Nonstationary Fluctuation Analysis Research Articles

Enhanced Synaptic Inhibition in the Dorsolateral Geniculate Nucleus in a Mouse Model of Glaucoma.

Elevated intraocular pressure (IOP) triggers glaucoma by damaging the output neurons of the retina called retinal ganglion cells (RGCs). This leads to the loss of RGC signaling to visual centers of the brain such as the dorsolateral geniculate nucleus (dLGN), which is critical for processing and relaying information to the cortex for conscious vision. In response to altered levels of activity or synaptic input, neurons can homeostatically modulate postsynaptic neurotransmitter receptor numbers, allowing them to scale their synaptic responses to stabilize spike output. While prior work has indicated unaltered glutamate receptor properties in the glaucomatous dLGN, it is unknown whether glaucoma impacts dLGN inhibition. Here, using DBA/2J mice, which develop elevated IOP beginning at 6-7 months of age, we tested whether the strength of inhibitory synapses on dLGN thalamocortical relay neurons is altered in response to the disease state. We found an enhancement of feedforward disynaptic inhibition arising from local interneurons along with increased amplitude of quantal inhibitory synaptic currents. A combination of immunofluorescence staining for the γ-aminobutyric acid (GABA)A-α1 receptor subunit, peak-scaled nonstationary fluctuation analysis, and measures of homeostatic synaptic scaling pointed to an ∼1.4-fold increase in GABA receptors at postsynaptic inhibitory synapses, although several pieces of evidence indicate a nonuniform scaling across inhibitory synapses within individual relay neurons. Together, these results indicate an increase in inhibitory synaptic strength in the glaucomatous dLGN, potentially pointing toward homeostatic compensation for disruptions in network and neuronal function triggered by increased IOP.

Mechanisms That Underlie Expression of Estradiol-Induced Excitatory Synaptic Potentiation in the Hippocampus Differ between Males and Females.

17β-estradiol (E2) is synthesized in the hippocampus of both sexes and acutely potentiates excitatory synapses in each sex. Previously, we found that the mechanisms for initiation of E2-induced synaptic potentiation differ between males and females, including in the molecular signaling involved. Here, we used electrical stimulation and two-photon glutamate uncaging in hippocampal slices from adult male and female rats to investigate whether the downstream consequences of distinct molecular signaling remain different between the sexes or converge to the same mechanism(s) of expression of potentiation. This showed that synaptic activity is necessary for expression of E2-induced potentiation in females but not males, which paralleled a sex-specific requirement in females for calcium-permeable AMPARs (cpAMPARs) to stabilize potentiation. Nonstationary fluctuation analysis of two-photon evoked unitary synaptic currents showed that the postsynaptic component of E2-induced potentiation occurs either through an increase in AMPAR conductance or in nonconductive properties of AMPARs (number of channels × open probability) and never both at the same synapse. In females, most synapses (76%) were potentiated via increased AMPAR conductance, whereas in males, more synapses (60%) were potentiated via an increase in nonconductive AMPAR properties. Inhibition of cpAMPARs eliminated E2-induced synaptic potentiation in females, whereas some synapses in males were unaffected by cpAMPAR inhibition; these synapses in males potentiated exclusively via increased AMPAR nonconductive properties. This sex bias in expression mechanisms of E2-induced synaptic potentiation underscores the concept of latent sex differences in mechanisms of synaptic plasticity in which the same outcome in each sex is achieved through distinct underlying mechanisms.SIGNIFICANCE STATEMENT Estrogens are synthesized in the brains of both sexes and potentiate excitatory synapses to the same degree in each sex. Despite this apparent similarity, the molecular signaling that initiates estrogen-induced synaptic potentiation differs between the sexes. Here we show that these differences extend to the mechanisms of expression of synaptic potentiation and result in distinct patterns of postsynaptic neurotransmitter receptor modulation in each sex. Such latent sex differences, in which the same outcome is achieved through distinct underlying mechanisms in males versus females, indicate that molecular mechanisms targeted for drug development may differ between the sexes even in the absence of an overt sex difference in behavior or disease.

