Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats.

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS+) and evaluate the synaptic relationship of NOS+ striatal neurons with those containing parvalbumin (PV+). In both the single-label and double-label studies, NOS+ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS+ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS+ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV+ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV+ dendrites, and on NOS+ soma and dendrites. In contrast, NOS+ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV+ or NOS+ neurons. These findings suggest that NOS+ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV+ or NOS+ striatal interneurons. NOS+ neurons do, however, receive synaptic input from PV+ neurons.

Light and electron microscopic immunohistochemical study of dopaminergic terminals in the striatal portion of the pigeon basal ganglia using antisera against tyrosine hydroxylase and dopamine.

A dopaminergic projection from the midbrain to the striatal portion of the basal ganglia is present in reptiles, birds, and mammals. Although the ultrastructure of these fibers and terminals within the striatum has been studied extensively in mammals, little information is available on the ultrastructure of this projection in nonmammals. In the present study, we used immunohistochemical labeling with antibodies against tyrosine hydroxylase (TH) or dopamine (DA) to study the dopaminergic input to the striatal portion of the basal ganglia in pigeons (i.e., lobus parolfactorius and paleostriatum augmentatum). At the light microscopic level, the anti-TH and anti-DA revealed a similar abundance and distribution of numerous labeled fine fibers and varicosities within the striatum. In contrast, the use of an antidopamine beta-hydroxylase antiserum (which labels only adrenergic and noradrenergic terminals) labeled very few striatal fibers, which were restricted to visceral striatum. These results demonstrate that anti-TH mainly labels dopaminergic terminals in the striatum. At the electron microscopic level, the anti-TH and anti-DA antisera labeled numerous axon terminals within the striatum (15-20% of all striatal terminals). These terminals tended to be small (with an average length of 0.6 microns) and flattened, and their vesicles tended to be small (35-60 nm in diameter) and pleomorphic. About 50% of the terminals were observed to make synaptic contacts in the planes of section examined, and nearly all of these synaptic contacts were symmetric. Both TH+ and DA+ terminals typically contacted dendritic shafts or the necks of dendritic spines, but a few contacted perikarya. No clear differences were observed between TH+ and DA+ terminals within medial striatum (whose neurons project to the nigra in birds) or between TH+ and DA+ terminals within lateral striatum (whose neurons project to the pallidum in birds). In addition, no differences were observed between medial and lateral striata in either TH+ or DA+ terminals. Thus, there is no evident difference in pigeons between striatonigral and striatopallidal neurons in their dopaminergic innervation. Our results also indicate that the abundance, ultrastructural characteristics, and postsynaptic targets of the midbrain dopaminergic input to the pigeon striatum are highly similar to those in mammals. This anatomical similarity is consistent with the pharmacologically demonstrable similarity in the role of the dopaminergic input to the striatum in birds and mammals.

[Animal experiment study of titanium with surface coatings of (Ti,Nb)ON and (Ti,Zr)O]. / Tierexperimentelle Prüfung von Titan mit Oberflächen-beschichtungen aus (Ti,Nb)ON und (Ti,Zr)O.

Titanium is considered to be biocompatible as long as the passive layer of TiO2 which is formed within the body, is not destroyed mechanically by the shearing forces acting on implants during function. Mechanically stable hard coatings on the basis of the so-called refractory metals render titanium wear-and-tear-resistant, with the added advantage for its biocompatibility of keeping its the physical and electrochemical properties constant, even in the event of relative movement against hard or soft tissue. Biological testing of coated and uncoated titanium in experimental animals shows that the deposition of new bone on (Ti,Zr)O or (Ti,Nb)ON surfaces takes place in the same way as on the surface of titanium.

An ultrastructural double-label immunohistochemical study of the enkephalinergic input to dopaminergic neurons of the substantia nigra in pigeons.

