A brainstem to hypothalamic arcuate nucleus GABAergic circuit drives feeding.

The obesity epidemic is principally driven by the consumption of more calories than the body requires. It is therefore essential that the mechanisms underpinning feeding behavior are defined. Neurons within the brainstem dorsal vagal complex (DVC) receive direct information from the digestive system and project to second-order regions in the brain to regulate food intake. Although γ-aminobutyric acid is expressed in the DVC (GABADVC), its function in this region has not been defined. In order to discover the unique gene expression signature of GABADVC cells, we used single-nucleus RNA sequencing (Nuc-seq), and this revealed 19 separate clusters. We next probed the function of GABADVC cells and discovered that the selective activation of GABADVC neurons significantly controls food intake and body weight. Optogenetic interrogation of GABADVC circuitry identified GABADVC → hypothalamic arcuate nucleus (ARC) projections as appetite suppressive without creating aversion. Electrophysiological analysis revealed that GABADVC → ARC stimulation inhibits hunger-promoting neuropeptide Y (NPY) neurons via GABA release. Adopting an intersectional genetics strategy, we clarify that the GABADVC → ARC circuit curbs food intake. These data identify GABADVC as a new modulator of feeding behavior and body weight and a controller of orexigenic NPY neuron activity, thereby providing insight into the neural underpinnings of obesity.

Modulation of GABA release by 5-HT1B receptors: An interplay with AMPA-receptors and voltage-gated Ca2+ channels.

Obesity has become a worldwide health challenge and commonly results from the intake of more calories than the body requires. The brain represents the master controller of food intake and as such has been the target of obesity medications. However, key mechanisms of druggable targets remain to be defined. Neurons within the arcuate nucleus of the hypothalamus co-expressing neuropeptide Y (NPY), agouti-related protein (AgRP) and GABA (NAG) are fundamental stimulators of hunger and food intake. NAG neurons also inhibit local satiety-promoting pro-opiomelanocortin (POMC) neurons. Agonists of the 1B subtype of metabotropic serotonin receptor (5-HT1BR) reduce food intake in part through the inhibition of hunger-promoting NAG neurons. We first confirmed that 5-HT1BR activation suppressed intake of a palatable Western diet in a mouse model of common dietary-induced obesity and genetically prone obesity. Next, we combined several electrophysiological approaches to analyse the effect of 5-HT1BRs in NAG neuron cell activity and GABA release. 5-HT1BR activation reduced NAG neuron action potential frequency and neurotransmitter release. We found that 5-HT1BR impact on GABA release from NAG neurons is mediated through voltage-gated Ca2+ channels with a critical input from glutamate receptors of AMPA subtype (AMPARs). As a fundamental outcome, this type of interplay provides an uncommon example of metabotropic action of AMPARs which regulates inhibitory signalling due to modulation of GABA release. As a translational outcome, our results provide a key mechanism through which 5-HT1BR drugs inhibit appetite-stimulating neurons within the brain to suppress food intake. This article is part of the Special Issue on "Ukrainian Neuroscience".

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons.

Mutations in the SNCA gene cause autosomal dominant Parkinson's disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small ß-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34-41 post differentiation. Once midbrain identity fully developed, at day 48-62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Multi-target action of ß-alanine protects cerebellar tissue from ischemic damage.

Brain ischemic stroke is among the leading causes of death and long-term disability. New treatments that alleviate brain cell damage until blood supply is restored are urgently required. The emerging focus of anti-stroke strategies has been on blood-brain-barrier permeable drugs that exhibit multiple sites of action. Here, we combine single-cell electrophysiology with live-cell imaging to find that ß-Alanine (ß-Ala) protects key physiological functions of brain cells that are exposed to acute stroke-mimicking conditions in ex vivo brain preparations. ß-Ala exerts its neuroprotective action through several distinct pharmacological mechanisms, none of which alone could reproduce the neuroprotective effect. Since ß-Ala crosses the blood-brain barrier and is part of a normal human diet, we suggest that it has a strong potential for acute stroke treatment and facilitation of recovery.

The regulatory role of GABAA receptor in Actinia equina nervous system and the possible effect of global ocean acidification.

Global warming and connected acidification of the world ocean attract a substantial amount of research efforts, in particular in a context of their impact on behaviour and metabolism of marine organisms, such as Cnidaria. Nevertheless, mechanisms underlying Cnidarians' neural signalling and behaviour and their (possible) alterations due to the world ocean acidification remain poorly understood. Here we researched for the first time modulation of GABAA receptors (GABAARs) in Actinia equina (Cnidaria: Anthozoa) by pH fluctuations within a range predicted by the world ocean acidification scenarios for the next 80-100 years and by selective pharmacological activation. We found that in line with earlier studies on vertebrates, both changes of pH and activation of GABAARs with a selective allosteric agonist (diazepam) modulate electrical charge transfer through GABAAR and the whole-cell excitability. On top of that, diazepam modifies the animal behavioural reaction on startle response. However, despite behavioural reactions displayed by living animals are controlled by GABAARs, changes of pH do not alter them significantly. Possible mechanisms underlying the species resistance to acidification impact are discussed.

Synaptic signalling in a network of dopamine neurons: what prevents proper intercellular crosstalk?

Human embryonic stem cell (hESC)-derived midbrain dopamine (DA) neurons stand out as a cell source for transplantation with their sustainability and consistency superior to the formerly used fetal tissues. However, multiple studies of DA neurons in culture failed to register action potential (AP) generation upon synaptic input. To test whether this is due to deficiency of NMDA receptor (NMDAR) coagonists released from astroglia, we studied the functional properties of neural receptors in hESC-derived DA neuronal cultures. We find that, apart from an insufficient amount of coagonists, lack of interneuronal crosstalk is caused by hypofunction of synaptic NMDARs due to their direct inhibition by synaptically released DA. This inhibitory tone is independent of DA receptors and affects the NMDAR coagonist binding site.

Extracellular GABA waves regulate coincidence detection in excitatory circuits.

KEY POINTS: Rapid changes in neuronal network activity trigger widespread waves of extracellular GABA in hippocampal neuropil. Elevations of extracellular GABA narrow the coincidence detection window for excitatory inputs to CA1 pyramidal cells. GABA transporters control the effect of extracellular GABA on coincidence detection. Small changes in the kinetics of dendritic excitatory currents amplify when reaching the soma. ABSTRACT: Coincidence detection of excitatory inputs by principal neurons underpins the rules of signal integration and Hebbian plasticity in the brain. In the hippocampal circuitry, detection fidelity is thought to depend on the GABAergic synaptic input through a feedforward inhibitory circuit also involving the hyperpolarisation-activated Ih current. However, afferent connections often bypass feedforward circuitry, suggesting that a different GABAergic mechanism might control coincidence detection in such cases. To test whether fluctuations in the extracellular GABA concentration [GABA] could play a regulatory role here, we use a GABA 'sniffer' patch in acute hippocampal slices of the rat and document strong dependence of [GABA] on network activity. We find that blocking GABAergic signalling strongly widens the coincidence detection window of direct excitatory inputs to pyramidal cells whereas increasing [GABA] through GABA uptake blockade shortens it. The underlying mechanism involves membrane-shunting tonic GABAA receptor current; it does not have to rely on Ih but depends strongly on the neuronal GABA transporter GAT-1. We use dendrite-soma dual patch-clamp recordings to show that the strong effect of membrane shunting on coincidence detection relies on nonlinear amplification of changes in the decay of dendritic synaptic currents when they reach the soma. Our results suggest that, by dynamically regulating extracellular GABA, brain network activity can optimise signal integration rules in local excitatory circuits.

Correction: Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alpha synuclein aggregation drives ferroptosis: an interplay of iron, calcium and lipid peroxidation.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors.

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

The Functional Role of Spontaneously Opening GABAA Receptors in Neural Transmission.

Ionotropic type of γ-aminobutyric acid receptors (GABAARs) produce two forms of inhibitory signaling: phasic inhibition generated by rapid efflux of neurotransmitter GABA into the synaptic cleft with subsequent binding to GABAARs, and tonic inhibition generated by persistent activation of extrasynaptic and/or perisynaptic GABAARs by GABA continuously present in the extracellular space. It is widely accepted that phasic and tonic GABAergic inhibition is mediated by receptor groups of distinct subunit composition and modulated by different cytoplasmic mechanisms. Recently, however, it has been demonstrated that spontaneously opening GABAARs (s-GABAARs), which do not need GABA binding to enter an active state, make a significant input into tonic inhibitory signaling. Due to GABA-independent action mode, s-GABAARs promise new safer options for therapy of neural disorders (such as epilepsy) devoid of side effects connected to abnormal fluctuations of GABA concentration in the brain. However, despite the potentially important role of s-GABAARs in neural signaling, they still remain out of focus of neuroscience studies, to a large extent due to technical difficulties in their experimental research. Here, we summarize present data on s-GABAARs functional properties and experimental approaches that allow isolation of s-GABAARs effects from those of conventional (GABA-dependent) GABAARs.

The role of tonic glycinergic conductance in cerebellar granule cell signalling and the effect of gain-of-function mutation.

KEY POINTS: A T258F mutation of the glycine receptor increases the receptor affinity to endogenous agonists, modifies single-channel conductance and shapes response decay kinetics. Glycine receptors of cerebellar granule cells play their functional role not continuously, but when the granule cell layer starts receiving a high amount of excitatory inputs. Despite their relative scarcity, tonically active glycine receptors of cerebellar granule cells make a significant impact on action potential generation and inter-neuronal crosstalk, and modulate synaptic plasticity in neural networks; extracellular glycine increases probability of postsynaptic response occurrence acting at NMDA receptors and decreases this probability acting at glycine receptors. Tonic conductance through glycine receptors of cerebellar granule cells is a yet undiscovered element of the biphasic mechanism that regulates processing of sensory inputs in the cerebellum. A T258F point mutation disrupts this biphasic mechanism, thus illustrating the possible role of the gain-of-function mutations of the glycine receptor in development of neural pathologies. ABSTRACT: Functional glycine receptors (GlyRs) have been repeatedly detected in cerebellar granule cells (CGCs), where they deliver exclusively tonic inhibitory signals. The functional role of this signalling, however, remains unclear. Apart from that, there is accumulating evidence of the important role of GlyRs in cerebellar structures in development of neural pathologies such as hyperekplexia, which can be triggered by GlyR gain-of-function mutations. In this research we initially tested functional properties of GlyRs, carrying the yet understudied T258F gain-of-function mutation, and found that this mutation makes significant modifications in GlyR response to endogenous agonists. Next, we clarified the role of tonic GlyR conductance in neuronal signalling generated by single CGCs and by neural networks in cell cultures and in living cerebellar tissue of C57Bl-6J mice. We found that GlyRs of CGCs deliver a significant amount of tonic inhibition not continuously, but when the cerebellar granule layer starts receiving substantial excitatory input. Under these conditions tonically active GlyRs become a part of neural signalling machinery allowing generation of action potential (AP) bursts of limited length in response to sensory-evoked signals. GlyRs of CGCs support a biphasic modulatory mechanism which enhances AP firing when excitatory input intensity is low, but suppresses it when excitatory input rises to a certain critical level. This enables one of the key functions of the CGC layer: formation of sensory representations and their translation into motor output. Finally, we have demonstrated that the T258F mutation in CGC GlyRs modifies single-cell and neural network signalling, and breaks a biphasic modulation of the AP-generating machinery.

Glutamate Receptor Probing with Rapid Application and Solution Exchange (RASE).

Probing of glutamate receptors at sub-millisecond time scale is a key element needed for understanding of their response kinetics, neural signal transduction, and, in a wider context, intercellular cross talk. One of the classical techniques used to obtain this type of data in electrophysiology is placing a recording pipette in front of a double-barrel solution application pipette, which provides a rapid switch between two solutions. Here we describe a modification of this classical technique, which utilizes a solution application pipette with several loading capillaries. Such a system is capable of replacing multiple application solutions within 7-12 s time intervals. This modified protocol enables ultrafast application of several solutions at the same set of receptors, thus allowing powerful paired-sample statistical approaches. In addition, the same experimental equipment fabricated for the ultra-resolution probing of receptor kinetics can also be applied in several other types of electrophysiological experiments.

Biphasic Modulation of NMDA Receptor Function by Metabotropic Glutamate Receptors.

A recently reported rapid potentiation of NMDA receptors by Group I metabotropic glutamate receptors (mGluRIs) via a Homer protein link is distinct from the classical, relatively slow inhibitory G-protein-associated signaling triggered by mGluRI activation. The relationship between these two mechanisms remains unknown. Here, we focused on the mGluRI-dependent modulation of NMDAR response in hippocampal dentate gyrus granule cells and cerebellar granule cells of C57BL6-J mice and found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. At a shorter time interval (units of millisecond), the Homer-mediated signal from mGluRIs prevails, causing upregulation of NMDAR function, in both dentate gyrus granule cells and cerebellar granule cells. Our results shed light on the possible mechanisms of anti-schizophrenia drugs that disrupt Homer-containing protein link.SIGNIFICANCE STATEMENT Here we study modulation of NMDA receptors triggered by activation of metabotropic glutamate receptors Group I via two distinct pathways: classical G-protein signaling system and newly discovered high-speed modulatory mechanism associated with Homer-protein-containing direct molecular link. We found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. We have also found that both crosstalk mechanisms cause significant changes in synaptic strength and plasticity. Our results resolve an apparent discrepancy between earlier studies that demonstrated contradictive effects of Homer-containing protein link disruption on NMDA receptor signaling. On top of that, our data provide a plausible explanation for unclear action mechanisms of anti-schizophrenia drugs.

Feature Article: Selective modulation of tonically active GABAA receptor functional subgroups by G-proteins and protein kinase C.

