Heterogeneous distribution of kir3 potassium channel proteins within dopaminergic neurons in the mesencephalon of the rat brain - PubMed (original) (raw)

Heterogeneous distribution of kir3 potassium channel proteins within dopaminergic neurons in the mesencephalon of the rat brain

Dirk Eulitz et al. Cell Mol Neurobiol. 2007 May.

Abstract

1. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of the ventral mesencephalon play an important role in the regulation of the parallel basal ganglia loops.2. We have raised affinity-purified polyclonal rabbit antibodies specific for all four members of the Kir3 family of inwardly rectifying potassium channels (Kir3.1-Kir3.4) to investigate the distribution of the channel proteins in the dopaminergic neurons of the rat mesencephalon at light and electron microscopic level. In addition, immunocytochemical double labeling with tyrosine hydroxylase (TH), a marker of dopaminergic neurons, were performed.3. All Kir3 channels were present in this region. However, the individual proteins showed differential cellular and subcellular distributions.4. Kir3.1 immunoreactivity was found in SNc fibers and some neurons of the substantia nigra pars reticulata (SNr). Few Kir3.3-positive neurons were found in the SNc. However, a strong Kir3.3 signal was identified in the SNr neuropil. Weak Kir3.4 staining was detected in neuronal somata as well as in dendritic fibers of both parts of the SN.5. In the VTA, Kir3.1, Kir3.3, and Kir3.4 showed only weak staining of neuropil structures. The distribution of the Kir3.2 channel protein was especially striking with strong labeling in the SNc and in the lateral but not central VTA.6. Our results suggest that the heterogeneously distributed Kir3.2 channel proteins could help to discriminate the dopaminergic neurons of VTA and SNc.

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Figures

Fig. 1.

Fig. 1.

Competitive ELISA assay of anti-Kir3 antibodies as exemplified by anti-Kir3.1 antibodies. Microtiter plates were coated with the respective GST-Kir3.x or GST-Kir6.2 fusion protein. Only preincubation of the antibodies with the cognate GST-Kir3 fusion protein decreased immunoreactivity in a concentration-dependent manner, whereas other GST-Kir fusion proteins had no effect.

Fig. 2.

Fig. 2.

Western Blot analysis of anti-Kir3 antibodies. Rat brain homogenates (10 μg/lane) were separated on a 10% SDS-PAGE and blotted onto nitrocellulose membranes. (A) Anti-Kir3.1 antibodies recognize three distinct bands of ∼50, ∼53, and ∼60 kD, all other anti-Kir3 antibodies recognize single bands of 48 kD (B, Kir3.2), 44 kD (C, Kir3.3), and 53 kD (D, Kir3.4). Preincubation with the cognate recombinant GST-Kir3 fusion proteins completely abolished detection by anti-Kir3 antibodies (right lanes).

Fig. 3.

Fig. 3.

Adjacent coronal sections of rat hippocampus were incubated with anti-Kir3 antibodies (left column, A–D) or with anti-Kir3 antibodies preincubated with their cognate GST-Kir3 fusion proteins (10 μg/mL, right column, a–d). Specific immunolabeling was totally repressed after preincubation. Inset in (D) shows cellular distribution of Kir3.4 channels at higher magnification in the area indicated with an arrow. Bar represents 1 mm.

Fig. 4.

Fig. 4.

Kir3 channels are differentially expressed in the ventral mesencephalon. Several subnuclei can be distinguished in SN and VTA; RLi, rostral linear raphe; IF, interfascicular nucleus; PBPM, parabrachial pigmented nucleus, medial part; PN, paranigral nucleus; PBPL, parabrachial pigmented nucleus, lateral part; SNr, substantia nigra pars reticulata; SNcd, substantia nigra pars compacta dorsal; SNcv, substantia nigra pars compacta, ventral part; SNcl, substantia nigra pars compacta, lateral part (A). Dopaminergic neurons are heterogeneously distributed in SN and VTA subnuclei (B). The Kir3.2 channel displays the most heterogeneous distribution pattern resembling the tyrosine hydroxylase staining with high expression in the SNc and the lateral VTA subnuclei and only weak immunostaining in the PN and RLi subnuclei of the VTA (D). The Kir3.1, Kir3.3, and Kir3.4 channels display a homogeneous distribution in both VTA and SN with predominant staining of the neuropil (C, E, F). Bar represents 500 μm, insets show 6-fold higher magnifications.

Fig. 5.

Fig. 5.

Kir3.2 channels are detected in dopaminergic neurons using fluorescence double labeling. Tyrosine hydroxylase-positive dopaminergic neurons are widely distributed in PN and PBPM subnuclei of the VTA (A1), substantia nigra (A2), and IF subnucleus (A3). Kir3.2 channels are similarly stained in the SN (B2) and some VTA subnuclei (B1) and thus show an extensive coexpression in the overlay image (C1, C2, indicated by arrows). However, some tyrosine hydroxylase-positive neurons of the PN and PBPM subnuclei of the VTA do not express the Kir3.2 channel (arrowhead in C1) and the IF subnucleus is even devoid of Kir3.2 despite many dopaminergic neurons (B3). Subnuclei are indicated in D1, D2 and D3. Bar represents 100 μm; * indicate blood vessels.

Fig. 6.

Fig. 6.

Subcellular expression of the Kir3.2 channel subunit. The plasma membrane (A, pm) as well as the golgi apparatus (B, g) of neurons in the IF subnucleus of the VTA lack Kir3.2 immunoreactivity, thus confirming the light microscopic data. Some diaminobenzidine deposits are seen in dendrites of neurons that originate outside the IF subnucleus (arrows in A). Double labeling experiments confirm that dopaminergic IF neurons do not express the Kir3.2 channel protein (C, D). No gold-label was detected on DAB-positive cells (C) or dendrites (D). In the PN subnucleus, however, Kir3.2 channels are extensively expressed in the cytoplasm (E, arrowheads) and plasma membrane (arrows in E, F) of dopaminergic neuronal somata and dendrites. All bars represent 500 nm; pm: plasma membrane; at: axon terminal;nuc: nucleus; den: dendrite; g: golgi apparatus; mit: mitochondria; *: synapse.

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