Size, Shape, and Distribution of Multivesicular Bodies in the Juvenile Rat Somatosensory Cortex: A 3D Electron Microscopy Study.

Multivesicular bodies (MVBs) are membrane-bound organelles that belong to the endosomal pathway. They participate in the transport, sorting, storage, recycling, degradation, and release of multiple substances. They interchange cargo with other organelles and participate in their renovation and degradation. We have used focused ion beam milling and scanning electron microscopy (FIB-SEM) to obtain stacks of serial sections from the neuropil of the somatosensory cortex of the juvenile rat. Using dedicated software, we have 3D-reconstructed 1618 MVBs. The mean density of MVBs was 0.21 per cubic micron. They were unequally distributed between dendrites (39.14%), axons (18.16%), and nonsynaptic cell processes (42.70%). About one out of five MVBs (18.16%) were docked on mitochondria, representing the process by which the endosomal pathway participates in mitochondrial maintenance. Other features of MVBs, such as the presence of tubular protrusions (6.66%) or clathrin coats (19.74%) can also be interpreted in functional terms, since both are typical of early endosomes. The sizes of MVBs follow a lognormal distribution, with differences across cortical layers and cellular compartments. The mean volume of dendritic MVBs is more than twice as large as the volume of axonic MVBs. In layer I, they are smaller, on average, than in the other layers.

A Quantitative Study on the Distribution of Mitochondria in the Neuropil of the Juvenile Rat Somatosensory Cortex.

Mitochondria play a key role in energy production and calcium buffering, among many other functions. They provide most of the energy required by neurons, and they are transported along axons and dendrites to the regions of higher energy demands. We have used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the somatosensory cortex of the juvenile rat. We have estimated the volume fraction occupied by mitochondria and their distribution between dendritic, axonal, and nonsynaptic processes. The volume fraction of mitochondria increased from layer I (4.59%) to reach its maximum in layer IV (7.74%) and decreased to its minimum in layer VI (4.03%). On average, 44% of mitochondrial volume was located in dendrites, 15% in axons and 41% in nonsynaptic elements. Given that dendrites, axons, and nonsynaptic elements occupied 38%, 23%, and 39% of the neuropil, respectively, it can be concluded that dendrites are proportionally richer in mitochondria with respect to axons, supporting the notion that most energy consumption takes place at the postsynaptic side. We also found a positive correlation between the volume fraction of mitochondria located in neuronal processes and the density of synapses.

Volume electron microscopy of the distribution of synapses in the neuropil of the juvenile rat somatosensory cortex.

Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87-94% were asymmetric and 6-13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80-91%) while symmetric synapses were mainly located on dendritic shafts (62-86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers.

Spaceflight induces changes in the synaptic circuitry of the postnatal developing neocortex.

The establishment of the adult pattern of neocortical circuitry depends on various intrinsic and extrinsic factors, whose modification during development can lead to alterations in cortical organization and function. We report the effect of 16 days of spaceflight [Neurolab mission; from postnatal day 14 (P14) to P30] on the neocortical representation of the hindlimb synaptic circuitry in rats. As a result, we show, for the first time, that development in microgravity leads to changes in the number and morphology of cortical synapses in a laminar-specific manner. In the layers II/III and Va, the synaptic cross-sectional lengths were significantly larger in flight animals than in ground control animals. Flight animals also showed significantly lower synaptic densities in layers II/III, IV and Va. The greatest difference was found in layer II/III, where there was a difference of 344 million synapses per mm(3) (15.6% decrease). Furthermore, after a 4 month period of re-adaptation to terrestrial gravity, some changes disappeared (i.e. the alterations were transient), while conversely, some new differences also appeared. For example, significant differences in synaptic density in layers II/III and Va after re-adaptation were no longer observed, whereas in layer IV the density of synapses increased notably in flight animals (a difference of 185 million synapses per mm(3) or 13.4%). In addition, all the changes observed only affected asymmetrical synapses, which are known to be excitatory. These results indicates that terrestrial gravity is a necessary environmental parameter for normal cortical synaptogenesis. These findings are fundamental in planning future long-term spaceflights.

Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy.

Impaired inhibition is thought to be important in temporal lobe epilepsy (TLE), the most common form of epilepsy in adult patients. We report that, in experimental TLE, spontaneous GABAergic inhibition was increased in the soma but reduced in the dendrites of pyramidal neurons. The former resulted from the hyperactivity of somatic projecting interneurons, whereas the latter was probably due to the degeneration of a subpopulation of dendritic projecting interneurons. A deficit in dendritic inhibition could reduce seizure threshold, whereas enhanced somatic inhibition would prevent the continuous occurrence of epileptiform activity.

Estimation of the number of synapses in the cerebral cortex: methodological considerations.

In the present work we discuss several sampling procedures commonly used for counting synapses in the cerebral cortex. We compare, within the same tissue, two frequently used sterereological methods for determining the numerical density of synapses per unit volume, using as an example the estimation of the number of types of synapses by layers in the neuropil of the adult human temporal neocortex. These two methods are a size-frequency method (formula N(A)/d) and the disector method (sigmaQ-/a x h). Since the size-frequency method is assumption-based and the disector method is considered to be an unbiased method, the latter is often recommended for the quantification of synapses and other objects. We obtained, however, similar estimates for the numerical density of the different types of synapses using both methods, although they presented different technical difficulties and statistical properties. In addition, we show that the size-frequency method is more efficient and easier to apply than the disector method. Nevertheless, there are other methods for quantification which may also be valid, depending on the aim of the research; but the data reported in many articles are often complicated, which makes it very difficult for the reader to follow all the steps of the calculation. If certain basic information were given, this would facilitate the interpretation and sharing of important information with other laboratories, regardless of the method used for quantification. Finally, based on our present results and previous literature, we propose a simple general protocol for estimating the numerical synaptic density by volume in the neuropil of the cerebral cortex.

Decalcification by ascorbic acid for immuno- and affinohistochemical techniques on the inner ear.

An ascorbic acid decalcifying solution was applied to immuno- and affinohistochemical studies on the inner ear. Rat inner ears fixed in 4% paraformaldehyde in PBS or in 2% acetic acid in ethanol solutions were adequately decalcified in an ascorbic acid solution, at a temperature of 4 degrees C. The decalcifying solution was prepared with 1% ascorbic acid and 0.84% sodium chloride in distilled water (pH 2.5-2.6). The decalcification time was in a direct relationship to the specimen calcification. In this study, two neuroactive substances (gamma-aminobutyric acid and calcitonin gene-related peptide), neurofilaments, and the galectine endogenous lectin were successfully detected immunohistochemically.

Ultrastructural differences among afferent synapses on cochlear hair cells: correlations with spontaneous discharge rate.

The major class of cochlear afferent fibers, the type-I or radial-fiber (RF) population, has been subdivided into three functional groups according to spontaneous discharge rate (SR): those with low SR have the highest acoustic thresholds, high SR fibers have the lowest thresholds and medium SR fibers are of intermediate sensitivity (Liberman [1978] J. Acoust. Soc. Amer. 63:442-455). Existing evidence from intracellular labeling studies at the light microscopic level (Liberman [1982a] Science 216:1239-1241) suggests that a single cochlear inner hair cell makes synaptic contact with representatives of all three functional groups; however, low and medium SR fibers are spatially segregated from high SR fibers around the hair cell circumference, and low and medium SR fibers are smaller in caliber than those with high SR. The present study extends to the ultrastructural level the structure-function correlations available via intracellular labeling. Analysis is based on serial section reconstruction of the synaptic contacts between 11 radial fibers of known SR and their target hair cells. Results suggest systematic differences in synaptic ultrastructure among fibers of the three SR groups: with decreasing SR, the size and complexity of the synaptic body (a presynaptic specialization characteristic of the peripheral afferent synapses in all hair cell systems and some other peripheral receptors) tend to increase, as does the associated number of synaptic vesicles. The possible functional significance of these trends is discussed in the context of other known structural and functional differences among the three SR groups.

