Calretinin as a Marker for Premotor Neurons Involved in Upgaze in Human Brainstem

Abstract

Eye movements are generated by different premotor pathways. Damage to them can cause specific deficits of eye movements, such as saccades. For correlative clinico-anatomical post-mortem studies of cases with eye movement disorders it is essential to identify the functional cell groups of the oculomotor system in the human brain by marker proteins. Based on monkey studies, the premotor neurons of the saccadic system can be identified by the histochemical markers parvalbumin (PAV) and perineuronal nets in humans. These areas involve the interstitial nucleus of Cajal (INC) and the rostral interstitial nucleus of the medial longitudinal fascicle (RIMLF), which both contain premotor neurons for upgaze and downgaze. Recent monkey and human studies revealed a selective excitatory calretinin (CR)-positive input to the motoneurons mediating upgaze, but not to those for downgaze. Three premotor regions were identified as sources of CR input in monkey: y-group, INC and RIMLF. These findings suggest that the expression pattern of parvalbumin and CR may help to identify premotor neurons involved in up- or downgaze. In a post-mortem study of five human cases without neurological diseases we investigated the y-group, INC and RIMLF for the presence of parvalbumin and CR positive neurons including their co-expression. Adjacent thin paraffin sections were stained for the aggrecan (ACAN) component of perineuronal nets, parvalbumin or CR and glutamate decarboxylase. The comparative analysis of scanned thin sections of INC and RIMLF revealed medium-sized parvalbumin positive neurons with and without CR coexpression, which were intermingled. The parvalbumin/CR positive neurons in both nuclei are considered as excitatory premotor upgaze neurons. Accordingly, the parvalbumin-positive neurons lacking CR are considered as premotor downgaze neurons in RIMLF, but may in addition include inhibitory premotor upgaze neurons in the INC as indicated by co-expression of glutamate decarboxylase in a subpopulation. CR-positive neurons ensheathed by perineuronal nets in the human y-group are considered as the homolog premotor neurons described in monkey, projecting to superior rectus (SR) and inferior oblique (IO) motoneurons. In conclusion, combined immunostaining for parvalbumin, perineuronal nets and CR may well be suited for the specific identification and subsequent analysis of premotor upgaze pathways in clinical cases of isolated up- or downgaze deficits.

Keywords: interstitial nucleus of Cajal; oculomotor nucleus; rostral interstitial nucleus of the medial longitudinal fascicle; saccadic burst neurons; y-group.

Figure 1

Overviews of neighboring transverse sections through the human oculomotor nucleus with Nissl-staining (A) and calretinin (CR) immunostaining (B). For clarity corresponding blood vessels are indicated by asterisks (A,B). In Nissl-staining the cytoarchitecture of nIII is demonstrated with its different motoneuronal subgroups (A). Only the central subgroup of nIII receives a strong input from CR-positive fibers and terminals (B, arrows). The detailed view of the inset in (B) shows CR-positive fibers (C, arrows) and putative terminals (C, arrowheads) targeting putative superior rectus and inferior oblique motoneurons (C, stars). A: A-group of medial rectus motoneurons; B: B-group of medial rectus motoneurons; SR: superior rectus muscle; IO: inferior oblique muscle. Scale bar in (B) = 500 μm (applies to A–B); in (C) = 50 μm.

Figure 2

Detailed views from transverse sections through the rostral midbrain at the level of the rostral interstitial nucleus of the medial longitudinal fascicle (RIMLF). The RIMLF is outlined by the presence of perineuronal nets here stained for aggrecan (ACAN) as a wing-shaped nucleus curving around the dorsomedial border of the red nucleus (RN) and bordered medially by the thalamo-subthalamic paramedian artery (A, stars). Magnifications of corresponding detailed views demonstrate examples of putative down-burst neurons, which are ensheathed by ACAN-based perineuronal nets (B, arrow), express parvalbumin (PAV)—immunoreactivity (D, arrow), but lack calretinin (CR; F, arrow). (C–G) Example of putative up-burst ACAN- and PAV-positive neurons (C,E, arrows) that express CR-immunostaining in addition (G, arrows). 3: third ventricle. Scale bar in (A) = 500 μm; in (G) = 100 μm (applies to B–G).

