Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Mar 1;20(5):1869-82.
doi: 10.1523/JNEUROSCI.20-05-01869.2000.

Expression of Kv1 Potassium Channels in Mouse Hippocampal Primary Cultures: Development and Activity-Dependent Regulation

Affiliations
Free PMC article

Expression of Kv1 Potassium Channels in Mouse Hippocampal Primary Cultures: Development and Activity-Dependent Regulation

G Grosse et al. J Neurosci. .
Free PMC article

Abstract

Excitability and discharge behavior of neurons depends on the highly variable expression pattern of voltage-dependent potassium (Kv) channels throughout the nervous system. To learn more about distribution, development, and activity-dependent regulation of Kv channel subunit expression in the rodent hippocampus, we studied the protein expression of members of the Kv1 subfamily in mouse hippocampus in situ and in primary cultures. In adult hippocampus, Kv1 (1-6) channel alpha-subunits were present, whereas at postnatal day 2, none of these proteins could be detected in CA1-CA3 and dentate gyrus. Kv1.1 was the first channel to be observed at postnatal day 6. The delayed postnatal expression and most of the subcellular distribution observed in hippocampal sections were mimicked by cultured hippocampal neurons in which Kv channels appeared only after 10 days in vitro. This developmental upregulation was paralleled by a dramatic increase in total K(+) current, as well as an elevated GABA release in the presence of 4-aminopyridine. Thus, the developmental profile, subcellular localization, and functionality of Kv1 channels in primary culture of hippocampus closely resembles the in situ situation. Impairing secretion by clostridial neurotoxins or blocking activity by tetrodotoxin inhibited the expression of Kv1.1, Kv1.2, and Kv1.4, whereas the other Kv1 channels still appeared. This activity-dependent depression was only observed before the initial appearance of the respective channels and lost after they had been expressed. Our data show that hippocampal neurons in culture are a convenient model to study the developmental expression and regulation of Kv1 channels. The ontogenetic regulation and the activity-dependent expression of Kv1.1, Kv1.2, and Kv1.4 indicate that neuronal activity plays a crucial role for the development of the mature Kv channel pattern in hippocampal neurons.

