Miniature Postsynaptic Currents Research Articles

Estrogen Receptor Beta and 2-arachidonoylglycerol Mediate the Suppressive Effects of Estradiol on Frequency of Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons of Metestrous Mice: An Acute Slice Electrophysiological Study.

Gonadotropin-releasing hormone (GnRH) neurons are controlled by 17β-estradiol (E2) contributing to the steroid feedback regulation of the reproductive axis. In rodents, E2 exerts a negative feedback effect upon GnRH neurons throughout the estrus-diestrus phase of the ovarian cycle. The present study was undertaken to reveal the role of estrogen receptor subtypes in the mediation of the E2 signal and elucidate the downstream molecular machinery of suppression. The effect of E2 administration at low physiological concentration (10 pM) on GnRH neurons in acute brain slices obtained from metestrous GnRH-green fluorescent protein (GFP) mice was studied under paradigms of blocking or activating estrogen receptor subtypes and interfering with retrograde 2-arachidonoylglycerol (2-AG) signaling. Whole-cell patch clamp recordings revealed that E2 significantly diminished the frequency of spontaneous postsynaptic currents (sPSCs) in GnRH neurons (49.62 ± 7.6%) which effect was abolished by application of the estrogen receptor (ER) α/β blocker Faslodex (1 μM). Pretreatment of the brain slices with cannabinoid receptor type 1 (CB1) inverse agonist AM251 (1 μM) and intracellularly applied endocannabinoid synthesis blocker THL (10 μM) significantly attenuated the effect of E2 on the sPSCs. E2 remained effective in the presence of tetrodotoxin (TTX) indicating a direct action of E2 on GnRH cells. The ERβ specific agonist DPN (10 pM) also significantly decreased the frequency of miniature postsynaptic currents (mPSCs) in GnRH neurons. In addition, the suppressive effect of E2 was completely blocked by the selective ERβ antagonist PHTPP (1 μM) indicating that ERβ is required for the observed rapid effect of the E2. In contrast, the ERα agonist PPT (10 pM) or the membrane-associated G protein-coupled estrogen receptor (GPR30) agonist G1 (10 pM) had no significant effect on the frequency of mPSCs in these neurons. AM251 and tetrahydrolipstatin (THL) significantly abolished the effect of E2 whereas AM251 eliminated the action of DPN on the mPSCs. These data suggest the involvement of the retrograde endocannabinoid mechanism in the rapid direct effect of E2. These results collectively indicate that estrogen receptor beta and 2-AG/CB1 signaling mechanisms are coupled and play an important role in the mediation of the negative estradiol feedback on GnRH neurons in acute slice preparation obtained from intact, metestrous mice.

Electrophysiology of Patterned Neuronal Networks on Monolayer Graphene

Graphene is an ideal candidate for next generation of electronic devices and of biosensors. Interfacing graphene with living cells and understanding its influence on their functionality is a keypoint for such applications. Here, we study the effect of a monolayer graphene surface onto the synaptogenesis of neural networks from primary neurons. Based on our previous work, we produced by UV single pulse laser ablation large patterned areas substrate, alternating microsized graphene stripes with and without graphene [1]. Interestingly, it was observed that neurons adhere evenly on the substrate but later they migrates and follow the patterned graphene region, suggesting a better anchorage of neurons to polylysine-coated graphene surface. Neurons functionality was measured by recording the miniature post-synaptic currents (mPSCs) by whole-cell patch-clamp recordings from DIV4 to DIV13. On control samples (i.e. on glass) synaptogenesis, indicated by an abrupt increase of both Glutamatergic and GABAergic mPSCs was first observed between DIV 8 and DIV 10. On SLG, instead, the mPSCs increase was observed already at DIV7 and became significant at DIV9, indicating that SLG has a boosting effect on the synaptogenesis. This result was confirmed on the stripes of patterned SLG, where synaptogenesis followed the same trend as on homogeneous SLG; no appreciable activity was recorded from neurons on the ablated areas.1. Matteo Lorenzoni, Fernando Brandi, Silvia Dante, Andrea Giugni, and Bruno Torre. “Simple and effective graphene laser processing for neuron patterning application.” Scientific reports: 2013; 3: 1954

Alterations of functional properties of hippocampal networks following repetitive closed-head injury

