MRZ-99030 - A novel modulator of Aß aggregation: II - Reversal of Aß oligomer-induced deficits in long-term potentiation (LTP) and cognitive performance in rats and mice.

ß-amyloid1-42 (Aß1-42) is a major endogenous pathogen underlying the aetiology of Alzheimer's disease (AD). Recent evidence indicates that soluble Aß oligomers, rather than plaques, are the major cause of synaptic dysfunction and neurodegeneration. Small molecules that suppress Aß aggregation, reduce oligomer stability or promote off-pathway non-toxic oligomerization represent a promising alternative strategy for neuroprotection in AD. MRZ-99030 was recently identified as a dipeptide that modulates Aß1-42 aggregation by triggering a non-amyloidogenic aggregation pathway, thereby reducing the amount of intermediate toxic soluble oligomeric Aß species. The present study evaluated the relevance of these promising results with MRZ-99030 under pathophysiological conditions i.e. against the synaptotoxic effects of Aß oligomers on hippocampal long term potentiation (LTP) and two different memory tasks. Aß1-42 interferes with the glutamatergic system and with neuronal Ca(2+) signalling and abolishes the induction of LTP. Here we demonstrate that MRZ-99030 (100-500 nM) at a 10:1 stoichiometric excess to Aß clearly reversed the synaptotoxic effects of Aß1-42 oligomers on CA1-LTP in murine hippocampal slices. Co-application of MRZ-99030 also prevented the two-fold increase in resting Ca(2+) levels in pyramidal neuron dendrites and spines triggered by Aß1-42 oligomers. In anaesthetized rats, pre-administration of MRZ-99030 (50 mg/kg s.c.) protected against deficits in hippocampal LTP following i.c.v. injection of oligomeric Aß1-42. Furthermore, similar treatment significantly ameliorated cognitive deficits in an object recognition task and under an alternating lever cyclic ratio schedule after the i.c.v. application of Aß1-42 and 7PA2 conditioned medium, respectively. Altogether, these results demonstrate the potential therapeutic benefit of MRZ-99030 in AD.

Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10µM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease.

Behavioural and cellular effects of exogenous amyloid-ß peptides in rodents.

A better understanding of Alzheimer's disease (AD) and the development of disease modifying therapies are some of the biggest challenges of the 21st century. One of the core features of AD are amyloid plaques composed of amyloid-beta (Aß) peptides. The first hypothesis proposed that cognitive deficits are linked to plaque-development and transgenic mice have been generated to study this link, thereby providing a good model to develop new therapeutic approaches. Since later it was recognised that in AD patients the cognitive deficit is rather correlated to soluble amyloid levels, consequently, a new hypothesis appeared associating the earliest amyloid toxicity to these soluble species. The purpose of this review is to give a summary of behavioural and cellular data obtained after soluble Aß peptide administration into rodents' brain, thereby showing that this model is a valid tool to investigate AD pathology when no plaques are present. Additionally, this method offers an excellent, efficient model to test compounds which could act at such early stages of the disease.

Evaluation of brain pharmacokinetics of (+)MK-801 in relation to behaviour.

The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)MK-801 is widely used in animal research (over 3000 publications), however its extracellular brain concentration has never been reported. Here, we show using in vivo microdialysis that systemic injection of (+)MK-801 at doses of 0.05, 0.1 or 0.2mg/kg resulted in peak brain ECF concentration of 6, 14 or 34 nM, respectively. Moreover, (+)MK-801 resulted in a dose-dependent learning impairment in the Morris water maze as well as hyperactivity in the open field. These data demonstrate for the first time that (+)MK-801 at doses producing behavioural alterations expected from NMDA receptor blockade reaches extracellular brain concentrations corresponding to the affinity at NMDA receptors.

Pharmacological and parametrical investigation of prepulse inhibition of startle and prepulse elicited reactions in Wistar rats.

