Cholinergic Neurons Research Articles

Noradrenergic current responses of neurons in rat oculomotor neural integrators.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are involved in the control of horizontal and vertical gaze, respectively. A previous study showed that PHN neurons exhibit depolarized or hyperpolarized responses to noradrenaline (NA). However, the adrenoceptor types that participate in NA-induced responses and the effects of NA on INC neurons have not yet been investigated. Furthermore, the relationship between NA-induced responses and neuron types defined by neurotransmitter phenotypes has not been determined. In this study, we investigated NA-induced current responses in PHN and INC neurons and the relationships between these responses and neuron types using whole cell recordings in wild-type and transgenic rat brainstem slices. Local application of NA to the cell soma induced slow inward (SI) and slow outward (SO) currents that were mainly mediated by α1 and α2 adrenoceptors, respectively. These current responses were observed in both PHN and INC neurons, although the proportion of INC neurons that responded to NA was low. Analyses of the distributions of the current responses revealed that in the PHN, all fluorescently identified inhibitory neurons exhibited SI currents, whereas glutamatergic and cholinergic neurons exhibited both SI and SO currents. In the INC, glutamatergic and inhibitory neurons preferentially exhibited SI and SO currents, respectively. When the PHN and INC neurons were characterized by their firing pattern, we found that the proportions of the currents depended on their firing pattern. These results suggest that various modes of noradrenergic modulation in horizontal and vertical neural integrators are dependent on neuron type.NEW & NOTEWORTHY Noradrenergic modulation of oculomotor neural integrators involved in gaze control has not been elucidated. Here, we report that noradrenaline (NA)-induced slow inward (SI) and outward (SO) currents are mediated mainly by α1 and α2 adrenoceptors in neurons that participate in horizontal and vertical gaze control. The NA-induced current responses differed depending on the neurotransmitter phenotype and firing pattern. These results suggest various modes of noradrenergic modulation in horizontal and vertical integrator neurons.

Pharmacologic blockade of nicotinic receptors in the suprachiasmatic nucleus increases ovarian atresia and inhibits follicular growth.

Reproduction in all mammalian species depends on the growth and maturation of ovarian follicles, that is, folliculogenesis. Follicular development can culminate with the rupture of mature follicles and the consequent expulsion of their oocytes (ovulation) or in atresia, characterized by the arrest of development and eventual degeneration. These processes are regulated by different neuroendocrine signals arising at different hypothalamic nuclei, including the suprachiasmatic nucleus (SCN). In the later, the activation of muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs) by acetylcholine is essential for the regulation of the pre-ovulatory signals that stimulate the rupture of mature follicles. To evaluate the participation of the nAChRs in the SCN throughout the oestrous cycle in the regulation of the hypothalamic-pituitary-ovarian axis. For this purpose, 90-day-old adult female rats in metoestrus, dioestrus, proestrus or oestrus were microinjected into the left- or right-SCN with 0.3 μL of saline solution as vehicle or with 0.225 μg of mecamylamine (Mec), a non-selective antagonist of the nicotinic receptors, diluted in 0.3 μL of vehicle. The animals were sacrificed when they presented vaginal cornification, indicative of oestrus stage, and the effects of the unilateral pharmacological blockade of the nAChRs in the SCN on follicular development, ovulation and secretion of oestradiol and follicle-stimulating hormone (FSH) were evaluated. The microinjection of Mec decreased the serum levels of FSH, which resulted in a lower number of growing and healthy follicles and an increase in atresia. The higher percentage of atresia in pre-ovulatory follicles was related to a decrease in the number of ova shed and abnormalities in oestradiol secretion. We also detected asymmetric responses between the left and right treatments that depended on the stage of the oestrous cycle. The present results allow us to suggest that during all the stages of the oestrous cycle, cholinergic signals that act on the nAChRs in the SCN are pivotal to modulate the secretion of gonadotropins and hence the physiology of the ovaries. Further research is needed to determine if such signals are generated by the cholinergic neurons in the SCN or by cholinergic afferents to the SCN.

