Frequency Of Miniature Postsynaptic Currents Research Articles

Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aβ synaptotoxicity

Synaptotoxic Aβ oligomers are thought to play a major role in the early pathology of Alzheimer´s disease (AD). However, the molecular mechanisms involved in Aβ-induced synaptic dysfunction and synapse damage remain largely unclear. Previously, Aβ synaptotoxicity has been reported to be enhanced by increased levels of a C-terminal fragment of the synaptic adhesion molecule N-cadherin that is generated by proteolytic shedding of the extracellular domains [1]. To address the molecular mechanisms involved in this process, we have now studied the functional synaptic changes induced by C-terminal fragments (CTF1) of synaptic adhesion proteins. We used synaptophysin-pHluorin (SypHy) fluorescence imaging to monitor synaptic vesicle exo- and endocytosis in cultures of mouse cortical neurons. We increased the levels of C-terminal fragments of synaptic adhesion proteins by pharmacologically inhibiting γ-secretase, which further degrades CTF1 fragments. We found that this intervention caused a delay in synaptic vesicle endocytosis. A similar effect was induced by overexpression of N-cadherin CTF1, but not by overexpression of Neurexin3β CTF1. Based on these observations, we further studied whether directly modulating synaptic vesicle endocytosis enhances Aβ synaptotoxicity. We pharmacologically induced a delayed synaptic vesicle endocytosis by a low concentration of the endocytosis inhibitor dynasore. This treatment enhanced synaptoxicity of Aβ oligomers as indicated by a reduced frequency of miniature postsynaptic currents. In conclusion, we propose that delayed endocytosis results in prolonged exposure of synaptic vesicle membranes to the extracellular space, thus enabling enhanced vesicle membrane binding of Aβ oligomers. This might in turn promote the endocytic uptake of toxic Aβ oligomers and might thus play an important role in intracellular Aβ-mediated synaptotoxicity in AD.

Acceleration of GABA-switch after early life stress changes mouse prefrontal glutamatergic transmission

Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in life. The EI-balance is (amongst others) influenced by the switch of GABAergic transmission from excitatory to inhibitory, the so-called “GABA-switch”.Here, we investigated how ELS affects the GABA-switch in mouse infralimbic Prefrontal Cortex layer 2/3 neurons, using the limited-nesting-and-bedding model. In ELS mice, the GABA-switch occurred already between postnatal day (P) 6 and P9, as opposed to P15–P21 in controls. This was associated with increased expression of the inward chloride transporter NKCC1, compared to the outward chloride transporter KCC2, both of which are important for the intracellular chloride concentration and, hence, the GABA reversal potential (Erev). Chloride transporters are not only important for regulating chloride concentration postsynaptically, but also presynaptically. Depending on the Erev of GABA, presynaptic GABAA receptor stimulation causes a depolarization or hyperpolarization, and thereby enhanced or reduced fusion of glutamate vesicles respectively, in turn changing the frequency of miniature postsynaptic currents (mEPSCs). In accordance, bumetanide, a blocker of NKCC1, shifted the Erev GABA towards more hyperpolarized levels in P9 control mice and reduced the mEPSC frequency. Other modulators of chloride transporters, e.g. VU0463271 (a KCC2 antagonist) and aldosterone -which increases NKCC1 expression-did not affect postsynaptic Erev in ELS P9 mice, but did increase the mEPSC frequency. We conclude that the mouse GABA-switch is accelerated after ELS, affecting both the pre- and postsynaptic chloride homeostasis, the former altering glutamatergic transmission. This may considerably affect brain development.

Structural and Functional Plasticity in the Dorsolateral Geniculate Nucleus of Mice following Bilateral Enucleation

Within the nervous system, plasticity mechanisms attempt to stabilize network activity following disruption by injury, disease, or degeneration. Optic nerve injury and age-related diseases can induce homeostatic-like responses in adulthood. We tested this possibility in the thalamocortical (TC) neurons in the dorsolateral geniculate nucleus (dLGN) using patch-clamp electrophysiology, optogenetics, immunostaining, and single-cell dendritic analysis following loss of visual input via bilateral enucleation. We observed progressive loss of vGlut2-positive retinal terminals in the dLGN indicating degeneration post-enucleation that was coincident with changes in microglial morphology indicative of microglial activation. Consistent with the decline of vGlut2 puncta, we also observed loss of retinogeniculate (RG) synaptic function assessed using optogenetic activation of RG axons while performing whole-cell voltage clamp recordings from TC neurons in brain slices. Surprisingly, we did not detect any significant changes in the frequency of miniature post-synaptic currents (mEPSCs) or corticothalamic feedback synapses. Analysis of TC neuron dendritic structure from single-cell dye fills revealed a gradual loss of dendrites proximal to the soma, where TC neurons receive the bulk of RG inputs. Finally, analysis of action potential firing demonstrated that TC neurons have increased excitability following enucleation, firing more action potentials in response to depolarizing current injections. Our findings show that degeneration of the retinal axons/optic nerve and loss of RG synaptic inputs induces structural and functional changes in TC neurons, consistent with neuronal attempts at compensatory plasticity in the dLGN.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABAA receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABAA receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

Gonadal Cycle-Dependent Expression of Genes Encoding Peptide-, Growth Factor-, and Orphan G-Protein-Coupled Receptors in Gonadotropin- Releasing Hormone Neurons of Mice.

