J. Physiol. (1981), 315, pp. 549-567
With 1 plate and 9 text-figures
Printed in Great Britain
549
ELECTROPHYSIOLOGY OF MAMMALIAN INFERIOR OLIVARY
NEURONES IN VITRO. DIFFERENT TYPES OF VOLTAGE-DEPENDENT
IONIC CONDUCTANCES
BY RODOLFO LLINAS AND YOSEF YAROM
From the Department of Physiology and Biophysics, New York University Medical
Center, 550 First Avenue, New York, NY 10016, U.S.A.
(Received 5 September 1980)
SUMMARY
The electrophysiological properties of guinea-pig inferior olivary (I.o.) cells have
been studied in an in vitro brain stem slice preparation.
1. Intracellular recordings from 185 neurones in this nucleus reveal that antidromic,
orthodromic or direct stimulation generates action potentials consisting of a fast spike
followed by an after-depolarizing potential (ADP). The ADP had an amplitude of
49 + 8 mV (mean + S.D.) and a duration which varied over a wide range with the level
of depolarization. This ADP is followed by an after-hyperpolarizing potential (AHP)
having an amplitude of 12 + 3 mV (mean + S.D.) from rest and lasting up to 250 msec.
The AHP shows a rebound depolarization wave.
2. Synaptic activation may be obtained by peri-olivary stimulation with a bipolar
electrode located in the immediate vicinity of the i.o. nucleus. These potentials are
a mixture of depolarizing and hyperpolarizing synaptic events which can be reversed
by direct membrane polarization.
3. Addition of tetrodotoxin (TTX) to the bath, or removal of extracellular Na,
abolishes the fast initial action potential but does not modify the ADP or the AHP.
Blockage of Ca conductance by Co, Mn, Cd or D600, or replacement of Ca by Mg,
abolishes the ADP-AHP sequence.
4. Hyperpolarization ofthe neurone uncovers a low-threshold Ca conductance which
is inactivated at rest and has similar pharmacological properties to the ADP. This
low-threshold spike plays a central role in the rebound potential following the AHP.
5. Simultaneous impalement of i.o. neurone pairs demonstrated the presence of
electrotonic coupling between neurones, which is especially prominent in the medial
accessory olive.
INTRODUCTION
Since the anatomical and physiological demonstration (Szentaigothai & Rajkovits,
1959; Eccles, Llinas & Sasaki, 1966) that the inferior olive is the site of origin of the
cerebellar climbing fibre system, many electrophysiological studies have been carried
out on the cells of this nucleus. Early research indicated that these neurones have
complex electrophysiological properties. Thus, intracellular recordings (Crill, 1970;
Armstrong, Eccles, Harvey & Matthews, 1968) showed that prolonged action
0022-3751/81/5130-1202 $07.5t C) 1981 The Physiological Society
R. LMNAS AND Y. YAROM
550
potentials could be evoked by direct or orthodromic stimulation. These action
potentials were characterized by a rapid initial spike and a prolonged afterdepolarizing potential. In a more recent set of experiments the early findings were
confirmed and expanded, and in addition electrotonic coupling between these
neurones was described (Llinas, Baker & Sotelo, 1974).
The findings reported in the present papers expand those results to include a
detailed analysis of the electrophysiological properties of inferior olivary (i.o.)
neurones in vitro. The ionic basis for these properties was studied further by using
agents known to block particular voltage-dependent ionic conductances and by
modifying the ionic composition of the bathing medium. Furthermore, because
simultaneous recordings from two i.o. neurones seemed feasible in the in vitro
preparation, a direct study of the problem of electrotonic coupling was undertaken.
Other aspects of the Ca-dependent action potentials will be dealt with in the
accompanying paper (Llinas & Yarom, 1981). Some of the results described here have
been presented in a preliminary fashion (Yarom & Llinas, 1979; Llina's & Yarom,
1979, 1980).
METHODS
Ti8sue preparation
Since this paper represents an extension of the in vitro work with the cerebellar cortex in our
laboratory, the techniques used here are quite similar to those reported previously (Llinas &
Sugimori, 1980a).
Adult guinea-pigs (400400g) were decapitated, after ether anaesthesia, using a small animal
guillotine. Immediately thereafter two longitudinal sections were made with rongeurs along the
lateral edge of the squamous portion of the occipital bone. The resulting bone slab was cut
transversely, rostral to the tentorium cerebelli, and pulled caudalward to expose the cerebellum
and brain stem. Following transaction of the cranial nerves and transverse section of the brain stem
at inferior collicular level rostrally and at the level of C1 caudally, the brain stem was swiftly
removed and placed in Ringer solution at close to 50C. It was then transacted longitudinally in
a parasagittal plane and fixed to a Vibratome plate in order to obtain thin longitudinal sections.
From a single brain stem, six 300,um slices could be obtained. Following sectioning the slices were
incubated in a Ringer solution for approximately 1 hr. The bathing medium was kept at room
temperature and a mixture of 95% 02 and 5 % C02 was bubbled into the bath during this period.
