RESUMEN
NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.
Asunto(s)
Antiinfecciosos/análisis , Células Asesinas Naturales/inmunología , Proteolípidos/análisis , Surfactantes Pulmonares/análisis , Linfocitos T/inmunología , Animales , PorcinosRESUMEN
Administration of monoclonal antibodies against acetylcholinesterase (AChE-mabs) to adult rats leads to a selective degeneration of the acetylcholine esterase-(AChE), choline acetyltransferase-(ChAT) and enkephalin-(ENK) positive preganglionic fibres of the splanchnic nerve innervating the adrenal gland. Here we used this approach of immunological sympathectomy, performed at postnatal day 2 (P2), in an attempt to study the development role of the preganglionic fibres in the adrenal medulla in more detail. Analysis was performed at P16 and revealed that the effect of this treatment varied considerably between animals, as judged by the number of remaining AChE-, ChAT- and ENK-positive fibres. The number and intensity especially of ENK fibres in the adrenal medulla correlated negatively with the number and staining intensity of ENK-immunoreactive chromaffin cells, suggesting a 'dose-response' relationship. Thus, the high early postnatal levels of ENK-like immunoreactivity generally persisted in chromaffin cells of adrenals with a successful immunosympathectomy, i.e. in those adrenals that lacked AChE-, ChAT- and ENK-positive nerves. In contrast, calcitonin gene-related peptide-like immunoreactivity in nerves and chromaffin cells was not affected. Large and strongly AChE-positive intra-adrenal ganglion neurones, recently termed type I ganglion neurones, were present also after AChE-mab treatment and had an apparently normal morphology. These results indicate a role for preganglionic fibres in the developmental regulation of ENK in the chromaffin cells. However, these fibres appear less important for the postnatal development of the type I ganglion neurones.
Asunto(s)
Médula Suprarrenal/embriología , Médula Suprarrenal/crecimiento & desarrollo , Neuropéptidos/biosíntesis , Simpatectomía , Acetilcolinesterasa/inmunología , Médula Suprarrenal/citología , Fibras Adrenérgicas/química , Fibras Adrenérgicas/enzimología , Factores de Edad , Animales , Anticuerpos Monoclonales , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Péptido Relacionado con Gen de Calcitonina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Células Cromafines/química , Células Cromafines/enzimología , Encefalinas/biosíntesis , Encefalinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Galanina/biosíntesis , Galanina/metabolismo , Ganglios Simpáticos/citología , Neuropéptidos/metabolismo , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Simpatectomía/métodosRESUMEN
The postnatal development of intraadrenal ganglion neurons was studied in rat by using indirect immunohistochemistry and in situ hybridization. The large neuropeptide tyrosine (NPY)-expressing ganglion neurons (type I ganglion neurons) matured postnatally, with marked increases in acetylcholinesterase (AChE)-, neurofilament 10 (NF10)-, and tyrosine hydroxylase (TH)-like immunoreactivities (LIs) paralleled by increasing levels of mRNAs encoding NPY, low-affinity neurotrophin receptor (LANR), and tropomyosin kinase receptor (trk). The smaller vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) ganglion neurons (type II ganglion neurons) expressed increasing levels of VIP mRNA postnatally and also contained immunoreactive nitric oxide synthase (NOS) and its mRNA. These type II ganglion neurons appeared to be relatively mature already at postnatal day (P2) and did not express detectable levels of LANR or trk mRNAs. The cell size of both the type I and type II ganglion neurons increased about 2.5-fold postnatally. The type I ganglion neurons formed more densely packed clusters with increasing age, whereas the type II ganglion neurons were spread out in small groups or individually, mainly in the peripheral parts of the medulla, and appeared to fulfill their migration into the medulla and/or to the inner regions of the cortex early postnatally, possibly after establishing contact with their cortical targets. We suggest that the type I ganglion neurons represent sympathetic ganglion neurons of the same origin as the chromaffin cells and that they mature mainly postnatally. The development of the type II (VIP/NOS) ganglion neurons takes place earlier; however, their phenotype remains more uncertain.