Rising serum estradiol triggers the surge release of gonadotropin-releasing hormone (GnRH) at late proestrus leading to ovulation. We hypothesized that proestrus evokes alterations in peptidergic signaling onto GnRH neurons inducing a differential expression of neuropeptide-, growth factor-, and orphan G-protein-coupled receptor (GPCR) genes. Thus, we analyzed the transcriptome of GnRH neurons collected from intact, proestrous and metestrous GnRH-green fluorescent protein (GnRH-GFP) transgenic mice using Affymetrix microarray technique. Proestrus resulted in a differential expression of genes coding for peptide/neuropeptide receptors including Adipor1, Prokr1, Ednrb, Rtn4r, Nmbr, Acvr2b, Sctr, Npr3, Nmur1, Mc3r, Cckbr, and Amhr2. In this gene cluster, Adipor1 mRNA expression was upregulated and the others were downregulated. Expression of growth factor receptors and their related proteins was also altered showing upregulation of Fgfr1, Igf1r, Grb2, Grb10, and Ngfrap1 and downregulation of Egfr and Tgfbr2 genes. Gpr107, an orphan GPCR, was upregulated during proestrus, while others were significantly downregulated (Gpr1, Gpr87, Gpr18, Gpr62, Gpr125, Gpr183, Gpr4, and Gpr88). Further affected receptors included vomeronasal receptors (Vmn1r172, Vmn2r-ps54, and Vmn1r148) and platelet-activating factor receptor (Ptafr), all with marked downregulation. Patch-clamp recordings from mouse GnRH-GFP neurons carried out at metestrus confirmed that the differentially expressed IGF-1, secretin, and GPR107 receptors were operational, as their activation by specific ligands evoked an increase in the frequency of miniature postsynaptic currents (mPSCs). These findings show the contribution of certain novel peptides, growth factors, and ligands of orphan GPCRs to regulation of GnRH neurons and their preparation for the surge release.

Neurosteroid dehydroepiandrosterone sulphate enhances pain transmission in rat spinal cord dorsal horn

BackgroundThe neurosteroid dehydroepiandrosterone sulphate (DHEAS) activates the sigma-1 receptor, inhibits gamma-aminobutyric acid A (GABAA) and glycine receptors, and induces hyperalgesic effects. Although its effects have been studied in various tissues of the nervous system, its synaptic mechanisms in nociceptive pathways remain to be elucidated. MethodsThe threshold of mechanical hypersensitivity and spontaneous pain behaviour was assessed using the von Frey test in adult male Wistar rats after intrathecal administration of DHEAS. We also investigated the effects of DHEAS on synaptic transmission in the spinal dorsal horn using slice patch-clamp electrophysiology. ResultsIntrathecally administered DHEAS elicited dose-dependent mechanical hyperalgesia and spontaneous pain behaviours (withdrawal threshold: saline; 51.0 [20.1] g, 3 μg DHEAS; 14.0 [7.8] g, P<0.01, 10 μg DHEAS; 6.9 [5.2] g, 15 min after administration, P<0.001). DHEAS at 100 μM increased the frequency of miniature postsynaptic currents in the rat dorsal spinal horn; this increase was extracellular Ca2+-dependent but not sigma-1 and N-methyl-d-aspartate receptor-dependent. DHEAS suppressed the frequency of miniature inhibitory postsynaptic currents in a GABAA receptor- and sigma-1 receptor-dependent manner. ConclusionsThese results suggest that DHEAS participates in the pathophysiology of nociceptive synaptic transmission in the spinal cord by potentiation of glutamate release and inhibition of the GABAA receptor.

Erratum to: 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.

Epileptiform curents in rat cortical neurons increase over time of the in vitro culture period

Here we show that in primary culture of rat cortical neurons the number of episodes of epileptiform curents (EC) provoked by extracellular magnesium removal increases over time. We demonstrate that NMDA receptor agonists in low concentrations induce an elevation of frequency of miniature postsynaptic currents followed by their synchronization resulting in EC. Ifenprodil did not block EC but strongly inhibited NMDA-evoked whole-cell currents, which say for a key role of the ifenprodil-resistant synaptic GluN2A-containing NMDA receptors in the generation of EC. We suppose that in cultured neurons the onset of EC and a gradual increase of the EC amplitude over the time of culture period is directed by an increase of synaptic connections density and displacement of the GluN2B subunit by GluN2A in synapses.

