Structural Correlates of CA2 and CA3 Pyramidal Cell Activity in Freely-Moving Mice.

Plasticity within hippocampal circuits is essential for memory functions. The hippocampal CA2/CA3 region is thought to be able to rapidly store incoming information by plastic modifications of synaptic weights within its recurrent network. High-frequency spike-bursts are believed to be essential for this process, by serving as triggers for synaptic plasticity. Given the diversity of CA2/CA3 pyramidal neurons, it is currently unknown whether and how burst activity, assessed in vivo during natural behavior, relates to principal cell heterogeneity. To explore this issue, we juxtacellularly recorded the activity of single CA2/CA3 neurons from freely-moving male mice, exploring a familiar environment. In line with previous work, we found that spatial and temporal activity patterns of pyramidal neurons correlated with their topographical position. Morphometric analysis revealed that neurons with a higher proportion of distal dendritic length displayed a higher tendency to fire spike-bursts. We propose that the dendritic architecture of pyramidal neurons might determine burst-firing by setting the relative amount of distal excitatory inputs from the entorhinal cortex.SIGNIFICANCE STATEMENT High-frequency spike-bursts are thought to serve fundamental computational roles within neural circuits. Within hippocampal circuits, spike-bursts are believed to serve as potent instructive signals, which increase the efficiency of information transfer and induce rapid modifications of synaptic efficacies. In the present study, by juxtacellularly recording and labeling single CA2/CA3 neurons in freely-moving mice, we explored whether and how burst propensity relates to pyramidal cell heterogeneity. We provide evidence that, within the CA2/CA3 region, neurons with higher proportion of distal dendritic length display a higher tendency to fire spike-bursts. Thus, the relative amount of entorhinal inputs, arriving onto the distal dendrites, might determine the burst propensity of individual CA2/CA3 neurons in vivo during natural behavior.

Multimodal Chemical Analysis of the Brain by High Mass Resolution Mass Spectrometry and Infrared Spectroscopic Imaging.

The brain functions through chemical interactions between many different cell types, including neurons and glia. Acquiring comprehensive information on complex, heterogeneous systems requires multiple analytical tools, each of which have unique chemical specificity and spatial resolution. Multimodal imaging generates complementary chemical information via spatially localized molecular maps, ideally from the same sample, but requires method enhancements that span from data acquisition to interpretation. We devised a protocol for performing matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MSI), followed by infrared (IR) spectroscopic imaging on the same specimen. Multimodal measurements from the same tissue provide precise spatial alignment between modalities, enabling more advanced image processing such as image fusion and sharpening. Performing MSI first produces higher quality data from each technique compared to performing IR imaging before MSI. The difference is likely due to fixing the tissue section during MALDI matrix removal, thereby preventing analyte degradation occurring during IR imaging from an unfixed specimen. Leveraging the unique capabilities of each modality, we utilized pan sharpening of MS (mass spectrometry) ion images with selected bands from IR spectroscopy and midlevel data fusion. In comparison to sharpening with histological images, pan sharpening can employ a plethora of IR bands, producing sharpened MS images while retaining the fidelity of the initial ion images. Using Laplacian pyramid sharpening, we determine the localization of several lipids present within the hippocampus with high mass accuracy at 5 µm pixel widths. Further, through midlevel data fusion of the imaging data sets combined with k-means clustering, the combined data set discriminates between additional anatomical structures unrecognized by the individual imaging approaches. Significant differences between molecular ion abundances are detected between relevant structures within the hippocampus, such as the CA1 and CA3 regions. Our methodology provides high quality multiplex and multimodal chemical imaging of the same tissue sample, enabling more advanced data processing and analysis routines.

AMIGO2 mRNA expression in hippocampal CA2 and CA3a.

AMIGO2, or amphoterin-induced gene and ORF (open reading frame) 2, belongs to the leucine-rich repeats and immunoglobulin superfamilies. The protein is a downstream target of calcium-dependent survival signals and, therefore, promotes neuronal survival. Here, we describe the mRNA distribution pattern of AMIGO2 throughout the mouse brain with special emphasis on the hippocampus. In the Ammon's horn, a detailed comparison between the subregional mRNA expression patterns of AMIGO2 and Pcp4 (Purkinje cell protein 4)--a known molecular marker of hippocampal CA2 (Cornu Ammonis 2)--revealed a prominent AMIGO2 mRNA expression level in both the CA2 and the CA3a (Cornu Ammonis 3a) subregion of the dorsal and ventral hippocampus. Since this CA2/CA3a region is particularly resistant to neuronal injury and neurotoxicity [Stanfield and Cowan (Brain Res 309(2):299­307 1984); Sloviter (J Comp Neurol 280(2):183­196 1989); Leranth and Ribak (Exp Brain Res 85(1):129­136 1991); Young and Dragunow (Exp Neurol 133(2):125­137 1995); Ochiishi et al. (Neurosci 93(3):955­967 1999)], we suggest that the expression pattern of AMIGO2 indeed fits with its involvement in neuroprotection.