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.     

Further evidence for the involvement of D2, but not D1 dopamine receptors in dopaminergic control of striatal cholinergic transmission

The relative involvement of D₁ (cyclase linked) and D₂ dopamine receptors in dopaminergic control of striatal cholinergic transmission has been investigated in the rat by comparing the effects of SKF 38393 and LY 141865 (which act as specific agonists at D₁ and D₂ dopamine receptors, respectively) on striatal acetylcholine and dopamine metabolite concentrations and on the potassium-evoked release of ³H-acetylcholine from rat striatal slices. LY 141865 given systemically produced a dose-dependent increase in acetylcholine concentrations and a concomitant reduction of homovanillic and dihydroxyphenylacetic acid levels in the striatum (ED₅₀ 0.1 mg/kg) whereas SKF 38393 (1–30 mg/kg) did not. SKF 38393 (30 mg/kg) also failed to modify the LY 141865 (1 mg/kg) induced alterations of striatal acetylcholine and dopamine metabolite levels when given concomitantly with the latter compound. In experiments $\text{in vitro}$, LY 141865 reduced (EC₅₀ 0.14 μM), whereas SKF 38393 (up to 100 μM) failed to affect, the potassium-evoked release of ³H-acetylcholine from striatal slices. When given concomitantly with LY 141865, SKF 38393 (10 μM) did not modify the ability of the former compound to diminish striatal ³H-acetylcholine release. Finally, SKF 38393 also failed to affect the release of striatal ³H-acetylcholine after chemical lesion of the nigro-striatal dopaminergic pathway. The present results provide evidence for the involvement of D₂ but not D₁ dopamine receptors in dopaminergic control of striatal cholinergic transmission and indicate that D₁ dopamine receptors do not exert any modulatory influence on D₂ dopamine receptor mediated dopaminergic transmission.     

Effects of nootropic drugs on brain cholinergic and dopaminergic transmission

High affinity choline uptake (HACU) in the hippocampus and striatal concentration of dopamine (DA) and homovanillic acid (HVA) as measures of the in vivo acetylcholine and DA turnover, respectively, were estimated in male rats, Long-Evans, following 6-day administration of various nootropics in clinically relevant doses: piracetam and its derivatives pramiracetam and oxiracetam (100 mg/kg/day), pyritinol (50 mg/kg/day). Piracetam treatment was without effect on HACU, but induced significant increase of HVA in the striatum leaving striatal DA concentration unchanged. On the contrary, pyritinol, pramiracetam and oxiracetam increased HACU, but did not change striatal DA and HVA levels.     

Evidence for a peripheral dopaminergic mechanism in the antihypertensive action of lergotrile

Lergotrile (0.5 mg/kg, i.p.) lowered blood pressure significantly in spontaneously hypertensive rats. This effect was antagonized by pretreatment with haloperidol, pimozide, or domperidone. In normotensive rats, administration of haloperidol or domperidone rapidly increased serum prolactin levels. Haloperidol also increased striatal levels of dihydroxyphenylacetic acid and homovanillic acid; however, domperidone did not, which confirms that this latter blocker probably acts primarily as a $\text{peripheral}$ dopamine antagonist. Taken together, these data suggest that lergotrile lowers blood pressure in hypertensive rats mainly by stimulating $\text{peripheral}$ dopamine receptors.     

Effects of Fetal Methylazoxymethanol Acetate Lesion on the Synaptic Neurochemistry of the Adult Rat Striatum

