406
297 3 June 1982
Third, growth cones in the periphery are not simply guided
by passive mechanisms. Active guidance mechanisms are sug-
gested because certain growth cones are confronted with several
possible choices and make cell-specific decisions of which way
to go. For example, the lE growth cones extend along the
epithelium until they near the 4B cell bodies. Rather than climb
onto the B pathway, they make a turn and grow along a tendon
to the 1D cells, thus completing the E pathway. In a similar
way, early motoneurone growth cones are confronted with the
B and C pathways, yet choose to grow laterally and pioneer
a new D pathway. The finding of specific choices made by
growth cones in the periphery is similar to the divergent
and specific choices made by identified growth cones in the
CNS 11 - 14.
Finally, we must re-evaluate the concept of 'pioneer
neurones'. Bate and Goodman have shown that other
peripheral nerves (such as the intersegmental nerve and the
median nerve) are pioneered by motoneurone growth cones
from neuroblast progeny in the CNS (manuscript in prep-
aration). Furthermore, pathways in the CNS are pioneered by
Received 30 November 1981; accepted 2 April 1982.
1. Harrison, R. G. J. exp. Zoo/. 9, 787-846 (1910).
2. Bate, C. M. Nature 21141, 54-56 (1976).
3. Keshishian, H. Devi Biol. 80, 388-397 (1980).
4. Keshishian, H. & Bentley, D. Soc. Neurosci. 7, 347 (1981).
5. Chang, S., Ho, R., Raper, J. A. & Goodman, C. S. Soc. Neurosci. 7,347 (1981).
6. Ho, R., Chang, S. & Goodman. C. S. Soc. Neurosci. 7, 348 (1981).
7. Goodman. C. S. & Spitzer, N. C. Nature 280, 208-214 (1979).
Electrophysiology of
mammalian thalamic neurones in vitro
RodoUo Llimis & Henrik Jahnsen
Department of Physiology and Biophysics,
New York University Medical Center,
550 First Avenue, New York, New York 10016, USA
Although much is known about the morphology and physiology
of thalamic neurones1, no information is available regarding
the ionic basis for the electrical excitabHity of these cells.
Furthermore, possible differences in the electrical properties
of the principal nerve cells in the various thalamic groups have
not been studied in sufficient detail to determine whether the
thalamus is, electrophysiologically, a unUorm set of neuronal
elements. Here we present evidence that thalamic neurones
have voltage-sensitive ionic conductances capable of generating
two distinct functional states-a repetitive and a burst-firing
mode. The neurones are switched from one state to the other
by membrane potential changes, each state being dominated
by different voltage-dependent ionic conductances. At mem-
brane potentials more positive than -60 mV, the neurones
respond to a depolarization with repetitive firing via Na+ -
dependent action potentials, whereas at potentials more nega-
tive than -65 mV, depolarization of the cell results in short
bursts of action potentials via an inactivating all-or-none Ca2 + -
dependent spike. This property, present in all the neurones
comprising the different thalamic nuclei, serves as the basis for
their oscillatory properties. Particularly, the inactivating Ca2 •
conductance represents the ionic basis for the post-anodal
exaltation, the mechanism most probably responsible for the
alpha rhythm.
The study was performed on 400-µ,m-thick thalamic slices
obtained by coronal section of guinea pig brain using a
vibratome cutting device (Oxford 0501 Vibratome). Typically,
six sections included most of the thalamus. The sections were
placed in a special lucite chamber under a flowing mammalian
Ringer's solution 2 • Electrophysiological analysis was perfor-
med, under direct microscopic observation, on neurones of the
anterior, medial and lateral thalamic nuclear groups, including
the lateral geniculate nucleus and neurones in the lamina
0028-0836/82/220406-03$01.00
Nature Vol.
progeny from two different classes of neuronal precursors-the
midline precursors and neuroblasts 12 ' 15 • Thus, there is no
specialized class of precursor cell which gives rise to cells having
the unique role of establishing axonal pathways throughout the
grasshopper embryo. Rather, neurones of diverse ectodermal
origin appear early in embryogenesis and pioneer pathways
when the distances are short and the terrain relatively simple.
