Autoradiographic localization and characterization of [125I]Lysergic acid diethylamide binding to serotonin receptors in Aplysia

doi:10.1016/0306-4522(88)90090-5

Neuroscience

Volume 24, Issue 3, March 1988, Pages 1089-1102

https://doi.org/10.1016/0306-4522(88)90090-5 Get rights and content

Abstract

The sensitive serotonergic radioligand 2-[¹²⁵I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [¹²⁵I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (K_d = 0.41nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [¹²⁵I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [³H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [¹²⁵I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein.

Images of serotonin receptor distribution in the Aplysia nervous system were obtained from auto-radiograms of [¹²⁵I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [¹²⁵I]lysergic acid diethylamide.

These studies indicate that [¹²⁵I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor subtypes can be identified by these methods and correlated with physiological responses in the same cell.

Reference (47)

BaileyC.H. et al.
Uptake of³H-serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation
Brain Res.
(1983)
DrummondA.H. et al.
Distribution of serotonin and dopamine receptors in Aplysia tissues: analysis by³H-LSD binding and adenylate cyclase stimulation
Brain Res.
(1980)
DrummondA.H. et al.
d-³H-LSD binding to serotonin receptors in molluscan nervous system
J. biol. Chem.
(1980)
GoldsteinR. et al.
Distribution of serotonin-immunoreactivity in juvenile Aplysia
Neuroscience
(1984)
HartigP.R. et al.
Microchemical synthesis of the serotonin receptor ligand,¹²⁵I-LSD
Analyt. Biochem.
(1985)
HartigP.R. et al.
¹²⁵I-LSD binding to serotonin and dopamine receptors in bovine caudate membranes
Neurochem. Int.
(1985)
LefkowitzR.J. et al.
Regulation of beta adrenergic receptors by guanyl-5′-yl imidodiphosphate and other purine nucleotides
J. biol. Chem.
(1976)
LevitanI.B.
Adenylate cyclase in isolated Helix and Aplysia neuronal cell bodies: stimulation by serotonin and peptide-containing extract
Brain Res.
(1978)
Moretti-RojasI. et al.
Serotonergic and adrenergic regulation of skeletal muscle metabolism in the rat. II. The use of [¹²⁵I] iodolysergic acid diethylamide and [¹²⁵I] iodopindolol as probes of sarcolemmal receptor function and specificity
J. biol. Chem.
(1983)
NewlinS.A. et al.
Separate serotonin and dopamine receptors modulate the duration of post-tetanic potentiation at an Aplysia synapse without affecting other aspects of synaptic transmission
Brain Res.
(1980)

OnoJ.K. et al.

Immunocytochemical localization and direct assays of serotonin-containing neurons in Aplysia

Neuroscience

(1984)

PazosA. et al.

The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site

Eur. J. Pharmac.

(1984)

S.-RozsaK.

The pharmacology of molluscan neurons

Prog. Neurobiol.

(1984)

ShimaharaT. et al.

Cyclic AMP induced by serotonin modulates the activity of an identified synapse in Aplysia by facilitating the active permeability to calcium

Brain Res.

(1977)

AndradeR. et al.

A G protein couples serotonin and GABA_B receptors to the same channels in hippocampus

Science, N.Y.

(1986)

BarnierL. et al.

Facilitory transmitter causes a selective and prolonged increase in cAMP in sensory neurons mediating the gill and siphon withdrawal reflex in Aplysia

J. Neurosci.

(1982)

BrunelliM. et al.

Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP

Science, N.Y.

(1976)

CarpenterD.O. et al.

Effect of curare on responses to different putative neurotransmitters in Aplysia neurons

J. Neurobiol.

(1977)

CedarH. et al.

cAMP in the nervous system of Aplysia californica. II. Effect of serotonin and dopamine

J. gen. Physiol.

(1972)

CoggeshallR.E.

The light and electron microscope study of the abdominal ganglion of Aplysia californica

J. Neurophysiol.

(1967)

DrummondA.H. et al.

LSD labels a novel dopamine receptor in molluscan nervous system

Nature

(1978)

DrummondA.H. et al.

Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP

EngelG. et al.

2-¹²⁵I-LSD, a new ligand for the characterization and localization of 5-HT₂ receptors

Naunyn-Schmiedebergs Arch. Pharmac.

