- ACSF: (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3,
1.25 NaH2PO4, 25 glucose bubbled with 95% O2, 5% CO2, PH 7.4.
- Intracellular solution: (in mM) potassium gluconate 115,
KCl 20, sodium phosphocreatine 10, HEPES 10, MgATP 2, NaGTP 0.3, and
0.1% biocytin for subsequent determination of morphology.
- Adult, male Wistar rats were anesthetized with halothane and perfused
through the heart with ice-cold, oxygenated, artificial cerebrospinal
fluid (ACSF). Brains were removed, and 300 µm transverse hippocampal
slices were made with a vibrating tissue slicer (Campden Instruments)
in ice-cold ACSF. Slices were then incubated at 35C for 30 min and kept
at room temperature until time of recording.
- Slices were placed on the stage of an Axioscop FS microscope (Zeiss)
where they were perfused with ACSF held at 34+-2C and visualized using
infrared differential interference video microscopy.
- Whole-cell current-clamp recordings were made using bridge amplifiers
- Patch-clamp electrodes were fabricated from thick walled borosilicate
glass (EN-1, Garner Glass) and had tip resistances of 4–11 MW
in saline. Bridge balance and capacitance compensation were performed
for all whole-cell recordings, which were terminated if series resistance
exceeded 80 MW.
- Electrophysiological traces were digitized via an ITC 16 or ITC18
digital-analog converter (Instrutech) under control of macros custom
programmed in IGOR Pro (Wavemetrics). Electrophysiological records were
filtered at 5 kHz and digitally sampled at 10-50 kHz.
- Data were acquired using Macintosh PowerPC computers using Pulse Control
software (Dr. Richard Bookman, University of Miami) or our own custom
macros, both written to run under Igor Pro (WaveMetrics).
- Analysis of electrophysiology was performed using IGOR Pro Software.
- Neurons were visualized using the DAB reaction (Vectastain ABC kit,
Vector Laboratories Inc., Burlingham, CA) using standard procedures.
No clearing or dehydration was used in order to prevent shrinkage of
slices. After histology, slices were mounted in aqueous mounting medium
(Moviol, Calbiochem, LaJolla, CA).
- The three-dimensional position and diameter of the dendritic branching
pattern were reconstructed using a Zeiss inverted microscope having
a 63x oil immersion objective fitted with semi-automated Neurolucida
hardware and software (version 2.1, MicroBrightField Inc., Colchester,
- The Neurolucida file was then converted to a NEURON geometry file
by the Neuroconvert program (version 2.0b4, D. Niedenzu and G. Klien,
MPI für medizinische Forschung, Heidelberg, Germany, 1998).