF or PTP, have profound impairments in learning tasks. In contrast, behavioral analysis did not reveal learning deficits in SyI-/- mice, which have increased PPF. CONCLUSIONS: Our results are consistent with models that propose a role for SLP in learning, as mice with decreased PPF or PTP, in the absence of known LTP deficits, also show profound learning impairments. Importantly, analysis of the SyI-/- mutants demonstrated that an increase in PPF does not disrupt learning.

Improving the quality of statistics in regulatory ecotoxicity tests.

: The results of an international workshop on the use of statistics in regulatory ecotoxicology are presented. There are currently many errors of omission in the recommendations on statistical analysis given in test guidelines. These are identified and advice is given on how to incorporate best statistical practice. The use of the no observed effect concentration (NOEC) as a summary statistic is questioned, and an alternative is suggested. Several areas of research that would resolve uncertainty in the design and analysis of ecotoxicity tests are also identified.

The alpha-Ca2+/calmodulin kinase II: a bidirectional modulator of presynaptic plasticity.

The alpha-Ca2+/calmodulin kinase II (alpha CaMKII) is required for long-term potentiation in the CA1 region of the hippocampus. Here, we report that this kinase also has a crucial role in presynaptic plasticity. Paired-pulse facilitation is blunted in the CA1 region of mice heterozygous for a targeted mutation of alpha CaMKII, confirming that this kinase can promote neurotransmitter release. Unexpectedly, field and whole-cell recordings of posttetanic potentiation show that the synaptic responses of mutants are larger than those of controls, indicating that alpha CaMKII can also inhibit transmitter release immediately after tetanic stimulation. Thus, alpha CaMKII has the capacity either to potentiate or to depress excitatory synaptic transmission depending on the pattern of presynaptic activation.

Inhibition of nitric oxide synthase does not impair spatial learning.

Nitric oxide (NO), a putative intercellular messenger in the CNS, may be involved in certain forms of synaptic plasticity and learning. This article reports a series of experiments investigating the effects of N omega-nitro-L-arginine methyl ester (L-NAME) upon various forms of learning and memory in the watermaze. L-NAME (75 mg/kg, i.p., sufficient to bring about > 90% inhibition of NO synthesis in brain) produced an apparent impairment in spatial learning when given to naive rats during acquisition (3 d, six training trials per day). This impairment was dose related, stereoselective, and attenuated by coadministration of L-arginine. A second study showed that L-NAME did not affect the retention of a previously learned spatial task. In addition, in a visual discrimination task, the rate at which criterion levels of performance were reached was unaffected by L-NAME. Thus, inhibition of NO synthase may cause a selective impairment of spatial learning without effect upon retention. However, analysis of the early training trials of the visual discrimination task revealed significantly elevated escape latencies in the L-NAME-treated rats, suggesting that inhibition of NO synthase may have more general effects. As normal rats learn the spatial task very rapidly, the possibility arises that the apparent deficit in learning is due to a disruption of some process other than learning per se. A further series of experiments investigated this possibility. L-NAME was found not to impair the learning of a new platform position in the same spatial environment. Surprisingly, L-NAME also had no effect on spatial learning in a second watermaze located in a novel spatial environment by rats well practiced with all aspects of watermaze training. Finally, L-NAME had no effect on spatial learning in naive rats trained with just one trial per day. Thus, systemic injection of an NO synthase inhibitor impairs behavioral performance in two tasks during their initial acquisition, but the basis of this functional disruption is unlikely to be due to any direct effect upon the mechanisms of spatial learning.

Inhibition of nitric oxide synthase does not prevent the induction of long-term potentiation in vivo.

