Cholinergic phytochemicals: From magic to medicine

The decline in cortical cholinergic activity in aging and degenerative diseases associated with dementia is, in conjunction with increasing genetic and molecular complexity, one of the most consistent observations in psychopathology. Numerous plants synthesize compounds, interacting with cholinergic systems which have been of value in exploring the role of the cholinergic system in aging and dementia. These chemicals deter predators by interfering with autonomic, central nervous or neuromuscular systems. The original hypothesis that memory loss in Alzheimer's disease relates to a central cholinergic deficiency was partly based on the experimental induction of related cognitive impairments in normal individuals exposed to the naturally occurring muscarinic antagonist scopolamine. Long before this, in ancient Greece, henbane (containing alkaloids such as atropine and scopolamine) was used to mimic 'dementia' and provoke prophesy. The tendency of such tropane alkaloids to induce hallucinations is consistent with low neocortical activities and the high incidence of psychotic features in Dementia with Lewy bodies (DLB). Other plant derived alkaloids such as physostigmine and galanthamine, inhibiting acetylcholinesterase, are among those including the synthetic chemical tacrine, being tested in dementia therapy. Cognitive enhancement, although significant, is limited and antipsychotic effects may be more prominent. The recent discovery of brain nicotinic receptors and the neuroprotective role of the tobacco alkaloid nicotine provides a further dimension to cholinergic therapy in retarding the neurodegenerative process. The potential for new cholinergic drug development based on the rich biodiversity of plant chemicals and principles of ethnobotany, in the context of modern pharmacology, is still largely unexplored in age-related mental disorders. Several plant species reputed to improve memory in European medicinal plant encyclopaedias have cholinergic activities which may be therapeutically relevant.     

Nicotinic receptors in dementia of Alzheimer, Lewy body and vascular types

Objectives - Comparisons were made of nicotinic receptors in 3 major forms of dementia in old age. Although it is well established the involvement of nicotinic receptors in Alzheimer's disease (AD), their status in the other two main causes of dementia in old age – dementia with Lewy bodies (DLB) and vascular dementia (VaD) is not widely reported. Methods – Temporal cortex was examined for epibatidine and α-bungarotoxin binding, and immunoreactivity of α4 and α7 nAChR subunits. Results – There were selective abnormalities in nicotinic receptor subtypes in the disorders examined. In AD there is a loss of high affinity receptor binding, reflecting a selective loss of α4 subunit, but no change in α7 subunits. Similar abnormalities in ligand binding are also apparent in DLB. In the VaD series, there was no overall loss of epibatidine binding or immunoreactivity for α4 or α7 subunits. Conclusions – Loss of cortical receptor α4 subunit appears to be a characteristic feature of neurodegenerative dementia but not dementia of vascular origin. Since nicotinic receptors control cerebral vasodilation, the relative integrity of the receptors in VaD may auger well for nicotinic therapy in this disorder in which there is a cholinergic abnormality, to judge by the loss of the presynaptic enzyme.     

A postmortem study of brain nicotinic receptors in Parkinson's and Alzheimer's disease

Brain nicotinic receptors were studied in the frontal cortex, temporal cortex, hippocampus and caudate nucleus in patients with Parkinson's disease (PD), Alzheimer's disease (AD) and controls. The B_max and K_d values of (−)-[³H]nicotine binding were determined with a Scatchard analysis. The number of nicotinic receptors declined both in PD and in AD patients in all brain areas examined. The K_d values were unchanged. There was a negative correlation between the degree of dementia in PD patients and the number of nicotinic receptors in the frontal cortex. A similar correlation was seen between the muscarinic/nicotinic receptor ratio in the frontal cortex and the degree of dementia in PD patients. The present findings indicate that nicotinic receptors are affected not only in AD, but also in PD and that dysfunction of the cholinergic system in the frontal cortex is involved in the dementia process in PD.     

