ChAT-Cre Mice Research Articles

Ngfr+ cholinergic projection from SI/nBM to mPFC selectively regulates temporal order recognition memory

Acetylcholine regulates various cognitive functions through broad cholinergic innervation. However, specific cholinergic subpopulations, circuits and molecular mechanisms underlying recognition memory remain largely unknown. Here we show that Ngfr+ cholinergic neurons in the substantia innominate (SI)/nucleus basalis of Meynert (nBM)-medial prefrontal cortex (mPFC) circuit selectively underlies recency judgements. Loss of nerve growth factor receptor (Ngfr−/− mice) reduced the excitability of cholinergic neurons in the SI/nBM-mPFC circuit but not in the medial septum (MS)-hippocampus pathway, and impaired temporal order memory but not novel object and object location recognition. Expression of Ngfr in Ngfr−/− SI/nBM restored defected temporal order memory. Fiber photometry revealed that acetylcholine release in mPFC not only predicted object encounters but also mediated recency judgments of objects, and such acetylcholine release was absent in Ngfr−/− mPFC. Chemogenetic and optogenetic inhibition of SI/nBM projection to mPFC in ChAT-Cre mice diminished mPFC acetylcholine release and deteriorated temporal order recognition. Impaired cholinergic activity led to a depolarizing shift of GABAergic inputs to mPFC pyramidal neurons, due to disturbed KCC2-mediated chloride gradients. Finally, potentiation of acetylcholine signaling upregulated KCC2 levels, restored GABAergic driving force and rescued temporal order recognition deficits in Ngfr−/− mice. Thus, NGFR-dependent SI/nBM-mPFC cholinergic circuit underlies temporal order recognition memory.

Differential cholinergic innervation of lemniscal versus non-lemniscal regions of the inferior colliculus

The inferior colliculus (IC), a midbrain hub for integration of auditory information, receives dense cholinergic input that could modulate nearly all aspects of hearing. A key step in understanding cholinergic modulation is to identify the source(s) and termination patterns of cholinergic input. These issues have not been addressed for the IC in mice, an increasingly important model for study of hearing. We examined cholinergic inputs to the IC in adult male and female mice. We used retrograde tracing and immunochemistry to identify three sources of cholinergic innervation of the mouse IC: the pedunculopontine tegmental nucleus (PPT), the laterodorsal tegmental nucleus (LDT) and the lateral paragigantocellular nucleus (LPGi). We then used Cre-dependent labeling of cholinergic neurons in normal-hearing ChAT-Cre mice to selectively label the cholinergic projections to the IC from each of the cholinergic sources. Labeling of cholinergic projections from the PPT and LDT revealed cholinergic axons and boutons terminating throughout the IC, with the ipsilateral projection being denser. Electron microscopic examination showed that these cholinergic axons can form traditional synaptic junctions with IC neurons. In separate experiments, selective labeling of cholinergic projections from the LPGi revealed bilateral projections to the IC. The LPGi axons exhibited relatively equal densities on ipsilateral and contralateral sides, but on both sides the terminations were largely restricted to the non-lemniscal regions of the IC (i.e., the dorsal cortex, lateral cortex and intercollicular tegmentum). We conclude first that cholinergic axons can form traditional synapses in the IC. In addition, lemniscal and non-lemniscal regions of the IC receive different patterns of cholinergic innervation. The lemniscal IC (IC central nucleus) is innervated by cholinergic neurons in the PPT and the LDT whereas the non-lemniscal “shell” areas of the IC are innervated by the PPT and LDT and by cholinergic neurons in the LPGi. Data AvailabilityData will be made available on request.

Structure and Function of Pancreatic Neural Circuitry in Obesity and Diabetes

INTRODUCTION: Pancreatic islets are richly innervated by autonomic nerves which contribute to physiological regulation of islet hormone release and blood glucose. Published studies suggest obesity disrupts pancreatic innervation and may contribute to metabolic dysfunction. However, these studies largely rely on 2D imaging of islets and experiments aimed at assessing the function of pancreatic nerves have not been organ specific. Determining how high-fat diet (HFD) impacts pancreatic nerve structure and function using 3D imaging and organ-specific neuromodulation may provide new insights into the pathophysiology of diabetes and identify novel therapeutic approaches to treat diabetes. METHODS: To test the hypothesis that HFD causes structural changes in pancreatic innervation, C57Bl6 mice were randomized to 1, 4 and 12 week high-fat (HFD) or low-fat diet (LFD) groups. Metabolic phenotyping was determined. Pancreata were cleared and immunolabeled using iDISCO+, imaged by lightsheet (4X) and confocal (10X) microscopy and images were analyzed using Imaris software. To test the effects of HFD on pancreatic nerve function, we used AAV8 delivery of cre-dependent neuromodulatory constructs to activate or silence pancreatic parasympathetic or sympathetic nerves. To modulate pancreatic parasympathetic nerves, we delivered cre-dependent hM3D(Gq) for activation, cre-dependent diphtheria toxin A subunit for silencing, or mCherry (control) into the pancreas of Chat-cre mice. To modulate pancreatic sympathetic nerves, we used pancreatic injection of retrograde AAV delivering cre and celiac injection of cre-dependent hM3Gq (activation), hM4Gi (silencing) or mCherry (control). Mice were fed LFD or HFD for up to 12 weeks and the effects of neuromodulation on glucose metabolism were examined. RESULTS: HFD significantly impaired glucose tolerance, insulin sensitivity and altered plasma insulin and glucagon after 1, 4 and 12 weeks of HFD consumption. HFD consumption significantly altered pancreatic neural structure. Within islets, 1 week of HFD significantly increased sympathetic neural volume. With prolonged HFD, there was significant loss of islet parasympathetic innervation. Reproducing loss of islet parasympathetic innervation impaired glucose tolerance but sympathetic activation did not alter glucose metabolism. Targeted silencing of pancreatic sympathetic innervation or activation of pancreatic parasympathetic innervation significantly improved glucose tolerance in hyperglycemic mice fed HFD. CONCLUSION: Short term HFD causes early rapid changes to islet sympathetic nerves while long term HFD consumption results in loss of pancreatic parasympathetic innervation of the islet. The effects of HFD consumption are improved by inhibiting pancreas-projecting sympathetic fibers or through activation of pancreatic parasympathetic nerves. These studies suggest modulating pancreatic innervation may improve glucose control in obesity. NIH F31 Predoctoral Fellowship (1F31DK129016-01A1, R.H.) ISMMS MSTP T32 (GM007280) NIH (R01NS097184, OT2OD024912, S.S.) American Diabetes Association (ADA #1-17-ACE-31, S.S) Department of Defense (W81XWH-20-1-0345, S.S). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholinergic interneurons in the shell region of the nucleus accumbens regulate binge alcohol consumption: A chemogenetic and genetic lesion study.

Binge drinking, characterized by heavy episodic alcohol consumption, poses significant health hazards and increases the likelihood of developing an alcohol use disorder (AUD). Given the growing prevalence of this behavior and its negative consequences, there is a need to explore novel therapeutic targets. Accumulating evidence suggests that cholinergic interneurons (CIN) within the shell region of the nucleus accumbens (NAcSh) play a critical role in reward and addiction. However, their specific involvement in binge alcohol administration remains unclear. We hypothesized that CIN in the NAcSh regulates binge alcohol consumption. To test this hypothesis, we used male ChAT-cre mice expressing Cre-recombinase in cholinergic neurons. We performed chemogenetic manipulation using Designer Receptor Exclusively Activated by Designer Drugs (DREADD) to examine the activity, and genetic ablation of CIN in the NAcSh to examine the amount of alcohol consumed in mice exposed to binge alcohol consumption using the 4-Days Drinking-in-Dark (DID) paradigm. The impact of CIN manipulations in the NAcSh on sucrose self-administration was used to control for taste and caloric effects. Additionally, in a separate group of mice, c-Fos immunofluorescence was employed to verify chemogenetic activation or inhibition. Histological and immunohistochemical techniques were used to verify microinfusion sites, DREADD expression in CINs, and genetic ablation. We found that, while chemogenetic activation of CIN in the NAcSh caused a significant increase in alcohol consumption, chemogenetic inhibition or genetic ablation of CIN significantly reduced the amount of alcohol consumed without affecting sucrose self-administration. The chemogenetic inhibition caused a significant reduction, whereas activation caused a significant increase, in the number of c-Fos-labeled CIN in the NAcSh. Our findings highlight the crucial involvement of CIN in the NAcSh in modulating binge alcohol consumption, suggesting that targeting these neurons could serve to modify alcohol-related behaviors.

