Figure 1.
Schematic representation of the injection/recording system presented in the study.
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.
Figure 2.
A. 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.
B. Sequential recording of EEG at different depths. The same three types of data are presented as in Figure 2A 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.
Figure 3.
Representative traces showing changes in the integrated value of the theta oscillation at different depths.
A. A theta oscillation integrated over 0.5 sec indicated the highest value presumably in the pyramidal layer of the hippocampus. After the first peak, the value declined towards the bottom of the stratum radiatum and then increased again.
B. Superimposition of the integrated values of the theta oscillation. The changes in the integrated values across seven independent experiments were superimposed. All of the data presented a similar pattern of peaks, and these peaks overlapped at a point that was assumed to be the CA1 pyramidal layer.
Figure 4.
Histological evaluations of injection accuracy and gene expression.
A. 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.
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.
C. 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.
Figure 5.
Comparison of distance from the cortical surface to the CA1 pyramidal layer in histological analyses and practical injections.
Figure 6.
Electrophysiological analysis of lentivirus-infected neurons.
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. A. 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, C. Pairing-induced LTP was not significantly different between the two types of neurons. EGFP(-) n=6, EGFP(+) n=7. D. The injection of ZsGreen lentivirus vector expressing GluA1 into hippocampus CA1 had partially rescued LTP in GluA1 knockout mice. (*P<0.05, **P<0.01) E. 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. (*P<0.05, **P<0.01).