This finding shifts the emphasis away from the special role
played by 'pioneers', and focuses our attention on the questions
of what cell-to-cell interactions and substrate features guide
the first peripheral growth cones. These questions can be
answered by manipulating the pioneer neurones, landmark
cells, and ectodermal epithelium in grasshopper embryo cul-
tures. In this way, we hope to establish by what mechanisms
these 'pioneering' growth cones are guided across short
distances of axon-less territory.
We thank Susannah Chang for the 1-5 antibody and Paul
Taghert and Michael Bastiani for the anti-LY antibody. This
work was supported by grants from the McKnight Foundation,
NSF, and Sloan Foundation to C.S.G.
8. Goodman. C. S., O'Shea, M., McCaman, R. E. & Spitzer. N. C. Science 204, 1219-1222
(1979).
9. Stewart. W.W. Cell 14, 741-759 (1978).
10. Taghert, P., Bastiani, M., Ho, R. K. & Goodman, C. S. Cell (submitted).
11. Raper, J. A. & Goodman, C. S. Soc. Neurosci. 7,347 (1981).
12. Goodman, C. S., Raper, J. A., Ho, R. & Chang, S. 40th Symp. Soc. dev. Biol. (in the press).
13. Raper, J. A., Bastiani, M. & Goodman, C. S. J. Nturosci. (in the press).
14. Raper, J. A., Bastiani, M. & Goodman, C. S. J. Neurosci. (in the press).
15. Bate, C. M. & Grunewald, E. B. J. Embryo/. Exp. Morphol. 61, 317-330 (1981).
interna. The techniques for sectioning and maintenance of the
slice have been described elsewhere 2 • Intracellular recordings
were made using potassium citrate-filled micropipettes having
a d.c. resistance of 50-90 Mn. Cells were activated either
antidromically or synaptically by means of bipolar stimulating
electrodes located on the midline region of the thalamic mass
(see arrow in Fig. lA) or directly through the microelectrode
using a bridge amplifier. Neurones were identified by their
antidromic and synaptic activation, and their thalamic site was
determined by direct observation. Although single somata were
often not visible, the exact outline of the groups of nuclei could
be easily determined (see Fig. lA). Intracellular recordings
from these neurones indicated that they could survive well for
>8 h after the slicing procedure. The stimulus amplitude
required for the antidromic activation of these cells (Fig. lB)
was often lower than that for the activation of synaptic inputs
(Figs lC, 2£, F). Considering the thickness of the slices, we
suspect that only monosynaptic connectivity can be reliably
studied in this material.
The electrical properties of the cells were examined by direct
stimulation via the recording electrode. As shown in Fig. 2A,
thalamic cells may be characterized by two distinct types of
electrical response. When the resting potential was more posi-
tive than -60 mV, thalamic neurones fired repetitively at
increasing frequencies with increasing amplitude of direct
depolarization. A plot of the instantaneous adapted firing
frequency, / (1 s pulse duration) against current injection (J)
is shown in Fig. 2C, which illustrates the graded nature of the
repetitive firing properties of these cells. However, in contrast
to other central neurones 3 , no primary firing range was obser-
ved: rather, the neurones fired with a typical minimal frequency
of -10 impulses s- 1 and reached firing frequencies of
165 impulsess- 1 • At this membrane potential level, we obser-
ved no after-depolarization comparable to that seen in
motoneurones4, and Purkinje2, inferior olivary5 and hippocam-
pal6 cells in vitro. On the other hand, when the cells had resting
potentials more negative than -60 mV, or were hyperpolarized
by direct inward current injections, rather than the continuous
firing, a single burst of action potentials was observed after a
step depolarization (see Fig. 2A, B). The lower record in Fig.
2A demonstrates a passive membrane depolarization produced
by a square current pulse that is subthreshold for spike initi-
ation. When, as shown in the upper record in Fig. 2A, the same
© 1982 Macmillan Journals Ltd
Nature Vol. 297 3 June 1982 407
Fig. 1 Neuronal localization in A B C
thalamic nuclei. A, photomicro-
graph of diencephalic slice (right)
and diagram illustrating the loca-
tion of thalamic nuclei (left). EL,
external medullary lamina; HN,
habenular nucleus; LG, late~al
geniculate nuclei; LN, lateral
nucleus; PN, parafascicular /2omv
nucleus; VN, ventral thalamic
nucleus; V, third ventricle. Arrow
indicates the location of the bipolar
stimulating electrode. B, anti-
v---
dromic invasion of VN cell after midline stimulation. C, synaptic activation from the same locus after increase in stimulus strength. The
voltage and time calibration are indicated.
