Histological Confirmation of Aplysia californica Neuron Structure Observed Using MR Microscopy

INTRODUCTION Since the first application of magnetic resonance microscopy (MRM) on large single cells (1), MRM has developed over the last 25 years as a complementary microimaging technique with a variety of applications ranging from materials to biological tissues (2). Several large single cells have been successfully imaged (3). Recently, using new surface microcoils to improve sensitivity, we performed the first MRM of neurons in mammalian tissue (4). Although it offers the potential to study tissues in vivo, the inherently low sensitivity of nuclear magnetic resonance (NMR) has limited MRM to the detection of only two cellular compartments—the cytoplasm and nucleus—in relatively large cells: i.e. frog ova (~1mm diameter) (1) and Aplysia californica neurons (~ 300-350 μm diameter) (5). However, it has so far been assumed that the structures observed in the MRM correspond to the nuclear and cytoplasmic compartments. In this work, we report the extension of the new microcoil technology to improve MRM of Aplysia californica neurons, and—for the first time—present correlative light microscopy including Nissl and fluorescent staining methods to label specific cellular substructures. These studies are also a precursor to the examination of the effects of collagenase on the MR signals in these neurons. METHODS MRM was performed using commercially available 500μm diameter (Serial # B6370) microcoils provided by Bruker Biospin. The MR studies were conducted on a 600 MHz (14.1T) Bruker Biospin system. In the standard protocol, extracted ganglia are placed in a collagenase solution to soften membranes thus aiding cell extraction (3). However, in the present study, collagenase digestion was omitted (see discussion). After isolation by gross dissection, L7 neurons (n=2) of Aplysia californica were fixed in a 4% Formaldehyde solution for no less than 24h, and then washed in PBS buffer (137mM NaCl; 2.7mM KCl; 10mM Na2HPO4; 1.8mM KH2PO4: pH 7.4) to remove fixative before imaging so that T2 relaxation is restored to pre-fixation levels for improved SNR. The slices were then embedded in 3% of agarose. Neurons were sectioned into 50 μm thick slices using a Vibratome. MRM was performed using a 2D diffusion-weighted image sequence (7.8 μm in-plane resolution, 200μm slice width, b= 1500 s/mm, TR/TE = 2000/20 ms, NEX=100, acquisition time = 7hrs8min). Following MRM, slices were stained with PKH 67 for detection of the plasma membrane, and DAPI for detection of the nucleus. Additionally, a separate slice adjacent to the MR imaged slice was stained with Nissl (0.5% cresyl violet, 0.3% glacial acetic acid, in ddH2O) to delineate the cytoplasm through labeling of the endoplasmic reticulum (ER).