The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Single-cell resolution mapping of neuronal damage in acute focal cerebral ischemia using thallium autometallography.

Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K(+)-probe thallium (Tl(+)) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl(+) after crossing the blood-brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl(+) uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl(+) uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl(+) uptake. At 24 hours, the areas of reduced Tl(+)uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of (201)TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K(+) metabolism and prediction of tissue viability in cerebral ischemia.

Insufficient endogenous redox buffer capacity may underlie neuronal vulnerability to cerebral ischemia and reperfusion.

Reactive oxygen species (ROS) are key players in ischemia-induced neurodegeneration. We investigated whether hippocampal neurons may lack sufficient redox-buffering capacity to protect against ROS attacks. Using organotypic hippocampal slice cultures (OHSCs) transiently exposed to oxygen and glucose deprivation (OGD) and gerbils suffering from a two-vessel occlusion (2VO) as complementary ex vivo and in vivo models, we have elucidated whether the intrinsic redox systems interfere with ischemia-induced neurodegeneration. Cell- type-specific immunohistological staining of hippocampal slice cultures revealed that pyramidal neurons, in contrast to astrocytes and microglia, express free thiols only weakly. In addition, free thiol levels were extensively decreased throughout the hippocampal formation immediately after OGD, but recovered within 24 hr after reperfusion. In parallel, progressive glia activation and proliferation were observed. Increased neuronal exposure to ROS was monitored by dihydroethidium oxidation in hippocampal pyramidal cell layers immediately after OGD. Coadministration of reduction equivalents (α-lipoic acid) and thiol-stimulating agents (enalapril, ambroxol) decreased ischemia-induced neuronal damage in OGD-treated OHSCs and in gerbils exposed to 2VO, whereas single drug applications remained ineffective. In summary, limited redox buffering capacities of pyramidal neurons may underlie their exceptional vulnerability to cerebral ischemia. Consistently, multidrug treatments supporting endogenous redox systems may offer a strategy to promote valid neuroprotection.

Early neuronal dysfunction by amyloid ß oligomers depends on activation of NR2B-containing NMDA receptors.

Several studies indicate that NMDA receptor signaling is involved in Aß oligomer-mediated impairment of neuronal function and morphology. Utilizing primary neuronal cell culture and hippocampal slices from rat and mouse, we found that Aß oligomer administration readily impairs long-term potentiation, reduces baseline synaptic transmission, decreases neuronal spontaneous network activity and induces retraction of synaptic contacts long before major cytotoxic effects are visible. Interestingly, all these effects can be blocked with the NR2B-containing NMDA-receptor antagonist ifenprodil or Ro 25-6981 suggesting that activation of downstream effectors of these receptors is involved in early detrimental actions of Aß oligomers. In line we found that Jacob, a messenger that can couple extrasynaptic NMDA-receptor activity to CREB dephosphorylation, accumulates in the nucleus after Aß oligomer administration and that the nuclear accumulation of Jacob can be blocked by a simultaneous application of ifenprodil. We conclude that Aß oligomers induce early neuronal dysfunction mainly by activation of NR2B-containing NMDA-receptors.

[De-novo generation of vascularized tissue using different configurations of vascular pedicles in perforated and closed chambers]. / De-novo Generierung von vaskularisiertem Gewebe mittels unterschiedlicher Gefässstielkonfigurationen in perforierten und geschlossenen Wachstumskammern.

Growing three-dimensional tissue within a chamber requires vigorous angiogenesis initiated by, for example, an arteriovenous fistula or a ligated vascular pedicle. Growth may also be enhanced by contact with the external environment. In this study tissue growth in a rat model, vascularized via an arteriovenous loop (AV Loop) or ligated pedicle, was compared in chambers that were either closed or perforated. Chambers were harvested at 4 weeks and tissue volume and histology compared. In perforated chambers, more tissue were generated using the ligated pedicle (0.75 ml+/-0.04) than the AV Loop (0.59 ml+/-0.01). Perforated chambers generated larger volumes of tissue than closed chambers because they encouraged tissue ingrowth through the perforations. Both vessel configurations supported tissue growth but, interestingly, the ligated pedicle resulted in significantly more tissue in the perforated chambers.

Functional interaction of metabotropic glutamate receptor 5 and NMDA-receptor by a metabotropic glutamate receptor 5 positive allosteric modulator.

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

The Na+/H+ exchanger modulates long-term potentiation in rat hippocampal slices.

