Topographic organization of the dorsal nucleus of the lateral lemniscus in the cat.

The dorsal nucleus of the lateral lemniscus (DNLL) is an auditory structure of the brainstem. It plays an important role in binaural processing and sound localization and it provides the inferior colliculus with an inhibitory projection. The DNLL is a highly conserved auditory structure across mammals, but differences among species in its detailed organization have been reported. The main goal of this study was to analyze the topographic organization of the cat DNLL. Single, small iontophoretic injections of biotinylated dextran amine were made at different loci in the central nucleus of the inferior colliculus (CNIC). The distribution of the labeled structures in the ipsi- and contralateral DNLL was computer reconstructed in three dimensions. In individual sections, a band of labeling is seen in the DNLL on both sides. These two labeled bands occupy symmetric locations and are made of retrogradely labeled neurons with flattened dendritic arbors oriented parallel to each other. Moreover, the ipsilateral labeled band contains labeled terminal fibers parallel to the labeled dendrites. With three-dimensional reconstructions, it becomes evident that the labeled band seen in each individual DNLL section represents a slice through a rostrocaudally oriented lamina. The shape, size, orientation, and location of this lamina change as the injection site is shifted along the tonotopic axis of the CNIC. An injection in the low-frequency region of the CNIC, produces a lamina that resembles a flattened tube located in the dorsolateral corner of the DNLL. An injection in the high-frequency region of the CNIC, by contrast, results in a lamina that is an elongated sheet located at the ventromedial surface of the DNLL. The laminae of the DNLL might constitute the structural basis for its tonotopical organization. Previous studies (Merchan MA, et al. 1994. J Comp Neurol 342:259-278) in conjunction with our current results suggest that the laminar organization in the DNLL might be common among mammals

Analysis of C-terminally substituted tachykinin-like peptide agonists by means of aequorin-based luminescent assays for human and insect neurokinin receptors.

Aequorin-based assays for stable fly, Stomoxys calcitrans, (STKR) and human (neurokinin receptor 1 (NK1), neurokinin receptor 2 (NK2)) neurokinin-like receptors were employed to investigate the impact of a C-terminal amino acid exchange in synthetic vertebrate ('FXGLMa') and invertebrate ('FX1GX2Ra') tachykinin-like peptides. C-terminally (Arg to Met) substituted analogs of the insect tachykinin-related peptide, Lom-TK I, displayed increased agonistic potencies in luminescent assays for human NK1 and NK2 receptors, whereas they showed reduced potencies in the STKR-assay. The opposite effects were observed when C-terminally (Met to Arg) substituted analogs of substance P were analysed. These substance P analogs proved to be very potent STKR-agonists, being more potent than Lom-TK I. On the other hand, Lom-TK-LMa, was shown to be a very potent NK1-agonist and was suggested to have more substance-P-mimetic than neurokinin-A-mimetic properties. NK1 and NK2 receptor agonists appeared to be more sensitive to changes at the penultimate amino acid position than STKR-agonists. This is also reflected in the sequence conservation that is observed in the naturally occurring tachykinin subgroups ('FXGLMa' vs. 'FX1GX2Ra'). The differential Arg-Met preference appears to be a major coevolutionary change between insect and human peptide-receptor couples. With regard to the peptide agonists, this change can theoretically be based on a single point mutation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Distinct recognition of OX1 and OX2 receptors by orexin peptides.

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor.

A few naturally occurring insect tachykinin-related peptides, such as stomoxytachykinin (Stc-TK), contain an Ala-residue instead of the highly conserved Gly-residue that is present in most other members of this peptide family. Stc-TK is a potent, partial agonist of the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR. By means of synthetic analogues, the Gly/Ala exchange, representing the addition of a single methyl group in the active core region of these peptides, was shown to be fully responsible for the generation of this partial agonism, which was also accompanied by an increase in agonistic potency. Surprisingly, this Ala-dependent reduction in maximal response levels was only observed for the agonist-induced cellular calcium rise. Stomoxytachykinin, Stc-TK, did not display partial agonism for the STKR-mediated cyclic AMP response. A possible explanation for this differential partial agonism is that the Gly-containing and Ala-replaced peptides recognize and stabilize active receptor conformations that differ in their functional coupling efficacies towards these response pathways. Drosotachykinins, Drm-TK, tachykinin-like peptides encoded in the fruit fly genome, were shown to be STKR-agonists. Interestingly, one of these peptides, which contains an Ala-residue instead of the conserved Gly-residue, also proved to be a potent, partial agonist for STKR.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe

Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines.

The activity of a series of synthetic tachykinin-like peptide analogs was studied by means of microscopic calcium imaging on recombinant neurokinin receptor expressing cell lines. A C-terminal pentapeptide (FTGMRa) is sufficient for activation of the stomoxytachykinin receptor (STKR) expressed in Schneider 2 cells. Replacement of amino acid residues at the position of the conserved phenylalanine (F) or arginine (R) residues by alanine (A) results in inactive peptides (when tested at 1microM), whereas A-replacements at other positions do not abolish the biological activity of the resulting insectatachykinin-like analogs. Calcium imaging was also employed to compare the activity of C-terminally substituted tachykinin analogs on three different neurokinin receptors. The results indicate that the major pharmacological and evolutionary difference between tachykinin-related agonists for insect (STKR) and human (NK1 and NK2) receptors resides in the C-terminal amino acid residues (R versus M). A single C-terminal amino acid change can turn an STKR-agonist into an NK-agonist and vice versa.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe