Glucose intolerance induces anxiety-like behaviors independent of obesity and insulin resistance in a novel model of nutritional metabolic stress.

OBJECTIVES: Type 2 diabetes (T2D) is a metabolic disease of major public health concern. It impacts peripheral tissues and the central nervous system, leading to systemic dysmetabolism and neurocognitive impairments, including memory deficits, anxiety, and depression. The metabolic determinants of these neurocognitive impairments remain unidentified. Here, we sought to address this question by developing a proprietary (P-) high-fat diet (HFD), in which glucose intolerance precedes weight gain and insulin resistance. METHODS: The P-HFD model was nutritionally characterized, and tested in vivo in mice that underwent behavioral and metabolic testing. The diet was benchmarked against reference models. . RESULTS: P-HFD has 42% kcal from fat, high monounsaturated/polyunsaturated fatty acid ratio, and 10% (w/v) sucrose in drinking water. When administered, from the early stages of glucose intolerance alone, animals exhibit anxiety-like behavior, without depression nor recognition memory deficits. Long-term P-HFD feeding leads to weight gain, brain glucose hypometabolism as well as impaired recognition memory. Using an established genetic model of T2D (db/db) and of diet-induced obesity (60% kcal from fat) we show that additional insulin resistance and obesity are associated with depressive-like behaviors and recognition memory deficits. DISCUSSION: Our findings demonstrate that glucose intolerance alone can elicit anxiety-like behavior. Through this study, we also provide a novel nutritional model (P-HFD) to characterize the discrete effects of glucose intolerance on cognition, behavior, and the physiology of metabolic disease.

Macrophages and the development and progression of non-alcoholic fatty liver disease.

The liver is the site of first pass metabolism, detoxifying and metabolizing blood arriving from the hepatic portal vein and hepatic artery. It is made up of multiple cell types, including macrophages. These are either bona fide tissue-resident Kupffer cells (KC) of embryonic origin, or differentiated from circulating monocytes. KCs are the primary immune cells populating the liver under steady state. Liver macrophages interact with hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells to maintain homeostasis, however they are also key contributors to disease progression. Generally tolerogenic, they physiologically phagocytose foreign particles and debris from portal circulation and participate in red blood cell clearance. However as immune cells, they retain the capacity to raise an alarm to recruit other immune cells. Their aberrant function leads to the development of non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a spectrum of conditions ranging from benign steatosis of the liver to steatohepatitis and cirrhosis. In NAFLD, the multiple hit hypothesis proposes that simultaneous influences from the gut and adipose tissue (AT) generate hepatic fat deposition and that inflammation plays a key role in disease progression. KCs initiate the inflammatory response as resident immune effectors, they signal to neighbouring cells and recruit monocytes that differentiated into recruited macrophages in situ. Recruited macrophages are central to amplifying the inflammatory response and causing progression of NAFLD to its fibro-inflammatory stages. Given their phagocytic capacity and their being instrumental in maintaining tissue homeostasis, KCs and recruited macrophages are fast-becoming target cell types for therapeutic intervention. We review the literature in the field on the roles of these cells in the development and progression of NAFLD, the characteristics of patients with NAFLD, animal models used in research, as well as the emerging questions. These include the gut-liver-brain axis, which when disrupted can contribute to decline in function, and a discussion on therapeutic strategies that act on the macrophage-inflammatory axis.

Neurotransmitters in Type 2 Diabetes and the Control of Systemic and Central Energy Balance.

Efficient signal transduction is important in maintaining the function of the nervous system across tissues. An intact neurotransmission process can regulate energy balance through proper communication between neurons and peripheral organs. This ensures that the right neural circuits are activated in the brain to modulate cellular energy homeostasis and systemic metabolic function. Alterations in neurotransmitters secretion can lead to imbalances in appetite, glucose metabolism, sleep, and thermogenesis. Dysregulation in dietary intake is also associated with disruption in neurotransmission and can trigger the onset of type 2 diabetes (T2D) and obesity. In this review, we highlight the various roles of neurotransmitters in regulating energy balance at the systemic level and in the central nervous system. We also address the link between neurotransmission imbalance and the development of T2D as well as perspectives across the fields of neuroscience and metabolism research.

