O. Häppölä

A new monoclonal antibody against the GABA-protein conjugate shows immunoreactivity in sensory neurons of the rat

A monoclonal antibody, 115AD5, was raised against GABA coupled to bovine serum albumin. The monoclonal antibody 115AD5 also reacted with other GABA-protein conjugates. The specificity of the monoclonal antibody was corroborated by enzyme-linked immunoassay, dot-immunobinding experiments and immunostaining of rat cerebellum sections. The monoclonal antibody 115AD5 could successfully be applied on Vibratome and cryostat sections using either indirect immunofluorescence or peroxidase techniques. In rat cerebellar cortex the monoclonal antibody 115AD5 gave an intense immunoreaction in stellate cells, in Golgi neurons, and in basket cells and their processes around Purkinje cell bodies. Purkinje cell dendrites showed GABA immunoreactivity while the cell bodies were non-reactive or only weakly reactive. There was labelling in some nuclei of Purkinje cells. GABA immunoreactivity was also found in dot-like structures in the granular layer. A large population of sensory neurons in rat thoracic and lumbar spinal dorsal root ganglia presented an intense immunoreactivity for the monoclonal antibody 115AD5. Nerve bundles immunoreactive for GABA were also seen in these ganglia. In the trigeminal ganglion, a major population of sensory neurons and some of their processes presented immunoreactivity for GABA. In the sensory nodose ganglion of the vagus nerve, many neuronal cell bodies and some fibres were immunoreactive for GABA. Ligation of the vagus nerve caudal to the ganglion resulted in an increased GABA immunoreactivity in neuronal somata of the ganglion, as well as in nerve fibres on the ganglionic side of the ligature. The present results suggest that in the rat, a population of sensory neurons in thoracic and lumbar spinal dorsal root ganglia, as well as in the trigeminal and nodose ganglia contain GABA. The presence of GABA immunoreactivity in these neurons raises the possibility of a neurotransmitter or modulator role.

[Met5]enkephalin-Arg6-Phe7- and [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibres and neurons in the superior cervical ganglion of the rat

[Met5]enkephalin-Arg6-Phe7-(MEAP-) and [Met5]enkephalin-Arg6-Gly7-Leu8-(MEAGL-) immunoreactivity was studied by indirect immunohistochemistry in the superior cervical ganglion of the rat with specific antisera produced in rabbits against the corresponding synthetic opioid peptides. Several MEAP- and a few MEAGL-immunoreactive principal nerve cells were observed in the ganglion, while the small intensely fluorescent cells appeared as non-reactive. The superior cervical ganglion also contained dense networks of MEAP- and MEAGL-immunoreactive nerve fibres, which often formed basket-like structures around the principal nerve cells and small intensely fluorescent cells. After ligation of the preganglionic nerve trunk with simultaneous transection of the main postganglionic trunks, a distinct accumulation of both MEAP- and MEAGL-immunoreactivity was observed on both sides of the ligature. Ligation of the preganglionic nerve trunk caused a marked decrease in the number of both MEAP- and MEAGL-immunoreactive nerve fibres in the ganglion. Ligation of the main postganglionic nerve trunks with simultaneous preganglionic nerve division resulted in accumulation of MEAP- and MEAGL-immunoreactive material on the ganglionic side of the ligature in both the external and internal carotid nerve. After division of both the pre- and postganglionic nerve trunks, some immunoreactive nerve fibres and principal nerve cells were still observed in the ganglion. A few immunoreactive neurons and nerve fibres were also observed in the ganglion stellatum. A large number of MEAP- and MEAGL-immunoreactive nerve fibres was detected in the spinal cord at the levels C6-Th6. A few neurons in the intermediolateral cell column of the spinal cord at levels C8-Th1 showed MEAP- but not MEAGL-immunoreactivity. The cultured superior cervical ganglion contained a few MEAP-immunoreactive neurons, and the fibre outgrowth showed immunoreactivity both to MEAP and MEAGL. In electron microscopy, MEAGL-immunoreactivity in the superior cervical ganglion was localized in nerve fibres containing neurotubules and in principal nerve cells. The present results demonstrate that the rat superior cervical ganglion contains both extrinsic and intrinsic MEAP- and MEAGL-immunoreactive nerve fibres. Most of these fibres are of preganglionic origin. Both the principal nerve and small intensely fluorescent cells are often surrounded by MEAP- or MEAGL-immunoreactive nerve fibres and may receive innervation by these fibres. Several ganglionic neurons projecting to the sympathetic target tissues show MEAP- and/or MEAGL-immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical Demonstration of Neuroactive Substances in the Inner Ear of Rat and Guinea Pig

