α4-1 Subunit mRNA of the nicotinic acetylcholine receptor in the rat olfactory bulb: cellular expression in adult, pre- and postnatal stages

In addition to their role in signal transduction, nicotinic acetylcholine receptors have been shown in vi-tro to be involved in neuronal growth cone regulation during development. This idea is supported by recent histochemical findings showing that iso- and archicortical nicotinic alpha4-1 receptor mRNA expression precedes cholinergic fiber ingrowth. To test whether this also holds true for rhinencephalic parts of the telencephalon, we have studied the olfactory bulb by digoxigenin-mediated in situ hybridization, using an alpha4-1 isoform-specific riboprobe and an alkaline-phosphatase-based detection system. Development is characterized by early intense alpha4-1 mRNA expression (embryonic d…

Neuropeptide Y (NPY)-like immunoreactivity in the guinea pig pineal organ.

Relatively little is known about mammalian pineal neuropeptides. In the present study neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the guinea pig pineal gland. NPY-LI was restricted to few intrapineal nerve fibers of faint fluorescence intensity. They showed no preferential localization with regard to the different pineal portions. As catecholaminergic fibers are abundant in the guinea pig pineal gland, the scarcity of NPY-LI fibers indicates that in the pineal colocalization of noradrenaline and NPY-LI is not a regular feature, in contrast to other organs. The possibility exists that in the pineal NPY-LI fibers are not of peripheral sympathetic but of central origin.

Modulatory control by non-competitive agonists of nicotinic cholinergic neurotransmission in the central nervous system

Abstract Several exogenous and endogenous compounds have been discovered that act on nicotinic acetylcholine receptors as non-competitive agonists (NCA) of low efficacy, and probably also as allosteric regulators of the receptor's sensitivity to acetylcholine. Nicotinic NCAs may be viewed as another facet in the increasingly complex picture of vertebrate brain organization, i.e. they may be part of a higher level ‘chemical’ network that overlays the neuronal network of the CNS. Furthermore, exogenous NCAs may provide a new therapeutic approach to Alzheimer's dementia.

Expression of the α4 isoform of the nicotinic acetylcholine receptor in the fetal human cerebral cortex

Nicotinic acetylcholine receptors are likely to play an important role in neuronal migration during development. Furthermore, the alpha4 receptor subunit gene is related to a hereditary juvenile form of epilepsy. Only little information is available, however, on the expression of cerebrocortical nicotinic acetylcholine receptors during human fetal development. Using non-isotopic in situ hybridization and immunohistochemistry, we have studied the distribution of the alpha4 subunit of the nicotinic acetylcholine receptor mRNA and protein in the human frontal cortex at middle (17-24 weeks of gestation) and late (34-42 weeks of gestation) fetal stages. Both, alpha4 receptor mRNA and alpha4 rece…

Immunohistochemistry of cholinergic receptors.

Acetylcholine and its receptors are involved in a variety of important signal transduction processes. As shown here paradigmatically for the human neuromuscular junction and the cerebral cortex, acetylcholine receptors can be visualized immunohistochemically at the cellular and subcellular level under physiological and pathological conditions. At normal motor endplates nicotinic cholinoceptors are localized at the surface of the postsynaptic junctional folds. In myasthenic syndromes investigation of muscle biopsies enables the diagnosis of receptor deficiencies at the ultrastructural level. In normal cerebral cortex pyramidal neurons are equipped with both nicotinic and muscarinic acetylcho…

Nicotinic receptor function in the mammalian central nervous system.

