Biochemical evidence for localization of AMPA-type glutamate receptor subunits in the dendritic raft

A low density Triton-insoluble fraction with characteristic lipid composition was prepared from synaptic plasma membrane from the rat forebrain. The fraction was named dendritic raft based on its absence of the presynaptic marker synaptophysin, the presence of postsynaptic Glutamate receptor (GluR) subunits, and its resemblance to raft, caveolae-like structure. We found a differential distribution of NMDA-type and AMPA-type GluR subunits in the dendritic raft and postsynaptic density (PSD) fractions; the latter type GluR subunits were localized to the dendritic raft as well as PSD fraction, whereas the former type was mostly localized to the PSD fraction. We also found the differential distribution of the components of ras/mitogen-activated protein kinase (MAPK) pathway to the dendritic raft and PSD fractions. Dendritic raft and PSD may possibly interact at the postsynaptic sites for efficient signal processing that is required for expression of synaptic plasticity.     

Cellular localization of AMPA type glutamate receptor subunits in the basal ganglia of pigeons (Columba livia)

Corticostriatal and thalamostriatal projections utilize glutamate as a neurotransmitter in mammals and birds. The influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1-4 subunits. Although the cellular localization of AMPA-type subunits has been well characterized in mammalian basal ganglia, their localization in avian basal ganglia has not. We thus carried out light microscopic single- and double-label and electron microscopic single-label immunohistochemical studies of GluR1-4 distribution and cellular localization in pigeon basal ganglia. Single-label studies showed that the striatal neuropil is rich in GluR1, GluR2, and GluR2/3 immunolabeling, suggesting the localization of GluR1, GluR2 and/or GluR3 to the dendrites and spines of striatal projection neurons. Double-label studies and perikaryal size distribution determined from single-label material indicated that about 25% of enkephalinergic and 25% of substance P-containing striatal projection neuron perikarya contained GluR1, whereas GluR2 was present in about 75% of enkephalinergic neurons and all substance-P -containing neurons. The perikaryal size distribution for GluR2 compared to GluR2/3 suggested that enkephalinergic neurons might more commonly contain GluR3 than do substance P neurons. Parvalbuminergic and calretininergic striatal interneurons were rich in GluR1 and GluR4, a few cholinergic striatal interneurons possessed GluR2, but somatostatinergic striatal interneurons were devoid of all subunits. The projection neurons of globus pallidus all possessed GluR1, GluR2, GluR2/3 and GluR4 immunolabeling. Ultrastructural analysis of striatum revealed that GluR1 was preferentially localized to dendritic spines, whereas GluR2/3 was found in spines, dendrites, and perikarya. GluR2/3-rich spines were generally larger than GluR1 spines and more frequently possessed perforated post-synaptic densities. These results show that the diverse basal ganglia neuron types each display different combinations of AMPA subunit localization that shape their responses to excitatory input. For striatal projection neurons and parvalbuminergic interneurons, the combinations resemble those for the corresponding cell types in mammals, and thus their AMPA responses to glutamate are likely to be similar.     

A developmental expression of AMPA-selective glutamate receptor subunits in human basal ganglia

The development of AMPA-selective glutamate receptors (GluR1 and GluR2–3) in human basal ganglia (BG) was investigated in 23 normal brains by means of an immunohistochemical method. Immunoreactivity to GluR1 and 2–3 was detected in the cytoplasm and dendrites of small and large neurons in the BG. GluR2–3 immunoreactivity-positive neurons were clearly observed at 23–24 gestational weeks (GW) in the globus pallidus and at 32 GW in the neostriatum, and reacted a peak at 32 GW and 39 GW, respectivelt. GluR2–3 positive neurons in the BG began to decrease at 1–4 months of age, reaching the low level of adults by 7 months of age. The developmental pattern of GluR1 was similar to that of GluR2–3 in the BG, but the immunoreactivity to GluR1 was a little weaker than that of GluR2–3 in the neostriatum. Furthermore, GluR1 and 2–3 subunit Western blotting confirmed the specificity of the immunohistochemistry. Our results suggest that the development of GluR1 and 2–3 in the BG is consistent with neuronal development in the BG, which supports further that GluR1 and 2–3 play an important role in neuronal differentiation and maturation of the BG.     

Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal ganglia

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.     

