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The pattern of expression of ionotropic glutamate receptor (GluR) subunits 1-4 in the medial nucleus of the trapezoid body (MNTB) has been reported to change during development. The aim of this study was to compare the distribution of the GluR1-4 subunits in the MNTB at postnatal day (P) 9, before high-frequency signal transmission in the auditory system has developed, with that observed in mature adult rats. GluR1-4 subunits were studied by preembedding and postembedding immunocytochemical methods. Increased levels of GluR1, 2/3, and 4 associated with development were evident only at postsynaptic sites of MNTB principal cell bodies receiving calyces of Held synapses, whereas receptor density at nonsynaptic sites was found to remain unaltered. Taken together, the expression pattern of GluR subunits and the density of immunoparticles in postsynaptic specializations are indicative of a compartmentalization of alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) subunits upon development. These developmental changes provide a morphological basis for establishment of the postsynaptic properties needed for high-frequency synaptic transmission of auditory signals to MNTB principal neurons., (Copyright 2006 Wiley-Liss, Inc.)

The localization of metabotropic glutamate receptors of groups II (mGluR2/3) and III (mGluR4a) and the subunits 2 and 3 of alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic glutamate receptors (GluR2/3) was investigated with immunocytochemical methods in the rat adrenal gland. MGluR2/3, mGluR4a and GluR2/3 immunoreactivities were observed in large-sized, centrally located type I adrenal medullary ganglion neurons. Furthermore, the small-sized type II adrenal ganglion neurons identified by their immunoreactivity to brain nitric oxide synthase (bNOS), also expressed mGluR2/3, mGluR4a and GluR2/3. These cells were disposed in the peripheral portion of the adrenal medulla. None of the type I neurons were positively labeled for bNOS. These morphological observations suggest that activation of glutamate receptors in ganglion neurons may be instrumental in the control of adrenal endocrine systems as well as blood regulation.

Several studies indicate that metabotropic glutamate receptors (mGluRs) participate in the transmission of visual stimuli in optic layers of the superior colliculus (SC). We examined the cellular and subcellular distribution of the group III mGluR4a in superficial layers of the rat SC by means of a specific antiserum and a preembedding immunogold method for electron microscopy. Deposits of mGluR4a immunoparticles were mostly observed on presynaptic membranes of large synaptic terminals, which made asymmetrical synapses and contained abundant spherical, clear synaptic vesicles and numerous electron translucent mitochondria. These characteristic ultrastructural features correspond to retinocollicular synaptic terminals. Also, chains of synaptic retinal terminals along dendrites were labeled for mGluR4a. About 70% of morphologically identified retinal terminals were mGluR4a immunopositive. Furthermore, mGluR4a immunoreactivity in SC greatly disappeared following retinal ablation. About 28% of cortical terminals identified by anterograde tracing showed mGluR4a labeling, whereas only 2% of collicular GABAergic profiles were labeled for mGluR4a. These results reveal that retinal terminals are the major contributors to the mGluR4a immunoreactivity observed in the superior collicular circuitry.

The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation., (Copyright 2003 Elsevier Science B.V.)

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.

The existence of a neuronal-glial signalling through the activation of neurotransmitter receptors expressed in glia is well-documented. In excitatory synapses, glutamate released from presynaptic terminals activates not only postsynaptic receptors, but also ionotropic and metabotropic glutamate receptors localized in the glia ensheathing the synapses. The medial nucleus of the trapezoid body of the auditory system is involved in the localization of sounds in the space. In this nucleus, the large excitatory synaptic terminals formed by the calyces of Held on the principal globular cell bodies are wrapped by astrocytic processes. Since these synapses are functional from early postnatal days, glia receiving excitatory synaptic signals from the calyces may participate in modulating the maturation and development of the system. Groups I and II of metabotropic glutamate receptors (mGluRs) have been localized in glial cells in different brain regions. To investigate whether group II mGluRs are present in the medial nucleus of the trapezoid body, we have studied the pattern of expression of mGluR2/3 in the developing and mature nucleus by means of immunocytochemichal methods. The most remarkable finding was the switch in the occurrence of mGluR2/3 from glial to neuronal compartments. Thus, a preferential localization of mGluR2/3 immunoreactivity was observed in astrocytic processes surrounding the calyces of Held during the early postnatal development. In contrast, the main feature in adult rats was the presence of the group II mGluRs in presynaptic calyces of Held and postsynaptic principal globular cells.From these observations we suggest a role for group II mGluRs in neuronal-glial signalling in the calyx of Held-principal globular neuron synapses. Activation of these receptors might be relevant to the maturation and modulation of synaptic transmission in the medial nucleus of the trapezoid body.

We have studied the presence and distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-selective glutamate receptor subunits (GluR1, 2, 3, and 4) in the adult cat visual cortical areas 17, 18, 19, and the lateral suprasylvian areas (LSA). Reverse transcription-polymerase chain reaction (RT-PCR) amplification indicated that the genes encoding GluR1, 2, 3, and 4 are expressed in these areas and Western blot analysis revealed that the size of the corresponding peptides is similar to those described in the rat brain. In situ hybridization (ISH) using digoxigenin-labeled riboprobes showed that mRNAs coding for GluR1 and GluR3 were located in cells in all layers of the areas examined and also in the underlying white matter. GluR1 mRNA was relatively abundant throughout layers II-VI while GluR3 mRNA revealed a more laminated pattern of expression, preferentially labeling cells in layers II, III, V, and VI. The distribution of AMPA-selective receptor subunit peptides was studied by immunohistochemistry using subunit specific antibodies and found to be consistent with ISH results. In addition, we observed that most of the cells strongly labeled by the anti-GluR1 antibody were non-pyramidal neurons and that intense GluR2/3 immunoreactivity was seen preferentially in pyramidal neurons. Interestingly, double-labeling experiments indicated that neurons expressing gamma-aminobutyric acid (GABA) as well as the GluR1 subunit were particularly abundant in deeper layers. The GluR4 peptide was predominantly found in a relatively low number of layer III and layer V neurons with either pyramidal or non-pyramidal morphology. Finally, the distribution of neurons expressing the various receptor subunits was similar in all the visual cortical areas studied. These findings indicate a high expression of GluR1-3 subunits in the cat visual cortex and that GluR1 and GluR2/3 subunits are particularly abundant in non-pyramidal and pyramidal neurons, respectively. In addition, the results described here provide a reference for future studies dealing with the effect of visual deprivation on the expression of this receptor type.

The superior colliculus (SC) is a laminar structure which can be separated into superficial and deep layers according to morphological and functional criteria. The superficial layers are interconnected with many nuclei, most of which process visual information. Also, these layers are known to project to the pontine grey. The topography of the projection is well established in many mammals but little is known about the morphology of the cells of origin of the projection. The pons of two adult albino rabbits were injected with Rhodamine latex microspheres and the retrogradely labeled cells in the SC were intracellularly filled with Lucifer yellow. The distribution of the labeled cells was organized into two bands. The upper one (71% of the cells) was located in the middle third of the stratum griseum superficiale (SGS). The lower band was less dense (29% of the cells) and located in the lower third of SGS and in the upper layers of the stratum opticum. Neurons corresponding to the upper band were preferentially vertically oriented. In particular, 47% were vertical fusiform, 23.5% globular/pyriform, 17.7% horizontal and 11.8% stellate in their morphology. Neurons corresponding to the lower band did not present so clear polarization. Thus, 57.1% were stellate, 28.6% were horizontal and 14.3% globular/pyriform. On the basis of their morphology, we consider that the cells of origin of the tectopontine projection form a particular population in the SC.

The morphological and morphometrical features of the ACTH-immunoreactive cells of the anterior pituitary gland were compared between adult rats with intact adrenals, adult rats without adrenals, and adult rats with regenerated adrenocortical nodules. The removal of the adrenal glands produced a great increase in the number and size of the cytoplasmic processes of the ACTH-immunoreactive cells that sometimes establish contact with those of the neighbouring immunoreactive cells. In addition, in this experimental group, the ACTH-cell percentage and the mean cell, cytoplasmic, and nuclear areas were greatly increased in comparison with those found in control animals. Several regenerated adrenocortical nodules were observed in the small-bowel segment of rats transplanted with neonatal adrenal glands. The percentage and morphometrical values of the pituitary ACTH-reactive cells of this experimental group, although slightly increased relative to the rats with intact adrenals, were greatly decreased in relation to those of the adrenalectomized animals. It is concluded that the neonatal adrenal tissue regenerated after its transplantation into adult rats, a process that modified the percentage, the morphological characteristics, and the morphometrical values of the ACTH-immunoreactive cells of the adrenalectomized rats.