The diversity of neuronal nicotinic receptors (nAChRs) in addition to their possible involvement in such pathological conditions as Alzheimer's disease have directed our research towards the characterization of these receptors in various mammalian brain areas. Our studies have relied on electrophysiological, biochemical, and immunofluorescent techniques applied to cultured and acutely dissociated hippocampal neurons, and have been aimed at identifying the various subtypes of nAChRs expressed in the mammalian central nervous system (CNS), at defining the mechanisms by which CNS nAChR activity is modulated, and at determining the ion permeability of CNS nAChR channels. Our findings can be sum…

Distribution of dopamine-beta-hydroxylase—like immunoreactivity in the rat pineal organ

The aim of the present investigation was to study the distribution in the rat pineal gland of dopamine-beta-hydroxylase (DBH) which is essential for the formation of the melatonin synthesis-regulating substance noradrenaline (NA). In 5- and 8-month-old male Sprague-Dawley rats DBH-like immunoreactivity (DBH-LI) was studied using polyclonal antibodies against DBH and the indirect immunofluorescent technique. DBH-LI was mainly located in pineal nerve fibres coming from the superior cervical ganglia. The intensity of the staining reaction was considerably lower than in non-pineal noradrenergic nerve fibres and the impression was gained by comparison of DBH-LI specimens with glyoxylic acid-trea…

The role of the hypothalamic paraventricular nuclei for the regulation of pineal melatonin synthesis: New aspects derived from the vasopressin-deficient Brattleboro rat

Abstract There is evidence for an involvement of the hypothalamic paraventricular nuclei (PVN) in the regulation of pineal melatonin synthesis in rats. Since electrical stimulation of the PVN or the systemic administration of arginine-vasopressin (AVP) result in a depression of the nocturnal melatonin surge, this neuropeptide appears to be pivotal for the transduction of PVN-efferent, pinealopetal signals. We therefore used an AVP-deficient animal model, the Brattleboro rat, to further investigate the mechanisms responsible for pineal regulation. Anesthetized adult male animals received 2 min of bilateral electrical stimulation of the PVN either during the day or at night. Thirty min later,…

Immunohistochemical evidence for the presence of peptides derived from proenkephalin, prodynorphin and proopiomelanocortin in the guinea pig pineal gland

By using a plethora of region-specific antisera, this light microscopic immunohistochemical study revealed that derivatives from the three opioid precursors, i.e. proenkephalin, prodynorphin and proopiomelanocortin are differentially distributed in the pineal gland of guinea pig. Various molecular forms of immunoreactive opioid peptides derived from proenkephalin or prodynorphin were present in a minority of pinealocytes as well as in nerves. In contrast to this dual distribution pattern of opioid-active peptides, the opioid-inactive derivative from proopiomelanocortin, alpha-melanocyte stimulating hormone, was exclusively present in a large proportion of pinealocytes. A multiple and differ…

Neuropeptide Y effects on pineal melatonin synthesis in the rat

Neuropeptide Y (NPY)-like immunoreactivity is present in the rodent pineal gland. To elucidate possible effects on pineal melatonin synthesis NPY (5 nmol/kg body wt.) was injected into the common carotid artery of male rats. Activities of the melatonin biosynthetic enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were determined by means of radioenzymatic methods. Intact light-exposed animal showed increased NAT activity at day- and at nighttime. Blinded animals showed a more than 10-fold reduction of NAT activity after nocturnal NPY injections. HIOMT activity was only slightly influenced at either time. These results are discussed in terms of the p…

Cellular Distribution and Expression of Cortical Acetylcholine Receptors in Aging and Alzheimer's Disease

Ligand binding studies show marked reductions of nicotinic, but not of muscarinic binding sites in Alzheimer's disease. Using monoclonal antibodies we studied immunohistochemically the expression of the respective receptor proteins in the frontal cortex of middle-aged (55 +/- 5 yr) controls, age-matched controls (73 +/- 6 yr), and patients with Alzheimer's disease (74 +/- 5 yr). Density of nicotinic cholinoceptive neurons was 8000/mm3 for middle-aged controls and 4000/mm3 for age-matched controls, but only 900/mm3 in Alzheimer's brains (p less than 0.0001). Densities of muscarinic cholinoceptive and of Nissl-stained neurons were not significantly different between the groups, pointing to a …

Neuronal markers in the rodent pineal gland ? an immunohistochemical investigation

