Investigating the transient and persistent effects of heat on Clostridium difficile spores.

Purpose. Clostridium difficile spores are extremely resilient to high temperatures. Sublethal temperatures are associated with the 'reactivation' of dormant spores, and are utilized to maximize C. difficile spore recovery. Spore eradication is of vital importance to the food industry. The current study seeks to elucidate the transient and persisting effects of heating C. difficile spores at various temperatures.Methods. Spores of five C. difficile strains of different ribotypes (001, 015, 020, 027 and 078) were heated at 50, 60 and 70-80 °C for 60 min in phosphate-buffered saline (PBS) and enumerated at 0, 15, 30, 45 and 60 min. GInaFiT was used to model the kinetics of spore inactivation. In subsequent experiments, spores were transferred to enriched brain heart infusion (BHI) broths after 10 min of 80 °C heat treatment in PBS; samples were enumerated at 90 min and 24 h.Results. The spores of all strains demonstrated log-linear inactivation with tailing when heated for 60 min at 80 °C [(x̄=7.54±0.04 log10 vs 4.72±0.09 log10 colony-forming units (c.f.u.) ml- 1; P<0.001]. At 70 °C, all strains except 078 exhibited substantial decline in recovery over 60 min. Interestingly, 50 °C heat treatment had an inhibitory effect on 078 spore recovery at 0 vs 60 min (7.61±0.06 log10 c.f.u. ml- 1 vs 6.13±0.05 log10 c.f.u. ml- 1; P<0.001). Heating at 70/80 °C inhibited the initial germination and outgrowth of both newly produced and aged spores in enriched broths. This inhibition appeared to be transient; after 24 h vegetative counts were higher in heat-treated vs non-heat-treated spores (x̄=7.65±0.04 log10 c.f.u. ml- 1 vs 6.79±0.06 log10 c.f.u. ml- 1; P<0.001).Conclusions. The 078 spores were more resistant to the inhibitory effects of higher temperatures. Heat initially inhibits spore germination, but the subsequent outgrowth of vegetative populations accelerates after the initial inhibitory period.

Biofilm-derived spores of Clostridioides (Clostridium) difficile exhibit increased thermotolerance compared to planktonic spores.

Biofilm-derived spores of strains of four ribotypes (001, 020, 027 & 078) of Clostridioides (Clostridium) difficile were found to exhibit increased thermotolerance compared to spores produced in planktonic culture. In addition, 'thick' and 'thin' exosporium morphotypes described previously were visualised by electron microscopy in both biofilm and planktonic spores.

Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars.

BACKGROUND: A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. METHODS: Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). RESULTS: CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ±â€¯0.02 vs 0.12 ±â€¯0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. CONCLUSIONS: We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.

Microbiologic factors affecting Clostridium difficile recurrence.

BACKGROUND: Recurrent Clostridium difficile infection (rCDI) places a huge economic and practical burden on healthcare facilities. Furthermore, rCDI may affect quality of life, leaving patients in an rCDI cycle and dependant on antibiotic therapy. AIMS: To discuss the importance of microbiologic factors in the development of rCDI. SOURCES: Literature was drawn from a search of PubMed from 2000 onwards with the search term 'recurrent Clostridium difficile infection' and further references cited within these articles. CONTENT: Meta-analyses and systematic reviews have shown that CDI and rCDI risk factors are similar. Development of rCDI is attendant on many factors, including immune status or function, comorbidities and concomitant treatments. Studies suggest that poor bacterial diversity is correlated with clinical rCDI. Narrow-spectrum gut microflora-sparing antimicrobials (e.g. surotomycin, cadazolid, ridinilazole) are in development for CDI treatment, while microbiota therapeutics (faecal microbiota transplantation, nontoxigenic C. difficile, stool substitutes) are increasingly being explored. rCDI can only occur when viable C. difficile spores are present, either within the gut lumen after infection or when reacquired from the environment. C. difficile spore germination can be influenced by gut environmental factors resulting from dysbiosis, and spore outgrowth may be affected stage by some antimicrobials (e.g. fidaxomicin, ramoplanin, oritavancin). IMPLICATIONS: rCDI is a significant challenge for healthcare professionals, requiring a multifaceted approach; optimized infection control to minimize reinfection; C. difficile-targeted antibiotics to minimize dysbiosis; and gut microflora restoration to promote colonization resistance. These elements should be informed by our understanding of the microbiologic factors involved in both C. difficile itself and the gut microbiome.

