In vivo binding of [125I]RTI-55 to dopamine transporters: pharmacology and regional distribution with autoradiography.

Previous studies have demonstrated that para-substituted WIN 35,065-2 analogs of cocaine show high binding affinity for dopamine uptake sites both in vitro and in vivo, and inhibit DA uptake in vitro. These analogs also produce potent cocaine-like behavioral effects in various procedures. The purpose of the present studies was to evaluate the iodinated WIN 35,065-2 analog [125I]RTI-55 as an in vivo ligand for the DA transporter. Following intravenous injection in mice, [125I]RTI-55 showed highest accumulation in areas with high densities of dopamine uptake sites. Light microscopic autoradiography was used to examine binding with higher resolution. Displacement studies demonstrated that [125I]RTI-55 binding in dopamine containing regions, striatum and olfactory tubercles, was saturable and inhibited by other cocaine analogs. GBR 12909 and WIN 35,428 significantly inhibited [125I]RTI-55 binding in striatum, while paroxetine significantly inhibited hypothalamic binding but had little effect in striatum. The latter finding suggests that [125I]RTI-55 also binds to the serotonin transporter. Haloperidol had no effect on [125I]RTI-55 binding in any brain region measured. In addition, treatment of animals with the dopamine neurotoxin MPTP caused significant reductions in striatal [125I]RTI-55 binding. The results of these studies indicate that [125I]RTI-55 binds primarily to the dopamine transporter in the mouse striatum in vivo.     

High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain.

RTI-55 (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester), one of the most potent inhibitors of dopamine uptake reported to date, was radioiodinated and tested as a probe for the cocaine receptor in Sprague-Dawley rat brain. Saturation and kinetic studies in the striatum revealed that [125I]RTI-55 bound to both a high- and low-affinity site. The Kd for the high-affinity site was 0.2 nM, while the Kd for the low-affinity site was 5.8 nM. The corresponding number of binding sites in the striatum was 37 and 415 pmol/g protein. The pharmacological profile of specific [125I]RTI-55 binding in the striatum was consistent with that of the dopamine transporter. Additionally, [125I]RTI-55 was found to bind with high affinity to the cerebral cortex. Scatchard analysis revealed a single high-affinity component of 0.2 nM with a density of 2.5 pmol/g protein. The pharmacological profile demonstrated by [125I]RTI-55 in the cerebral cortex matched that of the serotonin transporter. Autoradiographic analysis of sagittal brain sections with [125I]RTI-55 binding was consistent with these findings. Specific binding of [125I]RTI-55 was blocked by dopamine uptake inhibitors in areas rich in dopaminergic nerve terminals. Conversely, serotonin uptake inhibitors blocked the binding of [125I]RTI-55 in brain areas rich in serotonergic neurons. These results demonstrate that [125I]RTI-55 may be a very useful ligand for the dopamine and serotonin transporters.     

Mapping dopamine transporters in the human brain with novel selective cocaine analog [125I]RTI-121

The novel cocaine analog RTI-121[3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester] was evaluated as a probe for the in vitro labeling and localization of the dopamine transporter in the human brain. Saturation binding experiments conducted in sucrose-phosphate buffer (10 mM sodium phosphate, pH 7.4, 0.32 M sucrose) revealed high- and low-affinity binding components with affinity values (K_D) of 0.25 ± 0.04 and 4.9 ± 1.6 nM (mean ± SE) and densities (B_max) of 56.8 ± 13.8 and 147.7 ± 23.4 pmol/g tissue, respectively. In contrast, when saturation binding experiments were performed in phosphate-buffered saline (10 mM Na₂HP0₄, 1.8 mM KH₂Po₄, 136 mM NaCl,2.8 mM KC1, 10 mM NaI, pH 7.4), a 9-fold decrease in the density of the low-affinity component was noted, suggesting that the low-affinity RTI-121 binding site is associated with the region of the transporter involved in the ionic dependence of substrate recognition andor uptake. The rank order. of potency for inhibition of [1251]RTI-121 binding to human caudate membranes demonstrates that the radioligand selectively labels the dopamine transporter (GBR 12909 > RTI-121 > mazindol > nomifensine > (?)cocaine > desipramine > citalopram). Autoradiograpliic mapping of [¹²⁵1]RTI-121 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderate densities were also observed over the substantia nigra and the ventral tegmental area. Low-to-background labeling of [¹²⁵1]RTI- 121 was seen throughout the cerebral cortex, amygdaloid nuclei, globus pallidus, and thalamus. In comparison with the autoradiographic distribution of the cocaine analogs L3H1WIN 35,428 (or CFT) and [¹²⁵1]RTI-55 (or β-CIT), the labeling pattern for [¹²⁵1]RTI-121 was more restricted. These studies demonstrate that [¹²⁵1]RTI-121 labels dopamine-rich brain regions with greater selectivity than other currently available cocaine analogs, which makes it a potentially superior imaging probe for mapping the dopamine transporter in the human brain. © 1995 WiIey-Liss, Inc.     

