Enhance Neurotransmitter Release Research Articles

Glycine receptors support excitatory neurotransmitter release in developing mouse visual cortex

Glycine receptors (GlyRs) are found in most areas of the brain, and their dysfunction can cause severe neurological disorders. While traditionally thought of as inhibitory receptors, presynaptic-acting GlyRs (preGlyRs) can also facilitate glutamate release under certain circumstances, although the underlying molecular mechanisms are unknown. In the current study, we sought to better understand the role of GlyRs in the facilitation of excitatory neurotransmitter release in mouse visual cortex. Using whole-cell recordings, we found that preGlyRs facilitate glutamate release in developing, but not adult, visual cortex. The glycinergic enhancement of neurotransmitter release in early development depends on the high intracellular to extracellular Cl(-) gradient maintained by the Na(+)-K(+)-2Cl(-) cotransporter and requires Ca(2+) entry through voltage-gated Ca(2+) channels. The glycine transporter 1, localized to glial cells, regulates extracellular glycine concentration and the activation of these preGlyRs. Our findings demonstrate a developmentally regulated mechanism for controlling excitatory neurotransmitter release in the neocortex.

Age-Dependent MicroRNA Control of Synaptic Plasticity in 22q11 Deletion Syndrome and Schizophrenia

The 22q11 deletion syndrome (22q11DS) is characterized by multiple physical and psychiatric abnormalities and is caused by the hemizygous deletion of a 1.5-3 Mb region of chromosome 22. It constitutes one of the strongest known genetic risks for schizophrenia; schizophrenia arises in as many as 30% of patients with 22q11DS during adolescence or early adulthood. A mouse model of 22q11DS displays an age-dependent increase in hippocampal long-term potentiation (LTP), a form of synaptic plasticity underlying learning and memory. The sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2), which is responsible for loading Ca(2+) into the endoplasmic reticulum (ER), is elevated in this mouse model. The resulting increase in ER Ca(2+) load leads to enhanced neurotransmitter release and increased LTP. However, the mechanism by which the 22q11 microdeletion leads to SERCA2 overexpression and LTP increase has not been determined. Screening of multiple mutant mouse lines revealed that haploinsufficiency of Dgcr8, a microRNA (miRNA) biogenesis gene in the 22q11DS disease-critical region, causes age-dependent, synaptic SERCA2 overexpression and increased LTP. We found that miR-25 and miR-185, regulators of SERCA2, are depleted in mouse models of 22q11DS. Restoration of these miRNAs to presynaptic neurons rescues LTP in Dgcr8(+/-) mice. Finally, we show that SERCA2 is elevated in the brains of patients with schizophrenia, providing a link between mouse model findings and the human disease. We conclude that miRNA-dependent SERCA2 dysregulation is a pathogenic event in 22q11DS and schizophrenia.

Calcium-Dependent Isoforms of Protein Kinase C Mediate Glycine-Induced Synaptic Enhancement at the Calyx of Held

Depolarization of presynaptic terminals that arises from activation of presynaptic ionotropic receptors, or somatic depolarization, can enhance neurotransmitter release; however, the molecular mechanisms mediating this plasticity are not known. Here we investigate the mechanism of this enhancement at the calyx of Held synapse, in which presynaptic glycine receptors depolarize presynaptic terminals, elevate resting calcium levels, and potentiate release. Using knock-out mice of the calcium-sensitive PKC isoforms (PKC(Ca)), we find that enhancement of evoked but not spontaneous synaptic transmission by glycine is mediated primarily by PKC(Ca). Measurements of calcium at the calyx of Held indicate that deficits in synaptic modulation in PKC(Ca) knock-out mice occur downstream of presynaptic calcium increases. Glycine enhances synaptic transmission primarily by increasing the effective size of the pool of readily releasable vesicles. Our results reveal that PKC(Ca) can enhance evoked neurotransmitter release in response to calcium increases caused by small presynaptic depolarizations.

Contribution of EAG to excitability and potassium currents inDrosophilalarval motoneurons

Diversity in the expression of K(+) channels among neurons allows a wide range of excitability, growth, and functional regulation. Ether-à-go-go (EAG), a voltage-gated K(+) channel, was first characterized in Drosophila mutants by spontaneous firing in nerve terminals and enhanced neurotransmitter release. Although diverse functions have been ascribed to this protein, its role within neurons remains poorly understood. The aim of this study was to characterize the function of EAG in situ in Drosophila larval motoneurons. Whole cell patch-clamp recordings performed from the somata revealed a decrease in I(Av) and I(Kv) K(+) currents in eag mutants and with targeted eag RNAi expression. Spontaneous spike-like events were observed in eag mutants but absent in wild-type motoneurons. Thus our results provide evidence that EAG represents a unique K(+) channel contributing to multiple K(+) currents in motoneurons helping to regulate excitability, consistent with previous observations in the Drosophila larval muscle.

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

Neuronal integration and the depolarizing effects of axonal GABAA receptors

Despite their presence throughout the central nervous system, the impact of axonally expressed gamma-amino-butyric acid type-A receptors (GABA(A)Rs) on neuronal signaling is just beginning to be understood. A recently published article (Pugh JR and Jahr CE, J Neurosci 31: 565-574, 2011) tackled this important issue by investigating GABA(A)R-mediated function in axons of cerebellar granule cells. The results of Pugh and Jahr indicate parallel fiber GABA(A)Rs enhance neurotransmitter release probability and boost axonal and somatic excitability.

Currents for GPCRs

The muscarinic acetylcholine receptor exhibits voltage-dependent gating currents that may enhance neurotransmitter release at cholinergic synapses.

Protein kinase CK2 associates to lipid rafts and its pharmacological inhibition enhances neurotransmitter release

In the present work we report the presence of protein kinase CK2 in lipid raft preparations from rat brain synaptosomes, obtained after detergent extraction and subsequent isolation of detergent-resistant membranes using sucrose gradient ultracentrifugation. Moreover, the phosphorylation of syntaxin-1 at Ser14, a specific CK2 target, has been detected in lipid rafts, as assessed by a phospho-specific antibody. Treatment with DMAT, a specific CK2 inhibitor, results in a decrease of syntaxin-1 Ser14 phosphorylation in lipid rafts, while the glutamate release from synaptosomes is enhanced. In conclusion, CK2 might control neurotransmitter release by acting on SNARE proteins attached to cholesterol-enriched microdomains.

The Drosophila miR-310 Cluster Negatively Regulates Synaptic Strength at the Neuromuscular Junction

Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.

Dysregulation of Presynaptic Calcium and Synaptic Plasticity in a Mouse Model of 22q11 Deletion Syndrome

The 22q11 deletion syndrome (22q11DS) is characterized by cognitive decline and increased risk of psychiatric disorders, mainly schizophrenia. The molecular mechanisms of neuronal dysfunction in cognitive symptoms of 22q11DS are poorly understood. Here, we report that a mouse model of 22q11DS, the Df(16)1/+ mouse, exhibits substantially enhanced short- and long-term synaptic plasticity at hippocampal CA3-CA1 synapses, which coincides with deficits in hippocampus-dependent spatial memory. These changes are evident in mature but not young animals. Electrophysiological, two-photon imaging and glutamate uncaging, and electron microscopic assays in acute brain slices showed that enhanced neurotransmitter release but not altered postsynaptic function or structure caused these changes. Enhanced neurotransmitter release in Df(16)1/+ mice coincided with altered calcium kinetics in CA3 presynaptic terminals and upregulated sarco(endo)plasmic reticulum calcium-ATPase type 2 (SERCA2). SERCA inhibitors rescued synaptic phenotypes of Df(16)1/+ mice. Thus, presynaptic SERCA2 upregulation may be a pathogenic event contributing to the cognitive symptoms of 22q11DS.

The Mechanism for Prejunctional Enhancement of Neuromuscular Transmission by Ethanol in the Mouse

Ethanol has been shown to have both presynaptic and postsynaptic effects on synaptic transmission. However, the mechanisms by which ethanol affects evoked neurotransmitter release have not been studied at the mouse neuromuscular junction, a synapse at which binomial analysis of neurotransmitter release and measurements of prejunctional ionic currents can be made. Ethanol (400 mM) increased neurotransmitter release independently of both the cAMP and phorbol ester/Munc13 signaling pathways. Binomial analysis of neurotransmitter release revealed that ethanol increases the average probability of secretion without an effect on the immediately available store of the neurotransmitter. Application of ethanol also resulted in an inhibition of potassium currents in the motor nerve endings. These results suggest that the potentiating effects of ethanol on neurotransmitter release at the skeletal neuromuscular junction are mediated by an inhibition of the delayed rectifier potassium current, thus increasing both calcium entry into the nerve ending and the probability of neurotransmitter release. Identifying the mechanism through which ethanol enhances neurotransmitter release at the neuromuscular junction may be useful in determining the processes underlying the enhancement of neurotransmitter release at other synapses.

