Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy.

Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as 'lost caps'. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting 'cap angle' (ϕ), a size-independent measure of a particle's distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.

Different priming states of synaptic vesicles underlie distinct release probabilities at hippocampal excitatory synapses.

A stunning example of synaptic diversity is the postsynaptic target cell-type-dependent difference in synaptic efficacy in cortical networks. Here, we show that CA1 pyramidal cell (PC) to fast spiking interneuron (FSIN) connections have 10-fold larger release probability (Pv) than those on oriens lacunosum-moleculare (O-LM) interneurons. Freeze-fracture immunolabeling revealed that different nano-topologies and coupling distances between Ca2+ channels and release sites (RSs) are not responsible for the distinct Pv. Although [Ca2+] transients are 40% larger in FSINs innervating boutons, when [Ca2+] entry is matched in the two bouton populations, EPSCs in O-LM cells are still 7-fold smaller. However, application of a phorbol ester analog resulted in a ∼2.5-fold larger augmentation at PC - O-LM compared to PC - FSIN synapses, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct RS occupancies and demonstrate that incompletely primed, but docked, vesicles limit the output of PC - O-LM synapses.

Variability in the Munc13-1 content of excitatory release sites.

The molecular mechanisms underlying the diversity of cortical glutamatergic synapses are still incompletely understood. Here, we tested the hypothesis that presynaptic active zones (AZs) are constructed from molecularly uniform, independent release sites (RSs), the number of which scales linearly with the AZ size. Paired recordings between hippocampal CA1 pyramidal cells and fast-spiking interneurons in acute slices from adult mice followed by quantal analysis demonstrate large variability in the number of RSs (N) at these connections. High-resolution molecular analysis of functionally characterized synapses reveals variability in the content of one of the key vesicle priming factors - Munc13-1 - in AZs that possess the same N. Replica immunolabeling also shows a threefold variability in the total Munc13-1 content of AZs of identical size and a fourfold variability in the size and density of Munc13-1 clusters within the AZs. Our results provide evidence for quantitative molecular heterogeneity of RSs and support a model in which the AZ is built up from variable numbers of molecularly heterogeneous, but independent RSs.

Selective Enrichment of Munc13-2 in Presynaptic Active Zones of Hippocampal Pyramidal Cells That Innervate mGluR1α Expressing Interneurons.

Selective distribution of proteins in presynaptic active zones (AZs) is a prerequisite for generating postsynaptic target cell type-specific differences in presynaptic vesicle release probability (Pv) and short-term plasticity, a characteristic feature of cortical pyramidal cells (PCs). In the hippocampus of rodents, somatostatin and mGluR1α expressing interneurons (mGluR1α+ INs) receive small, facilitating excitatory postsynaptic currents (EPSCs) from PCs and express Elfn1 that trans-synaptically recruits mGluR7 into the presynaptic AZ of PC axons. Here we show that Elfn1 also has a role in the selective recruitment of Munc13-2, a synaptic vesicle priming and docking protein, to PC AZs that innervate mGluR1α+ INs. In Elfn1 knock-out mice, unitary EPSCs (uEPSCs) in mGluR1α+ INs have threefold larger amplitudes with less pronounced short-term facilitation, which might be the consequence of the loss of either mGluR7 or Munc13-2 or both. Conditional genetic deletion of Munc13-2 from CA1 PCs results in the loss of Munc13-2, but not mGluR7 from the AZs, and has no effect on the amplitude of uEPSCs and leaves the characteristic short-term facilitation intact at PC to mGluR1α+ IN connection. Our results demonstrate that Munc13-1 alone is capable of imposing low Pv at PC to mGluR1α+ IN synapses and Munc13-2 has yet an unknown role in this synapse.

A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses.

Elucidating the molecular mechanisms underlying the functional diversity of synapses requires a high-resolution, sensitive, diffusion-free, quantitative localization method that allows the determination of many proteins in functionally characterized individual synapses. Array tomography permits the quantitative analysis of single synapses but has limited sensitivity, and its application to functionally characterized synapses is challenging. Here, we aim to overcome these limitations by searching the parameter space of different fixation, resin, embedding, etching, retrieval, and elution conditions. Our optimizations reveal that etching epoxy-resin-embedded ultrathin sections with Na-ethanolate and treating them with SDS dramatically increase the labeling efficiency of synaptic proteins. We also demonstrate that this method is ideal for the molecular characterization of individual synapses following paired recordings, two-photon [Ca2+] or glutamate-sensor (iGluSnFR) imaging. This method fills a missing gap in the toolbox of molecular and cellular neuroscience, helping us to reveal how molecular heterogeneity leads to diversity in function.

