Pirenzepine Binding Research Articles

Lower levels of soluble β-amyloid precursor protein, but not β-amyloid, in the frontal cortex in schizophrenia

We identified a sub-group (25%) of people with schizophrenia (muscarinic receptor deficit schizophrenia (MRDS)) that are characterised because of markedly lower levels of cortical muscarinic M1 receptors (CHRM1) compared to most people with the disorder (non-MRDS). Notably, bioinformatic analyses of our cortical gene expression data shows a disturbance in the homeostasis of a biochemical pathway that regulates levels of CHRM1. A step in this pathway is the processing of β-amyloid precursor protein (APP) and therefore we postulated there would be altered levels of APP in the frontal cortex from people with MRDS. Here we measure levels of CHRM1 using [3H]pirenzepine binding, soluble APP (sAPP) using Western blotting and amyloid beta peptides (Aβ1–40 and Aβ1–42) using ELISA in the frontal cortex (Brodmann's area 6: BA 6; MRDS = 14, non-MRDS = 14, controls = 14). We confirmed the MRDS cohort in this study had the expected low levels of [3H]pirenzepine binding. In addition, we showed that people with schizophrenia, independent of their sub-group status, had lower levels of sAPP compared to controls but did not have altered levels of Aβ1–40 or Aβ1–42. In conclusion, whilst changes in sAPP are not restricted to MRDS our data could indicate a role of APP, which is important in axonal and synaptic pruning, in the molecular pathology of the syndrome of schizophrenia.

Lower levels of cortical [3H]pirenzepine binding to postmortem tissue defines a sub-group of older people with schizophrenia with less severe cognitive deficits

Multiple lines of evidence argue for lower levels of cortical muscarinic M1 receptors (CHRM1) in people with schizophrenia which is possibly due to a sub-group within the disorder who have a marked loss of CHRM1 (muscarinic receptor deficit sub-group (MRDS)). In this study we sought to determine if the lower levels of CHRM1 was apparent in older people with schizophrenia and whether the loss of CHRM1 was associated with symptom severity by measuring levels of cortical [3H]pirenzepine binding to CHRM1 from 56 people with schizophrenia and 43 controls. Compared to controls (173±6.3 fmol / mg protein), there were lower levels of cortical [3H]pirenzepine binding in the people with schizophrenia (mean±SEM: 153±6.0 fmol / mg protein; p=0.02; Cohen's d=- 0.46). [3H]pirenzepine binding in the people with schizophrenia, but not controls, was not normally distributed and best fitted a two-population solution. The nadir of binding separating the two groups of people with schizophrenia was 121 fmol / mg protein and levels of [3H]pirenzepine binding below this value had a 90.7% specificity for the disorder. Compared to controls, the score from the Clinical Dementia Rating Scale (CDR) did not differ significantly in MRDS but were significantly higher in the sub-group with normal radioligand binding. Positive and Negative Syndrome Scale scores did not differ between the two sub-groups with schizophrenia. Our current study replicates and earlier finding showing a MRDS within schizophrenia and, for the first time, suggest this sub-group have less severe cognitive deficits others with schizophrenia.

Pirenzepine Binding Sites in the Brain of the Honeybee Apis mellifera: Localization and Involvement in Non-Associative Learning.

Simple SummaryThe cholinergic system has been extensively studied in insects as the target of pesticides that affect the functioning of the nicotinic acetylcholine receptors. In the honeybee, the detrimental effects of pesticides are observed in behavior, including associative learning. The current work describes the expression of muscarinic acetylcholine receptors in the honeybee brain. We show that the muscarinic antagonist pirenzepine induces a slow-down of non-associative learning, the habituation of the proboscis extension reflex. We suggest that pirenzepine binding sites located on the neurons of the subesophagous ganglion are involved in the habituation of the reflex.Muscarinic acetylcholine receptors (mAChRs) play a central role in learning and memory in mammals as in honeybees. The results obtained in the honeybee Apis mellifera are based on the detrimental effects of the mAChR antagonists, atropine and scopolamine, on olfactory associative memory. Binding sites for the mAChR antagonist BODIPY® FL pirenzepine were localized in the brain of the honeybee forager. Pirenzepine binding sites were detected indifferently in several somata and neuropilar areas. The highest binding site densities were present in the central complex and in somata of the dorsomedial border of the antennal lobes. An additional binding pattern was found in somata of the subesophageal ganglion. By contrast, Kenyon cell (KC) somata were not stained. Pirenzepine (PZ) effects on non-associative learning were evaluated. Treated animals required more trials for the habituation of the proboscis extension reflex (PER) than controls, and the duration of the PER increased after PZ brain injection. These results suggest that the network mediating habituation of the PER involves PZ binding sites that are not necessarily present on the circuitry mediating olfactory conditioning of the PER.