Traumatic brain injury (TBI) is the leading cause of death for persons under the age of 45. Military service members who have served on multiple combat deployments and contact-sport athletes are at particular risk of sustaining repetitive TBI (rTBI). Cognitive and behavioral deficits resulting from rTBI are well documented. Optimal associative LTP, occurring in the CA1 hippocampal Schaffer collateral pathway, is required for both memory formation and retrieval. Surprisingly, ipsilateral Schaffer collateral CA1 LTP evoked by 100Hz tetanus was enhanced in mice from the 3× closed head injury (3× CHI) treatment group in comparison to LTP in contralateral or 3× Sham CA1 area, and in spite of reduced freezing during contextual fear conditioning at one week following 3× CHI. Electrophysiological activity of CA1 neurons was evaluated with whole-cell patch-clamp recordings. 3× CHI ipsilateral CA1 neurons exhibited significant increases in action potential amplitude and maximum rise and decay slope while the action potential duration was decreased. Recordings of CA1 neuron postsynaptic currents were conducted to detect spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs/sIPSCs) and respective miniature currents (mEPSCs and mIPSCs). In the 3× CHI mice, sEPSCs and sIPSCs in ipsilateral CA1 neurons had an increased frequency of events but decreased amplitudes. In addition, 3× CHI altered the action potential-independent miniature postsynaptic currents. The mEPSCs of ipsilateral CA1 neurons exhibited both an increased frequency of events and larger amplitudes. Moreover, the effect of 3× CHI on mIPSCs was opposite to that of the sIPSCs. Specifically, the frequency of the mIPSCs was decreased while the amplitudes were increased. These results are consistent with a mechanism in which repetitive closed-head injury affects CA1 hippocampal function by promoting a remodeling of excitatory and inhibitory synaptic inputs leading to impairment in hippocampal-dependent tasks.

Restoring GABA tone rescues hippocampal long-term depression impaired after maternal immune stress

Rat maternal immune challenge with lipopolysaccharide (LPS) led to an early disturbance of glutamatergic synaptic plasticity in the hippocampus of male offspring (Lante et al., 2007, 2008). This was evidenced by a premature loss of the ability to exhibit long-term depression (LTD) (Escobar et al., 2011), which occurred between the second and the third postnatal weeks instead of after the first postnatal month. We hypothesized this would be related to GABAergic system deficiency. Sprague Dawley rats received either 500 μg kg−1 LPS or 2 ml kg−1 isotonic saline ip on gestational day 19. Male offspring's hippocampus was studied between postnatal days 12–25. GABAergic interneuron density was determined by stereology. GABAergic transmission and the effect of tiagabine on glutamatergic synaptic plasticity were assessed with patch clamp and extracellular recordings respectively. Prenatal LPS triggered a clear deficit of GABAergic interneurons predominant in the CA3 area of the hippocampus, associated with presynaptic GABAergic transmission deficiency, as shown by reduced evoked inhibitory post synaptic currents and by a reduction of the frequency of miniature post synaptic currents. Of note, GABAA and GABAB receptors and GABA transporters appeared mainly unaffected. Increasing ambient GABA by impairing GABA re-uptake with tiagabine did not interact with the occurrence of LTD in normal animals in this developmental window, but rescued the impaired synaptic plasticity observed in LPS-challenged rats. Deficiency in tonic actions of GABA seems to be central to the dysregulation of synaptic plasticity observed after immune prenatal challenge. Modulating GABAergic tone may be a valuable therapeutic strategy for the cognitive impairment associated with this condition.

EFFECT OF HYPOXIA ON SYNAPTIC TRANSMISSION BETWEEN RETINAL GANGLION CELLS AND SUPERIOR COLLICULUS NEURONS IN COCULTURE

In this study we conducted a series of experiments to characterize the effect and define the mechanisms of hypoxia on synaptic transmission between retinal ganglion cells and superior colliculus (SC) neurons. Application of hypoxic solution leads to a long lasting potentiation (LTP) NMDA-mediated synaptic transmission. Analysis of the oxygen deficiency effect on the spontaneous and miniature postsynaptic currents (sPSC and mPSC respectively) revealed an increase in the frequency of their occurrence and the appearance of the second peak in the mPSC histogram distribution. The assessment of quantum and binomial parameters reflects the complex pre- and postsynaptic changes during the potentiation, independent of the release probability. Most likely this LTP can be caused by an increase in the total number of active synapses. Glutamatergic synaptic transmission mediated by non-NMDA activation receptor-channel complexes, responded to application of deoxygenated solution by the brief depression, which is the result of presynaptic dysfunction and associates with decrease in release probability and number of active zones. GABAergic synaptic transmission mediated by activation GABA(A)-receptor-channel complexes, responded to hypoxic action by long term depression (LTD). Analysis of sPSC and mPSC showed a significant decrease in the frequency of their occurrence and significant (P = 0.05) decrease in the quantum over a period of oxygen deficiency. In general, the effect of hypoxia-induced LTD of GABAergic synaptic transmission is based on complex changes of presynaptic (independent on the release probability) and postsynaptic (reduction sensitivity of receptors in postsynaptic membrane) mechanisms.

Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons

In rat thalamic paraventricular nucleus of thalamus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances excitability via concurrent decrease in G protein-coupled inwardly-rectifying potassium (GIRK)-like and activation of transient receptor potential cation (TRPC)4/5-like cationic conductances. An exploration of intracellular signaling pathways revealed the TRH-induced current to be insensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitors, but reduced by D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC). A corresponding change in the I–V relationship implied suppression of the cationic component of the TRH-induced current. Diacylglycerol (DAG) is a product of the hydrolysis of PC. Studies focused on the isolated cationic component of the TRH-induced response revealed a reduction by RHC80267, an inhibitor of DAG lipase, the enzyme involved in the hydrolysis of DAG to the endocannabinoid 2-arachidonoylglycerol (2-AG). Further investigation revealed enhancement of the cationic component in the presence of either JZL184 or WWL70, inhibitors of enzymes involved in the hydrolysis of 2-AG. A decrease in the TRH-induced response was noted in the presence of rimonabant or SR144528, membrane permeable CB1 and CB2 receptor antagonists, respectively. A decrease in the TRH-induced current by intracellular, but not by bath application of the membrane impermeable peptide hemopressin, selective for CB1 receptors, suggests a postsynaptic intracellular localization of these receptors. The TRH-induced current was increased in the presence of arachidonyl-2′-chloroethylamide (ACEA) or JWH133, CB1 and CB2 receptor agonists, respectively. The PI3-kinase inhibitor LY294002, known to inhibit TRPC translocation, decreased the response to TRH. In addition, a TRH-induced enhancement of the low-threshold spike was prevented by both rimonabant, and SR144528. TRH had no influence on excitatory or inhibitory miniature postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels.

Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat.

To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells.

Adenosine-mediated modulation of ventral horn interneurons and spinal motoneurons in neonatal mice.

Neuromodulation allows neural networks to adapt to varying environmental and biomechanical demands. Purinergic signaling is known to be an important modulatory system in many parts of the CNS, including motor control circuitry. We have recently shown that adenosine modulates the output of mammalian spinal locomotor control circuitry (Witts EC, Panetta KM, Miles GB. J Neurophysiol 107: 1925–1934, 2012). Here we investigated the cellular mechanisms underlying this adenosine-mediated modulation. Whole cell patch-clamp recordings were performed on ventral horn interneurons and motoneurons within in vitro mouse spinal cord slice preparations. We found that adenosine hyperpolarized interneurons and reduced the frequency and amplitude of synaptic inputs to interneurons. Both effects were blocked by the A1-type adenosine receptor antagonist DPCPX. Analysis of miniature postsynaptic currents recorded from interneurons revealed that adenosine reduced their frequency but not amplitude, suggesting that adenosine acts on presynaptic receptors to modulate synaptic transmission. In contrast to interneurons, recordings from motoneurons revealed an adenosine-mediated depolarization. The frequency and amplitude of synaptic inputs to motoneurons were again reduced by adenosine, but we saw no effect on miniature postsynaptic currents. Again these effects on motoneurons were blocked by DPCPX. Taken together, these results demonstrate differential effects of adenosine, acting via A1 receptors, in the mouse spinal cord. Adenosine has a general inhibitory action on ventral horn interneurons while potentially maintaining motoneuron excitability. This may allow for adaptation of the locomotor pattern generated by interneuronal networks while helping to ensure the maintenance of overall motor output.

