Analysis of injections made into the mitral cell (MOBmi) and granule (MOBgr) layers of the MOB and into the piriform cortical area (PIR) revealed novel topographic MOB projections to and within the lot, demonstrated different MOB connections with the anterior versus posterior PIR, and exposed novel characteristics of well-established cortical olfactory projections.
Projection roadmap of the MOB: route preferences of lateral versus medial MOB mitral cells
Double coinjections were made into the (1) dorsal, (2) middle, or (3) ventral MOBmi along the dorsal-ventral axis and into medial or lateral regions along the medial-lateral axis (Figures , , ).
Figure 4 Injections in the dorsal (A) and ventral (C) lateral MOB result in axons traveling directly toward the lot located on the same side. Within the lot, fibers from the dorsal lateral MOBmi travel predominantly in dorsal parts of the lot
(B), while those (more ...)
Figure 5 Schematic of lateral and medial MOB projections to lot. Colors of injections in (A) and lot fibers in (B) are linked. Axons from dorsal (blue) or ventral (purple) lateral MOB travel directly toward the lot on the same side (A) and remain approximately (more ...)
Figure 6 Axons from the dorsal medial MOB (A1) travel through lotd (A2) to lot and travel along the dorsolateral edge within the lot (A3,A4). Ventral medial MOB axons (B1,B2) travel ventrolaterally toward lot
(B2,B2') and within the ventromedial parts of lot
Regardless of their origin along the dorsal-ventral axis, axons from the lateral
MOB course through the granule layer headed toward the lot
, the main route from the olfactory bulb to olfactory cortical areas (Gloor, 1997
) (Figures , ). Within the lot
, axons from the dorsal lateral
region travel roughly in dorsal intermediate parts of the tract, while axons from the ventral lateral
MOBmi travel roughly in the ventral intermediate portion (Figures , ) before arborizing in the AON and PIR.
Axons from the medial MOB take different routes to join the lot depending on their origin along the dorsal-ventral axis. Those from dorsal medial MOBmi (Figure ) travel through the dorsal limb of the lot (lotd; Figure ) and through a distinct dorsolateral and lateral region of the lot (Figures ,). From the ventral medial MOBmi, axons travel ventrolaterally across the MOB toward the lot (Figures ) and extend caudally roughly through the ventromedial lot (Figures ). Axons from the middle medial MOBmi (Figure ) travel either dorsally via the lotd or go ventrolaterally toward the lot (Figure ). Axons from the lotd occupy the dorsolateral or lateral edge of the lot, while axons traveling ventrolaterally occupy more ventromedial parts (Figures ; see Figure for schematic).
Differential connectivity patterns of the anterior versus posterior PIR
Injections made into the MOBgr revealed unique connections of the MOB with the anterior and posterior PIR. FG injections encompassed within the dorsal deep MOBgr (Figure ) result in back-labeled neurons both in the posterior PIR (PIRp) and magnocellular nucleus (MA; Figure ). FG injections in the MOBmi that encroach slightly onto the superficial granular layer (Figure ) label neurons only in the MA, not the PIRp (Figure ). This suggests that the MA projects to both MOBgr and MOBmi, but that the PIRp projects only to the deep MOBgr. Corroborating this connection, PHAL/CTb injections in the PIRp (Figure ) result in labeled terminals solely in the deep MOBgr (Figure ), a pattern that is preserved in posterior MOBgr regions (Figures ). PIRp CTb injections confirm that cells in the entire MOBmi project back to the PIRp (Figures ). Together, the data suggest a connection chain from the MOBmi→PIRp→deep MOBgr. PHAL injections in the anterior PIR (PIRa) specifically innervate the superficial MOBgr layers and the MOBmi (Figures ). This pattern also is preserved in more posterior regions of the MOB (Figures ) and suggests a neural chain from MOBmi→PIRa→MOBmi/superficial MOBgr. Combined, these results demonstrate that (a) the MOBgr can be stratified into superficial and deep layers and (b) the PIRa and PIRp show differential connectivity patterns to the MOB, namely that the PIRa projects to the superficial MOBgr and MOBmi, while PIRp projects to deep MOBgr and avoids the MOBmi (Figure ). This distinct connectivity of the PIRa and PIRp possibly has important implications for their roles in MOB activation (see “Discussion”).
Figure 7 Stratification of MOBgr and differential connectivity patterns of PIRa and PIRp with MOB. Injections in deep MOBgr (A) retrogradely label neurons in both MA and PIRp (B), while injections in MOBmi (C) label only MA neurons and not PIRp (D). PHAL injections (more ...)