Asunto(s)
Médula Suprarrenal/inervación , Ganglios Simpáticos/crecimiento & desarrollo , Neuronas/química , Neuropéptido Y/análisis , Óxido Nítrico Sintasa/análisis , Péptido Intestinal Vasoactivo/análisis , Acetilcolinesterasa/análisis , Médula Suprarrenal/crecimiento & desarrollo , Animales , Dopamina beta-Hidroxilasa/análisis , Femenino , Ganglios Simpáticos/citología , Inmunohistoquímica , Hibridación in Situ , Masculino , Fenotipo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/análisisRESUMEN
Retrograde tract-tracing was used to study the projections of adrenal medullary ganglion neurons. The splanchnic nerve was cut close to the suprarenal ganglia and the retrograde tracer FluoroGold was applied at the site of nerve transection. Groups of adrenal medullary ganglion neurons exhibited FlurorGold- or Fast Blue-induced fluorescence restricted to the perikarya. Using immunohistochemistry most retrogradely labelled ganglion neurons showed immunoreactivity for neuropeptide Y. In addition, after splanchnicotomy most ganglion neurons expressed galanin and galanin message-associated peptide immunoreactivities which could not be observed in control adrenals. Taken together, the present results strongly indicate that adrenal medullary ganglion neurons project back into the splanchnic nerve perhaps representing feedback system modulating the preganglionic innervation of the adrenal gland.
Asunto(s)
Glándulas Suprarrenales/anatomía & histología , Vías Nerviosas/anatomía & histología , Nervios Esplácnicos/anatomía & histología , Médula Suprarrenal/anatomía & histología , Animales , Ganglios Simpáticos/anatomía & histología , Histocitoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Using in situ hybridization, immunohistochemistry and receptor binding methodology, the galanin messenger RNA levels, galanin binding and galanin-like immunoreactivity were examined in rats injected with carrageenan into the left hindpaw. Three days after injection, a distinct increase (63%) in galanin messenger RNA-positive neurons was observed in the medial laminae I and II of the ipsilateral dorsal horn (lumbar 4 and 5) as compared to the contralateral side. However, no alteration was found in galanin binding and galanin-like immunoreactivity in the dorsal horn. In dorsal root ganglia (lumbar 5), inflammation induced a significant decrease in galanin messenger RNA (39%) and galanin peptide (47%) on the ipsilateral side. Galanin binding was not detected in dorsal root ganglia, neither on the inflammatory nor on the control side. Increased levels of galanin-like immunoreactivity and galanin messenger RNA were seen in cells in the inflamed dermis and epidermis, especially in stratum granulosum. Most of the galanin-immunoreactive cells contained ED1-like immunoreactivity, a marker for macrophages. A strong galanin binding was seen in the inflamed dermis. Such binding sites may be targets for galanin released from local cells in inflamed dermis. Taken together, our results suggest that both neuronal and non-neuronal galanin or a galanin-like peptide is involved in the response to inflammation.
Asunto(s)
Sistema Nervioso Central/metabolismo , Galanina/biosíntesis , Inflamación/metabolismo , Nervios Periféricos/metabolismo , Animales , Autorradiografía , Carragenina , Ganglios Espinales/metabolismo , Inmunohistoquímica , Hibridación in Situ , Inflamación/inducido químicamente , Masculino , Microscopía Confocal , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Piel/metabolismo , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Neurotensin (NT) has been reported to have antinociceptive effects at the spinal level. In situ hybridization, electrophysiology, immunohistochemistry, and electronmicroscopy were used to investigate the distribution of NT receptors, possible effects of NT on primary sensory neurons, and the effect of nerve injury on the expression of NT receptors and NT. NT receptor (R) mRNA was observed in more than 25% of the small dorsal root ganglion (DRG) neurons, which lacked neuropeptide Y NPY-R mRNA and essentially other neuropeptide mRNAs. Intracellular recording using voltage-clamp mode showed that NT evokes an outward current in NPY-insensitive small neurons, and NPY an outward current in NT-insensitive small neurons. Both peptides lacked effect on several small DRG neurons. In the superficial dorsal horn NT immunoreactive (IR) terminals directly contacted primary afferent terminals without synaptic specializations. This new category (> 25%) of the small DRG neurons expressing NT-R mRNA was complementary to the around 60% of small neurons expressing NPY-R mRNA (and also substance P and calcitonin gene-related peptide mRNAs) and to the rest exhibiting somatostatin mRNA expression. The electrophysiological results support this classification, showing that NT and NPY have inhibitory effects on separate subpopulations of small DRG neurons. After sciatic nerve transection, a marked decrease was observed in (1) the number of NT-R mRNA-positive neurons in DRGs, (2) NT mRNA-positive neurons in the dorsal horn, and (3) NT-IR cell bodies and fibers in laminae I-II. Thus, axotomy causes downregulation of several NT systems at the spinal level, suggesting that the possible effects of NT on primary sensory neurons is attenuated after peripheral axotomy.