Abstract We used the cytotoxic properties of methylazoxymethanol acetate (MAM), which ablates mitotically active neuroblasts, to eliminate neurons in the fetal striatum to define the factors that regulate the development of the synaptic circuitry of this region. Adult rats whose mothers received a single intraperitoneal injection of 20 mg/kg of MAM on gestational days (DG) 14-17 were used in this study. MAM treatment at 14 DG caused a 49% decrease in striatal mass whereas treatment at 17 DG reduced the striatal weight by only 16%; MAM treatment on 15 or 16 DG gave intermediate results. Histologic analysis of Nissl-stained sections did not reveal an obvious disruption of striatal organization, although the region was clearly hypoplastic. The hypoplasia was associated with significant increases in the specific activities of choline acetyltransferase and tyrosine hydroxylase, although total activities of these enzymes per striatum were significantly depressed with the 14 or 15 DG treatments. In contrast, the specific activity of glutamate decarboxylase was unaffected by MAM treatment whereas the total activity of this enzyme was reduced commensurate with the degree of striatal hypoplasia. In rats lesioned at 15 DG, there was a similar 30% increase in the specific activities of all presynaptic dopaminergic markers studied. In contrast, the specific activity of the synaptosomal uptake process for [³H]choline was elevated by 60%, the specific activity of choline acetyltransferase was increased by only 30%, and the concentration of acetylcholine in the striatum was unchanged. Whereas the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]γ-aminobutyric acid ([³H]GABA) were unaffected by the 15 DG MAM treatment, the concentration of GABA was increased significantly by 20%. The specific binding of [³H]spiroperidol, [³H]quinuclidinyl benzilate ([³H]QNB). and [³H] muscimol to, respectively, dopamine, muscarinic, and GABA receptors was unchanged by the 15 DG MAM lesion. The nigral dopaminergic perikarya appeared unaffected by the 15 DG MAM lesion in that the tyrosine hydroxylase activity remained normal. Consistent with the loss of striatal GABAergic perikarya, the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]GABA were significantly reduced in the substantia nigra; however, the concentration of endogenous GABA was twofold greater than in control in this terminal region. The results of these studies indicate that the nigro-striatal dopaminergic pathway only partially compensates for the loss of neurons in its terminal field within the hypoplastic striatum. Striatal cholinergic and GABAergic neurons differ considerably in their responses to the MAM lesion, suggesting that they are derived from different neuroblast pools. Finally, the altered synaptic relationships induced by the fetal lesion may affect neurotransmitter turnover as evidenced by disparities in GABA and acetylcholine levels when compared with other presynaptic markers for the GABAergic and cholinergic neurons.     

LSD and related drugs as DA antagonists: Receptor-mediated effects on the synthesis and turnover of DA

We have earlier shown that d-lysergic acid diethylamide, LSD and its 2-bromo derivative, BOL like the dopamine (DA) antagonists haloperidol increased the rate of the $\text{in vivo}$ tyrosine hydroxylation in the striatum measured as the accumulation of DOPA after decarboxylase inhibition.Now we have found that several agents structurally similar to LSD increase the $\text{in vivo}$ tyrosine hydroxylation in the striatum. Psilocybin (50 mg/kg i.p.) and N,N-dimethyltryptamine (50 mg/kg i.p.) caused a short-lasting increase of DOPA accumulation, while mescaline (10 – 100 mg/kg i.p.) did not increase the DOPA accumulation. A marked increase of DOPA accumulation was observed after the 5-hydroxytryptamine (5-HT) antagonist cyproheptadine. The effects of LSD and structurally related drugs on the DOPA accumulation in the striatum appear to be mediated via DA antagonism at receptor level. However, these agents may control the DOPA accumulation via other receptors than DA receptors e.g. 5-HT receptors. A control of DOPA accumulation via receptors other than DA receptors appears to be predominant after treatment with N,N-dimethyltryptamine or psilocybin.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Effects of LSD and 2-bromo LSD on striatal DOPAC levels.

Administration of d-lysergic acid diethylamide (LSD) and its analogue 2-bromo lysergic acid diethylamide (BOL) resulted in a shortlasting increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the rat striatum. BOL was more potent than LSD in the dose range 0.5–4.0 mg/kg. Since there was a concomitant increase in the striatal $\text{in}$$\text{vivo}$ tyrosine hydroxylation as measured by DOPA accumulation after decarboxylase inhibition, our findings suggest that LSD and BOL increase the impulse flow in the nigro-neostriatal pathway probably by central dopamine (DA) receptor antagonism. However, 4 hrs after LSD the DOPAC level was decreased, while the DOPA accumulation was not. Thus the effect of LSD on the dopaminergic system appears not to be limited to a pure receptor antagonism. The possibility also exists that the effect of LSD on the nigro-neostriatal DA pathway is secondary to its effect on the central 5-hydroxytryptaminergic system.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

Stimulation of dopamine synthesis and release by morphine and D-Ala2-D-Leu5-enkephalin in the mouse striatum in vivo

The effects of opiates on dopamine (DA) release and synthesis were assessed in the mouse striatum $\text{in vivo}$ by simultaneously measuring 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels after inhibition of aromatic amino acid decarboxylase. This method was developed to assess stimulus-coupled changes in DA synthesis and release. Peripheral injections of morphine and intraventrcular injections of D-Ala²-Leu⁵-enkephalin elevated DOPAC levels, indicating that “opiates” stimulated DA release. Concomitantly, the rate of DA synthesis was increased. The effects were dose-dependent, saturable and antagonized by naloxone. When morphine and the enkephalin analog were given together in saturating doses, the effects of the two agents were not additive. Thus, the involvement of different receptors in the mediation of the effects of morphine and enkephalins could not be demonstrated.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Short Communication Occupation of Dopamine Receptors by N-n-Propylnorapomorphine or Spiperone and Acetylcholine Levels in the Rat Striatum

In an attempt to quantify the interactions between dopaminergic and cholinergic processes, the consequences of complete or partial activation (with N-n-propylnorapomorphine) or blockade (with spiperone) of dopamine receptors for the acetylcholine levels in the rat striatum were studied. The number of specific striatal binding sites (receptors) of spiperone was nearly three times that of N-n-propylnorapomorphine (76 and 26 pmol g-1 wet weight, respectively). The agonist produced a significant increase in the striatal levels of acetylcholine, but there was no simple relationship between receptor binding and these levels. A linear negative correlation was found between the striatal levels of acetylcholine and specific spiperone binding, showing that further receptor blockade induces a decrease in acetylcholine levels, which is independent of the receptors already occupied by the antagonist. The results of this study are evidence that one striatal dopamine receptor regulates the metabolism of at least 400 molecules of acetylcholine.     

Effect of butaclamol and its enantiomers upon striatal homovanillic acid and adenyl cyclase of olfactory tubercle in rats.

Butaclamol, a new neuroleptic agent, and its (+)- enantiomer caused a pronounced dose-related elevation of rat striatal homovanillic acid concentration $\text{in}$$\text{vivo}$. In addition, each blocked the dopamine-induced increase in adenyl cyclase activity of homogenates of the olfactory tubercle, a limbic area in the brain. The (-)-enantiomer of butaclamol did not exhibit these activities indicating a stereochemical specificity for dopamine receptor-blockade activity. The (+)-enantiomer was 2–3 times more potent than butaclamol, exhibiting activities similar to those of fluphenazine. The present findings are consistent with the existence of relationships between changes in dopamine turnover in the striatum and the production of extrapyramidal side effects and between changes in adenyl cyclase activity of olfactory tubercle and antipsychotic activity.     

Specific invivo binding of 77Br-brombenperidol in rat brain

The $\text{in vivo}$ binding of the radiobrominated neuroleptic brombenperidol in rat brain was studied. The accumulation of the radiolabeled neuroleptic was high in the striatum and relatively low in the cerebellum, cortex, and blood. Striatal binding of brombenperidol was saturable and displaced by subsequent administration of benperidol. The rationale for the development of ⁷⁵Br-brombenperidol as a radiopharmaceutical for the non-invasive imaging of cerebral dopamine receptor areas is presented.     

ONTOGENESIS OF DOPAMINERGIC-CHOLINERGIC INTERACTIONS IN THE RAT STRIATUM: A NEUROCHEMICAL STUDY

Abstract— In the striatum of the newborn rat, the activity of tyrosine hydroxylase, the concentration of dopamine and the activity of the synaptosomal high-affinity uptake process for dopamine is 10% of that of the adult; there is a linear and closely associated increase in all three parameters during maturation, achieving 75% of adult levels by 4 weeks after birth. In contrast, the specific activity of choline acetyltransferase exhibits a more delayed developmental rise commencing 1 week after birth; the concentration of acetylcholine is disproportionately high in the neonatal striatum and precedes the developmental increase in the activity of choline acetyltransferase. At birth, the specific activity of dopamine-sensitive adenylate cyclase is 20% of that of the adult striatum and achieves adult activity by 4 weeks after birth. Pretreatment with the neuroleptic, fluphenazinc. does not reduce the striatal content of acetylcholine until 8 days after birth. It is postulated that dopaminergic influences on cholinergic neuronal activity appear when the cholinergic neurons in the striatum cease dividing and start differentiating.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Striatal Slices Is Independent of Dopaminergic Tone