Role of Steroids in Hyperexcitatory Adverse and Anesthetic Effects of Sevoflurane in Neonatal Rats

Recent studies have demonstrated that long-term developmental effects of neonatal anesthesia were more prominent in males. We tested whether steroids, in general, and sex steroids, in particular, are involved in the mediation of sevoflurane-caused paradoxical cortical seizures during the early postnatal period. Methods: Cortical electroencephalograms, hippocampal synaptic activity, serum levels of steroids and the loss of the righting reflex (LORR), a marker of anesthetic effect, were measured on postnatal days 4-6 in Sprague Dawley rats of both genders exposed to 2.1% sevoflurane. Results: Episodes of seizures, persistent spikes in electroencephalograms and increases in serum corticosterone were similar in both genders. In the order of increasing potency, the corticosteroid receptor antagonist RU 28318, the estradiol receptor antagonist ICI 182780 and the estradiol synthesis inhibitor formestane decreased sevoflurane-induced seizures. Exogenous estradiol increased sevoflurane-caused seizures, spikes and serum levels of corticosterone. These estradiol-enhanced seizures and spikes were depressed by ICI 182780 and the NKCC1 inhibitor, bumetanide, while RU 28318 decreased seizures only. In hippocampal CA1 neurons, estradiol increased the amplitude, rise time and area under the curve of gamma-aminobutyric acid type A receptor (GABA_AR)-mediated miniature postsynaptic currents. Exogenous estradiol shortened, while ICI 182780 and formestane lengthened the time needed for sevoflurane to induce LORR. Conclusion: These findings provide evidence for gender-independent acute electroencephalographic effects of sevoflurane at this age. Corticosterone and estradiol are involved in the mediation of sevoflurane-induced seizures. Estradiol, but not corticosterone, also contributes to sevoflurane-caused spikes, by enhancing GABA_AR-mediated excitation in the cortex. By increasing GABA_AR-mediated inhibition in more mature caudal regions of the brain, estradiol contributes to sevoflurane-induced LORR.

Synthetic ciguatoxin CTX 3C induces a rapid imbalance in neuronal excitability.

Ciguatera is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs), sodium channel activator toxins. Symptoms of ciguatera include neurological alterations such as paraesthesiae, dysaesthesiae, depression, and heightened nociperception, among others. An important issue to understand these long-term neurological alterations is to establish the role that changes in activity produced by CTX 3C represent to neurons. Here, the effects of synthetic ciguatoxin CTX 3C on membrane potential, spontaneous spiking, and properties of synaptic transmission in cultured cortical neurons of 11-18 days in vitro (DIV) were evaluated using electrophysiological approaches. CTX 3C induced a large depolarization that decreased neuronal firing and caused a rapid inward tonic current that was primarily GABAergic. Moreover, the toxin enhanced the amplitude of miniature postsynaptic inhibitory currents (mIPSCs), whereas it decreased the amplitude of miniature postsynaptic excitatory currents (mEPSCs). The frequency of mIPSCs increased, whereas the frequency of mEPSCs remained unaltered. We describe, for the first time, that a rapid membrane depolarization caused by CTX 3C in cortical neurons activates mechanisms that tend to suppress electrical activity by shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition. Indeed, these results suggest that the acute effects of CTX on synaptic transmission could underlie some of the neurological symptoms caused by ciguatera in humans.

Carbon monoxide augments electrical signaling in cultured neural networks of hippocampal neurons partly through activation of BKCa channels.

Carbon monoxide (CO) is often viewed as a lethal gas in light of its capacity to prevent oxygen uptake in hemoglobin; however, it also functions to regulate a variety of proteins and physiological processes. Here we show that CO is an important chemical cue, to which neurons respond strongly, and this response is then integrated into neural network activity. In cultured mouse hippocampal neurons, CO enhanced synchronized spontaneous cytosolic Ca(2+) oscillations which arose from periodic action potentials through synaptic transmission. We used single-cell patch-clamp recording to investigate the neural network. Our results showed that the frequency of spontaneous and miniature post synaptic current was increased in neurons cultured for 14-18 days after addition of CO, with no change in current amplitude. BK channels have recently been demonstrated to be important in the action of CO. Our results showed that the effect of CO on neural network electrical activity was partly abolished after blocking the BK channels. Altogether, our results suggest that CO can influence neural network electrical activity and that BK channels participate in this regulation process.

Cellular plasticity induced by anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis antibodies.

ObjectiveAutoimmune-mediated anti–α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood.MethodsThe effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses.ResultsWe show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase.InterpretationThese results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder.

Multiple types of GABAA responses identified from zebrafish Mauthner cells.

