Cholinergic blockade under working memory demands encountered by increased rehearsal strategies: evidence from fMRI in healthy subjects

The connection between cholinergic transmission and cognitive performance has been established in behavioural studies. The specific contribution of the muscarinic receptor system on cognitive performance and brain activation, however, has not been evaluated satisfyingly. To investigate the specific contribution of the muscarinic transmission on neural correlates of working memory, we examined the effects of scopolamine, an antagonist of the muscarinic receptors, using functional magnetic resonance imaging (fMRI). Fifteen healthy male, non-smoking subjects performed a fMRI scanning session following the application of scopolamine (0.4 mg, i.v.) or saline in a placebo-controlled, repeated measure, pseudo-randomized, single-blind design. Working memory was probed using an n-back task. Compared to placebo, challenging the cholinergic transmission with scopolamine resulted in hypoactivations in parietal, occipital and cerebellar areas and hyperactivations in frontal and prefrontal areas. These alterations are interpreted as compensatory strategies used to account for downregulation due to muscarinic acetylcholine blockade in parietal and cerebral storage systems by increased activation in frontal and prefrontal areas related to working memory rehearsal. Our results further underline the importance of cholinergic transmission to working memory performance and determine the specific contribution of muscarinic transmission on cerebral activation associated with executive functioning.     

Habit learning dissociation in rats with lesions to the vermis and the interpositus of the cerebellum

After cerebellar tumors resection, patients show motor skill learning impairments but also cognitive deficits. However, their exact origins remain controversial. Using a rat model of cerebellar injury, we assessed the involvement of two structures often damaged during resection (vermis and interpositus nuclei) on habits development. During extended training of an instrumental task, rats develop response routines that are no longer voluntary or goal-directed but habit-based, evidenced by their insensitivity to changes in the value of the reward. Here we showed that, in contrast to sham or vermis lesioned rats, discrete lesions to interpositus nuclei prevented rats from developing habits with overtraining, without motor difficulties, nor alteration of the instrumental task acquisition. Our results suggest that the role of the cerebellum can be extended from motor skill learning to cognitive routines learning. Similar habit impairment could possibly account for some of the long-term outcome difficulties observed in cerebellar-damaged patients.     

Effect of oxotremorine on local cerebral glucose utilization in motor system regions of the rat brain

The [¹⁴C]2-deoxy-d-glucose technique³³ was used to test the effects of central muscarinic stimulation on local cerebral glucose utilization (LCGU) in rats. Systemic administration of the muscarinic agonist oxotremorine (OXO, 0.7 mg/kg, ip) increased LCGU in brain regions involved in motor function: the sensorimotor cortex; the extrapyramidal motor system, including the striatum, globus pallidus, red nucleus, substantia nigra, subthalamus and ventral nucleus of the thalamus; the cerebellar vermis, fastigial nucleus and nucleus interpositus; and the vestibular nucleus. No effects were observed in the fibers of the pyramidal tract, internal capsule, cerebellar white matter and the dentate nucleus. Increases were not affected by the methylatropine (1 mg/kg sc), but were completely antagonized by scopolamine (2.5 mg/kg, ip). The anatomic distribution and magnitude of the LCGU response to OXO were not simply correlated with the reported densities of muscarinic receptors.     

Effects of infusion of cholinergic drugs into the prefrontal cortex area on delayed matching to position performance in the rat

Rats trained on a Delayed Matching To Position (DMTP) task displayed mediating behavior during delays to solve the task. Infusion of the cholinergic antagonist scopolamine into the medial Prefrontal Cortex area (mPFC), dose dependently impaired performance independent of delay. These results indicate that scopolamine does not specifically affect working memory. Infusion of the cholinesterase inhibitor physostigmine, muscarinic subtype receptor antagonists, the dopamine (D₁) antagonist SCH23390, and of the GABA-A receptor antagonist bicuculline, did not affect performance in the DMTP task. In a post-hoc analysis scopolamine was found to impair discriminability in a delay-dependent manner only in animals that used mediating behavior in the majority of the trials. Furthermore, a time sampling method indicated that scopolamine infusions into the mPFC disrupted mediating behavior during the task. Results suggest that cholinergic systems in the mPFC play a role in directing attention to task relevant behavior.     

