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PubMed2001

Agonist Trapping by GABAA Receptor Channels.

GABAergic IPSCs have a relatively slow decay (deactivation) that appears to result from GABA(A) receptor channel openings that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and channel gating has not been possible. We developed a functional assay for occupancy of the GABA binding site or sites to test the GABA "trapping" hypothesis. Deactivation currents were compared in the absence and presence of bicuculline, a competitive antagonist that also allosterically inhibits GABA(A) receptors. This provided a model-independent, functional test of the hypothesis that GABA is trapped on the receptor during gating: bicuculline could only inhibit the channel if it was open but unbound by GABA. Although bicuculline inhibited spontaneous and neurosteroid-activated GABA(A) receptor currents, it failed to alter the deactivation time course of GABA-activated GABA(A) receptor currents. Protection of deactivation current from bicuculline block indicated that GABA remained bound to the receptors while the channel was open, thus suggesting that all open states, as well as all closed and desensitized states from which channel opening can occur, must be GABA liganded states. Trapping may be specific to agonists, because the positive allosteric modulator diazepam unbound from GABA(A) receptors independent of GABA binding and channel activity.

Animals↗
PubMed1997

Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Bötzinger complex: differential roles of glycinergic and GABAergic neural transmission.

A key distinction between neural pacemaker and conventional network models for the generation of breathing rhythm in mammals is whether phasic reciprocal inhibitory interactions between inspiratory and expiratory neurons are required. In medullary slices from neonatal rats generating respiratory-related rhythm, we measured the phasic inhibitory inputs to expiratory neurons with the use of whole cell patch clamp in the hypothesized rhythm generation site, the pre-Bötzinger complex (pre-BötC). Expiratory neurons, which generate tonic impulse activity during the expiratory period, exhibited inhibitory postsynaptic potentials (IPSPs) synchronized to the periodic inspiratory bursts of the hypoglossal nerve root (XIIn). Bath application of the glycine receptor antagonist strychnine (STR; 5-10 microM) reversibly blocked these inspiratory-phase IPSPs, whereas the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (BIC; 10-100 microM) had no effect on these IPSPs. Replacing the control in vitro bathing solution with a Cl(-)-free solution also abolished these IPSPs. Respiratory-related rhythmic activity was not abolished when inspiratory-phase IPSPs were blocked. The frequency and strength of XIIn rhythmic activity increased and seizurelike activity was produced when either STR, BIC, or Cl(-)-free solution was applied. Inspiratory-phase IPSPs were stable after establishment of whole cell patch conditions (patch pipettes contained 7 mM Cl-). Under voltage clamp, the reversal potential of inspiratory-phase inhibitory postsynaptic currents (IPSCs) was -75 mV. The current-voltage (I-V) curve for IPSCs shifted to the right when extracellular Cl- concentration was reduced by 50% (70 mM) and the reversal potential was reduced to -60 mV, close to the new Cl- Nernst potential. In tetrodotoxin (0.5 microM) under voltage clamp (holding potential = -45 mV), local application of glycine (1 mM) over pre-BötC induced an outward current and an increase in membrane conductance in expiratory neurons. The effect was blocked by bath application of STR (0.8-1 microM). Local application of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 mM) induced an outward current and an increase in membrane conductance that was blocked by BIC (10-100 mM). Under voltage clamp (holding potential = -45 mV), we analyzed spontaneous IPSCs during expiration in expiratory neurons. Bath application of BIC (10 microM) reduced the IPSC frequency (from 2.2 to 0.3 per s), whereas the inspiratory-phase IPSCs did not change. Bath application of STR (8-10 microM) abolished both IPSCs. These results indicate that 1) reciprocal inhibition of expiratory neurons is glycinergic and mediated by a glycine-activated Cl- channel that is not required for respiratory-related rhythm generation in neonatal rat medullary slices; 2) endogenous GABA and glycine modulate the excitability of respiratory neurons and affect respiratory pattern in the slice preparation; 3) both glycine and GABA(A) receptors are found on pre-BötC expiratory neurons, and these receptors are sensitive to STR and BIC, respectively; 4) glycine and GABA(A) inhibitory mechanisms play different functional roles in expiratory neurons: both glycine and GABA(A) receptors modulate neuronal excitability, whereas glycinergic transmission alone is responsible for reciprocal inhibition; and 5) intracellular Cl- concentration in these neonatal expiratory neurons is similar to that in adults.

Animals↗
PubMed2026

Induced pluripotent stem cell reprogramming: methodological evolution and challenges in clinical translation.

