Electron Microscopic Observation of Delta-Opioid Receptor-1 in the Rat Area Postrema

Guan, J.-L., Q.-P. Wang and Y. Nakai. Electron microscopic observation of delta-opioid receptor-1 in the rat area postrema. Peptides 18(10) 1623–1628, 1997.—The ultrastructural localization of delta-1-opioid-receptor in the rat area postrema was quantitatively studied by pre-embedding avidin-biotin-peroxidase-complex technique. Most of the immunoreactive profiles (67.4%) observed in the present study were axon terminals, whereas the immunopositive dendrites were less (28.3%). Within the axon terminals, the immunoreactivity was found stronger in the dense-cored vesicles than in the small, clear, and round vesicles. Almost 2/3 of the DOR-1 immunoreactive axon terminals had DAB reacted dense-cored vesicles. About half of the immunopositive axon terminals were found to make synapse to dendrites. The dendrites postsynaptic to DOR-1 immunoreactive axon terminals were identified as DOR-1 immunoreactive or not, mainly according to the immunoreactive appearance of the postsynaptic membrane. About half of the DOR-1 immunoreactive dendrites were observed to receive synapse; most of them have their immunoreactivity results at the postsynaptic membranes.     

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.     

Ultrastructural effects of colchicine on perikarya and initial segment of rat retinal ganglion cells.

Our ultrastructural study was focused on the perikaryal region and initial segment of the axon of rat retinal ganglion cells in controls and after intraocular injections of colchicine. In control rats that region contained, among other organelles, elements of the Golgi complex and, close to them, short isolated microtubules oriented preferentially toward the axon where they funnel and aggregate in bundles. One day after sufficient doses of colchicine to inhibit axoplasmic transport (2-20 micrograms) these cytoplasmic microtubules were absent, whereas some axonal microtubules were still present but reduced in number. In addition, colchicine induced an altered distribution of organelles, leaving empty spaces in the periphery and most organelles concentrated in the perinuclear region, especially around Golgi elements where numerous vesicles and tubules accumulate at the trans face of Golgi elements. These results suggest that the vesicles that leave the Golgi and have been directed towards axoplasmic transport may need the cytoplasmic microtubules located between Golgi elements and the axonal initial segments to reach the axon.     

The ultrastructural differentiation of synaptic sites in the inner plexiform layer of a teleostean retina.

The differentiation of axon terminals in the retina inner plexiform layer was studied by electron microscopy, with special reference to synaptic junctions, number and size of synaptic vesicles, dense core vesicles, biogenic amines and ATPase. Five types of synaptic junctions were found, including a ribbon type. They appear on different days of embryonic life and show different patterns of increase. The ultrastructural differentiation of the most frequent type is described in detail. The numbers of synaptic vesicles indicate the existence of three types of axon terminals which appear on different days. These and other data lead to the following model: The contacts between amacrine cells are the first to appear, followed by amacrine--bipolar cell contacts and amacrine--ganglion cell contacts. The latter are the most frequent ones and increase immediately after having appeared, while amacrine--bipolar cell contacts increase only some days later, which is also the case for bipolar--amacrine cell contacts. Biogenic amines and cytochemically detectable ATPase appear along with the formation of synaptic sites.     

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.     

Ultrastructure of the pineal organ of the killifish,Fundulus heteroclitus,with special reference to the secretory function

Summary The pineal organ of the killifish, Fundulus heteroclitus, was investigated by electron microscopy under experimental conditions; its general and characteristic features are discussed with respect to the photosensory and secretory function. The strongly convoluted pineal epithelium is usually composed of photoreceptor, ganglion and supporting cells. In addition to the well-differentiated photosensory apparatus, the photoreceptor cell contains presumably immature dense-cored vesicles (140–220 nm in diameter) associated with a well-developed granular endoplasmic reticulum in the perinuclear region and the basal process. These dense-cored vesicles appear rather prominent in fish subjected to darkness. The ganglion cell shows the typical features of a nerve cell; granular endoplasmic reticulum, polysomes, mitochondria and Golgi apparatus are scattered in the electron-lucent cytoplasm around the spherical or oval nucleus. The dendrites of these cells divide into smaller branches and form many sensory synapses with the photoreceptor basal processes. Lipid droplets appear exclusively in the supporting cell, which also contains well-developed granular endoplasmic reticulum and Golgi apparatus. Cytoplasmic protrusions filled with compact dense-cored vesicles (90–220 nm in diameter) are found in dark-adapted fish. The origin of these cytoplasmic protrusions, however, remains unresolved. Thus, the pineal organ of the killifish contains two types of dense-cored vesicles which appear predominantly in darkness. The ultrastructural results suggest that the pineal organ of fish functions not only as a photoreceptor but also as a secretory organ.We thank Dr. Grace Pickford for the fishes.     

