Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels.

The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 [11-2-[[2-[diethylaminomethyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one], hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of [3H]quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of [3H]-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.     

Influence of tissue integrity on pharmacological phenotypes of muscarinic acetylcholine receptors in the rat cerebral cortex

Distinct pharmacological phenotypes of muscarinic acetylcholine receptors (mAChRs) have been proposed. We compared the pharmacological profiles of mAChRs in intact segments and homogenates of rat cerebral cortex and other tissues by using radioligand binding assays with [(3)H]N-methylscopolamine ([(3)H]NMS). Recombinant M(1) and M(3) mAChRs were also examined. The density of mAChRs detected by [(3)H]NMS binding to rat cerebral cortex segments and homogenates was the same (approximately 1400 fmol/mg tissue protein), but the dissociation constant of [(3)H]NMS was significantly different (1400-1700 pM in segments and 260 pM in homogenates). A wide variation in [(3)H]NMS binding affinity was also observed among the segments of other tissues (ranging from 139 pM in urinary bladder muscle to 1130 pM in the hippocampus). The mAChRs of cerebral cortex were composed of M(1), M(2), M(3), and M(4) subtypes, which showed typical subtype pharmacology in the homogenates. However, in the cortex segments the M(3) subtype showed a low selectivity for M(3) antagonists (darifenacin, solifenacin) and was not distinguished by the M(3) antagonists from the other subtypes. Recombinant M(1) and M(3) mAChRs showed high affinity for [(3)H]NMS and subtype-specific pharmacology for each tested ligand. The present binding study under conditions where tissue integrity was kept demonstrates a wide variation in [(3)H]NMS binding affinity among mAChRs of many rat tissues and the presence of an atypical M(3) phenotype in the cerebral cortex, suggesting that the pharmacological properties of mAChRs are not necessarily constant, rather they may be significantly modified by tissue integrity and tissue type.     

Altered muscarinic receptor properties and function in the heart in diabetes

The cardiac cholinergic system was studied in streptozotocin (STZ)-diabetic and age-matched control rats. STZ-diabetic rats (8-10 weeks) were supersensitive to the negative chronotropic effects of acetylcholine, carbamylcholine and bethanechol; inotropic responses to these muscarinic agonists were unaltered. This phenomenon was associated with a decrease in acetylcholinesterase activity but no change in the rate and extent of neuronal choline uptake. [3H]N-methylscopolamine bound to muscarinic receptors in atria from both groups of rats with the same high affinity. The density of [3H]N-methylscopolamine binding sites, however, was 34% lower in atria from STZ-diabetic rats. Agonist binding affinity was lower in diabetes; carbamylcholine had a lower affinity for both the high- and low-affinity receptors. These results indicate that cardiac cholinergic supersensitivity in right atria in diabetes occurs before the development of autonomic neuropathy insofar as neuronal [3H]choline uptake is unaltered at this stage of STZ diabetes. Changes in agonist binding conformation, without a concomitant change in antagonist binding affinity, suggest that supersensitivity of right atria to muscarinic agonist may be a consequence of altered coupling of muscarinic receptor to transduction mechanisms involved in chronotropism in diabetes.     

Ontogeny of cortical muscarinic receptor subtypes and muscarinic receptor-mediated responses in rat

To determine the ontogenetic relationship of muscarinic receptor and effector systems in the central nervous system, the developmental time courses for binding sites with high (M1) and low (M2) affinity for pirenzepine as well as muscarinic receptor-mediated stimulation of phosphoinositide breakdown and inhibition of cyclic AMP accumulation were examined in rat cortex. M1 sites were 30% of adult levels at 1 week, 70% at 2 weeks, 90% at 3 weeks and equal to adult levels at 4 weeks postpartum. M2 sites, on the other hand, did not show a significant change between the ages of 1 and 6 weeks. Acetylcholine-stimulated phosphoinositide breakdown was detected at all ages tested (1, 2, 3, 4 and 6 weeks). The percentage of conversion of [3H] phosphoinositides to [3H]inositol phosphates, stimulated by 1 mM acetylcholine, increased with age until 3 weeks after birth and then decreased slightly at ages 4 and 6 weeks. Carbachol inhibition of [3H]cyclic AMP accumulation, on the other hand, was undetectable in tissues from 1- and 2-week-old rats, whereas in tissues from 3- and 4-week-old rats, the responses were at 6-week level. Thus, for carbachol inhibition of cyclic AMP accumulation, a time in development existed (2 weeks after birth) at which receptors appeared to be present but response to stimulation was absent. To examine indirectly the coupling of binding sites to second messenger systems via guanine nucleotide-binding regulatory proteins, the density of binding sites for the muscarinic receptor agonist, [3H]oxotremorine-M, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin regulates neuronal M2 muscarinic receptor function in the ileum of diabetic rats

