Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1µm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.

BACKGROUND AND PURPOSEMorphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [ KEY RESULTSWhen pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [ CONCLUSIONS AND IMPLICATIONSThese findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors.

Non-technical summaryRs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1β2γ2 receptors. We used two approaches to inhibit spontaneous GABA A R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)β2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1β2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [35 S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)β2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1β2γ2 receptor blockade (to 98% in both cases

The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT(3)) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT(3)B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.