Postsynaptic Sites Research Articles

Neurotrophin-3 from the dentate gyrus supports postsynaptic sites of mossy fiber-CA3 synapses and hippocampus-dependent cognitive functions.

At the center of the hippocampal tri-synaptic loop are synapses formed between mossy fiber (MF) terminals from granule cells in the dentate gyrus (DG) and proximal dendrites of CA3 pyramidal neurons. However, the molecular mechanism regulating the development and function of these synapses is poorly understood. In this study, we showed that neurotrophin-3 (NT3) was expressed in nearly all mature granule cells but not CA3 cells. We selectively deleted the NT3-encoding Ntf3 gene in the DG during the first two postnatal weeks to generate a Ntf3 conditional knockout (Ntf3-cKO). Ntf3-cKO mice of both sexes had normal hippocampal cytoarchitecture but displayed impairments in contextual memory, spatial reference memory, and nest building. Furthermore, male Ntf3-cKO mice exhibited anxiety-like behaviors, whereas female Ntf3-cKO showed some mild depressive symptoms. As MF-CA3 synapses are essential for encoding of contextual memory, we examined synaptic transmission at these synapses using ex vivo electrophysiological recordings. We found that Ntf3-cKO mice had impaired basal synaptic transmission due to deficits in excitatory postsynaptic currents mediated by AMPA receptors but normal presynaptic function and intrinsic excitability of CA3 pyramidal neurons. Consistent with this selective postsynaptic deficit, Ntf3-cKO mice had fewer and smaller thorny excrescences on proximal apical dendrites of CA3 neurons and lower GluR1 levels in the stratum lucidum area where MF-CA3 synapses reside but normal MF terminals, compared with control mice. Thus, our study indicates that NT3 expressed in the dentate gyrus is crucial for the postsynaptic structure and function of MF-CA3 synapses and hippocampal-dependent memory.

A Life-Threatening Neurotoxicity in Bungarus Candidus Snake Bite: A Case Report

Background: Bungarus Candidus, a snake inducing severe neurotoxic symptoms. Case: A 31-year-old female presented to the emergency department with complaints of pain in the whole body, limb weakness, shortness of breath after being bitten by a snake. Neurological examination, ptosis of both eyelids was found with normal movement of the eyeballs. Muscle strength in all extremities is 4 followed by decreased physiological reflexes. There are fang marks in the form of 2 slightly swollen and reddened black dots on the left foot. During observation, the patient had decreased consciousness, severe shortness of breath with desaturation without any signs of shock. Other clinical revealed bilateral dilated pupils, dysphonia, dysarthria, and muscle strength that was difficult to assess. The patient was intubated and subsequently admitted to the ICU. The patient regained consciousness but still had difficulty opening his eyes and weak muscle strength on the 2nd of ICU. The patient's condition improved on the 5th of ICU, extubating was performed and transferred to the non-ICU. The treatment obtained included 17 vials of SABU, analgesics, antibiotics, anticholinesterase, and anticholinergic. The patient was discharged after 11 days of hospitalization. Discussion: The neurotoxicity of snake venom is related to mechanisms of neuromuscular transmission blockade, pre-synaptic and post-synaptic. Bungarus Candidus has a peculiar type of toxin, candoxin that acts by inhibiting nicotinic acetylcholine receptors at the postsynaptic site. Recognition of symptoms and management interventions in snakebite cases are critical to patient outcomes and survival.

PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton, modulating emotional behavior.

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such as defects in the density and morphology of postsynaptic dendritic spines, underlie the pathology of various neuropsychiatric disorders. Protocadherin 17 (PCDH17) is associated with major mood disorders, including bipolar disorder and depression. However, the molecular mechanisms by which PCDH17 regulates spine number, morphology, and behavior remain elusive. In this study, we found that PCDH17 functions at postsynaptic sites, restricting the number and size of dendritic spines in excitatory neurons. Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety- and depression-like behaviors in mice. Mechanistically, PCDH17 interacts with actin-relevant proteins and regulates actin filament (F-actin) organization. Specifically, PCDH17 binds to ROCK2, increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3 (Ser3). Inhibition of ROCK2 activity with belumosudil (KD025) ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression, suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development. Hence, these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior, providing pathological insights into the neurobiological basis of mood disorders.

Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

A lack of drebrin causes olfactory impairment.

Olfactory deficit often occurs during the prodromal stage of Alzheimer's disease (AD). Although olfactory deficit is a useful measure for screening AD-related amnestic disorder, little is known about the cause of this deficit. Human and animal studies indicate that loss of the actin binding protein, drebrin, is closely related to cognitive dysfunction in AD. We hypothesized that the olfactory deficit in AD is caused by the loss of drebrin from the spine. To verify this hypothesis, we performed the buried food test in two types of drebrin knockout mice, such as drebrin-double (E and A) knockout (DXKO) mice, and drebrin A-specific knockout (DAKO) mice. The DXKO mice spent a significantly longer time to find food compared with the wild-type (WT) littermates. In contrast, the DAKO mice, in which drebrin E rather than drebrin A is expressed in the postsynaptic sites of mature neurons, spent an equivalent time trying to find food compared to that of the WT. The DXKO mice showed comparable food motivation and sensory functions other than olfaction, including visual and auditory functions. These results indicate that drebrin is necessary for normal olfactory function. Further study is needed to determine whether it is necessary for normal olfaction to express drebrin E during the developmental stage or to have drebrin (whether E or A) present after maturation.

ABHD6 drives endocytosis of AMPA receptors to regulate synaptic plasticity and learning flexibility

Trafficking of α‐Amino‐3–hydroxy‐5–methylisoxazole‐4–propionic acid (AMPA) receptors (AMPARs), mediated by AMPAR interacting proteins, enabled neurons to maintain tuning capabilities at rest or active state. α/β-Hydrolase domain-containing 6 (ABHD6), an endocannabinoid hydrolase, was an AMPAR auxiliary subunit found to negatively regulate the surface delivery of AMPARs. While ABHD6 was found to prevent AMPAR tetramerization in endoplasmic reticulum, ABHD6 was also reported to localize at postsynaptic site. Yet, the role of ABHD6 interacting with AMPAR at postsynaptic site, and the physiological significance of ABHD6 regulating AMPAR trafficking remains elusive. Here, we generated the ABHD6 knockout (ABHD6KO) mice and found that deletion of ABHD6 selectively enhanced AMPAR-mediated basal synaptic responses and the surface expression of postsynaptic AMPARs. Furthermore, we found that loss of ABHD6 impaired hippocampal long-term depression (LTD) and synaptic downscaling in hippocampal synapses. AMPAR internalization assays revealed that ABHD6 was essential for neuronal activity-dependent endocytosis of surface AMPARs, which is independent of ABHD6's hydrolase activity. The defects of AMPAR endocytosis and LTD are expressed as deficits in learning flexibility in ABHD6KO mice. Collectively, we demonstrated that ABHD6 is an endocytic accessory protein promoting AMPAR endocytosis, thereby contributes to the formation of LTD, synaptic downscaling and reversal learning.

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination.

During the development of the nervous system, there is an overproduction of neurons and synapses. Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening. We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway, at the neuromuscular junction, in the axonal development and synapse elimination process versus the synapse consolidation. The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination, in relation to other molecular pathways that we and others have found to regulate this process. In particular, we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors, coupled to downstream serine-threonine protein kinases A and C (PKA and PKC) and voltage-gated calcium channels, at different nerve endings in developmental competition. The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site, influence each other, and require careful studies to individualize the mechanisms of specific endings. We describe an activity-dependent balance (related to the extent of transmitter release) between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals. The downstream displacement of the PKA/PKC activity ratio to lower values, both in competing nerve terminals and at postsynaptic sites, plays a relevant role in controlling the elimination of supernumerary synapses. Finally, calcium entry through L- and P/Q- subtypes of voltage-gated calcium channels (both channels are present, together with the N-type channel in developing nerve terminals) contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination (the weakest in acetylcholine release and those that have already become silent). The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development. Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.

Postsynaptic cell type and synaptic distance do not determine efficiency of monosynaptic rabies virus spread measured at synaptic resolution.

Retrograde monosynaptic tracing using glycoprotein-deleted rabies virus is an important component of the toolkit for investigation of neural circuit structure and connectivity. It allows for the identification of first-order presynaptic connections to cell populations of interest across both the central and peripheral nervous system, helping to decipher the complex connectivity patterns of neural networks that give rise to brain function. Despite its utility, the factors that influence the probability of transsynaptic rabies spread are not well understood. While it is well established that expression levels of rabies glycoprotein used to trans-complement G-deleted rabies can result in large changes in numbers of inputs labeled per starter cell (convergence index [CI]), it is not known how typical values of CI relate to the proportions of synaptic contacts or input neurons labeled. And it is not known whether inputs to different cell types, or synaptic contacts that are more proximal or distal to the cell body, are labeled with different probabilities. Here, we use a new rabies virus construct that allows for the simultaneous labeling of pre- and postsynaptic specializations to quantify the proportion of synaptic contacts labeled in mouse primary visual cortex. We demonstrate that with typical conditions about 40% of first-order presynaptic excitatory synapses to cortical excitatory and inhibitory neurons are labeled. We show that using matched tracing conditions there are similar proportions of labeled contacts onto L4 excitatory pyramidal, somatostatin (Sst) inhibitory, and vasoactive intestinal peptide (Vip) starter cell types. Furthermore, we find no difference in the proportions of labeled excitatory contacts onto postsynaptic sites at different subcellular locations.

Enhanced TARP-γ8-PSD-95 coupling in excitatory neurons contributes to the rapid antidepressant-like action of ketamine in male mice

Ketamine produces rapid antidepressant effects at sub-anesthetic dosage through early and sustained activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), however, the exact molecular mechanism still remains unclear. Transmembrane AMPAR regulatory protein-γ8 (TARP-γ8) is identified as one of AMPAR auxiliary subunits, which controls assemblies, surface trafficking and gating of AMPARs. Here, we show that ketamine rescues both depressive-like behaviors and the decreased AMPARs-mediated neurotransmission by recruitment of TARP-γ8 at the postsynaptic sites in the ventral hippocampus of stressed male mice. Furthermore, the rapid antidepressant effects of ketamine are abolished by selective blockade of TARP-γ8-containing AMPAR or uncoupling of TARP-γ8 from PSD-95. Overexpression of TARP-γ8 reverses chronic stress-induced depressive-like behaviors and attenuation of AMPARs-mediated neurotransmission. Conversely, knockdown of TARP-γ8 in excitatory neurons prevents the rapid antidepressant effects of ketamine.

Brain-wide circuit-specific targeting of astrocytes

Astrocytes are integral components of brain circuitry. They enwrap synapses, react to neuronal activity, and regulate synaptic transmission. Astrocytes are heterogeneous and exhibit distinct features and functions in different circuits. Selectively targeting the astrocytes associated with a given neuronal circuit would enable elucidation of their circuit-specific functions but has been technically challenging to date. Recently, we constructed anterograde transneuronal viral vectors based on yellow fever vaccine YFV-17D. Among them, the replication-incompetent YFVΔNS1-Cre can selectively turn on reporter genes in postsynaptic neurons if the viral gene NS1 is expressed in postsynaptic neurons. Here we show that without exogenous expression of NS1 at the postsynaptic sites, locally injected YFVΔNS1-Cre selectively turns on reporter genes in astrocytes in downstream brain regions. The targeting of astrocytes can occur across the whole brain but is specific for the neuronal circuits traced. Therefore, YFVΔNS1-Cre provides a tool for selective genetic targeting of astrocytes to reveal their circuit-specific roles.

Phosphorylation of 4.1N by CaMKII Regulates the Trafficking of GluA1-containing AMPA Receptors During Long-term Potentiation in Acute Rat Hippocampal Brain Slices

ObjectiveGluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) inserted into postsynaptic membranes are key to the process of long-term potentiation (LTP). Some evidence has shown that 4.1N plays a critical role in the membrane trafficking of AMPARs. However, the underlying mechanism behind this is still unclear. We investigated the role of 4.1N-mediated membrane trafficking of AMPARs during theta-burst stimulation long-term potentiation (TBS-LTP), to illustrate the molecular mechanism behind LTP. MethodsLTP was induced by TBS in rat hippocampal CA1 neuron. Tat-GluA1 (MPR), which disrupts the association of 4.1N-GluA1, and autocamtide-2-inhibitory peptide, myristoylated (Myr-AIP), a CaMKII antagonist, were used to explore the role of 4.1N in the AMPARs trafficking during TBS-induced LTP. Immunoprecipitation (IP) and immunoblotting (IB)were used to detect protein expression, phosphorylation, and the interaction of p-CaMKII-4.1N-GluA1. ResultsWe found that Myr-AIP attenuated increases of p-CaMKII (T286), p-GluA1 (ser831), and 4.1N phosphorylation after TBS-LTP, and decreased the association of p-CaMKII-4.1N-GluA1, along with the expression of GluA1, at postsynaptic densities during TBS-LTP. We also designed interfering peptides to disrupt the interaction between 4.1N and GluA1, which showed that Tat-GluA1 (MPR) or Myr-AIP inhibited TBS-LTP and attenuated increases of GluA1 at postsynaptic sites, while Tat-GluA1 (MPR) or Myr-AIP had no effects on miniature excitatory postsynaptic currents (mEPSCs) in non-stimulated hippocampal CA1 neurons. ConclusionActive CaMKII enhanced the phosphorylation of 4.1N and facilitated the association of p-CaMKII with 4.1N-GluA1, which in turn resulted in GluA1 trafficking during TBS-LTP. The association of 4.1N-GluA1 is required for LTP, but not for basal synaptic transmission.

Role of the Glycine Receptor β Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and β subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRβ, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRβ that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRβ under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRβ was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRβ enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2β complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.

Effects of propofol on presynaptic synapsin phosphorylation in the mouse brain in vivo

The commonly used general anesthetic propofol can enhance the γ-aminobutyric acid-mediated inhibitory synaptic transmission and depress the glutamatergic excitatory synaptic transmission to achieve general anesthesia and other outcomes. In addition to the actions at postsynaptic sites, the modulation of presynaptic activity by propofol is thought to contribute to neurophysiological effects of the anesthetic, although potential targets of propofol within presynaptic nerve terminals are incompletely studied at present. In this study, we explored the possible linkage of propofol to synapsins, a family of neuron-specific phosphoproteins which are the most abundant proteins on presynaptic vesicles, in the adult mouse brain in vivo. We found that an intraperitoneal injection of propofol at a dose that caused loss of righting reflex increased basal levels of synapsin phosphorylation at the major representative phosphorylation sites (serine 9, serine 62/67, and serine 603) in the prefrontal cortex (PFC) of male and female mice. Propofol also elevated synapsin phosphorylation at these sites in the striatum and S9 and S62/67 phosphorylation in the hippocampus, while propofol had no effect on tyrosine hydroxylase phosphorylation in striatal nerve terminals. Total synapsin protein expression in the PFC, hippocampus, and striatum was not altered by propofol. These results reveal that synapsin could be a novel substrate of propofol in the presynaptic neurotransmitter release machinery. Propofol possesses the ability to upregulate synapsin phosphorylation in broad mouse brain regions.

Dopamine-Dependent Plasticity Is Heterogeneously Expressed by Presynaptic Calcium Activity across Individual Boutons of the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium influx and dopaminergic input in the presynaptic terminals of Kenyon cells (KCs), the principal neurons of the MB, triggers long-term depression (LTD), which plays a critical role in olfactory learning. However, it is controversial whether such synaptic plasticity is accompanied by a corresponding decrease in odor-evoked calcium activity in the KC presynaptic terminals. Here, we address this question by inducing LTD by pairing odor presentation with optogenetic activation of dopaminergic neurons (DANs). This allows us to rigorously compare the changes at the presynaptic and postsynaptic sites in the same conditions. By imaging presynaptic acetylcholine release in the condition where LTD is reliably observed in the postsynaptic calcium signals, we show that neurotransmitter release from KCs is depressed selectively in the MB compartments innervated by activated DANs, demonstrating the presynaptic nature of LTD. However, total odor-evoked calcium activity of the KC axon bundles does not show concurrent depression. We further conduct calcium imaging in individual presynaptic boutons and uncover the highly heterogeneous nature of calcium plasticity. Namely, only a subset of boutons, which are strongly activated by associated odors, undergo calcium activity depression, while weakly responding boutons show potentiation. Thus, our results suggest an unexpected nonlinear relationship between presynaptic calcium influx and the results of plasticity, challenging the simple view of cooperative actions of presynaptic calcium and dopaminergic input.

The Shank/ProSAP N-Terminal (SPN) Domain of Shank3 Regulates Targeting to Postsynaptic Sites and Postsynaptic Signaling

Members of the Shank family of postsynaptic scaffold proteins (Shank1–3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.

The human milk component myo-inositol promotes neuronal connectivity

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.

The intracellular C-terminal domain of mGluR6 contains ER retention motifs

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Ethanol depresses neurons in the lateral parabrachial nucleus by potentiating pre- and postsynaptic GABAA receptors.

As a psychoactive substance, ethanol is widely used in people's life. However, the neuronal mechanisms underlying its sedative effect remain unclear. In this study, we investigated the effects of ethanol on the lateral parabrachial nucleus (LPB), which is a novel component related to sedation. Coronal brain slices (280 μm thick) containing the LPB were prepared from C57BL/6J mice. The spontaneous firing and membrane potential of LPB neurons, and GABAergic transmission onto these neurons were recorded using whole-cell patch-clamp recordings. Drugs were applied through superfusion. The LPB neurons exhibited a regular spontaneous discharge at a rate of 1.5-3 Hz without burst firing. Brief superfusion of ethanol (30, 60, and 120 mM) concentration-dependently and reversibly suppressed the spontaneous firing of the neurons in LPB. In addition, when synaptic transmission was blocked by tetrodotoxin (TTX) (1 μM), ethanol (120 mM) caused hyperpolarization of the membrane potential. Furthermore, superfusion of ethanol markedly increased the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents, which were abolished in the presence of the GABAA receptor (GABAA-R) antagonist picrotoxin (100 μM). In addition, the inhibitory effect of ethanol on the firing rate of LPB neurons was completely abolished by picrotoxin. Ethanol inhibits the excitability of LPB neurons in mouse slices, possibly via potentiating GABAergic transmission onto the neurons at pre- and postsynaptic sites.

Subgenual and Hippocampal Pathways in Amygdala Are Set to Balance Affect and Context Processing.

The amygdala, hippocampus, and subgenual cortex area 25 (A25) are engaged in complex cognitive-emotional processes. Yet pathway interactions from hippocampus and A25 with postsynaptic sites in amygdala remain largely unknown. In rhesus monkeys of both sexes, we studied with neural tracers how pathways from A25 and hippocampus interface with excitatory and inhibitory microcircuits in amygdala at multiple scales. We found that both hippocampus and A25 innervate distinct as well as overlapping sites of the basolateral (BL) amygdalar nucleus. Unique hippocampal pathways heavily innervated the intrinsic paralaminar basolateral nucleus, which is associated with plasticity. In contrast, orbital A25 preferentially innervated another intrinsic network, the intercalated masses, an inhibitory reticulum that gates amygdalar autonomic output and inhibits fear-related behaviors. Finally, using high-resolution confocal and electron microscopy (EM), we found that among inhibitory postsynaptic targets in BL, both hippocampal and A25 pathways preferentially formed synapses with calretinin (CR) neurons, which are known for disinhibition and may enhance excitatory drive in the amygdala. Among other inhibitory postsynaptic sites, A25 pathways innervated the powerful parvalbumin (PV) neurons which may flexibly regulate the gain of neuronal assemblies in the BL that affect the internal state. In contrast, hippocampal pathways innervated calbindin (CB) inhibitory neurons, which modulate specific excitatory inputs for processing context and learning correct associations. Common and unique patterns of innervation in amygdala by hippocampus and A25 have implications for how complex cognitive and emotional processes may be selectively disrupted in psychiatric disorders.SIGNIFICANCE STATEMENT The hippocampus, subgenual A25, and amygdala are associated with learning, memory, and emotions. We found that A25 is poised to affect diverse amygdalar processes, from emotional expression to fear learning by innervating the basal complex and the intrinsic intercalated masses. Hippocampal pathways uniquely interacted with another intrinsic amygdalar nucleus which is associated with plasticity, suggesting flexible processing of signals in context for learning. In the basolateral (BL) amygdala, which has a role in fear learning, both hippocampal and A25 interacted preferentially with disinhibitory neurons, suggesting a boost in excitation. The two pathways diverged in innervating other classes of inhibitory neurons, suggesting circuit specificities that could become perturbed in psychiatric diseases.