Participation of acetylcholine and its receptors in the contractility of inflamed porcine uterus.

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10-5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10-5 M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10-5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10-5 M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10-7, 10-6 M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10-7 M) and ENDO/MYO (ACh: 10-5 M) than in the SAL group. MR2 or MR3 antagonists and ACh (10-5 M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10-6, 10-5 M) than in other groups. Both antagonists and ACh (10-5 M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.

Expression of alpha and beta adrenergic receptors in the pig uterus during inflammation.

Under physiological conditions, noradrenaline (NA) and adrenergic receptors (ARs) are implicated in the function of the uterus. The role of NA and the expression of ARs in the inflamed uterus is not fully understood. The aim of the present study was to determine the effect of inflammation on the levels of α1 (A, B, D)-, α2 (A, B, C)- and ß (1, 2, 3)-ARs mRNA and protein expression and the localization of these receptors in the porcine uterus. On Day 3 of the estrous cycle (Day 0 of the study), 50 ml of either saline (group SAL) or E. coli suspension (109 colony-forming units/ml, group E. coli) were injected into each uterine horn. In the control pigs (group CON), only laparotomy was performed. Eight days later, α1D-ARs mRNA (P < 0.001) and protein (P < 0.05) levels and α2A-ARs protein level (P < 0.05) were increased in the inflamed endometrium, while the α2C-ARs protein level (P < 0.001) was lowered, as compared to the SAL and CON groups. In the inflamed endometrium, ß2-ARs mRNA (P < 0.01) and protein (CON: P < 0.01, SAL: P < 0.001) expression was lower than in the other two groups, and ß1-ARs mRNA (P < 0.001) and protein (P < 0.01) expression was higher compared to the SAL group. After bacterial treatment, α2A- (P < 0.001) and α2B (P < 0.05) -ARs protein levels and ß2-ARs mRNA (CON: P < 0.01, SAL: P < 0.05) and protein (CON: P < 0.01, SAL: P < 0.05) expression in myometrium were found to be increased compared to both groups. In turn, in myometrium following E. coli infusion, the α2C-ARs protein level was lower (P < 0.01) than in the CON group. All studied receptors were present in the luminal and glandular epithelium, blood vessels and myometrial muscular cells of the gilt uteri in the E. coli, SAL and CON groups. The data show that inflammation changes the ARs expression in porcine uterus, suggesting their importance in the course/consequences of uterine inflammation. Those affected ARs may constitute a therapeutic target in an inflamed uterus.

Long-term estradiol-17ß exposure decreases the cholinergic innervation pattern of the pig ovary.

Elevated levels of endogenous estrogens in the course of pathological states of ovaries, as well as xenoestrogens, may lead to hyperestrogenism. It has previously been demonstrated that long-term estradiol-17ß (E2) administration in adult gilts affected the population of sympathetic intraovarian nerve fibers. The aim of this study has been to determine the effect of long-term E2 exposure on the cholinergic innervation pattern of porcine ovaries. Intraovarian distribution and the density of nerve fibers immunoreactive (IR) to vesicular acetylocholine transporter (VAChT) and/or neuronal isoform of nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) were determined. From day 4 of the first estrous cycle to day 20 of the second studied cycle, experimental gilts were intramuscularly injected with E2, while control gilts received corn oil. The ovaries were then collected and processed for double-labelling immunofluorescence. After E2 administration, the total number of fibers IR to VAChT, nNOS and VIP decreased significantly. The numbers of VAChT-, nNOS- and VIP-IR fibers within the ground plexus were significantly lower, while they were significantly higher around small or medium tertiary follicles. In the E2-affected ovaries, the numbers of nNOS- and VIP-IR fibers were significantly higher near secondary follicles and VAChT-IR in the vicinity of medullar blood vessels. In turn, around the latter structures there were significantly lowered populations of nNOS- and VIP-IR nerve fibers. These results suggest that the elevated E2 levels that occur during pathological states may affect the cholinergic innervation pattern of ovaries and their function(s).

