Postsynaptic Responses Research Articles

FMRP acts as a key messenger for visceral pain modulation.

Visceral pain is a common clinical symptom, which is caused by mechanical stretch, spasm, ischemia and inflammation. Fragile X syndrome (FXS) with lack of fragile X mental retardation protein (FMRP) protein is an inherited disorder that is characterized by moderate or severe intellectual and developmental disabilities. Previous studies reported that FXS patients have self-injurious behavior, which may be associated with deficits in nociceptive sensitization. However, the role of FMRP in visceral pain is still unclear. In this study, the FMR1 knock out (KO) mice and SH-SY5Y cell line were employed to demonstrate the role of FMRP in the regulation of visceral pain. The data showed that FMR1 KO mice were insensitive to zymosan treatment. Recording in the anterior cingulate cortex (ACC), a structure involved in pain process, showed less presynaptic glutamate release and postsynaptic responses in the FMR1 KO mice as compared to the wild type (WT) mice after zymosan injection. Zymosan treatment caused enhancements of adenylyl cyclase 1 (AC1), a pain-related enzyme, and NMDA GluN2B receptor in the ACC. However, these up-regulations were attenuated in the ACC of FMR1 KO mice. Last, we found that zymosan treatment led to increase of FMRP levels in the ACC. These results were further confirmed in SH-SY5Y cells in vitro. Our findings demonstrate that FMRP is required for NMDA GluN2B and AC1 upregulation, and GluN2B/AC1/FMRP forms a positive feedback loop to modulate visceral pain.

Intracellular Recordings of Postsynaptic Voltage Responses at the Drosophila Neuromuscular Junction.

Axonal damage can cause a loss of neural control of target peripheral muscles and other organs. The hallmark of complete recovery from severe axonal injury is a successful return of function. To assay the degree of functional loss or recovery from injury, a measurement of electrical communication at the nerve-target junction can be used. Drosophila larval neuromuscular junction (NMJ) provides a genetically tractable and easily accessible model to measure the electrophysiological properties of the synapse. To study the functional consequences of injuries to the peripheral nerve, we describe the procedure to measure the spontaneous and evoked response to neurotransmitter release at the NMJ.

Intracellular Acidification Suppresses Synaptic Vesicle Mobilization in the Motor Nerve Terminals.

Intracellular protons play a special role in the regulation of presynaptic processes, since the functioning of synaptic vesicles and endosomes depends on their acidification by the H+-pump. Furthermore, transient acidification of the intraterminal space occurs during synaptic activity. Using microelectrode recording of postsynaptic responses (an indicator of neurotransmitter release) and exo-endocytic marker FM1-43, we studied the effects of intracellular acidification with propionate on the presynaptic events underlying neurotransmitter release. Cytoplasmic acidification led to a marked decrease in neurotransmitter release during the first minute of a 20-Hz stimulation in the neuromuscular junctions of mouse diaphragm and frog cutaneous pectoris muscle. This was accompanied by a reduction in the FM1-43 loss during synaptic vesicle exocytosis in response to the stimulation. Estimation of the endocytic uptake of FM1-43 showed no disruption in synaptic vesicle endocytosis. Acidification completely prevented the action of the cell-membrane permeable compound 24-hydroxycholesterol, which can enhance synaptic vesicle mobilization. Thus, the obtained results suggest that an increase in [H+]in negatively regulates neurotransmission due to the suppression of synaptic vesicle delivery to the sites of exocytosis at high activity. This mechanism can be a part of the negative feedback loop in regulating neurotransmitter release.

Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

Non-synaptic Plasticity in Leech Touch Cells.

The role of Na+/K+-pumps in activity-dependent synaptic plasticity has been described in both vertebrates and invertebrates. Here, we provide evidence that the Na+/K+-pump is also involved in activity-dependent non-synaptic cellular plasticity in leech sensory neurons. We show that the resting membrane potential (RMP) of T cells hyperpolarizes in response to repeated somatic current injection, while at the same time their spike count (SC) and the input resistance (IR) increase. Our Hodgkin–Huxley-type neuron model, adjusted to physiological T cell properties, suggests that repetitive action potential discharges lead to increased Na+/K+-pump activity, which then hyperpolarizes the RMP. In consequence, a slow, non-inactivating current decreases, which is presumably mediated by voltage-dependent, low-threshold potassium channels. Closing of these putative M-type channels due to hyperpolarization of the resting potential increases the IR of the cell, leading to a larger number of spikes. By this mechanism, the response behavior switches from rapidly to slowly adapting spiking. These changes in spiking behavior also effect other T cells on the same side of the ganglion, which are connected via a combination of electrical and chemical synapses. An increased SC in the presynaptic T cell results in larger postsynaptic responses (PRs) in the other T cells. However, when the number of elicited presynaptic spikes is kept constant, the PR does not change. These results suggest that T cells change their responses in an activity-dependent manner through non-synaptic rather than synaptic plasticity. These changes might act as a gain-control mechanism. Depending on the previous activity, this gain could scale the relative impacts of synaptic inputs from other mechanoreceptors, versus the spike responses to tactile skin stimulation. This multi-tasking ability, and its flexible adaptation to previous activity, might make the T cell a key player in a preparatory network, enabling the leech to perform fast behavioral reactions to skin stimulation.

Erbin in Amygdala Parvalbumin-Positive Neurons Modulates Anxiety-like Behaviors

BackgroundAnxiety disorders are the most common psychiatric diseases, affecting 28% of people worldwide within their lifetime. The excitation-inhibition imbalance in the amygdala is thought to be an underlying pathological mechanism; however, the cellular and molecular control of amygdala excitation-inhibition balance is largely unknown. MethodsBy using mice expressing chemogenetic activator or inhibitor channel in amygdala parvalbumin (PV) neurons, Erbin mutant mice, and mice with Erbin specifically knocked down in amygdala PV neurons, we systematically investigated the role of amygdala PV neurons and Erbin expressed therein in the pathogenesis of anxiety disorders using the combined approaches of immunohistochemistry, electrophysiology, and behavior. ResultsIn naïve mice, chemogenetic inhibition of PV neurons produced anxiogenic effects, suggesting an essential role in the regulation of anxiety. In stressed mice with anxiety, excitatory postsynaptic responses on amygdala PV neurons were selectively diminished, accompanied by a decreased expression of Erbin specifically in amygdala PV neurons. Remarkably, both Erbin mutant mice and amygdala PV–specific Erbin knockdown mice exhibited impaired excitatory postsynaptic responses on amygdala PV neurons and increased anxiety-like behaviors. Furthermore, chemogenetic activation of amygdala PV neurons normalized anxiety behaviors in amygdala PV–specific Erbin knockdown mice and stressed mice. ConclusionsTogether, these results demonstrate that Erbin in PV neurons is critical for maintaining the excitation-inhibition balance in the amygdala and reveal a novel pathophysiological mechanism for anxiety disorders.

Estradiol Enhances the Depolarizing Response to GABA and AMPA Synaptic Conductances in Arcuate Kisspeptin Neurons by Diminishing Voltage-Gated Potassium Currents.