Prepulse inhibition (PPI) is the inhibition of an acoustic startle response (ASR) that is observed when a weak prepulse is presented shortly before a startling stimulus. Here we studied in Wistar rats the dependence of PPI on variations of the interstimulus interval (ISI; from 25-1020ms) after treatment with various drugs that are known to disrupt PPI. The motor response to the prepulse itself (prepulse elicited reaction, PER) was also studied. The direct dopamine receptor agonist apomorphine, the non-competitive NMDA glutamate receptor antagonist MK-801, and the cannabinoid CB1 receptor agonist WIN 55,212-2 all reduced PPI, depending on the ISI, with different effects on the PER and/or pulse alone. The serotonin 2A receptor agonist DOI tended to reduce PPI. The cannabinoid CB1 receptor antagonist AM 251 did neither affect PPI nor the responses to prepulses or startling noise pulses. Taken together this study supports the current notion of a pharmacologically complex pattern of regulation of PPI at different ISIs and suggests that the PER is a miniature ASR that does, however, not predict the level of PPI.

A new automated method to assess the rat recognition memory: validation of the method.

During the last decade, the rodent object recognition memory task had gained popularity in the field of pharmacological studies, and has been proposed as a useful method to evaluate the efficacy of memory enhancing compounds. In this context, it is important to establish reliable and automated methods with high throughput to evaluate recognition memory in the rat. When performed in a Y-maze apparatus, object recognition has been described to be less dependent on spatial information, and we here report a new method to assess novelty discrimination in a Y-maze apparatus recorded via automated video tracking. During the development of the method, many parameters were recorded and some were selected as being the most reliable, i.e. time spent in the distal half-arms during the first 2min of the test. The method was then validated under memory deficit conditions produced by a pharmacological treatment (scopolamine) and a long retention delay (72h) between the sample and the test. The time-induced deficit was prevented by administration of a M1 positive allosteric modulator, BQCA at a dose of 10mg/kg. Altogether, our data show that the novelty discrimination task performed in a Y-maze apparatus is a reliable method to assess the recognition memory of the rat. This new automated method is as sensitive as the classical object recognition test, and provides advantages such as easily definable parameters and the possibility to use an automated video tracking system that makes the measurement independent of the investigator.

Effects of 5-HT6 antagonists, Ro-4368554 and SB-258585, in tests used for the detection of cognitive enhancement and antipsychotic-like activity.

5-Hydroxytryptamine 6 (5-HT6) receptors are involved in learning and memory processes and are discussed as promising targets for the treatment of cognitive impairment in central nervous system disorders. A number of 5-HT6 antagonists are currently in the clinical development for schizophrenia and Alzheimer's disease (AD). There is some discrepancy regarding cognitive efficacy in subjects, and only limited data are available on the role of the 5-HT6 receptor in animal models of psychosis. The aim of this study was to investigate the efficacy of the selective 5-HT6 antagonists, Ro-4368554 (1-10 mg/kg, intraperitoneally) and SB-258585 (3-30 mg/kg, intraperitoneally), in animal models for schizophrenia and AD. Both compounds showed cognition-enhancing effects in object recognition, whereas only SB-258585 was able to prevent the scopolamine-induced deficit in the Morris water-maze test. Neither Ro-4368554 nor SB-258585 prevented scopolamine-induced impairment in contextual fear conditioning. Similarly, both compounds were ineffective on MK-801-induced deficits in contextual fear conditioning and spatial working memory. Ro-4368554, but not SB-258585 reversed the apomorphine-induced deficit in prepulse inhibition. Amphetamine-induced hyperlocomotion was not affected by either compound. Taken together, the overall efficacy of Ro-4368554 and SB-258585 in animal models for AD and schizophrenia is rather limited. These data show moderate efficacy in some models for AD but do not support the therapeutic potential of 5-HT6 antagonists for schizophrenia.

Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Abeta-bearing fragment is associated with reduced NMDA receptor density.