Distribution of cocaine recognition sites in monkey brain: II. Ex vivo autoradiography with [3H]CFT and [125I]RTI-55.

CFT [WIN 35,428, 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane] and its 4-iodinated analog RTI-55 have been proposed as imaging probes for cocaine recognition sites and for dopamine transporters. The central and peripheral distribution of radiolabeled forms of these compounds was studied 30 minutes after intravenous administration in squirrel monkey (Saimiri sciureus). Quantitative ex vivo autoradiography of brain tissue sections from animals receiving [3H]CFT (2.5 nmol/kg) revealed radioligand distribution primarily in dopamine-rich brain regions, and treatment with cocaine or mazindol substantially reduced [3H]CFT accumulation. The caudate nucleus, putamen, and nucleus accumbens/olfactory tubercle accumulated the highest levels of [3H]CFT, and within the caudate nucleus, medial-to-lateral and anterior-to-posterior gradients were observed. Low levels of [3H]CFT were found in the cortex, thalamus, hypothalamus, and background levels in white matter and in the cerebellum (striatal:cerebellar ratio > 3). The in vivo distribution of [3H]CFT closely paralleled its in vitro distribution (Synapse, 9:177-187). Although [125I]RTI-55 (0.2 nmol/kg) also distributed to dopamine-rich brain regions (striatal: cerebellar ratio of 3.6), high levels also were detected in cortex, thalamus, and in midbrain/brainstem structures. In the periphery, [125I]RTI-55 accumulation, measured as percent of administered dose, was higher than [3H]CFT in liver and lung, respectively. Of the radioactivity detected in soluble extracts of striatum, liver, and lung, greater than 90% comigrated with [3H]CFT or [125I]RTI-55 standards. The results support the hypothesis that dopamine-rich brain regions may be relevant to the effects of cocaine, and that both CFT and RTI-55 may be suitable imaging probes for these sites.     

Quantitative autoradiography of the dopamine uptake complex in rat brain using [H]GBR 12935: binding characteristics

The dopamine uptake complex was examined in the rat central nervous system using [³H]GBR 12935 and in vitro quantitative autoradiography to determine all binding data. [³H]GBR 12935 labels two unique binding sites, the dopamine uptake complex and a piperazine acceptor site. These two sites differ in their pharmacologic properties, anatomical distributions, densities, and response to lesions. Using appropriate binding conditions, [³H]GBR 12935 can be used to specifically label the dopamine uptake complex. [³H]GBR 12935 labeled a single binding site with characteristics of the dopamine uptake complex when mazindol (25 μM) was used as a blank. The specific binding and autoradiographic appearance of [³H]GBR 12935 to the dopamine uptake complex was improved by including trans-flupentixol (0.75 μM) to displace binding to a previously desrribed piperazine acceptor site, recently determined to be a site on cytochrome P450IID1. Binding was saturable and reversible to the dopamine uptake complex. The equilibrium dissociation constant (1.4 ± 0.7nM), maximal number of binding sites (6.0 ± 1.3pmol/mg protein), and Hill coefficient (1.1 ± 0.1) of [³H]GBR 12935 in rat striatum using mazindol to define non-specific binding was not significantly altered by the inclusion of trans-flupentixol (0.75 μM). Using GBR 12909 as a blank produced a greater maximal number of binding sites (8.4 ± 2.3pmol/mg protein), but no significant difference in the equilibrium dissociation constant (1.6 ± 0.3nM) or Hill coefficient (1.1 ± 0.1). A series of drugs that bind to the dopamine uptake complex displaced [³H]GBR 12935 in a rank order consistent with other binding and behavioral studies of this complex. The rank order of these drugs was GBR 12909 > mazindol > nomifensine > benztropine > desipramine > amphetamine > dopamine; all these drugs displayed a Hill coefficient near one and were best modeled as a single site. Cocaine and WIN 35, 428 (a cocaine congener) were unique in their competition for [³H]GBR 12935 binding, displaying biphasic curves, low Hill coefficients, and were best modeled as two site fits. Lesioning of the dopaminergic median forebrain bundle resulted in a dramatic loss of the dopamine uptake complex in the striatum, nucleus accumbens, olfactory tubercle, and substantia nigra. Other dopaminergic projection areas were decreased to a lesser extent. Striatal ibotenate lesions did not decrease the density of the dopamine uptake complex, despite a large decrease in the dopamine D₁ receptor. [³H]GBR 12935 can be used as an effective ligand to label the dopamine uptake complex for quantitative autoradiographic studies. It offers a number of advantages over previous autoradiographic assays for this complex including high specificity (> 95% specific binding in rat striatum), high sensitivity (detection of mazindol displaceable sites in the cerebral cortex), low background (comparable to film background), and low cost. This assay also supports the existence of two binding sites for cocaine on the dopamine uptake complex. The exact nature and differences between these two cocaine sites remains to be determined.     

Regional [H]acetylcholine and [H]nicotine binding in developing mouse brain

The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [³H]acetylcholine ([³H]ACh) and [³H]nicotine ([³H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [³H]NIC increased gradually with age in all brain regions studied. The [³H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [³H]ACh was found to be rather similar to that earlier described for [³H]alpha-bungarotoxin binding sites, whereas that for [³H]NIC differed from that described for other nicotinic ligands.     

[H]WIN 35,428 binding in the human brain

The binding of [³H]WIN 35,428 was studied in post-mortem human brain, including extrastriatal regions. In the putamen, dopamine almost completely inhibited the [³H]WIN 35,428 binding. Paroxetine inhibited the binding with similar affinity as cocaine, in the range 200–300 nM. In the frontal cortex, [³H]WIN 35,428 labelled cocaine- and alaproclate sensitive binding sites, of which a major fraction was of protein nature. The elucidation of the cocaine sensitive sites in the frontal cortex should be the subject of further research.     

Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients

We assessed muscarinic M₁, M₂ and M₄ receptor subtypes in the hippocampus of Alzheimer’s and control brains by receptor autoradiography using ligands such as [¹²⁵I]muscarinic toxin-1 ([¹²⁵I]MT-1, M₁ selective), [³H]AFDX-384 (M₂ partially selective) and [¹²⁵I]muscarinic toxin 4 ([¹²⁵I]M₄ toxin-1, M₄ selective). Our results revealed a significant decrease in muscarinic M₄ receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimer’s patients compared to controls. No changes in the density of M₁ or M₂ receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M₄ receptor could be the subtype which is selectively compromised in Alzeheimer’s disease (AD).     

Effect of dopaminergic drugs on the in vivo binding of [3H]WIN 35,428 to central dopamine transporters

[¹¹C]WIN 35,428 (also designated [¹¹C]CFT) is now being used in several positron emission tomography (PET) centers to image dopamine (DA) transporter sites in the mammalian brain. Whether and to what extent in vivo WIN 35,428 binding is influenced by intra- and extrasynaptic dopamine levels are largely unknown. The purpose of the present study was to evaluate the effects of various drugs, known to affect DA levels and release, on the binding of [³H]WIN 35,428 to central DA transporters in the mouse brain. D-Amphetamine, which releases DA from neurons and blocks the DA transporter directly, inhibited striatal [³H]WIN 35,428 binding in dose-dependent manner. Similarly, α-methyl-DL-p-tyrosine, an inhibitor of tyrosine hydroxylase, blocked [³H]WIN 35,428 binding, possibly via competitive inhibition by the metabolite p-hydroxyamphetamine. Specific binding of [³H]WIN 35,428 was insensitive to changes in synaptic DA levels caused by pretreatment of the animals with high doses of D₂ receptor agonists (apomorphine, bromocriptine), antagonists (haloperidol) or the inhibitor of dopaminergic neuron firing γ-butyrolactone (GBL). High doses (>50 mg/kg) of L-DOPA (in combination with benserazide), however, reduced [³H]WIN 35,428 binding significantly, yet for a relatively short time (approximately 2.5 h). Chronic treatment with L-deprenyl elicited no changes in in vivo [³H]WIN 35,428 accumulation in the striatum. Neurotoxic damage of DA neurons caused by administration of high doses of amphetamine was detected in the striatum by a significant reduction in [³H]WIN 35,428 binding 7 days after cessation of amphetamine treatment. Thus, [³H]WIN 35,428 binding was only affected by neurotoxic loss of neurons, by administration of uptake inhibitors, or by some treatments which significantly elevate DA levels. Compounds which inhibit DA release or deplete DA acutely do not increase [³H]WIN 35,428 binding, suggesting that normal or “resting” levels of DA are not sufficient to alter [³H]WIN 35,428 binding in vivo. These findings are important for our understanding of the function and regulation of the DA transporter, as well as the in vivo binding of the radioligand [³H/¹¹C]WIN 35,428. Moreover, they will be important for the interpretation of PET studies in which [¹¹C]WIN 35,428 is used to assess the integrity of dopaminergic neurons. © 1996 Wiley-Liss, Inc.     

