Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.     

重症肌无力中枢神经系统受损模型

目的近年研究结果表明，重症肌无力（ＭＧ）病变部位并不仅仅局限于神经肌接头（ＮＭＪ）处突触后膜烟碱型乙酰胆碱受体（ｎＡＣｈＲ），烟碱型乙酰胆碱受体抗体（ＡＣｈＲ－ａｂ）病理作用可能波及到中枢神经系统（ＣＮＳ）。因此，有必要建立模拟ＭＧ患者ＣＮＳ损害的动物模型，研究ＭＧ患者脑脊液中存在的ＡＣｈＲ－ａｂ引起ＣＮＳ损害的机制。方法从ＭＧ患者血中提取的ＡＣｈＲ－ａｂ经侧脑室穿刺注入到大鼠脑室系统，然后观察其症状和体征，以及用脑干听觉诱发电位仪（ＢＡＥＰ）检测鼠脑干听觉传导中枢功能。用免疫组化法（ＡＢＣ）研究ＡＣｈＲ－ａｂ与ＣＮＳ神经－ｎＡＣｈＲ之间免疫结合反应及其分布。结果大鼠除了出现脑干听觉传导中枢功能障碍外，还出现类似于ＭＧ动物模型表现的症状。免疫组化研究结果显示，神经－ｎＡＣｈＲ样阳性免疫反应广泛分布于ＣＮＳ许多部位。结论脑室内注入的ＡＣｈＲ－ａｂ与神经－ＡＣｈＲ结合引起ＣＮＳ功能障碍和出现ＭＧ动物模型样症状。我们首次建立的中枢受损的ＭＧ模型将有助于阐明ＡＣｈＲ－ａｂ引起中枢受损和ＣＮＳ下位运动神经元引起横纹肌收缩无力的机制。     

辣椒素对脊髓后角乙酰胆碱活度的影响

本文报道用乙酰胆碱（ＡＣｈ）微电极，连续记录了脊髓表面滴注辣椒素（Ｃａｐｓａｉｃｉｎ）时，脊髓后角内ＡＣｈ活度的动态变化，结果发现；脊髓表面滴注辣椒素可明显提高后角ＡＣｈ释放的作用，本研究为ＡＣｈ在脊髓后角水平参与痛觉信息调制提供了一个新的实验证据。     

Cholinergic modulation of basal and amphetamine-induced dopamine release in rat medial prefrontal cortex and nucleus accumbens

Behavioral evidence suggests that muscarinic/cholinergic inhibition of brain dopaminergic activity may be a useful principle for developing novel antipsychotic drugs (APDs). Thus, oxotremorine, a muscarinic agonist, attenuates amphetamine-induced locomotor activity in rodents, an effect also produced by a wide variety of proven APDs, whereas scopolamine, a muscarinic antagonist, has the opposite effect. Since atypical APDs such as clozapine, olanzapine, risperidone, ziprasidone and quetiapine, increase brain acetylcholine as well as dopamine (DA) release in a region-specific manner, their effects on cholinergic and dopaminergic neurotransmission may also contribute to various actions of these drugs. Oxotremorine (0.5-1.5 mg/kg) dose-dependently and preferentially increased DA release in rat medial prefrontal cortex (mPFC), compared to the nucleus accumbens (NAC). However, S-(-)-scopolamine (0.5-1.5 mg/kg) produced similar increases in DA release in the mPFC, but the effect was much less than that of oxotremorine. Whereas a dose of S-(-)-scopolamine of 0.5 mg/kg comparably increased DA release in the mPFC and NAC, 1.5 mg/kg had no effect on DA release in the NAC. Oxotremorine-M (0.5 mg/kg), a M(1/4)-preferring agonist, also increased DA release in the mPFC, but not the NAC, an effect completely abolished by telenzepine (3 mg/kg), a M(1/4)-preferring antagonist, which by itself had no effect on DA release in either region. Oxotremorine (0.5, but not 1.5, mg/kg) attenuated amphetamine (1 mg/kg)-induced DA release in the NAC, whereas S-(-)-scopolamine did not. Oxotremorine (1.5 mg/kg) and S-(-)-scopolamine (0.5 mg/kg) modestly but significantly potentiated amphetamine (1 mg/kg)-induced DA release in the mPFC. These results suggest that stimulation of muscarinic receptors, in particular M(1/4), as indicated by the effect of oxotremorine-M and telenzepine, may preferentially increase cortical DA release and inhibit amphetamine-induced DA release in the NAC.     

