The olfactory bulb, its layers and their functionality

The main olfactory bulb of all vertebrates is composed of multiple, and in birds arch-shaped, layers, the cytoarchitecture and functional organisation of which are conserved throughout all vertebrates (Rose 1914; Andres 2008; Su et al. 2009). Nieuwenhuys (1966) divided the main avian olfactory bulb into seven layers, which make up the so-called proper olfactory bulb, a region that neurophysiologi- cally analyses the input from the olfactory epithelium. From surface to deep these are: olfactory nerve fila (ONL), glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), internal plexiform layer (IPL), granule cell layer (GCL) and periventricular layer (PL). The size and structure of each layer is specific to each taxon and species according to its ecological and behavioural needs and possibly also its phylogeny (Wenzel 1987; Andres 2008). Birds with smaller olfactory bulbs also tend to have less distinct layers with some layers missing and others merged (Crosby & Schnitzlein 1982; Wenzel 1987). The associated olfactory bulb consists of the associated vegetative cells and is charac- terised by the ventricle and the associated ependym (McLean & Shipley 1992). Although not in- volved in olfactory processing as such, these layers are involved in tissue functions, which are impor- tant for the basic biological needs of the olfactory bulb, such as providing new granule cells in the early life of the animal and other vegetative functions (Meisami & Bhatnagar 1998).

Functionally, external odour molecules are sampled through breathing or actively “sniffing” (Hagelin

et al. 2003) and enter the nasal cavity via the nares. The transformation from an external signal to a neural response starts when these molecules encounter the olfactory sensory neurons embedded in the olfactory epithelium lining the nasal cavity (Gomez & Celii 2008). Unlike other neurons these re- ceptor neurons have a high turnover rate and are replaced at a constant pace, presumably to guar- antee full functionality at all times (Graziadei & Monti Graziadei 1978; Vollrath et al. 1985). The neu- ral coding of odour quality is thereby modulated by either a number of olfactory receptor cells, each responsible for particular properties of the odorous molecules (Buck & Axel 1991), or receptor cells might be responsive to a number of different odours (Nickell & Shipley 1992). A combination of both is likely and the intensity of an odour is meanwhile evoked by the number of receptors activated (Su

et al. 2009). Firing rates and odour responsiveness found in avian neural receptor cells are compara- ble to those found in reptiles and mammals and suggest a well developed olfactory system in birds (McKeegan 2002). Likewise, the high numbers of olfactory receptor cells found in birds (Steiger et al. 2008; Steiger et al. 2009b; Steiger et al. 2010) are important for a fully functional olfactory system in birds.

The most superficial layer of the olfactory bulb is the olfactory nerve layer (ONL). This layer is sur- rounded by axons of the olfactory sensory neurons and fed with unidirectional information (Vollrath

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et al. 1985; McLean & Shipley 1992). The olfactory filaments lead the information onto characteris- tic, spherical neuropils (Pinching & Powell 1971), the glomeruli, which make up the glomerular layer (GL). This cell-poor region sits between the olfactory fila and the external plexiform layer (EPL). Of ovoid shape, they are enclosed by a thin layer of neurons and glia cells (Kratskin 1995; Kosaka et al.

1998). Each glomerulus receives input from a single or a few olfactory receptor neurons (Shepherd 1991). This unidirectional connection and the physical arrangement of the glomeruli create the plat- form of an odorant receptor map (Uchida et al. 2000; Mori 2003; Mori et al. 2006). The glomeruli are also interconnected between each other and with the more dorsally situated mitral cells. Using a circuit of incoming and outgoing information the periglomerular cells are at the centre of informa- tion exchange and responsible for the creation of a topographic odour map while assessing quality and intensity of olfactory information (McLean & Shipley 1992; Su et al. 2009). The external plexi- form layer (EPL) and internal plexiform layer (IPL) are both thin layers with only a low density of cell bodies. The EPL is adjacent to the glomerular layer and the mitral cell layer (MCL). It has a large number of dendrites, which originate in the mitral and the more dorsally situated granule cell layer. The densely populated cell bodies of the granule layer are distinguished by clusters of thick somata, that are the cell bodies of the neuron (McLean & Shipley 1992). In some cases neurons of the gran- ule cell layer (GCL) fluently merge with the periventricular layer (PL). Each cell in the granule cell layer gives rise to short central dendrites and single, long apical dendrites. Long dendrites travel through the mitral cell body layer and terminate at the external plexiform layer, where they fuse with lateral dendrites of mitral and tufted cells. The dendrites receive synaptic input from mitral and tufted cells and create synaptic outputs through reciprocal dendrodendritic synapses (Kratskin 1995; Shepherd 1998). While in fish, amphibians and reptiles, the mitral cells connect with only one or a few glomeruli, in birds they send dendrites to more than eight glomeruli (Allison 1953).

