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Glutamate is widely distributed in the central nervous system (CNS) and is present in greater amounts than any other putative neurotransmitter. To study its distribution in the CNS, a monoclonal antibody was raised against gamma-L-glutamyl-L-glutamic acid (gamma-Glu-Glu) conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehydeborohydride....
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... Next, we examined whether CTB-labeled neurons in the PL, BLA, and vHIP are glutamatergic using monoclonal anti-gluta-mate antibody (GLU-4 for short), which has been used for labeling glutamatergic neurons [45,46]. In the PL region examined (AP, 1.98±0 mm), 89.8% of CTB-labeled neurons cells (743/829) were stained by GLU-4 antibody, a specific marker for labeling glutamatergic neurons, whereas 10.2% (86/829) were not labeled by GLU-4 (Fig. 2A~C). ...
The nucleus accumbens (NAc) is the major component of the ventral striatum that regulates stress-induced depression. The NAc receives dopaminergic inputs from the ventral tegmental area (VTA), and the role of VTA-NAc neurons in stress response has been recently characterized. The NAc also receives glutamatergic inputs from various forebrain structures including the prelimbic cortex (PL), basolateral amygdala (BLA), and ventral hippocampus (vHIP), whereas the role of those glutamatergic afferents in stress response remains underscored. In the present study, we investigated the extent to which descending glutamatergic neurons activated by stress in the PL, BLA, and vHIP project to the NAc. To specifically label the input neurons into the NAc, fluorescent-tagged cholera toxin subunit B (CTB), which can be used as a retrograde neuronal tracer, was injected into the NAc. After two weeks, the mice were placed under restraint for 1 h. Subsequent histological analyses indicated that CTB-positive cells were detected in 170~680 cells/mm² in the PL, BLA, and vHIP, and those CTB-positive cells were mostly glutamatergic. In the PL, BLA, and vHIP regions analyzed, stress-induced c-Fos expression was found in 20~100 cells/mm². Among the CTB-positive cells, 2.6% in the PL, 4.2% in the BLA, and 1.1% in the vHIP were co-labeled by c-Fos, whereas among c-Fos-positive cells, 7.7% in the PL, 19.8% in the BLA, and 8.5% in the vHIP were co-labeled with CTB. These results suggest that the NAc receives a significant but differing proportion of glutamatergic inputs from the PL, BLA, and vHIP in stress response.
... In 1984, Panula described that antibodies raised using an immunogen of histamine conjugated to hemocyanin with carbodiimide identify histaminergic neurons as being localized in the posterior hypothalamus [52]. In addition to its properties as a conjugant, 2% carbodiimide proved to be a good fixative for immunohistochemistry [53], but staining improved if tissues were fixed with carbodiimide at 4% followed by 4% or 5% paraformaldehyde as a post-fixative [54]. Irrespective of some differences in methodological procedures, neuroanatomical studies of mammalian taxa involving mouse, rat, guinea pig, and tree shrew show prominent histamine immunoreactivity for cell bodies in the posterior hypothalamus, now recognized as being the tuberomammillary nuclear (TM) complex [29,55,56]. ...
A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC), would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD) because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness.
... As mentioned above, the neurotransmitter involved in the excitatory input may be glutamate (Madl et al., 1986;Wu, 1998). Wu and Kelly (1996) and Fu et al. (1997aFu et al. ( , 1997b have shown that there are two separate excitatory mechanisms operating on the DLL neurons. ...
This chapter highlights the functional organization of the mouse auditory system and focuses on mouse as a useful and economical animal model for hearing research. Advances in gene targeting have led to a renaissance in the use of the mouse as a model for investigation of human disease mechanisms. Until recently, the most commonly used experimental animal for auditory research and many other fields in neuroscience was the cat; other species used to a lesser extent include the rat, guinea pig, chinchilla, gerbil, and ferret. The availability of strains of mice that carry specific genetic abnormalities affecting the brain, including the auditory system, has also proven useful to researchers. Understanding the diseases of the human auditory system and the underpinning cellular and molecular mechanisms is of primary interest in current hearing research. Comparative hearing research is important, because animal models can be developed, evaluated and eventually applied to clinical problems. The investigation of mouse mutants with hearing impairments is useful for elucidating the pathological processes underlying auditory defects, as well as for understanding the normal process of auditory development and sensory transduction. Deaf mouse mutants are also valuable for identifying the responsible genes by positional cloning, and are being used in the search for genes involved in human deafness.
