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Brain Research Apr 2024Swallowing is induced by a central pattern generator in the nucleus tractus solitarius (NTS). We aimed to create a medullary slice preparation to elucidate the neural...
Swallowing is induced by a central pattern generator in the nucleus tractus solitarius (NTS). We aimed to create a medullary slice preparation to elucidate the neural architecture of the central pattern generator of swallowing (Sw-CPG) and record its neural activities. Experiments were conducted on 2-day-old Sprague-Dawley rats (n = 46). The brainstem-spinal cord was transected at the pontomedullary and cervicothoracic junctions; the medulla was sliced transversely at thicknesses of 600, 700, or 800 μm. The rostral end of the slice was 100 μm rostral to the vagus nerve. We recorded hypoglossal nerve activity and electrically stimulated the vagus nerve or microinjected bicuculline methiodide (BIC) into the NTS. The 800-μm slices generated both rhythmic respiratory activity and electrically elicited neural activity. The 700-μm slices generated only respiratory activity, while the 600-μm slices did not generate any neural activity. BIC microinjection into the NTS in 800-μm slices resulted in the typical activity that closely resembled the swallowing activity reported in other experiments. This swallowing-like activity consistently lengthened the respiratory interval. Despite complete inhibition of respiratory activity, weak swallowing-like activity was observed under bath application of a non-NMDA receptor antagonist. Contrastingly, bath application of NMDA receptor antagonists resulted in a complete loss of swallowing-like activity and no change in respiratory activity. These results suggest that the 800-μm medullary slice preparation contains both afferent and efferent neural circuits and pattern generators of swallowing activity. Additionally, NMDA receptors may be necessary for generating swallowing activity. This medullary slice preparation can therefore elucidate Sw-CPG neural networks.
PubMed: 38679314
DOI: 10.1016/j.brainres.2024.148955 -
Journal of Neuroscience Methods Apr 2024Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in...
BACKGROUND
Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question.
NEW METHODS
We examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior.
RESULTS
We found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions.
COMPARISON WITH EXISTING METHODS
PAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures.
CONCLUSION
CFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.
PubMed: 38615721
DOI: 10.1016/j.jneumeth.2024.110127 -
Frontiers in Pharmacology 2024Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including... (Review)
Review
Neuroinflammation alters GABAergic neurotransmission in hyperammonemia and hepatic encephalopathy, leading to motor incoordination. Mechanisms and therapeutic implications.
Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including hyperammonemia and hepatic encephalopathy. Neuroinflammation is a main contributor to enhancement of GABAergic neurotransmission through increased activation of different pathways. For example, enhanced activation of the TNFα-TNFR1-NF-κB-glutaminase-GAT3 pathway and the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway in cerebellum of hyperammonemic rats enhances GABAergic neurotransmission. This is mediated by mechanisms affecting GABA synthesizing enzymes GAD67 and GAD65, total and extracellular GABA levels, membrane expression of GABA receptor subunits, of GABA transporters GAT1 and GAT three and of chloride co-transporters. Reducing neuroinflammation reverses these changes, normalizes GABAergic neurotransmission and restores motor coordination. There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and altogether modulate motor coordination and cognitive function. In this way, neuroinflammation may be also reduced by reducing GABAergic neurotransmission, which may also improve cognitive and motor function in pathologies associated to neuroinflammation and enhanced GABAergic neurotransmission such as hyperammonemia, hepatic encephalopathy or Parkinson's disease. This provides therapeutic targets that may be modulated to improve cognitive and motor function and other alterations such as fatigue in a wide range of pathologies. As a proof of concept it has been shown that antagonists of GABA receptors such as bicuculline reduces neuroinflammation and improves cognitive and motor function impairment in rat models of hyperammonemia and hepatic encephalopathy. Antagonists of GABA receptors are not ideal therapeutic tools because they can induce secondary effects. As a more effective treatment to reduce GABAergic neurotransmission new compounds modulating it by other mechanisms are being developed. Golexanolone reduces GABAergic neurotransmission by reducing the potentiation of GABA receptor activation by neurosteroids such as allopregnanolone. Golexanolone reduces neuroinflammation and GABAergic neurotransmission in animal models of hyperammonemia, hepatic encephalopathy and cholestasis and this is associated with improvement of fatigue, cognitive impairment and motor incoordination. This type of compounds may be useful therapeutic tools to improve cognitive and motor function in different pathologies associated with neuroinflammation and increased GABAergic neurotransmission.
PubMed: 38560359
DOI: 10.3389/fphar.2024.1358323 -
Frontiers in Pharmacology 2024The plant-based alkaloid muscimol is a potent agonist of inhibitory GABA-neurotransmitter receptors. GABA receptors are a heterogeneous family of pentameric complexes,...
