Novel and potent tacrine-related hetero- and homobivalent ligands for acetylcholinesterase and butyrylcholinesterase.

Based upon synthetic and biochemical results, a novel and potent tacrine analogue and heterobivalent analogues of tacrine, were designed. The role played by the amino groups of homo- and heterobivalent ligands in the interaction with the peripheral and catalytic sites of AChE and BuChE were investigated. The syntheses of these materials together with the results of AChE/BuChE inhibition assays are detailed.     

Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

人脑和人血清胆碱酯酶三维结构的计算机模拟研究

本文以同源的电鳐胆碱酯酶（Ｔ．ＡＣｈＥ）的三维结构为模板,模拟预测了人脑和血清胆碱酯酶（Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ）的三维结构和活力中心的组成。指Ｔ．ＡＣｈＥ,Ｈ．ＡＣｈＥ和Ｈ．ＢｕＣｈＥ宁间结构差异,并讨论了ＡＣｈ和Ｈ。ＡＣｈＥ的对接（ｄｏｃｋｉｎｇ）。Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ三维结构的确定将为进一步深入研究它的中毒机理和合理药物设计提供靶子。     

Experimental study of the effects of amiridin and tacrine on learning and memory

The authors studied the influence of amiridin and tacrine on learning and memory in mice and rat by passive avoidance conditioning test at norm and under scopolamine induced amnesia as well as of their effect on acetylcholine esterase (AChE) activity in brain cortex homogenates. Amiridin in doses 0.1 and 0.2 mg/kg showed a beneficial action on conditioning in untreated animals, its effect being comparable with that of piracetam. Tacrine was ineffective. In scopolamine treated animals amiridin and tacrine showed anti-amnestic action at dose of 0.1 mg/kg which was found ineffective with respect to AChE activity. The data suggests that the ameliorating effect of amiridin and tacrine on cognitive abilities in patients with senile dementia is not related their anticholinesterase properties.     

Butyrylcholinesterase in Human Brain and Acetylcholinesterase in Human Plasma: Trace Enzymes Measured by Two-Site Immunoassay

Enzyme-linked immunosorbent assays for acetylcholinesterase (AChE) and for butyrylcholinesterase (BuChE) were markedly more specific than conventional assays using selective enzyme inhibitors. The new assays were used with blood and brain samples containing traces of one enzyme dominated by large amounts of the other. The results showed that human plasma does contain AChE (8 ng/ml), even though its major cholinesterase is BuChE (3,300 ng/ml). BuChE immunoreactivity was not detected in human red blood cells but occurred in all brain regions. The cerebellum was the richest region tested (540 ng of BuChE/g of tissue), whereas the cerebral cortex was the poorest (240 ng of BuChE/g). However, because of the small local AChE content (99 ng/g), BuChE was the major cortical cholinesterase. The picture was reversed in the putamen, where BuChE immunoreactivity (340 ng/g) was far outweighed by that of AChE (6,100 ng/g).     

Design, synthesis and evaluation of isaindigotone derivatives as dual inhibitors for acetylcholinesterase and amyloid beta aggregation

A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. The synthetic compounds had IC(50) values at micro or nano molar range for cholinesterase inhibition, and some compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much better than the isaindigotone derivatives previously reported by our group. Most of these compounds showed higher self-induced Aβ aggregation inhibitory activity than a reference compound curcumin. The structure-activity relationship studies revealed that the derivatives with higher inhibition activity on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibition for both AChE and self-induced Aβ aggregation was further studied using CD, EM, molecular docking and kinetics.     