Asunto(s)
Vías Aferentes/fisiología , Ganglios Espinales/fisiología , Neuronas/fisiología , Receptores de Neuropéptido Y/biosíntesis , Receptores de Neurotensina/biosíntesis , Nervio Ciático/fisiología , Animales , Axones/fisiología , Axones/ultraestructura , Péptido Relacionado con Gen de Calcitonina/análisis , Potenciales Evocados/efectos de los fármacos , Lateralidad Funcional , Ganglios Espinales/citología , Expresión Génica , Hibridación in Situ , Masculino , Potenciales de la Membrana/efectos de los fármacos , Modelos Neurológicos , Terminaciones Nerviosas/fisiología , Terminaciones Nerviosas/ultraestructura , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptido Y/análisis , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Neurotensina/biosíntesis , Neurotensina/farmacología , Sondas de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/análisis , Receptores de Neurotensina/análisis , Sustancia P/análisis , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon gamma (IFN-gamma), interleukin 4 (IL-4) and transforming growth factor beta (TGF-beta) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63-88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-gamma, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.) and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-beta did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-gamma, while TGF-beta may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63-88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63-88-induced production of both IFN-gamma and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63-88 induced lymphoid cells to TGF-beta production. Thus, an autoantigen-specific production of TGF-beta occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically.
Asunto(s)
Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Tejido Linfoide/metabolismo , ARN Mensajero/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Secuencia de Aminoácidos , Animales , Inmunohistoquímica , Hibridación in Situ , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Tejido Linfoide/citología , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismoRESUMEN
To investigate transsynaptic effects on peptides of adrenal chromaffin cells in the rat, presynaptic sympathetic terminals were destroyed by intravenous injection of monoclonal antibodies to acetylcholinesterase. At several times thereafter, neuropeptide Y (NPY)-like immunoreactivity (NPY-IR) and methionine-enkephalin-like immunoreactivity (Met-Enk-IR) were measured by radioimmunoassay. Within 2 days of antibody injection, adrenal Met-Enk-IR increased five- to 10-fold and NPY-IR increased 50%. These effects were accompanied by large increases in proenkephalin A mRNA assayed by polymerase chain reaction. The peptide responses could reflect either an acute activation, as presynaptic terminals degenerated, or a chronic synaptic inactivation after terminal degeneration. To test the possibilities, muscarinic and nicotinic receptors were inhibited by repeated injection of atropine (1 mg/kg) and chlorisondamine (5 mg/kg). Measurements of urinary free catecholamine excretion showed that this treatment prevented the paroxysmal release of norepinephrine and reduced the release of epinephrine that normally followed injection of acetylcholinesterase antibodies. When the drugs were given alone for 2 or 4 days, adrenal Met-Enk-IR increased modestly and NPY-IR remained steady or declined. When given together with acetylcholinesterase antibodies, the cholinergic antagonists blocked the increase of NPY-IR but not Met-Enk-IR. Adding naloxone (1 mg/kg) to the treatment regimen enhanced the blockade of epinephrine excretion and largely prevented the antibody-induced increase in Met-EnK-IR. These findings indicate that adrenal NPY and enkephalin are not regulated identically. Adrenal NPY behaves as though controlled by transsynaptic cholinergic input. On the other hand, adrenal enkephalin may be regulated by additional or different mechanisms, possibly involving peptidergic transmission or synaptic inactivation.
