Connexin36 expression in major centers of the auditory system in the CNS of mouse and rat: Evidence for neurons forming purely electrical synapses and morphologically mixed synapses.

Electrical synapses formed by gap junctions composed of connexin36 (Cx36) are widely distributed in the mammalian central nervous system (CNS). Here, we used immunofluorescence methods to document the expression of Cx36 in the cochlear nucleus and in various structures of the auditory pathway of rat and mouse. Labeling of Cx36 visualized exclusively as Cx36-puncta was densely distributed primarily on the somata and initial dendrites of neuronal populations in the ventral cochlear nucleus, and was abundant in superficial layers of the dorsal cochlear nucleus. Other auditory centers displaying Cx36-puncta included the medial nucleus of the trapezoid body (MNTB), regions surrounding the lateral superior olivary nucleus, the dorsal nucleus of the medial lemniscus, the nucleus sagulum, all subnuclei of the inferior colliculus, and the auditory cerebral cortex. In EGFP-Cx36 transgenic mice, EGFP reporter was detected in neurons located in each of auditory centers that harbored Cx36-puncta. In the ventral cochlear nuclei and the MNTB, many neuronal somata were heavily innervated by nerve terminals containing vesicular glutamate transporter-1 (vglut1) and Cx36 was frequently localized at these terminals. Cochlear ablation caused a near total depletion of vglut1-positive terminals in the ventral cochlear nuclei, with a commensurate loss of labeling for Cx36 around most neuronal somata, but preserved Cx36-puncta at somatic neuronal appositions. The results suggest that electrical synapses formed by Cx36-containing gap junctions occur in most of the widely distributed centers of the auditory system. Further, it appears that morphologically mixed chemical/electrical synapses formed by nerve terminals are abundant in the ventral cochlear nucleus, including those at endbulbs of Held formed by cochlear primary afferent fibers, and those at calyx of Held synapses on MNTB neurons.

[Beta-lactamic antibiotics allergy in cataract surgery. Prevalence and preoperative characteristics of allergic patients]. / Alergia a antibióticos ß-lactámicos en cirugía de cataratas. Prevalencia y características preoperatorias de los pacientes alérgicos.

OBJECTIVE: To describe the proportion of patients allergic to ß-lactam antibiotics and the prevalence of preoperative conjunctival bacteria among those undergoing cataract surgery in our area. METHOD: Retrospective cross-sectional study of prevalence of ß-lactam allergic patients consecutively scheduled for cataract surgery from 11 July 2005 to November 2012. For studying the prevalence of conjunctival bacteria and clinical characteristics in the patients' preoperative examination, those under 18 years and those with cataract surgery combined with other eye surgeries were excluded. Data from the first preoperative examination of the remaining patients were selected. Clinical data were extracted from the database generated in the evaluation made for anesthetic purposes, and the microbiological data from the laboratory database. Both bases were linked through a patient history code. A comparison was made between the prevalence of conjunctival bacteria and clinical characteristics in allergic and non-allergic patients. RESULTS: From 12,409 adults selected for the bacteriological study, 862 (6.96%) were allergic to ß-lactams, their mean age (74.45 years) was higher than that of the non-allergic (P=.005). The proportion of women (71.4%) in the allergic patient group was much higher than that of men. The prevalence of pathogenic bacteria (especially Bacillus spp and Pseudomonas aeruginosa), lung disease and heart failure, was higher in allergic patients. CONCLUSIONS: The prevalence of allergy to ß-lactams in this study is within the range described in other populations. The higher prevalence of pathogenic bacteria and the predominance of women in those allergic to ß-lactams are useful data to guide their surgical prophylaxis.

[Spectrum and susceptibility of preoperative conjunctival bacteria]. / Espectro y susceptibilidad de las bacterias conjuntivales preoperatorias.

