Ann E. Stuart

From Fruit Flies to Barnacles, Histamine Is the Neurotransmitter of Arthropod Photoreceptors

to adapt, centering their operating range on the value of presynaptic voltage set by the background light intensity. Even when the photoreceptor is maintained in a depolarized state in bright lights, these hardy synapses do not fatigue (Hayashi et al., 1985). This intriguing synAnn E. Stuart* Department of Cell and Molecular Physiology University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599

Comparative sequence analysis and tissue localization of members of the SLC6 family of transporters in adult Drosophila melanogaster

SUMMARY The SLC6 family comprises proteins that move extracellular neurotransmitters, amino acids and osmolytes across the plasma membrane into the cytosol. In mammals, deletion of SLC6 family members has dramatic physiologic consequences, but in the model organism Drosophila melanogaster, little is known about this family of proteins. Therefore, in this study we carried out an initial analysis of 21 known or putative SLC6 family members from the Drosophila genome. Protein sequences from these genes segregated into either well-defined subfamilies, including the novel insect amino acid transporter subfamily, or into a group of weakly related sequences not affiliated with a recognized subfamily. Reverse transcription-polymerase chain reaction analysis and in situ hybridization showed that seven of these genes are expressed in the CNS. In situ hybridization revealed that two previously cloned SLC6 members, the serotonin and dopamine transporters, were localized to presumptive presynaptic neurons that previously immunolabelled for these transmitters. RNA for CG1732 (the putative GABA transporter) and CG15088 (a member of the novel insect amino acid transporter family) was localized in cells likely to be subtypes of glia, while RNA for CG5226, CG10804 (both members of the orphan neurotransmitter transporter subfamily) and CG5549 (a putative glycine transporter) were expressed broadly throughout the cellular cortex of the CNS. Eight of the 21 sequences were localized outside the CNS in the alimentary canal, Malpighian tubules and reproductive organs. Localization for six sequences was not found or not attempted in the adult fly. We used the Drosophila ortholog of the mammalian vesicular monoamine transporter 2, CG33528, to independently identify monoaminergic neurons in the adult fly. RNA for CG33528 was detected in a limited number of cells in the central brain and in a beaded stripe at the base of the photoreceptors in the position of glia, but not in the photoreceptors themselves. The SLC6 localization observations in conjunction with likely substrates based on phylogenetic inferences are a first step in defining the role of Na/Cl-dependent transporters in Drosophila physiology.

The distribution of histamine and serotonin in the barnacle's nervous system.

The use of antisera directed against conjugates of histamine and serotonin has revealed the locations of neurons labeling for these transmitters in the nervous system of barnacles. Photoreceptors label for histamine but not serotonin and also satisfy a number of other criteria indicating that histamine is their neurotransmitter. Photoreceptors also take up radioactively labeled histamine but not serotonin. Within the barnacles brain no somata are consistently found that label with antiserum against histamine, but one to three pairs of small cells, depending on species, label with antiserum against serotonin. The most impressive serotonin‐like immunoreactivity in the brain, however, is in a pair of large fibers ascending through the circumesophageal connectives and ramifying extensively. Within the ventral ganglion, the only other ganglion in the barnacle, ten pairs of cells label with antiserum against histamine. These neurons are confined to the posterior portion of the ganglion but ramify extensively throughout the ganglion. Antiserum against serotonin labels about 15 cell pairs, depending on species, located throughout the ganglion. The positions of the arbors of many of these cells suggest that these amines have a role in modulating either the motor pathways underlying feeding or the visual pathways responsible for the detection of shadows. Microsc. Res. Tech. 44:94–104, 1999.

Biochemical and physiological evidence that histamine is the transmitter of barnacle photoreceptors

We tested the hypothesis that histamine is the transmitter released by barnacle photoreceptors. Median and lateral ocelli were incubated with 3H-histidine and found to synthesize 3H-histamine, identified by high-voltage electrophoresis. Synthesis could be blocked by the histidine decarboxylase inhibitor (S)-alpha-fluoromethylhistidine. Histamine was applied to I-cells either by superfusion or by pressure ejection from a pipette (100 microM or 1 mM histamine) positioned close to the I-cells soma. When bath-applied at concentrations ranging from 5-100 microM, histamine hyperpolarized the I-cell in a dose-dependent fashion and increased its conductance. At 100 microM, histamine abolished the I-cells response to light. The response to a pulse of pressure-applied histamine was a hyperpolarization whose amplitude was graded with dose (determined by the duration of the pulse). This response persisted in concentrations of Co2+ and Cd2+ that blocked synaptic transmission from the photoreceptors. Cimetidine, an antagonist of mammalian H2 receptors, markedly decreased the cells responses both to HA and to light at 100 microM and blocked both responses at 1 mM. Pyrilamine and triprolidine, H1 antagonists, had a complex effect on the I-cells responses to histamine and to light. Neither H1 nor H2 antagonists, nor histamine itself, affected the voltage or light responses recorded in the presynaptic terminal region, or any phase of calcium-dependent action potentials induced in the terminal in the presence of tetraethylammonium ion. Thus, biochemical, immunocytochemical, and physiological evidence suggests that HA is the transmitter from these photoreceptors to the I-cells. Although gamma-aminobutyric acid (GABA) is also present in the photoreceptors, it did not affect the I-cells responses to light or to histamine when bath-applied at 100 microM. Thus, GABA does not appear to modulate transmission from the photoreceptor to the I-cell.

Immunocytochemical evidence for the presence of histamine and GABA in photoreceptors of the barnacle (Balanus nubilus).

