Organization of Afferents along the Anterior-posterior and Medial-lateral Axes of the Rat Orbitofrontal Cortex

Abstract

The orbitofrontal cortex (OFC) has been anatomically divided into a number of subregions along its medial-lateral axis, which behavioral research suggests have distinct functions. Recently, evidence has emerged suggesting functional diversity is also present along the anterior-posterior axis of the rodent OFC. However, the patterns of anatomical connections that underlie these differences have not been well characterized. Here, we use the retrograde tracer cholera toxin subunit B (CTB) to simultaneously label the projections into the anterior lateral (ALO), posterior lateral (PLO), and posterior ventral (PVO) portions of the rat OFC. Our methodological approach allowed us to simultaneously compare the density and input patterns into these OFC subdivisions. We observed distinct and topographically organized projection patterns into ALO, PLO, and PVO from the mediodorsal and the submedius nuclei of the thalamus. We also observed different levels of connectivity strength into these OFC subdivisions from the amygdala, motor cortex, sensory cortices and medial prefrontal cortical structures, including medial OFC, infralimbic and prelimbic cortices. Interestingly, while labelling in some of these input regions revealed only a gradient in connectivity strength, other regions seem to project almost exclusively to specific OFC subdivisions. Moreover, differences in input patterns between ALO and PLO were as pronounced as those between PLO and PVO. Together, our results support the existence of distinct anatomical circuits within lateral OFC along its anterior-posterior axis.

Keywords: amygdala; neuroanatomy; rodent; rostral–caudal; submedius nucleus; thalamus.

Graphical abstract

Fig. 1

Coronal sections depicting core of tracer deposits into ALO, PLO, or PVO, illustrated at the level of maximal tracer deposit. (A) Stars and oval shapes represent the intended and the observed injection sites core, respectively. Injections whose core fell outside the orbitofrontal cortex or in which the tracer did not diffuse are not represented here (see instead Fig. S1). n ALO = 6, n PLO = 5, n PVO = 4 injections. (B) Example injection sites of triple-labelled brain. Distances shown are distances from bregma in mm. AF: Alexa Fluor; AId: agranular insular cortex, dorsal part; AIv: agranular insular cortex, ventral part; ALO: lateral orbitofrontal cortex, anterior part; CTB: cholera toxin subunit B; DLO: orbitofrontal cortex, dorsolateral area; IL: infralimbic cortex; MO: orbitofrontal cortex, medial part; LO: orbitofrontal cortex, lateral part; M1: primary motor cortex; M2: secondary motor cortex; PL: prelimbic cortex; PLO: lateral orbitofrontal cortex, posterior part; PVO: ventral orbitofrontal cortex, posterior part; VO: orbitofrontal cortex, ventral part.

Fig. 2

Density of retrogradely-labelled cells following CTB injection into ALO, PLO, or PVO. Represented is the average density across brains discretized into four labelling levels (see Table S1 for exact average density values). Lighter or darker shades represent lower or higher labelling densities, respectively. This represents the average density of projections from multiple brains within a given structure but note that this does not indicate the precise location of these projections within the structure. Depiction of labelling in key areas of interest (amygdala, mediodorsal thalamus and submedius nucleus of the thalamus) was obtained by superimposing hand drawings of the labelling in each brain using an opacity value proportional to the density level of labelling observed. Star shapes represent the intended injection site cores. Distances shown are distances from bregma in mm. n ALO = 6, n PLO = 5, n PVO = 4. a24b: anterior cingulate cortex, area 24b; ALO: lateral orbitofrontal cortex, anterior part; AI, v, d, P: agranular insular cortex, ventral area, dorsal area, posterior area; Au1: primary auditory cortex; BLA: basolateral amygdala; CTB: cholera toxin subunit B; DLO: dorsolateral orbitofrontal cortex; GI: granular insular cortex; IL: infralimbic cortex; LEnt: lateral entorhinal cortex; max. deposit: maximal deposit; LO: lateral orbitofrontal cortex; M1: primary motor cortex; M2: secondary motor cortex; MD: mediodorsal nucleus of the thalamus; MO: medial orbitofrontal cortex; Pir: piriform cortex; PL: prelimbic cortex; PLO: lateral orbitofrontal cortex, posterior part; PVO: ventral orbitofrontal cortex, posterior part; PRh: perirhinal cortex; PT: paratenial nucleus of the thalamus; Re: nucleus reuniens of the thalamus; RS: retrosplenial cortex; S1BF: primary somatosensory cortex, barrel field; S1J: primary somatosensory cortex, jaw region; S1HL/FL: primary somatosensory cortex, hindlimb and forelimb regions; S1Tr: primary somatosensory cortex, trunk region; S2: secondary somatosensory cortex; Sub: submedius nucleus of the thalamus; V2: secondary visual cortex; VO: ventral orbitofrontal cortex.

Fig. 3

Example single-, double-, and triple-labelled cells. Represented is a plane of a z-stack obtained with a confocal microscope of the submedius nucleus of the thalamus in which are present: single-labelled cells after (1) ALO, (2) PLO, or (3) PVO injections; double-labelled cells after (4) ALO and PLO, or (5) after PLO and PVO injections; and (6) triple-labelled cells.

Fig. 4

Localization and density of retrogradely-labelled cells in the MD following CTB injection into ALO, PLO, or PVO. (A) Shaded areas correspond to superimposed hand drawings of the labelling in each brain included in the analysis, using an opacity value proportional to the labelling density level. n ALO = 6, n PLO = 5, n PVO = 4 injections. (B) Micrographs from representative brain with successful triple labelling. c: central; l: lateral; m: medial; AF: Alexa Fluor; ALO: lateral orbitofrontal cortex, anterior area; CTB: cholera toxin subunit B; MD: mediodorsal nucleus of the thalamus; PLO: lateral orbitofrontal cortex, posterior area; PVO: ventral orbitofrontal cortex, posterior area.

Fig. 5

Localization and density of retrogradely-labelled cells in Sub following CTB injection into ALO, PLO, or PVO. (A) Shaded areas correspond to superimposed hand drawings of the labelling in each brain included in the analysis, using an opacity value proportional to the labelling density level. n ALO = 6, n PLO = 5, n PVO = 4 injections. (B) Micrographs from representative brain with successful triple labelling. D: dorsal; V: ventral; AF: Alexa Fluor; ALO: lateral orbitofrontal cortex, anterior area; CTB: cholera toxin subunit B; PLO: lateral orbitofrontal cortex, posterior area; PVO: ventral orbitofrontal cortex, posterior area; Sub: submedius nucleus of the thalamus.

Fig. 6

Labelling strength in MD and Sub following injection of CTB into ALO, PLO, or PVO. (A) Average labelling strength in MD across coronal sections −1.88, −2.12, −2.30, and −2.56 mm from bregma.) (B) Average labelling strength in Sub across coronal sections −2.12, −2.30, and −2.56 mm from bregma. (C) Labelling strength in Sub after ALO, PLO, or PVO injections at coronal sections −2.12, −2.30, or −2.56 mm from bregma. n ALO = 6, n PLO = 5, n PVO = 4 injections. c: central; l: lateral; m: medial; D: dorsal; V: ventral; ALO: lateral orbitofrontal cortex, anterior area; CTB: cholera toxin subunit B; MD: mediodorsal nucleus of the thalamus; PLO: lateral orbitofrontal cortex, posterior area; PVO: ventral orbitofrontal cortex, posterior area; Sub: submedius nucleus of the thalamus.

Fig. 7

Labelling in the amygdala following CTB injection into ALO, PLO, or PVO. (A) Shaded areas correspond to superimposed hand drawings of the labelling in each brain included in the analysis, using an opacity value proportional to the labelling density level. n ALO = 6, n PLO = 5, n PVO = 4 injections. (B) Average labelling strength in regions of the amygdala across coronal sections −1.88, −2.12, −2.30 and −2.56 mm from bregma. n ALO = 6, n PLO = 5, n PVO = 4 injections. ALO: lateral orbitofrontal cortex, anterior area; CTB: cholera toxin subunit B; BLA: basolateral amygdala, anterior part; BLP: basolateral amygdala, posterior part; LaDL: lateral amygdala, dorsolateral part; LaML: lateral amygdala, mediolateral part; LaVL, lateral amygdala, ventrolateral part; PLO: lateral orbitofrontal cortex, posterior area; PVO: ventral orbitofrontal cortex, posterior area.

Fig. 8

Density of key inputs into ALO, PLO and PVO. The different number of circles represent the average density of retrogradely-labelled cells following CTB injection (i.e., absent, weak, moderate or strong) into ALO, PLO, and PVO. Panels depicting labelling in amygdala and in MD thalamus represent the average density labelling in all sections quantified and not at that particular coronal section. Distances shown are distances from bregma in mm. a24b: anterior cingulate cortex, area 24b; ACC: anterior cingulate cortex; AId: agranular insular cortex, dorsal part; AIv: agranular insular cortex, ventral part; ALO: lateral orbitofrontal cortex, anterior part; CTB: cholera toxin subunit B; DLO: orbitofrontal cortex, dorsolateral part; IL: infralimbic cortex; LaDL: lateral amygdala, dorsolateral part; LO: orbitofrontal cortex, lateral part; M1: primary motor cortex; M2: secondary motor cortex; MO: orbitofrontal cortex, medial part; PL: prelimbic cortex; PLO: lateral orbitofrontal cortex, posterior part; PVO: ventral orbitofrontal cortex, posterior part; VO: orbitofrontal cortex, ventral part; A: anterior; P: posterior; BL: basolateral; C: central; M: medial; L: lateral; D: dorsal; V: ventral.