Cell reprogramming can transform somatic cells into induced pluripotent stem cells providing a platform for patient-specific disease modeling, drug screening and regenerative medicine research. Since the advent of OKSM-mediated reprogramming, the system of technical approaches has evolved continuously - from integrated viral vectors to non-integrated episomal systems and, more recently, chemical reprogramming and CRISPR approaches. The simultaneous advances in single-cell multi-omics, biomaterials engineering, and artificial intelligence have further refined the controllability and precision of the reprogramming process. Despite these innovations, problems persist that hinder clinical translation: incomplete epigenetic resetting, ongoing clonal heterogeneity, genomic instability in long-term culture, and the lack of standardized Good Manufacturing Practice protocols for large-scale manufacturing. This review summarizes the trajectory of iPSC reprogramming technologies, with special emphasis on the translational applicability of each modality. We evaluated viral and nonviral delivery systems, chemical reprogramming, strategies that aid gene editing, and emerging engineering platforms, including microfluidics, smart biomaterials, and artificial-intelligence-driven process optimization. We further identify the core "translational triltrilas", namely, the inherent tradeoffs between security, homogeneity, and scalability, and propose a comprehensive strategy to overcome these bottlenecks. By linking basic mechanistic understandings with industrial and regulatory considerations, this review aims to provide a route for transitioning iPSC technology from a laboratory tool to a clinically viable manufacturing platform.

clinical translation↗
PubMed2023

Single-swap editing for the correction of common Duchenne muscular dystrophy mutations.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease of progressive muscle weakness and wasting caused by the absence of dystrophin protein. Current gene therapy approaches using antisense oligonucleotides require lifelong dosing and have limited efficacy in restoring dystrophin production. A gene editing approach could permanently correct the genome and restore dystrophin protein expression. Here, we describe single-swap editing, in which an adenine base editor edits a single base pair at a splice donor site or splice acceptor site to enable exon skipping or reframing. In human induced pluripotent stem cell-derived cardiomyocytes, we demonstrate that single-swap editing can enable beneficial exon skipping or reframing for the three most therapeutically relevant exons-DMD exons 45, 51, and 53-which could be beneficial for 30% of all DMD patients. Furthermore, an adeno-associated virus delivery method for base editing components can efficiently restore dystrophin production locally and systemically in skeletal and cardiac muscles of a DMD mouse model containing a deletion of Dmd exon 44. Our studies demonstrate single-swap editing as a potential gene editing therapy for common DMD mutations.

AAV↗
PubMed2001

Adenosine A(2A) receptor enhances GABA(A)-mediated IPSCs in the rat globus pallidus.

1. The actions of adenosine A(2A) receptor agonists were examined on GABAergic synaptic transmission in the globus pallidus (GP) in rat brain slices using whole-cell patch-clamp recording. GP neurones were characterized into two major groups, type I and type II, according to the degree of time-dependent hyperpolarization-activated inward rectification and the size of input resistance. 2. The A(2A) receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido- adenosine (CGS21680; 0.3-3 microM) enhanced IPSCs evoked by stimulation within the GP. The actions of CGS21680 were blocked by the A(2A) antagonists (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). 3. The CGS21680-induced increase in IPSCs was associated with a reduction in paired-pulse facilitation. CGS21680 (0.3 microM) increased the frequency of miniature IPSCs (mIPSCs) without affecting mIPSC amplitude. These observations demonstrated that the enhancement of IPSCs in the GP was attributable to presynaptic, but not postsynaptic, A(2A) receptors. 4. The results suggest that A(2A) receptors in the GP serve to inhibit GP neuronal activity, thereby disinhibiting subthalamic nucleus neurone activity. Thus, the A(2A) receptor-mediated presynaptic regulation in the GP, together with the A(2A) receptor-mediated intrastriatal presynaptic control of GABAergic neurotransmission described previously, may play a crucial role in controlling the neuronal functions of basal ganglia. This A(2A) receptor-mediated presynaptic dual control in the striatopallidal pathway could also afford the mode of action of A(2A) antagonists for ameliorating the symptoms of Parkinson's disease in an animal model.

Animals↗
PubMed2006

Cell domain-dependent changes in the glutamatergic and GABAergic drives during epileptogenesis in the rat CA1 region.