Critical Role of Presynaptic Nicotinic ACh Receptor in the Formation of Long-Term Potentiation: Implications for Development of Anti-Dementia Drugs

Background : Since neuronal nicotinic ACh receptors are involved in the cognitive function, they have been studied as a target of anti‐dementia drugs. The present study was designed to understand the role of nicotinic ACh receptors in the expression of long‐term potentiation (LTP), a cellular model of learning and memory. Methods : The ultrastructural localization of neuronal nicotinic ACh receptors in the rat hippocampus was examined electron‐immunohistochemically using an antibody against the α7 subunit, forming a brain‐type nicotinic ACh receptor. Miniature excitatory postsynaptic currents (mEPSCs) were monitored in cultured rat hippocampal neurons. Schaffer collateral‐CA1 LTP and perforant path LTP were analyzed by recording field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs) in the CA1 region and the dentate gyrus of rat hippocampal slices or in the intact mouse hippocampus. Results : α7 receptors are preferentially localized on presynaptic terminals, where the receptors are employed in the release of the excitatory neurotransmitter, glutamate. The probability of LTP development was markedly reduced in the presence of the neuronal nicotinic ACh receptor antagonists, α‐bungarotoxin and mecamylamine, in both the CA1 region and the dentate gyrus of rat hippocampal slices. Perforant path LTP was never induced in slices with selective cholinergic denervation using 192 IgG‐saporin, while it was not affected by atropine, a selective muscarinic ACh receptor antagonist, in normal slices. Nicotine facilitated hippocampal neurotransmission with the saturation occluding the potentiation induced by tetanic stimulation, and vice versa. A similar occlusion was also obtained with an intact mouse hippocampus. These types of LTP, which are dependent upon N‐methyl‐D‐aspartate (NMDA) receptors, were still induced by treatment with nicotine in the presence of D‐2‐amino‐5‐phosphonovaleric acid (APV), a selective NMDA receptor antagonist. Conclusion : The results of the present study suggest that presynaptic nicotinic ACh receptors play a critical role as a target of retrograde messengers in the formation of NMDA receptor‐dependent LTP. This may account for the involvement of nicotinic ACh receptors in cognitive function. Drugs enhancing the activity of neuronal nicotinic ACh receptors, therefore, are capable of expressing LTP, conversely, ameliorating dementia.     

Cholinergic therapy in dementia

After reviewing the evidence for cholinergic pathology in Alzheimer's disease and related disorders, this paper reviews strategies for treating dementia using cholinomimetic drugs. Special attention is paid to cholinesterase inhibitors, particularly tacrine, the drug recently approved by the FDA. New studies suggesting that muscarinic and nicotinic cholinergic receptor active drugs may be more effective will be reviewed. Brief mention will be made of strategies to slow the progression of Alzheimer's disease.     

Immunologic similarities between the hypothalamic α-bungarotoxin receptor and theTorpedo californica nicotinic cholinergic receptor

The solubilized rat central nervous system (hypothalamic) nicotinic cholinergic receptor and the Torpedo nicotinic cholinergic receptors are immunologically similar technique. Antibodies to the Electrophorus and Torpedo receptors also decrease the rate of α-bungarotoxin binding to these membraneous receptors. It is concluded that the Torpedo and hypothalamic nicotinic receptors are immunologically similar and that receptor binding sites for α-bungarotoxin and antibodies are physically close. These studies indicate that α-bungarotoxin can be used to study the nicotinic cholinergic receptor of the rat hypothalamus.     