Modelling affective pain in mice: Effects of inflammatory hypersensitivity on place escape/avoidance behaviour, anxiety and hedonic state.

BACKGROUND: The place escape/avoidance paradigm (PEAP) has been used to assess the affective component of pain in rats. Using the Complete Freund's Adjuvant (CFA) model of inflammatory pain, the current study aimed at developing a mouse version of PEAP and investigating the relation between PEAP and other behavioural responses, namely anxiety-like behaviour, locomotor activity, and hedonic state. NEW METHOD: A novel paradigm assessing the affective component of pain in mice was developed by modifying the setup known from rat studies: Animals were forced to stay 2 × 5 min in the light and the dark area of a box while being stimulated with a suprathreshold filament on the untreated or treated paw, respectively. This was followed by a 30-min test with unrestricted movement. Anxiety-like behaviour, locomotor activity, and hedonic state were assessed with the elevated zero maze (EZM), an open field setup, and a saccharin preference test, respectively, and correlated with the PEAP behaviour to examine potentially confounding parameters of the novel paradigm. RESULTS: In the PEAP, CFA-treated animals spent more time in the light area. CFA also increased anxiety-like behaviour significantly, whereas locomotor activity was unaffected. A significant, albeit modest, reduction in saccharin preference was observed. PEAP responses showed no significant correlations with any other behavioural measure. COMPARISON WITH EXISTING METHOD AND CONCLUSIONS: The PEAP results suggest that this paradigm might be successfully applied in mice to study affective pain. CFA treatment was associated with increased anxiety-like behaviour and anhedonia; however, this appeared unrelated to the PEAP responses.

Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R)-mediated neurotoxicity was studied in relation to subunit expression and the presence of Ca(2+)-permeable receptor channels. AMPA-mediated toxicity had two components: 1) a direct AMPA-R-mediated component, which was not due to Ca(2+) influx through voltage-gated Ca(2+) channels, reversal of the Na(+)/Ca(2+) exchanger or release of calcium from dantrolene-sensitive intracellular Ca(2+) stores, and 2) a minor, indirect component involving activation of NMDA receptor channels, because of glutamate release and removal of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during development. GluR2(R) levels also increased during development, and channel blockers of Ca(2+)-permeable AMPA-R lacking the GluR2(R) subunit (spermine and philanthotoxin) failed to prevent neurotoxicity or increases in [Ca(2+)](i). Thus, the direct AMPA-R-mediated toxicity may be explained by initiation of cell death by Ca(2+) fluxing through AMPA-R containing GluR2(R). The components of direct AMPA-R-mediated toxicity are proposed to be 1) toxicity mediated by GluR2(R)-lacking AMPA-R and 2) toxicity mediated by low-Ca(2+)-permeability AMPA-R containing GluR2(R).

Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization.

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2.

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.

Agonist discrimination between AMPA receptor subtypes.

The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H]AMPA radioligand binding experiments. (S)-4-bromohomoibotenic acid (BrHIBO) exhibited a 126-fold selectivity for GluR1o compared to GluR3o. Xenopus laevis oocytes were used to express functional homomeric and heteromeric recombinant AMPA-R and to determine BrHIBO potency (EC50) at these channels. (R,S)-BrHIBO exhibited a 37-fold selectivity range amongst the AMPA-R. It is hoped that BrHIBO can be used as a lead structure for the development of other subtype-selective compounds.

Depolarization-induced release of [(3)H]D-aspartate from GABAergic neurons caused by reversal of glutamate transporters.

Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to gamma-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [(3)H]D-aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [(3)H]D-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [(3)H]D-aspartate was decreased by about 40%. These results indicated that some of the released [(3)H]D-aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [(3)H]D-aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [(3)H]D-aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2, 4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [(3)H]D-aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [(3)H]D-aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [(3)H]D-aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [(3)H]D-aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.

Action of bicyclic isoxazole GABA analogues on GABA transporters and its relation to anticonvulsant activity.

