Manfred Gratzl

Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers

Syntaxin and SNAP‐25 (synaptosome‐associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP‐25 by the toxin, indicating that distinct pools of SNAP‐25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP‐25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP‐25 monomers, stable SNAP‐25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP‐25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP‐25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.

SNAP‐25 requirement for dendritic growth of hippocampal neurons

Structure and dimension of the dendritic arbor are important determinants of information processing by the nerve cell, but mechanisms and molecules involved in dendritic growth are essentially unknown. We investigated early mechanisms of dendritic growth using mouse fetal hippocampal neurons in primary culture, which form processes during the first week in vitro. We detected a key component of regulated exocytosis, SNAP‐25 (synaptosomal associated protein of 25 kDa), in axons and axonal terminals as well as in dendrites identified by the occurrence of the dendritic markers transferrin receptor and MAP2. Selective inactivation of SNAP‐25 by botulinum neurotoxin A (BoNTA) resulted in inhibition of axonal growth and of vesicle recycling in axonal terminals. In addition, dendritic growth of hippocampal pyramidal and granule neurons was significantly inhibited by BoNTA. In contrast, cleavage of synaptobrevin by tetanus toxin had an effect on neither axonal nor dendritic growth. Our observations indicate that SNAP‐25, but not synaptobrevin, is involved in constitutive axonal growth and dendrite formation by hippocampal neurons. J. Neurosci. Res. 56:539–546, 1999.

Rab3 Proteins and SNAP‐25, Essential Components of the Exocytosis Machinery in Conventional Synapses, are Absent from Ribbon Synapses of the Mouse Retina

GTP‐binding rab proteins, present in synaptic vesicles and endocrine secretory granules, have been shown to be involved in the control of regulated exocytosis. We found rab3 proteins in immunoblots of diverse areas of the mouse central nervous system (spinal cord, olfactory bulb, hippocampus, cerebellum and neocortex). Immunohistochemical observations at light‐ and electron‐microscopical levels in the hippocampus and other areas revealed rab3 proteins in virtually all synaptic fields and terminals of the areas investigated. In the retina, rab3A immunoreactivity was confined to the inner and outer plexiform layers. Ultrastructural examination revealed that rab3A was present in conventional terminals in the inner plexiform layer and in horizontal cell processes of the outer plexiform layer. In contrast ribbon synapses, which play a key role in transferring information from the photoreceptor cells to the central nervous system, were immunonegative. We also tested whether other proteins of the rab3 family are present in ribbon synapses. However, using an antibody recognizing rab3B and rab3C in addition to rab3A, we found no immunoreactivity in these synapses. Interestingly, we observed also no immunoreactivity for synaptosomal‐associated protein 25 (SNAP‐25) in ribbon synapses, but conventional synapses and horizontal cell processes were heavily stained. Our data show that the known rab3 and SNAP‐25 isoforms, which are components of the secretory apparatus of conventional synapses, are absent from ribbon synapses of the retina. Our observations suggest different mechanisms of transmitter exocytosis in conventional and ribbon terminals.

An Autocrine Role for Pituitary GABA: Activation of GABA-B Receptors and Regulation of Growth Hormone Levels

There is increasing evidence suggesting that the neurotransmitter γ-aminobutyric acid (GABA) is a local factor involved in the regulation of endocrine organs. Examples of such functions are documented in the pancreas, but recent results suggest that GABA may act in a similar way in the pituitary, in which GABA receptors are expressed and pituitary growth hormone (GH) cells provide a source of GABA. We hypothesised that GABA secreted in somatotropes may act as an autoregulatory signaling molecule. To test this hypothesis we first examined the nature of GABA receptors expressed by GH cells. RT-PCR analysis demonstrated that GABA-B receptor subunits R1 and R2 are present in the whole rat pituitary. Laser microdissection of immunostained GH cells, followed by RT-PCR as well as immunoelectron microscopy, showed that GABA-B receptors are expressed on somatotropes. To investigate GABA-B receptor function in somatotropes, we used rat GH3 adenoma cells, which, like pituitary GH cells, express GABA-B R1 and R2 (as assessed by RT-PCR and immunoelectron microscopy) and produce GABA (checked by high performance liquid chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by baclofen, a chromatography). After inhibition of endogenous GABA synthesis, GH production was stimulated by baclofen, a GABA-B receptor agonist. By contrast, blocking GABA-B receptors by an antagonist, phaclofen, decreased GH levels. We conclude that in GH-producing cells, GABA acts as an autocrine factor via GABA-B receptors to control GH levels.