PURPOSE: To describe the conjunctival bacterial spectrum of our patients undergoing intraocular surgery and their antibiotic sensitivity during the study period. METHODS: A retrospective study of preoperative conjunctival culture of patients consecutively scheduled for intraocular surgery from 21 February 2011 to 1 April 2013. Specimens were directly seeded onto blood-agar and MacConkey-agar (aerobiosis incubation, 2 days), and on chocolate-agar (6% CO2 incubation, 7 days). The identified bacteria were divided into 3 groups according to their origin; the bacteria susceptibility tests were performed on those more pathogenic and on some of the less pathogenic when more than 5 colonies were isolated. The sensitivity of the exigent growing bacteria was obtained with disk diffusion technique, and for of the non-exigent bacteria by determining their minimum inhibitory concentration. The Epidat 3.1 program was used for statistical calculations. RESULTS: A total of 13,203 bacteria were identified in 6,051 cultures, with 88.7% being typical colonizers of conjunctiva (group 1), 8.8% typical of airways (group 2), and the remaining 2.5% of undetermined origin (group 3). 530 cultures (8.8%) were sterile. The sensitivity of group 1 was: 99% vancomycin, 95% rifampicin, 87% chloramphenicol, 76% tetracycline. Levels of co-trimoxazole, aminoglycosides, quinolones, ß-lactams and macrolides decreased since 2007. The group 2 was very sensitive to chloramphenicol, cefuroxime, rifampicin, ciprofloxacin and amoxicillin/clavulanate. In group 3, to levofloxacin 93%, ciprofloxacin 89%, tobramycin 76%, but ceftazidime 53% and cefuroxime 29% decreased. CONCLUSIONS: None of the tested antibiotics could eradicate all possible conjunctival bacteria. Bacteria living permanently on the conjunctiva (group 1) have achieved higher resistance than the eventual colonizers.

Pathogenic conjunctival bacteria associated with systemic co-morbidities of patients undergoing cataract surgery.

PURPOSE: To identify the risk of patients undergoing cataract surgery of having pathogenic conjunctival bacteria associated with their systemic co-morbidities. METHODS: Retrospective study of consecutive patients undergoing their first cataract operation from July 2005 to April 2010. Their preoperative conjunctival bacteria were cultured, identified, and classified in bacterial groups. Their co-morbidities were defined from their clinical data and the answers to systematic questions asked in the anaesthetic evaluation. The Microsoft Access databases of the two data sets were merged for carrying out the statistical analysis. Univariate association of each bacterial group with each co-morbidity was studied by using χ(2)-test for categorical data and Student's t-test for continuous variables. Also, logistic regression models were used adjusting for age and sex. SPSS statistic programme, version 18 was used for all these analyses. Endophthalmitis cases in this surgical series were searched. RESULTS: In the 8333 selected patients, age was associated with increased conjunctival bacteria in all groups except for Streptococcus pneumoniae and Propionibacteriae. However, male sex was associated with these two groups and also with coagulase-negative Staphylococci, Corynebacterium xerosis, Staphylococcus aureus, and Gram-negative rods. After adjusting for age and sex, S. aureus was associated with diabetes, lung diseases, and renal and heart insufficiency; Gram-negative rods with smoking habit; Enterococci with diabetes; Streptococcus pneumoniae with kyphoscoliosis; and other Streptococci with diabetes and handicapped patients. CONCLUSION: The more pathogenic conjunctival bacteria were more likely associated with patients' co-morbidities, such as diabetes, lung diseases, renal and heart insufficiency, kyphoscoliosis, and smoking habit, than the less pathogenic ones.

Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits ß2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) ß2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Ultrastructure, synaptic organization, and molecular components of bushy cell networks in the anteroventral cochlear nucleus of the rhesus monkey.

Bushy cells (BCs) process auditory information in the ventral cochlear nucleus (VCN). Yet, most neuroanatomical findings come from studies in cats and rodents, and the ultrastructural morphological features of BCs in humans and higher nonhuman primates are unknown. In this study, we combined histological, immunocytochemical, and ultrastructural methods to examine the morphology and synaptic organization of BCs in the rhesus monkey VCN. We observed that BCs were organized in a complex neural network that appears to interconnect the cells. The fine structure of BC somata and dendrites, as well as their synaptic inputs, are similar to those in other mammals. We found that BCs received numerous endbulb-like VGLUT1- and VGLUT2-immunopositive endings. In addition, they expressed glutamate AMPA (GluR2/3 and GluR4), NMDA (NR1), delta1/2 receptor subunits, and the α1 subunit of the glycine receptor. These receptor types and subunits mediate fast excitatory synaptic transmission from the cochlea and inhibitory neurotransmission from noncochlear inputs. Parvalbumin immunostaining and semithin sections showed that BC dendrites are oriented toward neighboring BC somas to form neuronal clusters. Within the cluster, the incoming inputs established multiple, divergent synaptic contacts. Thus, BCs were connected by specialized dendrosomatic and somasomatic membrane junctions. Our results indicate that the cytoarchitectural organization of BCs is well conserved between primates and other mammalian species.

