Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.
),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:115-118.
RECORDING OF EXTRACELLULAR FIELD POTENTIAL
IN THE HIPPOCAMPAL SLICE
Shankaranarayana Rao BS and Raju TR
The mammalian brain slice preparation has
become a standard tool in neuroscience for
investigating the physiology and pharmacology
of neurons, as well as for the analysis of neuronal
circuits. This technique, which involves
maintaining a slice of brain tissue in physiological
saline solution and has become the technique of
choice for the study of synaptic mechanisms. It
can be used to investigate the effect of
neurotransmitters and drugs on neurons, various
internal and external perturbations on the brain,
the characteristics of ion channels and synaptic
plasticity. The hippocampal slice is used in our
laboratory to study synaptic plasticity
mechanisms; long-term potentiation (LTP) and
long-term depression (LTD).
The hippocampal slice preparation
A slice of brain tissue can be removed from the intact
brain and maintained in a physiological saline
(artificial cerebrospinal fluid, ACSF) solution with
appropriate temperature and oxygen conditions so
that neurons remain healthy and active. The
hippocampal slice is a particularly popular
preparation because the hippocampus is a layered
structure with a well-defined trisynaptic pathway,
where each individual synapse is easily accessible.
Figure 1. The position of the hippocampal formation in
the rat brain. The transverse slice of the dorsal
hippocampus is depicted (Amaral and Witter, 1989).
115
The hippocampus slice is most commonly used
slice preparation (Figure 1). Attraction of this slice
is due to its clearly layered-out cytoarchitecture,
where cell bodies lie in various clearly visible cell
bands, and dendrites make contact with fibers from
known origin. A lot is known about the histology,
as well as the pharmacology of the different areas
of the hippocampus. Even though the
hippocampus is the most widely used slice
preparation, many others have been established in
the last twenty years. It is theoretically possible to
cut any sort of slice from any region of the CNS.
Structure of the hippocampus
The hippocampus is composed of two gyri; the
hippocampal gyrus and the dentate gyrus (Figure 2).
In the hippocampal gyrus, the layer that is most
superficial is called the stratum oriens (OR), the thin
layer that contains the pyramidal cells is called the
pyramidal layer (PY) and the deepest layer is called
stratum radiatum (RD). In the dentate gyrus, the
major cell layer is the granule cell layer (stratum
granulosum). Superficial to the granular cell layer are
molecular and outer molecular layers (Figure 2).
Figure 2. Drawing of the hippocampus illustrating its
layered structure. OR, stratum oriens; PY, pyramidal layer;
RD, stratum radiatum; GC, granule cell layer; ML,
molecular layer; OL, outer molecular layer; CA4, hilus of
the facia dentate, FD, facia dentate; LM, lacunosum /
moleculare layer; FI, fimbria (Schopke et al. 1991).
Hippocampal trisynaptic circuitry recorded extracellularly and are therefore made up
Within the layers of the hippocampus, the of the response of many cells, often called a
neurons are arranged in three connecting population response. The response is a complex
pathways that allow information to flow in from response composed of the postsynaptic potentials
the entorhinal cortex, through the pathways and (PSPs) evoked in the dendrites and the action
out to either the subiculum or the fornix. These potentials recorded from the cell body or axons.
pathways are (see Figure 3); The dendritic response is referred to as a population
excitatory postsynaptic potential (p-EPSP) and the
1. Perforant path: Which consists of axons
action potential are called a population spike (p-
from cells in the entorhinal cortex (which is
spike). Both the p–EPSP and the p-spike are
adjacent to the hippocampus and receives inputs
recorded simultaneously and are superimposed
from neocortical association cortices) and synapses
upon each other. In addition, the directionality of
on the granule cells in the dentate gyrus.
the evoked potentials can be reversed depending
2. Mossy fiber pathway: Which consists of on whether the electrode is closer to the dendrites
axons that arise from the granule cells and project or to the cell bodies.
to the pyramidal cells of the CA3 region.
3. Schaffer collateral pathway: Which consists Field potential recordings
of axons that arise in pyramidal neurons in CA3
Electrophysiological recordings were carried out
and project to CA1 region of the hippocampus.
at room temperature in a submerged recording
Thus, the hippocampus provides three different chamber. Slices are perfused continuously with
synapses that can be studied to understand changes oxygenated (95% O2 / 5% CO2) ACSF at the rate
that occur at the synapse. of 2 ml/minute. ACSF used for incubation and for
subsequent recording were of the same
Preparation of hippocampal slices composition as used for slicing as mentioned above.
Synaptic responses are evoked by stimulating the
Rat is sacrificed by decapitation under deep
commissural/associational fibers in the CA3
anesthesia using halothane or sodium
pentabarbitone (60 mg/kg). Remove the brain as
quick as possible from the skull. Use plastic spatula
to cut the cranial nerves and submerge brain in
ice-cold (2-5ºC) oxygenated artificial cerebro-spinal
fluid (ACSF) for 1 minute before dissecting the
hippocampus from the brain. Hippocampus is
dissected out and immediately put into ACSF
chilled at 4º C. ACSF contained the following
concentration of salts: NaCl (118 mM), KCl (2.5
mM), NaHCO3 (2 mM), glucose (10 mM), NaH2PO4
(1.2 mM), MgCl2 (1.25 mM) and CaCl2 (2.5 mM) is
used. 400 µm thick transverse hippocampal slices
is prepared using a vibratome. Slices are incubated
in ACSF at room temperature for at least two hours
before starting field potential recordings.

