Márta Wilhelm

Amacrine cells of the anuran retina: Morphology, chemical neuroanatomy, and physiology

Amacrine cells are third‐order retinal interneurons, projecting their processes into the inner plexiform layer. Historically, they were not considered as neurons first. By the middle of the 20th century, their neuronal nature was confirmed, and their enermous diversity established. Amacrine cells have been most succesfully subdivided into morphological categories based on two parameters: diameter of the dendritic field and ramification pattern in the inner plexiform layer. Works combining anatomy, physiology, and neurochemistry are scarce and in the case of the anuran retina, the situation is even worse. Correlation between morphology, neurochemistry, and physiology is little studied. Here we try to build up a database and pinpoint some of the missing data. Obtaining those could help to better understand retinal function. Sporadic attempts did not make it possible to develop a comprehensive catalog of morphologically distinct amacrine cell types in the anuran retina. The number of morphologically identified amacrine cells currently stands at 16. The list of neurochemically identified distinct cell types can be given as follows: five types GABA‐contaning cell types with secondary markers and at least one without; two glycinergic cell types and one interplexiform cell where glycine colocalizes with somatostatin; one dopaminergic amacrine cell and also a variant of this with interplexiform morphology; two types of serotoninergic cells; three NADPHdiaphorase‐positive cells, one substance P‐positive cell type without identified second marker; one CCK‐positive cell type without identified second marker and the calbindin positive cells (at least one but potentially more types). This adds up to 19 cell types, out of which two are interplexiform in character. This is more than that could be identified by purely morphological means. Out of Cajals original 13 amacrine cell types described in the frog retina, 5 parallel unequivocally with neurons defined by neurochemistry. Three others have one close match each, but their exact identity is uncertain. The remaining amacrine cells have more than one potential matches. At the same time, on one hand the amacrine cell named two‐layered by Cajal so far has no match among the neurochemically identified amacrine cells. On the other hand, the interplexiform subtype of the dopaminergic cell, the somatostatin‐containing glycinergic interplexiform cell, the starburst cell, and the bistratified neuropeptide Y‐immunoreactive cell have no match among Cajals cells. All in all, the number of known amacrine and interplexiform cells now stands at at least 21 in the anuran retina. Physiological characterization of amacrine cells shows that their general features seem to be rather similar to those described in tiger salamander retina. In Xenopus retina, morphologically and physiologically identified amacrine cells responded to light stimulation most frequently with ON‐OFF characteristics. Immunhistochemical identification of the recorded and dye injected cells showed that amacrine cells of the “same physiological type” might have different morphology. In other words, amacrine cells with different morphology can respond similarly to illumination. Even so, small differences between almost identical responses may reflect that the cell they stem from indeed belongs to different cell types. Microsc. Res. Tech. 50:373–383, 2000.

Hippocampal “cholinergic interneurons” visualized with the choline acetyltransferase promoter: anatomical distribution, intrinsic membrane properties, neurochemical characteristics, and capacity for cholinergic modulation

Release of acetylcholine (ACh) in the hippocampus (HC) occurs during exploration, arousal, and learning. Although the medial septum-diagonal band of Broca (MS-DBB) is the major extrinsic source of cholinergic input to the HC, cholinergic neurons intrinsic to the HC also exist but remain poorly understood. Here, ChAT-tauGFP and ChAT-CRE/Rosa26YFP (ChAT-Rosa) mice were examined in HC. The HC of ChAT-tauGFP mice was densely innervated with GFP-positive axons, often accompanied by large GFP-positive structures, some of which were Neurotrace/DAPI-negative and likely represent large axon terminals. In the HC of ChAT-Rosa mice, ChAT-YFP cells were Neurotrace-positive and more abundant in CA3 and dentate gyrus than CA1 with partial overlap with calretinin/VIP. Moreover, an anti-ChAT antibody consistently showed ChAT immunoreactivity in ChAT-YFP cells from MS-DBB but rarely from HC. Furthermore, ChAT-YFP cells from CA1 stratum radiatum/stratum lacunosum moleculare (SR/SLM) exhibited a stuttering firing phenotype but a delayed firing phenotype in stratum pyramidale (SP) of CA3. Input resistance and capacitance were also different between CA1 SR/LM and CA3 SP ChAT-YFP cells. Bath application of ACh increased firing frequency in all ChAT-YFP cells; however, cholinergic modulation was larger in CA1 SR/SLM than CA3 SP ChAT-YFP cells. Finally, CA3 SP ChAT-YFP cells exhibited a wider AP half-width and weaker cholinergic modulation than YFP-negative CA3 pyramidal cells. Consistent with CRE expression in a subpopulation of principal cells, optogenetic stimulation evoked glutamatergic postsynaptic currents in CA1 SR/SLM interneurons. In conclusion, the presence of fluorescently labeled hippocampal cells common to both ChAT-tauGFP and ChAT-Rosa mice are in good agreement with previous reports on the existence of cholinergic interneurons, but both transgenic mouse lines exhibited unexpected anatomical features that departed considerably from earlier observations.

Functional anatomy of the photoreceptor and second-order cell mosaics in the retina of Xenopus laevis.

Abstract Mosaics of photoreceptors, and horizontal and bipolar cells of the Xenopus laevis retina were studied in whole-mount preparations applying lectin-cytochemical, immunocytochemical and intracellular labeling techniques. The combined density of all photoreceptor types was about 13700/mm2, of which rods represented 53%. Of the cones, the large long-wavelength-sensitive (86% of all cones) and the miniature ultraviolet-wavelength-sensitive (4%) ones could be labeled with peanut agglutinin, whereas the large short-wavelength-sensitive (10%) cones remained unlabeled. There were no significant regional differences in photoreceptor distribution. Bipolar cells were selectively labeled with antibodies against calretinin. Their density was between 4000 and 6000 cells/cm2, with slightly elevated numbers in the superior nasal quadrant. Two types of horizontal cell were injected intracellularly. The luminosity-type cells were more frequent (∼1000 cells/mm2) than the chromaticity cells (∼450 cells/mm2). The dendritic field size of the latter cell type was threefold bigger than that of the luminosity cells. The coverage factors were estimated to be 3.3 for the luminosity cells and 5.2 for the chromaticity cells. The luminosity cells contacted all photoreceptor types, whereas chromatic horizontal cells received their inputs from the short-wavelength-sensitive cones and from some, but not all, rods. Luminosity cells encounter about 50–60 potential synaptic partners within their dendritic fields, whereas chromatic horizontal cells only about 20. Chromatic horizontal cells form multiple synaptic contacts with the short-wavelength-sensitive cones. The results indicate that the overall photoreceptor to bipolar and bipolar to ganglion cell convergence in Xenopus retina is similar to that in the central retinal specialized regions of mammals, predicting comparable spatial resolutions.

Neurochemical characterization of nervous elements innervating the body wall of earthworms ( Lumbricus , Eisenia ): immunohistochemical and pharmacological studies

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological–physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus submuscularis is composed of nerve fibres only, whereas the plexus subepithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT3 and GABAB receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.

Comparison of blood and saliva lactate level after maximum intensity exercise

Several studies have described high correlation of salivary and blood lactate level during exercise. Measuring the effectiveness and intensity of training, lactate concentration in blood, and lately in saliva are used.The aim of our study was to evaluate the correlation between the concentration and timing of salivary and blood lactate level in endurance athletes and non-athletes after a maximal treadmill test, and to identify physiological and biochemical factors affecting these lactate levels.Sixteen volunteers (8 athletes and 8 non-athletes) performed maximal intensity (Astrand) treadmill test. Anthropometric characteristics, body composition and physiological parameters (heart rate, RR-variability) were measured in both studied groups. Blood and whole saliva samples were collected before and 1, 4, 8, 12, 15, 20 min after the exercise test. Lactate level changes were monitored in the two groups and two lactate peaks were registered at different timeperiods in athletes. We found significant correlation between several measured parameters (salivary lactate - total body water, salivary lactate - RR-variability, maximal salivary lactate - maximal heart rate during exercise, salivary- and blood lactate -1 min after exercise test). Stronger correlation was noted between salivary lactate and blood lactate in athletes, than in controls.

Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern.

Abstract Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm2 and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis.

Embryogenesis of the serotonergic system in the earthworm Eisenia fetida (Annelida, Oligochaeta): Immunohistochemical and biochemical studies