The aim of this study was to characterize APPC100.V717F transgenic (TgC100.V717F) mice which over-express a mutant C100 fragment of the amyloid precursor protein. The mice were compared to TgC100 wild type mice (TgC100.WT) and non-transgenic controls at 4-9 and 16-22 months of age. TgC100.V717F mice showed behavioural hyperactivity, particularly at a younger age, as shown by increased numbers of elevated plus maze arm entries and Y-maze arm entries, enhanced baseline locomotor activity in the open field, and enhanced amphetamine-induced hyperlocomotion. This hyperactivity was less pronounced in TgC100.WT which only displayed significant differences to non-transgenic controls at a younger age for the number of Y-maze arm entries and baseline locomotor activity in the open field. In addition, TgC100.V717F mice, but not TgC100.WT, demonstrated cognitive deficits, as shown by reduced spontaneous alternation in the Y-maze and markedly reduced retention in a passive avoidance test. At an older age, TgC100.V717F mice showed enhanced startle and increased immobility time in the forced swim test. In the TgC100.V717F mice, but not TgC100.WT, the behavioural changes were paralleled by a significant reduction in the expression of hippocampal NMDA receptor subunits types 1 and 2A. Concomitantly, we detected axonal disruption and apoptosis in the hippocampus of TgC100.V717F mice. In conclusion, these data demonstrate that the mutant C100 fragment is an effector of biochemical and both cognitive and non-cognitive behaviours. These transgenic mice may be a model for the psychotic features associated with early Alzheimer's disease.

Behavioural disturbances and altered Fos protein expression in adult rats after chronic pubertal cannabinoid treatment.

Cannabis is one of the world's most popular recreational drugs. However, little is known about long-lasting cellular and neurobehavioural effects of chronic cannabinoid intake, especially during puberty where cannabis use among humans is commonly initiated. This study in rats investigates the long-term effect of pubertal cannabinoid treatment on prepulse inhibition (PPI), locomotor activity and on anxiety in the elevated-plus maze during adulthood. Furthermore, changes in adult basic neuronal activity, assessed by c-Fos immunoreactivity (Fos IR), and a potentially altered Fos expression after acute treatment with dopaminergic drugs was evaluated. Chronic treatment with the synthetic cannabinoid full agonist WIN 55,212-2 (WIN; 1.2 mg/kg) was carried out over 25 days of the rats' puberty and subsequent behavioural testing was conducted in adult animals. Finally, Fos IR was evaluated in several brain regions under basal conditions and after acute administration of haloperidol (0.1 mg/kg) and apomorphine (2 mg/kg). Chronic WIN treated animals exhibited a lasting disruption of PPI. These rats were also more active in the open field and less anxious in the elevated-plus maze than their vehicle treated controls. Additionally, when comparing Fos IR in selected brain regions, these animals displayed altered basal neuronal activity and responded differently to acute application of haloperidol or apomorphine. Taken together, these results indicate that chronic stimulation of the cannabinoid receptor CB(1) during the rats' puberty not only leads to persistent behavioural changes but also to cellular long-term adaptations within brain regions critical for drug of abuse or neuropsychiatric diseases.

Effects of acute systemic and intra-cerebral stimulation of cannabinoid receptors on sensorimotor gating, locomotion and spatial memory in rats.

RATIONALE: Cannabinoid CB(1) receptors in the brain are targets of both endocannabinoid signalling and the psychoactive compounds of the hemp plant. They mediate neuronal effects of their ligands in various corticolimbic and striatal circuits by presynaptic regulation of transmitter release. OBJECTIVES/METHODS: This study investigates acute systemic effects of the full CB(1) receptor agonist WIN 55,212-2 (WIN) on prepulse inhibition (PPI) of the acoustic startle response (ASR), locomotor activity and spatial memory retrieval in an eight-arm radial-maze task. Furthermore, we tested the effect of local intra-cerebral micro-infusions of WIN into the nucleus accumbens (NAc), ventral tegmental area (VTA), dorsal (dHIP) and ventral (vHIP) hippocampus and medial prefrontal cortex (mPFC). RESULTS: Systemic WIN (1.2 mg/kg) reduced PPI without affecting ASR, had no effect on locomotion in the open field, but impaired retrieval of spatial memory. Infusions of 5 microg/0.3 microl WIN into either NAc (core or shell), dHIP or VTA did not affect PPI and locomotion immediately afterwards. However, PPI was significantly reduced after intra-mPFC and intra-vHIP infusion of WIN. Furthermore, WIN infusion into dHIP increased the number of reference memory errors in the maze, suggesting impairment of memory retrieval. CONCLUSIONS: Our data support the notion that CB(1) receptor stimulation impairs sensorimotor gating most likely by modulation of neurotransmitter release in mPFC and vHIP. The lack of effects of local WIN infusions in NAc and VTA might be due to low receptor abundance in these regions. Additionally, CB(1) receptor activation in dHIP impairs spatial memory retrieval. Taken together, cortico-hippocampal cannabinoid receptors play an essential role in the regulation of cognitive and behavioural processes.

