Target cell type-dependent differences in Ca2+ channel function underlie distinct release probabilities at hippocampal glutamatergic terminals

Target cell type-dependent differences in presynaptic release probability (Pr) and short-term plasticity are intriguing features of cortical microcircuits that increase the computational power of neuronal networks. Here we tested the hypothesis that different voltage-gated Ca2+ channel densities in presynaptic active zones (AZs) underlie different Pr Two-photon Ca2+ imaging, triple immunofluorescent labeling and three-dimensional electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed approximately 1.7 - 1.9-times higher Ca2+inflow per AZ area in high Pr boutons synapsing onto parvalbumin positive interneurons than in low Pr boutons synapsing onto mGluR1alpha positive interneurons. EM replica immunogold labeling, however, demonstrated only 1.15-times larger Cav2.1 and Cav2.2 subunit densities in high Pr AZs. Our results indicate target cell type-specific modulation of voltage-gated Ca2+ channel function or different subunit composition as possible mechanisms underlying the functional differences. In addition, high Pr synapses are also characterized by a higher density of docked vesicles, suggesting that a concerted action of these mechanisms underlies the functional differences. SIGNIFICANCE STATEMENT: Target cell type-dependent variability in presynaptic properties is an intriguing feature of cortical synapses. When a single cortical pyramidal cell establishes a synapse onto a somatostatin-expressing interneuron (IN), the synapse releases glutamate with low probability, whereas the next bouton of the same axon has high release probability when its postsynaptic target is a parvalbumin-expressing IN. Here we used combined molecular, imaging and anatomical approaches to investigate the mechanisms underlying these differences. Our functional experiments implied a approximately 2-fold larger Ca2+ channel density in high release probability boutons whereas freeze-fracture immunolocalization demonstrated only a 15% difference in Ca2+ channel subunit densities. Our results point toward a postsynaptic target cell type-dependent regulation of Ca2+ channel function or different subunit composition as the underlying mechanism

Presynaptic Calcium Channel Inhibition Underlies CB1 Cannabinoid Receptor-Mediated Suppression of GABA Release.

CB1 cannabinoid receptors (CB1) are located at axon terminals and effectively control synaptic communication and thereby circuit operation widespread in the CNS. Although it is partially uncovered how CB1 activation leads to the reduction of synaptic excitation, the mechanisms of the decrease of GABA release upon activation of these cannabinoid receptors remain elusive. To determine the mechanisms underlying the suppression of synaptic transmission by CB1 at GABAergic synapses, we recorded unitary IPSCs (uIPSCs) at cholecystokinin-expressing interneuron-pyramidal cell connections and imaged presynaptic [Ca(2+)] transients in mouse hippocampal slices. Our results reveal a power function with an exponent of 2.2 between the amplitude of uIPSCs and intrabouton [Ca(2+)]. Altering CB1 function by either increasing endocannabinoid production or removing its tonic activity allowed us to demonstrate that CB1 controls GABA release by inhibiting Ca(2+) entry into presynaptic axon terminals via N-type (Cav2.2) Ca(2+) channels. These results provide evidence for modulation of intrabouton Ca(2+) influx into GABAergic axon terminals by CB1, leading to the effective suppression of synaptic inhibition

Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells

Klinikai - immunológiai vizsgálatok szisztémás autoimmun kórképekben = Clinical-epidemiological studies in systemic autoimmune diseases

Klinikai - immunológiai vizsgálatok szisztémás autoimmun kórképekben Czirják László A populációs alapú vizsgálatainkat lezártuk, és a szisztémás sclerosis, Raynaud jelenség illetve rheumatoid arthritis prevalencia értékeit publikáltuk. A michrochimérismus vizsgálataink nem zárultak interpretálható eredményekkel. Megjegyzendő, hogy más nemzetközi munkacsoportoknak is problámát okozott a kimutathatósági határon levő vizsgálat reprodukálható elvégzése. Az indukált köpet vizsgálat nem bizonyult reprodukálhatónak. Más, alternatív módszert (surfactans D, KL-6) használunk a tüdőmanifesztáció vizsgálatára illetve követésére. Alapmunkánkat közlésre elfogadták, követéses vizsgálatunk publikálása folyamatban van. A hosszutávú követéses vizsgálataink eredményeit szisztémás sclerosisban publikáltuk, kimutattuk, hogy rossz prognozissal jár a papaneopláziás szindróma formájában jelentkező systemás sclerosis. Emellett kidolgoztunk és validáltunk egy a bőrfolyamat aktivitását mérő beteg önkitöltő kérdőívet. Az aktivitás vizsgálatához szükséges kérdőíveket (scleroderma-HAQ, DASH) magyar nyelven validáltuk. Az európai scleroderma aktivitási kritériumok validálása és egy új aktivitási kritériumrendszer kialakítása folyamatban van, már rendelkezésre áll egy igen nagyszámú betegből álló egyéves követéses vizsgálat adatsora (1. sz. melléklet). Gyulladásos izombetegségekben a prógnózist vizsgáló munkánat szintén lezártuk. | Clinical epidemiological studies on systemic autoimmune diseases We have finished our population-based studies on the prevalence of connective tissue diseases. With regard to rheumatoid arthritis, Raynaud?s phenomenon and systemic sclerosis we have published our results. Investigation of microchimesism in systemic sclerosis did not give conclusive results, we had detectability problems with this particular method, similar to other groups . The induced sputum investigations did not show reproducible results therefore we worked out other alternative methods for the evaluation of interstitial lung involvement in connective tissue diseases. Detection of surfactant D and KL-6 markers are good surrogate markers for the severity of lung disease. Our long term follow up studies on systemic sclerosis indicated that the presence of paraneoplastic syndrome and gastric antral ectasia (watermelon stomach) are poor prognostic signs besides the well known organ manifestations in systemic sclerosis. We worked out and validated a patient self assessment questionnaire measuring the skin thickness of scleroderma patients. We have also completed our clinical epidemiological studies on inflammatory myopathies. The presence of cancer associated myositis caused a poor prognosis. Conversely, interstitial lung disease did not cause an unfavourable prognosis in our cohort

Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca 2+ ] transients in weakly GCaMP6f expressing hippocampal pyramidal cells

Investigating neuronal activity using genetically encoded Ca2+ indicators in behaving animals is hampered by inaccuracies in spike inference from fluorescent tracers. Here we combine two‐photon [Ca2+] imaging with cell‐attached recordings, followed by post hoc determination of the expression level of GCaMP6f, to explore how it affects the amplitude, kinetics and temporal summation of somatic [Ca2+] transients in mouse hippocampal pyramidal cells (PCs). The amplitude of unitary [Ca2+] transients (evoked by a single action potential) negatively correlates with GCaMP6f expression, but displays large variability even among PCs with similarly low expression levels. The summation of fluorescence signals is frequency‐dependent, supralinear and also shows remarkable cell‐to‐cell variability. We performed experimental data‐based simulations and found that spike inference error rates using MLspike depend strongly on unitary peak amplitudes and GCaMP6f expression levels. We provide simple methods for estimating the unitary [Ca2+] transients in individual weakly GCaMP6f‐expressing PCs, with which we achieve spike inference error rates of ∼5%

Tonic endocannabinoid-mediated modulation of GABA release is independent of the CB1 content of axon terminals

The release of GABA from cholecystokinin-containing interneurons is modulated by type-1 cannabinoid receptors (CB1). Here we tested the hypothesis that the strength of CB1-mediated modulation of GABA release is related to the CB1 content of axon terminals. Basket cell boutons have on average 78% higher CB1 content than those of dendritic-layer-innervating (DLI) cells, a consequence of larger bouton surface and higher CB1 density. The CB1 antagonist AM251 caused a 54% increase in action potential-evoked [Ca2+] in boutons of basket cells, but not in DLI cells. However, the effect of AM251 did not correlate with CB1 immunoreactivity of individual boutons. Moreover, a CB1 agonist decreased [Ca2+] in a cell type- and CB1-content-independent manner. Replica immunogold labelling demonstrated the colocalization of CB1 with the Cav2.2 Ca2+ channel subunit. Our data suggest that only a subpopulation of CB1s, within nanometre distances from their target Cav2.2 channels, are responsible for endocannabinoid-mediated modulation of GABA release

Functional specification of CCK plus interneurons by alternative isoforms of Kv4.3 auxiliary subunits

CCK-expressing interneurons (CCK+INs) are crucial for controlling hippocampal activity. We found two firing phenotypes of CCK+INs in rat hippocampal CA3 area; either possessing a previously undetected membrane potential-dependent firing or regular firing phenotype, due to different low-voltage-activated potassium currents. These different excitability properties destine the two types for distinct functions, because the former is essentially silenced during realistic 8-15 Hz oscillations. By contrast, the general intrinsic excitability, morphology and gene-profiles of the two types were surprisingly similar. Even the expression of Kv4.3 channels were comparable, despite evidences showing that Kv4.3-mediated currents underlie the distinct firing properties. Instead, the firing phenotypes were correlated with the presence of distinct isoforms of Kv4 auxiliary subunits (KChIP1 vs. KChIP4e and DPP6S). Our results reveal the underlying mechanisms of two previously unknown types of CCK+INs and demonstrate that alternative splicing of few genes, which may be viewed as a minor change in the cells' whole transcriptome, can determine cell-type identity

Differential subcellular distribution of ion channels and the diversity of neuronal function

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells