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Proc. Natl. Acad. Sci. USAVol. 85, pp. 3221-3225, May
1988Neurobiology
Patch-clamp recording of amino acid-activated responses
in"organotypic" slice cultures
(cerebellum/hippocampus/y-aminobutyric acid-activated
channels/glutamate-activated channels)
I. LLANO*, A. MARTY*, J. W. JOHNSON*, P. ASCHER*, AND B. H.
GAHWILERt*Laboratoire de Neurobiologie, tcole Normale Supnrieure,
46 rue d'Ulm 75005 Paris, France; and tBrain Research Institute,
August-Forel-Strasse 1,8029 Zurich, Switzerland
Communicated by Rodolfo R. Llinds, December 18, 1987 (received
for review July 25, 1987)
ABSTRACT Patch-clamp recording techniques were usedto study the
properties of amino acid-activated channels incultured
"organotypic" slices from rat cerebellum and hippo-campus.
Hippocampal pyramidal cells responded to the threemain
glutamatergic agonists, N-methyl-D-aspartate (N-Me-D-Asp),
quisqualate, and kainate, whereas Purkinje cells re-sponded only to
quisqualate and kainate. Analysis of single-channel events recorded
in outside-out patches from hippo-campal neurons showed large
conductance events (50 pS),which occurred more frequently in the
presence of glycine.These events could be produced by N-Me-D-Asp
and also, atlow frequency, by quisqualate. On the other hand,
50-pSevents were never observed in Purkinje neurons. This sup-ports
the hypothesis that N-Me-D-Asp and "non-N-Me-D-Asp"receptors are
distinct molecular entities. Comparison ofwhole-cell and
outside-out patch recordings from Purkinjecells revealed a clear
spatial segregation of y-aminobutyricacid (GABA) and glutamate
receptors: although GABA recep-tors are found at high density in
somatic membrane, quisqua-late and kainate receptors are mostly
extrasomatic. The resultsshow that organotypic slice cultures are
amenable to patch-clamp methods. They also show that, in these
cultures, aminoacids receptors have specific distribution patterns
according tocell type and to region within a cell.
Our understanding of the basic membrane conductancesoperating in
vertebrate central neurons rests largely onstudies using two types
of in vitro preparations: primarycultures prepared from dissociated
neurons and brain slices.Both preparations have specific
advantages. One of the keyadvantages of primary cultures is that
the neuronal surface iswell exposed, allowing the use of
patch-clamp techniques(1). On the other hand, classical slices
permit the study ofidentified neurons connected by normal synapses.
A thirdtype of preparation, the "organotypic" culture of
brainslices, stands as an attractive intermediate between
dissoci-ated neurons and classical slices. In these cultures
(2-4)slices are placed for a few weeks in roller tubes, where
theyundergo a progressive thinning. When most successful,
thisprocess yields one or two layers of cells that retain
thegeneral organization of the original slice and the
mainmorphological and physiological properties of the
variousneurons present, including specific synaptic
connections(refs. 2-4; see also refs. 5 and 6). Furthermore,
organotypiccultures can be used to characterize synaptic
interactionsbetween anatomically remote brain areas, since
functionalsynaptic connections can be established between
coculturedslices derived from various brain regions (7).
In the present work, we show that patch-clamp recordingmethods
can be applied to organotypic cultures of brainslices. To explore
some of the potentials of this approach,
we have studied the responses of hippocampal and cerebel-lar
neurons to neuroactive amino acids. We have specificallyaddressed
three questions related to the spatial organizationand
developmental stage of neurons in organotypic cultures.(i) We have
examined whether Purkinje cells containedN-methyl-D-aspartate
(N-Me-D-Asp) receptors, since thesereceptors, which are absent in
in vitro slices of adult rats (8,9), have been reported to appear
transiently during develop-ment (10, 11) and to be present in
neurons presumed to bePurkinje cells from dissociated cultures
(12). (ii) We havestudied responses to glutamatergic agonists in
hippocampalpyramidal cells to compare the results with those
obtained inorganotypic cerebellar cultures, hippocampal cultures
(13),and hippocampal slices (14). (iii) We have investigated
iffunctional amino acid receptors in Purkinje cells are
differ-entially distributed in somatic vs. dendritic membranes.
METHODSOrganotypic cultures of rat cerebellar and
hippocampalslices were prepared from 1-day-old rats and 3- to
7-day-oldrats, respectively, and maintained in culture as described
(2).To reduce the number of nonneuronal cells, some of thecultures
were subjected to moderate doses of ionizing irra-diation [x-ramp,
250 kV, 1200 rads (1 rad = 0.01 gray)] at thetime of explantation.
