Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Modeling the selective growth advantage of genetically variant human pluripotent stem cells to identify opportunities for manufacturing process control.

BACKGROUND AIMS: The appearance of genetically variant populations in human pluripotent stem cell (hPSC) cultures represents a concern for research and clinical applications. Genetic variations may alter hPSC differentiation potential or cause phenotype variation in differentiated cells. Further, variants may have properties such as proliferative rate, or response to the culture environment, that differ from wild-type cells. As such, understanding the behavior of these variants in culture, and any potential operational impact on manufacturing processes, will be necessary to control quality of putative hPSC-based products that include a proportion of variant threshold in their quality specification. METHODS: Here we show a computational model that mathematically describes the growth dynamics between commonly occurring genetically variant hPSCs and their counterpart wild-type cells in culture. RESULTS: We show that our model is capable of representing the growth behaviors of both wild-type and variant hPSCs in individual and co-culture systems. CONCLUSIONS: This representation allows us to identify three critical process parameters that drive critical quality attributes when genetically variant cells are present within the system: total culture density, proportion of variant cells within the culture system and variant cell overgrowth. Lastly, we used our model to predict how the variability of these parameters affects the prevalence of both populations in culture.

Hyperspectral Mapping of Human Primary and Stem Cells at Cell-Matrix Interfaces.

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell-matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell-matrix interfaces.

Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) Cultures.

Cell-cell interactions are required for development and homeostasis in multicellular organisms from insects to mammals. A critical process governed by these interactions is cell competition, which functions throughout development to control tissue composition by eliminating cells that possess a lower fitness status than their neighbors. Human pluripotent stem cells (hPSCs) are a key biological tool in modeling human development and offer further potential as a source of clinically relevant cell populations for regenerative medicine applications. Recently, cell competition has been demonstrated in hPSC cultures and during induced pluripotent stem cell reprogramming. In turn, these findings suggest that hPSCs can be used as a tool to study and model cell-cell interactions during different stages of development and disease. Here, we provide a panel of protocols optimized for hPSCs to investigate the potential role that cell competition may have in determining the fate and composition of cell populations during culture. The protocols entail assessment of the competitive phenotype and the mode through which cell competition may lead to elimination of less-fit cells from mosaic cultures with fitter counterparts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Electroporation of hPSCs to establish a fluorescent reference cell line Support Protocol 1: Single-cell dissociation of hPSCs Support Protocol 2: Single-cell cloning of fluorescently labeled hPSCs Basic Protocol 2: Separate culture and co-culture proliferation assays Basic Protocol 3: Assessing levels of apoptosis in hPSC cultures using flow cytometry Basic Protocol 4: Transwell assay Support Protocol 3: Immunohistochemistry and image quantification of cleaved caspase-3 Basic Protocol 5: Cell confrontation assay Basic Protocol 6: Cell compression assay Basic Protocol 7: Time-lapse imaging to assess mechanical extrusion.

Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition.

The appearance of genetic changes in human pluripotent stem cells (hPSCs) presents a concern for their use in research and regenerative medicine. Variant hPSCs that harbor recurrent culture-acquired aneuploidies display growth advantages over wild-type diploid cells, but the mechanisms that yield a drift from predominantly wild-type to variant cell populations remain poorly understood. Here, we show that the dominance of variant clones in mosaic cultures is enhanced through competitive interactions that result in the elimination of wild-type cells. This elimination occurs through corralling and mechanical compression by faster-growing variants, causing a redistribution of F-actin and sequestration of yes-associated protein (YAP) in the cytoplasm that induces apoptosis in wild-type cells. YAP overexpression or promotion of YAP nuclear localization in wild-type cells alleviates their "loser" phenotype. Our results demonstrate that hPSC fate is coupled to mechanical cues imposed by neighboring cells and reveal that hijacking this mechanism allows variants to achieve clonal dominance in cultures.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus.

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.

Nociceptin/orphanin FQ suppresses the excitability of neurons in the ventromedial nucleus of the hypothalamus.

