MKC-231, a choline uptake enhancer: (2) Effect on synthesis and release of acetylcholine in AF64A-treated rats

The effect of MKC-231 on acetylcholine (ACh) synthesis and release was studied in the hippocampus of normal and AF64A-treated rats. AF64A (3 nmol/brain, i.c.v.) produced significant reduction of high-affinity choline uptake (HACU) and high K+-induced ACh release in hippocampal synaptosomes. Treatments with MKC-231 (10(-8) and 10(-7) M) showed significant reverse of the decrease in both HACU and ACh release. In hippocampal slices superfused with choline-containing artificial cerebro-spinal fluid (ACSF), high K+-induced ACh release was gradually decreased by repeated alteration of resting and high K+ stimulations in AF64A-treated rats. However, addition of MKC-231 (10(-8) to 10(-7) M) in the superfusate reduces this decrease. In vivo microdialysis studies indicate MKC-231 (10 mg/kg, p.o.) significantly reversed reduction of basal ACh concentrations in AF64A-treated rats, measured by radioimmunoassay without a cholinesterase inhibitor in the perfusate. These results indicate MKC-231 improves AF64A-induced cholinergic hypofunction by enhancing HACU, subsequently facilitating ACh synthesis and release in vitro and in vivo.     

MKC-231, a choline-uptake enhancer: (1) long-lasting cognitive improvement after repeated administration in AF64A-treated rats

MKC-231 is reported to increase high-affinity choline uptake (HACU) in vitro and improve learning impairment on a single oral administration in AF64A-treated rats. In this study, we investigated the effects of repeated administration of MKC-231 (1 and 3 mg/kg, p.o., 8 days) on learning impairment in the water-maze task in AF64A-treated rats 1, 24, 48, and 72 h after the last dose. Significant cognitive improvement was observed for 24 h, however, concentration measurement studies indicated MKC-231 was not detected in the brain by this time. We also studied the effects of 8-days repeated administration of MKC-231 on HACU 1, 24, 48, and 72 h after the last dose and observed an increase of HACU similar in time course with cognitive improvement. From these results, we discussed the possibility that MKC-231 could induce long-lasting procognitive effects by changing the choline transporter regulation system.     

MKC-231, a choline uptake enhancer,ameliorates working memory deficits and decreased hippocampal acetylcholine induced by ethylcholine aziridinium ion in mice

Summary The effects of acute and chronic administration of MKC-231, a new choline uptake enhancer, and two other nootropic agents, linopiridine (Dup 996) and tetrahydroaminoacridine (THA) on working memory deficits and decreased hippocampal acetylcholine (ACh) content were studied in a delayed non-matching to sample task, using a T-maze, in ethylcholine aziridinium ion (AF64A)-treated mice. Treatment with AF64A (3.5 nmol, i.c.v.) produced memory deficits and decreased hippocampal ACh content. In acute behavioral experiments, MKC-231 and THA had no significant effect on AF64A-induced memory deficits at any doses tested (0.3, 1.0 and 3.0mg/kg), whereas Dup 996, at a dose of 1.0mg/kg, significantly improved memory deficits. In chronic experiments, MKC-231 improved memory deficit at all doses tested (0.3, 1.0, or 3.0mg/kg p.o., once daily for 11 days) and Dup 996 did so only at a dose of 3.0 mg/kg, whereas THA did not improve memory deficit at any doses tested. In acute neurochemical experiments, MKC-231 and THA did not reverse the AF64A-induced hippocampal ACh depletion. Dup 996, however, further decreased hippocampal ACh content compared to that in the AF64A-treated group. In chronic experiments, MKC-231 significantly reversed hippocampal ACh depletion at doses of 0.3 and 1.0mg/kg, whereas neither Dup 996 nor THA reversed hippocampal ACh depletion at any doses tested. These results indicate that MKC-231 improved the AF64A-induced working memory deficit and hippocampal ACh depletion, probably by recovering reduced high-affinity choline uptake and ACh release.     

Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high‐affinity choline transporter and choline acetyltransferase

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non‐neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high‐affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112‐1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112‐1 cells showed increased binding of the CHT1 inhibitor [³H]hemicholinium‐3 (HC‐3) and greater [³H]choline uptake and ACh synthesis than NIH3T3ChAT 103‐1 cells, a CHT1‐negative control cell line. HC‐3 significantly inhibited ACh synthesis in NIH3T3ChAT 112‐1 cells but did not affect synthesis in NIH3T3ChAT 103‐1 cells. ACh synthesis in NIH3T3ChAT 112‐1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low‐affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112‐1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes. © 2009 Wiley‐Liss, Inc.     

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 (-44.7%) 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.     

Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: An autoradiographic study using3H-hemicholinium-3

Summary ³H-hemicholinium-3 (³H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of³H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I–III of the frontal cortex. By contrast, in the caudate-putamen the number of³H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (–40%), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD.     