The conjunctival bacterial pattern of diabetics undergoing cataract surgery.

PURPOSE: To ascertain the conjunctival bacterial pattern of diabetics undergoing cataract operation to reduce the risk of postoperative endophthalmitis (PE). METHODS: An observational retrospective study of the conjunctival bacteria of consecutive patients undergoing cataract surgery from July 2005 to November 2008. Records of patients having eye surgical prophylaxis in the 6 months before the culture and those patients having cataract operation combined with other surgical procedures were excluded. Aerobic and microaerobic cultures were carried out. Dade-Behring panels were used for bacterial identification. The database containing the isolated bacteria was linked to another Access database containing demographic and clinical data such as diabetes presence and baseline blood glucose and creatinine levels. The conjunctival bacteria of diabetics were compared with those of the non-diabetics. Epidat 3.1 program was used for statistical calculations. RESULTS: From 5922 selected patients, 1325 (22.37%) knew they were diabetics (higher prevalence than expected). Among self-reported non-diabetics, 900 (15.2%) could be 'unknown' diabetics; another 274 had an impaired renal function; and 3423 non-diabetics joined the control group. Diabetics have a significantly higher prevalence of Staphylococcus aureus, Enterococci, certain Streptococci, and Klebsiella sp. than non-diabetics. Diabetics and non-diabetics having a blood creatinine level above 105.2 mumol/l had an increased conjunctival bacterial prevalence; these groups had a higher mean age and men predominated. CONCLUSIONS: Diabetics have a conjunctival flora pattern whose increased bacteria are a predominant cause of many diabetic infections. An abnormally high blood creatinine level is an indicator of increased conjunctival colonisation in diabetics and non-diabetics.

Cochlear nucleus neurons redistribute synaptic AMPA and glycine receptors in response to monaural conductive hearing loss.

Neurons restore their function in response to external or internal perturbations and maintain neuronal or network stability through a homeostatic scaling mechanism. Homeostatic responses at synapses along the auditory system would be important for adaptation to normal and abnormal fluctuations in the sensory environment. We investigated at the electron microscopic level and after postembedding immunogold labeling whether projection neurons in the cochlear nucleus responded to modifications of auditory nerve activity. After unilaterally reducing the level of auditory inputs by approximately 20 dB by monaural earplugging, auditory nerve synapses on bushy cells somata and basal dendrites of fusiform cells of the ventral and dorsal cochlear nucleus, respectively, upregulated GluR3 AMPA receptor subunit, while inhibitory synapses decreased the expression of GlyRalpha1 subunit. These changes in expression levels were fully reversible once the earplug was removed, indicating that activity affects the trafficking of receptors at synapses. Excitatory synapses on apical dendrites of fusiform cells (parallel fibers) with different synaptic AMPA receptor subunit composition, were not affected by sound attenuation, as the expression levels of AMPA receptor subunits were the same as in normal hearing littermates. GlyRalpha1 subunit expression at inhibitory synapses on apical dendrites of fusiform cells was also found unaffected. Furthermore, fusiform and bushy cells of the contralateral side to the earplugging upregulated the GluR3 subunit at auditory nerve synapses. These results show that cochlear nucleus neurons innervated by the auditory nerve, are able to respond to small changes in sound levels by redistributing specific AMPA and glycine receptor subunits.

Neurochemistry of the afferents to the rat cochlear root nucleus: possible synaptic modulation of the acoustic startle.

