Quantitative autoradiography of serotonin uptake sites in rat brain using [H]cyanoimipramine

The binding of [³H]cyanoimipramine to serotonin uptake sites in rat brain slices was studied using quantitative autoradiography. Binding was of high affinity and was to a single class of binding site. This is in contrast to results previously obtained by others with [³H]imipramine where two binding sites were observed. The sites labeled by [³H]cyanoimipramine had properties consistent with this ligand labeling serotonin uptake sites, as: (1) binding is displaced by drugs which are potent inhibitors of serotonin uptake but not by drugs which are weak inhibitors of uptake; (2) binding is dependent on the presence of sodium ions as is the uptake of serotonin; (3) binding is almost completely eliminated in the brains of rats lesioned by the serotonin neurotoxin 5,7-dihydroxytryptamine; (4) the distribution of binding sites throughout the rat brain is highly correlated with that found previously for [³H]indalpine, a potent serotonin uptake inhibitor, and for [³H]imipramine. The properties of binding of [³H]cyanoimipramine make it an ideal ligand for the quantitative autoradiography of serotonin uptake sites.     

Characterization of [H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes

Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [³H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [³H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent K_d under optimal conditions of 35 nM and a B_max of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [³H]HCh-3 binding site. The specific binding of [³H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [³H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decar☐ylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaCl increased the affinity of the site for [³H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [³H]HCh-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.     

[H]Nipecotic acid binding to GABA uptake sites in human brain

The binding of [³H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with γ-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [³H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to high affinity site. The dissaciation constants (K_d) measured in kinetic and equilibrium experiments were in the same range (0.5–0.6 μM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [³H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.     

[H]Arginine vasopressin binding to rat brain: a homogenate and autoradiographic study

Arginine-vasopressin (AVP) has been implicated as a putative central neurotransmitter or neuromodulator in some brain functions. This study demonstrates binding of [³H]AVP to rat brain homogenates that is pH and temperature dependent, is saturable (K_d = 0.77 nM, B_max = 0.374 pmol/mg) and reversible. A number of AVP analogues competitively displaced the [³H]AVP binding, indicating that central AVP binding sites may have a resemblance to the peripheral (V₁) AVP vasopressor receptor. Homogenate binding occurred predominantly in the microsomal fraction (P₃) of the hypothalamus while in the hippocampus and septum binding was predominantly in the synaptosomal fraction (P₂). Autoradiographic methods showed displaceable [³H]AVP binding in the lateral septum, amygdala, supraoptic, paraventricular and suprachiasmatic nuclei of the hypothalamus supporting the results of homogenate binding in preparations of these regions.     

Rapid regulation of [H]hemicholinium-3 binding sites in the rat brain

The effects of various drug known to affect the sodium-dependent high-affinity choline-uptake system (SDHACU) in the brain were examined for their action upon the [³H]hemicholinium-3 ([³H]HCh-3) binding site, which is associated with the choline carrier. The [³H]HCh-3 binding sites are affected in a similar way to the SDHACU system. Thus, alterations in the velocity of choline-uptake are mediated through changes in the apparent number of available transport sites at cholinergic terminals.     

Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Characterization and autoradiographic distribution of hemicholinium-3 high-affinity choline uptake sites in mammalian brain

[3H]hemicholinium-3 (HC-3) binding characteristics have been investigated using membrane binding assays and in vitro receptor autoradiography. In rat brain membrane preparations, [3H]HC-3 binds with high affinity to an apparent single class of sites. [3H]HC-3 binding is Na+-dependent. The ligand selectivity pattern strongly suggests that [3H]HC-3 selectivity labels the high affinity choline uptake (HACU) in brain membranes (HC-3 greater than choline greater than carbamylcholine greater than acetylcholine). This hypothesis is also supported by quantitative autoradiographic data which demonstrate that the discrete distribution of [3H]HC-3 binding sites correlates very well with the known distribution of other cholinergic markers such as choline acetyltransferase (ChAT), acetylcholinesterase (AChE), HACU, and [3H]AH-5183 (blocker of the vesicular transport of acetylcholine). For example, high densities of labelling are observed for these different markers in the interpeduncular nucleus, anteroventral nucleus of the thalamus, striatum, basolateral nucleus of the amygdala, and an exquisite laminar distribution in the hippocampus. Similar autoradiographic distributions of [3H]HC-3 binding sites are observed in other mammalian species such as guinea pig and monkey. Finally, 7-day unilateral kainic acid lesions of the nucleus basalis magnocellularis (nbm) decrease cortical [3H]HC-3 binding and ChAT activity, although not to a similar extent. In summary, these results demonstrate that [3H]HC-3 is a selective ligand of the HACU in mammalian brain. Thus, it is now possible to characterize precisely various structural components of the cholinergic synapses using markers such as [3H]HC-3, ChAT, HACU, [3H]AH-5183, and selective muscarinic and nicotinic receptor radioligands.     

