Midsagittal surface anatomy
Figure 1 depicts the major subdivisions of the Göttingen minipig brain seen on a midsagittal section. The cingulate gyrus (CgA) curves anteriorly around the corpus callosum (cc) whereby it enters anteriorly and ventrally the subgenual cortical area (sgC) (Fig. 1b), which posteriorly is demarcated by the septum and more ventrally by the olfactory tubercle and the tenia tecta (Bjarkam et al. 2017a). The subgenual area continues anteriorly into more differentiated parts of the medial prefrontal cortex. Still, any macroscopic structure does not clearly mark this transition.
The Göttingen minipig subgenual cortical area (sgC)
The area identified as the Göttingen minipig subgenual cortical area (sgC) is, accordingly, located ventral to the rostral tip of the cc (Fig. 1). It is irregular-shaped and measures approximately 4.0 x 3.0 x 1.0 mm. It is subdivided into three distinct areas based on cytoarchitectural and laminar differentiation. These areas are in homology with human nomenclature (Brodmann 1909) named area 25 (BA25), area 33 (BA33), and indusium griseum (IG). Area 25 is located most anterior in the sgC and is both dorsally and posteriorly aligned by the more undifferentiated area 33 and indusium griseum, respectively, which thereby separate area 25 from the cc and the septum. The subgenual cortical area is laterally adjoined to the slit-formed anterior part of the lateral ventricle (LVa). The medial-lateral extent of the sgC is thereby restricted to no more than 1 mm at its widest point (Figs. 2c-h, 3ef).
Cytoarchitecture of the sgC and adjacent cortical areas
Area 25 (Figs. 2b-e, 3c-g) is a narrow cortex, approximately 1 mm thick. It has a poor laminar differentiation in the deeper layers due to a similar appearance of layer III and V neurons and a thin layer VI. Perpendicular to the surface cell, poor columns of white matter stretch deep into layers II and III, thereby segregating small groups of closely arranged neurons in the superficial layers (Figs. 2c-e, Fig. 4).
Layer I is thin and contains columns of white matter at regular intervals that stretch into the deeper layers. Layer II is well-defined and consists of two to three rows of small-sized neurons with sparse perinuclear cytoplasm and circular nuclei. The layer is compact and hence appears to be more intensely stained than the deeper layers. The pyramidal neurons in layer III have almost the same size and staining properties as the medium-sized (15-20 mm) neurons in layer V, and a clear separation between the two layers constituting 12 to 15 rows of irregularly oriented neurons is not possible. Layer VI is thin and consists of small, evenly stained neurons with smaller somata, and oval nuclei arranged parallel to the surface.
From the coronal and horizontal sections, it is evident that when approaching the cc and septum in the anterior-dorsal plane (Fig. 2b-e and Fig. 3c-g), area 25 becomes less differentiated with increasing cell sparse areas as well as an even more pronounced lack of laminar differentiation.
The anterior cingulate gyrus (CgA) (Fig. 2a and 3a-d) lies dorsal and anterior to area 25. It is characterized by a larger cortical thickness and densely arranged layer II cells. It is an agranular cortex where layer III and layer V inconspicuous fuses, and no explicit layer IV is evident. Layer VI contains similarly sized but more dispersed cells than layer V.
The medial prefrontal cortex (mPFC) (Fig. 2b-d, 3fg) lies anterior to the anterior cingulate cortex and ventral and anterior to BA25. The medial prefrontal cortex contains coarse cells with no apparent difference between layer III and V and is accordingly classified as the agranular cortex. Layer II is thin with lightly stained small neurons. Layer III and V have medium-sized and well-dispersed neurons which stain evenly. Layer VI is wide with smaller and more dispersed neurons.
Area 33 wraps around the rostral tip of the cc. It is less differentiated than area 25. Accordingly, the cortex is narrower and displays a complete lack of laminar differentiation due to diffusely arranged small, lightly stained neurons. These neurons become more intensely stained when approaching the septum (Fig. 2f). Area 33 abuts the indusium griseum, which is a neuron dense band of heavily stained small neurons, separating area 33 from the cc dorsally and the septum posterior.
