Cellular Mechanisms of Cognitive Enhancement: The In Vivo Modulation of the Firing Activity and the Responsiveness of Rat Hippocampal Neurons by Memantine and Alpha7 Nicotinic Acetylcholine Receptor Ligands.

Promising new pharmacological strategies for the enhancement of cognition target either nicotinic acetylcholine receptors (nAChR) or N-methyl-D-aspartate receptors (NMDAR). There is also an increasing interest in low-dose combination therapies co-targeting the above neurotransmitter systems to reach greater efficacy over the monotreatments and to reduce possible side effects of high-dose monotreatments. In the present study, we assessed modulatory effects of the α7 nAChR-selective agonist PHA-543613 (PHA), a novel α7 nAChR positive allosteric modulator compound (CompoundX) and the NMDAR antagonist memantine on the in vivo firing activity of CA1 pyramidal neurons in the rat hippocampus. Three different test conditions were applied: spontaneous firing activity, NMDA-evoked firing activity and ACh-evoked firing activity. Results showed that high but not low doses of memantine decreased NMDA-evoked firing activity, and low doses increased the spontaneous and ACh-evoked firing activity. Systemically applied PHA robustly potentiated ACh-evoked firing activity with having no effect on NMDA-evoked activity. In addition, CompoundX increased both NMDA- and ACh-evoked firing activity, having no effects on spontaneous firing of the neurons. A combination of low doses of memantine and PHA increased firing activity in all test conditions and similar effects were observed with memantine and CompoundX but without spontaneous firing activity increasing effects. Our present results demonstrate that α7 nAChR agents beneficially interact with Alzheimer's disease medication memantine. Moreover, positive allosteric modulators potentiate memantine effects on the right time and the right place without affecting spontaneous firing activity. All these data confirm previous behavioral evidence for the viability of combination therapies for cognitive enhancement.

Chemogenetic inhibition of the lateral hypothalamus effectively reduces food intake in rats in a translational proof-of-concept study.

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo efficacy assay in rats which could support chemogenetic drug discovery by providing a quick, simple and reliable animal model. Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation. Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts 1-3 h post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-N-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint. This study demonstrates that general chemogenetic silencing of the LH can be utilized as an optimal, fast and reliable in vivo experimental model for conducting preclinical proof-of-concept studies in order to validate the in vivo effectiveness of novel chemogenetic treatments. We also hypothesize based on our results that universal LH silencing with existing and human translatable genetic neuroengineering techniques might be a viable strategy to affect food intake and influence obesity.

Increased brain cytokine level associated impairment of vigilance and memory in aged rats can be alleviated by alpha7 nicotinic acetylcholine receptor agonist treatment.

Age-related neurocognitive disorders are common problems in developed societies. Aging not only affects memory processes, but may also disturb attention, vigilance, and other executive functions. In the present study, we aimed to investigate age-related cognitive deficits in rats and associated molecular alterations in the brain. We also aimed to test the effects of the alpha7 nicotinic acetylcholine receptor (nAChR) agonist PHA-543613 on memory as well as on the sustained attention and vigilance of aged rats. Short- and long-term spatial memories of the rats were tested using the Morris water maze (MWM) task. To measure attention and vigilance, we designed a rat version of the psychomotor vigilance task (PVT) that is frequently used in human clinical examinations. At the end of the behavioral experiments, mRNA and protein expression of alpha7 nAChRs, cytokines, and brain-derived neurotrophic factor (BDNF) were quantitatively measured in the hippocampus, frontal cortex, striatum, and cerebellum. Aged rats showed marked cognitive deficits in both the MWM and the PVT. The deficit was accompanied by increased IL-1beta and TNFalpha mRNA expression and decreased BDNF protein expression in the hippocampus. PHA-543613 significantly improved the reaction time of aged rats in the PVT, especially for unexpectedly appearing stimuli, while only slightly (non-significantly) alleviating spatial memory deficits in the MWM. These results indicate that targeting alpha7 nAChRs may be an effective strategy for the amelioration of attention and vigilance deficits in age-related neurocognitive disorders.

Aconitum Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0-3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0-5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications.

Potentiation of cognitive enhancer effects of Alzheimer's disease medication memantine by alpha7 nicotinic acetylcholine receptor agonist PHA-543613 in the Morris water maze task.