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II. Pre- and postnatal development

The pre- and postnatal development of cholinergic projections was investigated in the cat striate cortex by applying immunohistochemical methods based on a monoclonal antibody against choline acetyltransferase (ChAT). The earliest age investigated was gestational day 54. At this stage a sparse network of ChAT(+) fibers was distributed throughout the striate cortex. Subsequent postnatal maturation of ChAT(+) fibers was characterized by an increase in fiber density that started in layer VI and gradually progressed toward more superficial layers. By 4 weeks of age the density of ChAT(+) fibers and varicosities had reached adult levels in layers V and VI but was still subnormal in layers I-IV. The mature pattern of cholinergic innervation was established by 13 weeks of age. There was no evidence for developmental gradients in the anteroposterior and mediolateral directions within area 17. These results indicate that the cholinergic projection to striate cortex develops continuously in an inside-out sequence as is characteristic for most cortical maturation processes. There was no indication that striate cortex receives an especially dense cholinergic input during the critical period.     

Effect of protein synthesis inhibitors on the trophic action of the nerve stump

We report that protein synthesis inhibitors exert an inhibitory effect on the trophic action of the nerve stump. The sciatic nerve innervating the extensor digitorum longus muscles of mice was cut either as close to, or as far from, the muscle as possible. Denervation changes in the muscle were evaluated using the resting membrane potential and dose-response curves obtained by plotting acetylcholine-induced contractures. Actinomycin D (2 micrograms/kg, i.p.), ethidium bromide (10 micrograms/kg, i.p.), cycloheximide (1 or 5 mg/kg, i.p.), or chloramphenicol (100 mg/kg, p.o.) administration was immediately after neurotomy and continued daily until the day preceding muscle removal. Although denervation changes occurred significantly later in muscles with a long rather than a short nerve stump, the administrated antibiotics, excluding cycloheximide, accelerated the manifestation of denervation changes in muscles with long nerve stumps without affecting those in muscles with short nerve stumps.     

用AChR单克隆抗体建立大鼠重症肌无力被动转移模型的研究

目的利用乙酰胆碱受体(AChR)单克隆抗体建立重症肌无力(MG)被动转移模型。方法将AChR单克隆抗体mAb35注入3种品系大鼠腹腔，观察其临床症状并行药理学和电生理学及超微结构鉴定。结果被动转移mAb35后3种大鼠均可出现肌无力症状。其临床症状、药理学特点、电生理特点和超微结构变化均与MG患者相似。结论利用AChR单克隆抗体可在此3种品系大鼠成功建立获得性自身免疫性MG模型。     

Human cortical neurons contain both nicotinic and muscarinic acetylcholine receptors: an immunocytochemical double-labeling study

Using immunofluorescence histochemistry, in the human cerebral cortex neurons immunoreactive for both nicotinic and muscarinic acetylcholine receptor proteins could be demonstrated. Vibratome sections of biopsy and autopsy specimens of human temporal and occipital lobes were incubated with monoclonal antibodies specific for muscarinic (M 35) and nicotinic (WF 6) acetylcholine receptor protein. Immunoreactive sites were visualized using a biotin-streptavidin-phycoerythrin system (M 35, red fluorescence) and fluorescein-conjugated immunoglobulins (WF 6, green fluorescence). Immunofluorescence of both antibodies was preponderant in pyramidal neurons located in layers II/III and V and their apical dendrites. Some round and ovoid immunolabeled cells were encountered in layers VI and IV. About 30% of the cholinoceptive cortical neurons, in particular the pyramidal cells, displayed immunoreactivity for both receptor types. The present investigation shows a subpopulation of human cortical neurons to contain both nicotinic and muscarinic receptors. The coexistence of acetylcholine receptors may provide the morphological basis of simultaneous impact of acetylcholine on both receptor types in the same neuron of the human cerebral cortex.     

Decrease in acetylcholine receptor number correlated with increased naturally occurring trochlear motor neuron death during development

It has been observed that daily application of neostigmine onto the chorioallantoic membrane drastically reduced the total number of acetylcholine receptors in the superior oblique muscle of duck embryos. Here the effects of neostigmine on the magnitude of naturally occurring death of trochlear motor neurons during embryonic development were investigated. There was an enhanced loss of neurons in the neostigmine-treated embryos. Neostigmine neither affected the initial production of normal numbers of motor neurons nor had any direct toxic effect on their ultrastructure. The decrease in muscle activity did not always correlate with increased motor neuron survival. There may be a relationship between acetylcholine receptor distribution and naturally occurring neuronal death.     

