Enhanced long-term potentiation in the hippocampus of rats expressing mutant presenillin-1 is age related

Electrophysiological recordings were made from Fischer rats engineered to express the human presenilin 1 gene carrying the M146V mutation. Extracellular recordings of field excitatory post-synaptic potential (EPSPs) were made to investigate EPSP properties, paired pulse responses, posttetanic potentiation, and long-term potentiation in the stratum radiatum and dentate gyrus of hippocampal slices maintained in vitro. Transgenic rats aged approximately 6 months showed no differences from their wild-type littermates in any of these properties. However, at 18 months, long-term potentiation in the CA1 was facilitated in the transgenic rats with a different pattern of synaptic enhancement. No changes were observed in paired pulse facilitation (PPF) or post-tetanic potentiation (PPT) and no changes were seen in the dentate gyrus. Field potential amplitudes were significantly greater and PPF was enhanced in the CA1 of all older rats. Intracellular recordings from CA1 pyramidal cells of the older group of rats revealed no differences in the passive or active membrane properties of cells in the two groups, but intracellularly recorded EPSPs were significantly longer.     

Estrogen and progesterone treatment: effects on muscarinic M(4) receptor subtype in the rat brain

We investigated the effect of ovariectomy (OVX) and hormonal treatment for 10 weeks by estradiol and progesterone on muscarinic M₄ receptor subtype in different brain areas of female rats. Moreover, motor activity of OVX and hormone-treated rats was measured by automated open field exploration boxes. Receptor quantification in the hippocampus, frontal cortex, parietal cortex, amygdala and hypothalamus was done by receptor autoradiography using a selective ligand for muscarinic M₄ receptors. Ovariectomy up-regulated M₄ receptors in the dentate gyrus, CA1, CA3, frontal cortex and hypothalamus whereas the estrogen treatment restored M₄ binding to that of the sham group. Progesterone treatment had no effect on the ovariectomy-induced up-regulation of M₄ receptors. Ovariectomy significantly decreased the exploratory activity of the rats compared to the sham group. Estrogen treatment restored the exploratory behavior of the ovariectomized rats to that of the sham group whereas the progesterone-treated rats were less alert to the surrounding when compared to the sham and estrogen supplemented rats. The effect of estrogen on the hippocampal muscarinic M₄ receptor subtype is a novel finding and may have functional significance for cholinergic receptors especially in relation to postmenopausal memory problems and neurodegenerative disease like Alzheimer’s disease.     

Loss of muscarinic M4 receptors in spinal cord of arthritic rats: implications for a role of M4 receptors in pain response

Changes in the levels of muscarinic M4 receptors in spinal cord of acute and chronic arthritic rats (animal models of pain) were studied by receptor autoradiography using muscarinic M4 receptor subtype selective ligand. Arthritis was induced in female Lewis rats by single intradermal injection of heat-killed Mycobacterium butyricum and sacrificed 12 days (acute group) and 30 days (chronic and control groups) after induction of arthritis. Our results demonstrate significant reduction in the level of M4 receptors in the spinal cord (Rexed laminae I-X) of acute and chronic arthritic rats compared to controls. These findings suggest that the muscarinic M4 receptor subtype may be involved in cholinergic mechanisms of analgesia.     

Differential modification of muscarinic cholinergic receptors in the hippocampus of patients with Alzheimer''s disease: an autoradiographic study