Negative allosteric modulation of GluN1/GluN3 NMDA receptors

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

Learning Promotes Subfield-Specific Synaptic Diversity in Hippocampal CA1 Neurons

The hippocampus is functionally heterogeneous between the dorsal and ventral subfields with left–right asymmetry. To determine the possible location of contextual memory, we performed an inhibitory avoidance task to analyze synaptic plasticity using slice patch-clamp technique. The training bilaterally increased the AMPA/NMDA ratio at dorsal CA3–CA1 synapses, whereas the training did not affect the ratio at ventral CA3–CA1 synapses regardless of the hemisphere. Moreover, sequential recording of miniature excitatory postsynaptic currents and miniature inhibitory postsynaptic currents from the same CA1 neuron clearly showed learning-induced synaptic plasticity. In dorsal CA1 neurons, the training dramatically strengthened both excitatory and inhibitory postsynaptic responses in both hemispheres, whereas the training did not promote the plasticity in either hemisphere in ventral CA1 neurons. Nonstationary fluctuation analysis further revealed that the training bilaterally increased the number of AMPA or GABAA receptor channels at dorsal CA1 synapses, but not at ventral CA1 synapses, suggesting functional heterogeneity of learning-induced receptor mobility. Finally, the performance clearly impaired by the bilateral microinjection of plasticity blockers in dorsal, but not ventral CA1 subfields, suggesting a crucial role for contextual learning. The quantification of synaptic diversity in specified CA1 subfields may help us to diagnose and evaluate cognitive disorders at the information level.

AMPA-Type Glutamate Receptor Conductance Changes and Plasticity: Still a Lot of Noise

Twenty years ago, we reported from the Collingridge Lab that a single-channel conductance increase through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPARs) could mediate one form of plasticity associated with long-term potentiation (LTP) in the hippocampus (Benke et al., Nature 395:793-797, 1998). Revealed through peak-scaled non-stationary fluctuation analysis (PS-NSFA, also known as noise analysis), this component of LTP could be exclusively mediated by direct increases in channel conductance or by increases in the number of high conductance synaptic AMPARs. Re-evaluation of our original data in the light of the molecular details regarding AMPARs, conductance changes and plasticity suggests that insertion of high-conductance GluA1 homomers can account for our initial findings. Any potential cost associated with manufacture or trafficking of new receptors could be mitigated if pre-existing synaptic AMPARs also undergo a modest conductance change. The literature suggests that the presence of high conductance AMPARs and/or GluA1 homomers confers an unstable synaptic state, suggesting state transitions. An experimental paradigm is proposed to differentiate these possibilities. Validation of this state diagram could provide insight into development, disease pathogenesis and treatment.

Differential Somatic Ca2+ Channel Profile in Midbrain Dopaminergic Neurons.

Dopaminergic (DA) neurons located in the ventral midbrain continuously generate a slow endogenous pacemaker activity, the mechanism of which is still debated. It has been suggested that, in the substantia nigra pars compacta (SNc), the pacemaking relies more on Ca(2+) channels and that the density of L-type Ca(2+) channels is higher in these DA neurons than in those located in the ventral tegmental area (VTA). This might lead to a higher Ca(2+) load in SNc DA neurons and explain their higher susceptibility to degeneration. However, direct evidence for this hypothesis is lacking. We found that the L-type current and channel density are indeed higher in the somata of rat SNc DA neurons and that this current undergoes less inactivation in this region. Nonstationary fluctuation analysis measurements showed a much higher number of L-type channels in the soma of SNc DA neurons, as well as a smaller single-channel conductance, pointing to a possible different molecular identity of L-type channels in DA neurons from the two areas. A major consequence of this is that pacemaking and, even more so, bursting are associated with a larger Ca(2+) entry through L-type channels in SNc DA neurons than in their VTA counterparts. Our results establish a molecular and functional difference between two populations of midbrain DA neurons that may contribute to their differential sensitivity to neurodegeneration. Dopamine neurons from the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are involved in various brain functions, such as movement initiation and goal directed behavior, respectively. This work shows that, although both neurons fire in a similar regular and slow pacemaker mode, this firing activity is supported by different calcium channel landscapes. Indeed, the L-type calcium current is larger in the soma of dopamine neurons of the SNc, leading to a higher charge transfer through L-type channels during pacemaking and bursting. Therefore, these neurons may be physiologically exposed to a larger stress than their neighbors from the VTA.

