Neural processing in the primary auditory cortex following cholinergic lesions of the basal forebrain in ferrets.

Cortical acetylcholine (ACh) release has been linked to various cognitive functions, including perceptual learning. We have previously shown that cortical cholinergic innervation is necessary for accurate sound localization in ferrets, as well as for their ability to adapt with training to altered spatial cues. To explore whether these behavioral deficits are associated with changes in the response properties of cortical neurons, we recorded neural activity in the primary auditory cortex (A1) of anesthetized ferrets in which cholinergic inputs had been reduced by making bilateral injections of the immunotoxin ME20.4-SAP in the nucleus basalis (NB) prior to training the animals. The pattern of spontaneous activity of A1 units recorded in the ferrets with cholinergic lesions (NB ACh-) was similar to that in controls, although the proportion of burst-type units was significantly lower. Depletion of ACh also resulted in more synchronous activity in A1. No changes in thresholds, frequency tuning or in the distribution of characteristic frequencies were found in these animals. When tested with normal acoustic inputs, the spatial sensitivity of A1 neurons in the NB ACh- ferrets and the distribution of their preferred interaural level differences also closely resembled those found in control animals, indicating that these properties had not been altered by sound localization training with one ear occluded. Simulating the animals' previous experience with a virtual earplug in one ear reduced the contralateral preference of A1 units in both groups, but caused azimuth sensitivity to change in slightly different ways, which may reflect the modest adaptation observed in the NB ACh- group. These results show that while ACh is required for behavioral adaptation to altered spatial cues, it is not required for maintenance of the spectral and spatial response properties of A1 neurons.

Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Sildenafil Reverses the Neuropathological Alzheimer's Disease Phenotype in Cholinergic-Like Neurons Carrying the Presenilin 1 E280A Mutation.

Background: Familial Alzheimer's disease (FAD) presenilin 1 E280A (PSEN 1 E280A) is characterized by functional impairment and the death of cholinergic neurons as a consequence of amyloid-ß (Aß) accumulation and abnormal phosphorylation of the tau protein. Currently, there are no available therapies that can cure FAD. Therefore, new therapies are urgently needed for treating this disease. Objective: To assess the effect of sildenafil (SIL) on cholinergic-like neurons (ChLNs) harboring the PSEN 1 E280A mutation. Methods: Wild-type (WT) and PSEN 1 E280A ChLNs were cultured in the presence of SIL (25µM) for 24 h. Afterward, proteinopathy, cell signaling, and apoptosis markers were evaluated via flow cytometry and fluorescence microscopy. Results: We found that SIL was innocuous toward WT PSEN 1 ChLNs but reduced the accumulation of intracellular Aß fragments by 87%, decreased the non-physiological phosphorylation of the protein tau at residue Ser202/Thr205 by 35%, reduced the phosphorylation of the proapoptotic transcription factor c-JUN at residue Ser63/Ser73 by 63%, decreased oxidized DJ-1 at Cys106-SO3 by 32%, and downregulated transcription factor TP53 (tumor protein p53), BH-3-only protein PUMA (p53 upregulated modulator of apoptosis), and cleaved caspase 3 (CC3) expression by 20%, 32%, and 22%, respectively, compared with untreated mutant ChLNs. Interestingly, SIL also ameliorated the dysregulation of acetylcholine-induced calcium ion (Ca2+) influx in PSEN 1 E280A ChLNs. Conclusions: Although SIL showed no antioxidant capacity in the oxygen radical absorbance capacity and ferric ion reducing antioxidant power assays, it might function as an anti-amyloid and antiapoptotic agent and functional neuronal enhancer in PSEN 1 E280A ChLNs. Therefore, the SIL has therapeutic potential for treating FAD.

Loss of Cholinergic and Monoaminergic Afferents in APPswe/PS1ΔE9 Transgenic Mouse Model of Cerebral Amyloidosis Preferentially Occurs Near Amyloid Plaques.

Alzheimer's disease (AD) is characterized by a loss of neurons in the cortex and subcortical regions. Previously, we showed that the progressive degeneration of subcortical monoaminergic (MAergic) neurons seen in human AD is recapitulated in the APPswe/PS1ΔE9 (APP/PS) transgenic mouse model. Because degeneration of cholinergic (Ach) neurons is also a prominent feature of AD, we examined the integrity of the Ach system in the APP/PS model. The overall density of Ach fibers is reduced in APP/PS1 mice at 12 and 18 months of age but not at 4 months of age. Analysis of basal forebrain Ach neurons shows no loss of Ach neurons in the APP/PS model. Thus, since MAergic systems show overt cell loss at 18 months of age, the Ach system is less vulnerable to neurodegeneration in the APP/PS1 model. We also examined whether the proximity to Aß deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aß deposits exhibit a more severe loss of afferents than the areas that are more distal to Aß deposits. Collectively, the results indicate that the APP/PS model recapitulates the degeneration of multiple subcortical neurotransmitter systems, including the Ach system. In addition, the results indicate that Aß deposits cause global as well as local toxicity to subcortical afferents.