Effects of aging on nerve sprouting and regeneration

We examined the effects of aging on nerve terminal sprouting and regeneration in the peripheral nervous system. Motor end-plates were demonstrated in rat soleus muscles by means of a cholinesterase-silver stain which permitted measurement of end-plate length and nerve terminal branching. Terminal sprouting was elicited by “pharmacological denervation” brough about by botulinum toxin treatment. In the youngest (2-month-old) animals, the end-plates were small, and the terminals had simple branch patterns. Botulinum toxin treatment elicited a striking increase in end-plate length and terminal arborization. In the older (10- and 18-month-old) animals, the end-plate length was greater, and nerve terminal branching patterns were more complex. Botulinum toxin elicited a sprouting response that was less marked than in the youngest animals. In the oldest (28-month-old) animals, the end-plates were shorter and the terminals less complex. Botulinum failed to elicit any significant sprouting response. Regeneration of motor and sensory axons after a crush lesion of the sciatic nerve was measured using radiolabeled proteins carried by axonal transport as a marker of axonal outgrowth. Our results showed that the average rate of regeneration slowed with increasing age, although a small population of axons continued to regenerate rapidly regardless of age. Evidence implicating inherent defects in the ability of nerves to sprout and regenerate with increasing age is presented. Our findings in the peripheral nervous system may shed light on similar functional changes occurring in the central nervous system.     

Impaired synaptic functions with aging as characterized by decreased calcium influx and acetylcholine release

Age-related alterations of presynaptic functions were studied in terms of acetylcholine (ACh) synthesis and release using synaptosomes isolated from mouse brain cortices. The following three findings were obtained: (1) Choline acetyltransferase activity and ACh production rate remained constant throughout all ages tested. This observation, obtained with synaptosomes, was not consistent with data reported for brain slices (Gibson GE, Peterson C: J Neurochem 37:978–984, 1981). Various conditions, such as low glucose or membrane depolarization, modulated ACh synthesis to similar extents in young and aged synaptosomes. (2) Depolarization-induced release of ACh from synaptosomes significanty decreased in the senescent stage. The fraction of ACh released from aged synaptosomes was less than that released from young synaptosomes, although the ACh contents in the synaptosomes did not change with age. (3) Calcium influx induced by depolarization was lower in the synaptosomal preparations from aged mice than in those from young mice. A strong positive correlation was observed between the amounts of ACh released and the increased calcium levels when the data for all preparations, both from young and aged mice, were plotted. This indicates that diminished calcium influx may cause the reduced ACh release by aged synapses. The present study provides evidence for an age-related decrease in presynaptic functions, that is, a reduction in calcium influx via voltage-dependent calcium channels followed by a decreased ACh release from synapses despite an abundance of ACh within the synapses. © 1996 Wiley-Liss, Inc.     

δ1-OPIOID receptor-mediated controlofacetylcholine (ACh) release in human neocortex slices

In slices of human neocortex, prelabelled with [³H]-choline, the release of [³H]-acetylcholine reflects the evoked release of endogenous acetylcholine whichwaselicited by the same electrical stimulation paradigm. [³H]-Acetylcholine releasewasdepressed by the δ-opioid receptor agonist -Pen₂- -Pen₅-enkephalin. When the nerve endings were depolarized byelevatingextracellular potassium the evoked [³H]-acetylcholine release was similarlydepressed by -Pen₂- -Pen₅-enkephalin in theabsence, but notin the presence, of tetrodotoxin which blocks action potential propagation.Therefore, the δ-opioid receptor inhibiting [³H]-acetylcholine release should notbe located tocholinergic nerve terminals, but rather to interneurons. The somatostatin₂receptorpartial agonist octreotide per se did not influence action potential-evoked [³H]-acetylcholine release, but prevented the inhibition of release of [³H]-acetylcholine by -Pen₂- -Pen₅-enkephalin.Similarly, the δ₁-opioid receptor antagonist 7-benzylidenenaltrexon perse did notinfluence [³H]-acetylcholine release, but prevented of the inhibition ofrelease by -Pen₂- -Pen₅-enkephalin.