Presynaptic localization of an AMPA-type glutamate receptor in corticostriatal and thalamostriatal axon terminals

The neostriatum is known to receive glutamatergic projections from the cerebral cortex and thalamic nuclei. Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) are located on axon terminals of corticostriatal and thalamostriatal afferents, respectively, whereas VGluT3 is found in axon terminals of cholinergic interneurons in the neostriatum. In the present study, the postsynaptic localization of ionotropic glutamate receptors was examined in rat neostriatum by the postembedding immunogold method for double labelling of VGluT and glutamate receptors. Immunoreactive gold particles for AMPA receptor subunits GluR1 and GluR2/3 were frequently found not only on postsynaptic but also on presynaptic profiles immunopositive for VGluT1 and VGluT2 in the neostriatum, and GluR4-immunoreactive particles were observed on postsynaptic and presynaptic profiles positive for VGluT1. Quantitative analysis revealed that 27-45% of GluR1-, GluR2-, GluR2/3- and GluR4-immunopositive particles found in VGluT1- or VGluT2-positive synaptic structures in the neostriatum were associated with the presynaptic profiles of VGluT-positive axons. In contrast, VGluT-positive presynaptic profiles in the neostriatum showed almost no immunoreactivity for NMDA receptor subunits NR1 or NR2A/B. Furthermore, almost no GluR2/3-immunopositive particles were observed in presynaptic profiles of VGluT3-positive (cholinergic) terminals that made asymmetric synapses in the neostriatum, or in those of VGluT1- or VGluT2-positive terminals in the neocortex. The present results indicate that AMPA receptor subunits but not NMDA receptor subunits are located on axon terminals of corticostriatal and thalamostriatal afferents, and suggest that glutamate released from these axon terminals controls the activity of the terminals through the presynaptic AMPA autoreceptors.     

Immunohistochemical localization of metabotropic glutamate receptors mGluR1a and mGluR2/3 in the rat basal ganglia

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule-associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2-ir or SV2-ir and was not altered by decortication or unilateral 6-hydroxydopamine (6-OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a-ir was tightly clustered along large MAP2-ir dendrites. In contrast to the variations in mGluR1a-ir staining, similar punctate neuropil mGluR2/3-ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3-ir puncta colocalized with SV2-ir, and striatal mGluR2/3-ir puncta were markedly reduced in number after decortication. Neither mGluR1a-ir nor mGluR2/3-ir could be detected in TH-ir soma within substantia nigra pars compacta, or in TH-ir striatal terminals. Overall, our observations suggest that mGluR1a and mGluR2/3 receptors have distinct cellular localizations in different components of the basal ganglia circuitry and are likely to subserve distinct functions. Our data support the presence of mGluR2/3 on the terminals of corticostriatal afferents, where they may regulate glutamate release. In contrast, mGluR1a appears to be a postsynaptic receptor of neurons in the neostriatum, globus pallidus, and substantia nigra pars reticulata. J. Comp. Neurol. 390:5–19, 1998. © 1998 Wiley-Liss, Inc.     

AMPA-type glutamate receptor subunits are expressed in the avian cochlear hair cells and ganglion cells.

The cellular localization of AMPA-type glutamate receptor subunits was examined in the pigeon inner ear using subunit specific polyclonal antibodies (GluR1-4). In the auditory ganglion cell bodies immunoreactivity for the subunits GluR2/3 and GluR4, but not for GluR1 was detected. The hair cells showed diffuse immunoreactivity for GluR4. Additionally, immunostaining for the subunits GluR2/3 and GluR4 was present below the hair cells. These results indicate that the AMPA type glutamate receptors play a role in neurotransmission at the hair cell afferent synapse in the avian auditory system.     

Expression of the AMPA-type glutamate receptor subunits in the chick optic tectum changes biphasically after retinal deafferentation

Effects of retinal lesions on the expression of AMPA-type glutamate receptor (GluR) subunits in the chick optic tectum were evaluated with immunohistochemistry and immunoblotting. Expression of GluR1 and GluR2/3 subunits decreased in the deafferented tectum after 2 days and increased after 7 days postlesion. These results suggest biphasic effects of retinal lesions upon the expression of GluR subunits, possibly due to removal of the glutamatergic input from the retina.     

Adenosine A2A receptor in the monkey basal ganglia: ultrastructural localization and colocalization with the metabotropic glutamate receptor 5 in the striatum

The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.     

Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat basal ganglia

The differential expression of specialized voltage-gated potassium (Kv) channel subtypes in the nervous system probably reflects the wide range of functions. Although there have been previous reports in the cellular and subcellular localizations of various Kv mRNAs and proteins, the comprehensive study described here is the first in which the expression of six Kv1 channel subunits have been directly compared in the rat basal ganglia. In the present study, we have found that staining patterns of the six Kv1 channel subunits overlap in some areas of the basal ganglia, but each has a unique pattern of expression. It was noted that Kv 1.4 subunit had a strikingly high level of expression in the globus pallidus compared to the caudate-putamen. This distinct distribution formed the clear demarcations between caudate-putamen and globus pallidus. The dot-like staining pattern of Kv1 subunits was observed through the accumbens nucleus. Strong staining for Kv1.4 was observed in the cerebral peduncle, not in the subthalamic nucleus. In the substantia nigra, immunoreactivity for Kv1.4 subunit was prominent in the pars reticulata of the substantia nigra. The staining intensity for Kv1.2 was high in the pars compacta of the substantia nigra. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the basal ganglia.     

Co-localization of AMPA-type glutamate receptor immunoreactivity in neurons of the rat subthalamic nucleus

In order to determine the precise cellular localization of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunit immunoreactivity in the rat subthalamic nucleus, single and double immunofluorescence was performed. Intense level of GluR1, GluR2, GluR2/3 and GluR4 immunoreactivity was found in almost all neurons of the subthalamic nucleus. By double immunofluorescence, the subthalamic neurons in the same sections that displayed a strong immunoreactivity for GluR1 were found to display a robust GluR2 immunoreactivity and the subthalamic neurons that displayed GluR2 immunoreactivity were also found to express GluR4 immunoreactivity. The present results thus demonstrate that individual neurons of the subthalamic nucleus are likely to co-express GluR1 and GluR2, and GluR2 and GluR4 immunoreactivity. The native AMPA channels in the subthalamic neurons may, therefore, be composed of heteromeric subunits. The present results provide information of the neuroanatomical localization of AMPA receptor subunits in neurons of the subthalamic nucleus. The localization of AMPA receptor subunits may be related to functional characteristics of AMPA channels in the subthalamic neurons.     

alpha-actinin-2 in rat striatum: localization and interaction with NMDA glutamate receptor subunits

Alpha-actinin (alpha-actinin-2) is a protein which links the NR1 and NR2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptors to the actin cytoskeleton. Because of the importance of NMDA receptors in modulating the function of the striatum, we have examined the localization of alpha-actinin-2 protein and mRNA in striatal neurons, and its biochemical interaction with NMDA receptor subunits present in the rat striatum. Using an alpha-actinin-2-specific antibody, we found intense immunoreactivity in the striatal neuropil and within striatal neurons that also expressed parvalbumin, calretinin and calbindin. Conversely, alpha-actinin-2 immunoreactivity was not detected in neurons expressing choline acetyltransferase and neuronal nitric oxide synthase. Dual-label in situ hybridization revealed that the highest expression of alpha-actinin-2 mRNA is in substance P-containing striatal projection neurons. The alpha-actinin-2 mRNA is also present in enkephalinergic projection neurons and interneurons expressing parvalbumin, choline acetyl transferase and the 67-kDa isoform of glutamic acid decarboxylase, but was not detected in somatostatin-expressing interneurons. Immunoprecipitation of membrane protein extracts showed that alpha-actinin-2 is present in heteromeric complexes of NMDA subunits, but is not associated with AMPA receptors in the striatum. A subunit-specific anti-NR1 antibody co-precipitated major fractions of NR2A and NR2B subunits, but only a minor fraction of striatal alpha-actinin-2. Conversely, alpha-actinin-2 antibody immunoprecipitated only modest fractions of striatal NR1, NR2A and NR2B subunits. These data demonstrate that alpha-actinin-2 is a very abundant striatal protein, but exhibits cellular specificity in its expression, with very high levels in substance-P-containing projection neurons, and very low levels in somatostatin and neuronal nitric oxide synthase interneurons. Despite the high expression of this protein in the striatum, only a minority of NMDA receptors are linked to alpha-actinin-2. This interaction may identify a subset of receptors with distinct anatomical and functional properties.     

