Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo.

Previous reports indicate that alterations of activity of cholinergic neurons $\text{in vivo}$ are followed by parallel changes in sodium-dependent high affinity choline uptake $\text{in vitro}$. These results are consistent with the proposal that this portion of choline uptake is regulatory in the synthesis of ACh. These results also suggest the possibility of utilizing sodium-dependent high affinity choline uptake as a measure of the relative state of cholinergic activity $\text{in vivo}$. In this study, we administer a number of drugs reported to alter turnover and release of ACh (both are measures of cholinergic activity $\text{in vivo}$, and subsequently examine sodium-dependent high affinity choline uptake $\text{in vitro}$. Administration of pentobarbital, chloral hydrate, morphine, physostigmine, Δ⁹ THC, hemicholinium-3 and oxotremorine, drugs which decrease ACh turnover and release, caused a reduction in choline uptake. Conversely, administration of pentylenetetrazol, atropine, scopolamine, and haloperidol, drugs which increase ACh turnover and release, caused an increase in choline uptake $\text{in vitro}$. These findings support the proposal that sodium-dependent high affinity choline uptake can be used as a relative measure of the activity of cholinergic neurons $\text{in vivo}$.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

Exogenous Choline Enhances the Synthesis of Acetylcholine Only Under Conditions of Increased Cholinergic Neuronal Activity

Abstract: The effect of choline (60 mg/kg, i.p.) on fluphenazine- and pentylenetetrazol-induced alterations in the concentration of acetylcholine (ACh) and/or the rate of sodium-dependent high-affinity choline uptake (HACU) in rat striatum and hippocampus was studied. Systemic administration of the dopamine receptor blocking agent fluphenazine hydrochloride (0.5 mg/kg, i.p.) decreased the concentration of ACh in the striatum; this effect was prevented by the prior administration of choline. The central nervous system stimulant pentylenetetrazol (30 mg/kg, i.p.) reduced the concentration of ACh in both striatum and hippocampus and increased the velocity of HACU in the hippocampus. Pretreatment with choline totally prevented the depletion of ACh induced by pentylenetetrazol in the striatum. In the hippocampus, prior administration of choline prevented the pentylenetetrazol-induced increase in the rate of HACU and attenuated the effect of pentylenetetrazol on the levels of ACh. Results indicate that the acute administration of choline antagonizes pharmacologically induced alterations in cholinergic activity as assessed by the rate of HACU and the steady-state concentration of ACh. Furthermore, data support the hypothesis that the administration of choline increases the ability of central cholinergic neurons to synthesize ACh under conditions of increased neuronal activity.     

CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN CORPUS STRIATUM OF RESERPINISED RATS1

Rats treated with reserpine show increased V_max for the high affinity uptake of choline into small slices of corpus striatum. The choline acetyltransferase activity of whole homogenates of striatum is also increased. These changes are consistent with increased cholinergic neuronal activity in the striatum and seem likely to be adaptations mediating increased rates of synthesis of acetylcholine. The maximal increases found occurred concurrently, consistent with coupling of the high affinity uptake of choline and its acetylation in cholinergic nerve terminals of the rat. That increased high affinity uptake is accompanied by increased choline acetyltransferase activity, suggests the input of choline is not the sole determinant of rates of synthesis of acetylcholine, in spite of the large Vmas for striatal choline acetyltransferase, compared with that for high affinity uptake. These results seem best explained by kinetic coupling, in the rat, of the high affinity uptake of choline with a limited pool of choline acetyltransferase preferentially localised at the nerve terminal plasma membrane.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

The effects of various cognition-enhancing drugs on in vitro rat hippocampal synaptosomal sodium dependent high affinity choline uptake

The purpose of the present study was to compare the effect of seven drugs, that have been reported to enhance cognitive functions, on rat hippocampal cholinergic neuronal activity. The latter was assessed by measuring the effects of the drugs on in vitro sodium-dependent high affinity choline uptake (HACU) into rat hippocampal synaptosomes 30 minutes after their in vivo administration. 3,4-Diaminopyridine (0.1 mg/kg IP), like pramiracetam (44 and 88 mg/kg IP), increased HACU with higher or lower doses being ineffective. Centrophenoxine (100 mg/kg IP) decreased HACU. Piracetam (100 and 500 mg/kg IP), aniracetam (10-200 mg/kg PO), lysine vasopressin (0.005-0.05 mg/kg IM) and 4-aminopyridine (0.01-3.0 mg/kg IP) were ineffective. The results indicate that 3,4-diaminopyridine and centrophenoxine, like pramiracetam may be increasing cognitive function in part by affecting hippocampal cholinergic neuronal activity. In addition, the findings indicate the usefulness of using in vitro HACU as a biochemical measurement to assess the potential effect of cognitive-enhancing drugs on cholinergic neuronal activity in vivo.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Oral Administration of Gintonin Attenuates Cholinergic Impairments by Scopolamine,Amyloid-β Protein,and Mouse Model of Alzheimer’s Disease

