Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.     

Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to "turn down" neuronal circuits controlling locomotion.     

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.     

Impaired striatum-dependent behavior in GASP-1-knock-out mice

G protein-coupled receptor (GPCR) associated sorting protein-1 (GASP-1) is suspected to play a key role in recycling and degradation of several GPCRs. In a previous study, we have shown that GASP-1-knock-out (GASP-1-KO) mice displayed deficits in acquiring a cocaine self-administration task, associated with an exacerbated down-regulation of striatal dopaminergic and cholinergic receptors. Among several possibilities, GASP-1 deficiency could have impaired memory processes underlying the acquisition of the operant conditioning task. Therefore, the present study investigated cognitive performances of GASP-1-KO mice and their wild-type littermates (WT) in a broad variety of memory tasks. Consistent with a deficit in procedural memory, GASP-1-KO mice showed delayed acquisition of a food-reinforced bar-press task. During water-maze training in hidden- or visible-platform paradigms, mutant and WT mice acquired the tasks at the same rate. However, GASP-1 mice exhibited persistent thigmotaxic swimming, longer distance to the platform, and reduced swim speed. There was no deficit in several tasks requiring simple behavioral responses (Barnes maze, object recognition and passive avoidance tasks). Thus, the ability to acquire and/or express complex responses seems affected in GASP-1-deficient mice. Hippocampal functions were preserved, as the retention of an acquired memory in spatial tasks remained unaffected. The pattern of behavioral deficits observed in GASP-1-KO mice is coherent with current knowledge on the role of striatal GPCRs in acquisition/expression of skilled behavior and in motivation. Together with the previous findings, the so far established phenotype of GASP-1-KO mice makes them a potentially exciting tool to study striatal functions.     

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.     

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha

Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRbeta-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRbeta- or CNTFRalpha-containing receptors.     

VAChT knock-down mice show normal prepulse inhibition but disrupted long-term habituation

The neurotransmitter acetylcholine (ACh) plays a crucial role in both the central and peripheral nervous system. Central cholinergic transmission is important for cognitive functions and cholinergic disruptions have been associated with different neural disorders. We here tested the role of cholinergic transmission in basic cognitive functions, i.e. in prepulse inhibition (PPI) and short-term habituation (STH) as well as long-term habituation (LTH) of startle using mice with a 65% knockdown (KD) of the vesicular ACh transporter (VAChT). These mice are slow in refilling cholinergic synaptic transmitter vesicles, leading to a reduced cholinergic tone. Prepulse inhibition has been assumed to be mediated by cholinergic projections from the midbrain to the reticular formation. Surprisingly, PPI and STH were normal in these mice, whereas LTH was disrupted. This disruption could be rescued by pre-testing injections of the ACh esterase inhibitor galantamine, but not by post-testing injections. The lack of a PPI deficit might be because of the fact that VAChT KD mice show disruptions mainly in prolonged cholinergic activity, therefore the transient activation by prepulse processing might not be sufficient to deplete synaptic vesicles. The disruption of LTH indicates that the latter depends on a tonic cholinergic inhibition. Future experiments will address which cholinergic cell group is responsible for this effect.     

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.     

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.     

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.     

Dihydrotestosterone modulates spatial working-memory performance in male mice

Androgens affect cognitive processes in both humans and animals. The effects of androgens may be limited to certain cognitive domains, specifically spatial memory, but this hypothesis remains elusive. Here, we tested castrated and sham-operated mice in various behavioral tasks to ask whether androgens affect multiple or specific cognitive domains in male mice. Castration impaired spatial working memory performance in the delayed matching to place water maze task following a 1-h, but not a 1-min, retention interval, as has been reported for rats. In contrast, castration had no effect on novel object recognition memory, spatial reference memory in the water maze, motor coordination, or passive avoidance memory. Castration increased anxiety-like behavior in the open field test, but not the elevated zero maze. Finally, we assessed the effects of androgen replacement with non-aromatizable dihydrotestosterone on spatial working memory following various retention intervals. Dihydrotestosterone recovered spatial memory performance following a 24-h, but not a 1-h retention interval, and had no effect at other retention intervals. These data support that in male mice androgens specifically affect spatial working memory performance, and that the neurobiological processes underlying spatial memory formation may be differentially affected by androgens.     

