A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse

Abstract

The hippocampus plays a critical role in learning and memory and higher cognitive functions, and its dysfunction has been implicated in various neuropathological disorders. Electrophysiological recording undertaken in live brain slices is one of the most powerful tools for investigating hippocampal cellular and network activities. The plane for cutting the slices determines which afferent and/or efferent connections are best preserved, and there are three commonly used slices: hippocampal-entorhinal cortex (HEC), coronal and transverse. All three slices have been widely used for studying the major afferent hippocampal pathways including the perforant path (PP), the mossy fibers (MFs) and the Schaffer collaterals (SCs). Surprisingly, there has never been a systematic investigation of the anatomical and functional consequences of slicing at a particular angle. In the present study, we focused on how well fiber pathways are preserved from the entorhinal cortex (EC) to the hippocampus, and within the hippocampus, in slices generated by sectioning at different angles. The postmortem neural tract tracer 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) was used to label afferent fibers to hippocampal principal neurons in fixed slices or whole brains. Laser scanning confocal microscopy was adopted for imaging DiI-labeled axons and terminals. We demonstrated that PP fibers were well preserved in HEC slices, MFs in both HEC and transverse slices and SCs in all three types of slices. Correspondingly, field excitatory postsynaptic potentials (fEPSPs) could be consistently evoked in HEC slices when stimulating PP fibers and recorded in stratum lacunosum-moleculare (sl-m) of area CA1, and when stimulating the dentate granule cell layer (gcl) and recording in stratum lucidum (sl) of area CA3. The MF evoked fEPSPs could not be recorded in CA3 from coronal slices. In contrast to our DiI-tracing data demonstrating severely truncated PP fibers in coronal slices, fEPSPs could still be recorded in CA1 sl-m in this plane, suggesting that an additional afferent fiber pathway other than PP might be involved. The present study increases our understanding of which hippocampal pathways are best preserved in the three most common brain slice preparations, and will help investigators determine the appropriate slices to use for physiological studies depending on the subregion of interest.

Distinct Synchronous Network Activity During the Second Postnatal Week of Medial Entorhinal Cortex Development

“…Although DiI injections were relatively superficial and therefore prone to be affected by slicing, we observed significant innervation of CA1 and dentate gyrus by fibers originating from entorhinal cortex (Supplementary Figure S2). This indicates that entorhinal-hippocampal connectivity in our slice preparations is not affected more than entorhinal-NeoC connectivity, in line with previous results (Xiong et al, 2017).…”

Direct Medial Entorhinal Cortex Input to Hippocampal CA3 Is Crucial for eEF2K Inhibitor-Induced Neuronal Oscillations in the Mouse Hippocampus

“…hippocampus in vitro (Rafiq et al, 1993;Xiong et al, 2017). In this way, afferents from the entorhinal cortex to the CA3 neurons (Neves et al, 2008) remained functional in acute slices.…”

“…Acute hippocampal slices were prepared from 4-to 8-week-old male mice as described previously (Huang et al, 2015;Weng et al, 2016;Wang et al, 2017;Yun et al, 2018;Li et al, 2019) with slight modifications to ensure the connectivity of the hippocampal formation with the entorhinal cortex in vitro according to Xiong et al (2017). Briefly, after anesthesia with isoflurane, the brains were isolated and immersed in pre-carbogenated (95% O 2 /5% CO 2 ) ice-cold slicing salt buffer solution (composition in mM: 92 NaCl, 2.5 KCl, 0.5 CaCl 2 •2H 2 O, 10 MgSO 4 , 1.25 NaH 2 PO 4 , 25 glucose, 30 NaHCO 3 , 3 sodium pyruvate, 20 HEPES, titrated to pH 7.4; Pan et al, 2015).…”

A comprehensive knowledge base of synaptic electrophysiology in the rodent hippocampal formation

“…Extrapolating these in vitro manipulations to in vivo brain function is especially challenging because severed neuronal processes during sectioning alter connection probability and neuronal reconstruction integrity. For instance, one half of CA1 stratum oriens or stratum pyramidale neurons are partially axotomized in typical electrophysiological preparations, which affects morphological identification (Halasy, Buhl, Lorinczi, Tamas, & Somogyi, 1996; Kogo et al, 2004) and synaptic activity (Gulyas et al, 2010) differentially in longitudinal and transverse slices (Couey et al, 2013; Surmeli et al, 2015; Xiong, Metheny, Johnson, & Cohen, 2017). Homeostatic mechanisms also change synaptic strength after slicing in order to compensate for axonal loss (Dumas et al, 2018; Kirov, Sorra, & Harris, 1999), implying that the time elapsed since slice preparation (which is hardly ever reported in peer-reviewed publications) may be a key determinant of synaptic amplitude.…”