Correlated network activity is important in the development of many neural circuits. Purkinje cells are among the first neurons to populate the cerebellar cortex, where they sprout exuberant axon collaterals. We used multiple patch-clamp recordings targeted with two-photon microscopy to characterize monosynaptic connections between the Purkinje cells of juvenile mice. We found that Purkinje cell axon collaterals projected asymmetrically in the sagittal plane, directed away from the lobule apex. On the basis of our anatomical and physiological characterization of this connection, we constructed a network model that robustly generated waves of activity that traveled along chains of connected Purkinje cells. Consistent with the model, we observed traveling waves of activity in Purkinje cells in sagittal slices from young mice that require GABA(A) receptor-mediated transmission and intact Purkinje cell axon collaterals. These traveling waves are absent in adult mice, suggesting they have a developmental role in wiring the cerebellar cortical microcircuit.

Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

Cerebellar Golgi cells (GoCs) efficiently control the spiking activity of granule cells through GABAA receptor-mediated tonic and phasic inhibition. Recent experiments provided compelling evidence for the extensive interconnection of GoCs through electrical synapses, but their chemical inhibitory synaptic inputs are debated. Here, we investigated the GABAergic synaptic inputs of GoCs using in vitro electrophysiology and quantitative light microscopy (LM) and electron microscopy (EM). We characterized GABAA receptor-mediated IPSCs in GoCs and Lugaro cells (LuCs), and found that IPSCs in GoCs have lower frequencies, smaller amplitudes, and much slower decay kinetics. Pharmacological and LM immunolocalization experiments revealed that GoCs express α3, whereas LuCs express α1 subunit-containing GABAA receptors. The selective expression and clustered distribution of the α3 subunit in GoCs allowed the quantitative analysis of GABAergic synapses on their dendrites in the molecular layer (ML). EM and LM experiments in rats, and wild-type and GlyT2-GFP transgenic mice revealed that only one third of axon terminals establishing GABAergic synapses on GoC dendrites contain GlyT2, ruling out LuCs, globular cells, and any noncortical glycinergic inputs as major inhibitory sources. We also show that axon terminals of stellate/basket cells very rarely innervate GlyT2-GFP-expressing GoCs, indicating that only a minority of the inhibitory inputs to GoCs in the ML originates from local interneurons, and the majority of their inhibitory inputs exclusively releases GABA.

AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.

Excitatory neurotransmission and its activity-dependent plasticity are largely determined by AMPA-receptors (AMPARs), ion channel complexes whose cell physiology is encoded by their interactome. Here, we delineate the assembly of AMPARs in the endoplasmic reticulum (ER) of native neurons as multi-state production line controlled by distinct interactome constituents: ABHD6 together with porcupine stabilizes pore-forming GluA monomers, and the intellectual-disability-related FRRS1l-CPT1c complexes promote GluA oligomerization and co-assembly of GluA tetramers with cornichon and transmembrane AMPA-regulatory proteins (TARP) to render receptor channels ready for ER exit. Disruption of the assembly line by FRRS1l deletion largely reduces AMPARs in the plasma membrane, impairs synapse formation, and abolishes activity-dependent synaptic plasticity, while FRRS1l overexpression has the opposite effect. As a consequence, FRSS1l knockout mice display severe deficits in learning tasks and behavior. Our results provide mechanistic insight into the stepwise biogenesis of AMPARs in native ER membranes and establish FRRS1l as a powerful regulator of synaptic signaling and plasticity.

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.

The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.

Information processing and storage in the brain rely on AMPA-receptors (AMPARs) and their context-dependent dynamics in synapses and extra-synaptic sites. We found that distribution and dynamics of AMPARs in the plasma membrane are controlled by Noelins, a three-member family of conserved secreted proteins expressed throughout the brain in a cell-type-specific manner. Noelin tetramers tightly assemble with the extracellular domains of AMPARs and interconnect them in a network-like configuration with a variety of secreted and membrane-anchored proteins including Neurexin1, Neuritin1, and Seizure 6-like. Knock out of Noelins1-3 profoundly reduced AMPARs in synapses onto excitatory and inhibitory (inter)neurons, decreased their density and clustering in dendrites, and abolished activity-dependent synaptic plasticity. Our results uncover an endogenous mechanism for extracellular anchoring of AMPARs and establish Noelin-organized networks as versatile determinants of constitutive and context-dependent neurotransmission.

Synapses exhibit different short-term plasticity patterns and this behaviour influences information processing in neuronal networks. We tested how the short-term plasticity of excitatory postsynaptic currents (EPSCs) depends on the postsynaptic cell type, identified by axonal arborizations and molecular markers in the hippocampal CA1 area. Three distinct types of short-term synaptic behaviour (facilitating, depressing and combined facilitating-depressing) were defined by fitting a dynamic neurotransmission model to the data. Approximately 75 % of the oriens-lacunosum-moleculare (O-LM) interneurones received facilitating EPSCs, but in three of 12 O-LM cells EPSCs also showed significant depression. Over 90 % of the O-LM cells were immunopositive for somatostatin and mGluR1alpha and all tested cells were decorated by strongly mGluR7a positive axon terminals. Responses in eight of 12 basket cells were described well with a model involving only depression, but the other cells displayed combined facilitating-depressing EPSCs. No apparent difference was found between the plasticity of EPSCs in cholecystokinin- or parvalbumin-containing basket cells. In oriens-bistratified cells (O-Bi), two of nine cells showed facilitating EPSCs, another two depressing, and the remaining five cells combined facilitating-depressing EPSCs. Seven of 10 cells tested for somatostatin were immunopositive, but mGluR1alpha was detectable only in two of 11 tested cells. Furthermore, most O-Bi cells projected to the CA3 area and the subiculum, as well as outside the hippocampal formation. Postsynaptic responses to action potentials recorded in vivo from a CA1 place cell were modelled, and revealed great differences between and within cell types. Our results demonstrate that the short-term plasticity of EPSCs is cell type dependent, but with significant heterogeneity within all three interneurone populations.

Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD) for gamma-aminobutyric acid (GABA) is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A) and GABA(C)) or metabotropic receptors (GABA(B)) while it is terminated by the re-uptake of GABA through transporters (GATs).

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.

The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons.

Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K(+) channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K(+) channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K(+) channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.

Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA(A)Rγ2(−/−) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ((AU1)γ2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)γ2(77F) subunit (Cre-2A-(AU1)γ2(77F)) into the neocortex of GABA(A)Rγ2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)γ2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2(77I) and (AU1)γ2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.

Local circuit GABAergic interneurons comprise the most diverse cell populations of neuronal networks. Interneurons have been characterized and categorized based on their axo-somato-dendritic morphologies, neurochemical content, intrinsic electrical properties and their firing in relation to in-vivo population activity. Great advances in our understanding of their roles have been facilitated by their selective identification. Recently, we have described three major subtypes of deep short-axon cells (dSACs) of the main olfactory bulb (MOB) based on their axo-dendritic distributions and synaptic connectivity. Here, we investigated whether dSACs also display pronounced molecular diversity and whether distinct dSAC subtypes selectively express certain molecules. Multiple immunofluorescent labeling revealed that the most commonly used molecular markers of dSACs (e.g. vasoactive intestinal polypeptide, calbindin and nitric oxide synthase) label only very small subpopulations (< 7%). In contrast, voltage-gated potassium channel subunits Kv2.1, Kv3.1b, Kv4.3 and the GABA(A) receptor alpha1 subunit are present in 70-95% of dSACs without showing any dSAC subtype-selective expression. However, metabotropic glutamate receptor type 1alpha mainly labels dSACs that project to the glomerular layer (GL-dSAC subtype) and comprise approximately 20% of the total dSAC population. Analysing these molecular markers with stereological methods, we estimated the total number of dSACs in the entire MOB to be approximately 13,500, which is around a quarter of the number of mitral cells. Our results demonstrate a large molecular heterogeneity of dSACs and reveal a unique neurochemical marker for one dSAC subtype. Based on our results, dSAC subtype-specific genetic modifications will allow us to decipher the role of GL-dSACs in shaping the dynamic activity of the MOB network.

In many brain regions, hyperpolarization-activated cationic currents (Ih) are involved in the generation of rhythmic activities, but the role of Ih in olfactory oscillations remains unclear. Knowledge of the cellular and subcellular distributions of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) subunits is necessary for understanding the role of Ih in olfactory network activities. Using light microscopic immunocytochemistry, we demonstrate strong HCN1 labelling of the glomerular layer and moderate staining of granule cell, internal and external plexiform layers of the rat main olfactory bulb. In the glomerular layer, among many unlabelled neurons, two distinct subpopulations of juxtaglomerular cells are labelled. Approximately 10% of the juxtaglomerular cells strongly express HCN1. These small diameter cells are immunoreactive for GABA and comprise a subpopulation of periglomerular cells. An additional subset of juxtaglomerular cells ( approximately 1%) expresses low levels of HCN1. They are large in diameter, GABA immunonegative but immunopositive for vesicular glutamate transporter 2, characterizing them as external tufted cells. Quantitative immunogold localization revealed that the somatic plasma membranes of periglomerular cells contain approximately four times more HCN1 labelling than those of external tufted cells. Unlike in cortical pyramidal cells, immunogold density for HCN1 does not significantly differ in somatic and dendritic plasma membranes of external tufted cells, indicating that post-synaptic potentials arriving at proximal and distal dendrites are modulated by the same density of Ih. Our results demonstrate a cell type-dependent expression of HCN1 in the olfactory bulb and predict a differential contribution of distinct juxtaglomerular cell types to network oscillations.

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.

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.

Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content.