The inhibitory action of bicyclic isoxazole gamma-aminobutyric acid (GABA) analogues and their 4,4-diphenyl-3-butenyl (DPB) substituted derivatives has been investigated in cortical neurones and astrocytes as well as in human embryonic kidney (HEK 293) cells transiently expressing either mouse GABA transporter-1 (GAT-1), GAT-2, -3 or -4. It was found that 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol (THPO) and 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) displayed some inhibitory activity on GAT-1 and GAT-2, where the compounds exhibited a slightly lower potency on GAT-2 compared to GAT-1. DPB substituted THPO displayed higher inhibitory potency than the parent compound regarding the ability to inhibit GABA uptake via GAT-1 and GAT-2. Concerning the inhibitory mechanism, THPO, THAO and DPB-THPO were competitive inhibitors on GAT-1 transfected HEK 293 cells and the same mechanism was observed for THPO in GAT-3 transfected cells. Regarding GABA uptake into neurones and astroglia cells THAO and DPB-THAO both displayed competitive inhibitory action. The observations that THPO, THAO as well as their DPB derivatives act as competitive inhibitors together with earlier findings such as potent anticonvulsant activity, lack of proconvulsant activity and the ability of THPO to increase extracellular GABA concentration, indicate that these bicyclic isoxazole GABA analogues and their DPB derivatives may be useful lead structures in future search for new antiepileptic drugs.

Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures.

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors.

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.

Development of calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured neocortical neurons visualized by cobalt staining.

The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 microM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7%+/-0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 microM cyclothiazide, the developmental profile of cobalt uptake mediated by 25 microM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4%+/-0.2% at 5 DIV to 21.7%+/-1.6% at 12 DIV. The effect of 200 microM kainate was similar to that seen with 25 microM AMPA plus 50 microM cyclothiazide, resulting in 17.7%+/-0.3% stained neurons at 12 DIV. The cobalt uptake was specific to AMPA and kainate receptors because NMDA receptors and voltage-gated calcium channels were found not to mediate any cobalt staining. In addition, 10 microM 6-nitro-7-sulphamoylbenzo-[f]-quinoxaline-2,3-dione (NBQX) was able to prevent all staining at 5 and 8 DIV and most of the staining at 12 DIV, indicating that the non-NMDA ionotropic glutamate receptors are involved in cobalt uptake into the neurons. The AMPA receptor-selective antagonist GYKI 53655 was used to differentiate between cobalt influx through AMPA- or kainate-preferring receptors. After pretreatment with concanavalin A (con A), an inhibitor of kainate receptor desensitization, cobalt uptake was assessed after stimulation by 200 microM kainate in the presence of 25 microM GYKI 53655. No cobalt staining was observed under these conditions, indicating that most if not all of the cobalt influx induced by kainate was mediated through AMPA receptor channels.

AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) excitotoxicity was examined in cultured neocortical neurons using the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. Neurons were exposed to AMPA at different culture periods during development of the neurons. In order to describe the pharmacology of AMPA-mediated toxicity, several glutamate receptor antagonists were used: MK-801, NS 394, NBQX, GYKI 52466, GYKI 53405 and GYKI 53655. Increased excitotoxicity was observed when cortical neurons cultured for 5, 8 and 12 days in vitro (DIV) were exposed to a high concentration of AMPA (500 microM) for 6 h. However, only at DIV 12 was part of the toxicity mediated directly through AMPA receptors since 10 microM MK-801 blocked all AMPA toxicity at DIV 5 and 8, but only some of the AMPA response at DIV 12. This indicated that NMDA receptors were being activated, causing some of the observed toxicity. The high dose of AMPA was not sufficient to damage all neurons since 59% remained viable after exposure to AMPA even for neurons that were cultured for 12 DIV. Since it is known that both glutamate and AMPA activate AMPA receptors with a fast and rapidly desensitizing response, this could explain the relatively low toxicity produced by 500 microM AMPA. This was investigated by blocking AMPA receptor desensitization with cyclothiazide. Using a lower concentration (25 microM) of AMPA, addition of 50 microM cyclothiazide increased the AMPA induced excitotoxicity in cultured cortical neurons at all DIV except for DIV 2. This combination of AMPA + cyclothiazide yielded 77% cell death for DIV 12 cultures. In contrast to the results observed with 500 microM AMPA, the neurotoxicity mediated directly by AMPA receptors when desensitization was blocked was seen as early as 5 DIV since 10 microM MK-801 did not completely block the response whereas 10 microM NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity with the following rank order: GYKI 53655 > GYKI 52466 > or = GYKI 53405, which is in agreement with their published potencies.

