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.     

Striatal dopamine innervation and receptor density: regional effects of the weaver mutation

Mice homozygous for the autosomal recesive gene weaver (wv) exhibit a regionally specific depletion of forebrain dopamine (DA). DA is reduced approximately 70% in the dorsal striatum of homozygotes (wv/wv) relative to heterozygous (+/wv) controls while DA content in ventral striatum is relatively unchanged. The goal of the present study was to determine the regional effects of the weaver mutation on striatal DA receptors and DA uptake sites using quantitative autoradiography. Catecholamine histofluorescence was used to examine midbrain DA-containing cell bodies. Compared to behaviorally normal (+/-) littermates, the binding of [³H]spiroperidol to D₂ sites was significantly increased in the dorsal but not ventral striatum of wv/wv mice. Binding of the D₁ ligand, [³H]SCH23390, was significantly decreased throughout the striatum of wv/wv mice. The binding of [³H]mazindol to DA uptake sites was dramatically reduced in all wv/wv striatal regions except the ventrolateral portion. Compared to +/-littermates, wv/wv mice had far fewer fluorescent cell bodies in the substantia nigra and a less pronounced reduction of ventral tegmental area fluorescent somata. These findings support the hypothesis that heterogeneities exist in the genetic control of the mesotelencephalic DA system. The results underscore the usefulness of the weaver mouse in the study of mesostriatal sub-systems, receptor regulation, and potentially as a model of human neuropathologies that affect distinct populations of cells in the mesotelecephalic system.     

The effects of chronic continuous versus intermittent levodopa treatments on striatal and extrastriatal D1 and D2 dopamine receptors and dopamine uptake sites in the 6-hydroxydopamine lesioned rat — an autoradiographic study

The effects of chronic ‘continuous’ infusion and ‘intermittent’ modes of levodopa/carbidopa administration on apomorphine induced circling behaviour, DA uptake sites (labelled with [³H]mazindol) and D₁ and D₂ DA receptor binding (labelled with [³H]SCH 23390 and [³H]sulpiride, respectively) were investigated in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. The circling behaviour in response to apomorphine was greatly enhanced following chronic ‘intermittent’ but not ‘continuous’ levodopa treatments. Following the ‘intermittent’ regime, the lower dose of apomorphine induced a period of intense circling with delayed onset and rapid offset, than in rats given either ‘continuous’ infusion of levodopa or saline. The 6-OHDA lesion itself induced gross depletion of [³H]mazindol binding in all striatal subregions, NAc and OT, but not frontal cortex. [³H]Sulpiride binding in the ventrolateral striatal quadrant was increased on the denervated side and this correlated with the peak contralateral turns in response to 0.5 mg/kg apomorphine challenge. This asymmetry in striatal [³H]sulpiride binding was reduced in both groups of rats receiving levodopa. [³H]sulpiride binding in the NAc and OT and [³H]SCH 23390 binding in the striatum, NAc, OT and SNr were unaffected by DA denervation or either regime of levodopa treatments. ‘Continuous’ infusion and not ‘intermittent’ injections of levodopa reduced [³H]mazindol binding in the striatal subregions and the frontal cortex on both the denervated and intact sides. The potentiation of the behavioural response to apomorphine by chronic ‘intermittent’ levodopa treatment does not correspond with the levodopa induced alterations in striatal or extrastriatal DA receptors. In the same group of animals the narrowing of the duration of response to the lower dose of apomorphine may mimic the fluctuations in response to levodopa, seen clinically in long-term levodopa treated parkinsonian patients.     

Regulation of neurotensin-containing neurons in the rat striatum. Effects of unilateral striatal lesions with quinolinic acid and ibotenic acid on neurotensin content and its binding site density