Regional distribution of [H]imipramine binding in rat brain

[³H]imipramine binding was measured in 23 microdissected areas of the rat brain and compared to published values for the endogenous levels of serotonin, noradrenaline and dopamine in the same areas.

The density of [³H]imipramine binding sites appears to be highly correlated with the distribution of endogenous serotonin especially where the serotonin is located mainly in nerve terminals. A weak but still significant correlation also exists with the distribution of endogenous noradrenaline whereas no such correlation could be detected for endogenous dopamine.     

In vivo imaging of baboon and human dopamine transporters by positron emission tomography using [11C]WIN 35,428

[¹¹C]WIN 35,428 was evaluated as a specific in vivo radioligand for the dopamine transporter site by PET scanning in nonhuman primates and humans. In studies with a baboon (Papio anubis), [¹¹C]WIN 35,428 accumulated in brain regions containing dopamine transporters, i.e., the striata. This accumulation was Partially blocked by prior administration of (?)cocaine (4 mg/kg, i.v. ). Placement of a unilateral lesion of dopamine-containing nerve terminals with MPTP resulted in a unilateral reduction in [¹¹C]WIN 35,428 accumulation in the striatum on the side of the lesion. Imaging of D₂ dopamine receptors with [¹¹C]NMSP in the same MPTP-treated animals showed much less reduction in the postsynaptic D₂ dopamine receptors as compared to the much larger reduction in the dopamine transporters labeled with [¹¹C]WIN 35,428. A total of ten normal human volunteers (five males and five females) with ages ranging from 19 to 81 years were studied. The caudate/cerebellar and putamen/cerebellar ratios ranged from 4.4 to 5.7 90 min after injection of the tracer. Preliminary kinetic modeling with arterial plasma sampling resulted in an average binding potential (K₃/K₄) of 4.98 in the caudate nucleus and 5.13 in putamen. To demonstrate in vivo blockade with dopamine reuptake inhibitors, two subjects received prior oral doses of 6 mg mazindol. Subject 5 had significant reductions of 29% in the caudate/cerebellar ratio at 90 min, 35% in the putamen/cerebellar ratio at 90 min, 45% in the caudate k³/k⁴ ratio from 6.7 to 3.7, and 46% in the putamen k³/k⁴ from 4.7 to 2.5. Subject 8 had significant reductions of 20% in both the caudate/cerebellar ratio and the putamen/cerebellar ratio at 90 min. During the human PET studies, a number of neuropsychological tests and physiological measurements were performed. No significant changes were found after administration of the [¹¹C]WIN 35,428 alone. Taken together, these data indicate that [¹¹C]WIN 35,428 is a promising radioligand for future studies of neuropsychiatric disorders that involve the dopamine transporter site. © 1993 Wiley-Liss, Inc.

1 This article is a US government work and as such, is in the public domain in the United States of America.

     

Endogenously released dopamine inhibits the binding of dopaminergic PET and SPECT ligands in superfused rat striatal slices

Pharmacologically induced changes in synaptic levels of dopamine (DA) have been found, in some studies, to affect the in vivo binding of dopaminergic radioligands. In the present study we used a superfused brain slice preparation to examine the effect of synaptically released dopamine on the binding of some commonly used PET and SPECT radioligands under more controlled conditions than those present in vivo. The release of DA was evoked by electrical stimulation of striatal slices and the sensitivity of binding of the D₁ receptor ligand, [³H]SCH 23390, the D₂ receptor ligands [³H]raclopride and [¹²³I]epidepride, and the DA uptake transporter ligands, [³H]WIN 35,428 and [¹²³I]RTI-55, to the frequency of stimulation examined. Most affected by stimulation was the specific binding of [³H]SCH 23390, which was fully inhibited at 2.5 Hz. This was followed by [³H]raclopride and [¹²³I]epidepride, respectively, the binding of the latter showing only a 50% reduction at the highest frequency of 10 Hz. [³H]WIN 35,428 and [¹²³I]RTI-55 binding was unaffected by stimulation. The effects of stimulation on [³H]raclopride binding were prevented by reserpine pretreatment of the rat, when combined with inclusion of the dopamine synthesis inhibitor, α-methyl-p-tyrosine, in the superfusate medium. We conclude that, in brain slices, the binding of D₁ and D₂ receptor ligands but not that of DA uptake transporter ligands is readily inhibited by DA released into the synaptic cleft. Brain slices may prove to be a useful model system for the investigation of factors affecting competition between radioligand binding and endogenous neurotransmitters. © 1996 Wiley-Liss, Inc.     