Porcine dorsal root ganglia ovarian neurons are affected by long lasting testosterone treatment.

We studied the effect of testosterone overdose on the number, distribution and chemical coding of ovarian neurons in the dorsal root ganglia (DRGs) in pigs. On day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde tracer Fast Blue. From day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with testosterone, while the control gilts received oil. After the completion of the protocol the Th16-L5 DRGs were collected. Injections of testosterone increased the testosterone (~3.5 fold) and estradiol-17beta (~1.6 fold) levels in the peripheral blood, and reduced the following in the DRGs: the total number of the Fast Blue-positive perikarya, the population of perikarya in the L2-L4 ganglia, and the numbers of SP(+)/CGRP(+), SP(+)/PACAP(+), SP(+)/nNOS(+) and SP(-)/nNOS(+) perikarya. In the testosterone-injected gilts, the populations of SP(+)CGRP(-), small and large androgen receptors-expressing perikarya were increased. These results suggest that elevated androgen levels during pathological states may regulate the transmission of sensory modalities from the ovary to the spinal cord, and antidromic regulation of the ovarian functions.

Neuropeptide profile changes in sensory neurones after partial prepyloric resection in pigs.

This report details the first identification of the sources of sensory innervation of the porcine stomach prepyloric region. The Fast Blue (FB) retrograde tracing technique detected the sensory prepyloric neurons in the bilateral nodose ganglia (NGs) as well as thoracic dorsal root ganglia (DRGs). Double-labelling immunofluorescence demonstrated expression of substance P (SP), calcitonin gene-related peptide (CGRP), neuronal isoform of nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and galanin (GAL) in both NGs and DRGs. Additionally, we found that partial resection of the stomach prepyloric area increased expression of the SP, CGRP, NOS, VIP and GAL in the prepyloric sensory neurons. In the control left NGs, both a higher total number of FB-positive perikarya as well as a higher percentage of the peptides expressing prepyloric neurons were visualized than in the right NGs. However, compared to the control group, prepyloric resection evoked greater increases in peptide expression in the right-side NGs sensory neurons. In the ganglia of this side, the proportion of the SP-IR perikarya increased by approximately 15%, while CGRP-IR increased by 28%, NOS-IR 14%, VIP-IR 43% and GAL-IR 13%. On the opposite left side, the ganglia proportion of the CGRP-IR perikarya increased by approximately 10%, while NOS-IR increased by 3%, VIP-IR 36% and GAL-IR by 2%. The only decrease (by 5%) was observed in the case of SP expression. We also found that 92% of the sensory neurons originated from NGs and 8% from DRGs. Our results indicate that, in the pig, SP, CGRP, NOS, VIP and GAL participate in the vagal sensory transduction from the stomach prepyloric area. Moreover, increased expression of the peptides and neuronal isoform of nitric oxide synthase in the sensory neurons following transection of their peripheral dendrites suggests their possible participation in the neuronal recovery and/or reinnervation process.

Acetylsalicylic acid-induced changes in the chemical coding of extrinsic sensory neurons supplying the prepyloric area of the porcine stomach.

Acetylsalicylic acid is a popular drug that is commonly used to treat fever and inflammation, but which can also negativity affect the mucosal layer of the stomach, although knowledge concerning its influence on gastric innervation is very scarce. Thus, the aim of the present study was to study the influence of prolonged acetylsalicylic acid supplementation on the extrinsic primary sensory neurons supplying the porcine stomach prepyloric region. Fast Blue (FB) was injected into the above-mentioned region of the stomach. Acetylsalicylic acid was then given orally to the experimental gilts from the seventh day after FB injection to the 27th day of the experiment. After euthanasia, the nodose ganglia (NG) and dorsal root ganglia (DRG) were collected. Sections of these ganglia were processed for routine double-labelling immunofluorescence technique for substance P (SP), calcitonine gene related peptide (CGRP), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP). Under physiological conditions within the nodose ganglia, the percentage of the FB-labeled neurons immunoreactive to particular substances ranged between 17.9 ± 2.7% (VIP-like immunoreactive (LI) neurons in the right NG) and 60.4 ± 1.7% (SP-LI cells within the left NG). Acetylsalicylic acid supplementation caused a considerable increase in the expression of all active substances studied within both left and right NG and the percentage of neurons positive to particular substances fluctuated from 47.2 ± 3.6% (GAL-LI neurons in the right NG) to 67.2 ± 2.0% (cells immunoreactive to SP in the left NG). All studied substances were also observed in DRG neurons supplying the prepyloric region of the stomach, but the number of immunoreactive neurons was too small to conduct a statistical analysis. The obtained results show that ASA may influence chemical coding of the sensory neurons supplying the porcine stomach, but the exact mechanisms of this action still remain unknown.