Septal stimulation attenuates hippocampal seizure with subregion specificity.

Deep brain stimulation (DBS) is a promising approach for the treatment of epilepsy. However, the optimal target for DBS and underlying mechanisms are still not clear. Here, we compared the therapeutic effects of DBS on distinct septal subregions, aimed to find the precise targets of septal DBS and related mechanisms for the clinical treatment. Assisted by behavioral test, electroencephalography (EEG) recording and analyzing, selectively neuronal manipulation and immunohistochemistry, we assessed the effects of DBS on the three septal subregions in kainic acid (KA)-induced mouse seizure model. DBS in the medial septum (MS) not only delayed generalized seizure (GS) development, but reduced the severity; DBS in the vertical diagonal band of Broca (VDB) only reduced the severity of GS, while DBS in the horizontal diagonal band of Broca (HDB) subregion showed no anti-seizure effect. Notably, DBS in the MS much more efficiently decreased abnormal activation of hippocampal neurons. EEG spectrum analysis indicated that DBS in the MS and VDB subregions mainly increased the basal hippocampal low-frequency (delta and theta) rhythm. Furthermore, ablation of cholinergic neurons in the MS and VDB subregions blocked the anti-seizure and EEG-modulating effects of septal DBS, suggesting the seizure-alleviating effect of DBS was dependent on local cholinergic neurons. DBS in the MS and VDB, rather than HDB, attenuates hippocampal seizure by activation of cholinergic neurons-augmented hippocampal delta/theta rhythm. This may be of great therapeutic significance for the clinical treatment of epilepsy with septal DBS. The optical target of deep brain stimulation in the septum is still not clear. This study demonstrated that stimulation in the medial septum and vertical diagonal band of Broca subregions, but not the horizontal diagonal band of Broca, could alleviate hippocampal seizure through cholinergic neurons-augmented hippocampal delta/theta rhythm. This study may shed light on the importance of precise regulation of deep brain stimulation therapy in treating epileptic seizures.

Neuronal PAS domain 1 identifies a major subpopulation of wakefulness-promoting GABAergic neurons in the basal forebrain

Here, we describe a group of basal forebrain (BF) neurons expressing neuronal Per-Arnt-Sim (PAS) domain 1 (Npas1), a developmental transcription factor linked to neuropsychiatric disorders. Immunohistochemical staining in Npas1-cre-2A-TdTomato mice revealed BF Npas1+ neurons are distinct from well-studied parvalbumin or cholinergic neurons. Npas1 staining in GAD67-GFP knock-in mice confirmed that the vast majority of Npas1+ neurons are GABAergic, with minimal colocalization with glutamatergic neurons in vGlut1-cre-tdTomato or vGlut2-cre-tdTomato mice. The density of Npas1+ neurons was high, five to six times that of neighboring cholinergic, parvalbumin, or glutamatergic neurons. Anterograde tracing identified prominent projections of BF Npas1+ neurons to brain regions involved in sleep-wake control, motivated behaviors, and olfaction such as the lateral hypothalamus, lateral habenula, nucleus accumbens shell, ventral tegmental area, and olfactory bulb. Chemogenetic activation of BF Npas1+ neurons in the light period increased the amount of wakefulness and the latency to sleep for 2 to 3 h, due to an increase in long wake bouts and short NREM sleep bouts. NREM slow-wave and sigma power, as well as sleep spindle density, amplitude, and duration, were reduced, reminiscent of findings in several neuropsychiatric disorders. Together with previous findings implicating BF Npas1+ neurons in stress responsiveness, the anatomical projections of BF Npas1+ neurons and the effect of activating them suggest a possible role for BF Npas1+ neurons in motivationally driven wakefulness and stress-induced insomnia. Identification of this major subpopulation of BF GABAergic neurons will facilitate studies of their role in sleep disorders, dementia, and other neuropsychiatric conditions involving BF.

Expression of ChAT, Iba-1, and nNOS in the Central Nervous System following Facial Nerve Injury.

Facial nerve injury can cause significant functional impairment, impacting both the peripheral and central nervous systems. The present study evaluated changes in facial motor function, numbers of cholinergic neurons and microglia, and nNOS levels in the facial nucleus of the central nervous system (CNS) following peripheral facial nerve injury. Facial nerve function, as determined by eyeblink and whisker-movement reflexes, was evaluated at baseline and 1, 2, 3, 4, 8, and 12 weeks after inducing facial nerve injury through compression or axotomy. The expression of choline acetyltransferase (ChAT), ionized calcium-binding adaptor molecule 1 (Iba-1), and neuronal nitric oxide synthase (nNOS) in the facial nucleus of the CNS was analyzed 2, 4, and 12 weeks after peripheral facial nerve injury. Compression-induced facial nerve injury was found to lead to temporary facial motor impairment, whereas axotomy resulted in persistent impairment. Moreover, both compression and axotomy reduced ChAT expression and increased Iba-1 and nNOS expression in the facial nucleus, indicating upregulation of an inflammatory response and neurodegeneration. These results indicate that, compared with compression-induced injury, axotomy-induced facial nerve injury results in greater facial motor dysfunction and more persistent microglial and nitric oxide activation in the facial nucleus of the CNS.

Roles and Mechanisms of Choline Metabolism in Nonalcoholic Fatty Liver Disease and Cancers.

Choline participates in three major metabolic pathways: oxidation, phosphorylation, and acetylation. Through oxidation, choline is converted to betaine and contributes to methyl metabolism and epigenetic regulation. Through phosphorylation, choline participates in phospholipid metabolism, and serves as the precursor of phosphocholine, phosphatidylcholine, glycerophosphocholine, and other essential compounds, thereby modulating lipid metabolism and transport. Through acetylation, choline is transformed into acetylcholine in cholinergic neurons, playing a vital role in neurotransmission. Moreover, gut microbiota can metabolize choline into trimethylamine-N-oxide, and be involved in the pathogenesis of various diseases such as nonalcoholic fatty liver disease (NAFLD), cancer, cardiovascular disease, etc. Since choline metabolism is implicated in the development of NAFLD and diverse cancers, including liver cancer, it may serve as a therapeutic target for these diseases in the future. Currently, there are numerous therapeutic agents targeting choline metabolism to treat NAFLD and cancers, but most of them are ineffective and some even have adverse effects that lead to a series of complications. Therefore, further research and clinical validation are required to obtain safe and efficacious drugs. This review comprehensively summarizes the choline metabolic pathway and its regulatory mechanisms, elucidates the roles and mechanisms of choline metabolism in the aforementioned diseases, and provides a discussion of the current advances and immense potential of this field.

The Serotonin 4 Receptor Subtype: A Target of Particular Interest, Especially for Brain Disorders.

In recent years, particular attention has been paid to the serotonin 4 receptor, which is well expressed in the brain, but also peripherally in various organs. The cerebral distribution of this receptor is well conserved across species, with high densities in the basal ganglia, where they are expressed by GABAergic neurons. The 5-HT4 receptor is also present in the cerebral cortex, hippocampus, and amygdala, where they are carried by glutamatergic or cholinergic neurons. Outside the central nervous system, the 5-HT4 receptor is notably expressed in the gastrointestinal tract. The wide distribution of the 5-HT4 receptor undoubtedly contributes to its involvement in a plethora of functions. In addition, the modulation of this receptor influences the release of serotonin, but also the release of other neurotransmitters such as acetylcholine and dopamine. This is a considerable asset, as the modulation of the 5-HT4 receptor can therefore play a direct or indirect beneficial role in various disorders. One of the main advantages of this receptor is that it mediates a much faster antidepressant and anxiolytic action than classical selective serotonin reuptake inhibitors. Another major benefit of the 5-HT4 receptor is that its activation enhances cognitive performance, probably via the release of acetylcholine. The expression of the 5-HT4 receptor is also altered in various eating disorders, and its activation by the 5-HT4 agonist negatively regulates food intake. Additionally, although the cerebral expression of this receptor is modified in certain movement-related disorders, it is still yet to be determined whether this receptor plays a key role in their pathophysiology. Finally, there is no longer any need to demonstrate the value of 5-HT4 receptor agonists in the pharmacological management of gastrointestinal disorders.

Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Cholinergic Interneurons in the Accumbal Shell Region Regulate Binge Alcohol Self-Administration in Mice: An In Vivo Calcium Imaging Study.

Recently, we and others have shown that manipulating the activity of cholinergic interneurons (CIN) present in the NAc can modulate binge alcohol consumption. The present study is designed to examine the relationship between binge alcohol consumption and the activity of the CIN in real time by using an in vivo microendoscopic technique. We hypothesized that mice exposed to Drinking in the Dark (DID)-a recognized mouse model for binge drinking-would exhibit increased activity in the accumbal shell region (NAcSh). To test this hypothesis, male mice expressing Cre-recombinase in the cholinergic neurons were exposed to binge alcohol consumption (alcohol group), employing the DID method, and utilized in vivo calcium imaging to observe CIN activity in real time during alcohol consumption. The control (sucrose) group was exposed to 10% (w/v) sucrose. As compared to sucrose, mice in the alcohol group displayed a significant increase in the frequency and amplitude of discharge activity, which was measured using calcium transients in the CIN present in the NAcSh. In summary, our findings suggest that the activity of CIN in the NAcSh plays a crucial role in alcohol self-administration. These results emphasize the potential significance of targeting CIN activity as a therapeutic approach for addressing AUD.

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

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-β (Aβ) 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. To assess the effect of sildenafil (SIL) on cholinergic-like neurons (ChLNs) harboring the PSEN 1 E280A mutation. 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. We found that SIL was innocuous toward WT PSEN 1 ChLNs but reduced the accumulation of intracellular Aβ 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. 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.

De novo transcriptome analysis of Protohermes xanthodes Navás (Megaloptera: Corydalidae) reveling the effects of sublethal chlorpyrifos on the expression of cholinergic neuronal genes

The insect cholinergic neuron system is the target for various pesticides, including organophosphate, carbamate and neonicotinoid pesticides. In this study, we conducted a de novo transcriptome analysis on the aquatic insect Protohermes xanthodes and identified for the first time presenting sixteen genes encoding cholinergic neuronal proteins (PxanChaT, PxanVAChT, PxanmAChR, PxannAChRs, and PxanAChEs), which are candidates for acetylcholine synthesis, transport, reception and degradation in cholinergic synapse. FPKM analysis revealed that these genes are primarily expressed in head and nerve cord of P. xanthodes larvae, and some of them are also abundant in hindgut, malpighian tubules and tracheae. After exposed to different concentrations of sublethal chlorpyrifos (CPF), expression of these cholinergic neuronal genes are generally increased and then decreased with the increase of CPF concentration, except PxannAChRα9 which is increased in both 4.2 and 8.4 μg/L CPF groups. Short-term (96 h) CPF exposure resulted in significant up-regulation of PxanAChE1 expression in P. xanthodes larvae exposed to 0.42 and 4.2 μg/L CPF concentrations, while PxanAChE2 was up-regulated only in 0.42 μg/L CPF group. After long-term (14 d) CPF exposure, PxanAChE1 expression was down-regulated in 0.168 and 0.42 μg/L CPF groups. PxanAChE2 expression was dramatically decreased in all CPF groups. Moreover, acetylcholinesterase (AChE) activity was significantly decreased across all long-term CPF exposure groups. These results suggested that sublethal exposure to CPF can disrupt the expression of cholinergic neuronal genes in P. xanthodes larvae, and implied that long-term sublethal CPF exposure may cause toxic effects on P. xanthodes larvae by inhibiting AChE activity. Furthermore, identification of cholinergic neuronal genes in P. xanthodes provided candidate molecular markers for study the toxic effects of environmental pollutants on the neuron system of an aquatic predatory insect with ecological importance.