In some cases, the antimitotic agentsuridine, 5-fluorodeoxyuridine,
and cytosine P-D-arabinofu-ranoside were added to the culture tubes
at equal concen-trations (1-0.1 ,uM) starting on day 3 or 4 of
culture and wereremoved after 20 hr. Exposure to ionizing radiation
andapplication of antimitotic agents can be expected not only
toreduce the number of nonneuronal cells but also to reducethe
number of neuroblasts still undergoing division and/ormigration.
However, Bodian silver staining indicated thatintermediate-size
neurons (presumed interneurons) andsmall neurons (tentatively
identified as granule cells) werestill numerous in cultures
subjected to these manipulations(B.H.G., unpublished observations).
The slices were usedwithin 2-5 wk after explantation.During
experimental recordings, the slices were main-
tained at room temperature while being perfused with asolution
containing (in mM) 140 NaCI, 2.5 KCl, 1 CaC12, and10 Hepes (sodium)
(pH 7.2). During most experiments 200nM tetrodotoxin was added and
in some 5.5 mM glucose wasadded. Variations in the composition of
this solution areindicated in the figure legends. The whole-cell
configurationand the outside-out patch configuration of the
patch-clamptechnique (1) were used. The composition of the
internalsolutions was as follows (in mM): (a) KCI: 120 KCI/10EGTA
(potassium)/5 Hepes (potassium); (b) CsCl: 120CsCI/10 EGTA
(cesium)/5 Hepes (cesium); (c) CsF: 120CsF/10 CsCI/10 EGTA
(potassium)/10 Hepes (potassium)
Abbreviations: GABA, y-aminobutyric acid; N-Me-D-Asp,
N-methyl-D-aspartate.
3221
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 85 (1988)
(internal pH, 7.2). Electrode resistance was 1.5-5 M1i.During
the recording of large whole-cell currents, the seriesresistance
led to errors in the imposed membrane potentialof up to 10 mV. In
addition, some of our results indicate lackof voltage control of
neurites (see Results). These errorswere left uncorrected.
Purkinje cells were recognized in the living state by theirlarge
size and their characteristic dark cytoplasm as visual-ized with
phase-contrast microscopy. They were alwayslocalized in peripheral
parts of the culture and could, there-fore, easily be distinguished
from neurons derived from thedeep cerebellar nuclei, which were
localized in the center ofthe cerebellar culture. Observation of
dendritic processes in20 Purkinje cells from cultures treated with
antimitotics andx-rays and intrasomatically injected either with
horseradishperoxidase or Lucifer yellow (B.H.G., unpublished
obser-vations) showed that their dendritic trees were very
similarto those described in control cultures (4). The
identificationof the Purkinje cells was confirmed (J. Mariani,
personalcommunication) by the fact that the cells identified
asdescribed above were all selectively stained by an antibodyto
calbindin (28-kDa calcium-binding protein) (15).
In hippocampus, pyramidal cells were recognized by theirlarge
size and location in or near cell layer CA1 or CA3. Onlyin those
cultures with a clearly visible pyramidal cell layerwas
identification considered possible (data from pyramidalcells are
specified as such; in other cases, data from uniden-tified
hippocampal neurons are also included). In all casesthe cell under
study was identified as a neuronal cell by thepresence of large
currents activated by depolarization.
In some cultures seal formation and the progression towhole-cell
recording proceeded smoothly with the majorityof neurons chosen for
study. In other slices, however, ahigh-resistance seal was formed
but following patch break-age, the response to voltage steps
resembled the response ofa partly obstructed pipette. Voltage- and
time-dependentcurrents were not elicited by depolarizing voltage
com-mands. A negative resting potential (-60 to - 80 mV)
wastypically recorded in these cases, even with
Cs'-containingpipette solutions. A possible explanation of these
observa-tions may be that seals were made on nerve or glial
cellprocesses that ran on the exposed surface of neuronal
cellbodies. This problem arose more frequently in cultures
thatremained several cell layers thick, and it was more severewith
hippocampal slices than with cerebellar slices. Al-though this odd
behavior was systematically observed insome of the slices, other
slices yielded success rates com-parable to those normally obtained
in dissociated neuronalcultures. It will obviously be important to
understand theorigin of these differences to increase the
proportion ofusable recordings in the future.
Fast microperfusion of agonists was achieved either with aU tube
(16) or with a double-barreled tube (17).Noise analysis and
single-channel analysis were per-
formed as described by Ascher et al. (18).