Asunto(s)
Glándulas Suprarrenales/inervación , Encefalinas/genética , Encefalinas/metabolismo , Neuropéptido Y/metabolismo , Precursores de Proteínas/genética , Acetilcolinesterasa/inmunología , Glándulas Suprarrenales/metabolismo , Animales , Atropina/farmacología , Secuencia de Bases , Clorisondamina/farmacología , Antagonistas Colinérgicos , Cartilla de ADN/química , Epinefrina/orina , Masculino , Datos de Secuencia Molecular , Norepinefrina/orina , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We have applied in situ hybridization histochemistry, Northern analysis, and immunocytochemistry to study the regulation of the immediate-early gene (IEG) c-fos, c-jun, and junB mRNAs and the respective proteins in the rat adrenal medulla. Electrophoresis mobility shift assay was used to examine changes in AP-1 DNA binding activity. In nontreated rats the mRNA and protein levels for these three IEGs were low. Reflex stimulation of adrenal medulla elicited by a single capsaicin injection induced a rapid and marked elevation in the mRNA levels for these IEGs. Stimulation with nicotine also caused a drastic increase in the mRNA levels, whereas muscarine only induced moderate elevations. c-fos and c-jun were induced strongly in adrenaline cells and only weakly in noradrenaline cells. junB was upregulated mainly in adrenaline cells. The AP-1 DNA binding activity was low in control adrenals, whereas a marked increase was observed after nicotine treatment. Treatment of the animals with a nicotinic (chlorisondamine) or a muscarinic (atropine) receptor antagonist did not change the expression of IEGs studied. The combination of the two drugs, however elevated the mRNA levels for all three IEGs, especially for junB. Pretreatment of the rats with chlorisondamine alone or in combination with atropine diminished the capsaicin-induced increase in c-fos, whereas atropine alone was less efficient. Increase in c-jun mRNA was not affected by these drugs. The capsaicin-induced elevation of junB mRNA levels was not influenced by chlorisondamine or atropine alone, whereas both combined potentiated the effect of capsaicin. The present results demonstrate that neurotransmitters released from splanchnic nerve terminals induce expression of c-fos, c-jun, and junB in adrenal chromaffin cells which results in increased AP-1 DNA binding activity. Although stimulation of both nicotinic and muscarinic cholinergic receptors may mediate the induction of these IEGs, it is possible that also another neurotransmitter(s), in addition to ACh, released from splanchnic nerve terminals is involved in the regulation of their expression, especially of c-jun and junB.
Asunto(s)
Médula Suprarrenal/fisiología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/metabolismo , Animales , Capsaicina/farmacología , ADN/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Neuronal markers, peptides and enzymes were analyzed in the rat adrenal medulla during the postnatal period, i.e., when the 'functional' splanchnic innervation is assumed to 'mature'. Nerve fibers were present on day 2 as indicated by neurofilament 10 (NF10)- and growth associated protein 43 (GAP43)-like immunoreactivities (LIs). Acetylcholinesterase (AChE)- and enkephalin (ENK)-immunoreactive (IR) fibers, presumably of preganglionic nature, increased in number and intensity during the postnatal period. In contrast, calcitonin gene-related peptide (CGRP)- and galanin (GAL)-IR fibers were almost fully developed on day 2. Thus, the presumably sensory innervation of the adrenal gland seems to precede the development of the autonomic nerves. The AChE- and ENK-IR fibers may exert a suppressive effect on ENK-, CGRP- and neurotensin (NT)-LIs in chromaffin cells, since the levels of these peptides were high in the early postnatal period and then decreased. On the other hand, GAL-LI in chromaffin cells was low also in young rats, while GAP43-IR cells were observed at all stages. Neuropeptide tyrosine (NPY) was expressed in many chromaffin cells at all stages and its turnover rate seemed to decrease towards the adult stage. The expression of the catecholamine synthezising enzymes changed only marginally during development. These results indicate that the preganglionic fibers, but not the sensory axons, in the splanchnic nerve are involved in the developmental control of expression of some, but not all, peptides in the chromaffin cells and that these changes thus may reflect the maturation of a 'functional' transmission.
Asunto(s)
Médula Suprarrenal/crecimiento & desarrollo , Médula Suprarrenal/inervación , Envejecimiento/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/fisiología , Neuropéptidos/biosíntesis , Acetilcolinesterasa/análisis , Acetilcolinesterasa/biosíntesis , Médula Suprarrenal/citología , Animales , Animales Recién Nacidos , Biomarcadores/análisis , Péptido Relacionado con Gen de Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Encefalinas/análisis , Encefalinas/biosíntesis , Femenino , Técnica del Anticuerpo Fluorescente , Proteína GAP-43 , Galanina , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/biosíntesis , Fibras Nerviosas/fisiología , Fibras Nerviosas/ultraestructura , Proteínas del Tejido Nervioso/análisis , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/biosíntesis , Neuronas/citología , Neuropéptido Y/análisis , Neuropéptido Y/biosíntesis , Neuropéptidos/análisis , Biosíntesis de Péptidos , Péptidos/análisis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within 1 h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.