Brain stem slices were removed from the incubation bath after 1 hr but could be kept in good
condition in the bath for periods up to 24 hr. The recording chamber was similar to that previously
used in this laboratory (Llinas & Sugimori, 1980a), as were the Ringer solutions and oxygenation
methods.
Recording techniques
I.O. cells were recorded intra- and extracellularly by means of K-acetate-filled micropipettes
with an average d.c. resistance of 60-80MQ. Although the high degree of visualization obtained
in cerebellar slices could not be obtained for the brain stem, a clear definition of the outline of the
subnuclei of the inferior olive could be achieved by trans-illumination of the tissue with a
conventional fibre optics system. When simultaneous multiple penetrations were attained, the
micro-electrodes approached the tissue from the right and left side of the recording chamber and
were carefully aligned above the inferior olive before cell impalement.
A typical brain stem slice is seen in Plate 1 A. In this case the slice was stained with cresyl violet
for photographic purposes. During the experiment the tissue was held on the Sylgard surface at
the bottom of the recording chamber by means of surface adhesion and further stabilized by the
local stimulating electrode near the inferior olive (see arrow in Plate 1 A). Electrical stimulus at
that point provided antidromic as well as orthodromic activation of the olivary neurones. Electrical
properties of neurones were determined by means of a high-input impedance bridge amplifier
permitting current injections of the order of 0 5-10 nA. Because long-lasting current injections were
INFERIOR OLI VAR Y CELLS IN VITRO
551
often attempted in these cells, both the reference electrode and the metallic contact with the
micro-electrode electrolyte were selected for minimum polarization.
The exact location and identification of the cells was determined following intracellular injection
of horseradish peroxidase (HRP), which allowed their positive identification as i.o. cells (Ramon
y Cajal, 1911) (see Plate 1 B). The techniques used for the HRP staining were the same as those
reported previously (Llina's & Sugimori, 1980a).
RESULTS
The results described in these papers were obtained from 185 neurones in the
principal and the medial accessory olivary subnuclei. Three paradigms were used to
provoke neuronal activity: antidromic, orthodromic and direct stimulation. Because
the olivo-cerebellar was transacted in preparing the slices, electrical activation of
this pathway could not be used to elicit antidromic invasion (Crill, 1977; Llinas et al.
1974). Thus, while a clear separation could be made between antidromic and chemical
synaptic activation following pharmacological or ionic blockage of Ca conductance,
it was not always possible to distinguish between antidromic and electrotonic invasion. This ambiguity arose because these cells are electrotonically coupled and,
thus, a given neurone may be activated either antidromically from its own axon or
orthodromically via its electrotonic coupling to neighbouring cells, themselves being
antidromically invaded. In many cases, however, a clear all-or-none invasion of cells
could be obtained in the absence of short latency depolarizations (SLD) (Llina's et al.
1974), indicating antidromic invasion.
Action potentials in inferior olivary neurones
Antidromic activation
Examples of intra- and extracellular recordings from I.o. cells following antidromic
activation are seen in Fig. 1. As reported previously (Crill, 1970; Llinas et al. 1974),
the antidromic activation of these cells is characterized by an initial sharp action
potential having a duration of approximately 1 msec and an 80-90 mV amplitude
(Fig. 1 A and B, Table 1). This spike is followed by a prolonged after-depolarizing
potential (ADP) and a long-lasting large after-hyperpolarizing potential (AHP). The
ADP lasts for 10-15 msec at an amplitude of approximately 50 mV, and is studded
with small wavelets (arrows in Fig. 1 A; see also Fig. 2 A-D). Its termination is rather
abrupt and merges directly into the AHP. The AHP has an overall duration of
approximately 150-200 msec and an amplitude of 10-15 mV, depending on the
resting potential level and on the magnitude of the ADP (Table 1). The AHP usually
terminates rather sharply (arrow in Fig. 1 B), which, as will be seen in detail below
(and in Llinas & Yarom, 1981), indicates that an active rebound response follows the
AHP.
Extracellular single unit recordings obtained before impalement are shown in Fig.
1 C and D. The initial response to antidromic invasion is a positive-negative wave
which, as shown in Fig. 1 C, is followed by a positivity corresponding in time to the
ADP seen intracellularly, and by a negative wave (Fig. 1 C and D) which corresponds
to the AHP as seen in records A and B. Given that this type of extracellular recording
was obtained just before somatic impalement, the polarity of the extracellular action
potential waveform suggests that the active current sink for the ADP and the active
R. LLINAS AND Y. YAROM
552
current source for the AHP are both located remotely from the recording site. Indeed
these potentials have opposite polarity to that expected from the immediate vicinity
of an active response, which should be a negativity corresponding to the ADP and
a positivity to the AHP. These points will be treated in detail in the accompanying
paper (Llina's & Yarom, 1981).
B
A
J
~~~~~20OmV
+D
C
5 mV
10 msec
40 msec
F
E
20 mV
=
0~~~~~-------10 msec
-------30 msec
Fig. 1. Antidromic activation of i.o. neurones. A and B, antidromically activated action
potentials recorded intracellularly. Initial fast depolarization followed by ADP and a
prolonged AHP can be seen in A. In B, at slower sweep speed, the AHP is seen to terminate
sharply (arrow). C and D, extracellular recording from the same cell before impalement.
