Department of Neurobiology, State University of New York at Stony Brook, Stony Brook, New York 11794
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ABSTRACT |
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Ramcharan, E. J., C. L. Cox, X. J. Zhan, S. M. Sherman, and J. W. Gnadt. Cellular Mechanisms Underlying Activity Patterns in the Monkey Thalamus During Visual Behavior. J. Neurophysiol. 84: 1982-1987, 2000. We show for the first time with in vitro recording that burst firing in thalamic relay cells of the monkey is evoked by activation of voltage-dependent, low threshold Ca2+ spikes (LTSs), as has been described in other mammals. Due to variations in LTS amplitude, the number of action potentials evoked by an LTS could vary between 1 and 8. These data confirm the presence of two modes of firing in the monkey for thalamic relay cells, tonic and burst, the latter related to the activation of LTSs. With these details of the cellular processes underlying burst firing, we could account for many of the firing patterns we recorded from the lateral geniculate nucleus of the thalamus in behaving monkeys. In particular, we found clear evidence of burst firing during alert wakefulness, which had been thought to occur only during sleep or certain pathological states. This makes it likely that the burst firing seen in awake humans has the same cellular basis of LTSs, and this supports previous suggestions that burst firing represents an important relay mode for visual processing.
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INTRODUCTION |
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From studies of rodents and
carnivores, we know that thalamic relay cells respond in one of
two modes: tonic and burst. In tonic mode, a
cell's firing rate fairly faithfully and linearly reflects the
amplitude and duration of an excitatory input. In contrast, burst
firing is characterized by high-frequency clusters of 10 action
potentials and represents a less linear relay of its input. The switch
in firing modes is dependent on the inactivation state of a
voltage-dependent, transient, low threshold Ca2+
current known as IT (Bal et al. 1995
; Crunelli
et al. 1989
; Deschênes et al. 1984
; Jahnsen and Llinàs
1984a
,b
). At depolarized membrane potentials, IT
is inactivated and the neuron responds in tonic mode. Hyperpolarization
maintained for
50-100 ms removes this inactivation, and a
sufficiently large depolarizing current will then activate
IT. This, in turn, leads to a large, triangular, all-or-none Ca2+ spike that typically evokes a
burst of action potentials riding its crest.
Although bursting tends to be rhythmic at 1-10 Hz during
slow wave sleep or certain pathological states, like epilepsy
(Steriade et al. 1993), recent studies in cats indicate
that bursting appears arrhythmically during alert wakefulness and may
provide an important form of information transfer to cortex that is
less linear than tonic firing, but provides better signal
detectability. In this regard, burst firing could provide a sort of
"wake-up call" to cortex signaling some potentially important
change in the environment. Evidence for burst mode firing
during wakefulness has now been reported for monkeys and humans
(Lenz et al. 1998
; Radhakrishnan et al.
1999
; Ramcharan et al. 2000
; Tsoukatos et
al. 1997
). Using in vitro recordings, we sought here to confirm
at a cellular level that burst-like activity recorded extracellularly
from thalamic relay cells in behaving monkeys also is related to a
mechanism involving IT.
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METHODS |
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recordings in thalamic slices.
Intracellular recordings were obtained in vitro from relay
neurons of dorsal thalamus and the lateral geniculate nucleus of adult
and juvenile macaque monkeys (2 rhesus and 3 cynomologus) in compliance
with approved animal protocols (e.g., the National Institutes of Health
Guide for the Care and Use of Laboratory Animals). Briefly, animals
were deeply anesthetized with pentobarbital sodium and a block of
tissue containing the dorsal thalamus was removed, sliced (400-500
µm), stabilized, and prepared for intracellular recording according
to standard protocols (Cox and Sherman 1999). Intracellular recordings were made in current clamp mode using fine
tipped recording pipettes (40-100 M
filled with 1 M KAc and 2-3%
neurobiotin). An Axoclamp 2A amplifier (Axon Instruments, Foster City,
CA) was used in bridge mode to record voltage signals. Throughout the
recordings, an active bridge circuit was adjusted to balance the drop
in potential produced by passing current through the recording
electrode. Apparent input resistance was calculated from the slope of
the linear portion of the current/voltage relationship. In some
experiments, the Na+ channel blocker tetrodotoxin
(TTX; 1µM) was added to the bath to block action potentials.
Following the recording, the slices were placed in 4% paraformaldehyde
for overnight fixation, and standard protocols were used to reveal the
neurobiotin and assess the morphology of the labeled cell with the
light microscope.
recordings in the behaving monkey.
