The speaker declares no conflicts of interest regarding the presentation. The presentation will discuss functional MRI (fMRI) and manganese-enhanced MRI (MEMRI) studies of the mouse brain. It will cover issues with BOLD fMRI in mice such as spatial resolution requirements and anesthesia effects. It will also discuss applications of MEMRI for mapping neural activity and connectivity in olfactory and auditory systems of mouse models.
1. Declaration of Conflict of Interest or Relationship Speaker Name: Xin Yu Kai-Hsiang Chuang I have no conflicts of interest to disclose with regard to the subject matter of this presentation.
2. Functional MRI of the Mouse Brain Kai-Hsiang Chuang L aboratory of Molecular Imaging Singapore Bioimaging Consortium Agency for Science, Technology and Research Singapore Xin Yu Laboratory of Functional and Molecular Imaging National Institute of Neurological Disorders and Stroke National Institues of Health
28. The central auditory system (CAS) Cochlea CN: Cochlear Nucleus SoC: Superior Olive Complex LL: Lateral Leminiscus IC: Inferior colliculus MGN: Medial Geniculate Nuclues AC: Auditory cortex 2mm VIII Nerve CN SoC LL IC MGN AC
29. Dorsal Ventral IC Low Frequency < 1 kHz High Frequency 60kHz The tonotopic organization of the mouse inferior colliculus (IC) 16 kHz 32 kHz 40 kHz Romand and Ehret, 1990 Dorsal Ventral
30. Longitudinal imaging studies over 3 days in 24 h time intervals Mn Inj. 0.4mmol/kg Mn Inj. 0.2mmol/kg 20-50 kHz 40 kHz No Stimulation Clearance Time Post 24 hr Post 48 hr Post 72 hr 0 255 n=4
31. MEMRI detected pure tone stimulated neuronal activity in the mouse IC 16 kHz 40 kHz Coronal IC image IC Tonotopic Map (Electrophysiology) 16kHz 40kHz n=8 for each group 0 255
32. 2D coronal IC slices along the caudal-rostral axis 40 kHz 0 255 Rostral Caudal 16 kHz
33. 3D contour of frequency specific activity patterns by voxel-wise t statistic analysis 16 kHz 40 kHz P<0.05 16 kHz 40 kHz n 8 for each group
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36. Longitudinal studies of 16 and 40 kHz pure tone stimulation in Fgf17 mutant mice Mn Inj. 0.4mmol/kg Mn Inj. 0.2mmol/kg 40 kHz 16 kHz No Stimulation Clearance Time P21 Two days P23 P24 P20
37. Altered tonotopic organization of the IC in Fgf17 -/- mice Fgf17 +/- Fgf17 -/- 16 kHz 40 kHz SI threshold =Mean+1.5*SD n=7 n=10 Maximal intensity maps 0 200 400 600 800 16 40 16 40 Fgf17 +/- Fgf17 -/- * Activity center to IC center distance ( m) Fgf17 +/- Fgf17 -/- 0 200 300 400 500 100 16 & 40 kHz activity center distance ( m) * P<0.01 * P<0.01
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Notes de l'éditeur
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Catheterization
Mn ion is a Ca analog. It can enter neurons thru voltage-gated Ca channel. So, it can be used to detect neural activation by suitable delivery and stimulation. After entering neurons, Mn can be transported to the projecting neurons by microtubules in the axons and can cross 2-3 synapses. Therefore, it can be used as an anterograde tract tracer. It is also an excellent T1 relaxation agent that can be detected by T1-w MRI. Based on these properties, the activity it detects is more related to neural firing rather than vascular response. Since it will stay in the cell for at least 1 day, we can perform experiment outside the magnet when the animal is awake and take images afterward. After Mn is released from the cell, we can repeat the same experiment again in the same animal. So it will be suitable to follow the change in neural plasticity. However, too much Mn will cause neurotoxicity, it is not yet good for human imaging. Besides, Mn can not penetrate BBB, there are limited ways to deliver Mn to targeted regions, either by breaking the BBB or not.
(add one of my slices)
Another way to detect neural function by Mn does not need to break the blood-brain barrier. Combining the activity dependent uptake and tract tracing properties of Mn, a previous study by Pautler and Koretsky detected odorant activation in the mouse OB by exposing an odor together with vaporized Mn. The sensory neurons activated by the odorant will uptake more Mn and transport more to their projected regions in the OB. As can be seen here, without odorant stimulation, there is no specific enhancement, but with amyl acetate, the lateral and ventral side of the bulb accumulate more Mn than other regions.
Olfactory bulb is the first stage of olfactory processing in the brain. There are only about one thousand different types of sensory neurons in the epithelium of the nostrils. How the neural circuits are organized to identify and discriminate millions of different odors? An odorant activates a combination of different kinds of receptors. The activated receptors will project the info to round-shaped structures, called glomeruli, in the OB. These glomeruli are about 50 to 100 microns in diameter in mice and are where the receptors form synapses with the mitral cells in OB. As shown in this pseudo-colored coronal section of a mouse OB, the glomeruli and mitral cells are organized well in two different layers. Especially, axons from the same type of receptors are projected to 2 of the glomeruli in a stereotypic way. For example, in this cartoon the red type of receptor will merge all of their axons to the red gloermulus and the location of this glomerulus is quite fixed in the OB among individuals. Therefore, the olfactory coding by a combination of activated receptors is transformed to a spatial pattern of a group of activated glomeruli. Each glomerulus will have synapse with 10s of mitral and tufted cells underneath. Then the info is further refined in the mitral cells by lateral inhibition and projected to the primary olfac cortex. Due to this modular organization, a glomerulus and the column underneath is regarded as a functional unit of olfactory processing.
To understand the coding of olfac information in the bulb, it is important to map the glomerular activation. Several imaging methods have been applied, including traditional tracer, 2-DG autoradiography, cFos mRNA experssion, optical imaging of intrinsic signal or Ca-ch dye, and even functional MRI. However, these methods suffered from limited spatial resolution, field-of-view and invasive procedures that cannot allow longitudinal study in the same animal
For each animal, the time series data was realigned to the last image to minimize sub-voxel movement. As can be seen from a time series of a coronal slice of the OB after Mn injection, Mn enhancement increase in some regions in the glomerular layer, and then in the mitral cell layer. Also can be seen in a ROI in glomerular signal keeps increasing, but in a deeper region, there is no change. It should be noted that the enhancement in the bulb is not due to injection but tract tracing. Because the activated glomeruli will accumulate more Mn and have higher enhancement, the area under the intensity time course was calculated to represent the level of activity. The calculated area map was shown as pseudo-color from red to yellow overlaid on the T1w image. We can also see high signal change in the olfactory nerve layer, because this is where Mn get into the bulb.
rate of 1–6 mm/h in axons Three kinds of odorants, acetophenone, carvone, octanal, were tested in different mice. The brighter color represents higher signal increase. There are common enhancement among the three different odorants, but it is mainly in the olfactory nerve layer. In the glomerular layer, enhancement pattern specific to each odorant can be identified. For exmaple, Acetophenone activated more medial and dorsal, carvone more ventral, and octanal more lateral.
To better visualize the enhancement pattern in the glomerular layer, this layer was flattened to create a 2D odor map. The flattening was done in coronal view. A vertical line crossing the center of the blub was defined. Then moving from the dorsal center to the ventral, along the medial or lateral, the glomerular layer was divided into several partitions with a fixed arc length. The activity in each partition was averaged and arranged into a straight line. By repeating the same procedure slice-by-slice, the glomerular layer was flattened and a dorsal-centered odor map was created.
Because no odor condition is not perfectly no odor, and there might be some enhancement due to the smell of the anesthetic during the MRI scanning, the residual activity and other individual variation could bias the result was eliminated by comparing the odor stimulation group to the control group using t-test. The glomerular activation patterns specific the an odorant can be easily compared. Acetophenone activated more dorsal & medial. Carvone activated more ventral. Octanal activated more dorsal lateral and medial. Besides, the symmetrical activation patterns in the medial and lateral sides are consistent with the bilateral arrangement of similar glomeruli.