Recently, we reported bilateral increases in striatal neurotensin (NT) levels following unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway. In the present study, the effect of unilateral striatal lesions with quinolinic acid (QA, 300 nmol) or ibotenic acid (IBO, 130 nmol) on striatal NT levels and binding site densities were analyzed in order to investigate other possible regulations of NT systems. QA and IBO injection decreased γ-aminobutyric acid (GABA) levels and [¹²⁵I]iodosulpride (a specific D2 receptor antagonist) binding site densities in the lesioned striatum, indicating degeneration of striatal intrinsic neurons. Striatal dopaminergic terminals were not altered by QA as shown by the lack of changes in [³H]dihydrotetrabenazine ([³H]TBZOH, a specific ligand of the vesicular monoamine transporter) binding site densities. Moreover, QA lesion induced an increase in NT levels and a decrease in NT binding sites in the lesioned striatum without any change in the contralateral structure. In contrast to QA, IBO might destroy a certain proportion of dopaminergic terminals in the lesioned striatum, as shown by a 54% decrease in [³H]TBZOH binding. Furthermore, IBO lesion enhanced striatal NT levels bilaterally, while NT binding sites decreased in the lesioned striatum and increased in the contralateral side. The present results suggest that not only dopaminergic neurons but also striatal intrinsic neurons may control NT systems in the striatum.     

Effects of chronic cocaine administration on dopamine transporter mRNA and protein in the rat

Male Sprague–Dawley rats were administered cocaine (10, 15 or 25 mg/kg) or vehicle, i.p., once daily for 8 consecutive days and killed 1 h after the last injection. Acute cocaine administration produced dose-dependent increases in spontaneous locomotor activity. These levels of activity were further enhanced by 8 days of chronic treatment, indicating the emergence of behavioral sensitization. Chronic cocaine administration resulted in dose-dependent decreases in the density of dopamine transporter (DAT) mRNA in both the substantia nigra pars compacta and ventral tegmental area as shown by in situ hybridization histochemistry. Changes in DAT binding sites were assessed using [³H]mazindol quantitative autoradiography. In contrast to the levels of mRNA, there were few changes in the number of [³H]mazindol binding sites. Although the density of binding sites was unaltered in most regions, [³H]mazindol binding was increased in the anterior nucleus accumbens. This study extends previous findings by demonstrating the dose-dependent nature of the changes in DAT mRNA that accompanies chronic cocaine administration. The levels of DAT binding sites within the dorsal and ventral striatum, however, were largely unchanged. This mismatch suggests that cocaine may differentially influence the gene expression of DAT in the ventral midbrain as compared to the density of DAT binding sites in the basal forebrain. © 1997 Elsevier Science B.V. All rights reserved.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Neurotrophic factor for serotonergic neurons prevents degeneration of grafted raphe neurons in the cerebellum.

[³H]SCH 23390 binds stereospecifically and with high affinity to D₁ dopaminergic receptors in the developing chick retina. Autoradiographic experiments revealed that in retinas from 3-day-old chicken and embryos with 12, 14 and 16 days of development, specific labeling of [³H]SCH 23390 was mainly observed over the plexiform layers of the tissue, showing that dopaminergic D₁ receptors are localized in retina cell neurites since the initial stages of neurite formation. The total number of [³H]SCH 23390 binding sites increased 5-fold during the differentiation of the retina, while the dopamine-dependent cyclic adenosine monophosphate (AMP) accumulation was significantly decreased. Consequently, the ratio between dopamine-dependent cyclic AMP accumulation and [³H]SCH 23390 binding sites decreased 10-fold as retina differentiated, indicating that a significant portion of D₁ receptors in retinas from adult chicken are not effectively coupled to adenylate cyclase molecules.     

A selective lesion of striatonigral neurons decreases presynaptic binding of [H]hemicholinium-3 to striatal interneurons

We have used the suicide transport agent, volkensin, to produce selective lesions of striatal efferent neurons projecting to the substantia nigra in the rat. In order to evaluate potential trans-synaptic effects, we examined cholinergic interneurons intrinsic to the striatum following destruction of striatonigral projection neurons by nigral injection of volkensin. There was no change in the number of large interneurons identified either by Nissl stain or by immunocytochemistry for choline acetyltransferase, indicating that volkensin was not directly toxic to this group of neurons. However, [³H]hemicholinium-3 binding to the choline re-uptake site on the presynaptic cholinergic terminals decreased. No change in [³H]hemicholinium-3 binding was seen after destruction of dopaminergic afferents with 6-hydroxydopamine. Striatonigral afferents to the cholinergic interneurons contain substance P which has been shown to stimulate acetylcholine release. The decrease in [³H]hemicholinium-3 binding may reflect loss of this afferent input. However, striatonigral neurons are an efferent target of the cholinergic interneuron as well, and a presynaptic effect due to loss of target neurons also may contribute.     

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [³H]AMPA, [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [³H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [³H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [³H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [³H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation.     

5-HT1B receptor binding in degenerative movement disorders

Using [³H]sumatriptan as a radioligand, 5-hydroxytryptamine (5-HT)_1B receptors were examined in posterior striatum and midbrain post-mortem tissue sections of 12 patients who had died from representative degenerative movement disorders as compared to nine controls. In the control human basal ganglia, the highest densities of [³H]sumatriptan binding were observed in the globus pallidus and substantia nigra. No significant change in the density of [³H]sumatriptan binding sites was found in the striatum and substantia nigra of the six Parkinson's disease brains. In the two brains from patients with progressive supranuclear palsy an increase was found in the densities of [³H]sumatriptan binding sites, most marked in the substantia nigra. In contrast, [³H]sumatriptan labelling was almost absent in the striatonigral degeneration brain and was markedly reduced in the three Huntington's disease brains. This study indicates that the status of 5-HT_1B receptors is different in each degenerative movement disorder and suggests that human 5-HT_1B receptors are located somatodendritically on GABAergic and peptidergic caudate-putamen neurons which project to the substantia nigra and globus pallidus, where these receptors are presynaptic.     

Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand, [H]TCP

Receptor binding sites for the phencyclidine (PCP) analogue, [³H]TCP, have been localized in the rat and guinea pig central nervous systems by in vitro autoradiography. Quantitation of [³H]TCP binding site densities in rat brain reveals highest levels in the forebrain, in particular the strata oriens and radiatum of the hippocampus, the molecular layer of the dentate gyrus and superficial layers of the cerebral cortex. Moderate levels of binding occur in the amygdala, thalamus, anterior olfactory nucleus external plexiform layer of the olfactory bulb, olfactory tubercle, geniculate nuclei and deep layers of the cortex. Low levels of binding occur throughout most of the septum, diagonal band, hypothalamus, pons-medulla and cerebellum. Spinal cord grey matter also has low levels binding. Excitotoxin lesions of the hippocampal formation, which destroy the pyramidal and granule cells, reduce the binding of [³H]TCP to strata radiatum and oriens and the molecular layer of the dentage gyrus by 60% suggesting that [³H]TCP labels intrinsic neurons in these regions. Residual binding is probably on afferent terminals. Ibotenic acid lesions of the caudate-putamen reduce [³H]TCP binding by 70%, indicating that binding sites are localized on intrinsic striatal neurons. 6-Hydroxydopamine lesions do not alter [³H]TCP binding levels the caudate, suggesting the absence of binding sites on dopaminergic terminals in the caudate.     

Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [³H]quinuclidinyl benzilate (QNB), M₁ receptors were directly labeled with [³H]pirenzepine (PZ) and non-M₁ receptors were labeled by the competition of 100 nM PZ with [³H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [³H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [³H]QNB. Subtype analysis showed that there was a reduction of both M₁ (−26%) and non-M₁ (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (B_max) and not affinity (K_d). There was no significant change in [³H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.     

In vitro effects of melatonin on [H]muscimol binding in rat brain

1. The possible interaction of melatonin with the GABAergic system at the receptor level was investigated.

2. The effect of melatonin on the binding of the GABA agonist, [³H]muscimol, in crude synaptic membrane preparations was assayed.

3. In fresh membrane preparations, melatonin increased [³H]muscimol binding in the rat striatum and frontal cortex. Treatment of membrane preparations with Triton X-100 appeared to abolish depress the enhancing effect of melatonin. The use of Tris HCl as buffer appeared to be more effective than Tris-Citrate buffer suggesting a possible important ionic effect.

4. The present findings suggest that melatonin is capable of enhancing [³H]muscimol binding , perhaps at the benzodiazepine-GABA-receptor-ionophore complex.     

Detection of dopamine receptors in homogenates of rat hippocampus and other brain areas

The binding of [³H]spiperone (24–26pM) specifically to the dopaminergic (DAergic) D₂ site has been investigated in homogenates of rat hippocampus, pineal gland and cerebellum. It was found that specific binding in hippocampal homogenates was heat-labile and for a part pH-sensitive. Like striatal tissue, binding to the dopaminergic D₂ site in hippocampal and cerebellar homogenates was greatly reduced by (pre)incubation with the iron chelatoro-phenanthroline. The binding characteristics in hippocampus and cerebellum have been compared to those in the striatum. In all tissues tested, low (74–138 times fewer than in striatum) but significant numbers of DAergic D₂ sites were found. The DAergic D₂site in the hippocampus, as far as measured in cerebellum and pineal gland exhibited similar binding characteristics to that in the striatum.     