Cholinesterases: New Roles in Brain Function and in Alzheimer's Disease

The most important therapeutic effect of cholinesterase inhibitors (ChEI) on approximately 50% of Alzheimer's disease (AD) patients is to stabilize cognitive function at a steady level during a 1-year period of treatment as compared to placebo. Recent studies show that in a certain percentage (approximately 20%) of patients this cognitive stabilizing effect can be prolonged up to 24 months. This long-lasting effect suggests a mechanism of action other than symptomatic and cholinergic. In vitro and in vivo studies have consistently demonstrated a link between cholinergic activation and APP metabolism. Lesions of cholinergic nuclei cause a rapid increase in cortical APP and CSF. The effect of such lesions can be reversed by ChEI treatment. Reduction in cholinergic neurotransmission–experimental or pathological, such as in AD–leads to amyloidogenic metabolism and contributes to the neuropathology and cognitive dysfunction. To explain the long-term effect of ChEI, mechanisms based on -amyloid metabolism are postulated. Recent data show that this mechanism may not necessarily be related to cholinesterase inhibition. A second important aspect of brain cholinesterase function is related to enzymatic differences. The brain of mammals contains two major forms of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The two forms differ genetically, structurally, and for their kinetics. Butyrylcholine is not a physiological substrate in mammalian brain, which makes the function of BuChE of difficult interpretation. In human brain, BuChE is found in neurons and glial cells, as well as in neuritic plaques and tangles in AD patients. Whereas, AChE activity decreases progressively in the brain of AD patients, BuChE activity shows some increase. To study the function of BuChE, we perfused intracortically the rat brain with a selective BuChE inhibitor and found that extracellular acetylcholine increased 15-fold from 5 nM to 75 nM concentrations with little cholinergic side effect in the animal. Based on these data and on clinical data showing a relation between cerebrospinal fluid (CSF) BuChE inhibition and cognitive function in AD patients, we postulated that two pools of cholinesterases may be present in brain, the first mainly neuronal and AChE dependent and the second mainly glial and BuChE dependent. The two pools show different kinetic properties with regard to regulation of ACh concentration in brain and can be separated with selective inhibitors. Within particular conditions, such as in mice nullizygote for AChE or in AD patients at advanced stages of the disease, BuChE may replace AChE in hydrolizing brain acetylcholine.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

New multipotent tetracyclic tacrines with neuroprotective activity

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5–13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca²⁺ overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of β-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70 mM K⁺ and 60 μM H₂O₂. Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.     

Altered Levels of Acetylcholinesterase in Alzheimer Plasma

Background

Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem.

Principal Findings

Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were ∼20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G₁+G₂ forms and not G₄ tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer''s disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G₁+G₂ forms, the subset of AChE species which are increased in Alzheimer''s brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer''s plasma, attributed in part to AChE-T subunits common in brain and CSF.

Conclusion

Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation.     

2-(2-indolyl-)-4(3H)-quinazolines derivates as new inhibitors of AChE: design,synthesis, biological evaluation and molecular modelling

We recently reported that synthetic derivatives of rutaecarpine alkaloid exhibited high acetyl cholinesterase (AChE) inhibitory activity and high selectivity for AChE over butyrylcholinesterases (BuChE). To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), in this paper, further research results were presented. Starting from a structure-based drug design, a series of novel 2-(2-indolyl-)-4(3H)-quinazolines derivates were designed and synthesized as the ring-opened analogues of rutaecarpine alkaloid and subjected to pharmacological evaluation as AChE inhibitors. Among them, derivates 3a–c and 3g–h exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE. The structure–activity relationships were discussed and their binding conformation and simultaneous interactions mode were further clarified by kinetic characterization and the molecular docking studies.     

Inhibition of two different cholinesterases by tacrine

Kinetic parameters of the effect of tacrine as a cholinesterase inhibitor have been studied in two different sources: snake venom (Bungarus sindanus) acetylcholinesterase (AChE) and human serum butyrylcholinesterase (BChE). Tacrine inhibited both venom acetylcholinesterase (AChE) as well as human serum butyrylcholinesterase (BChE) in a concentration-dependent manner. Kinetic studies indicated that the nature of inhibition was mixed for both enzymes, i.e. Km values increase and Vmax decrease with the increase of the tacrine concentration. The calculated IC50 for snake venom and for human serum were 31 and 25.6 nM, respectively. Ki was observed to be 13 nM for venom acetylcholinesterase (AChE) and 12 nM for serum butyrylcholinesterase (BChE). KI (constant of AChE-ASCh-tacrine complex into AChE-ASCh complex and tacrine) was estimated to be 20 nM for venom and 10 nM for serum butyrylcholinesterase (BChE), while the gammaKm (dissociation constant of AChE-ASCh-tacrine complex into AChE-tacrine complex and ASCh) were 0.086 and 0.147 mM for snake venom AChE and serum BChE, respectively. The present results suggest that this therapeutic agent used for the treatment of Alzheimer's disease can also be considered an inhibitor of snake venom and human serum butyrylcholinesterase. Values of Ki and KI show that tacrine had more affinity with these enzymes as compared with other cholinesterases from the literature.     

The effects of some porphyrinogenic drugs on the brain cholinergic system.