Immunohistochemical evidence of the co-localisation of cocaine and amphetamine regulatory peptide with neuronal isoform of nitric oxide synthase, vasoactive intestinal peptide and galanin within the circular muscle layer of the human caecum.

The enteric nervous system consists of about one hundred million of neurons. In big mammals (including humans) intestinal enteric neuronal cells are grouped into three types of intramural ganglia located within myenteric, as well as outer and inner submucosal plexuses, which are connected by numerous nerve fibres. Both nerve fibres and cell bodies located in the gastrointestinal tract utilise a broad spectrum of active substances. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). The goal of the current study was to determinate the distribution and degree of co-localisation of CART with substances taking part in intestinal motor activity by double labelling immunofluorescence technique. During the study CART-, neuronal isoform of nitric oxide synthase (nNOS)-, vasoactive intestinal peptide (VIP)- and/or galanin (GAL) - like immunoreactive (LI) nerve fibres in the circular muscle layer of the human caecum were observed in all patients studied. The degree of co-localisation of particular substances with CART depended on their type. The majority of CART-LI fibres contained simultaneously nNOS, slightly lower degree of co-localisation was observed in the case of the VIP, while simultaneously CART- and GAL-positive nerve fibres were observed less often.

Expression of oxytocin receptors is greatly reduced in the placenta of heavy mares with retained fetal membranes due to secondary uterine atony.

REASONS FOR PERFORMING STUDY: Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. OBJECTIVES: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membranes or retain them because of secondary uterine atony. STUDY DESIGN: Controlled study using archived tissues. METHODS: Placental biopsies (containing the endometrium and allantochorion) were taken from 8 heavy draught mares during parturition. Four mares expelled fetal membranes shortly after foaling (control mares) and 4 mares retained them (expulsion time was >3 h from delivery). The 4 mares that retained fetal membranes had secondary atony of the uterus. The amount of oxytocin receptors was estimated by measuring the intensity of western blot bands. The presence and location of oxytocin receptors were determined by immunocytochemistry. RESULTS: Oxytocin receptor expression was nearly 50 times less intense in mares with placenta retention due to secondary atony of the uterus and immunocytochemical staining was barely visible. In the control mares, oxytocin receptors were found in both epithelial and endothelial cells of the placenta and staining was most intense where the endometrium contacts the allantochorion. CONCLUSIONS: Inadequate expression of oxytocin receptors may be a cause of uterine atony leading to fetal membrane retention.

Co-expression of PACAP with VIP, SP and CGRP in the porcine nodose ganglion sensory neurons.

Our previous study revealed the expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase-activating polypeptide-immunoreactive (PACAP-IR) porcine nodose perikarya. Co-expression of these substances was examined using a double-labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan-neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right-side ganglion and 66.5% in the left side, respectively, co-expressed PACAP-IR. Moreover, 60.6% of the PACAP-IR cells in the right-side ganglion and 62.1% in the left, respectively, co-expressed VIP. SP-IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co-expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.

Long-term testosterone administration affects the number of paracervical ganglion ovary-projecting neurons in sexually mature gilts.

