Peter C. Brunjes

The neocortex and olfactory cortices share many features including their laminar organization, developmental sequences, and cell types. Previous work indicates that neocortical pyramidal cells exhibit a gradient of dendritic size: cells involved in the initial processing of information are less complex than those in subsequent, higher processing areas. Results presented here confirm that the same is true for the olfactory cortex: pyramidal cells in the region closest to the olfactory bulb, the anterior olfactory nucleus, have smaller total dendritic length and occupy less neural space than those in the posterior piriform cortex. These findings add to the evidence for general rules of development, organization, and function across forebrain cortices.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age‐related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh‐like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age‐related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system.

Both the lateral olfactory tract (LOT) and anterior limb of the anterior commissure (AC) carry olfactory information. The LOT forms the projection from the olfactory bulb to the ipsilateral olfactory cortices, while the AC carries odor information across the midline to the contralateral olfactory cortex and bulb. The LOT and AC differ on a number of dimensions, including early development and functional onset. The present work, examining their myelination in mice, reveals additional important differences. For example, the LOT initiates myelination 3–4 days earlier than the AC, evidenced by both an earlier increase in myelin basic protein staining seen with immunohistochemistry and an earlier appearance of myelinated fibers using electron microscopy. While both exhibit a period of rapid myelination, it occurs 4–5 days earlier in the LOT than the AC. The tracts also respond differently to early sensory restriction. Unilateral naris occlusion from the day after birth to postnatal day 30 had no consistent effects on the AC but resulted in significantly thinner myelin sheaths relative to axon caliber in the LOT. Finally, the two tracts differ structurally (the LOT contains larger, more densely packed axons with significantly thicker myelin sheaths resulting in a conduction velocity that is more than twice as fast as the AC). The findings indicate that these two large, accessible tracts provide an important means for studying brain maturation due to basic differences in both the timing of their maturation and general organization.

Despite the fact that pigs are reputed to have excellent olfactory abilities, few studies have examined regions of the pig brain involved in the sense of smell. The present study provides an overview of the olfactory bulb, anterior olfactory nucleus, and piriform cortex of adult pigs using several approaches. Nissl, myelin, and Golgi stains were used to produce a general overview of the organization of the regions and confocal microscopy was employed to examine 1) projection neurons, 2) GABAergic local circuit neurons that express somatostatin, parvalbumin, vasoactive intestinal polypeptide, or calretinin, 3) neuromodulatory fibers (cholinergic and serotonergic), and 4) glia (astrocytes and microglia). The findings revealed that pig olfactory structures are quite large, highly organized and follow the general patterns observed in mammals.

Neurons in the cerebral cortex stratify on the basis of their time of origin, axonal terminations and the molecular identities assigned during early development. Olfactory cortices share many feature with the neocortex, including clear lamination and similar cell types. The present study demonstrates that the markers differentially expressed in the projection neurons of the cerebral cortex are also found in olfactory areas. Three of the four regions examined (pars principalis of the anterior olfactory nucleus: AONpP, anterior and posterior piriform cortices: APC, PPC, and the olfactory tubercle) expressed transcription factors found in deep or superficial neurons in the developing neocortex, though large differences were found between areas. For example, while the AONpP, APC and PPC all broadly expressed the deep cortical marker CTIP2, NOR1 (NR4a3) levels were higher in AONpP and DAARP-32 was more prevalent in the APC and PPC. Similar findings were encountered for superficial cortical markers: all three regions broadly expressed CUX1, but CART was only observed in the APC and PPC. Furthermore, regional variations were observed even within single structures (e.g., NOR1 was found primarily in in the dorsal region of AONpP and CART expression was observed in a discrete band in the middle of layer 2 of both the APC and PPC). Experiments using the mitotic marker EDU verified that the olfactory cortices and neocortex share similar patterns of neuronal production: olfactory cells that express markers found in the deep neocortex are produced earlier than those that express superficial makers. Projection neurons were filled by retrograde tracers injected into the olfactory bulb to see if olfactory neurons with deep and superficial markers had different axonal targets. Unlike the cerebral cortex, no specificity was observed: neurons with each of the transcription factors examined were found to be labelled. Together the results indicate that olfactory cortices are complex: they differ from each other and each is formed from a variable mosaic of neurons. The results suggest that the olfactory cortices are not merely a remnant architype of the primordial forebrain but varied and independent regions.

The present series of studies was designed to provide a general overview of the development of the region connecting the olfactory bulb to the forebrain. The olfactory peduncle contains several structures involved in processing odor information with the anterior olfactory nucleus (cortex) being the largest and most studied. Results indicate that considerable growth occurs in the peduncle from postnatal day (P)10-P20, with reduced expansion from P20-P30. No evidence was found for the addition of new projection or interneurons during the postnatal period. GABAergic cells decreased in both number and density after P10. Glial populations exhibited different patterns of development, with astrocytes declining in density from P10-P30, and both oligodendrocytes and microglia increasing through the interval. Myelination in the anterior commissure emerged between P11-14. Dense cholinergic innervation was observed at P10 and remained relatively stable through P30, while considerable maturation of serotonergic innervation occurred through the period. Unilateral naris occlusion from P1-P30 resulted in about a 30% reduction in the size of the ipsilateral peduncle but few changes were observed on the contralateral side. The ipsilateral peduncle also exhibited higher densities of GAD67- containing interneurons and cholinergic fibers suggesting a delay in normal developmental pruning. Lower densities of interneurons expressing CCK, somatostatin and NPY and in myelin basic protein staining were also observed. Understanding variations in developmental trajectories within the olfactory peduncle may be an important tool for unravelling the functions of the region.

