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.

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.

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.

A-type K(+) current (I(A)) plays a critical role in controlling the excitability of pyramidal cell (PC) dendrites. In vitro dendritic patch-pipette recordings have demonstrated a prominent, sixfold increase in I(A) density along the main apical dendrites of rat hippocampal CA1 PCs. In these cells, I(A) is mediated by Kv4.2 subunits, whose precise subcellular distribution and densities in small-diameter oblique dendrites and dendritic spines are still unknown. Here we examined the densities of the Kv4.2 subunit in 13 axo-somato-dendritic compartments of CA1 PCs using a highly sensitive, high-resolution quantitative immunogold localization method (sodium dodecyl sulphate-digested freeze-fracture replica-labelling). Only an approximately 70% increase in Kv4.2 immunogold density was observed along the proximo-distal axis of main apical dendrites in the stratum radiatum with a slight decrease in density in stratum lacunosum-moleculare. A similar pattern was detected for all dendritic compartments, including main apical dendrites, small-diameter oblique dendrites and dendritic spines. The specificity of the somato-dendritic labelling was confirmed in Kv4.2(-/-) tissue. No specific immunolabelling for the Kv4.2 subunit was found in SNAP-25-containing presynaptic axons. Our results demonstrate a novel distribution pattern of a voltage-gated ion channel along the somato-dendritic surface of CA1 PCs, and suggest that the increase in the I(A) along the proximo-distal axis of PC dendrites cannot be solely explained by a corresponding increase in Kv4.2 channel number.

The glomeruli of the main olfactory bulb are the first processing station of the olfactory pathway, where complex interactions occur between sensory axons, mitral cells and a variety of juxtaglomerular neurons, including external tufted cells (ETCs). Despite a number of studies characterizing ETCs, little is known about how their morphological and functional properties correspond to each other. Here we determined the active and passive electrical properties of ETCs using in vitro whole-cell recordings, and correlated them with their dendritic arborization patterns. Principal component followed by cluster analysis revealed two distinct subpopulations of ETCs based on their electrophysiological properties. Eight out of 12 measured physiological parameters exhibited significant difference between the two subpopulations, including the membrane time constant, amplitude of spike afterhyperpolarization, variance in the interspike interval distribution and subthreshold resonance. Cluster analysis of the morphological properties of the cells also revealed two subpopulations, the most prominent dissimilarity between the groups being the presence or absence of secondary, basal dendrites. Finally, clustering the cells taking all measured properties into account also indicated the presence of two subpopulations that mapped in an almost perfect one-to-one fashion to both the physiologically and the morphologically derived groups. Our results demonstrate that a number of functional and structural properties of ETCs are highly predictive of one another. However, cells within each subpopulation exhibit pronounced variability, suggesting a large degree of specialization evolved to fulfil specific functional requirements in olfactory information processing.

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.