The influence of testosterone (T) overdose on the number and distribution of ovarian neurons in the paracervical ganglion (PCG) in pigs was examined. To identify the ovarian neurons, on day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue. From next day to the expected day 20 of the second studied cycle, experimental gilts were injected with T, while control gilts received oil. The PCG was then collected and processed for double-labeling immunofluorescence. T injections increased the T (∼3.5-fold) and estradiol-17ß (∼1.6-fold) levels in the peripheral blood, and reduced the following in the PCG: the total number of Fast Blue-positive neurons, the number of perikarya in the lateral part of the PCG, the numbers of VAChT(+)/SOM(+), VAChT(+)/VIP(+), VAChT(+)/nNOS(+), VAChT(+)/VIP(-), VAChT(+)/DßH(-), VAChT(-)/SOM(-), VAChT(-)/VIP(-), VAChT(-)/nNOS(-) and VAChT(-)/DßH(-) perikarya, In the T-affected PCG, the populations of ovarian perikarya coded VAChT(-)/SOM(+), VAChT(-)/VIP(+) and VAChT(-)/DßH(+), and expressing androgen receptor were increased. After T treatment within the PCG dropped the density of nerve fibers expressing VAChT and/or SOM, VIP, DßH. Obtained data suggest that elevated androgen levels occurring during pathological processes may regulate ovary function(s) by affecting the PCG gonad-supplying neurons.

Reduction of the number of neurones in the caudal mesenteric ganglion innervating the ovary in sexually mature gilts following testosterone administration.

The effect of testosterone on the morphological and chemical plasticity of the porcine caudal mesenteric ganglion (CaMG) ovary-projecting neurones was investigated. To identify the neurones on day 3 of the oestrous cycle, the ovaries of both the control and experimental gilts were injected with Fast Blue retrograde neuronal tracer. From next day until day 20 of the anticipated second studied cycle, experimental gilts were injected with testosterone, whereas control gilts received oil. Testosterone injections increased testosterone (by approximately 3.5-fold) and 17ß-oestradiol (by approximately 1.6-fold) levels in the peripheral blood and decreased the following in the CaMG: the total number of Fast Blue-positive perikarya (including small ones); the population of small perikarya in the caudal, ventral and dorsal ganglional regions; the numbers of dopamine-ß-hydroxylase (DßH) and/or neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) small and large perikarya; the numbers of small perikarya containing DßH (but not NPY, SOM, GAL); and the density of DßH and/or NPY, SOM nerve fibres. A disappearance of small and large non-noradrenergic perikarya and an increase in the total number of androgen receptor-immunoreactive perikarya was noted. Our results suggest that elevated androgen levels occurring during pathological states may regulate ovary function(s) by affecting the CaMG gonad-supplying neurones.

Immunohistochemical characterization of neurons and neuronal processes in the dorsal vagal nucleus of the pig.

The vagus nerve is responsible for efferent and afferent innervation of the gastrointestinal tract. The efferent fibres originate from neurons located in the dorsal motor nucleus of the vagus (DMX) and control mainly the gastric motor and secretory function. The main goal of our study was to examine expression of ChAT, SP, LENK, NOS and CART in the neuronal matrix of the porcine DMX. Double-labeling immunofluorescence revealed plenty of ChAT-IR neuronal cell bodies and fibres distributed throughout the nuclear matrix. Between the cholinergic somata and processes numerous SP-, LENK- and CART-IR neuronal protrusions forming dense networks were identified. While net of the NOS-IR fibres presented moderate density, the SP- and LENK-IR processes were often observed to form a basket-like structure closely surrounding the cholinergic parasympathetic neurons. Individual CART-IR basket-like formations were also encountered. A few double labeled ChAT-/NOS-IR perikarya in the rostral segment of the nucleus were also found. We confirm expression of studied antigens in the porcine DMX and provide morphological foundations for a possible regulatory role of SP, LENK, NOS and CART in porcine vago-visceral signaling.

A population of nesfatin 1-like immunoreactive (LI) cells in the mucosal layer of the canine digestive tract.