We have used quantitative light microscopic autoradiographic techniques to analyze changes in muscarinic cholinergic receptors in the hippocampus in Alzheimer type dementia (ATD). The density and distribution of muscarinic cholinergic receptors has been correlated with the density of neurons, neuritic plaques and neurofibrillary tangles in the CA1 subfield of the hippocampus of control and ATD patients. The number of pyramidal cells per mm² in the CA1 sector was significantly decreased in ATD cases as compared to controls, although there were large variations among cases. The most marked reductions in cell counts were observed in patients with a history of profound dementia. The densities of muscarinic receptors, as well as the proportions of M₁ and M₂ subtypes, in the CA1 sector and dentate gyrus were not significantly different between ATD and old non-demented patients. Neuritic plaques, even in high numbers, did not affect the density of muscarinic receptors; moreover, the densities of receptors over the neuritic plaques did not differ from the surrounding neuropil. However, in some ATD cases there was a marked decrease in the concentration of these receptors in the CA1 sector and subiculum, with no change in the proportions of muscarinic receptor subtypes. These patients exhibited frequent extracellular remnants of neurofibrillary tangles (ghost tangles), but scarce neuritic plaques, and were those showing severe losses of pyramidal cells. There was a significant positive correlation between the total concentration of muscarinic receptors in the CA1 and the density of pyramidal cells, suggesting that decreases in receptor concentration result from a severe neuronal loss. We observed that the ratio of muscarinic receptors per pyramidal cell was significantly increased in ATD patients. This might indicate a possible upregulatory mechanism for muscarinic receptors in the population of remaining neurons in ATD. However, decreases of receptor numbers following severe neuronal fall out suggest that compensatory mechanisms are no longer possible in such cases. The question is raised whether these differences between cases reflect different diseases or different stages of the same disease.     

The effect of cholinergic denervation and hippocampal sympathetic ingrowth on the internalization of muscarinic receptors in rat hippocampus

Following cholinergic denervation of the hippocampus by medial septal (MS) lesions, an unusual neuronal reorganization occurs in which peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the hippocampus (hippocampal sympathetic ingrowth; HSI). Previously, we have found that with MS lesions, animals with (the HSI⁽⁺⁾ group) and without (HSI⁽⁻⁾ group) ingrowth differed in carbachol stimulated PI hydrolysis, in PKC activity, and in muscarinic cholinergic receptors (mAChR). In this study, performed in hippocampal slices obtained four weeks after MS lesions, we utilized the hydrophilic muscarinic antagonist [³H] N-methylscopolamine ([³H]NMS) and hydrophobic muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) in the presence of either 4-α-phorbol or phorbol 12,13-dibutyrate (PDBu) to determine the effect of MS lesions with and without ingrowth on PKC-mediated mAChR internalization. In the presence of PDBu, a group effect was observed in [³H]NMS binding, with control groups > HSI⁽⁺⁾ group > HSI⁽⁻⁾ group. However, [³H]QNB binding was similar across groups. These results suggest that the cholinergic denervation of the hippocampus enhances the internalization of mAChRs, which is modified in the presence of HSI.     

Interaction of anandamide with the M(1) and M(4) muscarinic acetylcholine receptors

The M(1) and M(4) muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M(1) and M(4) receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [(3)H]N-methylscopolamine and [(3)H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M(4) (40-45%) than at the M(1) receptor (17-20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [(3)H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB(1) receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.     

Receptor reserve of phosphoinositide-coupled muscarinic receptors in mouse hippocampus in vivo

The ability of the partial muscarinic agonist pilocarpine to increase in vivo phosphoinositide (PI) hydrolysis in mouse brain was compared to two full agonists. Pilocarpine increased in vivo phosphoinositide (PI) hydrolysis in cortex, striatum, and to the greatest extent in the hippocampus. Pilocarpine injected either subcutaneously or intracerebroventricularly robustly increased in vivo PI hydrolysis in hippocampus up to 500% of control levels and the increases were blocked by the muscarinic antagonist scopolamine. The increases in vivo PI hydrolysis induced by pilocarpine were 60-75% of the magnitude of the full muscarinic agonists oxotremorine-M and cis-dioxolane. The muscarinic M(1) preferring antagonist pirenzepine potently blocked pilocarpine-induced increases in in vivo PI hydrolysis, consistent with the increase being mediated by M(1) receptors. Since pilocarpine is a relatively weak partial agonist, these data suggest a substantial level of receptor reserve for the PI response in mouse hippocampus.     

