Characterization of GABA(A) and glycine receptors in neurons of the developing rat inferior colliculus

GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) expressed in developing neurons (P3-8) of the rat inferior colliculus (IC) were investigated using fast application of transmitters on nucleated patches and whole-cell patch-clamp recordings. At a holding potential of -60 mV and an E(Cl) close to 0 mV GABA and glycine activated inward currents with a half-maximal activation concentration of 127 microM and 353 microM, respectively. GABA(A)Rs activated and deactivated significantly faster than GlyRs (20-80% rise-time: 0.71 ms for GABA(A)Rs and 1.31 ms for GlyRs; amplitude-weighted decay time constant, tau(w), 22.4+/-1.3 ms for GABA(A)Rs and 108.7+/-32.9 ms for GlyRs). The tau(w) values for receptor desensitization were 26.8+/-1.9 ms for GABA(A)Rs and 177.9+/-47.2 ms for GlyRs. Recovery from desensitization was significantly faster for GlyRs (tau=334 ms) than for GABA(A)Rs (tau1=82 ms; tau2=3783 ms). The tau(w) values of GABA(A)R-mediated spontaneous and miniature inhibitory postsynaptic currents were not significantly different from those of GABA(A)Rs activated by fast application, but significantly different to the tau(w) of GlyRs activated by fast application. The different properties of GABA(A)Rs and GlyRs expressed in the same IC neuron suggest a distinct functional contribution to the development of inhibitory synapses in the IC.     

The inferior colliculus of the rat: a quantitative analysis of monaural frequency response areas

Frequency response areas (FRAs) were measured for 237 single units in the inferior colliculus (IC) of urethane-anesthetized pigmented rats using monaural pure-tone stimulation. Based on qualitative criteria [J Neurosci 21 (2001) 7303], FRAs were classified as V-shaped in 69% of neurons, non-V-shaped in 29%, and unclassifiable in the remaining 2%. Non-V-shaped FRAs were heterogeneous, comprising a number of subtypes including narrow, closed, low- and high-tilt, multipeaked, U-shaped, mosaic and inhibitory. To complement this subjective classification, we applied quantitative measures used by others (e.g. [J Neurophysiol 84 (2000) 1012]), including the inverse slope of the upper and lower FRA borders, Q-values, and other measures of bandwidth. The results suggest that FRAs in the rat IC are best described as forming a continuous distribution among subtypes, rather than clustering into discrete categories. Moreover, there is a broad range of frequency tuning characteristics and FRA types across the entire frequency spectrum. Within this general pattern, however, there are some frequency-specific differences in FRA type distribution. The relative proportion of V-shaped FRAs was greatest at the high and low ends of the auditory range, with the highest proportion of non-V-shaped FRAs in the mid-range from 6 to 12 kHz. For most neurons with multipeaked FRAs, the peak frequencies were not harmonically related. Frequency tuning in the pigmented rat IC is generally similar to that in other species. Comparison of Q values across auditory nuclei shows little evidence that FRAs are sharpened at levels above the auditory nerve. Rather, there is a broad range of frequency tuning properties at each level.     

Postembedding immunocytochemical GABA labeling in rat neocortex cultures: applicability in quantitative studies

The value of gamma-aminobutyric acid (GABA) immunogold labeling in the quantification of nerve endings was studied in dissociated rat neocortex cultures. Adjacent sections were processed in 3 series according to the same staining protocol. Of all nerve ending profiles examined, 20% appeared to be GABA-positive, while 7% could not be classified. In cultures pretreated with gabaculine, GABA-positive endings were labeled more heavily. Cell bodies could always be discriminated as GABA-positive or GABA-negative. Of all neuronal cell profiles, 14% appeared to be GABA-positive. These neurons could also be identified by light microscopy using the peroxidase-anti-peroxidase method.     

