Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm

Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.     

A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers

Central cholinergic systems have been visualized by immunohistochemistry using antibodies to choline acetyltransferase (ChAT). Peripheral cholinergic cells and fibers, however, have been hardly detectable with most of these antibodies. This phenomenon suggests that a different form of ChAT may exist in peripheral tissues. Here we report two types of mRNA for ChAT expressed by alternative splicing in rat pterygopalatine ganglion. One is exactly identical with ChAT mRNA reported in the central nervous system (ChAT of a common type; cChAT). The other lacks exons 6, 7, 8 and 9, which was detected only in the pterygopalatine ganglion (ChAT of a peripheral type; pChAT). The peculiarity of pChAT in chemical structure, possessing a splice joint of the exons 5 and 10, led us to produce rabbit antisera against a recombinant peptide of 41 amino acids which spans over the splice joint. On Western blots using a successfully obtained antiserum, an intense band of about 50 kDa, corresponding to the expected molecular weight of pChAT, was detected in the pterygopalatine ganglion but not in the brain. Immunohistochemistry using the antiserum failed to reveal positive staining of known brain cholinergic structures, while it permitted us to observe peripheral, probably cholinergic, nerve cells and fibers including those in the pterygopalatine ganglion and enteric nervous system.     

Expression and localization of pChAT as a novel method to study cholinergic innervation of rat adrenal gland

Cholinergic innervation of the rat adrenal gland has been analyzed previously using cholinergic markers including acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). In the present study, we demonstrate putative cholinergic neurons in the rat adrenal gland using an antibody to pChAT, which is the product of a splice variant of ChAT mRNA that is preferentially localized in peripheral cholinergic nerves. Most of the ganglionic neurons as well as small single sporadic neurons in the adrenal gland were stained intensely for pChAT. The density of pChAT-immunoreactive (IR) fibers was distinct in the adrenal cortex and medulla. AChE-, cChAT- and VAChT-immunoreactivities were also observed in some cells and fibers of the adrenal medulla, while the cortex had few positive nerve fibers. These results indicate that ganglionic neurons of the adrenal medulla and nerve fibers heterogeneously express cholinergic markers, especially pChAT. Furthermore, the innervation of the adrenal gland, cortex and medulla, by some cholinergic fibers provides additional morphological evidence for a significant role of cholinergic mechanisms in adrenal gland functions.     

Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats

Using a recently developed antiserum against a splice variant (pChAT) of choline acetyltransferase, the enzyme which synthesizes acetylcholine, we carried out an immunohistochemical examination in the digestive canal of rats. Positive staining was exclusively localized to neuronal cells and fibers. Positive somata were distributed widely in the intramural ganglia throughout the digestive tract from the esophagus to the rectum. Double staining indicated that, in the rat, virtually all pChAT immunoreactive somata exhibited histochemical activity for acetylcholinesterase but not for NADPH-diaphorase. In the guinea pig, however, there were a few neurons possessing both pChAT and NADPH-diaphorase. We also found a few neuronal somata which were positive for acetylcholinesterase but not for pChAT. The results suggest that pChAT immunohistochemistry is useful for studying the enteric cholinergic system.     

Co-expression pattern of neuronal nitric oxide synthase and two variants of choline acetyltransferase in myenteric neurons of porcine ileum