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study

Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-DNA interaction studies are of great significance as it helps in the development of new therapeutic drugs. It provides a deeper understanding into the mechanism through which therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA (Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The values of binding constant from various experiments were calculated to be in the order of 103 M−1 which is consistent with the groove binding mode. Various spectrofluorimetric experiments like competitive displacement of well known dyes with drug, iodide quenching experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established by viscometric, circular dichroic and molecular modelling studies. Thermodynamic parameters obtained from isothermal titration calorimetric studies suggest that the interaction of pirenzepine with Ct-DNA is enthalpically driven. The value of TΔS and ΔH calculated from calorimetric studies were found to be 4.3 kcal mol−1 and -2.54 kcal mol−1 respectively, indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction and hydrogen bonding. The binding energy calculated was −7.5 kcal mol−1 from modelling studies which was approximately similar to that obtained by isothermal titration calorimetric studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-DNA cannot be precluded.

Muscarinic receptor binding changes in postmortem Parkinson's disease.

Parkinson's disease (PD) is a devastating disorder, affecting approximately 2% of people aged 60 and above. It is marked by progressive neurodegeneration that has long been known to impact dopaminergic cells and circuits, but more recently the acetylcholine system has also been implicated in the complex aetiology and symptomatology of the disease. While broad changes in cholinergic markers have been described, insight into the contribution of specific acetylcholine receptors is less clear. To address this important unknown, in this study we performed [3H] pirenzepine, [3H] 4DAMP, and [3H] AF-DX 384 in situ radioligand binding on postmortem tissues from Brodmann's area 6, 9, 46, and the caudate putamen, from PD and matched controls to detect muscarinic M1, M3, and M1/2/4 receptors, respectively. We found no difference in [3H] pirenzepine binding between PD and controls across all regions assessed. [3H] 4DAMP binding was found to be higher in PD CPu and BA9 than in controls. [3H] AF-DX 384 was higher in BA9 of PD compared with controls. In sum, we show selective increase in M3 receptors in cortical and subcortical regions, as well as increased M2/M4 in cortical area BA9, which together support a role for cholinergic dysfunction in PD.

Higher levels of different muscarinic receptors in the cortex and hippocampus from subjects with Alzheimer's disease.

It has been suggest that drugs specifically targeting muscarinic receptors will be useful in treating Alzheimer's disease. We decided to determine if the response to such drugs may be altered, because of changes in the levels of muscarinic receptors in the CNS from subjects with the disorder. We used in situ radioligand binding with autoradiography to measure the levels of [3H]pirenzepine binding to muscarinic M1 receptors, [3H]AF-DX 386 binding to muscarinic M1, M2, and M4 receptors, and [3H]4-DAMP binding to muscarinic M1 and M3 receptors in the dorsolateral prefrontal cortex and hippocampus from subjects with Alzheimer's and age/sex-matched controls. Compared with controls, [3H]pirenzepine binding was higher in the dentate gyrus from subjects with Alzheimer's disease. [3H]AF-DX 386 binding was higher in the subiculum and parahippocampal gyrus from subjects with the disorder. In Alzheimer's disease, [3H]-DAMP binding was higher in the dorsolateral prefrontal cortex but not different in the hippocampus. Our data show complex changes in the levels of muscarinic receptors in the CNS from subjects with Alzheimer's disease which may affect clinical response to treatment with drugs-targeting these receptors.