Anatomy of the inferior colliculus in rat

Summary This paper defines the pattern of subdivision of the inferior colliculus in rat. It is based on serial sections of brains of albino and hooded rats cut in the frontal, sagittal and horizontal planes using Golgi, Nissl and a combined cell-myelin method. In rat, like in other mammals, the inferior colliculus consists of a central nucleus, an external cortex, and a dorsal cortex. The central nucleus is flattened in the frontal plane and confined to the caudomedial part of the inferior colliculus. It is characterized by a lamellar organization of disc-shaped neurons interspersed with multipolar cells. The cells are small to medium-sized. Although there is a dorsoventral gradient in size and packing density of cells within the nucleus, the overall size is smaller and the packing density larger than in adjacent subdivisions. The two cortices each consists of three layers. The outer-most layer is common to the two cortices, forming a fibro-cellular capsule continuous along most of the circumference of the inferior colliculus. The external cortex is located lateral, rostral, ventral and ventrocaudal to the central nucleus. Its second layer, deep to the superficial capsule, is characterized by clusters of many small and a few medium-sized neurons in a myelin-dense neuropil. Layer 3, which constitutes the major portion of the subdivision, consists of relatively scattered, small, medium and large cells, the most characteristic element being large multipolar neurons with coarse Nissl granules. The dorsal cortex is located dorsocaudal and dorsomedial to the central nucleus. Its second layer is composed of small neurons, while the third, deep layer in addition contains medium-sized neurons. The cell density is intermediate to that of the central nucleus and the deep part of the external cortex. We have tried to facilitate the parcellation by reference to easily recognizable, nearby structures and to standard stereotaxic coordinates.     

Histogenesis of the inferior colliculus in rat

Summary The early histogenesis of the inferior colliculus from embryonic day 11 to 18 (E11 to E18) has been studied in the rat by analysis of Golgi impregnated material and plastic sections.This analysis has shown that the pseudo-stratified columnar neuroepithelium observed at E11 is followed by the appearance at E12 of three zones: marginal, intermediate and ventricular. Signs of cell differentiation are first observed in the intermediate zone. Secondary rearrangements occur within this zone, and by E16 a thin cortical plate (the cortex of the inferior colliculus) develops at the junction of the intermediate and marginal zones giving rise to the external and pericentral nuclei of this structure, which has a cortical organization in adults. The remainder of the intermediate zone (the nucleus of the inferior colliculus), invaded by axons, expands dramatically by E16–E17 and gives rise to the central and dorso-medial nuclei of the inferior colliculus which have a nuclear organization in adults. The morphogenetic events which take place in these two regions differ and can be identified by the study of cell migration and differentiation.In the nucleus of the inferior colliculus, neuronal migration begins with detachment of the ventricularly directed process, or trailing process, of the primitive epithelial cell from the ventricular surface. This is followed by the ascent of the cell nucleus through the pially directed, or leading, process by a mechanism identical to the perikaryal translocation already described in other regions of the nervous system. This mechanism of cell migration is characteristic of a first type of migratory young neuron (type I). Axons initiate from the leading process of these cells during migration and dendrites grow out in various directions giving these cells a bipolar or a multipolar appearance. Dendritic differentiation occurs first in the outermost cells of the nucleus and proceeds inwards.In the cortex of the inferior colliculus, neuronal migration also begins with detachment of the ventricular process, which occurs by E12, immediately followed by the detachment and retraction of the apical or leading process. Within the intermediate zone, migratory cells become rounded and sprout numerous processes. One of these processes is tipped by a growth cone and displays all the characteristics of an axon. It is directed tangentially in the intermediate layer. Dendritic growth and differentiation starts when the cells reach their final position in the cortical plate, and proceeds from the innermost cells outward. Due to the inadequacy of our methods for identifying radial glial fibers, the mechanism of migration of this type of cell (type II) remains unclear.Our results confirm that the inferior colliculus of the rat is organized as a central nuclear mass surrounded by a thin cortex. As previously observed in other regions of the nervous system, the modes of cell migration and differentiation in the cortical and non-cortical structures of the inferior colliculus appear different.     