Estrogen receptor alpha-immunoreactive astrocytes are increased in the hippocampus in Alzheimer's disease

Postmenopausal estrogen use may decrease the risk, and delay the onset and progression, of Alzheimer's disease (AD). By means of fluorescence immunocytochemistry, the present study investigated the distribution of estrogen receptor alpha (ERalpha) in the human hippocampus in controls and in AD cases. ERalpha immunoreactivity was observed in neurons and glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the hippocampus both in controls and AD cases. The number and density of GFAP- and ERalpha-positive astrocytes was increased in AD. The number of GFAP-immunoreactive astrocytes, the number of nuclear ERalpha-staining astrocytes, and cytoplasmic ERalpha-staining astrocytes per unit area (1 mm(2)) significantly increased (P < 0.001, P < 0.05, P < 0.05, respectively) in CA1 in AD patients, while the percentage of ERalpha-immunoreactive astrocytes of the two groups did not differ (P > 0.05). These data suggest an important role for ERalpha-mediated effects of estrogens on neurons and astrocytes in the hippocampus of human and AD patients.     

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.     

GABAergic neurons of the rat dorsal hippocampus express muscarinic acetylcholine receptors

The expression of muscarinic acetylcholine receptors (mAChRs) in glutamic acid decarboxylase (GAD)-positive cells in the different strata of CA1, CA3, and the dentate gyrus (DG) of the dorsal hippocampus is examined by way of quantitative immunofluorescent double labeling employing M35, the monoclonal antibody raised against purified mAChR protein. Of all GAD-positive neurons, 97.5% express mAChRs. Conversely, 92.9% of the muscarinic cholinoceptive nonpyramidal neurons express GAD. These results indicate that the vast majority of the γ-aminobutyric acid (GABA)ergic neurons express mAChRs. In addition to GAD, parvalbumin (PARV) and somatostatin (SOM) are two neurochemical substances notably expressed in GABAergic neurons. In order to examine whether the entire muscarinic cholinoceptive nonpyramidal cell group can be characterized by these three GABAergic markers, a cocktail of GAD, PARV, and SOM was used in a fluorescent double-labeling experiment with M35. These results show that 97.2% of all muscarinic cholinoceptive nonpyramidal neurons can be neurochemically characterized by the content of GAD, PARV, and SOM. In conclusion, nearly all GABAergic cells express mAChRs and, conversely, virtually the entire muscarinic cholinoceptive nonpyramidal cell group belongs to the GABAergic cell population. This study, therefore, provides anatomical evidence for an extensive neuronal connectivity of the hippocampal muscarinic cholinoceptive nonpyramidal system and the inhibitory GABAergic circuitry.     

Neuroprotection by memantine against neurodegeneration induced by β-amyloid(1–40)

Progressive neuronal loss and cognitive decline in Alzheimer's disease (AD) might be aggravated by beta-amyloid-enhanced excitotoxicity. Memantine is an uncompetitive NMDA receptor antagonist under clinical development for the treatment of AD. Memantine has neuroprotective actions in several in vitro and in vivo models. In the present study, we determined whether memantine protected against beta-amyloid induced neurotoxicity and learning impairment in rats. Twenty Sprague-Dawley rats received vehicle or vehicle plus memantine (steady-state plasma concentrations of 2.34+/-0.23 microM, n=10) s.c. by osmotic pump for 9 days. After 2 days of treatment, 2 microl of water containing beta-amyloid 1-40 [Abeta(1-40)] were injected into the hippocampal fissure. On the ninth day of treatment, animals were sacrificed, and morphological and immunohistochemical techniques were used to determine the extent of neuronal degeneration and astrocytic and microglial activation in the hippocampus. Psychomotor activity and spatial discrimination were tested on the eighth day of treatment. Abeta(1-40), but not water, injections into hippocampus led to neuronal loss in the CA1 subfield, evidence of widespread apoptosis, and astrocytic and microglial activation and hypertrophy. Memantine treated animals had significant reductions in the amount of neuronal degeneration, pyknotic nuclei, and GFAP immunostaining as compared with vehicle treated animals. These data suggest that memantine, at therapeutically relevant concentrations, can protect against neuronal degeneration induced by beta-amyloid.     

