Effect of transient neonatal hypothyroidism on the free aspartic acid, glutamic acid, and GABA content of different central auditory regions in the rat

The aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) contents were determined in four central auditory system regions in rats with transient neonatal hypothyroidism compared with control ones: the ventral and dorsal parts of the cochlear nucleus, the central nucleus of the inferior colliculus, the auditory cortex, and in an extra-auditory structure, the substantia nigra pars reticulata. The animals were sacrificed at 50 days of age, brain tissue samples were taken out by microdissection, and the free amino acids were extracted. The amino acid content was assessed by double-isotope labelling following two-dimensional thin-layer chromatography separation. GABA content was significantly decreased in both cochlear nucleus regions and glutamic acid was elevated in the inferior colliculus. Neonatal hypothyroidism had no significant effect on the aspartic acid levels in the regions studied. The results suggest an effect of neonatal hypothyroidism on regional contents of free amino acids known as candidate neurotransmitters in the auditory system.     

The effect of lesions of the dorsal acoustic stria on the levels of glutamic acid decarboxylase and GABA transaminase in the inferior colliculus and cochlear nucleus of the guinea-pig

The levels of glutamic acid decarboxylase (GAD) and γ-amino butyric acid-α-oxoglutarate transaminase (GABA-T) have been investigated in the cochlear nucleus and inferior colliculus of the guinea-pig after hemilateral section of the dorsal acoustic stria. Animals were cerebellectomised and the stria on one side cut. Eleven days later the animals were killed and GAD and GABA-T assayed in the respective nuclei. There was no change in the enzyme levels of the inferior colliculi showing no direct GABA-ergic fibres ascending through the stria and terminating in the inferior colliculi. The levels of both GAD and GABA-T in the cochlear nucleus on the operated side decreased significantly. It is concluded that about 30% of the GAD containing terminals in the cochlear nucleus arise from fibres descending through the dorsal acoustic stria. The bulk of the GABA-ergic transmission in the cochlear nucleus is assumed to involve an intrinsic system of short axoned neurones, the majority of which have their nerve endings in the dorsal part of the nucleus.     

Cytology of large neurons in the guinea pig dorsal cochlear nucleus contacting the inferior colliculus

Large neurons in the dorsal cochlear nucleus of the guinea pig which project to the inferior colliculus were identified after injections of the neural tracer WGA-HRP. Retrograde labelled cells (pyramidal and giant neurons) in the dorsal cochlear nucleus were glycine and GABA immunonegative and showed a similar ultrastructure. Between 30 and 60% of their perimeter was covered by axo-somatic boutons, most of which (>50%) contained pleomorphic synaptic vesicles. Other boutons (about 40% of total) contained flat vesicles and few (5-6%) contained round vesicles, a characteristic of the excitatory cells innervating the inferior colliculus. Immunogold-cytochemistry, coupled to silver intensification, showed that more than 50% of axo-somatic pleomorphic boutons and over 90% of boutons containing flat and pleomorphic vesicles store glycine. Rare WGA-HRP labelled axo-somatic boutons containing flat-pleomorphic vesicles were seen on pyramidal and giant neurons. This suggests that a few inhibitory collicular terminals contact the excitatory large neurons in the dorsal cochlear nucleus.     

Ascending and descending projections to the inferior colliculus in the rat

The ascending and descending projections to the central nucleus of the inferior colliculus (IC) were studied with the aid of retrograde transport of horseradish peroxidase (HRP). HRP-labelled cells were found in contralateral cochlear nuclei, where the majority of different cell types was stained. Few labelled cells were observed in the ipsilateral cochlear nuclei. HRP-positive neurones were found in all nuclei of the superior olivary complex on the ipsilateral side with the exception of the medial nucleus of the trapezoid body, which was never labelled either ipsilaterally or contralaterally. The largest concentration of HRP-labelled cells was usually observed in the ipsilateral superior olivary nucleus. Smaller numbers of labelled cells were present in contralateral nuclei of the superior olivary complex. Massive projections to the inferior colliculus were found from the contralateral and ipsilateral dorsal nucleus of the lateral lemniscus and ipsilateral ventral nucleus of the lateral lemniscus. Many neurones of the central and external nuclei of the contralateral inferior colliculus were labelled with HRP. Topographic organisation of the pathways ascending to the colliculus was expressed in the cochlear nuclei, lateral superior olivary nucleus and in the dorsal nucleus of the lateral lemniscus. HRP--positive cells were found in layer V of the ipsilateral auditory cortex, however, the evidence for topographic organisation was lacking.     