As the main input and output port of the olfactory bulb, the mitral cells in the mitral cell layer essen- tially are responsible for all reactive responses to olfactory cues (Nieuwenhuys 1966). They project signals, by communicating through the usual electrochemical process of signal conduction via their axons (Banich & Compton 2011), from the olfactory bulb to higher brain compartments (Nickell & Shipley 1992). They are also the biggest neurons in the olfactory bulb, although of non-uniform size and shape. Usually the mitral cell layer appears in well-defined lines of one or two layers but some- times, this layer also infiltrates neighbouring layers (Crosby & Schnitzlein 1982; Halasz 1990; McLean & Shipley 1992). Apart from their interconnection with the glomerular layer and the external plexi- form layer within the olfactory bulb, efferent axons of the mitral cell layer leaving the olfactory bulb target three higher brain regions in vertebrates: the piriform cortex, the hippocampus (HP) and the

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amygdala (in mammals) and the nucleus taeniae in birds. There is considerable behavioural evidence for the involvement of olfaction in homing, such as in pigeons, swifts and petrels (See Chapter One and Benvenuti et al. 1973; Baldaccini et al. 1974; Fiaschi et al. 1974; Papi 1991; Benvenuti et al.

1993; Benvenuti & Ranvaud 2004; Bonadonna et al. 2004; Gagliardo et al. 2011). Ablation experi- ments of the piriform cortex in pigeons showed a lessened ability to navigate through unfamiliar ar- eas, suggesting that the piriform cortex must play a role in homing using olfactory cues (Papi & Casini 1990; Gagliardo et al. 2000).

Mitral axons also project into the hippocampus (HP) in pigeons (Atoji & Wild 2006), which has been shown to be involved in spatial memory tasks (Broadbent et al. 2004). Nocturnal Leach storm petrels (Oceanodroma leucorhoa) for example had larger hippocampi volumes when nesting in forest, com- pared to birds nesting on flat meadows. Presumably, birds living in the forest had larger spatial de- mands associated with returning to their nest sites at night in the darker, navigationally more de- manding forests (Abbott et al. 1999). Leach’s and other nocturnal petrels are known to use olfactory cues to home (Grubb 1974; Bonadonna & Bretagnolle 2002), hence a correlation between homing and the involvement of the hippocampus is likely. Food storing birds also have been found to have a relatively large hippocampus and it is likely, that olfaction plays a part in the proper relocation of hidden food items (Buitron & Nuechterlein 1985; Harriman & Berger 1986; Krebs et al. 1989; Shet- tleworth 2003). The nucleus taenia is located close to the striatopallidal complex (SPC) and deemed to function much in the same way as the amygdala in mammals (Reiner & Karten 1985; Cheng et al.

1999). The amygdala processes, among other things, associative learning based on olfactory cues (Schoenbaum et al. 1998).

Owing to these large and comprehensive connections between the mitral cell layer and higher brain areas, it can be assumed that the relative volume of the mitral cell layer as well as the density of cells with which it is populated, can provide information about the overall importance of the olfac- tory system in birds. Larger bulbs for example should have larger numbers of mitral cells. Or if they had a comparatively lower number of mitral cells, they should have comparatively larger cells that are able to process more information at the same time. Here and for the first time, I uncover the histological organisation of the brain (see Chapter Three) and the olfactory bulb of the kakapo and discuss its delineation against the forebrain. In a comparative approach, I analyse the overall size of the olfactory bulb of the kakapo and examine the different layers encompassing the olfactory bulb, particularly the mitral cell layer.

68 4.3 Methods