(1984)

Cited by (18)

A 5-HT<inf>1A</inf>-like receptor is involved in the regulation of the embryonic rotation of Lymnaea stagnalis L.
2010, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Cilia driven rotation of the pond snail Lymnaea stagnalis embryos is regulated by serotonin (5-HT). In the present study, physiological and biochemical assays were used to identify the 5-HT receptor type involved in rotation. The 5-HTergic agonists applied stimulated the rotation by 180–400% and their rank order potency was as follows: LSD > 5-HT > 8-OH-DPAT > WB4101 ≫ 5-CT. The applied antagonists, spiperone, propranalol and mianserin inhibited the 5-HT or 8-OH-DPAT stimulated rotation of the embryos by 50–70%. ³H-5-HT was bound specifically to the washed pellet of the embryo homogenates. The specific binding of ³H-5-HT was saturable and showed a single, high affinity binding site with K_d 7.36 nM and B_max 221 fmol/mg pellet values. This is the first report demonstrating the high affinity binding of ³H-5-HT to the native receptor in molluscs. All of the pharmacons that stimulated the rotation or inhibited the 5-HT or 8-OH-DPAT evoked stimulation displaced effectively the binding of ³H-5-HT. 5-HT resulted in the inhibition of forskolin stimulated cAMP accumulation, showing that 5-HT is negatively coupled to adenylate cyclase. Our results suggest that in the 5-HTergic regulation of the embryonic rotation in L. stagnalis a 5-HT_1A-like receptor of the vertebrate type is involved.
Structure and function of invertebrate 5-HT receptors: A review
2001, Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology
Over the last decade, knowledge of invertebrate serotonin receptors has expanded greatly. The first 5-HT receptor from Drosophila was cloned 10 years ago, and subsequently, 11 additional receptor genes have been cloned from Drosophila, molluscs (Lymnaea and Aplysia) and nematodes (Caenorhabditis and Ascaris). Information has also accumulated from physiological and biochemical studies that have used vertebrate serotonergic ligands to characterize endogenous invertebrate receptors. Although the endogenous receptors are often classified according to mammalian-based categories, in many cases the pharmacological properties of vertebrate and invertebrate receptors differ significantly and the actual identity of the latter is questionable. By providing information on the gene structure and amino acid sequence, molecular cloning studies offer a more definitive way to identify and classify invertebrate 5-HT receptors. This review summarizes information on the pharmacological and transductional properties of cloned invertebrate 5-HT receptors, and considers recent studies of endogenous receptors in the light of this new data.
Distribution of serotonin uptake and binding sites in the cnidarian Renilla koellikeri: An autoradiographic study
1998, Tissue and Cell
Using [³H]-serotonin ([³H]-5-HT) as radiolabel and autoradiography, we have mapped the distribution of 5-HT uptake and binding sites in the sea pansy Renilla koellikeri in order to identify potential cellular pathways of 5-HT inactivation and to identify cellular substrates for the previously characterized 5-HT receptor involved in the modulation of peristaltic behavior. Uptake measured in fresh polyp tissues occurred via two processes: a high affinity (uptake₁), clomipramine-sensitive process with a K_m of 0.45 μM, and another of lower affinity (uptake₂) with a Km of 11.6 μM. Autoradiograms of high affinity uptake sites revealed a diffuse distribution of label with higher density in the ectoderm and endoderm, and lower density in the mesoglea. No subsets of cells, including serotonergic neurons, appeared to retain label preferentially, thus suggesting that removal of 5-HT and its chemical conversion is a general property of sea pansy tissues. Under incubation conditions identical to those used in a previous radiobinding analysis, autoradiograms of binding sites were generated on sections from lightly fixed and cryosectioned polyps. In contrast to uptake sites, binding sites appeared as aggregations of label around neurons of the subectodermal, mesogleal and endodermal nerve nets. In the endoderm where the myoepithelia subtend peristalsis, myoepithelial cells appeared unlabeled, suggesting that 5-HT exerts its modulatory effect on peristalsis principally via neurons. Taken together, these results indicate that 5-HT is released as a neurohormone in the sea pansy and that it may act as a broad-range neuromodulator.
Age dependent changes in serotonin and dopamine receptors in Aplysia californica
1997, Comparative Biochemistry and Physiology - C Pharmacology Toxicology and Endocrinology
Age related changes in dopaminergic and serotonergic receptors were examined in Aplysia californica. In this study dopamine (DA) and serotonin (5-HT) receptor levels were examined for animals belonging to 4-, 5-, 6-, 8-, 9- and 12-month age groups. Receptors analysis was performed using radio-labeled d-[³H] lysergic acid diethylamide (LSD) as the specific ligand. Specific binding for 5-HT was found to be significantly greater than that for DA in the young (4-month post-hatch) animals. The total DA and 5-HT receptor levels changed significantly with age. Dopamine levels increased from 5.34 fmol/mg of protein at 4 months to 19.11 fmol/mg at 12 months. Serotonin receptor levels increased from 7.35 fmol/mg at 4 months to 20.45 at 12 months.
Characterization of serotonin binding sites in insect (Locusta migratoria) brain
1993, Insect Biochemistry and Molecular Biology
Radioligand binding studies using [³H]serotonin, [³H]ketanserin and [³H]mianserin were used to characterize 5HT receptor sites in membrane preparations from the brain of the locust, Locusta migratoria migratorioides. The [³H]serotonin binds reversibly to brain membrane preparations with high affinity and the receptor has 5HT₁ characteristics. However, studies with specific antagonists suggest that the receptor differs pharmacologically from the 5HT₁ receptor subtypes which have been identified in vertebrate brain. Scatchard analysis indicates that the binding sites have a single component with a dissociation constant of 2.98±0.19nM and B_max value of 14.45±1.12 pmol/g tissue. Serotonin, bufotenin, N,N-dimethyltryptamine, d-LSD, BOL are equipotent displacers of [³H]serotonin binding. Several other potential agonists and antagonists were tested and shown to effect varying degrees of inhibition.
Ca and Mg ions have no significant effect on the [³H]serotonin binding; however, guanine nucleotides modulate the binding suggesting that G-protein is involved in the serotonin action.
No specific, saturable ketanserin binding was found, thus indicating lack of a vertebrate-like 5HT₂ receptor.
[³H]Mianserin binds to the brain membrane; however, the pharmacology of mianserin binding suggests that mianserin binds to an octapamine-rather than a serotonin-receptor.
Pharmacological characterization of a 5-HT receptor in locust nervous tissue
1992, European Journal of Pharmacology
A 5-HT receptor in the nervous tissue of the desert locust (Schistocerca gregaria Forsk.) was investigated, using [³H]LSD (lysergic acid diethylamide) as the radioligand. [³H]LSD labels in addition a putative dopamine receptor whose specific [³H]LSD binding nevertheless could easily be diminished by co-incubation with 1 μM dopamine. The binding site was characterized by a K_D of 1.64 nM, and a maximal concentration of binding sites of 79.8 fmol/mg protein. Pharmacological investigation revealed a relatively low affinity for the putative natural agonist, serotonin (K₁ = 0.209 μM). In contrast to the high affinity of classical serotonergic antagonists (e.g. dihydroergotamine or (+)-butaclamol) substances with subtype specificity such as 8-OH-DPAT (8-hydroxy-1-(N,N-dipropyl)-aminotetralin) or ketanscrin have only moderate affinities. Quantitative comparison of the pharmacological data demonstrated that there is obviously no pharmacological homology with vertebrate 5-HT receptors characterized so far. The only receptors with a close pharmacological relationship to the 5-HT receptor of locusts are the 5-HT_drol receptor expressed in Drosophila nervous tissue and a 5-HT receptor in snail nervous tissue which might be homologous to that of locusts. The 5-HT receptor investigated, was shown to be G-protein-coupled, as addition of stable GTP analogues or depletion of Mg²⁺ ions from the incubation medium led to agonist-specific lowering of the affinity.