Comparison of the agonist binding site of homomeric, heteromeric, and chimeric GluR1(o) and GluR3(o) AMPA receptors.

A series of AMPA [(R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid] analogues were evaluated for activity at homomeric, heteromeric, and chimeric rat GluR1(o) and GluR3(o) receptors expressed in Xenopus oocytes, using the two-electrode voltage clamp technique. The formation of heteromeric receptor complexes was demonstrated by cross-immunoprecipitation of both subunits from solubilized oocyte membranes. The AMPA analogue ACPA [(R,S)-2-amino-3(3-carboxy-5-methyl-4-isoxazolyl)propionic acid] was the most potent and selective agonist tested at GluR1(o) and GluR3(o), with a 10-fold selectivity for GluR3(o). ACPA showed an intermediate potency at both the GluR1(o) + 3(o) heteromeric complex as well as at the homomeric chimeric receptors. These experiments suggest that for receptor activation, agonist binding occurs between the interface of the GluR1 and GluR3 subunits in the heteromeric channel complex, perhaps between the S1 region of one subunit and the S2 region of another. Also, it seems that 1) electronegative group substitutions on the isoxazole ring of AMPA and 2) decreasing the pKa of the sub stituent at position 3 play a major role in determining the degree of receptor activation under steady-state conditions. Future studies will examine the effects of single amino acid mutations in these receptors, giving a more precise localization of the agonist binding site.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.

Characterization of two alternatively spliced forms of a metabotropic glutamate receptor in the central nervous system of the rat.

Amplification of complementary DNA by the polymerase chain reaction and anti-peptide antibodies were used to characterize the expression of two alternatively spliced forms of a metabotropic glutamate receptor (mGluR1 alpha and mGluR1 beta) in the central nervous system of the rat. Polymerase chain reaction analysis showed that mGluR1 alpha was the predominate of the two forms in the cerebellum, diencephalon, mesencephalon, olfactory bulb and brainstem, while mGluR1 beta was the major form present in the hippocampus. Approximately equal amounts of the two receptors were expressed in the cerebral cortex, septum and striatum. Immunochemical analyses of the two receptors were conducted in the rat cerebellum and hippocampus. An mGluR1 alpha-specific antibody labelled a protein with a relative molecular weight of 146,000 on immunoblots of the hippocampus and cerebellum. Immunoblot analysis of the developmental expression of mGluR1 alpha in the hippocampus and cerebellum demonstrated that in both structures, the levels of mGluR1 alpha were at or near their maximum levels in the adult brain. In contrast, two mGluR1 beta-specific antibodies failed to detect mGluR1 beta on immunoblots of brain tissue, thus precluding an immunocytochemical analysis of this receptor. Although low levels of a higher-molecular weight protein, possibly a dimeric form of mGluR1 beta were seen with one of the mGluR1 beta-specific antibodies, we hypothesize that some of the mGluR1 beta present in brain tissue may undergo proteolytic cleavage of the carboxy terminus. Immunocytochemical analysis of mGluR1 alpha showed that very high levels of this receptor were expressed in Purkinje cell bodies and dendrites. In the granule cell layer, some Golgi neurons were immunostained. The granule cells were not labelled. In the hippocampus, mGluR1 alpha immunoreactivity was present in interneurons of the stratum oriens and the dentate hilar region. Double-labelling studies demonstrated that these interneurons were also immunopositive for the neuropeptide somatostatin. The presence of mGluR1 alpha in cells of the hippocampus that are associated with the release of somatostatin, suggest that this receptor could play a role in regulating hippocampal excitability in both normal and epileptic tissues.

Expression of functional metabotropic and ionotropic glutamate receptors in baculovirus-infected insect cells.

An ionotropic glutamate receptor of the kainate subtype (GluR6) and a G-protein coupled metabotropic glutamate receptor (mGluR1 alpha) were expressed and studied in two insect cell lines: sf9 cells from Spodoptera frugiperda and MG1 cells from Trichoplusia ni. Application of kainate to GluR6-infected MG1 cells produced kainate-activated currents. Glutamate activation of mGluR1 alpha in MG1- and sf9-infected cells caused rapid, transient increases in intracellular calcium levels. This effect was more pronounced in MG1 cells compared to sf9 cells. These results indicate that functional glutamate receptors can be expressed in the baculovirus system, and that MG1 cells may have several advantages over the widely used sf9 cells for studying the functional properties of receptors and channels.