Dopaminergic Dependency of Cholinergic Pallidal Neurons.

We employed the whole-cell patch-clamp method and ChAT-Cre mice to study the electrophysiological attributes of cholinergic neurons in the external globus pallidus. Most neurons were inactive, although approximately 20% displayed spontaneous firing, including burst firing. The resting membrane potential, the whole neuron input resistance, the membrane time constant and the total neuron membrane capacitance were also characterized. The current-voltage relationship showed time-independent inward rectification without a "sag". Firing induced by current injections had a brief initial fast adaptation followed by tonic firing with minimal accommodation. Intensity-frequency plots exhibited maximal average firing rates of about 10Hz. These traits are similar to those of some cholinergic neurons in the basal forebrain. Also, we examined their dopamine sensitivity by acutely blocking dopamine receptors. This action demonstrated that the membrane potential, excitability, and firing pattern of pallidal cholinergic neurons rely on the constitutive activity of dopamine receptors, primarily D2-class receptors. The blockade of these receptors induced a resting membrane potential hyperpolarization, a decrease in firing for the same stimulus, the disappearance of fast adaptation, and the emergence of a depolarization block. This shift in physiological characteristics was evident even when the hyperpolarization was corrected with D.C. current. Neither the currents that generate the action potentials nor those from synaptic inputs were responsible. Instead, our findings suggest, that subthreshold slow ion currents, that require further investigation, are the target of this novel dopaminergic signaling.

Targeting a vulnerable septum-hippocampus cholinergic circuit in a critical time window ameliorates tau-impaired memory consolidation

BackgroundAbnormal tau accumulation and cholinergic degeneration are hallmark pathologies in the brains of patients with Alzheimer’s disease (AD). However, the sensitivity of cholinergic neurons to AD-like tau accumulation and strategies to ameliorate tau-disrupted spatial memory in terms of neural circuits still remain elusive.MethodsTo investigate the effect and mechanism of the cholinergic circuit in Alzheimer's disease-related hippocampal memory, overexpression of human wild-type Tau (hTau) in medial septum (MS)-hippocampus (HP) cholinergic was achieved by specifically injecting pAAV-EF1α-DIO-hTau-eGFP virus into the MS of ChAT-Cre mice. Immunostaining, behavioral analysis and optogenetic activation experiments were used to detect the effect of hTau accumulation on cholinergic neurons and the MS-CA1 cholinergic circuit. Patch-clamp recordings and in vivo local field potential recordings were used to analyze the influence of hTau on the electrical signals of cholinergic neurons and the activity of cholinergic neural circuit networks. Optogenetic activation combined with cholinergic receptor blocker was used to detect the role of cholinergic receptors in spatial memory.ResultsIn the present study, we found that cholinergic neurons with an asymmetric discharge characteristic in the MS-hippocampal CA1 pathway are vulnerable to tau accumulation. In addition to an inhibitory effect on neuronal excitability, theta synchronization between the MS and CA1 subsets was significantly disrupted during memory consolidation after overexpressing hTau in the MS. Photoactivating MS-CA1 cholinergic inputs within a critical 3 h time window during memory consolidation efficiently improved tau-induced spatial memory deficits in a theta rhythm-dependent manner.ConclusionsOur study not only reveals the vulnerability of a novel MS-CA1 cholinergic circuit to AD-like tau accumulation but also provides a rhythm- and time window-dependent strategy to target the MS-CA1 cholinergic circuit, thereby rescuing tau-induced spatial cognitive functions.Graphical

Tracking the Neurodegeneration and Behavioral Changes in Mice Model of Prodromal Phase Alpha-Synucleinopathy


 
 