From the presentfindings we conclude : (1) The evoked release of [³H]-acetylcholine from humanneocortex slices reflects the release of endogenous acetylcholine.(2) It is inhibited in an indirectmanner by opioid receptors of the δ₁-subtype,which (3) are not localized oncholinergic axon terminals but on soma and dendrites ofsomatostatin-containing interneurons,where they inhibit somatostatin release. (4) Theseinterneurons innervate cholinergic nerveendings in the human neocortex and appear to facilitateacetylcholine release via somatostatin₂ receptors.     

Effects of 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183) on rat cortical synaptosome choline uptake, acetylcholine storage and release

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.     

Muscarinic cholinergic receptor M1 in the rat basolateral amygdala: Ultrastructural localization and synaptic relationships to cholinergic axons

Muscarinic neurotransmission in the anterior basolateral amygdalar nucleus (BLa) mediated by the M1 receptor (M1R) is critical for memory consolidation. Although knowledge of the subcellular localization of M1R in the BLa would contribute to an understanding of cholinergic mechanisms involved in mnemonic function, there have been no ultrastructural studies of this receptor in the BLa. In the present investigation, immunocytochemistry at the electron microscopic level was used to determine which structures in the BLa express M1R. The innervation of these structures by cholinergic axons expressing the vesicular acetylcholine transporter (VAChT) was also studied. All perikarya of pyramidal neurons were labeled, and about 90% of dendritic shafts and 60% of dendritic spines were M1R+. Some dendrites had spines suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of interneurons. M1R immunoreactivity (M1R‐ir) was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M1R+ terminals forming asymmetrical synapses were dendritic spines, most of which were M1R+. The main targets of M1R+ terminals forming symmetrical synapses were M1R+ perikarya and dendritic shafts. About three‐quarters of VAChT+ cholinergic terminals formed synapses; the main postsynaptic targets were M1R+ dendritic shafts and spines. In some cases M1R‐ir was seen near the postsynaptic membrane of these processes, but in other cases it was found outside of the active zone of VAChT+ synapses. These findings suggest that M1R mechanisms in the BLa are complex, involving postsynaptic effects as well as regulating release of neurotransmitters from presynaptic terminals. J. Comp. Neurol. 521:1743–1759, 2013. © 2012 Wiley Periodicals, Inc.     

乙酰胆碱受体抗体与大鼠中枢神经元烟碱型乙酰胆碱受体之间免疫结合反应

本研究旨在探讨长期以来无定论的重症肌无力（MG）患者血和脑脊液（CSF）中的乙酰胆碱受体抗体（AChRab）能否与中枢神经元烟碱型乙酰胆碱受体（神经-nAChR）结合，并引起中枢神经系统（CNS）功能障碍。用免疫亲和层析法从AChRab阳性的全身型MG患者血中提取纯化AChRab，然后用免疫组化法探讨AChRab与大鼠中枢神经-nAChR之间的免疫结合反应。结果首次表明，AChRab与神经-nAChR之间的阳性免疫结合反应广泛分布于大鼠大脑皮层、脑干颅神经运动核团、脊髓前角运动神经元等部位，提示MG患者AChRab不仅可与神经肌接头（NMJ）处肌-nAChR结合引起肌无力等症状，还可与CNS神经-nAChR结合，并可能引起CNS功能障碍。     

重症肌无力CNS损害和神经细胞关系

目的　探讨神经细胞凋亡与重症肌无力 ( MG)中枢神经系统 ( CNS)损害之间的关系。方法　MG患者血中提取纯化的 Ig G注入 SD大鼠脑室系统 ,然后用透射电镜观察大鼠中枢神经细胞形态学变化。结果　侧脑室注射 MG患者 Ig G后 ,在大鼠大脑皮层、海马神经细胞中观察到神经细胞凋亡现象。结论　 MG中枢损害过程中 ,神经细胞凋亡可能起重要的作用。 MG患者乙酰胆碱受体抗体 ( ACh RAb)与 CNS神经 -ACh R结合 ,诱发神经细胞凋亡 ,从而可能致 MG CNS功能障碍。     

Large cholinergic nerve terminals on subsets of motoneurons and their relation to muscarinic receptor type 2

The cholinergic C-bouton is a large nerve terminal found exclusively apposing motoneuron cell somata and proximal dendrites. The origin and function of the C-bouton is not known. An antiserum against the vesicular acetylcholine transporter was used to identify large cholinergic nerve terminals putatively of the C-type in close apposition to motoneuron cell somata. This type of nerve terminal was present in the rat spinal cord ventral horn, but only in some cranial motor nuclei. Fluoro-Gold tracing showed that subsets of spinal motoneuron cell somata were contacted by different numbers of putative C-boutons. Thus, motoneurons innervating an intrinsic foot muscle were contacted by about half the number of cholinergic terminals found on motoneurons of the predominantly fast-twitch gastrocnemius muscle. Slow-twitch soleus motoneurons showed an intermediate innervation. There was a strong correlation between the presence of putative C-boutons and muscarinic receptor 2 (m2)-like immunoreactivity (-LI) within a motor nucleus. By using confocal laser microscopy, the m2-LI appeared to be confined to the motoneuron cell membrane and strongly enriched beneath the C-type nerve terminal. Thus, our results suggested a differential distribution of large cholinergic C-boutons, depending on motoneuron type, and that the presence of this nerve terminal type is associated with m2-LI in the postsynaptic membrane.     

Contribution of valine 7' of TMD2 to gating of neuronal alpha3 receptor subtypes

The second transmembrane domain (TMD2) of the Cys-loop family of ligand-gated ion channels forms the channel pore. The functional role of the amino acid residues contributing to the channel pore in neuronal nicotinic alpha3 receptors is not well understood. We characterized the contribution of TMD2 position V7' to channel gating in neuronal nicotinic alpha3 receptors. Site-directed mutagenesis was used to substitute position alpha3 (V7') with four different amino acids (A, F, S, or Y) and coexpressed each mutant subunit with wild-type (WT) beta2 or beta4 subunits in Xenopus oocytes. Whole-cell voltage clamp experiments show that substitution for an alanine, serine, or phenylalanine decreased by 2.3-6.2-fold the ACh-EC(50) for alpha3beta2 and alpha3beta4 receptor subtypes. Interestingly, mutation V7'Y did not produce a significant change in ACh-EC(50) when coexpressed with the beta2 subunit but showed a significant approximately two-fold increase with beta4. Similar responses were obtained with nicotine as the agonist. The antagonist sensitivity of the mutant channels was assessed by using dihydro-beta-erythroidine (DHbetaE) and methyllycaconitine (MLA). The apparent potency of DHbetaE as an antagonist increased by approximately 3.7- and 11-fold for the alpha3beta2 V7'S and V7'F mutants, respectively, whereas no evident changes in antagonist potency were observed for the V7'A and V7'Y mutants. The V7'S and V7'F mutations increase MLA antagonist potency for the alpha3beta4 receptor by approximately 6.2- and approximately 9.3-fold, respectively. The V7'A mutation selectively increases the MLA antagonist potency for the alpha3beta4 receptor by approximately 18.7-fold. These results indicate that position V7' contributes to channel gating kinetics and pharmacology of the neuronal nicotinic alpha3 receptors.     

Multiple actions of acetylcholine at a muscarinic receptor studied in the rat hippocampal slice

Responses of hippocampal pyramidal cells to topical application of acetylcholine (ACh) were measured in the in vitro hippocampal slice preparation.ACh but not cyclic GMP produced a short-latency hyperpolarization associated with a decrease in input resistance. This was followed by a long-latency but long duration depolarization associated in some cells with an increase in input resistance. This change in resistance followed the depolarization and outlasted it by 5–20min, until complete recovery. During the depolarization there was a reduction in magnitude of EPSPs produced by activation of the Schaffer collateral excitatory afferents. The reversal potential for the hyperpolarization was about —95 mV, and it was blocked by 4-aminopyridine. The depolarization, but not the hyperpolarization was markedly attenuated in slices maintained in low (25°C) temperature. The responses to ACh were blocked by atropine but not byd-tubocurarine. The hyperpolarization as well as the depolarization were present in slices treated with tetrodotoxin (TTX)., but were reduced in slices superfused with a low Ca²⁺-high Mg²⁺ medium, and in slices treated with Mn²⁺ and Co²⁺ ions. It is suggested that ACh causes a fast increase in gK⁺, followed by along-lasting energy-dependent depolarization associated with action potential discharges, a decrease in conductance and a suppression of EPSPs.