current pulse was superimposed on a d.c. depolarization, the the amplitude of this response. With larger hyperpolarizations
cell fired repetitively. This is expected if the sum of the two the slow component reached the threshold for fast spike activa-
depolarizations reaches the firing level of the cell. However, tion. Note that the hyperpolarization had a slow recovery time
when the membrane potential was hyperpolarized from rest, suggesting the presence of an 'early Na conductance'. The
another type of electroresponsiveness was observed. Indeed, increased excitability that occurred with hyperpolarization was
the excitability of the cell was increased by membrane hyper- also demonstrated after synaptic activation. Figure 2E shows
polarization. Thus, single burst responses having an abrupt that smoothly graded excitatory synaptic potentials which
onset and a smooth falling phase were obtained by current reached the firing level were obtained in a thalamic neurone
pulses as in Fig. 2A when the cell was previously hyperpolarized having a resting potential of -60 mV after midline thalamic
by 7 mV (Fig. 2B). Note the marked difference between the stimulation of increasing amplitude. When the neurone was
firing level for the tonic firing (arrow in Fig. 2A) and that of hyperpolarized by 15 mV (Fig. 2F), the same synaptic activation
the burst response (arrow in Fig. 2B). The burst response had generated all-or-none slow depolarization, which in turn acti-
two distinct components: (1) a slowly rising all-or-none vated fast spikes. Note once again the different firing levels for
depolarization, the amplitude of which is directly related to the the activation of the slow response and the fast action potentials.
level of membrane hyperpolarization; and (2) one or more fast These two modes of electroresponsiveness were found to
spikes activated from the slow depolarization. have different ionic mechanisms as demonstrated by the addi-
Such a burst was also observed as a rebound response at the tion of specific ionic channel blockers to the bathing solution.
break of a hyperpolarizing current pulse (Fig. 2D). In these The fast action potentials, which seem to arise from the soma
conditions the two components could be easily separated. In and axon areas (judging from the extracellular negativity they
fact, with low-amplitude hyperpolarizing pulses only the slow generate) can be blocked by tetrodotoxin (TIX) indicating that
component was observed, emphasizing the graded nature of they are sodium-dependent8 • As shown in Fig. 2G, a step
Fig, 2 Electrophysiological and
pharmacological properties of
A B C
thalamic neurones. A, a subthresh-
JOO •••
••
old depolarization of the cell at
resting level (broken line) pro-
••
duces, after a d.c. depolarization,
repetitive firing of the cell during
the same current pulse. B, after •
d.c. hyperpolarization, similar cur- •
rent pulses as in A produce a single
high frequency spike burst. C, plot ••
of the instantaneous adapted ~ •
frequency (/) of the repetitive -1-------------- ' ~ j0.5nA
firing of the cells as seen in A for
different levels of current injection 50ms 0.5 1.0
(/). D, rebound burst response I (nA)
D E F
after hyperpolarizing pulses of
different amplitudes. E, in another
cell, excitatory synaptic potentials
obtained from rest show their
graded character and the firing
level of the neurone. F, synaptic
potentials of similar amplitude as
in E produce, following hyper- ____________ j20mv
polarization, long-lasting all-or-
none responses on which fast
spikes are generated at the same 20ms
firing level as in E. G, slow all-or- G H I
none response generating fast
spike, from a cell slightly hyper- TTX TTX+Co 2 +
polarized from rest potential. H,
after blockage of Na+ conductance
with TIX, the fast action potential
is blocked but the slow response
remains unchanged. I, addition of ltomv
CoC1 2 to the bathing solution
lo.5nA
abolishes completely the slow
response seen in H, even when the 20ms
current pulse is increased in ampli-
tude by 2.5 times.

© 1982 Nature Publishing Group

408
depolarization from a holding level of -65 m V produced a large
all-or-none depolarization and a secondary fast spike. Addition
of TIX to the bath at 1 µg mi- 1 completely blocked the fast
action potential, leaving the underlying all-or-none slow
depolarization unaltered (Fig. 2H). By contrast, addition of
Ca2 +-conductance blockers such as CoCh (ref. 9) or CdCh (ref.