Cognitive performance and cholinergic transmission: influence of muscarinic and nicotinic receptor blockade

The cholinergic system is essential in mediating cognitive processes. Although there has been extensive research regarding cholinergic receptor subsystems, the specific contribution of the muscarinic and nicotinic receptor system to cognitive processes still has not been sufficiently explored. In the present study, we examined the selective contribution of muscarinic and nicotinic antagonism to cognitive performance in healthy human subjects. A single-blind, double-dummy, time-elapsed, repeated measures cross-over design was used on 15 healthy males. Subjects completed a neuropsychological test battery assessing a wide range of cognitive domains after 0.4 mg scopolamine (intravenous), 0.2 mg/kg mecamylamine (max. 15 mg; oral) or placebo. Subjects were tested under three conditions: placebo/placebo (PP), scopolamine/placebo (SP) and mecamylamine/placebo (MP). Results show that scopolamine significantly impaired the free recall and recognition performance in the verbal learning test. No other cognitive domain was affected, neither by scopolamine nor by mecamylamine. In line with the existing literature, antagonism of muscarinic receptors resulted in specific cognitive impairments, predominantly memory performance.     

p53 inactivation leads to impaired motor synchronization in mice

We have combined genetic and pharmacological approaches to investigate the behavioural consequences of inactivation of the murine p53 protein. Our behavioural analysis revealed that p53-null mice (p53KO) exhibit a very specific and significant motor deficit in rapid walking synchronization. This deficit, observed using the rotarod test, was the only behavioural defect of p53KO mice. We demonstrated that it was not due to an increase in neuronal number or abnormal connectivity in the olivo-cerebellar system, thought to control motor synchronization. In order to test the role of p53 in the central nervous system, we injected a pharmacological inhibitor of p53 activation, pifithrin-alpha, into the cerebellum of wild-type mice. This treatment mimicked the walking synchronization deficit of p53KO mice, suggesting that presence of p53 protein in the cerebellum is necessary to execute this synchronization of walking. Our investigation reveals a functional role of cerebellar p53 protein in adult walking synchronization.     

Medial septal galanin and acetylcholine: influence on hippocampal acetylcholine and spatial learning

Neurochemical and behavioral studies in the rat have provided evidence for the view that galanin impairs learning via an inhibitory modulation of cholinergic neurons in the septohippocampal projection, believed to be important for learning and memory. To test this hypothesis, galanin was microinjected via a unilateral chronic cannula located in MS/dBB of rats. Infusion of galanin in the MS/dBB, which contains a high number of 125I-galanin binding sites, did not impair spatial acquisition or memory. On the contrary, spatial acquisition tended to be facilitated by 1 and 3 nmoles of galanin, while the 0.3 nmol dose had no effect. Intraseptal injections of scopolamine (10 microg/rat), a non-specific muscarinic antagonist, also failed to alter learning performance. In contrast, co-injections of galanin (3 nmol) and scopolamine (10 microg) resulted in a marked impairment of spatial acquisition. The effect of intraseptal galanin on basal acetylcholine release in the ventral hippocampus was examined by in vivo microdialysis and high-performance liquid chromatography. Both galanin (3 nmol/rat) and scopolamine (10 microg/rat) infused into the MS/dBB increased basal acetylcholine release in the ventral hippocampus. The combined injections of galanin and scopolamine resulted in an excessive increase in acetylcholine release. These results indicate, that galanin activates septohippocampal cholinergic neurons, suggesting that septal galanin may have a facilitatory role in spatial learning. Moreover, the level of muscarinic activity within the septal area appears to be critical for the effects of galanin on cognitive functions, since the combination of galanin and scopolamine produced a marked impairment in spatial learning, despite a marked increase in hippocampal acetylcholine release. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

Cognitive performance and cholinergic transmission: influence of muscarinic and nicotinic receptor blockade

The cholinergic system is essential in mediating cognitive processes. Although there has been extensive research regarding cholinergic receptor subsystems, the specific contribution of the muscarinic and nicotinic receptor system to cognitive processes still has not been sufficiently explored. In the present study, we examined the selective contribution of muscarinic and nicotinic antagonism to cognitive performance in healthy human subjects. A single-blind, double-dummy, time-elapsed, repeated measures cross-over design was used on 15 healthy males. Subjects completed a neuropsychological test battery assessing a wide range of cognitive domains after 0.4 mg scopolamine (intravenous), 0.2 mg/kg mecamylamine (max. 15 mg; oral) or placebo. Subjects were tested under three conditions: placebo/placebo (PP), scopolamine/placebo (SP) and mecamylamine/placebo (MP). Results show that scopolamine significantly impaired the free recall and recognition performance in the verbal learning test. No other cognitive domain was affected, neither by scopolamine nor by mecamylamine. In line with the existing literature, antagonism of muscarinic receptors resulted in specific cognitive impairments, predominantly memory performance.

     

Differential roles for nicotinic and muscarinic cholinergic receptors in sustained visuo-spatial attention? A study using a 5-arm maze protocol in mice.

A 5-arm maze was used to measure sustained visuo-spatial attention in C57Bl/6 mice and test the hypothesis of differential functional roles for central nicotinic and muscarinic receptors in mediating task performance. Mice were first trained to acquire the basic visual discrimination task in which, on each trial, one randomly chosen arm among the five open arms was baited and remained lit until an arm-choice was made. Mice were then submitted to attentional testing in which trials using light signals of 2, 1 or 0.5 s were intermixed to evaluate the decrement in correct responses as a function of the decrease in light signal duration and thus, to construct a reference curve for the attentional performance of C57Bl/6 mice. Mice were then divided into four groups and received, in rotation, over four pharmacological sessions according to a Latin-square design, i.p. injections of either mecamylamine (4.0 mg/kg), scopolamine (0.8 mg/kg), the combination of mecamylamine and scopolamine or saline, 20 min before re-testing. Injection of cholinergic antagonists produced decreases in percentage of correct responses, which were systematically associated with significant increases in choice latencies. Mecamylamine produced slight disruption, whereas scopolamine and the combined treatment both produced severe disruption. In conclusion, whereas both nicotinic and muscarinic cholinergic antagonists disrupt performance in the attentional task, the increase in response latencies entails that correct responding becomes more dependent on the working memory processes and thus compromises conclusions as to a selective disruption of attention.     

Combined blockade of cholinergic receptors shifts the brain from stimulus encoding to memory consolidation

High central nervous system levels of acetylcholine (ACh) are commonly regarded as crucial for learning and memory, and a decline in cholinergic neurotransmission is associated with Alzheimer's dementia. However, recent findings revealed exceptions to this rule: The low ACh tone characterizing slow wave sleep (SWS) has proven necessary for consolidation of hippocampus-dependent declarative memories during this sleep stage. Such observations, together with recent models of a hippocampal-neocortical dialogue underlying systems memory consolidation, suggest that high levels of ACh support memory encoding, whereas low levels facilitate consolidation. We tested this hypothesis in human subjects by blocking cholinergic neurotransmission during wakefulness, starting 30 min after learning. Subjects received the muscarinic antagonist scopolamine (4 microg/kg bodyweight intravenously) and the nicotinic antagonist mecamylamine (5 mg orally). Compared to placebo, combined muscarinic and nicotinic receptor blockade significantly improved consolidation of declarative memories tested 10 hr later, but simultaneously impaired acquisition of similar material. Consolidation of procedural memories, which are not dependent on hippocampal functioning, was unaffected. Neither scopolamine nor mecamylamine alone enhanced declarative memory consolidation. Our findings support the notion that ACh acts as a switch between modes of acquisition and consolidation. We propose that the natural shift in central nervous system cholinergic tone from high levels during wakefulness to minimal levels during SWS optimizes declarative memory consolidation during a period with no need for new memory encoding.     

Effects of Intracortical Infusion of Anticholinergic Drugs on Neuronal Plasticity in Kitten Striate Cortex

During a critical period of postnatal development the mammalian visual cortex is highly susceptible to experience-dependent alterations of neuronal response properties. These modifications are facilitated by the neuromodulators noradrenalin and acetylcholine. To identify the cholinergic mechanisms responsible for this facilitation, muscarinic and nicotinic antagonists were infused into the visual cortex of kittens while the animals were subject to monocular deprivation. Subsequently the ocular dominance of cortical cells was assessed by single-unit recording. Ocular dominance changes were suppressed by scopolamine and pirenzepine but not by gallamine, hexamethonium and mecamylamine. This blocking effect was concentration-dependent, and control experiments revealed that it was not due to suppression of neuronal responses to light. It is concluded from these results that acetylcholine facilitates neuronal plasticity in the visual cortex through mechanisms activated by muscarinic M₁ receptors.     