Afferents to the primary startle circuit are essential for the elicitation and modulation of the acoustic startle reflex (ASR). In the rat, cochlear root neurons (CRNs) comprise the first component of the acoustic startle circuit and play a crucial role in mediating the ASR. Nevertheless, the neurochemical pattern of their afferents remains unclear. To determine the distribution of excitatory and inhibitory inputs, we used confocal microscopy to analyze the immunostaining for vesicular glutamate and GABA transporter proteins (VGLUT1 and VGAT) on retrogradely labeled CRNs. We also used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to detect and localize specific neurotransmitter receptor subunits in the cochlear root. Our results show differential distributions of VGLUT1- and VGAT-immunoreactive endings around cell bodies and dendrites. The RT-PCR data showed a positive band for several ionotropic glutamate receptor subunits, M1-M5 muscarinic receptor subtypes, the glycine receptor alpha1 subunit (GlyRalpha1), GABAA, GABAB, and subunits of alpha2 and beta-noradrenergic receptors. By immunohistochemistry, we confirmed that CRN cell bodies exhibit positive immunoreaction for the glutamate receptor (GluR) 3 and NR1 GluR subunits. Cell bodies and dendrites were also positive for M2 and M4, and GlyRalpha1. Other subunits, such as GluR1 and GluR4 of the AMPA GluRs, were observed in glial cells neighboring unlabeled CRN cell bodies. We further confirmed the existence of noradrenergic afferents onto CRNs from the locus coeruleus by combining tyrosine hydroxylase immunohistochemistry and tract-tracing experiments. Our results provide valuable information toward understanding how CRNs might integrate excitatory and inhibitory inputs, and hence how they could elicit and modulate the ASR.

Revealing the molecular layer of the primate dorsal cochlear nucleus.

In nonprimate mammals, the dorsal cochlear nucleus (DCN) is thought to play a role in the orientation of the head toward sounds of interest by integrating acoustic and somatosensory information. Humans and higher primates might not use this system because of reported phylogenetic changes in DCN cytoarchitecture [Moskowitz N (1969) Comparative aspects of some features of the central auditory system of primates. Ann N Y Acad Sci 167:357-369; Moore JK, Osen KK (1979) The cochlear nuclei in man. Am J Anat 154:393-418; Moore JK (1980) The primate cochlear nuclei: loss of lamination as a phylogenetic process. J Comp Neurol 193:609-629]. In this study, we re-evaluated this question from a comparative perspective and examined the rhesus monkey (cercopithecoid primate) using more sensitive probes and higher resolution imaging methods. We used electron microscopy to identify parallel fibers and their synapses, and molecular markers to determine that primates exhibit the main components of excitatory neurotransmission as other mammals. We observed that characteristics of the monkey molecular layer resembled what has been reported for nonprimates: (1) immunohistochemistry revealed many unmyelinated, thin axons and en passant glutamatergic synapses on dendritic spines; (2) immunohistochemistry for phosphodiesterase (PDE10A) showed the nuclei of granule cells distributed in the external molecular layer and the deep layers in the DCN; (3) antibodies for the inositol trisphosphate receptor (IP3r) and calbindin immunostained cartwheel cells; (4) postembedding immunogold labeling revealed synaptic expression of AMPA and delta glutamate receptor subunits on spines in parallel fiber endings; and (5) parallel fibers use vesicular glutamate transporter 1 (VGLUT1) to package glutamate into the synaptic vesicles and to mediate glutamate transport. These observations are consistent with the argument that the rhesus monkey DCN has neuronal features similar to those of other nonprimate mammals.

Neurotrophins act at presynaptic terminals to activate synapses among cultured hippocampal neurons.

We have recently demonstrated that embryonic E16 hippocampal neurons grown in cultures are unable to form fast synaptic connections unless treated with BDNF or NT-3. This experimental system offers an opportunity to define the roles of neurotrophins in processes leading to formation of functional synaptic connections. We have used ultrastructural and electrophysiological methods to explore the cellular locations underlying neurotrophin action on synaptic maturation. The rate of spontaneous miniature excitatory postsynaptic currents (mEPSCs) evoked by hyperosmotic stimulation was 7-16-fold higher in neurotrophin-treated cells than in controls. In addition, the potent neurotransmitter-releasing drug alpha-latrotoxin was virtually ineffective in the control cells while it stimulated synaptic events in neurotrophin-treated cells. Likewise, the membrane-bound dye FM1-43 was taken up by terminals in neurotrophin-treated cultures five-fold more than in controls. Both the total number and the number of docked synaptic vesicles were increased by neurotrophin treatment. Activation of synaptic responses by neurotrophins occurred even when postsynaptic glutamate receptors and action potential discharges were pharmacologically blocked. These results are consistent with a presynaptic locus of action of neurotrophins to increase synaptic vesicle density which is critical for rapid synaptic transmission. They also suggest that neurotrophins can activate synapses in the absence of pre- and postsynaptic neuronal activity.