Nipecotic acid, an uptake blocker, prevents fading of the γ-aminobutyric acid effect

In rats under urethane, ionotophoretic applications of GABA (30–60 nA) in the str. pyramide of CA1, showed a rapidly fading inhibitory effect. By contrast, GABA had a well-maintained inhibitory effect in str. radiatum. During iontophoresis of nipecotic acid (30–85 nA) identical applications of GABA in str. pyramidale caused a more prominent depression without fading, which suggests that removal of GABA, by uptake, can at least in part account for ‘fading. Nipecotic acid also prolonged the paired-pulse inhibition, presumably by prolonging the duration of inhibitory postsynaptic potentials.     

Extended action of MKC-242, a selective 5-HT(1A) receptor agonist, on light-induced Per gene expression in the suprachiasmatic nucleus in mice

We reported previously that (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole hydrochloride (MKC-242) (3 mg kg(-1), i.p.), a selective 5-HT(1A) receptor agonist, accelerated the re-entrainment of hamster wheel-running rhythms to a new 8 hr delayed or advanced light-dark cycle, and also potentiated the phase advance of the wheel-running rhythm produced by light pulses. The molecular mechanism underlying MKC-242-induced potentiation of this phase shift, however, has not yet been elucidated. We examined the effects of MKC-242 on light-induced mPer1 and mPer2 mRNA expression in the suprachiasmatic nucleus (SCN) of mice. MKC-242 (5 mg kg(-1), i.p.) potentiated light-induced mPer1 and mPer2 expression in the SCN of mice housed in constant darkness for 2 days, when mRNA levels were observed 3 hr after light-exposure. More potentiating action of MKC-242 on mPer2 expression in the SCN was observed in mice housed in constant darkness for 9-10 days. This facilitatory action of MKC-242 on mPer1 expression was antagonized by WAY100635, a selective 5-HT(1A) receptor blocker, indicating that MKC-242 activated 5-HT(1A) receptors. Other drugs such as 8-hydroxy-dipropylaminotetralin (10 mg kg(-1), i.p.), paroxetine (10 mg kg(-1), i.p.), buspirone (10 mg kg(-1), i.p.), and diazepam (10 mg kg(-1), i.p.) did not display a potentiating action on light-induced mPer1 and mPer2 expression in the SCN. In the behavioral experiments, we found that MKC-242 (5 mg kg(-1), i.p.) potentiated light-induced phase delays of free-running rhythm in mice. The present results suggest that prolonged increase of mPer1 or mPer2 expression in the SCN by MKC-242 may be involved in the potentiation of photic entrainment by MKC-242 in mice.     

Binding of the [125I]3β-(Iodophenyl)tropan-2β-carboxylic acid isopropyl ester to the dopamine transporter at a physiologically relevant temperature: Mutually exclusive binding and different ionic requirements for various uptake blockers and substrates

The present study describes the binding of cocaine analog [¹²⁵I]3β-(iodophenyl)tropan-2β-carboxylic acid isopropyl ester ([¹²⁵I]RTI-121), a highly selective ligand for the dopamine transporter (DAT), to rat striatal synaptosomal membranes at 37°C. Saturation analysis of [¹²⁵I]RTI-121 binding revealed a single binding site with similar affinity for RTI-121 at both 50 and 134 mM NaCl. However, the density of binding sites was reduced at 134 mM NaCl. Various uptake blockers and substrates of the DAT monophasically inhibited the specific binding of [¹²⁵I]RTI-121. Increasing the NaCl concentration from 50 mM to 134 mM enhanced the affinity of the substrate dopamine and amphetamine for the DAT, without affecting that of the uptake blockers. At 134 mM NaCl, the copresence of GBR12935, BTCP, cocaine, amphetamine, or dopamine decreased the affinity of RTI-121 to the extent predicted by a model in which the binding of all compounds is mutually exclusive. This, along with a different NaCl sensitivity for blockers and substrates, suggests that the two categories of compounds recognize nonidentical but overlapping binding domains on the DAT. In contrast, the mutually exclusive binding with similar NaCl sensitivity for RTI-121 and the other uptake blockers tested here suggests the involvement of common binding domains in the recognition of these blockers. Synapse 25:155–162, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of monoamine oxidase enzymes in the action of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, a selective neurotoxin, in the squirrel monkey: Binding and biochemical studies

