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. 2000 Aug 29;97(18):10260-5.
doi: 10.1073/pnas.160037097.

Connexin expression in electrically coupled postnatal rat brain neurons

L Venance  1 , A Rozov, M Blatow, N Burnashev, D Feldmeyer, H Monyer
Affiliations

Connexin expression in electrically coupled postnatal rat brain neurons

L Venance et al. Proc Natl Acad Sci U S A. .

Abstract

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription-PCR revealed a differential expression pattern of connexins in these identified neurons.

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Figures

Figure 1
Figure 1
Electrical coupling between cell pairs in different regions of the postnatal rat brain. (a) Electrical coupling between two bipolar GABAergic interneurons of layer 2/3 visual cortex. (b) Electrical coupling between layer 4 fusiform interneuron and spiny stellate neuron. (c) Electrical coupling between two hippocampal dentate gyrus basket cells. In all cases, the voltage response of cell 1 after current injection is also detectable in cell 2 and vice versa, although with a significantly reduced amplitude. Fast signals like action potentials showed a clearly distorted time course. This is most visible in the bottom traces of a, where a single reflected voltage response in cell 2 was scaled to the amplitude of the action potential in the current-injected cell 1 and vice versa. The recordings shown here were made in the presence of 5 μM 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) and 10 μM bicuculline to block chemical synapses.
Figure 2
Figure 2
Anatomical reconstruction and firing pattern of electrically coupled neurons. IR-DIC images, action potential firing patterns, and camera lucida-type reconstructions of biocytin-injected neurons are shown for all cell types. (a) Bipolar interneuron in layer 2/3 of P14 rat visual cortex. (b and c) Fusiform interneuron (b) and spiny stellate cell (c) in layer 4 of P14 rat somatosensory cortex. (d) Hippocampal dentate gyrus basket cell. In the reconstructions, the axon is red and the soma and dendrites are black. (The scale bar is 10 μm for all IR-DIC images, 100 μm for the reconstructions in a–c, and 50 μm for the reconstruction in d.)
Figure 3
Figure 3
Biochemical characterization of electrically coupled neurons. (a1) Biocytin-labeled bipolar interneuron in the visual cortex. (a2) In situ hybridization of the same section with a digoxigenin-labeled somatostatin riboprobe. (b1) Biocytin-labeled fusiform interneuron in the barrel cortex. (b2) In situ hybridization of the same section with a digoxigenin-labeled somatostatin riboprobe. (c1) Biocytin-labeled hippocampal dentate gyrus basket cell. (c2) Immunocytochemistry of the same section with a parvalbumin-specific antibody. (Scale bar, 20 μm.)
Figure 4
Figure 4
Identification of Cx expression by single-cell RT-PCR. (a) Location of PCR primers (arrows) and hybridization probes (bars) for the analysis of Cx expression in the cortical and hippocampal cell types. M1 to M4 denote the putative transmembrane regions. For Cx36 amplification the 5′ primer (P5′) was located on exon 1; the 3′ primer (P3′) and nested 3′ primer (P3′n) were on exon 2. The expression analysis of the other Cx was a two-step procedure. First, generic Cx primers (white arrows) located on conserved coding region (exon 2) were used, and the constituents in the RT-PCR product were evaluated by Southern analysis, using subunit-specific probes (white bar) and direct DNA sequencing. Because the common, generic primers did not span an intron, the origin of the PCR amplicon was verified in separate reactions by use of Cx subunit-specific primers (black arrows), with the 5′ primer located in the 5′ untranslated region (exon 1) and the 3′ and nested 3′ primers located in the coding region (exon 2). The identity of the PCR products obtained with the specific primers was also confirmed by Southern analysis with Cx-specific oligonucleotide probes (black bar) and direct DNA sequencing. (b) Representative experiment from bipolar neurons in P14 rat visual cortex. Gel electrophoresis (Upper) was carried out for RT-PCR products, as was a corresponding Southern blot (Lower) for six bipolar neurons after amplification with specific Cx36 and Cx32 primers, respectively. An RT-PCR product was obtained for three of six neurons with Cx36 primers and for one of another set of six cells with Cx32 primers. The upper band seen after amplification with Cx36 primers has been verified by sequencing to represent a single strand that runs at a height different from that of a double strand. (c) Representative experiment from basket cells of P14 rat hippocampus. Gel electrophoresis was carried out for RT-PCR products, as was a corresponding Southern blot for six basket cells after amplification with Cx36 and Cx26 primers, respectively. An RT-PCR product was obtained in three of six neurons with both primer pairs. M, size marker.
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