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The Journal of Neuroscience : the... Mar 2024Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to...
Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.
Topics: Mice; Animals; Male; Acetylcholine; Gonadotropin-Releasing Hormone; Carbachol; Neurons; Cholinergic Agents; Nicotine; Luteinizing Hormone; gamma-Aminobutyric Acid
PubMed: 38320853
DOI: 10.1523/JNEUROSCI.1780-23.2024 -
Journal of Comparative Physiology. B,... Feb 2024In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca] to activate the apical exit of Cl through TMEM16A Cl channels, which acts as a...
In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca] to activate the apical exit of Cl through TMEM16A Cl channels, which acts as a driving force for fluid secretion. To sustain the Cl secretion, [Cl] must be maintained to levels that are greater than the electrochemical equilibrium mainly by Na-K-2Cl cotransporter-mediated Cl entry in basolateral membrane. Glucose transporters carry glucose into the cytoplasm, enabling the cells to produce ATP to maintain Cl and fluid secretion. Sodium-glucose cotransporter-1 is a glucose transporter highly expressed in acinar cells. The salivary flow is suppressed by the sodium-glucose cotransporter-1 inhibitor phlorizin. However, it remains elusive how sodium-glucose cotransporter-1 contributes to maintaining salivary fluid secretion. To examine if sodium-glucose cotransporter-1 activity is required for sustaining Cl secretion to drive fluid secretion, we analyzed the Cl currents activated by the cholinergic agonist, carbachol, in submandibular acinar cells while comparing the effect of phlorizin on the currents between the whole-cell patch and the gramicidin-perforated patch configurations. Phlorizin suppressed carbachol-induced oscillatory Cl currents by reducing the Cl efflux dependent on the Na-K-2Cl cotransporter-mediated Cl entry in addition to affecting TMEM16A activity. Our results suggest that the sodium-glucose cotransporter-1 activity is necessary for maintaining the oscillatory Cl secretion supported by the Na-K-2Cl cotransporter activity in real time to drive fluid secretion. The concerted effort of sodium-glucose cotransporter-1, Na-K-2Cl cotransporter, and apically located Cl channels might underlie the efficient driving of Cl secretion in different secretory epithelia from a variety of animal species.
Topics: Animals; Mice; Acinar Cells; Carbachol; Chlorides; Glucose; Phlorhizin; Sodium; Sodium-Potassium-Chloride Symporters
PubMed: 38308715
DOI: 10.1007/s00360-024-01532-w -
Neurobiology of Disease Mar 2024Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing...
BACKGROUND
Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing refractory epilepsy. However, the optimal stimulation targets and effective parameters of DBS to reduce seizures remian unidentified.
OBJECTIVES
This study endeavors to scrutinize the therapeutic potential of DBS within the zona incerta (ZI) across diverse seizure models and elucidate the associated underlying mechanisms.
METHODS
We evaluated the therapeutic potential of DBS with different frequencies in the ZI on kainic acid (KA)-induced TLE model or M1-cortical seizures model, pilocarpine-induced M1-cortical seizure models, and KA-induced epilepsy model. Further, employing calcium fiber photometry combined with cell-specific ablation, we sought to clarified the causal role of ZI GABAergic neurons in mediating the therapeutic effects of DBS.
RESULTS
Our findings reveal that DBS in the ZI alleviated the severity of seizure activities in the KA-induced TLE model. Meanwhile, DBS attenuated seizure activities in KA- or pilocarpine-induced M1-cortical seizure model. In addition, DBS exerts a mitigating influence on KA induced epilepsy model. DBS in the ZI showed anti-seizure effects at low frequency spectrum, with 5 Hz exhibiting optimal efficacy. The low-frequency DBS significantly increased the calcium activities of ZI GABAergic neurons. Furthermore, selective ablation of ZI GABAergic neurons with taCasp3 blocked the anti-seizure effect of low-frequency DBS, indicating the anti-seizure effect of DBS is mediated by the activation of ZI GABAergic neurons.
CONCLUSION
Our results demonstrate that low-frequency DBS in the ZI attenuates seizure via driving GABAergic neuronal activity. This suggests that the ZI represents a potential DBS target for treating both hippocampal and cortical seizure through the activation of GABAergic neurons, thereby holding therapeutic significance for seizure treatment.