Effects of indapamide on atherosclerosis development in cholesterol-fed rabbits.

We examined the effect of indapamide (IND) on the development of atherosclerosis lesions in rabbits maintained on a 1% (wt/wt) cholesterol-enriched diet for a 16-week period. IND was supplemented to the diet at three different levels to correspond to doses of 0.1, 0.3, and 1 mg/kg/day. Throughout the treatment, dietary consumption and body weight gains were comparable among the experimental and control groups. IND had no significant effect on plasma cholesterol, triglycerides, or phospholipids concentrations. Despite the lack of effect of the drug on these parameters, its administration produced a tendency toward a reduction in the aortic content of cholesterol and a dose-dependent and significant decrease in aortic damage. In the aortic arch, the extent of intimal aortic surface covered by grossly discernible atherosclerotic lesions was decreased by IND 1 mg/kg/day from 11.02 +/- 1.10 to 6.00 +/- 1.00% (p < 0.05) and from 9.72 +/- 1.39 to 5.37 +/- 1.20% (p < 0.05) in the remaining thoracic sections. In addition, the former dose also reduced the number of lesions per square centimeter from 3.69 +/- 0.68 to 1.72 +/- 0.53% (p < 0.05), and from 3.37 +/- 0.85 to 1.55 +/- 0.46% (p < 0.05) in aortic arch and in thoracic sections, respectively. The possibility that IND reduces the development of atherosclerotic lesions produced by diet-induced hypercholesterolemia through a mechanism involving its calcium antagonist and/or its antioxidant activity is discussed.

Choline-acetyltransferase-like immunoreactivity in the organ of Corti of the rat during postnatal development.

The mammalian cochlea receives efferent innervation from neurons located in the superior olivary complex. This efferent olivocochlear innervation is divided in two separate systems, lateral and medial, which mainly innervate afferent dendrites connected to inner hair cells and the cell body of outer hair cells, respectively. Besides other substances, lateral and medial efferent terminals of the adult cochlea use acetylcholine (ACh) as a neurotransmitter. In this study, we have used immunocytochemistry to detect the presence of choline acetyltransferase (ChAT), the synthesizing enzyme of ACh, in efferent olivocochlear terminals during the development of the rat. The appearance and distribution of immunoreactivity to ChAT has been studied in developing rat cochleas from birth (postnatal day 1, P1) to adulthood. Attention was paid to the temporal relationships between the expression of ChAT, the presence of other putative neuroactive substances, the onset of hearing and other developmental phenomena. Our results indicate that ChAT-like immunoreactivity is already present at birth (P1) in the region of inner hair cells, that it appears at P3 in the outer hair cell area and that it reaches an adult pattern of distribution by P15. ACh may thus be present early in the developing cochlea, before the onset of hearing, as it also occurs with other putative transmitters/modulators such as enkephalins, CGRP or GABA. It is suggested that ACh could be involved in the modulation of sound-evoked potentials as soon as they appear, and in the regulation of other developmental phenomena such as neurite outgrowth or synaptogenesis.

Effects of oxodipine and nitrendipine on the size of experimental myocardial infarct in the rat.

The protective effect of either oxodipine or nitrendipine (4 mg/kg/twice daily, orally), two dihydropyridines exhibiting calcium channel-blocking properties, against induced infarct-like lesions was examined. Experimental myocardial infarct-like lesions were induced in rats by subcutaneous injection of isoproterenol (0.5 mg/kg). The lesions were mainly located in subendocardial areas of the free wall of the left ventricle, and were intramural at the apex and ventricular septum. Pretreatment with oxodipine significantly reduced the infarct size in the apical and basal myocardium (P < 0.05). Possible explanations for the lack of effect of nitrendipine pretreatment are discussed.

