Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice

Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.     

Molecular forms of acetylcholinesterases in Alzheimer's disease

In this study, we examined 26 cases of Alzheimer's disease (AD) and 14 age-matched controls. In Brodmann area 21 cerebral cortex of the AD cases, there was no change in soluble G1 and G4 acetylcholinesterase (AChE) (EC 3.1.1.7), a significant 40% decrease in membrane-associated G4 AChE, significant 342 and 406% increases in A12 and A8 AChE, and a significant 71% decrease in choline acetyltransferase (ChAT) (EC 2.3.1.6). Our working hypothesis to account for these changes postulates that soluble globular forms are unchanged because they are primarily associated with intrinsic cortical neurons that are relatively unaffected by AD, that ChAT and membrane-associated G4 AChE decrease because they are primarily associated with incoming axons of cholinergic neurons that are abnormal in AD, and that asymmetric forms of AChE increase because of an acrylamide-type impairment of fast axonal transport in diseased incoming cholinergic axons. In the nucleus basalis of Meynert (nbM) of the 26 AD cases, there was a significant 61% decrease in the number of cholinergic neurons, an insignificant 23% decrease in nbM ChAT, a significant 298% increase in nbM ChAT per cholinergic neuron, and a significant 7% increase in the area of cholinergic perikarya. To account for the increased ChAT in cholinergic neurons and the enlargement of cholinergic perikarya, we propose that slow axonal transport may be impaired in nbM cholinergic neurons in AD.     

TRH analog MK-771 reverses neurochemical and learning deficits in medial septal-lesioned rats

Microinjection of ibotenic acid into medial septum of rats decreased choline acetyltransferase (CAT) and high-affinity choline uptake (HACU) activities in hippocampus and retarded the learning of a spatial memory task in the radial-arm maze. Administration of MK-771, a stable TRH analog, to such animals restored HACU activity in hippocampus to normal levels. Daily treatment of rats with MK-771 prior to maze running also restored the animals' learning ability. MK-771 did not enhance hippocampal HACU activity or maze performance in sham-lesioned rats. These results suggest that MK-771 reversed the ibotenic acid-induced memory deficit by restoring septohippocampal cholinergic function. MK-771 and other TRH analogs may represent novel agents for improving memory deficits produced by cholinergic insufficiency in Alzheimer's disease.     

Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis

We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10- to 12-fold increase in the release of endogenous ACh above spontaneous release. Potassium-evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 +/- 13.0% decrease in spontaneous ACh release and a 60.4 +/- 8.2% decrease in potassium-evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4-diaminopyridine (3,4-DAP) whereas 10 mM 3,4-DAP caused a sevenfold increase. The increase in ACh release caused by 3,4-DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high-affinity choline uptake and this could be inhibited by hemicholinium-3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium-dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.     

M1 Muscarinic Agonist Treatment Reverses Cognitive and Cholinergic Impairments of Apolipoprotein E-Deficient Mice

Abstract: Recent studies suggest that apolipoprotein E (apoE) plays a specific role in brain cholinergic function and that the E4 allele of apoE (apoE4), a major risk factor for Alzheimer's disease (AD), may predict the extent of cholinergic dysfunction and the efficacy of cholinergic therapy in this disease. Animal model studies relevant to this hypothesis revealed that apoE-deficient (knockout) mice have working memory impairments that are associated with distinct dysfunction of basal forebrain cholinergic neurons. Cholinergic replacement therapy utilizing M₁-selective muscarinic agonists has been proposed as effective treatment for AD patients. In the present study, we examined whether the memory deficits and brain cholinergic deficiency of apoE-deficient mice can be ameliorated by the M₁-selective agonist 1-methylpiperidine-4-spiro-(2'-methylthiazoline), [AF150(S)]. Treatment of apoE-deficient mice with AF150(S) for 3 weeks completely abolished their working memory impairments. Furthermore, this reversal of cognitive deficit was associated with a parallel increase of histochemically determined brain choline acetyltransferase and acetylcholinesterase levels and with the recovery of these cholinergic markers back to control levels. These findings show that apoE deficiency-related cognitive and cholinergic deficits can be ameliorated by M₁-selective muscarinic treatment. They also provide a novel model system for development and evaluation of therapeutic strategies directed specifically at the AD patients whose condition is attributed to the apoE genotype.     

Microwave irradiation affects radial-arm maze performance in the rat

After 45 min of exposure to pulsed 2450 MHz microwaves (2 μsec pulses, 500 pps, 1 mW/cm², average whole body SAR 0.6 W/kg), rats showed retarded learning while performing in the radial-arm maze to obtain food rewards, indicating a deficit in spatial “working memory” function. This behavioral deficit was reversed by pretreatment before exposure with the cholinergic agonist physostigmine or the opiate antagonist naltrexone, whereas pretreatment with the peripheral opiate antagonist naloxone methiodide showed no reversal of effect. These data indicate that both cholinergic and endogenous opioid neurotransmitter systems in the brain are involved in the microwave-induced spatial memory deficit. © 1994 Wiley-Liss, Inc.     

Some cholinergic themes related to Alzheimer's disease: Synaptology of the nucleus basalis,location of m2 receptors,interactions with amyloid metabolism,and perturbations of cortical plasticity

Cholinergic neurons in the nucleus basalis of Meynert (nbM) receive cholinergic, GABAergic and monoaminergic synapses. Only few of these neurons display the sort of intense m2 immunoreactivity that would be expected if they were expressing m2 as their presynaptic autoreceptor. The depletion of cortical m2 in Alzheimer's disease (AD) appears to reflect the loss of presynaptic autoreceptors located on incoming axons from the nucleus basalis of Meynert (nbM) and also the loss of postsynaptic receptors located on a novel group of nitric oxide producing interstitial neurons in the cerebral cortex. The defect of cholinergic transmission in AD may enhance the neurotoxicity of amyloid β, leading to a vicious cycle which can potentially accelerate the pathological process. Because acetylcholine plays a critical role in regulating axonal growth and synaptic remodeling, the cholinergic loss in AD can perturb cortical plasticity so as to undermine the already fragile compensatory reserve of the aging cerebral cortex.     

Sexually dimorphic responses to neonatal basal forebrain lesions in mice: I. Behavior and neurochemistry

The nucleus basalis magnocellularis (nBM) provides the primary source of cholinergic input to the cortex. Neonatal lesions of the nBM produce transient reductions in cholinergic markers, persistent abnormalities in cortical morphology, and spatial navigation impairments in adult mice. The present study examined sex differences in the effects of an electrolytic nBM lesion on postnatal day 1 (PND 1) in mice on behavior and neurochemistry in adulthood. Mice were lesioned on PND 1 and tested at 8 weeks of age on a battery of behavioral tests including passive avoidance, cued and spatial tasks in the Morris water maze, simple and delayed nonmatch to sample versions of an odor discrimination task, and locomotor activity measurements. Following behavioral testing, mice were sacrificed for either morphological assessment or neurochemical analysis of a cholinergic marker or catecholamines. There were no lesion or sex differences in acquisition or retention of passive avoidance, performance of the odor discrimination tasks, or activity levels. Control mice showed a robust sex difference in performance of the spatial water maze task. The lesion produced a slight cued but more dramatic spatial navigation deficit in the water maze which affected only the male mice. Neurochemical analyses revealed no lesion-induced changes in either choline acetyltransferase activity or levels of norepinephrine or serotonin at the time of testing. The subsequent report shows a sex difference in lesion-induced changes in cortical morphology which suggests that sexually dimorphic cholinergic influences on cortical development are responsible for the behavioral deficits seen in this study. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 582–594, 1998     

Reversal of heroin neurobehavioral teratogenicity by grafting of neural progenitors

A major objective in identifying the mechanisms underlying neurobehavioral teratogenicity in an animal model is the possibility of designing therapies that reverse or offset teratogen-induced neural damage. In our previous studies, we identified deficits in hippocampal muscarinic cholinergic receptor-induced translocation of protein kinase C (PKC) γ as the likely central factor responsible for the adverse behavioral effects of pre-natal heroin exposure. Neural progenitors (NP) have the ability to recover behavioral deficits after focal hippocampal damage. Therefore, we explored whether behavioral and synaptic defects could be reversed in adulthood by neural progenitor grafting. Pregnant mice were injected daily with 10 mg/kg of heroin on gestational days 9–18. In adulthood, offspring showed deficits in the Morris maze, a behavior dependent on the integrity of septohippocampal cholinergic synaptic function, along with the loss of the PKCγ and PKCβII responses to cholinergic stimulation. Mice that were exposed pre-natally to heroin and vehicle control mice were then grafted in adulthood with NP. Importantly, most grafted cells differentiated to astrocytes. NP reversed the behavioral deficits ( p  =   0.0043) and restored the normal response of hippocampal PKCγ and PKCβII ( p  =   0.0337 and p  =   0.0265 respectively) to cholinergic receptor stimulation. The effects were specific as the PKCα isoform, which is unrelated to the behavioral deficits, showed almost no changes. Neural progenitor grafting thus offers an animal model for reversing neurobehavioral deficits originating in septohippocampal cholinergic defects elicited by pre-natal exposure to insults.     

Passive avoidance deficits following lesions of the posteroventral hippocampo-subiculo-entorhinal area in the developing rat

Young rats, 13, 16, and 20 days of age, underwent discrete bilateral electrolytic lesions of the posteroventral hippocampo-subiculo-entorhinal area, and were trained on a cool-draft-stimulus passive avoidance task 20 min later. Significant deficits in passive avoidance learning were observed at all ages studied following either small or more extended damage as compared to performance of sham-lesioned animals. The impairment was dependent upon the size of the lesion. Extended bilateral lesions of the parietal cortex overlying hippocampus induced no deficit. These results confirm that this part of the hippocampal complex plays a role in passive avoidance learning in the rat. They also show that this control of behavior is already established by the second week of life, thus supporting our previous experiments that demonstrate a cholinergic nicotinic involvement of this region in acquisition of passive avoidance as early as the 11th day of age.     

Spatial learning deficit in the rat after exposure to a 60 Hz magnetic field

Rats were trained in ten daily sessions to perform in a 12-arm radial maze, which is a behavioral test for spatial memory functions. Exposure to a 60 Hz magnetic field (45 min, 0.75 mT) immediately before each training session retarded learning significantly. Pretreatment with the cholinergic agonist physostigmine before magnetic field exposure reversed the field's effect on spatial learning. Data from this experiment indicate that magnetic field-induced spatial learning deficit is caused by the effect of the field on cholinergic systems. © 1996 Wiley-Liss, Inc.     

Lesions of the Basal Forebrain Cholinergic System in Mice Disrupt Idiothetic Navigation

Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer’s disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer’s disease patients perform poorly on both real space and computerized cued (allothetic) or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer’s disease.     

Effect of losartan and enalapril on cognitive deficit caused by Goldblatt induced hypertension

The present study was designed to evaluate the learning and memory, in an altered physiological state associated with increased blood pressure and activated renin angiotensin system in Wistar rats. The role of angiotensin in cognitive function was assessed by treatment with angiotensin converting enzyme (ACE) inhibitor enalapril (2 mg/kg), angiotensin 1 receptor (AT(1)) antagonist losartan (5 mg/kg) and their combination. The experimental renal hypertension was induced by the method of Goldblatt. Learning and memory was assessed using the radial arm maze test. Acetylcholine esterase (AChE) levels in the pons medulla, hippocampus, striatum and frontal cortex were measured as a cholinergic marker of learning and memory. Results indicate that in comparison to normotensive rats, renal hypertensive rats committed significantly higher number of errors and took more trials and days to learn the radial arm maze learning and exhibited memory deficit in the radial arm maze retrieval after two weeks of retention interval, indicating impaired acquisition and memory. Treatment with enalapril, losartan and their combination attenuated the observed memory deficits indicating a possible role of renin angiotensin system in cognitive function. AChE level was reduced in hippocampus and frontal cortex of renal hypertensive rats which could be attributed to the observed memory deficit in hypertensive rats. It can be concluded that, renal hypertensive rats had a poor acquisition, retrieval of the learned behavior, perhaps a possible disturbance in memory consolidation process and that this state was reversed with ACE inhibitor enalapril and AT 1 receptor antagonist losartan.     

