Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

Ultracytochemical study on the localization of superoxide producing sites in stimulated rat neutrophils.

Superoxide anion production in neutrophils plays an important role in the microbicidal defense system in the body. In this study, isolated rat neutrophils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocation during different cell stimulation time periods. Blood and peritoneal neutrophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of PMA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O(2)(-) was performed with the 3, 3'-diaminobenzidine-manganese (DAB/Mn) cytochemical method and cationized ferritin (CF) particles were added to stimulation media to monitor endocytotic events that occurred during neutrophil stimulation. Unstimulated neutrophils were devoid of O(2)(-) activity in cytoplasmic granules and at the plasma membrane surface. After 5 min stimulations with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the neutrophils. CF particles which were added to the stimulation media became internalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O(2)(-) activity. At 10 min stimulation with PMA, O(2)(-)-positive granules subsequently fused with each other and translocated to sub-plasma membrane regions where they either contacted the plasma membrane or fused with CF-containing endocytotic vesicles. Little reaction product was observed on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rates and yields of O(2)(-) production. Results from this study suggest that the O(2)(-)-producing sites of rat neutrophils originate intracellularly and translocate to the plasma membrane surface following stimulation with PMA, PMA + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore, these compartments appear to possess the ability to fuse with endocytotic vesicles, a process that may be linked to intracellular microbicidal activity in circulating and tissue neutrophils.

Structure, distribution and innervation of muscle spindles in avian fast and slow skeletal muscle.

Muscle spindles in 2 synergistic avian skeletal muscles, the anterior (ALD) and posterior (PLD) latissimus dorsi, were studied by light and electron microscopy to determine whether morphological or quantitative differences existed between these sensory receptors. Differences were found in the density, distribution and location of muscle spindles in the 2 muscles. They also differed with respect to the morphology of their capsules and intracapsular components. The slow ALD possessed muscle spindles which were evenly distributed throughout the muscle, whereas in the fast PLD they were mainly concentrated around the single nerve entry point into the muscle. The muscle spindle index (number of spindles per gram wet muscle weight) in the ALD was more than double that of its fast-twitch PLD counterpart (130.5+/-2.0 vs 55.4+/-2.0 respectively, n = 6). The number of intrafusal fibres per spindle ranged from 1 to 8 in the ALD and 2 to 9 in the PLD, and their diameters varied from 5.0 to 16.0 microm and 4.5 to 18.5 microm, respectively. Large diameter intrafusal fibres were more frequently encountered in spindles of the PLD. Unique to the ALD was the presence of monofibre muscle spindles (12.7% of total spindles observed in ALD) which contained a solitary intrafusal fibre. In muscle spindles of both the ALD and PLD, sensory nerve endings terminated in a spiral fashion on the intrafusal fibres in their equatorial regions. Motor innervation was restricted to either juxtaequatorial or polar regions of the intrafusal fibres. Outer capsule components were extensive in polar and juxtaequatorial regions of ALD spindles, whereas inner capsule cells of PLD spindles were more numerous in juxtaequatorial and equatorial regions. Overall, muscle spindles of the PLD exhibited greater complexity with respect to the number of intrafusal fibres per spindle, range of intrafusal fibre diameters and development of their inner capsules. It is postulated that the differences in muscle spindle density and structure observed in this study reflect the function of the muscles in which they reside.

Ultrastructure of the larval tentacle and its skeletal muscle in Xenopus laevis.

During premetamorphic development, tadpoles of Xenopus laevis possess a transitory pair of long, slender, mobile tentacles situated at the corners of the mouth. Microscopic examination of the larval tentacle typically reveals three distinct compartments: a central core of cartilage, a laterally situated skeletal muscle, and a nerve supply medially. Along the length of each tentacle, the epidermis is supplied by many unmyelinated nerve fibers, presumably sensory in nature, which terminate as naked axons in close association with the epidermal cells. The striated tentacular muscle, in the proximal region of the lateral compartment, consists of extrafusal muscle fibers of varying size which range in number from 36 to 48 per tentacle (n = 10). Using morphometric criteria, we have classified the skeletal muscle fibers of the larval tentacular muscle into three types: large (30-50 microns), intermediate (20-30 microns), and small (10-20 microns). By electron microscopy, each type displays characteristic sarcomeric banding patterns, sarcotubular and mitochondrial disposition, and motor endplate ultrastructure. Our morphological observations indicate that the tentacles of the Xenopus tadpole are complex mobile facial extensions which may play roles in mechanoreception and/or chemoreception during the waterborne stages of development. Because of its transitory nature, the Xenopus tentacle may be a useful experimental model in future studies of neuromuscular development and subsequent regression in a relatively short period of time.

An ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils.

Alkaline phosphatase (ALPase) activity was examined by cerium-based ultracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 ng/ml PMA or 10(-7) M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.

Quantitative morphology of mast cells in skeletal muscle of normal and genetically dystrophic mice.