Although present in great variety in the brain, the role of Na(+)/H(+) exchangers (NHEs) in hippocampal plasticity is still unknown and the effect of NHE inhibition on long-term potentiation (LTP) has not been studied yet. As it is conceivable that NHE inhibitors may severely affect mechanisms that are considered to underlie learning and memory we investigated whether the broad-spectrum NHE inhibitor 5'-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 microM) influences LTP induced by different stimuli based on a theta burst in interface hippocampus slices from 7-8-week-old Wistar and 30-month-old Fischer 344/Brown-Norway F1 hybrid (F344/BN) rats. EIPA did not affect basal synaptic transmission, paired pulse inhibition, or LTP induced by a weak stimulus, but improved the maintenance of the LTP of the population spike induced by a strong tetanus. Our data suggest that NHE activity serves as a negative feedback mechanism to control neuronal excitability and plasticity in both young and senescent animals.

Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.

Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction.

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.

The novel selective PDE9 inhibitor BAY 73-6691 improves learning and memory in rodents.

The present study investigated the putative pro-cognitive effects of the novel selective PDE9 inhibitor BAY 73-6691. The effects on basal synaptic transmission and long-term potentiation (LTP) were investigated in rat hippocampal slices. Pro-cognitive effects were assessed in a series of learning and memory tasks using rodents as subjects. BAY 73-6691 had no effect on basal synaptic transmission in hippocampal slices prepared from young adult (7- to 8-week-old) Wistar rats. A dose of 10 microM, but not 30 microM, BAY 73-6691 enhanced early LTP after weak tetanic stimulation. The dose effective in young adult Wistar rats did not affect LTP in hippocampal slices prepared from young (7- to 8-week-old) Fischer 344 X Brown Norway (FBNF1) rats, probably reflecting strain differences. However, it increased basal synaptic transmission and enhanced early LTP after weak tetanic stimulation in hippocampal slices prepared from very old (31- to 35-month-old) FBNF1 rats. BAY 73-6691 enhanced acquisition, consolidation, and retention of long-term memory (LTM) in a social recognition task and tended to enhance LTM in an object recognition task. Bay 73-6691 attenuated the scoplamine-induced retention deficit in a passive avoidance task, and the MK-801-induced short-term memory deficits in a T-maze alternation task. The mechanism of action, possibly through modulation of the NO/cGMP-PKG/CREB pathway, is discussed. Our findings support the notion that PDE9 inhibition may be a novel target for treating memory deficits that are associated with aging and neurodegenerative disorders such as Alzheimer's disease.

Inhibitors of cation-chloride-cotransporters affect hypoxic/hypoglycemic injury in hippocampal slices.

Electroneutral cation-chloride cotransporters are abundantly expressed in the brain and are involved in the regulation of the intracellular Cl(-) concentration and thus gamma-aminobutyric acid-dependent inhibition of neuronal excitability. As yet there is little evidence whether or not Na(+)-K(+)-2Cl(-) or K(+)-Cl(-) cotransporters are involved in neuronal hyperexcitability and death in cerebral ischemia. In this study, by measuring propidium iodide staining in organotypic hippocampal slice cultures from young rats and population spike recovery in acutely isolated hippocampal slices from adult rats after a hypoxic/hypoglycemic insult, we were able to assess if cation-chloride cotransport inhibitors reduce neuronal injury. The Na(+)-K(+)-2Cl(-) cotransport inhibitor bumetanide in the range of 1-10 microM reduced neuronal damage in the slice cultures by 25%, but did not affect population spike recovery in acutely isolated slices. In contrast the K(+)-Cl(-) cotransport inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA, 100 microM) significantly diminished the restitution of the population spikes from 33% before to 8% after hypoxia/hypoglycemia and increased the damage in the slice cultures by 60%. Consequently, our data suggest that the Na(+)-K(+)-2Cl(-) cotransporter may contribute to neuronal injury and that the activity of the K(+)-Cl(-) cotransporters is an intrinsic protective mechanism of neurons against ischemic damage.

The ATP-regulated K+-channel inhibitor HMR-1372 affects synaptic plasticity in hippocampal slices.

Long-term potentiation (LTP) and long-term depression of synaptic transmission in the hippocampus are widely studied models of learning and memory processes. The role of ATP-regulated K+ channels (K(ATP)+ channels), which are abundant in the brain, has not yet been studied in long-term potentiation or long-term depression. We investigated whether K(ATP)+ channel inhibition by the highly selective K(ATP)+-channel blocker 1-[[5-[2-(5-tert-butyl-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (HMR-1372), a novel putative class III antiarrhythmic, affects long-term potentiation or the long-term depression induced by 3,5-dihydroxyphenylglycine (30 microM) in submerged rat hippocampal slices. HMR-1372 (10 microM) did not affect basal synaptic transmission, paired pulse inhibition, long-term depression or long-term potentiation elicited by a weak (weak long-term potentiation) tetanus, but significantly amplified the long-term efficacy of long-term potentiation elicited by a strong tetanus (strong long-term potentiation). The K(ATP)+-channel inhibitor glibenclamide (20 microM) also ameliorated only strong long-term potentiation. Our data suggest that K(ATP)+ channels are activated during or after induction of long-term potentiation and play a role in controlling synaptic excitability.