GABA innervation in adult rat oculomotor nucleus: A radioautographic and immunocytochemical study

Summary GABA innervation in the adult rat oculomotor nucleus (n.III) was investigated using two complementary approaches: radioautography after incubation of brain slices with tritiated GABA ([³H]GABA) along with localin vivo microinjections of the tracer, and GABA immunocytochemical procedures involving antibodies directed against a GABA-glutaraldehyde-protein conjugate. As determined by radioautography afterin vitro orin vivo labelling, the [³H]GABA uptake sites in the n.III mainly involved axon terminals. These were distributed throughout the neuropil and were often closely apposed to unlabelled motoneuron somata. A small number of glial cells also showed preferential accumulation of the tracer. The GABA-immunostaining likewise involved axon terminals throughout the nucleus, but no glial cells were immunopositive. In the dorsal region of the structure, occasional GABA-immunostained internuclear neurons were observed among unstained motoneuron cell bodies. Electron microscopic examination of [³H]GABA-labelled or GABA-immunostained profiles in n.III revealed axon terminals of around 1 m in diameter, always filled with small, round synaptic vesicles homogeneously distributed throughout the axoplasm. These boutons frequently contained mitochondria and one or more large granular vesicles. In single thin sections, 35% of [³H]GABA-labelled, and 19% of GABA-immunostained varicose profiles exhibited a synaptic differentiation, suggesting the existence of a predominantly if not entirely junctional innervation. These synapses mostly involved dendritic trunks or dendritic branches and were usually of the symmetrical type. A few, which were always symmetrical, were also observed on large somata of motoneurons. Some of the dendrites synaptically contacted by GABA-immunostained axon terminals were themselves GABA immunoreactive. These data substantiate the idea that GABA is involved in the control of motoneuron activity in n.III, and provide a structural basis for the inhibitory role of this transmitter in oculomotor function.     

Local circuit neurons in the rat ventrobasal thalamus--a GABA immunocytochemical study

The ventrobasal thalamus of seven rats was processed for immunocytochemistry using antisera to glutamate decarboxylase or gamma-aminobutyrate (GABA). Glutamate decarboxylase-stained sections showed a network of stained fibers and terminals but no stained cell bodies. GABA-stained sections had fewer stained fibers and terminals but did show a few stained cell bodies. Cell bodies were especially apparent when carbazole was used for a chromogen for the peroxidase-antiperoxidase visualization. The GABA-stained cells were found to be distributed throughout the ventrobasal complex, to have smaller soma cross-sectional areas than most other cells (81 +/- 34 microns vs 105 +/- 36 microns for all cells) and to make up 0.4 +/- 0.3% of the neuronal population of the ventrobasal complex. Injections of horseradish peroxidase into the somatosensory cortex (SI) retrogradely filled many neurons in the ventrobasal thalamus, but none of these labeled neurons were double labeled with GABA. These results indicate that the GABA-labeled cells probably represent a small population of local circuit neurons in the rat ventrobasal thalamus.     

The development of the extracellular space in neonatal rat inferior colliculus: An electron microscopic study,

Rat inferior colliculi were fixed by intracardiac perfusion of 4% methanol-free formalin in an isotonic phosphate-sucrose buffer, dehydrated in ethanol and studied during the first two weeks of postnatal life. The extracellular space during the first week was made up of intercellular gaps about 100–150 Å wide and large intercellular lakes (1–3 μ long) which diminished in size and number as maturation proceeded. Similar intercellular lakes were found in tissue fixed by immersion in glutaraldehyde containing ruthenium red. In these tissues an intense ruthenium red reaction product, distributed extracellularly, suggested an intercellular substance comparable to that described in adult brain. Intercellular lakes were also found in tissue dehydrated in acetone. Morphometric estimations indicated the extracellular space of neuropil during the first week to be about twice that of the adult, which was approached progressively.     

The neocortical projection to the inferior colliculus in the albino rat

Summary The purpose of the present study was to define the field of termination of the neocortical projection to the inferior colliculus in rat. The study was based on fiber degeneration following large lesions of the cerebral cortex, and anterograde transport of wheat germ agglutinin horseradish peroxidase ejected inotophoretically into more restricted neocortical loci. Neocortical fibers were found to supply the dorsal and external cortices of the inferior colliculus. The central nucleus, in contrast, did not receive such fibers. The results speak in favor of three separate projections, one partly bilateral to the deeper part of the dorsal collicular cortex, a second ipsilateral to the superficial part of this subdivision, and a third ipsilateral to the external collicular cortex.     