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.

Down-regulation of endogenous KLHL1 decreases voltage-gated calcium current density

The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ∼68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α1A and α1H protein levels. Biophysical analysis and western blot experiments suggest CaV3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.

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.

Differential involvement of perineuronal astrocytes and microglia in synaptic stripping after hypoglossal axotomy

Following peripheral axotomy, the presynaptic terminals are removed from lesioned neurons, that is synaptic stripping. To elucidate involvement of astrocytes and microglia in synaptic stripping, we herein examined the motoneuron perineuronal circumference after hypoglossal nerve transection. As reported previously, axotomy-induced slow cell death occurred in C57BL/6 mice but not in Wistar rats. Synaptophysin labeling in the hypoglossal nucleus exhibited a minor reduction in both species after axotomy. Slice patch recording showed that the mean frequency of miniature postsynaptic currents in axotomized motoneurons was significantly lower in rats than in mice. We then estimated the relative coverage of motoneuron perineuronal circumference by line profile analysis. In the synaptic environment, axotomy-induced intrusion of astrocytic processes was significantly more extensive in rats than in mice, whereas microglial intrusion into the synaptic space was significantly more severe in mice than in rats. Interestingly, in the extrasynaptic environment, the prevalence of contact between astrocytic processes and lesioned motoneurons was significantly increased in rats, while no significant axotomy-induced alterations in astrocytic contact were observed in mice. These findings indicate that astrocytic, but not microglial, reaction may primarily mediate some anti-apoptotic effects through synaptic stripping after hypoglossal nerve axotomy. In addition, enlargement of astrocytic processes in the extrasynaptic environment may also be involved in neuronal protection via the increased uptake of excessive glutamate.

Acute Enhancement of Synaptic Transmission and Chronic Inhibition of Synaptogenesis Induced by Perfluorooctane Sulfonate through Mediation of Voltage-Dependent Calcium Channel

Perfluorooctane sulfonate (PFOS) is a persistent and bioaccumulative pollutant ubiquitous in wildlife and humans. Although the distribution and fate of PFOS have been widely studied, its potential neurotoxicity remains largely unknown. In the present study, the acute and chronic effects of PFOS on the development and synaptic transmission of hippocampal neurons was examined. Perfusion with PFOS markedly increased the frequency of miniature postsynaptic currents (mPSCs) and slightly elevated the amplitude of mPSCs in cultured hippocampal neurons. Perfusion with PFOS also increased the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded in the CA1 region of hippocampal slices. Both of these effects were largely blocked by the L-type Ca2+ channel antagonist nifedipine. Further studies showed that PFOS enhanced inward Ca2+ currents and increased intracellular Ca2+ in cultured neurons; these effects were also substantially inhibited by nifedipine. Moreover, prolonged treatment with PFOS moderately inhibited neurite growth and dramatically suppressed synaptogenesis in cultured neurons in a nifedipine-sensitive manner. Thus, through enhancement of Ca2+ channels, PFOS may exhibit both acute excitotoxic effects on synaptic function and chronically inhibit synaptogenesis in the brain.

Silencing-Induced Metaplasticity in Hippocampal Cultured Neurons

Silencing-induced homeostatic plasticity is usually expressed as a change in the amplitude or the frequency of miniature postsynaptic currents. Here we report that, prolonged (approximately 24 h) silencing of mature (20-22 days in vitro) cultured hippocampal neurons using the voltage-gated sodium channel blocker tetrodotoxin (TTX) produced no effects on the amplitude or frequency of the miniature excitatory postsynaptic currents (mEPSCs). However, the silencing changed the intrinsic membrane properties of the neurons, resulting in an increased excitability and rate of action potentials firing upon TTX washout. Allowing neurons to recover in TTX-free recording solution for a short period of time after the silencing resulted in potentiation of mEPSC amplitudes. This form of activity-dependent potentiation is different from classical long-term potentiation, as similar potentiation was not seen in nonsilenced neurons treated with bicuculline to raise their spiking activity to the same level displayed by the silenced neurons during TTX washout. Also, the potentiation of mEPSC amplitudes after the recovery period was not affected by the N-methyl-d-aspartate receptor blocker d-2-amino-5-phosponopentanoic acid or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 but was abolished by the L-type calcium channel blocker nifedipine. We thus conclude that the potentiation of mEPSC amplitudes following brief recovery of spiking activity in chronically silenced neurons represents a novel form of metaplasticity that differs from the conventional models of homeostatic synaptic plasticity.