Although some embryological and morphological features speak in favour of a neuronal character of rodent pinealocytes, histochemistry and ultrastructure let this issue appear controversial. Using antibodies to different neurofilaments, the neural adhesion molecule L1, synaptophysin and tubulin as neuronal markers, the pineal glands of rat and guinea-pig were studied by means of immunofluorescence. Neurofilament-immunoreactivity was present in some rat pineal nerve fibers and in the majority of guinea-pig pinealocytes, L1 decorated rat intrapineal nerve fibers, synaptophysin was almost ubiquitously distributed in the pineal of both species, while tubulin-immunofluorescence was seen in nerve …

Expression of α4-1 and α5 Nicotinic Cholinoceptor mRNA in the Aging Rat Cerebral Cortex

Although important in neurodegeneration, systematic studies of nicotinic acetylcholine receptor expression in normal aging human brains are difficult to perform. We have studied the expression of nicotinic receptor alpha 4-1 and alpha 5 mRNA in the frontal and parietal isocortex of 3- (young adult), 24- (late middle aged), and 33-month-old (old) rats by nonisotopic in situ hybridization. In all groups transcripts were mainly present in layer II/III and V pyramidal neurons. The numerical densities of alpha 4-1 mRNA-containing neurons with respect to those of cresyl violet-stained neurons decreased with aging in the rat frontal and parietal cortex, while those of alpha 5 mRNA-containing neuro…

Effects of electrical stimulation of the superior cervical ganglia on the number of ?synaptic? ribbons and the activity of melatonin-forming enzymes in the rat pineal gland

Melatonin metabolism in the mammalian pineal gland is under the clear influence of sympathetic fibers originating in the superior cervical ganglia (SCG). Previous studies suggested that pineal "synaptic" ribbons (SR) as well are regulated by the gland's sympathetic innervation. To gain more insight into the mechanisms involved, we examined the effects of sympathetic stimulation on SR number and on the activity of melatonin forming enzymes, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The SCG in adult male rats were stimulated electrically during daytime for either 15 or 120 min. Immediately following stimulation, the glands were removed and processed fo…

Nicotinic cholinoceptors in the rat pineal gland as analyzed by Western blot, light- and electron microscopy

Abstract The monoclonal antibody WF6, raised against purified Torpedo nicotinic acetylcholine receptor (nAChR) was used to study the distribution of cholinoceptors in the rat pineal gland by means of Western blot analysis, light- and electron microscopy. The immunoblot analysis using homogenized pineal gland revealed a labeled protein band of apparent molecular weight 40 kDa which was identified as α-subunits of a nAChR. In the light microscope, approximately one-fourth of the pinealocytes exhibited cytoplasmic immunoreactivity (IR) of varying density. In the electron microscope, IR was seen as patchy staining of cell membranes of pinealocyte somata and processes. Presynaptic IR material wa…

Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ hybridization.

The pharmacology of telencephalic nicotinic acetylcholine receptors (nAChRs) has become an important issue in recent years. While in the human brain a direct pharmacological assessment is difficult to achieve the visualization of nAChRs has been enabled by histochemical techniques providing an ever increasing and improving resolution. Receptor autoradiography was used to visualize binding sites on the level of cortical layers whereas immunohistochemistry has allowed for the cell type-specific and ultrastructural localization of receptor protein. Further investigations have to elucidate the cellular sites of NAChR biosynthesis by visualizing subunit-specific transcripts. Using autopsy sample…

Forebrain specialization and the olfactory system in anseriform birds. An architectonic and tracing study.

In anseriform birds the mediodorsal part of the rostral forebrain is covered by a corticoid (= layered) structure, establishing a unique feature of this avian group since in other birds the non-cortical accessory or dorsal hyperstriatum occupies the corresponding surface area of the hemisphere. The efferents of the olfactory bulb are shown to reach this region, which thus can be identified as a heavily enlarged retrobulbar area. The large expansion of this olfactory representation may indicate an important biological function. In comparison to the mammalian olfactory system the three stratified olfactory projection centers of birds should be regarded as retrobulbar, prepiriform and periamyg…

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation.

Inforimation on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve f…

Cellular expression of α4 subunit mRNA of the nicotinic acetylcholine receptor in the developing rat telencephalon

By contrast to adult brain, little is known on the development of nicotinic acetylcholine receptor (nAChR) expression. Using a digoxigenin-labeled riboprobe for in situ hybridization, alpha 4 nAChR subunit mRNA expression was studied in embryonic and postnatal rat neocortex and hippocampus where it was transiently increased in neuronal subpopulations and preceded cholinergic fiber ingrowth. alpha 4 expression was increased in neocortical layer VIb between E20 and P2 and, about birth, in dentate gyrus granule cells subsequently decreasing to adult levels. nAChR mRNA expression is increased at the developing neuromuscular endplate preceding cholinergic innervation which triggers changes in no…

Noradrenergic innervation of the pineal gland — histochemical basis of scintigraphic imaging?

There is first evidence that the adult human pineal gland may be equipped with noradrenergic fibers as is the case in a variety of mammalian species. It therefore appears worthwhile to investigate the capacity of pineal noradrenergic nerves to take up 123I-MIBG, a prerequisite for scintigraphic imaging.

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiolog…

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer's disease: histotopographical correlation with amyloid plaques and hyperphosphorylated-tau protein

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the p…

Expression of inhibitory glycine receptors in postnatal rat cerebral cortex.

The developmental expression of inhibitory glycine receptors was analyzed in postnatal rat cerebral cortex using the specific monoclonal antibody, MAb 4a. This antibody defines an epitope common to all known glycine receptor alpha-subunits. At birth, high levels of immunoreactivity were found, which transiently increased during the second postnatal week, but subsequently declined to low adult levels. Biochemical analysis of the MAb 4a antigen from parietal areas indicates that cortical glycine receptors correspond to the neonatal receptor isoform previously identified in spinal cord of newborn animals. Immunocytochemistry showed that, within 2 weeks after birth, MAb 4a-reactive glycine rece…

Cellular distribution in the rat telencephalon of mRNAs encoding for the alpha 3 and alpha 4 subunits of the nicotinic acetylcholine receptor.

Pharmacological and electrophysiological studies provide evidence for the involvement of different nicotinic acetylcholine receptor isoforms in rat neocortical and hippocampal signal transduction. Yet, rather little is known on the cellular localization of these isoforms. With the availability of isoform specific nucleic acid probes and sensitive non-isotopic detection systems, nicotinic receptors can be studied on the mRNA level in individual neurons. In this way, we have paradigmatically studied the distribution of the alpha 3 and alpha 4 isoform mRNAs of the nicotinic receptor in the rat telencephalon. In the cerebral cortex, alpha 3 transcripts were mainly located in pyramidal neurons o…

Cellular Acetylcholine Receptor Expression in the Brain of Patients with Alzheimer’s and Parkinson’s Dementia

Binding studies and receptor autoradiography reveal the overall changes of acetylcholine receptors (AChR) in Alzheimer’s and Parkinson’s dementia cortices.2,5 A detailed account of these changes requires a study of neurochemical phenotype of individual neurons as basic elements of networks constituting the substrate of cortical functions.4 Examples will be given for cell-type specific AChR localization in normal and diseased human cerebral cortex.

In vitro synthesis of dopamine and noradrenaline in the isolated rat pineal gland: day-night variations and effects of electrical stimulation.

: Isolated rat pineal glands were incubated in vitro in a medium containing [I4C]dopamine or [14C]tyrosine, and the tissue contents of l4C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [l4C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [l4C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [l4C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine β-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyro…

Expression of nicotinic acetylcholine receptors in the rat superior cervical ganglion on mRNA and protein level.

