Selective injection system into hippocampus CA1 via monitored theta oscillation.

Methods of cell biology and electrophysiology using dissociated primary cultured neurons allow in vitro study of molecular functions; however, analysis of intact neuronal circuitry is often preferable. To investigate exogenous genes, viral vectors are most commonly injected using a pipette that is inserted from the top of the cortex. Although there are few reports that describe the success rate of injection in detail, it is sometimes difficult to locate the pipette tip accurately within the CA1 pyramidal cell layer because the pyramidal layer is only 0.1 mm thick. In the present study, we have developed a system to inject viral vectors accurately into the mouse hippocampal CA1 pyramidal cell layer using a stereotaxic injection system with simultaneous electrophysiological monitoring of theta oscillation. The pipette tip was positioned reliably based on integrated values of the theta oscillation in the hippocampal CA1 pyramidal cell layer. This approach allows accurate injection of solutions and provides an efficient method of gene transfer using viral vectors into the hippocampus, which can be a useful tool for studies involving the molecular mechanisms of neuronal functions

Electrophysiological analysis of lentivirus-infected neurons.

<p>EGFP-expressing lentivirus vectors were stereotaxically injected into the CA1 pyramidal neurons, and patch clamp analyses were carried out on infected and non-infected neurons. <b>A</b>. <b>B</b>. Analysis of the mEPSCs showed comparable frequency, amplitude, and decay time compared to the non-infected neurons. Non-infected cells: EGFP(-) n=7, infected cells: EGFP(+) n=8, <b>C</b>. Pairing-induced LTP was not significantly different between the two types of neurons. EGFP(-) n=6, EGFP(+) n=7. <b>D</b>. The injection of ZsGreen lentivirus vector expressing GluA1 into hippocampus CA1 had partially rescued LTP in GluA1 knockout mice. (*<i>P<0</i>.<i>05</i>, **<i>P<0.01</i>) <b>E</b>. Statistical analysis of LTP in wild (EGFP (-)), rescued, and GluA1 knockout mice in 10 min and 30 min after LTP induction. LTP was partially rescued by GluA1-expressing lentivirus infection in GluA1 knockout mouse. (*<i>P<0</i>.<i>05</i>, **<i>P<0.01</i>).</p

Schematic representation of the injection/recording system presented in the study.

<p>A glass pipette containing copper or Ag/AgCl EEG electrode is attached to a micro-syringe via a connection tube and is controlled by a manipulator. The electrode is connected to a personal computer to analyze the recorded signals and to calculate the real-time integrated value of the theta oscillation.</p

Comparison of distance from the cortical surface to the CA1 pyramidal layer in histological analyses and practical injections.

<p>Comparison of distance from the cortical surface to the CA1 pyramidal layer in histological analyses and practical injections.</p

Representative EEG data.

<div><p><b>A</b>. Raw EEG data collected from a recording electrode (top) and filtered EEG after selecting for theta oscillations (4-8 Hz) (middle). An isolated theta oscillation in a 10 sec and 20 sec bin was analyzed using integrated values (0.5 sec and 10 sec durations) at a depth of 0.95mm from the cortex. </p> <p><b>B</b>. Sequential recording of EEG at different depths. The same three types of data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083129#pone-0083129-g002" target="_blank">Figure 2A</a> with changes at different depths indicated at the bottom. Twenty seconds of recording data at each depth were collected, analyzed, and integrated over 0.5 sec and 10 sec.</p></div

Histological evaluations of injection accuracy and gene expression.

<div><p><b>A</b>. Dye was injected to confirm the position of the glass electrode tip after identification of the CA1 pyramidal layer based on the analysis of the theta oscillation. Pontamine skyblue dye was injected, and the slice was counter-stained with cresyl violet. The dye-injected area was shown as a dark area corresponding to the CA1 pyramidal layer. Scale bar, 200µm.</p> <p><b>B</b>. Latex microspheres demonstrated the precise injection sites in pyramidal cell layer. Injected fluorescent-coated latex microspheres accumulated at the bottom layer in stratum pyramidale. Scale bar, 200µm. Or: stratum orience, Py: stratum pyramidale, Rad: stratum radiatum, LM: stratum lacunosum-moleculare.</p> <p><b>C</b>. Herpes simplex virus infected-neurons expressing EGFP and GluA1 in the hippocampus. A herpes simplex virus vector expressing EGFP and GluA1 simultaneously was injected into the hippocampus of GluA1 knockout mice. The scheme of the expression cassette was indicated in the top left. Three days after the injection, a brain slice from the injected brain was stained with anti-GluA1 antibody (violet). Note that EGFP-positive neurons (green) expressed exogenous GluA1 at various levels in the picture taken in the same focus plane (top: low magnification, scale bar, 100µm; bottom: high magnification, scale bar, 20µm). DAPI: nuclear staining. Scale bar, 100µm.</p></div