10) to the bathing solution at a concentration of 1 mM, or
substitution of MnCh (3.5 mM) for CaCb, abolished this all-or-
none response (Fig. 2/). These results indicate that the slow
spike is generated by a voltage-dependent change. in Ca 2 +
conductance 1 1. Furthermore, the nature of this response is
similar to the low-threshold Ca2 +-dependent spikes described
in the inferior olive 5 •12 •
A more detailed study of the switching between a Ca 2 +-
dominated and a Na+ -dominated electroresponsiveness
revealed that this change occurs in almost an all-or-none man-
ner. Figure 3 shows that when the excitability of a cell was
challenged with a series of brief depolarizing pulses (of sufficient
amplitude to fire the cell), and simultaneously the membrane
A Furthermore, as observed in inferior olivary neurones 5 , these
\. iill IIOmV
d membrane potential level, the calcium conductance which gen-
B troresponsive properties observed in thalamic recordings in vivo
a b
IIOmV
lo.5nA
50'ms
Fig. 3 Voltage-dependent burst-to-tonic switching of thalamic
cell activity. A, response of a thalamic cell after short current
pulses delivered from a slowly rising ramp depolarization pulse.
The cell switched abruptly from a burst response to tonic firing
as the d.c. potential decreased by -10 mV from the initial value.
B, records obtained at a higher sweep speed at the times indicated
by a to d in A. Note the transition from burst response (a and
b) to tonic response (c), followed by the abrupt return to a burst
response (d).
© 1982 Nature Publishing Group
Nature Vol. 297 3 June 1982
potential was gradually decreased by a slow ramp depolarizing
current, the response to the pulse changed from the burst mode
to tonic firing. In this case the bursts had a duration of 30-
130 ms, and generated a set of fast action potentials at a
frequency of 150-320 impulses s-1, depending on the level of
membrane hyperpolarization. As the membrane was slowly
depolarized, the test pulses produced, rather than the burst, a
continuous repetitive spiking for the duration of the pulse
(compare b and c in Fig. 3B). The frequency of this repetitive
firing was related to the total level of depolarization as indicated
in the f-1 plot in Fig. 2C.
The mechanism for the switching between these two excitabil-
ity states was clarified by further studying the properties of the
Ca 2 +-dependent electroresponsiveness. As shown in Fig. 2D,
the amplitude and the rate of rise of this Ca 2 +-dependent
response varies with membrane potential in a very steep man-
ner. In addition, as in the case of the inferior olivary cells, these
responses completely inactivate at membrane potential levels
more positive than -60 mV. This voltage-dependent inactiva-
tion explains both the presence of burst responses when the
cell is hyperpolarized and the rebound activation of these cells.
rebound calcium spikes show a long refraction, indicating that
the kinetics of recovery from the inactivated state are slow.
This slow recovery also explains why such responses are unitary
as their activation is followed by a rather prolonged refractory
period of 80-150 ms.
In conclusion, these findings indicate that thalamic neurones
have special voltage-sensitive ionic conductance properties
which allow them to change from a phasic bursting response
(followed by neuronal silence) to graded repetitive firing, and
this switching is modulated by membrane potential. When the
cells have a resting level more negative than -60 mV, a low
threshold calcium response is obtained. The other integrative
state is characterized by tonic firing brought about by depolariz-
ation of the cell to levels more positive than -60 mV. At this
erates the slow response is inactivated and the cell fires repeti-
tively. Although the precise distribution of these conductances
over the soma-dendritic area of these neurones has not been
determined, by analogy with inferior olivary cells 5 · 12 , we suggest
that the Ca 2 +-deinactivated response probably resides at the
somatic level, as does the Na+-dependent action potential.
These electrical properties further explain many of the elec-
where such switching of tonic to phasic firing has in fact been
described 13 •14 • Moreover, in conjunction with the recurrent
inhibition observed in vivo, this newly described Ca 2 + conduct-
ance represents the functional basis of the so-called 'post-
anodal exaltation' underlying the recruiting responses 12 at cor-
tical level and most probably the a rhythm 15
This research was supported by USPHS grant NS13742 from
the National Institute of Neurological and Communicative
Disorders and Stroke. H.J. was supported in part by grants
from the University of Copenhagen, the Danish MRC and the
Weimann Foundation, and by an Albert Cass Travelling
Fellowship.
Received 21 January; accepted 29 March 1982.
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