Paul R. Hartig

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex

The anatomic relationship between serotonergic (5-HT) axons and 5-HT2 receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT2 receptors, localized by the ligand N1-methyl-2-125I-LSD (125I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT2 receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT2 receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT2 receptors in layer Va of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT2 receptors and fine axons in other areas of the forebrain, suggesting that 5-HT2 receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT2 receptors may mediate the effects of dorsal but not median raphe projections.

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus†

Abstract: The serotonin 5‐HT2C receptor (formerly designated the 5‐HT1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5‐HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five‐fold basal level, with an EC50 of 11 nM. This response was not inhibited by muscarinic or β‐adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent Ki values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin‐stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin‐stimulated cGMP formation was calcium, phospholipase A2, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A2 or lipoxygenase inhibitors p‐bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5‐HT2C receptors trigger cGMP formation in a calcium‐sensitive manner.

Characterization of 125I-lysergic acid diethylamide binding to serotonin receptors in rat frontal cortex.

Abstract: 125I‐Lysergic acid diethylamide (125I‐LSD) is the first 125I‐labeled ligand for serotonin receptor studies. Its binding to rat frontal cortex membranes is saturable, reversible, and stereospecific. Specific binding is linearly dependent on tissue concentration and represents 70–80% of the total binding. Scatchard plots of the binding data are linear with a KD of 1.5 nM, a Bmax of 12.4 fmol/mg wet weight tissue, and a Hill slope of 1.02. The binding kinetics are highly temperature‐dependent. At 37°C the bimolecular association rate constant is 1.28 × 108 min−1M−1 and the dissociation rate constant is 0.087 min−1 (t1/2= 8.0 min). At 0°C < 4% dissociation occurs over 40 min and the association rate is similarly depressed. Inhibition of 125I‐LSD binding by a variety of serotonergic, dopaminergic, and adrenergic ligands reveals a 5‐hydroxytryptamine2 (5‐HT2) serotonergic profile for this binding site. Regional distribution studies of 125I‐LSD binding in rat brain show that areas with the highest levels of binding include the cortex and striatum. Iodinated radioligands can be synthesized with specific activities exceeding 2,000 Ci/mmol, which makes them approximately 75‐fold more sensitive than tritiated radioligands. This high specific activity, coupled with the selectivity of 125I‐LSD for 5‐HT2 sites, makes this ligand a sensitive new probe for 5‐HT2 serotonin receptors.

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Abstract: Methylation of 2‐125I‐lysergic acid diethylamide (125I‐LSD) at the N1 position produces a new derivative, N1‐methyl‐2‐125I‐lysergic acid diethylamide (125I‐MIL), with improved selectivity and higher affinity for serotonin 5‐HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I‐MIL represents 80–90% of total binding, and the apparent dissociation constant (KD) for serotonin 5‐HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I‐MIL also displays a high affinity for serotonin 5‐HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I‐MIL exhibits at least 60‐fold higher affinity for serotonin 5‐HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I‐MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0°C. Autoradiographic experiments confirm the improved specificity of 125I‐MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I‐MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I‐MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.

Serotonin 5-HT1C receptors are expressed at high density on choroid plexus tumors from transgenic mice

Choroid plexus tumors develop spontaneously in adult transgenic mice carrying integrated copies of SV40 early region genes. In this communication, we report that these tumors exhibit the highest density of serotonin receptors (6600 fmol/mg protein) found in any tissue. 125I-LSD binding to choroid plexus tumors displays a pharmacological profile that matches the properties of 5-HT1C receptors in normal choroid plexus tissue. Autoradiographic localization of 125I-LSD binding in brain sections from transgenic mice shows high levels of labelling in the tumors, in correlation with immunohistochemical staining for SV40 large T antigen expression. Choroid plexus tumors from these transgenic mice provide an excellent model system for the study of serotonin 5-HT1C receptors.

In vivo binding of 125I-LSD to serotonin 5-HT2 receptors in mouse brain

The binding of 125I-LSD (2-[125I]-lysergic acid diethylamide) was studied in various mouse brain regions following intravenous injection of the radioligand. The high specific activity of 125I-LSD enabled the injection of low mass doses (14 ng/kg), which are well below the threshold for induction of any known physiological effect of the probe. The highest levels of 125I-LSD binding were found in the frontal cortex, olfactory tubercles, extra-frontal cortex and striatum while the lowest level was found in the cerebellum. Binding was saturable in the frontal cortex but increased linearly in the cerebellum with increasing doses of 125I-LSD. Serotonergic compounds potently inhibited 125I-LSD binding in cortical regions, olfactory tubercles, and hypothalamus but had no effect in the cerebellum. Dopaminergic compounds caused partial inhibition of binding in the striatum while adrenergic compounds were inactive. From these studies we conclude that 125I-LSD labels serotonin 5-HT2 receptor sites in cortical regions with no indication that other receptor sites are labeled. In the olfactory tubercles and hypothalamus, 125I-LSD labeling occurs predominantly or entirely at serotonin 5-HT2 sites. In the striatum, 125I-LSD labels approximately equal proportions of serotonergic and dopaminergic sites. This data indicates that 125I-LSD labels serotonin receptors in vivo and suggests that appropriate derivatives of 2I-LSD may prove useful for tomographic imaging of serotonin 5-HT2 receptors in the mammalian cortex.

Synthesis and in vivo characterization of d-(+)-(N1-[11C]methyl)-2-Br-LSD: a radioligand for positron emission tomographic studies of serotonin 5-HT2 receptors

D-(+)-N1-Methyl-2-Br-LSD (MBL), which displays high affinity and selectivity for serotonin receptors in vitro, has been labeled with carbon-11 for localization of cerebral serotonin 5-HT2 receptors by positron emission tomography. [11C]MBL was prepared from [11C]iodomethane and D-(+)-2-Br-LSD within 20 min from end of bombardment. The average specific activity of [11C]MBL was 2300 mCi/mumol and the average radiochemical yield was 17%, both at end of synthesis. The in vivo regional distribution of radioactivity in brain after i.v. administration of [11C]MBL to mice paralleled the known density of serotonin 5-HT2 receptors. The maximum specific binding, defined by a frontal cortex to cerebellum radioactivity concentration ratio of 5.4 to 1, was reached 30 min postinjection. Administration of ketanserin, a potent serotonin 5-HT2 receptor antagonist, markedly blocked radioligand binding in all brain regions examined except cerebellum.

Photolabeling of brain membrane proteins by lysergic acid diethylamide

3H-Lysergic acid diethylamide (3H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a subpopulation of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.