The ventral aspect of medulla oblongata presents two elevations, the pyramid raised by the corticospinal tract and the olive raised by the inferior olivary complex. On the ventral surface of the pyramid, lie a group of neurons collectively known as the arcuate nuclei [1]. Arcuate nuclei are one of the pre-cerebellar group of nuclei, whose role is being speculated in regulation of breathing and respiration in humans by mediating the ventilator responsiveness to carbon dioxide [2-4] as aplasia of these nuclei was seen in brains of babies with sudden infant death syndrome [5]. On the contrary, there are also studies documenting aplasia of these nuclei in adult brains suggesting that they are not critical to survival [4, 6]. Recently, changes in the pre-cerebellar brainstem structures are being associated with their role in cognitive functions [3, 7]. Furthermore, lack of definitive arcuate nuclei in other species [8, 9] has also contributed to scarcity of information regarding their precise function. Embryologically, arcuate nuclei develop from the rhombic lip and are considered ectopic pontine nuclei, which project to the cerebellum [1, 10, 11]. The efferents from the arcuate nuclei follow two routes: one fiber system runs dorsally in the midline to reach the floor of fourth ventricle and courses laterally as the stria medullares; and the other fiber system passes anteriorly on the surface of medulla oblongata as the anterior external arcuate fibers (AEAF) [1]. These fibers project mainly to the contralateral cerebellum via the restiform body, however the exact site of termination of these fibers in human cerebellum has not yet been documented; although efferent fibers from nucleus pararaphales (which lies in the midline of medulla) in macaque brains follow the course taken by AEAF to reach the floccular region of the cerebellum and are documented to play a role in vertical eye movements [12]. Although AEAF are considered efferents of arcuate nuclei, recent studies have suggested dorsal aspect of the inferior olivary nucleus as the source of hypertrophic AEAF based on the similar histo-cytochemical staining pattern of the neurons and the fibers [13] as well as common embryonic origin from the rhombic lip [11]. All these aspects have brought back interest in the development, connections and functions of this small group of neurons.

Hypertrophy (marked enlargement) of AEAF [13] and prevalence of circumolivary and preolivary bundles in human brain [14] has recently arisen interest in the variant anatomy of the human brainstem. Although recent studies have reported variable patterns of fibers seen on the ventral aspect of the medulla oblongata, albeit some vagueness still exists in their identification and classification. Hence, the objective of this work was to observe the detailed morphology of AEAF in the Indian population.

This study was performed on 50 formalin fixed adult human brains (100 brainstem sides) of unknown sex, age, and medical history, in the Department of Anatomy, All India Institute of Medical Sciences, New Delhi. The national capital region of India is a large area where people from various parts of the country reside. The institutional guidelines for procurement of human cadavers and their use for medical teaching and research were strictly adhered. The anonymised body donors included in this study all gave informed consent during their lifetime to be part of research projects and student training. The ethics committee declared the use of these corpses for scientific studies as legal. Therefore, no separate vote of the local ethics committee was required.

The brainstems were observed macroscopically for the presence of prominent transversely running fibers on the ventral aspect of the medulla oblongata. For evaluation, two independent observers (PK, DB) noted the brainstem specimens for AEAF. The prevalence, laterality, and the morphology of fibers was noted. Since these fibers were present in relation to the olive, AEAF were further classified based on this parameter. Fibers appearing at the anterior midline fissure, running on the ventral aspect of pyramids, continuing above the superior pole of olive were called the supraolivary fibers. Based on the number of fiber bundles, they were further sub-classified as single, double, or multiple (Fig. 1A–C). Fibers appearing at the anterior midline fissure, running transversely, superficial to the ventral surface of olives were called the preolivary fibers. Some of them were observed to present additional prominent bundles (Fig. 1D–F). The fibers winding around the inferior pole of the olive were called the circumolivary fibers and they were sub-classified as single or double bundles (Fig. 1G–I). Circumolivary and few preolivary fibers were seen forming a U shaped structure: with descending limb as the lateral part of pyramid and ascending limb merging with the inferior cerebellar peduncle in both the bundles. However, the two were distinguished based on the location of genu: in preolivary fibers, the genu was seen running across the ventral olivary surface with parts of olive seen on either side (Fig. 1F) while in circumolivary bundle the genu was seen winding below the lower pole of the olive (Fig. 1G). The thickness of supraolivary and circumolivary fiber bundles (≥0.1 mm) was also measured using vernier callipers (mm).