Development of high-affinity choline transport sites in rat forebrain: a quantitative autoradiography study with [3H]hemicholinium-3.

The development of cholinergic terminals in rat brain has been quantitatively analyzed by [3H]hemicholinium-3 autoradiography. [3H]Hemicholinium-3 binds to high affinity choline transport sites, a specific marker for cholinergic neurons. In neonatal animals, kinetic and pharmacologic binding characteristics and regional distribution of [3H]hemicholinium-3 sites are consistent with specific cholinergic localization, as in the adult. The distribution of cholinergic terminals is described in the adult rat brain and during development, including heterogeneity of binding within several regions such as the striatum, nucleus accumbens, olfactory tubercle, cortex, and hippocampus. Early development and maturation vary greatly between brain regions. At embryonic day E18 and day 0, specific binding density is high only in the medial habenula. Development occurs primarily during the postnatal period in most brain regions examined. Many brain regions exhibit a lull in development between days 5 and 10, although the rate of development is highly region specific. Specific binding increases 2-12-fold between day 5 and adult animals, with adult density being achieved anywhere from day 15 to after day 21. The ontogeny of [3H]hemicholinium-3 binding sites generally occurs in a rostral to caudal direction. In the striatal body the characteristic lateral to medial gradient of binding site density is apparent by day 5, and development is more rapid in the lateral striatum. Patches of dense [3H]hemicholinium-3 binding coincident with acetylcholinesterase are observed on day 5 in the caudal striatum. The various patterns of cholinergic terminal development suggest that factors regulating cholinergic development are regional and complex.     

Quantitative light microscopic localization of [H]naltrindole binding sites in the rat brain

The binding of radiolabeled naltrindole ([³H]NTI), a selective δ-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent K_d of 0.25 ± 0.08nM (B_max of 597.5 fmol/mg protein). Autoradiographic localization of [³H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1–3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.     

Autoradiography of [H]cytochalasin B binding in rat brain

A technique is described for the autoradiographic localization of -glucose inhibitable cytochalasin B binding in fresh frozen sections of rat brain. The technique has been used to study the regional distribution of glucose transporter proteins. Binding sites within the cerebellum and hippocampus were more concentrated in the synaptic zones, which would be expected to display relatively high metabolic rates for glucose. However, a general inter-regional correlation of cytochalasin B binding with metabolic rates was not apparent.     

Quantitative autoradiographic distribution of [3H]AF-DX 116 muscarinic-M2 receptor binding sites in rat brain

The distribution of muscarinic-M2 receptors in rat brain was investigated by in vitro autoradiography using [3H]AF-DX 116, a putative probe for the muscarinic-M2 receptor subtype. Incubation of rat brain coronal sections with 10 nM [3H]AF-DX 116 showed highest binding site densities in discrete areas such as the superior colliculus and certain thalamic and brainstem nuclei, similar to the distribution reported for [2H]acetylcholine/M2 sites. [3H]AF-DX 116 site densities were markedly lower in forebrain areas such as cortex, striatum, and hippocampus, in contrast to the distribution seen for [3H]pirenzepine-M1 binding sites, which were concentrated in these forebrain areas; however, differential patterns of labeling were observed for the two muscarinic-M2 probes, [3H]AF-DX 116 and [3H]acetylcholine, in the hippocampal formation. Although [3H]AF-DX 116 binding was broadly distributed in multiple subfields of the hippocampus, [3H]acetylcholine binding was discretely distributed in a manner resembling that of acetylcholinesterase staining. This suggests the existence of muscarinic-M2 subtypes in the CNS, especially in the hippocampal formation.     