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 values. Two-photon Ca2+ imaging, triple immunofluorescent labeling, and 3D electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed ∼1.7-1.9 times higher Ca2+ inflow per AZ area in high Pr boutons synapsing onto parvalbumin-positive interneurons (INs) than in low Pr boutons synapsing onto mGluR1α-positive INs. 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 an approximately twofold 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.

Human pyramidal to interneuron synapses are mediated by multi-vesicular release and multiple docked vesicles.

Classic theories link cognitive abilities to synaptic properties and human-specific biophysical features of synapses might contribute to the unparalleled performance of the human cerebral cortex. Paired recordings and multiple probability fluctuation analysis revealed similar quantal sizes, but 4-times more functional release sites in human pyramidal cell to fast-spiking interneuron connections compared to rats. These connections were mediated on average by three synaptic contacts in both species. Each presynaptic active zone (AZ) contains 6.2 release sites in human, but only 1.6 in rats. Electron microscopy (EM) and EM tomography showed that an AZ harbors 4 docked vesicles in human, but only a single one in rats. Consequently, a Katz's functional release site occupies ~0.012 µm(2) in the human presynaptic AZ and ~0.025 µm(2) in the rat. Our results reveal a robust difference in the biophysical properties of a well-defined synaptic connection of the cortical microcircuit of human and rodents.

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 [Ca(2+)] 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 [Ca(2+)] in a cell type- and CB1-content-independent manner. Replica immunogold labelling demonstrated the colocalization of CB1 with the Cav2.2 Ca(2+) 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.

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.

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone.

Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca(2+)] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca(2+) imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca(2+)] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.

The effects of an Echinacea preparation on synaptic transmission and the firing properties of CA1 pyramidal cells in the hippocampus.

Traditionally, Echinacea preparations are used as antiinflammatory agents and immune-enhancers. In addition to these effects, their anxiolytic potency has been recognized recently in laboratory tests. Our aim in this study was to uncover the potential effects of an Echinacea preparation on neuronal operations in the hippocampus, a brain region that is involved in anxiety and anxiety-related behaviors. Using in vitro electrophysiological techniques, we observed that excitatory synaptic transmission in hippocampal slices was significantly suppressed by an Echinacea extract found to be effective in anxiety tests. In contrast, no change in inhibitory synaptic transmission could be detected upon application of this extract. In addition, our experiments revealed that at low concentration the Echinacea extract reduced the spiking activity of CA1 pyramidal cells, while at high concentration increased it. This latter observation was parallel to the reduction in the magnitude of the h-current-mediated voltage responses in pyramidal cells. At any concentrations, the passive membrane properties of CA1 pyramidal cells were found to be unaltered by the Echinacea extract. In summary, the Echinacea extract can significantly regulate excitatory, but not inhibitory, synaptic transmission in the hippocampus, and this action might be involved in its anxiolytic effects observed in behaviour tests.

Cannabinoids attenuate hippocampal γ oscillations by suppressing excitatory synaptic input onto CA3 pyramidal neurons and fast spiking basket cells.

CB(1) cannabinoid receptor (CB(1)R) activation by exogenous ligands can impair memory processes, which critically depend on synchronous neuronal activities that are temporarily structured by oscillations. In this study, we aimed to reveal the mechanisms underlying the cannabinoid-induced decrease in gamma oscillations. We first verified that cannabinoids (CP55,940 and WIN55,212-2) readily suppressed carbachol-induced gamma oscillations in the CA3 region of hippocampal slices via activation of CB(1)Rs. The cannabinoid-induced decrease in the peak power of oscillations was accompanied by reduced and less precise firing activity in CA3 pyramidal cells and fast spiking basket cells. By examining the cannabinoid sensitivity of synaptic inputs we found that the amplitude of evoked excitatory postsynaptic currents was significantly suppressed upon CB(1)R activation in both CA3 pyramidal cells and fast spiking basket cells. In contrast, evoked inhibitory postsynaptic currents in CA3 pyramidal cells were unaltered. Furthermore, we observed that a CB(1)R agonist-induced decrease in the oscillation power at the beginning of the drug application was accompanied primarily by the reduced discharge of fast spiking basket cells, while pyramidal cell firing was unaltered. This result implies that the dampening of cholinergically induced gamma oscillations in the hippocampus by cannabinoids can be explained by a reduced excitatory input predominantly onto fast spiking basket cells, which leads to a reduction in neuronal firing frequency and precision, and thus to smaller field potentials. In addition, we uncovered that the spontaneously occurring sharp wave-ripple activities in hippocampal slices could also be suppressed by CB(1)R activation suggesting that cannabinoids profoundly reduce the intrinsically generated oscillatory activities at distinct frequencies in CA3 networks by reducing synaptic neurotransmission.