Brain histamine improves colonic hyperpermeability through the basal forebrain cholinergic neurons, adenosine A2B receptors and vagus nerve in rats.

Intestinal barrier dysfunction, leaky gut, is implicated in various diseases, including irritable bowel syndrome (IBS) and neurodegenerative conditions like Alzheimer's disease. Our recent investigation revealed that basal forebrain cholinergic neurons (BFCNs), critical for cognitive function, receive signals from butyrate and orexin, playing a role in regulating intestinal barrier function through adenosine A2B signaling and the vagus. This study explores the involvement and function of brain histamine, linked to BFCNs, in the regulation of intestinal barrier function. Colonic permeability, assessed by quantifying absorbed Evans blue in rat colonic tissue, showed that histamine did not affect increased colonic permeability induced by LPS when administered subcutaneously. However, intracisternal histamine administration improved colonic hyperpermeability. Elevating endogenous histamine levels in the brain with SKF91488, a histamine N-methyltransferase inhibitor, also improved colonic hyperpermeability. This effect was abolished by intracisternal chlorpheniramine, an histamine H1 receptor antagonist, not ranitidine, an H2 receptor antagonist. The SKF91488-induced improvement in colonic hyperpermeability was blocked by vagotomy, intracisternal pirenzepine (suppressing BFCNs activity), or alloxazine (an adenosine A2B receptor antagonist). Additionally, intracisternal chlorpheniramine injection eliminated butyrate-induced improvement in colonic hyperpermeability. These findings suggest that brain histamine, acting via the histamine H1 receptor, regulates intestinal barrier function involving BFCNs, adenosine A2B signaling, and the vagus. Brain histamine appears to centrally regulate intestinal barrier function influenced by butyrate, differentiating its actions from peripheral histamine in conditions like IBS, where mast cell-derived histamine induces leaky gut. Brain histamine emerges as a potential pharmacological target for diseases associated with leaky gut, such as dementia and IBS.

Neuroprotection of Cholinergic Neurons with a Tau Aggregation Inhibitor and Rivastigmine in an Alzheimer's-like Tauopathy Mouse Model.

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer's disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets.

The following article for this Special Issue was previously published and can be found in its respective issue online: "Whole-brain neural connectivity to cholinergic neurons in the nucleus basalis of Meynert".

Whole-brain neural connectivity to cholinergic neurons in the nucleus basalis of Meynert (Published in JNC 166.2 issue) https://onlinelibrary.wiley.com/doi/10.1111/jnc.15873.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Distinct subpopulations of ventral pallidal cholinergic projection neurons encode valence of olfactory stimuli.

To better understand the function of cholinergic projection neurons in the ventral pallidum (VP), we examined behavioral responses to appetitive (APP) and aversive (AV) odors that elicited approach or avoidance, respectively. Exposure to each odor increased cFos expression and calcium signaling in VP cholinergic neurons. Activity and Cre-dependent viral vectors selectively labeled VP cholinergic neurons that were activated and reactivated in response to either APP or AV odors, but not both, identifying two non-overlapping populations of VP cholinergic neurons differentially activated by the valence of olfactory stimuli. These two subpopulations showed differences in electrophysiological properties, morphology, and projections to the basolateral amygdala. Although VP neurons are engaged in both approach and avoidance behavioral responses, cholinergic signaling is only required for approach behavior. Thus, two distinct subpopulations of VP cholinergic neurons differentially encode valence of olfactory stimuli and play distinct roles in approach and avoidance behaviors.

Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Cell-type-specific optogenetic fMRI on basal forebrain reveals functional network basis of behavioral preference.

The basal forebrain (BF) is a complex structure that plays key roles in regulating various brain functions. However, it remains unclear how cholinergic and non-cholinergic BF neurons modulate large-scale functional networks and their relevance in intrinsic and extrinsic behaviors. With an optimized awake mouse optogenetic fMRI approach, we revealed that optogenetic stimulation of four BF neuron types evoked distinct cell-type-specific whole-brain BOLD activations, which could be attributed to BF-originated low-dimensional structural networks. Additionally, optogenetic activation of VGLUT2, ChAT, and PV neurons in the BF modulated the preference for locomotion, exploration, and grooming, respectively. Furthermore, we uncovered the functional network basis of the above BF-modulated behavioral preference through a decoding model linking the BF-modulated BOLD activation, low-dimensional structural networks, and behavioral preference. To summarize, we decoded the functional network basis of differential behavioral preferences with cell-type-specific optogenetic fMRI on the BF and provided an avenue for investigating mouse behaviors from a whole-brain view.