Prenatal hippocampal granule cells in primary cell culture form mossy fiber boutons at pyramidal cell dendrites.

Mossy fiber boutons are the sites of synaptic signalling between hippocampal granule and pyramidal neurons. We studied the formation and localization of these terminals during development of prenatal hippocampal neurons in primary culture. Using the synaptic vesicle membrane proteins synaptophysin and synaptoporin as markers we observed that both proteins were mainly localized in perikarya and processes of fetal hippocampal neurons during the first days in vitro (DIV). Following DIV 6 synaptophysin was present in small terminals. After DIV 20 in addition large terminals immunoreactive for synaptophysin and synaptoporin were found, which were identified by electron microscopy as mossy fiber boutons impinging on pyramidal neuron dendrites. Synaptic vesicles and endosomes in the mossy fiber boutons were labeled when incubated with exogenous horseradish peroxidase, indicating that they were competent for exo‐endocytosis. Taken together, our data show that hippocampal granule neurons grown in dissociated primary cultures form mossy fiber boutons containing synaptophysin and synaptoporin at pyramidal cell dendrites. Since the composition and the characteristic morphology of mossy fiber boutons formed in vitro is the same as observed in vivo we conclude that their development follows an intrinsic program. J. Neurosci. Res. 51:602–611, 1998.

Expression of histamine receptors and effect of histamine in the rat carotid body chemoafferent pathway.

Chemosensory information from peripheral arterial oxygen sensors in the carotid body is relayed by petrosal ganglion neurons to the respiratory networks in the medulla oblongata. Biogenic amines, including histamine, released from glomus (type I) cells of the carotid body are considered to be primary transmitters in hypoxic chemosensitivity. Immunocytochemistry at light‐and electron‐microscopical levels, and RT‐PCR, revealed the expression of histamine receptors 1 and 3 as well as histidine decarboxylase in the rat carotid body glomus cells and petrosal ganglion neurons. Histamine receptors 1 and 3, but not histidine decarboxylase, were also observed in the ventrolateral, intermediate and commissural subnuclei of the nucleus tractus solitarii in the medulla oblongata. In order to examine the possible role of histamine in the afferent branch of the respiratory system, we applied histamine receptor 1 and 3 agonists to the carotid body, which caused a mildly increased phrenic nerve activity in a working heart–brainstem preparation. Moreover, microinjection of antagonists of histamine receptors 1 and 3 into the nucleus tractus solitarii caused significant changes in the inspiratory timing and the chemoreceptor response. Our data show that histamine acting via histamine receptors 1 and 3 plays an important neuromodulatory role in the afferent control of chemosensitivity.

Selective expression of histamine receptors in rat mesencephalic trigeminal neurons.

The perikarya of sensory neurons of the mesencephalic trigeminal nucleus (MTN) receive dense histaminergic hypothalamic innervation. In this study, we examine the yet unknown expression and localization of histamine receptors in the rat MTN using immunohistochemistry with subtype-specific antibodies. Same as the masticatory muscle spindle somata H1 receptors were located along the entire MTN, whereas H3 receptors were detected in the caudal pontine part of the nucleus, which receives input from periodontal afferents. Most of the immunostained cell bodies were surrounded by histidine decarboxylase-, histamine- or vesicular monoamine transporter 2-containing pericellular varicose fibers and terminals in a basket-like manner. Our results suggest that rat MTN neurons are directly influenced by histaminergic descending projections from the hypothalamus. It can be inferred that processing of proprioceptive information at the level of the MTN is controlled via histamine H1 and H3 receptors through different postsynaptic mechanisms.

Histaminergic and dopaminergic traits in the human carotid body.