Impaired spatial navigation and age-dependent hippocampal synaptic dysfunction are associated with chronic inflammatory response in db/db mice.

Type 2 diabetes mellitus (T2DM) increases the risk of developing Alzheimer's disease (AD), which has been proposed to be driven by an abnormal neuroinflammatory response affecting cognitive function. However, the impact of T2DM on hippocampal function and synaptic integrity during aging has not been investigated. Here, we investigated the effects of aging in T2DM on AD-like pathology using the leptin receptor-deficient db/db mouse model of T2DM. Our results indicate that adult T2DM mice exhibited impaired spatial acquisition in the Morris water maze (MWM). Morphological analysis showed an age-dependent neuronal loss in the dentate gyrus. We found that astrocyte density was significantly decreased in all regions of the hippocampus in T2DM mice. Our analysis showed that microglial activation was increased in the CA3 and the dentate gyrus of the hippocampus in an age-dependent manner in T2DM mice. However, the expression of presynaptic marker protein (synaptophysin) and the postsynaptic marker protein [postsynaptic density protein 95 (PSD95)] was unchanged in the hippocampus of adult T2DM mice. Interestingly, synaptophysin and PSD95 expression significantly decreased in the hippocampus of aged T2DM mice, suggesting an impaired hippocampal synaptic integrity. Cytokine profiling analysis displayed a robust pro-inflammatory cytokine profile in the hippocampus of aged T2DM mice compared with the younger cohort, outlining the role of aging in exacerbating the neuroinflammatory profile in the diabetic state. Our results suggest that T2DM impairs cognitive function by promoting neuronal loss in the dentate gyrus and triggering an age-dependent deterioration in hippocampal synaptic integrity, associated with an aberrant neuroinflammatory response.

Chemogenetic activation of VGLUT3-expressing neurons decreases movement.

Vesicular glutamate transporters (VGLUTs) are responsible for the storage of glutamate into secretory vesicles. The VGLUT3 isoform is mainly expressed in neurons that secrete other classical neurotransmitters, including the cholinergic interneurons in the striatum, and VGLUT3-expressing neurons often secrete two distinct neurotransmitters. VGLUT3 is discretely distributed throughout the brain and is found in subpopulations of spinal cord interneurons, in subset of neurons in the dorsal root ganglion, and in Merkel cells. Mice with a global loss of VGLUT3 are hyperactive and the modulation of specific VGLUT3-expressing circuits can lead to changes in movement. In this study, we tested the hypothesis that increased activity of VGLUT3-expressing neurons is associated with decreased movement. Using a mouse line expressing excitatory designer receptor exclusively activated by designer drugs (hM3Dq-DREADD) on VGLUT3-expressing neurons, we showed that activation of hM3Dq signalling acutely decreased locomotor activity. This decreased locomotion was likely not due to circuit changes mediated by glutamate nor acetylcholine released from VGLUT3-expressing neurons, as activation of hM3Dq signalling in mice that do not release glutamate or acetylcholine from VGLUT3-expressing neurons also decreased locomotor activity. This suggests that other neurotransmitters are likely driving this hypoactive phenotype. We used these mouse lines to compare the effects of DREADD agonists in vivo. We observed that clozapine-N-oxide (CNO), clozapine, compound 21 and perlapine show small differences in the speed at which they prompt behavioural responses but the four of them are selective DREADD ligands.

Early monocyte modulation by the non-erythropoietic peptide ARA 290 decelerates AD-like pathology progression.