The mammalian superior colliculus (SC) may be divided into distinct layers on the basis of cytoarchitectonic criteria. These layers have different functions and patterns of connectivity. The superficial layers (stratum zonale, stratum griseum superficiale (SGS) and stratum opticum) are intimately associated with the processing of visual information. In the present study, we have investigated the morphology of the SC neurons that project ipsilaterally to the dorsal lateral geniculate nucleus (dLGN). Rhodamine latex microspheres were injected into the dLGN of nine adult rabbits. Retrogradely labeled cells were then intracellularly injected with Lucifer Yellow. The somata of the cells from which the projection originated were primarily located in a band occupying the medial third of the SGS and were found to reside at an average depth of 373 microns. Morphological analysis of these neurons revealed that 37% had a stellate shape, 27% were vertical fusiform, 18% were globular/pyriform, 14% were oriented horizontally and 4% were pyramidal in their morphology. Within each morphological class, we have examined the different subtypes with respect to incidence, localisation and characteristics of the dendritic arborizations.

The neuronal projection between the superficial layers of the mammalian superior colliculus (SC) and the ipsilateral dorsal lateral geniculate nucleus (dLGN) is well established. However, the participation of the contralateral SC remains controversial. In this study we have presented evidence for the existence of SC neurons which project to the contralateral dLGN and also performed a detailed statistical analysis of such SC cells. The dLGN of nine adult rabbits was injected with Rhodamine latex microspheres and the retrogradely labeled cells of the SC were intracellularly injected with Lucifer yellow. The number of labeled cells is less than that found in the ipsilateral SC, but some are always present. The somata of these cells are located mainly in the lower half of the stratum griseum superficiale at a mean depth of 738 microns. 40% of the cells were horizontal, 34% stellate, 23% globular or pyriform and 3% vertical fusiform in their morphology. On the basis of morphology and localization, this population is clearly distinct from the corresponding ipsilateral cell population.

The aim of this study is to describe the morphological features of the origin cells of the visual corticopontine projection in the cat. To this end, the pontine grey matter of adult cats was injected with Rhodamine Latex Microspheres. The resulting, retrogradely marked cells of the visual cortex, were subsequently injected intracellularly with Lucifer Yellow. The majority of these cells (68%) were layer V pyramidal cells. However, a significant proportion of these retrogradely-labeled cells were atypical (26.5%) and non-pyramidal (5.5%) in their morphology. The majority of the labeled cells were found at the interhemispheric surface. The somatic dimensions of the visual corticopontine cells were on average 22.8 x 17.0 microns. The mean number of basal dendrites of pyramidal cells was 5.3 and their average tangential spread was 296.1 microns. For non-pyramidal cells, the values were 3.2 and 195.6 microns respectively. The main bifurcation of apical dendrites was located on average at 95.0 microns from the soma and their mean tangential spread was 193.9 microns. The depth of the soma was on average 1086.9 microns from the pial surface.

The percentage, distribution, shape, intensity of staining and morphometrical parameters of the pituitary TSH immunoreactive cells and the histological features of the thyroid glands, were compared between adult rats with intact adrenals, without adrenals and biadrenalectomized animals with neonatal adrenal grafts. After the removal of the adrenal glands, TSH immunoreactive cells increased in percentage and exhibited a higher complexity of the cellular outline, than that of the intact animals. The nuclear, cytoplasmic and cell areas were significantly increased. However the bigger enhancement of the cytoplasmic area relative to the nuclear area, produced a decrease in the nuclear/cell area ratio. The thyroid glands showed some histological evidences of activation. After the transplantation of neonatal adrenal glands to adult rats, several adrenocortical nodules were present in the lumen of the small bowel segment. These adrenal masses induced a great decrease in the TSH cell area, which coupled with a smaller but significant variation of the nuclear area, led to an increase in the nuclear/cell area ratio relative to that observed in adrenalectomized animals. In addition, the distribution, shape and intensity of the immunoreactive material was similar to that observed in intact animals. In this experimental group, thyroid histology was observed to be similar to that of the intact animals.

The feasibility of excluded and everted small intestine segments as reservoirs for adrenal tissue transplantation was compared between biadrenalectomized adult rats with isogenic transplants of adrenal quarters or allogenic transplants of neonatal adrenal glands. Small clusters of adrenocortical cells, located between the adipocytes and the mesenteric vessels of the segments' lumen, were observed following isogenic transplantation. Several large regenerated adrenocortical nodules, surrounded by a connective capsule, were present in each of the rats transplanted with neonatal adrenal glands. Medullary tissue did not regenerate. It was concluded that the excluded and everted segments of small intestine were optimal sites for adrenal tissue transplantation and that the regeneration process of the adrenocortical tissue takes place more quickly after the transplantation of neonatal adrenal glands than after the transplantation of adrenal quarters.

Injections of the low diffusion retrograde tracer rhodamine latex microspheres were made in the pontine grey matter of the cat in order to study the cortical convergence to the pons. We found a different distribution of cells in the convex surface of the brain hemisphere making ventral or dorsolateral injections. In the first case, cells were grouped in the top of the gyri. In the second case, cells were more frequent in the bottom of the sulci. Our results show a possible retinotopic organization of this projection.

The characteristics and distribution pattern of retinal afferent terminals making synaptic contacts on narrow field vertical neurons in the stratum griseum superficiale of the rabbit superior colliculus were studied using the Golgi-gold substitution technique in combination with either autoradiographic or degenerative methods. At the level of light microscopy, identified gold-toned vertical neurons showed similar features to those previously described by others. Although their axons were frequently seen rising from the basal dendritic tuft, they could also emerge from an apical dendrite, but rarely from the cell body. The electron microscopic study revealed that these neurons received radiolabelled or degenerated profiles with typical features of retinal terminals, while more proximal parts seemed to receive fewer terminals but of larger size. Axo-somatic synaptic junctions were very rare and, interestingly, retinal terminals were not seen forming synapses on the basal dendritic arborization. The present results indicate that the narrow field vertical cells are targets for retinotectal projection.

After WGA-HRP injections in the pontine grey involving the dorsolateral pontine nucleus, a great number of labeled cells were found in the superficial layers of the ipsilateral superior colliculus. The majority of these cells were located in the stratum griseum superficiale (SGS). Few labeled cells were found in the stratum opticum, and the stratum zonale (SZ) showed no labeled cells. Labeled cells in the SGS formed a rather homogeneous population as most of them had fusiform somata with an upper dendritic process which runs vertically to reach the SZ. These cells were mainly located in the middle third of the SGS, forming a sublamina in this layer. These results demonstrate the participation of the superficial tectal layers in the ipsilateral descending pathway of the superior colliculus, allowing visual information to reach precerebellar stations at the dorsolateral pontine nucleus.

In the method described, the head of a fixed-stage microscope and the attached micromanipulator are simultaneously displaced during focussing movements of the microscope. This design allows axial displacements of the microscope towards the cell to be injected. This arrangement makes the filling of neurons in brain slices by intracellular iontophoresis injection of Lucifer yellow considerably easier.

This study examines the thalamo-cortical projections to the most superficial neocortical layers in the hedgehog (Erinaceus europaeus) after small injections of horseradish peroxidase and horseradish peroxidase conjugated to wheat germ agglutinin in the somato-sensory cortex. The injections were limited to layers I, II and upper parts of layer III/IV. Retrogradely labeled cells were plotted in serial sections through the thalamus. Injections in the somato-sensory cortex gave a pattern of elongated columns of labeled cells, extending rostro-caudally in the nucleus ventralis thalami. In the neocortex, labeled fibers extended for considerable distances running horizontally in layer I. Complementary observations demonstrate the thalamic origin of certain, coarse ascending bundles observed previously in Golgi preparations of the hedgehog. It is concluded that a major cortical input to layer I originates in the hedgehog in the principal thalamic (relay) nuclei. After injections in the somato-sensory cortex, retrogradely labeled cells were also found in the nucleus ventro-medialis thalami and very few in a zone medial to the nucleus ventralis thalami corresponding to the intralaminar thalamic nuclei. The contributions of this latter system seem to be limited in comparison with other mammals.