Presynaptic localization of Kv1.4-containing A-type potassium channels near excitatory synapses in the hippocampus

Abstract

Mammalian Shaker voltage-gated potassium channels that contain the Kv1.4 subunit exhibit rapid activation and prominent inactivation processes, which enable these channels to integrate brief (approximiately milliseconds) depolarizations over time intervals of up to tens of seconds. In the hippocampus, Kv1.4 immunoreactivity is detected at greatest density in two regions: (1) the middle molecular layer (MML), where perforant path axons synapse with dentate granule cells, and (2) the stratum lucidum (SL) of CA3, where the mossy fibers travel in tight fasciculi and form en passante synapses onto CA3 pyramidal cells. We have studied the localization of Kv1.4 within these regions in detail. First, we compared the distribution of Kv1.4 and synaptophysin (a synaptic vesicle protein primarily localized near termini) under confocal immunofluorescence microscopy. In the MML, Kv1.4 and synaptophysin immunofluorescence appeared to overlap. In the SL, however, Kv1.4 and synaptophysin staining was detected in nonoverlapping, irregular patches ( approximately 5-10 micro m in diameter). Ultrastructural studies of these two regions revealed that Kv1.4 immunoreactivity was absent from the surface membranes of cell bodies and dendrites and occurred prominently on axons, including axonal "necks" near termini. Small excitatory synaptic boutons also were labeled in the MML; by contrast, the mossy fiber synaptic expansions in the SL were not stained. These localizations may enable Kv1.4-containing channels to regulate the process of neurotransmitter release at these excitatory synapses.

Fig. 1.

Kv1.4 is localized to the dentate gyrus middle molecular layer and to stratum lucidum. A, View of the dorsal hippocampus at low magnification after immunoperoxidase staining for Kv1.4. Reaction product is highest in the middle molecular layer, the polymorphic layer, and stratum lucidum. B, Drawing indicating the major tissue layers, the principal excitatory neurons, and their synapses. Pathways labeled are pp, the perforant (entorhinal–hippocampal) path; mf, the mossy fiber tract; com, the commissural path; andsc, the Schaffer collateral path. Layers of the dentate gyrus are labeled ml, the molecular layer;gcl, the granule cell layer; and pml, the polymorphic, or hilar, layer. Layers of the hippocampus are labeledso, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare; and sl (in CA3 only), stratum lucidum. C, View of a portion of dentate gyrus at higher magnification. Layers are labeled as in B, but the molecular layer is divided into inner, middle, and outer regions (iml, mml, andoml). Kv1.4 staining is present in several layers, and is highest in the mml and pml. Mml staining has a fine, homogeneous appearance. D, View of a portion of CA3 at higher magnification. Kv1.4 staining within sl has a patchy or punctate appearance (arrows) different from that seen in the mml.E, View of a portion of distal CA3 at higher magnification. In this region, Kv1.4 immunoreactivity (arrow) exhibits a fibrillar appearance different from that seen in D. Scale bars: A, 0.25 mm;C, E, 30 μm; D, 50 μm.

Fig. 2.

A–F, Localization of Kv1.4 and synaptophysin by confocal microscopy. A, Synaptophysin immunoreactivity in CA3. Immunofluorescence occurs at high density in patches within sl. Expression is lower in sr and sp.B, Kv1.4 immunoreactivity in CA3. Within sl, pattern appears similar to that observed for synaptophysin. C, Superimposition of A and B. Kv1.4 and synaptophysin patches in sl are largely noncoinciding.D, Synaptophysin expression in the dentate. Immunoreactivity occurs in fine puncta throughout the molecular layer. Signal intensity is oml > mml, iml > gcl. E, Kv1.4 expression in the dentate. Signal intensity is mml > oml > iml > gcl. F, Superimposition ofD and E. Scale bar, 25 μm.

Fig. 3.

Immunoelectron microscopic localization of Kv1.4 on axons within the dentate gyrus. A,B, Schematic drawings indicating the area depicted in electron micrographs in C–E and Figure4. Box within A contains a portion of the dentate molecular and granule cell layers and is expanded asB; box within B encloses the middle molecular layer alone and includes representations of the main features contained within the accompanying electron micrographs: dendrites of granule cells that flank smaller profiles of perforant path-derived axons, their termini, and spines. C, Axon sectioned longitudinally exhibits membrane-associated puncta of immunoreaction product at three locations (arrowheads). Also notable within the stained axon are microtubules and a dense core granule (arrow). D, An axon (course indicated by white arrowheads) in longitudinal section synapses with an unlabeled spine. Label (dark arrowhead) is present in an axon near the synapse. Two small profiles in cross section are also labeled (dark arrowheads).E, Small labeled and unlabeled profiles in cross section crowded between two longitudinally sectioned dendrites. Labeled profiles (1–4) are of variable size, contain synaptic vesicles, and may represent portions of axons at various distances from termini. F, A small myelinated axon sectioned obliquely contains dense reaction product associated with the cell membrane (polymorphic layer). Den, Dendrite;mit, mitochondrion; s, spine;sv, synaptic vesicles. Scale bars, 0.5 μm.

Fig. 4.

Kv1.4 localization within presynaptic termini in the middle molecular layer. A, Drawing based on the micrograph shown in B. B, Single, complex terminal forming three synapses with two spines. Immunoreaction product (arrowhead) appears to arise from portion of terminal membrane opposite from synaptic active zones. C, Membrane-associated stain is detected within two presynaptic termini. Within one (T1), reaction product is near, but apparently not within an active zone. The second labeled terminal (T2) synapses with two spines. Reaction product (arrowheads) also is noted within an axon (ax) seen in longitudinal section and associated with the membrane of another profile that contains clear synaptic vesicles and dense core granules.D, Cluster of three synapses. In one terminal (T1), reaction product appears to arise from the membrane at the active zone. E, Stain in a small profile of a presynaptic terminal. Scale bars, 0.2 μm. Abbreviations are as in Figure 3.

Fig. 5.

Kv1.4 localization in the stratum lucidum of CA3. A, B, Schematic drawings indicating the area depicted in electron micrographs. Box withinA encloses a portion of stratum pyramidale and stratum lucidum and is expanded as B; box withinB encloses representations of main features of the accompanying electron micrographs: large proximal dendrites of pyramidal cells are flanked either by fasciculi of mossy fiber axons or by synaptic structures formed by mossy fiber expansions and complex pyramidal cell spines. C, Labeled axons within a mossy fiber fasciculus (MF fasc) pass between a CA3 pyramidal cell soma (soma) and the proximal dendrite (den) of another pyramidal cell. D, Lower magnification view of stratum lucidum containing a mossy fiber fasciculus with transverse and obliquely sectioned mossy fibers, a pyramidal cell dendritic branch (den), and a “synaptic complex” consisting of multiple spine branches (s) derived from a pyramidal cell “thorny excrescence” and synaptic vesicle-filled, multilobulated mossy fiber expansions (MFE). Numerous stained axons in cross section are noted (solid arrowheads). Another axon, sectioned lengthwise, is stained heavily near its transition into a mossy fiber expansion (open arrowheads).E, A longitudinally sectioned axon within a mossy fiber fasciculus contains immunoreaction product near the site of origin of a presynaptic expansion. F, Stained small axon sectioned longitudinally. MT, Microtubule. Scale bars:C, D, 0.5 μm; (0.25 μm inD, inset); E, 0.2 μm;F, 0.2 μm.