Biomedical subjects

J R Wolff

Publications and source records attributed to J R Wolff.

At least 37 records · Page 2Linked to original sources

PubMed1996

Cholinoceptive neurons without acetylcholinesterase activity and enzyme-positive neurons without cholinergic synaptic innervation are present in the main olfactory bulb of adult rat.

Light and electron microscopic histochemistry revealed acetylcholinesterase-positive and acetylcholinesterase-negative neurons in the main olfactory bulb of adult rat. Their distribution patterns on various neuron types have been analysed in detail. (1) No acetylcholinesterase staining could be demonstrated in the granule cells which receive a large number of the cholinergic synapses. (2) In contrast, enzyme activity was present in the soma and dendrites in most of the non-cholinergic and non-cholinoceptive relay cells (mitral cells and tufted cells) and in a subset of short-axon interneurons, where cholinergic synapses could not be detected. (3) Within the neuropil of glomeruli, two compartments were present, one of which was free of acetylcholinesterase-positive structures, while many enzyme-positive neuronal elements were seen in the other. (4) Characteristically, cholinergic and non-cholinergic neuronal structures showed triadic arrangements. (5) The axonal release of acetylcholinesterase from cholinergic axons is probable. It is suggested that, in the olfactory bulb, acetylcholinesterase is release by cholinergic afferent axons, and it is the cholinergic synapses that determine which postsynaptic neurons are cholinoceptive rather than the intraneuronal presence of acetylcholinesterase. In the main olfactory bulb, the acetylcholinesterase present in the relay cells therefore appears to have functions other than the hydrolysis of acetylcholine.

Acetylcholinesterase↗

PubMed1996

Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

Animals↗

PubMed1996

Prenatal exposure to sodium bromide affects the postnatal growth and brain development.

Previous experiments suggest that bromide ions interfere with trophic interactions of neurons in intact adult rats and may modify reactive neuroplasticity during postnatal development. Here we report on bromide effects on normal development. Rat embryos were exposed to sodium bromide (NaBr) by providing an aqueous solution of 250 mg/% NaBr in the drinking water, ad libitum, to their dams. Controls received either tap water or saline (250 mg%NaCl) to drink. Application to dams was restricted to the 5th to 15th days of gestation. Measurements of bromide concentrations in samples of blood and brain homogenates revealed, however, that bromide transfer to embryos was not restricted to the treatment period. Because of delayed excretion dams provided the offspring with bromide via placenta and milk up to 10 days after birth, although at decreasing concentrations. Significant delays in postnatal development were observed in all bromide-treated animals. Permanent deficits were recorded for body weight, brain weight and the protein content of brain tissue. Additionally, there were some changes in brain structure, e.g. the laminar structure of the neocortex was modified. In contrast to developmental deficits, the size of olfactory glomeruli was consistently larger in bromide-treated rats during postnatal and glomeruli attained a mean diameter that was 30 percent larger than in controls at 3 months of age. Results suggest that pre- and perinatal exposure of rats to moderate concentrations of NaBr may interfere with postnatal development including that of brain. Since after complete excretion of bromide developmental deficits persist and show periods of partial compensation and decompensation, induction of these bromide effects is probably indirect. The exact mechanism of bromide action on developmental processes remains to be elucidated.

Age Factors↗

PubMed1995

Expression of S100 protein in the vestibular nuclei during compensation of unilateral labyrinthectomy symptoms.

In adult guinea pigs, unilateral labyrinthine lesions were inflicted by chloroform injections into the middle ear. Immunoreactivity for S100 protein (S100) in the vestibular nuclei was studied during compensation of lesion-induced postural asymmetry symptoms, i.e., nystagmus, asymmetrical head position. 1 h after unilateral labyrinthectomy, increased levels of astroglial S100 immunoreactivity were found in the superior vestibular nucleus and in the medial/lateral vestibular nucleus border region on the side contralateral to the deafferentation. Bilaterally, the astrocytic S100 immunoreaction increased in the lateral vestibular nuclei around Deiters neurons. Maximal expression of S100 was noted 3 h after the lesion. Subsequently, it diminished. Our data reveal that transsynaptically altered neuronal activity induces an astrocytic reaction which provides increased levels of S100 to the local neuropil. Calcium and zinc binding S100 proteins may play a functional role for the neuroplasticity during vestibular compensation.