... The immunostaining procedure was performed using a monoclonal antiserum directed against the carbodiimide conjugate of glutamate, as described earlier (Madl et al, 1986). Mean intensities were calculated from the pixel intensity histograms generated from image mosaics. ...
... Several other neurochemicals, including myo-inositol, lactate, and GABA, were altered in the HIPP of GLS1 het mice (Supplementary Information,Figure S1b). To verify the decreases in glutamate further, tissue sections were immunostained with Glu2, a mouse monoclonal antibody directed against carbodiimide-conjugated glutamate (Madl et al, 1986). The experiment included 21 sections from FC, 26 from HIPP, and 18 from THAL. ...
Dysregulated glutamatergic neurotransmission has been strongly implicated in the pathophysiology of schizophrenia (SCZ). Recently, presynaptic modulation of glutamate transmission has been shown to have therapeutic promise. We asked whether genetic knockdown of glutaminase (gene GLS1) to reduce glutamatergic transmission presynaptically by slowing the recycling of glutamine to glutamate, would produce a phenotype relevant to SCZ and its treatment. GLS1 heterozygous (GLS1 het) mice showed about a 50% global reduction in glutaminase activity, and a modest reduction in glutamate levels in brain regions relevant to SCZ pathophysiology, but displayed neither general behavioral abnormalities nor SCZ-associated phenotypes. Functional imaging, measuring regional cerebral blood volume, showed hippocampal hypometabolism mainly in the CA1 subregion and subiculum, the inverse of recent clinical imaging findings in prodromal and SCZ patients. GLS1 het mice were less sensitive to the behavioral stimulating effects of amphetamine, showed a reduction in amphetamine-induced striatal dopamine release and in ketamine-induced frontal cortical activation, suggesting that GLS1 het mice are resistant to the effects of these pro-psychotic challenges. Moreover, GLS1 het mice showed clozapine-like potentiation of latent inhibition, suggesting that reduction in glutaminase has antipsychotic-like properties. These observations provide further support for the pivotal role of altered glutamatergic synaptic transmission in the pathophysiology of SCZ, and suggest that presynaptic modulation of the glutamine-glutamate pathway through glutaminase inhibition may provide a new direction for the pharmacotherapy of SCZ.
... no. G9282; Sigma, St. Louis, MO) raised by Madl et al. [63]. The specificity of both antibodies in fly tissues is revealed because both gave similar labeling patterns in Drosophila to an antibody against vGluT (above), and both immunolabeled the same cells in two other species of fly (Musca, Calliphora) that they labeled in Drosophila. ...
Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA(B) type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA(B) receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA(A) receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in alpha-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.
... Controls for nonspecific labeling. The specificity of all antibodies used in this study has been tested previously by ELISA, by immunoblotting, and with immunohistochemical procedures (7,13,14,16,22,24,25). As additional controls in the present study, we substituted either normal rabbit serum or normal mouse serum for the primary antisera and also omitted primary antibodies. ...
... in the present experiments produce specific labeling of GABAergic and glutamatergic neurons, respectively (7,13,14,16,22,24,25). As additional controls, we conducted immunoperoxidase analyses of the labeling of neurons in the cerebellar cortex whose phenotype is established. ...
... The patterns of labeling achieved with glutamate immunohistochemical localizations are illustrated in Fig. 1, B and C. Note that granule cells are densely stained, whereas they did not express GAD immunoreactivity. This pattern conforms to that documented in prior immunocytochemical localizations of glutamate (20,24). A very few Purkinje cells expressed a low level of glutamate-LI, presumably reflecting an intracellular pool of glutamate employed for the synthesis of GABA or glutamate-containing proteins. ...