The aim of this study was to investigate for the first time nesfatin-1-like immunoreactive (LI) cells in the mucosal layer of the canine digestive tract. Distribution of nesfatin-1 - LI cell bodies and the co-localization of nesfatin-1 with somatostatin (SOM), cocaine-and amphetamine-regulated transcript peptide (CART), protein gene product 9.5 (PGP 9.5) and vasoactive intestinal peptide (VIP) were studied by immunohistochemistry in the selected parts of the canine digestive tract. Evaluation of the nesfatin 1-LI cells number has been based on the counting of cell bodies per observation field. Nesfatin-1-LI cells amounted to 0.87±0.06, 1.06±0.02, 0.85±0.11 and 0.56±0.13 in gastric fundus, duodenum, jejunum and descending colon, respectively. The co-localization of nesfatin-1 with the other substances studied was not observed. Functions of nesfatin-1 - LI cells in the canine digestive tract are unknown.

Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from follicular phase but decreased it in isthmus from early II luteal phase. SNP differentially modulated oviductal contraction depending on the type of muscular strips and period examined. These results showed the estrous phase-dependent changes related to endogenous NO system which might be of physiological importance to the oviduct for secretory and ciliary functions involved in gametes and embryo(s) transportation.

The relationship and co-localization of vasoactive intestinal peptide (VIP)- and Leu5-enkephalin (LENK)-immunoreactivity in the female genital tract of the pig.

The vasoactive intestinal peptide (VIP) and opioid family member Leu5-enkephalin (LENK) have already been established as playing independently significant roles in the functioning of the female genital tract. However, the mutual influence of both neuropeptides on female genital function has not been examined until now. Therefore, the aim of this study was to compare the distribution of VIP- and/or LENK-immunoreactive (IR) structures throughout the female genital tract of the pig. Immunohistochemical examination revealed that the great majority of the immunopositive structures co-expressed both peptides. Nevertheless, a small population of exclusively VIP- or LENK-IR processes and perikarya were also distinguished. The muscular layer of the organs examined revealed the greatest density of VIP- and/or LENK-IR nerve fibers. The mucosa of the ampulla, isthmus, cervix and vagina was supplied with a moderate number of single labeled LENK-IR processes, while exclusively VIP-IR fibers were found mainly in vaginal mucosa. The infundibulum was found to be poorly supplied with single labeled VIP- or LENK-IR fibers. The paracervical ganglion (PCG), the expected source of VIP- and/or LENK-IR nerve fibers innervating the organs under investigation, has been found to contain double labeled LENK-/VIP-IR as well as single labeled VIP-IR perikarya. The great number of specific co-localization between VIP and LENK in nerve processes of the porcine female genital organs may indicate a functional regulatory interaction between the neuropeptides studied, requiring further study.

Evidence for coexistence of choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-immunoreactivity in the thoracolumbar and sacral spinal cord neurons of the pig.

Previous immunocytochemical studies provided conflicting data concerning occurrence of the CGRP-immunoreactive neuronal cell bodies in the porcine spinal cord. In the present study, we have investigated expression of the CGRP and its possible coexpression with ChAT in the gray matter of the thoracic, lumbar and sacral spinal cord of the pig. Our study revealed a large number of CGRP-immunoreactive cells in the motor nucleus of the ventral horn, and less and singe perikarya intermediolateral and intermediomedial nuclei, respectively. Double staining immunocytochemistry, depending on the cross-section level, disclosed the highest ChAT/CGRP colocalization subsequently in the motor nucleus of the ventral horn, then in the intermediolateral and intermediomedial nucleus. Our data provide morphological evidence confirming expression of CGRP in the porcine spinal nuclei while its coexpression in cholinergic neurons suggests that CGRP may play a role in modulation of the spinal cholinergic transmission.

Distribution of cocaine- and amphetamine-regulated transcript in the hippocampal formation of the guinea pig and domestic pig.