An increased ratio of the glutamatergic drive to the overall glutamatergic/GABAergic drive characterizes the chronic stage of temporal lobe epilepsy (TLE), but it is unclear whether this modification is present during the latent period that often precedes the epileptic stage. Using the pilocarpine model of TLE in rats, we report that this ratio is decreased in hippocampal CA1 pyramidal cells during the early phase of the latent period (3-5 days post pilocarpine). It is, however, increased during the late phase of the latent period (7-10 days post pilocarpine), via cell domain-dependent alterations in synaptic current properties, concomitant with the occurrence of interictal-like activity in vivo. During the late latent period, the glutamatergic drive was increased in somata via an enhancement in EPSC decay time constant and in dendrites via an increase in EPSC frequency and amplitude. The GABAergic drive remained unchanged in the soma but was decreased in dendrites, since the drop off in IPSC frequency was more marked than the increase in IPSC kinetics. Theoretical considerations suggest that these modifications are sufficient to produce interictal-like activity. In epileptic animals, the ratio of the glutamatergic drive to the overall synaptic drive was not further modified, despite additional changes in synaptic current frequency and kinetics. These results show that the global changes to more glutamatergic and less GABAergic activities in the CA1 region precede the chronic stage of epilepsy, possibly facilitating the occurrence and/or the propagation of interictal activity.

Algorithms↗
PubMed1995

Endogenous opioids regulate long-term potentiation of synaptic inhibition in the dentate gyrus of rat hippocampus.

Long-term potentiation (LTP) of excitatory transmission in the hippocampus has been extensively studied as a synaptic model of learning and memory. Here we report a new form of LTP in which inhibitory synaptic signals are potentiated following tetanic stimulation of an opioid-containing excitatory pathway in the presence of opioid antagonists. The lateral perforant path (LPP) was stimulated at the dentate outer molecular layer of hippocampal slices. Evoked synaptic currents were recorded from dentate granule cells using whole-cell voltage-clamp techniques. A high-frequency stimulus train (100 Hz, 1 sec) delivered to the LPP in the presence of naloxone (1 microM) was found to induce a long-lasting potentiation (20 min to 2 hr) in the amplitude of gamma-aminobutyric acidA (GABAA) receptor-mediated inhibitory postsynaptic currents (IPSCs) of granule cells. Such a potentiation was not observed when tetanizing the LPP in control medium. Naloxone-revealed LTP of LPP-evoked IPSCs did not depend upon the presence of granule cell discharge, and was not accompanied by potentiation of mossy fiber-evoked IPSCs, indicating that feedforward, but not feedback, inhibitory circuits were involved. Induction of this LTP could be completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). However, it was not significantly affected by hyperpolarization of granule cells. These results suggest that LTP may occur at the excitatory synapses between LPP terminals and GABAergic interneurons, rather than at the inhibitory synapses between interneurons and granule cells. Further examination using selective opioid antagonists demonstrated that blocking delta, but not mu and kappa, receptors is critical for inducing LTP of IPSCs in granule cells.

2-Amino-5-phosphonovalerate↗
PubMed2006

Optimizing fluctuation analysis of GABAAergic IPSCs for accurate unitary currents.

Nonstationary fluctuation analysis of synaptic currents requires division of currents into bins of time, with little agreement on how to select an optimal bin width. We used simulated inhibitory postsynaptic currents (simIPSCs) in an empirical approach to establish the optimal bin width needed for estimation of the unitary current, ie. We found acceptable accuracy (< or = 5%) at bin widths shorter than the length of the stationary segment of simIPSCs that persisted when Gaussian noise was added to the simulated currents. We also studied evoked and spontaneous IPSCs mediated by receptors for gamma-aminobutyrate (GABA) in thalamic neurons. Similar to simIPSCs, analysis of the IPSCs yielded saturating relationships between bin width and accuracy of unitary current estimate. Whereas standard error decreased, the accuracy of ie estimates increased with decreasing bin width, forming a plateau at bins below 2-3 ms in duration. Using this approach, one can reliably determine the optimal bin width for nonstationary noise analysis.

Algorithms↗
PubMed2005

Mice lacking the major adult GABAA receptor subtype have normal number of synapses, but retain juvenile IPSC kinetics until adulthood.

There is a large variation in structurally and functionally different GABA(A) receptor subtypes. The expression pattern of GABA(A) receptor subunits is highly regulated, both temporarily and spatially. Especially during development, profound changes in subunit expression have been described. In most brain areas, the GABA(A) receptor alpha1 subunit replaces the alpha2 and/or alpha3 subunit as major alpha subunit. This is accompanied by a marked decrease in the open time of GABA(A) receptors and hence in the duration of postsynaptic responses. We describe here the development of GABAergic, synaptic transmission in mice lacking the alpha1 subunit. We show that alpha1 is to a large extent--but not entirely--responsible for the relatively short duration of postsynaptic responses in the developing and the mature brain. However, alpha1 already affects GABAergic transmission in the neonatal cerebral cortex when it is only sparsely expressed. It appears that the alpha1 -/- mice do not show a large reduction in GABAergic synapses but do retain long-lasting postsynaptic currents into adulthood. Hence, they form a good model to study the functional role of developmental GABA(A) receptor subunit switching.