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is a new neurotoxin that causes degeneration of the dopaminergic nigrostriatal neurons and induces a Parkinson-like state in several species, including humans and monkeys. The present study was designed to better characterize the properties of [3H]MPTP binding sites and to evaluate the interaction of MPTP with the oxidation of dopamine by monoamine oxidase (MAO) in an animal species (Saimiri Sciureus) shown to be lesioned by MPTP. Our data confirm the presence of high affinity and saturable binding sites for [3H]MPTP in the squirrel monkey. Specific binding with analogous characteristics also occurs in peripheral tissues. Various substances failed to inhibit the [3H]MPTP binding, whereas only MAO inhibitors (MAOI) were able to antagonize this binding to brain and peripheral tissues. In particular, deprenyl, a selective inhibitor of MAO type B enzyme, was relatively more potent as a displacer of [3H]MPTP from its binding sites both in brain and in peripheral tissues. Our results further suggest a correspondence between [3H]MPTP sites and MAO, particularly MAO-B, in monkey brain. Moreover, our data show that the oxidative deamination of dopamine is inhibited by MPTP in vitro. In conclusion, these data are consistent with the hypothesis of the involvement of MAO in the neurotoxic effects of MPTP, even though further experiments are necessary to better clarify the molecular mechanism of MPTP neurotoxicity.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Distribution of Na/K-ATPase alpha 3 isoform, a sodium-potassium P-type pump associated with rapid-onset of dystonia parkinsonism (RDP) in the adult mouse brain

The Na(+)/K(+)-ATPase1 alpha subunit 3 (ATP1α(3)) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid-onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1α(3) in the adult mouse brain. Our results show that localization of ATP1α(3) is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1α(3) in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1α(3) isoform. Other structures with high expression of ATP1α(3) included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1α(3) in projections and cell bodies in hippocampus; most of these ATP1α(3)-positive cell bodies showed colocalization to GABAergic neurons. ATP1α(3) expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1α(3) is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms.     

In vitro characterization of SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolone, monohydrochloride): a novel partial dopamine D2 and D3 receptor agonist and serotonin 5-HT1A receptor agonist

Present Parkinson's disease treatment strategies are far from ideal for a variety of reasons; it has therefore been suggested that partial dopamine receptor agonism might be a potential therapeutic approach with potentially fewer side effects. In the present study, we describe the in vitro characterization of the nonergot ligand SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolonemonohydrochloride). SLV308 binds to dopamine D(2), D(3), and D(4) receptors and 5-HT(1) (A) receptors and is a partial agonist at dopamine D(2) and D(3) receptors and a full agonist at serotonin 5-HT(1) (A) receptors. At cloned human dopamine D(2,L) receptors, SLV308 acted as a potent but partial D(2) receptor agonist (pEC(50) = 8.0 and pA(2) = 8.4) with an efficacy of 50% on forskolin stimulated cAMP accumulation. At human recombinant dopamine D(3) receptors, SLV308 acted as a partial agonist in the induction of [(35)S]GTPgammaS binding (intrinsic activity of 67%; pEC(50) = 9.2) and antagonized the dopamine induction of [(35)S]GTPgammaS binding (pA(2) = 9.0). SLV308 acted as a full 5-HT(1) (A) receptor agonist on forskolin induced cAMP accumulation at cloned human 5-HT(1) (A) receptors but with low potency (pEC(50) = 6.3). In rat striatal slices SLV308 concentration-dependently attenuated forskolin stimulated accumulation of cAMP, as expected for a dopamine D(2) and D(3) receptor agonist. SLV308 antagonized the inhibitory effect of quinpirole on K(+)-stimulated [(3)H]-dopamine release from rat striatal slices (pA(2) = 8.5). In the same paradigm, SLV308 had antagonist properties in the presence of quinpirole (pA(2) = 8.5), but the partial D(2) agonist terguride had much stronger antagonistic properties. In conclusion, SLV308 combines high potency partial agonism at dopamine D(2) and D(3) receptors with full efficacy low potency serotonin 5-HT(1) (A) receptor agonism and is worthy of profiling in in vivo models of Parkinson's disease.     

Anticonvulsant effect of enhancement of noradrenergic transmission in the superior colliculus in genetically epilepsy-prone rats (GEPRs): a microinjection study

An expanding body of data has indicated that the seizure prone state in genetically epilepsy-prone rats (GEPRs) is partially caused by deficits in central nervous system noradrenergic transmission. Several lines of evidence suggest that the noradrenergic terminals in the superior colliculus (SC) may act as determinants of seizure predisposition in the GEPR. In order to assess the role of the noradrenergic transmission in the SC in the regulation of seizure severity, several drugs with different mechanisms of enhancing noradrenergic transmission were bilaterally microinfused into the SC of GEPR-9s (severe seizure GEPRs). The rats were tested for audiogenic seizure intensity at 0.25, 1, 2, 3, and 4 h after treatments. Bilateral infusion of vehicle produced no reduction in the severity of the audiogenic seizure. Desipramine (2, 4, 8 μg/side), nisoxetine (2, 4, 8 μg/side), and idazoxan (0.25, 1, 4 μg/side) all decreased the seizure severity in a dose-dependent fashion. Significant decreases in the seizure severity were also observed after administration of methoxamine (0.15 μg/side) or phenylephrine (0.15 μg/side). Pretreatment with prazosin (1 μg/side) significantly diminished the anticonvulsant effectiveness of methoxamine and nisoxetine while prazosin, by itself, had no effects on the seizure intensity. These results suggest that noradrenergic transmission in the SC may be involved in the seizure regulation in GEPR-9s, and that this regulation may be mediated, at least in part, through α₁ receptors.     

