Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis

We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10- to 12-fold increase in the release of endogenous ACh above spontaneous release. Potassium-evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 +/- 13.0% decrease in spontaneous ACh release and a 60.4 +/- 8.2% decrease in potassium-evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4-diaminopyridine (3,4-DAP) whereas 10 mM 3,4-DAP caused a sevenfold increase. The increase in ACh release caused by 3,4-DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high-affinity choline uptake and this could be inhibited by hemicholinium-3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium-dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.     

Choline Acetyltransferase Activity in the Rat Brain Cortex Homogenate, Synaptosomes, and Capillaries After Lesioning the Nucleus Basalis Magnocellularis

Stereotaxic lesions of the nucleus basalis magnocellularis were made unilaterally in male Wistar rats with either kainic or ibotenic acid, using the contralateral side as control. Differences in behavior, body weight, and survival were observed between the kainic and ibotenic acid-treated rats. One week after surgery, the rats were sacrificed and the effect of the lesions on choline acetyltransferase activity was measured in brain cortex homogenate, synaptosomes, and capillaries. In kainic acid-lesioned rats, choline acetyltransferase activity decreased in homogenate and synaptosomes of the ipsilateral side with respect to that of the contralateral side; but the ibotenic acid lesion, which also reduced the ipsilateral choline acetyltransferase activity in homogenate, showed a rather different effect on the enzymatic activity of the synaptosomes. There were also differences between the effect of kainic and ibotenic acid lesions on choline acetyltransferase activity in the capillaries of the ipsilateral side with respect to that of the contralateral one. However, capillary choline acetyltransferase activity of the treated rats was in both sides three times higher than that of unoperated rats.     

Ionic and Metabolic Requirements for High-Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction

Abstract: We have shown previously that in the chick ciliary nerve-iris muscle preparation Na⁺-dependent high-affinity choline uptake was confined to the nerve terminals. In this paper the sodium-dependent high-affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [³H]choline and its conversion to [³hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na⁺-dependent choline (Ch⁺) uptake was shown to be linear for 16 min and to reach an equilibrium before Na⁺-independent Ch⁺ uptake, which continued to increase for 60 min. Elevated [K⁺]₀ concentrations inhibited Ch⁺ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K⁺]₀. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K⁺]₀. Reduction of SDHACU and sodium-dependent ACh synthesis by depolarization with high [K⁺]₀ or by inhibition of Na, K-ATPase implies that the electrochemical gradients for Ch⁺ and Na⁺ are important in providing a driving force for high-affinity Ch⁺ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K-ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch⁺ uptake and ACh synthesis, in some cases Ch⁺ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K⁺]₀ and ouabain. Na⁺-dependent Ch⁺ uptake and ACh synthesis were found to be temperature-dependent with a Q₁₀ (20–30°) of 3.6 and 6.6, respectively and a Q₁₀ (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch⁺ taken up which is converted to ACh. ACh did not reduce Ch⁺ transport when present at 100 μM.     

Effects of GABAergic Drugs In Vivo on High-Affinity Choline Uptake In Vitro in Mouse Hippocampal Synaptosomes

The effects of several gamma-aminobutyric acid (GABA)-ergic drugs on sodium-dependent high-affinity choline uptake (HACU) were investigated in the hippocampus. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by those drugs that enhance GABA transmission. The convulsant 3-mercaptopropionic acid, which decreases GABA levels, stimulated HACU. From these results and previous findings, it appears that GABA mediates a tonic inhibitory effect on the septal-hippocampal cholinergic system.     