Immunocytochemical localization of glutamate receptor subunits in the brain stem and cerebellum of the turtle Chrysemys picta

The regional distribution of ionotropic (AMPA and NMDA) and metabotropic (mGluR1alpha) glutamate receptor subunits was examined in the brain stem and cerebellum of the pond turtle, Chrysemys picta, by using immunocytochemistry and light microscopy. Subunit-specific antibodies that recognize NMDAR1, GluR1, GluR4, and mGluR1alpha were used to identify immunoreactive nuclei in the brain stem and cerebellum. Considerable immunoreactivity in the turtle brain stem and cerebellum was observed with regional differences occurring primarily in the intensity of staining with the antibodies. The red nucleus, lateral reticular nucleus and cerebellum labeled intensely for NMDAR1 and moderately for GluR1. The cerebellum also labeled strongly for mGluR1alpha. All of the cranial nerve nuclei labeled intensely for NMDAR1 and to varying degrees for GluR1, GluR4, and mGluR1alpha. Counterstaining revealed the presence of neuronal somata where there were no immunoreactive neurons in individual nuclei. This finding suggests that there are subpopulations of immunoreactive neurons within a given nucleus that bear different glutamate receptor subunit compositions. The results suggest that the glutamate receptor subunit distribution in the brain stem and cerebellum of turtles is similar to that reported for rats. Additionally, there is considerable colocalization of NMDA and AMPA receptors as revealed by light microscopy. These results have implications for the organization of neural circuits that control motor behavior in turtles, and, generally, for the function of brain stem and cerebellar neural circuits in vertebrates.     

Localization of metabotropic glutamate receptor 7 mRNA and mGluR7a protein in the rat basal ganglia.

Metabotropic glutamate receptors (mGluRs) coupled to G-proteins have important roles in the regulation of basal ganglia function. We have examined the localization of the mGluR7 mRNA and mGluR7a protein in the basal ganglia of the rat. Strong mGluR7 hybridization signals are found in cerebral cortex and striatum, but much less intense signals are present in other components of the basal ganglia. Abundant mGluR7a immunoreactivity was found in striatum, globus pallidus (GP), and substantia nigra pars reticulata (SNr). Examination using confocal microscopy together with dendritic and presynaptic markers as well as studies in lesion models provided evidence for the presence of mGluR7a on presynaptic terminals in all three structures. Electron microscopic studies confirmed the presence of mGluR7a in axon terminals in both the striatum and the GP and also revealed the presence of mGluR7a at postsynaptic sites in both of these regions. Our data demonstrate that mGluR7a is located not only on presynaptic glutamatergic terminals of the corticostriatal pathway, where it may serve as an autoreceptor, but also on terminals of striatopallidal and striatonigral projections, where it may modulate the release of gamma-aminobutyric acid (GABA). The presence of mGluR7 at these multiple sites in the basal ganglia suggests that this receptor has a particularly crucial role in modulating neurotransmitter release in major basal ganglia pathways.     

Self-induced accumulation of glutamate in striatal astrocytes and basal ganglia excitotoxicity

Excitotoxicity induced by high levels of extracellular glutamate (GLU) has been proposed as a cause of cell degeneration in basal ganglia disorders. This phenomenon is normally prevented by the astrocytic GLU‐uptake and the GLU‐catabolization to less dangerous molecules. However, high‐GLU can induce reactive gliosis which could change the neuroprotective role of astrocytes. The striatal astrocyte response to high GLU was studied here in an in vivo rat preparation. The transient striatal perfusion of GLU (1 h) by reverse microdialysis induced complex reactive gliosis which persisted for weeks and which was different for radial‐like glia, protoplasmic astrocytes and fibrous astrocytes. This gliosis was accompanied by a persistent cytosolic accumulation of GLU (immunofluorescence quantified by confocal microscope), which persisted for weeks (self‐induced glutamate accumulation), and which was associated to a selective decrease of glutamine synthetase activity. This massive and persistent self‐induced glutamate accumulation in striatal astrocytes could be an additional factor for the GLU‐induced excitotoxicity, which has been implicated in the progression of different basal ganglia disorders. © 2012 Wiley Periodicals, Inc.     

Ontogeny of subunits 2 and 3 of the AMPA-type glutamate receptors in Purkinje cells of the developing chick cerebellum

Several molecules, involved in cellular communication in the mature nervous system, appear to play important roles during neural development. These roles include neuronal growth, morphological changes of neurites, and neuronal survival. Such plasticity processes seem to be in part the result of activation of different receptor subtypes, which could cause Ca(2+) influx, a major candidate to be an outgrowth promoter. In this context, we performed immunohistochemical and in situ hybridization experiments to examine the following aspects of the development of chick cerebellum Purkinje cells: (i) expression of AMPA-type glutamate receptor GluR2/3 proteins; (ii) the levels of mRNAs coding for the GluR2 and GluR3 flip/flop isoforms; and (iii) expression of calbindin (CB) and parvalbumin (PV). Expression of GluR2/3 proteins, CB, PV, and the mRNAs coding for GluR2 and GluR3 splice variants all revealed a differential expression during development in chick Purkinje cells. GluR2/3 proteins and the GluR3 flop variant start to be expressed at E10, while the expression of CB, PV, the GluR3 flip isoform and the splice variants of GluR2 all started around E12-E14. All proteins showed an increasing expression from embryonic stages into the posthatching period. These results reveal a developmentally regulated expression of GluR2/3 proteins, including their splice variants, and of CB and PV in Purkinje cells. These findings may suggest a relationship between these proteins and specific cerebellar developmental processes.     

