Insects and Flowers — The Biology of a Partnership

Abstract

1. Brain local field potentials, which are recorded extra-cellularly, are caused by the superposition of extra-cellular currents generated by the activation of trans-membrane voltage-gated and (mainly) synaptic con-ductances in nearby neurons. Experiments were carried out in vivo with adult locusts of both sexes. The brain was supported by a wax-coated plafform inserted between the connectives and was superfused with physiological saline [140 mM NaCI,5 mM KCI,5 mM CaCI2,4 mM NaHCO3, 1 mM MgCI2, 6.3 mM Hepes (pH 7.1)] at room temperature. Local field potentials were recorded with patch pipettes filled with saline (1 megohm) that were connected to a dc amplifier and low-pass filtered on-line with an eight-pole digital Butterworth filter (Model 9002, Frequency Devices, Haverhill, MA) or off-line with a Savitsky-Golay algorithm (13). Local field potentials were recorded in the mushroom body from two sites that are usually separated by 200 to 400 Km. Intracel-lular recordings from antennal lobe neurons were made with potassium acetate-filled microelectrodes (80 to 120 megohm) and an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Airborne odors were delivered to the antenna with five Teflon-coated steel tubes in gentle pressure pulses controlled by electrical and pneumatic valves. 12. A variable delay (up to several hundred milliseconds) was usually observed between the onset of the current pulse triggering the odor delivery and the onset of the physiological response in the antennal lobe or mushroom body. This delay is explained by the lag time separating the openings of the electrical and pneumatic valves and by the distance separating the odor delivery tubes from the antenna (up to 5 cm), which caused long transport times. Such long distances were chosen to minimize stimulation of an-tennal mechanoreceptors. 13. Field potential and intracellular data were digitized at 5 kHz with a TL1-DMA board (Axon Instruments). To produce the cross-correlograms, the electrophysi-ological traces typically were broken up into about 240 (or 120) windows of 1024 points. Each window overlapped the next one by 896 (or 768) points. The data were digitally filtered with a fourth-degree Savitsky-Golay smoothing algorithm (using 257 or 513 points) (W. The neuromodulator serotonin (5-hydroxytryptamine, 5-HT) has been associated with mood disorders such as depression, anxiety, and impulsive violence. To define the contribution of 5-HT receptor subtypes to behavior, mutant mice lacking the 5-HT1B receptor were generated by homologous recombination. These mice did not exhibit any obvious developmental or behavioral defects. However, the hyperlocomotor effect of the 5-HT1A/1B agonist RU24969 was absent in mutant mice, indicating that this effect is mediated by 5-HT1 B receptors. Moreover, when confronted with an intruder, mutant mice attacked the intruder faster and more intensely than did wild-type mice, suggesting the participation of 5-HT1 B receptors in aggressive behavior. Serotonergic drugs are used to treat migraine , depression, and anxiety, and a sero-tonin deficit has been associated with behaviors such as suicide, impulsive violence, depression, and alcoholism (1). The multiple actions of serotonin are mediated by the interaction of this amine with at least 14 receptors (2), most of which belong to the GTP-binding protein (G protein)-coupled receptor family. The 5-HT1B receptor, which is the rodent homolog of the human 5-HTlD3 receptor , is expressed in a variety of brain regions, including the basal ganglia, central gray, hippocampus, and raphe nuclei (3, 4). Pharmacological studies with weak specific SCIENCE * VOL. 265 * 23 SEPTEMBER 1994 agonists have suggested that activation of 5-HTlB receptors might lead to an increase in anxiety and locomotion and to a decrease in food intake, sexual activity, and aggressive behavior (5). The consequences of a blockade of 5-HTlB receptors or of their human counterpart are unknown because there are no specific antagonists for these receptors. To study the function of the 5-HTIB receptor, we have generated by homologous recombination in embryonic stem (ES) cells homozygous mutant mice lacking both copies of the gene encoding the 5-HTlB receptor (6, 7). Four positive ES cell clones were obtained with both the JA and the JB targeting vectors (Fig. 1 and Table 1). Southern (DNA) blot analyses with Xba I digests and the E2A1 probe or the neo probe confirmed that accurate targeting occurred and that no additional integration took place. Cells from the positive clones JA7 and JB13 were microinjected into 3.5-day C57BL/6 mouse blastocysts. The two clones gave rise to highly chimeric mice, which were bred with C57BL/6 females to test for germline transmission of the mutated 5-HTIB receptor gene. The positive chime-ras were bred with females from the 1 29/Sv-ter inbred strain to obtain heterozygotes on the 1 29/Sv-ter genetic background. Ho-mozygous animals were generated by het-erozygote crossings, and the expected 1: 2:1 ratio of wild-type (WT), heterozygous, and 1875 1