Figures

Fig. 1.
Fig. 1.
Differential localization of Kv1 channel subtypes in mouse hippocampus. a, Coronal vibratome sections (50 μm) of hippocampi from adult mice were incubated with antisera against Kv1.1 (A), Kv1.2 (B), Kv1.3 (C, C′), Kv1.4 (E), Kv1.5 (D,D′), or Kv1.6 (F) as outlined in Materials and Methods. The arrow in Emarks the intense staining of the mossy fibers by the antiserum against Kv1.4. The differential staining in the various layers of hippocampal CA3 region are outlined in detail for Kv1.3 (compare Cwith C′). Kv1.3 occurs in high amounts in stratum pyramidale (sp), whereas lesser amounts are present in stratum radiatum (sr), and almost no channel protein is found in stratum oriens (so). Kv1.5 appears to be localized to stratum pyramidale (sp) of CA3 and to a lesser extent of CA1 in which it preferentially occurs in the neuropil as given in detail in D′. Scale bar:A–F, 500 μm; C′, D′, 130 μm. b, Coronal vibratome sections (70 μm) of hippocampi from mice pups of postnatal day 2 or 6 were incubated with antisera against Kv1.1 (A, B), Kv1.2 (C), Kv1.3 (D), Kv1.4 (E), Kv1.5 (F), or Kv1.6 (G) or without the first antiserum (H) as outlined in Materials and Methods. Kv1.1 is the earliest channel to be detected in some pyramidal neurons of CA3 and dentate gyrus at postnatal day 6 (B), which is clearly distinguishable when comparing with the respective control section (H). However, no specific immunoreactivity can be observed with the Kv1.1 antiserum at postnatal day 2 (compare A with H). The incubation of postnatal day 6 hippocampal section with Kv1.2 to Kv1.6 (C–G) only gives an unspecific staining closely resembling the one of the control section (H).
Fig. 1.
Fig. 1.
Differential localization of Kv1 channel subtypes in mouse hippocampus. a, Coronal vibratome sections (50 μm) of hippocampi from adult mice were incubated with antisera against Kv1.1 (A), Kv1.2 (B), Kv1.3 (C, C′), Kv1.4 (E), Kv1.5 (D,D′), or Kv1.6 (F) as outlined in Materials and Methods. The arrow in Emarks the intense staining of the mossy fibers by the antiserum against Kv1.4. The differential staining in the various layers of hippocampal CA3 region are outlined in detail for Kv1.3 (compare Cwith C′). Kv1.3 occurs in high amounts in stratum pyramidale (sp), whereas lesser amounts are present in stratum radiatum (sr), and almost no channel protein is found in stratum oriens (so). Kv1.5 appears to be localized to stratum pyramidale (sp) of CA3 and to a lesser extent of CA1 in which it preferentially occurs in the neuropil as given in detail in D′. Scale bar:A–F, 500 μm; C′, D′, 130 μm. b, Coronal vibratome sections (70 μm) of hippocampi from mice pups of postnatal day 2 or 6 were incubated with antisera against Kv1.1 (A, B), Kv1.2 (C), Kv1.3 (D), Kv1.4 (E), Kv1.5 (F), or Kv1.6 (G) or without the first antiserum (H) as outlined in Materials and Methods. Kv1.1 is the earliest channel to be detected in some pyramidal neurons of CA3 and dentate gyrus at postnatal day 6 (B), which is clearly distinguishable when comparing with the respective control section (H). However, no specific immunoreactivity can be observed with the Kv1.1 antiserum at postnatal day 2 (compare A with H). The incubation of postnatal day 6 hippocampal section with Kv1.2 to Kv1.6 (C–G) only gives an unspecific staining closely resembling the one of the control section (H).
Fig. 2.
Fig. 2.
Expression of Kv1 channel proteins in developing neurons in primary cultures. Hippocampal neurons cultivated for 18 d (18 DIV) were incubated with the antisera against Kv1.1–Kv1.6 (large panels). All Kv1 channel α-subunits are present in the somatodendritic compartment of pyramidal neurons. Kv1.1, Kv1.2, and Kv1.6 were also detected in axons, as indicated by thearrowheads. After 6 DIV, none of the Kv1 channel proteins showed up in the neuronal cultures (insets) analyzed by phase contrast. Scale bars, 20 μm.
Fig. 3.
Fig. 3.
Presence of Kv1.4 in axons of cultivated granule cells mirrors its occurrence in adult mossy fibers in vivo. Hippocampal neurons cultivated for 18 d (18 DIV) were incubated with the antisera against Kv1.4. A granule cell (G) characterized by its smaller and more globular perikaryon compared with pyramidal neurons is shown inA. Besides the somatodendritic compartment, the axon (ax) with its branching collaterals is also stained.B gives a detail of the hippocampal CA3 area. The granule cell-derived mossy fibers (mf) contacting proximal dendrites of pyramidal neurons (stratum pyramidale,sp) contain high amounts of Kv1.4 α-subunit. Scale bars: A, 20 μm; B, 250 μm.
Fig. 4.
Fig. 4.