Intraventricular administration of antivasopressin serum inhibits retention in mice

Elevations and decrements in the levels of the posterior pituitary hormone vasopressin result in facilitations and deficits in retention, respectively, in rats. Despite the frequent use of mice in studies of pharmacological influences on retention, there is a paucity of information regarding the role of endogenous peptides, particularly vasopressin, in the memory processes of mice. In the present experiment, mice suffering from acute inactivation of central vasopressin, induced by an immediately posttraining, 2 microliters, intracerebroventricular injection of antivasopressin serum, displayed a retention deficit for passive avoidance training. The results of this experiment suggest that endogenous vasopressin modulates the memory processes of mice, as well as rats.     

Cortical modulation of cholinergic neurons in the striatum

Previous reports suggest the existence of a cortico-striatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (1 hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striatal cholinergic neurons can be influenced by this cortico-striatal pathway.     

Aluminum-Induced Cholinergic Deficits in Different Brain Parts and Its Implications on Sociability and Cognitive Functions in Mouse

Aluminum is associated with etiology of many neurodegenerative diseases specially Alzheimer’s disease. Chronic exposure to aluminum via drinking water results in aluminum deposition in the brain that leads to cognitive deficits. The study aimed to determine the effects of aluminum on cholinergic biomarkers, i.e., acetylcholine level, free choline level, and choline acetyltransferase gene expression, and how cholinergic deficit affects novel object recognition and sociability in mice. Mice were treated with AlCl₃ (250 mg/kg). Acetylcholine level, free choline level, and choline acetyltransferase gene expression were determined in cortex, hippocampus, and amygdala. The mice were subjected to behavior tests (novel object recognition and social novelty preference) to assess memory deficits. The acetylcholine level in cortex and hippocampus was significantly reduced in aluminum-treated animals, as compared to cortex and hippocampus of control animals. Acetylcholine level in amygdala of aluminum-treated animals remained unchanged. Free choline level in all the three brain parts was found unaltered in aluminum-treated mice. The novel object recognition memory was severely impaired in aluminum-treated mice, as compared to the control group. Similarly, animals treated with aluminum showed reduced sociability compared to the control mice group. Our study demonstrates that aluminum exposure via drinking water causes reduced acetylcholine synthesis in spite of normal free choline availability. This deficit is caused by reduced recycling of acetylcholine due to lower choline acetyltransferase level. This cholinergic hypofunction leads to cognitive and memory deficits. Moreover, hippocampus is the most affected brain part after aluminum intoxication.     

Retrograde amnesia produced by intrastriatal atropine and its reversal by choline

A number of studies have shown that cholinergic blockade of the striatum produces amnesia. In the present experiment it was predicted that by increasing the synthesis of striatal acetylcholine such amnesic state would be prevented. Atropine was injected into the striatum of rats before training of passive avoidance; some of these rats were also injected, intrastriatally, with choline before testing the retention of the task. Atropine alone produced amnesia while the combination of treatments reversed this effect.     