Acetylcholine release from cholinergic nerves in the gastrointestinal tract is limited by neuronal M(2) muscarinic receptors. In diabetic animals, M(2) muscarinic receptor function in the ileum is increased, leading to decreased acetylcholine release and smooth muscle contraction in response to nerve stimulation. The mechanisms responsible for increased M(2) muscarinic receptor function are unknown but may contribute to the gastrointestinal dysmotility that occurs frequently in diabetics. In this study, we investigated whether insulin modulates M(2) muscarinic receptor function in the gastrointestinal tract of diabetic rats. M(2) muscarinic receptor function was tested by measuring the ability of an agonist, pilocarpine, to inhibit and an antagonist, methoctramine, to potentiate electrical field stimulation (EFS)-induced contraction of ileum in vitro. Insulin administration (0.2, 0.6, and 2 U s.c. daily for 7 days) reversed the diabetes-induced increase in M(2) muscarinic receptor function and restored normal contractions to EFS. Insulin had no effect on the function of postjunctional M(3) muscarinic receptors, determined by measuring contractile responses to acetylcholine. These data suggest that insulin tonically inhibits neuronal M(2) muscarinic receptors. Thus, loss of insulin removes this inhibition and increases M(2) muscarinic receptor function leading to decreased acetylcholine release and contraction to EFS. In nondiabetic rats, there was a trend that higher insulin doses (0.6 and 2 U) increased M(2) muscarinic receptor function, suggesting a bell-shaped concentration-response relationship for insulin. In conclusion, lack of insulin or excess insulin increases M(2) muscarinic receptor function in rat ileum. This mechanism may contribute to decreased acetylcholine release in the gastrointestinal tract of diabetics, resulting in dysmotility.     

Functional evidence for multiple gamma-aminobutyric acidB receptor subtypes in the rat cerebral cortex.

The aim of this work was the identification of pharmacologically distinct subtypes of gamma-aminobutyric acidB (GABAB) receptors in the central nervous system. Inasmuch as GABAB receptors are often sited on axon terminals where they mediate inhibition of transmitter release, we chose as models the GABAB receptors mediating inhibition of release of 1) endogenous GABA; 2) endogenous glutamate; and 3) somatostatin-like immunoreactivity (SRIF-LI). The experimental set up consisted of rat cerebrocortical synaptosomes depolarized in superfusion with 12 or 15 mM KCl. Endogenous GABA and glutamate were measured by high-performance liquid chromatography and SRIF-LI by radioimmunoassay. The selective GABAB receptor agonist (-)-baclofen inhibited in a concentration-dependent manner the K(+)-evoked release of GABA, glutamate and SRIF-Ll with similar potencies and efficacies [EC50 values, 1.1-1.5 microM; maximal inhibition, 45-50% at about 10 microM (-)-baclofen]. The GABAB receptor antagonist phaclofen concentration-dependently reduced the effects of (-)-baclofen on the release of GABA and SRIF-Ll but not on the release of glutamate, where it was ineffective up to 1000 microM. The rank order of potency (Ki values are shown in parentheses) are: SRIF-Ll (7.8 microM); GABA (10.4 microM); and glutamate (greater than 115 microM). The novel GABAB receptor antagonist 3-aminopropyl(diethoxymethyl) phosphinic acid (CGP 35348) displayed a different pattern on the three release systems examined (Ki values are shown in parentheses): SRIF-Ll (0.38 microM); glutamate (0.48 microM); and endogenous GABA (greater than 115 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Central muscarinic receptor subtypes and carrageenin-induced paw oedema in rats

In an earlier report from this laboratory, it was demonstrated that the central cholinergic system exerted a modulatory pro-inflammatory effect on carrageenin-induced paw inflammation in rats. In this study, the role of the central muscarinic receptors in cholinergic modulation of peripheral inflammation was investigated by using several M₁ and M₂ receptor agonists and antagonists. The M₁ receptor agonists aceclidine and arecholine and the nonspecific muscarinic receptor agonist oxotremorine augmented carrageenin oedema, an effect attenuated by the M₁ receptor antagonist scopolamine. Physostigmine behaved like a M₁ receptor agonist at all dose levels. However, the other M₂ receptor agonist, carbachol, produced a dose-dependent dual effect, with lower doses attenuating the oedema and higher doses augmenting the inflammation. While the former action appeared to be due to M₂ receptor stimulation, because it was blocked by AF-DX 116—a M₂ receptor antagonist, the latter action appeared to be induced by M₁ receptor stimulation, because it was inhibited by scopolamine. The pro-inflammatory effect of the M₂ receptor antagonists AF-DX 116 and gallamine appeared to be induced by enhanced neuronal release of acetyl-choline, because the effects were not evident following pretreatment with hemicholinium, which attenuates synthesis of the amine. Muscarinic receptor binding studies with (³H)-QNB indicated that the corpus striatum has substantially higher population of M₁ receptors compared with the cerebellum. In the corpus striatum, (³H)-QNB binding indicated initial up-regulation followed by down-regulation of M₁ receptors during peak inflammation, which appeared to persist even after a decrease in the inflammation. In contrast, the M₁ receptors in the cerebellum appeared to be down-regulated very transiently during the early phase of the inflammation. While these receptor alterations may be due to the inflammation, it is equally possible that they represent changes induced by the stress of pain and inflammation induced by carrageenin.     

Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist [3H]pirenzepine [( 3H]PZ) and the classical muscarinic antagonist [3H](-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for [3H](-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of [3H](-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Synthesis and pharmacological evaluation of dimeric muscarinic acetylcholine receptor agonists

Two dimeric analogs of the muscarinic acetylcholine receptor (mAChR) agonist phenylpropargyloxy-1,2,5-thiadiazole-quinuclidine (NNC 11-1314) were synthesized and pharmacologically evaluated. In radioligand binding assays on Chinese hamster ovary (CHO) cell membranes expressing the individual human M(1) to M(5) mAChR subtypes, both dimers [(3S)-1,4-bis-(3-[(3-azabicyclo[2.2.2]octanyl)-1,2,5-thiadiazol-4-yloxy]-1-propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1607) and (3S)-1,3-bis-(3-[(3-azabicyclo[2.2.2]octanyl)-1,2,5-thiadiazol-4-yloxy]-1-propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1585)] exhibited higher binding affinities than the monomeric NNC 11-1314. Only NNC 11-1585, however, displayed significant selectivity for the M(1) and M(2) mAChRs relative to the other subtypes. Although binding studies in rat brain homogenates supported the selectivity profile of NNC 11-1585 observed in the CHO membranes, rat heart membrane experiments revealed complex binding behavior for all three agonists that most likely reflected differences in species and host cell environment between the heart and CHO cells. Subsequent functional assays with phosphatidylinositol hydrolysis revealed that all three novel ligands were partial agonists relative to the full agonist oxotremorine-M at the CHO M(1), M(3), and M(5) mAChRs, with NNC 11-1607 displaying the highest functional selectivity. In the CHO M(2) and M(4) mAChR cells, agonist-mediated effects on forskolin-stimulated cAMP accumulation were characterized by bell-shaped concentration-response curves, with the exceptions of NNC 11-1607, which had no discernible effects at the M(2) mAChR, and NNC 11-1585, which could only inhibit cAMP accumulation at the M(4) mAChR. Thus, we identified NNC 11-1607 as a novel functionally selective M(1)/M(4) mAChR agonist. Our data suggest that dimerization of mAChR agonists is a viable approach in designing more potent and functionally selective agonists, as well as in providing novel tools with which to probe the nature of agonism at these receptors.     

Allosteric interactions at the m1, m2 and m3 muscarinic receptor subtypes

The purpose of our study was to investigate the interactions of allosteric antagonists at the individual m1, m2 and m3 muscarinic receptor subtypes. This was achieved through the use of transformed Chinese hamster ovary cells stably expressing the rat m1 or m3 receptor genes. A homogeneous population of the m2 subtype was obtained from rat heart tissue. Our data indicate that the cardioselective antagonists (gallamine, methoctramine, AF-DX 116 and himbacine) display the following rank order of potency for both displacing ligand binding to the primary site on the receptor and allosterically decelerating ligand dissociation: m2 greater than m1 greater than m3. Schild analysis showed the following rank order of the magnitude of gallamine's cooperative interactions with the three receptor subtypes: m3 greater than m1 greater than m2. By comparison, the ion-channel blockers (verapamil, phencyclidine and quinidine) exhibited a rank order of potency for cooperative effects similar to that of cardioselective antagonists; however, these blockers did not show appreciable specificity in their interaction with the receptor primary binding site. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the allosteric antagonists at each of the three muscarinic receptor subtypes, thus revealing the complex nature of interaction (both competitive and allosteric) between many of these compounds with the muscarinic receptor. Despite the fact that the majority of allosteric muscarinic antagonists are also K+ channel blockers, the use of pertussis toxin did not support the notion that this channel represents the allosteric site coupled to the receptor.     