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study.

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABAA receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABAA receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABAA receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABAA receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y11 purinoceptor. Thus, our findings reveal that postsynaptic GABAA receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.

Regulation of Spermatogenic Cell T-Type Ca(2+) Currents by Zn(2+): Implications in Male Reproductive Physiology.

Zn(2+) is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis, and prostate contain high Zn(2+) concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV 3.2-encoded T-type Ca(2+) currents (ICaT) which are positively or negatively modulated by Zn(2+) in other tissues. To explore whether ICaT could be regulated by Zn(2+) and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn(2+) concentrations. Zn(2+) decreased ICaT in a concentration-dependent manner (IC50 = 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn(2+) . Further analysis showed that Zn(2+) did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady-state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non-stationary fluctuation analysis, we found that Zn(2+) mainly reduced the number of available Ca(2+) channels without changing the single channel current amplitude. ICaT modulation by Zn(2+) could be relevant for spontaneous Ca(2+) oscillations during spermatogenesis and in pathophysiological conditions such as diabetes.

Zebrafish TARP Cacng2 is required for the expression and normal development of AMPA receptors at excitatory synapses.

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor-related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non-mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12-h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles, smaller tail velocities and aberrant C-bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non-stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non-functional with respect to the development of AMPA synaptic transmission.

GABAergic transmission to kisspeptin neurons is differentially regulated by time of day and estradiol in female mice.

Gonadotropin-releasing hormone (GnRH) secretion is regulated by estradiol feedback. This feedback switches from negative to positive in females; this switch depends on time of day in many species. Estradiol feedback is likely conveyed via afferents. Kisspeptin neurons of the arcuate nucleus and anteroventral-periventricular region (AVPV) may differentially regulate GnRH neurons during negative and positive feedback, respectively. We tested estradiol and time of day regulation of GABAergic transmission and postsynaptic response to GABA in these two populations using transgenic mice with GFP-identified kisspeptin neurons. Ovariectomized (OVX) mice treated or not with estradiol (E) were studied in the AM (negative feedback) or PM (positive feedback). GABAA receptor reversal potential was unaffected by time of day or estradiol. GABA depolarized the membrane potential of arcuate neurons from OVX+E mice; this response was blunted in cells from OVX mice. GABA hyperpolarized AVPV kisspeptin neurons, except in the OVX PM group in which GABA did not alter membrane potential attributable to a PM hyperpolarization of baseline membrane potential. In both kisspeptin neuron populations from OVX mice, the frequency of GABAergic spontaneous postsynaptic currents was increased in the PM; this increase was blunted by estradiol. In arcuate, but not AVPV, kisspeptin neurons, estradiol reduced miniature postsynaptic current amplitude independent of time of day. Using nonstationary fluctuation analysis and diazepam to manipulate GABAA receptor apparent affinity, the decrease in arcuate miniature postsynaptic current amplitude was attributed to decreased number of receptors bound by GABA. Time of day and estradiol feedback thus both target presynaptic and postsynaptic mechanisms to differentially regulate kisspeptin neurons via GABAergic transmission.

Maximum likelihood estimation of biophysical parameters of synaptic receptors from macroscopic currents.

Dendritic integration and neuronal firing patterns strongly depend on biophysical properties of synaptic ligand-gated channels. However, precise estimation of biophysical parameters of these channels in their intrinsic environment is complicated and still unresolved problem. Here we describe a novel method based on a maximum likelihood approach that allows to estimate not only the unitary current of synaptic receptor channels but also their multiple conductance levels, kinetic constants, the number of receptors bound with a neurotransmitter, and the peak open probability from experimentally feasible number of postsynaptic currents. The new method also improves the accuracy of evaluation of unitary current as compared to the peak-scaled non-stationary fluctuation analysis, leading to a possibility to precisely estimate this important parameter from a few postsynaptic currents recorded in steady-state conditions. Estimation of unitary current with this method is robust even if postsynaptic currents are generated by receptors having different kinetic parameters, the case when peak-scaled non-stationary fluctuation analysis is not applicable. Thus, with the new method, routinely recorded postsynaptic currents could be used to study the properties of synaptic receptors in their native biochemical environment.