Mice with selective elimination of striatal acetylcholine release are lean,show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)^{D2‐Cre‐flox/flox} mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT^{D2‐Cre‐flox/flox} mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT^{D2‐Cre‐flox/flox} mice showed increased physical activity. Interestingly, VAChT^{D2‐Cre‐flox/flox} mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.     

Regulation of Tyrosine Phosphatase STEP61 by Protein Kinase A during Motor Skill Learning in Mice

Recently, striatal-enriched protein tyrosine phosphatase (STEP) and its upstream regulator protein kinase A (PKA) have been suspected to play a role in the intracellular mechanisms of fear conditioning and spatial memory. However, whether they contribute to the learning and memory of motor skills is totally unknown. In this study, we have investigated the role of STEP and PKA activities during motor skill learning associated with the accelerating rotarod task. We observed that learning the rotarod task differentially modulated the levels of phosphorylated STEP61 at serine 221, a site directly regulated by PKA, in the hippocampus, motor cortex and striatum. In a second set of experiments, we have pharmacologically inhibited PKA by the injection of Rp-cAMPS directly into the dorsal striatum of mice before rotarod trainings. PKA phosphorylation of STEP prevents the dephosphorylation of STEP substrates, whereas inhibition of PKA promotes STEP activity. Striatal PKA inhibitions dose-dependently impaired mice performances on the accelerating rotarod task. General motor abilities testing revealed an intact motor control in mice treated with 5 and 20 µg of Rp-cAMPS, but not at the highest dose of 40 µg. This suggested that motor learning was selectively affected by PKA inhibition at lower doses. Most notably, striatal inhibition of PKA reduced the levels of phosphorylated STEP61 at serine 221. Our data support that inactivation of STEP61 by the PKA activity is part of the molecular process associated with motor skill learning.     

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission

Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.     

Cognitive Dysfunction Precedes the Onset of Motor Symptoms in the MitoPark Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder primarily characterized by progressive loss of dopamine neurons, leading to loss of motor coordination. However, PD is associated with a high rate of non-motor neuropsychiatric comorbities that often develop before the onset of movement symptoms. The MitoPark transgenic mouse model is the first to recapitulate the cardinal clinical features, namely progressive neurodegeneration and death of neurons, loss of motor function and therapeutic response to L-DOPA. To investigate whether MitoPark mice exhibit early onset of cognitive impairment, a non-motor neuropsychiatric comorbidity, we measured performance on a spatial learning and memory task before (∼8 weeks) or after (∼20 weeks) the onset of locomotor decline in MitoPark mice or in littermate controls. Consistent with previous studies, we established that a progressive loss of spontaneous locomotor activity began at 12 weeks of age, which was followed by progressive loss of body weight beginning at 16–20 weeks. Spatial learning and memory was measured using the Barnes Maze. By 20 weeks of age, MitoPark mice displayed a substantial reduction in overall locomotor activity that impaired their ability to perform the task. However, in the 8-week-old mice, locomotor activity was no different between genotypes, yet MitoPark mice took longer, traveled further and committed more errors than same age control mice, while learning to successfully navigate the maze. The modest between-day learning deficit of MitoPark mice was characterized by impaired within-day learning during the first two days of testing. No difference was observed between genotypes during probe trials conducted one or twelve days after the final acquisition test. Additionally, 8-week-old MitoPark mice exhibited impaired novel object recognition when compared to control mice. Together, these data establish that mild cognitive impairment precedes the loss of motor function in a novel rodent model of PD, which may provide unique opportunities for therapeutic development.