RATIONALE: There are controversial pieces of evidence whether combination therapies using memantine and cholinesterase inhibitors are beneficial over their monotreatments. However, results of preclinical studies are promising when memantine is combined with agonists and allosteric modulators of the alpha7 nicotinic acetylcholine receptor (nAChR). OBJECTIVES: Here, we tested the hypothesis that cognitive enhancer effects of memantine can be potentiated through modulating alpha7 nAChRs in a scopolamine-induced amnesia model. METHODS: Monotreatments, as well as co-administrations of selective alpha7 nicotinic acetylcholine receptor agonist PHA-543613 and memantine were tested in the Morris water maze task in rats. The efficacy of the co-administration treatment was observed on different domains of spatial episodic memory. RESULTS: Low dose of memantine (0.1 mg/kg) and PHA-543613 (0.3 mg/kg) successfully reversed scopolamine-induced short-term memory deficits both in monotreatments and in co-administration. When recall of information from long-term memory was tested, pharmacological effects caused by co-administration of subeffective doses of memantine and PHA-543613 exceeded that of their monotreatments. CONCLUSION: Our results further support the evidence of beneficial interactions between memantine and alpha7 nAChR ligands and suggest a prominent role of alpha7 nAChRs in the procognitive effects of memantine.

Converging Evidence on D-Amino Acid Oxidase-Dependent Enhancement of Hippocampal Firing Activity and Passive Avoidance Learning in Rats.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptor activation requires the binding of a co-agonist on the glycine-binding site. D-serine is the main endogenous co-agonist of NMDA receptors, and its availability significantly depends on the activity of the metabolic enzyme D-amino acid oxidase (DAAO). Inhibition of DAAO increases the brain levels of D-serine and modulates a variety of physiological functions, including cognitive behavior. METHODS: Here, we examined the effects of a novel 4-hydroxypyridazin-3(2H)-one derivative DAAO inhibitor, Compound 30 (CPD30), on passive avoidance learning and on neuronal firing activity in rats. RESULTS: D-serine administration was applied as reference, which increased cognitive performance and enhanced hippocampal firing activity and responsiveness to NMDA after both local and systemic application. Similarly to D-serine, CPD30 (0.1 mg/kg) effectively reversed MK-801-induced memory impairment in the passive avoidance test. Furthermore, local iontophoretic application of CPD30 in the vicinity of hippocampal pyramidal neurons significantly increased firing rate and enhanced their responses to locally applied NMDA. CPD30 also enhanced hippocampal firing activity after systemic administration. In 0.1- to 1.0-mg/kg doses, CPD30 increased spontaneous and NMDA-evoked firing activity of the neurons. Effects of CPD30 on NMDA responsiveness emerged faster (at 10 minutes post-injection) when a 1.0-mg/kg dose was applied compared with the onset of the effects of 0.1 mg/kg CPD30 (at 30 minutes post-injection). CONCLUSIONS: The present results confirm that the inhibition of DAAO enzyme is an effective strategy for cognitive enhancement. Our findings further facilitate the understanding of the cellular mechanisms underlying the behavioral effects of DAAO inhibition in the mammalian brain.

Long-term cognitive impairment without diffuse axonal injury following repetitive mild traumatic brain injury in rats.

Repetitive mild traumatic brain injuries (TBI) impair cognitive abilities and increase risk of neurodegenerative disorders in humans. We developed two repetitive mild TBI models in rats with different time intervals between successive weight-drop injuries. Rats were subjected to repetitive Sham (no injury), single mild (mTBI), repetitive mild (rmTBI - 5 hits, 24 h apart), rapid repetitive mild (rapTBI - 5 hits, 5 min apart) or a single severe (sTBI) TBI. Cognitive performance was assessed 2 and 8 weeks after TBI in the novel object recognition test (NOR), and 6-7 weeks after TBI in the water maze (MWM). Acute immunohistochemical markers were evaluated 24 h after TBI, and blood biomarkers were measured with ELISA 8 weeks after TBI. In the NOR, both rmTBI and rapTBI showed poor performance at 2 weeks post-injury. At 8 weeks post-injury, the rmTBI group still performed worse than the Sham and mTBI groups, while the rapTBI group recovered. In the MWM, the rapTBI group performed worse than the Sham and mTBI groups. Acute APP and RMO-14 immunohistochemistry showed axonal injury at the pontomedullary junction in the sTBI, but not in other groups. ELISA showed increased serum GFAP levels 8 weeks after sTBI, while no differences were found between the injury groups in the levels of phosphorylated-tau and S100ß. Results suggest that the rmTBI protocol is the most suitable model for testing cognitive impairment after mild repetitive head injuries and that the prolonged cognitive impairment after repetitive mild TBI originates from different structural and molecular mechanisms compared to similar impairments after single sTBI.