RESULTS
Purkiije Cells and Pyramidal Hippocampal Cells
RespondDifferently to Excitatory Amino Acids. At least three
differenttypes of glutamate receptors can be distinguished in
thevertebrate nervous system (reviewed in ref. 19). One recep-tor
type is selectively activated by N-Me-D-Asp; the corre-sponding ion
channel is blocked by Mg2+ ions (20). Theother two receptor types
are selectively activated by quis-qualate and kainate,
respectively. Adult Purkinje cells areknown to respond with an
increase in cationic conductanceto quisqualate and kainate but to
have a low sensitivity toN-Me-D-Asp (8, 9). In contrast, adult
hippocampal pyrami-
dal cells and, in particular, those of the CA1 region respondto
all three compounds (14, 19).Under whole-cell voltage-clamp, all
Purkinje cells tested
responded to applications of quisqualate and kainate with alarge
conductance increase. At a holding potential of -60mV, 2 ,uM
quisqualate elicited inward currents that ranged inamplitude from
0.6 to 4.2 nA (mean = 1.32; n = 8), whereasapplication of 10 ,tM
kainate led to currents ranging from 0.5to 1.9 nA (mean = 1.07; n =
7). In contrast, none of the cellstested (n = 6) responded to
N-Me-D-Asp (100 ttM), evenwhen glycine, known to potentiate the
response to N-Me-D-Asp in dissociated cultures from mouse brain
(17), wasadded at concentrations (1-10 jLM) that saturate the
poten-tiating effect in those cultures. The selective sensitivity
to"non-N-Me-D-Asp" agonists is illustrated in Fig. 1, whichshows
current records obtained from a Purkinje cell uponapplication of
each of the three compounds. In the experi-ment shown in Fig. 1,
Cd2+ was included in the bath solutionto block synaptic
transmission. The same result was ob-tained in the absence of Cd2 ,
which was previously shownnot to block N-Me-D-Asp receptors (19).
As illustrated inFig. 1, when pipettes filled with the KCl internal
solutionwere used, the initial inward current elicited by
quisqualateor kainate was often followed by an outward current.
Noattempt was made to identify the ionic mechanism of thiscomponent
of the response. A similarly activated outwardcurrent in other
neuronal types has been attributed by someauthors to activation of
the Na/K pump and by others to theactivation of Ca-dependent K
currents (reviewed in ref. 19).
In contrast to the N-Me-D-Asp insensitivity of
cerebellarPurkinje cells, hippocampal neurons responded to
N-Me-D-Asp as well as to quisqualate and kainate (Fig. 2).
Theresponses usually had a much slower time course than
thoserecorded in dissociated cultures from mouse brain with
asimilar perfusion system: the half-time of the onset and theoffset
was in the range of tens of seconds in organotypicallycultured
hippocampal neurons instead of hundreds of milli-seconds in
dissociated cultures. The response kinetics didnot depend on drug
perfusion pipette position but did varyfrom agonist to agonist
(Fig. 2) and cell to cell. A possibleexplanation of these
observations is that many of the recep-tors are on distant
dendritic branches, partially buried withinthe slice. The existence
of accessible somatic receptors andmore distant dendritic receptors
is expected to give rise torather complicated current kinetics. A
second differencewith respect to the responses obtained in
dissociated neuro-nal cultures was the absence of a large effect of
glycine onthe N-Me-D-Asp response recorded in the whole-cell
mode.The response to 10 ,uM N-Me-D-Asp was barely affected by1 ,M
glycine in five of six whole-cell recordings fromhippocampal
neurons of cultured slices (mean augmentation,1.1; range,
0.97-1.2). In one cell, however, faster response
100pM NMDA10pM Gly 10pM Kai 2pM Quis
FIG. 1. Whole-cell current responses of a Purkinje cell
toglutamatergic agonists. Holding potential, -60 mV. Agonists
wereapplied during the time indicated by the bars above each
record. Theslice was bathed in Mg2"-free external medium, which was
supple-mented with 5.5 mM glucose, 200 nM tetrodotoxin, and 250
uMCd2 . Internal solution, KCL. Quis, quisqualate; Kai,
kainate;NMDA, N-Me-D-Asp.