Figure 3. Schematic of transverse hippocampal brain slice

Evoked field potentials preparation from the rat depicting the trisynaptic circuitry
Evoked field potentials are recorded of the hippocampus. Also illustrated is an extracellular
electrode placed in the stratum radiatum and intracellular
postsynaptically in response to an evoked stimulus
electrode in the stratum pyramidale (modified from
in presynaptic cells. Evoked potentials are usually Barrionuevo and Brown, 1993).
116
stratum radiatum layer using a 2-conductor cluster
Platinum-Iridium electrode (tip diameter 25 mm
each). The extracellular recording electrodes are
made of a glass micropipette filled with 2 M NaCl
(tip-resistance of 2-4 MW). In case of the CA1 field
potential recordings, Schaffer collateral inputs were
stimulated with concentric bipolar Tungsten
electrode (Outer Diameter 125 mm, FHC) has
shown schematically in Figure 3. Data acquisition
was done using LTP software (Anderson and
Collingridge, 2001).
A single current pulse applied to afferent inputs
to either CA1 or CA3 area (Schaffer collateral or
commissural associational, respectively) evoked a
short latency negative potential (Figure 4) in the
stratum radiatum. This potential is termed as the
“extracellular field excitatory post synaptic
potential (fEPSP)”. The stimulus artifact that
appears before the post synaptic potential is a
reflection of the current stimulus applied to the field
and this is followed by the fiber volley, which is an
extracellular reflection of the presynaptic action
potential generated by the stimulus pulse. The
initial slope of the fEPSP (mV/ms) is the most
widely used measure of synaptic strength in
experiments on synaptic physiology (Figure 4).
Figure 4. A field excitatory post synaptic potential (fEPSP)
depicting stimulus artifact, fiber volley (a reflection of
presynaptic action-potential) and initial slope. The initial
slope is an index of synaptic strength.
Stimulation paradigms used to induce synaptic
plasticity
A test pulse (100 msec in duration) when
delivered alone evoked a fEPSP in stratum
radiatum. For generating a stable baseline, two such
pulses, spaced 50 ms apart were delivered at 0.033
117
Hz (once every 30 seconds). Typically, that was
half of the maximal value obtained for the input/
output (I/O) curve.
Different patterns of stimulus trains were used
to evoke various forms of synaptic plasticity
studied. The temporal structure of the high
frequency stimulation used to induce LTP. This
LTP-inducing stimulus is typically called a ‘theta
burst stimulation’ (TBS). It consisted of two trains
of bursts, given at 10 seconds intervals. Each train
contained 10 bursts. Each burst consisted of 10
pulses (each pulse was of 100 msec duration) at a
frequency of 100 Hz, with an interburst interval of
200 ms. Thus, each conditioning train lasted for 2
seconds, with a total of 100 pulses per train. The
stimulus intensity during the TBS was twice of that
used during pre and post TBS baseline.
While measuring the maximum attainable
potentiation (i.e., saturation of LTP), same TBS was
applied to the input pathway multiple times. Once
a post-TBS stable baseline was obtained for at least
10 minutes, the next episode of TBS was presented.
The capacity of a pathway to undergo further
synaptic potentiation, was considered to have
saturated, when further application of TBS failed
to yield a potentiation greater than 15% over
baseline obtained following the previous tetanus.
To obtain the threshold for the induction of LTP
and depotentiation (LTD), input fibers were
stimulated with 900 pulses at 10 Hz . To induce
depotentiation, first a stable post-TBS baseline was
obtained (as described above), and following this,
low- frequency stimuli (LFS) of 900 pulses at 1 Hz
(i.e., 1 pulse per second) was presented to the same
pathway.
Data acquisition and analysis
The recorded extracellular evoked responses
were amplified by a high-impedance differential
AC amplifier and digitized (10 kHz A/D rate). A
personal computer equipped with acquisition and
analysis software developed by Anderson
(Anderson and Collingridge, 2001) was used for
storage and analysis. The LTP software package
was programmed to generate the necessary
stimulus triggers for specific experimental protocol
and acquire as well as display the resultant
waveforms on-line. It was also programmed to
carry out on-line analysis of relevant waveform
parameters (e.g. fEPSP slope, population spike
amplitude, peak latency etc.) and graphically
display the variations in these parameters with
time (e.g. fEPSP slope versus time etc.). This
allowed an efficient on-line monitoring of the
stability of slice recording and the outcome of
experimental manipulations being performed. All
the information gathered from an experiment, i.e.
the original responses (fEPSP), the calculated
parameters (fEPSP slope, peak amplitude, etc.), or
the variation with time (fEPSP slope versus time,
etc.) were stored electronically for off-line analysis.
118
References
1. Amaral DG and Witter MP (1989) The three
dimentional organization of the hippo-campal
formation. A review of anatomical data. Neuroscience,
31:571-591.
2. Anderson WW and Collingridge GL (2001) The LTP
Program: A data acquisition program for on-line
analysis of long-term potentiation and other synaptic
events. J. Neurosci. Methods, 108:71-83, 2001.
3. Barrionuevo G and Brown TH (1993) Associative
long-term potentiation in hippocampal slices. Proc
Natl Acad Sci USA 80:7347-7351.
4. Schopke R, Wolfer DP, Lipp H-P, and Lesinger-
Trigona M-C (1991) Swimming navigation and
structural variations of the infrapyramidal mossy
fibers in the hippocampus of the mouse.
Hippocampus 1: 315-328.