Extracortical descending projections to the rat inferior colliculus

Feedback controlling is an important element in the sensory processing in the auditory system. It has been long recognized that the inferior colliculus (IC) sends direct ascending projections to the medial geniculate body (MGB), but receives feedback regulation from the auditory cortex. In the present study we probed the shorter extracortical projections to the IC, including the direct descending pathway from the MGB. In the rat, the fluorescence retrograde tracers Fluorogold, True Blue or Rhodamine latex microspheres were injected into the IC, and the auditory thalamus and surrounding regions were examined for fluorescent neurones. We did not find any retrograde labelling in the ventral division of the MGB. However, retrogradely labelled neurones were found in the medial and suprageniculate nuclei of the MGB. We also observed densely packed groups of fluorescent neurones in the peripeduncular nucleus and numerous labelled neurones in the nucleus of the brachium of the IC. The existence of a direct descending pathway to the IC from at least some auditory thalamic nuclei challenges the perception of the colliculo-thalamic relationship as one-way traffic and suggests more direct involvement of the auditory thalamus in the feedback regulation of the incoming acoustic signals.     

Pallidotectal projection to the inferior colliculus of the rat

Summary After injection of fluorescent tracer into the inferior colliculus (IC), retrogradely labeled cells were observed not only in the temporoauditory cortex (ACx) and the substantia nigra pars lateralis, but also in the globus pallidus (GP). These labeled GP cells were localized exclusively in the caudal portion of the GP, which has been known to project to the ACx. Employing a retrograde fluorescent double labeling technique, the GP-IC neurons were found to be distributed in a separate manner from the GP-ACx neurons within the caudal GP. The present study provides further anatomical evidence that the caudal GP has a functional role in auditory processing.Abbreviations ACx temporoauditory cortex - BC Brachium conjunctivum - CP cerebral peduncle - CPu caudate putamen - DY Diamidino Yellow - EP entopeduncular nucleus - FG Fluoro-Gold - GP globus pallidus - I internal capsule - IC inferior colliculus - OT optic tract - SC superior colliculus - SN1 substantia nigra pars lateralis - T thalamus - TB True Blue - TPC nucleus tegmenti pedunculopontinus pars compacta     

A GABA immunocytochemical study of rat motor thalamus: light and electron microscopic observations.

A light and electron microscopic study of GABA-immunoreactive neurons and profiles in the ventroanterior-ventrolateral and ventromedial nuclei of rat dorsal thalamus was conducted using antiserum raised against GABA. Less than 1% of the neurons in these motor-related nuclei exhibited GABA immunoreactivity, confirming previous reports that these nuclei are largely devoid of interneurons. Immunoreactive neurons in the ventral anterior-ventral lateral complex and ventromedial nucleus were bipolar or multipolar in shape, and tended to be smaller than non-immunoreactive neurons. GABA immunoreactivity in the neuropil consisted of labeled axon terminals and myelinated and unmyelinated axons, and was lower in the ventral anterior-ventral lateral complex and ventromedial nucleus than in neighboring thalamic nuclei. The density of neuropil immunolabeling was slightly higher in ventral anterior-ventral lateral complex than in ventromedial nucleus. GABA-immunoreactive axon terminals, collectively termed MP boutons for their medium size and pleomorphic vesicles (and corresponding to "F" profiles of some previous studies of thalamic ultrastructure), formed symmetric synapses and puncta adhaerentia contacts predominantly with large and medium-diameter (i.e. proximal) non-immunoreactive dendrites. Approximately 12 and 18% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, were GABA-immunopositive. Many of these immunoreactive profiles probably arose from GABAergic neurons in the thalamic reticular nucleus, substantia nigra pars reticulata and entopeduncular nucleus. Two types of non-immunoreactive axon terminals were distinguished based on differences in morphology and synaptic termination sites. Boutons with small ovoid profiles and round vesicles that formed prominent asymmetric synapses onto small-diameter dendrites were observed. Mitochondria were rarely observed within these boutons, which arose from thin unmyelinated axons. These boutons composed approximately 82 and 74% of boutons in the ventral anterior-ventral lateral complex and ventromedial nucleus, respectively, and were considered to arise predominantly from neurons in the cerebral cortex. In contrast, boutons with large terminals that contained round or plemorphic vesicles and formed multiple asymmetric synapses predominantly with large-diameter dendrites were also observed. Puncta adhaerentia contacts were also common. Mitochondria were numerous within large boutons with round vesicles, which arose from myelinated axons. Many of the large boutons were likely to have originated from neurons in the cerebellar nuclei. Approximately 6% of the boutons in the ventral anterior-ventral lateral complex and 8% in ventromedial nucleus were of the large type.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inferior colliculus of the mouse. A Nissl and Golgi study