 Background:
 The aggregation of misfolded alpha-synuclein (αSyn) has been shown to cause neurodegeneration in a mouse model and lead to behavioral changes. While others have examined end-stage (after neurodegeneration has set in) behavioral changes in mice after αSyn preformed fibrils (PFF) seeding, the precise timeline of circuit changes and associated behavioral deficits during the prodromal phase of synucleinopathic spread is poorly understood. We set out to measure and characterize the extent to which behavior is affected by primary motor cortex (M1) PFF seeding in mice.
 Experimental Design:
 We quantified the neuronal cell body density at M1 layers 2/3 and 5 using immunohistochemistry cell assays to determine if there were neurodegenerative effects under the PFF seeding strategy. Furthermore, using Bonsai, a visual reactive programming language, we tracked the behavior of the PFF seeded mice for exploratory motives, decreases in which can be used to show cognitive decline. To further ascertain the effect of αSyn on vulnerable excitatory pathways in the cortex, we performed prodromal injections of αSyn PFF into the M1 of ChATCre mice along with projection tracing in the basal forebrain.
 Results:
 During the first 3 months following PFF seeding, the density of neuronal cell bodies in M1 layers 2/3 and 5 remains unchanged in PFF seeded mice compared to control mice. The exploratory index of PFF seeded mice was comparable to that of control mice. These results suggest insignificant neurodegeneration and behavioral change in the first 3 months following PFF seeding.
 Conclusion:
 Work done previously in this lab tracking the spread of PFF in M1 shows significant spread of αSyn for 3 months following prodromal injection. The results from this work show that there is little neurodegeneration or behavioral change in that timeframe despite the significant spread of αSyn. Future work hinges on further probing this phenomenon.
 
 

Basal Forebrain Cholinergic Innervation Induces Depression-Like Behaviors Through Ventral Subiculum Hyperactivation

Malfunction of the ventral subiculum (vSub), the main subregion controlling the output connections from the hippocampus, is associated with major depressive disorder (MDD). Although the vSub receives cholinergic innervation from the medial septum and diagonal band of Broca (MSDB), whether and how the MSDB-to-vSub cholinergic circuit is involved in MDD is elusive. Here, we found that chronic unpredictable mild stress (CUMS) induced depression-like behaviors with hyperactivation of vSub neurons, measured by c-fos staining and whole-cell patch-clamp recording. By retrograde and anterograde tracing, we confirmed the dense MSDB cholinergic innervation of the vSub. In addition, transient restraint stress in CUMS increased the level of ACh in the vSub. Furthermore, chemogenetic stimulation of this MSDB-vSub innervation in ChAT-Cre mice induced hyperactivation of vSub pyramidal neurons along with depression-like behaviors; and local infusion of atropine, a muscarinic receptor antagonist, into the vSub attenuated the depression-like behaviors induced by chemogenetic stimulation of this pathway and CUMS. Together, these findings suggest that activating the MSDB-vSub cholinergic pathway induces hyperactivation of vSub pyramidal neurons and depression-like behaviors, revealing a novel circuit underlying vSub pyramidal neuronal hyperactivation and its associated depression.

The organization of cholinergic projections in the visual thalamus of the mouse.

Cholinergic projections from the brainstem serve as important modulators of activity in visual thalamic nuclei such as the dorsal lateral geniculate nucleus (dLGN). While these projections have been studied in several mammals, a comprehensive examination of their organization in the mouse is lacking. We used the retrograde transport of viruses or cholera toxin subunit B (CTB) injected in the dLGN, immunocytochemical labeling with antibodies against choline acetyltransferase (ChAT), brain nitric oxide synthase (BNOS), and vesicular acetylcholine transporter (VAChT), ChAT-Cre mice crossed with a reporter line (Ai9), as well as brainstem virus injections in ChAT-Cre mice to examine the pattern of thalamic innervation from cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus (LDTg), and the parabigeminal nucleus (PBG). Retrograde tracing demonstrated that the dLGN receives input from the PPTg, LDTg, and PBG. Viral tracing in ChAT-Cre mice and retrograde tracing combined with immunocytochemistry revealed that many of these inputs originate from cholinergic neurons in the PBG and PPTg. Most notable was an extensive cholinergic projection from the PBG which innervated most of the contralateral dLGN, with an especially dense concentration in the dorsolateral shell, as well as a small region in the dorsomedial pole of the ipsilateral dLGN. The PPTg was found to provide a sparse somewhat diffuse innervation of the ipsilateral dLGN. Neurons in the PPTg co-expressed ChAT, BNOS, and VAChT, whereas PBG neurons expressed ChAT, but not BNOS or VAChT. These results highlight the presence of distinct cholinergic populations that innervate the mouse dLGN.