Ontogeny of gamma-aminobutyric acid in efferent fibers to the rat cochlea.

Cochlear efferent innervation originates in two different groups of neurons located in the superior olivary complex. A first group of olivocochlear neurons (lateral efferent neurons) lies in the lateral superior olive. They send axons to the organ of Corti, where they synapse with radial afferent dendrites of primary auditory neurons, postsynaptic to the inner hair cells. The second group of neurons (medial efferent neurons) is found in medial subnuclei of the superior olivary complex and sends axons to synapse with outer hair cells. Subpopulations of both medial and lateral olivocochlear neurons probably use gamma-aminobutyric acid (GABA) as a neurotransmitter. We have used an immunoperoxidase technique to detect GABA-like immunoreactivity (GABA-LI) in postnatal maturing rat cochleas. The GABA-LI appeared in the inner hair cell region by P3 (P1 = birth) and reached a mature appearance by P15-P16. In the outer hair cell region, GABA-like immunoreactive fibers and terminals could not be identified until P9 and they were only found in the apical end of the cochlea. There was a dual gradient of maturation of GABA-LI in the cochlea. The GABA-LI appeared first at the cochlear base and then extended towards the apex. It also appeared earlier (about a week) in the inner hair cell region than in the outer hair cell region. This dual gradient of maturation is in close agreement with previous data concerning the maturation of the cochlea.

Astrocyte cytoarchitecture in cochlear nuclei of the rat: an immunocytochemical study.

Astrocytes play important roles in the development, maintenance and function of neural circuits. We have studied the astrocytic cytoarchitecture of the adult rat cochlear nuclei using a monoclonal antibody against glial fibrillary acidic protein, a well-known intermediate filament of the cytoskeleton of the glial cells. The cochlear nuclear complex is the first central step in the ascending auditory pathway. The morphology and distribution of astrocytes, as well as the relationship of astroglial processes with neurons, have been found to be different in the three main subdivisions of the cochlear nuclei and could be related to their function.

Synaptophysin immunoreactivity in the cochlear nuclei of mammals: a comparative study.

Synaptophysin is a Ca(2+)-binding glycoprotein located in the presynaptic membrane and synaptic vesicles. This glycoprotein has been also involved in the release of neurotransmitters. The presence of synaptophysin within the cochlear nuclear complex of several animal species was analyzed. Differences in size and neuronal density of the cochlear nuclei were observed between the animal species studied. In fact, cat and rat cochlear nuclei showed a very similar morphology and neuronal distribution, while mouse, hamster and guinea pig exhibited important differences. The morphology and arrangement of synaptic boutons, for each neuronal type considered, were similar among species.

Expression of GAP-43 in growing efferent fibers during cochlear development.

The development of olivocochlear efferent fibers has been studied by means of the immunocytochemical detection of the growth-associated protein GAP-43. This study has been carried out in pre- and postnatal, developing, pigmented rats. Results indicate that olivocochlear efferents reach the developing auditory epithelium from embryonic day 18 on. GAP-43-like immunoreactivity persists in the organ of Corti until the beginning of the second postnatal month. The relationships between the expression of GAP-43 in olivocochlear efferent fibers and other plasticity-related phenomena are discussed.

Ontogeny of glutamate decarboxylase and gamma-aminobutyric acid immunoreactivities in the rat cochlea.

gamma-Aminobutyric acid (GABA)-ergic efferent nerve fibers were studied during the postnatal development of the rat cochlea, using light microscopic immunocytochemical techniques. Antibodies against GABA and its synthesizing enzyme, glutamate decarboxylase (GAD), were used. Immunoreactivity to GAD is already present at birth (postnatal day 1) and could be found below the inner hair cells of the basal turn. Immunoreactivity progressively extends toward the apical turn until day 3. GAD-like immunoreactivity appears under the outer hair cells on postnatal day 15 and is only found in the upper part of the second turn and in the apical turn. The distribution of GABA-like immunoreactivity closely corresponds to that observed with the anti-glutamate decarboxylase antibody. However, the GABA-like immunoreactivity appears about 1-2 days after GAD-like immunoreactivity. At the beginning of the 3rd postnatal week, an adult pattern of GABA- and GAD-like immunoreactivity is established. These results suggest that GABA, which appears under the inner hair cells largely before the onset of hearing, may play a neurotrophic function during cochlear maturation and participate in the regulation of the first cochlear potentials as soon as they appear.

Immunocytochemical detection of glutamate decarboxylase in the postnatal developing rat organ of Corti.

Using a fluorescent avidin-biotin technique, we have immunolocalized the GABA synthesizing enzyme, glutamate decarboxylase, in postnatal developing and adult rat organs of Corti. At birth, the glutamate decarboxylase-like immunoreactivity is already present in the basal turn below the inner hair cells, i.e. within lateral olivocochlear efferent fibers of the inner spiral bundle. In the apical turn, the inner spiral bundle displays an immunoreactivity as early as postnatal day 3. Only the outer hair cells of the upper second turn and apex receive fibers immunostained for glutamate decarboxylase that most probably belong to the medial olivocochlear efferent innervation. They first appear at this level at postnatal day 15. Within these two regions of the organ of Corti, the glutamate decarboxylase-like immunoreactivity reaches an adult-like pattern at postnatal days 17-18. These results strengthen the hypothesis that GABA is a putative neurotransmitter that could be used by subpopulations of the two olivocochlear innervations. They also suggest that GABA either plays a neurotrophic function or participates in the regulation of the first cochlear potentials at the level of lateral efferent synapses.

Immunocytochemical detection of calcitonin gene-related peptide in the postnatal developing rat cochlea.

Using the immunofluorescence technique, three populations of fibers staining for calcitonin gene-related peptide are identified in postnatal developing cochleas and in adult rat cochleas. During the maturation of the cochlea, the immunostaining first appears in the basal turn and then extends toward the apex of the cochlea. The first population of immunostained fibers belongs to the lateral olivocochlear innervation. It is observed at postnatal day 4 within the inner spiral bundle of the organ of Corti. The second population of calcitonin gene-related peptide-like immunostained fibers belongs to the medial olivocochlear innervation. It is first identified at postnatal day 6 under the outer hair cells, generally in the first row. In the older stages, this population of fibers progressively extends toward the external row of outer hair cells. Finally, the third population of immunostained fibers belongs to the sympathetic supply of the cochlea. They can be identified at postnatal day 6 around the cochlear artery and its branches, and also within the entire modiolus. Our results confirm the presence of calcitonin gene-related peptide in fibers of the lateral and medial efferent innervations of the cochlea. They indicate an early appearance of calcitonin gene-related peptide in the developing cochlea, before the onset of the cochlear function, suggesting for this peptide either a neurotrophic function or a regulation of the early cochlear potentials at the level of the lateral efferent synapses.

A morphometrical method to estimate isoproterenol-induced infarct size in the rat.

A morphometrical method to quantify the geometrical distribution of ventricular necrosis induced in rat following the s.c. injection of isoproterenol was developed and tested. Each ventricle was divided into two portions, apical and basal, and then 10 microns thick sections of both portions were cut. Necrotic areas were detected with nitro blue tetrazolium. Total and necrotic areas were measured for each section. Thereafter, the total and necrotic volume were calculated assuming that the ventricles form a cone-like structure composed of successive frustum of a cone crowned by an apical cone. The study of volumes, necrotic and whole cardiac could be relevant for a more actual experimental comparison of the anti-ischemic drugs activity. Our results indicated that necrotic volume is unevenly distributed, especially at lower doses of isoproterenol treatment, being greater at the apical than at the basal portions of the ventricles. The importance of careful study of the ventricular apex is stressed. Potential sources of error in determining necrotic volume are discussed.