Statistical mapping of functional olfactory connections of the rat brain in vivo

The olfactory pathway is a unique route into the brain. To better characterize this system in vivo, rat olfactory functional connections were mapped using magnetic resonance (MR) imaging and manganese ion (Mn²⁺) as a transport-mediated tracer combined with newly developed statistical brain image analysis. Six rats underwent imaging on a 1.5-T MR scanner at pre-administration, and 6, 12, 24, 36, 48, and 72 h and 5.5, 7.5, 10.5, and 13.5 days post-administration of manganese chloride (MnCl₂) into the right nasal cavity. Images were coregistered, pixel-intensity normalized, and stereotactically transformed to the Paxinos and Watson rat brain atlas, then averaged across subjects using automated image analysis software (NEUROSTAT). Images at each time point were compared to pre-administration using a one-sample t statistic on a pixel-by-pixel basis in 3-D and converted to Z statistic maps. Statistical mapping and group averaging improved signal to noise ratios and signal detection sensitivity. Significant transport of Mn²⁺ was observed in olfactory structures ipsilateral to site of Mn²⁺ administration including the bulb, lateral olfactory tract (lo) by 12 h and in the tubercle, piriform cortex, ventral pallidum, amygdala, and in smaller structures such as the anterior commissure after 24 h post-administration. MR imaging with group-wise statistical analysis clearly demonstrated bilateral transsynaptic Mn²⁺ transport to secondary and tertiary neurons of the olfactory system. The method permits in vivo investigations of functional neuronal connections within the brain.

Introduction

The rat olfactory system consists of olfactory receptor neurons connected through the bulb to cells whose axons project through the lateral olfactory tract to regions of the olfactory cortex including the anterior olfactory nucleus, tubercle, and piriform cortex as well as the amygdala and entorhinal cortex (Shipley, 1985). To explore this system, classic tract tracing studies of the CNS have used substances, such as wheat germ agglutinin conjugated to horseradish peroxidase that could only be visualized in post-mortem tissue (Itaya, 1987). In the last decade, several groups have used radioisotopes of metal ions, such as cadmium, nickel, mercury and Mn²⁺ to study the olfactory system as a direct route for exposure to the brain (Brenneman et al., 2000, Tjalve and Henriksson, 1999). In particular, Mn²⁺ has been shown to be taken up by olfactory receptor neurons and, because it can transverse synapses, has direct access to the brain (Gianutsos et al., 1997, Tjalve et al., 1996). Although Mn²⁺ is essential for the function of several proteins, including mitochondrial products, it is known to have toxic effects as it accumulates in the brain (Gavin et al., 1990, Sloot and Gramsbergen, 1994). These original studies of radiolabeled ⁵⁴Mn applied to the olfactory system showed transient accumulation of the metal in regions that were synaptically connected to primary olfactory neurons. Briefly, Mn²⁺ acts as a calcium analog and enters neurons through Ca²⁺ channels (Narita et al., 1990, Pautler and Koretsky, 2002, Takeda et al., 1998), then it has been indicated to be packaged into vesicles and transported down the axon in a microtubule-dependent manner. Once released from presynaptic neurons, Mn²⁺ crosses the synapse, enters post-synaptic neurons, and thus distributed through interconnecting regions by selectively anterograde transport (Pautler and Koretsky, 2002, Sloot and Gramsbergen, 1994). Based on these early investigations, several groups have proposed to exploit the paramagnetic properties of Mn²⁺, which has a T1-shortening effect on MRI and develop a technique that allows in vivo imaging of neuroanatomical connectivity. These studies have shown enhancement of the mouse olfactory and visual systems (Pautler et al., 1998), the rat optic pathway (Watanabe et al., 2001), functional plasticity in the songbird brain (Van der Linden et al., 2002), as well as monkey striatal connections (Saleem et al., 2002), and rat somatosensory cortical projections (Leergaard et al., 2003).

The development of this unique in vivo imaging technique will facilitate greatly a better understanding of neuroanatomical connectivity. Also, the mechanisms of uptake (calcium channels) and transport (microtubular dependency) permit investigations of more physiological properties, which, in turn can lead to inferences on the functional nature of the neuronal connection. Investigations of Mn²⁺ transport within the brain are also critical for better understanding of manganese-induced toxicity, particularly in the research field of environmental and occupational health (Brenneman et al., 2000, Henriksson and Tjalve, 2000, Henriksson et al., 1999, Sloot and Gramsbergen, 1994). In addition, more recent studies indicated the olfactory pathway as a potential transport system for other larger molecules such as neurotrophic factors (Thorne and Frey, 2001). Even though exact mechanisms of such transport are not fully understood, the investigations of functional connections of the olfactory system are becoming increasingly important.

Although this unique imaging technique has been presented as a novel method for in vivo tract tracing, analysis methods used in these studies were limited to observation of individual animals or use of conventional region of interest (ROI) analysis. Limitations of these methods include potential effects from observer bias and inaccurate ROI placement as well as inability to discern smaller, less obvious enhancements. In this study, we present an image analysis method to define statistically the degree and extent of Mn²⁺ transport in the brain using stereotactic parametric mapping techniques and its application to investigate functional neuronal connection of the rat stereotactic system. Techniques for stereotactic anatomic standardization combined with statistical analysis have been developed and applied in humans, both PET (Fox et al., 1985, Friston et al., 1989, Minoshima et al., 1993a) and MRI (Friston et al., 1995) for more than a decade. A recent study by Leergaard et al. (2003) applied a stereotactic approach to facilitate anatomic localization of Mn²⁺ transport from the rat somatosensory cortex. We extend these previous research studies to include not only 3-D stereotactic image standardization, but also group-wise statistical analysis and serial imaging to show the functional connections within the rat olfactory system using manganese-enhanced magnetic resonance imaging. This approach can elucidate the functional connections of the olfactory tract without the necessity of predefined regions of interest. The method also improves substantially the signal to noise ratio through averaging of multiple subjects in a common coordinate system.