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 Aβ deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aβ deposits exhibit a more severe loss of afferents than the areas that are more distal to Aβ 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 Aβ deposits cause global as well as local toxicity to subcortical afferents.

Normal Skeletal Muscle Growth and Function Require Insulin Receptors Expressed by Cholinergic Motor Neurons

It is well known that insulin stimulates skeletal muscle growth. However, it is currently unknown how insulin does so since it was previously shown that insulin receptors expressed by skeletal muscles are dispensable in this regard. In this study, we focused on insulin receptors expressed by the cholinergic motor neurons and generated conditional knockout mice which lack insulin receptors specifically in the cholinergic neurons. We found that insulin activates cholinergic motor neurons and that insulin receptors expressed by the cholinergic neurons are required for normal growth of skeletal muscle. We also noted that the conditional knockout mice have underdeveloped neuromuscular junction and compromised skeletal muscle function. Together, our study suggests that insulin stimulates and activates cholinergic motor neurons to promote normal skeletal muscle growth and function. This work was supported by the National Research Foundation of Korea (NRF-2022R1A2C3005613 to Jong-Woo Sohn) funded by the Korean Ministry of Science and ICT. 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.

The laterodorsal tegmental nucleus improves breathing and sleep in a Parkinson’s disease animal model

Parkinson's disease (PD) is a neurological disorder resulting from the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). PD is characterized by classical and non-classical symptoms including respiratory instability and sleep disturbances. An important region for the control of sleep and possibly respiration is the laterodorsal tegmental nucleus (LDTg), a nucleus composed of cholinergic, glutamatergic, and GABAergic neurons. We hypothesize that stimulation of cholinergic neurons in the LDTg can lead to improvements in sleep and respiratory disturbances in a murine model of PD. Adult mice (CEUA: 6641200919 and SCRI: 18819) that express the fluorescent green protein in cholinergic cells (Chat-cre, ai6) received bilateral injections of AAV-hM3Dq-mCherry or AAV-mCherry vectors into LDTg. After ten days, the animals received nigrostriatal injections of vehicle or 6-hydroxydopamine (6-OHDA, 10 μg/μl). Eight days later, we implanted EEG and EMG electrodes. In 6-OHDA-injected mice, there was a 79% and 29% reduction in the number of dopaminergic neurons in SNpc and cholinergic neurons in LDTg, respectively. This reduction was associated with an increase in the number of apneas and a 17% and 26% reduction in respiratory frequency during non-REM (NREMs) and REM (REMs) sleep, respectively. In the same animals, there was also a decrease in wakefulness time, and an increase in total sleep time. Selective pharmacogenetic stimulation of ChAT+ neurons of the LDTg rescued the respiratory frequency during REMs and reduced the number of apneas both in NREMs and REMs in 6-OHDA mice. These results demonstrate that in this PD model the LDTg plays a significant role in respiratory changes associated with sleep regulation. FAPESP, CAPES, CNPq and NIH. 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.

Basal forebrain cholinergic neurons have specific characteristics during the perinatal period.