In central nervous system, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyse acetylcholine. Diminished cholinesterase activity is known to alter several mental and psychomotor functions. The symptoms of cholinergic crisis and those observed during acute attacks of acute intermittent porphyria are very similar. The aim of this study was to investigate if there could be a link between the action of some porphyrinogenic drugs on brain and the alteration of the cholinergic system. To this end, AChE and BuChE activities were assayed in whole and different brain areas. Muscarinic acetylcholine receptor (mAChR) levels were also measured. Results obtained indicate that the porphyrinogenic drugs tested affect central cholinergic transmission. Quantification of mAChR gave quite different levels depending on the xenobiotic. Veronal administration inhibited 50% BuChE activity in whole brain, cortex and hippocampus; concomitantly cortex mAChR was 30% reduced. Acute and chronic isoflurane anaesthesia diminished BuChE activity by 70-90% in whole brain instead cerebellum and hippocampus mAChR levels were only altered by chronic enflurane anaesthesia. Differential inhibition of cholinesterases in the brain regions and their consequent effects may be of importance to the knowledge of the mechanisms of neurotoxicity of porphyrinogenic drugs.     

Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors

The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedl?nder reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.     

Thermodynamic analysis of human retinal acetylcholinesterase inhibition using an anti-Alzheimer's drug, tacrine, through the development of a dual substrate and temperature model

The present study determines the energy parameters, such as the Gibb's free energy change (deltaG), enthalpy change (deltaH), heat of activation (deltaH*), entropy change (deltaS), temperature coefficient (Q10) and activation energy (Ea), of human retinal acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by tacrine. The stereo-frequency collisions factor (PZ, the number of sterically and energetically favorable collisions occurring between tacrine and AChE) was also studied in this investigation. Tacrine significantly increased the value of deltaG, deltaH, deltaH*, Q10, Ea and PZ factor, and decreased the value of deltaS for AChE. Since there is no known report on the inhibition of human retinal AChE by tacrine, these results were compared with the reported values for the energy parameters of camel retinal and chicken brain AChE inhibition by an anti-cancer drug, cyclophosphamide. The uniqueness of this approach lies in the development of the 'dual substrate and dual temperature' model, which may open up a new, more efficient avenue for the study of various enzyme catalyzed reactions.     

Effect of anti-dementia drugs on LPS induced neuroinflammation in mice

Inflammation has been recently implicated in pathogenesis of dementia disorders. Effect of anti-dementia (Acetylcholinesterase inhibitor) drugs tacrine, rivastigmine and donepezil were studied on neuroinflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) in mice. Interleukin-2 (IL-2) and isoforms of acetylcholinesterase (AChE) were estimated in different brain areas as marker for neuroinflammation and cholinergic activity respectively. LPS significantly increased the level of IL-2 in all the brain areas while enhancement of AChE activity varied in brain areas. It was found that administration of tacrine, rivastigmine and donepezil in mice significantly attenuated the LPS induced increased levels of IL-2 along with the significant reduction of AChE activity predominantly in salt soluble (SS) fraction as compared to the detergent soluble (DS) fraction in a dose dependent manner. In vitro effect of LPS was also studied in different brain areas. LPS significantly increased the AChE activity in SS fractions but the significant increase was not found in DS fractions. The present study indicate that cholinesterase inhibitor anti-dementia drugs are effective against LPS induced neuroinflammation that may be linked to enhanced cholinergic activity.     

Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.     

Gender-related differences in circadian rhythm of rat plasma acetyl- and butyrylcholinesterase: Effects of sex hormone withdrawal

The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2–30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.     

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.     

Immunochemistry of mammalian cholinesterases

Advances in the study of cholinesterase biology have been facilitated by the development of polyclonal and monoclonal antibodies to acetylcholinesterase (AChE) (EC 3.1.1.7) and butyrylcholinesterase (BuChE) (EC 3.1.1.8) in several laboratories. Our work has focused on murine monoclonal antibodies to the mammalian enzymes. Two dozen antibodies are now in hand, with primary specificity for the AChE of human red blood cells, rabbit brain, and rat brain, and for the BuChE of human plasma. These antibodies exhibit a restricted but useful range of affinities for other mammalian cholinesterases of corresponding types. Several applications are described, including an analysis of BuChE phylogeny within the higher primates, an immunodisplacement assay of AChE in normal human red blood cells and cells from patients with paroxysmal nocturnal hemoglobinuria, a study of immunochemical differences between membrane-associated and soluble AChE of rabbit brain, and initial work on the immunofluorescence cytochemistry of the rat brain.