Autoradiographic localization of cocaine binding sites by [3H]CFT ([3H]WIN 35,428) in the monkey brain

The cocaine analog [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]CFT or [3H]WIN 35,428 binds with high affinity and selectivity to cocaine receptors in the monkey caudate-putamen. [3H]CFT was used to map the regional distribution of cocaine binding sites in slide-mounted sections of monkey brains using autoradiographic techniques. Hemicoronal brain sections were incubated with [3H]CFT (3 nM) alone or in the presence of excess (-)-cocaine (30 microM) to mask the binding sites. High densities of [3H]CFT binding sites were detected in the caudate nucleus, putamen, and nucleus accumbens. In all three regions, binding was markedly reduced by coincubation with unlabeled (-)-cocaine, indicating low levels of nonspecific binding. Little or no binding was observed in the cortex, thalamus, globus pallidus, or white matter tracts at the levels studied. In order to characterize binding sites for [3H]CFT in tissue sections, competition experiments were conducted using a fixed concentration of [3H]CFT (3 nM) and a range of concentrations of (-)-cocaine, (+)-cocaine, CFT, Lu 19-005, GBR 12909, bupropion, and citalopram. The IC50 values for the drugs in tissue sections corresponded closely with their reported IC50 values in monkey caudate-putamen membranes (r = 0.99, p less than 0.001), suggesting that [3H]CFT binding is similar in the two preparations. These findings support the view that cocaine receptors labeled by [3H]CFT are localized predominantly in dopamine-rich brain regions implicated in the behavioral effects and abuse of cocaine.     

Blood-brain barrier transport of choline is reduced in the aged rat

An age-related impairment in choline transport across the blood-brain barrier (BBB) may contribute to the cholinergic mechanisms of geriatric memory dysfunction. To test this hypothesis, the brain choline uptake in male Fisher 344 rats at 2, 18 and 24 months of age was studied using the Oldendorf technique. The V_max of cholie transport in the 24-month-old rats (0.05 ± 0.04 nmol/min/g) was significantly lower than that in the 2-month-old rat (2.5 ± 1.0 nmol/min/g) (P < 0.05). The K_m of transport in old rats (13 ± 35 μM) was also significantly smaller than the value in 24-month-old rats (450 ± 195 μM), while the constant of the non-saturable component of the transport, K_d, was not significantly different in older rats (1.2 ± 0.1 μl/min/g). These results indicate that the carrier in old rats has reduced capacity and increased affinity to choline. The reduced choline carrier capacity explains the significant decrease in BBB choline transport in aged rats.     

Regional distribution of high-affinity [H]somatostatin binding sites in the human brain

The distribution of high-affinity binding sites for [³H]somatostatin has been studied in membrane preparations from a number of regions of normal human brain. The highest densities of binding sites (> 48 fmol/mg protein) were found in the cerebral and cerebellar cortices and the hippocampus, with intermediate binding densities (30–46 fmol/mg protein) being present in the basal ganglia, amygdala, septum and claustrum. The lowest densities of binding sites (<14 fmol/mg protein) were observed in the hypothalamus, thalamus and substantia nigra. The binding of [³H]somatostatin in both the frontal cortex and cerebellar cortex demonstrated pharmacological specificity, since somatostatin-28, but not somatostatin-28_1–12 or Des AA^{1,2,4,5,12,13}, -Trp⁸-somatostatin, competed for the binding sites. Scatchard analysis of the binding in both frontal cortex and cerebellar cortex revealed the presence of two classes of high-affinity binding sites.     

Corticotropin-releasing factor antagonist blocks microwave-induced decreases in high-affinity choline uptake in the rat brain

Acute (45-min) irradiation with pulsed low-level microwaves (2450-MHz, 2 μsec pulses at 500 pps, average power density of 1 mW/cm², whole-body average specific absorption rate of 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake (HACU) activity in the frontal cortex and hippocampus of the rat. These effects were blocked by pretreating the animals before exposure with intracerebroventricular injection of the specific corticotropin-releasing factor (CRF) receptor antagonist, α-helical-CRF_9–41 (25 μg). Similar injection of the antagonist had no significant effect on HACU in the brain of the sham-exposed rats. These data suggest that low-level microwave irradiation activates CRF in the brain, which in turn causes the changes in central HACU.     

[H]MK-801 binding sites in neonate rat brain

[³H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [³H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg²⁺ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, theN-methyl-d-asparatate-type glutamate receptor and MK-801 binding site are functionally coupled.     