Synaptic and intrinsic properties interact to sculpt neuronal output. Kisspeptin neurons in the hypothalamic arcuate nucleus help convey homeostatic estradiol feedback to central systems controlling fertility. Estradiol increases membrane depolarization induced by GABAA receptor activation in these neurons. We hypothesized that the mechanisms underlying estradiol-induced alterations in postsynaptic response to GABA, and also AMPA, receptor activation include regulation of voltage-gated potassium currents. Whole-cell recordings of arcuate kisspeptin neurons in brain slices from ovariectomized (OVX) and OVX+estradiol (OVX+E) female mice during estradiol negative feedback revealed that estradiol reduced capacitance, reduced transient and sustained potassium currents, and altered voltage dependence and kinetics of transient currents. Consistent with these observations, estradiol reduced rheobase and action potential latency. To study more directly interactions between synaptic and active intrinsic estradiol feedback targets, dynamic clamp was used to simulate GABA and AMPA conductances. Both GABA and AMPA dynamic clamp-induced postsynaptic potentials (PSPs) were smaller in neurons from OVX than OVX+E mice; blocking transient potassium currents eliminated this difference. To interrogate the role of the estradiol-induced changes in passive intrinsic properties, different Markov model structures based on the properties of the transient potassium current in cells from OVX or OVX+E mice were combined in silico with passive properties reflecting these two endocrine conditions. Some of tested models reproduced the effect on PSPs in silico, revealing that AMPA PSPs were more sensitive to changes in capacitance. These observations support the hypothesis that PSPs in arcuate kisspeptin neurons are regulated by estradiol-sensitive mechanisms including potassium conductances and membrane properties.SIGNIFICANCE STATEMENT Kisspeptin neurons relay estradiol feedback to gonadotropin-releasing hormone neurons, which regulate the reproductive system. The fast synaptic neurotransmitters GABA and glutamate rapidly depolarize arcuate kisspeptin neurons and estradiol increases this depolarization. Estradiol reduced both potassium current in the membrane potential range typically achieved during response to fast synaptic inputs and membrane capacitance. Using simulated GABA and glutamate synaptic inputs, we showed changes in both the passive and active intrinsic properties induced by in vivo estradiol treatment affect the response to synaptic inputs, with capacitance having a greater effect on response to glutamate. The suppression of both passive and active intrinsic properties by estradiol feedback thus renders arcuate kisspeptin neurons more sensitive to fast synaptic inputs.

Self-powered, wireless-control, neural-stimulating electronic skin for in vivo characterization of synaptic plasticity

Synaptic plasticity underlies brain changes when learning or memory takes place. Long-term memory requires modification of synaptic strengths between neurons. The characterization of the synaptic changes therefore reflects the long term storage of information in the brain structures. Traditionally, electrical neural-stimulating technique for characterizing synaptic plasticity requires external power source and steer-by-wire system. Here, a novel self-powered, wireless-control, neural-stimulating electronic skin (e-skin) for in vivo characterization of synaptic plasticity has been presented. The e-skin is composed of flexible photosensitive-triboelectric MAPbI3/PDMS units. The outputting electrical neural-stimulating signal of the e-skin is generated by human body activities without any batteries, and the neural-modulation can be controlled by photo illumination on/off as wireless switch. To demonstrate the application of the e-skin in characterizing synaptic changes, we connect the device to the hippocampus of the mouse brain. The e-skin neural stimulating in the CA3 of mouse hippocampus and simultaneous recording field excitatory postsynaptic potentials (fEPSP) in the CA1 demonstrate that the e-skin can successfully elicit post-synaptic responses for in vivo characterization of synaptic plasticity. This self-powered photo-operate e-skin can provoke a new research direction for realizing battery-free, wireless-control, electrical neural-stimulating systems with implications in biomedical engineering and neural science.

Early-Stage Ocular Hypertension Alters Retinal Ganglion Cell Synaptic Transmission in the Visual Thalamus.

Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma, where elevated intraocular pressure (ocular hypertension, OHT) stresses retinal ganglion cell (RGC) axons as they exit the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy, transport inhibition, and gliosis. Importantly, many of these changes appear to occur prior to irreversible neuronal loss, making them promising points for early diagnosis of glaucoma. It is unknown whether OHT has similarly early effects on the function of RGC output to the brain. To test this possibility, we elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. Five weeks post-injection, bead-injected eyes showed a modest RGC loss in the peripheral retina, as evidenced by RBPMS antibody staining. Additionally, we observed reduced dendritic complexity and lower spontaneous spike rate of On-αRGCs, targeted for patch clamp recording and dye filling using a Opn4-Cre reporter mouse line. To determine the influence of OHT on retinal projections to the brain, we expressed Channelrhodopsin-2 (ChR2) in melanopsin-expressing RGCs by crossing the Opn4-Cre mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 460 nm light. The use of a Opn4-Cre reporter system allowed for expression of ChR2 in a narrow subset of RGCs responsible for image-forming vision in mice. Five weeks following OHT induction, paired pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability at retinogeniculate synapses was elevated. Additionally, miniature synaptic current frequency was slightly reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons, assessed using a Sholl analysis, showed a reduced complexity. Strikingly, these changes occurred prior to major loss of RGCs labeled with the Opn4-Cre mouse, as indicated by immunofluorescence staining of ChR2-expressing retinal neurons. Thus, OHT leads to pre- and post-synaptic functional and structural changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport changes, these retinogeniculate synaptic changes are among the earliest signs of glaucoma.