Note the initial positive-negative potential followed by a slow positive wave and long
negativity. E and F, effect of direct current hyperpolarization on ADP and AHP. Note
that the amplitude and duration of the AHP are related to the duration of the ADP.
Dashed lines in A and B correspond to resting potential; in E and F lower dashed line
indicates level of membrane polarization from rest.
Intracellular recordings from another cell show the effect of small d.c. changes
in membrane potential on excitability (Fig. 1 E and F). A small hyperpolarization
of the impaled neurone had little effect on the initial antidromic spike but produced
a diminution and ultimately a complete disappearance of the ADP and of the AHP
(Fig. 1 E). The ADP itself appeared to be composed of several all-or-none components,
which is evidenced by the waveform breaks that are often seen in the rising and falling
phase of this after-potential (Fig. 1 E). In all cells recorded, the amplitude of the AHP
was related to that of the ADP. Indeed, these two responses are concomitant in that
a reduction in the ADP is invariably accompanied by a reduction ion the AHP (see
Fig. I E and F).
INFERIOR OLI VAR Y CELLS IN VITRO
553
Direct and synaptic activation
A set of records obtained by direct stimulation of an olivary cell is illustrated in
Fig. 2A and B. This form of stimulation generates a sequence of potentials similar
to that seen in Fig. 1 for antidromic activation. Thus, once the cell reaches a firing
level, a rather stereotyped action potential pattern consisting of a fast spike followed
by the ADP-AHP sequence described above is observed as well as the rebound wave
TABLE 1. Values for resting potential, Na and Ca spike height, threshold and input resistance for
twenty representative neurones
Amplitude
AHP amplitude
Threshold
Cell
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15*
16*
17*
18*
Na
spike
(mV)
ADP
From
rest
(mV)
(mV)
Absolute
Na
spike
ADP
(mV)
(mV)
(mV)
Resting
poten-
Input
tial
tance
(mV)
(Me)
resis-
62
10
-80
22
70
15
58
-75
60
40
11
10
-69
59
18
49
12
-80
5
19
75
12
55
10
-74
21
62
12
51
-71
15
19
59
50
8
-70
62
60
14
12
-79
65
48
-71
16
55
46
13
-74
61
54
10
14
-74
64
22
50
8
-73
13
65
20
48
12
-80
8
20
68
50
13
10
-78
21
65
35
15
-73
28
28
58
40
10
-75
32
32
65
75
40
18
26
21
77
40
14
-81
10
28
16
67
19t
95
45
-78
13
8
30
18
70
94
20t
60
20
21
35
Mean
49
82
12
-75
12
21
64
30
+ S.D.
+7
+8
+4
+3
+3
+3
+4
+5
Spike heights were measured from the resting potential to the peak of the spike. Input resistances
were determined with the hyperpolarizing current pulses (0-5 nA) in order to avoid delayed
rectification. *, with TTX added; t, Na-free. Threshold for ADP in cells 15 to 20 obtained in the
absence of Na spike.
88
80
70
81
83
89
78
90
74
80
88
78
80
80
at the end of the AHP (arrow, Fig. 2B). Note the difference in the voltage step at
the 'make' and 'break' of the current pulse in the presence of an action potential.
The smaller amplitude and the fast rate of fall of this potential following a spike
indicates an increase in membrane conductance during the AHP (see also Fig. 4A
and C).
Synaptic activation of I.o. cells was often observed following stimulation of the
peripheral white matter near the caudo-dorsal aspect of the nucleus (arrow in Plate
1 A). Frequently, as shown in Fig. 2C and D, action potentials similar to those in
Fig. 1 could be evoked by such stimulation. In these cases the spike, rather than
554
R. LLINAS AND Y. YAROM
arising directly from the resting level, was generated from a graded depolarization.
However, here it was difficult to determine to what extent this potential was due to
chemical synaptic transmission and to what extent to electrotonic coupling or
decremental electroresponsiveness from dendrites. For instance, as seen in C and D,
some of the synaptic potentials had a rather slow onset and were followed by a
plateau-like waveform giving them a rather angular shape with a fast decay at the
20 mV
50 msec
20 msec
DI
C
20 mV
20 msec
50 msec
Fig. 2. Intracellular recording of response to direct and orthodromic stimulation. A, direct
stimulation activates an action potential similar to that in Fig. 1. Note that firing level
is the same for two amplitudes of current injection (bottom trace). B, same records as A
displayed at slower sweep speed to show the rebound (arrow) and the small amplitude
of the voltage step at the current pulse break. C and D, synaptic activation generated
by peri-olivary stimulus. Note the slow Tate of rise and prolonged duration of these
subthreshold potentials. D is same record as C at slower sweep speed.
end reminiscent of dendritic local responses in other vertebrate neurones (Spencer
& Kandel, 1961; Llinas & Nicholson, 1971; Barrett & Barrett, 1976; Llinas & Hess,
1976; Schwartzkroin & Slawsky, 1977; Wong, Prince & Basbaum, 1979; Llinas &
Sugimori, 1980a, b). However, because it was often possible to reverse some of these
potentials, as seen in Fig. 3, we conclude that at least in part they were generated
by chemical synaptic transmission.