The plant-based alkaloid muscimol is a potent agonist of inhibitory GABA-neurotransmitter receptors. GABA receptors are a heterogeneous family of pentameric complexes, with 5 out of 19 subunits assembling around the central anion pore. Muscimol is considered to bind to all receptor subtypes at the orthosteric drug binding site at the β+/α- interface. Recently, we observed that the antipsychotic drugs clozapine (CLZ), loxapine (LOX) and chlorpromazine (CPZ) although exerting functional inhibition on multiple GABA receptor subtypes showed diverging results in displacing 3H-muscimol. While a complete displacement could be observed in hippocampal membranes by bicuculline (BIC), and no displacement with CPZ, the compounds CLZ and LOX competed partially. Non-sigmoidal, complex dose response curves were indicative of multiple sites. In the current study we now aimed to investigate more extensively this heterogeneity of bicuculline sensitive muscimol sites in rat brain. We tested membranes from four different brain regions (hippocampus, cerebellum, thalamus and striatum) and selected recombinantly expressed subunit combinations with displacement assays. 3H-muscimol displacement was tested with BIC, LOX, CLZ and CPZ. ligand structural analysis and computational docking was performed. We observed a unique pharmacology of each tested compound in the studied brain regions. Combining two of the tested ligands suggests that in striatum all CLZ sites are contained in the pool of LOX sites, while the CPZ sites may in part be non-overlapping with LOX sites. Experiments on recombinantly expressed receptors indicate, that BIC can displace 3H-muscimol from all tested receptors, while LOX and CLZ display different and variable competition indicative of multiple sites. Molecular docking produced structural correlates of the observed diversity of muscimol sites on the basis of bicuculline bound experimental structures. These findings indicate that 3H-muscimol binding sites in rat brain are heterogeneous, with different populations of receptors, which are CPZ, LOX or CLZ sensitive or insensitive. These binding sites show a varying distribution in different rat brain regions. Molecular docking suggests that the so-called loop F region of α subunits drives the observed differences.
PubMed: 38549678
DOI: 10.3389/fphar.2024.1368527 -
Scientific Reports Mar 2024Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine...
Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3β-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABA receptor increased after ALLO treatment. To evaluate if the ovarian GABA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.
Topics: Pregnancy; Female; Rats; Animals; Pregnanolone; Progesterone; Proliferating Cell Nuclear Antigen; Bicuculline; Receptors, GABA-A; Corpus Luteum
PubMed: 38493224
DOI: 10.1038/s41598-024-57102-1 -
Biological Psychiatry Global Open... Mar 2024Mutations in predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical...
BACKGROUND
Mutations in predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical hyperexcitability, such as seizures. Human stem cell-derived null excitatory neurons have smaller soma size and reduced synaptic connectivity but are also hyperexcitable due to higher input resistance. Paradoxically, networks of null neurons show a decrease in the frequency of network bursts consistent with a hypoconnectivity phenotype. Here, we examine this issue.
METHODS
We reanalyzed multielectrode array data from 3 isogenic cell line pairs recorded over 6 weeks ( = 144). We used a custom burst detection algorithm to analyze network events and isolated a phenomenon that we termed reverberating super bursts (RSBs). To probe potential mechanisms of RSBs, we conducted pharmacological manipulations using bicuculline, EGTA-AM, and DMSO on 1 cell line ( = 34).
RESULTS
RSBs, often misidentified as single long-duration bursts, consisted of a large-amplitude initial burst followed by several high-frequency, low-amplitude minibursts. Our analysis revealed that null networks exhibited increased frequency of RSBs, which produced increased bursts compared with isogenic controls. Bicuculline or DMSO treatment did not affect RSBs. EGTA-AM selectively eliminated RSBs and rescued network burst dynamics.
CONCLUSIONS
During early development, null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hypersynchronic states that potentially translate into seizures. Network hyperexcitability depends on asynchronous neurotransmitter release that is likely driven by presynaptic Ca and can be rescued by EGTA-AM to restore typical network dynamics.
PubMed: 38420187
DOI: 10.1016/j.bpsgos.2024.100290 -
IBRO Neuroscience Reports Jun 2024There is evidence that both the GABAergic system and serotonin reuptake inhibitor (SSRI) such as citalopram are involved in the modulation of anxiety and depression...
BACKGROUND
There is evidence that both the GABAergic system and serotonin reuptake inhibitor (SSRI) such as citalopram are involved in the modulation of anxiety and depression processes. In this research, we examined the effects of GABA receptor agents and citalopram on anxiety- and depression-related behaviors and their interaction in male mice.
METHODS
For intracerebroventricular (i.c.v.) infusion, a guide cannula was implanted in the left lateral ventricle. Anxiety and depression behaviors were evaluated using the elevated plus-maze (EPM) and forced swimming test (FST).
