Neuromechanical effects of pyrethroids, allethrin, cyhalothrin and deltamethrin on the cholinergic processes in rat brain

Our previous microdialysis study of freely moving rats demonstrated that 3 pyrethroids, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) differentially modulate acetylcholine (ACh) release in the hippocampus. To better understand the mechanisms of their modulatory effects and also other effects on the cholinergic system in the brain, the activities of ACh hydrolyzing enzyme acetylcholinesterase (AChE), ACh synthesizing enzyme choline acetyltransferase (ChAT) and ACh synthesizing rate-limiting step, high-affinity choline uptake (HACU) were examined in the present study. The pyrethroids studied had no effect on AChE activity in the cortex, hippocampus and striatum. These pyrethroids had no significant effect on ChAT in the cortex and hippocampus, but striatal ChAT was increased at higher dosage (60 mg/kg) by all three compounds. Lineweaver-Burk analysis of hippocampal HACU revealed that the pyrethroids did not alter the Michaelis-Menten constant (Km) value but caused alteration of maximal velocity (Vmax). Allethrin (60 mg/kg) and cyhalothrin (20 and 60 mg/kg) decreased while deltamethrin (60 mg/kg) increased the Vmax for HACU. In vitro study showed that at higher concentrations (> or = 10(-) (6) M) allethrin and cyhalothrin reduced the hippocampal HACU but deltamethrin increased it. These results suggest that mechanisms of ACh synthesis are involved in the modulatory effects of the pyrethroids on ACh release and other cholinergic activities.     

Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.     

胆碱转运体与阿尔茨海默病

阿尔茨海默病主要病理学特征是在脑中形成大量的老年斑和神经元纤维缠结以及出现弥漫性脑萎缩.胆碱能系统的失调与阿尔茨海默病的发生机制关系密切.具体表现为基底前脑的胆碱能系统紊乱,胆碱乙酰化酶、乙酰胆碱含量显著减少,以及大量胆碱能神经元退化.胆碱转运体是胆碱能系统中用于转运胆碱进入细胞的关键蛋白体,有三种类型:高亲和力胆碱转运体、胆碱转运体类蛋白及非特异性有机阳离子转运体.近年,很多研究表明胆碱转运体的异常与一系列神经退行性紊乱有关.本文简要综述胆碱能系统中胆碱转运体的生理作用及其在阿尔茨海默病中异常代谢和可能机制的研究进展,以期为防治阿尔茨海默病提供进一步的理论和实验依据.     

Early memory deficits precede plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction

A large body of evidence has shown that cognitive deficits occur early, before amyloid plaque deposition, suggesting that soluble amyloid-β protein (Aβ) contributes to the development of early cognitive dysfunction in Alzheimer disease (AD). However, the underlying mechanism(s) through which soluble Aβ exerts its neurotoxicity responsible for cognitive dysfunction in the early stage of AD remains unclear so far. In this study, we used preplaque APPswe/PS1dE9 mice ages 2.5 and 3.5 months to examine alterations in cognitive function, oxidative stress, and cholinergic function. We found that only soluble Aβ, not insoluble Aβ, was detected in these preplaque APPswe/PS1dE9 mice. APPswe/PS1dE9 mice 2.5 months of age did not show any significant changes in the measures of cognitive function, oxidative stress, and cholinergic function, whereas 3.5-month-old APPswe/PS1dE9 mice exhibited spatial memory impairment in the Morris water maze, accompanied by significantly decreased acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) as well as increased malondialdehyde (MDA) and protein carbonyls. In 3.5-month-old preplaque APPswe/PS1dE9 mice, correlational analyses revealed that the performance of impaired spatial memory was inversely correlated with soluble Aβ, MDA, and protein carbonyls, as well as being positively correlated with ACh, ChAT, SOD, and GSH-px; soluble Aβ level was inversely correlated with ACh, ChAT, SOD, and GSH-px, as well as being positively correlated with MDA and protein carbonyls; ACh level showed a significant positive correlation with ChAT, SOD, and GSH-px, as well as a significant inverse correlation with MDA and protein carbonyls. Collectively, this study provides direct evidence that increased oxidative damage and cholinergic dysfunction may be early pathological responses to soluble Aβ and involved in early memory deficits in the preplaque stage of AD. These findings suggest that early antioxidant therapy and improving cholinergic function may be a promising strategy to prevent or delay the onset and progression of AD.     

Exogenous Choline Enhances the Synthesis of Acetylcholine Only Under Conditions of Increased Cholinergic Neuronal Activity

Abstract: The effect of choline (60 mg/kg, i.p.) on fluphenazine- and pentylenetetrazol-induced alterations in the concentration of acetylcholine (ACh) and/or the rate of sodium-dependent high-affinity choline uptake (HACU) in rat striatum and hippocampus was studied. Systemic administration of the dopamine receptor blocking agent fluphenazine hydrochloride (0.5 mg/kg, i.p.) decreased the concentration of ACh in the striatum; this effect was prevented by the prior administration of choline. The central nervous system stimulant pentylenetetrazol (30 mg/kg, i.p.) reduced the concentration of ACh in both striatum and hippocampus and increased the velocity of HACU in the hippocampus. Pretreatment with choline totally prevented the depletion of ACh induced by pentylenetetrazol in the striatum. In the hippocampus, prior administration of choline prevented the pentylenetetrazol-induced increase in the rate of HACU and attenuated the effect of pentylenetetrazol on the levels of ACh. Results indicate that the acute administration of choline antagonizes pharmacologically induced alterations in cholinergic activity as assessed by the rate of HACU and the steady-state concentration of ACh. Furthermore, data support the hypothesis that the administration of choline increases the ability of central cholinergic neurons to synthesize ACh under conditions of increased neuronal activity.     

Exogenous Nerve Growth Factor Increases the Activity of High-Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats

The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium-sensitive high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats. In 24-month-old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4-month-old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4-month-old animals, NGF treatment at 1.2 micron/day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle-treated 4-month-old animals; vehicle treatment had no effect on the activity of either marker. In 24-month-old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24-month-old control animals. Synaptosomal HACU in 24-month-old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post-ischemic regional changes in acetylcholine synthesis following transient forebrain ischemia in gerbils

The synthesis rate of brain acetylcholine (ACh) was estimated 30 min and 5 days following transient forebrain ischemia performed by 10 min bilateral carotid occlusion in gerbils. ACh synthesis was evaluated from the conversion of radiolabeled choline (Ch) into ACh after an i.v. administration of [methyl-³H]Ch. Endogenous and labeled Ch and ACh were quantified by HPLC. The synthesis rate of ACh was significantly decreased following 30 min of recirculation. The reductions reached 55.4% in the hippocampus, 51.2% in the cerebral cortex and 44.4% in the striatum. Five days after ischemia, the values returned to normal in the cerebral cortex and in the striatum, while ACh synthesis remained selectively lowered (–30.4%, p<0.01) in the hippocampus. These cholinergic alterations may account for both early and delayed post-ischemic behavioral and mnesic deficits.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Cerebral Cortex: Effect of Quinolinic Acid Lesions of the Basal Forebrain

The effects of neurotensin (NT) on endogenous acetylcholine (ACh) release from basal forebrain, frontal cortex, and parietal cortex slices were tested. The results show that NT differentially regulates evoked ACh release from frontal and parietal cortex slices without altering either spontaneous or evoked ACh release from basal forebrain slices. In the frontal cortex, NT significantly inhibited evoked ACh release by a tetrodotoxin (TTX)-insensitive mechanism, suggesting an action directly on cholinergic terminals. In the parietal cortex, NT enhanced evoked ACh release by a TTX-sensitive mechanism, suggesting an action of NT on the cholinergic neuron or in close proximity to the cholinergic neuron. The effects of NT on ACh release were confined to evoked ACh release; that is, spontaneous ACh release was not affected. NT did not affect spontaneous or potassium-evoked ACh release from occipital cortex slices. The second set of experiments tested the effects of quinolinic acid (QUIN) lesions of the basal forebrain cell bodies on the NT-induced regulation of evoked ACh release in the cerebral cortex. QUIN lesions of basal forebrain cell bodies caused decreases in choline acetyltransferase activity (27 and 28%), spontaneous ACh release (14 and 21%), and evoked ACh release (38 and 44%) in frontal and parietal cortex, respectively. In addition, 11 days following QUIN lesions of basal forebrain cell bodies, the action of NT to regulate evoked ACh release in frontal cortex or parietal cortex was no longer observed. The results suggest that in the rat frontal and parietal cortex, NT differentially regulates the activity of cholinergic neurons by decreasing and increasing evoked ACh release, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic innervation of the retrosplenial cortex via the fornix pathway as determined by high affinity choline uptake,choline acetyltransferase activity,and muscarinic receptor binding in the rat

The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC.     