An example of such reversal is seen in Fig. 3A where electrical stimulation of the
peri-olivary white matter generated an orthodromic activation of the cell (Fig. 3A,
0 0 nA). Membrane hyperpolarization via the recording electrode produced an
INFERIOR OLI VARY CELLS IN VITRO
555
increase in the amplitude of this potential (07 and 1'2 nA). A clear reversal of the
later part of this potential was observed as the cell was depolarized (1-3 to 2-9 nA).
The properties of the reversal indicate, however, that two components may be
present, an early depolarization followed by a late inhibition (i.p.s.p.). The presence
of the late i.p.s.p. is most likely reponsible for the reversal seen at the depolarized
levels in Fig. 3A, given the late onset of this change in polarity and the relatively
low level of direct current injection (13 nA) required to invert this late component
of the orthodromic response.
B
A
29
I 1~~~~~~~~~~~~~~~~~~~~~~
2>4
2o0 mV
r~~~~~~~~~~~~~
_
C
20 msec
+055-
00
*
00
-7
I10 mV
2A
(nA)
------j 5 nA
it
1 0mselC
V5m
o
~~~~~~~5
mV
-1 *3
(nA)
30 msec
Fig. 3. Synaptic potentials recorded from three olivary neurones following stimulation of
peri-olivary white matter. A, synaptic potential composed of excitatory post-synaptic
potential (e.p.s.p.) followed by inhibitory post-synaptic potential (i.p.s.p.) recorded with
different levels of polarization. Note that, due to high resting level, the action potential
generated by the synaptic potential at rest consists of Na conductances only (level of
polarization is given to left of each trace). B, reversal of e.p.s.p. C, i.p.s.p. reversed by
direct current injection.
A second example is shown in Fig. 3 B using square current pulses rather than direct
current injection. In this case the reversal does not show the latency shift seen in
panel A and thus may represent the reversal of a chemically mediated synaptic
potential. In some cells white matter stimulation produced mainly hyperpolarizing
potentials as illustrated in Fig. 3 C. Their duration and time course are similar to those
reported in previous studies (Llinas et al. 1974) and may be reversed by membrane
hyperpolarization. However, as elaborated in the Discussion of this paper, it is highly
likely that none of these potentials is due to the activation of only excitatory or only
inhibitory terminals but, rather, they represent both types of synaptic responses
acting together and in varying degrees upon the I.o. neurones.
R. LLINAS AND Y. YAROM
556
Na- and Ca-dependent components of the inferior olivary action potential
The fast Na spike
One of the obvious advantages of the in vitro preparation is the possibility of
modifying the membrane conductance by changing the ionic composition of the
Ringer solution or by adding drugs known to block certain ionic conductances.
A
C
TTX
E
8 msec
D
-
120mV
~12nA
F
120mV
50 msec
100 msec
Fig. 4. TTX-resistant action potential. A and B, control olivary response to direct
stimulation. C and D, action potential recorded after adding 105g/ml. TTX. E,
superimposed spikes shown in A and C displayed at faster sweep speed. Note reduction
in rise time after TTX and increased threshold. F, same records as E at slower sweep speed,
demonstrating that the ADP and AHP are unaffected by TTX.
Following the classical assumptions that action potentials are generated by either
Na or Ca conductances (Hodgkin & Huxley, 1952; Hagiwara, 1973), experiments
were designed to determine the contribution of these ions to the I.o. spikes
Initially action potentials were evoked in normal Ringer solution (Fig. 4A and B)
and then Na was replaced with tris or choline, or tetrodotoxin (TTX) at 1O-5 g/ml.
was added to the Ringer solution (Llinas & Sugimori, 1980a). All these manipulations
produce a blockage of the early component of the action potential (Fig. 4C). This
is particularly clear in Fig. 4E where the superposition of the two action potentials
allows a direct comparison of their rise time and firing level. These records show that
the Na conductance, which underlies the initial component of the action potential,
has a lower threshold and faster rise time than the TTX-resistant component (see
Table 1). The complementary experiment, that is the removal of the late component
of the action potential, was obtained by eliminating the Ca current either by removal
of Ca from the Ringer solution or by blocking Ca conductance as will be described
in detail below. In this case, as shown in Fig. 5B and D, the ADP and the long AHP
were blocked leaving only the early Na-dependent component. Reduction in the
amplitude of this spike was observed in most experiments and probably relates to
the decreased excitability of the neurone in the presence of an increased divalent ion
INFERIOR OLI VAR Y CELLS IN VITRO
557
concentration (Frankenhaeuser & Hodgkin, 1957). In short, the initial part of the
i.o. action potential is produced by a transient increase in the Na conductance and
resembles very closely the action potentials seen in other central neurones (cf. Eccles,
1964).