The central core of the olfactory peduncle (the tissue connecting the olfactory bulb to the forebrain) includes a white matter tract that extends caudally to the anterior commissure. The purpose of the present study was to examine this “anterior limb of the anterior commissure” (ALAC) to determine if the axons that progress through it segregate on the basis of their point of origin, neurotransmitter type, size or shape. While local differences in axon density were observed in the ALAC, they were not consistent between samples of the anterior and posterior peduncle, and no other compartmentalization within the tract was observed. The innervation of the caudal olfactory peduncle by neuromodulatory fibers was examined to determine if they enter the region via the ALAC. Cholinergic fibers densely filled the peduncle, followed in order by serotonergic, noradrenergic, histaminergic and orexinergic processes. Differences in the distribution of the fibers were noted for each system. While each axon type could be observed in the ALAC, it is probable that they enter the peduncle though several routes. Data for axon caliber in the ALAC was compared to information previously collected on the peduncle’s other white matter region, the lateral olfactory tract. Axons in the ALAC were smaller, suggesting that the olfactory system is organized with a fast system for distributing incoming sensory information and a more economical, distributed system for subsequent processing.

The olfactory peduncle, the region connecting the olfactory bulb with the basal forebrain, contains several neural areas that have received relatively little attention. The present work includes studies that provide an overview of the region in the mouse. An analysis of cell soma size in pars principalis (pP) of the anterior olfactory nucleus (AON) revealed considerable differences in tissue organization between mice and rats. An unbiased stereological study of neuron number in the cell‐dense regions of pars externa (pE) and pP of the AON of 3‐, 12‐, and 24‐month‐old mice indicated that pE has about 16,500 cells in 0.043 mm³and pP about 58,300 cells in 0.307 mm³. Quantitative Golgi studies of pyramidal neurons in pP suggested that mouse neurons are similar to although smaller than those of the rat. An immunohistochemical analysis demonstrated that all peduncular regions (pE, pP, the dorsal peduncular cortex, ventral tenia tecta, and anterior olfactory tubercle and piriform cortex) have cells that express either calbindin, calretinin, parvalbumin, somatostatin, vasoactive intestinal polypeptide, neuropeptide Y, or cholecystokinin (antigens commonly co‐expressed by subspecies of γ‐aminobutyric acid [GABA]ergic neurons), although the relative numbers of each cell type differ between zones. Finally, an electron microscopic comparison of the organization of myelinated fibers in lateral olfactory tract in the anterior and posterior peduncle indicated that the region is less orderly in mice than in rats. The results provide a caveat for investigators who generalize data between species, as both similarities and differences between the laboratory mouse and rat were observed. J. Comp. Neurol. 519:2870–2886, 2011.

Several lines of evidence indicate that complex odorant stimuli are parsed into separate data streams in the glomeruli of the olfactory bulb, yielding a combinatorial “odotopic map.” However, this pattern does not appear to be maintained in the piriform cortex, where stimuli appear to be coded in a distributed fashion. The anterior olfactory nucleus (AON) is intermediate and reciprocally interconnected between these two structures, and also provides a route for the interhemispheric transfer of olfactory information. The present study examined potential coding strategies used by the AON. Rats were exposed to either caproic acid, butyric acid, limonene, or purified air and the spatial distribution of Fos‐immunolabeled cells was quantified. The two major subregions of the AON exhibited different results. Distinct odor‐specific spatial patterns of activity were observed in pars externa, suggesting that it employs a topographic strategy for odor representation similar to the olfactory bulb. A spatially distributed pattern that did not appear to depend on odor identity was observed in pars principalis, suggesting that it employs a distributed representation of odors more similar to that seen in the piriform cortex. J. Comp. Neurol. 519:277‐289, 2011.

The anterior olfactory nucleus (AON) occupies a crucial position within the olfactory circuit, as it is able to influence function in nearly every major synaptic processing stage of both the ipsilateral and the contralateral pathways. Nevertheless, very little is known about the region's internal organization and circuitry. The present study provides basic quantitative and qualitative data on the morphology of several cell types within the two major regions of the AON, pars externa and pars principalis. In pars externa two types of cells are analyzed, the “classical” cell (type I), containing only apically directed dendrites with large spines, and a previously unreported cell with basilar dendrites and complex, spiny apical processes (type II). In pars principalis the characteristic pyramidal cell is described both on the basis of the depth of the cell bodies in the cell layer comprising the structure and on the basis of their radial location. Several other nonpyramidal neurons are also described. The findings provide useful basic information necessary for understanding and modeling the circuitry of the AON. J. Comp. Neurol. 518:1603–1616, 2010.