The presence and localization of different neuropeptides and other putative neurotransmitters or -modulators were examined by immunohistochemistry in the cochleovestibular end organs and in neurons innervating them in rats and guinea pigs. In the organ of Corti neural elements beneath inner hair cells showed immunoreactivity for enkephalin (ENK), calcitonin gene-related peptide (CGRP), L-glutamate decarboxylase (GAD), substance P (SP) and tyrosine hydroxylase (TH). Nerve chalices of type I vestibular hair cells contained SP and GAD, but not consistently. SP was only occasionally observed in neuronal cell bodies of the 8th cranial nerve but fine fibers with different neuroactive substances were seen in the nerve trunk in the following relative numbers: TH greater than SP greater than CGRP greater than ENK. The present data demonstrate the presence of several different neuroactive substances in the rat and guinea pig inner ear suggesting a multiplicity of neurotransmitters or -modulators in this system.

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat.

SummaryIndirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland.The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland.The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs.

SummaryThe localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.

Pre- and postnatal development of 5-hydroxytryptamine-immunoreactive cells in the superior cervical ganglion of the rat

The pre- and postnatal development of 5-hydroxytryptamine (5-HT)-immunoreactive cells in the superior cervical ganglion of the rat was studied by indirect immunofluorescence method with an antibody to a conjugate of bovine serum albumin and 5-HT. The superior cervical ganglia from 11-day-old embryos to 90-day-old postnatal rats were examined. 5-HT-immunoreactive cells were first detected in the superior cervical ganglion on the 12th day of gestation. At this stage of development the ganglionic cells formed a condensed group, and most of them showed 5-HT-immunoreactivity. During later prenatal development the relative number of the 5-HT-immunoreactive cells in the ganglion decreased and most ganglionic cells appeared as non-reactive. 5-HT-immunoreactive cells showed some variation in size and fluorescence intensity during the whole prenatal development. In the ganglia of newborn rats a wide range of 5-HT-immunoreactive cell sizes was detected. Two types of 5-HT-immunoreactive cells were distinguished in the ganglia of 7- to 35-day-old rats: small (5-15 microns in diameter) cells and large (15-30 microns in diameter) cells, resembling in size small intensely fluorescent (SIF) cells and principal nerve (PN) cells, respectively. The relative number of the large 5-HT-immunoreactive cells gradually decreased after the first postnatal week, and these cells were not detected in the ganglia of 90-day-old rats. Small 5-HT-immunoreactive cells constantly formed clusters in the ganglia of postnatal rats. A marked, statistically significant increase in the density of 5-HT-immunoreactive cells occurred during the 4th postnatal week. The 5-HT-immunoreactive cells appeared in the prenatal superior cervical ganglion of the rat approximately at the same time as the cells showing formaldehyde-induced catecholamine fluorescence. Transient occurrence of the large 5-HT-immunoreactive cells during the first postnatal weeks of development indicates, that in the superior cervical ganglion of the rat, 5-hydroxytryptamine may be expressed in some developing PN cells early postnatally. After the 5th postnatal week, the large 5-HT-immunoreactive neuronal cells cannot be detected, and 5-HT is expressed only in the SIF cells.

Localization of l-glutamate decarboxylase and GABA transaminase immunoreactivity in the sympathetic ganglia of the rat