Nociceptin or orphanin FQ (N/OFQ) stimulates food intake when injected into the ventromedial nucleus of the hypothalamus (VMN). The VMN negatively regulates energy balance in part by tonically activating proopiomelanocortin arcuate neurons, thereby suppressing food intake. However, it is not clear how orexigenic neurotransmission within the VMN can stimulate food intake. We tested the hypothesis that the orexigenic action of N/OFQ results from its inhibition of anorexigenic VMN neurons. We studied the effects of N/OFQ on the electrical properties of anorexigenic VMN neurons in acute brain slices. Ionic mechanisms underlying the actions of N/OFQ were studied using whole cell patch-clamp recordings from VMN neurons expressing the anorexigenic leptin receptor (LepRb). Bath application of N/OFQ to LepRb-expressing VMN neurons elicited a robust, reversible membrane hyperpolarization that suppressed neuronal excitability by raising the action potential firing threshold and cell rheobase. N/OFQ activated a postsynaptic, G-protein coupled, inwardly rectifying potassium (GIRK) current that was sensitive to G-protein inactivation, blocked by the GIRK blocker SCH23390, and occluded by the GABAB agonist and potent GIRK activator, baclofen. Application of the selective N/OFQ receptor antagonist SB-612111 blocked the inhibitory effects of N/OFQ. We concluded that N/OFQ directly inhibited VMN neurons by activating a GIRK. These results implicate the site-specific contributions of orexigenic neuropeptides at VMN neurons to suppress anorexigenic output. This study thus advances our understanding regarding the contributions of the VMN to hypothalamic regulation of energy balance.

Neuropeptide Y suppresses anorexigenic output from the ventromedial nucleus of the hypothalamus.

Output from the hypothalamic ventromedial nucleus (VMN) is anorexigenic and is supported by the excitatory actions of leptin. The VMN is also highly sensitive to the orexigenic actions of Neuropeptide Y (NPY). We report that NPY robustly inhibits VMN neurons by hyperpolarizing them and decreasing their ability to fire action potentials. This action was mediated by Y(1) receptors coupled to the activation of GIRKs (G-protein-coupled inwardly rectifying potassium channels). Approximately 80% of VMN neurons expressing leptin receptors were sensitive to the actions of NPY, whereas 75% of NPY-sensitive neurons in VMN also responded to glucose by being uniformly inhibited by elevations in glucose. Interestingly, only approximately 36% of NPY-sensitive, leptin receptor b-expressing neurons were also glucosensitive. We suggest that NPY inhibits VMN neurons that are excited by leptin, thereby arresting the anorexigenic tone exerted by VMN neurons. The results further suggest a dynamic interplay between anorexigenic and orexigenic neuromodulators within the VMN to directly affect energy balance.

National survey of management of transient ischaemic attack in Australia: take immediate action.

OBJECTIVE: To understand the current organisation of services for people with transient ischaemic attack (TIA) and the processes of assessment and management across Australian hospitals. DESIGN AND SETTING: Cross-sectional survey in 2008 of 134 Australian hospitals, mostly urban centres that treat large numbers of stroke patients. MAIN OUTCOME MEASURES: Survey questions covered assessment, early management and follow-up practices, as well as organisation of services for TIA. RESULTS: Seventy-four hospitals (55%) responded: 47 (64%) reported access to a stroke unit, and 19 (26%) to a specialist clinic for TIA. Initial assessment included blood tests, electrocardiogram and brain computed tomography at most sites (92%-94%), and carotid imaging at more than half (65%), but magnetic resonance imaging at only 3% of sites. A tool to stratify the risk of subsequent stroke was used at 38 sites (51%), more commonly in hospitals with a stroke unit than in those without such a unit (64% v 30%; P = 0.005). Treatment was initiated at the initial assessment at 42 sites (58%), more commonly at stroke unit than non-stroke unit sites (68% v 37%; P = 0.007). Formalised policies for management of TIA patients were used at 38 sites (54%), with clear differences between sites with a stroke unit and those without (70% v 25%; P < 0.001). CONCLUSION: Access to rapid assessment and management services for TIA varies considerably between Australian hospitals. The presence of organised stroke care at a hospital leads to improved processes of care for patients presenting with TIA.

Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions.

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.

Nesfatin-1 inhibits NPY neurons in the arcuate nucleus.

Although the novel satiety factor nesfatin-1 has been shown to influence feeding behavior through effects on melanocortin signaling, the specific hypothalamic neuronal substrates through which such effects are mediated have yet to be elucidated. To identify neuronal cell types potentially important in mediating nesfatin-1's effects, whole cell current clamp recordings were made from hypothalamic arcuate nucleus neurons and the effects of bath applied nesfatin-1 on membrane excitability determined. Neurons then underwent phenotypic identification post hoc using single cell RT-PCR. Nesfatin-1 (10 nM) had effects on the majority of arcuate nucleus neurons tested, with most responsive cells hyperpolarizing following exposure. Furthermore, 9 of 11 identified NPY neurons hyperpolarized in response to nesfatin-1 exposure. Pharmacological experiments revealed that glibenclamide (500 nM), an ATP-sensitive potassium conductance antagonist, prevented nesfatin-1-induced hyperpolarization. Therefore, nesfatin-1-induced inhibition of feeding may be mediated through the inhibition of orexigenic NPY neurons.

GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

Neuropeptide W influences the excitability of neurons in the rat hypothalamic arcuate nucleus.

Neuropeptide W (NPW) is a ligand of the recently deorphaned receptor GPR7. Intracerebroventricular injection of this peptide results in reduced serum growth hormone concentration. Using whole-cell patch clamp recordings from somatostatin (SS) neurons in the hypothalamic arcuate nucleus, identified post-hoc using single-cell RT-PCR, we investigated the effects of NPW on membrane excitability. NPW application in acute slices of the arcuate nucleus resulted in the depolarization of the majority (62.5%) of the SS neurons tested, while smaller proportions of cells showed hyperpolarization or no response. Both the depolarization and hyperpolarization of arcuate SS neurons were preserved during recordings where voltage-gated sodium channels were blocked with tetrodotoxin, suggesting direct effects of NPW on the excitability of SS neurons. The observed depolarization of the majority of the SS neurons tested suggests that the central effects of NPW to inhibit growth hormone release results from activation of arcuate SS neurons, which could result in an inhibition of GHRH-releasing neurons.

The area postrema: a brain monitor and integrator of systemic autonomic state.

The area postrema is a medullary structure lying at the base of the fourth ventricle. The area postrema's privileged location outside of the blood-brain barrier make this sensory circumventricular organ a vital player in the control of autonomic functions by the central nervous system. By virtue of its lack of tight junctions between endothelial cells in this densely vascularized structure and the presence of fenestrated capillaries, peptide and other physiological signals borne in the blood have direct access to neurons that project to brain areas with important roles in the autonomic control of many physiological systems, including the cardiovascular system and systems controlling feeding and metabolism. However, the area postrema is not simply a conduit through which signals flow into the brain, but it is now being recognized as the initial site of integration for these signals as they enter the circuitry of the central nervous system.

Local relationships between restricted water diffusion and oxygen consumption in the ischemic human brain.

BACKGROUND AND PURPOSE: MR is widely used to depict still ischemic but viable tissue in acute stroke. However, the relationship between the apparent diffusion coefficient (ADC) and energy failure from reduced oxygen supply are unknown in man. METHODS: Acute carotid-territory stroke patients were studied prospectively with both diffusion tensor-imaging and back-to-back steady-state 15O-PET. Substantial numbers of voxels with oxygen extraction fraction >0.70 (ie, significant ongoing hypoxia) were identified in 3 patients (imaged at 7, 16 and 21 hours after stroke onset). In this voxel population, the quantitative relationships between the ADC and cerebral metabolic rate of oxygen (CMRO2), and ADC and cerebral blood flow (CBF), were assessed. RESULTS: The ADC remained essentially unchanged until CBF reached values approximately 20 mls/100g per min, beyond which it declined linearly. In contrast, except when severely reduced, the ADC was a poorer predictor of CMRO2. For both CBF and CMRO2, however, the relationship with ADC became steeper with longer times since onset, ie, the same ADC reflected lower perfusion and CMRO2 with elapsed time. CONCLUSIONS: Despite the small sample and late times from stroke onset, the findings indicate that the degree of restricted water diffusion reliably reflects the severity of oxygen deprivation below the penumbral threshold but is less strongly related to metabolic disruption, which may explain why the ADC does not reliably predict tissue outcome. However, the same degree of diffusion restriction may correspond to greater severity of tissue disruption with elapsing time, which has relevance for stroke therapy. Time elapsed since stroke onset should be taken into account when interpreting ADC declines and in voxel-based infarct prediction models.

Intrinsic activated microglia map to the peri-infarct zone in the subacute phase of ischemic stroke.