Intrahippocampal transplants of septal cholinergic neurons: high-affinity choline uptake and spatial memory function

Recent studies have demonstrated that intrahippocampal cholinergic septal grafts can ameliorate deficits in spatial memory function and hippocampal cholinergic neurochemical activity in animals with disruptions of the septohippocampal system. However, no study has determined if the restoration of spatial memory function is correlated to the restoration of cholinergic activity, as measured by high-affinity choline uptake (HACU). The present study was designed to determine if such a correlation between behavioral and neurochemical restoration exists. Male Sprague-Dawley rats received either sham lesions (SHAM), bilateral lesions of the septohippocampal pathway (LES), or bilateral lesions along with intrahippocampal septal grafts (SG). After 8 months, rats were tested for their ability to perform spatial reference, spatial navigation and working memory tasks. Upon completion of the behavioral testing, neurochemical activity of the hippocampus was measured by HACU. The results indicate that animals in the SG group had significantly higher behavioral scores and hippocampal HACU rates than animals in the LES group. Regression analysis indicates that a significant correlation exists between performance on each behavioral task and HACU rates. These results demonstrate that hippocampal cholinergic activity, as measured by HACU, correlates significantly with performance on tests of spatial memory function.     

Glycine receptor distribution in mouse CNS: autoradiographic localization of [H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [³H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Evaluation of [3H]paroxetine as an in vivo ligand for serotonin uptake sites: A quantitative autoradiographic study in the rat brain

Paroxetine, a selective inhibitor of serotonin uptake and an antidepressant, was used in conjunction with quantitative ex vivo autoradiography to study the feasibility of imaging serotonin terminals in the living brain. Tritiated paroxetine was injected in the rat tail vein, and the brain was processed for quantitative autoradiography 3 hours later. Animals received either [³H]paroxetine alone (100 μCi/animal) or a mixture of labeled paroxetine (100 μCi) and an excess of unlabeled drug (0.5 or 2 mg/kg intravenously [i.v.]). Computerized image analysis of the resulting autoradiograms revealed high densities of radioactivity in brain regions known to contain high densities of serotonergic terminals and high specific binding of [³H]paroxetine in vitro, such as the raphe nuclei, interpeduncular nucleus, basolateral amygdala, substantia nigra, and some hypothalamic nuclei. Radioactivity uptake in these brain regions was effectively blocked (50-72%) by coadministration of excess unlabeled paroxetine. However, cortical and hippocampal binding of paroxetine in vivo was moderately high, in contrast to the relatively sparse serotonergic innervation in these regions. Only a relatively small proportion of cortical and hippocampal binding (20-40%) could be blocked by excess unlabeled paroxetine, indicating that most of the radioactivity in these regions is not associated with serotonin terminals or uptake sites. The usefulness of [³H]paroxetine as an in vivo ligand for imaging serotonin terminals in the human brain is limited by these nonserotonergic binding sites. © 1993 Wiley-Liss, Inc.     

Distribution of catecholamine uptake sites in human brain as determined by quantitative [3H] mazindol autoradiography

Because of the importance of the catecholamine system in Parkinson's disease and its relevance to a variety of clinical movement disorders, catecholamine uptake sites were mapped in the human brain using [3H] mazindol autoradiography. Displacement studies with known dopamine (DA) and noradrenaline (NA) uptake blockers showed that binding in the striatum was to dopamine uptake sites; binding in the locus coeruleus was to noradrenergic uptake sites. By using the selective noradrenergic uptake blocker desmethylimipramine (DMI), a comprehensive map of both DA and NA uptake sites was generated. In general, catecholamine uptake sites were better seen in terminals than in cells of origin or axonal projections. In some areas, such as the locus coeruleus, punctate binding could be seen over individual pigmented cells. A variegated pattern of binding was seen in caudate nucleus and putamen and some correspondence of patches of low binding with striosomes was observed in the caudate. The highest levels of binding to DA uptake sites was observed in the striatum, where regional differences in binding occurred. The most dense binding was seen in the ventral striatum, and a rostral-to-caudal decrement in binding levels in caudate nucleus and putamen was evident. Binding was more intense in the putamen compared to the caudate and within the caudate lower values were seen laterally. The highest levels of binding to noradrenergic uptake sites were in the locus coeruleus and dorsal raphé, although these sites may be on terminals from other projections. Whereas uptake sites were more often evident in known catecholamine pathways, [3H] mazindol binding was seen in some areas where catecholamine neurons or terminals had not been identified previously. These maps of the catecholamine uptake system add further information concerning the nature of the distribution of catecholamines in human brain and provide an important baseline for the study of disease and ageing processes.