Electron microscopic immunohistochemical double-label studies were carried out in pigeons to characterize the ultrastructural organization and postsynaptic targets of enkephalinergic (ENK+) striatonigral projection. ENK+ terminals in the substantia nigra were labeled with antileucine-enkephalin antiserum by using peroxidase-antiperoxidase methods, and dopaminergic neurons were labeled with anti-tyrosine hydroxylase antiserum by using silver-intensified immunogold methods. ENK+ terminals on dopaminergic neurons were equal in abundance to ENK+ terminals on nondopaminergic neurons, although the former were typically somewhat smaller than the latter (mean size: 0.50 vs. 0.75 micron, respectively). ENK+ terminals were evenly distributed on the cell bodies and dendrites of dopaminergic neurons, and they were evenly distributed on dendrites but rare on perikarya of nondopaminergic neurons. Transection of the basal telencephalic output revealed that 75% of the nigral ENK+ terminals were of basal telencephalic origin. These telencephalic ENK+ terminals included over 80% of those smaller than 0.80 micron on dopaminergic neurons and smaller than 1.0 micron on nondopaminergic neurons, and none greater than this in size. Both telencephalic and the nontelencephalic ENK+ nigral terminals made predominantly symmetric synapses on nigral neurons. Although the basal telencephalic ENK+ terminals uniformly targeted dendrites and perikarya, nontelencephalic ENK+ terminals seemed to avoid perikarya. The results indicate that ENK+ striatonigral neurons in birds may directly influence both dopaminergic and nondopaminergic neurons of the substantia nigra. Based on similar data for substance P-containing striatonigral terminals, the roles of enkephalin and substance P in influencing nigral dopaminergic neurons may differ slightly, as they appear to target preferentially different portions of dopaminergic neurons. The overall results in pigeons are similar to those for ENK+ terminals in the ventral tegmental area in rats, suggesting that the synaptic organization of the ENK+ input to the tegmental dopaminergic cell fields is similar in mammals and birds.

Thalamostriatal projection neurons in birds utilize LANT6 and neurotensin: a light and electron microscopic double-labeling study.

Based on its location, connectivity and neurotransmitter content, the dorsal thalamic zone in birds appears to be homologous to the intralaminar, midline, and mediodorsal nuclear complex in the thalamus of mammals. We investigated the neuroactive substances used by thalamostriatal projection neurons of the dorsal thalamic zone in the pigeon. Single-labeling experiments showed that many neurons in the dorsal thalamic zone are immunoreactive for neurotensin and the neurotensin-related hexapeptide, (Lys8,Asn9)NT(8-13) (LANT6). Double-labeling experiments, using the retrograde fluorescent tracer, FluoroGold, combined with fluorescence immunocytochemistry for either LANT6 or neurotensin, showed that neurotensin- and LANT6-containing neurons in the dorsal thalamic zone project to the striatum of the basal ganglia. Immunofluorescence double-labeling experiments showed that neurotensin and LANT6 are often (possibly always) co-expressed in neurons in the dorsal thalamic zone. Electron microscopic immunohistochemical double-labeling showed that LANT6 terminals in the striatum make asymmetric contacts with heads of spines labeled for substance P and heads of spines not labeled for substance P, suggesting that these terminals synapse with both substance P-containing and non-substance P-containing medium spiny striatal projection neurons. These findings indicate that LANT6 and neurotensin may be utilized as neurotransmitters in thalamostriatal projections in birds and raise the possibility that this may also be the case in other amniotes.

Dopaminergic terminals form synaptic contacts with enkephalinergic striatal neurons in pigeons: an electron microscopic study.

Medium spiny projection neurons of the striatum consist of two major neuropeptide-specific types, one type containing substance P and another type containing enkephalin. Both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites. However, whether each of these types receives direct dopaminergic input onto distal dendritic shafts and onto dendritic spines has not been explored in depth. In the present study, we used electron microscopic immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto enkephalin-positive (ENK+) striatal neurons in pigeons, in whom ENK+ striatal perikarya, dendritic shafts and spines can be readily labeled. Antibodies against tyrosine hydroxylase were used to label dopaminergic terminals using a silver-intensified immunogold method. ENK+ neurons were labeled using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts, and dendritic spine necks of ENK+ striatal neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto ENK+ striatal neurons in a manner similar to that described previously by us for substance P-positive striatal medium spiny neurons.

A pre-embedding triple-label electron microscopic immunohistochemical method as applied to the study of multiple inputs to defined tegmental neurons.

