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First published online November 2, 2007
Journal of Experimental Biology 210, 3962-3969 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006577
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Histamine operates Cl–-gated channels in crayfish neurosecretory cells

Jorge Cebada and Ubaldo García*

Department of Physiology, Biophysics and Neuroscience, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco, 07360 México City, México


Figure 1
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  		Fig. 1. HA inhibits the excitability of the XO-SG system in the isolated eyestalk preparation. (A) Schematic representation of the experimental array used to obtain simultaneous recordings from an X-organ cell body and the XO-SG tract. (B) Fluorescence micrography of the X-organ region obtained after retrograde staining of the tract OX-SG with Calcium Green-dextran. (C) Effect of HA on the spontaneous electrical activity propagated along the XO-SG tract (upper trace); note that during the HA superfusion (50 µmol l–1) most of the electrical activity was inhibited. This effect is due to the hyperpolarization evoked by HA on the X-organ cells (intracellular recording, bottom trace). (D) Hyperpolarization evoked by HA is associated with an increase in membrane conductance, indicated by reduction of the input resistance. The dotted lines indicate zero membrane potential.

 

Figure 2
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  		Fig. 2. HA activates a Cl– conductance in X-organ neurons. (A) Current-clamp recordings from an X-organ neuron in culture. Note that during the application of HA, the membrane potential (Em) reached –60 mV at all holding potentials explored. (B,C) HA-evoked currents (IHA) obtained at different holding potentials from –80 to –20 mV; the interval between each HA pulse was 3 min. All the traces were obtained from the same neuron at two Cl– equilibrium potentials (–62.5 mV for B and –32 mV for C). (D) Current–voltage relationship for the experimental conditions described in B and C. Both the solid circle and open circle curves correspond to the average ± s.e.m. of 12 cells, and the Cl– equilibrium potential corresponded to –62.5 mV or –32 mV, respectively.

 

Figure 3
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  		Fig. 3. Cl– ligand-gated currents in X-organ neurons. (A) Representative current traces obtained from the same neuron in response to the EC50 concentrations for Glu, GABA and HA. (B) Glu-evoked currents obtained after, during and before the superfusion of a desensitizing Glu concentration (3 µmol l–1, upper traces); note that the HA-evoked currents modified neither the amplitude nor the time course during the Glu superfusion (bottom traces). (C) As in B, GABA superfusion (1 µmol l–1) did not modify the HA-evoked current (bottom traces), but desensitized the GABA response (upper traces). Arrows mark the current traces obtained during the superfusion of Glu or GABA.

 

Figure 4
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  		Fig. 4. X-organ neuron sensitivity to HA. (A) Representative HA-evoked currents obtained at –40 mV holding potential, during application of HA pulses at the indicated concentrations. (B) Peak currents (mean ± s.e.m.) versus HA concentration (3–5 observations per point). The solid line correspond to a non-linear regression using Imax=1/[1+(EC50/HA)n], where HA=molar HA concentration, n=the Hill coefficient and EC50=HA concentration giving half-maximal effect, being the free parameters. The fit yielded EC50=3.3±1 µmol l–1 and n=2.6±0.4.

 

Figure 5
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  		Fig. 5. Effects of Cl– channel blockers on the HA response in X-organ neurons. (A,B) Superimposed current traces obtained after, during and before the superfusion of Cl– channel blockers. Neither picrotoxin nor strychnine at 100 µmol l–1 modified the HA-evoked current. (C) The cholinergic antagonist, d-tubocurarine (dTC; 20 µmol l–1) blocked the HA-evoked current reversibly. (D) Average current–voltage curves (5 observations per point); solid circles, control conditions; open circles, blockage exerted by dTC. (E) Inhibition curve. Each point represents the average value for five observations. The solid line corresponds to a non-linear regression giving an adjusted IC50=21±2 µmol l–1.

 

Figure 6
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  		Fig. 6. Effects of HAergic antagonists on the HA evoked-current in X-organ neurons. (A–D) Current traces evoked by HA (5 µmol l–1, 10 s) after, during and before the superfusion of H1 and H2 antagonists at the indicated concentrations; all the records were obtained at a holding potential of –40 mV, and the interval between pulses was 3 min. Note that in all cases the blockage was reversible. (E) Normalized peak currents (mean ± s.e.m.) versus log molar concentration of H1 and H2 antagonists (4–6 observations per point; see the IC50 values in the text). Squares, tiotidine; diamonds, cimetidine; circles, ranitidine; triangles, mepyramine.

 

Figure 7
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  		Fig. 7. Distribution of HA immunoreactivity in the crayfish eyestalk. (A) Schematic representation of a dorsal view of the eyestalk and the relative position of HAergic like single neurons. LG, lamina ganglionaris; ME, medulla externa; GS, sinus gland; MI, medulla interna; MT, medulla terminalis; HB, hemielpsoidal body; ON, optic nerve. (B,C) FITC fluorescence for HA immunoreactive neurons observed by confocal microscopy; image compositions were done by merging 200 slides (1 µm section thickness). Scale bars, 20 µm.

 




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