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Praca oryginalna Original paper
Initially acetylcholinesterase (AChE) (8) and later choline acetyltransferase (ChAT) have been used as enzymatic markers for studying cholinergic neurons. ChAT catalyses synthesis of acetylcholine (ACh) from its two components acetyl coenzyme A and choline, while AChE is responsible for degradation of acetyl-choline. Quantitative measurements of ChAT and ACh in the brain revealed regional correlation, suggesting that ChAT is a highly specific marker for cholinergic neurons (3, 5, 11). Antisera raised to purified ChAT preparations have been used successfully for histo-chemical localization of cholinergic nerve structures (9). Recently, vesicular acetylcholine transporter (VAChT), an additional, specific marker for choliner-gic neurons enabled immunocytochemical identifica-tion of the neurons (1). Although, both ChAT- and VAChT-immunoreactivity specifically identify choli-nergic neurons they represent different subcellular localization. While ChAT as a cytoplasmic enzyme preferentially labels neuronal somata, VAChT is as-sociated with ACh-containing synaptic vesicles thus constituting prevailing marker for neural processes.
Over the last years cholinergic neurons have been studied mostly on laboratory animals (12, 18). Never-theless, due to inter-species differences simple
trans-fer of experimental data acquired from small labora-tory animals to large husbandry mammals, and espe-cially to human bears certain risk. In fact, pig due to its embryological, anatomical and physiological simi-larities to human constitutes especially valuable spe-cies for bio-medical research (15). Hitherto, however, morphology and distribution of the porcine hypo-thalamic cholinergic neurons have been poorly studied, since only report of Ca³ka et al. (2) is available.
The present study was designed to identify and com-pare the localization and distribution of ChAT- and VAChT-immunoreactive neurons in preoptic, supra-optic and tuberal nuclei of the pig hypothalamus. The results were obtained by application of staining methods for immunocytochemical detection of ChAT and VAChT.
Material and methods
Handling of animals followed the Principles of labora-tory animal care (NIH publication No. 86-23, rev. 1985) and the specific national laws on experimental animal handling.
Five sexually mature gilts of the Large White Polish race (body weight ca. 130 kg) were used for the study. The animals originated from a commercial fattening farm. They
Distribution and morphology
of ChAT- and VAChT-immunoreactive neurons
in the tuberal hypothalamus of the pig
JAROS£AW CA£KA, JUDYTA JURANEK, KRZYSZTOF W¥SOWICZ, JERZY KALECZYC, MIROS£AW £AKOMY
Division of Animal Anatomy, Department of Functional Morphology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego 13, 10-719 Olsztyn, Poland
Ca³ka J., Juranek J., W¹sowicz K., Kaleczyc J., £akomy M.
Distribution and morphology of ChAT- and VAChT-immunoreactive neurons in the tuberal hypothalamus of pigs
Summary
Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are currently accepted specific markers for cholinergic neurons. Current knowledge concerning the organization of the hypo-thalamic cholinergic system has been derived predominantly from laboratory animals. In the present study, applying immunocytochemistry the authors investigated the morphology and distribution of the ChAT- and VAChT-immunoreactive nerve cells and terminals in the porcine hypothalamic preoptic, supraoptic and tuberal nuclei. ChAT-immunoreactive perikarya were present in the tuberal arcuate nucleus. Numerous VAChT--positive terminals were found in the external layer of the median eminence, while perikarya occupied the perifornical, dorso-caudal and dorsal hypothalamic nuclei. These results provide morphological indications that the cholinergic hypothalamic system may affect the secretory function of the median eminence in pigs.
Medycyna Wet. 2007, 63 (4) 413
were kept under standard conditions with free access to food and water. Prior to the main anesthesia the pigs were pretreated with propionylpromazine (Combelen, Bayer, Germany; 0.4 mg/kg b.w., i.m.). The animals were deeply anaesthetized with pentobarbital (Vetbutal, Biowet, Poland; 25 mg/kg, b.w., i.v.) and perfused transcardially with 4% solution of paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The skulls were then opened and the brains removed, the hypothalami were dissected out, postfixed in the same fixative as used for perfusion (2 hours), rinsed in PB overnight and transferred to 30% solution of sucrose in PB. After 5 days the tissues were cut with a cryostat into 20 µm frozen sections. Tissue sections were stored in cryoprotectant solution in the freezer (19) until they were processed for immunocytochemistry.
Selected sections containing preoptic, supraoptic and tuberal hypothalamic nuclei were subjected to single immunostaining for ChAT or VAChT. The slides were air-dried, hydrated in phosphate-buffered saline (PBS) and blocked with mixture containing 0.25% Triton X-100, 1% bovine serum albumin, and 10% normal goat serum in PBS for 1 h at room temperature. After rinsing in PBS (3 × 10 min.) selected sections were incubated with a rabbit poly-clonal anti-ChAT antibody (Chemicon International Inc.) diluted to 1 : 5000. Other sections were incubated with a rabbit polyclonal anti-VAChT antibody (Phoenix Phar-maceuticals Inc.) in dilution 1 : 4000. Incubation was car-ried out overnight at room temperature and the following morning the slides were rinsed in PBS (3 × 10 min.). Bio-tinylated secondary antisera (Vector Laboratories Inc.) directed against the host of primary antisera in dilution 1 : 400, were then incubated for 1 h at room temperature. After rinsing in PBS (3 × 10 min.) sections were incubated with CY3-conjugated streptavidin (Jackson Immuno--Research Laboratories Inc.) in dilution 1 : 4000 for 1 h at room temperature. Finally the slides were rinsed in PBS (3 × 10 min.) and then coverslipped with carbonate-buf-fered glycerol (pH 8.6).
The slides were then analyzed and photographed under a fluorescent microscope (Axiophot, Zeiss, Germany).
Omission of the primary antisera as well as their re-placement with normal rabbit serum proved the specificity of the immunoreaction.
Results and discussion
All hypothalamic nuclei examined in this study were identified according to an atlas of the stereotaxic con-figuration of hypothalamic nerve centers in the pig of Szteyn et al. (16) and Welento (21).
ChAT-immunocytochemistry. In the region under study positive ChAT-immunoreactive staining was found in tuberal arcuate and ventromedial hypothala-mic nuclei. In the arcuate nucleus concentration of stained cells was observed in the ventral part of the nuclear region occupying the area located over the separation of the median eminence (fig. 1a) and exten-ded towards ventrolateral aspect of the nucleus.
In the ventromedial nucleus only scattered weakly stained neurons were encountered (fig. 1b). Labeled perikarya were oval, fusiform or triangular in shape
and approximately 15-20 µm in diameter. Between stained cell bodies ChAT-positive processes penetra-ted tissue matrix. A granular reaction product that com-pletely filled the cytoplasm of the perikaryon and often one or more of its processes visualized ChAT immu-noreactivity. Cellular nuclei were not stained.
The nuclei of the preoptic area and supraoptic region were devoid of the ChAT-immunospecific staining.
VAChT-immunocytochemistry. Immunoreactive axons were observed throughout the rostro-caudal extent of the median eminence. The VAChT-immu-nostaining was distributed mainly in the external layer of the organ with the greatest concentrations adjacent to the blood vessels (fig. 1c). The stained processes formed a ring-shaped concentration of the more inten-sive VAChT-immunolabeled profiles surrounding the blood vessels and filled the outer border of the exter-nal layer. Between blood vessels of the exterexter-nal zone the moderately or weakly stained, densely packed, VAChT-immunolabeled beads were more or less uni-formly distributed.
The VAChT immunostaining was found in neurons of the perifornical nucleus (fig. 1d). The immunoreac-tivity was observed in loosely arranged, intensely stained, spindle-shaped cells measuring along longi-tudinal axis from 25 to 50 µm. The fusiform perikarya lain parallelly to the surface of the fornix. The VAChT--immunoreactive material appeared as clumps distributed within the cytoplasm.
Dorsal hypothalamic nucleus was found to contain loosely arranged the VAChT-immunoreactive peri-karya and processes (fig. 1e). Round, multipolar and fusiform cell bodies measured 10 to 25 µm in dia-meter. Between the stained perikarya profiles of the immunolabeled processes were observed.
