1328
LONG-TERM POTENTIATION: PHYSIOLOGY AND PHARMACOLOGY PI1
533.1
SlMULTANEOUS PATCH-CLAMP RECORDING AND CONFOCAL
IMAGING OF CA'+ INFLUX INTO CAI NEURONES OF THE RAT
HIpPOCAMPAL SLICE. -,*
B.G. Frenmelli* and G.L. ~ o l l i n e r i d ~ . ~
~
~of Pharmacology,
~
t
. The Medical School, Birmingham University,
BirminghamU.K. (Span: European Neuroscience Association)
(LTP) is a long lasting increase in synaptic efficacy
Long-term
induced by tetanic stimulation. In CAI pyramidal cells of the hippocampus, the
induction of LTP is initiated by the activation of NMDA receptors and a
postsynaptic caZ+ influx (Collingridgeet al 1983; 1. Physio1334, 33-46: Lynch el
a! 1983; Nature 305, 719-721). ca2+ entry as a consequence of tetanic stimulation
has been imaged using FURA-2 hut the relative contributionsof voltageoperated and ligand-gated ca2" entry has not been separated (Regehr and Tank
1990; Nature 345, 807-810). By w m b i i g whole-cell patch-clamp techniques in
slices wilh confocal laser scanning microscopy of a ca2+ fluorescent dye FLUO3, introduced into cells lh~onghthe patch pipette, we have attempted to
differentiate, in various subcellularlocations, between activation of voltageoperated and ligand-gated ca2+ channels during the te!anus.Tetanic stimulation
increases the fluorescence in voltage-clamped denddtes.This effect is blocked by
the NMDA receptor antagonist AP5 and shows anomalous voltage-dependence
consistent with NMDA receptor activation in the presence of extracellularM~".
These data indicate tetanus-induced ca2+ entry into dendrites directly though
NMDA receptor channels. In contrast, ~ a ' ' enters the soma during the tetanus
under current- but not voltage-clamp, indicating that the caZ+ entry into the
soma is through voltage-operated caZCchannels. In support of this, depolarising
voltage steps from -70 to -10 mV caused a marked increase in somatic but not
dendritic [caZ+]. The analysis of small areas of dendrite (approx 4 gm long)
shows the decay in intracellular [caZ+] is heterogeneous and oscillates as it returns
to baseline levels. This may be due to (?a2+-induced ca2+ release.
THURSDAY PM
533.2
Confocal Imaging of Calcium Transients and WholeCell Recording from Pyramidal Cells in Area CAI of
Rat Hippocampal Slices. R.J.Adams and IAS&u&L
Computational Neurobiology Laboratoly, The Salk Institute. La Jolia, Ca
92037.
Calcium transients are considered to be important mediators of
synaptic plasticity in the hippocampus and elsewhere in the nervous System.
We are using confocal microscopy to image changes in intracellular calcium
ion concentration in pyramidal cells in area CA1 of 400)lm thick Slices Of rat
hippocampus. Cells about 100)lm below the surface of the slice are recorded
from using whole-cell techniques using electrodes of about 5Mn reSlStanCe.
The recording electrode filling solution includes fluo-3 as
a fluorescent
indicator of calcium ion concentration. Low noise electrical recording with
simultaneous high temporal- and spatial-resolution imagining can be
achieved with this technique.
Synaptic stimulation from axons of the Schaffer collateral1
commissural pathway produces calcium transients in the dendrites and soma
a single
a single action
of these cells. Changes in intracellular calcium in response to
excitatory postsynaptic potential, causing the firing of
potential, can be detected in the proximal dendrites and soma.
533.3
CONFOCAL IMAGING O F CHANGES I N SYNAPTIC
STRUCTURE I N L I V I N G HIPPOCAMPAL S L I C E S . A_
F i n e , T. Hosakawa* a n d T . V . P . B l i s s . D e p t .
P h y s i o l . & Biophys., Dalhousie Univ., Halifax,
N . S . , C a n a d a B3H 4H7 a n d D i v . N e u r o p h y s i o l . &
N e u r o p h a r m . , NIMR, M i l l H i l l , L o n d o n UK.
L a s e r - s c a n n i n g confocal m i c r o s c o p y c a n b e
u s e d t o o b t a i n s u b m i c r o n - r e s o l u t i o n i m a g e s of
s y n a p t i c morphology i n l i v i n g hippocampal
slices. We have i n v e s t i g a t e d w h e t h e r s u c h
m o r p h o l o g i c c h a n g e s occur i n a s s o c i a t i o n w i t h
changes i n physiological function. Synaptic
s t r u c t u r e s a r e l a b e l l e d b y m i c r o i n j e c t i o n of a
c o n c e n t r a t e d o i l s o l u t i o n o f D i l i n t o the
s u p e r f u s e d s l i c e ; d y e d r o p l e t s are r e m o v e d
a f t e r 3 0 m i n . A n t e r o g r a d e d i f f u s i o n of d y e
p e r m i t s r a p i d , a n d often i n t e n s e , l a b e l l i n g o f
d e n d r i t i c s p i n e s and a x o n t e r m i n a l s . V i a b i l i t y
of l a b e l l e d n e u r o n s i s assessed b y e t h i d i u m
bromide exclusion. F i e l d p o t e n t i a l s from
l a b e l l e d r e g i o n s are n o r m a l , a n d L T P c a n b e
i n d u c e d b y electrical or c h e m i c a l s t i m u l a t i o n .
B r i e f e x p o s u r e t o h i g h c o n c e n t r a t i o n s of k a i n i c
a c i d c a n i n d u c e large changes i n d e n d r i t i c
s h a f t and s p i n e morphology; s m a l l e r and
i n c o n s i s t e n t changes can be seen i n c o n t r o l
slices a n d a f t e r i n d u c t i o n o f LTP b y e l e v a t e d
e x t r a c e l l u l a r calcium.
533.4
ANALYSIS O F THE VARIABILITY O F EVOKED SYNAWIC CURRENTS
DURING LONG-TERM POTENTIATION (LTP) m HIPPOCAMPAL
SLICES P. Renner*, T. Manabe*. D.M. Kullmann*. R.A. Zalutskv & R.A. Nicoll
Depts. Pharmacol. and Physiol., UCSF, San Francisco CA 94143.
Quantal analysis of synaptic transmission was originally restricted to a small
number of different synapses where the quanta1 amplitude could be easily
resolved. The development of high resolution whole-cell recording techniques
raises the possibility of extending this analysis to CNS synapses with small quantal size. These techniques have recently been used to analyze changes in the variability of evoked excitatory postsynaptic currents during LTP (Malinow and
Tsien, Nature 346177, 1990; Bekkers and Stevens, Nature 346724, 1990). This
analysis revealed a decrease in the coefficient of variation (CV), which was interpreted as an increase in transmitter release. We have repeated this analysis using
different conditioning paradigms to elicit LTP. We first checked whether the CV
analysis could detect some well known pre- and postsynaptic changes in transmission. Manipulations which affect the presynaptic site (applying baclofen,
changing the Ca/Mg ratio, paired pulse facilitation), showed the expected change
in CV, and the postsynaptically acting glutamate receptor antagonist CNQX had
no effect. LTP was induced using low frequency stimulation (0.2 Hz) paired with
postsynaptic depolarization (pairing). In 14 cells showing an average two-fold
potentiation we saw no change in CV, whereas paired pulse facilitation, which
was monitored in the same experiments gave the expected decrease in CV. When
a tetanus was used instead of pairing lo induce LTP, the results were more variable and, on average, a decrease in CV was observed (15 cells). Since the CV was
unchanged, our results favor the hypothesis that LTP induced by low frequency
pairing is expressed postsynaptically. This interpretation however depends on
several assumptions which may well not apply for synapses onto CAI pyramidal
cells (Korn et al., Nature 350: 282).
533.5
THE EFFECT O F LONG-TERM POTENTIATION ON MINIATURE
SYNAPTIC CURRENTS IN HIPPOCAMPAL SLICES. T.Manabe'.
P.Renner*..
Depts. Pharmacol. and Physiol., UCSF,
CA 94143.
Brief repetitive activation of excitatory synapses in the CA1 region
results in long-term potentiation (LTP) of synaptic transmission in pyramidal
cells. While there is general agreement that the induction of LTP occurs in
the postsynaptic cell, the site of LTP expression remains controversial. We
have used whole-cell recording from guinea pig slices to analyze
spontaneousexcitatory postsynaptic currents (sEPSCs) before and after LTP.
Cells were voltage-clamped at -80 mV and EPSCs were evoked with
electrical stimulation of afferent fibers at 0.2 Hz. LTP was induced by
pairing 0.2 Hz stimulation vjjth depolarization of the postsynaptic cell to 0
mV for 2.5 min. F'resynaptic stimulation alone caused the frequency of
sEPSCs to nearly double wilh little change in size. Following LTP induction,
however, a significant increase in the size of sEPSCs was detected in 6 of 7
cells. In a second set of experiments we used a glass pipette, filled with 0.5
M sucrose in normal Ringer, to evoke miniature EPSCs (mEPSCs). This
pipette was also used for focal elecnical stimulation of afferents. Control
mEPSCs and EPSCs were recorded in the absence of tetrodotoxin F X ) .
FollowingLTP induction l T X was added to abolish spontaneouspresynaptic
action potentials and mEPSCs were again recorded. A significant increase in
the size of mEPSCs was observed in 6 of 7 cells. Although we cannot
exclude an increase in the amount of transmitter per quantum, a change in
W S C size is usually interpreted as reflecting a change in postsynaptic
sensitivity to the transmitter.
533.6
SOCIETY FOR NEUROSCIENCE ABSTRAmS, VOLUME 17,1991
CHANGES I N NEURONAL EXCITABILITY DURING LONGT E R M P O T E N T I A T I O N . W.D.A. G a r n e r & R.F. T h o m ~ s o n .
Neurosciences P r o g r a m , University o f S o u t h e r n Californiai L o s
Angeles, C A 90089-2520.
High-frequency stimulation of the Schaffer axons, often inducing
long-term potentiation, decreased the afterhyperpolarization (aAHP and
mAHP) associated with the A current and M current. Inaacellular
recordings from C A I neurons (Vm = -77 + 2 m V SD; n = 102 cells)
showed that the decrease in the a A H P lasted about 30 s and was
enhanced b y application o f 4-aminopyridine and 3,4-diaminopyridine,
a n d by depolarization o f the postsynaptic cell immediately prior t o highfrequency stimulation. Conversely, when the postsynaptic cell was
hyperpolarized immediately prior to stimulation, the a A H P increased in
magnitude and the A P duration decreased slightly relative to controls.
T h e decrease in the mAHP lasted significantly longer (at least 75 min).
We postulate that suppression o f the A current transiently increases
[ca2+lh which, i n turn, enhances activation of ca2+-activated K
channels. Overall, C A I cell excitability decreases during and
immediately following high-frequency stimulation o f Schaffer axons.
Suppression o f the M current c a n increase cell excitability by a
mechanism yet t o be determined.
[Supported by N S F BNS8718300, O N R N0001488K0112, and
McKnight grants to R.F. Thompson, and an A P A Graduate Fellowship
t o W.D.A. Gamer.]