Post-transcriptional control of catalase expression in garlic-treated rats.

Regulation of catalase (CAT) expression, a major antioxidant enzyme that detoxifies H2O2, is very complex. Garlic is effective to prevent or ameliorate oxidative stress probably through its intrinsic antioxidant properties and/or to its ability to modify antioxidant enzyme expression. In this paper we studied the effect of a 2% garlic diet on the renal and hepatic CAT expression (mRNA levels, and enzyme activity, content, synthesis, and degradation). The study was made 2 weeks after feeding rats with a 2% garlic diet. CAT activity and content were measured by a spectrophotometric method and Western blot, respectively. CAT mRNA levels and CAT synthesis (k(s)) and degradation (kD) in vivo were measured by Northern blot and kinetic of reappearance of CAT activity after aminotriazole injection, respectively. Garlic-treatment decreased CAT activity and content, and CAT mRNA levels were unchanged in both tissues. k(s) decreased and kD remained unchanged in kidney and liver. The decrease in k(s) without changes in kD and CAT mRNA levels could explain the low CAT expression in garlic-fed rats. In vivo H2O2 generation in kidney and liver was markedly decreased in garlic-fed rats which could be due to a direct antioxidant effect of garlic. This may be the initial event in the garlic-fed rats that leads to the decreased CAT expression. Our data strongly suggest that the diminished renal and hepatic CAT expression in garlic-fed rats is mediated by post-transcriptional changes (mainly low translational efficiency) which could be an adaptation to the low H2O2.

Distinct Localization of P2X receptors at excitatory postsynaptic specializations.

ATP mediates fast excitatory synaptic transmission in some regions of the central nervous system through activation of P2X receptors. Nonetheless, the functional significance of ATP-mediated neurotransmission is not yet understood. Using postembedding immunocytochemistry, we describe the distribution of P2X(2), P2X(4), and P2X(6) subunits in cerebellum and in the CA1 region of the hippocampus. Dendritic spines of cerebellar Purkinje cells showed immunogold labeling for all three subunits when apposed to parallel fiber (PF) terminals. In contrast, no immunogold labeling was observed on dendritic spines or cell bodies receiving inputs from climbing fibers and basket cells, respectively. In CA1 pyramidal cells, postsynaptic membranes apposed to terminals of Schaffer collaterals were immunogold-labeled for P2X(2), P2X(4), and P2X(6) subunits. Immunolabeling was also observed perisynaptically and intracellularly in relation to membranes of the endoplasmic reticulum. The analysis of the tangential distribution of gold particles showed that they were preferentially located at the peripheral portion of the postsynaptic specialization of both parallel fiber and Schaffer collateral synapses. By double imunogold labeling using antibodies for P2X receptor subunits and GluR2/3 subunits of the AMPA glutamate receptors, we show that synapses expressing P2X receptors are also glutamatergic. The present study shows for the first time qualitatively and quantitatively the precise localization of P2X receptors in brain. Moreover, our data indicate that P2X receptors may play a significant role at glutamatergic synapses.

Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes.

