DL-3-n-butylphthalide enhances synaptic plasticity in mouse model of brain impairments

Synaptic impairment results in cognitive dysfunction of Alzheimer’s disease (AD). As a plant extract, it is found that DL-3-n-butylphthalide (L-NBP) rescues abnormal cognitive behaviors in AD animals. However, the regulatory effects of L-NBP on synaptic plasticity remains unclear. APP/PS1 mice at 12 months old received oral L-NBP treatment for 12 weeks. A water maze test assessed cognitive performances. In vitro patch-clamp recordings and in vivo field potential recordings were performed to evaluate synaptic plasticity. The protein expression of AMPA receptor subunits (GluR1 and GluR2) and NMDA receptor subunits (NR1, NR2A, and NR2B) was examined by Western blot. In addition, glutaminase activity and glutamate level in the hippocampus were measured by colorimetry to evaluate presynaptic glutamate release. L-NBP treatment could significantly improve learning and memory ability, upregulate NR2A and NR2B protein expressions, increase glutaminase activity and glutamate level in the hippocampus, and attenuate synaptic impairment transmission in the AD mice. L-NPB plays a beneficial role in AD mice by regulating NMDA receptor subunits’ expression and regulating presynaptic glutamate release.

Dynamic Mechanism of Epilepsy Generation and Propagation After Ischemic Stroke

Epilepsy is the second largest neurological disease which seriously threatens human life and health. The one important reason of inducing epilepsy is ischemic stroke which causes insufficient oxygen supply from blood vessels to neurons. However, few studies focus on the underlying mechanism of the generation and propagation of epilepsy after ischemic stroke by utilizing modeling methods. To explore the mechanism, this paper establishes a coupled network model consisting of neurons and astrocytes, and introduces a blood vessel to simulate the condition of ischemic stroke. First we study the effect of the degree of vascular blockage on the generation of epilepsy. The results demonstrate that the important reason of epilepsy after ischemic stroke is the disruption of ion concentration gradient. Then we study three factors that influence the epileptic propagation after ischemic stroke: massive glutamate release, excessive receptor activation and high extracellular potassium concentration. The results demonstrate that massive glutamate acting on postsynaptic neurons and the excessive activation of glutamate receptors on postsynaptic neurons promote the epileptic propagation in neuronal population, and massive glutamate acting on astrocytes and excessive activation of metabotropic glutamate receptors on presynaptic neurons inhibit the epileptic propagation, and the potassium uptake by astrocytes suppresses the epileptic propagation. The results are consistent with the experimental phenomena. The simulation results also shed light on the fact that astrocytes have neuroprotective effect. Our results on the generation and propagation of epilepsy after ischemic stroke could offer theoretical guidelines for the treatment of epilepsy after ischemic stroke.

A Conditional Glutamatergic Synaptic Vesicle Marker for Drosophila

Glutamate is a principal neurotransmitter used extensively by the nervous systems of all vertebrate and invertebrate animals. It is primarily an excitatory neurotransmitter that has been implicated in nervous system development as well as a myriad of brain functions from the simple transmission of information between neurons to more complex aspects of nervous system function including synaptic plasticity, learning, and memory. Identification of glutamatergic neurons and their sites of glutamate release are thus essential for understanding the mechanisms of neural circuit function and how information is processed to generate behavior. Here we describe and characterize smFLAG-vGlut, a conditional marker of glutamatergic synaptic vesicles for the Drosophila model system. smFLAG-vGlut is validated for functionality, conditional expression, and specificity for glutamatergic neurons and synaptic vesicles. The utility of smFLAG-vGlut is demonstrated by glutamatergic neurotransmitter phenotyping of 26 different central complex neuron types of which nine were established to be glutamatergic. This illumination of glutamate neurotransmitter usage will enhance the modeling of central complex neural circuitry and thereby our understanding of information processing by this region of the fly brain. The use of smFLAG for glutamatergic neurotransmitter phenotyping and identification of glutamate release sites can be extended to any Drosophila neuron(s) represented by a binary transcription system driver.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

Volatile anesthetics alter presynaptic function including effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including unconsciousness and amnesia. The nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling pathway has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated NO/cGMP signaling in relation to volatile anesthetic actions in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors of cGMP levels. Using the fluorescent biosensor cGull we found that electrical stmulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited -33.2% by isoflurane (0.51 mM) and -23.8% by sevoflurane (0.57 mM) (p<0.0001) compared to a stimulation without anesthetic. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, were reduced by in neurons expressing the cGMP scavenger sponGee. This reduction in anesthetic effect was recapitulated by inhibiting HCN channels, a cGMP-modulated effector that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify a novel mechanism by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway.

An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats

The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebrocortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca2+ level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca2+ channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine‐rich C kinase substrate (MARCKS), in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q‐type Ca2+ channels and PKC/MARCKS pathways.

Endocannabinoid-Mediated Control of Neural Circuit Excitability and Epileptic Seizures

Research on endocannabinoid signaling has greatly advanced our understanding of how the excitability of neural circuits is controlled in health and disease. In general, endocannabinoid signaling at excitatory synapses suppresses excitability by inhibiting glutamate release, while that at inhibitory synapses promotes excitability by inhibiting GABA release, although there are some exceptions in genetically epileptic animal models. In the epileptic brain, the physiological distributions of endocannabinoid signaling molecules are disrupted during epileptogenesis, contributing to the occurrence of spontaneous seizures. However, it is still unknown how endocannabinoid signaling changes during seizures and how the redistribution of endocannabinoid signaling molecules proceeds during epileptogenesis. Recent development of cannabinoid sensors has enabled us to investigate endocannabinoid signaling in much greater spatial and temporal details than before. Application of cannabinoid sensors to epilepsy research has elucidated activity-dependent changes in endocannabinoid signaling during seizures. Furthermore, recent endocannabinoid research has paved the way for the clinical use of cannabidiol for the treatment of refractory epilepsy, such as Dravet syndrome, Lennox-Gastaut syndrome and tuberous sclerosis complex. Cannabidiol significantly reduces seizures and is considered to have comparable tolerability to conventional antiepileptic drugs. In this article, we introduce recent advances in research on the roles of endocannabinoid signaling in epileptic seizures and discuss future directions.

Chronic Alcohol Dysregulates Glutamatergic Function in the Basolateral Amygdala in a Projection- and Sex-Specific Manner

Chronic intermittent ethanol (CIE) produces alcohol dependence, facilitates anxiety-like behavior, and increases post-CIE alcohol intake. The basolateral amygdala (BLA) is one of several brain regions that regulates anxiety-like behavior and alcohol intake through downstream projections. The BLA receives information from two distinct input pathways. Afferents from medial structures like the thalamus and prefrontal cortex enter the BLA through the stria terminalis whereas lateral cortical structures like the anterior insula cortex enter the BLA through the external capsule. CIE induces input- and sex-specific adaptations to glutamatergic function in the BLA. Previous studies sampled neurons throughout the BLA, but did not distinguish between projection-specific populations. The current study investigated BLA neurons that project to the NAC (BLA-NAC neurons) or the BNST (BLA-BNST neurons) as representative "reward" and "aversion" BLA neurons, and showed that CIE alters glutamatergic function and excitability in a projection- and sex-specific manner. CIE increases glutamate release from stria terminalis inputs only onto BLA-BNST neurons. At external capsule synapses, CIE increases postsynaptic glutamatergic function in male BLA-NAC neurons and female BLA-BNST neurons. Subsequent experiments demonstrated that CIE enhanced the excitability of male BLA-NAC neurons and BLA-BNST neurons in both sexes when glutamatergic but not GABAergic function was intact. Thus, CIE-mediated increased glutamatergic function facilitates hyperexcitability in male BLA-NAC neurons and BLA-BNST neurons of both sexes.

Opioid withdrawal abruptly disrupts amygdala circuit function by reducing peptide actions

Opioid withdrawal drives relapse and contributes to compulsive drug use through disruption of endogenous opioid dependent learning circuits in the amygdala. Normally, endogenous opioids control these circuits by inhibiting glutamate release from basolateral amygdala principal neurons onto GABAergic intercalated cells. Using patch-clamp electrophysiology in rat brain slices, we reveal that opioid withdrawal dials down this endogenous opioid inhibition of synaptic transmission. Peptide activity is dialled down due to a protein kinase A dependent increase in the activity of the peptidase, neprilysin. This disrupts peptidergic control of both GABAergic and glutamatergic transmission through multiple amygdala circuits, including reward-related outputs to the nucleus accumbens. This likely disrupts peptide-dependent learning processes in the amygdala during withdrawal. and may direct behaviour towards compulsive drug use. Restoration of endogenous peptide activity during withdrawal may be a viable option to normalise synaptic transmission in the amygdala and restore normal reward learning.

Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release, however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release. In non-neuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of non-photobleached probes within 10-seconds. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.