Age Factors↗
PubMed2002

Short-term effects of pilocarpine on rat hippocampal neurons in culture.

PURPOSE: Status epilepticus (SE) has been considered an epileptogenic factor in humans. In the pilocarpine (PILO) model, after a brief period marked by SE, the rats exhibit recurrent spontaneous seizures, mimicking the clinical features of temporal lobe epilepsy. The aim of our study was to identify the molecular actions of PILO that could account for its ability to induce SE. METHODS: Whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons (2-3 weeks old) in the absence and in the presence of PILO (1-10 microM), to study the spontaneous activity, the evoked, and the miniature postsynaptic currents. The postsynaptic currents were isolated pharmacologically. RESULTS: PILO (1 and 10 microM) caused an initial increase followed by a decrease in the frequency of spontaneous activity. The increase in the frequency of excitatory postsynaptic currents (EPSCs) and inhibitory PSCs (IPSCs) was blocked by atropine (1 microM), indicating that this effect is mediated through muscarinic receptors. PILO also promoted a brief increase of the amplitude of IPSCs indirectly evoked by stimulation of a neuron synaptically connected to the neuron under study. Conversely, PILO promoted a sustained increase on the amplitude of electrically evoked EPSCs. In presence of tetrodotoxin (TTX; 300 nM), PILO (1 microM) increased the frequency of miniature EPSCs and IPSCs without changing their amplitude during the first 3 min of application. CONCLUSIONS: These results indicate that PILO acting through muscarinic receptor causes an imbalance between excitatory and inhibitory transmission that can result in the generation of SE observed in animals acutely treated with PILO.

Animals↗
PubMed2026

PGM1 deficiency is linked to sarcomeric and mitochondrial dysfunction in patient-derived iPSC-cardiomyocytes.

BACKGROUND: PGM1-congenital disorder of glycosylation (PGM1-CDG) is frequently associated with cardiomyopathy. Although galactose therapy corrects glycosylation defects, cardiac dysfunction typically persists, suggesting a glycosylation-independent mechanism. Recent evidence of mitochondrial abnormalities in PGM1-deficient human and murine heart, together with the association of PGM1 with the Z-disk protein LDB3 (ZASP/Cypher), suggests a critical role for PGM1 in cardiomyocyte structural and energetic homeostasis. We hypothesized that PGM1-related cardiomyopathy arises from a glycosylation-independent disruption of Z-disk-mitochondrial coupling driven by loss of PGM1-LDB3 interactions, resulting in mitochondrial energy failure and impaired contractile function. METHODS: Induced pluripotent stem cell-derived cardiomyocytes (iCMs) were generated from PGM1-deficient patient fibroblasts. Multielectrode array (MEA) recordings, untargeted (glyco)proteomics, and pathway analysis were performed to assess functional and molecular changes. Key findings were validated using tracer metabolomics and mitochondrial respiration assays. RESULTS: PGM1-deficient iCMs exhibited reduced beating frequency, impaired contractility, and prolonged contraction kinetics. Proteomic analyses revealed depletion of Z-disk components, including LDB3. AlphaFold3 structural modeling predicted a direct interaction between PGM1 and LDB3, implicating PGM1 in Z-disk integrity, which was confirmed in vitro. In addition, mitochondrial proteins were severely depleted, prompting us to investigate mitochondrial function. Functional validation confirmed extensive metabolic rewiring, energy depletion, and severely impaired mitochondrial respiration. Finally, the in silico drug repurposing identified possible therapeutic options that could target PGM1-deficient cardiomyopathy. CONCLUSION: Our data suggests PGM1 is key regulator of cardiomyocyte function, linking sarcomeric Z-disk integrity with mitochondrial metabolism. These mechanistic insights offer a foundation for developing targeted therapies for PGM1-CDG and potentially other cardiomyopathies involving Z-disk dysfunction.

Humans↗
PubMed2007

Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy.