Apparent absence of hypothalamic cholinergic neurons in the common ostrich and emu: Implications for global brain states during sleep.

We examined the presence/absence and parcellation of cholinergic neurons in the hypothalami of five birds: a Congo grey parrot (Psittacus erithacus), a Timneh grey parrot (P. timneh), a pied crow (Corvus albus), a common ostrich (Struthio camelus), and an emu (Dromaius novaehollandiae). Using immunohistochemistry to an antibody raised against the enzyme choline acetyltransferase, hypothalamic cholinergic neurons were observed in six distinct clusters in the medial, lateral, and ventral hypothalamus in the parrots and crow, similar to prior observations made in the pigeon. The expression of cholinergic nuclei was most prominent in the Congo grey parrot, both in the medial and lateral hypothalamus. In contrast, no evidence of cholinergic neurons in the hypothalami of either the ostrich or emu was found. It is known that the expression of sleep states in the ostrich is unusual and resembles that observed in the monotremes that also lack hypothalamic cholinergic neurons. It has been proposed that the cholinergic system acts globally to produce and maintain brain states, such as those of arousal and rapid-eye-movement sleep. The hiatus in the cholinergic system of the ostrich, due to the lack of hypothalamic cholinergic neurons, may explain, in part, the unusual expression of sleep states in this species. These comparative anatomical and sleep studies provide supportive evidence for global cholinergic actions and may provide an important framework for our understanding of one broad function of the cholinergic system and possible dysfunctions associated with global cholinergic neural activity.

Functional GnRH receptor signaling regulates striatal cholinergic neurons in neonatal but not in adult mice.

Reproduction in mammals is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Recent studies from our laboratory established that the basal ganglia of the human brain contain additional large populations of GnRH synthesizing neurons which are absent in adult mice. Such extrahypothalamic GnRH neurons mostly occur in the putamen where they correspond to subsets of the striatal cholinergic interneurons (ChINs) and express GnRHR autoreceptors. In an effort to establish a mouse model for functional studies of striatal GnRH/GnRHR signaling, we carried out electrophysiological experiments on acute brain slices from male transgenic mice. Using PN4-7 neonatal mice, half of striatal ChINs responded with transient hyperpolarization and decreased firing rate to 1.2 µM GnRH, whereas medium spiny projection neurons remained unaffected. GnRH acted on its specific receptor because no response was observed in the presence of the GnRHR antagonist Antide. Addition of the membrane-impermeable G protein-coupled receptor inhibitor GDP-ß-S to the internal electrode solution eliminated the effect of GnRH. Further, GnRH was able to inhibit ChINs in presence of tetrodotoxin which blocked action potential mediated events. Collectively, these data indicated that the receptor underlying the effects of GnRH in neonatal mice is localized within ChINs. GnRH responsiveness of ChINs was transient and entirely disappeared in adult mice. These results raise the possibility to use neonatal transgenic mice as a functional model to investigate the role of GnRH/GnRHR signaling discovered earlier in adult human ChINs.

Functionally refined encoding of threat memory by distinct populations of basal forebrain cholinergic projection neurons.

Neurons of the basal forebrain nucleus basalis and posterior substantia innominata (NBM/SIp) comprise the major source of cholinergic input to the basolateral amygdala (BLA). Using a genetically encoded acetylcholine (ACh) sensor in mice, we demonstrate that BLA-projecting cholinergic neurons can 'learn' the association between a naive tone and a foot shock (training) and release ACh in the BLA in response to the conditioned tone 24 hr later (recall). In the NBM/SIp cholinergic neurons express the immediate early gene, Fos following both training and memory recall. Cholinergic neurons that express Fos following memory recall display increased intrinsic excitability. Chemogenetic silencing of these learning-activated cholinergic neurons prevents expression of the defensive behavior to the tone. In contrast, we show that NBM/SIp cholinergic neurons are not activated by an innately threatening stimulus (predator odor). Instead, VP/SIa cholinergic neurons are activated and contribute to defensive behaviors in response to predator odor, an innately threatening stimulus. Taken together, we find that distinct populations of cholinergic neurons are recruited to signal distinct aversive stimuli, demonstrating functionally refined organization of specific types of memory within the cholinergic basal forebrain of mice.

Basal forebrain cholinergic systems as circuits through which traumatic stress disrupts emotional memory regulation.