Carotid body (CB) chemoreceptors are the main sensors detecting systemic hypoxia. Studies in animals revealed that dopamine and histamine may serve as transmitters between the chemoreceptor cells and the afferent nerve. To gain insight whether histamine and dopamine could play a role in the human CB and thus be important for the understanding of breathing disorders, we have investigated the chemosensory traits in human CBs from nine subjects of different ages obtained at autopsy. Immunohistochemistry revealed expression of histidine decarboxylase, vesicular monoamine transporter 2, histamine receptors 1 and 3 in virtually all chemosensory cells within the glomeruli of different ages. By contrast, catecholaminergic traits (tyrosine hydroxylase and vesicular monoamine transporter 1) were only detected in a subset of CB chemosensory cells at each age group while dopamine D2 receptors were expressed in the great majority of them. Our data suggest that histamine along with catecholamines may serve as transmitters between chemoreceptor cells and the afferent nerve in humans as well.

Identification of Voltage‐Activated Na+ and K+ Channels in Human Steroid‐Secreting Ovarian Cells

ells communicate via extracellular molecules, including neurotransmitters, hormones, and growth factors. Depending on their nature, distinct signal transduction mechanisms exist, including receptor-mediated generation of second messengers (e.g., cAMP, cGMP), phosphorylation/dephosphorylation of receptors and channels, or changes in intracellular Ca concentrations. Increased intracellular Ca levels result from their release from intracellular Ca stores or from the influx of extracellular Ca through channels activated by the binding of a variety of ligands, or via voltage-activated Ca channels. Different types of cells have specific receptor types and signal transduction mechanisms tailored to their needs. In neurons, fast changes in membrane potential, carried along dendritic and axonal processes, are brought about mainly by activation of voltage-activated Na and K channels. In nerve terminals, arriving action potentials lead to subsequent activation of Ca channels and finally to Ca triggered release of neurotransmitters. In aminergic and peptidergic endocrine cells, similar mechanisms underlies the exocytosis of hormones. Thus their cell membranes contain voltage-activated Na, K, and Ca channels. In contrast, channels, receptors, and transmembrane signaling in steroidproducing cells have not been studied very often so it is unknown whether these cells contain such a set of channels. There is evidence, however, that voltage-activated K and Ca channels are present in avian and porcine ovarian cells. During the last few years we have focused our attention on a human steroidogenic ovarian cell type, namely luteinized granulosa cells (GCs). Human GCs are obtained from in vitro fertilization patients, and can be cultivated for several days and used for functional studies. Like their counterparts in the corpus luteum, these cells produce progesterone, which is crucial for maintaining pregnancy. As shown previously, these cells possess receptors for various hormones and neurotransmitters (e.g., oxytocin, relaxin, catecholamines, acetylcholine), and activation of these receptors is linked to increased Ca, which is mainly, though not exclusively derived from intracellular stores. In an attempt to further characterize the signal transduction machinery of these cells, we have now started to examine their as yet unknown electrophysiological properties.

Expression of the endocytic proteins dynamin and amphiphysin in rat gastric enterochromaffin-like cells

Dynamin and amphiphysin play crucial roles in a variety of endocytic processes. Previous investigations of expression and functions of these proteins were performed mostly on neurons. The aim of this study was to investigate the presence and interaction of dyn and amph in gastric enterochromaffin-like cells. These endocrine cells of the gastric mucosa play a pivotal role in the regulation of acid secretion. Exocytosis of histamine-containing secretory vesicles has been described in detail. However, the mechanisms of endocytosis are unknown in this neuroendocrine cell type. Using RT-PCR and western blotting, we detected dynamin-1, -2 and -3 in highly enriched isolated enterochromaffin-like cells. Dynamin-1 and -2 were expressed at similar high levels, whereas dynamin-3 was of low abundance. Immunofluorescence microscopy located dynamin-1 and -2 to the cytoplasm and cell surface, whereas dynamin-3 was distributed differently in the perinuclear area. The presence of amphiphysin-1 and -2 RNAs was revealed by RT-PCR and a new splice variant of amphiphysin-2 was detected. Amphiphysin-1 and -2 were also detected in enterochromaffin-like cells by immunohistochemistry in the same locations as dynamin-1 and -2. Amphiphysin-1 and dynamin-1 co-immunoprecipitated with amphiphysin-2. In addition, dynamin-1 and amphiphysin-2 partially colocalized at the plasma membrane. Our results confirm the interaction of dynamin and amphiphysin and imply a role in endocytosis in enterochromaffin-like cells. To our knowledge, this is the first demonstration of the co-expression of all three dynamin isoforms in a non-tumor cell.