Parvalbumin-containing fast-spiking basket cells generate the field potential oscillations induced by cholinergic receptor activation in the hippocampus.

Gamma frequency oscillations in cortical regions can be recorded during cognitive processes, including attention or memory tasks. These oscillations are generated locally as a result of reciprocal interactions between excitatory pyramidal cells and perisomatic inhibitory interneurons. Here, we examined the contribution of the three perisomatic interneuron types--the parvalbumin-containing fast-spiking basket cells (FSBCs) and axo-axonic cells (AACs), as well as the cholecystokinin-containing regular-spiking basket cells (RSBCs) to cholinergically induced oscillations in hippocampal slices, a rhythmic activity that captures several features of the gamma oscillations recorded in vivo. By analyzing the spiking activities of single neurons recorded in parallel with local field potentials, we found that all three cell types fired phase locked to the carbachol-induced oscillations, although with different frequencies and precision. During these oscillations, FSBCs fired the most with the highest accuracy compared with the discharge of AACs and RSBCs. In further experiments, we showed that activation of µ-opioid receptors by DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin acetate), which significantly reduced the inhibitory, but not excitatory, transmission, suppressed or even blocked network oscillations both in vitro and in vivo, leading to the desynchronization of pyramidal cell firing. Using paired recordings, we demonstrated that carbachol application blocked GABA release from RSBCs and reduced it from FSBCs and AACs, whereas DAMGO further suppressed the GABA release only from FSBCs, but not from AACs. These results collectively suggest that the rhythmic perisomatic inhibition, generating oscillatory fluctuation in local field potentials after carbachol treatment of hippocampal slices, is the result of periodic GABA release from FSBCs.

Distinct synaptic properties of perisomatic inhibitory cell types and their different modulation by cholinergic receptor activation in the CA3 region of the mouse hippocampus.

Perisomatic inhibition originates from three types of GABAergic interneurons in cortical structures, including parvalbumin-containing fast-spiking basket cells (FSBCs) and axo-axonic cells (AACs), as well as cholecystokinin-expressing regular-spiking basket cells (RSBCs). These interneurons may have significant impact in various cognitive processes, and are subjects of cholinergic modulation. However, it is largely unknown how cholinergic receptor activation modulates the function of perisomatic inhibitory cells. Therefore, we performed paired recordings from anatomically identified perisomatic interneurons and pyramidal cells in the CA3 region of the mouse hippocampus. We determined the basic properties of unitary inhibitory postsynaptic currents (uIPSCs) and found that they differed among cell types, e.g. GABA released from axon endings of AACs evoked uIPSCs with the largest amplitude and with the longest decay measured at room temperature. RSBCs could also release GABA asynchronously, the magnitude of the release increasing with the discharge frequency of the presynaptic interneuron. Cholinergic receptor activation by carbachol significantly decreased the uIPSC amplitude in all three types of cell pairs, but to different extents. M2-type muscarinic receptors were responsible for the reduction in uIPSC amplitudes in FSBC- and AAC-pyramidal cell pairs, while an antagonist of CB(1) cannabinoid receptors recovered the suppression in RSBC-pyramidal cell pairs. In addition, carbachol suppressed or even eliminated the short-term depression of uIPSCs in FSBC- and AAC-pyramidal cell pairs in a frequency-dependent manner. These findings suggest that not only are the basic synaptic properties of perisomatic inhibitory cells distinct, but acetylcholine can differentially control the impact of perisomatic inhibition from different sources.

Fast synaptic subcortical control of hippocampal circuits.

Cortical information processing is under state-dependent control of subcortical neuromodulatory systems. Although this modulatory effect is thought to be mediated mainly by slow nonsynaptic metabotropic receptors, other mechanisms, such as direct synaptic transmission, are possible. Yet, it is currently unknown if any such form of subcortical control exists. Here, we present direct evidence of a strong, spatiotemporally precise excitatory input from an ascending neuromodulatory center. Selective stimulation of serotonergic median raphe neurons produced a rapid activation of hippocampal interneurons. At the network level, this subcortical drive was manifested as a pattern of effective disynaptic GABAergic inhibition that spread throughout the circuit. This form of subcortical network regulation should be incorporated into current concepts of normal and pathological cortical function.

Unique clustering of A-type potassium channels on different cell types of the main olfactory bulb.

Theoretical and functional studies predicted a highly non-uniform distribution of voltage-gated ion channels on the neuronal surface. This was confirmed by recent immunolocalization experiments for Na+, Ca2+, hyperpolarization activated mixed cation and K+ channels. These experiments also indicated that some K+ channels were clustered in synaptic or non-synaptic membrane specializations. Here we analysed the subcellular distribution of Kv4.2 and Kv4.3 subunits in the rat main olfactory bulb at high resolution to address whether clustering characterizes their distribution, and whether they are concentrated in synaptic or non-synaptic junctions. The cell surface distribution of the Kv4.2 and Kv4.3 subunits is highly non-uniform. Strong Kv4.2 subunit-immunopositive clusters were detected in intercellular junctions made by mitral, external tufted and granule cells (GCs). We also found Kv4.3 subunit-immunopositive clusters in periglomerular (PGC), deep short-axon and GCs. In the juxtaglomerular region some calretinin-immunopositive glial cells enwrap neighboring PGC somata in a cap-like manner. Kv4.3 subunit clusters are present in the cap membrane that directly contacts the PGC, but not the one that faces the neuropil. In membrane specializations established by members of the same cell type, K+ channels are enriched in both membranes, whereas specializations between different cell types contain a high density of channels asymmetrically. None of the K+ channel-rich junctions showed any of the ultrastructural features of known chemical synapses. Our study provides evidence for highly non-uniform subcellular distributions of A-type K+ channels and predicts their involvements in novel forms of intercellular communication in the olfactory pathway.

Release probability-dependent scaling of the postsynaptic responses at single hippocampal GABAergic synapses.

The amount of neurotransmitter released after the arrival of an action potential affects the strength and the trial-to-trial variability of postsynaptic responses. Most studies examining the dependence of synaptic neurotransmitter concentration on the release probability (P(r)) have focused on glutamatergic synapses. Here we asked whether univesicular or multivesicular release characterizes transmission at hippocampal GABAergic synapses. We used multiple probability functional analysis to derive quantal parameters at inhibitory connections between cannabinoid receptor- and cholecystokinin (CCK)-expressing interneurons and CA3 pyramidal cells. After the recordings, the cells were visualized and reconstructed at the light-microscopic level, and the number of boutons mediating the IPSCs was determined using electron microscopy (EM). The number of active zones (AZs) per CCK-immunopositive bouton was determined from three-dimensional EM reconstructions, thus allowing the calculation of the total number of AZs for each pair. Our results reveal an approximate fivefold discrepancy between the numbers of functionally determined release sites (17.4 +/- 3.2) and structurally identified AZs (3.7 +/- 0.9). Channel modeling predicts that a fivefold to sevenfold increase in the peak synaptic GABA concentration is required for the fivefold enhancement of the postsynaptic responses. Kinetic analysis of the unitary IPSCs indicates that the increase in synaptic GABA concentration is most likely attributable to multivesicular release. This change in the synaptic GABA concentration transient together with extremely low postsynaptic receptor occupancy permits a P(r)-dependent scaling of the postsynaptic response generated at a single hippocampal GABAergic synaptic contact.

Novel subcellular distribution pattern of A-type K+ channels on neuronal surface.

Potassium channels comprise the most diverse family of ion channels. In nerve cells, their critical roles in synaptic integration and output generation have been demonstrated. Here, we provide evidence for a distribution that predicts a novel role of K+ channels in the CNS. Our experiments revealed a highly selective clustering of the Kv4.3 A-type K+ channel subunits at specialized junctions between climbing fibers and cerebellar GABAergic interneurons. High-resolution ultrastructural and immunohistochemical experiments demonstrated that these junctions are distinct from known chemical and electrical (gap junctions) synapses and also from puncta adherentia. Each cerebellar interneuron contains many such K+ channel-rich specializations, which seem to be distributed throughout the somatodendritic surface. We also show that such K+ channel-rich specializations are not only present in the cerebellum but are widespread in the rat CNS. For example, mitral cells of the main olfactory bulb establish Kv4.2 subunit-positive specializations with each other. At these specializations, both apposing membranes have a high density of K+ channels, indicating bidirectional signaling. Similar specializations with pronounced coclustering of the Kv4.2 and 4.3 subunits were observed between nerve cells in the medial nucleus of the habenula. Based on our results and on the known properties of A-type K+ channels, we propose that strategically clustered K+ channels at unique membrane specializations could mediate a novel type of communication between nerve cells.