Immune electron microscopic subcellular localization of Kv1.2 in hippocampal neurons. Hippocampal neurons (15 DIV) were processed for electron microscopy as described in Materials and Methods. Immunoreactive staining can be detected at the plasma membrane (filled arrows) of a dendrite (de) (A), an axon (ax) with the ending presynaptic (pre) terminal (D), and the perikaryon (B). In addition, Kv1.2 α-subunit is found to be localized in vesicular structures (open arrows) in presynaptic and postsynaptic (C) areas and in dendrites (A). Rarely, large dense core vesicles (asterisk in A) and clathrin-coated vesicles (cl in C) were observed. Scale bar: A, 0.9 μm; B–D, 0.5 μm.
Fig. 5.
Fig. 5.
Developmental increase in K+currents in hippocampal neurons. Left traces show superimposed series of currents after voltage jumps (top panels) from −70 to +50 mV (150 msec) with a preceding hyperpolarization to −120 mV (100 msec). Current responses show a transient (IA) and a sustained (IK) component in a cell at 8 DIV (middle), as well as in a more mature cell at 16 DIV.Right traces show similar voltage steps without a hyperpolarizing prepulse, which isolates the sustainedIK. In the younger cell, maximal total outward current was 3317 pA and IK was 1753 pA, whereas at 16 DIV total current amplitude was 9292 pA andIK was 2986 pA.
Fig. 6.
Fig. 6.
Expression of Kv1.1–Kv1.6 channels is differentially downregulated by toxins impairing synaptic activity. Hippocampal neurons received solvent (control), tetrodotoxin (Ttx; 1 μm), tetanus toxin (TeNt; 1 nm), or botulinum A toxin (BoNt/A; 20 nm) at 13 DIV as indicated on the right. Neurons were fixed 5 d later and processed for immunocytochemistry using the Kv1 antisera indicated below each row of panels. The arrowsdenote the axonal staining observed with the antisera against Kv1.6, Kv1.2, and Kv1.1. Note the complete disappearance of Kv1.4 and the reduced levels of Kv1.1 and Kv1.2 channel proteins in toxin-treated neurons, whereas the expression of Kv1.6 channel subunit remained unchanged. Comparable data were obtained in at least three different experiments. Scale bar, 20 μm.
Fig. 7.
Fig. 7.
Axonal localization of Kv1.1 and Kv1.2 and their disappearance after BoNt/A treatment. The protocol followed in principle the one outlined in Figure 5, but started at 7 DIV and lasted 11 d up to 18 DIV. After fixation neurons were double-labeled using the indicated antisera against Kv1.1 and Kv1.2 (red) and a monoclonal antibody against MAP2 (green). The arrows indicate axons that are only immunopositive for Kv1.1 (top panels) or Kv1.2, respectively, but exhibit no staining for the dendritic marker MAP2. Pretreatment with BoNt/A only impairs the expression of the Kv1 channel proteins but does not influence MAP2. The earlier application of BoNt/A (7 DIV) may explain why no residual immunoreactivity was found in axons (compare with Fig. 6, last row)
Fig. 8.
Fig. 8.
Immunoreplica analysis of control and toxin-treated hippocampal primary cultures. Hippocampal neurons were poisoned at 13 (A) or 18 (B) DIV with the following toxins: TTX 1 μm, TeNt 1 nm, BoNt/A 20 nm, and BoNt/B 20 nm as indicated. The cultures were harvested 5 d later and subjected to SDS-PAGE and immunoblotting using monoclonal antibodies against SNAP-25 and synaptobrevin, as well as antisera against the indicated Kv1 channel proteins. As expected, treatment with TeNt and BoNt/B completely abolished synaptobrevin and treatment with BoNt/A cleaved SNAP-25, whereas treatment with TTX did not change either of these proteins. Note that the amount of Kv1.1 and Kv1.2 channel protein was reduced in toxin-treated cultures and poisoned at 13 DIV (A) but not at 18 DIV (B), whereas Kv1.3, Kv1.5, and Kv1.6 (C) remained unchanged irrespective of toxin treatment. Comparable data were obtained in at least three different experiments from different preparations of primary cultures.
Fig. 9.
Fig. 9.
Differential localization of Kv1.4 in neurons and glial cells with or without toxin treatment. Hippocampal neurons received solvent (Con), tetanus toxin (TeNt; 1 nm), or botulinum A toxin (BoNt/A; 20 nm) at 13 DIV as given in Figure6. Five days later, cultures were either fixed and processed for immunocytochemistry (top two panels) or harvested and subjected to SDS-PAGE and immunoblotting using the Kv1.4 antiserum or the antibodies against SNAP-25 or synaptobrevin as indicated. Although synaptobrevin and SNAP-25 are clearly cleaved by the respective toxin, no reduction in the total protein content of Kv1.4 can be observed. As revealed by the immunocytochemical analysis, Kv1.4 is only reduced in neurons (N) but still present in glial cells, probably astrocytes (asterisks). Scale bar, 25 μm.

Similar articles

See all similar articles

Cited by 40 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback