Hemicholinium-3 Binding Research Articles

Deficits in development of central cholinergic pathways caused by fetal nicotine exposure: Differential effects on choline acetyltransferase activity and [ 3H]hemicholinium-3 binding

Nicotine has been hypothesized to induce neurobehavioral teratology by mimicking prematurely the natural developmental signals ordinarily communicated by the ontogeny of cholinergic synaptic transmission. In the current study, the effects of fetal nicotine exposure (2 mg/kg/day or 6 mg/kg/day) on development of central cholinergic pathways were examined in striatum and hippocampus of animals exposed from gestational days 4 through 20, using maternal infusions with osmotic minipumps. Brain region weights and choline acetyltransferase activity, an enzymatic marker for development of cholinergic nerve terminals, were within normal limits in the nicotine-exposed animals. However, development of [ 3H]hemicholinium-3 binding which labels the presynaptic high affinity cholinergic transporter, was deficient in both striatum and hippocampus. Abnormalities occurred during two distinct phases; in the early neonatal period, when [ 3H]hemicholinium-3 binding sites are transiently overexpressed, and during or after the period of rapid synaptogenesis, when binding in controls is rising consequent to the increase in nerve impulse activity. These data thus indicate that fetal nicotine exposure, even at doses that do not cause overt signs of maternal/fetal/neonatal toxicity or growth impairment, influences both specific gene expression of cholinergic nerve terminal markers, as well as indices of neuronal function. Comparison of regional selectivity at the two dose levels indicated greater sensitivity of the striatum, a region with a prenatal peak of neuronal mitosis, as compared to hippocampus, where mitosis peaks postnatally; the regional differences are consistent with vulnerability to nicotine during a critical phase of cell development.

Actions of ginsenoside Rb 1 on choline uptake in central cholinergic nerve endings

The ginsenoside Rb 1 has previously been reported to improve memory deficits induced by anticholinergic drug treatment, and to facilitate acetylcholine (ACh) release from rat brain hippocampal slices. The increase in ACh release was not associated with an increase in calcium uptake into nerve terminals, but was associated with an increase in uptake of the precursor choline. In the present studies, analysis of choline uptake kinetics indicated that Rb 1 increased the maximum velocity of choline uptake, while the affinity of the choline uptake carrier for choline ( K m ) was not significantly altered. Acute treatment with Rb 1 did not alter the number of [ 3H]hemicholinium-3 (HC-3) binding sites in any of three cholinergic brain regions examined, suggesting that the increase in the maximum velocity of choline uptake was not associated with an increase in the number of choline carriers. However, chronic (3 day) administration of Rb 1 did increase the number of choline uptake sites in the hippocampus, and to a lesser extent in the cortex.

High‐Affinity Choline Transport Regulation by Drug Administration During Postnatal Development

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.

Quantitative autoradiography of hemicholinium-3 binding sites in human amygdala

We report the binding properties and subnuclear localization of [ 3H]hemicholinium-3 binding sites in human amygdala using quantitative autoradiography. Specific binding was saturable and high affinity (apparent K d 2–11 nM). Binding was highest in the basolateral nucleus which receives dense cholinergic innervation from the basal forebrain. Binding closely approximated acetylcholinesterase reactivity. These data support [ 3H]hemicholinium-3 as a quantitative marker for cholinergic terminals in human brain.

Cholinergic markers in ALS spinal cord

We analyzed binding sites for quinuclidinyl benzilate (QNB) and hemicholinium-3 (HC-3) by quantitative slice autoradiography and the activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in spinal cord of 5–7 patients with amyotrophic lateral sclerosis (ALS). In the ventral horn, QNB binding sites were markedly reduced (38% of controls; P <0.001), whereas HC-3 binding sites were only moderately affected (76%, P <0.01). Losses in cholinergic marker enzymes were inconsistent. The loss of muscarinic binding sites in the ventral horn was the most reliable cholinergic disease marker in ALS.

Regulation of hemicholinium binding sites in isolated nerve terminals.

High-affinity uptake of choline, the rate-limiting, regulatory step for the synthesis of acetylcholine is regulated via presynaptic auto- and heteroreceptors. Binding studies using tritiated hemicholinium-3 ([3H]HCh-3) as the specific ligand for the choline carrier revealed that the number of hemicholinium binding sites in nerve terminals isolated from insect brain changes corresponding to the activity of synaptosomal kinase A and kinase C. Activation of kinase A apparently increases the total number of hemicholinium binding sites by recruiting additional occult carriers, whereas the effect of kinase C activity is most appropriately explained by preventing a down-regulation of carrier proteins. The kinase-mediated regulation of choline transporters is obviously due to a phosphorylation of the carrier protein itself.

Autoradiographic analysis of muscarinic receptors and choline-uptake mechanisms within foetal basal forebrain transplants

The pharmacological characteristics of both muscarinic receptors and high-affinity choline uptake sites were examined within intracerebral implants of foetal basal forebrain cell suspensions. Approximately 12 weeks after implantation, the transplants were identified by acetylcholinesterase histochemistry. Muscarinic receptors were labelled by [3H]quinuclidinyl benzylate (QNB) autoradiography. The M1 and M2 receptor components of QNB binding were differentiated by pirenzepine competition. The distribution of the high-affinity choline uptake site was examined using [3H]hemicholinium-3 (HC3) autoradiography. Unilateral lesion of the nucleus basalis reduced [3H]QNB (8-25%) and [3H]HC3 (19-43%) binding throughout host frontoparietal cortex ipsilateral to the lesion but did not significantly alter these cholinergic markers within cingulate cortex, subcortical white matter, striatum or septum. Saturation analysis of the implanted tissue revealed the presence of a single population of [3H]QNB and [3H]HC3 binding sites with affinities similar to those of the host tissue (KD = 0.43 nM and 20.4 nM respectively). However, the receptor profile which developed appeared to be intrinsic to the implant; it was unaffected by the site of implantation and was dissimilar to that which ultimately developed in the donor tissue when left in situ.

Decreased [ 3H]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

Effect of Unilateral Perinatal Hypoxic‐Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High‐Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study

The binding characteristics and distribution of M1 and M2 muscarinic cholinergic receptors and high-affinity choline uptake sites were studied in the striatum of the rat at 3-4 and 9-12 weeks of age after exposure to unilateral perinatal hypoxic-ischemic brain injury. High-affinity choline uptake sites were labeled with [3H]hemicholinium-3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AF-DX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine-labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium-3-labeled high-affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF-DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium-3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that striatal cholinergic systems are resistant to hypoxic-ischemic injury.

Further studies on the biochemical characterization and autoradiographic distribution of [ 3H]hemicholinium-3 binding sites in rat brain: a presynaptic cholinergic marker

Hemicholinium-3 (HC-3) is a potent inhibitor of the high-affinity choline uptake system (NACU). Here we report on the biochemical characterization and autoradiographic distribution of [3H]hemicholinium-3 binding sites in rat brain, confirming and expanding results from previous studies. The binding of [ 3H]HC-3 to striatal membranes was specific, to a single site, sodium-dependent, saturable, and of high-affinity, K d values being about 3 n m for striatum, 5 n m for the hippocampus and 12 n m for neocortex. [ 3H]HC-3 specific binding exhibited a pharmacological profile suggestive of physiologically relevant interactions and fully comparable to that reported for HACU. The uneven distribution of [ 3H]HC-3 binding sites exhibited a high degree of correspondence with the reported distribution of HACU and other enzymatic presynaptic cholinergic markers. The punctual differences between our study and previous works on [ 3H]HC-3 binding are analysed. We conclude that [3H]HC-3 labelling may be used as a selective and quantifiable marker of the cholinergic presynaptic terminals in close relationship with HACU.

Differential response of striatal dopamine and muscarinic cholinergic receptor subtypes to the loss of dopamine: I. Effects of intranigral or intracerebroventricular 6-hydroxydopamine lesions of the mesostriatal dopamine system

Quantitative autoradiography was utilized to examine the response of the dopamine (DA) and muscarinic cholinergic system within the striatum to lesions of the mesostriatal DA system following intranigral 6-hydroxydopamine (6-OHDA) injections. In addition, the response of DA system was examined in the striatum of animals treated with low, medium, or high doses of 6-OHDA made intracerebroventricularly (icv). Three weeks following removal of the mesostriatal DA fibers with intranigral 6-OHDA, there was an almost complete depletion of DA and [ 3H]mazindol binding throughout the striatum. The resulting increase in D2 receptors labeled with [ 3H]spiroperidol (27%) was most evident in the lateral striatum and topographically correlated with an increase in choline uptake sites labeled with [ 3H]hemicholinium-3 (20%). There was a smaller but significant decrease in D1 receptors labeled with [ 3H]SCH 23390 (15–18%) that was not topographically related to changes in [ 3H]spiroperidol or [ 3H]hemicholinium-3 binding. All doses of icv 6-OHDA produced a significant loss of DA and of [ 3H]mazindol binding as compared to vehicle injections that was more pronounced in the medial than in the lateral striatum. No increase in D1 receptors was observed with any dose of 6-OHDA and greater than 90% loss of DA and [ 3H]mazindol resulted in an increase in D2 receptors in the lateral striatum and a reduction in D1 receptors in the dorsal striatum. These data are consistent with the evidence that there is independent regulation of the two subtypes of the DA receptor. Moreover, the distribution and regulation of the subtypes of the muscarinic receptor were independent. Muscarinic M2 receptors ([ 3H]- N-methylscopolamine in presence of excess pirenzepine) showed a lateral to medial gradient (highest laterally) that was related to the pattern of choline uptake sites and D2 receptors. Loss of DA resulted in a reduction in M2 receptors (24–30%) that was correlated with the increase in choline uptake sites. In contrast, M1 ([ 3H]pirenzepine) receptors showed a reverse gradient from the M2 receptor and a smaller reduction following loss of DA.

Ontogeny of dopamine D 1 and D 2 receptor subtypes in rat basal ganglia: a quantitative autoradiographic study

The ontogeny of D 1 and D 2 dopamine (DA) receptors in rat basal ganglia was examined by quantitative autoradiography using the iodinated ligands [ 125I]SCH 23982 and [ 125I]iodobenzamide ([ 125I]IBZM), respectively. Temporal and spatial differences in the development of the receptor subtypes were observed. Scatchard transformation of saturation isotherms conducted at postnatal day 10 (P10) and P60, showed that there was no age-related change in the affinity of [ 125I]SCH 23982 binding to D 1 receptors ( K d = 2.6 nM) but there was a significant increase in the B max (771 compared to 2032 fmol/mg protein, P = 0.002). A statistically significant difference in K d was noted between ages P10 and P60 for [ 125I]IBZM labelling of D 2 receptors (0.62 vs 1.00 nM, respectively, P < 0.01). A significant increase in the B max (211 and 721 fmol/mg protein, P < 0.01) was also observed. D 1 receptors were visible as distinct patches at P1. The highest density was found in the ventrolateral caudate-putamen (CPu). By P5 the patches were found in all subregions of the CPu and nucleus accumbens. Between P7 and P10 the binding became distinctly less patchy due to a marked increase in the density of D 1 receptors in non-patch (matrix) regions. Adult levels of receptor were seen by P30. The concentration of DA (measured by HPLC) and binding of [ 3H]mazindol to DA uptake sites in whole striatum showed similar and nonlinear increases with age. The age-related change in the topography of binding sites for [ 3H]mazindol was similar to that of D 1 receptors at the same ages. Both D 2 receptors and [ 3H]hemicholinium-3 (HC-3) binding to high affinity transport sites for choline developed initially in the dorso-lateral CPu. Their topography was largely overlapping but distinct from that of the D 1 receptor. D 2 receptors were not consistently observed until P3 in the CPu, and zones of enriched binding were aligned with zones of low density for D 1 receptors. The density of D 2 receptors reached adult levels by P30. The differential development of the DA receptors was also evident in the substantia nigra (SN) and globus pallidus (GP). D 1 receptors were found in SN prior to the appearance of D 2 receptors and throughout development the density was greater in pars reticulata than in pars compacta, whereas the density of D 2 receptors was higher in the pars compacta. At all ages the density of D 1 receptors was greater than the density of D 2 receptors in the GP and reached adult levels before reaching it in the CPu or SN. These findings suggest that D 1 and D 2 receptors develop independently of each other in the basal ganglia. Moreover, their ontogeny is related to different phases of the development of the patch and matrix compartments: (1) an increase in D 1 receptors in the patch compartment related to an increase in the density of [ 3H]mazindol sites, (2) an increase in D 2 receptors in the matrix related to maturation of [ 3H]HC-3 binding, (3) a later phase in which an increase in D 1 receptors in the matrix correlated with an increase in the density of [ 3H]mazindol sites in that compartment.

High-affinity choline uptake carrier in Alzheimer's disease: implications for the cholinergic hypothesis of dementia

We examined the density and the state of affinity of [ 3H]hemicholinium-3 ([ 3H]HC-3) binding sites, a marker of the presynaptic high-affinity choline uptake (HACU) carrier, in 4 representative regions of 13 postmortem Alzheimer's disease (AD) brains, as well as in 12 matched control brains. Significant reductions in the densities of [ 3H]HC-3 binding sites were found both in frontal cortex (−44%) and hippocampus (−36.5%) of AD brains in comparison to controls. On the other hand the densities of [ 3H]HC-3 binding sites in AD brains in caudate-putamen and cerebellar cortex showed no significant differences when compared to controls. No significant change in the state of affinity of these sites could be observed in the saturation assays carried out in hippocampus and frontal cortex. Our findings concur with the reported data by using other presynaptic cholinergic markers in AD and confirm that some degree of cholinergic degeneration, highly specific for the basal forebrain neurons, occurs in AD. However, these results, obtained in a group of AD brains belonging to severely demented patients, do not show a dramatic loss of the HACU in many AD brains. Although this fact could be due to the existence of a compensatory mechanism, our results probably suggest that dementia in AD cannot be explained only by the loss of neocortical cholinergic presynaptic terminals arising from the basal forebrain and also may clarify as to why the acetylcholine precursors or the muscarinic agonists are not effective in AD dementia.

3H]Hemicholinium-3 binding in rats with status epilepticus induced by lithium chloride and pilocarpine

The specific binding of [ 3H]hemicholinium-3 ([ 3H]HCh-3) and high-affinity [ 3H]choline uptake were measured in rats with status epilepticus induced by lithium and pilocarpine. The specific binding of [ 3H]HCh-3 in cortex and hippocampus from rats with status epilepticus increased to 2- to 3-fold of control while the striatal [ 3H]HCh-3 binding increased minimally. Scatchard analyses revealed that the observed changes resulted from an increase in B max of [ 3H]HCh-3 binding. High-affinity [ 3H]choline uptake remained unchanged. These results further implicate phospholipase A 2 in the regulation of [ 3H]HCh-3 binding sites.

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), M 1 receptors were directly labeled with [ 3H]pirenzepine (PZ) and non-M 1 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 M 1 (−26%) and non-M 1 (−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.

Development of D1 and D2 dopamine receptors and associated second messenger systems in fetal striatal transplants

Stereotactic implantation of fetal brain regional anlage into adult host brain (“brain transplantation”) appears to be an increasingly viable strategy for therapy of neurodegenerative diseases. We have studied implantation of fetal striatum into adult striatum, previously lesioned by neurotoxic amino acid injection, as a model for transplantation therapy of Huntington's disease. The beginning of behavioral recovery to apomorphine is not apparent until 6.5 months after implantation. By 4 months after implantation cerebral blood flow through the implants appears equal to that in the intact contralateral striatum. At this time, cerebral glucose utilization is reduced in the implants but increases following apomorphine treatment. The development of D1 and D2 dopamine (DA) receptors is markedly deficient in the striatal grafts at both 4 and 6.5 months after implantation. Very little D2 radioligand binding was observed in the grafts at either time point; D1 receptors appeared in a patchy fashion by 6.5 months at densities approaching normal striatum. In situ hybridization of D2 dopamine receptor mRNA demonstrated robust hybridization signal in normal striatum and accumbens but no signal in 6.5-month-old striatal grafts. Adenylate cyclase (AC) activity, examined with high-affinity [ 3H]forskolin binding, also appeared in patches similar to D1 receptors at 6.5 months. In contrast, protein kinase C activity, labeled with [ 3H]phorbol ester, was very apparent in the grafts at both time points. Higher and generally homogenous densities of muscarinic cholinergic receptors, assessed with [ 3H]QNB binding, develop in the grafts, but there appear to be few functioning cholinergic terminals, as measured by [ 3H]hemicholinium binding. Our data demonstrate that very few D1 or D2 DA receptors develop in fetal striatal neurons removed from E15–16 embryos and subsequently implanted into adult ibotenic acid-lesioned striata. The lack of D2 receptor development appears to derive from lack of transcription of the D2 receptor gene. By 6.5 months, D1 receptor sites and AC activity both appear in discrete patches, consistent with the known in vitro association of D1 receptors with stimulation of AC activity. In this model, the majority of graft protein kinase C activity, to the extent that it is labeled with [ 3H]phorbol ester, is not associated with DA receptors. DA receptor development in fetal striatal implants may require coimplantation of DA neurons.

