Reticular Formation Connections Underlying Horizontal Gaze: The Central Mesencephalic Reticular Formation (cMRF) as a Conduit for the Collicular Saccade Signal

Abstract

The central mesencephalic reticular formation (cMRF) occupies much of the core of the midbrain tegmentum. Physiological studies indicate that it is involved in controlling gaze changes, particularly horizontal saccades. Anatomically, it receives input from the ipsilateral superior colliculus (SC) and it has downstream projections to the brainstem, including the horizontal gaze center located in the paramedian pontine reticular formation (PPRF). Consequently, it has been hypothesized that the cMRF plays a role in the spatiotemporal transformation needed to convert spatially coded collicular saccade signals into the temporally coded signals utilized by the premotor neurons of the horizontal gaze center. In this study, we used neuroanatomical tracers to examine the patterns of connectivity of the cMRF in macaque monkeys in order to determine whether the circuit organization supports this hypothesis. Since stimulation of the cMRF produces contraversive horizontal saccades and stimulation of the horizontal gaze center produces ipsiversive saccades, this would require an excitatory cMRF projection to the contralateral PPRF. Injections of anterograde tracers into the cMRF did produce labeled terminals within the PPRF. However, the terminations were denser ipsilaterally. Since the PPRF located contralateral to the movement direction is generally considered to be silent during a horizontal saccade, we then tested the hypothesis that this ipsilateral reticuloreticular pathway might be inhibitory. The ultrastructure of ipsilateral terminals was heterogeneous, with some displaying more extensive postsynaptic densities than others. Postembedding immunohistochemistry for gamma-aminobutyric acid (GABA) indicated that only a portion (35%) of these cMRF terminals are GABAergic. Dual tracer experiments were undertaken to determine whether the SC provides input to cMRF reticuloreticular neurons projecting to the ipsilateral pons. Retrogradely labeled reticuloreticular neurons were predominantly distributed in the ipsilateral cMRF. Anterogradely labeled tectal terminals were observed in close association with a portion of these retrogradely labeled reticuloreticular neurons. Taken together, these results suggest that the SC does have connections with reticuloreticular neurons in the cMRF. However, the predominantly excitatory nature of the ipsilateral reticuloreticular projection argues against the hypothesis that this cMRF pathway is solely responsible for producing a spatiotemporal transformation of the collicular saccade signal.

Keywords: PPRF; eye movement; gaze; oculomotor; saccade; superior colliculus.

Figure 1

Distribution of central mesencephalic reticular formation (cMRF) terminals labeled by biotinylated dextran amine (BDA). The distribution of labeled terminals (stipple) and axons (lines) observed following a BDA injection into the cMRF (A,B) is charted on a rostral to caudal series of sections through the midbrain (A–F), pons (A–G) and medulla (G–I).

Figure 2

BDA labeled reticuloreticular axons at the level of the abducens nucleus. The pattern of reticuloreticular axons (lines) and terminals (stipple) is illustrated for the region shown by a box in (C) at higher magnification in (A). The center of the BDA injection within the cMRF that produced the labeling is shown in (B). Note the ipsilateral predominance of the terminal labeling in the pontine reticular formation (PRF) and medullary reticular formation (MdRF).

Figure 3

Morphology of reticuloreticular axons labeled by BDA. (A) Low magnification photomicrograph of a section through the abducens nucleus showing the location of the higher magnification plates in this figure (labeled boxes). Some of the BDA labeled axons displayed close associations with counterstained somata (arrowheads), while others terminated in the neuropil (arrows). The images were taken from the ipsilateral PRF (B,C), contralateral PRF (D,E), ipsilateral MdRF (F) and contralateral MdRF (G). The case shown is the same as illustrated in Figure 2. (Number of 1.0 μm Z axis planes merged: B,C,E = 3, D,F = 1, G = 4).

Figure 4

Distribution of cMRF terminals labeled by Phaseolus vulgaris leucoagglutinin (PhaL). The distribution of labeled terminals (stipple) and axons (lines) observed following a PhaL injection into the cMRF (A,B) is charted on a rostral to caudal series of sections through the midbrain (A–F), pons (A–G) and medulla (H–I).

Figure 5

PhaL labeled reticuloreticular axons at the level of the abducens nucleus. The pattern of reticuloreticular axons (lines) and terminals (stipple) is illustrated for the region shown by a box in the inset. The injection site is illustrated in Figure 4. Note the ipsilateral predominance of the terminal labeling in the PRF and MdRF.