Colchicine effects on neurosecretory neurons and other hypothalamic and hypophysial cells,with special reference to changes in the cytoplasmic membranes

Summary The morphological effects of colchicine on the entire neurosecretory (NS) tract and on various hypothalamic nuclei have been studied. The perturbation in axonal flow, indicated by the accumulation of NS material, coincide with fragmentation of the cytoplasmic membranes, i. e. the Golgi apparatus and the endoplasmic reticulum, whereas the neurotubules remain relatively well preserved. Autophagic destruction of NS material is observed along the entire length of the NS fibres. The rapid and systematic changes in the axoplasmic reticulum, known to store calcium, lead us to envisage a role for this system — similar to that of the sarcoplasmic reticulum — in controlling the transport of NS vesicles. The junctional zone between the stalk and the neural lobe seems to play a particular rôle in the transport of NS material to the posthypophysial terminals of the NS axons. Colchicine provokes an increase in dense-cored vesicles in most of the neurons of the other hypothalamic nuclei studied: arcuate, suprachiasmatic, periventricular and ventromedial. Membranous alterations are also observed in these sites. Colchicine administered to animals which were hypothyroid, castrated or adrenalectomized, reveals stimulated neurons, identified by their excessive content of dense-cored vesicles. These neurons display no specific localization, for they occur in all hypothalamic nuclei, irrespective of the stimulation. The frequency of stimulation of neurons of the periventricular nucleus is striking.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

Monoamine precursor uptake in ganglia of a mollusc Anodonta cygnea L. (Bivalvia)

Summary In vitro-uptake and localization of ³H-5-hydroxytryptophan and ³H-dihydroxyphenylalanine have been investigated by means of light microscopic autoradiography in the central nervous system of the freshwater mussel, Anodonta cygnea L. Accumulation of the labelled monoamine precursors could be observed both over neuronal perikarya and neuropil in the ganglia. ³H-5-hydroxytryptophan and ³H-dihydroxyphenylalanine were taken up by neurons characterized by a small size (10–20 m), a polygonal form and an intense staining with toluidine blue. Labelled axon branches situated either in the neuropil or between nerve cells in the cortex exhibited only a moderate activity, frequently of a diffuse character.After pretreatment for 24 h with colchicine, silver grains were also seen over the neuronal perikarya and some axon profiles. This shows that local uptake of the monoamine precursors and, presumably, the synthesis of the monoamines takes place both at perikaryal and axonal/terminal level.The nerve cells accumulating the monoamine precursors contain a nucleus with rich chromatin content and a deeply invaginated nuclear envelope. Further ultrastructural characteristics of these neurons are their well-developed rough endoplasmic reticulum system, a great number of mitochondria and glycogen granules, and the presence of dense-cored vesicles of different types. All these features are responsible for the electron-dense appearance of the cytoplasm.     

Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra

The presence of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the peduncle of Hydra was demonstrated by immunogold electron microscopy. Thin sections of Lowicryl-embedded tissue labeled with antisera to RFamide and 5-nm gold-conjugated, secondary antibody and of Epon-Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms/ml RFamide. These results are the first demonstration that the dense-cored vesicles of Hydra neurons contain a neuropeptide.     

Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

Fine structure and composition of the submucous nerve plexus of the guinea-pig trachea

Summary The ultrastructure of the nerves forming the submucous plexus of cervical and thoracic parts of the trachea was studied in the guinea-pig. Specimens were obtained from 6 animals perfused with warm fixative and from 6 animals in which pieces of trachea were incubated in buffer containing 5-hydroxydopamine before being immersed in cold fixative. Of the two types of axonal terminal identified in the nerves, one contained mainly large dense-cored vesicles, and the second contained numerous small vesicles. In specimens incubated in 5-hydroxydopamine, the small vesicles of the latter terminals exhibited the electron-dense cores which are characteristic of adrenergic axonal terminals. Counts made on perfused specimens showed that, in both the thoracic and cervical parts of the trachea, the density of adrenergic terminals was higher than that of terminals containing mainly large dense-cored vesicles. Overall terminal density was, however, higher in the thoracic than in the cervical part of the trachea, and estimates of nerve size showed that this was associated with the presence in the thoracic plexus of a substantially greater proportion of nerves with less than 6 axons. The possible function of the nerves in the control of the calibre of the submucous blood vessels was discussed.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

Structure of anterior dorsal ventricular ridge in snakes.

Anterior dorsal ventricular ridge (ADVR) is a major subcortical, telencephalic nucleus in snakes. Its structure was studied in Nissl, Golgi, and electron microscopic preparations in several species of snakes. Neurons in ADVR form a homogeneous population. They have large nuclei, scattered cisternae of rough endoplasmic reticulum in their cytoplasm, and bear dendrites from all portions of their somata. The dendrites have a moderate covering of pedunculated spines. Clusters of two to five cells with touching somata can be seen in Nissl, Golgi, and electron microscopic preparations. The area of apposition may contain a series of specialized junctions which resemble gap junctions. Three populations of axons can be identified in rapid Golgi preparations of snake ADVR. Type 1 axons course from the lateral forebrain bundle and bear small varicosities about 1 mu long. Type 2 axons arise from ADVR neurons and bear large varicosities about 5 mu long. The origin of the very thin type 3 axons is not known; they bear small varicosities about 1 mu long. The majority of axon terminals in ADVR are small (1 mu to 2 mu long), contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates on dendritic spines and shafts and on somata. A small percentage of terminals are large, 5 mu in length, contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates only on dendritic spines. A small percentage of terminals are small, contain pleomorphic synaptic vesicles, and form symmetric active zones. This type of axon terminates on dendritic shafts and on somata.     

Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).     

Electron microscopy of the arcuate nucleus of normal and 5-hydroxydopamine treated cats

The arcuate nucleus of normal cats and of cats treated with 5-hydroxydopamine (5-OHDA) was investigated by electron microscopy. The neurons of the arcuate nucleus were classified into three types, clear, intermediate and dark, according to their fine structure. The clear type contained numerous dense-cored vesicles and well developed cell organelles. All three types were frequently seen to be partially surrounded by glial processes. Many axo-somatic and axo-dendritic synapses mostly small in diameter were also observed around the neurons. Synaptic contacts were demonstrated between axon endings and axonal processes which contained elementary granules. After administration of 5-OHDA small and large dense-cored vesicles appeared in the nerve endings surrounding the neurons. The relationship between the dense-cored vesicles in the perikarya and dopamine was briefly discussed.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Catecholamine-containing nerve terminals of the eccrine sweat glands of macaques

Summary A loose network of catecholamine-containing nerves was demonstrated with a fluorescence histochemical method (Falck-Hillarp) in the coiled portion of eccrine sweat glands in the digital pads of macaques after the injection of nialamide and noradrenaline. In the skin of untreated control animals, fluorescent fibers appear only in some of the glands. A systemic administration of reserpine and a local injection of 6-hydroxydopamine (6-OHDA) or 5-hydroxydopamine (5-OHDA) into the digital pad cause a complete disappearance of fluorescent fibers around the glands and blood vessels. Electron micrographs reveal many unmyelinated varicose axon profiles outside the basement membrane of secretory tubules. Most of these profiles contain many small agranular vesicles and a few large dense-cored vesicles (cholinergic terminal), and some have numerous small granular and a few large densecored vesicles (adrenergic terminal).The local injection of 6-OHDA causes various degenerative changes in the adrenergic terminals but the cholinergic ones and the rest of the cellular structure remain intact. The injection of 5-OHDA induces a significant increase of electron-dense granules in the vesicles of adrenergic terminals.The presence of catecholamine and the effects of 6-OHDA and 5-OHDA in the nerve terminals indicate that the innervation of the eccrine sweat glands of macaques consists of cholinergic as well as adrenergic terminals.Publication No. 783 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grant RR 00163 of the Animal Resources Branch, Division of Research Resources.We acknowledge the excellent assistance of Tsutomu Yoshida, Tsuneka zu Fuse, John Ochsner, and Nickolas Roman.     

Diurnal variations in the photoreceptor synaptic terminals of the newt retina

Newt photoreceptor synaptic terminals undergo a variety of morphological changes over a 24-hr (LD 12:12) cycle. During the day, dense-cored synaptic vesicles were found to increase in number and accumulate near the synaptic lamellae; during the dark phase, the dense-cored vesicles decreased in number, while large clear vesicles and profiles of smooth endoplasmic reticulum increased in frequency. The most marked change in photoreceptor synaptic terminal morphology occurred after 10 hr of darkness, at 0730 hr. At this time, photoreceptor synaptic terminal cross-sectional area was found to increase dramatically. Morphometric analysis showed that the number of synaptic vesicles in these terminals remained constant throughout the day, as did the perimeter of photoreceptor terminal profiles. The observed increase in area of synaptic terminals at 0730 hr was found to be due to a decrease in the folding of the terminal plasma membrane. Qualitative observations showed endocytosis to be occurring at a rapid rate at this time as well; and since the number of synaptic vesicles and terminal perimeter did not change, exocytosis of synaptic vesicles was assumed to be occurring at an equally rapid rate. These findings support an extension to the hypothesis of Monaghan and Osborne (1975), suggesting that photoreceptor synaptic vesicles become "supercharged" with transmitter substance in the light.     

The regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm

Summary Electron microscopic observations have been made on the regeneration of neuromuscular junctions during spontaneous re-innervation of the rat diaphragm, following unilateral transsection of the phrenic nerve. 3 and 4 weeks after denervation motor end plates displayed the pattern of almost complete degeneration, i.e. persisting subneural foldings, deprived of neural contact and covered with collagen fibrils and fibrocytes. From observations at 5, 10 and 24 weeks after denervation the following sequence of events could be established: a few small axon terminals, accompanied by Schwann cells, became apposed to subneural folds, while most foldings were covered initially by Schwann cells or still by collagen fibrils. Gradually an increasing number of subneural folds came into contact with axon terminals. At 24 weeks all junctions displayed the pattern of a mature motor end plate. In the majority of regenerating neuromuscular junctions single dense-cored vesicles of approximately 900–1200 Å were present in axon terminals.It is concluded that under the present conditions restoration of neuromuscular transmission is accomplished by a re-innervation of the preserved subneural apparatuses of former junctions by regenerating axons. The significance of the occurrence of dense-cored vesicles in regenerating motor end plates is discussed.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.