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer’s disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced [Ca²⁺]_i transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated [Ca²⁺]_i transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 2 weeks) also significantly attenuated amyloid-β protein (Aβ)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to Aβ and could be utilized for AD prevention or therapy.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

High-Affinity Choline Transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na⁺- and Cl^–-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.     

Treatment with Oxiracetam or Choline Restores Cholinergic Biochemical and Pharmacological Activities in Striata of Decorticated Rats

Interruption of the corticostriatal pathway by undercutting the frontal cortex resulted after 2 weeks in a 40% reduction of basal acetylcholine (ACh) release in vivo, and in inhibition of the striatal sodium-dependent high-affinity uptake of choline (SDHACU) to the same extent. The lesion, too, completely prevented the rise (about 35%) in striatal ACh content induced by oxotremorine and apomorphine acting at muscarine and dopamine receptors, respectively. Acute intraperitoneal injections of 100 mg/kg of either oxiracetam or choline chloride resulted in time-dependent recovery of ACh output from the striata of decorticated rats to control levels. Oxiracetam also normalized the ex vivo striatal SDHACU activity of decorticated rats 2 h after administration without any effect in sham-operated rats. Oxiracetam or choline chloride administered before oxotremorine (0.8 mg/kg, i.p.) or apomorphine (1 mg/kg, i.p.) reinstated the ACh-increasing effect of these agonists. It is suggested that choline chloride acts directly simply by being the precursor for ACh, whereas oxiracetam may act indirectly, possibly by increasing the availability of choline chloride for ACh synthesis. Furthermore, the frontally decorticated rat could constitute a useful model for studying means to restore the deficit in striatal cholinergic neurotransmission.     

Postnatal Development of the Acetylcholine System in Different Parts of the Rat Cerebellum

Abstract: The components of the cholinergic nervous system, i.e., choline acetyltransferase, acetylcholinesterase, sodium-dependent high-affinity choline uptake, acetylcholine, and the muscarinic acetylcholine receptors, in the developing archi- and paleocerebellum of the rat have been investigated by biochemical methods. A close correlation between the development of the different elements of the system has been demonstrated in the two areas. The cholinergic structure develops first in the archicerebellum, which displays high levels of choline acetyltransferase, acetylcholinesterase, acetylcholine, and sodium-dependent high-affinity choline uptake. The paleocerebellum receives a sparser cholinergic innervation during development. The differences in the values for these components in the cerebellum as a whole may reflect the development of cholinergic and noncholinergic neuronal structures. It is concluded that the development of the cholinergic system cannot be analyzed in the cerebellum as a whole; rather specific regions such as the archi-, paleo-, or neocerebellum must be examined.     

Dopaminergic modulation of the septo-hippocampal cholinergic system activity under stress

The effects of the dopaminergic agonist apomorphine or the antagonist sulpiride on high affinity choline uptake and newly synthesized acetylcholine release by hippocampal synaptosomal preparations, were examined in rats subjected to immobilization stress. Increased dopamine uptake by septal synaptosomal preparations was taken as evidence for increased mesoseptal dopaminergic activity in response to stress. While apomorphine treatment failed to alter choline uptake or acetylcholine release in unhandled rats, it did however prevent the stress-induced increase in these cholinergic parameters. In contrast, after treatment with sulpiride both choline uptake and acetylcholine release were increased in unhandled rats, as they were after acute stress. Acute stress of sulpiride treated rats however resulted in changes similar to those produced by administration of either sulpiride or stress separately. We conclude that the mesoseptal dopaminergic system plays an important role in modulating the activity of the septo-hippocampal cholinergic system under stress.     

Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108-15

The neuroblastoma X glioma hybrid clone NG108-15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108-15 cells. The uptake system appears to bear little if any resemblance to the Na⁺-dependent high-affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high- and low- affinity choline uptake systems, neither system is dependent on Na⁺ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na⁺, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca²⁺ or Mg²⁺ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium-3, but only at high concentrations of the drug (IC₅₀= 30–80 μm ). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30-40%. Growth of the cells in N⁶,O^2′ dibutyryladenosine-3′,5′-cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108-15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high-affinity choline uptake system normally associated with cholinergic neurons.     

Increase in rat striatal acetylcholine content by bromocriptine: Evidence for an indirect dopaminergic action

Bromocriptine, at the optimal dose and time of 4 mg/kg, 90 min, increased the content of acetylcholine in the rat striatum by about 30% without affecting the acetylcholine content in other brain regions. Striatal choline acetyltransferase and acetylcholinesterase activities and sodium-dependent high affinity choline uptake were not affected by the $\text{in vivo}$ administration or the $\text{in vitro}$ incubation with even high amounts of the drug. The increase in striatal acetylcholine by bromocriptine was mediated through the dopaminergic system since pretreatment with pimozide or penfluridol, powerful dopamine receptor antagonists, completely prevented the effect while parachlorophenylaline and phenoxybenzene pretreatment were ineffective. The action of bromocriptine, differently from that of apomorphine, was also blocked upon inhibition of tyrosine hydroxylase by alphamethylparatyrosine, suggesting that intact catecholamine synthesis is necessary for the drug to act. The requirement of dopamine by bromocriptine was further indicated when no potentiation of the cholinergic response to bromocriptine occurred following induction of dopamine receptor supersensitivity by long-term 6-hydroxydopamine lesion of the nigroneostriatal pathway. On the other hand, evidence is presented to show that bromocriptine acts in synergism with dopamine as the latency period for the onset of bromocriptine's cholinergic action was significantly decreased when it was administered in combination with a subthreshold dose of L-dopa, the dopamine precursor. There also was no summation of bromocriptine's increase with apomorphine's increase in striatal acetylcholine content at supramaximal doses possibly indicating that the same population of intrastriatal cholinergic neurons is the common target of both drugs.It is proposed that bromocriptine exerts an inhibitory effect on the striatal cholinergic neurons through a stimulation of the dopaminergic system but, differently from apomorphine, it requires the presence of endogenous dopamine for its action.     

beta-Endorphin administration increases hippocampal acetylcholine levels.

The contents of acetylcholine and choline were determined in rat cortex, striatum, and hippocampus following intraventricular injection of β-endorphin or D-Ala²-enkephalinamide, a synthetic enkephalin analog, in doses known to produce analgesia in experimental animals. These opiate polypeptides produced significant increases in acetylcholine levels in the hippocampus, a subcortical structure rich in cholinergic terminals. The acetylcholine content of the hippocampus (but not the cortex or striatum) was significantly elevated 15, 30, and 60 minutes after a single intraventricular injection of β-endorphin (10 μg/brain) or D-Ala²-enkephalinamide (10 μg/brain). Peak alterations in regional acetylcholine concentrations and in analgetic effectiveness both occurred 30 minutes after peptide administration. Choline concentrations were unchanged by any of the experimental treatments. Naloxone hydrochloride (1 mg/kg, subcutaneously) affected neither brain acetylcholine concentrations, nor the response latencies of rats placed on a hot-plate; it did, however, antagonize the changes in these parameters caused by β-endorphin or D-Ala²-enkephalinamide. These data suggest that endorphins may normally regulate the physiologic activity of some cholinergic neurons.     

Time course of decline of radiolabeled acetylcholine formed following intracerebroventricular administration of tritiated choline: Effects of oxotremorine and scopolamine

Rats were injected intracerebroventricularly with 5 Ci of [methyl-³H]choline. The time course of decline of the rediolabeled acetylcholine (ACh) formed was estimated in the ispilateral cerebral cortex and striatum. The [³H]ACh levels declined biphasically from the cerebral tissue. The initial decline proceeded rapidly, after which labeled ACh declined more slowly. Scopolamine (1 mg/kg, i.v.) caused a significant increase in the rat of [³H]ACh disappearance, which can be interpreted as an enhancement of ACh release. By contrast, oxotremorine (0.8 mg/kg, i.v.) markedly reduced the [³H]ACh disappearance. The results show that drug-induced changes in cholinergic neuronal activities can be estimated from the disappearance of radioactive ACh after labeling the endogenous transmitter through intracerebroventricular administration of labeled choline.