An ultrastructural study of central mesencephalic reticular formation connections with the superior colliculus, nucleus raphe interpositus and paramedian pontomedullary reticular formation in the macaque monkey

The central mesencephalic reticular formation (cMRF) is believed to play a role in horizontal gaze control due to its connections with the superior colliculus (SC), which generates saccadic signals, nucleus raphe interpositus (RIP), which gates saccades, and the paramedian pontomedullary reticular formation (PPRF), which directs horizontal gaze. However, the mechanism by which the cMRF affects these structures is still unknown. In the present study, terminals were labeled by injecting the neuronal tracer, biotinylated dextran amine (BDA) into the cMRF of Macaca fascicularis monkeys. Their relationships with the target neurons in SC, RIP and PPRF were examined with the light and electron microscope. To test whether these projections are inhibitory, postembedding gamma-aminobutyric acid (GABA) immunochemistry was used to identify putative inhibitory synaptic profiles.;In the first study, BDA labeled reticulotectal profiles in the ipsi- and contralateral intermediate gray layer (SGI) of SC were examined. Nearly all (94.7%) of the ipsilateral BDA labeled terminals were GABA-positive (GABA +), but profiles postsynaptic to these labeled terminals were exclusively GABA-negative (GABA-). In addition, BDA labeled terminals were observed to contact BDA labeled dendrites, indicating the presence of a monosynaptic feedback loop connecting the cMRF and ipsilateral SC. In contrast, within the contralateral SGI, half of the BDA labeled terminals were GABA +, while more than a third were GABA. All of the postsynaptic profiles were GABA-. Thus, the cMRF is part of an inhibitory feedback loop to the ipsilateral SC which presumably tunes the "winner-take-all" processes defining the location of activity within that SC, in this way modifying the saccade vector. The presence of both excitatory and inhibitory projections to the contralateral SC is surprising, as this side is presumably silent during horizontal sacades. We propose that this projection may coordinate activity in the opposite SC in more complex situations, such as series of saccades.;The second study analysed the reticuloraphe terminals in RIP. GABA postembedding revealed that approximately half (51.4%) could be categorized as GABA +, and approximately a third (35.5%) as GABA-. Nearly all (98.4 %) of the profiles postsynaptic to the BDA-labeled terminals were GABA-, in agreement with the known glycinergic character of RIP. Among 61 BDA-labeled terminals with an evident synaptic density, a tenth (11.5%) contacted somata, half (54.1%) contacted proximal dendritic profiles, and a third (37.7%) contacted distal dendritic profiles. This suggests that cMRF provides robust input to RIP, when compared to the cMRF's collicular projection, which mainly terminates distally (67.7%). Dual tracer studies in which BDA was injected into the SC and wheatgerm agglutinin conjugated horse radish pperoxidase (WGA-HRP) was injected into RIP revealed that tectal axons terminate on cMRF reticuloraphe neurons. Thus, a trans-cMRF pathway is present, such that the tectal axons that activate burst neurons in the PPRF, also activate inhibitory reticuloraphe neurons in the cMRF. These, in turn, gate the activity of RIP omnipause neurons (OPNs), releasing PPRF burst neurons from their inhibitory gating. The excitatory input to RIP may come from cMRF fixation cells that receive their inputs from SC fixation zone.;In the third study, labeled reticuloreticular axons from cMRF were observed in the PPRF, primarily ipsilaterally. The density of this projection was not as robust as cMRF's SC and RIP projections. Furthermore, the labeled terminals mostly contacted distal dendrites. GABA postembedding immunohistochemistry revealed that a third of these terminals were GABA+ (35.93%), and half were GABA- (53.13%). The minor inhibitory projection may suppress ipsilateral burst neurons during horizontal saccades. The excitatory projection may be artifact, or it may contact reticulospinal neurons subserving gaze-related head turns.;In summary, cMRF's most important projections are inhibitory: (1) inhibitory feedback to the ipsilateral SC to help produce a unified saccade signal by "winner-take-all" mechanisms and (2) gating OPN activity. Three of the cMRF projections may contain excitatory axons as well: (1).The cMRF's contralateral SC projections contain both excitatory and inhibitory components, which may contribute to the coordination of activity between the two colliculi. Similarly, cMRF sends both excitatory and inhibitory efferents; to (2) RIP; and (3) PPRF. This suggests that instead of simply relaying saccadic signals to these targets, the cMRF both up- and down-regulates their activity.