Cholinergic enteric neurons were demonstrated immunohistochemically so far by using antibodies staining the common choline acetyltransferase (cChAT) in neurons of the central nervous system. The results of staining in the enteric nervous system of various species were, however, not satisfactory. We describe here findings obtained with a newly raised antibody against a peripheral variant of choline acetyltransferase (pChAT) in myenteric neurons of the pig small intestine. Triple labelling for pChAT/cChAT/neuronal nitric oxide synthase (nNOS) revealed 19.7% of 1664 neurons (within 40 ganglia) to be immunoreactive exclusively for pChAT whereas 29.6% were positive for cChAT alone and 18.8% were reactive only for nNOS. Colocalization of pChAT and cChAT was found in 22.4%, of pChAT and nNOS in 8.1% and of cChAT and nNOS in 1.4%. All three markers were simultaneously found in only 1 of 1664 neurons. To investigate the presence and possible colocalization of the above markers within morphologically defined neuron types, triple labelling of cChAT or nNOS with pChAT and a neurofilament (NF) antibody pool was applied and the coexpression patterns of pChAT and cChAT as well as of pChAT and nNOS in 120 neurons of each type were recorded. All type I, II, IV and V neurons displayed immunoreactivity either for one or both cholinergic markers. These neuron types were considered to be cholinergic. All type VI neurons, a descending neuron population, were negative for cChAT but positive for nNOS. However, 95% were immunoreactive for both pChAT and nNOS. The physiological significance of the possible co-existence of acetylcholine and nitric oxide within type VI neurons remains to be clarified. It is concluded that the pChAT and cChAT antibodies used here recognize partly different populations of enteric neurons in the pig. Thus, for total immunohistochemical characterization of cholinergic enteric neurons both forms of choline acetyltransferase have to be considered.     

Peripheral type of choline acetyltransferase: biological and evolutionary implications for novel mechanisms in cholinergic system

The peripheral type of choline acetyltransferase (pChAT) is an isoform of the well-studied common type of choline acetyltransferase (cChAT), the synthesizing enzyme of acetylcholine. Since pChAT arises by exons skipping, its amino acid sequence is similar to that of cChAT, except the lack of a continuous peptide sequence encoded by all the four exons from 6 to 9. While cChAT expression has been observed in both the central and peripheral nervous systems, pChAT is preferentially expressed in the peripheral nervous system.pChAT appears to be a reliable marker for the visualization of peripheral cholinergic neurons and their processes, whereas other conventional markers including cChAT have not been used successfully for it. In mammals like rodents, pChAT immunoreactivity has been observed in most, if not all, physiologically identified peripheral cholinergic structures such as all parasympathetic postganglionic neurons and most neurons of the enteric nervous system. In addition, pChAT has been found in many peripheral neurons that are derived from the neural crest. These include sensory neurons of the trigeminal ganglion and the dorsal root ganglion, and sympathetic postganglionic neurons. Recent studies moreover indicate that pChAT, as well as cChAT, appears ubiquitously expressed among various species not only of vertebrate mammals but also of invertebrate mollusks. This finding implies that the alternative splicing mechanism to generate pChAT and cChAT has been preserved during evolution, probably for some functional benefits.     

微透析技术在建立灵长类帕金森病模型中的应用

为了检测单侧注射ＭＰＴＰ制备的帕金森病恒河猴模型纹状体内多巴胺及其代谢产物的含量变化,本研究采用脑内微透析技术和高效液相色谱－ 电化学方法检测了双侧尾状核头部多巴胺及其代谢产物３,４－二羟基苯乙酸和高香草酸的含量。结果证明,ＭＰＴＰ注射侧与注射对侧相比,多巴胺、３,４－二羟基笨乙酸和高香草酸的含量分别降低８５．７％ , ９５．１％ 和６７．８％ 。这与动物呈现单侧帕金森病症状,核磁共振检查显示单侧黑质区域密度减低、面积缩小以及经抗酪氨酸羟化酶免疫组化方法显示脑切片单侧多巴胺能神经元明显减少等现象一致。以上结果表明,脑内微透析技术是活体检测脑内神经递质含量变化的有效方法,可以反映模型的病程变化,可用于评价帕金森病的治疗效果。     

Immunohistochemical localization of choline acetyltransferase of a peripheral type in the rat larynx

As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy: and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P.     

Choline acetyltransferase (ChAT) immunoelectron microscopy distinguishes at least three types of efferent synapses in the organ of Corti