Topics: Humans; Drug Resistant Epilepsy; Zona Incerta; Pilocarpine; Calcium; Deep Brain Stimulation; GABAergic Neurons; Epilepsy; Kainic Acid; Seizures
PubMed: 38290566
DOI: 10.1016/j.nbd.2024.106424 -
BMC Complementary Medicine and Therapies Jan 2024The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza...
Anti-rheumatic colchicine phytochemical exhibits potent antiviral activities against avian and seasonal Influenza A viruses (IAVs) via targeting different stages of IAV replication cycle.
BACKGROUND
The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications.
METHODS
In this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC > 200 µg/ml), permitting the screening for their anti-influenza potential.
RESULTS
Herein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC values was further depicted by testing the three highly effective alkaloids, based on their potent IC values against seasonal influenza A/H1N1 virus, showing comparable IC values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle.
CONCLUSION
This study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents.
Topics: Humans; Colchicine; Influenza A virus; Influenza A Virus, H5N1 Subtype; Pilocarpine; Influenza A Virus, H1N1 Subtype; Influenza, Human; Molecular Docking Simulation; Seasons; Phytochemicals; Atropine; Antiviral Agents
PubMed: 38254071
DOI: 10.1186/s12906-023-04303-2 -
International Journal of Medical... 2024Our aim was to evaluate the effect of prophylactic pilocarpine on acute salivary symptoms after radioactive iodine (RAI) therapy in patients with differentiated thyroid...
Our aim was to evaluate the effect of prophylactic pilocarpine on acute salivary symptoms after radioactive iodine (RAI) therapy in patients with differentiated thyroid cancer. We enrolled 88 patients (76 women and 12 men; mean age: 47 years; range: 20-74 years) with differentiated thyroid cancer who received RAI. Patients were divided into pilocarpine (51 patients) and control (37 patients) groups. Pilocarpine was given orally, at a dose of 5 mg three times a day, from 2 days before and 12 days after RAI therapy. Symptoms and signs of acute sialadenitis within 3 months of RAI therapy were recorded. During the 3 months after RAI therapy, 13 of the 88 patients (14.7%) developed acute symptomatic sialadenitis (swelling or pain of salivary glands). Acute salivary symptoms were reported by 4 (7.8%) and 9 (24.3%) patients in the pilocarpine and control groups, respectively. Acute salivary symptoms were less frequent in the pilocarpine than control group ( = 0.04), but did not differ by age, sex, or RAI dose ( = 0.3357, = 0.428, and = 0.2792). Pilocarpine reduced the likelihood of acute sialadenitis after RAI therapy in patients with differentiated thyroid cancer.
Topics: Male; Humans; Female; Middle Aged; Thyroid Neoplasms; Iodine Radioisotopes; Pilocarpine; Sialadenitis; Acute Disease; Adenocarcinoma
PubMed: 38250604
DOI: 10.7150/ijms.84590 -
Clinical Therapeutics Feb 2024This study was undertaken to evaluate the safety and efficacy of CSF-1 (0.4% pilocarpine hydrochloride ophthalmic solution) for use in individuals with presbyopia. (Randomized Controlled Trial)
Randomized Controlled Trial
PURPOSE
This study was undertaken to evaluate the safety and efficacy of CSF-1 (0.4% pilocarpine hydrochloride ophthalmic solution) for use in individuals with presbyopia.