Dopaminergic and cholinergic antagonism in a novel-object detection task with rats.

In a free-choice test, rats display a tendency to interact more with a novel object than a familiar object. In the present report, we assessed the role of the dopaminergic and cholinergic systems in the expression of this novelty detection. Rats were injected with a dopaminergic antagonist (sulpiride, U-99194A, clozapine, or L-745,870) or a cholinergic antagonist (mecamylamine or scopolamine) prior to the free-choice novel-object test. The dopamine antagonists did not block novel-object detection. In contrast, scopolamine, but not mecamylamine, reliably blocked the expression of novelty detection, indicating a role for muscarinic receptors.     

The cerebellum and sensorimotor coupling: Looking at the problem from the perspective of vestibular reflexes

Cerebellar modules process afferent information and deliver outputs relevant for both reflex and voluntary movements. The response of cerebellar modules to a given input depends on the whole array of signals impinging on them. Studies on vestibular reflexes indicate that the response of the cerebellar circuits to the vestibular input is modified by the integration of multiple visual, vestibular and somatosensory afferent signals. In this way the cerebellum slowly adapts these reflexes when they are not adequate to the behavioural condition and allows their fast modifications when the relative position of the body segments and that of the body in space are changed. Studies on voluntary movements indicate that the cerebellum is responsible for motor learning that consists of the development of new input-output associations. Several theoretical, anatomical and clinical studies are consistent with the hypothesis that the cerebellum allows the delivery of motor commands which vary according to the condition of the motor apparatus. Finally, the cerebellum could change the relation between visual information and aimed reaching movements according to the position of the eyes in the orbit and of the neck over the body. We propose that, due to the large expansion of its cortex, an important function of the cerebellum could be that of expanding the range of sensorimotor associations according to all the factors characterizing the behavioural condition. Indeed, following cerebellar lesion, learning is often lost, the movement results impaired and requires an increased attention. In the light of the recently discovered connections of the cerebellum with the rostral regions of the frontal lobe, it can be suggested that the ability of cerebellar circuits to modify the rules of input-output coupling according to a general context is a fundamental property allowing the cerebellum to control not only motor but also cognitive functions.     

Cholinergic modulation of visuospatial responding in central thalamus

Central thalamus has extensive connections with basal ganglia and frontal cortex that are thought to play a critical role in sensory-guided goal-directed behavior. Central thalamic activity is influenced by cholinergic projections from mesopontine nuclei. To elucidate this function we trained rats to respond to lights in a reaction time (RT) task and compared effects of muscarinic (2.4, 7.3, 22 nmol scopolamine) and nicotinic (5.4, 16, 49, 98 nmol mecamylamine) antagonists with the GABA_A agonist muscimol (0.1, 0.3, 1.0 nmol) in central thalamus. We compared this with subcutaneous (systemic) effects of mecamylamine (3.2, 9.7, 29 µmol/kg) and scopolamine (0.03, 0.09, 0.26 µmol/kg). Subcutaneous scopolamine increased omissions (failure to respond within a 3-s response window) at the highest dose tested. Subcutaneous mecamylamine increased omissions at the highest dose tested while impairing RT and per cent correct at lower doses. Intrathalamic injections of muscimol and mecamylamine decreased per cent correct at doses that did not affect omissions or RT. Intrathalamic scopolamine increased omissions and RT at doses that had little effect on per cent correct. Anatomical controls indicated that the effects of mecamylamine were localized in central thalamus and those of scopolamine were not. Drug effects did not interact with attention-demanding manipulations of stimulus duration, proximity of stimulus and response locations, or stimulus array size. These results are consistent with the hypothesis that central thalamus mediates decisional processes linking sensory stimuli with actions, downstream from systems that detect sensory signals. They also provide evidence that this function is specifically influenced by nicotinic cholinergic receptors.     