The effects of neurotensin (NT) alone or in combination with the dopamine antagonist sulpiride were tested on the release of endogenous acetylcholine (ACh) from striatal slices. NT enhanced potassium (25 mM)-evoked ACh release from striatal slices in a dose-dependent manner. This effect was tetrodotoxin-insensitive, suggesting an action directly on cholinergic elements. The dopamine antagonist sulpiride (5 x 10(-5) M) significantly increased (63%) potassium-evoked ACh release from striatal slices; potassium-evoked ACh release was further increased (90%) in the presence of NT (10(-5) M) and sulpiride (5 x 10(-5) M). The second set of experiments tested the effects of 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra on NT-induced increases of potassium-evoked ACh release. These lesions did not alter the NT regulation of potassium-evoked ACh release from striatal slices, but did significantly increase spontaneous (33%) and potassium-evoked (40%) ACh release from striatal slices. Striatal choline acetyltransferase activity was not affected by 6-OHDA lesions. In addition, following 6-OHDA lesions, sulpiride was ineffective in altering ACh release from striatal slices. Furthermore, evoked ACh release in the presence of the combination of NT and sulpiride was not different from that in the presence of NT alone. These results suggest that in the rat striatum, NT regulates cholinergic interneuron activity by interacting with NT receptors associated with cholinergic elements. Moreover, the NT modulation of cholinergic activity is independent of either an interaction of NT with D2 dopamine receptors or the sustained release of dopamine.     

Effect of aging on recovery of striatal dopamine receptors following n-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) blockade

The recovery rate of striatal dopamine receptors following blockade by N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) $\text{in vivo}$ is reduced by 25–35% in striata from senescent Wistar rats when compared to mature counterparts. No differences in binding affinity for [³H]-spiperone were observed for the different age groups at various times after EEDQ injection. These results suggest that loss of striatal dopamine receptors during aging may be due to a decreased biosynthetic rate.     

Effects of atropine treatment on cortical,striatal and hippocampal high-affinity uptake of choline in the mouse

Changes in high-affinity uptake of choline (H.A._Ch) were studied in synaptosomes from different mouse brain regions following intravenous (i.v.) administration of atropine (0.3–30 mg/kg body weight) $\text{in vivo}$. The Ch-uptake was expressed as a Ch-uptake index, defined as the ratio between H.A._Ch and the corresponding choline acetylt-ransferase (ChAt) activity. The Ch-uptake index was highest in the hippocampus and lowest in the striatum. In the hippocampus a dose-dependent increase in this index was found following atropine treatment, while the striatal Ch-uptake index was unaffected by atropine. Atropine given i.v. in a dose of 10 mg/kg induced a 86% increase in V_max in synaptosomes from the hippocampus.     

Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3Hcatecholamine synthesis in discrete brain regions of adult rats

Endogenous noradrenaline levels are elevated in medulla oblongata, mesencephalon, pons and thalamus of adult rats which had been treated with 6-hydroxydopamine on days 1, 2, 8 and 15 after birth. Levels in spinal cord, cerebellum, hippocampus/amygdala and cortex are depressed, whereas no significant changes are observed in striatum, hypothalamus and medulla spinalis. The rate at which medulla oblongata synthesizes tritiated noradrenaline and dopamine from tritiated tyrosine $\text{in}$$\text{vitro}$ is markedly enhanced. No effect was apparent on catecholamine synthesis in hypothalamus. Tritiated noradrenaline synthesis, but not tritiated dopamine synthesis, in the cortex is depressed. These results support the view that neonatal 6-hydroxydopamine treatment causes a degeneration of noradrenaline nerve terminals in the cortex and induces an increase in noradrenaline terminals in the medulla oblongata.     

Cytidine and Uridine Increase Striatal CDP-Choline Levels Without Decreasing Acetylcholine Synthesis or Release

Summary Aims: Treatments that increase acetylcholine release from brain slices decrease the synthesis of phosphatidylcholine by, and its levels in, the slices. We examined whether adding cytidine or uridine to the slice medium, which increases the utilization of choline to form phospholipids, also decreases acetylcholine levels and release. Methods: We incubated rat brain slices with or without cytidine or uridine (both 25–400 μM), and with or without choline (20–40 μM), and measured the spontaneous and potassium-evoked release of acetylcholine. Results: Striatal slices stimulated for 2 h released 2650±365 pmol of acetylcholine per mg protein when incubated without choline, or 4600±450 pmol/mg protein acetylcholine when incubated with choline (20 μM). Adding cytidine or uridine (both 25–400 μM) to the media failed to affect acetylcholine release whether or not choline was also added, even though the pyrimidines (400 μM) did enhance choline`s utilization to form CDP-choline by 89 or 61%, respectively. The pyrimidines also had no effect on acetylcholine release from hippocampal and cortical slices. Cytidine or uridine also failed to affect acetylcholine levels in striatal slices, nor choline transport into striatal synaptosomes. Conclusion: These data show that cytidine and uridine can stimulate brain phosphatide synthesis without diminishing acetylcholine synthesis or release.