The septal area (Fig. 2h, 3h) is delimited anteriorly by the indusium griseum. The septal area is easily recognized by diffusely arranged neurons with oval somata and clear nuclei distributed in a magnocellular medial septal complex and a parvocellular and a magnocellular lateral septal complex (Bjarkam et al., 2017a).
NeuN immunohistochemical staining was performed in order to strengthen the above given Nissl based characterization of the sgC and is accordingly shown in figure 4. The poor laminar differentiation of area 25 is readily appreciable with intermixed layer III and V, and layer II and VI are readily distinguishable. The cell poor columns of white matter are likewise evident.
Neuronal tract tracing
Due to the limited area size and nearby location to the lateral ventricle and longitudinal cerebral fissure, only 3/8 animals received sgC injections. The location of these (? trajectories) and their retrograde distribution are illustrated in figure 5 and presented in the overview in table I.
Retrograde tracing
The FluroGold tracing was visible as pale, white/yellow labelled fibres and perikarya (Fig. 6). The intensity of the retrograde tracing varied from almost invisible with a few neurons weakly labelled to widespread tracing with numerous neurons almost filled with FG. Schematic illustrations depicting areas with definite tracing are shown in figure 5 and schematically listed in Table 1.
Cortical retrograde tracing was seen primarily to the neighbouring ipsilateral ventral and medial prefrontal cortical areas with some contralateral labelling as well. Tracing was most widespread in layer III, but with some labelling of layer V neurons. The densest cortical labelling was found bilaterally in the medial prefrontal cortex (mPFC) (Fig. 6d) with a primarily laminar distribution to layer V. Furthermore, moderate tracing to cortical areas ventral to area 25 was seen bilaterally as well as weak to moderate tracing to the cingulate cortex. Interestingly, ipsilateral retrograde tracing to the insular cortex is clearly seen. Prominent projections were furthermore observed to the medial aspect of the amygdala and the hippocampal formation. Tracing to the hippocampus was seen in the ventral and intermediate parts, but with the heaviest labelling in the dorsal part. The entorhinal cortex (Ent) and subiculum (S) also showed moderate retrograde connections.
Subcortical retrograde tracing
The basal ganglia received only sparse retrograde tracing. A few neurons in the caudate nucleus (Cd) were labelled, but no tracing was found in the accumbens nucleus (Acb) or the putamen (Pu).
The diencephalon received distinct retrograde tracing primarily to the thalamus (Th). Tracing from anterior and ventral parts of the sgC (RT4 and RT7) resulted in tracing to the anterior and mediodorsal parts of the thalamus, whereas tracing from the posterior and dorsal parts of sgC (RT8) mainly showed tracing to the mediodorsal thalamus. In the hypothalamus (Hyp), weak to moderate tracing was demonstrated in the dorsal and posteriorparts.
Brainstem tracing was evident in distinct areas. Heavy labelling appeared bilaterally in the ventral tegmental area (VTA) and ipsilateral in the lateral substantia nigra (SN). Weak projections to the periaqueductal grey (PAG) in the mesencephalon and more caudal to the midline raphe nuclei in pons could also be identified. No retrograde tracing was seen caudal to the pontine area.
Anterograde tracing
BDA development revealed a moderate distinct brown/black labelling in certain areas, enabling identification of tracer-filled nerve fibers and terminals (fig. 7).
Cortical projections were observed to the adjoining medial and ventral prefrontal cortex and the cingulate cortex. Dense labelling was observed in the mid-portion of the cingulate gyrus bilaterally as well as in more dorsal and lateral located cortical areas. The hippocampus showed moderate labelling both in the ventral and dorsal parts. Some anterograde tracing was also found in the ventral piriform cortex.
Subcortical projections were less extensively seen. Moderate tracing to the caudate nucleus and the putamen were, however, evident. Just a diffuse weak tracing appeared in the medial aspects of the thalamus and the hypothalamus.
Table 1. Summarizes the retro- and anterograde tracing results.
Table 1. Tracing results. +++ heavy, ++ moderate, + weak, 0 no tracing, -- inconclusive, (b) bilateral