Frequency tuning and response latencies at three levels in the brainstem of the echolocating bat,Eptesicus fuscus

To determine the level at which certain response characteristics originate, we compared monaural auditory responses of neurons in ventral cochlear nucleus, nuclei of lateral lemniscus and inferior colliculus. Characteristics examined were sharpness of frequency tuning, latency variability for individual neurons and range of latencies across neurons.Exceptionally broad tuning curves were found in the nuclei of the lateral lemniscus, while exceptionally narrow tuning curves were found in the inferior colliculus. Neither specialized tuning characteristic was found in the ventral cochlear nuclei.All neurons in the columnar division of the ventral nucleus of the lateral lemniscus maintained low variability of latency over a broad range of stimulus conditions. Some neurons in the cochlear nucleus (12%) and some in the inferior colliculus (15%) had low variability in latency but only at best frequency.Range of latencies across neurons was small in the ventral cochlear nucleus (1.3–5.7 ms), intermediate in the nuclei of the lateral lemniscus (1.7–19.8 ms) and greatest in the inferior colliculus (2.9–42.0 ms).We conclude that, in the nuclei of the lateral lemniscus and in the inferior colliculus, unique tuning and timing properties are built up from ascending inputs.Abbreviations AVCN anteroventral cochlear nucleus - BF best frequency - CV coefficient of variation - DCN dorsal cochlear nucleus - FM frequency modulation - IC inferior colliculus - NLL nuclei of lateral lemniscus - PSTH post stimulus time histogram - PVCN posteroventral cochlear nucleus - SD standard deviation - SPL sound pressure level - VCN ventral cochlear nuclei - VNLLc ventral nucleus of the lateral lemniscus, columnar division     

Identification of tuberculo-ventral neurons in the polymorphic layer of the rat dorsal cochlear nucleus

The tuberculo-ventral tract represents a short nervous circuit within the auditory cochlear nuclei. Tuberculo-ventral neurons of the dorsal cochlear nucleus send isofrequency inhibitory inputs to bushy cells of the ventral cochlear nucleus. Injection of wheat germ agglutinin conjugated to horseradish peroxidase into the rat ventral cochlear nucleus, labelled tuberculo-ventral neurons retrogradely in the deep polymorphic layer of the ipsilateral dorsal cochlear nucleus. Five to 20% of the perimeter of these cells was covered by synaptic boutons, most of which contained flat and pleomorphic vesicles. These boutons contained glycine and sometimes GABA. Occasional small axo-somatic boutons contained round vesicles and were immunonegative for both glycine and GABA. This study shows that the synaptic profile of tuberculo-ventral neurons is different from that of other medium-size glycinergic neurons within the polymorphic layer or more superficial regions of the dorsal cochlear nucleus like cartwheel neurons. In fact the latter mostly receive boutons that contain pleomorphic vesicles.     

Alpha-neo-endorphin-like immunoreactivity in the cat brain stem

This paper examines the distribution of fibers and cell bodies containing alpha-neo-endorphin in the cat brain stem by using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive cell bodies was found in the superior central nucleus, nucleus incertus, dorsal tegmental nucleus, nucleus of the trapezoid body, and in the laminar spinal trigeminal nucleus, whereas a low density of such perikarya was observed in the inferior colliculus, nucleus praepositus hypoglossi, dorsal nucleus of the raphe, nucleus of the brachium of the inferior colliculus, and in the nucleus of the solitary tract. The highest density of immunoreactive fibers was found in the substantia nigra, dorsal motor nucleus of the vagus, nucleus coeruleus, lateral tegmental field, marginal nucleus of the brachium conjunctivum, and in the inferior and medial vestibular nuclei. These results indicate that alpha-neo-endorphin is widely distributed in the cat brain stem and suggest that the peptide could play an important role in several physiological functions, e.g., those involved in respiratory, cardiovascular, auditory, and motor mechanisms.     