The expression of nicotinic acetylcholine receptors (nAChR) in the rat superior cervical ganglion was investigated by Western blotting, immunohistochemistry and non-radioactive in situ hybridization applying probes for the alpha 4-1 and beta 2 subunit mRNA. Immunoblot analysis of homogenized ganglia using the anti-nAChRs antibody WF6 revealed a labeled protein band of apparent molecular weight of 40 kDa which is typical for the alpha subunit of nAChRs. Applying double-labeling immunofluorescence with antibodies against tyrosine hydroxylase, nAChR-like molecules were identified in most postganglionic neurons and in a subpopulation of small intensely fluorescent (SIF) cells. alpha 4-1 and bet…

Principal neurons projecting to the pineal gland in close association with small intensely fluorescent cells in the superior cervical ganglion of rats

The localization in the superior cervical ganglia (SCG) of small, intensely fluorescent (SIF) cells and of principal nerve (PN) cells innervating the pineal gland was examined in adult male Sprague-Dawley rats. PN cells were demonstrated by means of the retrograde neuron-tracing method using the fluorescent tracer Fluoro-Gold (FG) injected into the pineal gland. SIF cells were visualized by the formaldehyde-induced fluorescence method. Twenty-nine percent of the FG-labeled PN cells were found closely associated with SIF cells. In the rostral half of the ganglion, 43% of the SIF cells were situated in juxtaposition to one or several labeled neurons. The possible influence of SIF cells on the…

Cellular and subcellular distribution of receptors in the entorhinal—hippocampal system: Morphologic and biochemical aspects

Stimulation of Serotonin-N-Acetyltransferase Activity in the Pineal Gland of the Mongolian Gerbil (Meriones unguiculatus) by Intracerebroventricular Injection of Vasoactive Intestinal Polypeptide

There is ever-increasing evidence that intrapineal peptides have an important role in the modulation of pineal melatonin synthesis. In the pineal gland of the Mongolian gerbil (Meriones unguiculatus), we have previously shown the presence of VIP-immunoreactive nerve fibers as well as pinealocytic VIP receptors. To assess the functional significance of these findings, 10 microliters of a 1 microM or 1 nM solution of VIP were injected into the lateral ventricle of gerbils over a period of 10 min. Animals were killed 1.5 hr after injection, and the superficial pineal glands were excised and assayed for N-acetyltransferase (NAT) activity. Injection of the 1 microM VIP solution stimulated the NA…

Nicotinic Receptors in Human Brain

A vast knowledge is currently available on the molecular biology and the pharmacology of nicotinic acetylcholine receptors (nAChR) in the mammalian central nervous system (CNS) (Sargent, 1993). Only few attempts have been made to approach the expression of nAChRs at the level of functional systems, considering the different cell types involved and their connectivity. This aspect is of particular importance in order to evaluate nAChR expression under pathological conditions. Histochemical techniques have proven to be useful since immunohistochemistry and in situ hybridization can be performed on human autopsy tissue and allow for a cell type-specific localization of nAChR proteins and nAChR …

Molecular Histochemistry of Nicotinic Receptors in Human Brain

Only a decade ago the existence and functional significance of central nervous nicotinic acetylcholine receptors (nAChR) was still a subject of controversy. Today, the importance of this receptor class for signal transduction in human brain in normal and pathological conditions has become quite evident. nAChRs have turned out to be important pharmacological targets in disorders like Alzheimer’s disease (AD) (Arneric et al., 1994). One prerequisite to understand nAChR function is a detailed study of the cellular distribution of nAChR subtypes. In recent years several human-specific data have been made available. This paper attempts to show actual developments in this field, summarizing the e…

Characterization of rodent pineal astrocytes by immunofluorescence microscopy using a monoclonal antibody (J1-31).

In previous studies pineal astrocytes have been characterized immunohistochemically mainly by use of antisera to glial fibrillary acidic protein. Because of the recent demonstration of this protein in non-astrocytic cells the question of its specificity as an astrocytic marker has been raised. A possible alternative tool for characterizing pineal astrocytes is the J1-31 monoclonal antibody, which is directed against a 30 000 dalton astrocytic protein clearly distinguishable from glial fibrillary acidic protein. Immunofluorescence microscopy of this antibody in the pineal gland of rat and guinea-pig revealed a staining pattern similar to that obtained by glial acidic fibrillary protein antis…