Reactive oxygen species are involved in gentamicin (GM) nephrotoxicity, and garlic is effective in preventing or ameliorating oxidative stress. Therefore, the effect of garlic on GM nephrotoxicity was investigated in this work. Four groups of rats were studied: (i) fed normal diet (CT), (ii) treated with GM (GM), (iii) fed 2% garlic diet (GA), and (iv) treated with GM and 2% garlic diet (GM + GA). Rats were placed in metabolic cages and GM nephrotoxicity was induced by injections of GM (75 mg/kg every 12 h) for 6 d. Lipoperoxidation and enzyme determinations were made in renal cortex on day 7. GM nephrotoxicity was made evident on day 7 by (i) tubular histological damage, (ii) enhanced BUN and urinary excretion of N-acetyl-beta-D-glucosaminidase, and (iii) decreased creatinine clearance. These alterations were prevented or ameliorated in GM + GA group. The rise in lipoperoxidation and the decrease in Mn-SOD and glutathione peroxidase (GPx) activities observed in the GM group, were prevented in the GM + GA group. Cu, Zn-SOD activity and Mn-SOD and Cu,Zn-SOD content did not change. CAT activity and content decreased in the GM, GA, and GM + GA groups. CAT mRNA levels decreased in the GM group. The protective effect of garlic is associated with the prevention of the decrease of Mn-SOD and GPx activities and with the rise of lipoperoxidation in renal cortex.

Differential distribution of glutamate receptors in the cochlear nuclei.

Glutamate receptors are the major excitatory neurotransmitter receptors of the mammalian central nervous system, and include AMPA, kainate, delta, NMDA, and metabotropic types. In the cochlear nucleus (CN), the AMPA receptor subunits GluR2-4 are found in major kinds of neurons, while GluR1 subunit distribution is more restricted. GluR2 is low in the anteroventral CN, suggesting that many AMPA receptors here are calcium-permeable. Delta receptors are most prevalent in cartwheel cells in the dorsal CN. Of the NMDA receptors, NR1 is widespread while the NR2 subunits show more restricted distributions. Of the metabotropic glutamate receptors, mGluR1alpha is most prevalent in the dorsal CN, and mGluR2 is concentrated in Golgi cells and unipolar brush cells. AMPA receptors in endbulb synapses in the anteroventral CN are mainly GluR3+4 complexes: probably an adaptation for rapid auditory neurotransmission. Glutamate receptors are differentially distributed in synapses of fusiform cells of the dorsal CN; GluR4 and mGluR1alpha are present only at basal dendrite synapses (auditory nerve), while other glutamate receptors occupy both apical and basal synapses. Analysis of cytoplasmic distribution suggests that a selective targeting mechanism may restrict movement of GluR4 and mGluR1alpha to basal dendrites, although other targeting mechanisms may be present.

Selective targeting of glutamate receptors in neurons.

Glutamate receptors mediate the majority of excitatory responses in the central nervous system (CNS). Neurons express multiple subtypes and subunits of glutamate receptors, which are differentially distributed at pre- and postsynaptic sites. This allows the cell to respond differentially depending on the subunit composition of receptors at the postsynaptic membrane. The process by which receptors are targeted selectively to the appropriate synapse is poorly understood. Evidence exists that targeting of glutamate receptors to the different neuronal compartments is regulated at multiple levels involving a general targeting step; a local step where receptor-containing organelles are moved to the synapse; and a step where the receptors are stabilized at the synapse, which may involve interaction with an anchoring protein.

Calnexin and the immunoglobulin binding protein (BiP) coimmunoprecipitate with AMPA receptors.

To identify proteins that interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, we carried out coimmunoprecipitation analyses on detergent-solubilized rat forebrain membranes. Membranes were solubilized with Triton X-100, and immunoprecipitation was done using subunit-specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein Aagarose. Proteins bound to the antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and western blotting. With solubilization in low ionic strength buffer, several coimmunoprecipitating proteins, with Mr = 17,000-100,000, were identified in silver-stained gels. Western blots were then probed with antibodies to a series of candidate proteins that were chosen based on the molecular masses of the copurifying proteins. Two of these were identified as the molecular chaperones calnexin (90 kDa) and the immunoglobulin binding protein (BiP; 78 kDa). Immunoprecipitation with antibodies to calnexin and BiP demonstrated that glycosylated AMPA receptor subunits were associated. The relationship between AMPA receptors and calnexin and BiP was further studied with immunocytochemistry of the hippocampus. Both calnexin and BiP labeling was present not only in the cell body but also in dendrites of hippocampal pyramidal neurons, where double-label immunofluorescence also showed the presence of AMPA receptor subunits.