BACKGROUND AND PURPOSE: Microglial activation is an important component of the neuroinflammatory response to ischemic stroke. Experimental studies have outlined such patterns temporally and spatially. In vivo studies in stroke patients have relied on positron emission tomography and (R)-PK11195, a ligand that binds peripheral benzodiazepine binding sites. In this study we sought to establish temporal and spatial patterns of microglial activation in ischemic stroke with particular emphasis on a defined peri-infarct zone. METHODS: Using this technique, we studied carotid territory ischemic stroke patients in 3 time windows up to 30 days after ictus. Controls were studied in a single session. [11C](R)-PK11195 injection was followed by 3-dimensional acquisition over 60 minutes. Cerebral blood volume (CBV) was measured afterward with the use of standard C15O paradigms. Analysis employed the reference tissue model in which ipsilateral cerebellum was used to generate parametric binding potential maps corrected for CBV. Data were coregistered to T1-based MRI. Using control data to identify 99% confidence limits, a region of interest analysis was applied to identify significant binding in core infarction, contralateral hemisphere, and within a defined peri-infarct zone. RESULTS: Four patients (mean age, 66 years) were imaged across 9 sessions. Four age-matched controls were studied. Within this model, ipsilateral cerebellum was validated as a reference tissue. With the use of control-derived confidence limits and correction for CBV, significant binding potential rises were identified beyond 72 hours and extending to 30 days in core infarction, contralateral hemisphere, and peri-infarct zone. CONCLUSIONS: In ischemic stroke patients, minimal activation of microglia is seen before 72 hours. Beyond this, binding potential rises in core infarction, peri-infarct zone, and contralateral hemisphere to 30 days. This may represent a therapeutic opportunity that extends beyond time windows traditionally reserved for neuroprotection.

Group II and III mGluRs-mediated presynaptic inhibition of EPSCs recorded from hippocampal interneurons of CA1 stratum lacunosum moleculare.

We have studied the effects of groups II and III metabotropic glutamate receptor (mGluR) activation on excitatory responses recorded from hippocampal interneurons of CA1 stratum lacunosum moleculare (SLM). Excitatory postsynaptic currents (EPSCs) evoked by stimulation of the perforant pathway were reduced either by the group II mGluR agonist LY354740 (50-100 nM, 49.1+/-5.7% of control) or by the group III mGluR agonist l-2-amino-4-phosphonobutyric acid (l-AP4) (50 microM, 36.8+/-4.4% of control). Both drugs significantly enhanced paired-pulse facilitation of the EPSCs. Furthermore, both 100 nM LY354740 and 50 microM l-AP4 reduced the frequency, but not the amplitude, of miniature excitatory synaptic currents (mEPSCs), recorded in the presence of 1 microM TTX and 50 microM picrotoxin, or EPSCs evoked by perforant pathway stimulation in the presence of 2.5 mM Sr2+. The broad-spectrum mGluR antagonist LY341495 (10-50 microM) did not affect test EPSCs elicited 210 ms after stimulation at 100 Hz. At network level, 1-5 microM LY354740 significantly reduced the power of gamma frequency oscillations induced by 20 microM carbachol, 600 nM kainate and 5 mM K+ in hippocampal CA1 area. Our results show powerful modulation of excitatory transmission impinging on interneurons of CA1 SLM by presynaptic group II or III mGluRs.

Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area.

We studied neurogliaform neurons in the stratum lacunosum moleculare of the CA1 hippocampal area. These interneurons have short stellate dendrites and an extensive axonal arbor mainly located in the stratum lacunosum moleculare. Single-cell reverse transcription-PCR showed that these neurons were GABAergic and that the majority expressed mRNA for neuropeptide Y. Most neurogliaform neurons tested were immunoreactive for alpha-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed alpha-actinin-2 and neuropeptide Y. Neurogliaform neurons received monosynaptic, DNQX-sensitive excitatory input from the perforant path, and 40 Hz stimulation of this input evoked EPSCs displaying either depression or initial facilitation, followed by depression. Paired recordings performed between neurogliaform neurons showed that 85% of pairs were electrically connected and 70% were also connected via GABAergic synapses. Injection of sine waveforms into neurons during paired recordings resulted in transmission of the waveforms through the electrical synapse. Unitary IPSCs recorded from neurogliaform pairs readily fatigued, had a slow decay, and had a strong depression of the synaptic response at a 5 Hz stimulation frequency that was antagonized by the GABA(B) antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845). The amplitude of the first IPSC during the 5 Hz stimulation was also increased by CGP55845, suggesting a tonic inhibition of synaptic transmission. A small unitary GABA(B)-mediated IPSC could also be detected, providing the first evidence for such a component between GABAergic interneurons. Electron microscopic localization of the GABA(B1) subunit at neurogliaform synapses revealed the protein in both presynaptic and postsynaptic membranes. Our data disclose a novel interneuronal network well suited for modulating the flow of information between the entorhinal cortex and CA1 hippocampus.