Extension of the proboscis was conditioned in restrained honeybees with odor as the conditioned stimulus (CS) and sucrose solution-delivered to the antenna (to elicit extension of the proboscis) and then to the proboscis itselfas the unconditioned stimulus (US). In a first series of experiments, acquisition was found to be very rapid, both in massed and in spaced trials; its associative basis was established by differential conditioning and by an explicitly unpaired control procedure (which produced marked resistance to acquisition in subsequent paired training); and both extinction and spontaneous recovery in massed trials were demonstrated. In a series of experiments on the nature of the US, eliminating the proboscis component was found to lower the asymptotic level of performance, whereas eliminating the antennal component was without effect; reducing the concentration of sucrose from 20% to 7% slowed acquisition but did not lower the asymptotic level of performance; and secondorder conditioning was demonstrated. In a series of experiments on the role of the US, an omission contingency designed to eliminate adventitious response-reinforcer contiguity was found to have no adverse effect on acquisition. In a series of experiments designed to analyze the resistance to acquisition found after explicitly unpaired training in the first experiments, no significant effect was found of prior exposure either to the CS alone or to the US alone, although the unpaired procedure again produced substantial resistance that was shown to be due to inhibition rather than to inattention; extinction after paired training was found to be facilitated by unpaired presentations of the US, The relation between these results for honeybees and those of analogous experiments with vertebrates is considered.

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdm1 activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.

The distribution of GABA-like immunoreactivity in the brain of the honeybee was investigated with antisera generated against GABA protein conjugates. The binding of the antisera in paraffin serial sections was studied with the peroxidase-antiperoxidase method. GABA-like immunoreactive fibers appeared in all main neuropile areas. The staining of the optic lobes showed pronounced stratification. The receptor cells of compound eyes, ocelli, and antennae were not labelled. Several prominent fiber tracts showed GABA-like immunoreactivity, whereas other tracts were devoid of staining. There are no major immunoreactive commissures linking the two brain hemispheres with the exception of small commissures that bridge short distances between the beta-lobes and the antennal lobes. Several fibers in the cervical connective were also labelled; some of those may descend from the suboesophageal ganglion to the thoracic ganglia. The dense reactivity seen in the optic and antennal neuropiles implies that GABA is more important in mediating local rather than more distant neural interactions.

The distribution of dopamine in the brain and suboesophageal ganglion of the honeybee Apis mellifera was investigated by means of immunocytochemistry with a well-characterized antiserum against dopamine. The binding of the antiserum in paraffin serial sections was studied with the peroxidase-antiperoxidase method. Dopamine-like immunoreactive neurons are present in most parts of the brain and in the suboesophageal ganglion. Only the optic lobes are devoid of label. There are ca. 330 dopamine immunoreactive somata in each brain hemisphere plus respective suboesophageal hemiganglion, which is less than 0.1% of the entire neuronal population. Most of the labelled somata are situated within three clusters: one below the lateral calyx and two in the anterior-ventral protocerebrum. Other labelled somata lie dispersed or in small groups around the protocerebral bridge, below the optic tubercles, proximal to the ventral rim of the lobula, and in the lateral and ventral somatal rind of the suboesophageal ganglion. Similar to neurons that react with an antiserum against serotonin, the fine processes of dopamine immunoreactive fibers have a varicose appearance which is typical for aminergic neurons. In addition to the neuronal staining, dopamine-like immunoreactivity is also present in the sheath surrounding the brain and in the retina, where it is not restricted to any particular cell type. A detailed account is given for those neurons and groups of neurons that could be traced and reconstructed in some detail. A common feature of all dopamine immunoreactive fibers is that each fiber invades large volumes of neuropil, suggesting that dopamine is more important in mediating distant rather than local neural interactions.

The mushroom bodies have been suggested to be essentially involved in learning and memory in insects. In the honeybee Apis mellifera they are composed of about 340,000 intrinsic elements, called Kenyon cells, which can be easily separated from all other neurons of the brain. Here we describe a preparation in which we studied ionic currents in the isolated Kenyon cell somata, using tight-seal whole-cell recording. Several outward and inward currents were identified and investigated by the use of pharmacological agents and in ion substitution experiments: a rapidly inactivating A-type potassium current that is completely blocked with 5 mM 4-aminopyridine; a calcium-activated potassium current that is blocked by 1-100 nM charybdotoxin; a delayed rectifier-type potassium current that is only weakly sensitive to tetraethylammonium but is blocked by 100 microM quinidine; a rapidly activating and inactivating, TTX-sensitive sodium current; a persistent sodium current that is both TTX and cadmium sensitive; and a calcium current that is completely blocked at 50 microM cadmium and is affected by verapamil and nifedipine only at high concentrations (100 microM). The currents described here are very similar to currents found in other insect neurons or muscle cells. This preparation will not only facilitate studies concerning the action of transmitters and neuromodulators that are contained within neurons converging onto the Kenyon cells, but will also allow a study of the role of the adenylyl cyclase pathway, elements of which are expressed in Kenyon cells, and are known to be essential for learning in invertebrates.