Differential ontogeny of putative M1 and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart

Studies with [3H]pirenzepine [( 3H]PZ) suggest that this nonclassical muscarinic antagonist selectively identifies putative M1 muscarinic receptors. We now compare the ontogeny of these putative M1 sites, identified by high-affinity [3H]PZ binding, with sites identified by the classical antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) in murine cerebral cortical and cardiac homogenates. Dissociation constants (Kd) for [3H]PZ (2.1-6 nM in the cortex and 2.0-21 nM in the heart) and for (-)-[3H]QNB (10-28 pM in the cortex and 10-39 pM in the heart) are similar in adult and neonatal tissues, whereas receptor density (maximum binding, femtomoles per milligram of protein) varies significantly. Cerebral cortical [3H]PZ binding rises from 14% at birth, to 88% of adult levels by day 14, peaks at 128% at day 28 and falls to the mean adult level of 606 fmol/mg of protein. Cerebral cortical (-)-[3H]QNB binding parallels [3H]PZ binding. Conversely, parallel studies show cardiac (-)-[3H]QNB density is 3- to 17-fold greater than the comparable density of high-affinity [3H]PZ binding sites throughout ontogeny. We conclude that: 1) the high ratio of [3H]PZ binding to (-)-[3H]QNB binding identifies the murine cerebral cortex as a tissue which contains predominantly putative M1 muscarinic binding sites; 2) the relatively low ratio of [3H]PZ binding to (-)-[3H]QNB binding throughout ontogeny identifies the murine heart as a tissue which contains primarily the putative M2 muscarinic binding site; and 3) M1 and M2 receptor binding sites show distinct developmental curves in the cerebral cortex and heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha adrenergic receptor subtypes in human, monkey and dog cerebral arteries

In helical strips of human and monkey cerebral arteries, norepinephrine produced a greater contraction than that in dog cerebral arteries. In monkey cerebral arteries, phenylephrine and norepinephrine produced a similar magnitude of maximum contractions, although the ED50 value of phenylephrine was approximately 5.6 times greater than that of norepinephrine. Clonidine (up to 10(-5) M) did not produce contractions. Dog cerebral arteries responded to phenylephrine in high concentrations with a greater contraction than that induced by norepinephrine and to clonidine with significant contractions. Contractions induced by norepinephrine of human and monkey cerebral arteries were attenuated by low concentrations of prazosin but were not influenced by yohimbine in concentrations up to 10(-8) M. In contrast, norepinephrine-induced contractions of dog cerebral arteries were attenuated by yohimbine but were unaffected by prazosin. It appears that norepinephrine-induced contractions are mediated by alpha-2 adrenoceptors in dog cerebral arteries and by alpha-1 receptors in human and monkey cerebral arteries as well as monkey and dog mesenteric arteries. The relative unresponsiveness of monkey and dog cerebral arteries to adrenergic nerve stimulation may not be explained by a paucity of alpha adrenoceptors in neuroeffector junction.     

Identification of multiple muscarinic binding site subtypes in cat and human cerebral vasculature.

The binding characteristics of the nonselective muscarinic antagonist [3H]N-methyl scopolamine ([3H]NMS) have been studied in membrane fractions of cat and human cerebral blood vessels. A computer-fitting method was used to analyze the data obtained from association/dissociation, saturation and competition experiments. Specific binding of [3H]NMS to membrane preparations from cat and human pia-arachnoid vessels was found to be saturable (respective Bmax values of 98 +/- 15 and 67 +/- 7 fmol/mg protein) and of high affinity (KD values of 165 +/- 28 and 125 +/- 12 pM, respectively). Competition studies, in the presence of various well-characterized M1, M2 or M3 putative muscarinic antagonists, performed against the binding of [3H]NMS, revealed the heterogeneity of muscarinic binding sites in these vascular tissues. A population of M1 sites was clearly identified in both human and cat pial vessel membranes and accounted for approximately 40 (human) and 20% (cat) of the total population of cerebrovascular muscarinic binding sites labeled with [3H]NMS. Such observation was further supported by saturation studies of [3H]NMS binding performed under M1 blocking conditions (75 nM pirenzepine). Competition and saturation (in the presence of M2 antagonists) studies suggested the presence of M2 sites (approximately 35% of total sites) in cat pial vessels. However, under the same conditions, no M2 binding sites could be detected in human cerebrovascular membranes. A small population of M3 sites (approx. 20%) was found in both human and cat cerebrovascular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of subtypes of the muscarinic receptor to adenylate cyclase in the corpus striatum and heart