Biochemical evidence indicates that GABA and histamine may both be synthesized by barnacle photoreceptors (Koike & Tsuda, 1980; Timpe & Stuart, 1984; Callaway & Stuart, 1989b). We used antisera against GABA- and histamine-protein conjugates to determine whether the photoreceptors contain either or both of these antigens. Both antisera labeled all of the photoreceptors in each of the three ocelli. Histamine-like immunoreactivity was found throughout each photoreceptor cell but was most intense at their presynaptic terminals. Histamine-like immunoreactivity was blocked by preincubation of the antibody either with histamine or with a histamine-protein conjugate. GABA-like immunoreactivity was found in all parts of the photoreceptors including the cell body, axon, rhabdomeric dendrites, and presynaptic terminals. GABA-protein conjugates blocked the GABA-like labeling of the photoreceptors, while protein conjugates with histamine, L-glutamate, L-glutamine, beta-alanine, and taurine did not. Histamine-like immunoreactivity in the supraesophageal ganglion was confined to the photoreceptor terminals and a second, loose plexus of endings in the main neuropil. GABA-like immunoreactivity, in contrast, was found in approximately twenty-five pairs of neurons of this ganglion. In the cirral nerves, which are expected to contain inhibitory motoneurons, unidentified axons also labeled with the GABA antiserum.

Excitatory and Inhibitory Motoneurons in the Central Nervous System of the Leech

The locomotion and reflex responses of the leech are brought about by inmuscles that are arranged in a regular, simple pattern in the body wall and that flatten, shorten, lengthen, or bend the animal. In the segmental ganglia, it is possible to recognize by morphological and physiological criteria the individual motoneurons that cause contractions and relaxations of these muscles.

Uptake of the Neurotransmitter Histamine into the Eyes of Larvae of the Barnacle (Balanus amphitrite)

The photoreceptors of adult barnacles use histamine as their neurotransmitter and take up 3H-histamine selectively from the extracellular medium. We assayed for the uptake of 3H-histamine into the eyes of the free-swimming (nauplius) and settling (cyprid) larval stages of Balanus amphitrite. The extracellular space of nauplii proved permeable to dyes below about 800 molecular weight (MW), indicating that 3H-histamine (MW 111) introduced into seawater would have access to internal structures. 3H-Histamine was taken up into nauplii by a process with a KD of 0.32 μM. Uptake was antagonized by chlorpromazine, which also blocks uptake of 3H-histamine into adult photoreceptors. In autoradiographs of serial sections of nauplii and cyprids incubated in 3H-histamine, the ocelli and compound eyes were labeled; other structures in the animal were not. No eyes or other structures were labeled with 3H-serotonin, a related amine whose transporter commonly transports histamine as well. These experiments show that a histamine-specific transporter similar to that found in the adult is expressed in all of the eyes of barnacle larvae. In the ocelli, where photoreceptors and pigment cells may be distinguished in the light microscope, label was unexpectedly concentrated far more over the pigment cells than over the photoreceptors.

Does the neurotransmitter transporter underlie adaptation at a histaminergic photoreceptor synapse

Using autoradiographic and biochemical techniques, we studied the sodium-dependent forward and reverse transport of the neurotransmitter histamine in an arthropod photoreceptor in order to test whether the transporter plays a central role in visual signal transfer at this synapse. In particular, we asked whether the histamine transporter might be the important factor in synaptic adaptation, the process by which the operating range of the synapse adapts to increasing depolarizations of the photoreceptor in increasing background light. Drugs known from electrophysiological observations to interfere with synaptic adaptation blocked the uptake of [3H]histamine into photoreceptors. These drugs also blocked the sodium (Na)-triggered efflux of [3H]histamine, previously loaded into photoreceptors, via the histamine transporter. Several lines of evidence showed that efflux of [3H]histamine did not occur via calcium-dependent exocytosis. First, efflux occurred when the preparation was bathed in calcium (Ca)-free/EGTA salines or in cobalt (Co)-containing salines. Even more importantly, efflux could be elicited from axons, whose membranes must contain the transporter protein since they take up [3H]histamine independently from the presynaptic terminals. Since both adaptation and the histamine transporter are blocked by the same agents, the transporter may underlie adaptation by maintaining the cleft histamine concentration in a particular range independent of light intensity. We also characterized the transporter further and found that it is partially dependent on chloride ions, and that neither [3H]norepinephrine nor [3H]dopamine are transported (at 20 microM), adding to evidence that the transporter is highly selective for histamine.

Currents in the Synaptic Terminals of Barnacle Photoreceptors

The photoreceptors of the giant barnacle are distinguished anatomically by large axons, visible under the dissecting microscope even to their presynaptic terminal arborizations. Intracellular recordings made from these cells near their presynaptic arbor have shown that the ocellar response to light is conducted with little decrement to the presynaptic terminals, and have demonstrated the presence of voltage-sensitive calcium channels in this region of the cell (Hudspeth & Stuart, 1977; Ross & Stuart, 1978; Edgington & Stuart 1979,1981). Using optical methods, Ross and colleagues have studied the time course of the resulting changes in intracellular calcium concentration (Stockbridge & Ross, 1984).

Vision in barnacles

Abstract The large photoreceptors and ganglion cells of barnacles have invited study of how receptors transduce light to electrical energy, and how these receptor potentials are subsequently processed by higher-order neurons. This primitive visual system is particularly likely to increase our understanding of the transmission of very small signals along a sensory pathway. This article focuses on the fate of the graded signals generated in the photoreceptors: their conduction to the receptors terminals, their modification by second- and third-order cells of the visual pathway, and their influence on motoneurons.