169-OR: Magnetogenetic Stimulation of Pancreatic Nerves Regulates Blood Glucose

There is substantial evidence that neural regulation plays a critical role in glucose metabolism and hormone release. Targeting neural populations innervating the pancreas could provide an alternative therapy for diabetes. However, our understanding of the precise physiological roles of pancreatic nerves is incomplete because we lack tools to study these populations in a non-invasive, targeted, temporally controlled manner. Here, we developed and validated a novel magnetogenetic neuromodulatory construct in vitro and in vivo then targeted defined pancreatic nerves to assess their roles in glucose metabolism and hormone secretion. The construct, coding for TRPV1 ion channel fused to a nanobody binding endogenous ferritin (NbTRPV1), was transfected into neural cell lines and primary neurons. Magnetic stimulation significantly increased intracellular calcium in cells expressing NbTRPV1. For in vivo delivery, the construct was packaged into AAV and infused into the pancreas through the pancreatic duct, using serotypes and promoters to target β cells or pancreatic nerves. Ex vivo calcium imaging confirmed magnet activation of pancreas-projecting neurons expressing NbTRPV1. In vivo, targeting NbTRPV1 to β cells improved glucose tolerance in male and female mice, validating the efficacy of this construct to regulate cell activity in the pancreas of freely moving mice. Magnet treatment of ChAT-CRE mice expressing NbTRPV1 in parasympathetic pancreatic neurons significantly improved glucose tolerance and increased insulin secretion. Conversely, targeting vagal sensory nerves from the pancreas in Advillin-iCRE mice impaired glucose tolerance with no significant effect on hormone secretion. These data demonstrate for the first time a method for remote, temporally controlled, targeted modulation of pancreatic islets and nerves to regulate blood glucose. This approach can be used to study the contribution of pancreatic nerves in a wide range of diseases, from diabetes to pancreatic cancer. Disclosure R. Li: None. M. Jimenez gonzalez: None. L. E. Pomeranz: None. G. J. Schwartz: None. S. Stanley: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-ACE-31 to S.S.); National Institutes of Health (R01NS097184, OT2OD024912)

A New Mouse Strain to Study Motoneurons in Pompe Disease

Pompe disease is a lysosomal storage disorder resulting from a deficiency in the lysosomal enzyme acid α‐glucosidase (GAA). The most severe form of the disease is associated with complete or near complete absence of functional GAA which results in cardiorespiratory failure and death within the first year of life. The only approved treatment for Pompe disease is human recombinant GAA (enzyme replacement therapy (ERT)) which improves overall survival rates but the recombinant protein does not reach the central nervous system. Respiratory impairment in Pompe disease has historically been attributed to muscle weakness but an accumulation of studies of Pompe patients and animal models indicate that motoneuron pathology is functionally relevant. In an effort to better understand the role of motoneurons in Pompe disease, we used breeding strategies to create a new mouse model with motoneuron histopathology features seen in Pompe disease while also allowing easy visualization of motoneurons via a fluorescent tag. Chat‐cre mice (Jackson) were crossed with Gaa−/− mice (Taconic). The resulting heterozygous strain were crossed with eYFP+/+ mice (courtesy Dr. Brian Harfe). Further breeding resulted in homozygous mice which were Chat‐cre+/+/Gaa−/−/eYFP+/+. These mice were then crossed to a Flox‐Gaa+/+ (Taconic) to create a heterozygous strain (Chat‐cre+/−/Gaa+/−/eYFP+/−/Flox‐Gaa+/−). Further inbreeding produced mice that were Chat‐cre+/+/Gaa+/−/eYFP+/+/Flox‐Gaa+/+. The intent of this final cross was to produce mice in which alpha motoneurons express eYFP but do not express GAA. Histological evaluation of the brainstem and spinal cord of these mice showed robust eYFP expression in the brainstem including the hypoglossal motor nucleus, nucleus ambiguous, and facial motor nucleus. In the cervical spinal cord, alpha motoneurons throughout the ventral horn also had robust fluorescence. This included putative phrenic motoneurons as determined based on location and size. The robust eYFP expression was restricted to motoneurons which indicates that motoneurons are Gaa−/− (based on the breeding scheme). Ongoing studies are evaluating GAA protein expression in peripheral tissues, glycogen accumulation in motoneurons, and also brainstem and spinal cord motoneuron loss over the lifespan. In addition, the eYFP signal in motoneurons will permit rigorous evaluation of motoneuron transduction efficacy in gene therapy experiments.Support or Funding Information2R01HD052682‐11A1 (DDF/BJB)

Generation of a ChATCre mouse line without the early onset hearing loss typical of the C57BL/6J strain

The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChATCre mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23753A mutation (a.k.a., the ahl mutation). To develop ChATCre mice without accelerated hearing loss, we backcrossed ChATIRES-Cre mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChATIRES-Cre and the wild-type allele at the Cdh23 locus (ChATCre,Cdh23WT). In the new line, auditory brainstem response thresholds were ∼20 dB lower than those in 9 month old ChATIRES-Cre mice at all frequencies tested (4–31.5 kHz). These thresholds were stable throughout the period of testing (3–12 months of age).We then bred ChATCre,Cdh23WT animals with Ai14 reporter mice to confirm the expression pattern of ChATCre. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei.We conclude that the backcrossed ChATCre,Cdh23WT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChATCre,Cdh23WT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.

Multiparametric assessment of the impact of opsin expression and anesthesia on striatal cholinergic neurons and auditory brainstem activity.

Recent developments in genetic engineering have established murine models that permit the selective control of cholinergic neurons via optical stimulation. Despite copious benefits granted by these experimental advances, the sensory physiognomy of these organisms has remained poorly understood. Therefore, the present study evaluates sensory and neuronal response properties of animal models developed for the study of optically induced acetylcholine release regulation. Auditory brainstem responses, fluorescence imaging, and patch clamp recording techniques were used to assess the impact of viral infection, sex, age, and anesthetic agents across the ascending auditory pathway of ChAT-Cre and ChAT-ChR2(Ai32) mice. Data analyses revealed that neither genetic configuration nor adeno-associated viral infection alters the early stages of auditory processing or the cellular response properties of cholinergic neurons. However, anesthetic agent and dosage amount profoundly modulate the response properties of brainstem neurons. Last, analyses of age-related hearing loss in virally infected ChAT-Cre mice did not differ from those reported in wild type animals. This investigation demonstrates that ChAT-Cre and ChAT-ChR2(Ai32) mice are viable models for the study of cholinergic modulation in auditory processing, and it emphasizes the need for prudence in the selection of anesthetic procedures.

Chemogenetic manipulation of parasympathetic neurons (DMV) regulates feeding behavior and energy metabolism

Parasympathetic nervous system (PNS) innervates with several peripheral organs such as liver, pancreas and regulates energy metabolism. However, the direct role of PNS on food intake has been poorly understood. In the present study, we investigated the role of parasympathetic nervous system in regulation of feeding by chemogenetic methods. Adeno associated virus carrying DREADD (designer receptors exclusively activated by designer drugs) infused into the target brain region by stereotaxic surgery. The stimulatory hM3Dq or inhibitory hM4Di DREADD was over-expressed in selective population of dorsal motor nucleus of the vagus (DMV) neurons by Cre-recombinase-dependent manners. Activation of parasympathetic neuron by intraperitoneal injection of the M3-muscarinic receptor ligand clozapine-N-oxide (CNO) (1 mg/kg) suppressed food intake and resulted in body weight loss in ChAT-Cre mice. Parasympathetic neurons activation resulted in improved glucose tolerance while inhibition of the neurons resulted in impaired glucose tolerance. Stimulation of parasympathetic nervous system by injection of CNO (1 mg/kg) increased oxygen consumption and energy expenditure. Within the hypothalamus, in the arcuate nucleus (ARC) changed AGRP/POMC neurons. These results suggest that direct activation of parasympathetic nervous system decreases food intake and body weight with improved glucose tolerance.

A cell autonomous torsinA requirement for cholinergic neuron survival and motor control

Cholinergic dysfunction is strongly implicated in dystonia pathophysiology. Previously (Pappas et al., 2015;4:e08352), we reported that Dlx5/6-Cre mediated forebrain deletion of the DYT1 dystonia protein torsinA (Dlx-CKO) causes abnormal twisting and selective degeneration of dorsal striatal cholinergic interneurons (ChI) (Pappas et al., 2015). A central question raised by that work is whether the ChI loss is cell autonomous or requires torsinA loss from neurons synaptically connected to ChIs. Here, we addressed this question by using ChAT-Cre mice to conditionally delete torsinA from cholinergic neurons ('ChAT-CKO'). ChAT-CKO mice phenocopy the Dlx-CKO phenotype of selective dorsal striatal ChI loss and identify an essential requirement for torsinA in brainstem and spinal cholinergic neurons. ChAT-CKO mice are tremulous, weak, and exhibit trunk twisting and postural abnormalities. These findings are the first to demonstrate a cell autonomous requirement for torsinA in specific populations of cholinergic neurons, strengthening the connection between torsinA, cholinergic dysfunction and dystonia pathophysiology.

Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity.

The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs) in dopamine D1 receptor (D1R) expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT), during various reward-enforced behaviors and in a “waiting”-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP) paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs) in the 5-choice-serial-reaction-time-task (5CSRTT) than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG) expression (cFos and FosB) induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

Abstract P249: Melanocortin-4 Receptors in Cholinergic Preganglionic Neurons of the Hindbrain and Spinal Cord are Important in Mediating Cardiovascular Responses to Acute Stress

Previous studies suggest that melanocortin-4 receptor (MC4R) activation in areas outside of the hypothalamus may regulate appetite but the role of MC4R in specific neuronal populations of the hindbrain in regulating cardiovascular function are still unknown. We examined the impact of total body MC4R deficiency (LoxTB-MC4R mice) and restoration of MC4R specifically in the brainstem and/or spinal cord on blood pressure (BP) response to acute stress. We selectively rescued MC4R only in preganglionic parasympathetic neurons of the dorsal motor of the vagus (DMV) and in the nucleus tractus solitarius (NTS) (LoxTB-MC4R/Phox2B-cre mice, n=5) or in cholinergic preganglionic neurons of the hindbrain and spinal cord (LoxTB-MC4R/Chat-cre mice, n=4). Mice were implanted with telemetry probes for measurement of mean arterial pressure (MAP) and heart rate (HR). After a 10-day recovery period, MAP and HR were continuously measured for 30 minutes before, during and 30 minutes after an air jet stress test. Acute air jet stress significantly increased MAP by 33±3 in WT mice and 31±2 in LoxTB-MC4R/Chat-cre mice compared to only 18±4 or 20±3 mmHg in LoxTB-MC4R/Phox2B-cre mice and LoxTB-MC4R mice, respectively. HR was increased by 180±20, 110±20, 100±23, and 127±15 bpm in response to air jet stress in WT, LoxTB-MC4R, LoxTB-MC4R/Phox2B and LoxTB-MC4R/Chat-cre mice, respectively. These results indicate that MC4Rs in cholinergic preganglionic neurons of the hindbrain and spinal cord play a key role in the BP responses to acute stress. (NHLBI-PO1HL51971, NIGMS- P20GM104357, and AHA-SDG5680016)

Characterization of Channelrhodopsin and Archaerhodopsin in Cholinergic Neurons of Cre-Lox Transgenic Mice