Presynaptic Miniature Gabaergic Currents in Developing Interneurons

Miniature synaptic currents have long been known to represent random transmitter release under resting conditions, but much remains to be learned about their nature and function in central synapses. In this work, we describe a new class of miniature currents ("preminis") that arise by the autocrine activation of axonal receptors following random vesicular release. Preminis are prominent in gabaergic synapses made by cerebellar interneurons during the development of the molecular layer. Unlike ordinary miniature postsynaptic currents in the same cells, premini frequencies are strongly enhanced by subthreshold depolarization, suggesting that the membrane depolarization they produce belongs to a feedback loop regulating neurotransmitter release. Thus, preminis could guide the formation of the interneuron network by enhancing neurotransmitter release at recently formed synaptic contacts.

Presynaptic roles of intracellular Ca2+ stores in signalling and exocytosis

The signalling roles of Ca(2+)(ic) (intracellular Ca(2+)) stores are well established in non-neuronal and neuronal cells. In neurons, although Ca(2+)(ic) stores have been assigned a pivotal role in postsynaptic responses to G(q)-coupled receptors, or secondarily to extracellular Ca(2+) influx, the functions of dynamic Ca(2+)(ic) stores in presynaptic terminals remain to be fully elucidated. In the present paper, we review some of the recent evidence supporting an involvement of Ca(2+)(ic) in presynaptic function, and discuss loci at which this source of Ca(2+) may impinge. Nerve terminal preparations provide good models for functionally examining putative Ca(2+)(ic) stores under physiological and pathophysiological stimulation paradigms, using Ca(2+)-dependent activation of resident protein kinases as sensors for fine changes in intracellular Ca(2+) levels. We conclude that intraterminal Ca(2+)(ic) stores may, directly or indirectly, enhance neurotransmitter release following nerve terminal depolarization and/or G-protein-coupled receptor activation. During conditions that prevail following neuronal ischaemia, increased glutamate release instigated by Ca(2+)(ic) store activation may thereby contribute to excitotoxicity and eventual synaptopathy.

Central Cholinesterase Inhibition Enhances Glutamatergic Synaptic Transmission

Central cholinergic overstimulation results in prolonged seizures of status epilepticus in humans and experimental animals. Cellular mechanisms of underlying seizures caused by cholinergic stimulation remain uncertain, but enhanced glutamatergic transmission is a potential mechanism. Paraoxon, an organophosphate cholinesterase inhibitor, enhanced glutamatergic transmission on hippocampal granule cells synapses by increasing the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in a concentration-dependent fashion. The amplitude of mEPSCs was not increased, which suggested the possibility of enhanced action potential-dependent release. Analysis of EPSCs evoked by minimal stimulation revealed reduced failures and increased amplitude of evoked responses. The ratio of amplitudes of EPSCs evoked by paired stimuli was also altered. The effect of paraoxon on glutamatergic transmission was blocked by the muscarinic antagonist atropine and partially mimicked by carbachol. The nicotinic receptor antagonist α -bungarotoxin did not block the effects of paraoxon; however, nicotine enhanced glutamatergic transmission. These studies suggested that cholinergic overstimulation enhances glutamatergic transmission by enhancing neurotransmitter release from presynaptic terminals.

High cortical spreading depression susceptibility and migraine‐associated symptoms in Cav2.1 S218L mice

The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

Episodic ataxia type 1 mutations differentially affect neuronal excitability and transmitter release

Heterozygous mutations of KCNA1, the gene encoding potassium channel Kv1.1 subunits, cause episodic ataxia type 1 (EA1), which is characterized by paroxysmal cerebellar incoordination and interictal myokymia. Some mutations are also associated with epilepsy. Although Kv1.1-containing potassium channels play important roles in neuronal excitability and neurotransmitter release, it is not known how mutations associated with different clinical features affect the input-output relationships of individual neurons. We transduced rat hippocampal neurons, which were cultured on glial micro-islands, with lentiviruses expressing wild-type or mutant human KCNA1, and injected either depolarizing currents to evoke action potentials or depolarizing voltage commands to evoke autaptic currents. alpha-Dendrotoxin and tetraethylammonium allowed a pharmacological dissection of potassium currents underlying excitability and neurotransmission. Overexpression of wild-type Kv1.1 decreased both neuronal excitability and neurotransmitter release. By contrast, the C-terminus-truncated R417stop mutant, which is associated with severe drug-resistant EA1, had the opposite effect: increased excitability and release probability. Another mutant, T226R, which is associated with EA1 that is complicated by contractures and epilepsy, had no detectable effect on neuronal excitability; however, in common with R417stop, it markedly enhanced neurotransmitter release. The results provide direct evidence that EA1 mutations increase neurotransmitter release, and provide an insight into mechanisms underlying the phenotypic differences that are associated with different mutations.

Presynaptic Presenilins Promote Potentiation

Presynaptic, but not postsynaptic, presenilins enhance neurotransmitter release and long-term potentiation at the hippocampal CA3-CA1 synapse.

Use of Phosphatide Precursors to Promote Synaptogenesis

New brain synapses form when a postsynaptic structure, the dendritic spine, interacts with a presynaptic terminal. Brain synapses and dendritic spines, membrane-rich structures, are depleted in Alzheimer's disease, as are some circulating compounds needed for synthesizing phosphatides, the major constituents of synaptic membranes. Animals given three of these compounds, all nutrients-uridine, the omega-3 polyunsaturated fatty acid docosahexaenoic acid, and choline-develop increased levels of brain phosphatides and of proteins that are concentrated within synaptic membranes (e.g., PSD-95, synapsin-1), improved cognition, and enhanced neurotransmitter release. The nutrients work by increasing the substrate-saturation of low-affinity enzymes that synthesize the phosphatides. Moreover, uridine and its nucleotide metabolites activate brain P2Y receptors, which control neuronal differentiation and synaptic protein synthesis. A preparation containing these compounds is being tested for treating Alzheimer's disease.

PKC-dependent phosphorylations modify the action of deltamethrin on rat brain N-type (Ca V2.2) voltage-sensitive calcium channel

Voltage clamp electrophysiological studies using wild type Ca V2.2 and its β 3 subunit coexpressed in Xenopus oocytes revealed that deltamethrin increased the rate of activation, prolonged inactivation and reduced peak current. Site-directed mutagenesis of threonine 422 to glutamic acid (T422E, one of five protein kinase C (PKC)-dependent phosphorylation sites) resulted in a channel that acted as if it were permanently phosphorylated. This resulted in an increased probability of opening during depolarization and a reduced inhibition by the βγ subunit of heterotrimeric G-protein. Deltamethrin treatment of T422E Ca V2.2 enhanced peak current ∼50% over ethanol-treated controls with an EC 50 of 9.8 × 10 −11 M. Phosphorylation of wild type Ca V2.2 is evoked by the phorbol ester, phorbol 12-myristrate, 13 acetate (PMA), by activating endogenous protein kinase C (PKC) in oocytes. PKC-dependent phosphorylation activated by PMA of wild type Ca V2.2 has been previously shown to slow channel deactivation and increased Ca 2+ influx and subsequent neurotransmitter release. Following PMA-activated phosphorylation, deltamethrin significantly increased peak current and slowed deactivation of the phosphorylated channel, which would be consistent with slower channel closure, greater Ca 2+ influx and enhanced neurotransmitter release seen in vivo. Deltamethrin treatment in the absence of PMA-activated phosphorylation resulted in no effect on the deactivation kinetics of unphosphorylated Ca V2.2 or the T422E mutant. Thus, Ca V2.2 is modified by deltamethrin but the resulting perturbations are dependent upon its PKC-dependent phosphorylation state.

Suppressed Ca 2+ /CaM/CaMKII-dependent K ATP channel activity in primary afferent neurons mediates hyperalgesia after axotomy

Painful axotomy decreases K(ATP) channel current (IK(ATP)) in primary afferent neurons. Because cytosolic Ca(2+) signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca(2+)-calmodulin (CaM)-Ca(2+)/CaM-dependent kinase II (CaMKII) regulates IK(ATP) in large DRG neurons. Immunohistochemistry identified the presence of K(ATP) channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200-positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IK(ATP) by ionomycin, a Ca(2+) ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal K(ATP) channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca(2+)-evoked K(ATP) channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IK(ATP) by glybenclamide, a selective K(ATP) channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective K(ATP) channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IK(ATP) inhibition by AIPm. AIPm also depolarized RMP in control neurons via K(ATP) channel inhibition. Unitary current conductance and sensitivity of K(ATP) channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.