All in vivo experimental procedures were carried out according
to standard protocols (e.g., Gnadt and Mays 1995).
Chronic transcranial recording cylinders and scleral eye coils for
recording eye position were surgically implanted under general
anesthesia followed by several weeks recovery time. Eye movements were
recorded relative to a fixed head position. As inducement to work, the daily intake of water was restricted to that earned as reward for
successful completion of fixation trials. Each session, subjects were
allowed to work to satiation. Tungsten microelectrodes (0.5-1.0 M
)
were lowered through the dura mater into the thalamus. Individual spikes, monitored on an oscilloscope and isolated through a
time/amplitude window discriminator, were recorded by computer at a
sampling time of 0.1 ms while eye position was monitored with a
resolution of 2 ms temporally and 0.2° spatially.
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RESULTS |
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Properties of thalamic neurons in vitro
We recorded intracellularly from 17 neurons in an in vitro slice
preparation of the monkey thalamus; 7 were from the lateral geniculate
nucleus and 10 from other, unspecified dorsal thalamic nuclei. All
neurons exhibited a resting membrane potential more negative than 50
mV (
62.1 ± 6.1 mV; mean ± SD) and overshooting action
potentials, and the input resistance of the population was 26.5 ± 14.2 M
. Several of these neurons were injected with neurobiotin
following recording, and three well stained cells were recovered
histologically (see Fig. 2A).
Figure 1A shows that
hyperpolarizing current pulses injected into a geniculate neuron
produced a long latency depolarizing sag in the voltage trace. This is
also seen in thalamic cells of rodents and cats, where the sag is due
to activation of the hyperpolarization-activated mixed cation
conductance, Ih. At the conclusion of the
hyperpolarizing current step, the cell passively repolarized, and this
led to a short, high-frequency burst of action potentials. Depolarizing
current pulses from the resting membrane potential of 63 mV produced
an ohmic response for smaller, subthreshold pulses, and with increasing
stimulus intensity produced tonic firing (Fig. 1A). When the
membrane potential of this cell was held at the more hyperpolarized
level of
92 mV, depolarizing current pulses evoked a burst of action
potentials (Fig. 1A). Similar properties were observed in
recordings from other thalamic nuclei as well (e.g., Fig.
1B). Thus we see that thalamic neurons in the monkey can
discharge action potentials in both burst and tonic firing modes, and
that the burst response requires initial hyperpolarization of the
resting membrane potential.
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In other mammalian species, burst firing is clearly
voltage-dependent, requiring a relatively hyperpolarized membrane
potential to de-inactivate IT before a low
threshold Ca2+ spike (LTS) can be activated.
Figure 1C, taken from another geniculate neuron, shows this
sort of voltage dependency for IT and the
LTS. From a depolarized membrane potential of 67 mV at which
IT is inactivated, a depolarizing pulse
evokes tonic firing. With initial hyperpolarization of the membrane
potential to
72,
76, and
82 mV, subsequent depolarizing current
pulses evoked a characteristic LTS on which a high-frequency burst of
action potentials rode. In addition, as has been documented for
geniculate cells in the cat (Zhan et al. 2000
), the
number of action potentials per burst is related to the initial
membrane potential: the more hyperpolarized this potential, the more
IT is de-inactivated, leading to a larger evoked LTS, which in turn evokes a larger burst of action potentials. Figure 1D summarizes this relationship. It is important to
emphasize here that the relatively small LTS evoked from less
hyperpolarized membrane potentials may activate a single action
potential (e.g., the
72 mV example of Fig. 1C).
Figure 2 shows data from another
geniculate relay neuron that was labeled with neurobiotin after
recording. The camera-lucida reconstruction (Fig. 2A) shows
soma and dendritic morphology characteristic of thalamic relay cells
described in other species. At resting levels, this cell responded to
depolarizing current pulses with tonic firing but did produce burst
firing in response to the termination of hyperpolarizing pulses (Fig.
2B, Control). TTX was then added to block action
potentials and thus reveal the LTS more clearly (Fig. 2B,
TTX). Figure 2C shows that the amplitude of the
LTS depends on the initial membrane potential (cf. Fig. 1, C
and D). At 57 mV, only an ohmic response is seen to a
depolarizing step, because IT is largely
inactivated at this membrane potential. However, from relatively
hyperpolarized membrane potentials (
61,
76, and
83 mV), adequate
depolarizing current steps evoke LTSs. There are two points to note
about the LTSs evoked in Fig. 2C: 1) amplitude is
greater for LTSs activated from more hyperpolarized initial membrane
potentials; and 2) for any given initial membrane potential,
the sizes of LTSs evoked are fairly constant. The latter point
indicates that these LTSs are activated in a nearly "all-or-none" manner, as has been shown for thalamic relay cells in the cat (Zhan et al. 1999
). The experiment shown in Fig.