Nitric oxide (NO), a putative intercellular messenger in the CNS, may be involved in certain forms of synaptic plasticity and learning. This article reports a series of experiments investigating whether an inhibitor of NO synthase, N omega-nitro-L-arginine methyl ester (L-NAME), affects long-term potentiation (LTP) in vivo, as the results of recent in vitro experiments would predict. L-NAME, given as an acute injection at a dose sufficient to inhibit hippocampal NO synthase (> 90%), had no effect on perforant path-dentate gyrus LTP induced by a strongly suprathreshold tetanus, but appeared to impair LTP induced by a weak near-threshold tetanus that may be more physiologically relevant. However, subsequent studies revealed that chronic L-NAME treatment (> 95% inhibition of NO synthase) had no effect upon LTP induction, and that acute (but not chronic) treatment resulted in a gradual but significant reduction in nontetanized baseline field potentials. The baseline shift appeared to be of a magnitude sufficient to account for the apparent impairment of weak tetanus-induced LTP. This possibility was further examined in a two-hemisphere experiment in which the time course of changes in the field EPSP of the nontetanized pathway served as the within-subject control for the tetanized pathway. No impairment of LTP induction was observed; indeed, if anything, there was a trend for greater potentiation with L-NAME. Because NO has also been implicated in the control of vasodilation, the effect of L-NAME on cerebrovascular function was also investigated. Peripheral blood pressure was significantly increased by L-NAME at the same dose that affected the field EPSP. Local cerebral glucose utilization was unchanged, while local cerebral blood flow decreased significantly in various brain regions, including the hippocampus, indicating an uncoupling of cerebral metabolism and blood flow. Thus, while NO synthase inhibition does not appear to limit the induction of LTP in vivo, it does reduce the size of baseline field EPSPs and affect local cerebrovascular function.

Nitric oxide synthase inhibitors block long-term potentiation induced by weak but not strong tetanic stimulation at physiological brain temperatures in rat hippocampal slices.

Nitric oxide synthase (NOS) inhibitors have been shown to block long-term synaptic enhancements in the mammalian hippocampus. This effect has been somewhat controversial, however, showing sensitivity to both temperature and stimulus strength. We have demonstrated a differential effect of the NOS inhibitor L-NG-nitroarginine (NOArg) on long-term potentiation (LTP) induced by weak and strong tetanic stimulation in slices of rat hippocampus. NOArg prevented LTP induction by a weak tetanus that produced stable potentiation in control slices, while the NOS inhibitor was without effect when strong tetani were used. These results suggest that nitric oxide (NO) produced as a result of tetanic stimulation plays a role in adjusting the threshold of LTP induction, but is not necessary for establishing synaptic enhancement under conditions of strong synaptic activation.

Induction of long-term potentiation in the basolateral amygdala does not depend on NMDA receptor activation.

Long-term potentiation (LTP) can be induced in the lateral and basolateral amygdala by stimulating synaptic afferents in the external capsule (EC). We examined the sensitivity of amygdaloid LTP to the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5), which is known to block LTP induction in the Schaffer collateral/CA1 synapses in the hippocampus. While relatively high concentrations (100 microM) of DL-AP5 were effective in preventing LTP induction in the lateral and basolateral amygdala in vitro, the same concentrations also significantly depressed synaptic responses to low-frequency stimulation. Furthermore, at 50 microM, a concentration sufficient to block both synaptic responses mediated by NMDA receptors and LTP induction in the hippocampus and neocortex, AP5 did not affect the probability of inducing LTP in the amygdala. Application of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which blocks non-NMDA excitatory amino acid receptors, reduced the monosynaptic response to EC stimulation by 85%. The remaining CNQX-insensitive response did not appear to be mediated by NMDA-type receptors, since it was not reduced by 50 or 100 microM AP5, and showed none of the voltage sensitivity characteristic of NMDA responses. These data suggest that while the induction of LTP in the amygdala produced by EC stimulation is blocked by high doses of AP5, plasticity at these synapses probably does not require activation of NMDA receptors.

Inhibition of nitric oxide synthesis impairs two different forms of learning.

Nitric oxide (NO), an intercellular messenger in the central nervous system of vertebrates, plays an important role in the establishment of synaptic plasticity. In order to investigate the role of NO and synaptic plasticity in learning, we injected rats and rabbits with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) prior to training on two tests of learning. Rats treated with L-NAME were impaired in learning a spatial learning task, while rabbits given the NO synthase inhibitor demonstrated learning deficits in the conditioned eyeblink response. The results support the hypothesis that NO plays a critical role in acquisition of two different forms of learning.