Serial sections of cell- and fiber-stained and Golgi-impregnated material from adult mice were used to study the cytoarchitectonics, fiber and neuronal architecture of the inferior colliculus. The size of the cells, the pattern of dendritic branching, and the appearance of the neuropil were the features used to delineate the three main regions of the auditory tectum: the central mass of cells or central nucleus, the cortex, and the paracentral nuclei. The central nucleus contains two major cell types: the bipolar cells, which are the most abundant, and the multipolar cells. The dendrites of the bipolar cells are oriented in the same direction and the afferent axons of the lateral lemniscus run along them, contributing to form fibrodendritic strips: the laminae of the central nucleus. The orientation of these laminae differs in the various parts of the central nucleus and delineates four subdivisions. In these four subdivisions, the laminae maintain the same relative position throughout the anteroposterior axis of the central nucleus, but they stop abruptly at the periphery of the nucleus. The cortex surrounds the central nucleus dorsally and caudally. The lamination in four layers concentric to the surface, the increasing gradient of size from the periphery to the deep tissue, the existence of two major types of cells, stellate and pyramidal, permit this structure to be considered as a true cortex. The paracentral nuclei are scattered around the central nucleus. The commissural nucleus is composed of cells with a simple dendritic branching pattern perpendicular or parallel to the fibers of the intercollicular commissure. The dorsomedial and ventrolateral nuclei are characterized by the presence of large multipolar cells. The nucleus of the rostral pole, distinct from the anterior pole of the central nucleus, is composed of small and medium-sized multipolar cells. The lateral nucleus appears as an extension of the dorsal cortex with only two or three layers of cells. The neuronal organization in the central nucleus appears similar in the mouse and in the cat, suggesting an identical processing of auditory information in the two species. Our results seem to establish definitely the cortical nature of the sheet of cells covering the central nucleus.     

The cholinergic innervation develops early and rapidly in the rat cerebral cortex: a quantitative immunocytochemical study.

A recently developed method for determining the length of cholinergic axons and number of cholinergic axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase was used to estimate the areal and laminar densities of the cholinergic innervation in rat frontal (motor), parietal (somatosensory) and occipital (visual) cortex at different postnatal ages. This cortical innervation showed an early beginning, a few immunostained fibers being already present in the cortical subplate at birth. In the first two postnatal weeks, it developed rapidly along three parameters: a progressive increase in the number of varicosities per unit length of axon, and a lengthening and branching of the axons. Between postnatal days 4 and 16, the number of varicosities increased steadily from two to four per 10 microm of cholinergic axon. The mean densities of cholinergic axons increased from 1.4 to 9.6, 1.7 to 9.3 and 0.7 to 7.2 m/mm(3), and the corresponding densities of varicosities from 0.4 to 3.9, 0.4 to 3.5, and 0.2 to 2.6x10(6)/mm(3) in the frontal, parietal and occipital areas, respectively. The rate of growth was maximal during these first two weeks, after which the laminar pattern characteristic of each area appeared to be established. Adult values were almost reached by postnatal day 16 in the parietal cortex, but maturation proceeded further in the frontal and particularly in the occipital cortex.These quantitative data on the ingrowth and maturation of the cholinergic innervation in postnatal rat cerebral cortex substantiate a role for acetylcholine in the development of this brain region and emphasize the striking growth capacity of individual cholinergic neurons.     