Organization of the serotonergic system and changes of the serotonin (5‐HT) content were studied during the embryogenesis of the earthworm Eisenia fetida, using immunocytochemistry and HPLC. A gradual emergence of 5‐HT immunoreactive (IR) cells and their axon projections in the several ganglia of the central (CNS) and peripheral nervous system are described in the context of a staged time‐scale of development. The first 5‐HT‐IR neurons appear in the subesophageal ganglion at an early embryonic stage (E2), followed by neurons in some rostrally located ventral ganglia. In the cerebral ganglion, 5‐HT‐IR cells can be detected only from stage E5. The number of labeled cells in each ganglion of the embryo increases until hatching, when it is still considerably lower than that observed in adults. This shows that the development of the 5‐HTergic system is far from complete by the end of embryogenesis. Organization of 5‐HT‐IR innervation of the body wall starts by stages E3 to E4. In the stomatogastric nervous system the first 5‐HT‐IR fibers can be detected by stage E5. By stage E9 5‐HT immunopositive neurons can be observed in both the stomatogastric ganglia and the enteric plexus. Both 5‐HT levels and the numbers of the labeled cells show a significant increase before hatching, which indicate a functional maturation of the 5‐HTergic system. Based on the early appearance of 5‐HT, we suppose that it may play a regulatory role in both the gangliogenesis and the maturation of peripheral functions necessary during postembryonic life. J. Comp. Neurol. 497:451–467, 2006.

Neuro-immune interactions in the dove brain.

Mast cells (MC) are of hematopoetic origin. Connective tissue type MCs are able to function in IgE dependent and independent fashion, change their phenotype according to the tissue environment. They are able to enter the brain under normal physiological conditions, and move into this compact tissue made of neurons. In doves MCs are found only in the medial habenula (MH) and their number is changing according to the amount of sex steroids in the body. MCs are able to synthesize and store a great variety of biologically active compounds, like transmitters, neuromodulators and hormones. They are able to secrete GnRH. With the aid of electron microscopy we were able to describe MC-neuron interactions between GnRH-positive MCs and neurons. Piecemeal degranulation (secretory vesicles budding off swollen and active granules) seems to be a very efficient type of communication between MCs and surrounding neurons. Different types of granular and vesicular transports are seen between GnRH-immunoreactive MCs and neurons in the MH of doves. Sometimes whole granules are visible in the neuronal cytoplasm, in other cases exocytotic vesicles empty materials of MC origin. Thus MCs might modulate neuronal functions. Double staining experiments with IP3-receptor (IP3R), Ryanodine-receptor (RyR) and serotonin antibodies showed active MC population in the habenula. Light IP3R-labeling was present in 64-97% of the cells, few granules were labeled in 7-10% of MCs, while strong immunoreactivity was visible in 1-2% of TB stained cells. No immunoreactivity was visible in 28-73% of MCs. According to cell counts, light RyR-positivity appeared in 27-52%, few granules were immunoreactive in 4-19%, while strong immunopositivity was found only in one animal. In this case 22% of MCs were strongly RyR-positive. No staining was registered in 44-73% of MCs. Double staining with 5HT and these receptor markers proved that indeed only a part of MCs is actively secreting. Resting cells with only 5HT-immunopositivity are often visible. The activational state of MCs is changing at higher estrogen/testosterone level, thus with the secretion of neuromodulators they might alter sexual and parental behavior of the animals.

Structure and function of photoreceptor and second-order cell mosaics in the retina of Xenopus

The structure, physiology, synaptology, and neurochemistry of photoreceptors and second-order (horizontal and bipolar) cells of Xenopus laevis retina is reviewed. Rods represent 53% of the photoreceptors; the majority (97%) are green light-sensitive. Cones belong to large long-wavelength-sensitive (86%), large short-wavelength-sensitive (10%), and miniature ultraviolet wavelength-sensitive (4%) groups. Photoreceptors release glutamate tonically in darkness, hyperpolarize upon light stimulation and their transmitter release decreases. Photoreceptors form ribbon synapses with second-order cells where postsynaptic elements are organized into triads. Their overall adaptational status is regulated by ambient light conditions and set by the extracellular dopamine concentration. The activity of photoreceptors is under circadian control and is independent of the central body clock. Bipolar cell density is about 6000 cells/mm2 They receive mixed inputs from rods and cones. Some bipolar cell types violate the rule of ON-OFF segregation, giving off terminal branches in both sublayers of the inner plexiform layer. The majority of them contain glutamate, a small fraction is GABA-positive and accumulates serotonin. Luminosity-type horizontal cells are more frequent (approximately 1,000 cells/mm2) than chromaticity cells (approximately 450 cells/mm2). The dendritic field size of the latter type was threefold bigger than that of the former. Luminosity cells contact all photoreceptor types, whereas chromatic cells receive their inputs from the short-wavelength-sensitive cones and rods. Luminosity cells are involved in generating depolarizing responses in chromatic horizontal cells by red light stimulation which form multiple synapses with blue-light-sensitive cones. Calculations indicate that convergence ratios in Xenopus are similar to those in central retinal regions of mammals, predicting comparable spatial resolution.

Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.