Hippocampal sympathetic ingrowth and cholinergic denervation uniquely alter muscarinic receptor subtypes in the hippocampus

Following cholinergic denervation of the hippocampus by medial septal lesions, an unusual neoronal reorganization occurs, in which peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the hippocampus. Previously, we have found that both hippocampal sympathetic ingrowth (HSI) and cholinergic denervation (CD), alone, altered the total number and affinity of muscarinic cholinergic receptors (mAChR). In this study, we utilized the muscarinic antagonist [³H]Pirenzepine, in combination with membrane radioligand binding techniques, to determine the effects of HSI and CD on hippocampal M₁ and M₁ + M₃ mAChR subtypes, 4 weeks after MS lesions. In both the dorsal and ventral hippocampus, HSI was found to markedly diminish the number of M₁ AChRs, while CD was found to increase the number of M₁ AChRs. Neither treatment affected the affinity of the M₁ AChR. However, when M₁ + M₃ binding was assessed, CD was found to decrease the affinity in both hippocampal regions, without altering the number of receptors. Neither affinity nor number of M₁ + M₃ receptors was altered by HSI. The results of this study suggest that both cholinergic denervation and hippocampal sympathetic ingrowth uniquely affect hippocampal muscarinic receptors.     

Visual assessment of medial temporal lobe atrophy in demented and healthy control subjects: correlation with volumetry

The present study evaluated the validity of visual rating of medial temporal lobe atrophy on coronal magnetic resonance imaging scans in a population of demented and non-demented individuals. Medial temporal lobe atrophy in 194 subjects was visually rated from hard copies, using a 0–4 rating scale, and a comparison was made with the absolute volumes (ccm) of the medial temporal lobe as estimated with volumetry, using a stereological method. We found a highly significant correlation between the estimated and stereologically measured volumes. There was a 10-fold difference in time spent on rating medial temporal lobe atrophy (1–2 min) vs. time spent calculating the medial temporal lobe volume (10–12 min) on a single subject. The diagnostic accuracy of both methods showed that visual rating was more efficient than volumetry in differentiating Alzheimer’s disease from control subjects, We conclude that visual rating is a reliable and fast method to estimate medial temporal lobe atrophy in demented subjects in a clinical setting.     

M1 muscarinic receptor signaling in mouse hippocampus and cortex

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.     

Galantamine and behavior in Alzheimer disease: analysis of four trials

Kavanagh S, Gaudig M, Van Baelen B, Adami M, Delgado A, Guzman C, Jedenius E, Schäuble B. Galantamine and behavior in Alzheimer disease: analysis of four trials.
Acta Neurol Scand: 2011: 124: 302–308.
© 2011 John Wiley & Sons A/S. Objectives – Many individuals with Alzheimer’s disease (AD) experience behavioral and neuropsychiatric symptoms, which may cause caregiver distress and lead to the institutionalization of the patient. This analysis characterized behavioral symptoms and caregiver distress in trials of galantamine and their response to treatment. Materials and methods – Data were pooled from four randomized, placebo‐controlled clinical trials of galantamine in patients with mild to moderate AD (three studies) or AD plus cerebrovascular disease (one study) (n = 2177). Behavior and associated caregiver distress were assessed in each study using the Neuropsychiatric Inventory (NPI) and NPI distress (NPI‐D), respectively. Results – After 5/6 months, but not after 3 months, NPI score was significantly improved with galantamine vs placebo (P = 0.013). The benefit was particularly pronounced in patients categorized as having advanced moderate AD. At 5/6 months, there was a numerical benefit of galantamine over placebo in terms of caregiver distress; the difference was statistically significant in patients with moderate or advanced moderate AD. Conclusions – Galantamine reduces behavioral symptoms in patients with mild to moderate AD, leading to reduced caregiver burden. The reductions were greatest in patients with moderate or advanced moderate disease.     