For many neural regions it is of interest to know the identity of the target structures of two different types of inputs to that neural region. Such studies require use of a triple-label immunohistochemical method to differentially label the class of target structure and the two types of input so that they can be visualized at the electron microscopic (EM) level. We describe here a procedure for combining three different markers (diaminobenzidine, benzidine dihydrochloride, and silver-intensified immunogold) for triple-label EM immunohistochemical pre-embedding labeling. All three markers are distinct at the LM and EM levels. An example of this approach as applied to studying striatal input to the ventral tegmental area is presented and the advantages of this approach are discussed.

[Model for immunologic testing of biomaterials]. / Modell zur immunologischen Prüfung von Biomaterialien.

Corrosion products and electric fields are capable of changing proteins to antigens, thus permitting the immunological system to identify the biomaterial as foreign. The reaction between corrosion products and a macro-molecule also leads to an antigen (carrier antigen), such as conformational changes of a macro-molecule, e.g. a protein, caused by the electric field at the implant surface (modified macro-molecule antigen). While the sensitivity to corrosion and the effectiveness of galvanic elements is measurable by electrochemical methods, suitable methods of determining the field strength in the vicinity of biomaterial surfaces are still unavailable. The influence of the double layer of uncoated and coated titanium surfaces on the conformation of proteins and their conversion to antigens are investigated with polyclonal antibodies capable of identifying the unchanged protein despite adsorption to the surface. 14C-marked Bovine Serum Albumin serves as a model protein. Determination of the total number of protein molecules adsorbed is effected via the detection of the emitted electrons. The quotient of the concentration of natural proteins to the concentration of adsorbed molecules gives the biocompatibility index, which is independent of the surface area, and gives an indication of the expected biocompatibility of the material. The results of the biological tests of titanium and two coating materials on titanium were confirmed in an animal experiment. It is possible that in the future immunological tests may replace experiments in animals.

Ultrastructural double-labeling demonstrates synaptic contacts between dopaminergic terminals and substance P-containing striatal neurons in pigeons.

Immunohistochemical studies in rats have demonstrated dopaminergic input onto medium spiny neurons of the striatum. Medium spiny neurons, however, are known to consist of two major neuropeptide-specific types, those containing substance P (SP) and those containing enkephalin. Although both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites, the extent to which both types also receive direct dopaminergic input onto distal dendritic shafts or onto dendritic spines is uncertain. In the present study, we used EM immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto SP+ striatal neurons. We examined the striatum of pigeons, in whom SP+ striatal neurons, including their dendritic shafts and spines, can be readily labeled. Antibodies against tyrosine hydroxylase (TH) were used to identify dopaminergic terminals, which were labeled using silver-intensified immunogold. The SP+ neurons were labeled immunohistochemically using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts and dendritic spines of SP+ neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto SP+ striatal neurons in a manner similar to that described for dopaminergic input onto striatal medium spiny neurons in general.

Ultrastructural single- and double-label immunohistochemical studies of substance P-containing terminals and dopaminergic neurons in the substantia nigra in pigeons.