Neurotrophins differentially enhance acetylcholine release, acetylcholine content and choline acetyltransferase activity in basal forebrain neurons

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K⁺‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm ), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca²⁺ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm ) abolished increases in ChAT activity and prevented enhancement of K⁺‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

CHOLINE: SELECTIVE ACCUMULATION BY CENTRAL CHOLINERGIC NEURONS

Abstract— Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [³H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [³H]choline, suggesting that cholinergic nerve endings were mainly responsible for [³H]choline uptake. At 0.1 mm concentrations of [³H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.     

Effects of MK-771, A TRH analog,on pentobarbital-induced alterations of cholinergic parameters in discrete regions of rat brain

MK-771 (l-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) was administered intraventricularly to conscious and pentobarbital-narcotized rats. In the conscious rats MK-771 did not affect the regional levels of acetylcholine (ACh) or the rate of sodium-dependent high-affinity choline uptake (HACU). MK-771 was found to antagonize pentobarbital-induced elevations of ACh levels in the cortex, hippocampus and striatum. MK-771 also reversed the depressant effects of pentobarbital on the HACU of the cortex and hippocampus. Striatal HACU was unaltered by the administration of pentobarbital or the combination of pentobarbital and MK-771.     

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE-Tg mice as compared with controls. Also, the high-affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE-Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE-Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus-maze test. Systemic application of a low dose of physostigmine (100 microgram/kg i.p.) normalized all of the enhanced parameters in hAChE-Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE-Tg mice, hippocampal ACh release is up-regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.     

Time course of decline of radiolabeled acetylcholine formed following intracerebroventricular administration of tritiated choline: Effects of oxotremorine and scopolamine

Rats were injected intracerebroventricularly with 5 Ci of [methyl-³H]choline. The time course of decline of the rediolabeled acetylcholine (ACh) formed was estimated in the ispilateral cerebral cortex and striatum. The [³H]ACh levels declined biphasically from the cerebral tissue. The initial decline proceeded rapidly, after which labeled ACh declined more slowly. Scopolamine (1 mg/kg, i.v.) caused a significant increase in the rat of [³H]ACh disappearance, which can be interpreted as an enhancement of ACh release. By contrast, oxotremorine (0.8 mg/kg, i.v.) markedly reduced the [³H]ACh disappearance. The results show that drug-induced changes in cholinergic neuronal activities can be estimated from the disappearance of radioactive ACh after labeling the endogenous transmitter through intracerebroventricular administration of labeled choline.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used ¹⁵N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before ¹H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.     

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.     

Cholinergic Surface Antigen Chol-1 Is Present in a Subclass of VIP-Containing Rat Cortical Synaptosomes

The colocalization of vasoactive intestinal polypeptide (VIP) with the cholinergic specific surface antigen Chol-1 was investigated in synaptosomes derived from the rat cerebral cortex. Immunoaffinity purification of cortical synaptosomes using antisera to Chol-1 resulted in the copurification of VIP and cholinergic nerve terminals. VIP was purified with a yield of 75% of that of choline acetyltransferase (ChAT). These results suggest that approximately 53% of the cortical cholinergic terminals contain VIP, whereas 75% of the cortical VIP content is present in these cholinergic terminals. Both hypotonic lysis and depolarization of the nerve terminals resulted in the differential release of VIP and acetylcholine (ACh), indicating the different compartmentalization in the same nerve terminal. Complement-mediated lysis of cholinergic nerve terminals, using antisera to Chol-1, resulted in the release of 64% of the ChAT, 71% of ACh, and 27% of the VIP. The application of our method enables quantifying and mapping, with a fast, efficient, and specific technique, the coexisting peptides in cholinergic neurons of distinct brain areas.