Towards a high-affinity allosteric enhancer at muscarinic M1 receptors

Loss of forebrain acetylcholine (ACh) is an early neurochemical lesion in Alzheimer’s Disease (AD), and muscarinic receptors for ACh are involved in memory and cognition, so a muscarinic agonist could provide ‘replacement therapy’ in this disease. Muscarinic receptors, which couple to G-proteins, occur throughout the CNS, and in the periphery they mediate the responses of the parasympathetic nervous system, so selectivity is crucial. The five subtypes of muscarinic receptor, M₁–M₅, have a distinct regional distribution, with M₂ and M₃ mediating most of the peripheral effects, M₂ predominating in hindbrain areas, and M₁ predominating in the cortex and hippocampus—the brain regions most associated with memory and cognition, which has lead to a search for a truly M₁-selective muscarinic agonist. That search has so far been unsuccessful, but acetylcholinesterase inhibitors such as donepezil (Aricept), which potentiate cholinergic neurotransmission, have a therapeutic role in the management of AD; so the M₁ receptor remains a therapeutic target. Our approach is to develop allosteric enhancers—compounds which bind to the receptor at an ‘allosteric’ site which is distinct from the ‘primary’ site to which the endogenous ligand binds, and which enhance the affinity (or efficacy) of the endogenous ligand. We have developed radioligand binding assays and analyses for the detection and quantitatitation of allosteric interactions of a test agent with labelled and unlabelled ‘primary’ ligands, and we report here some results of the initial phase of a chemical synthesis project to develop potent and selective allosteric enhancers at muscarinic M₁ receptors.     

The effects of aging on muscarinic receptor/G-protein coupling in the rat hippocampus and striatum

In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age which has been traced to events early in the signal transduction pathway. Our laboratory has therefore focussed on investigations at this level. The current experiments investigate the effects of age on G-protein/receptor interactions by using competitive binding assays to measure the ability of GppNHp to decrease the proportion of receptors bound to G-proteins in the absence and the presence of added Mg2+. L-[3H]Quinuclidinyl benzilate was used as a nonselective ligand and [3H]pirenzepine as an M1 selective ligand. We find that: (1) muscarinic receptors and G-proteins in the striatum appear to become loosely coupled with age, with no change in Mg2+ sensitivity. (2) M1-receptor/G-protein complexes in the hippocampus display increased sensitivity to the presence of Mg2+ with age, with those from old but not young tissue requiring added Mg2+ in order to uncouple. This effect, however, may not be M1 specific.     

Brain muscarinic cholinergic receptors in Huntington's disease

Summary Muscarinic cholinergic receptors and choline acetyltransferase (ChAT) activity were studied in postmortem brain tissue from patients with Huntington's disease and matched control subjects. In comparison with controls, reductions in ChAT activity were found in the hippocampus, but not in the temporal cortex in Huntington's disease. Patients with Huntington's disease showed reduced densities of the total number of muscarinic receptors and of M-2 receptors in the hippocampus while the density of M-1 receptors was unaltered. Muscarinic receptor binding was unchanged in the temporal cortex. These results indicate a degeneration in Huntington's disease of the septo-hippocampul cholinergic pathway, but no impairment of the innominato-cortical cholinergic system.     

Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types

Summary Cholinergic nicotinic and muscarinic receptor binding were measured in post mortem human brain tissue, using low (nM) concentrations of (³H)-nicotine to detect predominately the high affinity nicotinic site and (³H)-N-methylscopolamine in the presence and absence of 3×10^–4 M carbachol to measure both the low and high affinity agonist subtypes of the muscarinic receptor group. Consistent with most previous reports, the nicotinic but not muscarinic binding was reduced in the different forms of dementia associated with cortical cholinergic deficits, including Alzheimer's and Parkinson's disease, senile dementia of Lewy body type (SDLT) and Down's syndrome (over 50 years). Analysis of (³H)-nicotine binding displaced by a range of carbachol concentrations (10^–9–10^–3 M) indicated 2 binding sites for nicotine and that the high affinity rather than low affinity site was reduced in Alzheimer's disease. In all 3 cortical areas investigated (temporal, parietal and occipital) there were increases in the low affinity muscarinic site in Parkinson's disease and SDLT but not Alzheimer's disease or middle-aged Down's syndrome. This observation raised the question of whether the presence of neurofibrilalry tangles (evident in the latter but not former 2 disorders) is incompatible with denervation-induced muscarinic supersensitivity in cholinoceptive neurons which include cortical pyramids generally affeced by tangle formation.