Contextual and spatial systems facilitate changes in emotional memory regulation brought on by traumatic stress. Cholinergic basal forebrain (chBF) neurons provide input to contextual/spatial systems and although chBF neurons are important for emotional memory, it is unknown how they contribute to the traumatic stress effects on emotional memory. Clusters of chBF neurons that project to the prefrontal cortex (PFC) modulate fear conditioned suppression and passive avoidance, while clusters of chBF neurons that project to the hippocampus (Hipp) and PFC (i.e. cholinergic medial septum and diagonal bands of Broca (chMS/DBB neurons) are critical for fear extinction. Interestingly, neither Hipp nor PFC projecting chMS/DBB neurons are critical for fear extinction. The retrosplenial cortex (RSC) is a contextual/spatial memory system that receives input from chMS/DBB neurons, but whether this chMS/DBB-RSC circuit facilitates traumatic stress effects on emotional memory remain unexplored. Traumatic stress leads to neuroinflammation and the buildup of reactive oxygen species. These two molecular processes may converge to disrupt chBF circuits enhancing the impact of traumatic stress on emotional memory.

Comparing tonic and phasic dendritic calcium in cholinergic pedunculopontine neurons and dopaminergic substantia nigra neurons.

Several brainstem nuclei degenerate in Parkinson's disease (PD). In addition to the well-characterized dopaminergic neurons of the substantia nigra pars compacta (SNc), the cholinergic neurons of the pedunculopontine nucleus (PPN) also degenerate in PD. One leading hypothesis of selective vulnerability is that pacemaking activity and the activation of low-threshold L-type calcium current are major contributors to tonic calcium load and cellular stress in SNc dopaminergic neurons. However, it is not yet clear whether the vulnerable PPN cholinergic neurons share this property. Therefore, we used two-photon dendritic calcium imaging and whole-cell electrophysiology to evaluate the role of L-type calcium channels in tonic and phasic dendritic calcium signals in PPN and SNc neurons. In addition, we investigated N- and P/Q-type calcium channel regulation of firing properties and dendritic calcium in PPN neurons. We found that blocking L-type channels reduces tonic firing rate and dendritic calcium levels in SNc neurons. By contrast, the tonic calcium load in PPN neurons did not depend on L-, N- or P/Q-type channels. However, we found that blocking either L-type (with nifedipine) or N- and P/Q-type (with omega-conotoxin MVIIC) channels reduces phasic calcium influx in PPN dendrites. Together, these findings show that L-type calcium channels play different roles in the activity of SNc and PPN neurons, and suggest that low-threshold L-type channels are not responsible for tonic calcium levels in PPN cholinergic neurons and are therefore not likely to be a source of selective vulnerability in these cells.

Cholinergic Basal Forebrain Connectivity to the Basolateral Amygdala Modulates Food Intake.

Obesity results from excessive caloric input associated with overeating and presents a major public health challenge. The hypothalamus has received significant attention for its role in governing feeding behavior and body weight homeostasis. However, extrahypothalamic brain circuits also regulate appetite and consumption by altering sensory perception, motivation, and reward. We recently discovered a population of basal forebrain cholinergic (BFc) neurons that regulate appetite suppression. Through viral tracing methods in the mouse model, we found that BFc neurons densely innervate the basolateral amygdala (BLA), a limbic structure involved in motivated behaviors. Using channelrhodopsin-assisted circuit mapping, we identified cholinergic responses in BLA neurons following BFc circuit manipulations. Furthermore, in vivo acetylcholine sensor and genetically encoded calcium indicator imaging within the BLA (using GACh3 and GCaMP, respectively) revealed selective response patterns of activity during feeding. Finally, through optogenetic manipulations in vivo, we found that increased cholinergic signaling from the BFc to the BLA suppresses appetite and food intake. Together, these data support a model in which cholinergic signaling from the BFc to the BLA directly influences appetite and feeding behavior.

Reward contingency gates selective cholinergic suppression of amygdala neurons.

Basal forebrain cholinergic neurons modulate how organisms process and respond to environmental stimuli through impacts on arousal, attention, and memory. It is unknown, however, whether basal forebrain cholinergic neurons are directly involved in conditioned behavior, independent of secondary roles in the processing of external stimuli. Using fluorescent imaging, we found that cholinergic neurons are active during behavioral responding for a reward - even prior to reward delivery and in the absence of discrete stimuli. Photostimulation of basal forebrain cholinergic neurons, or their terminals in the basolateral amygdala (BLA), selectively promoted conditioned responding (licking), but not unconditioned behavior nor innate motor outputs. In vivo electrophysiological recordings during cholinergic photostimulation revealed reward-contingency-dependent suppression of BLA neural activity, but not prefrontal cortex. Finally, ex vivo experiments demonstrated that photostimulation of cholinergic terminals suppressed BLA projection neuron activity via monosynaptic muscarinic receptor signaling, while also facilitating firing in BLA GABAergic interneurons. Taken together, we show that the neural and behavioral effects of basal forebrain cholinergic activation are modulated by reward contingency in a target-specific manner.