Functional dissociation of behavioral effects from acetylcholine and glutamate released from cholinergic striatal interneurons

In the striatum, cholinergic interneurons (CINs) have the ability to release both acetylcholine and glutamate, due to the expression of the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter 3 (VGLUT3). However, the relationship these neurotransmitters have in the regulation of behavior is not fully understood. Here we used reward-based touchscreen tests in mice to assess the individual and combined contributions of acetylcholine/glutamate co-transmission in behavior. We found that reduced levels of the VAChT from CINs negatively impacted dopamine signalling in response to reward, and disrupted complex responses in a sequential chain of events. In contrast, diminished VGLUT3 levels had somewhat opposite effects. When mutant mice were treated with haloperidol in a cue-based task, the drug did not affect the performance of VAChT mutant mice, whereas VGLUT3 mutant mice were highly sensitive to haloperidol. In mice where both vesicular transporters were deleted from CINs, we observed altered reward-evoked dopaminergic signalling and behavioral deficits that resemble, but were worse, than those in mice with specific loss of VAChT alone. These results demonstrate that the ability to secrete two different neurotransmitters allows CINs to exert complex modulation of a wide range of behaviors

Cholinergic Modulation of Behaviour

The cholinergic system is one of the most influential and essential signalling systems in the body. In the brain, cholinergic neurons innervate many brain regions where they influence a wide variety of behaviours. However, the precise role of each cholinergic region on distinct types of behaviour is not well known. Furthermore, in recent years there has been evidence that many cholinergic neurons in the brain have a capacity for co-transmission. Yet the functional significance of secreting two classical neurotransmitters from the same neuron is still largely unidentified. In this thesis, we investigated how different cholinergic nuclei modulate behavioural functions. To do that we selectively eliminated acetylcholine (ACh) release from cholinergic neurons of the striatum, brainstem and basal forebrain in mice. We then evaluated cognitive and non-cognitive behaviours using classical behavioural tests as well as sophisticated automated touchscreens tasks. In the striatum cholinergic interneurons are known to co-release ACh and glutamate (Glu), so we focused our investigation on how the individual neurotransmitters modulate striatal-dependent behaviours. We demonstrated that ACh modulates cognitive behaviours such as cognitive flexibility, extinction and cue detection. Glu released from striatal cholinergic interneurons also affects striatal-dependent behaviours but usually in an opposing manner to ACh, so, a balance between ACh and Glu is critical to regulating behaviours. As dopaminergic signalling in the striatum is widely influenced by ACh and Glu released by cholinergic interneurons, we also investigated how dopaminergic signalling changes while animals are performing a striatal-dependent cognitive task. In the brainstem, we showed that ACh influences motor functions and stress but does not have a major impact on cognition. However, stress induced by brainstem ACh-deficiency can interfere with results from cognitive tasks. In the forebrain, we find that ACh signalling is essential for maintaining social memory. Decreased cholinergic signalling in the hippocampus and cortex lead to deficits in social recognition. In conclusion, we demonstrate the complexity that ACh brings into behavioural regulation and how changes in its release can contribute to the pathophysiology of diseases such as Parkinson’s disease and Alzheimer’s disease. Ultimately, this data helps define novel pharmacological mechanisms tailored to improve specific cholinergic-mediated symptoms

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

Functional dissociation of behavioral effects from acetylcholine and glutamate released from cholinergic striatal interneurons

International audienceIn the striatum, cholinergic interneurons (CINs) have the ability to release both acetylcholine and glutamate, due to the expression of the vesicular acetylcholine transporter (VAChT) and the vesicular glutamate transporter 3 (VGLUT3). However, the relationship these neurotransmitters have in the regulation of behavior is not fully understood. Here we used reward-based touchscreen tests in mice to assess the individual and combined contributions of acetylcholine/glutamate co-transmission in behavior. We found that reduced levels of the VAChT from CINs negatively impacted dopamine signalling in response to reward, and disrupted complex responses in a sequential chain of events. In contrast, diminished VGLUT3 levels had somewhat opposite effects. When mutant mice were treated with haloperidol in a cue-based task, the drug did not affect the performance of VAChT mutant mice, whereas VGLUT3 mutant mice were highly sensitive to haloperidol. In mice where both vesicular transporters were deleted from CINs, we observed altered reward-evoked dopaminergic signalling and behavioral deficits that resemble, but were worse, than those in mice with specific loss of VAChT alone. These results demonstrate that the ability to secrete two different neurotransmitters allows CINs to exert complex modulation of a wide range of behaviors

Figure 3- Motor Behaviour

2-5 month old male and female mice in which hM3Dq signalling is activated on VGLUT3-expressing neurons on a wild-type background or on VGLUT3-expressing neurons null for VGLUT3 or VAChT. Mouse lines were treated with saline or CNO and assessed on the rotarod, treadmill, metal bar catalepsy or Y-maze tasks.</p

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

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity

© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. The ability to directly measure acetylcholine (ACh) release is an essential step toward understanding its physiological function. Here we optimized the GRABACh (GPCR-activation-based ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the subsecond response kinetics, physiologically relevant affinity and precise molecular specificity for ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice performing a variety of behaviors