Because a glomerulus is the functional unit in the OB, can MRI detect individual glomeruli? The sensitivity of this technique was tested by a kind of transgenic mouse where the M71 olfactory receptors were replaced by rat I7 receptors and were colabeled with green fluorescent protein. Under fluorescent microscope, we can easily see that there are two rI7 glomeruli on the dorsal surface of the bulb. Because this receptor is known to respond to octanal, we performed the Mn odor mapping experiment using octanal stimulation. From the Mn map, two focal enhancement have very good correspondence with the rI7 glomeruli in the fluorescent image. There are also some other Mn enhanced spots in the bulb, this is because an odorant will activate not just one but also other different types of receptors, and those spots could be the corresponding glomeruli.
Left image, show you the major auditory nuclei and their virtual location in the whole brain structure. Right show the primary excitatory projections between the major nuclei.
Here, we focused on the auditory midbrain, inferior colliculus. The tonotopic organization in the IC has been established by electrophysiology. it showed that the dorsal area of the IC represented the low frequency sound less than 1 kHz and ventral area represented the high frequency sound up to 60kHz. It also revealed that neurons most sensitive to a particular frequency are located in an iso-frequency band, such as the 16 kHz, 32 kHz and 40 kHz iso-frequency bands, which align from dorsal to ventral IC.
Next, we performed a longitudinally studies on the frequency associated activity mapping over three days with 24hr time interval.. Mice are first exposed to 20-50kHz, acquired images, keep 24hr in quiet environment for the clearance (non-specific diffusion of Mn from the active area), exposed to 40khz sound for 24hr and acquire the image again. We observed the similar activity maps to previous studies.
We also compared the activity maps by different pure tones, 40kHz and 16kHz with peak SPL 89db. The left figure shows the slice orientation in the whole brain. Here we display a five consecutive coronal slice of the IC from caudal to rostral. Comparing with the electrophysiological iso frequency contour, signal enhancement by 16khz is more dorsal to 40khz induced signal enhancement, which is at the ventral IC.
We also compared the activity maps by different pure tones, 40kHz and 16kHz with peak SPL 89db. The left figure shows the slice orientation in the whole brain. Here we display a five consecutive coronal slice of the IC from caudal to rostral. Comparing with the electrophysiological iso frequency contour, signal enhancement by 16khz is more dorsal to 40khz induced signal enhancement, which is at the ventral IC.
Fibroblast growth factors play critical role during the embryonic brain development. Fgf8 and Fgf17 are expressed in the mid-hind brain border and work as morphogens to direct the formation of the mid hind brain. Recently, questions have been raised whether these morphogens could also work as guidance cues to direct the axonal targeting during the circuit formation and enventually contribute to the functional development. However, Fgf8 mutation is lethal. We cannot study the effects of Fgf8 mutation on the functional development. While Fgf17 knockout mice can survive to adulthood. We can therefore examine whether there are altered functional patterns in Fgf17 mutant mice, which could indicate some misformed neuronal connections. Kelly Matthew NIH Anil Lawalni NYU otolaryngology Besides studying the functional alteration induced by environmental manipulation, we also studied functional alteration in the genetic mutant mice. We selected fgf17 mutant mice. Since Fgf17 is involved in the early mid-hind brain development. Early study reported that they have smaller inferior colliculus. Our recent studies also show that they have a normal peripheral auditory system. They have normal inner ear morphology, the auditory brainstem response test doesn’t show difference from the wild type. Therefore, it is a perfect mouse model for us to study the functional patterns in the anatomically altered IC. FGF-8 and FGF-17 are tightly localized to specific regions of the developing brain and are only expressed in the embryo during the early phases of proliferation and neurogenesis.
Kelly Matthew NIH Anil Lawalni NYU otolaryngology Besides studying the functional alteration induced by environmental manipulation, we also studied functional alteration in the genetic mutant mice. We selected fgf17 mutant mice. Since Fgf17 is involved in the early mid-hind brain development. Early study reported that they have smaller inferior colliculus. Our recent studies also show that they have a normal peripheral auditory system. They have normal inner ear morphology, the auditory brainstem response test doesn’t show difference from the wild type. Therefore, it is a perfect mouse model for us to study the functional patterns in the anatomically altered IC. FGF-8 and FGF-17 are tightly localized to specific regions of the developing brain and are only expressed in the embryo during the early phases of proliferation and neurogenesis.
We also want to examine the variability of the activity patterns from individual mice. since we can