Hyperactivity of spinal dorsal horn neurons plays an important role in the development of diabetic neuropathic pain. However, little is known as to whether synaptic input to spinal dorsal horn neurons is altered in diabetic neuropathy. Also, the function of GABAB receptors in the control of synaptic input to dorsal horn neurons in diabetes remains poorly understood. To determine the changes in synaptic input to dorsal horn neurons and the GABAB)receptor function in streptozotocin-induced diabetes, we performed whole-cell recording (GDP-beta-S included in the internal solution) on lamina II neurons in rat spinal cord slices. The frequency of glutamatergic mEPSCs and the amplitude of monosynaptic EPSCs evoked from the dorsal root were significantly higher in diabetic than in control rats. On the other hand, the basal frequency and amplitude of GABAergic spontaneous IPSCs and mIPSCs and those of glycinergic spontaneous IPSCs and mIPSCs did not differ significantly between control and diabetic rats. The GABAB agonist baclofen produced a significantly greater reduction in dorsal root-evoked EPSCs and the frequency of mEPSCs in control than in diabetic rats. However, the inhibitory effect of baclofen on GABAergic and glycinergic spontaneous IPSCs and mIPSCs was not significantly different in the two groups. These findings suggest that increased glutamatergic input from primary afferents to dorsal horn neurons may contribute to synaptic plasticity and central sensitization in diabetic neuropathic pain. Furthermore, the function of presynaptic GABAB receptors at primary afferent terminals, but not that on GABAergic and glycinergic interneurons, in the spinal cord is reduced in diabetic neuropathy.

Animals↗
PubMed2004

Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons.

The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABA(A)R subunits alpha2 and gamma2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABA(A)R alpha2 and gamma2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrin-/- mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABA(A)R alpha1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrin-/- neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABA(A)R alpha2 and gamma2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABA(A)Rs, synaptic clustering of glycine receptors was completely abolished in gephyrin-/- neurons. Finally, artificial extrasynaptic aggregation of GABA(A)R was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABA(A)Rs cluster at synapses by a gephyrin-independent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABA(A)Rs, and other signal-transducing components.

Animals↗
PubMed2001

Dopamine D2 receptor mediated presynaptic inhibition of striatopallidal GABA(A) IPSCs in vitro.

The modulation of GABA release within the globus pallidus (GP) by dopamine was studied using whole-cell patch clamp recordings from visually identified neurones. In sagittal slices, single shock electrical stimulation in the striatum evoked GABA(A) inhibitory postsynaptic currents (IPSCs), which were inhibited by dopamine in a dose-dependent manner (0.3-30 microM) with an IC(50) value of 0.7 microM. The inhibition was accompanied by an increase in paired pulse facilitation, indicative of a presynaptic effect. In coronal slices, stimulation within the GP adjacent to the recording site evoked GABA(A) IPSCs which were relatively unaffected by dopamine indicating the lack of modulation of GABA release from terminals of local GP axon collaterals. No consistent changes in holding current, membrane potential, firing rate or the frequency of spontaneous IPSCs was observed.Tetrodotoxin-resistant miniature (m)IPSCs were recorded in chloride-loaded cells. Dopamine (3-30 microM) reduced the frequency of mIPSCs, but was without effect on mIPSC amplitude, confirming a presynaptic effect. The addition of the "D2 like" agonist quinpirole (3 microM), but not the "D1 like" agonist SKF 38393 (10 microM), mimicked these effects. The "D2 like" antagonist sulpiride (10 microM), while having no effect alone, blocked the action of dopamine. In contrast the dopamine D4 selective antagonist L745, 870 (1 microM) or D1 antagonist SCH 23390 (10 microM) were without effect. These results indicate that dopamine acts on presynaptic D2 receptors on striatopallidal terminals to reduce the release of GABA in the GP. Attenuation of this mechanism following the depletion of dopamine may contribute to the changes in GP neuronal activity observed in animal models of Parkinson's disease.

Animals↗
PubMed1999

Prostaglandin E2 inhibits spontaneous inhibitory postsynaptic currents in rat supraoptic neurones via presynaptic EP receptors.