Animals↗

PubMed1995

Identification of the Ulex europaeus agglutinin-I-binding protein as a unique glycoform of the neural cell adhesion molecule in the olfactory sensory axons of adults rats.

Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.

Agglutinins↗

PubMed1995

Essential functions of synapsins I and II in synaptic vesicle regulation.

Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.

Animals↗

PubMed1995

S100 immunoreactivity in a subpopulation of oligodendrocytes and Ranvier's nodes of adult rat brain.

The Ca(2+)- and Zn(2+)-binding S-100 proteins (S100) are predominantly localized in astrocytes of adult mammalian brain. In addition, light and electron microscopic immunocytochemistry revealed S100 in a small subpopulation of oligodendrocytes. By nuclear morphology and abundance of rough ER and Golgi fields, these cells resembled actively myelinating oligodendrocytes. S100 immunoreactivity was also found in paranodal loops and outer mesaxons of isolated of myelin sheaths. Data suggests that oligodendroglial content of S100 relates to cell turnover and/or myelin repair in the adult rat brain, and that S100 is present during myelin compaction.

Animals↗

PubMed1995

Structural dynamics of synapses and synaptic components.

Learning and memory formation are apparently based on cascades of molecular and cellular processes with increasing time constants (ms to days and weeks), but even the most long-lasting effects are transient. Memory traces may permanently modify the behavior (activity patterns, gene expression) of neurons and neuronal networks. Therefore the question is raised whether our current view on the stability of synapses under normal conditions is tenable. Evidence is reviewed suggesting that as direct or indirect effects of modifications in bioelectrical activity and chemical trophicity, synapses may be remodeled and removed within days and weeks, and possibly within hours. Accordingly, species-specific connectivity patterns are not restricted to the standard architecture of the CNS, but (morpho-)genetics allow for a considerable number of alternative wiring patterns, which appear under unusual conditions during ontogenesis and in adulthood. Our present knowledge suggests that, rather than the formation of synapses, they are a selective process. Until now there is no direct method of measuring either synaptic reorganization or the average life span of synapses. Specific cases, however, allow to estimate synapse turnover during ontogenesis, at its lowest possible level. Such data suggest that each synapse is on average remodeled or replaced several to many times during normal developmental, e.g. in the cerebral cortex of Marmoset monkeys at the very least 5 to 10 times (corresponding to 250 million synapses eliminated per hour in area 17!). It is discussed how the consequences of synapse turnover could be utilized by learning processes. Conclusions are followed by an outlook.

Animals↗

PubMed1995

Modifications of S100-protein immunoreactivity in rat brain induced by tissue preparation.

Immunocytochemistry using antibodies against various molecular forms of the Ca++ and Zn(++)-binding S100 proteins predominantly labelled astrocytes. However, especially in the neocortex the staining pattern is variable. Methods of tissue preparation have been evaluated with the aim to preserve as much S100 immunoreactivity as possible. Optimal results were obtained after perfusion fixation with 4-5% aldehydes, 0.1 M sodium cacodylate, 0.1% CaCl2, pH 7.3. In such preparations, astrocytes were completely labelled including their lamellar compartments in large parts of the central nervous system. Ca(++)-withdrawal had adverse affects on S100 immunoreactivity. Cryostat sections treated with EDTA-containing solutions before fixation showed that Ca(++)-free S100 can apparently not be fixed to the tissue. Perfusion fixatives containing EDTA resulted in inhomogeneous loss of S100 staining, indicating a differential susceptibility of astrocytic subpopulations. A different type of reduction in S100 immunoreactivity occurred around large neocortical blood vessels. Perivascular defects in immunostaining occasionally appeared even after optimal fixation, but could be regularly provoked by mildly acidic fixation (pH 6.6) or prolonged barbiturate anaesthesia. These defects might be based on S100 release into the cerebrospinal fluid. Presumably under none of the conditions studied can the immunoreactivity of all S100-forms and -fractions be completely preserved in the tissue. However, recommendations are presented for optimizing tissue preparation, to the extent that premortal modifications affecting the stainability of astrocytes may be detected by S100 immunohistochemistry in fixed brain tissue.