Asunto(s)
Acetilcolinesterasa/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Ganglios Simpáticos/efectos de los fármacos , Ganglionectomía/métodos , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/metabolismo , Animales , Sistema Cromafín/citología , Sistema Cromafín/metabolismo , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Hibridación in Situ , Masculino , Neuropéptidos/genética , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The distribution of neurons in the rat CNS that synthesize mRNA for the alpha 1A/D and alpha 1B adrenoceptors was revealed by the in situ hybridization method. Forty-eight-mer DNA probes were synthesized to two different and unique regions of both the alpha 1A/D and alpha 1B mRNAs. Tissue sections from all levels of the CNS and some peripheral ganglia were incubated in a hybridization cocktail containing one of these four probes. The two mRNAs were expressed in a discrete and often complementary manner to each other, and identical hybridization patterns were seen for the probes directed against the same mRNA. The alpha 1A/D probes hybridized heavily with neurons in the internal granular and internal plexiform layers of the olfactory bulb, in layers II-V of most areas of the cerebral cortex, and in the lateral aspect of the lateral amygdaloid nucleus, with pyramidal neurons of CA1-CA4 regions, hilar and granular neurons of the dentate gyrus, and neurons in the reticular thalamic nucleus, cranial and spinal motor nuclei, and the inferior olivary nucleus. Light labeling was seen in a variety of other regions in the brain and spinal cord. The alpha 1B probes hybridized heavily with neurons in the mid layers of cerebral cortex and with virtually all neurons in the thalamus, except the reticular and habenular nuclei. In addition, labeling was seen in the lateral and central amygdaloid nuclei, in brainstem and spinal motor nuclei, over most neurons of the dorsal and medullary raphe nuclei and neurons of the intermediolateral cell column in the spinal cord. Light labeling was seen in the septal nucleus, the horizontal limb of the diagonal band, the paraventricular and lateral hypothalamic nuclei, the pontine and medullary reticular formation, and in most laminae in the spinal cord. The patterns of labeling obtained with the alpha 1B probes resemble the labeling seen in previous autoradiographic ligand binding studies utilizing "general" alpha 1 ligands, while the labeling patterns seen with the alpha 1A/D probes do not correspond to any published alpha 1 receptor distribution pattern, indicating that this mRNA likely encodes for a novel adrenoceptor. The present findings further expand the heterogeneity of adrenoceptor mRNAs presented in two accompanying studies (Nicholas et al., 1993a,b). This differential distribution of adrenoceptors subtypes provides a framework for the functional diversity to the apparently widespread, diffuse, and rather homogeneous noradrenergic innervation of the CNS.
Asunto(s)
Encéfalo/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Animales , Hibridación in Situ , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/genética , Distribución TisularRESUMEN
The olfactory bulbs of young and adult normal rats and of colchicine-treated rats and of some other species were analysed for the presence of neuropeptide Y and neuropeptide Y messenger RNA, using immunohistochemistry at the light- and electron-microscopic levels and with in situ hybridization. In the rat and mouse, but not in monkey and guinea-pig, neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA were observed in ensheathing cells in the olfactory nerve layer of the olfactory bulb and within nerve bundles in the olfactory mucosa. Double staining experiments revealed that neuropeptide Y-like immunoreactivity was often present in a restricted compartment, mainly the Golgi apparatus, of S-100 protein-positive ensheathing cells. After colchicine treatment a different distribution of neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA was observed. Thus, in the outer olfactory nerve layer both neuropeptide Y-like immunoreactivity and neuropeptide Y messenger RNA disappeared, whereas in the inner part messenger RNA levels remained high and neuropeptide Y-like immunoreactivity was observed in many granule-like structures distributed diffusely in the cytoplasm. The present findings suggest that neuropeptide Y may be involved in the control of regeneration, growth and/or guiding of the axons of the olfactory sensory neurons, the only mammalian neurons known to have a continuous renewal and growth during adult life.