Prostaglandin E2 (PGE2) has been implicated in the excitatory regulation of magnocellular neurones in the supraoptic nucleus (SON). We have recently reported that PGE2 excited SON neurones by directly activating postsynaptic PGE2 receptors (EP receptors) of a subclass other than EP1-3, but did not affect excitatory postsynaptic currents (EPSCs). In the present study, we examined presynaptic effects of PGE2 on rat SON neurones by measuring spontaneous inhibitory postsynaptic currents (IPSCs) by a slice patch-clamp technique. PGE2 inhibited spontaneous IPSCs in a dose-dependent and reversible manner. PGE2 selectively suppressed the frequency of IPSCs without affecting the amplitude of IPSCs in the presence of tetrodotoxin, a blocker of Na+ channels, indicating that the effects were presynaptic. The inhibitory effects of PGE2 on the frequency of IPSCs were mimicked by the EP1/EP3 agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, whereas the EP2 agonist, butaprost, or the FP agonist, fluprostenol, had little effect. The effects of PGE2 on IPSCs were unaffected by the selective EP1 antagonist, SC-51322. They were unaffected also by antagonists of GABAB and alpha2 adrenergic receptors, which are present at presynaptic terminals of GABA neurones in the SON and cause suppression of spontaneous IPSCs. The inhibitor of PG synthesis, indomethacin, had little effect on spontaneous IPSCs and on the inhibitory effects of PGE2 as well as of the GABAB agonist, baclofen, and noradrenaline. These results suggest that PGE2 inhibits release of GABA from the GABAergic terminals innervating SON neurones by activating presynaptic EP receptors, presumably of the EP3 subclass, and that such a presynaptic mechanism may play a role in the excitatory regulation of SON neurones by PGE2.

Alprostadil↗
PubMed2025

Interspecies Organoids Reveal Human-Specific Molecular Features of Dopaminergic Neuron Development and Vulnerability.

The disproportionate expansion of telencephalic structures during human evolution involved tradeoffs that imposed greater connectivity and metabolic demands on midbrain dopaminergic neurons. Despite the central role of dopaminergic neurons in human-enriched disorders, molecular specializations associated with human-specific features and vulnerabilities of the dopaminergic system remain unexplored. Here, we establish a phylogeny-in-a-dish approach to examine gene regulatory evolution by differentiating pools of human, chimpanzee, orangutan, and macaque pluripotent stem cells into ventral midbrain organoids capable of forming long-range projections, spontaneous activity, and dopamine release. We identify human-specific gene expression changes related to axonal transport of mitochondria and reactive oxygen species buffering and candidate cis- and trans-regulatory mechanisms underlying gene expression divergence. Our findings are consistent with a model of evolved neuroprotection in response to tradeoffs related to brain expansion and could contribute to the discovery of therapeutic targets and strategies for treating disorders involving the dopaminergic system.

Brain evolution↗
PubMed2006

Acute injury to superficial cortex leads to a decrease in synaptic inhibition and increase in excitation in neocortical layer V pyramidal cells.

Injury to the superficial layers of cerebral cortex produces alterations in the synaptic responses of local circuits that promote the development of seizures. To further delineate the specific changes in synaptic strength that are induced by this type of cortical injury, whole cell voltage-clamp recordings were used to examine evoked and spontaneous synaptic events from layer V pyramidal cells in coronal slices prepared from surgically traumatized rat neocortices in which the superficial third of the cortex (layers I, II, and part of III) was removed. Slices from intact neocortices were used as controls. Examinations of fast inhibitory postsynaptic currents (IPSCs) indicated that traumatized slices were disinhibited, exhibiting evoked IPSCs (eIPSCs) with lower peak amplitudes. Measurements of spontaneous IPSCs (sIPSCs) revealed no difference in the mean amplitudes of sIPSCs recorded in traumatized versus control slices. However, the mean sIPSC frequency was lower in traumatized slices, indicative of a decrease in GABA release at these inhibitory synapses. Traumatized slices also displayed an increase in synaptic excitation, exhibiting spontaneous EPSCs (sESPCs) with larger peak amplitudes and higher frequencies. Peak-scaled nonstationary fluctuation analysis of sEPSCs and sIPSCs was used to obtain estimates of the unit conductance and number of functional receptor channels. EPSC and IPSC channel numbers and IPSC unit conductance did not differ between traumatized and intact slices. However, the mean unit conductance of EPSCs was higher (+25%) in traumatized slices. These findings suggest that acute injury to the superficial neocortical layers results in a disinhibition of cortical circuits that stems from a decline in GABA release likely due to the loss of superficial inhibitory interneurons and an enhancement of synaptic excitation consequent to an increase in the AMPA receptor unit conductance.

Acute Disease↗
PubMed2022

Suppression of trinucleotide repeat expansion in spermatogenic cells in Huntington's disease.

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington's disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10&#xa0;nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, only FAN1, OGG1, and PCNA showed significant suppression. When the methylation profile of HD cells was analyzed, FAN1 and OGG1 showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

Male↗