The VAChT-immunoreactive structures were en-countered in dorso-caudal hypothalamic nucleus (fig. 1f). The regular, stained cell bodies, measuring 15-25 µm in diamter, were irregularly spread through-out the nuclear area. The VAChT-immunoreactive ma-terial filled their multipolar, round and spindle-shaped perikarya together with proximal parts of neuronal pro-cesses. The nuclear matrix was penetrated by modera-tely and weakly stained fibers. Between the stained somata single, multipolar, larger perikarya measuring 30 to 40 µm in diameter were observed. Their cyto-plasm was filled with clumps of the immunoreac-tive material. The nuclei were devoid of the VAChT--immunoreaction product.
The results of our investigation show differential distribution of ChAT- and VAChT-immunoreactive neurons throughout porcine hypothalamus.
Differential visualization of cholinergic perikarya and processes by ChAT and VAChT immunoreacti-vity was the most spectacular in the arcuate/median eminence (ARC/ME) complex. Prominent ChAT-im-munoreactivity of the arcuate neurons was
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nied by lack of ChAT-positive staining in the median eminence. On the other hand VAChT-immunoreactive terminals were found in the median eminence, while the arcuate nucleus was devoid of VAChT-immuno-reactivity. Our results corroborate with observations of Weihe et al. (20) showing that cholinergic nerve terminals contain more VAChT- than ChAT-immuno-reactivity, consistent with concentration of VAChT within terminal neuronal arborizations in which secre-tory vesicles are clustered. Thus, ChAT-immunoreac-tivity is more prominent in cholinergic cell bodies and proximal processes reflecting cytosolic localization of ChAT. Distribution of both cholinergic markers in the porcine ARC/ME complex is in good agreement with findings of Ichikawa et al. (6) who showed ChAT-im-munopositive perikarya in the rat arcuate nucleus, while both ChAT- and VAChT-immunoreactivities were dis-closed in the median eminence. Likewise, presence of the ChAT-immunoreactive arcuate perikarya sending their processes towards the external layer of the median eminence was reported in the rat and monkey (17). Despite of the established differences in the ChAT and VAChT distribution in the pig it becomes evident that there exists a great similarity in the organization of the cholinergic centers of the ARC/ME complex in the mammalian species studied.
The exceptionally high representation of choli-nergic terminals in the external layer of the median eminence indicates locally confined capacity for ace-tylcholine production. Although the functional signi-ficance of those fibers remains still unclear there are suggestions that they may affect the secretory func-tion of the neuroendocrine organ. Cholinergic stimu-lation has been implicated in CRH release from the rat hypothalamus (7). Acetylcholine has been ascribed to induce LH-RH release from the rat medial basal hypo-thalamus (14), while atropine inhibited ovulation (13). Moreover, ChAT was present in the highest concen-tration in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH (10). Cholinergic drugs inhibited prolactin secretion (4). At present however, the precise role of cholinergic neu-rons in the pig ARC/ME complex awaits confirma-tion, and deserves further study.
Our investigation revealed concentration of the VAChT-immunoreactive neurons in the perifornical, dorso-caudal and dorsal hypothalamic nuclei. Although VAChT-immunoreactive the parikarya have not stained for ChAT. The reason for that is unclear though earlier application of AChE histochemistry in the porcine
hypothalamus revealed group of neurons of similar distribution and morphology in those nuclei of the pig (2). The significance of the cholinergic cell group is unknown and further morphological and functional analyses are required.
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Authors address: dr hab. Jaros³aw Ca³ka, prof. UWM, ul. Oczapow-skiego 13, bl. 105 J, 10-719 Olsztyn; e-mail: calkaj@uwm.edu.pl
Fig. 1. (a) ChAT-immunopositive cell bodies (large arrows) and fibers (small arrows) in the arcuate nucleus. (b) Spindle shaped cell (arrow) of the ventromedial nucleus with moderately stained cytoplasm. (c) Dense plexus of the VAChT immuno-positive terminals (arrows) surrounding blood vessels of the external layer of the median eminence. (d) VAChT-immuno-reactive perikarya (arrows) in the perifornical nucleus, F-fornix. (e) Concentration of the VAChT-immunoVAChT-immuno-reactive cell bodies (arrows) in the dorsal hypothalamic nucleus. (f) The dorso-caudal hypothalamic nucleus was composed of regular VAChT--immunoreactive neurons (large arrows) and larger neurons filled with clumps of the immunoreactive material (small arrow). Bar = 40 µm.
