Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity

Abstract

The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.

Keywords: ToF-SIMS; lipids; mass spectrometry imaging; membranes; neurons.

Figure 1

Comparison of sample preparation for hippocampal neurons using ToF-SIMS imaging. From left to right: Ion images of total ion count (TIC), InO₂ at m/z 114.91, phosphatidylcholine (PC) headgroup at m/z 184.07, overlay of these ions, and spectra obtained from (A) chemically fixed, (B) frozen hydrated, and (C) freeze-dried samples. Scale bars: 100 μm.

Figure 2

Different lipid distribution across neuronal plasma membranes shown by independent component analysis in positive ion mode. (A) Color map of ICs showing their contributions to the cell body, neurites, and background. Color scale represents the lowest (blue) to the highest (red) levels of contribution. IC-images of the first nine ICs dominating in the (B) cell body and the (C) neurites in three different samples (in three rows). (D) Overlay of the nine IC-images of the cell body (green) and neurites (red). (E) TIC images of the three samples. Scales bars: 200 μm.

Figure 3

Cross-correlation coefficient differences analysis. (A) Comparison of a pair of spectra with and without a particular peak to determine the contribution of the peak to the spectral difference. CCD of the pair decreases if peak 1 is included but increases if peak 3 is included. (B) Comparison of CCD within the groups (basal) and between treatment and control groups (change) to determine which peaks significantly contribute to the difference between these groups based on (C) criteria.

Figure 4

Alterations of the lipid compositions of the plasma membranes of hippocampal neurons following drug treatments (TTX, BIC). The trend of alteration within individual lipid subclasses was obtained by comparing their peak intensities from the treatments to those from the controls. The trends (increase as ↑ or decrease as ↓) in regions (cell body or neurites) under treatments (TTX or BIC) are presented in the X axis. Lipid subclasses are presented in the Y axis. The number of lipid compounds having a similar trend within their lipid subclass are expressed as percentages in the Z axis.