Asunto(s)
Neuroglía/química , Neuropéptido Y/biosíntesis , Bulbo Olfatorio/citología , Nervio Olfatorio , Animales , Colchicina/farmacología , Técnica del Anticuerpo Fluorescente , Cobayas , Técnicas para Inmunoenzimas , Hibridación in Situ , Masculino , Ratones , Mucosa Olfatoria/citología , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Using immunohistochemistry the expression of galanin (GAL) and galanin message-associated peptide (GMAP) in the superior cervical ganglion (SCG) was investigated 2, 4, 7, and 14 days after unilateral transection of the cervical sympathetic trunk (decentralization) or after cutting the external and internal carotid nerves (axotomy), as well as 7 days after removal of parotid gland tissue. In control SCGs including the sympathetic trunk and the carotid nerves, very few neurons and fibers were GAL/GMAP-positive. Two and 7 days after decentralization, about 5% of all counted neuron profiles in the ipsilateral SCG were GAL/GMAP immunoreactive. Immunoreactive cell bodies were distributed throughout the SCG, with a greater number in the most caudal portion of the ganglion. Many GAL/GMAP-positive nerve fibers were observed in the whole SCG, with strongly fluorescent bundles of immunoreactive fibers accumulated at the caudal end of the SCG. Several GAL/GMAP-immunoreactive nerve fibers were seen ipsilaterally in the external carotid nerve, whereas only a few positive fibers could be observed in the internal carotid nerve. About 2% of all counted neuron profiles in SCGs ipsilateral to decentralization still contained GAL/GMAP-immunoreactivity 14 days after the operation. The number of GAL/GMAP-positive cell bodies was at least doubled in the contralateral SCGs after decentralization compared to controls. After axotomy, about 50% of all counted neuron profiles were GAL/GMAP-positive in the ipsilateral SCG and distributed throughout the SCG. A strong accumulation of immunoreactive nerve fibers was observed in both the internal and external carotid nerves. The number of GAL/GMAP-positive cell bodies was slightly increased in the contralateral SCGs. After unilateral removal of parotid gland tissue, many GAL/GMAP-positive cell bodies and some fibers were observed in the caudal half of the ipsilateral SCG. The number of immunoreactive nerve fibers was increased also in the external carotid nerve, but not in the internal carotid nerve. In situ hybridization revealed prepro GAL mRNA in about 5% of all SCG neuron profiles in decentralized SCGs, paralleling the increase seen in GAL/GMAP peptide content. There was also a small increase in prepro VIP mRNA-positive cells in the caudal part of the SCG. The present results indicate that both pre- and postganglionic lesions increase the content of GAL/GMAP in the SCG, with a much more pronounced increase after transection of the carotid nerves.
Asunto(s)
Neuronas/metabolismo , Biosíntesis de Péptidos , Médula Espinal/fisiología , Ganglio Cervical Superior/metabolismo , Sistema Nervioso Simpático/fisiología , Animales , Especificidad de Anticuerpos , Técnica del Anticuerpo Fluorescente , Lateralidad Funcional , Galanina , Inmunohistoquímica , Hibridación in Situ , Masculino , Fibras Nerviosas/metabolismo , Neuronas/citología , Neuropéptidos/biosíntesis , Sondas de Oligonucleótidos , Péptidos/análisis , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/citologíaRESUMEN
A novel method to selectively lesion preganglionic sympathetic neurones has been combined with immunohistochemistry to study the expression of peptides in the rat superior cervical ganglion (SCG). Thus, systemic administration of monoclonal antibodies against acetylcholinesterase (AChE) caused a marked reduction in the number of enkephalin (ENK)-positive fibres and a total disappearance of fibres immunoreactive for calcitonin gene-related peptide (CGRP) and AChE in the SCG. A marked increase in the number of galanin/galanin message-associated peptide (GAL/GMAP)-immunoreactive cell bodies was also observed. The present results indicate that probably all CGRP and most ENK containing fibres in the rat SCG are of preganglionic origin and that peptides not normally expressed in SCG neurones, e.g. GAL and GMAP, can be upregulated after deafferentation.