Anesthesia↗

PubMed1995

Chloride is preferentially accumulated in a subpopulation of dendrites and periglomerular cells of the main olfactory bulb in adult rats.

GABA is predominantly an inhibitory transmitter. Mediated by GABAA receptors, GABA opens chloride channels, induces a passive flux of chloride ions, which is usually directed from extracellular to intracellular space, and hyperpolarizes postsynaptic neurons. Recent electrophysiological data suggested that GABA may also depolarize neurons and exert excitatory actions. However, it remained unclear whether excitatory GABA effects are based on reversed transmembrane chloride gradient due to modifications in extracellular or intracellular chloride concentrations. Here, the first histochemical evidence is provided for local redistribution of chloride in the CNS of healthy adult rats. Olfactory bulbs were examined using freeze substitution, silver trapping of chloride and intensification techniques at light and electron microscopic level. The chloride content of precipitates was evidenced by electron spectroscopic imaging using a CEM 902 (Zeiss) electron microscope. Chloride concentration was high in a subpopulation of some periglomerular cell bodies and isolated dendritic profiles, while it seemed to be very low in certain parts of the glomerular neuropil including intercellular clefts. Data suggest that reversed chloride gradients can be demonstrated by cytochemical methods, and may be responsible for excitatory GABA effects on selected periglomerular neurons and dendrites in the olfactory glomeruli. Conditions leading to chloride redistribution in the CNS of normal adult rats remain to be determined.

Animals↗

PubMed1995

S100 protein expression in subpopulations of neurons of rat brain.

Available data are conflicting as regards the occurrence of Ca2+ and Zn2+ binding S100 proteins in neurons of mammalian brain. Here the localization and expression of S100 was re-investigated using several different antibodies and in situ hybridization. A map is provided for the distribution of two classes of S100-positive neuron populations in the adult rat CNS. "Persistently S100-positive" neurons had large size, were strongly immunoreactive and were mainly distributed in the nuclei of the lower brainstem and cerebellum. "Variably S100-positive" neurons were preferentially found in the forebrain of rats older than 90 days and were especially numerous in limbic regions. The S100-immunoreactivity in these neurons was moderately intense, occurred with high interindividual variation and appeared related to function as suggested by variations due to anesthesia. The expression of S100 mRNA in neurons was re-investigated at high spatial resolution with non-radioactive in situ hybridization using an oligonucleotide specific for S100 beta-mRNA. Expression of S100 was demonstrated in astrocytes and in those neuron populations which were also strongly S100-immunoreactive. No expression of S100 beta message was seen in weakly immunoreactive neurons, b but this may be due to low sensitivity of the techniques used. The data suggest that the S100 proteins are synthesized in all astrocytes and in distinct subpopulations of neurons in rat brain. These neurons show a characteristic topography and vary in S100 expression probably due to their function and maturation.

Animals↗

PubMed1995

Synaptic and non-synaptic cholinergic innervation of the various types of neurons in the main olfactory bulb of adult rat: immunocytochemistry of choline acetyltransferase.

The cholinergic neuronal structures and their synaptic connections in the main olfactory bulb of adult rats were analysed by using choline acetyltransferase immunocytochemistry. Within the glomeruli, cholinergic nerve fibers were restricted to strands which subdivided the neuropil into small compartments, the interior of which contained sensory axons but was devoid of cholinergic axons. Small numbers of choline acetyltransferase neurons were detected in all layers. Ultrastructural analysis revealed selective triadic synaptic relationships with different neuron classes in the intraglomerular area and in the external plexiform layer. These triads were made up of (i) a cholinergic axon, (ii) one or several periglomerular or granule cell dendrites, and (iii) usually one relay cell dendrite. In these triads, asymmetric cholinergic synapses were selectively focused on dendrites (gemmules and spines) of periglomerular or granule cells. Within the glomerulus, mitral and tufted cell dendrites were closely apposed to some cholinergic axon varicosities, most abundantly near arborizations of the apical dendrites. However, cholinergic synapses were never seen on any relay cell dendrite. In the external plexiform layer, cholinergic synapses were present on all parts of the superficial short-axon cells. In the internal plexiform layer and the granule cell layer, cholinergic axon varicosities exhibited close apposition or asymmetric synapses with granule cell gemmules. The data suggest that cholinergic projections from the basal forebrain to the main olfactory bulb focus synaptic innervation on interneurons. On relay cells, direct acetylcholine effects may occur, but these must be based on non-synaptic acetylcholine release at the surface of their dendrites.

Animals↗

PubMed1995

Expression of protein kinase C family members in the cerebral endothelial cells.

The protein kinase C (PKC) family is composed of at least four conventional (alpha, beta I, beta II, and gamma) and several related novel (delta, epsilon, eta, and zeta) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (alpha, beta, gamma, delta, epsilon, eta, and zeta) by using reverse transcriptase-polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC-gamma isoform could not be detected. Primary cultures of endothelial cells expressed PKC-alpha, -beta, -delta, -eta, and -epsilon isoenzymes, whereas the immortalized cell line expressed PKC-alpha, -delta, -epsilon, and -eta. The rat aortic endothelium contained only PKC-alpha and -delta isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood-brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.

Animals↗

PubMed1995

Distribution of Bergmann glial somata and processes: implications for function.

We have used immunocytochemistry for glial fibrillary adidic protein and glutamine synthetase to selectively label Bergmann glia in the adult rat cerebellum. From measurements of radial, tangential and en face sections we provide new data on the distribution and disposition of these glial cells. Specifically, Bergmann glia were found to have a mean areal packing density of 8,269 somata/mm2, their radial processes are packed at a mean density of 39,000/mm2, and their endfeet at the pial surface have a mean density of 19,973/mm2. Each Bergmann glial cell ist "responsible" for the equivalent of a column of cerebellar cortex having a base of 11 microns x 11 microns, a height of 170 microns, and a volume of 20,559 microns3. There are 8.1 Bergmann glia for each Purkinje cell, and each glial cell ensheaths between 2,142 and 6,358 Purkinje cell synapses. We use these data to offer insights on the roles of Bergmann glia during development and in the adult brain.

Animals↗

PubMed1994

Postnatal development of glial fibrillary acidic protein, vimentin and S100 protein in monkey visual cortex: evidence for a transient reduction of GFAP immunoreactivity.

In the cerebral cortex of some species, the gradual appearance of glial fibrillary acidic protein (GFAP) is often interpreted as reflecting the parallel maturation of neuronal connectivity. We studied the postnatal maturation of astrocytes in the primary visual cortex of Callithrix jacchus using antibodies against GFAP, vimentin and S100 protein as immunohistochemical markers. In the cortical grey matter of this species, the overall GFAP-immunoreactivity (IR) as measured by image analysis is high at birth (130% of the adult value), decreases until about 3 months (80%) and increases again towards adult values (100%). Vimentin-IR was high at birth, and declined towards 3 months and later. In contrast, S100-IR augmented postnatally in neuropil, and showed a laminar shift of maximum IR from layer IV to supragranular layers during ontogenesis. The decrease of GFAP-IR is predominantly due to changes in density of GFAP-positive (+) astrocytes within cortical tissue (newborn: 18,600 GFAP+astrocytes/mm3; 1 month: 11,600/mm3; 3 months: 5,700/mm3; adult: 10,200/mm3), while the overall number of astrocytes remained relatively constant as shown by the number of S100-positive astrocytic cell bodies. At times of low GFAP-IR a reduced area density of intermediate filaments was found in astrocytes by electron microscopy. The period of reduced GFAP-expression coincides with the time of prominent synapse remodeling in the visual cortex of marmosets. These data suggest that GFAP-expression may depend on functional conditions rather than time-dependent maturation.

Aging↗

PubMed1994

Astrocytes as rapid sensors of peripheral axotomy in the facial nucleus of rats.