Summary Using anatomical criteria, the olivo-cochlear fibers ending in the organ of Corti (efferent fibers) have recently been separated into two systems: a lateral system innervating principally the inner hair cell (IHC) area and a medial system innervating mainly the outer hair cells (OHCs). Electrophysiological and biochemical experiments suggest that acetylcholine may be a neurotransmitter of these efferent fibers. However, efferent synapses that use acetylcholine as neurotransmitter have not yet been identified at the electron microscopic level. Using a pre-embedding immunoelectron microscopic technique with a monoclonal antibody against choline acetyltransferase (ChAT), we localized ChAT-immunostained fibers below both the IHCs and OHCs. In the inner spiral bundle, one type of ChAT-immunostained fibers was vesiculated and formed axo-dendritic synapses with the afferent auditory dendrites contacting the inner hair cells. A second type of ChAT-immunostained fibers seen in the inner spiral bundle was unvesiculated. Unstained vesiculated varicosities synapsing with the auditory dendrites were also seen in the inner spiral bundle. At the OHC level, ChAT immunostaining was found in nearly all the terminals synapsing with the OHCs. The finding of two types of ChAT-immunostained efferent synapses in the organ of Corti, i.e. axodendritic synapses in the inner spiral bundle and axosomatic synapses with the OHCs, supports the hypothesis that both the lateral and the medial olivocochlear systems use acetylcholine as a neurotransmitter. The finding of numerous unstained synapses in the inner spiral bundle, and some below OHCs, together with previous data about putative cochlear neurotransmitters, suggests the possibility of additional non-cholinergic olivo-cochlear systems. It might soon appear useful to reclassify efferents according to the nature of the different neurotrans-mitters/ co-transmitters found in the various efferent synapses of the organ of Corti.     

Immunoelectron microscopy identifies several types of GABA-containing efferent synapses in the guinea-pig organ of Corti

Using an immunoperoxidase technique, we have localized by light and electron microscopy GABA-immunostained fibers within a component of the efferent innervation of the organ of Corti. At the light microscopic level, GABA-immunostained fibers were observed within the inner spiral bundle (below the inner hair cells) and the tunnel spiral bundle. The immunostaining was clearly more intense in the upper turns than in the basal turns. Mostly in the upper turns, GABA-immunostained fibers were seen crossing the tunnel of Corti to reach the outer hair cells where they formed large immunostained patches at the base of the cells. Unevenly distributed throughout these upper turns, immunostained fibers were seen climbing along the outer hair cells and traveling near the non-sensorineural Hensen's cells. The electron microscopic observations of GABA-immunostained fibers in the upper turns allowed us to identify within the inner spiral bundle vesiculated varicosities synapsing with radial dendrites connected to the inner hair cells. In the outer hair cell area, the GABA-immunostained fibers made several kinds of synaptic contacts. They included a minor population of the large axosomatic synapses with the basal pole of the outer hair cells and many axodendritic synapses with the spiral dendrites connected to these cells. Occasionally, the GABA-immunostained climbing fibers also synapsed with the outer hair cells at a supranuclear level. These result confirm previous light microscopic data dealing with the projection of the GABA-immunostained fibers along the cochlear partition. Moreover, they extend them in characterizing several kinds of GABA-immunostained synapses. These latter findings agree with previous neurochemical electrophysiological data which suggests an efferent neurotransmitter role for GABA. Nevertheless, such an existence of an efferent innervation predominantly projecting to the upper turns of the cochlea adds another criterion distinguishing the "apical" from the "basal" cochlea.     

ChAT-like immunoreactivity of olivocochlear fibres on rat outer hair cells during the postnatal development

Summary Several studies present a great deal of information about putative efferent neurotransmitters and their distribution in the adult and developing cochlea. Anatomical mapping of outer hair cell efferent fibres during ontogeny is still not available. Using quantitative electron microscopy in combination with immunocytochemistry, the distribution of ChAT-like immunoreactivity in the developing rat was investigated. Adult-like immunoreactivity in the whole cochlea is first observed in 30-day-old rats. We localized the adult-like immunoreactivity in all efferent fibres and synapses of the outer hair cells along the entire cochlear duct. An adult-like reaction in the whole cochlea could be observed on the 25th day after birth in two out of three cases. On the 20th postnatal day, no adult-like ChAT immunoreactivity was found, with the exception of one case where labelling was seen in the basal region only. The adult-like ChAT immunoreactivity on the 30th day, 2–3 weeks after the onset of hearing, is the latest maturation of all features of the organ of Corti so far investigated. Synaptogenesis of the outer hair cell efferents reaches an adult-like appearance already on the 16th day after birth.     

Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat

A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component.     

Projections of auditory nerve in the cat as seen by anterograde transport methods.