The high-threshold Ca action potential
Since, as described above, elimination of the Na current does not alter most of the
i.o. action potential, the properties of this remaining spike were analysed independently from Na electroresponsiveness. Direct depolarization of i.o. neurones under
A
B
C
Mn2+
D
Cd2+
20 msec
Fig. 5. Effect of Mn and Cd. A and C, control. In B the Ca has been replaced by Mn (2 mM).
In D, 1 mM-Cd has been added to the normal solution. In both experiments the ADP and
the AHP disappeared and the Na-dependent spike is revealed.
these conditions produces a large depolarization showing very little change from the
control ADP (Fig. 4C and D). Note, however, that as stated above the level of
depolarization required to activate this potential is higher than in the presence of
the Na spike, indicating that in normal Ringer solution the Na component plays an
important role in activating the ADP-AHP components of the action potential. The
rather slow rate of rise contrasts clearly with the control spike and indicates that this
late action potential, besides having a higher threshold, is generated by a conductance
having slower onset kinetics and/or an origin remote from the impalement site.
Detailed analysis of the ADP indicates that it is composed of many all-or-none
components which can be separated by hyperpolarizing the cell (as shown in Fig. 1 E).
As mentioned above, the ADP is studded with small wavelets which in part reflect
axonal events in accordance with the well-known fact that I.o. cells often generate
R. LLINAS AND Y. YAROM
spike trains that can be observed as multiple excitatory post-synaptic potentials at
the Purkinje cell level (Eccles et al. 1966; Llinas & Volkind, 1973). On the other hand,
some of these potentials are TTX-resistant (see Fig. 7), indicating a Ca dependence.
This multi-component property supports the conclusion that the ADP is generated,
as in other neurones, in a non-continuous manner at dendritic level, where each
dendrite may contribute one or several portions of the potential.
As reported above, the third component of the i.o. spike, the AHP, is also unaltered
by blockage of Na conductance (gNa). Furthermore, as indicated in Fig. 1 E and F, the
amplitude of this rather long-lasting potential is directly related to the amplitude
and duration of the ADP. By analogy with other systems (Meech & Standen, 1975;
Heyer & Lux, 1976a, b; Barrett & Barrett, 1976; Meech, 1978; Hoston & Prince,
1980), the most likely explanation for this hyperpolarization is that the AHP is
produced by a Ca-dependent K conductance. This possibility was tested by replacing
extracellular Ca with Mn (Fig. 5B). This results in a disappearance of the ADP and
the AHP which follows (Fig. 5B). Indeed, as described above, all that remains after
the Ca blockage is the fast Na-dependent action potential which is followed by a small
AHP lasting for a shorter period. A similar finding was obtained after the addition
oft mM-Cd to the bathing solution (compare Fig. 5 C and D). Mn and Cd were selected
because they are known to block voltage-dependent Ca conductances in other cells
(Baker, Hodgkin & Ridgway, 1971; Hagiwara, 1973; Kostyuk & Krishtal, 1977).
Similar findings were obtained by removing Ca altogether or by adding Co or D600
to the medium.
558
Low-threshold Ca electroresponsiveness following membrane hyperpolarization
A rather unexpected finding was the fact that membrane excitability in I.O cells is
increased by both d.c. membrane depolarization and hyperpolarization. Indeed, if
subthreshold synaptic or direct stimulation is superimposed upon a depolarized or
hyperpolarized membrane, the previously subthreshold stimuli will now elicit a
regenerative response. Such an experiment is shown in Fig. 6. Thus subthreshold
depolarization by a short current pulse (Fig. 6B) will add with a maintained d.c.
depolarization to generate the full action potential (Fig. 6A) described above.
Furthermore, d.c. hyperpolarization of the cell (as shown in C) will also produce an
increase in excitability, as demonstrated by the generation of an action potential by
the previously subthreshold current pulse. This increased excitability is present
regardless of the fact that, as evident in C, the input resistance of the cell is reduced
during membrane hyperpolarization due to anomalous rectification. Note that under
both conditions the Na spikes are quite similar in amplitude and duration, but the
depolarization which follows in C is much smaller, its falling phase slower, and its
AHP reduced. This reduction in AHP is probably due to both a decrease in the K
driving force and to a reduction in the magnitude of the Ca action potential.
A similar set of records obtained from another cell following synaptic activation
may be seen in Fig. 6D, E and F. It is important to emphasize at this stage that
the d.c. voltage requirement for the occurrence of these changes in membrane
excitability is quite small in both the depolarizing and hyperpolarizing directions.
Depolarizations of 5-6 mV from rest, added to the current pulse, will activate the
large Na-Ca spike typical of the I.o. cell. Also, hyperpolarizations of 6-8 mV from
INFERIOR OLI VAR Y CELLS IN VITRO
559
D
A
0
L--
B
E
C
F
-
/_-_
15
-----------------------------------
15 mV
mV
nA
I_
nA
30 msec
20 msec
Fig. 6. Effect of membrane potential on excitability of i.o. neurones. A to C, direct
stimulation of cell by curreent pulse. D to F, orthodromic stimulation. B and E, at resting
potential both stimuli are subthreshold for spike initiation. A and D, 4 mV membrane
depolarization. The same stimuli as in B and E now elicit a typical olivary response. C
and F, 6 and 4 mV hyperpolarization respectively. The same stimuli as in B and E elicit
a new type of response. Note that the ADP and AHP are smaller in C and F than in A
and D, and that during hyperpolarization there is a reduction in input resistance. (Resting
membrane potential is given by continuous line.)
I
TTX
A
J1
c
-
I
F
ALL',
_
25 msec
120 mV
nA
1 0 msec
Fig. 7. Effect of Co and TTX on two types of i.o. action potentials. A to C, control,
dependence of action potentials on membrane potential level. D to F, effect of Co. Note
that both high-threshold (D) and low-threshold (F) Ca spikes are blocked. G to I, effect
of TTX. Fast spike components (A and C) are blocked and both types of Ca spikes remain.
(Resting membrane potential is given by continuous line.)
R. LLMNAS AND Y. YAROM
560
rest are sufficient to modify the excitability of the cell such that a depolarization
which does not exceed the previous resting potential can activate the cell (Fig. 6C).
When voltage-dependent Ca conductance change is modified by the addition of Co
or Cd (Fig. 7), or by the removal of Ca from the perfusion fluid, only the Na-dependent
action potential remains. In addition, the electroresponsiveness produced following
d.c. hyperpolarization is also lost (Fig. 7 F), indicating that the initial potential
obtained following membrane hyperpolarization is carried by Ca ions. (This spike will
be referred to henceforth as a low-threshold Ca spike.)
In a second set of experiments, TTX was added to the bath. Under these conditions,
as shown in Fig. 7 C-I, the membrane electroresponsiveness remained in both the
depolarized and the hyperpolarized condition. Furthermore, the firing level for the
low-threshold Ca spike was lower than that for the Na spike (compare E and I). This
indicates that the spike in C was brought about initially by the low-threshold Ca spike
which then triggered the Na action potential.
It is clear that the rise times of the Ca-dependent spikes obtained from hyperpolarized and depolarized levels are different, as are also the amplitude of the AHPs
and the speeds of their falling phase. Furthermore, while the high-threshold Ca spike
is composed of several all-or-none components, the low-threshold spike does not show
such fractionation, suggesting it is generated at a single site. As will be seen in the
accompanying paper (Llinas & Yarom, 1981), these two Ca-dependent spikes seem
to be generated by different sets of voltage-dependent Ca conductances distributed
differently over the cell surface.
Electrotonic coupling between inferior olivary neuroses
In a previous set of papers it was suggested from indirect evidence that the short
latency depolarization (SLD) which may be seen in I.o. cells following antidromic
invasion is produced by electrotonic coupling (Llinis et al. 1974). Such coupling was
assumed to occur between dendrites where distinct gap junctions are observed
(Sotelo, Llina's & Baker, 1974). Two paradigms were used in the present study to test
the physiological findings. As illustrated in Fig. 8A, antidromic stimulation of an i.o.
cell generates the typical i.o. action potential. In B hyperpolarization of the cell (via
a short inward pulse) revealed a graded type of depolarizing potential generated by
a mixture of electrotonic coupling, Ca electroresponsiveness, and chemical synaptic
transmission. In D, E and F, a similar set of records was obtained from the same
cell following the addition of Mn to the bath. This ion blocks both types of Ca action
potentials and also chemical synaptic transmission. Note that the SLD can be clearly
seen in the absence of Ca spikes or chemical transmission, indicating direct charge
transfer between I.o. neurones.
A more direct demonstration of electrotonic coupling is given in Fig. 9, where
simultaneous recordings from a pair of neighbouring neurones show non-rectifying
electrotonic coupling. The neurone pairs demonstrating coupling were generally
10-100,m apart. These spikes could he independently activated by white matter
stimulation. In C the generation of an action potential in cell a produced a clear
depolarization in cell b. The depolarization was generated across the membrane in
cell b since after removing the micro-electrode from this cell, no potential could be
observed when cell a was activated. A second approach to this problem is illustrated
INFERIOR OLI VARY CELLS IN VITRO
D
A
120 mV
E
B
FI
-~
, |20mV
40 msec
i10 mV
CF
20 msec
Fig. 8. Mn-resistant subthreshold potentials elicited by peri-olivary white matter stimulation. A to C, control. In B hyperpolarization reveals subthreshold responses. C, same
records as B at high gain and faster sweep speed. D to F, records obtained as in A-C,
after addition of 5 mM-Mn to bath. Note persistence of graded response (E, F) indicating
electrotonic coupling of i.o. neurones.
A
A
0
B
D
I20 mV
C )
a
b b
a
b
|
~~E
I20mV b
1
t
-~~~
|1
nA
1
10mV
5mV
25 msec
Fig. 9. Electrotonic coupling between I.o. neurones. A, diagram of neurones to indicate
possible site of dendritic coupling (arrow). B, action potentials obtained from two
simultaneously recorded neurones following peri-olivary white matter stimulation. C,
direct stimulation of cell a elicits typical olivary spike in a and short-latency response in
b. D and E, second cell pair. Negative (D) or positive (E) current pulse in cell a elicits
hyperpolarization (D) or depolarization (E) of both cells. Note decreased amplitude and
time course of response of cell b. Last trace in E was recorded after removing electrode
from cell b only.