The binding properties of a series of muscarinic antagonists were compared with their ability to antagonize muscarinic receptor mediated inhibition of adenylate cyclase activity in homogenates of the corpus striatum and heart of rats. When measured by the competitive inhibition of the binding of the muscarinic antagonist N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists in the corpus stratum and cerebral cortex were consistent with a model incorporating a minimum of three populations of muscarinic receptors, a high affinity site for pirenzepine (M1), a high affinity site for AF-DX 116 [11] [2-[ (diethylamino)methyl]-1-piperidinyl] acetyl] -5, 11-dihydro-6H-pyrido [2,3-b] 1,4] benzodiazepine-6-one (M2) and a third population (non-Ml, non-M2 sites) displaying low affinity for the latter antagonists. The results of similar experiments on the heart showed that this tissue contained a uniform population of M2 muscarinic receptors. The binding properties of the M2 receptor in cerebral cortex and corpus stratum were also investigated directly in antagonist [3H] AF-DX 116 competition experiments and, although the high affinity AF-DX 116 site in brain (M2) exhibited selectivity for the cardioselective antagonists AF-DX 116 and gallamine, some differences were noted between M2 sites in brain and heart. The muscarinic adenylate cyclase response in the corpus striatum was relatively insensitive to the M2 selective antagonists AF-DX 116 and gallamine as well as the M1 selective antagonist pirenzepine, suggesting that non-M1, non-M2 sites inhibit adenylate cyclase activity in the corpus striatum. In contrast, the effects of muscarinic antagonists on the muscarinic adenylate cyclase response in the heart were consistent with the postulate that M2 receptors inhibit adenylate cyclase activity in this tissue.     

人食管下括约肌M2和M3受体的基因表达

目的 探讨人食管下括约肌的胆碱能受体基因表达和分布的情况，从分子水平揭示食管下括约肌的抗返流机制。方法 应用RT-PCR方法，研究钩状纤维和套索纤维中M2、M3的分布与表达。结果 钩状纤维、套索纤维M2和M3均高表达，M2的表达水平分别为0．411±0．023和0．409±0．044，M3的表达水平分别为0．141±0．016和0．145±0．023，而且M2比M3有较高的表达，两者差异有统计学意义（P〈0．01）．结论 在钧状纤维、套索纤维中．存在丰富的M2、M3,同时与M3相比，M2处于优势。     

Faecal peritonitis causes oedema and neuronal injury in pig cerebral cortex

Encephalopathy is a common complication of sepsis. However, little is known about the morphological changes that occur in the brain during sepsis. Faecal peritonitis was induced in pigs that were killed 8 h later and frontal cortex samples were taken immediately after death. The tissue was investigated using light and electron microscopy and compared with frontal cortex samples taken from sham-operated controls. Septic pigs had 49.5% more perimicrovessel oedema than sham pigs. However, the tight junctions between cerebral microvessel endothelial cells appeared morphologically intact in both septic and sham pigs. Sepsis also resulted in neuronal injury, disruption of astrocytic end-feet and swollen, rounded erythrocytes. These morphological changes may be sufficient to underlie the clinical features seen in septic encephalopathy.     

Analysis of neurotransmitter receptor distribution patterns in the cerebral cortex

The concentration of transmitter receptors varies between different locations in the human cerebral cortex, but also between the different cortical layers within the same area. Analyzing the regional differences in the laminar distribution patterns of various neurotransmitter receptor binding sites by means of quantitative receptor autoradiography may thus provide a functionally relevant insight into the organization of the cortex. Here we introduce an approach to the analysis of in vitro receptor autoradiographic data, providing a framework for the assessment of differences in both mean concentration and laminar distribution patterns across multiple subjects. Initially, laminar receptor distribution patterns for cortical areas are quantified by sampling density profiles in a series of regions of interest (ROIs) from digitalized autoradiographs and computing a mean profile per ROI. These ROI mean profiles are then corrected for distortions in the laminar pattern introduced by cortical folding and averaged to yield a mean profile per area, receptor and hemisphere. Differences in mean binding site concentration between areas are quantified by the asymmetry coefficient which is the difference of the mean concentrations divided by their sum. To quantify differences in laminar receptor distribution patterns between areas, the effects of absolute binding site concentration are first removed by dividing each profile by its mean value. Differences in the laminar pattern were then quantified by calculating the Euclidean distance between these mean corrected profiles. For single subject analysis, we propose a permutation test, comparing the differences between the mean profiles for two areas to differences between groups of profiles randomly assembled from all ROIs sampled in either area. Group inference can then be based on a between-subject conjunction analysis after correcting p-values to control for multiple comparisons. The feasibility of the presented approach is demonstrated by an exemplary analysis of the neurochemical differences between the ventral parts of the second and the third visual area.