Figure 6

Morphology of reticuloreticular axons labeled by PhaL. (A) Low magnification photomicrograph of a section through the abducens nucleus showing the location of the higher magnification plates (labeled boxes) in this uncounterstained section. The images were taken from the ipsilateral and contralateral supragenual region (SG; B,C, respectively), ipsilateral and contralateral abducens nucleus (VI) (D,E, respectively), and ipsilateral and contralateral PRF (F,G, respectively). Note the larger number of boutons ipsilaterally in the PRF. The case shown is the same as illustrated in Figures 4, 5. (Number of 1.0 μm Z axis planes merged: B,E,G = 10, C,F = 13, D = 4).

Figure 7

Location of electron microscopic (EM) samples from the PRF. The distribution of labeled terminals (stipple) and axons (lines) observed following a small BDA injection into the cMRF (A,B) is charted on a rostral to caudal series of sections through the midbrain (A–E) and pons (A–F). The location of areas sampled for electron microscopy is indicated by blue boxes.

Figure 8

Ultrastructure of reticuloreticular axon terminals. Axon terminals that were labeled with BDA (At*) following the injection of the cMRF shown in Figure 7 were electron dense. Most of these terminals contacted (arrowheads) small dendrites (Den). These labeled terminals were heterogeneous: some were packed with clear spherical vesicles (A–C) and displayed asymmetric synaptic densities (C). Others contained pleomorphic vesicles and made symmetric contacts (D–F). Postembedding immunohistochemistry for gamma-aminobutyric acid (GABA) labeled a portion of the BDA labeled axon terminals (At^*+), as well as some terminals not labeled with BDA (At⁺). The BDA labeled terminals that were overlain with numerous gold particles indicating they were GABAergic contained pleomorphic vesicles and made symmetric synaptic contacts (D–F).

Figure 9

Distribution of midbrain reticuloreticular neurons charted on a rostral to caudal series of sections. An injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) centered in the PRF (G–I) labeled reticuloreticular neurons (red dots) in the cMRF (C–F) and the piMRF (A,B), as well as other midbrain cells (blue dots). Note the primarily ipsilateral distribution. Adjacent insets show the level of the selected sections.

Figure 10

Injection sites for a dual tracer case indicated on a rostral to caudal series of sections. BDA was injected into the superior colliculus (SC; Blue area in A–E) and WGA-HRP was injected into the lateral PRF (Red area in F–H).

Figure 11

Overlapping distribution of tectoreticular terminals and reticuloreticular neurons. The distribution of tectal terminals (stipple) labeled by BDA and reticuloreticular neurons (red diamonds) labeled by WGA-HRP that resulted from the injections illustrated in Figure 10 are shown for the ipsilateral midbrain reticular formation (MRF). Note the overlap in their distributions within the cMRF (B–F) and the piMRF (A). The reticuloreticular cells were scattered amongst BDA labeled reticulotectal neurons (black dots) in the cMRF. Other WGA-HRP labeled cells are indicated by black diamonds.

Figure 12

The relationship between tectal inputs and reticuloreticular neurons in the cMRF. Drawings reveal the presence of close associations (arrowheads) between BDA labeled tectoreticular terminals and reticuloreticular cells (B–J) labeled from a PRF injection of WGA-HRP. The location of the example cells within the cMRF is shown in (A). While a number of close associations are present, the labeled axons rarely (E,H) follow the contours of the cell, and the number of contacts varied.

Figure 13

Pattern of contacts between tectoreticular terminals and cMRF cells. Photomicrographs show examples of close associations (arrowheads) between brown, retrogradely labeled reticuloreticular neurons and black, anterogradely labeled tectoreticular terminals (A–E). Individual axons display one or two boutons associated with the cell in most (B,D,E), but not all (A,C), cases. In contrast, black, BDA labeled tectoreticular terminals line up along the dendrites of the black, BDA labeled reticulotectal neurons in the same case (F).

Figure 14

Circuit diagram showing the cMRF as a conduit for collicular influence on lower brainstem centers. Connections from the left side of the midbrain that would produce rightward (contraversive) saccades are shown. Neurons located in the cMRF receive direct input from the SGI of the SC and forward a signal to the horizontal gaze center, the brainstem center that controls horizontal saccadic eye movements. The horizontal gaze center contains both excitatory burst neurons (EBNs) and inhibitory burst neurons (IBNs) that are premotor cells projecting to the ipsilateral and contralateral abducens nucleus, respectively. Axons of collicular neurons (blue pentagon) that travel via the predorsal bundle, provide collaterals to the ipsilateral cMRF before crossing and terminating in the horizontal gaze center. The cMRF contains both inhibitory (red circle) and excitatory (blue circle) cells that project to the ipsilateral horizontal gaze center. In addition, it contains a population of cells (purple circle) that project to the contralateral horizontal gaze center. The cMRF also provides feedback to the SC, which is purely inhibitory (red circle), ipsilaterally, and both inhibitory and excitatory (red and blue circles, respectively), contralaterally. The activity of cMRF neurons for rightward (R) and leftward (L) saccades is indicated by small arrows.