Enhancement of the antipsychotic effect of risperidone by sodium nitroprusside in rats

Recently, a single injection of the nitric oxide donor sodium nitroprusside (SNP) was found to induce a rapid and sustained antipsychotic effect in treatment-resistant schizophrenia (TRS). Moreover, a single i.p. injection of SNP in rats was found to generate both rapid and persisting changes in brain synaptic plasticity, including enhanced excitatory postsynaptic current responses and spine morphology in layer V pyramidal cells in the medial prefrontal cortex (mPFC) brain slices. Here we used the conditioned avoidance response (CAR) test in rats to investigate the antipsychotic-like efficacy of SNP in combination with low-dose risperidone. In addition, we performed microdialysis experiments in freely moving rats to measure neurotransmitter efflux in the mPFC and the nucleus accumbens (NAc). Risperidone caused only 20% suppression of CAR, which is far below the degree of CAR suppression required to predict a significant clinical antipsychotic effect. Addition of a low dose of SNP to risperidone dramatically enhanced the antipsychotic-like effect to a clinically relevant level. SNP significantly enhanced the risperidone-induced dopamine output in the mPFC but not in the NAc. The increased prefrontal dopamine release induced by the drug combination may also improve cognition as indicated by previous preclinical and clinical studies and, furthermore, via enhanced synaptic spine function and morphology in mPFC generate a both rapid and prolonged antipsychotic and pro-cognitive effect. Our results delineate SNP as a promising new treatment option for schizophrenia, including TRS, when added to currently available antipsychotic medication in order to improve efficacy at maintained or even reduced dosage.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long‐range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement‐related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike‐time arrivals through long‐range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Energetic substrate availability regulates synchronous activity in an excitatory neural network.

Neural networks are required to meet significant metabolic demands associated with performing sophisticated computational tasks in the brain. The necessity for efficient transmission of information imposes stringent constraints on the metabolic pathways that can be used for energy generation at the synapse, and thus low availability of energetic substrates can reduce the efficacy of synaptic function. Here we study the effects of energetic substrate availability on global neural network behavior and find that glucose alone can sustain excitatory neurotransmission required to generate high-frequency synchronous bursting that emerges in culture. In contrast, obligatory oxidative energetic substrates such as lactate and pyruvate are unable to substitute for glucose, indicating that processes involving glucose metabolism form the primary energy-generating pathways supporting coordinated network activity. Our experimental results are discussed in the context of the role that metabolism plays in supporting the performance of individual synapses, including the relative contributions from postsynaptic responses, astrocytes, and presynaptic vesicle cycling. We propose a simple computational model for our excitatory cultures that accurately captures the inability of metabolically compromised synapses to sustain synchronous bursting when extracellular glucose is depleted.

Glutamate/GABA co-release selectively influences postsynaptic glutamate receptors in mouse cortical neurons

Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance.

Experience-Dependent Structural Plasticity of Adult-Born Neurons in the Aging Hippocampus.