The ultrastructure of the central nucleus of the inferior colliculus of the Sprague-Dawley rat

Previous studies from this laboratory have indicated that increased numbers of GABAergic neurons, as well as total neurons, occur in the central nucleus of the inferior colliculus (IC) of genetically epilepsy-prone rats (GEPRs) as compared to non-seizuring Sprague-Dawley rats. Since electron microscopic studies of the IC have not been reported for rats, we wanted to determine the ultrastructure of neurons and their processes in this brain region to serve as a basis for future studies on neuronal circuitry in the GEPRs. Both disc-shaped and stellate types were found for each of three size categories: large, medium and small. Thus, six types of neuron were distinguished by differences in somatic size, shape, organelles and dendritic orientation. Large neurons (longest diameter greater than 25 micron), which are the least frequent cell type, contained vast perikaryal cytoplasm, eccentrically located nuclei and abundant granular endoplasmic reticulum (GER) adjacent to the nucleus as well as clustered in the cytoplasm; many axosomatic, symmetric synapses were present. Medium-sized neuronal somata (15-25 micron in diameter) had smooth as well as infolded nuclear membranes and clusters of GER in their cytoplasm but no GER adjacent to the nucleus; synapses were sparse along the surface of their somata. Small neurons (10-15 micron in diameter), which are the most frequent cell type, had scant perikaryal cytoplasm, usually infolded nuclei, frequently two nucleoli, and few or no stacked cisternae of GER in the perikaryal cytoplasm; only infrequent axosomatic synapses were found. Based on previous retrograde and immunocytochemical studies, most large disc-shaped and stellate cells project to the medial geniculate body and are probably excitatory, but some large stellate neurons have been shown to be GABAergic and it is doubtful that such neurons participate in this projection. A dense plexus of terminals that form symmetric synapses covers the soma and proximal dendrites of large neurons, and may provide a strong GABAergic inhibition of this type of projection neuron. Small and medium-sized disc-shaped cells also project to the thalamus but they lack this dense axosomatic plexus. The stellate cells from these same two size categories probably do not project to the thalamus and may be GABAergic local circuit neurons. Other ultrastructural features of IC neurons that were analysed include dendrites, dendritic spines, axon hillocks, initial segments and terminals, as well as the laminae of myelinated axons. Dendrites were either beaded or smooth and few spines were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of glycine receptor alpha subunit in cultivated rat spinal neurons: an immunocytochemical study.

Ontogenesis of the inhibitory glycine receptor was studied up to 12 days in vitro in spinal neurons placed previously in culture at embryonic day 14. The alpha subunit of the receptor was detected using standard and confocal immunofluorescence and a specific monoclonal antibody. The immunostaining was compared to that of synaptophysin, a synaptic vesicle antigen, which was taken as an index of synaptic maturity. Glycine receptors could be detected intracellularly, and not at the cellular surface in some cells as early as 2-3 days in vitro (DIV) prior to any synaptic contact. At 4-5 DIV, the number of cells which expressed the immunoreactivity and the fluorescence intensity increased. At this stage, spherical fluorescent blobs started to migrate in the neurites. From 6 DIV, the glycine receptor alpha subunit was detected at the neuronal surface and was organized in clusters whose number increased progressively with time. From 7 DIV, the intrasomatic immunoreactivity decreased, and at day 12, the pattern of labelling was similar to that observed in the adult spinal cord. A diffuse presence of the receptor at the surface of neurons could never be visualised, and when detected, the glycine receptors were always clustered. Thus, the increasing expression of clusters of glycine receptors at the neuronal surface was paralleled by that of synaptophysin in neuritic varicosities. These data suggest that transport of glycine receptors to the plasmamembrane and the formation of aggregates occurs simultaneously to synaptogenesis.