Changes in cholinergic and glutamatergic markers in the striatum from a sub-set of subjects with schizophrenia

Background/objectivesHaving separated a sub-group of people with schizophrenia based on a marked loss of cortical [3H]pirenzepine binding (MRDS); we wished to determine if MRDS had lower levels of [3H]pirenzepine and other muscarinic receptor antagonist binding to the striatum and if this was due to loss of pre- or post-synaptic neurons or glia measured using surrogate markers (25kilodalton synaptosomal-associated protein (SNAP 25), postsynaptic density protein 95 (PSD 95), glial fibrillary acidic protein (GFAP) 41/43) of cell number. Methods[3H]pirenzepine, [3H]AF-DX 384 and [3H]4-DAMP binding to the striatum from 37 subjects with schizophrenia (19 MRDS) and 20 controls as well as SNAP 25, PSD 95 and GFAP 41/43 in crude particulate membrane were measured. Results[3H]pirenzepine and [3H]AF-DX 384 binding to the striatum were significantly lower in schizophrenia due to lower binding of both radioligands in the striatum from MRDS. Levels of PSD 95 were higher in schizophrenia, predominantly due to higher levels in MRDS. ConclusionsOur data suggest muscarinic M1 ([3H]pirenzepine) and M2 and/or M4 receptors ([3H]AF-DX 384) are lower in the striatum from MRDS which could mediate inappropriate adaption to internal and external cues which, in turn, would affect motivation, cognition and motor control. Increased levels of PSD 95 could indicate increased post-synaptic boutons or changes in NMDA receptor-mediated signalling in MRDS.

Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

An investigation of the factors that regulate muscarinic receptor expression in schizophrenia

We previously identified a group of subjects with schizophrenia who, on average, have a 75% decrease in cholinergic receptor, muscarinic 1 (CHRM1) in Brodmann's area (BA) 9. To extend this finding, we determined i) if the decrease in CHRM1 was present in another functionally related CNS region (BA6), ii) whether the marked decrease in CHRM1 was accompanied by changes in levels of other CHRMs and iii) potential factors responsible for the decreased CHRM1 expression. We measured CHRM1 and CHRM3 using in situ radioligand binding with [3H]pirenzepine and [3H]4-DAMP respectively in BA6 from 20 subjects with schizophrenia who had low levels of CHRM1 in BA9 (SzLow[3H]PZP), 18 subjects with schizophrenia whose levels of CHRM1 were similar to controls (SzNormal[3H]PZP) and 20 control subjects. Levels of CHRM1, 3 and 4 mRNA were measured using qPCR and levels of the transcription factors, SP1 and SP3, were determined using Western blots. In BA6, the density of [3H]pirenzepine binding was decreased in subjects with SzLow[3H]PZP (p<0.001) compared to controls. The density of [3H]4-DAMP binding, levels of CHRM1, 3 and 4 mRNA and levels of SP1 and SP3 was not significantly different between the three groups. This study shows that the previously identified decrease in CHRM1 expression is not confined to the dorsolateral prefrontal cortex but is present in other cortical areas. The effect shows some specificity to CHRM1, with no change in levels of binding to CHRM3. Furthermore, this decrease in CHRM1 does not appear to be associated with low levels of CHRM1 mRNA or to simply be regulated by the transcription factors, SP1 and SP3, suggesting that other mechanisms are responsible for the decreased CHRM1 in these subjects.

Simvastatin reverses the downregulation of M1/4 receptor binding in 6-hydroxydopamine-induced parkinsonian rats: The association with improvements in long-term memory

BackgroundIt is believed that muscarinic M1/4 receptors are closely correlated to the dopaminergic system and are strongly involved in the pathogenesis of Parkinson’s disease (PD). In addition to regulating lipid metabolism and protection from stroke, statins have been used to regulate the declined cognition. We aimed to explore the regional changes in M1/4 receptors in the 6-hydroxydopamine (6-OHDA)-lesioned rat brain. MethodsPD rat model was set up by injecting 6-OHDA into the unilateral medial forebrain bundle; while simvastatin (10mg/kg/day) or saline was orally administrated for 3weeks, respectively. Long-term memory was measured using the Morris water maze. [3H]pirenzepine binding autoradiography was applied to investigate the alterations of M1/4 receptors in the PD rat brains. Results6-OHDA induced long-term memory deficits along with downregulation of M1/4 receptors in the hippocampus, the medial amygdala, the posteromedial cortical and the piriform cortex; simvastatin administration significantly ameliorated the impaired memory and reversed the downregulation of M1/4 receptors in the examined brain regions. Profound positive correlations were verified between the decline in long-term memory activity and the restoration of M1/4 receptors in different brain regions after simvastatin treatment. Conclusions/significanceOur results provide strong evidence that M1/4 receptor modulation after simvastatin administration did, at least partially, contribute to the improvement in the long-term memory in 6-OHDA-induced PD rats. These results provide a possible mechanism for simvastatin treatment in psycho-neurological diseases such as PD via M1/4 receptors.