Autoradiographic comparison of cholinergic and other transmitter receptors in the normal human hippocampus

The vulnerability of the human hippocampal complex to disease, trauma, and aging indicates the necessity to target this area therapeutically. The distribution and density of transmitter receptors provide a rational basis for this approach, and in this study the topography of 11 different pharmacological sites is compared with the cholinergic innervation, which is partcularly vulnerable in dementia. The regional distribution of cholinergic innervation to the normal adult human hippocampus and adjacent cortex, marked by acetylcholinesterase (AChE) fiber and terminal reactivity, is notable for its concentration in CA2/3 of Ammon's horn and the dentate fascia. Neither nicotinic (high-affinity nicotine binding) nor muscarinic (“M₁” or “M₂”) cholinergic receptor binding paralleled this distribution. In Ammon's horn, 5-HT₂ and kainate receptor binding more closely resembled the pattern of AChE, being concentrated in CA2–4 compared with CA1. By contrast, muscarinic M₁ and M₂, 5-HT_1A, benzodiazepine (including zolpidem-insensitive binding), NMDA (MK801), and AMPA/QUIS receptors were higher in CA1 and/or subiculum. Kainate binding, like AChE, was high in CA4. 5-HT₂ and nicotinic binding partially mimicked the pattern of AChE around the granule layer. In the subicular complex and parahippocampal gyrus, where cholinergic activity is relatively lower, muscarinic, 5-HT_1A, and benzodiazepine binding were relatively high and the nicotinic receptor was remarkable for its highest density compared to other areas examined. In stratum lacunosum-moleculare of CA1, which was relatively low in AChE activity, there was a dense band of nicotinic, M₂, and benzodiazepine receptor binding. These observations, while reflecting the anatomical complexity of chemical signaling in the hippocampal region, indicate a unique distribution for the nicotinic receptor and suggest that the cholinergic input may specifically interact with 5-HT and excitatory amino acid systems via 5-HT₂ and kainate receptor subtypes in governing signaling to the dentate and CA3 regions.     

Colocalization of muscarinic and nicotinic receptors in cholinoceptive neurons of the suprachiasmatic region in young and aged rats

In the present study muscarinic and nicotinic cholinergic receptors in the SCN region were demonstrated and analyzed, employing monoclonal antibodies to purified muscarinic and nicotinic cholinergic receptor proteins. A near-total colocalization of the two acetylcholine receptor subclasses in cholinoceptive neurons of the SCN area was found. The antibodies applied to aging rat brain (at 30–34 months) revealed a clear decrease in immunoreactivity in senescence albeit with a high level of individual variability. Furthermore, in 8 out of 10 aged animals examined a considerable increase of astrocytes possessing muscarinic cholinergic receptors was observed.     

Upregulation of neuronal nicotinic receptor subunits alpha4, beta2, and alpha7 in transgenic mice overexpressing human acetylcholinesterase

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits α4, β2, and α7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [³H]epibatidine and [¹²⁵I]αbungarotoxin binding in the cortex and the caudate putamen of these mice. Quantitative in situ hybridization showed significant upregulation of mRNA corresponding to the nicotinic receptor subunits α4, β2, and α7 in various brain regions in the transgenic mice compared to nontransgenic controls. Our results suggest that disruption of balanced cholinergic transmission by constitutive overexpression of acetylcholinesterase is accompanied by variable upregulation of several nicotinic receptor subtypes, in particular these associated with cholinergic terminals participating in compensatory response.     

Combined blockade of cholinergic receptors shifts the brain from stimulus encoding to memory consolidation

High central nervous system levels of acetylcholine (ACh) are commonly regarded as crucial for learning and memory, and a decline in cholinergic neurotransmission is associated with Alzheimer's dementia. However, recent findings revealed exceptions to this rule: The low ACh tone characterizing slow wave sleep (SWS) has proven necessary for consolidation of hippocampus-dependent declarative memories during this sleep stage. Such observations, together with recent models of a hippocampal-neocortical dialogue underlying systems memory consolidation, suggest that high levels of ACh support memory encoding, whereas low levels facilitate consolidation. We tested this hypothesis in human subjects by blocking cholinergic neurotransmission during wakefulness, starting 30 min after learning. Subjects received the muscarinic antagonist scopolamine (4 microg/kg bodyweight intravenously) and the nicotinic antagonist mecamylamine (5 mg orally). Compared to placebo, combined muscarinic and nicotinic receptor blockade significantly improved consolidation of declarative memories tested 10 hr later, but simultaneously impaired acquisition of similar material. Consolidation of procedural memories, which are not dependent on hippocampal functioning, was unaffected. Neither scopolamine nor mecamylamine alone enhanced declarative memory consolidation. Our findings support the notion that ACh acts as a switch between modes of acquisition and consolidation. We propose that the natural shift in central nervous system cholinergic tone from high levels during wakefulness to minimal levels during SWS optimizes declarative memory consolidation during a period with no need for new memory encoding.     

Nicotinic receptor abnormalities in Alzheimer''s and Parkinson''s diseases.

The status of cholinergic receptors in dementia is related to the question of potential cholinergic therapy. Whilst muscarinic receptor binding is generally reported to be normal or near normal, findings are reported which indicate substantial reductions of hippocampal nicotinic (high affinity nicotine) binding (occurring in conjunction with decreased choline acetyltransferase) in both Alzheimer's and Parkinson's but not Huntington's disease. A further indication that nicotinic receptor function may be abnormal in Alzheimer's disease is the extensive loss of an endogenous compound, detected for the first time in human brain, which inhibits normal nicotinic binding. Both receptor binding and the inhibitor are also substantially decreased with increasing age in the normal hippocampus.     

Nicotinic Receptors, Amyloid-β, and Synaptic Failure in Alzheimer’s Disease

Dysfunctional cholinergic transmission is thought to underlie, at least in part, memory impairment and cognitive deficits in Alzheimer’s disease (AD). However, it is still unclear whether this is a consequence of the loss of cholinergic neurons and elimination of nicotinic acetycholine receptors (nAChRs) in AD brain or of a direct impact of molecular interactions of the amyloid-β (Aβ) peptide with nAChRs, leading to dysregulation of receptor function. This review examines recent progress in our understanding of the roles of nicotinic receptors in mechanisms of synaptic plasticity, molecular interactions of Aβ with nAChRs, and how Aβ-induced dysregulation of nicotinic receptor function may underlie synaptic failure in AD.     

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.     

Impairment of cholinergic neurotransmission in adult and aged transgenic Tg2576 mouse brain expressing the Swedish mutation of human beta-amyloid precursor protein

To address the question of whether beta-amyloid peptides also affect cholinergic neurotransmission in vivo, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages ranging from 7 to 24 months were examined by immuno- and histochemical staining for choline acetyltransferase (ChAT) and acetycholinesterase (AChE), by assaying cholinergic enzyme activities and high-affinity choline uptake as well muscarinic and nicotinic cholinergic receptor binding levels by quantitative autoradiography. Cortical and hippocampal activities of AChE and ChAT were not different between transgenic mice and non-transgenic littermates regardless of the postnatal ages examined. However, high-affinity choline uptake was reduced in the hippocampus of 21-month-old transgenic mice. In brains of 8-month-old transgenic mice which do not yet demonstrate cortical beta-amyloids, reduced binding levels of cortical and hippocampal M1-muscarinic cholinergic receptors were observed, which were still reduced in 17-month-old transgenic mouse brains with high plaque load as compared to non-transgenic littermates. M2-muscarinic cholinergic receptor binding was hardly affected in brains from 8-month-old transgenic mice, but in 17-month-old transgenic mice reduced cortical and hippocampal binding levels were observed as compared to non-transgenic controls. Decreased cortical nicotinic cholinergic receptor binding was detected in 17-month-old transgenic mice. The development of changes in cholinergic synaptic markers in transgenic Tg2576 mouse brain before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cholinergic neurotransmission and may be referred to the deficits in learning and memory also observed in these mice before significant plaque load.     

Aromatherapy as a safe and effective treatment for the management of agitation in severe dementia: the results of a double-blind,placebo-controlled trial with Melissa

BACKGROUND: Behavioral and psychological symptoms in dementia are frequent and are a major management problem, especially for patients with severe cognitive impairment. Preliminary reports have indicated positive effects of aromatherapy using select essential oils, but there are no adequately powered placebo-controlled trials. We conducted a placebo-controlled trial to determine the value of aromatherapy with essential oil of Melissa officinalis (lemon balm) for agitation in people with severe dementia. METHOD: Seventy-two people residing in National Health Service (U.K.) care facilities who had clinically significant agitation in the context of severe dementia were randomly assigned to aromatherapy with Melissa essential oil (N = 36) or placebo (sunflower oil) (N = 36). The active treatment or placebo oil was combined with a base lotion and applied to patients' faces and arms twice a day by caregiving staff. Changes in clinically significant agitation (Cohen-Mansfield Agitation Inventory [CMAI]) and quality of life indices (percentage of time spent socially withdrawn and percentage of time engaged in constructive activities, measured with Dementia Care Mapping) were compared between the 2 groups over a 4-week period of treatment. RESULTS: Seventy-one patients completed the trial. No significant side effects were observed. Sixty percent (21/35) of the active treatment group and 14% (5/36) of the placebo-treated group experienced a 30% reduction of CMAI score, with an overall improvement in agitation (mean reduction in CMAI score) of 35% in patients receiving Melissa balm essential oil and 11% in those treated with placebo (Mann-Whitney U test; Z = 4.1, p < .0001). Quality of life indices also improved significantly more in people receiving essential balm oil (Mann-Whitney U test; percentage of time spent socially withdrawn: Z = 2.6, p = .005; percentage of time engaged in constructive activities: Z = 3.5, p = .001). CONCLUSION: The finding that aromatherapy with essential balm oil is a safe and effective treatment for clinically significant agitation in people with severe dementia, with additional benefits for key quality of life parameters, indicates the need for further controlled trials.     

Nicotinic acetylcholine binding sites in Alzheimer''s disease

In Alzheimer's disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.     

Involvement of the cholinergic system in conditioning and perceptual memory

The cholinergic systems play a pivotal role in learning and memory, and have been the centre of attention when it comes to diseases containing cognitive deficits. It is therefore not surprising, that the cholinergic transmitter system has experienced detailed examination of its role in numerous behavioural situations not least with the perspective that cognition may be rescued with appropriate cholinergic ‘boosters’.Here we reviewed the literature on (i) cholinergic lesions, (ii) pharmacological intervention of muscarinic or nicotinic system, or (iii) genetic deletion of selective receptor subtypes with respect to sensory discrimination and conditioning procedures. We consider visual, auditory, olfactory and somatosensory processing first before discussing more complex tasks such as startle responses, latent inhibition, negative patterning, eye blink and fear conditioning, and passive avoidance paradigms.An overarching reoccurring theme is that lesions of the cholinergic projection neurones of the basal forebrain impact negatively on acquisition learning in these paradigms and blockade of muscarinic (and to a lesser extent nicotinic) receptors in the target structures produce similar behavioural deficits. While these pertain mainly to impairments in acquisition learning, some rare cases extend to memory consolidation. Such single case observations warranted replication and more in-depth studies. Intriguingly, receptor blockade or receptor gene knockout repeatedly produced contradictory results (for example in fear conditioning) and combined studies, in which genetically altered mice are pharmacological manipulated, are so far missing. However, they are desperately needed to clarify underlying reasons for these contradictions.Consistently, stimulation of either muscarinic (mainly M₁) or nicotinic (predominantly α7) receptors was beneficial for learning and memory formation across all paradigms supporting the notion that research into the development and mechanisms of novel and better cholinomimetics may prove useful in the treatment of neurodegenerative or psychiatric disorders with cognitive endophenotypes.     

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [³H]quinuclidinyl benzilate to membranes and the binding of [³H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [¹²⁵I]α-bungarotoxin or [³H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.