Statistical analysis was done using STATA (Stata Co.). Laterality and co-existence of the different fiber bundles was compared using Fischer’s exact test while rank sum test was used to compare the thickness of the various fiber bundles. The P-value less than 0.05 was considered statistically significant.

Distinct transversely running AEAF were observed in 13 out of 50 brains (26%). Based on the course of these fibers on the ventral surface of medulla oblongata, the AEAF were further classified and their prevalence noted in 100 brainstem sides (Table 1).

Several types of fibre bundles superficial to the pyramids have been described in early and mid 20th century [15-18]. Due to the varied nomenclature used, there was perplexity in understating the course and connections of these aberrant fibers. Hence, nomenclature of AEAF with respect to the olive, which is an important landmark of the region is proposed in the present study. Circumolivary fibers were initially described as aberrant cortico-pontine fasciculus as they were seen arising from the lateral aspect of the pyramids [16]. These fibers were accordingly suggested to terminate in various cranial nerve nuclei especially: facial, vagus, and hypoglossal [15]; nucleus ambiguus; lateral reticular nucleus [17]. Majority agree about their termination in the cerebellum via the restiform body [16, 17, 19]. Swank [17] suggested that the fibres can be detected microscopically even if not seen grossly. The prevalence of circumolivary fibers has been documented to be 7.65% [17] and 6.14% [14] as compared to 9% observed in the present study. Prevalence of preolivary fibers was noted to be higher in the Indian population as compared to the German population (18% vs. 2.45%) [14]. Supraolivary fibers have not been described in literature, although these were the most prevalent (96.15%) type of AEAF found in the brain specimens in the present study. Reports of previous studies documenting thicker circumolivary bundle on the left side [14, 17] corroborates with the findings of the present study. On the contrary, preolivary fibers were reported to be thicker on the right side [14].

With respect to the laterality analysis, supraolivary and preolivary fibers were noted bilaterally in the majority of the brains while circumolivary fibers seen unilaterally in the majority of brain specimens. The observations of the present study corroborate with the previous studies wherein circumolivary fibers have been reported to occur predominantly unilaterally, with left as the dominant side [14, 17]. The observations of presence of unilateral preolivary fibers on the left side also corroborates with Brendel et al. [14]. Variations in the prevalence, laterality and morphometry of the AEAF could be due to the possible differences in the ethnicity of the study population. Additionally, shrinkage (11%–13%) of the brainstem by embalming procedure and storage in formalin based solution [20] has to be kept in mind when extrapolating the bundle thickness data. Furthermore, lack of information such as exact age, sex, and medical history of the deceased are the limitations of the study.

Embryologically, the expression of Wnt1, PAX6, and BMP are critical for maintaining the neuronal cell population of the rhombic lip [21, 22]. Expression of a plethora of factors such as Ntn1 by the floorplate [23]; repulsive diffusive guidance molecules SLIT2 and 3 by the facial nucleus [24] at specific ontogenetic periods govern the migration of pontine nuclei [25]. Since arcuate nuclei are considered ectopic pontine nuclei, any derangement in the molecular regulation in the migratory pathway of these nuclei may result in an ectopic group of neurons, giving rise to aberrant fiber bundles. Correspondingly, ectopic nuclear masses have been documented in the ventral aspect of medulla oblongata [19] and across the dorsolateral aspect of the inferior cerebellar peduncle as corpus pontobulbare (nucleus of the circumolivary bundle) [11] in the brains presenting circumolivary fibers [17].

The study is intriguing because it highlights the asymmetries found in the brainstem, which are unique to human species [6, 26]. Arcuate nuclei have been implicated in regulation of ventilation [2-4, 20] and their functions have been reported to change markedly with age, being critical during earliest developmental stages [27]. There is a call to further explore their role in other breathing related conditions. Knowledge of the variations in the pattern of AEAF is important for neuroradiologists and neurosurgeons as it can aid in better understanding the fiber bundle presentation for diagnosis and while performing surgeries in this crucial region respectively.

To conclude, the observations of the present study opens avenues for further research focusing on the connections and functions of the additional fibers and to elucidate developmental factors involved in the migration of pre-cerebellar neurons in humans.