Kinetic properties and open probability of α7 nicotinic acetylcholine receptors

The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of α7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of α7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat α7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 ± 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 μM), it was increased to 0.599 ± 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics.

The Permeation Pathway Mechanism in Ciona Intestinalis Hv Channel

Voltage-gated proton (HV) channels are expressed by different cells type including immune cells, microglia, among others. In other voltage-gated cation channels, the pore-forming domains are S5, S6, and the P loop connecting S5 and S6. Since Hv channels do not contain a S5-S6 region, other parts of the channel must form the pore domain. Interestingly, Hv channel present three gating charges in the S4 followed by an asparagine, N264, highly conserved among all Hv channel. Using non-stationary fluctuation analysis we establish the conductance for the dimer and the monomer in 200 and 100 fS, respectively. Mutations at position S191 (S2) and N264 (S4) modified the unitary conductance of Hv channel. Furthermore, mutants S191E/D and N264R removed the H+ current remaining the S4 functioning unaltered, according to voltage-clamp fluorometry experiments. The introduction of an arginine or lysine at position 264 or negative residue at position S191, drastically reduced or abolished the proton currents. Interestingly, mutations of the analogue position S191 of Ciona VSOP, in the voltage sensor domain of a non-conductor Shaker K+ channel produce fully functional voltage gated H+ channels. In the same way, mutation resembling the position N264 of Ciona Hv in the VSD of the voltage-dependent phosphate Ci-VSP spontaneously entail the formation of a voltage-gated H+ channel. Thus, S191 and N264 form the molecular determinants of permeation pathway in Hv channel. Supported by Fondecyt grants ACT 1104 and Fondecyt 1120802 to CG and 1110430 to R.L.

C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells

The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

Will the Real Single HCN Channel Please Stand Up?

Will the Real Single HCN Channel Please Stand Up?

Alpha7 Nicotinic Receptors: Intrinsic Kinetics and Modulation by PNU 120596

We investigated the kinetics of choline evoked currents, and the effect of the positive modulator PNU 120596 on alpha7 nicotinic acetylcholine receptors expressed by GH4C1 cells (obtained from Siena Biotech SpA) in whole-cell and outside-out patch-clamp experiments. using a theta-tube system for rapid application of agonists, we measured the dependence of current kinetics on the solution exchange rate. By extrapolation and by kinetic modeling we estimated the intrinsic kinetics of the receptor: what would be the amplitude and kinetics of choline-evoked currents at instantaneous agonist application. In the presence of PNU 120596 the single channel mean open time is drastically prolonged, this allowed us to determine the ratio of simultaneously open channels using nonstationary fluctuation analysis. By determining the approximate number of channels in a patch, we could determine the peak open probability of 10 mM choline-evoked currents in the absence of the modulator (0.0333 ± 0.0056), as well as the open probability in the presence of 10 mM choline and 10 μM PNU 120596 (0.632 ± 0.065). We performed kinetic experiments to determine the affinity of PNU 120596 to three different conformational states of the receptor: resting state, desensitized state and a slowly developing second desensitized state. We found that PNU 120596 was ineffective at both the resting and the slow desensitized states, while it bound to the desensitised state and re-activated the receptors. We investigated the nature of cooperativity between the agonist and the modulator. We found that while the agonist increases the apparent affinity of the modulator only by inducing desensitized conformation (which is preferred by the modulator), the modulator induces a true increase of agonist affinity probably by allosterically affecting the conformation of the agonist binding site itself.

