Vesicular acetylcholine transporter (VAChT) over-expression induces major modifications of striatal cholinergic interneuron morphology and function

International audienceStriatal cholinergic interneurons (CIN) are pivotal for the regulation of the striatal network. Acetylcholine (ACh) released by CIN is centrally involved in reward behavior as well as locomotor or cognitive functions. Recently, BAC transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT–ChR2) and displaying almost 50 extra copies of the VAChT gene were used to dissect cholinergic circuit connectivity and function using optogenetic approaches. These mice display over-expression of the vesicular acetylcholine transporter (VAChT) and increased cholinergic tone. Consequently, ChAT–ChR2 mice are a valuable model to investigate hypercholinergic phenotypes. Previous experiments established that ChAT–ChR2 mice display an increased sensitivity to amphetamine induced-locomotor activity and stereotypes. In the present report, we analyzed the impact of VAChT over-expression in the striatum of ChAT-ChR2 mice. ChAT-ChR2 mice displayed increased locomotor sensitization in response to low dose of cocaine. In addition, we observed a dramatic remodeling of the morphology of CIN in ChAT-ChR2 transgenic mice. VAChT immunolabeling was markedly enhanced in the soma and terminal of CIN from ChAT-ChR2 mice as previously shown (Crittenden et al. 2014). Interestingly, the number of cholinergic varicosities was markedly reduced (−87%) whereas their size was significantly increased (+177%). Moreover, VAChT over-expression dramatically modified its trafficking along the somatodendritic and axonal arbor. These findings demonstrate that ChAT–ChR2 mice present major alterations of CIN neuronal morphology and increased behavioral sensitization to cocaine, supporting the notion that the increased levels of VAChT observed in these mice make them fundamentally different from wild-type mice

Behavioral effect of selective deletion of beta2* nicotinic acetylcholine receptors in neuropeptide Y expressing interneurons

Neuropeptide Y (NPY) is an abundant neuropeptide in the neocortex involved in numerous processes. NPY-expressing neurons differ in electrophysiological, molecular and morphological properties but overall, they are GABAergic interneurons (GINs). All cortical layers receive cholinergic inputs from the basal forebrain, which are crucial for cognition. Cholinergic receptors are differentially distributed across the cortex and the activation of nicotinic acetylcholine receptors (nAChRs) located on principal neurons and GINs modulates synaptic plasticity and behavior

Evaluating Sequential Response Learning in the Rodent Operant Touchscreen System

Sequential and cue-directed response learning in rodents have been previously shown to depend on intact striatal signaling. In particular, these behaviors rely on striatal dopamine and acetylcholine release, with an impairment of sequential response learning evident in animal models with alterations in the two systems. Here we provide a protocol for testing sequential response/response chain learning using the rodent touchscreen system. Specifically, the present protocol is designed to implement the heterogeneous sequence task, adapted from Keeler et al. (2014), in the rodent touchscreen apparatus. This task has been used previously to assess complex motor learning and response selection in mice. In the following protocol, the task is performed in touchscreen-based automated chambers with five response locations using food reinforcers to maintain performance. The sequence task requires the subject to make five nose pokes to white square stimuli appearing in five different locations sequentially from left to right. © 2021 Wiley Periodicals LLC. Basic Protocol: Implementation of the heterogeneous sequence task. Support Protocol: Creation of the heterogeneous sequence task ABET II touchscreen schedule

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

© FASEB 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. FASEB J. 33, 7018–7036 (2019). www.fasebj.org

Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors

International audienceAcetylcholine is an important modulator of striatal activity, and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nAChRs expressed by striatal interneurons. In the present study, we used FISH to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely, to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning, and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes that, despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by cholinergic interneurons and play a significant role in behavio

AAV-mediated neuronal expression of an scFv antibody selective for Aβ oligomers protects synapses and rescues memory in Alzheimer models

International audienceThe accumulation of soluble oligomers of the amyloid-β peptide (AβOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AβOs and shows minimal reactivity to Aβ monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AβOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AβO binding to neurons and AβO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AβOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AβO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease

NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1*

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5–30 μm, ws-LYNX1 competed with 125I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μm ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μm caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding