Identification and localization of the nicotinic acetylcholine receptor alpha3 mRNA in the brain of the honeybee, Apis mellifera.

The nicotinic acetylcholine receptors are ligand-gated ion channels responsible for rapid neurotransmission and are target sites for pesticides in insects. In the honeybee Apis mellifera, pharmacological and electrophysiological studies have shown that different nicotinic acetylcholine receptor subtypes may exist in the brain. Here, we have identified a honeybee cDNA that encodes a 537 amino acid protein with features typical of nicotinic acetylcholine receptor alpha subunit, and sequence homology to human alpha3. In situ hybridization on cryosections shows that the Apisalpha3 mRNA is differently expressed in larvae and adult. In larvae, Apisalpha3 mRNA expression is restricted to the suboesophageal ganglia. In adult, it is further expressed in the optic lobes, the dorsal lobes, the antennal lobes and the calyces of mushroom bodies. Together our results suggest that Apisalpha3 shows a controlled expression pattern during development.

Imidacloprid-induced facilitation of the proboscis extension reflex habituation in the honeybee.

Imidacloprid is a new insecticide from the family of the neonicotinoids, which interact with the insect nicotinic acetylcholine receptor. The effects of imidacloprid at a dose that does not affect sensory or motor functions are studied on non-associative learning abilities in the honeybee. The behavioral procedure is the habituation of the proboscis extension reflex (PER). Imidacloprid topically applied on the thorax (1 microl) at the doses of 5, 10, and 20 ng/bee induces an increase of the gustatory threshold defined as the lowest concentration of a sucrose solution applied to the antennae able to elicit the PER. The ability of the honeybee to move in an open-field-like apparatus is impaired at the doses of 2.5, 5, 10, and 20 ng/bee. These effects are amplified with time and reach a maximum 60 min after application. The lowest dose of 1.25 ng/bee has no effect on the gustatory function but increases the motor activity and facilitates the PER habituation independently of time. This result fits our hypothesis that a slight activation of the cholinergic system with a low dose of imidacloprid can facilitate a simple form of learning in the honeybee.

Memory impairment induced by cholinergic antagonists injected into the mushroom bodies of the honeybee.

The role of honeybee central brain structures, suspected to be cholinergic, has been studied in learning and memory. The nicotinic antagonist mecamylamine and the muscarinic antagonist scopolamine were locally injected into the calyces and the alpha-lobes of mushroom bodies, and their effects on memory acquisition and retrieval were investigated using one-trial olfactory conditioning of the proboscis extension reflex. A strong impairment of the olfactory learning was noticed following mecamylamine injection into the mushroom body calyces. Mecamylamine and scopolamine disturbed retrieval processes when injected into the alpha-lobes of mushroom bodies but remain without effect on these processes when injected into the mushroom body calyces. These results emphasise the role of the cholinergic networks of the mushroom bodies in the formation and recall of memory in the honeybee. They suggest that the role of the brain structures in these processes is sequential. Mushroom body calyces involved in the associative process of olfactory learning could be relayed by the alpha-lobes for information retrieval.

Nicotinic acetylcholine receptor ligands differently affect cytochrome oxidase in the Honeybee brain.

The objective of this study was to determine if nicotinic receptor antagonists known for their ability to impair memory in the honeybee could induce changes in brain metabolism. We tested the effect of antagonists [hexamethonium, mecamylamine, alpha-bungarotoxin (alpha-BTX)] and agonist (nicotine) brain injections on cytochrome oxidase (CO) histochemistry. Within as little as 30 min following nicotine injection, an increase of the staining was observed in almost all the structures analyzed. The increase was limited to the alpha-lobe after alpha-BTX injection. In contrast, the antagonists hexamethonium and mecamylamine reduced CO staining in this structure that seems to be involved in information retrieval. These results suggest that the decrease of metabolism in the alpha-lobe obtained with hexamethonium and mecamylamine injections could be related to the impairment of retrieval processes previously observed with these drugs.

Functional cytochrome oxidase histochemistry in the honeybee brain.