561
R. LLINAS AND Y. YAROM
562
in Fig. 9D and E in another cell pair. In this case direct hyperpolarization of one
cell (a) generated a hyperpolarization in a second cell (b). As expected from the
electrotonic spread of this potential, the amplitude in cell b was much smaller than
that in cell a and the time course was slower. However, there was no obvious delay
between the two potentials. A reversal of the direction of current injection produced
a depolarization of both cells, indicating non-rectifying coupling between these
neurones. The absence of a response following removal of the electrode from cell b
(bottom trace in Fig. 9E) showed that the previously recorded potentials were
generated across the membrane of cell b. A more detailed analysis of the electrotonic
coupling and its possible modulation by synaptic input will be considered in a later
publication.
DISCUSSION
The data presented above illustrate the types ofresults which may be obtained from
mammalian brain stem in vitro. This is in itself encouraging since obtaining stable
recordings from this portion of the mammalian central nervous system (c.N.s.) has
been technically rather difficult. Indeed, protracted-recordings from the inferior olive
and from vagal motoneurones (Yarom, Sugimori & Llinas, 1980) suggest that most
of the brain stem may be successfully studied using the slice preparation.
In general terms the viability of the brain stem slice is as good as that of the
cerebellum or hippocampus slice and permits, because of its sturdiness and lack of
obvious stability problems, such approaches as simultaneous recording from two or
more neurones. Moreover, comparing the degree of difficulty in obtaining satisfactory
intracellular recordings in vivo with the present experience indicates that the in vitro
technique is a vastly superior approach to the study of the biophysical properties of
these cells. Indeed, intracellular recordings could be maintained for as long as 4 hr,
which is often necessary in testing the influence of various ionic milieux on cell
function or when determining the pharmacological properties of given drugs.
General electrophysiology
The most outstanding feature of the electrical properties of i.o. neurones is the
complexity of their action potentials. In contrast to other neurones in the C.N.s.,
antidromic or orthodromic activation of the i.o. cell at rest generates, in addition to
the usual fast Na action potential, a prolonged ADP-AHP complex. These afterpotentials underlie the rhythmic firing properties described for olivary cells (Armstrong & Harvey, 1966; Armstrong et at. 1968; Llinas & Volkind, 1973; de Montigny
& Lamarre, 1973; Lamarre & Puil, 1974; Headley, Lodge & Duggan, 1976). The Ca
action potential (which follows the initial Na spike), triggers a Ca-dependent K
conductance and thus this neurone tends to oscillate at a rather low frequency,
usually 4-10/sec. While the manner in which these different ionic conductances
interlock to generate this rhythmic behaviour will be treated in the accompanying
paper, the three conductances described here represent the most salient electrophysiological properties of these neurones.
INFERIOR OLI VARY CELLS IN VITRO
563
Na action potentials
Activation of the Na action potential in the absence of a voltage-dependent Ca
conductance was observed following the blockage of Ca conductance with Mn, Co,
Cd, D600 or by simply replacing Ca with Mn. The properties of this Na spike
were indeed quite similar to those in other C.N.S. cells. Thus, the action potential had
a rapid rate of rise, lasted for about 1 msec, was followed by a short AHP and could
be blocked by TTX. As far as Na conductance is concerned, therefore, the i.o. neurone
seems quite similar to other C.N.s. neurones.
Ca conductance
High-threshold Ca spike. Because the prolonged ADP which follows the initial Na
spike is eliminated by the addition of agents known to block Ca conductances, and
because it requires the presence of extracellular Ca for its generation, we conclude
that it must be generated by an inward Ca current. As will be seen in the
accompanying paper (Llinas & Yarom, 1981), this Ca current is most probably
restricted to the dendrites of the i.o. cell, in keeping with a similar distribution of
Ca conductances in both cerebellar Purkinje cells (Llinas & Sugimori, 1978, 1980a)
and hippocampal pyramidal cells (Schwartzkroin & Slawsky, 1977; Wong et al. 1979).
The high-threshold Ca-dependent spike is best observed following blockage of Na
current. As shown in Fig. 4, under these circumstances direct depolarization of the
i.o. neurone to an appropriate level (30 mV from rest) produces an action potential
very similar to the ADP seen in the presence of a Na conductance. Because this
response requires a larger depolarization than is necessary to activate the Na spike,
the term 'high-threshold' is used. This finding implies that the initial Na spike serves
as an electrical stimulus to this response and that this conductance is probably
located remote from the recording point.
Because the ADP-AHP complex shows no obvious change following Na blockage
it must be concluded that the high-threshold Ca conductance, besides producing a
prolonged ADP, serves to activate a very powerful and long-lasting conductance
change, most probably to K ions. Since the termination of the AHP is generally
abrupt, the presence of an active repolarizing phenomenon, akin to the post-anodal
exaltation observed in nerve and in other central neurones (Purpura, 1967), must
operate in i.o. neurones. In the present case, this post-anodal exaltation is produced
by a rapid increase in Ca conductance which is normally inactivated at rest.