Alzheimer's disease (AD) pathology is characterized by amyloid-ß (Aß) deposition and tau hyper-phosphorylation, accompanied by a progressive cognitive decline. Monocytes have been recently shown to play a major role in modulating Aß pathology, and thereby have been pointed as potential therapeutic targets. However, the main challenge remains in identifying clinically relevant interventions that could modulate monocyte immune functions in absence of undesired off-target effects. Erythropoietin (EPO), a key regulator of erythrocyte production, has been shown to possess immunomodulatory potential and to provide beneficial effects in preclinical models of AD. However, the transition to use recombinant human EPO in clinical trials was hindered by unwanted erythropoietic effects that could lead to thrombosis. Here, we used a recently identified non-erythropoietic analogue of EPO, ARA 290, to evaluate its therapeutic potential in AD therapy. We first evaluated the effects of early systemic ARA 290 administration on AD-like pathology in an early-onset model, represented by young APP/PS1 mice. Our findings indicate that ARA 290 early treatment decelerated Aß pathology progression in APP/PS1 mice while improving cognitive functions. ARA 290 potently increased the levels of total monocytes by specifically stimulating the generation of Ly6CLow patrolling subset, which are implicated in clearing Aß from the cerebral vasculature, and subsequently reducing overall Aß burden in the brain. Moreover, ARA 290 increased the levels of monocyte progenitors in the bone marrow. Using chimeric APP/PS1 mice in which Ly6CLow patrolling subset are selectively depleted, ARA 290 was inefficient in attenuating Aß pathology and ameliorating cognitive functions in young animals. Interestingly, ARA 290 effects were compromised when delivered in a late-onset model, represented by aged APP1/PS1. In aged APP/PS1 mice in which AD-like pathology is at advanced stages, ARA 290 failed to reverse Aß pathology and to increase the levels of circulating monocytes. Our study suggests that ARA 290 early systemic treatment could prevent AD-like progression via modulation of monocyte functions by specifically increasing the ratio of patrolling monocytes.

Cholinergic transmission from the basal forebrain modulates social memory in male mice.

Disruptions in social behaviour are prevalent in many neuropsychiatric disorders such as schizophrenia, bipolar disorder and autism spectrum disorders. However, the underlying neurochemical regulation of social behaviour is still not well understood. The central cholinergic system has been proposed to contribute to the regulation of social behaviour. For instance, decreased global levels of acetylcholine release in the brain leads to decreased social interaction and an impairment of social memory in mice. Nonetheless, it has been difficult to ascertain the specific brain areas where cholinergic signalling influences social preference and social memory. In this study, we investigated the impact of different forebrain cholinergic regions on social behaviour by examining mouse lines that differ in their regional expression level of the vesicular acetylcholine transporter-the protein that regulates acetylcholine secretion. We found that when cholinergic signalling is highly disrupted in the striatum, hippocampus, cortex and amygdala mice have intact social preference but are impaired in social memory, as they cannot remember a familiar conspecific nor recognize a novel one. A similar pattern emerges when acetylcholine release is disrupted mainly in the striatum, cortex, and amygdala; however, the ability to recognize novel conspecifics is retained. In contrast, cholinergic signalling of the striatum and amygdala does not appear to significantly contribute to the modulation of social memory and social preference. Furthermore, we demonstrated that increasing global cholinergic tone does not increase social behaviours. Together, these data suggest that cholinergic transmission from the hippocampus and cortex are important for regulating social memory.

Role of Microglia in Modulating Adult Neurogenesis in Health and Neurodegeneration.

Microglia are the resident immune cells of the brain, constituting the powerhouse of brain innate immunity. They originate from hematopoietic precursors that infiltrate the developing brain during different stages of embryogenesis, acquiring a phenotype characterized by the presence of dense ramifications. Microglial cells play key roles in maintaining brain homeostasis and regulating brain immune responses. They continuously scan and sense the brain environment to detect any occurring changes. Upon detection of a signal related to physiological or pathological processes, the cells are activated and transform to an amoeboid-like phenotype, mounting adequate responses that range from phagocytosis to secretion of inflammatory and trophic factors. The overwhelming evidence suggests that microglia are crucially implicated in influencing neuronal proliferation and differentiation, as well as synaptic connections, and thereby cognitive and behavioral functions. Here, we review the role of microglia in adult neurogenesis under physiological conditions, and how this role is affected in neurodegenerative diseases.

