Technical Reference #215

215.

Abundant GFP Expression and LTP in Hippocampal Acute Slices by In Vivo Injection of Sindbis Virus Massimo D'Apuzzo; Georgia Mandolesi; Gerald Reis; Erin M. Schuman, California Institute of Technology, The Journal of Neuroscience, 86(215), (2001)
Link To Paper

Abstract:
Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organot

Materials & Methods:
Virus construction Sindbis virus was generated using the sindbis expression kit (Invitrogen). The fragments coding for EGFP and EGFP-actin were released from pEGFP-C1 and pEGFP-Actin (Clontech) respectively using NheI and ApaI and subcloned into the XbaI and ApaI sites of SinRep5. Capped RNA was generated and cotransfected with the DH (26S) helper RNA (invitrogen) into baby hamster kidney cells. After 36 h the supernatant was collected and concentrated by differential centrifugation (200000 g for 3 h at 4°C). In vivo injection Adult male Sprague-Dawley rats (48–52 days old) were anesthetized with a ketamine (90 mg/kg)/xylazine (10 mg/kg) mixture by intraperitoneal injection and placed in a stereotaxic frame (Kopf Instrument). Occasionally additional doses of anesthetic were delivered to keep the anesthesia level stable throughout the experiment. Two holes were drilled in the skull to gain access to the pyramidal cell layer of area CA1 in the hippocampus. The injection coordinates were established using the point of intersection of the sagittal suture with the best fit along the coronal suture (Bregma) as a reference for the lateral and antero-posterior coordinates (AP: 25.5 mm; L: 65.2 mm). The dura mater was carefully removed to expose the brain surface which was used as reference point for the vertical coordinates (V: 22.8 mm). To obtain reproducible injections quartz micropipettes with a microfilament (1 mm OD 0.7 OD 10-cm-length Sutter Instrument) were beveled (K. T. Brown Type Sutter Instrument). Viroid solution was delivered in short pulses (20–400 ms) supplied by a picospritzer apparatus (General Valve Picospritzer II). To obtain a reproducible volume per pressure pulse the injection pressure was varied between 20 and 40 PSI. Multiple pulses were applied up to a total volume of 0.50 6 0.10 ml. The surgery was completed by suturing the scalp and allowing the rat to recover with analgesic treatment (ibuprofen PO). Hippocampal slice preparation Injected rats were killed 16–20 h after the in vivo injection. Rats were anesthetized with halothane and decapitated and the brain was rapidly removed to ice-cold oxygenated artificial cerebrospinal fluid [ACSF which contained (in mM) 119 NaCl 2.5 KCl 1.3 MgSO4 2.4 CaCl2 1.0 NaH2PO4 26.2 NaHCO3 and 22 glucose]. Hippocampal slices were prepared using a Stoelting tissue chopper. In experiments requiring high-magnification imaging the rats were anesthetized with a ketamine (40 mg/kg)/xylazine (5 mg/kg) mixture and the slices were prepared with a cooled oscillating tissue slicer (OTS-300–04; FHC Brunswick ME). Serial sections of 500 mm were cut. The extraneous cortical and subcortical tissue was gently dissected away with the small end of a spatula. The injected slices were identified by fluorescence microscopy and then allowed to recover in an interface chamber at room temperature for at least 2 h. Electrophysiology Hippocampal slices were perfused with oxygenated ACSF at room temperature in a submerged chamber. The injected area was identified with an epifluorescence microscope. Field excitatory postsynaptic potentials (fEPSPs) measured in stratum radiatum were evoked by stimulation of the Schaffer collateral-commissural afferents (1 stimulus every 15 s). The initial slope was measured. The extracellular recording electrodes were filled with 3 M NaCl. Input-output (I/O) relations were monitored by measuring the EPSP slope in response to increasing stimulation currents. Paired-pulse facilitation was measured at two different intervals: 50 and 100 ms. Tetanic stimulation was delivered at the test intensity in 1-s trains at 100 Hz with four trains 30 s apart or four trains 5 min apart for short- and long-term potentiation respectively. Ensemble average plots represent group means of each EPSP for all the experiments aligned with respect to the time of the LTP induction. Error bars indicate the standard error of the mean (SE) calculated for the entire data set for a given time point. Paired or unpaired t-tests were used to calculate the statistical significance of within group or between group comparisons respectively. A one-way ANOVA was used to calculate the statistical significance of potential differences between the I/O curves.

Microscopic Technique
Fluorescence Microscopy, Epifluorescence, Confocal Microscopy

Cell Type(s)
CHO