Striatal dopaminergic terminals in type 1 and type 2 alcoholics measured with [H]dihydrotetrabenazine and human whole hemisphere autoradiography

A number of studies have pointed to the importance of dopamine system in the context of alcoholism. Previous studies have shown lower dopamine transporter levels on late-onset Cloninger type 1 alcoholics. However, whether this lower level is due to a lower level of dopamine transporter protein or a lower level of dopaminergic nerve terminals remains unclear. The aim of this study was to compare putative alterations of dopaminergic terminals in caudate, putamen and nucleus accumbens of type 1 and type 2 alcoholics and healthy controls by using [³H]dihydrotetrabenazine as a radioligand in postmortem human whole hemisphere autoradiography. We compared the present results with the findings of our earlier studies on the dopamine transporter in these same subjects, demonstrating that alcoholics do not differ significantly from controls in striatal [³H]dihydrotetrabenazine binding. Although type 1 alcoholics have been reported to have up to 36% lower striatal dopamine transporter levels than controls, the results suggest that the density of their dopaminergic nerve terminals is not altered.     

Transport of [H]mazindol binding sites in mesostriatal dopamine axons

6-Hydroxydopamine injections along mesostriatal dopaminergic axons can be used to interrupt axonal transport from cell bodies in the substantia nigra pars compacta to terminal fields in the striatum. Such lesions produce accumulations of high-affinity dopamine uptake sites (as measured by [³H]mazindol binding) and acetylcholinesterase proximal to the injection, suggesting that at least a portion of the [³H]mazindol binding and acetylcholinesterase activity seen in the striatum is located presynaptically on the mesostriatal dopaminergic fibers.     

123/125I]RTI-55, an in vivo label for the serotonin transporter.

[123I]RTI-55, an iodinated derivative of the cocaine analog 3 beta-phenyltropane-2 beta-carboxylic acid methyl ester, was evaluated as an agent for in vivo labeling of the serotonin transporter. Labeling of the precursor of RTI-55 with I-123 was efficient and yielded a high specific activity product. After intravenous injection of [123I]RTI-55 into rats, the tracer accumulated in regions with high densities of serotonin and dopamine uptake sites. The distribution of [123I]RTI-55 binding in areas rich in serotonin uptake sites correlated with [3H]serotonin uptake measured in vitro in the same regions. Specific [123I]RTI-55 binding to serotonin uptake sites was inhibited by paroxetine but not by GBR 12,909. Treatment of rats with neurotoxic doses of fenfluramine caused decreases of 66% (in the hypothalamus) to 83% (in the superior colliculi) of specific [125I]RTI-55 binding in all areas except in the striatum and the olfactory tubercles (regions rich in dopamine transporters). These results indicate that [123/125I]RTI-55 binds, although not selectively, to the serotonin transporter in vivo. Furthermore, they suggest that [123I]RTI-55 holds promise as a SPECT imaging agent for the study of the serotonin transporter in humans in health and disease.     

Photoperiodic effects on puberty and specific 2-[I]iodomelatonin binding sites in Siberian hamsters

