Identification and localization of a neuron-specific isoform of TAF1 in rat brain: implications for neuropathology of DYT3 dystonia.

The neuron-specific isoform of the TAF1 gene (N-TAF1) is thought to be involved in the pathogenesis of DYT3 dystonia, which leads to progressive neurodegeneration in the striatum. To determine the expression pattern of N-TAF1 transcripts, we developed a specific monoclonal antibody against the N-TAF1 protein. Here we show that in the rat brain, N-TAF1 protein appears as a nuclear protein within subsets of neurons in multiple brain regions. Of particular interest is that in the striatum, the nuclei possessing N-TAF1 protein are largely within medium spiny neurons, and they are distributed preferentially, though not exclusively, in the striosome compartment. The compartmental preference and cell type-selective distribution of N-TAF1 protein in the striatum are strikingly similar to the patterns of neuronal loss in the striatum of DYT3 patients. Our findings suggest that the distribution of N-TAF1 protein could represent a key molecular characteristic contributing to the pattern of striatal degeneration in DYT3 dystonia.

In vivo detection of amyloid ß deposition using ¹9F magnetic resonance imaging with a ¹9F-containing curcumin derivative in a mouse model of Alzheimer's disease.

Amyloid ß (Aß) deposition in the brain is considered the initiating event in the progression of Alzheimer's disease (AD). Amyloid imaging is widely studied in diagnosing AD and evaluating the disease stage, with considerable advances achieved in recent years. We have developed a novel ¹9F-containing curcumin derivative (named FMeC1) as a potential imaging agent. This compound can exist in equilibrium between keto and enol tautomers, with the enol form able to bind Aß aggregates while the keto form cannot. This study investigated whether FMeC1 is suitable as a ¹9F magnetic resonance imaging (MRI) probe to detect Aß deposition in the Tg2576 mouse, a model of AD. In ¹9F nuclear magnetic resonance (NMR) spectra obtained from the whole head, a delayed decreased rate of F ¹9F signal was observed in Tg2576 mice that were peripherally injected with FMeC1 in comparison to wild-type mice. Furthermore, ¹9F MRI displayed remarkable levels of ¹9F signal in the brain of Tg2576 mice after the injection of FMeC1. Histological analysis of FMeC1-injected mouse brain showed penetration of the compound across the blood-brain barrier and binding to Aß plaques in peripherally injected Tg2576 mice. Moreover, the distribution of Aß deposits in Tg2576 mice was in accordance with the region of the brain in which the ¹9F signal was imaged. FMeC1 also exhibited an affinity for senile plaques in human brain sections. These findings suggest the usefulness of FMeC1 as a ¹9F MRI probe for the detection of amyloid deposition in the brain. Furthermore, the properties of FMeC1 could form the basis for further novel amyloid imaging probes.

NPR-A regulates self-renewal and pluripotency of embryonic stem cells.

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.

Axonal ligation induces transient redistribution of TDP-43 in brainstem motor neurons.

Nuclear exclusion of TAR DNA binding protein 43 (TDP-43) and formation of cytosolic aggregates are a pathological characteristic of amyotrophic lateral sclerosis (ALS). However, the molecular basis of the aberrant distribution of TDP-43 remains elusive. Here, we show evidence that axonal ligation induced transient nuclear exclusion and peripheral accumulation of TDP-43, without apparent cytosolic aggregates in hypoglossal neurons in mice. Immunohistochemistry showed marked loss of nuclear TDP-43 7-14 days after ligation, which was accompanied by reduction of choline acetyltransferase (ChAT). TDP-43 staining was restored in the nucleus on day 28 exclusively in the neurons with normalized ChAT expression. We also showed that importin beta, which was shown to mediate nuclear transport of TDP-43 was downregulated transiently by nerve ligation. The analysis of the peripheral nerves proximal to the ligation revealed that TDP-43 markedly accumulated with a concomitant decrease in active autophagosome. Moreover, we showed that TDP-43 was present in the microsome fraction containing endoplasmic reticulum (ER) or autophagosomes in the brainstem section, indicating that TDP-43 is axonally transported with vesicles. These results indicate that axonal damage is associated with redistribution of TDP-43 through the combination of defective axonal autophagy periphery and the impaired nuclear transport system in the soma. Moreover, it was also shown that transient redistribution of TDP-43 does not prevent motor neurons from axonal regeneration. Therefore, our data suggest that the subcellular distribution of TDP-43 correlates to the innervation status of motor neurons, which may be governed by unidentified cause of ALS.

Distribution of natriuretic peptide receptor-C immunoreactivity in the rat brainstem and its relationship to cholinergic and catecholaminergic neurons.

The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides.

Low expression of FGF1 (fibroblast growth factor-1) in rat parasympathetic preganglionic neurons.

Fibroblast growth factor-1 (FGF1), a member of the FGF family of growth factors, is localized in cholinergic neurons where it has trophic activity. We recently reported that cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) contain little FGF1, raising the possibility that FGF1 is not localized to parasympathetic preganglionic cholinergic neurons. To clarify this issue, we investigated the co-localization of FGF1 with cholinergic neuron markers in the Edinger-Westphal nucleus (EWN), salivatory nucleus, DMNV, and sacral parasympathetic nucleus by double immunofluorescence using antibodies to FGF1 and choline acetyltransferase (ChAT). The neurons in the EWN were devoid of FGF1. In the salivatory nucleus, 13% of ChAT-positive neurons were also positive for FGF1. In the DMNV, only 8% of ChAT-positive neurons contained FGF1, and in the sacral parasympathetic nucleus, 18% of ChAT-positive neurons were FGF1-positive. We also confirmed that a large number of ChAT-positive motor neurons in the oculomotor nucleus, facial nucleus, hypoglossal nucleus, and spinal motor neurons contained FGF1. The results confirmed that parasympathetic preganglionic neurons are largely devoid of FGF1, which is a unique feature among cholinergic neurons.

Immunohistochemical mapping of natriuretic peptide receptor-A in the brainstem of Macaca fascicularis.

Natriuretic peptide receptor-A (NPR-A) mediates the biological effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and is involved in maintaining cardiovascular homeostasis. In this immunohistochemical study we examined the distribution of NPR-A in the brainstem of the cynomolgus monkey. NPR-A immunoreactivity was localized to neurons in specific brainstem regions. NPR-A-immunoreactive perikarya were found in the red nucleus and the oculomotor nucleus in the midbrain, the parabrachial nucleus and the locus coeruleus in the pons, and the dorsal motor nucleus of the vagus, the hypoglossal nucleus, the cuneate nucleus, the gracile nucleus, the nucleus ambiguus, the lateral reticular nucleus, the reticular formation, and the inferior olivary nucleus in the medulla oblongata. Extensive networks of immunoreactive fibers were apparent in the red nucleus, the oculomotor nucleus, the principal sensory trigeminal nucleus, and the parabrachial nucleus. Double immunostaining revealed NPR-A immunoreactivity in cholinergic neurons of the parabrachial nucleus, the dorsal motor nucleus of vagus, the hypoglossal nucleus, and the nucleus ambiguus. However, there was no colocalization of NPR-A and tyrosine hydroxylase in the locus coeruleus. The wide anatomical distribution of NPR-A-immunoreactive structures suggests that natriuretic peptides, besides having a role in the central regulation of endocrine and cardiovascular homeostasis, may also mediate diverse physiological functions.

Comparison of MR images and histochemical localization of intra-arterially administered microglia surrounding beta-amyloid deposits in the rat brain.

The therapeutic use of microglial cells has recently received some attention for the treatment of Alzheimer disease (AD), but few non-invasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technique for tracking microglia injected intra-arterially in vivo. We micro-injected Abeta42 into the left hippocampus and saline into the right hippocampus of rats. We then administered microglia, which were labeled with enhanced green fluorescent protein (EGFP) gene and Resovist, into the carotid artery. After monitoring exogenously administered microglia using MRI, we compared the MR images and the histochemical localization of administered microglia. MRI revealed clear signal changes attributable to Resovist-containing microglia in Abeta-injected areas. Histochemistry demonstrated that EGFP-positive microglia accumulated around Abeta deposits and internalized the peptide. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for AD.

Immunohistochemical study of TAFII250 in the rat laryngeal nervous system.

The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TAFII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was down-regulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.

Fate of (D-Ala2)-deltorphin-I-like immunoreactive neurons in 6-hydroxydopamine lesioned rat brain.

The use of a polyclonal antiserum specific to C-terminal tetrapeptide amide of (D-Ala2)deltorphin-I, a naturally occurring amphibian skin opioid peptide, has already demonstrated the presence of immunoreactive neurons in rat midbrain. Double immunostaining identified these neurons as a subpopulation of the mesencephalic dopaminergic neurons that were also tyrosine hydroxylase-immunopositive and calbindin-D28kD- negative, namely, the neurons predominantly affected in Parkinson disease. We followed the fate of these neurons after a monolateral injection of 6-hydroxy-dopamine into rat brain. Almost all the immunopositive neurons and their nigrostriatal, mesolimbic and mesocortical projections on the side ipsilateral to the lesion disappeared. Only a few scattered immunopositive neurons within the substantia nigra, pars compacta, and those of supramammillary nucleus remained unaffected. The consistent overlap of dopamine and this new molecule provides a further key to identifying the mammalian counterpart of these amphibian skin opioid peptides.