Figure 3

(A) Schematic transverse section through the rostral interstitial nucleus of the medial longitudinal fascicle (RIMLF) indicating the plane (see cutting angle in sagittal view in inset) and area of the plots shown in B–E (rectangle). Schematic drawings from the RIMLF at four different planes from caudal to rostral (B–E) demonstrating putative up-burst neurons identified by simultaneous expression of parvalbumin (PAV) and calretinin (CR; red dots) and down-burst neurons expressing PAV only (black dots). The thalamo-subthalamic artery serves as a useful landmark (B–E, dotted lines). Note that both populations are intermingled. CP, cerebral peduncle; INC, interstitial nucleus of Cajal; PO, principal inferior olive; MLF, medial longitudinal fascicle; nIII, oculomotor nucleus; nIV, trochlear nucleus; nVI, abducens nucleus; PC, posterior commissure; MB, mammillary body; MT, mammillothalamic tract; RN, red nucleus; SC, superior colliculus; ST, subthalamus; TR, tractus retroflexus. Scale bar in (E) = 1 mm (applies to B–E).

Figure 4

Detailed views from transverse sections through the rostral midbrain at the level of the interstitial nucleus of Cajal (INC). The INC is highlighted by the presence of aggrecan (ACAN) -based perineuronal nets as a compact oval nucleus (A). Neighboring sections were stained for parvalbumin (PAV), calretinin (CR) and glutamate decarboxylase (GAD; B,D,F). Rectangles indicate the areas for detailed views in (C,E,G), which demonstrate examples of putative up-burst neurons, which contain parvalbumin (PAV) and calretinin (CR; C,E, arrows) and putative down-burst neurons, which express PAV, but lack CR (C,E, open arrows). In one example the additional lack of GAD could be demonstrated (C,E,G open arrow head) . An example of a putative inhibitory down-burst neuron, which expresses immunoreactivity for GAD and PAV, but lacks CR is indicated by an arrow head (C,E,G). For clarity corresponding blood vessels are marked by asterisks. 3: third ventricle. Scale bar in (A) = 500 μm; in (F) = 500 μm (applies to B,D,F); in (G) = 50 μm (applies to C,E,G).

Figure 5

(A) Schematic transverse section through the interstitial nucleus of Cajal (INC) indicating the plane (inset) and area (rectangle) of the plots shown in (B,C). Drawings of INC at two different planes from caudal and rostral INC (B,C) demonstrating putative upgaze neurons identified by simultaneous expression of parvalbumin (PAV) and calretinin (CR, red dots). Neurons expressing PAV only (B,C, black dots) may include excitatory and inhibitory downgaze neurons (compare Figures 4C,E,G, solid arrow head). Note that both populations are intermingled. EWcp: central projecting Edinger-Westphal nucleus; PO: principal inferior olive; MB: mammillary body; MLF: medial longitudinal fascicle; MT: mammillothalamic tract; nIII: oculomotor nucleus; nIV: trochlear nucleus; nVI: abducens nucleus; PAG: periaqueductal gray; PC: posterior commissure; RN: red nucleus; SC: superior colliculus; SN: substantia nigra.; ST: subthalamus; TR: tractus retroflexus. Scale bar in (C) = 200 μm (applies to B,C).

Figure 6

(A,B) Schematic drawings of a human brain stem, where the dotted line indicates the level of the neighboring transverse sections in (C–H), (C,E,G) show overviews of transverse sections in cresyl violet staining to demonstrate the cytoarchitecture (C) immunostaining for aggrecan (ACAN) to identify the y-group (Y) (E) and for calretinin (CR) (G). Rectangles indicate the detailed views shown in (D,F,H). Note that most CR-positive neurons are associated with ACAN-based perineuronal nets as indicated by arrows pointing to corresponding neurons (F,H). ARC, arcuate nucleus; DCN, dorsal cochlear nucleus; DEN, dentate nucleus; DVN, descending vestibular nucleus; ICP, inferior cerebellar peduncle; INC, interstitial nucleus of Cajal; PO, principal inferior olive; MLF, medial longitudinal fascicle; MVN, medial vestibular nucleus; nIII, oculomotor nucleus; nIV, trochlear nucleus; nVII, facial nucleus; PC, posterior commissure; PPH, prepositus nucleus; PT, pyramidal tract; RIMLF, rostral interstitial nucleus of the MLF; RN, red nucleus; SC, superior colliculus; SOL, solitary nucleus; SpV, spinal trigeminal nucleus; TR, tractus retroflexus; VCN, ventral cochlear nucleus; VN, vestibular nuclei. Scale bar in (G) = 500 μm (applies to C,E,G); in (H) = 100 μm (applies to D,F,H).