METHODS
Two Phase 3 multicenter, randomized, double-masked, vehicle-controlled, parallel-group clinical trials were conducted in 35 private ophthalmology clinics in the United States from October 2020 to February 2022. Key inclusion criteria were the following: (1) age 45-64 years, (2) distance-corrected near visual acuity (DCNVA) at 40 cm ≥0.40 and ≤0.90 logarithm of the minimum angle of resolution (logMAR, approximately 20/50-20/160 Snellen) in at least 1 eye, (3) manifest refraction (MR) between -4.50 and +2.00 diopter (D) sphere in each eye with ≤2.00D difference between eyes, (4) <2.00D of cylinder MR in each eye, (5) ≤0.04 logMAR (20/20-2 or better) corrected distance visual acuity (CDVA) at 4 m in each eye. Key exclusion criteria were the following: (1) >0.14 logMAR (7 letters) improvement in post-vehicle treatment in monocular DCNVA in either eye at visit 1, (2) introcular pressure (IOP) <9 or >22 mm Hg, (3) average dark-adapted pupillometry <3.5 mm in either eye, (4) prior refractive surgery or intraocular lens (IOL) implantation. Participants applied CSF-1 or vehicle twice per day for 2 weeks. Efficacy and safety assessments were performed at several times on days 1, 8, and 15. Response was defined as ≥3-line gain in DCNVA without loss of ≥1-line in CDVA in the study eye under mesopic room lighting conditions. The primary efficacy endpoint was measured 1 hour post-dose 1 on day 8. Key secondary endpoints were 2 hours post-dose 1, and 1 and 2 hours post-dose 2, also on day 8. Safety endpoints were ocular and non-ocular treatment-related adverse events (TRAE), conjunctival redness, drop comfort, slit-lamp biomicroscopy, intraocular pressure, indirect fundoscopy, and CDVA at 4 m.
FINDINGS
Six hundred thirteen participants were randomized to CSF-1 (n = 309) or vehicle (n = 304). Participants were predominantly White (80.8%) and female (62.0%), with mean age (standard deviation) of 54.7 (4.8). CSF-1 met the primary and key secondary endpoints. At the primary endpoint, 40.1% of the CSF-1 group achieved response versus 19.1% of the vehicle group (P < 0.0001). The percentage of responders was significantly greater in CSF-1 compared with vehicle at all tested times. Changes from baseline in all safety endpoints were comparable between groups. Most adverse events (AEs) were mild and transient. Neither serious nor severe AEs were reported with CSF-1.
IMPLICATIONS
CSF-1, a low-dose pilocarpine ophthalmic solution, demonstrated superiority to vehicle in improving near vision in individuals with presbyopia without compromising distance vision. CSF-1 demonstrated a favorable safety profile.
CLINICALTRIALS
gov identifier: NCT04599933 (NEAR-1), NCT04599972 (NEAR-2).
Topics: Female; Humans; Middle Aged; Lens Implantation, Intraocular; Lenses, Intraocular; Macrophage Colony-Stimulating Factor; Ophthalmic Solutions; Pilocarpine; Presbyopia
PubMed: 38216351
DOI: 10.1016/j.clinthera.2023.12.005 -
JCI Insight Jan 2024Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of...
Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.
Topics: Animals; Nestin; Pilocarpine; Seizures; Status Epilepticus; Hippocampus; Neurogenesis; Epilepsy
PubMed: 38193536
DOI: 10.1172/jci.insight.175627 -
Brain Research Bulletin Feb 2024In temporal lobe epilepsy (TLE), the epileptogenic zones, such as the temporal lobe structure, could generate pathological high-frequency oscillations (pHFOs,...
In temporal lobe epilepsy (TLE), the epileptogenic zones, such as the temporal lobe structure, could generate pathological high-frequency oscillations (pHFOs, 250-500 Hz) before the ictal period. These pHFOs have also been observed during the process of seizures in both TLE patients and animals, exhibiting a critical role as promising biomarkers for TLE seizures. TLE seizures could be modulated via regulating the neural excitability in epileptogenic zones, for that TLE is primarily associated with the excitation-inhibition imbalance. However, whether these kinds of modulations could also impact the pHFOs characteristics during TLE seizures is still unclear. For this purpose, we pharmaco-genetically inhibited the principal cells (PCs) in the mouse CA3 region and tracked the difference in the behavioral and electrophysiological features during LiCl-pilocarpine-induced TLE seizure between the hM4Di+CNO (experimental) mice and mCherry+CNO (control) mice. Delayed latency, decreased averaged duration, and reduced counts of the generalized seizure were observed in the experimental mice. Besides, the electrophysiological characteristics, such as the firing rate of PCs and the count of pHFO, exhibited significant decline in the CA3 and CA1 regions. During TLE seizure, there existed strong phase-coupling between pHFO and PCs spike timing in the control mice, while it was abolished in the experimental mice. In addition, we also found that the counts of pHFO were significantly associated with the behavioral features, indicating the close relationships within them. Collectively, our findings suggested that alterations in pHFO and the retardation of seizures may be attributed to disruptions in neuronal excitability, and the variations of electrophysiological features were related to seizure severity during TLE seizures. These results provide valuable insights into the role of pHFOs in TLE and shed light on the underlying mechanisms involved.