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     

Levodopa or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in dopamine and GABAA, receptors in the striatopallidal complex

Dopamine D1 and D2 receptors as well as the GABA/benzodiazepine receptor complex in the striatum and the globus pallidus (internal: GPi and external: GPe) were studied by autoradiography using [³H]SCH 23390, [³H]spiperone, and [³H]flunitrazepam ([³H]FNZ) respectively, in five groups of cynomolgus monkeys. These included (i) untreated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-monkeys; (ii) MPTP monkeys treated chronically with levodopa injections; (iii) MPTP monkeys treated chronically with injections of the novel D2 agonist U91356A; (iv) MPTP monkeys treated chronically with U91356A delivered through an osmotic mini-pump; and (5) naive controls. Animals treated in a pulsatile mode with U91356A or levodopa injections showed progressive sensitization to their respective drug and developed choreic dyskinesia. In contrast, animals treated in a continuous mode with U91356A showed behavioral tolerance but did not develop dyskinesia. A trend for a down-regulation of putaminal D2 receptors was observed following D2 agonist stimulation with U913356A. Striatal [³H]FNZ binding was significantly decreased only in animals treated in a continuous mode with U91356A. The dopamine receptor decrease in the striatum could be implicated with the development of tolerance but cannot explain the appearnce of dyskinesia. Denervation by MPTP was associated with a decrease of the GPe/GPi [³H]FNZ binding ratio which reflects an imbalance of striatal output pathways; this ratio was not reversed by any of the treatments although changes were observed in the GPe and GPi. Indeed, pulsatile U91356A treatment restored the decreased [³H]FNZ binding in the GPe near control values and levodopa showed a similar tendency. A significant increase of [³H]FNZ binding in the GPi only of dyskinetic monkeys, namely those treated with pulsatile U91356A or levodopa was seen compared to untreated MPTP or naive controls. This GABA_A receptor up-regulation might lead to a supersensitive state of the GPi to gabaergic input which may be involved in the mechanism underlying the development of dopaminomimetic-induced dyskinesia.     

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [³H]kainate, [³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) and [³H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [³H]cyclohexyladenosine ([³H]CHA) binding to presynaptic adenosine A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [³H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [³H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [³H]AMPA and [³H]CHA binding at 20 days post-enucleation. [³H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [³H]kainate and [³H]AMPA binding at this time point indicated a selective increase in the b_max value for the high affinity [³H]kainate binding site and a concomitant decrease in the b_max value for the high affinity [³H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [³H]AMPA or [³H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [³H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.     

The effects of serotonergic and dopaminergic lesions on sodium-sensitive [3H]mazindol binding in rat hypothalamus and corpus striatum

The effects of intracerebroventricular administration of 6-hydroxydopamine (6-OHDA) and 5,7-dihydroxytryptamine (5,7-DHT) on sodium-sensitive [3H]mazindol binding were investigated in the rat hypothalamus and corpus striatum. In the hypothalamus, specific [3H]mazindol binding was inhibited by low concentrations of sodium and stimulated by high-sodium concentrations, whereas in the corpus striatum, only a sodium-dependent stimulation of [3H]mazindol binding was observed. Lesions with 6-OHDA significantly reduced sodium-dependent [3H]mazindol binding in the corpus striatum, but had no effect on the binding of [3H]mazindol in the absence of sodium. Lesions of serotonergic neurons with 5,7-DHT, however, had no effect on [3H]mazindol binding in the striatum, but resulted in a significant increase in the number of [3H]mazindol binding sites in the hypothalamus. These data suggest that [3H]mazindol may bind to two anatomically distinct binding sites, one that is stimulated and the other inhibited by sodium. The sodium-stimulated binding sites appear to be located on dopaminergic terminals in the striatum, and in the hypothalamus, the sodium-inhibited sites appear to be regulated by serotonergic neuronal activity.     

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.     

Localization and quantification of the dopamine transporter: comparison of [H]WIN 35,428 and [I]RTI-55

Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [³H]WIN 35,428 and [¹²⁵I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/1 was used to block [¹²⁵I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [¹²⁵I]RTI-55 binding was significantly higher than [³H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [³H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.