Clozapine enhances disruption of prepulse inhibition after sub-chronic dizocilpine- or phencyclidine-treatment in Wistar rats.

Sensitisation (i.e. progressive enhancement) of behavioural abnormalities induced by repeated treatment with non-competitive NMDA receptor antagonists in animals is considered an animal model for schizophrenia. Here, male Wistar rats were treated for 11 days with either dizocilpine (0.1 mg/kg), phencyclidine (PCP, 2 mg/kg), or saline and tested for prepulse inhibition (PPI) of the acoustic startle response (ASR). The aims of this study were twofold: First, we tested whether sensitisation of PPI deficits previously found in Sprague-Dawley rats were also found in Wistar rats, and, second, whether these effects can be ameliorated by the atypical antipsychotic clozapine. PPI is a paradigm for the assessment of sensorimotor gating (and its deficits) and is impaired in schizophrenic patients. After the sub-chronic treatment the rats were tested drug-free (day 12), and on the following days after drug challenge by PCP (2 mg/kg), combinations of PCP (2 mg/kg) and clozapine (5 and 10 mg/kg), or clozapine (5 mg/kg) alone. PPI was significantly reduced by both NMDA receptor antagonists. This effect was not further enhanced by the daily treatment. Startle magnitude was increased after eight days of dizocilpine-treatment only, indicating sensitisation of startle-potentiation by this drug. Testing the rats drug-free on day 12 revealed enhanced PPI and reduced startle (compared to the matching test on day 0) irrespective of previous treatment. Drug challenge with PCP (2 mg/kg) again reduced PPI in all groups. Clozapine (5 and 10 mg/kg) failed to antagonise the PPI-disruptive effects of PCP and even enhanced the PCP-induced PPI-deficits in rats pretreated with PCP or dizocilpine. These findings suggest: (1) that PPI and startle are influenced differently by non-competitive NMDA receptor antagonists, (2) that PCP and dizocilpine reduce PPI in Wistar rats, but do not lead to a sensitisation of this effect; and (3) that under the present schedule of treatments, the antipsychotic compound clozapine does not antagonise but rather enhances PPI-disruptive effects of non-competitive NMDA receptor antagonists, pointing towards a complex interaction of the brain processes underlying the action of psychotomimetic and atypical antipsychotic drugs.

Involvement of serotonin1A receptors in cardiovascular responses to stress: a radio-telemetry study in four rat strains.

We studied the effect of treatment with the serotonin-1A (5-HT1A) receptor ligands buspirone, 8-hydroxy-di-propyl-aminotetralin (8-OH-DPAT), and (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl-methylamino)ethyl]-8-azaspiro[4,5]decane-7,9-dione methyl sulphonate (MDL73,005EF) on blood pressure and heart rate increases to open field stress. We compared Spontaneously Hypertensive Rats (SHR), Fawn-Hooded (FH) rats, Wistar-Kyoto (WKY) rats, and Sprague-Dawley (SD) rats instrumented with radio-telemetry probes. Buspirone treatment reduced the blood pressure increase in SHR, FH rats, and WKY rats and heart rate increase in FH rats and WKY rats. 8-OH-DPAT treatment reduced the blood pressure increase in FH rats and WKY rats, but had no effect in SHR and enhanced the pressor response in SD rats. This treatment reduced the heart rate increase in FH rats and WKY rats only. Similarly, MDL73,005EF treatment reduced the blood pressure increase in FH rats and WKY rats, but had no effect in SHR and enhanced this response in SD rats. Little effect of this treatment was seen on heart rate changes. For comparison, diazepam treatment abolished the pressor response in SD rats and reduced it in FH rats and WKY rats, but not SHR. Differential effects of the treatments were also seen between strains for locomotor activity in the open field, although behavioural changes could not explain the effects of the drugs on cardiovascular responses. These data suggest that 5-HT1A receptors are involved in cardiovascular stress responses; however, the extent of this involvement differs between rat strains and the drugs used. These results could be important for our understanding of possible anxiolytic properties of antipsychotic drugs with affinity for the 5-HT1A receptor.