When juvenile male Siberian hamsters are transferred from a long photoperiod to a short photoperiod, sexual maturation is greatly delayed by a pineal-dependent process. We hypothesized that the eventual onset of puberty during short photoperiod exposure may be caused by a loss of receptors for the pineal hormone, melatonin. This study quantitated specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei and pars tuberalis of Siberian hamsters exposed to short photoperiod (10 h light per day) for either 12 or 30 weeks and in hamsters exposed to long photoperiod (16 h light per day) for the same time intervals. Photoperiodic exposure significantly affected testes weight. The hamsters exposed to long photoperiod for either 12 or 30 weeks had mean testes weights > 700 mg, in contrast to hamsters in short photoperiod for 12 weeks (mean testes weights < 30 mg) or 30 weeks (mean testes weights approximately 350 mg). The affinity of specific 2-[¹²⁵I]iodomelatonin binding sites in both regions was significantly lower in hamsters exposed to short photoperiod as compared to hamster exposed to long photoperiod, at either 12 or 30 weeks. In contrast, there were no effects of photoperiod or duration of exposure on the density of specific 2-[¹²⁵I]iodomelatonin binding sites in either the suprachiasmatic nuclei or the pars tuberalis. Furthermore, a change in the affinity of the specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei was observed between the hamsters housed in short photoperiod for 12 weeks (sexually immature) and the hamsters housed in short photoperiod for 30 weeks (undergoing puberty). These results demonstrate that although the onset of puberty after long-term exposure to short photopoeriod does not involve a loss of specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei or pars tuberalis, it is associated with a decrease in the affinity of specific 2-[¹²⁵I]iodomelatonin binding sites in these regions.     

Decreased [H]hemicholinium binding to high-affinity choline uptake sites in aged rat brain

The binding of [³H]hemicholinium ([³H]HCh-3) to sodium-dependent high-affinity choline uptake sites provides a useful neuroanatomical and functional marker of the cholinergic system. We examined the autoradiographic distribution of [³H]HCh-3 binding sites in the forebrain of young (4–6 months) and old (32 months) rats. There was a widespread reduction of [³H]HCh-3 binding site density in the aged rat brain. This loss presented regional differences with maximal reduction in the medial and posterior striatum (55%) and in the dentate gyrus (47%), in limbic areas such as basolateral amygdala, tubercle olfactorium and piriform cortex the autoradiographic signal was about 25–30% lower. In aged hippocampus and cerebral cortex the density of [³H]HCh-3 binding sites was about 40% lower, the difference between young and senescent animals being less evident in the medial septum and basal nucleus. No significant alterations were observed in interpeduncular nucleus from old rats. These data are in agreement with the functional results obtained by measuring other cholinergic parameters in the aged rat and confirm the vulnerability of cholinergic system during aging     

A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428.

The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.     

Multiple binding sites for [125I]RTI-121 and other cocaine analogs in rat frontal cerebral cortex

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high-affinity and selective ligand [¹²⁵I]RTI-121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high- and low-affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the binding of 15 pM [¹²⁵I]RTI-121 in a biphasic fashion following a two-site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [¹²⁵I] RTI-121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [¹²⁵]RTI-82 and subsequent analysis of SDS-page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI-121 and the presence of several known transporters in the tissue studied. Synapse 30:9–17, 1998. Published 1998 Wiley-Liss, Inc.     

Altropane,a SPECT or PET imaging probe for dopamine neurons: I. dopamine transporter binding in primate brain

Increasing evidence suggests that the dopamine transporter is an important marker for physiological and pathological changes in dopamine neurons. Potent dopamine transport inhibitors of the phenyltropane series (e.g., WIN 35,428 or CFT) are particularly suitable for PET (positron emission tomography) or SPECT (single photon emission computed tomography) imaging of the dopamine transporter in living brain. We investigated whether altropane, an N-iodoallyl analog of WIN 35,428 (IACFT:E-N-iodoallyl-2β-carbomethoxy-3β-(4-fluorophenyl)tropane), displayed in vitro properties suitable for evaluation as a SPECT imaging agent. In brain striatum of cynomolgus monkey (Macaca fascicularis), the unlabeled E-isomer (IC₅₀: 6.62 ± 0.78 nM) was more potent than the Z-isomer (IC₅₀: 52.6 ± 0.3 nM) and displayed a relatively high dopamine:serotonin transporter selectivity (28-fold). In radiolabeled form, [¹²⁵I]altropane bound to sites in the striatum with a single high affinity (K_D: 5.33 ± 0.55 nM) and with a site density (B_MAX: 301 pmol/g original wet tissue weight) that was within the density range reported previously for the dopamine transporter in striatum. Drugs inhibited [¹²⁵I]altropane binding with a rank order of potency that corresponded closely to their potencies for inhibiting [³H]WIN 35,428 binding (r²: 0.99; P < 0.0001) to the blocking dopamine transport. The favorable binding properties of altropane, together with its rapid entry into primate brain and highly localized distribution in dopamine-rich brain regions, suggest it is a suitable iodinated probe for monitoring the dopamine transporter in vitro and in vivo by SPECT or PET imaging. Synapse 29:93–104, 1998. © 1998 Wiley-Liss, Inc.