Synaptic modification in cortical structures underlies the acquisition of novel information that results in learning and memory formation. In the adult dentate gyrus, circuit remodeling is boosted by the generation of new granule cells (GCs) that contribute to specific aspects of memory encoding. These forms of plasticity decrease in the aging brain, where both the rate of adult neurogenesis and the speed of morphological maturation of newly generated neurons decline. In the young-adult brain, a brief novel experience accelerates the integration of new neurons. The extent to which such degree of plasticity is preserved in the aging hippocampus remains unclear. In this work, we characterized the time course of functional integration of adult-born GCs in middle-aged mice. We performed whole-cell recordings in developing GCs from Ascl1CreERT2;CAGfloxStopTom mice and found a late onset of functional excitatory synaptogenesis, which occurred at 4 weeks (vs. 2 weeks in young-adult mice). Overall mature excitability and maximal glutamatergic connectivity were achieved at 10 weeks. In contrast, large mossy fiber boutons (MFBs) in CA3 displayed mature morphological features including filopodial extensions at 4 weeks, suggesting that efferent connectivity develops faster than afference. Notably, new GCs from middle-aged mice exposed to enriched environment for 7 days showed an advanced degree of maturity at 3 weeks, revealed by the high frequency of excitatory postsynaptic responses, complex dendritic trees, and large size of MFBs with filopodial extensions. These findings demonstrate that adult-born neurons act as sensors that transduce behavioral stimuli into major network remodeling in the aging brain.

P2X2 Receptor Expression and Function Is Upregulated in the Rat Supraoptic Nucleus Stimulated Through Refeeding After Fasting.

Magnocellular neurons in the supraoptic nucleus (SON), which synthesize and release arginine vasopressin (AVP) and oxytocin (OT), express several subtypes of ATP-stimulated purinergic P2X receptors (P2XR) that modulate neuronal activity as well as neurotransmitter and hormone release. However, the physiological impact of this modulation is not well understood. Here, we tested a hypothesis that P2XRs play a role in the sustained release of hormones from SON neurons stimulated through fasting/refeeding. We studied the effect of 2 h of refeeding after 48 h of fasting on P2XR and P2YR mRNA expression and ATP-induced presynaptic and postsynaptic responses in the SON of 30-day-old rats. Quantitative real-time PCR revealed that the expression of P2X2R and AVP mRNA was upregulated, whereas P2X4R, P2X7R, P2Y2R, and OT mRNA levels were not significantly changed and P2Y1R mRNA expression was decreased. Whole-cell patch clamp recordings performed on isolated rat brain slices showed that the amplitude of the ATP-stimulated somatic current and the ATP-induced increases in the frequency of spontaneous GABAergic inhibitory postsynaptic currents were significantly higher in SON neurons from fasted/refed rats than in SON neurons from normally fed rats. No evidence was found for changes in the presynaptic effect of ATP in SON neurons not expressing somatic P2XRs. These results suggest that the increased activity of SON neurons synthesizing AVP is associated with enhanced expression of P2X2Rs on neuronal cell bodies and their GABAergic presynaptic nerve terminals.

Infection-induced endothelial amyloids impair memory.

Patients with nosocomial pneumonia exhibit elevated levels of neurotoxic amyloid and tau proteins in the cerebrospinal fluid (CSF). In vitro studies indicate that pulmonary endothelium infected with clinical isolates of either Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus produces and releases cytotoxic amyloid and tau proteins. However, the effects of the pulmonary endothelium-derived amyloid and tau proteins on brain function have not been elucidated. Here, we show that P. aeruginosa infection elicits accumulation of detergent insoluble tau protein in the mouse brain and inhibits synaptic plasticity. Mice receiving endothelium-derived amyloid and tau proteins via intracerebroventricular injection exhibit a learning and memory deficit in object recognition, fear conditioning, and Morris water maze studies. We compared endothelial supernatants obtained after the endothelia were infected with P. aeruginosa possessing an intact [P. aeruginosa isolated from patient 103 (PA103) supernatant] or defective [mutant strain of P. aeruginosa lacking a functional type 3 secretion system needle tip complex (ΔPcrV) supernatant] type 3 secretion system. Whereas the PA103 supernatant impaired working memory, the ΔPcrV supernatant had no effect. Immunodepleting amyloid or tau proteins from the PA103 supernatant with the A11 or T22 antibodies, respectively, overtly rescued working memory. Recordings from hippocampal slices treated with endothelial supernatants or CSF from patients with or without nosocomial pneumonia indicated that endothelium-derived neurotoxins disrupted the postsynaptic synaptic response. Taken together, these results establish a plausible mechanism for the neurologic sequelae consequent to nosocomial bacterial pneumonia.-Balczon, R., Pittet, J.-F., Wagener, B. M., Moser, S. A., Voth, S., Vorhees, C. V., Williams, M. T., Bridges, J. P., Alvarez, D. F., Koloteva, A., Xu, Y., Zha, X.-M., Audia, J. P., Stevens, T., Lin, M. T. Infection-induced endothelial amyloids impair memory.