In Vitro Effects of Arachidonic and L-Glutamic Acids on the High-Affinity Choline Transport in Rat Hippocampus

A second messenger role for arachidonic acid (AA) in the regulation of the high-affinity choline uptake (HACU) was suggested. It was repotted that micromolar concentrations of AA applied in vitro decreased the HACU values and increased the specific binding of [³H]hemicholinium-3 ([³H]HCh-3). It was published that L-glutamic acid (GA) applied in vivo produced a fall in the HACU values. In addition, GA liberates free AA. In this study, an ability of GA to influence in vitro the activity of presynaptic cholinergic nerve terminals via its effect on the release of AA is investigated in hippocampal synaptosomes of young Wistar rats. Millimolar concentrations of GA decrease both the high- and low-affinity choline uptake, the specific as well as nonspecific binding of [³H]HCh-3 and the activity of Na⁺,K⁺-ATPase. Kinetic analysis (Lineweaver-Burk and Scatchard plots) reveals a change in V_max and B_max, but not in K_M and K_D. It appears very likely that under normal conditions GA applied in vitro is not able to change markedly the choline transport via its effect on the release of AA. Results confirm the hypothesis about an indirect inhibitory role for glutamatergic receptors on cholinergic cells.     

High-Affinity Choline Uptake in the Hippocampus: Its Relationship to the Physiological State Produced by Administration of Barbiturates and Other Treatments

Abstract: Choline uptake in hippocampal synaptosomes was not inhibited by pentobarbital administration when rats were decapitated immediately upon loss of the righting reflex (3–4 min) even though it was inhibited at later times post-injection, when the rats were still unable to right themselves. Choline uptake was increased when the animals were decapitated at convulsion after an injection of picrotoxin, high doses of bicuculline, or one of the convulsant barbiturates. However, another convulsant barbiturate, as well as strychnine and lower doses of bicuculline, did not increase choline uptake even though the animals also convulsed. Thus loss of righting reflex or convulsion is not directly correlated with changes in choline uptake. At 7 min after injection, levels of pentobarbital in the hippocampus (and other brain regions) were correlated with the degree of inhibition of choline uptake up to about 50% inhibition; however, greater inhibition could not be achieved with much higher brain levels of the drug. Although hippocampal uptake was partially inhibited at 1 h after septal lesions, 3 h after the lesion the inhibition was no longer apparent. Inhibition was almost complete 10–12 days after the lesion. These results suggest that other factors in addition to impulse flow influence choline uptake.     

Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

High-Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.     

Effects of K+-Depolarization,Arachidonic Acid,Ethanol, and Aging on the High-Affinity Choline Transport in Rat Hippocampus

The Na⁺-dependent high-affinity choline uptake (HACU) transport and the [³H]hemicholinium-3 ([³H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3–6 months) and old (22 months) Wistar rats. In vitro effects of 100–300 M arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [³H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K⁺-depolarization effect on HACU and the decreased influence of AA on [³H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.     

Effect of Unilateral Decortication on Choline Acetyltransferase Activity in the Nucleus Basalis and Other Areas of the Rat Brain

Acetyl-coenzyme A: choline O-acetyltransferase (EC 2.3.1.6) (ChAT) enzyme activity was measured in the nucleus basalis and other microscopically identified brain areas at various times after unilateral cortical lesions were made in the rat. Initially, a significant decrease in ChAT activity was detected in the nucleus basalis ipsilateral to the lesion. However, after 120 days ChAT activity had apparently recovered, as levels of the enzyme at that time were not significantly different from control values. No changes in ChAT activity could be detected in any of the other brain areas similarly studied. The significance of these findings and their relationship to the morphological changes seen in neurones of the nucleus basalis after cortical lesions are discussed.     

Quinolinic Acid Stimulates Somatostatin Gene Expression in Cultured Rat Cortical Neurons

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphonovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p less than 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoethanolamine Enhances High-Affinity Choline Uptake and Acetylcholine Synthesis in Dissociated Cell Cultures of the Rat Septal Nucleus

Dissociated rat septal nucleus cells cultured in defined medium exhibited twofold increases in the maximal rates of sodium-dependent, high-affinity choline uptake and acetylcholine formation when grown in the presence of phosphoethanolamine. The effect was concentration-dependent (EC50 = 15 microM) and appeared to be associated with in vitro maturation of cholinergic neurons rather than with enhanced survival. Choline acetyltransferase, acetylcholinesterase, and choline kinase activities were unaffected by this treatment. The effect of phosphoethanolamine was specific for cholinergic neurons, because treatment with this compound did not alter the kinetic constants for high-affinity neuronal uptake of gamma-aminobutyric acid or dopamine. The action appeared to be mediated primarily through activation of the sodium-dependent, high-affinity transport mechanism for choline as opposed to alterations in the storage and release of acetylcholine.     

High-Affinity Uptake of Neurotransmitters in Rat Neostriatum: Effects of Aging

High-affinity uptake of dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) was determined in crude synaptosomal preparations from neostriatal regions of rats 7, 17, and 27 months of age. Dopamine uptake was highest in rostral neostriatum, but no age-related differences were detected. On the other hand, the high-affinity uptake of both GABA and glutamate was increased with age. This may reflect astrocytic hypertrophy or hyperplasia, which have been reported to occur in the neostriatum during the aging process.     

Neurochemical Characterization of an Antimony-Choline Analog in Rat Cortical Synaptosomes

Abstract: An analog of choline, in which nitrogen was replaced by antimony, was neurochemically characterized in rat cortical synaptosomes. It was found to be a substrate for several cholinergic enzymes, transported by a Na⁺-dependent, hemicholinium-3-sensitive process, acetylated, and subsequently released by depolarization in a calcium-dependent manner. Sb-choline also competed with choline for Na⁺-dependent uptake and for acetylation by [¹⁴C]acetyl-CoA in synaptosomes. These results suggest that Sb-choline and its acetylated product should be useful substrates for the x-ray microanalytical localization of cholinergic pools in intact nerve terminals.     

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Active uptake of 3,4-dihydroxyphenylethylamine (dopamine) is sodium- and temperature-dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+-dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low- and high-affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.     

Intra-Retrosplenial Cortical Grafts of Cholinergic Neurons: Functional Incorporation and Restoration of High Affinity Choline Uptake

Fetal septal neurons transplanted into the deafferented retrosplenial cortex (RSC) of rats have been shown to reinnervate the host brain and ameliorate spatial memory deficits. In the present study we examined the effects of implanting cholinergic neurons on high affinity choline uptake (HACU) in the denervated RSC and the correlational relationship between this cholinergic parameter and the level of behavioral recovery. Three groups of animals were used: 1) normal control rats (NC), 2) rats with lesions of the fornix and cingulate pathways (FX), and 3) lesioned rats with fetal septal grafts in the RSC (RSCsep-TPL). We found that intra-RSC septal grafts produced significant increases in HACU, and that recovery of HACU was significantly correlated with the improvements in the performance of spatial reference memory, spatial navigation, and spatial working memory tasks. We have also investigated the ability of the host brain to modulate the activity of the implanted neurons. In particular we evaluated the effect of the animals' performance in a 6-arm radial maze task on high affinity choline uptake (HACU). Animals in each of the NC, FX, and RSCsep-TPL groups were randomly assigned one of the following subgroups: 1) rats that performed the maze task before the determination of HACU (BEH), or 2) rats that did not perform the maze task before the determination of HACU (NON-BEH). Significant increases were observed in the NC and RSCsep-TPL groups, but not in the FX animals, indicating that fetal septal grafts in the RSC can become functionally incorporated with the host neural circuitry, and that the activity of the implanted cholinergic neurons can be modulated by the host brain.     

Changes in Choline Acetyltransferase Activity and High-Affinity Choline Uptake,but Not in Acetylcholinesterase Activity and Muscarinic Cholinergic Receptors,in Rat Somatosensory Cortex after Sciatic Nerve Injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [³H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.     

Cyclic AMP-Mediated Enhancement of High-Affinity Choline Transport and Acetylcholine Synthesis in Brain

Abstract: Intracerebroventricular administration of N⁶, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time-dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent V_max without a change of the K_m for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.