The vast majority of striatonigral projection neurons in pigeons contain substance P (SP), and the vast majority of SP-containing fibers terminating in the substantia nigra arise from neurons in the striatum. To help clarify the role of striatonigral projection neurons, we conducted electron microscopic single- and double-label immunohistochemical studies of SP+ terminals and/or dopaminergic neurons (labeled with either anti-dopamine, DA, or anti-tyrosine hydroxylase, TH) in pigeons to determine: (1) the synaptic organization of SP+ terminals, (2) the synaptic organization of TH+ perikarya and/or dendrites, and (3) the synaptic relationship between SP+ terminals and TH+ neurons in the substantia nigra. Tissue single-labeled for SP revealed numerous SP+ terminals contacting thin unlabeled dendrites in the substantia nigra, but few SP+ terminals were observed contacting perikarya or large-diameter dendrites. SP+ terminals contained round, densely packed, clear vesicles, and often contained one or more dense-core vesicles. Synaptic junctions between SP+ terminals and their targets were more often symmetric (86%) than asymmetric. In tissue single-labeled for DA, we observed few terminals contacting DA+ perikarya, whereas terminals contacting DA+ dendrites were more abundant. Terminals contacting DA+ structures comprised at least four different morphologically distinct types based on the morphology of the clear synaptic vesicles and the type of synaptic junction. One type of terminal contained round clear vesicles and made symmetric synapses, and thus resembled the predominant type of SP+ terminal. The second type contained round clear vesicles and made asymmetric synapses, the third type contained medium-size pleomorphic clear vesicles and made symmetric synapses, and the fourth type contained small pleomorphic clear vesicles and made symmetric synapses. The presence of contacts between SP+ terminals and dopaminergic dendrites in the substantia nigra was directly demonstrated in tissue double-labeled for SP (by the peroxidase-antiperoxidase procedure, or PAP, with diaminobenzidine) and TH (by either the silver-intensified immunogold procedure or the PAP procedure with benzidine dihydrochloride). SP+ terminals commonly contacted thin TH+ dendrites in the substantia nigra, but few SP+ terminals contacted large-diameter TH+ dendrites or perikarya. Synapses between SP+ terminals and TH+ neurons were always symmetric. TH+ dendrites also were contacted by terminals not labeled for SP, which were more abundant than were SP+ terminals. Non-TH+ neurons were also contacted by both SP+ terminals and non-SP+ terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

[Inhibition of caries by Na-saccharin]. / Zur kariostatischen Wirkung von Na-Saccharin.

The inhibition of dental caries by Na-saccharin was studied in an optimized and standardized experiment with cara rats. The animals were given a cariogenic diet ad libitum as well as under controlled frequency feeding conditions. The addition of 0.5% Na-saccharin to the cariogenic diet produced no cariostatic effects. Ancilliary microbiological in vitro and in vivo experiments showed no appreciable bacteriostatic effects on the cariogenic bacteria flora.

[Microbiological in vitro and animal experiments assessing the cariogenic potential of various snacks]. / Zur Einschätzung des kariogenen Potentials verschiedener Zwischenmahlzeiten.

Microbiological in vitro and animal experiments were performed to determine the cariogenic potential of different snacks. The determination of the oral salivary sugar clearance time by the aid of a cariogenic S. mutans strain as standard for the cariogenic potential resulted in a relatively high clearance time when a praline was consumed, according to a high cariogenic potential. When consuming a "safe for teeth"-product no easily fermenting substances in the saliva can be shown, suggesting a lacking cariogenic potential. The cariogenic potential of banana, milk-chocolate, Milchschnitte, Müsli-Frucht-Schnitten (cereals and fruits) is between that of the praline and the "safe for teeth"-product. The animal experiments showed in increasing strength of fissure caries after programmed feeding of the following snacks: Basic-diet----milk-chocolate----positive control-diet/Milchschnitte Müsli-Frucht-Schnitten----banana. The highest caries values occurred after having fed banana, the lowest after having fed milk-chocolate. Milchschnitte and Müsli-Frucht-Schnitten showed similar caries values which were between the values after feeding of milk-chocolate and banana.

[Sweetening agent, Palatinit under specific consideration as to microbiological and caries-prophylactic aspects]. / Der Saccharoseautauschstoff Palatinit unter besonderer Berücksichtigung mikrobiologischer und kariesprophylaktischer Aspekte.

Several cariogenic bacterial species, a mixed streptococci flora composed of nine species, as well as the plaque- and saliva-mixed flora from human beings were tested for acid production from the sugar substitute palatinit, which was compared to sorbitol, mannitol, xylitol and the low molecular carbohydrates sucrose, glucose and fructose. Complementary experiments with gnotobiotic and conventionally fed rats were performed in order to test the cariogenic properties of palatinit and palatinit-chocolate in comparison to xylitol, sorbitol and sucrose and the corresponding chocolate substances. Palatinit and palatinit-chocolate show highly significant lower cariogenic properties than sucrose and sucrose-chocolate. On the basis of these microbiological and animal experiments, the use of palatinit as sugar substitute for caries prophylaxis can be recommended.