Cholinergic neurons of the basal forebrain (BF) represent the main source of cholinergic innervation of large parts of the neocortex and are involved in adults in the modulation of attention, memory, and arousal. During the first postnatal days, they play a crucial role in the development of cortical neurons and cortical cytoarchitecture. However, their characteristics, during this period have not been studied. To understand how they can fulfill this role, we investigated the morphological and electrophysiological maturation of cholinergic neurons of the substantia innominata-nucleus basalis of Meynert complex (SI/NBM) in the perinatal period in mice. We show that cholinergic neurons, whether or not they express γ-aminobutyric acid (GABA) as a co-transmitter, are already functional at embryonic day 18 (E18). Until the end of the first postnatal week, they constitute a single population of neurons with a well-developed dendritic tree, a spontaneous activity including bursting periods, and a short latency response to depolarizations (early-firing). They are excited by both their GABAergic and glutamatergic afferents. During the second postnatal week, a second, less excitable, neuronal population emerges, with a longer delay response to depolarizations (late-firing), together with the hyperpolarizing action of GABAAR-mediated currents. This classification into early-firing (40%) and late-firing (60%) neurons, is again independent of the co-expression of GABAergic markers. These results strongly suggest that during the first postnatal week, the specific properties of developing SI/NBM cholinergic neurons allow them to spontaneously release acetylcholine, or acetylcholine and GABA, into the developing cortex.Significance statement During early postnatal days, basal forebrain cholinergic neurons from the substantia innominata-nucleus basalis of Meynert complex (SI/NBM) play a crucial role in cortical development. However, their morphological and electrophysiological characteristics have not been studied during this period. Here we show that perinatal SI/NBM cholinergic, whether they still/have co-expressed GABAergic markers, belong to a single, homogeneous population of spontaneously active, early-firing, bursting neurons, activated by both glutamatergic and GABAergic afferents until the GABAARs-mediated current polarity shift from excitatory to inhibitory has occurred. This early high excitability suggests that they can release acetylcholine or acetylcholine and GABA into the developing cortical layers and fulfill their well-described role on the early development of cortical neurons and networks.

Conserved expression of the zebrafish syt4 gene in GABAergic neurons in the cerebellum of adult fishes revealed by mammalian SYT4 immunoreactive-like signals

Synaptotagmin 4 (syt4) belongs to the synaptotagmin protein family, which has 17 and 28 family members in human and zebrafish, respectively. In zebrafish and rodents, syt4 is known to express abundantly in the entire central nervous system in the early developmental stages. In adult rodents, the gene expression shifts to be predominant in the cerebellum, mostly in Purkinje cells, a type of GABAergic neurons. However, there is no report of the expression pattern of syt4 in the adult zebrafish brain. Therefore, we hypothesize that the expression of syt4 is conserved in adult zebrafish and is specific to the GABAergic neurons, likely Purkinje cells, in the cerebellum. To examine the hypothesis, we first show that only one copy of syt4 gene remains in the zebrafish genome, and it is orthologous to the gene in other vertebrates. We further observe mammalian SYT4 antibody immunoreactive-like (mSYT4-ir) signals in several structures in the hindbrain including the medial divisions of the valvula cerebelli and the corpus cerebelli. In addition, our observations indicate the presence of mSYT4-ir signals in GABAergic neurons, most notably in the Purkinje cell layer of the molecular layer in the aforementioned structures. Conversely, mSYT4-ir signals are not observed in glutamatergic or cholinergic neurons. Therefore, we deduce that the syt4 gene in zebrafish exhibits a homologous expression pattern to those of previously studied vertebrate species, which is revealed by the positive immunoreactive-like signals of mammalian SYT4 antibodies.

Electroacupuncture regulates Rab5a-mediating NGF transduction to improve learning and memory ability in the early stage of AD mice.