大鼠脑内代谢型谷氨酸受体5亚型(mGluR5)定位分布的免疫细胞化学研究

本研究用免疫细胞化学技术观察了大鼠脑内参与兴奋性突触传递的代谢型谷氨酸受体5亚型(mGluR5)的精确定位分布.mGluR5阳性浓染的神经元胞体和纤维密集地分布于大脑皮质浅层、嗅球、伏核、尾壳核、前脑基底部、隔区、苍白球、腹侧苍白球、海马CA1和CA2区、下丘中央核、被盖背侧核和三叉神经脊束核尾侧亚核浅层;淡染而稀疏的mGluR5阳性神经元胞体和纤维见于屏状核、终纹床核、杏仁中央核、丘脑部分核团、上丘浅灰质层、外侧丘系背侧核和延髓中央灰质.     

The disc electrophoretic separation of proteins from various parts of the guinea pig brain

—Disc electrophoresis was used to study the saline-soluble and detergent-soluble proteins of various parts of the auditory pathway in the guinea pig brain. The five areas studied were the cochlear nucleus, olivary complex, inferior colliculus, medial geniculate and the auditory cortex. A study was also made of the proteins extracted from subcellular fractions obtained from guinea pig cerebral cortex. The electrophoretic patterns showed small but significant differences in the five areas of the brain, both in the fast-running acidic proteins and in the very slow-moving proteins. The higher levels of the auditory pathway, to which the more complex information processing and storage is normally attributed, showed more complex electrophoretic patterns than the lower areas. Gross differences also occurred in the patterns of both the saline-soluble and T-X-100-soluble proteins of the subcellular fractions obtained by gradient density centrifugation. The simplest patterns were obtained from the myelin-rich fraction and the mitochondrial fraction whilst the most complex patterns were given by the proteins of the nerve ending fraction and the cell soluble fraction.     

Muscarinic Receptors in the Cochlear Nucleus and Auditory Nerve of the Guinea Pig

The specific-binding properties of l-[3H]quinuclidinyl benzilate, a muscarinic acetylcholine-receptor antagonist, were investigated in synaptic and other membrane preparations of the guinea pig cochlear nucleus and auditory nerve. Binding parameters for all experiments were consistent with a single binding site with a Hill coefficient of 1.0. The binding of the ligand was specific and of high affinity, with values of KD in the range of 30-80 pM. Bmax was 0.352 +/- 0.023 pmol/mg protein for the dorsal cochlear nucleus and 0.215 +/- 0.011 pmol/mg protein for the ventral cochlear nucleus. The dorsal cochlear nucleus/ventral cochlear nucleus ratio for density of muscarinic receptors (1.6/1.0) was maintained across two different buffer systems, which varied with respect to the inclusion of proteolysis inhibitors. The results for auditory nerve indicated a level of binding much below that of the cochlear nucleus, with Bmax = 0.052 +/- 0.011 pmol/mg protein. The results of specific-binding experiments for l-[3H]quinuclidinyl benzilate support a role for acetylcholine as a neurotransmitter in the cochlear nucleus. The greater density of muscarinic receptors in the dorsal cochlear nucleus may indicate greater cholinergic activity in the dorsal relative to the ventral cochlear nucleus.     

THE EFFECT OF THE CONVULSANT 3-MERCAPTOPROPIONIC ACID ON ENZYMES OF THE γ-AMINO-BUTYRATE SYSTEM IN THE RAT CEREBRAL CORTEX

—The activities of GABA enzymes in rat cerebral cortex were studied after the administration of the convulsant, 3-mercaptopropionic acid (MP). We found that MP markedly inhibited glutamate decarboxylase (EC 4.1.1.15) and activated GABA-aminotransferase (EC 2.6.1.c). The level of GABA appeared to be decreased during convulsions but thereafter returned to normal. The study of the subcellular distribution of GABA enzymes after the administration of MP indicated that the glutamate decarboxylase present in the nerve endings was not affected, while GABA aminotransferase in the mitochondria was activated to a similar extent to that observed in the original homogenate. These results together with the recovery of glutamate decarboxylase activity in cortical homogenates by dialysis suggested a reversible type of inhibition, whereas the effect on GABA-aminotransferase seemed to be more permanent.     