BACKGROUND: Mast cells are indigenous connective tissue cells that function in the process of inflammation and edema. Their numbers were studied in a quantitative morphological study of the soleus muscles from 32-week-old and 56-week-old normal and genetically dystrophic dy2J and mdx mice to determine the incidence of mast cells in muscle to increasing age and to normal and myopathic conditions. METHODS: Soleus muscles from normal C57B1/J and from dystrophic C57B1/SnJ (dy2J/dy2J) and C57BL/10ScSn mdx mice were processed for examination by light and electron microscopy. Quantitation of mast cells was performed on semi-thick sections and expressed as an average of cells per millimeter squared of muscle tissue. RESULTS: Mast cells were observed in the connective tissue interstitium that normally separates skeletal muscle into fascicles. Their cytoplasmic granules stained metachromatically with toluidine blue and often obscured the single, centrally placed nucleus. They occurred singly or in small groups and were most frequently seen adjacent to neurovascular elements within the muscle, and in many cases were closely associated with the outer capsular regions of muscle spindles. Between the 32- and 56-week-old groups in each strain, an age-related increase in mast cell numbers was observed. In the dystrophic conditions, the dy2J and mdx skeletal muscles exhibited a two- to four-fold increase in mast cells when compared to normals in both age groups. Extensive connective tissue proliferation and sites of necrotic and regenerating muscle were common features in both myopathies. CONCLUSIONS: Results of this study indicate that a significantly higher number of mast cells which exist in dy2J and mdx murine skeletal muscles may be related to the high amount of connective tissue infiltration and extensive muscle fiber remodelling in these conditions. Moreover, the close proximity of mast cells to muscle spindles and nerve fascicles suggests that these cells may play a role in modulating their activities.

Ultracytochemical localization of alkaline phosphatase (ALPase) activity in neutrophils of the rat lung following injection of lipopolysaccharide.

Alkaline phosphatase (ALPase) activity in neutrophils was examined by enzyme ultracytochemistry at sites of experimentally induced acute inflammation in the rat lung and compared with those in non-inflammatory regions. Neutrophil accumulations in the lung were stimulated by intraperitoneal (IP) injection and intratracheal (IT) instillation of lipopolysaccharide (LPS), an endotoxin from Escherichia coli. Microsliced sections of fixed lung were incubated in a cerium-based reaction medium for the detection of ALPase activity. As cytochemical controls, sections were incubated in a substrate-free medium or in a medium containing 2 mM levamisole for inhibition of ALPase activity. Both IP and IT injections resulted in a significant accumulation of neutrophils in the lung, however, their histological distributions differed, the former stimulating high accumulations within the capillaries and interalveolar spaces, and the latter within the confines of the alveolar spaces. In neutrophils from controls and non-inflamed regions of the lung, ALPase activity was detected as an electron-dense reaction product localized predominantly to small spherical and tubular membrane-bounded cytoplasmic compartments. In the IP-injected rats, prominent ALPase activity was observed at their plasma membrane surfaces, most notably at sites of endothelial cell contact. Following IT instillation of LPS, neutrophils suspended in the alveolar spaces showed a reduced plasma membrane reactivity, however, at type I pneumocyte contact regions, enzyme activity was significantly increased. In all cases, ALPase activity was not detected at the endothelial plasma membranes. Some reaction, however, was seen on the microvilli of the type II surfactant-producing cells. These results indicate that ALPase activity of rat neutrophils in the lung can be increased by LPS injection and that its activity may also be related to the sites of cell-cell interaction and cell surroundings.

Distribution of dystrophin and neurofilament protein in muscle spindles of normal and Mdx-dystrophic mice: an immunocytochemical study.

Dystrophin is a high molecular weight protein localized under the sarcolemma of normal extrafusal muscle fibers but absent in skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. Muscle spindles in the soleus of 32-week-old normal and age-matched mdx mice were examined by immunocytochemical methods to determine the localization of dystrophin in polar and equatorial regions of the intrafusal fibers. Spindles were serially sectioned in transverse and longitudinal planes, and were double-labelled with an antibody to dystrophin and with an antibody to a 200 kD neurofilament protein, which revealed their sensory innervation. By fluorescence microscopy, intrafusal fibers in the soleus of mdx mice were deficient in dystrophin throughout their lengths, whereas their sensory nerve terminals stained intensely with the nerve-specific antibody and appeared unaltered in dystrophy. In the normal soleus, intrafusal fibers displayed a regional variability in the distribution of dystrophin. Polar regions of bag and chain fibers exhibited a peripheral rim of sarcolemmal staining equivalent to that seen in the neighboring extrafusal fibers. Dystrophin labelling in equatorial regions of normal intrafusal fibers, however, showed dystrophin-deficient segments alternating in a spiral fashion with positive-staining domains along the sarcolemma. Double-labelling for dystrophin and neurofilament protein showed that these dystrophin-deficient sites were subjacent to the annulospiral sensory nerve wrappings terminating on the intrafusal fibers. These findings suggest that dystrophin is not an integral part of the subsynaptic sensory membrane in equatorial regions of normal intrafusal fibers and thus is not directly related to sensory signal transduction. The complete absence of this protein in mdx intrafusal fibers indicates that these fibers exhibit the same primary defect in muscular dystrophy as seen in the extrafusal fibers. However, because of their small diameters, capsular investment, and relatively low tension outputs, dystrophic intrafusal fibers may be less prone to the sarcolemmal membrane disruption that is characteristic of extrafusal fibers in this disorder.