Neurochemical stimulation of the rat substantia innominata increases cerebral blood flow (but not glucose use) through the parallel activation of cholinergic and non-cholinergic pathways

Neurochemical activation of the substantia innominata (SI) in the rat, through the direct injection of the cholinergic agonist carbachol, has been reported to induce large increases in cerebral blood flow (CBF) throughout cortical and subcortical projection regions. The present study aimed to determine whether the vasomotor responses to cholinergic stimulation of the SI were, or were not, the consequence of an increase in metabolic activity. To this end, coupled measurements of CBF and cerebral glucose use (CGU) were undertaken during carbachol-elicited stimulation of the SI. Infusion of carbachol into the basal forebrain induced significant CBF increases in several ipsilateral cortical and subcortical areas including the amygdala. In contrast, CGU increased only in the ipsilateral amygdala and SI. Thus, we tested the hypothesis of a direct neurogenic, rather than metabolic, contribution of the basalocortical system. In this respect, carbachol-elicited stimulation resulted in significant increases in extracellular acetylcholine concentrations in the ipsilateral parietal cortex; systemic pretreatment with the muscarinic receptor antagonist scopolamine completely abolished the increase in cortical CBF elicited by cholinergic stimulation of the SI in the ipsilateral frontoparietal motor cortex while it failed to affect the increase observed in the ipsilateral temporal cortex. Several conclusions can be drawn from the present study. The stimulation of the SI by carbachol induces an increase in CBF that can be dissociated from changes in underlying glucose metabolism. Secondly, these induced changes in cortical CBF are paralleled by an increase in acetylcholine release. Lastly, the failure of scopolamine to block the flow response in all cortical regions would suggest that SI stimulation will evoke the release of vasodilatatory neurotransmitter(s) as well as acetylcholine itself.     

Altered emotionality, spatial memory and cholinergic function in caveolin-1 knock-out mice

Neurological phenotypes associated with loss of caveolin 1 (cav-1) (the defining structural protein in caveolar vesicles, which regulate signal transduction and cholesterol trafficking in cells) in mice have been reported recently. In brain, cav-1 is highly expressed in neurons and glia. We investigated emotional and cognitive behavioural domains in mice deficient in cav-1 (CavKO mice). CavKO mice were more anxious and spent more time in self-directed grooming behaviour than wild-type (wt) mice. In a spatial/working memory task, CavKO mice failed to recognize the object displacement, thus showing a spatial memory impairment. CavKO mice showed higher locomotor activity than wt mice, thus suggesting reduced inhibitory function by CNS cholinergic systems. Behavioural response to the cholinergic muscarinic antagonist, scopolamine (2 mg/Kg), was decreased in CavKO mice. Few behavioural sex differences emerged in mice; whereas the sex differences were generally attenuated or even reverted in the null genotype. Our data confirm a distinct behavioural phenotype in CavKO mice and indicate a selective alteration in central cholinergic function.     

Selective Immunolesioning of the Basal Forebrain Cholinergic System Disrupts Short-term Memory in Rats

Selective depletion of nerve growth factor receptor-bearing neurons in the basal forebrain cholinergic nuclei by the immunotoxin 192 IgG-saporin offers a new and highly useful tool for the study of the role of the forebrain cholinergic system in cognitive functions. In the present study, we have tested the effects of 192 IgG—saporin in an operant delayed matching-to-position task which has previously been used to discriminate between delay-dependent learning impairments and delay-independent disturbances of non-mnemonic processes. Rats were first trained to criterion performance and then received intraventricular injections of 5 μg of 192 IgG—saporin 4 weeks prior to a second testing session. Rats with 192 IgG—saporin lesions displayed a significant delay-dependent decline in performance compared to normal controls, indicating a deficit in short-term memory. Administration of the muscarinic blocker scopolamine (0.5 mg/kg, i.p.) produced more pronounced impairment in the performance of the normal control rats across all delays, and induced further impairment also in animals with 192 IgG-saporin lesions. These effects were not observed following control injections of methyl scopolamine, suggesting that the impairment induced by scopolamine was due to the blockade of central muscarinic receptors. No improvement in performance was observed in either group following systemic treatment with the muscarinic cholinergic agonist arecoline (1.0 mg/kg). Biochemical and morphological analyses confirmed the selective and severe (>90–95%) depletion of cholinergic neurons throughout the septal-diagonal band area and the nucleus basalis region by the intraventricular 192 IgG—saporin treatment. Although the immunotoxin was observed to produce additional damage to the cerebellar Purkinje cells, no gross motor abnormalities were observed that could contribute to the effects on accuracy in the task used here. In conclusion, the results show that selective combined lesions of the basal forebrain cholinergic neurons in the septal—diagonal band area and nucleus basalis produce long-lasting impairments in short-term memory, thus providing further support for a role of this system in cognitive functions.     