The study of cholinergic signaling in the mammalian CNS has been greatly facilitated by the advent of mouse lines that permit the expression of reporter proteins, such as opsins, in cholinergic neurons. However, the expression of opsins could potentially perturb the physiology of opsin-expressing cholinergic neurons or mouse behavior. Indeed, the published literature includes examples of cellular and behavioral perturbations in preparations designed to drive expression of opsins in cholinergic neurons. Here we investigate expression of opsins, cellular physiology of cholinergic neurons and behavior in two mouse lines, in which channelrhodopsin-2 (ChR2) and archaerhodopsin (Arch) are expressed in cholinergic neurons using the Cre-lox system. The two mouse lines were generated by crossing ChAT-Cre mice with Cre-dependent reporter lines Ai32(ChR2-YFP) and Ai35(Arch-GFP). In most mice from these crosses, we observed expression of ChR2 and Arch in only cholinergic neurons in the basal forebrain and in other putative cholinergic neurons in the forebrain. In small numbers of mice, off-target expression occurred, in which fluorescence did not appear limited to cholinergic neurons. Whole-cell recordings from fluorescently-labeled basal forebrain neurons revealed that both proteins were functional, driving depolarization (ChR2) or hyperpolarization (Arch) upon illumination, with little effect on passive membrane properties, spiking pattern or spike waveform. Finally, performance on a behavioral discrimination task was comparable to that of wild-type mice. Our results indicate that ChAT-Cre x reporter line crosses provide a simple, effective resource for driving indicator and opsin expression in cholinergic neurons with few adverse consequences and are therefore an valuable resource for studying the cholinergic system.

Optogenetic activation of septal cholinergic neurons suppresses sharp wave ripples and enhances theta oscillations in the hippocampus.

Theta oscillations in the limbic system depend on the integrity of the medial septum. The different populations of medial septal neurons (cholinergic and GABAergic) are assumed to affect different aspects of theta oscillations. Using optogenetic stimulation of cholinergic neurons in ChAT-Cre mice, we investigated their effects on hippocampal local field potentials in both anesthetized and behaving mice. Cholinergic stimulation completely blocked sharp wave ripples and strongly suppressed the power of both slow oscillations (0.5-2 Hz in anesthetized, 0.5-4 Hz in behaving animals) and supratheta (6-10 Hz in anesthetized, 10-25 Hz in behaving animals) bands. The same stimulation robustly increased both the power and coherence of theta oscillations (2-6 Hz) in urethane-anesthetized mice. In behaving mice, cholinergic stimulation was less effective in the theta (4-10 Hz) band yet it also increased the ratio of theta/slow oscillation and theta coherence. The effects on gamma oscillations largely mirrored those of theta. These findings show that medial septal cholinergic activation can both enhance theta rhythm and suppress peri-theta frequency bands, allowing theta oscillations to dominate.

Electrophysiological biomarkers in spinal muscular atrophy: proof of concept

ObjectivePreclinical therapies that restore survival motor neuron (SMN) protein levels can dramatically extend survival in spinal muscular atrophy (SMA) mouse models. Biomarkers are needed to effectively translate these promising therapies to clinical trials. Our objective was to investigate electrophysiological biomarkers of compound muscle action potential (CMAP), motor unit number estimation (MUNE) and electromyography (EMG) using an SMA mouse model.MethodsSciatic CMAP, MUNE, and EMG were obtained in SMNΔ7 mice at ages 3–13 days and at 21 days in mice with SMN selectively reduced in motor neurons (ChATCre). To investigate these measures as biomarkers of treatment response, measurements were obtained in SMNΔ7 mice treated with antisense oligonucleotide (ASO) or gene therapy.ResultsCMAP was significantly reduced in SMNΔ7 mice at days 6–13 (P < 0.01), and MUNE was reduced at days 7–13 (P < 0.01). Fibrillations were present on EMG in SMNΔ7 mice but not controls (P = 0.02). Similar findings were seen at 21 days in ChATCre mice. MUNE in ASO-treated SMNΔ7 mice were similar to controls at day 12 and 30. CMAP reduction persisted in ASO-treated SMNΔ7 mice at day 12 but was corrected at day 30. Similarly, CMAP and MUNE responses were corrected with gene therapy to restore SMN.InterpretationThese studies confirm features of preserved neuromuscular function in the early postnatal period and subsequent motor unit loss in SMNΔ7 mice. SMN restoring therapies result in preserved MUNE and gradual repair of CMAP responses. This provides preclinical evidence for the utilization of CMAP and MUNE as biomarkers in future SMA clinical trials.