Non-invasive imaging of neuroanatomical structures and neural activation with high-resolution MRI

Abstract

Several years ago, manganese-enhanced magnetic resonance imaging (MEMRI) was introduced as a new powerful tool to image active brain areas and to identify neural connections in living, non-human animals. Primarily restricted to studies in rodents and later adapted for bird species, MEMRI has recently been discovered as a useful technique for neuroimaging of invertebrate animals. Using crayfish as a model system, we highlight the advantages of MEMRI over conventional techniques for imaging of small nervous systems. MEMRI can be applied to image invertebrate nervous systems at relatively high spatial resolution, and permits identification of stimulus-evoked neural activation non-invasively. Since the selection of specific imaging parameters is critical for successful in vivo micro-imaging, we present an overview of different experimental conditions that are best suited for invertebrates. We also compare the effects of hardware and software specifications on image quality, and provide detailed descriptions of the steps necessary to prepare animals for successful imaging sessions. Careful consideration of hardware, software, experiments, and specimen preparation will promote a better understanding of this novel technique and facilitate future MEMRI studies in other laboratories.

Keywords: crayfish; manganese-enhanced magnetic resonance imaging; neural activity; neuroanatomy.

Figure 1

(A) 600 MHz (14.1 T) NMR spectrometer located at Georgia State University. The spectrometer is equipped with a MICRO 5 probe for imaging. (B) Juvenile crayfish (3.6–3.7 cm lengths from rostrum to telson) were used in all studies. (C) MnCl₂ was injected into the pericardial chamber after crayfish were cannulated by inserting a short piece of fused silica connected to polyethylene tubing (arrowhead). To measure functional Mn²⁺ uptake in the brain, animals were implanted with stimulating silver wire electrodes placed around the left antenna II nerve (arrows). (D) Animals were gently moved into NMR tubes (1 cm diameter) prior to imaging where there remained motionless during the session. All photos were taken by Jens Herberholz.

Figure 2

(A) A single coronal section through the head and thorax of a crayfish injected with 45 μl of 50 mM MnCl₂ and imaged with a T₁-weighted MSME sequence. Mn²⁺ uptake into the central brain areas and into the right eyestalk can be seen. (B) Magnified view of the right eyestalk. Individual anatomical regions are visible. (C) Magnified view of the central brain areas. Olfactory and accessory lobe (arrows) and the rostral parts of the brain connectives (arrowheads) can be identified. (D) A single sagittal section from a 3D stack acquired with a RARE imaging sequence after the animal was injected with 100 μl of 20 mM MnCl₂. Mn²⁺ uptake into the entire ventral nerve cord as well as individual thoracic and abdominal ganglia can be detected.

Figure 3

(A) A single axial section through the head acquired using a T₁-weighted MSME imaging sequence. Note that in these axial slices, the animal's left side corresponds to the viewer's right side. After the animal was injected with 40 μl of 25 mM MnCl₂ and stimulated with electric shocks delivered to the left antenna II nerve, Mn²⁺ uptake into different areas of the central brain can be detected. More Mn²⁺ uptake can be seen on the left (i.e., stimulated) side of the brain, especially in the left antenna II nerve (arrow) and the left accessory lobe (arrowhead). (B) Another axial section from the same scan located 200 μm more posterior. Clear differences between the two sides of the brain are still visible in this section. (C) The same animal and axial section shown in (A) but imaged with a T₂-weighted RARE imaging sequence. More overall signal intensity and contrast can be seen but differential Mn²⁺ uptake between the two sides of the brain is less discrete. (D) The same animal and axial section as shown in (B) but imaged with a T₂-weighted RARE imaging sequence. More overall signal intensity and contrast can be seen but differences in Mn²⁺ uptake between the two sides of the brain are less pronounced. A schematic of the main structures of the crayfish brain is shown on the right (modified from Sandeman et al., 1988). The left antenna II nerve (arrow) and the left accessory lobe (arrowhead) are labeled in the schematic for comparison.

Figure 4

Stacks of four single brain slices obtained from one stimulated and one unstimulated animal. T₁- and T₂-weighted images from both animals were used to measure grayscale values for each brain slice (Table 1). The regions of interest (ROIs) for the two sides of the brain were selected using a single T₂-weighted image from each animal and kept identical for all slices. The ROIs are shown as rectangular boxes in the respective images; the red box in the image of the stimulated animal indicates the side of the animal that received electric stimulation of the antenna II nerve. The imaging parameters for each scan are given in the results.

Figure 5

(A) A single coronal section through the thorax and abdomen illustrating Mn²⁺ uptake into the ventral nerve cord. Images were acquired using a T₁-weighted MSME sequence after the animal was injected with 10 μl of 50 mM MnCl₂ and the ventral nerve cord was repeatedly stimulated to elicit escape tail-flips mediated by the lateral giant neurons. Most of the nerve cord is visible, and ganglia as well as ganglionic nerve roots can be seen. (B) Using a slightly modified T₁-weighted MSME sequence in the same animal, a single axial section of the abdomen at the location of the third abdominal ganglion is shown. The boundaries of the abdominal ganglion and localized Mn²⁺ uptake within the ganglion are visible. (C) Magnified view of the third abdominal ganglion. By highlighting the most intensely labeled areas, Mn²⁺ uptake is seen in discrete areas of the ganglion.