Developmental Switch in Neuropeptide Y and Melanocortin Effects in the Paraventricular Nucleus of the Hypothalamus

Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.

Synapse Loss in Cortex of Agrin-Deficient Mice after Genetic Rescue of Perinatal Death

Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling.

Synaptic vesicles recycling spontaneously and during activity belong to the same vesicle pool

Recently, it has been claimed that vesicles recycling spontaneously and during activity belong to different pools. Here we simultaneously measured, using spectrally separable styryl dyes, the release kinetics of vesicles recycled spontaneously or upon stimulation and the effects of the v-ATPase blocker folimycin on the frequency of miniature postsynaptic currents in rat hippocampal neurons. Our results provide evidence as to the identities of the vesicle pools recycling at rest and during stimulation.

α1-Adrenergic modulation of synaptic input to Purkinje neurons in rat cerebellar brain slices

The inhibitory activity in the cerebellar network, as investigated in acute brain slices from 14-20 days old rats, is modulated by alpha1-adrenergic stimulation. The specific alpha1-adrenoceptor agonist phenylephrine (PhE; 10 microM) or the alpha-adrenoceptor agonist 6-fluoronoradrenaline (10 microM) increases the frequency and the amplitude of spontaneous postsynaptic currents (sPSC) in Purkinje neurons. The effects are sensitive to the alpha1-adrenoceptor antagonists prazosin (30 microM) and phentolamine (10 microM). The PhE-induced augmentation is suppressed when phospholipase C is blocked by preincubation with U73122 (10 microM) but is not affected by inhibition of protein kinases with H7 (10 microM) or GF109203X (10 microM). Involvement of intracellular Ca(2+) stores was shown by a reduced PhE effect after blocking of SERCA pumps with cyclopiazonic acid (30 microM) and thapsigargin (1 microM). The persistence of the PhE effect on the frequency of miniature postsynaptic currents, as recorded in presence of tetrodotoxin, indicates a presynaptic localization of the alpha1-adrenoceptors. A block of voltage-gated Ca(2+) channels with nifedipine, verapamil, or omega-conotoxin MVIIC did not suppress the PhE-induced increase of the frequency and amplitude of sPSC. The results suggest that alpha1-adrenoceptors at presynaptic terminals mediate an increase of the spontaneous synaptic inhibition of Purkinje neurons in the cerebellar cortex via release of Ca(2+) from intracellular stores.

Synchronous and asynchronous exocytosis induced by subthreshold high K+ at Cs(+)-loaded terminals of rat hippocampal neurons.

Transmitter release at Cs(+)-loaded autaptic terminals was selectively activated by the subthreshold concentration of external K+, and Ca(2+) channel types and transmitter pools involved in synchronous and asynchronous exocytosis were studied. When a neuron was depolarized to +30 mV by applying a current through a pipette containing Cs(+) for >30 s, a rapid external K+ jump to 3.75-10 mM, otherwise ineffective, produced an outward current (K10 response). K10 responses were initially graded (type-1) and then became a spike and plateau-shape with (type-2) or without a latency (type-3). On repolarization to -60 mV, a high K+ jump induced inward currents (called also K10 response) similar to those at +30 mV, whose shape changed from that of type-3, then type-2 and finally type-1 over 30 min. During a period favorable for inducing a type-3 response, a current similar to this response was generated by a voltage pulse (+ 80 or 90 mV, 20 or 30 ms) to the cell soma. Currents similar to K10 responses were rarely induced by a high K+ jump without a conditioning depolarization except for some cells, but consistently produced when 3 mM Cs(+) and 50 microM 4-aminopyridine were externally applied for tens of minutes. Picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione with 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid or Cd(2+) in, or Ca(2+) removal from, a high-K+ solution blocked all the K10 responses, while a plateau remaining after a high K+ jump was not blocked by Ca(2+) removal immediately after the K+ jump. Thus Cs(+) loading and decreased K+ concentration in autaptic terminals by a conditioning depolarizing current selectively sensitize the terminals to a subthreshold high K+ jump for depolarization to activate synchronous or asynchronous transmitter release. Nicardipine (5-10 microM) blocked type-1 and -2 responses but not type-3 responses, while omega-conotoxin (10 microM) blocked all the types of K10 response in the presence of nicardipine. Increasing the interval of high K+ jumps biphasically increased the magnitude of K10 response, preferentially in the postjump fraction reflecting purely the asynchronous activation of exocytotic machinery, and decreased the reduction of miniature postsynaptic current frequency after a K10 response. These results suggest the roles of N(P/Q)-type Ca(2+) channels in synchronous exocytosis at the terminals, L-type Ca(2+) channels in initiating a Ca(2+) action potential at the parent axon and both types in asynchronous exocytosis and also suggest the different releasable pools of transmitter for two modes of exocytosis in cultured hippocampal neurons.