Widespread decreases in cortical muscarinic receptors in a subset of people with schizophrenia

These studies were undertaken to investigate the selectivity of cortical muscarinic receptor radioligand binding in muscarinic M(1) and M(4) receptor knockout mice and to determine whether a marked decrease in [(3)H]pirenzepine binding in Brodmann's area (BA) 9 from a subset of people with schizophrenia was predictive of decreased muscarinic receptors in other central nervous system (CNS) regions. Our data show that, under the conditions used, [(3)H]pirenzepine binding was highly selective for the muscarinic M(1) receptor whereas both [(3)H]AF-DX 386 and [(3)H]4DAMP had less discriminatory power. In addition, the data suggest that a marked decrease in [(3)H]pirenzepine binding in BA 9 from a subset of people with schizophrenia is predictive of decreases in muscarinic receptors in other CNS regions. However, there were some region-specific decreases in muscarinic receptors in tissue from people with schizophrenia who were outside this subset. These data add to a growing body of evidence suggesting there are widespread decreases in muscarinic receptors in the CNS of some subjects with schizophrenia, as demonstrated by neuroimaging. Our data have implications for understanding the potential clinical utility of drugs directed at the orthosteric and allosteric sites of muscarinic receptors to treat schizophrenia.

Levels of Ionotropic Glutamate and Muscarinic Receptors in Three Animal Models of Schizophrenia

There are well validated rodent paradigms of schizophrenia which are based on environmental manipulation (e.g. altered rearing conditions) or drug challenges. These manipulations induce behavioural changes in rodents that are thought to involve neuronal circuitry similar to the ones that are affected by the pathophysiology of the disorder. This study has investigated whether three such rodent paradigms (isolation rearing, neonatal PCP treatment or sub-chronic PCP treatment) are associated with changes in muscarinic receptors (CHRMs) or ionotropic glutamate receptors, some of which have been reported to be altered in the CNS of subjects with schizophrenia. ( 3 H)pirenzepine (CHRM1), ( 3 H)4DAMP (CHRM1/CHRM3), ( 3 H)MK801 (NMDA receptors) and ( 3 H)kainate (kainate receptors; KAR) binding were measured using in situ radioligand binding and autoradiography. Isolation rearing caused widespread decreases in ( 3 H)4DAMP (p = 0.01) and ( 3 H)kainate binding (p = 0.03). Neonatal PCP caused widespread increases in ( 3 H)4DAMP binding (p <0.0001), whereas sub-chronic PCP treatment caused widespread decreases in the binding of that radioligand (p < 0.002) and widespread increases in (3H)MK801 binding (p < 0.0001). There were no changes in ( 3 H)pirenzepine binding to CHRM1 receptors in any paradigm or no significant within region changes in the binding of any radioligand. In conclusion, in the absence of any changes in CHRM1 receptors, our ( 3 H)4DAMP and the binding of (3H)MK801 data would suggest that different rodent paradigms cause variable changes in levels of CHRM3 and KAR in the rat CNS. Our data raises the possibility that such changes may, in part, modulate the behavioural differences that have been observed after isolation rearing, neonatal PCP treatment or sub-chronic PCP treatment.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

Pirenzepine Promotes the Dimerization of Muscarinic M1 Receptors through a Three-step Binding Process

Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.

