Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis.

BACKGROUND:Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings. METHODS:Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG35-55) or with bovine myelin basic protein (MBP), in TCR2D2 mice immunized with MOG35-55, and in SJL/J mice immunized with myelin proteolipid lipoprotein (PLP139-151). Strain-matched control mice were sham-immunized. RGC density was counted on retinal flatmounts at the end of each experiment. RESULTS:Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG35-55-immunized TCR2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy. CONCLUSIONS:Retinal neuroaxonal damage develops quickly during EAE. Changes in retinal thickness mirror neuronal loss and clinical severity. Monitoring of the IRL thickness after immunization against MOG35-55 in C57Bl/6J mice seems the most convenient model to study retinal neurodegeneration in EAE

Group I mGluRs increase locomotor network excitability in Xenopus tadpoles via presynaptic inhibition of glycinergic neurotransmission

The group I metabotropic glutamate receptor agonist (S)-3,5-dihyroxyphenylglycine (DHPG) increases the frequency of rhythmic swimming activity in Xenopus tadpoles. This study explores the possibility that group I receptor modulation occurs in part via depression of inhibitory synaptic transmission. Applications of the glycine receptor antagonist strychnine occluded the effects of DHPG, providing preliminary evidence that group I receptors affect motor network output by reducing glycinergic transmission. This evidence was supported further by intracellular and whole-cell patch-clamp recordings from presumed motorneurons. DHPG applications produced two prominent effects: (i) during swimming activity, glycinergic mid-cycle IPSPs were reduced in amplitude; and (ii) during quiescent periods, the frequency of spontaneous miniature IPSPs was also reduced. No change in membrane potential or input resistance following group I receptor activation was detected. The reduction in fast synaptic inhibition provides a plausible explanation for the increased excitability of the locomotor network, although other contributory mechanisms activated in parallel by group I receptors cannot be discounted. Aspects of this work have been published previously in abstract form [R. J. Chapman &amp; K. T. Sillar (2003) SFN Abstracts 277.8].</p

Combined imaging and chemical sensing of L-glutamate release from the foregut plexus of the Lepidopteran, Manduca sexta

A new combined imaging and chemical detection sensor for the measurement of localized L-glutamate release at the insect neuromuscular junction (NMJ) is presented. The sensor is comprised of an L-glutamate-sensitive fluorescent gel, spin-coated onto the tip of an optical imaging fiber. The gel is composed of L-glutamate oxidase (GLOD); a pH-sensitive fluorescent dye, SNAFL; and poly(acrylamide-co-N-acryloxysuccinimide) (PAN). NH3 is liberated from the interaction of L-glutamate with GLOD, which reversibly reduces the emitted fluorescence signal from SNAFL. This sensor has a spatial resolution of 3-4 μm, and an L-glutamate detection limit of between 10 and 100 μM. L-glutamate release and re-uptake from the foregut plexus of Manduca sexta was detected by the sensor in the presence of the L-glutamate re-uptake blocker dihydrokainate, and the post-synaptic L-glutamate receptor antagonist CNQX.</p

Myotropic effects of new proctolin analogues modified in the position 5 of peptide chain in insects

To explain the role of the Thr5 residue of proctolin (Arg-Tyr-Leu-Pro-Thr) in the myotropic activity of this insect neuropeptide, we synthesized two groups of its analogues: 1) Arg-Tyr-Leu-Pro-X-OH with X = Val (1), D-Val (2), Ile (3), D-Ile (4), Ala (5), D-Ala (6), Asn (7), Gln (8), Ser (9), Pro (10), Phe (11), Asp (12), Glu (13), Arg (14), D-Arg (15), Lys (16) and Gly (17) and 2) Arg-Tyr-Leu-Pro-R', where R' = isobutylamine (18), S-l-methyl-1-phenylmethylamine (19), R-1-methyl-1-phenylmethylamine (20), R-2-amino-1-propanol (21), S-2-amino-1-propanol (22), R-1-amino-2-propanol (23), S-2-amino-1-propanol (24), 3-amino-1-propanol (25). Decapeptide proctolylproctolin (H-Arg-Tyr-Leu-Pro-Thr-Arg-Tyr-Leu-Pro-Thr-OH) (26) was synthesized. Syntheses of these peptides were carried out by solid-phase method. All peptides were bioassayed in vitro on the semi-isolated hearts of Tenebrio molitor using a cardioexcitatory test and on the foregut of locust (Schistocerca gregaria). Peptides 1, 3, 5, 9, 13, 14, 16, 22, and 23 retained about 30-50% of the cardioexcitatory activity in T. molitor. Analogues 1 and 3 preserved about 50% and analogue 8 about 80% of the myotropic activity, whereas compound 4 and 9 showed a very weak contractile activity in S. gregaria.</p

Muscarinic acetylcholine and proctolin receptors in the foregut of the locust Schistocerca gregaria:Role of inositol phosphates, protein kinase C and calcium in second messenger effects

Acetylcholine (ACh) induced dose-dependent contraction of the locust (Schistocerca gregaria) foregut was antagonised by 4-DAMP (1,1-dimethyl-4- diphenylacetoxypiperidinium; 10 nM-10 μM) and Li+ (50- 100 mM). The inhibition by Li+, of ACh-induced gut contraction suggests that this muscarinic ACh receptor is linked to phosphatidylinositol second messenger systems. ACh (1 μM and 0.5 mM) stimulated concentration-dependent production of [3H]-inositol phosphates, including [3H]-inositol trisphosphate, from foregut homogenates incubated in Tris buffer containing [3H]-myo-inositol. Pre-incubation of homogenates with Li+ (50 mM) or atropine (10 μM) reduced ACh-stimulated inositol phosphate production by 95%. The contractile effects of ACh, 1-oleoyl-2-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu), but not those of proctolin, on the foregut, were reduced by the protein kinase C (PKC) inhibitor H7 (10 nM-10 μM) and the L-type Ca2+ channel blocker verapamil (1 μM-0.1 mM). In comparison, the contractile effects of proctolin, but not those of ACh, OAG and PDBu, were potentiated by H7 (10 nM- 10 μM) and attenuated by the inositol 1,4,5-trisphosphate (IP3) receptor blocker decavanadate (10 μM-0.5 mM) and ryanodine (1 μM-0.1 mM), an inhibitor of intracellular Ca2+ release. Decavanadate and ryanodine had no effect on ACh-induced tissue contraction. Proctolin-induced contraction was unaffected by the presence of verapamil at concentrations as high as 1 mM but was abolished by 10 mM Co2+ . These data suggest, that in the locust foregut, stimulation of ACh receptors causes activation of PKC, thereby promoting Ca2+ entry via the opening of L-type Ca2+ channels. However, the function of IP3 produced following activation of tissue muscarinic receptors is unclear. In contrast, activation of proctolin receptors causes verapamil-insensitive entry of extracellular Ca2+ as well as the generation of IP3 which causes release of Ca2+ from intracellular stores to bring about an increase in gut contractility.</p