This study provides a detailed description concerning the distribution of cocaineand amphetamine-regulated transcript (CART) subunits - CART(61-102) and rhCART(28-116) - in the hippocampal formation (HF) of the guinea pig and domestic pig, focussing on the dentate gyrus (DG) and hippocampus proper (HP). Although in both studied species CART-immunoreactive (CART-IR) neuronal somata and processes were present generally in the same layers, some species-specific differences were still found. In the granular layer (GL) of both species, the ovalshaped neurons and some thick varicose fibres were encountered. In the guinea pig there was an immunoreactive "band of dots", probably representing crosssectioned terminals within the DG molecular layer (MOL), whereas in the domestic pig, some varicose fibres were detected, thus suggesting a different orientation of, at least, some nerve terminals. Furthermore, some CART-positive cells and fibres were observed in the hilus (HL) of the guinea pig, whereas in the analogical part of the domestic pig only nerve terminals were labelled. In both species, in the pyramidal layer (PL) of the hippocampus proper, CART-IR triangular somata were observed in the CA3 sector, as well as some positive processes in MOL; however, a few immunoreactive perikarya were found only in the CA1 sector of the guinea pig. As regards the localization patterns of two isoforms of CART in the guinea pig, both peptide fragments were present simultaneously in each of the labelled neurons or fibres, whereas in the domestic pig three types of fibres may be distinguished within the area of the DG. In the hilus and MOL of the dentate gyrus, there were fibres expressing both isoforms of CART in their whole length (fibres of the first type). Fibres of the second type (in GL) coexpressed both peptides only on their short segments, and the last ones (in MOL) expressed solely rhCART(28-116). These results indicate that the distribution of the two CART isoforms are specifically related, thus the relationship between the two CART isoforms may imply different metabolic profiles of CART-expressing neurons.

Distribution and chemical coding of calretinin- and calbindin-expressing enteric neurons in the duodenum of the sheep.

Recent decades has brought significant advances in our knowledge of the chemical coding and function of enteric neurons. Calcium ions are important second messenger involved in many aspects of neuron physiology. In the present study, we analyzed immunohistochemically the presence of calcium binding proteins (calretinin and calbindin) in various subpopulations of enteric neurons from the ovine duodenum. Ten percent of submucous neurons were immunoreactive (IR) to calretinin. The presence of calretinin was not detected in myenteric neurons. Calretinin-expressing nerve fibres were found in both myenteric and submucous ganglia, between the circular and longitudinal smooth muscle layers and in the lamina muscularis mucosae. Calretinin-IR submucous neurons did not exhibit the presence of SP, NPY and VIP. Co-localization of calretinin and serotonin was found only in a small number of submucous neurons. Calbindin was expressed in 35% of myenteric neurons and in 60% of submucous neurons. Nerve fibres containing calbindin were localized in myenteric and submucous ganglia where they frequently formed basket-like formations. Calbindin-positive nerve fibres emerging from myenteric ganglia ran between the circular and longitudinal smooth muscle layers. Immunoreactivity to calbindin was also visualized in the lamina muscularis mucosae, around mucosal glands and blood vessels. None of calbindin-IR myenteric neurons revealed immunoreactivity to SP, NPY, VIP and serotonin. Virtually all calbindin-expressing submucous neurons were SP-positive. In moderate numbers of submucous perikarya, co-incidence of calbindin and NPY, calbindin and VIP or calbindin and serotonin was observed. We conclude that in the ovine duodenum, the expression of calretinin and calbindin is species specific. Co-localization studies and distribution patterns indicate that in the duodenum of the sheep, calretinin and calbindin may be present in several functional subclasses of enteric neurons.

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma.

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.

Re-examination of the topographical localization of facial nucleus in the pig.

Previous publications have provided different descriptions of the topographical organization of the facial nucleus of the pig. Since swine is used in biomedical research due to its embryological, anatomical and physiological similarities to human, we have reinvestigated the anatomical organization of the facial nucleus with application of fluorescent retrograde tracer Fast Blue, antibody to choline acetyltransferase and acetylcholinesterase histochemistry. Our findings demonstrate that in the porcine medulla facial motoneurons constitute a large cellular group occupying the ventro-lateral medulla. The neuronal group is interposed rostro-caudally between the superior and inferior olive, and located ventro-medially to the spinal nucleus of the trigeminal nerve. The present results clarify the anatomical description of this important brain stem nucleus in the pig.