Fustin flavonoid attenuates β‐amyloid (1–42)‐induced learning impairment

Natural flavonoids ameliorate amyloid-beta peptide (Abeta)-induced neurotoxicity. We examined whether the fustin flavonoid affects Abeta-induced learning impairment in mice. Repeated treatment with fustin significantly attenuated Abeta (1-42)-induced conditioned fear and passive avoidance behaviors. This effect was comparable to that of EGb761, a standard extract of ginkgo. Fustin treatment significantly prevented decreases in acetylcholine (ACh) levels, choline acetyltransferase (ChAT) activity, and ChAT gene expression induced by Abeta (1-42). Fustin also consistently suppressed increases in acetyl cholinesterase (AChE) activity and AChE gene expression induced by Abeta (1-42). In addition, fustin significantly attenuated Abeta (1-42)-induced selective decreases in muscarinic M1 receptor gene expression and muscarinic M1 receptor binding activity (as determined by [(3)H]pirenzepine binding) by modulating extracellular signal-regulated kinase 1/2 (ERK 1/2) and cAMP response-element binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) expression. These effects of fustin were reversed by treatment with dicyclomine, a muscarinic M1 receptor antagonist, and SL327, a selective ERK inhibitor, but not by chelerythrine, a pan-protein kinase C (PKC) inhibitor. Taken together, our results suggest that fustin attenuates Abeta (1-42)-impaired learning, and that the ERK/CREB/BDNF pathway is important for the M1 receptor-mediated cognition-enhancing effects of fustin.

Altered M1 Muscarinic Acetylcholine Receptor (CHRM1)-Gαq/11 Coupling in a Schizophrenia Endophenotype

Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M(1) subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Galpha(q/11)-[35S]-GTPgammaS binding was significantly decreased in the MRDS group (pEC(50) (M)=5.69+/-0.16) compared with the control group (6.17+/-0.10) and the non-MRDS group (6.05+/-0.07). The levels of Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPgammaS binding to Galpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with schizophrenia displaying markedly decreased PZP binding (MRDS) we have shown that although agonist potency may decrease, the efficacy of CHRM1-Galpha(q/11) coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia.

Decreased muscarinic receptor binding in the frontal cortex of bipolar disorder and major depressive disorder subjects

Background Dysfunction of the cholinergic muscarinic receptors has been implicated in the pathology of bipolar disorder and major depressive disorder. However, there is conflicting evidence regarding the association between individual muscarinic receptors and the two disorders. Methods We used the muscarinic receptor selective radioligands [ 3H]pirenzepine, [ 3H]AFDX-384 and [ 3H]4-DAMP to measure the levels of muscarinic 1 (CHRM1) and muscarinic 4 (CHRM4) receptors, muscarinic 2 (CHRM2) and muscarinic 4 (CHRM4) receptors and muscarinic 3 (CHRM3) receptor, respectively. Radioligand binding was measured in Brodmann's area (BA) 10 of the rostral prefrontal cortex, BA 46 of the dorsolateral prefrontal cortex and BA 40 of the parietal cortex in the post-mortem CNS from subjects with bipolar disorder or major depressive disorder and control subjects. Results [ 3H]AFDX-384 binding was decreased in BA 46 in both bipolar disorder ( p < 0.01) and major depressive disorder ( p < 0.05). [ 3H]4-DAMP binding was decreased in BA 10 in bipolar disorder ( p < 0.05) but not major depressive disorder ( p > 0.05). [ 3H]AFDX-384 and [ 3H]4-DAMP binding were unaltered in any other cortical region examined for either disorder ( p > 0.05). [ 3H]pirenzepine binding was not significantly altered in either disorder in any cortical region examined ( p > 0.05). Limitations 9 bipolar disorder, 9 major depressive disorder and 19 control subjects were used in the study. Conclusion Our data is consistent with previously published data implicating a role for CHRM2 receptors in the pathology of bipolar and major depressive disorder. The demonstration of a novel association between decreased CHRM3 receptor expression and bipolar disorder suggests bipolar and major depressive disorder differs in the underlying nature of their cholinergic dysfunction.

Perinatal PCP treatment alters the developmental expression of prefrontal and hippocampal muscarinic receptors

Perinatal phencyclidine (PCP) treatment has been used to model brain pathological processes that may be present in schizophrenia such as increased apoptosis during early brain development, and long-term alterations in expression of parvalbumin-containing interneurons and glutamatergic N-methyl- d-aspartate (NMDA) receptors. We report that this treatment also affects receptor expression of another excitatory neurotransmitter receptor, the muscarinic receptor. Female rat pups received injections of the NMDA receptor antagonist PCP (10 mg/kg, s.c.) or saline on postnatal days (PN)7, 9 and 11. [ 3H]Pirenzepine binding to M1/4 receptors was examined at four time-points (PN12, 18, 32 and 96) following treatment cessation. Significant effects of treatment on [ 3H]pirenzepine binding were evident immediately after treatment cessation with a decrease in PCP-treated rats at PN12 in the prefrontal cortex (− 24%, p < 0.05) and hippocampus (− 19%, p < 0.05). After this initial decrease, binding subsequently increased to 47% above control levels in the prefrontal cortex of adolescent animals, which remained elevated in adulthood (+ 10%, p < 0.05), while in the hippocampus there was a trend towards increased binding in adolescent animals and no change thereafter. This work adds to findings demonstrating that perinatal PCP exposure leads to long-term imbalance of excitatory and inhibitory neurotransmitter systems, supporting its relevance as a developmental model of schizophrenia pathology. Alterations in muscarinic receptor expression may contribute specifically to the cognitive impairments reported to occur after perinatal NMDA receptor antagonist treatment.

Levels of [ 3H]pirenzepine binding in Brodmann's area 6 from subjects with schizophrenia is not associated with changes in the transcription factor SP1 or BACE1

Decreased muscarinic M1 receptor (CHRM1) mRNA has been reported in Brodmann's area (BA) 6 from subjects with schizophrenia. We have extended this study by measuring levels of CHRM1 ([ 3H]pirenzepine binding), CHRM3 ([ 3H]4-DAMP binding), the transcription factor SP1 and the CHRM1 downstream target beta-site APP-cleaving enzyme 1 (BACE1) in BA 6 from 19 subjects with schizophrenia and 19 control subjects. Radioligand binding was quantified using either in situ radioligand binding with autoradiography or, in cohorts of 10 control subjects and 10 subjects with schizophrenia, membrane enriched fraction (MEF) CNS ([ 3H]pirenzepine binding only). Levels of SP1 and BACE1 were measured by Western blotting. [ 3H]pirenzepine binding to tissue sections was in two layers, binding to tissue sections (Binding layer 1: p < 0.01; Binding layer 2: p < 0.001) and MEF ( p < 0.05) were decreased in schizophrenia. Levels of [ 3H]4-DAMP binding, SP1 and BACE1 were not altered in subjects with the disorder. This study shows a decrease in levels of CHRM1 in BA 6 from subjects with schizophrenia; as CHRM1 and BA 6 are important in maintaining normal cognitive function, these data support the hypothesis that decreased levels of cortical CHRM1 may contribute to the cognitive deficits associated with schizophrenia. Our findings on BACE1 suggest that the schizophrenia phenotype reported in BACE −/− mice is not simply due to lack of that protein in the cortex.

GLUTAMATERGIC MODULATION OF PARVALBUMIN MESSENGER RNA-CONTAINING NEURONS VIA THE N-METHYL D-ASPARTATE RECEPTOR IN THE PREFRONTAL CORTEX IN SCHIZOPHRENIA

Decreased muscarinic M1 receptor (CHRM1) mRNA has been reported in Brodmann's area (BA) 6 from subjects with schizophrenia. We have extended this study by measuring levels of CHRM1 ([3H]pirenzepine binding), CHRM3 ([3H]4-DAMP binding), the transcription factor SP1 and the CHRM1 downstream target beta-site APP-cleaving enzyme 1 (BACE1) in BA 6 from 19 subjects with schizophrenia and 19 control subjects. Radioligand binding was quantified using either in situ radioligand binding with autoradiography or, in cohorts of 10 control subjects and 10 subjects with schizophrenia, membrane enriched fraction (MEF) CNS ([3H]pirenzepine binding only). Levels of SP1 and BACE1 were measured by Western blotting. [3H]pirenzepine binding to tissue sections was in two layers, binding to tissue sections (Binding layer 1: p < 0.01; Binding layer 2: p < 0.001) and MEF (p < 0.05) were decreased in schizophrenia. Levels of [3H]4-DAMP binding, SP1 and BACE1 were not altered in subjects with the disorder. This study shows a decrease in levels of CHRM1 in BA 6 from subjects with schizophrenia; as CHRM1 and BA 6 are important in maintaining normal cognitive function, these data support the hypothesis that decreased levels of cortical CHRM1 may contribute to the cognitive deficits associated with schizophrenia. Our findings on BACE1 suggest that the schizophrenia phenotype reported in BACE−/− mice is not simply due to lack of that protein in the cortex.