γ-Aminobutyric acid (GABA) binds to ionotropic GABAA receptors to mediate fast inhibitory synaptic transmission in the central nervous system (CNS). GABAA receptors are pentameric structures composed of receptor subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3) with various stoichiometries. They play important roles in the control of neural networks and are the pharmacological targets for the treatment of diseases such as epilepsy, autism, and schizophrenia. Thus far, there has been no report on GABA synaptic transmission in developing zebrafish. Here we used whole-cell patch-clamp electrophysiology to record GABAA-mediated miniature postsynaptic currents from the Mauthner cells of embryonic zebrafish. Spontaneous GABAA currents occurred infrequently and were low in amplitude (27.2 ± 0.9 pA). Analysis of their kinetics suggested the existence of three main types of events: the first (group I) is mediated by a single type of receptor with decay kinetics of 54 ± 1.6 ms; the second (group II) is also mediated by a single receptor type, but exhibits significantly longer decay kinetics (151 ± 7.2 ms); and the third type of synapse (group III) contains multiple receptor types with fast (τ1=28.7 ± 2.5 ms) and slow (τ2=153 ± 11 ms) kinetics. Thus, for the first time, we report the properties of GABA synaptic currents associated with the Mauthner cells of zebrafish.

Modulation of network activity and induction of homeostatic synaptic plasticity by enzymatic removal of heparan sulfates.

Heparan sulfates (HSs) are complex and highly active molecules that are required for synaptogenesis and long-term potentiation. A deficit in HSs leads to autistic phenotype in mice. Here, we investigated the long-term effect of heparinase I, which digests highly sulfated HSs, on the spontaneous bioelectrical activity of neuronal networks in developing primary hippocampal cultures. We found that chronic heparinase treatment led to a significant reduction of the mean firing rate of neurons, particularly during the period of maximal neuronal activity. Furthermore, firing pattern in heparinase-treated cultures often appeared as epileptiform bursts, with long periods of inactivity between them. These changes in network activity were accompanied by an increase in the frequency and amplitude of miniature postsynaptic excitatory currents, which could be described by a linear up-scaling of current amplitudes. Biochemically, we observed an upregulation in the expression of the glutamate receptor subunit GluA1, but not GluA2, and a strong increase in autophosphorylation of α and β Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), without changes in the levels of kinase expression. These data suggest that a deficit in HSs triggers homeostatic synaptic plasticity and drastically affects functional maturation of neural network.

Adrenoreceptor modulation of oromotor pathways in the rat medulla.

Regulation of feeding behavior involves the integration of multiple physiological and neurological pathways that control both nutrient-seeking and consummatory behaviors. The consummatory phase of ingestion includes stereotyped oromotor movements of the tongue and jaw that are controlled through brain stem pathways. These pathways encompass not only cranial nerve sensory and motor nuclei for processing feeding-related afferent signals and supplying the oromotor musculature but also reticular neurons for orchestrating ingestion and coordinating it with other behaviors that utilize the same musculature. Based on decerebrate studies, this circuit should be sensitive to satiety mechanisms mediated centrally by A2 noradrenergic neurons in the caudal nucleus of the solitary tract (cNST) that are potently activated during satiety. Because the first observable phase of satiety is inhibition of oromotor movements, we hypothesized that norepinephrine (NE) would act to inhibit prehypoglossal neurons in the medullary reticular formation. Using patch-clamp electrophysiology of retrogradely labeled prehypoglossal neurons and calcium imaging to test this hypothesis, we demonstrate that norepinephrine can influence both pre- and postsynaptic properties of reticular neurons through both α1- and α2-adrenoreceptors. The α1-adrenoreceptor agonist phenylephrine (PE) activated an inward current in the presence of TTX and increased the frequency of both inhibitory and excitatory miniature postsynaptic currents. The α2-adrenoreceptor agonist dexmedetomidine (DMT) inhibited cNST-evoked excitatory currents as well as spontaneous and miniature excitatory currents through presynaptic mechanisms. The diversity of adrenoreceptor modulation of these prehypoglossal neurons may reflect their role in a multifunctional circuit coordinating both ingestive and respiratory lingual function.

The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer™5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.