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice.

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres by microglia in vitro. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain. No effect of CNO on cytokine expression was observed in DREADD-negative mice. Interestingly, CNO activation of M3Dq in microglia was able to attenuate LPS-mediated decrease in social interactions. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, can trigger an inflammatory-like response that preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases.

MouseBytes, an open-access high-throughput pipeline and database for rodent touchscreen-based cognitive assessment.

Open Science has changed research by making data accessible and shareable, contributing to replicability to accelerate and disseminate knowledge. However, for rodent cognitive studies the availability of tools to share and disseminate data is scarce. Automated touchscreen-based tests enable systematic cognitive assessment with easily standardised outputs that can facilitate data dissemination. Here we present an integration of touchscreen cognitive testing with an open-access database public repository (mousebytes.ca), as well as a Web platform for knowledge dissemination (https://touchscreencognition.org). We complement these resources with the largest dataset of age-dependent high-level cognitive assessment of mouse models of Alzheimer's disease, expanding knowledge of affected cognitive domains from male and female mice of three strains. We envision that these new platforms will enhance sharing of protocols, data availability and transparency, allowing meta-analysis and reuse of mouse cognitive data to increase the replicability/reproducibility of datasets.

Forebrain Cholinergic Signaling Regulates Innate Immune Responses and Inflammation.

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.

Regulation of Cognitive Processing by Hippocampal Cholinergic Tone.

Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric diseases. Here we tested how information processing is regulated by cholinergic tone in genetically modified mice targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine release. We measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of paired associates learning task (PAL). Acquisition of information in the mouse PAL task correlated to levels of hippocampal VAChT, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signaling. Disrupted forebrain cholinergic signaling also affected working memory, a result reproduced by selectively decreasing VAChT in the hippocampus. In contrast, spatial memory was relatively preserved, whereas reversal spatial memory was sensitive to decreased hippocampal cholinergic signaling. This work provides a refined roadmap of how synaptically secreted acetylcholine influences distinct behaviors and suggests that distinct forms of cognitive processing may be regulated in different ways by cholinergic activity.

Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology.

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

Deletion of the vesicular acetylcholine transporter from pedunculopontine/laterodorsal tegmental neurons modifies gait.

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox ). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.

Cholinergic circuits in cognitive flexibility.

Cognitive flexibility, the ability to adjust behavior in response to new and unexpected conditions in the environment, is essential for adaptation to new challenges and survival. The cholinergic system is an important modulator of this complex behavior however, the exact cholinergic circuits involved in this modulation and the precise influence of acetylcholine (ACh) in the process is still not fully understood. Here we review the role of different cholinergic circuits in cognitive flexibility. Strong evidence indicates that cholinergic interneurons (CINs) from the dorsomedial striatum are essential for facilitating the establishment of a new selected strategy; an effect that seems to depend mainly on activation of muscarinic receptors. Cholinergic neurons from the nucleus basalis magnocellularis (nBM), which project to the prefrontal cortex, seem to modulate the initial inhibition of a previously learned strategy, however, this concept is still controversial. Additionally, some studies suggest that basal forebrain cholinergic neurons projecting to the hippocampus, basolateral amygdala, and posterior parietal cortex may also participate on the modulation of cognitive flexibility. We highlight the fact that when investigating effects of ACh on behavioral flexibility, or any other behavior, one has to keep in mind two important particularities of the cholinergic system: (1) Many cholinergic neurons in the brain co-release glutamate or GABA with ACh. Methodologies that rely on neuronal silencing or ablation lead to simultaneous elimination of both neurotransmitters, making interpretation of results complex. (2) The cholinergic gene locus has a unique organization, with the vesicular acetylcholine transporter (VAChT) gene present within the intron between the first and second exons of the choline acetyltransferase (ChAT) gene. Thus, behavioral studies using transgenic animals generated with ChAT bacterial artificial chromosome (BAC) clones should be considered carefully, taking into consideration that these mice may overexpress VAChT and therefore, present a hypercholinergic tone that can be a confounder in behavioral studies.