Nerve growth factor (NGF) loss is a potential factor for the degeneration of basal forebrain cholinergic neurons (BFCNs) in Alzheimer's disease (AD), and Rab5a is a key regulatory molecule of NGF signaling transduction. Here, we investigated the changes of Rab5a in 5 × FAD mice and further explored the mechanism of Electroacupuncture (EA) treatment in improving cognition in the early stage of AD. The total Rab5a and Rab5a-GTP in 5-month-old 5 × FAD mice and wild-type mice were detected using WB and IP technologies. 5 × FAD mice were treated with EA at the Bai hui (DU20) and Shen ting (DU24) acupoints for 4 weeks and CRE/LOXP technology was used to confirm the role of Rab5a in AD mediated by EA stimulation. The Novel Object Recognition and Morris water maze tests were used to evaluate the cognitive function of 5 × FAD mice. The Nissl, immunohistochemistry, and Thioflavin S staining were used to observe pathological morphological changes in the basal forebrain circuit. The Golgi staining was used to investigate the synaptic plasticity of the basal forebrain circuit and WB technology was used to detect the expression levels of cholinergic-related and NGF signal-related proteins. The total Rab5a was unaltered, but Rab5a-GTP increased and the rab5a-positive early endosomes appeared enlarged in the hippocampus of 5 × FAD mice. Notably, EA reduced Rab5a-GTP in the hippocampus in the early stage of 5 × FAD mice. EA could improve object recognition memory and spatial learning memory by reducing Rab5a activity in the early stage of 5 × FAD mice. Moreover, EA could reduce Rab5a activity to increase NGF transduction and increase the levels of phosphorylated TrkA, AKT, and ERK in the basal forebrain and hippocampus, and increase the expression of cholinergic-related proteins, such as ChAT, vAchT, ChT1, m1AchR, and m2AchR in the basal forebrain and ChAT, m1AchR, and m2AchR in the hippocampus, improving synaptic plasticity in the basal forebrain hippocampal circuit in the early stage of 5 × FAD mice. Rab5a hyperactivation is an early pathological manifestation of 5 × FAD mice. EA could suppress Rab5a-GTP to promote the transduction of NGF signaling, and enhance the synaptic plasticity of the basal forebrain hippocampal circuit improving cognitive impairment in the early stage of 5 × FAD mice.

Electrophysiology and 3D-imaging reveal properties of human intracardiac neurons and increased excitability with atrial fibrillation.

Altered autonomic input to the heart plays a major role in atrial fibrillation (AF). Autonomic neurons termed ganglionated plexi (GP) are clustered on the heart surface to provide the last point of neural control of cardiac function. To date the properties of GP neurons in humans are unknown. Here we have addressed this knowledge gap in human GP neuron structure and physiology in patients with and without AF. Human right atrial GP neurons embedded in epicardial adipose tissue were excised during open heart surgery performed on both non-AF and AF patients and then characterised physiologically by whole cell patch clamp techniques. Structural analysis was also performed after fixation at both the single cell and at the entire GP levels via three-dimensional confocal imaging. Human GP neurons were found to exhibit unique properties and structural complexity with branched neurite outgrowth. Significant differences in excitability were revealed between AF and non-AF GP neurons as measured by lower current to induce action potential firing, a reduced occurrence of low action potential firing rates, decreased accommodation and increased synaptic density. Visualisation of entire GPs showed almost all neurons are cholinergic with a small proportion of noradrenergic and dual phenotype neurons. Phenotypic distribution differences occurred with AF including decreased cholinergic and dual phenotype neurons, and increased noradrenergic neurons. These data show both functional and structural differences occur between GP neurons from patients with and without AF, highlighting that cellular plasticity occurs in neural input to the heart that could alter autonomic influence on atrial function. KEY POINTS: The autonomic nervous system plays a critical role in regulating heart rhythm and the initiation of AF; however, the structural and functional properties of human autonomic neurons in the autonomic ganglionated plexi (GP) remain unknown. Here we perform the first whole cell patch clamp electrophysiological and large tissue confocal imaging analysis of these neurons from patients with and without AF. Our data show human GP neurons are functionally and structurally complex. Measurements of action potential kinetics show higher excitability in GP neurons from AF patients as measured by lower current to induce action potential firing, reduced low firing action potential rates, and decreased action potential accommodation. Confocal imaging shows increased synaptic density and noradrenergic phenotypes in patients with AF. Both functional and structural differences occur in GP neurons from patients with AF that could alter autonomic influence on atrial rhythm.

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.

Acetylcholine Engages Distinct Amygdala Microcircuits to Gate Internal Theta Rhythm.

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOM→CCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOM→CCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.