Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts

J. B. Dean, R. Q. Huang, J. S. Erlichman, T. L. Southard, D. T. Hellard

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 μm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential- like activity, which had peak-to-peak amplitudes of ≤ 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade: and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip (≤3 μm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, was averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording, which typically lasted for 1-3 h, Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (< 1 min) steady-state perforation at 22-25°C. This adaptation of conventional perforated patch recording was termed 'rapid perforated patch recording'. The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].

Original languageEnglish
Pages (from-to)21-40
Number of pages20
JournalNeuroscience
Volume80
Issue number1
DOIs
StatePublished - 25 Jun 1997

Fingerprint

Artifacts
Amphotericin B
Neurons
Synaptic Potentials
Staining and Labeling
Video Microscopy
Medulla Oblongata
Solitary Nucleus
Gap Junctions
Carisoprodol
Neurosciences
Action Potentials
lucifer yellow
Coloring Agents
Injections
biocytin

Keywords

  • Brain slice
  • Dorsal motor nucleus of vagus
  • Electrotonic coupling
  • Gap junction
  • Nucleus tractus solitarius
  • Perforated patch recording

Cite this

Dean, J. B. ; Huang, R. Q. ; Erlichman, J. S. ; Southard, T. L. ; Hellard, D. T. / Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts. In: Neuroscience. 1997 ; Vol. 80, No. 1. pp. 21-40.
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Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts. / Dean, J. B.; Huang, R. Q.; Erlichman, J. S.; Southard, T. L.; Hellard, D. T.

In: Neuroscience, Vol. 80, No. 1, 25.06.1997, p. 21-40.

Research output: Contribution to journalArticle

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T1 - Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts

AU - Dean, J. B.

AU - Huang, R. Q.

AU - Erlichman, J. S.

AU - Southard, T. L.

AU - Hellard, D. T.

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N2 - Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 μm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential- like activity, which had peak-to-peak amplitudes of ≤ 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade: and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip (≤3 μm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, was averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording, which typically lasted for 1-3 h, Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (< 1 min) steady-state perforation at 22-25°C. This adaptation of conventional perforated patch recording was termed 'rapid perforated patch recording'. The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].

AB - Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 μm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential- like activity, which had peak-to-peak amplitudes of ≤ 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade: and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip (≤3 μm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, was averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording, which typically lasted for 1-3 h, Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (< 1 min) steady-state perforation at 22-25°C. This adaptation of conventional perforated patch recording was termed 'rapid perforated patch recording'. The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].

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