Distribution of some enzymes associated with the metabolism of glutamate, aspartate, gamma-aminobutyrate and glutamine in cat spinal cord

The activities of glutamine synthetase, glutaminase, glutamate decarboxylase, GABA aminotransferase, glutamate dehydrogenase, and aspartate aminotransferase were measured in four areas of the cat spinal cord and in dorsal and ventral roots. Five of the six enzymes showed identical distribution patterns; i.e. the activities in the dorsal and ventral gray matter were equal and those of dorsal and ventral white matter were equal. No statistical differences in the mean enzyme activities in the dorsal and ventral roots were found. Glutamate decarboxylase was the only enzyme which had a different pattern. The enzyme activity in dorsal gray was twice that of ventral gray; the same pattern as the GABA concentration in both these areas. The glutamine synthetase activities in the cord areas and roots correlated with the glutamine distribution reported earlier. Thus, the distribution of glutamine (not a transmitter) and GABA (questionable transmitter) in gray matter are dictated by their synthesizing enzymes, whereas the distribution of glutamate and aspartate (likely transmitter suspects) cannot be explained on the basis of enzyme activities. Therefore, the enzyme activities may be related to the amino acid levels primarily in metabolic compartments, whereas the excess of certain amino acids in specific areas of the cord and roots may be related to functional compartments accumulated for use in synaptic transmission.     

Distribution of myo-Inositol in the Cat Cochlear Nucleus

Abstract: The distribution of myo -inositol, a substance that has been implicated in synaptic transmission, has been mapped within sections of the cat cochlear nucleus as well as some nearby regions. Highest values in the cochlear nucleus were found in regions of granule cells along the periphery of the anteroventral subdivision of the nucleus. Highest values overall were found in the molecular layer of the cerebellar flocculus. A fairly good correlation was found between myo -inositol levels and activities of the enzymes of acetyl-choline metabolism in the cat cochlear nucleus, supporting the possibility that myo -inositol may be involved in cholinergic synaptic transmission. No positive correlation was found between myo -inositol levels and the levels of glutamate, aspartate, glycine, or γ-aminobutyric acid (GABA). The most striking gradient of myo -inositol levels within a region was found in the auditory nerve, where different myo -inositol levels might be related to nerve fibers innervating different parts of the cochlea. The distribution of scyllo -inositol, a stereoisomer of myo -inositol, was also examined, and found to parallel closely the distribution of myo -inositol, with levels 4–5% as high.     

Content of components GABA metabolism in the brain of cats and rats

The activity of glutamate decarboxylase (L-glutamate carboxy-1-lyase; EC 4.1.1.15), GABA-transaminase (GABA-alpha-ketoglutarate aminotransferase, EC 2.6.1.19), content of gamma-aminobutyric, glutamic and aspartic acids were studied in different parts and subcellular particles of the cat and rat brain. It is shown that regional and subcellular distribution of the GABA metabolic components in the cat and rat brain are mainly similar, but quantitative indices are different.     

Postnatal development of GABA- and glycine-like immunoreactivity in the cochlear nucleus of the Mongolian gerbil (Meriones unguiculatus)

The maturation of the morphological substrate for inhibitory interactions was investigated in the cochlear nucleus of the gerbil with immunocytochemistry for gamma aminobutyric acid (GABA) and glycine on alternating vibratome sections. The patterns of immunostaining obtained with both antibodies in the adult closely conformed to the general mammalian scheme. Qualitative analyses revealed an age-related increase in staining intensity and in the relative numbers of immunolabelled cells after birth up to the age of 3–4 weeks. As early as birth and in all subdivisions of the cochlear nucleus, a few labelled cells and puncta in the sections were stained either with the GABA or the glycine antibody. Immunoreactive puncta and cells were, however, far less abundant than in the adult, and the staining intensity of cells was only weak. The most strikingly GABA-immunolabelled cells at birth were the Golgi cells of the granule-cell domains. The numbers of weakly GABA- and glycine-immunostained cells of the dorsal cochlear nucleus clearly increased between birth and the third postnatal week. At approximately the onset of hearing (postnatal day 12–14), some cells of the dorsal cochlear nucleus and small cells of the ventral cochlear nucleus gained adult-like GABA-staining properties. Almost adult-like labelling intensity was observed in glycine-immunoreactive cells of the deep dorsal cochlear nucleus and in some small cells of the ventral cochlear nucleus. Puncta staining to both antibodies appeared adult-like throughout the cochlear nucleus. About 2 weeks after the onset of hearing (at the latest), adult-like staining of all subsets of immunoreactive cells occurred throughout the cochlear nucleus in all specimens. Received: 25 March 1997 / Accepted: 15 March 1998     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.     