Ascending projections of the auditory nerve of the cat were studied by autoradiographic methods after injection of labeled precursors into the cochlea. In animals in which [³H]leucine was injected, transport to the cochlear nucleus was observed after survival periods of 2–7 days, but there was no evidence of transport to more central structures in the auditory pathways. In these cases the pattern of labeling around cell bodies depended on the type of cell and on the location of the cell within subdivisions of the cochlear nucleus. For example, there was dense perisomatic labeling around spherical cells in the anteroventral cochlear nucleus and around octopus cells in the posteroventral cochlear nucleus. Some other types of cells in subdivisions of both anteroventral and posteroventral cochlear nucleus had little perisomatic labeling. In the dorsal cochlear nucleus, the most densely labeled area consisted of a band just central to the layer of fusiform cells. Labeled fibers were not found peripheral to this band in the granular layer, molecular layer and peripheral part of the layer of fusiform cells. In the dorsal nucleus there was no evidence of dense perisomatic labeling such as was found around some cells in the ventral divisions of the cochlear nucleus. Following injections of a mixture of [³H]proline and [³H]fucose into the cochlea, labeling was seen in the superior olivary complex. Transneuronal transport was suggested as the explanation for this finding because of (1) the choice of the labeled precursors, (2) the length of the survival periods (14 or 20 days), and (3) the evidence that isotope was within neurons in the cochlear nucleus.We concluded that under appropriate conditions, unique patterns of silver grains are found around different types of cells in the various subdivisions of the cochlear nucleus, apparently because fibers and terminals of the auditory nerve become saturated with the radioisotope. As a result, a highly detailed survey of projections of the auditory nerve to each subdivision of the cochlear nucleus is revealed at the light-microscopic level. Under different experimental conditions labeled fibers are found in the superior olivary complex, but these conditions are those most conducive to transneuronal transport and so such experiments cannot be used as evidence of projection of auditory nerve fibers beyond the cochlear nucleus.     

Mechanics of the mammalian cochlea.

In mammals, environmental sounds stimulate the auditory receptor, the cochlea, via vibrations of the stapes, the innermost of the middle ear ossicles. These vibrations produce displacement waves that travel on the elongated and spirally wound basilar membrane (BM). As they travel, waves grow in amplitude, reaching a maximum and then dying out. The location of maximum BM motion is a function of stimulus frequency, with high-frequency waves being localized to the "base" of the cochlea (near the stapes) and low-frequency waves approaching the "apex" of the cochlea. Thus each cochlear site has a characteristic frequency (CF), to which it responds maximally. BM vibrations produce motion of hair cell stereocilia, which gates stereociliar transduction channels leading to the generation of hair cell receptor potentials and the excitation of afferent auditory nerve fibers. At the base of the cochlea, BM motion exhibits a CF-specific and level-dependent compressive nonlinearity such that responses to low-level, near-CF stimuli are sensitive and sharply frequency-tuned and responses to intense stimuli are insensitive and poorly tuned. The high sensitivity and sharp-frequency tuning, as well as compression and other nonlinearities (two-tone suppression and intermodulation distortion), are highly labile, indicating the presence in normal cochleae of a positive feedback from the organ of Corti, the "cochlear amplifier." This mechanism involves forces generated by the outer hair cells and controlled, directly or indirectly, by their transduction currents. At the apex of the cochlea, nonlinearities appear to be less prominent than at the base, perhaps implying that the cochlear amplifier plays a lesser role in determining apical mechanical responses to sound. Whether at the base or the apex, the properties of BM vibration adequately account for most frequency-specific properties of the responses to sound of auditory nerve fibers.     

Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiation

Cyclin A2 plays a major role in cell cycle modulation. Cyclin A2 level has not been determined in the inner ear yet. RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry were used to measure cyclin A2 expression in rat cochlea tissues of different ages, isolated cochlear stem cells and stem cell-derived differentiated cells. The results indicated that cyclin A2 level in cochlea tissues decreased gradually from newborn to adult. Furthermore, cyclin A2 level fell down after differentiation of cochlear stem cells. It was suggested that cyclin A2 might be involved in the modulation of rat cochlea development and cochlear stem cell differentiation.