Intact Acquisition and Short-Term Retention of Non-Motor Procedural Learning in Parkinson’s Disease

Procedural learning is a form of memory where people implicitly acquire a skill through repeated practice. People with Parkinson’s disease (PD) have been found to acquire motor adaptation, a form of motor procedural learning, similarly to healthy older adults but they have deficits in long-term retention. A similar pattern of normal learning on initial exposure with a deficit in retention seen on subsequent days has also been seen in mirror-reading, a form of non-motor procedural learning. It is a well-studied fact that disrupting sleep will impair the consolidation of procedural memories. Given the prevalence of sleep disturbances in PD, the lack of retention on following days seen in these studies could simply be a side effect of this well-known symptom of PD. Because of this, we wondered whether people with PD would present with deficits in the short-term retention of a non-motor procedural learning task, when the test of retention was done the same day as the initial exposure. The aim of the present study was then to investigate acquisition and retention in the immediate short term of cognitive procedural learning using the mirror-reading task in people with PD. This task involved two conditions: one where triads of mirror-inverted words were always new that allowed assessing the learning of mirror-reading skill and another one where some of the triads were presented repeatedly during the experiment that allowed assessing the word-specific learning. People with PD both ON and OFF their normal medication were compared to healthy older adults and young adults. Participants were re-tested 50 minutes break after initial exposure to probe for short-term retention. The results of this study show that all groups of participants acquired and retained the two skills (mirror-reading and word-specific) similarly. These results suggest that neither healthy ageing nor the degeneration within the basal ganglia that occurs in PD does affect the mechanisms that underpin the acquisition of these new non-motor procedural learning skills and their short-term memories.     

An in vivo cholinergic influence on the retention of [3H]noradrenaline in regional brain synaptosomes

Rats were intraventricularly (icv) injected with [³H]noradrenaline and the retention of the amine was determined in synaptosomes obtained from cerebral cortex, hypothalamus and brain stem. Previous icv administration of hemicholinium-3, effective enough to markedly decrease brain acetylcholine levels, increased the retention of synaptosomal [³H]noradrenaline in hypothalamus and cerebral cortex; this increased retention did not occur in the brain stem. The increased retention of [³H]noradrenaline, produced by hemicholinium-3, was reversed by a concomitant icv dose of choline, which in turn reversed the decrease of acetylcholine caused by hemicholinium-3. These results are interpreted as brain cholinergic activity having an influence on the turnover of noradrenaline in some brain regions.     

Nicotinic receptor subtypes and cognitive function

Nicotinic receptor systems are involved in a wide variety of behavioral functions including cognitive function. Nicotinic medications may provide beneficial treatment for cognitive dysfunction such as Alzheimer's disease, schizophrenia, and attention deficit hyperactivity disorder (ADHD). Nicotine has been shown to improve attentional performance in all of these disorders. Better efficacy with fewer side effects might be achieved with novel nicotinic ligands selective for particular nicotinic subtypes. To develop these novel selective nicotinic ligands it is important to use animal models to determine the critical neurobehavioral bases for nicotinic involvement in cognitive function. Nicotine-induced cognitive improvement in rats is most consistently seen in working memory tasks. We have found that both acute and chronic nicotine administration significantly improves working memory performance of rats in the radial-arm maze. The pharmacologic and anatomic mechanisms for this effect have been examined in our laboratory in a series of local drug infusion studies. Both alpha 4 beta 2 and alpha 7 nicotinic receptors in the ventral hippocampus and basolateral amygdala are involved in working memory function. Working memory impairments were caused by local infusion of either alpha 4 beta 2 or alpha 7 antagonists. Ventral hippocampal alpha 4 beta 2 blockade-induced working memory deficits are reversed by chronic systemic nicotine treatment, while ventral hippocampal alpha 7 blockade-induced working memory deficits were not found to be reversed by the same nicotine regimen. Interestingly, alpha 4 beta 2 and alpha 7 induced deficits were not found to be additive in either the ventral hippocampus or the basolateral amygdala. In fact, in the amygdala, alpha 7 antagonist cotreatment actually reversed the working memory impairment caused by alpha 4 beta 2 antagonist administration. These studies of the neural nicotinic mechanisms underlying cognitive function are key for opening avenues for development of safe and effective nicotinic treatments for cognitive dysfunction.