AMPA-Selective glutamate receptor subunits in astroglial cultures

We analysed AMPA ionotropic receptor subunits at the mRNA level (GluR-1 to -4) and at the protein level (GluR-1 and GluR-2/3/4C) in “primary astroglial cultures” (non-neuronal cell cultures highly enriched in glial fibrillary acidic protein [GFAP] positive cells) prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocamps, and striatum and in “brain non-neuronal cell cultures” (low percentage of GFAP positive cells) prepared from cerebellum, brainstem, mesencephalon, and hypothalamus. For comparison, we also determined ampa subunit mRNA and protein levels in different brain regions. By Northern blot analysis mRNAs for the AMPA receptor subunits (glur-1,-2,-3,-4) were detected in primary rat cerebral hemispheres astroglial cultures. Immunoblotting analysis with anti-GluR-1 and anti GluR-2/3/4C polyclonal antibodies confirmed the presence of low leve of immunoreactive proteins of the same size of those identifice in vivo as GluR subunits. Expression of GluR genes varied depending on the brain area used as starting material for the preparation of the cultures: GluR-1, -2, and -3 were mailly expressed in cortical cultures, while GluR-4 expression predominated in brainstem derived cultures. Interestingly this pattern of expression correlates with that observed in the intact brain, where high levels of GluR-4 mRNA and low levels of the other GluR subunits were found in the brainstem. In conclusion our results confirm the existence of glutmate ionotropic receptors of the AMPA type in primary astroglial cultrues and suggest that GluR-4 is the main AMPA receptor subunit expressed in non neuronal cells of the central nervous system. © 1993 Wiley-Liss, Inc.     

Immunohistochemical localization of nicotinic acetylcholine receptor subunits in the mesencephalon and diencephalon of the chick (Gallus gallus).

Monoclonal antibodies against two alpha-bungarotoxin-binding subunits (alpha 7 and alpha 8) of the nicotinic acetylcholine receptors (nAChRs) were used as immunohistochemical probes to map their distribution in the chick diencephalon and mesencephalon. The distribution of the alpha 7 and alpha 8 nAChR subunits was compared to the distribution of immunoreactivity produced by a monoclonal antibody against the beta 2 structural subunit of the nAChRs. Structures that contained high numbers of alpha 7-like immunoreactive (LI) somata included the intergeniculate leaflet, nucleus intercalatus thalami, nucleus ovoidalis, organum paraventricularis, nucleus rotundus, isthmic nuclei, nucleus trochlearis, oculomotor complex, nucleus interstitio-pretecto-subpretectalis, stratum griseum centrale of the optic tectum, and nucleus semilunaris. Neuropil staining for alpha 7-LI was intense in the nucleus dorsomedialis hypothalami, nucleus geniculatus lateralis ventralis, griseum tecti, isthmic nuclei, nucleus lentiformis mesencephali, nucleus of the basal optic root, and stratum griseum et fibrosum superficiale of the tectum. High numbers of alpha 8-LI somata were found in the stratum griseum et fibrosum superficiale of the tectum and the nucleus interstitio-pretecto-subpretectalis, and intense neuropil staining for alpha 8-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griseum et fibrosum superficiale of the tectum. High numbers of beta 2-LI somata were found only in the nucleus spiriformis lateralis, whereas neuropil staining for beta 2-LI was intense in the nucleus geniculatus lateralis ventralis, nucleus suprachiasmaticus, nucleus lateralis anterior, nucleus habenularis lateralis, area pretectalis, griseum tecti, nucleus lentiformis mesencephalis, nucleus externus, and nucleus interpeduncularis, and in the stratum griseum centrale, stratum griseum et fibrosum superficiale, and stratum opticum of the tectum. These results indicate that there are major disparities in the localization of the alpha-bungarotoxin-binding alpha 7 and alpha 8 nAChR subunits and the beta 2 structural nAChR subunit in the chick diencephalon and mesencephalon. These nAChR subunits appear, however, to coexist in several regions of the chick brain.     

Chemokine receptor CXCR2 regulates the functional properties of AMPA-type glutamate receptor GluR1 in HEK cells

Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.