Cholinergic Regulation of hnRNPA2/B1 Translation by M1 Muscarinic Receptors.

UNLABELLED: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD. SIGNIFICANCE STATEMENT: In Alzheimer's disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer's disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels.

α7 nicotinic ACh receptor-deficient mice exhibit sustained attention impairments that are reversed by ß2 nicotinic ACh receptor activation.

BACKGROUND AND PURPOSE: Disruptions of executive function, including attentional deficits, are a hallmark of a number of diseases. ACh in the prefrontal cortex regulates attentive behaviour; however, the role of α7 nicotinic ACh receptor (α7nAChR) in attention is contentious. EXPERIMENTAL APPROACH: In order to probe attention, we trained both wild-type and α7nAChR knockout mice on a touch screen-based five-choice serial reaction time task (5-CSRT). Following training procedures, we then tested sustained attention using a probe trial experiment. To further differentiate the role of specific nicotinic receptors in attention, we then tested the effects of both α7nAChR and ß2nAChR agonists on the performance of both wild-type and knockout mice on the 5-CSRT task. KEY RESULTS: At low doses, α7nAChR agonists improved attentional performance of wild-type mice, while high doses had deleterious effects on attention. α7nAChR knockout mice displayed deficits in sustained attention that were not ameliorated by α7nAChR agonists. However, these deficits were completely reversed by the administration of a ß2nAChR agonist. Furthermore, administration of a ß2nAChR agonist in α7nAChR knockout mice elicited similar biochemical response in the prefrontal cortex as the administration of α7nAChR agonists in wild-type mice. CONCLUSIONS AND IMPLICATIONS: Our experiments reveal an intricate relationship between distinct nicotinic receptors to regulate attentional performance and provide the basis for targeting ß2nAChRs pharmacologically to decrease attentional deficits due to a dysfunction in α7nAChRs.

Forebrain deletion of the vesicular acetylcholine transporter results in deficits in executive function, metabolic, and RNA splicing abnormalities in the prefrontal cortex.

One of the key brain regions in cognitive processing and executive function is the prefrontal cortex (PFC), which receives cholinergic input from basal forebrain cholinergic neurons. We evaluated the contribution of synaptically released acetylcholine (ACh) to executive function by genetically targeting the vesicular acetylcholine transporter (VAChT) in the mouse forebrain. Executive function was assessed using a pairwise visual discrimination paradigm and the 5-choice serial reaction time task (5-CSRT). In the pairwise test, VAChT-deficient mice were able to learn, but were impaired in reversal learning, suggesting that these mice present cognitive inflexibility. Interestingly, VAChT-targeted mice took longer to reach criteria in the 5-CSRT. Although their performance was indistinguishable from that of control mice during low attentional demand, increased attentional demand revealed striking deficits in VAChT-deleted mice. Galantamine, a cholinesterase inhibitor used in Alzheimer's disease, significantly improved the performance of control mice, but not of VAChT-deficient mice on the 5-CSRT. In vivo magnetic resonance spectroscopy showed altered levels of two neurochemical markers of neuronal function, taurine and lactate, suggesting altered PFC metabolism in VAChT-deficient mice. The PFC of these mice displayed a drastic reduction in the splicing factor heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), whose cholinergic-mediated reduction was previously demonstrated in Alzheimer's disease. Consequently, several key hnRNPA2/B1 target transcripts involved in neuronal function present changes in alternative splicing in VAChT-deficient mice, including pyruvate kinase M, a key enzyme involved in lactate metabolism. We propose that VAChT-targeted mice can be used to model and to dissect the neurochemical basis of executive abnormalities.