Slow presynaptic mechanisms that mediate adaptation in the olfactory pathway of Drosophila

The olfactory system encodes odor stimuli as combinatorial activity of populations of neurons whose response depends on stimulus history. How and on which timescales previous stimuli affect these combinatorial representations remains unclear. We use in vivo optical imaging in Drosophila to analyze sensory adaptation at the first synaptic step along the olfactory pathway. We show that calcium signals in the axon terminals of olfactory receptor neurons (ORNs) do not follow the same adaptive properties as the firing activity measured at the antenna. While ORNs calcium responses are sustained on long timescales, calcium signals in the postsynaptic projection neurons (PNs) adapt within tens of seconds. We propose that this slow component of the postsynaptic response is mediated by a slow presynaptic depression of vesicle release and enables the combinatorial population activity of PNs to adjust to the mean and variance of fluctuating odor stimuli.

A Tripartite Interaction Among the Calcium Channel α1- and β-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion.

Neurotransmitter release is initiated by the influx of Ca2+ via voltage-gated calcium channels. The accessory β-subunit (CaVβ) of these channels shapes synaptic transmission by associating with the pore-forming subunit (CaVα1) and up-regulating presynaptic calcium currents. Besides CaVα1, CaVβ interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of CaVβ/F-actin association on synaptic transmission has not yet been explored. We here study how CaVβ4, the major calcium channel β isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous CaVβ4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to CaVα1. We found that exogenously expressed wild-type CaVβ4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. CaVβ4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by CaVβ4 is abolished upon disruption of the actin cytoskeleton. The CaVα1 association-deficient CaVβ4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between CaVβ4 and F-actin also support the recruitment of SVs to the RRP in a CaVα1-independent manner. Our studies show an emerging role of CaVβ in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.

Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses

AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing, we show that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated,but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1β and neurexin-3β SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses.

Postsynaptic Mechanisms Render Syn I/II/III Mice Highly Responsive to Psychostimulants.

BackgroundSynapsins are encoded by SYN I, SYN II, and SYN III, and they regulate neurotransmitter release by maintaining a reserve pool of synaptic vesicles.MethodsPresynaptic dopamine responses to cocaine were examined by microdialysis, and postsynaptic responses were evaluated to various dopamine receptor agonists in the open field with SynI/SynII/SynIII triple knockout mice.ResultsTriple knockout mice showed enhanced spontaneous locomotion in a novel environment and were hyper-responsive to indirect and direct D1 and D2 dopamine agonists. Triple knockout animals appeared sensitized to cocaine upon first open field exposure; sensitization developed across days in wild-type controls. When mutants were preexposed to a novel environment before injection, cocaine-stimulated locomotion was reduced and behavioral sensitization retarded. Baseline dopamine turnover was enhanced in mutants and novel open field exposure increased their striatal dopamine synthesis rates. As KCl-depolarization stimulated comparable dopamine release in both genotypes, their readily releasable pools appeared indistinguishable. Similarly, cocaine-induced hyperlocomotion was indifferent to blockade of newly synthesized dopamine and depletion of releasable dopamine pools. Extracellular dopamine release was similar in wild-type and triple knockout mice preexposed to the open field and given cocaine or placed immediately into the arena following injection. Since motor effects to novelty and psychostimulants depend upon frontocortical-striatal inputs, we inhibited triple knockout medial frontal cortex with GABA agonists. Locomotion was transiently increased in cocaine-injected mutants, while their supersensitive cocaine response to novelty was lost.ConclusionsThese results reveal presynaptic dopamine release is not indicative of agonist-induced triple knockout hyperlocomotion. Instead, their novelty response occurs primarily through postsynaptic mechanisms and network effects.