Figure 8 Pyramidal cells of PIRa (green) send excitatory projections (indicated by +) to MOB mitral cells (mi; purple). PIRa (green) also directly projects to superficial MOB granule cells (gr; orange), which in turn inhibit (indicated by –) tufted cells (more ...)
Differential PIRa and PIRp connectivity is substantiated by FG injections in the medial and lateral MOBmi that back-label neurons only in the PIRa and not PIRp (Figures ). This pattern holds true regardless of the dorsal-ventral position of the MOB injections (data not shown). Our data also suggests that PIRa receives more inputs from MOBmi than the PIRp. From a sagittal view, MOB fiber ramifications decrease as they progress from PIRa to PIRp (Figures ).
Figure 9 Connectional differences between PIRa and PIRp. FG injections in dorsal medial
(A) and ventral lateral
(D) MOBmi back-label neurons in PIRa (B,E), but not in PIRp (C,F). Layer Ia of PIR, labeled with PHAL (see Figure for PHAL injection), appears to (more ...)
A rough topography within PIRa also exists where more dorsal neurons project to dorsal MOBmi and more ventral PIRa neurons project to ventral MOBmi (Figures ). This coarse organization is observed more clearly when CTb and FG are double injected in the dorsal and ventral MOBmi, respectively (Figures ). CTb neurons roughly cluster in more dorsal parts of the PIRa, while FG neurons occupy more ventral regions (Figures ).
MOB cortical projections
Double coinjections were made into the dorsal medial (BDA; Figure ) and ventral lateral (PHAL; Figure ) MOBmi and fibers from the rostral to caudal regions of the brain were examined. Regardless of their origin, all axons extensively arborize along the molecular Ia sublayer of the olfactory cortex without any spatial topographic specificity. From the lot, fibers from dorsal medial and ventral lateral MOBmi first arborize in the AON, extending across its external, dorsal, lateral, and posterior ventral divisions (Figures , ). Caudally, fibers continue into the Ia layer of the PIR, dorsal/ventral taenia tecta (TTd, TTv), but do not project as far mediodorsal as the dorsal peduncular area (DP; Figures , ). Axons densely ramify at the juncture between the PIR and the olfactory tubercle (OT; Figures , ). Several cases substantiate this PIR-OT junction labeling where it appears that axons from different parts of the MOB, including the MOBmi and glomerular layer (MOBgl; Figures ), extend into layer II and wrap around pyramidal OT neurons (Figures ).
Figure 10 The ventral lateral MOB projects to olfactory cortical structures from the AON to the ENT (2.345 to –4.28 mm from bregma). Axons ramify in AONd, AONm, AONpv (A), TTd, TTv (B,C), PIR (A–I), OTl (D), AAA (E), NLOT (F), COAa (F–H) (more ...)
Figure 11 The dorsal medial MOB projects to olfactory cortical structures from the AON to the ENT (2.345 to –3.98 mm from bregma). Axons ramify in AONd, AONm, AONpv (A), TTd, TTv (B,C), PIR (A–I), OTl (D), AAA (E), NLOT (F), COAa (F–H), (more ...)
Figure 12 Dense projections from different regions of the MOB including the MOBgl and MOBmi (A,E,F) to the junction between the PIR and OT (B–D,G–I). (C) is magnified in (D) to demonstrate how axons wrap around pyramidal OT neurons (H,I). Scale (more ...)
Fibers continue caudally and terminate in the lateral OT, avoiding its medial portion across the rostral-caudal extent of the structure (Figures , ). PHAL and BDA MOBmi axons also terminate in the anterior amygdalar area (AAA; Figures , ), nucleus of the olfactory tract (NLOT; (Figures , ), bed nucleus of accessory olfactory tract (BA; Figures , ), anterior cortical amygdalar area (COAa; Figures , ), posterior lateral COA (COApl; Figures , ), anterior ventral part of the medial amygdalar nucleus (MEAav; Figures , ), piriform-amygdalar area (PAA; Figures , ), and postpiriform transition area (TR; Figures , ). Terminal boutons are not present in the posterior medial COA (COApm; Figures , ). Similarly, arborizations are observed in the Ia layer of the lateral entorhinal cortical area (ENTl), but not the medial ENT (ENTm; Figures , ).
The number of axons progressively decreases not only as they project medially, but also caudally. For example, massive axons in the AON and PIR dramatically decrease in number after the PIR-OT juncture (Figures , ), an observation that is more evident in sagittal sections (Figure ).