Pipecolic acid: a new type of α-amino acid possessing bicuculline-sensiti action in the mammalian brain

Using unit recording and electrophoretic techniques, pharmacological properties of pipecolic acid (PA) were studied in the brain neurons of rats. PA response was blocked by bicuculline more effectively than GABA response but not blocked by strychnine. Stereochemical findings obtained using the HGS-model demonstrated that PA structure is almost the same as a part of bicuculline structure. The present results suggest that PA might be a new type of substance possessing bicuculline-sensitive action. The site of the action of PA was also discussed.     

Cholinergic projections from the parabigeminal nucleus (Ch8) to the superior colliculus in the mouse: a combined analysis of horseradish peroxidase transport and choline acetyltransferase immunohistochemistry

Choline acetyltransferase (ChAT) immunocytochemistry, acetylcholinesterase histochemistry and muscarinic receptor autoradiography demonstrated a cholinergic innervation within the superior colliculus. A method for the concurrent visualization of ChAT and transported horseradish peroxidase showed that a major extrinsic source for this cholinergic input is in the parabigeminal nucleus. We have designated these cholinergic neurons as the Ch8 cell group.     

In vivo labeling of phencyclidine (PCP) receptors with 3H-TCP in the mouse brain

The phencyclidine (PCP) derivative N-[1-(2-thienyl)cyclohexyl]-piperidine (3H-TCP) was used to label in vivo the N-methyl-D-aspartate (NMDA) receptor-associated ionic channel in the mouse brain. After the injection of a tracer dose of 3H-TCP, a spread labeling throughout the brain was observed, but was the highest in the cerebellum. Preadministration of unlabeled TCP (30 mg/kg) resulted in a 90% reduction of 3H-TCP binding. PCP, TCP, MK-801, dexoxadrol, ketamine, and SKF 10,047 isomers dose-dependently prevented the in vivo 3H-TCP binding. ID50 determined in the cerebrum and the cerebellum were respectively correlated with K0.5 for 3H TCP high (rat cortex) and low affinity (rat cerebellum) sites in vitro. The pharmacological specificity of the 3H-TCP binding site in the cerebellum was significantly different from that in the cerebrum. ID50 values were generally higher than in the cerebrum and, particularly, MK-801, the most potent drug in the cerebrum, was without significant effect in the cerebellum, at any time and at doses as high as 30 mg/kg. N-[1-(2-benzo(b) thiophenyl)cyclohexyl]piperidine (BTCP), desipramine, and atropine showed a more efficient prevention of 3H-TCP binding in the cerebellum than in the cerebrum. The prevention of the binding by TCP or PCP, at doses close to their ID50 values, was rapid and then decreased slowly. The effect of MK-801 was long-lasting. This study confirm previous in vitro studies: 3H-TCP is an efficient tool for the labeling of the NMDA receptor-associated ionic channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACTH- and α-MSH-induced grooming, stretching, yawning and penile erection in male rats: Site of action in the brain and role of melanocortin receptors

The effect of adrenocorticotropin (ACTH)(1-24) and α-melanocyte stimulating hormone (α-MSH) on grooming, stretching, yawning and penile erection was studied after injection into different brain areas. Both peptides induce the above responses when injected into the hypothalamic periventricular region of the third ventricle. This region includes the paraventricular nucleus, the dorsomedial nucleus, the ventromedial nucleus and the anterior hypothalamic area. The minimal effective dose of both peptides was 0.5 μg and the maximal effect was seen with 2 μg, the highest dose tested. Irrespective of the injection site, grooming started 5–7 min after injection of either peptide, while stretching, yawning and penile erection started only after 15–35 min and lasted for 90–120 min. In contrast both peptides were ineffective when injected into the preoptic area, the caudate nucleus or the CA1 field of the hippocampus. Grooming, stretching and yawning, but not penile erection, were prevented by cyclic[AcCys¹¹, D-Nal¹⁴, Cys¹⁸, AspNH₂²²]-β-MSH (11-22) (HS014), a selective melanocortin 4 receptor antagonist, injected into the same periventricular area 10 min before of ACTH(1-24) or α-MSH. The results show that ACTH(1-24) and α-MSH act in the hypothalamic periventricular region to induce the above responses and that grooming, stretching and yawning, but not penile erection, are mediated by melanocortin 4 receptors.