Asunto(s)
Neuropéptidos/metabolismo , Ganglio Cervical Superior/metabolismo , Simpatectomía , Acetilcolinesterasa/inmunología , Acetilcolinesterasa/metabolismo , Animales , Anticuerpos Monoclonales , Blefaroptosis/fisiopatología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Piloerección/fisiología , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/enzimologíaRESUMEN
The effect of acute subcutaneous administration of methamphetamine on the expression of neurotensin mRNA was investigated in the adult rat striatum. At different time points (2, 6 and 24 h) following drug administration rats were killed, and mRNA levels were quantified both on films and emulsion-dipped tissue sections from two striatal levels. Two hours after methamphetamine injection, a dramatic increase in neurotensin mRNA levels was detected in different areas of the striatum at both rostral and caudal levels. Numerous positive cells were observed in the dorsomedial, dorsolateral and ventrolateral parts of the striatum. This up-regulation reflected an increase both in the number of cells expressing neurotensin mRNA and in the mean mRNA levels. This increase was still present after 6 h and was similar to the 2 h treated group at the rostral level of the striatum, but lower at the caudal one. Twenty-four hours after methamphetamine injection, neurotensin mRNA levels were back to control values, or in some areas even below. A strong increase in neurotensin mRNA-expressing cells was also seen in the olfactory tubercle, and the time-course was similar to the one observed in the striatum. In a second set of experiments, the effect of methamphetamine was evaluated on adjacent striatal sections hybridized with probes directed against neurotensin and substance P mRNAs, respectively. Two hours after drug administration, a significant increase in the levels of both peptide mRNAs was observed (+190% for neurotensin, +80% for substance P). These results demonstrate that methamphetamine is able to induce a dramatic, rapid and transient increase in striatal neurotensin mRNA levels, which may partly account for the elevation in neurotensin peptide levels observed in the striatonigral pathway after methamphetamine. The different anatomical localization of neurotensin mRNA-expressing cells observed after haloperidol and methamphetamine treatments, as well as the fact that the D1 receptor antagonist SCH-23390 is able to counteract the effect of methamphetamine but not that of haloperidol on neurotensin mRNA expression, suggests that there are at least two different subpopulations of neurotensin cells in the striatum. One population is regulated via D1 receptors and projects to the substantia nigra pars reticulata. The second is sensitive to D2 receptor stimulation and may project to the globus pallidus and/or may represent interneurons.
Asunto(s)
Cuerpo Estriado/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Metanfetamina/farmacología , Neurotensina/biosíntesis , Bulbo Olfatorio/metabolismo , Animales , Autorradiografía , Cafeína/farmacología , Cuerpo Estriado/efectos de los fármacos , Haloperidol/farmacología , Masculino , Bulbo Olfatorio/efectos de los fármacos , Sondas de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sustancia P/biosíntesis , Radioisótopos de Azufre , Factores de TiempoRESUMEN
The distribution of mRNA for the rat alpha-1 A/D, alpha-1B, alpha-2A/D (RG20), alpha-2B (RNG), alpha-2C (RG10), beta-1 and beta-2 adrenergic receptors were studied in the rat kidney using in situ hybridization. After hybridized sections were exposed to autoradiography film or dipped in photographic emulsion and counterstained with hematoxylin and eosin, specific and selective labeling patterns characteristic for each probe in the kidney were observed. Labeling with the probe to the alpha-1A/D receptor was only observed in vessels in the renal parenchyma and in the ureter. Alpha-1B receptor mRNA was demonstrated in the outer and inner stripe of the outer medulla, corresponding to segment S3 of proximal tubules and the thick ascending limb of loop of Henle. Alpha-2A/D receptor mRNA was distributed in the inner stripe of the outer medulla and in the inner medulla, corresponding to collecting tubules, and in the ureter. The strongest signal in the kidney was obtained with the alpha-2B receptor probe, showing labelling in the outer stripe of the outer medulla with tubular rays radiating into the cortex, coinciding with segment S3 of proximal tubules. Weak labeling obtained with the alpha-2C receptor probe was present in the renal medulla. Labeling obtained with the probe to the beta-1 receptor was seen in the entire cortex and to a lesser extent also in the outer medulla. In addition, beta-1 receptor mRNA was shown in perirenal adipose tissue and in the ureter. Labeling obtained with the probe to the beta-2 receptor was demonstrated in the outer and inner stripe of the outer medulla.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Riñón/metabolismo , ARN Mensajero/biosíntesis , Receptores Adrenérgicos/genética , Animales , Autorradiografía , Northern Blotting , Hibridación in Situ , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos/clasificaciónRESUMEN
Oligonucleotide probes were designed to sequences of the rat alpha 1B- and alpha 2B-adrenergic receptor mRNA and used for in situ hybridization histochemistry on tissue sections of kidneys from control and thyroidectomized rats. Both alpha 1B- and alpha 2B-receptor mRNA labelling was demonstrated in proximal tubule cells in the outer stripe of the outer medulla, with tubular rays radiating into the cortex. Thyroidectomy induced a more than 4-fold increase in mRNA for the alpha 1B-receptor in the kidney, whereas no change in alpha 2B-receptor mRNA levels could be demonstrated in thyroidectomized rats as compared to control animals. The results suggest that thyroid hormone plays an important role in regulating expression of alpha 1B-receptors in renal tubule cells.