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Proc. Natl. Acad. Sci. USA 85 (1988) 3223
10 pM NMDA1 pM Gly 10 pM Kai 2 pM Quis
|0.5 nAlos
FIG. 2. Whole-cell current responses of a hippocampal pyrami-dal
cell to glutamatergic agonists. Holding potential, -50 mV.Agonists
were applied during the times indicated by the bars aboveeach
record. The slice was bathed in Mg2'-free external mediumwith 200
nM tetrodotoxin. Internal solution, CsF. See Fig. 1 legendfor
abbreviations.
kinetics and a significant augmentation by glycine
(2.4-fold)were observed (see Discussion).
Single-Channel Analysis of Excitatory Amino
Acid-InducedCurrents. Fig. 3A (upper trace) illustrates the
response of anoutside-out patch from a hippocampal pyramidal cell
to 10,uM N-Me-D-Asp with 1 ,uM glycine. The mean amplitude ofthe
most commonly observed single-channel event at -50mV was 2.3 pA in
this patch. With a reversal potential of 0mV, the corresponding
value of the single-channel conduc-tance is 46 pS. The mean
conductance (± SD) estimated innine different patches was 48 ± 4
pS. The influence of 1 uMglycine on the response to 5 or 10 puM
N-Me-D-Asp wasmeasured in seven patches. In five of these patches,
the totalchannel open time was increased by glycine by a factor of
4.9± 3.3 (mean ± SD). This was due predominantly to anincrease in
the frequency of channel opening. There wasonly a slight increase
in channel mean open time from avalue of 7.5 ± 1.8 ms (mean ± SD; n
= 5) with N-Me-D-Asp
alone to a value of 9.1 + 1.2 ms (mean + SD; n = 7) in
thepresence of glycine. In the remaining two patches glycineclearly
augmented the response to N-Me-D-Asp, but theeffect could not be
quantified because too few channelopenings were observed with
N-Me-D-Asp alone.The lower trace of Fig. 3A presents some of the
single-
channel events recorded during application of quisqualate (2,uM)
in the same patch as the upper trace. In this case themain event
was a 12-pS channel, but many 6-pS events (ofwhich only one is
illustrated) were also recorded. In addi-tion, there were a few
large openings having the sameconductance as the main event
observed after addition ofN-Me-D-Asp. These large-conductance
events were ob-served, albeit at low frequency, in all three
patches studied.They carried 5% of the quisqualate-induced
current.The responses to kainate (data not shown) were
associated
with current fluctuations in which individual events couldnot be
resolved. The ratio of the noise variance to the totalcurrent
yielded a "mean" single-channel conductance of 2.1pS. When fitted
with a single Lorentzian, the noise spectrayielded time constants
of about 1-3 ms, values comparableto those found in dissociated
cultures (21).The single-channel records obtained from Purkinje
cells
differed markedly from those obtained in hippocampal neu-rons by
the (expected) absence of responses to N-Me-D-Asp.As discussed
below, none of the patches studied showed adetectable response to 2
AM quisqualate (n = 5). With ahigher concentration (10 PM),
responses to quisqualate wereobtained in four of nine patches
tested. These responsesconsisted of an initial inward current of
3-4 pA, whichdesensitized within 30-50 ms to a level of activity
wheresingle-channel events could be clearly resolved. Over
theentire period of recording analyzed (13 min),
transitionscorresponding to the N-Me-D-Asp-induced conductance
A10 PM NMDA
1 pM Gly
2pMQuisv C -
B10PM Quis w I9 1 n 1~~~~~~~~~~~~~~IJ %~50 pM Kai
FIG. 3. (A) Selected records obtained from an outside-out patch
of a hippocampal pyramidal cell during application of the indicated
agonists.Holding potential, -50 mV; Mg2'-free external medium;
pipette solution, CsF. The arrows mark the current level
corresponding to the meansingle-channel current of
N-Me-D-Asp-activated channels in this patch at -50 mV. (B) Selected
records from an outside-out patch of acerebellar Purkinje cell
during application of the indicated agonists. Holding potential, -
60 mV. The dotted lines indicate the expected currentlevel for an
N-Me-D-Asp-activated channel at this potential. In the case of
kainate, the beginning of the dotted line marks the time of
agonistapplication. Mg2'-free external medium, 5.5 mM glucose;
pipette solution, CsCI. All records were filtered with an 8-pole
Bessel filter at a cutofffrequency of 1 kHz. See Fig. 1 legend for
abbreviations.
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Proc. Natl. Acad. Sci. USA 85 (1988)(45-50 pS) were never
observed. To examine more rigor-ously the possibility that 45- to
50-pS channels may never-theless have contributed to the response,
3 min of recordingsat low mean current level (
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Proc. Natl. Acad. Sci. USA 85 (1988) 3225
ochloride (2.5-10 gM) in a partially reversible manner (n =6).
Analysis of single-channel records, obtained in smalleroutside-out
patches, indicated that there are multiple con-ductance states. The
most frequent state had a conductanceof 28-30 pS, which compares
well with the value of 30 pSreported by Bormann et al. (26) for
dissociated cultures ofspinal neurons. A less frequent state with a
conductance of19-20 pS was also observed, but the 40-pS events
describedby these authors were not found in the patches
studied.
DISCUSSION
N-Me-D-Asp induced responses in hippocampal neurons butnot in
cerebellar Purkinje cells. The sensitivity of hippo-campal neurons
was expected since all previous studiesexcept one (28) have
indicated that pyramidal cells bearN-Me-D-Asp receptors and are
excited by N-Me-D-Asp(reviewed in ref. 19). The pattern of
sensitivity to excitatoryamino acids and, in particular, the
absence of N-Me-D-Aspsensitivity in Purkinje cells suggest at first
sight that theneurons present in organotypic slices have the
properties ofadult neurons. This should be qualified, however,
since ithas been suggested that the sensitivity of rat Purkinje
cells toN-Me-D-Asp is also absent at birth, that it develops in
thedays after birth, and that it then disappears again about 1month
later (10, 11).The properties of N-Me-D-Asp responses recorded
in
outside-out patches from hippocampal cells are similar tothose
reported in cultures of dissociated neurons from mam-malian brain
(12, 13, 18, 20). The observation here of anaugmentation by glycine
suggests that glycine sensitivity (17)is a general property of
N-Me-D-Asp channels, although theeffect is not of uniform
magnitude.The whole-cell responses of the hippocampal neurons
to
N-Me-D-Asp, however, differ from those of dissociatedcultures in
their slow time course and in their insensitivity toglycine. A link
between these properties is suggested by thefact that the only
whole-cell recording exhibiting a signifi-cant augmentation of the
N-Me-D-Asp response by glycine(2.4-fold) also had the most rapid
responses to applicationand removal of agonists. The results could
be explained bythe presence of a diffusional barrier between the
bathingsolution and neuronal receptors. Such a barrier could
delaythe access of exogenous agonists to their receptors andpermit
the accumulation of glycine in the intercellular spaceup to a
concentration sufficient to mask any potentiatingeffect of
exogenous glycine (see ref. 17).
In outside-out patches from cerebellar and hippocampalneurons,
quisqualate opened channels of several differentconductances. The
main conductance state, defined as theconductance state observed
most frequently among single-channel events, had a value of 12-14
pS. In the case ofkainate, analysis of the ratio of the current
noise variance tothe total agonist-induced current yielded a value
of 2.1-2.3pS for the main conductance state. Both of these values
arein agreement with what has been found in dissociatedcultures
from various brain regions (12, 13, 21).The study of the "minor"
states elicited by non-N-Me-D-
Asp agonists, however, revealed a marked difference be-tween
Purkinje cells and hippocampal neurons. In the caseof hippocampal
neurons, the minor states included somelarge single-channel events
with a conductance very similarto that of the main event activated
by N-Me-D-Asp agonists.The activation of these large conductance
events by non-N-Me-D-Asp agonists, which have also been observed in
dis-sociated cultures from hippocampus (13), cerebellum (12),and
cortex (21), has been interpreted by two types of theories:one in
which the 50-pS openings represent a "substate" of a
channel that can be activated through separate receptors
byN-Me-D-Asp and non-N-Me-D-Asp agonists (12, 13) and onein which
the large conductance openings observed withnon-N-Me-D-Asp agonists
indicate that these compounds areweak agonists for the N-Me-D-Asp
receptor (21). The fact thatthe non-N-Me-D-Asp agonists did not
induce any 50-pSopenings in Purkinje cells supports the second
ty'pe of inter-pretation, in which the 50-pS conductance is
exclusivelylinked with the N-Me-D-Asp receptor.
We thank Ms. L. Riestchin for her technical assistance in
prepar-ing the cultures. This work was supported by the Centre
National dela Recherche Scientifique (Unite Associde 295), the
Institut Nationalde la Sante et de la Recherche Mddicale (CRE
856001), and theUniversitd Pierre et Marie Curie. I.Ll. was
supported by the CentreNational de la Recherche Scientifique and
the Fondation pour laRecherche Mddicale. J.W.J. was supported by
the National ScienceFoundation/Centre National de la Recherche
Scientifique exchangeprogram and by National Research Service Award
MH09313 fromthe National Institute of Mental Health.
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