Developmental regulation of GABAergic signalling in the hippocampus of neuroligin 3 R451C knock-in mice: an animal model of Autism

Autism Spectrum Disorders (ASDs) comprise an heterogeneous group of neuro-developmental abnormalities, mainly of genetic origin, characterized by impaired social interactions, communications deficits, and stereotyped behaviors. In a small percentage of cases, ASDs have been found to be associated with single mutations in genes involved in synaptic function. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. NLs interact with presynaptic neurexins (Nrxs) to ensure a correct cross talk between post and presynaptic specializations. Here, transgenic mice carrying the human R451C mutation of Nlgn3, were used to study GABAergic signaling in the hippocampus early in postnatal life. Whole cell recordings from CA3 pyramidal neurons in slices from NL3R451C knock-in mice revealed an enhanced frequency of Giant Depolarizing Potentials (GDPs), as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAA-mediated (GPSCs), but not AMPA-mediated postsynaptic currents (EPSCs). Changes in frequency of mGPSCs were associated with an acceleration of their decay kinetics, in the absence of any change in unitary synaptic conductance or in the number of GABAA receptor channels, as assessed by peak scaled non-stationary fluctuation analysis. The enhanced GABAergic but not glutamatergic transmission early in postnatal life may change the excitatory/inhibitory balance known to play a key role in the construction and refinement of neuronal circuits during postnatal development. This may lead to behavioral deficits reminiscent of those observed in ASDs patients.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Cocaine sensitization inhibits the hyperpolarization-activated cation current Ih and reduces cell size in dopamine neurons of the ventral tegmental area

The progressive augmentation of motor activity that results from repeated cocaine administration is termed behavioral sensitization. This phenomenon is thought to be a critical component in compulsive drug taking and relapse. Still, the cellular mechanisms that underlie sensitization remain elusive. Cocaine abuse, nonetheless, is known to evoke neuroplastic adaptations in dopamine (DA) neurotransmission originating from the midbrain's ventral tegmental area (VTA). Here, we report that concomitant with the development of locomotor sensitization to cocaine the hyperpolarization-activated cation current (I(h)) amplitude is depressed by ∼40% in VTA DA cells. Such effect did not result from a negative shift in I(h) voltage dependence. Nonstationary fluctuation analysis indicates that this inhibition was caused by an ∼45% reduction in the number of h-channels with no change in their unitary properties. The cocaine-induced I(h) depression was accompanied by a reduction in cell capacitance of similar magnitude (∼33%), leaving h-current density unaltered. Two implications follow from these data. First, I(h) inhibition may contribute to cocaine addiction by increasing bursting probability in DA cells and this effect could be intensified by the decrease in cell capacitance. Second, the cocaine-induced diminution of DA cell capacitance may also lead to reward tolerance promoting drug-seeking behaviors.

Probing TARP Modulation of AMPA Receptor Conductance with Polyamine Toxins

The properties of synaptic AMPA receptors (AMPARs) depend on their subunit composition and association with transmembrane AMPAR regulatory proteins (TARPs). Although both GluA2 incorporation and TARP association have been shown to influence AMPAR channel conductance, the manner in which different TARPs modulate the mean channel conductance of GluA2-containing AMPARs is unknown. Using ultrafast agonist application and nonstationary fluctuation analysis, we found that TARP subtypes differentially increase the mean channel conductance, but not the peak open probability, of recombinant GluA2-containing AMPARs. TARP γ-8, in particular, enhances mean channel conductance to a greater degree than γ-2, γ-3, or γ-4. We then examined the action of a use-dependent antagonist of GluA2-containing AMPARs, philanthotoxin-74 (PhTx-74), on recombinant AMPARs and on GluA2-containing AMPARs in cerebellar granule neurons from stargazer mice transfected with TARPs. We found that the rate and extent of channel block varies with TARP subtype, in a manner that correlates linearly with mean channel conductance. Furthermore, block of GluA2-containing AMPARs by polyamine toxins varied depending on whether channels were activated by the full agonist glutamate or the partial agonist kainate, consistent with conductance state-dependent block. Block of GluA2-lacking AMPARs by PhTx-433 is also modulated by TARP association and is a function of agonist efficacy. Our data indicate that channel block by polyamine toxins is sensitive to the mean channel conductance of AMPARs, which varies with TARP subtype and agonist efficacy. Furthermore, our results illustrate the utility of polyamine toxins as sensitive probes of AMPAR channel conductance and suggest the possibility that TARPs may influence their channel properties by selectively stabilizing specific channel conformations, rather than altering the pore structure.