The location of L-glutamate decarboxylase and gamma-aminobutyrate (GABA)-transaminase immunoreactivity in the superior cervical ganglion and in the coeliac-superior mesenteric ganglion complex of the rat was studied by an indirect immunofluorescence method and by immunoelectron microscopy, with specific antisera raised in rabbits against the corresponding enzymes. In light microscopy, several glutamate decarboxylase- or GABA-transaminase-immunoreactive principal nerve cells were detected in the superior cervical ganglion and coeliac-superior mesenteric ganglion complex. In addition, numerous small cells in both the superior cervical ganglion and coeliac-superior mesenteric ganglion complex showed intense immunoreactivity to glutamate decarboxylase or GABA-transaminase. The small cells were 10-20 micron in diameter and resembled in size and morphology the small intensely fluorescent cells. In consecutive sections, the small glutamate decarboxylase-immunoreactive cell clusters also showed immunoreactivity to tyrosine hydroxylase, suggesting that these cells contain the enzymes for both GABA and catecholamine synthesis. In the superior cervical ganglion and in the coeliac-superior mesenteric ganglion complex, GABA-transaminase immunoreactivity was also localized in fibre-like processes around and between the principal nerve cells, in nerve trunks traversing the ganglia, and around or in close contact with ganglionic blood vessels. Furthermore, GABA-transaminase immunoreactivity was observed in fibre-like structures close to the capsule of the ganglia. Division of the preganglionic nerve trunk of the superior cervical ganglion caused no detectable change in GABA-transaminase immunoreactivity in the ganglion. In immunoelectron microscopy of the superior cervical ganglion, GABA-transaminase immunoreactivity was localized in nerve fibres in association with neurotubules. A large number of GABA-transaminase labelled principal nerve cells were detected, containing immunoreactivity evenly distributed in their cytoplasm. GABA-transaminase immunoreactivity was also observed in satellite cells and their processes in the superior cervical ganglion. The present immunocytochemical results provide evidence that the rat sympathetic ganglia contain an intrinsic neuronal system showing histochemical markers for GABA synthesis and inactivation, but its functional role in the modulation of ganglionic neurotransmission remains to be established.

Histamine-immunoreactive cells in the superior cervical ganglion and in the coeliac-superior mesenteric ganglion complex of the rat.

SummaryHistamine-immunoreactive small cells were detected in the superior cervical ganglion and in the coeliac-superior mesenteric ganglion complex of the rat with a specific antiserum produced in rabbits. Most histamine-immunoreactive cells were arranged in clusters, often around small blood vessels. Solitary immunoreactive small cells were also observed and they were easily distinguished from mast cells. The principal nerve cells showed no immunore-activity to histamine and no histamine-containing nerve fibers were detected in the ganglia by the present method. Due to close morphological similarities, it is concluded that the small immunoreactive cells observed in the present study represent small intensely fluorescent (SIF) cells first detected by formaldehyde-induced catecholamine fluorescence.

Histamine-immunoreactive endocrine cells in the adrenal medulla of the rat

Histamine is widely distributed in various mammalian tissues and it has been shown that histamine is located in mast cells as well as in other structures. Biochemical evidence has been presented that histamine acts as a neurotransmitter in the central nervous system. Immunohistochemical studies have demonstrated the location of histamine-immunoreactive neuronal cells in both the central and peripheral nervous system. Biochemical studies have shown that histamine is present in the adrenal gland, while the location of histamine in the adrenal medulla is not known. There is pharmacological and biochemical evidence that exogenous histamine affects the catecholamine secretion of the adrenal medulla. The present study was undertaken to examine the location of histamine in the rat adrenal medulla by an indirect immunofluorescence method using a specific histamine antiserum. We now report the presence of histamine-immunoreactive endocrine cells in the adrenal medulla of the rat and suggest that histamine is located in the noradrenaline-secreting cells.

Peptide YY-like immunoreactivity in sympathetic neurons of the rat.

The occurrence of peptide YY-like peptides in parts of the sympathetic nervous system of the rat was studied by immunocytochemistry and immunochemistry plus analysis by high performance liquid chromatography. Peptide YY-immunoreactive neurons and nerve fibers were detected in the superior cervical ganglion. Co-localization studies indicated that peptide YY and neuropeptide Y immunoreactivities co-exist in a subpopulation of neurons of the superior cervical ganglion. Immunochemical analysis revealed peptide YY-immunoreactive material, distinct from neuropeptide Y, in extracts of the superior cervical ganglion. On reverse-phase high performance liquid chromatography, extracts of superior cervical ganglion revealed several peaks of peptide YY-like immunoreactive material, one of which eluted close to the position of authentic porcine peptide YY. Peptide YY-immunoreactive nerve fibers were also present in sympathetic target tissues including the auricula and atria of the heart, carotid body, submandibular salivary gland and the adrenal cortex. It is suggested that peptide YY and/or peptide YY-like peptides are present not only in endocrine cells, but also in a subpopulation of cell bodies and fibers of the peripheral sympathetic nervous system.