Modulation of cue‐triggered reward seeking by cholinergic signaling in the dorsomedial striatum

The dorsomedial striatum (DMS) has been strongly implicated in flexible, outcome‐based decision making, including the outcome‐specific Pavlovian‐to‐instrumental transfer effect (PIT), which measures the tendency for a reward‐predictive cue to preferentially motivate actions that have been associated with the predicted reward over actions associated with different rewards. Although the neurochemical underpinnings of this effect are not well understood, there is growing evidence that striatal acetylcholine signaling may play an important role. This study investigated this hypothesis by assessing the effects of intra‐DMS infusions of the nicotinic antagonist mecamylamine or the muscarinic antagonist scopolamine on expression of specific PIT in rats. These treatments produced dissociable behavioral effects. Mecamylamine infusions enhanced rats’ tendency to use specific cue‐elicited outcome expectations to select whichever action was trained with the predicted outcome, relative to their performance when tested after vehicle infusions. In contrast, scopolamine infusions appeared to render instrumental performance insensitive to this motivational influence of reward‐paired cues. These drug treatments had no detectable effect on conditioned food cup approach behavior, indicating that they selectively perturbed cue‐guided action selection without producing more wide‐ranging alterations in behavioral control. Our findings reveal an important role for DMS acetylcholine signaling in modulating the impact of cue‐evoked reward expectations on instrumental action selection.     

Cholinergic modulation in the olfactory bulb influences spontaneous olfactory discrimination in adult rats

Cholinergic neuromodulation in the olfactory bulb has been hypothesized to regulate mitral cell molecular receptive ranges and the behavioral discrimination of similar odorants. We tested the effects of cholinergic modulation in the olfactory bulb of cannulated rats by bilaterally infusing cholinergic agents into the olfactory bulbs and measuring the rats' performances on separate spontaneous and motivated odor-discrimination tasks. Specifically, 6 microL/bulb infusions of vehicle (0.9% saline), the muscarinic antagonist scopolamine (7.6 mM and 38 mM), the nicotinic antagonist mecamylamine hydrochloride (3.8 mM and 19 mM), a combination of both antagonists, or the acetylcholinesterase inhibitor neostigmine (8.7 mM) were made 20 min prior to testing on an olfactory cross-habituation task or a rewarded, forced-choice odor-discrimination task. Spontaneous discrimination between chemically related odorants was abolished when nicotinic receptors were blocked in the olfactory bulb, and enhanced when the efficacy of cholinergic inputs was increased with neostigmine. Blocking muscarinic receptors reduced but did not abolish odor discrimination. Interestingly, no behavioral effects of modulating either nicotinic or muscarinic receptors were observed when rats were trained on a reward-motivated odor-discrimination task. Computational modeling of glomerular circuitry demonstrates that known nicotinic cholinergic effects on bulbar neurons suffice to explain these results.     

Scopolamine impairs eyeblink conditioning in cerebellar LTD-deficient mice

We investigated the effect of the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine on eyeblink conditioning in glutamate receptor subunit 62 null mice, which have severe impairments in cerebellar long-term depression (LTD). Mice were injected intraperitoneally with scopolamine (0.5 mg/kg) or saline, and conditioned using a delay paradigm with tone and periorbital shock but with no overlap between them. The saline-injected mutant mice learned this paradigm normally, as predicted from our previous study. When scopolamine was injected, learning was impaired more severely in the mutant mice than in the wild type mice. Basic sensory and motor performances were not affected. These results suggest that eyeblink conditioning in cerebellar LTD deficient mice depends largely on neural functions susceptible to blocking of mAChRs.