Irina Fedorova

M2 Muscarinic Acetylcholine Receptor Knock-Out Mice Show Deficits in Behavioral Flexibility, Working Memory, and Hippocampal Plasticity

Muscarinic acetylcholine receptors are known to play key roles in facilitating cognitive processes. However, the specific roles of the individual muscarinic receptor subtypes (M1-M5) in learning and memory are not well understood at present. In the present study, we used wild-type (M2+/+) and M2 receptor-deficient (M2-/-) mice to examine the potential role of M2 receptors in learning and memory and hippocampal synaptic plasticity. M2-/- mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation tests, respectively. The behavioral deficits of M2-/- mice were associated with profound changes in neuronal plasticity studied at the Schaffer-CA1 synapse of hippocampal slices. Strikingly, short-term potentiation (STP) was abolished, and long-term potentiation (LTP) was drastically reduced after high-frequency stimulation of M2-/- hippocampi. Treatment of M2-/- hippocampal slices with the GABAA receptor antagonist, bicuculline, restored STP and significantly increased LTP. Whole-cell recordings from CA1 pyramidal cells demonstrated a much stronger disinhibition of GABAergic than glutamatergic transmission in M2-/- hippocampi, which was particularly prominent during stimulus trains. Increased strength of GABAergic inhibition is thus a likely mechanism underlying the impaired synaptic plasticity observed with M2-/- hippocampi. Moreover, the persistent enhancement of excitatory synaptic transmission in CA1 pyramidal cells induced by the transient application of a low concentration of a muscarinic agonist (referred to as LTPm) was totally abolished in M2-/- mice. Because impaired muscarinic cholinergic neurotransmission is associated with Alzheimers disease and normal aging processes, these findings should be of considerable therapeutic relevance.

Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1–M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5−/− mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5−/− mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5−/− mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.

Role for M5 muscarinic acetylcholine receptors in cocaine addiction.

Muscarinic cholinergic receptors of the M5 subtype are expressed by dopamine‐containing neurons of the ventral tegmentum. These M5 receptors modulate the activity of midbrain dopaminergic neurons, which play an important role in mediating reinforcing properties of abused psychostimulants like cocaine. The potential role of M5 receptors in the reinforcing effects of cocaine was investigated using M5 receptor‐deficient mice in a model of acute cocaine self‐administration. The M5‐deficient mice self‐administered cocaine at a significantly lower rate than wild‐type controls. In the conditioned place preference procedure, a classic test for evaluating the rewarding properties of drugs, M5‐deficient mice spent significantly less time in the cocaine‐paired compartment than control mice. Moreover, the severity of the cocaine withdrawal syndrome (withdrawal‐associated anxiety measured in the elevated plus‐maze) was significantly attenuated in mice lacking the M5 receptor. These results demonstrate that M5 receptors play an important role in mediating both cocaine‐associated reinforcement and withdrawal.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration.

Abstract1. The potential neuroprotective actions of the A3 adenosine receptor (A3AR) were investigated using mice with functional deletions of the A3AR (A3AR−/−) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival.2. Untreated A3AR−/− mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques.3. A3AR−/− mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A3AR−/− mice also showed evidence of decreased nociception in the hot-plate, but not tail-flick tests. Further, A3AR−/− mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A3AR+/+) and A3AR−/− mice. However, the extent of neuronal death in the CA2–3 subfields was less pronounced in A3AR+/+ than A3AR−/− mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A3AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.).4. These results indicate that pharmacologic or genetic suppression of A3AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A3AR agonists, A3AR−/− mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.

An n-3 fatty acid deficiency impairs rat spatial learning in the Barnes maze.

In this study, the authors demonstrate that rats with n-3 fatty acid deficiency display spatial learning deficits in the Barnes circular maze. Dams were deprived of n-3 fatty acids during pregnancy and lactation, and their offspring were weaned to the same deficient diet. There was a 58% loss of brain docosahexaenoic acid (DHA) in the n-3 fatty acid-deficient rats in comparison to n-3 fatty acid-adequate rats. At 8 weeks of age, deficient rats demonstrated moderate impairment in Barnes maze performance compared with the n-3 fatty acid-adequate rats during the initial training. In the reversal learning task, the n-3 fatty acid-deficient rats showed a profound deficit in performance: They required more time to find a new position of the escape tunnel, which was accompanied by a higher number of errors and perseverations. The n-3 fatty acid-deficient rats had reduced tissue levels of dopamine in the ventral striatum and enhanced levels of the metabolite 3,4-dihydroxyphenylacetic acid in frontal cortex and hypothalamus. In summary, this study demonstrates that rats with low brain DHA have a deficit in spatial reversal learning that could be related to changes in dopamine transmission in critical brain circuits.

Effects of Docosahexaenoic Acid in Preventing Experimental Choroidal Neovascularization in Rodents

BACKGROUND The purpose of this study is to evaluate the effects of docosahexaenoic acid (DHA), a major omega-3-polyunsaturated fatty acid (ω-3-PUFAs), in the development of experimental choroidal neovascularization (CNV) in rodents. METHODS Experimental second generation Long Evans rats fed with diets of varying ω-3-PUFA content designed to produce significantly different retinal DHA levels were used in our studies. A transgenic mouse model (fat-1) engineered to over-produce DHA was also studied. CNV was induced by rupture of Bruchs membrane using laser photocoagulation. At 7 days after induction, animals were euthanatized, and eyes were collected. RPE/choroid flatmounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and high-performance 3D imaging software. RESULTS The median of CNV complex volumes of animals with DHA-adequate diets was lower by 63% relative to that of animals with DHA-deficient diets. The median of CNV complex volumes in fat-1 transgenic mice was decreased by 59% relative to that of wild type controls. CONCLUSIONS Dietary intake or genetic manipulation to increase the sources of DHA significantly diminished the volume of induced CNV lesions in rodents. They suggest that consumption of ω-3-PUFAs may serve to prevent CNV.