RESULTS
The results revealed that i.c.v. microinjection of muscimol (1 µg/mouse) enhanced % OAT (open arm time) and % OAE (open arm entries) in the EPM test and decreased immobility time in the FST without affecting locomotor activity, presenting anxiolytic- and antidepressant-like behaviors in the EPM and FST, respectively. On the other hand, i.c.v. microinjection of bicuculline (1 µg/mouse) reduced % OAT and % OAE without affecting locomotor activity and immobility time, presenting an anxiogenic-like effect. Moreover, i.p. administration of citalopram (8 mg/kg) increased %OAT and %OAE and reduced immobility time with no effect on locomotor activity, showing anxiolytic- and antidepressant-like responses in male mice. Furthermore, i.c.v. infusion of an ineffective dosage of muscimol potentiated the anxiolytic- and antidepressant-like responses induced by i.p. injection of citalopram in male mice. When citalopram and bicuculline were co-injected, a non-significant dose of bicuculline reversed the anxiolytic-like effect of citalopram in male mice. Also, the data revealed synergistic anxiolytic- and antidepressant-like behaviors between citalopram and muscimol in male mice.
CONCLUSIONS
The results suggested an interaction between citalopram and GABAergic agents on the modulation of anxiety and depression behaviors in male mice.
PubMed: 38415182
DOI: 10.1016/j.ibneur.2024.02.003 -
Nutrients Dec 2023exhibits various biological activities; however, their sleep-promoting effects have not been previously reported. In this study, we evaluated the hypnotic effects and...
exhibits various biological activities; however, their sleep-promoting effects have not been previously reported. In this study, we evaluated the hypnotic effects and sleep-wake profiles of root (KS-126) using a pentobarbital-induced sleep-acceleration test and polysomnographic recordings. Additionally, we investigated the molecular mechanism of KS-126 through patch-clamp electrophysiology. Our polysomnographic recordings revealed that KS-126 not only accelerated the onset of non-rapid eye movement sleep (NREMS) but also extends its duration. Considering the temporal dynamics of the sleep-wake stages, during the initial and subsequent periods KS-126 extended NREMS duration and decreased wakefulness, thereby enhancing sleep-promoting effects. Furthermore, the assessment of sleep quality via analysis of electroencephalogram power density indicated that KS-126 did not significantly alter sleep intensity. Finally, we found that KS-126 enhanced GABA receptor-mediated synaptic responses in primary hippocampal neurons, leading to an increase in the percentage of the GABA current. This effect was not affected by the selective benzodiazepine receptor antagonist flumazenil, but was entirely inhibited by the GABA receptor antagonist bicuculline. In conclusion, KS-126 extends the duration of NREMS without altering its intensity by prolonging GABAergic synaptic transmission, which modulates GABA receptor function.
Topics: Receptors, GABA-A; Aralia; Eye Movements; Sleep; gamma-Aminobutyric Acid
PubMed: 38140279
DOI: 10.3390/nu15245020 -
Frontiers in Cellular Neuroscience 2023While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and...
While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity , functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.
PubMed: 38026689
DOI: 10.3389/fncel.2023.1287089 -
Pharmaceuticals (Basel, Switzerland) Nov 2023Cleomin, a 1,3-oxazolidine-2-thione, was recently isolated from , a species traditionally used for treating painful conditions. Reports about the pharmacological...
Cleomin, a 1,3-oxazolidine-2-thione, was recently isolated from , a species traditionally used for treating painful conditions. Reports about the pharmacological activities of cleomin are lacking. Here, the antinociceptive effects of cleomin were investigated using mice models of pain, namely the formalin, the cold plate, and the tail flick tests. Motor integrity was assessed in the rota-rod test. Antagonism assays and in silico docking analyses were performed to investigate the putative mechanisms of action. Cleomin (12.5-25 mg/kg), at doses that did not induce motor impairment, induced dose-dependent antinociception in both early and late phases of the formalin test and reduced nociceptive behaviors in both the cold plate and tail flick tests. Pretreatments with phaclofen and atropine attenuated the antinociceptive effects of cleomin, implicating the involvement of GABA and muscarinic receptors. In silico docking studies suggested satisfactory coupling between cleomin and GABA and M receptors, hence corroborating their role in cleomin's activity. Pretreatments with naloxone, yohimbine, bicuculline, and methysergide did not affect the antinociception of cleomin. In silico pharmacokinetics prediction showed a good drug ability profile of cleomin. In conclusion, cleomin promoted antinociception mediated by GABA and muscarinic receptors. These findings support further investigation of the analgesic potential of cleomin.
PubMed: 38004413
DOI: 10.3390/ph16111547