In prior studies that used transneuronal transport of isogenic recombinants of pseudorabies virus, we established that medial medullary reticular formation (MRF) neurons sent collateralized projections to both diaphragm and abdominal muscle motoneurons. Furthermore, inactivation of MRF neurons in cats and ferrets increased the excitability of diaphragm and abdominal motoneurons, suggesting that MRF neurons controlling respiratory activity are inhibitory. To test this hypothesis, the present study determined the neurochemical phenotypes of MRF premotor respiratory neurons in the ferret by using immunohistochemical procedures. Dual-labeling immunohistochemistry combining pseudorabies virus injections into respiratory muscles with the detection of glutamic acid decarboxylase-like immunoreactive and glutamate-like immunoreactive cells showed that both GABAergic and glutamatergic MRF neurons project to respiratory motoneurons, although the latter are more common. These data suggest that the role of the MRF in respiratory regulation is multifaceted, as this region provides both inhibitory and excitatory influences on motoneuron activity.
... The major concern of the present study was to detect a possible participation of GABAergic inhibition in the cerebellar projection to the PN and the NRTP. We used antibodies against both GABA and glutamate to label the BDA-traced terminals-assuming that these are the transmitters used by LN projection neurons (Chan-Palay, 1977;Madl et al., 1986Madl et al., , 1987Monaghan et al., 1986;Kumoi et al., 1988;De Zeeuw et al., 1989). Ideally, a homogeneous population of projection neurons using only one transmitter should show no overlap in the densities of staining with the two antisera. ...
The pontine nuclei (PN) and the nucleus reticularis tegmenti pontis (NRTP) are sources of an excitatory projection to the cerebellar cortex via mossy fibers and a direct excitatory projection to the cerebellar nuclei. These precerebellar nuclei, in turn, receive a feedback projection from the cerebellar nuclei, which mostly originate in the lateral nucleus (LN). It has been suggested that the feedback projection from the LN partially uses gamma-aminobutyric acid (GABA) as a transmitter. We tested this hypothesis by using a combination of anterograde tracing (biotinylated dextran amine injection into the LN) and postembedding GABA and glutamate immunogold histochemistry. The pattern of labeling in the PN and the NRTP was compared with that of cerebellonuclear terminals in two other target structures, the parvocellular part of the nucleus ruber (RNp) and the ventromedial and ventrolateral thalamus (VM/VL). The projection to the inferior olive (IO), which is known to be predominantly GABAergic, served as a control. A quantitative analysis of the synaptic terminals labeled by the tracer within the PN, the NRTP, and the VL/VM revealed no GABA immunoreactivity. Only one clearly labeled terminal was found in the RNp. In contrast, 72% of the terminals in the IO were clearly GABA immunoreactive, confirming the reliability of our staining protocol. Correspondingly, glutamate immunohistochemistry labeled the majority of the cerebellonuclear terminals in the PN (88%), the NRTP (90%), the RNp (93%), and the VM/VL (63%) but labeled only 5% in the IO. These data do not support a role for GABAergic inhibition either in the feedback systems from the LN to the PN and the NRTP or within the projections to the RNp and the VM/VL.
... Several body regions were excised, although the tentacles and oral disk were investigated in the greatest detail. The immunocytochemical procedure is based on the use of a monoclonal antibody characterized by Madl et a!. ( 1986). The antibody, purchased from INCSTAR, was raised in mouse against the dipeptide ‘¿ y-L-glutamyl-L-glu tamic acid (-y-Glu-Glu) conjugated to keyhole limpet he mocyanin via glutaraldehyde-borohydride. ...
... Control procedures indicated that the observed im munostaining represents specific antibody binding to ‘¿ y Glu-Glu. The absence of effect or small effect of pread sorption tests with glutamate, N-Ac-Glu, and Asp-Glu on immunostaining in our preparations is largely consistent with the results ofcorresponding tests elsewhere (Madl et al., 1986). Moreover, the absence of immunostaining in preparations fixed without carbodiimide all but excludes the presence of endogenous y-Glu-Glu in the tissues. ...
The distribution of glutamate in the tentacles and oral disk of the sea anemone Metridium senile was investigated by wholemount immunohistochemistry with the use of a monoclonal antibody raised against the derivatization product gamma-L-glutamyl-L-glutamic acid. Immunoreactivity was localized in one class of tentacle nematocysts and on their associated threads. These nematocysts were concentrated at the distal end of tentacles, none being found at the base of tentacles or in the oral disk. Muscle end-feet of epitheliomuscular cells also stained in the longitudinal muscle of tentacle ectoderm. In contrast, immunostaining in the oral disk was confined to ectodermal granule-containing cells overlying the radial muscle. These results support a role for glutamate as an osmolyte precursor in nematocysts but provide little clue as to the functional significance of this amino acid in muscle and gland cells.
... A variety of biochemical and autoradiographic techniques have been used to study the distribution of these amino acids in the CNS. Antibodies prepared against amino acids conjugated to antigenic proteins can be quite specific; immunocytochemistry using these antibodies has proved to be a sensitive tool for both light and electron microscopic studies (Van den Pol et al., 1990;Chagnaud et al., 1989;Liu et al., 1989;Ottersen, 1989;Hepler et al., 1988;Aoki et al., 1987;Campistron et al., 1986;Mad1 et al., 1986;Matute and Streit, 1986;Somogyi et al., 1985;Storm-Mathisen et al., 1983). The most persuasive immunocytochemical evidence for identification of neurotransmitters in a given pathway is their demonstration in axon terminals (Zhang et al., 1990), which can only be achieved with electron microscopy (EM). ...
We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.
... They reported that in the cerebral cortex immunoreactivity is localized in pyramidal, as well as nonpyramidal neurons of all cortical layers. Since then, efforts in a number of other laboratories have resulted in the production of antibodies directed against excitatory amino acids conjugated to protein carriers, which specifically demonstrate Glu and Asp in perfusion-fixed tissue (Campistron et al., 1986;Madl et al., 1986Madl et al., , 1987Aoki et al., 1987;Hepler et al., 1988). Using these antibodies, investigators have provided descriptions of the morphology and distribution of amino acid-containing neurons in the neocortex of a variety of mammalian species. ...
... For example, some authors reported that in the somatosensory cortex of rats, cats and monkeys Glu-immunoreactivity is localized mainly in pyramidal neurons distributed in layers II, ID, V, and VI (Conti et al., 1987b(Conti et al., , 1989. Others described Glu-like immunoreactivity chiefly in pyramidal cells of layer in of cortical area 18 in the rat, while labelled cells were not detected in area 18a (Madl et al., 1986). The presence of Asp-containing neurons, the majority of which are pyramidal cells, has been shown in various areas of the rat cortex, but again reports on the distribution of these cells in the neocortex vary between studies (Campistron et al., 1986;Aoki et al., 1987;Conti et al., 1987a;Madl et al., 1987). ...
The distribution and morphology of neurons containing glutamate (Glu) or aspartate (Asp) were examined in the developing and adult rat visual cortex using the peroxidase-antiperoxidase technique at the light and electron microscopic levels. In the adult, Glu-labelled neurons were pyramidal cells distributed in layers El-VI with a preponderance in layers II-IIL Asp-positive neurons were distributed throughout layers I-VI, with the highest density in layers Il-in and the lowest in layer IV and the upper part of layer V. The majority of labelled neurons were pyramidal, whilst a small number of nonpyramidal cells was also observed. Glu- and Asp-immunoreactive neurons were first observed in the immature visual cortex on posmatal day 3, but did not demonstrate their adult pattern of distribution until the fourth posmatal week. Both populations attained their mature ultrastructural features by the end of the third posmatal week. The combination of immunohistochenistry with wheat germ agglutinin- horseradish peroxidase (WGA-HRP) histochemistry was used to examine the Glu- and Asp-containing cortical neurons which give rise to corticofugal and callosal projections. Injections of WGA-HRP into the pons resulted in retrograde labelling of large cells in layer V. Of these cells, 42% were also Glu-immunoreactive while 51% were Asp- positive. Injections in the superior colliculus labelled large and medium-sized neurons in layer V, of these cells, 46% were also stained for Glu and 66% for Asp. Injections in the dorsal lateral geniculate nucleus labelled cells throughout layer VI. Of these, 60% were also Glu-immunoreactive and 61% Asp-positive. Finally, after injections in the visual cortex, labelled cells were found throughout layers II-VI in the contralateral visual cortex. Of these cells, 38% were also labelled for Glu while 49% were also Asp- immunoreactive.