Low-threshold Ca conductance change following hyperpolarization. A conductance
change not seen previously in either vertebrate or invertebrate neurones is that
generating the rebound Ca potential observed in these cells. Thus, following membrane
hyperpolarizations above 70 mV (Llina's & Yarom, 1981), i.o. cells may produce
Ca-dependent action potentials following direct or synaptic stimulation. The time
course of this Ca spike is normally of the order of 20-25 msec, has a fast rate of rise
and a slow decay. In the hyperpolarized cell this spike has the lowest threshold and
normally activates a Na spike which in turn generates the dendritic Ca and K
response. This Ca conductance, which is inactivated at rest membrane potential, is
de-inactivated by hyperpolarizing the membrane, very much as described for the K
A current in invertebrates (Hagiwara, Kusano & Saito, 1961; Connor & Stevens,
564
R. LLINAS AND Y. YAROM
1971). In contrast to the ADP, this Ca spike generates a sharp negative wave
extracellularly (Llina's & Yarom, 1980), indicating a somatic localization in accordance
with the rather low threshold. The implications of the coupling between dendritic
Ca-K and the low-threshold somatic Ca conductance will be discussed in the
accompanying paper in relation to other central neurones capable of oscillatory
behaviour.
Chemical and electrical synaptic transmission
Chemical transmission. The present in vitro approach has allowed the demonstration
of both chemical and electrical synaptic input to the i.o. neurones. Unfortunately,
the peri-olivary stimulation which evokes chemical synaptic transmission does not
permit separation of excitatory and inhibitory synaptic afferents to these cells. As
seen in Figs. 2 and 3, synaptic potentials capable of reaching threshold for spike
generation are easily obtained. However, in reversing these potentials with current
injections it became clear that, due to the low reversing level (-30 to -20 mV), the
synaptic potential must be a mixture of excitatory and inhibitory inputs. Probably
a different plane of slicing or the utilization of the en bloc brain stem approach (Yarom
& Llinas, 1979; Llinas, Yarom & Sugimori, 1980) will be required to determine the
site of origin for these synaptic inputs and to allow the separation of the excitatory
and inhibitory synapses to the neurones.
Electrical transmission. The in vitro preparation has allowed a direct demonstration
of electrical coupling between neurones in the mammalian C.N.S. Indeed, while
indirect results were obtained in these neurones (Llinas et al. 1974) and in other
regions of the mammalian brain (Baker & Llinas, 1971; Korn, Sotelo & Crepel, 1973),
this is the first successful demonstration of electrotonic synaptic transmission in
mammals using dual simultaneous penetrations. Coupling could be demonstrated in
only 10 % of the cell pairs studied (six out of sixty). We suspect this low yield is due
to (a) the species studied (the number of gap junctions in guinea-pig is small compared
with, for instance, that in rat (C. Sotelo, personal communication), and (b) a possible
reduction in the total number of coupled neurones due to uncoupling by either
increased intracellular Ca or pH (Rose & Loewenstein, 1975; Baux, Simmoneau, Tauc
& Segundo, 1978; Stern, Spray, Harris & Bennett, 1980) following neuronal damage
during slicing. Nevertheless, in those cases where electrotonic coupling was observed,
both an action potential (Fig. 9B and C) and direct depolarization or hyperpolarization
of one of the cells (Fig. 9D and E) produced a distinct potential in the coupled
neurone. Because of the uncertainty produced by the bridge circuit, the coupling
coefficient (Bennett, 1966) cannot be properly determined. It is clear nevertheless that
in the observed pairs the coupling was approximately 0-25, somewhat in keeping with
coupling coefficients found in lower vertebrates (Bennett, 1966).
Amongst the properties of the coupling which we aimed to study was its possible
modification by synaptic input. This research was very much hampered by the
difficulty of distinguishing the contribution of a change in resting membrane
resistance from the possible shunting of the coupling site. However, since the coupling
sites are enveloped by synaptic terminals (Sotelo et al. 1974; King, 1976), it is clear
that such a possibility must be kept in mind in future studies (Spira & Bennett, 1972;
Llinas, 1974).
INFERIOR OLI VARY CELLS IN VITRO
565
In conclusion then, i.o. neurones are capable of generating Na action potentials
and two classes of Ca-activated spikes, and are electrotonically coupled.
Research was supported by United States Public Health Service program grant NS-13742 from
the National Institute of Neurological and Communicative Disorders and Stroke.
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The Journal of Physiology, Vol. 315
Plate 1
-:'
R. LLINAS AND Y. YAROM
N. I...
(Facing p. 567)
INFERIOR OLI VARY CELLS IN VITRO
567
EXPLANATION OF PLATE
Light micrographs of brain stem slice and horseradish peroxidase (HRP)-filled i.o. neurones. A,
350psm thick parasagittal section of guinea-pig brain stem stained with cresyl violet. Inferior olivary
nucleus is clearly discernible near ventral surface of brain stem rostral to cervical spinal cord.
Arrowhead indicates area of stimulation. Most recordings were obtained from caudal region of the
nucleus. B, two olivary neurones stained following intracellular injection with HRP.