Cholinergic dysfunction in the dorsal striatum promotes habit formation and maladaptive eating.

Dysregulation of habit formation has been recently proposed as pivotal to eating disorders. Here, we report that a subset of patients suffering from restrictive anorexia nervosa have enhanced habit formation compared with healthy controls. Habit formation is modulated by striatal cholinergic interneurons. These interneurons express vesicular transporters for acetylcholine (VAChT) and glutamate (VGLUT3) and use acetylcholine/glutamate cotransmission to regulate striatal functions. Using mice with genetically silenced VAChT (VAChT conditional KO, VAChTcKO) or VGLUT3 (VGLUT3cKO), we investigated the roles that acetylcholine and glutamate released by cholinergic interneurons play in habit formation and maladaptive eating. Silencing glutamate favored goal-directed behaviors and had no impact on eating behavior. In contrast, VAChTcKO mice were more prone to habits and maladaptive eating. Specific deletion of VAChT in the dorsomedial striatum of adult mice was sufficient to phenocopy maladaptive eating behaviors of VAChTcKO mice. Interestingly, VAChTcKO mice had reduced dopamine release in the dorsomedial striatum but not in the dorsolateral striatum. The dysfunctional eating behavior of VAChTcKO mice was alleviated by donepezil and by l-DOPA, confirming an acetylcholine/dopamine deficit. Our study reveals that loss of acetylcholine leads to a dopamine imbalance in striatal compartments, thereby promoting habits and vulnerability to maladaptive eating in mice.

Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT-associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y-maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety-like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT-mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.-Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains.

Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.

Elimination of the vesicular acetylcholine transporter in the forebrain causes hyperactivity and deficits in spatial memory and long-term potentiation.

Basal forebrain cholinergic neurons, which innervate the hippocampus and cortex, have been implicated in many forms of cognitive function. Immunolesion-based methods in animal models have been widely used to study the role of acetylcholine (ACh) neurotransmission in these processes, with variable results. Cholinergic neurons have been shown to release both glutamate and ACh, making it difficult to deduce the specific contribution of each neurotransmitter on cognition when neurons are eliminated. Understanding the precise roles of ACh in learning and memory is critical because drugs that preserve ACh are used as treatment for cognitive deficits. It is therefore important to define which cholinergic-dependent behaviors could be improved pharmacologically. Here we investigate the contributions of forebrain ACh on hippocampal synaptic plasticity and cognitive behavior by selective elimination of the vesicular ACh transporter, which interferes with synaptic storage and release of ACh. We show that elimination of vesicular ACh transporter in the hippocampus results in deficits in long-term potentiation and causes selective deficits in spatial memory. Moreover, decreased cholinergic tone in the forebrain is linked to hyperactivity, without changes in anxiety or depression-related behavior. These data uncover the specific contribution of forebrain cholinergic tone for synaptic plasticity and behavior. Moreover, these experiments define specific cognitive functions that could be targeted by cholinergic replacement therapy.

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.

Stanniocalcin 2 expression is regulated by hormone signalling and negatively affects breast cancer cell viability in vitro.

Stanniocalcin 1 (STC1) and STC2 are secreted, homodimeric glycoproteins that share 30% amino acid sequence identity. Breast tumour gene profiling studies have demonstrated significantly upregulated STC2 expression in hormone-responsive positive breast tumours; therefore, the purpose of this study was to investigate STC2 hormonal regulation and function in breast cancer cells. Here we report that STC2 is expressed in a number of human breast cancer cell lines, regardless of their oestrogen (E(2)) and progesterone (P4) receptor status, and its expression is readily detectable in human and mouse mammary gland tumours. Besides E(2), retinoic acid (RA) and P4 play an important role in the regulation of STC2 expression, not only in MCF-7 but also in other breast cancer and non-breast cell lines. The expression of the related hormone, STC1, is not affected by the above hormones in breast and endometrial cancer cell lines implying a fundamental difference in regulation in cancer cell lines. The induction of STC2 expression by E(2) and RA occurs at the transcriptional level but through intermediary transcription factors. The STC2 proximal promoter region is not responsible for hormonal induction, but exhibits a high basal transcriptional activity. Constitutive STC2 expression in human breast cancer cell lines resulted in significant impairment of cell growth, migration and cell viability after serum withdrawal. In conclusion, STC2 is a downstream target of E(2), P4 and RA signalling pathways. In hormone receptor negative cell lines it can function in a paracrine/autocrine fashion to reduce cell proliferation.

Human stanniocalcin-2 exhibits potent growth-suppressive properties in transgenic mice independently of growth hormone and IGFs.

Stanniocalcin (STC)-2 was discovered by its primary amino acid sequence identity to the hormone STC-1. The function of STC-2 has not been examined; thus we generated two lines of transgenic mice overexpressing human (h)STC-2 to gain insight into its potential functions through identification of overt phenotypes. Analysis of mouse Stc2 gene expression indicates that, unlike Stc1, it is not highly expressed during development but exhibits overlapping expression with Stc1 in adult mice, with heart and skeletal muscle exhibiting highest steady-state levels of Stc2 mRNA. Constitutive overexpression of hSTC-2 resulted in pre- and postnatal growth restriction as early as embryonic day 12.5, progressing such that mature hSTC-2-transgenic mice are approximately 45% smaller than wild-type littermates. hSTC-2 overexpression is sometimes lethal; we observed 26-34% neonatal morbidity without obvious dysmorphology. hSTC-2-induced growth retardation is associated with developmental delay, most notably cranial suture formation. Organ allometry studies show that hSTC-2-induced dwarfism is associated with testicular organomegaly and a significant reduction in skeletal muscle mass likely contributing to the dwarf phenotype. hSTC-2-transgenic mice are also hyperphagic, but this does not result in obesity. Serum Ca2+ and PO4 were unchanged in hSTC-2-transgenic mice, although STC-1 can regulate intra- and extracellular Ca2+ in mammals. Interestingly, severe growth retardation induced by hSTC-2 is not associated with a decrease in GH or IGF expression. Consequently, similar to STC-1, STC-2 can act as a potent growth inhibitor and reduce intramembranous and endochondral bone development and skeletal muscle growth, implying that these tissues are specific physiological targets of stanniocalcins.