Decrease in [ 3H]hemicholinium binding to high-affinity choline uptake sites in deafferented striatum: restoration by oxiracetam

Frontal cortical deafferentation of the rat striatum reduces the tone of striatal cholinergic neurons. We used biochemical and autoradiographic techniques to investigate whether the [ 3H]hemicholonium-3 ([ 3H]HCh-3) binding to sodium-dependent high-affinity choline uptake sites was influenced by this lesion. Frontal deafferentation produced a reduction of about 30% in the number of [ 3H]HCh-3 binding sites ( B rmmax) in striatum, with no significant changes in the binding affinity ( K d). Autoradiography showed a significant reduction of [ 3H]HCh-3 binding sites in the anteromedial portion of the striatum, but not in the posterior part of frontal deaffetented rats. Oxiracetam (100 mg/kg), a nootropic drug, did not affect the distribution of [ 3H]HCh-3 binding sites in sham-operated rats but completely overcame the reduction in the number of [ 3H]HCh-3 binding sites in deafferented striatum.

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M 1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [ 3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M 1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [ 3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (B max) in non-M 1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M 1 subtype.

Intrastriatal grafts derived from fetal striatal primordia: II. Reconstitution of cholinergic and dopaminergic systems

Reconstitution of striatal cholinergic and dopaminergic systems was studied in intrastriatal grafts derived from embryonic day 15 rat striatal primordia and implanted into adult host rats in which unilateral ibotenic acid lesions had previously been made in the striatum. Histochemical, immunohistochemical, and ligand binding autoradiographic techniques were applied to analyze different constituents of these two systems and to study their locations relative to each other in grafts allowed to grow for 9-17 months following transplantation. For the cholinergic system, a modular organization was found in the striatal grafts with stains for choline acetyltransferase and acetylcholinesterase, respectively the synthetic and degradative enzymes for cholinergic neurons; by autoradiographic [3H]hemicholinium binding, specific for high affinity choline uptake sites associated with cholinergic terminals; and by autoradiographic [3H]pirenzepine binding, selective for M1 receptors. For the dopaminergic system, a comparable modular organization was found in the grafts by immunostaining for tyrosine hydroxylase, the catecholamine synthetic enzyme; by autoradiographic [3H]mazindol binding for dopamine uptake sites; and by [3H]SCH23390 binding for dopamine D1 receptors and [3H]sulpiride binding for dopamine D2 receptors. The results indicate that the distributions of the cholinergic and dopaminergic markers in striatal grafts are in close anatomical register. These markers for intracellular and membrane-associated components of the cholinergic and dopaminergic systems were preferentially localized in the acetylcholesterase-rich patches of the grafts in which cortical and thalamic fibers have also been found in striatal grafts, and in which output neurons projecting to the pallidum are located. This anatomical correlation suggests that the substrates for cholinergic-dopaminergic interactions typical of the normal striatum may be reinstated in the grafts both in relation to efferent neurons establishing connections with the host brain that are typical of normal striatofugal connections, and in relation to major afferent fiber systems from the host brain originating in regions known to project densely to the normal striatum. Accordingly, the cholinergic and dopaminergic systems in such grafts may regulate the functional influence of the grafts on the behavior of host animals.

Characterization of [3H]Hemicholinium‐3 Binding Sites in Human Brain Membranes: A Marker for Presynaptic Cholinergic Nerve Terminals

We report here on the binding properties of [3H]hemicholinium-3, a selective inhibitor of the high-affinity choline uptake process, to human brain membranes. Under the assay conditions described, the binding of [3H]hemicholinium-3 exhibited a dependency of physiological conditions on pH, temperature, and NaCl concentrations. Striatal binding proved to be specific, to a single site, saturable, and reversible, with an apparent KD of 10 nM and a Bmax of 82 fmol/mg of protein. [3H]Hemicholinium-3 specific binding exhibited a pharmacological profile and an ionic dependency suggestive of physiologically relevant interactions and comparable with those reported for the high-affinity choline uptake. Moreover, specific [3H]hemicholinium-3 binding exhibited an uneven regional distribution: striatum much greater than nucleus basalis greater than spinal cord much greater than midbrain = cerebellum greater than or equal to hippocampus greater than neocortex = anterior thalamus greater than posterior thalamus much much greater than white matter. This distribution closely corresponds to the reported activity of both enzymatic cholinergic presynaptic markers and high-affinity choline uptake in mammalian brain. There are no significant differences between these results and those previously found in the rat brain using this radioligand. Our results demonstrate, for the first time, the presence of [3H]hemicholinium-3 binding sites in human brain and strongly support the proposal that this radioligand binds to the carrier site mediating the high-affinity choline uptake process on cholinergic neurons. Thus, [3H]hemicholinium-3 binding may be used in postmortem human brain as a selective and quantifiable marker of the presynaptic cholinergic terminals.