Uptake and Release of γ-Aminobutyric Acid in the Guinea Pig Cochlear Nucleus After Axotomy of Cochlear and Centrifugal Fibers

Abstract: This study attempts to determine if γ-aminobutyric acid (GABA) may be a transmitter of cochlear nerve fibers projecting from the cochlea to the cochlear nucleus, and of centrifugal fibers projecting to the cochlear nucleus via the trapezoid body and the acoustic striae of the medulla. The uptake and the electrically evoked release of exogenous [¹⁴C]GABA were measured, in vitro, in the three major subdivisions of the guinea pig cochlear nucleus: the anteroventral, posteroventral, and dorsal cochlear nuclei. These activities were compared using unlesioned animals, animals with bilateral cochlear ablations, and animals whose trapezoid body and acoustic striae were interrupted on the right side of the medulla. Subdivisions from unlesioned animals took up [¹⁴C]GABA, achieving concentrations in the tissues that were 11–19 times that in the medium. Electrical stimulation evoked a Ca²⁺-dependent release of [¹⁴C]GABA from each subdivision. Bilateral cochlear ablation, which presumably destroyed the cochlear nerve fibers, had no effect on [¹⁴C]GABA uptake and release. Section of the trapezoid body and the acoustic striae on the right side of the medulla typically severed all known connections of the right posteroventral and dorsal cochlear nuclei with the rest of the brain, but left intact many connections involved with the right anteroventral cochlear nucleus. This lesion partially depressed [¹⁴C]GABA uptake and release in the right posteroventral and dorsal cochlear nuclei, but not in the right anteroventral cochlear nucleus. These findings suggest that one or more of the centrifugal tracts projecting to the cochlear nucleus may be GABAergic, 88% or more of the cochlear nerve fibers probably are not GABAergic, and some neurons of the cochlear nucleus are probably GABAergic.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

Evidence for a Glutamatergic Pathway from the Guinea Pig Auditory Cortex to the Inferior Colliculus

Abstract: We attempt to provide evidence that the projection from the guinea pig auditory cortex (AC) to the inferior colliculus (IC) may contain glutamatergic or GABAergic fibers. Seven days after unilateral AC aspiration, histological studies indicated almost complete AC destruction and preterminal degeneration of fibers and terminal fields in the dorsal cortex (DCIC), external cortex (ECIC), and central nucleus (CNIC) of the IC ipsilateral to the ablated AC. Contralaterally, degeneration appeared in the DCIC. AC ablation depressed the electrically evoked Ca²⁺-dependent release of d -[³H]aspartate ( d -[³H]Asp) in the ipsilateral DCIC, ECIC, and CNIC, and d -[³H]Asp uptake in the CNIC. Together with other evidence that the corticocollicular pathway is excitatory, these findings suggest that this projection may contain glufamatergic and/or aspartatergic (Glu/Asp-ergic) fibers. Glutamic acid decarboxylase immunoreactivity was not apparent in presumed pyramidal cells of layer V of the AC retrogradely labeled with biotinylated dextran injected into the ipsilateral IC. Thus, corticocollicular neurons probably do not synthesize GABA and may not be GABAergic. However, AC ablation depressed [¹⁴C]GABA release from the ipsilateral DCIC and ECIC, and [¹⁴C]GABA uptake in the DCIC. These findings are consistent with the atrophy or down-regulation of some subcortical neurons that mediate GABAergic transmission in the IC.     

Neonatal deafening causes changes in Fos protein induced by cochlear electrical stimulation

The influence of neonatal deafness on cochlear electrically evoked Fos expression in the auditory brainstem was examined. Newborn rats were deafened by systemic injection of kanamycin, 1 mg/g daily for 12 days. At 4, 5, 6 or 8 weeks of age, these animals received cochlear electrical stimulation with a basal monopolar electrode for 90 minutes. Age-matched untreated control animals received similar stimulation. Experimental and control animals were assessed for spiral ganglion cell densities and Fos immunoreactive staining in the central nucleus of the inferior colliculus. Spiral ganglion cell assessments showed significant decreases in spiral ganglion cell densities in deafened rats compared to age-matched controls, at 5 weeks of age in lower turns and 6 and 8 weeks in all turns. Cochlear electrical stimulation induced Fos immunoreactive staining in the nucleus of auditory brain stem neurons in treatment and control groups. A significantly greater number of Fos immunoreactive neurons was found in the contralateral central nucleus of inferior colliculus in 5, 6 and 8 week old deafened animals compared to age-matched controls. The increases were larger with a longer duration of deafness. These results suggest that there are changes in auditory processing as a consequence of neonatal deafness.