Lateral Mobility of Presynaptic α7-Containing Nicotinic Receptors and Its Relevance for Glutamate Release

Surface diffusion of postsynaptic receptors shapes synaptic transmission. Presynaptic receptors also influence transmission, but the relevance of their mobility for synaptic function is unknown. Using single-particle tracking with quantum dots, we show that calcium-permeable α7-containing nicotinic acetylcholine receptors (α7-nAChRs), capable of promoting transmitter release, are mobile on presynaptic terminals but constrained in synaptic space on rat hippocampal neurons in culture. Additional immobilization of presynaptic α7-nAChRs by antibody crosslinking increases glutamate release capacity as seen in the frequency of spontaneous miniature postsynaptic currents and the size of the readily releasable pool of transmitter. Conversely, blocking glutamate release by targeting tetanus toxin to individual synapses increases α7-nAChR dwell time at presynaptic sites. The effects on release require functional α7-nAChRs and may to depend on CAST/ELKS (calpastatin/glutamine, leucine, lysine, and serine-rich protein), which an unbiased proteomic screen yielded. The results support a new homeostatic regulatory mechanism in which α7-nAChR restrain may be adjusted as needed at presynaptic sites via active zone proteins to maintain transmitter release capability.

Long-lasting distortion of GABA signaling in MS/DB neurons after binge-like ethanol exposure during initial synaptogenesis

Using a well-established model of binge-like ethanol treatment of rat pups on postnatal days (PD) 4–9, we found that maturation of GABAA receptor (GABAAR) miniature postsynaptic currents (mPSCs) was substantially blunted for medial septum/diagonal band (MS/DB) neurons in brain slices on PD 11–16. Ethanol reduced mPSC amplitude, frequency, and decay kinetics, while attenuating or exaggerating allosteric actions of zolpidem and allopregnanolone, respectively. The impact of ethanol in vivo was long lasting as most changes in MS/DB GABAAR mPSCs were still observed as late as PD 60–85. Maturing MS/DB neurons in naïve brain slices PD 4–16 showed increasing mPSC frequency, decay kinetics, and zolpidem sensitivity that were nearly identical to our earlier findings in cultured septal neurons (DuBois et al., 2004, 2006). These rapidly developing mPSC parameters continued to mature through the first month of life then stabilized throughout the remainder of the lifespan. Finally, equivalent ethanol-induced alterations in GABAAR mPSC signaling were present in MS/DB neurons from both male and female animals. Previously, we showed ethanol treatment of cultured embryonic day 20 septal neurons distorts the maturation of GABAAR mPSCs predicting that early stages of GABAergic transmission in MS/DB neurons are vulnerable to intoxication injury (DuBois et al., 2004, 2006). Since the overall character, timing, and magnitude of GABAergic mPSC developmental- and ethanol-induced changes in the in vivo model so closely mirror chronologically equivalent adaptations in cultured septal neurons, this suggests that such parallel models of ethanol impairment of GABAergic synaptic development in vivo and in vitro should be useful for translational studies exploring the efficacy and mechanism of action of potential therapeutic interventions from the cellular to whole animal level.

In VivoSynaptic Scaling Is Mediated by GluA2-Lacking AMPA Receptors in the Embryonic Spinal Cord

When spiking activity within a network is perturbed for hours to days, compensatory changes in synaptic strength are triggered that are thought to be important for the homeostatic maintenance of network or cellular spiking activity. In one form of this homeostatic plasticity, called synaptic scaling, all of a cell's AMPAergic miniature postsynaptic currents (mEPSCs) are increased or decreased by some scaling factor. Although synaptic scaling has been observed in a variety of systems, the mechanisms that underlie AMPAergic scaling have been controversial. Certain studies find that synaptic scaling is mediated by GluA2-lacking calcium receptors (CP-AMPARs), whereas others have found that scaling is mediated by GluA2-containing calcium-impermeable receptors (CI-AMPARs). Spontaneous network activity is observed in most developing circuits, and in the spinal cord this activity drives embryonic movements. Blocking spontaneous network activity in the chick embryo by infusing lidocaine in vivo triggers synaptic scaling in spinal motoneurons; here we show that AMPAergic scaling occurs through increases in mEPSC conductance that appear to be mediated by the insertion of GluA2-lacking AMPA receptors at the expense of GluA2-containing receptors. We have previously reported that in vivo blockade of GABAA transmission, at a developmental stage when GABA is excitatory, also triggered AMPAergic synaptic scaling. Here, we show that this form of AMPAergic scaling is also mediated by CP-AMPARs. These findings suggest that AMPAergic scaling triggered by blocking spiking activity or GABAA receptor transmission represents similar phenomena, supporting the idea that activity blockade triggers scaling by reducing GABAA transmission.

Asymmetric N-cadherin expression results in synapse dysfunction, synapse elimination, and axon retraction in cultured mouse neurons.

Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an in vivo asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.