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Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N -ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.

Cognitive functions are increasingly understood to involve coordinated activity patterns between multiple brain regions, and their disruption by neuropsychiatric disorders is similarly complex. Closed-loop neurostimulation can directly modulate neural signals with temporal and spatial precision. How to leverage such an approach to effectively identify and target distributed neural networks implicated in mediating cognition remains unclear. We review current conceptual and technical advances in this area, proposing that devices that enable large-scale acquisition, integrated processing, and multiregion, arbitrary waveform stimulation will be critical for mechanistically driven manipulation of cognitive processes in physiological and pathological brain networks., Competing Interests: Declaration of interests The authors declare no competing interests in relation to this work., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recording from the human brain at the spatiotemporal resolution of action potentials provides critical insight into mechanisms of higher cognitive functions and neuropsychiatric disease that is challenging to derive from animal models. Here, organic materials and conformable electronics are employed to create an integrated neural interface device compatible with minimally invasive neurosurgical procedures and geared toward chronic implantation on the surface of the human brain. Data generated with these devices enable identification and characterization of individual, spatially distribute human cortical neurons in the absence of any tissue penetration (n = 229 single units). Putative single-units are effectively clustered, and found to possess features characteristic of pyramidal cells and interneurons, as well as identifiable microcircuit interactions. Human neurons exhibit consistent phase modulation by oscillatory activity and a variety of population coupling responses. The parameters are furthermore established to optimize the yield and quality of single-unit activity from the cortical surface, enhancing the ability to investigate human neural network mechanisms without breaching the tissue interface and increasing the information that can be safely derived from neurophysiological monitoring., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Implantable devices for the wireless modulation of neural tissue need to be designed for reliability, safety and reduced invasiveness. Here we report chronic electrical stimulation of the sciatic nerve in rats by an implanted organic electrolytic photocapacitor that transduces deep-red light into electrical signals. The photocapacitor relies on commercially available semiconducting non-toxic pigments and is integrated in a conformable 0.1-mm 3 thin-film cuff. In freely moving rats, fixation of the cuff around the sciatic nerve, 10 mm below the surface of the skin, allowed stimulation (via 50-1,000-μs pulses of deep-red light at wavelengths of 638 nm or 660 nm) of the nerve for over 100 days. The robustness, biocompatibility, low volume and high-performance characteristics of organic electrolytic photocapacitors may facilitate the wireless chronic stimulation of peripheral nerves., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca 2+ -induced increase in presynaptic cAMP that is mediated by Ca 2+ -sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by β-adrenergic receptors (βARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that βARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these βARs occluded PF-PC LTP, while the β1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2 -/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the βAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP. SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to β-adrenergic receptor (βAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by βARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this βAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of βARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon., (Copyright © 2020 the authors.)

Bioelectronic devices must be fast and sensitive to interact with the rapid, low-amplitude signals generated by neural tissue. They should also be biocompatible and soft, and should exhibit long-term stability in physiologic environments. Here, we develop an enhancement-mode, internal ion-gated organic electrochemical transistor (e-IGT) based on a reversible redox reaction and hydrated ion reservoirs within the conducting polymer channel, which enable long-term stable operation and shortened ion transit time. E-IGT transient responses depend on hole rather than ion mobility, and combine with high transconductance to result in a gain-bandwidth product that is several orders of magnitude above that of other ion-based transistors. We used these transistors to acquire a wide range of electrophysiological signals, including in vivo recording of neural action potentials, and to create soft, biocompatible, long-term implantable neural processing units for the real-time detection of epileptic discharges. E-IGTs offer a safe, reliable and high-performance building block for chronically implanted bioelectronics, with a spatiotemporal resolution at the scale of individual neurons.

Key Points: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu 7 ) receptors that reduce Ca 2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu 7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu 7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission., Abstract: Neurotransmitter release is driven by Ca 2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu 7 ) receptors provoke a reduction in Ca 2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu 7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca 2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu 7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu 7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu 7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The vast majority of neurons within the striatum are GABAergic medium spiny neurons (MSNs), which receive glutamatergic input from the cortex and thalamus, and form two major efferent pathways: the direct pathway, expressing dopamine D1 receptor (D1R-MSNs), and the indirect pathway, expressing dopamine D2 receptor (D2R-MSNs). While molecular mechanisms of MSN degeneration have been identified in animal models of striatal damage, the molecular factors that dictate a selective vulnerability of D1R-MSNs or D2R-MSNs remain unknown. Here, we combined genetic, chemogenetic, and pharmacological strategies with behavioral and neurochemical analyses, and show that the pool of cannabinoid CB1 receptor (CB1R) located on corticostriatal terminals efficiently safeguards D1R-MSNs, but not D2R-MSNs, from different insults. This cell-specific response relies on the regulation of glutamatergic signaling, and is independent from the CB1R-dependent control of astroglial activity in the striatum. These findings define cortical CB1R as a pivotal synaptic player in dictating a differential vulnerability of D1R-MSNs versus D2R-MSNs, and increase our understanding of the role of coordinated cannabinergic-glutamatergic signaling in establishing corticostriatal circuits and its dysregulation in neurodegenerative diseases., (© The Author 2017. Published by Oxford University Press.)

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing., (© 2017 International Society for Neurochemistry.)

The co-existence of presynaptic G protein coupled receptors, GPCRs, has received little attention, despite the fact that interplay between the signaling pathways activated by such receptors may affect the neurotransmitter release. Using immunocytochemistry and immuhistochemistry we show that mGlu7 and β-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. mGlu7 receptors readily couple to pathways that inhibit glutamate release. We found that when mGlu7 receptors are also coupled to pathways that enhance glutamate release by prolonged exposure to agonist, and β-adrenergic receptors are also activated, a cross-talk between their signaling pathways occurs that affect the overall release response. This interaction is the result of mGlu7 receptors inhibiting the adenylyl cyclase activated by β adrenergic receptors. Thus, blocking Gi/o proteins with pertussis toxin provokes a further increase in release after receptor co-activation which is also observed after activating β-adrenergic receptor signaling pathways downstream of adenylyl cyclase with the cAMP analog Sp8Br or 8pCPT-2-OMe-cAMP (a specific activator of the guanine nucleotide exchange protein directly activated by cAMP, EPAC). Co-activation of mGlu7 and β-adrenergic receptors also enhances PLC-dependent accumulation of IP1 and the translocation of the active zone protein Munc13-1 to the membrane, indicating that release potentiation by these receptors involves the modulation of the release machinery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.

Increased nitric oxide (NO) production seems to play a key role in cyclophosphamide (CYP)-induced cystitis, although the underlying mechanisms and the relative involvement of the different NO synthase (NOS) isoforms remain to be elucidated. Moreover, the role of the urethra in this process is also unclear. In this study, we have analyzed the changes in the expression and distribution of the inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms of NOS, and the alterations in nerve-mediated contractility in the bladder and urethra of CYP-treated rats. Accordingly, Wistar rats were treated with 150 mg kg(-1) CYP for 4 (acute treatment) or 48 h (intermediate treatment), or with 70 mg kg(-1) CYP every 3 days for 10 days (chronic treatment), and the changes in protein expression were assessed by immunohistofluorescence and in Western blots, while mRNA expression was assessed by conventional and quantitative PCR. Similarly, nerve-mediated contractility was analyzed in vitro. Unexpectedly, no iNOS expression was detected in CYP-treated animals, while a transient downregulation of nNOS expression and a progressive upregulation of eNOS was observed, although the eNOS accumulated was not in the active phosphorylated form. Qualitative changes in mRNA expression were also observed in the bladder and urethra, although contractility only diminished in the bladder and this change was not dependent on NOS activity. These findings suggest that spatiotemporal alterations in NO production by constitutive NOS may be involved in the pathogenicity of CYP. Further studies will be necessary to understand the contribution of eNOS to the increases in NO associated with bladder inflammation, or that of free radicals., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.

Calcium channels that mediate glutamate release (N-type and P/Q-type) are expressed in distinct populations of cerebrocortical nerve terminals in adult mice. mGlu7 receptors are exclusively expressed in nerve terminals containing N-type Ca(2+) channels, which are less tightly coupled to glutamate release than P/Q-type Ca(2+) channels. We recently reported that in addition to inhibit, mGlu7 receptors can also potentiate glutamate release via phosphatidyl inositol (4,5)-bisphosphate hydrolysis and activation of the non-kinase diacylglycerol binding protein Munc13-1, a protein that primes synaptic vesicles for exocytosis. Here, we assessed whether mGlu7 receptor-mediated potentiation of glutamate release is restricted to nerve terminals expressing N-type Ca(2+) channels to compensate for their weak coupling to release. In the hippocampus, mGlu7 receptors are expressed both in nerve terminals containing N-type Ca(2+) channels and in nerve terminals containing P/Q-type Ca(2+) channels. When analyzed, we observed potentiation of mGlu7 receptor mediated release in wild type hippocampal nerve terminals at physiological (1.3 mM) and low (0.1 mM) concentrations of external Ca(2+). By contrast, in nerve terminals from mice lacking the α1B subunit of N-type channels (Ca(v)2.2), in which evoked release is mediated by P/Q-type channels only, no release potentiation was observed at 1.3 mM Ca(2+). We conclude that release potentiation at 1.3 mM [Ca(2+)](e) occurs in nerve terminals expressing N-type channels, whereas that which occurs at low 0.1 mM [Ca(2+)](e) represents the release from nerve terminals containing P/Q-type Ca(2+) channels. Although, mGlu7 receptor mediated potentiation is independent of Ca(2+) channel activity, as it was induced by the Ca(2+) ionophore ionomycin, release potentiation is influenced by the Ca(2+) channel type and/or the associated release machinery., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Modern radiotherapy uses complex treatments that necessitate more complex quality assurance procedures. As a continuous medium, GafChromic EBT films offer suitable features for such verification. However, its sensitometric curve is not fully understood in terms of classical theoretical models. In fact, measured optical densities and those predicted by the classical models differ significantly. This difference increases systematically with wider dose ranges. Thus, achieving the accuracy required for intensity-modulated radiotherapy (IMRT) by classical methods is not possible, plecluding their use. As a result, experimental parametrizations, such as polynomial fits, are replacing phenomenological expressions in modern investigations. This article focuses on identifying new theoretical ways to describe sensitometric curves and on evaluating the quality of fit for experimental data based on four proposed models., Methods: A whole mathematical formalism starting with a geometrical version of the classical theory is used to develop new expressions for the sensitometric curves. General results from the percolation theory are also used. A flat-bed-scanner-based method was chosen for the film analysis. Different tests were performed, such as consistency of the numeric results for the proposed model and double examination using data from independent researchers., Results: Results show that the percolation-theory-based model provides the best theoretical explanation for the sensitometric behavior of GafChromic films. The different sizes of active centers or monomer crystals of the film are the basis of this model, allowing acquisition of information about the internal structure of the films. Values for the mean size of the active centers were obtained in accordance with technical specifications. In this model, the dynamics of the interaction between the active centers of GafChromic film and radiation is also characterized by means of its interaction cross-section value., Conclusions: The percolation model fulfills the accuracy requirements for quality-control procedures when large ranges of doses are used and offers a physical explanation for the film response.

N-13A malignant ascitic hepatoma, induced by 4-dimethylaminoazobenzene in Wistar rats, does not produce distant mestastases when transplanted in vivo. The mixed co-cultures of N-13A tumor cells and several tissue explants of newborn Wistar rats show selective adhesivity between tumoral and hepatic epithelial cells, but not with fibroblast-like cells of nervous tissue, kidney or diaphragm. In short-term co-cultures, N-13A cells in contact with rat hepatocytes prepared by the collagenase perfusion technique, display a selective adherence capacity with the production of abundant microvilli and fingerlike protrusions (microspikes) which are elaborated by the neoplastic cells. Groups of tumoral cells tightly envelop the free surface of the cultured hepatocytes. Tight-junction formations are observed, and immature desmosomes and polydesmosomic systems are also seen between both tumoral and normal cells.