The variations of neural metabolism induced by surgical and chemical treatments were studied in the honeybee brain by the means of cytochrome oxidase (CO) histochemistry. CO staining is considerably reduced in the alpha-lobe by antennal input deprivation. Chemical stimulation (50 mM K(+)) was linked to an increase of CO staining in antennal lobes (AL) and to a decrease in the basal ring of calyces (Cal). Application of the nicotinic ligand imidacloprid (10(-4) M) resulted in increased CO labelling within 30 min in all the structures analysed. Treatment with a lower concentration (10(-8) M) resulted in reduced staining in Cal and central body (CB). We conclude that CO histochemistry can be used to identify the target structures of cholinergic ligands in the honeybee brain.

Effect of potassium channel modulators on the release of glutamate induced by ischaemic-like conditions in rat hippocampal slices.

The effects of the potassium channel openers lemakalim, RP 52891 and galanin and the potassium channel blockers glibenclamide and gliquidone were evaluated by the release of endogenous glutamate from rat hippocampal slices subjected to a brief period of ischaemia (2-10 min). Ischaemia was mimicked by incubating slices in a glucose free medium equilibrated with 95% N2/5% CO2. These conditions evoked a release of glutamate which was insensitive to tetrodotoxin and Ca2+ indicating a non-vesicular origin. The release of glutamate evoked by a 6- or 8-min period of ischaemia was reduced by 25-40% in the presence of lemakalim (10 microM), RP 52891 (10 microM) or galanin (0.3 microM), whereas it was enhanced by 60 to 100% in the presence of glibenclamide (1 microM) and gliquidone (2 microM). These observations suggest that cellular damage resulting from ischaemia induced excessive release of glutamate in the hippocampus may be partly reduced by potassium channel openers, and conversely increased by sulfonylureas.

Fate of the catecholamine stores in the rabbit carotid body superfused in vitro.

In rabbit carotid bodies (CBs) superfused during 1-5 h, with an air-equilibrated medium containing no tyrosine (TYR), the dopamine (DA) content decreased by approximately 60% after 1 h and remained constant afterwards. TYR and 3,4-dihydroxyphenylacetic acid (DOPAC) decreased with the same time course. Noradrenaline (NA) content exhibited a biphasic decrease of lesser magnitude than that of DA. Superfusions with a TYR-containing medium did not prevent the reduction in DA and TYR. Large amounts of DA and DOPAC were recovered in the effluent during the first hour of superfusion but after 90 min the two substances had declined below the detection limits (i.e. 0.5 and 1 pmol/5 min, respectively). The DA efflux decreased exponentially during the first hour and was not altered by changing the oxygen partial pressure (PO2) of the medium. The DOPAC efflux declined after 40 min of superfusion and was modulated by PO2. The DA and the DOPAC effluxes were not suppressed by omitting calcium ions from the superfusing medium. In 4 cat CBs equal amounts of DA and NA were recovered from the effluent during the first hour of superfusion.

Comparison of the monoamine and catabolite content in the cat and rabbit carotid bodies.

The monoamine and catabolite contents of a large number of rabbit (n = 95) and cat (n = 32) carotid bodies (CBs) have been measured by high performance liquid chromatography with electrochemical detection (HPLC-ED). The dopamine (DA) content as well as that of its precursors tyrosine (TYR), dihydroxyphenylalanine (DOPA) and catabolites dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA) were approximately equal in both species. The noradrenaline (NA) content was 10 times larger in the cat than in the rabbit CBs. Twenty-nine out of the 32 cat CBs contained more NA than DA while the reverse was true in 92 out of 95 rabbit CBs. In 11 cats the right CB was sympathectomized and its DA and NA contents were compared to those of intact contralateral organs.

Changes in monoamine content of the rabbit carotid body following prolonged electrical stimulation of the carotid sinus nerve.

In the anaesthetized, paralysed and artificially ventilated rabbit the carotid sinus nerve (CSN) has been stimulated electrically for 6 h. At the end of the stimulation period, the carotid body (CB) has been removed, frozen and processed for measurement of the monoamines (MA) and of their catabolites with high-pressure liquid chromatography and electrochemical detection (HPLC-ECD). Results show a significant increase of dopamine (DA) and adrenaline (A) content and of all the metabolites. Besides an important augmentation of DA metabolism suggests that CSN efferent activity exerts some regulation on the MA content and turnover of the CB.