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.

Acidic fibroblast growth factor (FGF-1) in the anterior horn cells of ALS and control cases.

The expression and localization of acidic fibroblast growth factor (aFGF; FGF-1) were examined in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and controls by reverse transcription-polymerase chain reaction (RT-PCR) method and immunohistochemistry. The RT-PCR experiments demonstrated that aFGF amplification products were clearly detected in all control cases but could be scarcely seen in ALS patients. aFGF immunoreactivity was detected in the anterior horn cells of the spinal cord. Double immunostaining for aFGF and choline acetyltransferase revealed that the majority (95.9%) of cholinergic neurons expressed aFGF. In ALS cases, the number and the staining intensity of aFGF-positive neurons were markedly decreased. These results suggest that aFGF is involved in ALS pathology.

Fetal alveolar epithelial cells contain [D-Ala(2)]-deltorphin I-like immunoreactivity: delta- and mu-opiate receptors mediate opposite effects in developing lung.

Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-LI-positive cells were alveolar cells negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-LI-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta-receptor-specific) versus dermorphin (mu-receptor-specific). Type II pneumocyte differentiation, assessed by [(3)H]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [(3)H]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorphin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta-blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu-blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by binaltorphimine, dermorphin-inhibited thymidine incorporation was reversed by delta, kappa-, or mu-blockers. The delta- and mu-receptor messenger RNAs occurred pre- and postnatally, whereas kappa-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from proSP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.

Correlation of the expression level of C1q mRNA and the number of C1q-positive plaques in the Alzheimer Disease temporal cortex. analysis of C1q mrna and its protein using adjacent or nearby sections.

We compared the expression level of C1q mRNA and the number of C1q-positive plaques in adjacent or nearby brain sections from Alzheimer disease (AD) and control cases. Small blocks of temporal cortex were fixed with 4% paraformaldehyde for 2 days at 4 degrees C. After cryoprotection with solutions containing 10-20% glycerol and 2% dimethylsulfoxide, 40-microm sections were cut from the tissue blocks. A section from each case was stained by immunohistochemistry using a C1q antibody, while RNA was purified from adjacent or nearby sections using a combination of proteinase K pretreatment followed by extraction using Trizol reagent. The expression of C1q B chain mRNA was analyzed in these samples by the reverse-transcription polymerase chain reaction (RT-PCR). The intensities of the PCR products were measured by an image analyzer. The expression of C1q B chain mRNA was significantly more abundant in AD than in control cases (p < 0.05). Immunohistochemical analysis showed that C1q protein was localized in senile plaques in the AD brain. The number of C1q-positive plaques correlated with the expression level of C1q gene (p < 0.05). The present results suggest that C1q protein in senile plaques originates is endogenously produced in the AD brain.

2-buten-4-olide, an endogenous feeding suppressant, improves spatial performance through brain acidic fibroblast growth factor in mice.

Endogenous sugar acid 2-buten-4-olide, a satiety substance, has been shown to increase the blood glucose, norepinephrine, and glucocorticoid concentrations that are known to modulate learning and memory processes. The glucose-induced release of acidic fibroblast growth factor facilitated the hippocampus-dependent memory function. In the present study, we investigated the effect of 2-buten-4-olide on the spatial performance of male DDY mice undergoing the water maze task. The intraperitoneal injection of 2-buten-4-olide (5 mg/kg) facilitated the spatial performance, which was indicated by a reduction in the escape latency in which the mouse finds and climbs the goal platform in comparison to the vehicle-injected control mice. In the probe test after removing the platform, the 2-buten-4-olide-treated mice stayed a longer time in the quadrant where the platform was originally located and crossed more frequently at the platform location than did the control mice. The pretreatment of acidic fibroblast growth factor antibody injected into the lateral ventricle eliminated the effect of 2-buten-4-olide both during the training sessions and during the probe test. Therefore, 2-buten-4-olide was found to improve the spatial performance, and this effect is mediated, at least in part, by acidic fibroblast growth factor.

Production and immunohistochemical application of antiserum against Tyr-D-Ala-Phe, a N-terminal tripeptide common to dermorphin/deltorphin family.

Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 microg of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin.

Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons.

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.

Ontogeny of (D-Ala(2))-deltorphin I-like immunoreactive neurones in foetal rat brain.

Foetal rat brain from embryonic day (ED) 12-22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of (D-Ala(2))-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15-17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.

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.

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.