View all citing articles on Scopus

^†: Current address: National Institute on Drug Abuse, Addiction Research Center, Baltimore, Maryland, U.S.A.

View full text

Autoradiographic localization and characterization of [125I]Lysergic acid diethylamide binding to serotonin receptors in Aplysia

Abstract

Brain Res.

Brain Res.

J. biol. Chem.

Neuroscience

Analyt. Biochem.

Neurochem. Int.

J. biol. Chem.

Brain Res.

J. biol. Chem.

Brain Res.

Neuroscience

Eur. J. Pharmac.

Prog. Neurobiol.

Brain Res.

A G protein couples serotonin and GABAB receptors to the same channels in hippocampus

Science, N.Y.

Facilitory transmitter causes a selective and prolonged increase in cAMP in sensory neurons mediating the gill and siphon withdrawal reflex in Aplysia

J. Neurosci.

Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP

Science, N.Y.

Effect of curare on responses to different putative neurotransmitters in Aplysia neurons

J. Neurobiol.

cAMP in the nervous system of Aplysia californica. II. Effect of serotonin and dopamine

J. gen. Physiol.

The light and electron microscope study of the abdominal ganglion of Aplysia californica

J. Neurophysiol.

LSD labels a novel dopamine receptor in molluscan nervous system

Nature

Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP

2-125I-LSD, a new ligand for the characterization and localization of 5-HT2 receptors

Naunyn-Schmiedebergs Arch. Pharmac.

Autoradiographic localization and characterization of [¹²⁵I]Lysergic acid diethylamide binding to serotonin receptors in Aplysia

A G protein couples serotonin and GABA_B receptors to the same channels in hippocampus

2-¹²⁵I-LSD, a new ligand for the characterization and localization of 5-HT₂ receptors