Topics: Humans; Mice; Animals; Epilepsy, Temporal Lobe; Seizures; Temporal Lobe; Pilocarpine; Electroencephalography
PubMed: 38184151
DOI: 10.1016/j.brainresbull.2024.110869 -
BMC Research Notes Jan 2024Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption...
OBJECTIVES
Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption increases susceptibility to silica-induced pulmonary inflammation and fibrosis. This study investigated the potential of HFWD to alter silica-induced effects on airway epithelial ion transport and smooth muscle reactivity.
METHODS
Six-week-old male F344 rats were fed a HFWD or standard rat chow (STD) and exposed to silica (Min-U-Sil 5, 15 mg/m, 6 h/day, 5 days/week, for 39 d) or filtered air. Experimental endpoints were measured at 0, 4, and 8 weeks post-exposure. Transepithelial potential difference (V), short-circuit current (I) and transepithelial resistance (R) were measured in tracheal segments and ion transport inhibitors [amiloride, Na channel blocker; NPPB; Cl- channel blocker; ouabain, Na, K-pump blocker] identified changes in ion transport pathways. Changes in airway smooth muscle reactivity to methacholine (MCh) were investigated in the isolated perfused trachea preparation.
RESULTS
Silica reduced basal I at 4 weeks and HFWD reduced the I response to amiloride at 0 week compared to air control. HFWD + silica exposure induced changes in ion transport 0 and 4 weeks after treatment compared to silica or HFWD treatments alone. No effects on airway smooth muscle reactivity to MCh were observed.
Topics: Male; Rats; Animals; Amiloride; Silicon Dioxide; Diet, Western; Rats, Inbred F344; Epithelium; Ion Transport; Methacholine Chloride; Muscle, Smooth
PubMed: 38172968
DOI: 10.1186/s13104-023-06672-w -
Neurologia 2024The growth hormone (GH) has been reported as a crucial neuronal survival factor in the hippocampus against insults of diverse nature. Status epilepticus (SE) is a...
INTRODUCTION
The growth hormone (GH) has been reported as a crucial neuronal survival factor in the hippocampus against insults of diverse nature. Status epilepticus (SE) is a prolonged seizure that produces extensive neuronal cell death. The goal of this study was to evaluate the effect of intracerebroventricular administration of GH on seizure severity and SE-induced hippocampal neurodegeneration.
METHODOLOGY
Adult male rats were implanted with a guide cannula in the left ventricle and different amounts of GH (70, 120 or 220ng/3μl) were microinjected for 5 days; artificial cerebrospinal fluid was used as the vehicle. Seizures were induced by the lithium-pilocarpine model (3mEq/kg LiCl and 30mg/kg pilocarpine hydrochloride) one day after the last GH administration. Neuronal injury was assessed by Fluoro-Jade B (F-JB) staining.
RESULTS
Rats injected with 120ng of GH did not had SE after 30mg/kg pilocarpine, they required a higher number of pilocarpine injections to develop SE than the rats pretreated with the vehicle, 70ng or 220ng GH. Prefrontal and parietal cortex EEG recordings confirmed that latency to generalized seizures and SE was also significantly higher in the 120ng group when compared with all the experimental groups. FJ-B positive cells were detected in the hippocampus after SE in all rats, and no significant differences in the number of F-JB cells in the CA1 area and the hilus was observed between experimental groups.
CONCLUSION
Our results indicate that, although GH has an anticonvulsive effect in the lithium-pilocarpine model of SE, it does not exert hippocampal neuroprotection after SE.
Topics: Animals; Male; Rats; Anticonvulsants; Growth Hormone; Lithium; Neuroprotective Agents; Pilocarpine; Seizures; Status Epilepticus
PubMed: 38161069
DOI: 10.1016/j.nrleng.2023.12.003