Study Of Neurotransmitters Pathway From Conjugate-Horizontal-Eye-Movements-Related Nuclei To Oculomotor Nucleus In Rats

ObjectiveTo examine the localization and distribution of brain-stem afferent neurons to the ratmedial rectus subdivision of oculomotor nucleus, and establish the neurofibra pathwayfrom conjugate-horizontal- eye-movements-related nuclei to oculomotor nucleus in ratbrain. On the basis of that, to further investigate the possible excitatory and inhibitoryneurotransmitters involved in these pathways, and then establish the excitatory andinhibitory pathway from these conjugate-horizontal-eye-movements-related nuclei tooculomotor nucleus. Thereby to provide the theory basis for the occurrence and treatmentof conjugate horizontal eye movements disturbances.Methods30 Healthy and mature Wister rats, 220～250g, randomly divided into 2 groups. 14 Rats were inthe first group: After anesthetized, rats were placed on brain stereotaxic apparatus. 0.1μlof 50% horseradish peroxidase (HRP) solution was slowly injected into one side of themedial rectus subgroup of oculomotor nucleus of the rat. After survival for 48h, ratswere perfused and fixed, and brains were removed and were cut in serial coronal sections(25～40μm) and collected in 5 groups. To reveal HRP, sections were treated with TMBreaction. One group of sections were used to observe injection sites and distributions ofHRP retrogradely labeled neurons in the brain stem. The other 4 groups were used forimmunohistochemical staining for glutamate (Glu),γ-amino-butyric acid (GABA),glutamic acid decarboxylase (GAD) and Glutamate Receptor 1(GluR1) to detect thepossible neurotransmitters in these nuclei.16 rats were in the 2nd group: After anesthetized,rats were placed on brain stereotaxic apparatus, 0.1μl of 3% fluorogold (FG) wasslowly injected into one side of the medial rectus subgroup of oculomotor nucleus of rats.After survival for 5～7days, rats were perfused and fixed, and the brains were removedand were cut in serial coronal sections (25μm) and collected in 5 groups. 1 group ofsections were used to observe injection sites and distributions of FG retrogradely labeled neurons in the brain stem. The other 4 groups were used for immunofluorescence stainingfor glutamate, GABA, GAD and GluR1 to observe distributions of various of labeledneurons in the brain stem and also their projections to oculomotor nucleus.Results(1) Retrogradely tracing results: After injection of HRP or FG into one side of medialrectus subdivision in oculomotor nucleus, retrogradely labeled neurons and neurofibrawere found in the contralateral abducens nucleus (ABN), bilateral medial vestibularnucleus(MVN) and pontine paramedian reticular formation(PPRF) and also in theipsilateral superior vestibular nucleus and nucleus of prepositus hypoglosal (PH).(2) On the immunohistochemical (or immunofluorescence) staining sections of GABA,GAD, glutamate and GIuR1, three types of labeled neurons were found respectively,including retrogradely labeled neurons, the immunoreactive neurons of correspondingprimary antibody and retrolabeled/immunoreactive double labeled neurons.Retrolabeled neurons with glutamate or GluR1 immunoreactive were mainly found incontralateral abducens nucleus and ipsilateral nucleus of prepositus hypoglosal, whileretrogradely labeled neurons with GABA or GAD immunoreactive were scarce in bothnuclei; In ipsilateral medial vestibular nucleus, retrogradely labeled neurons withglutamate or GluR1 immunoreactive were predominantly found; but in the contralateralmedial vestibular nucleus, retrolabeled neurons with GABA or GAD immunopositivewere also found. Also, retrogradely labeled neurons with GABA, GAD, Glu or GluR1immunoreactive neurons distributed relatively evenly in the PPRF.Conclusions(1) Contralateral abducens nucleus, bilateral medial vestibular nucleus and PPRF,ipsilateral superior vestibular nucleus and prepositus hypoglosal nucleus all have efferentconnections with medial rectus subgroup of oculomotor nucleus, and were possiblyinvolved in the coordination of conjugate horizontal eye movements.(2) Internuclear neurons of abducens nulens project contralaterally to oculomotor nucleusmedial rectus subgroup, and use glutamate as excitatory neurotransmitter, partly throughthe activation of GluR1 receptor; while GABA was probably not the mainneurotransmitter in the inhibitory projection. The nucleus of prepositns hypoglosal hasefferent connection mainly with the ipsilateral medial rectus subdivision of oculomotornucleus, and its excitatory and inhibitory projections were similar to that of abducensnucleus.. (3)The projections from medial vestibular nucleus to ipsilateral medial rectus subgroup ofOMN were mainly excitatory and use glutamate as neurotransmitter, partly through theactivation of GluR1 receptor. However, the excitatory and inhibitory projections, frommedial vestibular nucleus to contralaterally OMN medial rectus subdivision and fromPPRF to bilaterally OMN medial rectus subdivision, use Glu and GABA as excitatory andinhibitory neurotransmitters respectively. It need to be further determined if there is theco-existence of each other or other neurotransmitters in these projections.