IMPACT STATEMENT: Here we study intracellular mechanisms which regulate inhibitory signaling delivered through continuously (tonically) open ionotropic receptors of γ-aminobutyric acid (GABA) of dentate gyrus granule cells (DGCs). We found that, apart of classical GABA-A receptors (GABAARs) which can be activated by GABA binding, a significant part of tonic inhibitory current is delivered by newly discovered spontaneously opening GABAARs (s-GABAARs), which enter active state without binding of GABA. We have also found that conventional GABAARs and s-GABAARs are regulated by different intracellular mechanisms, which may overlap and thus induce various signaling repercussions. Our results demonstrate that s-GABAARs play a key role in the mechanism that implements DGCs functional role in the brain. On top of that, since regulatory mechanisms under study are affected in a number of pathological states, our results may have broad implications for treatment of neurological disorders.

Spontaneously opening GABAA receptors play a significant role in neuronal signal filtering and integration.

Continuous (tonic) charge transfer through ionotropic receptors of γ-aminobutyric acid (GABAARs) is an important mechanism of inhibitory signalling in the brain. The conventional view has been that tonic GABA-ergic inhibitory currents are mediated by low concentrations of ambient GABA. Recently, however, it was shown that the GABA-independent, spontaneously opening GABAARs (s-GABAARs), may contribute significantly to the tonic GABAAR current. One of the common approaches to temporal lobe epilepsy (TLE) therapy is an increase of GABA concentration in the cerebrospinal fluid to augment tonic current through GABAARs. Such an increase, however, generates multiple side effects, which impose significant limitations on the use of correspondent drugs. In contrast, activation/deactivation of s-GABAARs in a GABA-independent manner may provide a mechanism of regulation of tonic conductance without modification of extracellular GABA concentration, thus avoiding connected side effects. Although s-GABAARs have been detected in our earlier work, it is unclear whether they modulate neural signalling, or, due to their independence from the neurotransmitter, they provide just a stable background effect without much impact on neural crosstalk dynamics. Here, we focused on the causal relationship between s-GABAAR activity and signal integration in the rat's dentate gyrus granule cells to find that s-GABAARs play an important role in neural signal transduction. s-GABAARs shape the dynamics of phasic inhibitory responses, regulate the action potential generation machinery and control the coincidence detection window pertinent to excitatory input summation. Our results demonstrate that tonic inhibition delivered by s-GABAARs contributes to the key mechanisms that ensure implementation of neural signal filtering and integration, in a GABA-independent manner. This makes s-GABAAR a new and important actor in the regulation of long-term neural plasticity and a perspective target for TLE therapy.

GABAA receptors activate fish feeding behaviour via two distinct functional pathways.

Benzodiazepines, acting through ionotropic receptors of γ-aminobutyric acid (GABAA receptors, GABAR), have been shown to modify feeding behaviour and increase appetite in humans and non-human subjects. However, the cellular and molecular mechanisms that underlie connected short-term behavioural fluctuations are still unclear. In the present study, we used Carassius gibelio (Prussian carp) as a model organism to research the impact of scantily explored benzodiazepine phenazepam (PNZ) on feeding behaviour and the related molecular mechanisms of PNZ action at single-cell and single-receptor levels. We found that the feeding activity of C. gibelio is under control of GABARs via two distinct mechanisms: orthosteric (triggered by GABA binding site) and allosteric (triggered by benzodiazepine binding site). PNZ displayed clear stimulatory effects on both mechanisms in a GABA-dependent manner. In addition, orthosteric and allosteric effects were found to be partially competitive, which leads to complex behavioural repercussions of conjoint effects of GABAR ligands.

Hemichannel-mediated release of lactate.

In the central nervous system lactate contributes to the extracellular pool of readily available energy substrates and may also function as a signaling molecule which mediates communication between glial cells and neurons. Monocarboxylate transporters are believed to provide the main pathway for lactate transport across the membranes. Here we tested the hypothesis that lactate could also be released via opening of pannexin and/or functional connexin hemichannels. In acute slices prepared from the brainstem, hippocampus, hypothalamus and cortex of adult rats, enzymatic amperometric biosensors detected significant tonic lactate release inhibited by compounds, which block pannexin/connexin hemichannels and facilitated by lowering extracellular [Ca(2+)] or increased PCO2 Enhanced lactate release triggered by hypoxia was reduced by ∼50% by either connexin or monocarboxylate transporter blockers. Stimulation of Schaffer collateral fibers triggered lactate release in CA1 area of the hippocampus, which was facilitated in conditions of low extracellular [Ca(2+)], markedly reduced by blockade of connexin hemichannels and abolished by lactate dehydrogenase inhibitor oxamate. These results indicate that lactate transport across the membranes may occur via mechanisms other than monocarboxylate transporters. In the central nervous system, hemichannels may function as a conduit of lactate release, and this mechanism is recruited during hypoxia and periods of enhanced neuronal activity.

Sub-millisecond ligand probing of cell receptors with multiple solution exchange.

The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10-15 s. This protocol, which has been validated in our recent studies and takes 20-60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.