Facial nerve transection leads to functional and structural reactions in lesioned motor neurones and surrounding glial cells. Data from this study provide evidence that the most rapid reaction described so far consists of an increase in immunoreactivity of connexin-43 (cx-43), the predominant gap junction protein in astrocytes. The ipsilateral facial nucleus is selectively marked as early as 0.75 to 1.5 hours after axotomy, while the unlesioned side as well as the unoperated controls remain faintly stained. Thus, enhanced coupling capacity of astrocytes by gap junctions appears to be a sensitive indicator of modified neuronal-glial interaction in the CNS.

Animals↗

PubMed1993

Distribution of GABA-immunoreactive nerve fibers and cells in the cervical and thoracic paravertebral sympathetic trunk of adult rat: evidence for an ascending feed-forward inhibition system.

Neurochemical and immunohistochemical evidence suggests that the superior cervical ganglion (SCG) contains all components of a gamma-aminobutyric acid (GABA)ergic transmission system, which includes GABAergic axons of unknown origin. The number of nerve fibers with and without GABA-like immunoreactivity was determined in interganglionic connectives at all cervical and thoracic levels of the paravertebral sympathetic trunk. In addition, the distribution of GABA-immunoreactive (IR) neurons was established within the ganglion chain and compared with the relative frequency of principal neurons richly innervated by GABA-IR axon terminals. The following results were obtained: 1) the total number of nerve fibers in cross sections did not significantly vary between the cervical levels, but it increased steadily from upper to lower thoracic segments; 2) in contrast, the number of GABA-IR fibers decreased from the cervical sympathetic trunk below the SCG (approximately 300 fibers) down to the seventh to tenth thoracic ganglion, below which no such fiber was seen; 3) GABA-IR nerve fibers originate from a subclass of GABA-IR cells; these are small, bipolar neurons with predominantly ascending, unmyelinated axon-like processes; 4) the number of principal neurons richly innervated by GABA-IR nerve fibers decreased from the SCG to the upper thoracic ganglia, and was very small below; and 5) apart from basket-like innervation, GABA-IR axons also formed diffuse networks around GABA-negative principal neurons predominantly in cervical and upper thoracic ganglia. These data suggest that the GABAergic innervation of paravertebral sympathetic ganglia is more complex than previously suspected. What appears as preganglionic afferents from several spinal segments (C8-Th7) innervate GABAergic neurons in the sympathetic trunk which have ascending axons and focus their inhibitory effects on the cervical sympathetic ganglia, predominantly the SCG. These data suggest that GABAergic small interganglionic neurons form a feed-forward inhibition system, which may be driven by multisegmental spinal input in the paravertebral sympathetic ganglion chain.

Afferent Pathways↗

PubMed1993

Pre- and postnatal development of the primary visual cortex of the common marmoset. I. A changing space for synaptogenesis.

The primary visual cortex of Callithrix jacchus occupies a large portion of the occipital neocortex and can be safely delineated from fetal stages onwards. In 20 animals ranging in age from fetal to adult age the morphological development of area 17 was evaluated and compared with the growth of whole brain, skull, and head size. Cortical thickness, surface area, and volume of the area were determined in addition to predominant growth directions. The volume of area 17 approximately doubles between birth (241 mm3) and three months of age (506 mm3). This maximum value marks an overshoot in growth (volume: 180%, surface area: 150%, thickness: 122%), which is followed by a considerable reduction before adult values (100%) are reached. Although these values seem to indicate that the overall reduction in size is fairly isometric, growth and regression are locally anisometric. For example, layers II-IVc contribute disproportionately to the overshoot; thickening is less pronounced than tangential growth and follows a slightly different time course. These data suggest that the developing visual cortex represents a highly dynamic distribution space for the developing synaptic junctions which should be taken into account in studies on synaptogenesis. By comparison it is suggested that this growth dynamic is not restricted to area 17 but also occurs in some other parts of the cerebral cortex. In contrast, most subcortical brain regions apparently do not undergo overshoot growth. Structural changes of the skull compensate the overshoot in cortex growth, so that head size increases steadily.

Animals↗