Facilitation and inhibition of firing activity and N-methyl-D-aspartate-evoked responses of CA1 hippocampal pyramidal cells by alpha7 nicotinic acetylcholine receptor selective compounds in vivo.

Alpha7 nicotinic acetylcholine receptors (nAChRs) are promising novel targets for the treatment of neurocognitive disorders. Although the cognitive enhancer potential of alpha7 nAChR agonists and positive allosteric modulators (PAMs) has been confirmed in several preclinical animal models, there are only sparse in vivo electrophysiological data on their effects on the firing activity and excitability of neurons. The present study investigated and compared local effects of alpha7 nAChR agonist PHA-543613 and PAMs PNU-120596 and NS-1738 on the spontaneous and N-methyl-D-aspartate-evoked (NMDA-evoked) firing rate of rat CA1 hippocampal pyramidal cells, in vivo. Furthermore, effects of alpha7 nAChR antagonist methyllycaconitine (MLA) and GABA were also tested. Results showed substantially different effects of the alpha7 nAChR agonist and PAMs. While PNU-120596 and NS-1738 predominantly and significantly increased both spontaneous and NMDA-evoked firing rate of the neurons, application of PHA-543613 resulted in almost equal distribution of facilitatory and inhibitory effects. The increase of the NMDA-evoked firing rate exerted by NS-1738 was superadditive over the sum of the single effects of NMDA and NS-1738. The simultaneous application of alpha7 nAChR agonist PHA-543613 and PAM NS-1738 resulted in additive increase of both spontaneous and NMDA-evoked firing rate. However, NS-1738 counteracted inhibitory effects of PHA-543613 in 5 out of 6 neurons, resulting in a synergistic potentiation of their firing responses to NMDA. Our results suggest that alpha7 nAChR PAMs increase neuronal excitability more potently than agonists, while the remarkable occurrence of inhibitory effects of PHA-543613 (possibly originating from receptor desensitization) implies that agonists may exert neuroprotective effects.

Cognitive Enhancer Effects of Low Memantine Doses Are Facilitated by an Alpha7 Nicotinic Acetylcholine Receptor Agonist in Scopolamine-Induced Amnesia in Rats.

Alpha7 nicotinic acetylcholine receptors (nAChRs) play an important role in learning and memory and are promising targets for pharmacological cognitive enhancement. Memantine, an approved substance for Alzheimer's disease treatment, is an antagonist of the N-Methyl-D-aspartate receptor (NMDAR) and also acts as an alpha7 nAChR antagonist. Here, we tested the interaction between an alpha7 nAChR agonist (PHA-543613) and memantine. Efficacy of memantine, PHA-543613, and their co-administration were investigated on the spatial working memory of rats using the spontaneous alternation paradigm in T-maze. Scopolamine-induced transient amnesia was used to model cognitive impairment. First, the dose-response relationship was assessed for memantine, and its lowest effective dose was found to be 0.1 mg/kg. Then, co-administration treatments with subeffective doses of the alpha7 nAChR agonist PHA-543613 and different doses of memantine were tested. The co-administration of subeffective drug doses significantly improved memory performance of the rats and reversed scopolamine-induced deficits. Interestingly, a higher than effective (0.3 mg/kg) dose of memantine did not increase performance in monotreatment, only in co-administration with PHA-543613. However, the co-administration of PHA-543613 did not further increase the efficacy of the previously effective monotreatment doses of memantine. Thus, the efficacy of memantine monotreatment and its co-administration with PHA-543613 converged to create a common ceiling effect, with an additive interaction found in the behavioral effects. These results suggest that memantine and PHA-543613 may exert their cognitive enhancer effects on the same target, possibly on the alpha7 nAChRs. Results also suggest possible benefits of a combination therapy with memantine and alpha7 nAChR agonists.