2C was also performed on this cell before TTX was applied
(data not shown), and Fig. 2D summarizes the dependence on
initial membrane potential of both LTS amplitude (
) and number of
action potentials in a burst (
). These data clearly indicate that
the number of action potentials in a burst depends on the magnitude of
the LTS.
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Properties of thalamic neurons in the behaving monkey
We studied a total of 21 geniculate neurons in vivo (9 magnocellular and 12 parvocellular). For all in vivo recordings
illustrated, the monkey actively fixated a small spot. During fixation,
we quickly plotted the neuronal receptive field manually. All cells reported here were located 10-15° from the fovea. We then applied a
computer-generated, drifting sinusoidal grating onto the receptive field. Figure 3 shows data from three
representative neurons in response to the grating. For each cell, we
show a scatter plot of time intervals before each action potential
versus the corresponding interval after each spike. To interpret the
scatter plots, it is useful to keep in mind the temporal constraints
for IT: de-inactivation of
IT follows a complex function of time and
membrane potential (Lu et al. 1992) which generally
requires
50-100 ms of hyperpolarization. Consequently, a silent
period precedes each LTS. On the other hand, tonic firing has no such
timing constraints since a tonic action potential can follow or precede
another by any interval greater than a refractory period of ~1 ms.
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As we have shown previously (Ramcharan et al. 2000),
there are often three distinct clusters in the scatter plots for
geniculate relay cells. The large, middle cluster of data points
reflects tonic firing. A second cluster is a horizontal strip of points with postspike intervals
100 ms. Many of these points represent the
last action potential before an LTS mediated burst. The third and
particularly interesting cluster appears as a vertical band with
prespike intervals
100 ms. Studies of cat geniculate cells have shown
that virtually all action potentials with a prespike interval
100 ms
and a postspike interval
4 ms are the first action potential in a
burst, with all subsequent action potentials having a postspike
interval
4 ms being other members of the burst (Lu et al.
1992
). However, these criteria are conservative and misidentify
as tonic firing many action potentials that occur in bursts. We had
suggested elsewhere (Ramcharan et al. 2000
) that many of
these spikes in the third cluster may represent LTSs that activate a
single action potential. We have now confirmed in vitro that LTSs
indeed may activate a single action potential following a prespike
hyperpolarization, which could fall anywhere in the vertical cluster
with a postspike interval >4 ms. We cannot be certain from the
extracellular recording employed in vivo whether these action
potentials represent single spikes riding a small LTS, tonic firing, or
a mixture, but the point here is that they have a similar functional
implication for signal detection and thalamocortical transmission.
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DISCUSSION |
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The vertical band of action potentials following a preceding one
by 100 ms has at least two functional correlates. First, the
appearance of the preceding silent period indicates that the action
potential appears on a background of very low spontaneous activity and
will thus be more readily detectable than action potentials in the
broad cluster of tonic firing that occur on a background of higher
spontaneous activity (Guido et al. 1995
; Sherman
1996
). Second, thalamocortical synapses appear to have the
property of paired-pulse depression (Agmon and
Connors 1992
; Stratford et al. 1996
). That is,
for an interval lasting for 10 s of ms, the second action
potential in a pair in a thalamocortical axon evokes a reduced
excitatory postsynaptic potential (EPSP) compared with that evoked by
the first. This means that action potentials in the vertical cluster,
appearing after a silent period long enough for paired-pulse depression
to wear off, will evoke a maximum EPSP, whereas those in the broad
central cluster will occur while the depression is still in force and
will thus activate a smaller EPSP.
We also point out that a single action potential riding an LTS is
functionally quite similar to a more traditional multiple spike burst.
As noted above, both occur after a requisite silent period and would
thus reflect comparable signal detectability (Guido et al.
1995; Sherman 1996
). Furthermore, paired-pulse
depression in the thalamocortical synapse implies that a single action
potential on an LTS and the first spike in a burst will evoke a
comparable EPSP, whereas succeeding action potentials in a burst will
evoke depressed EPSPs. Thus the overall postsynaptic response may not be so different for these two conditions of burst mode firing. Finally,
in terms of information theory, a burst behaves like a single event
regardless of how many action potentials it contains (Reinagel
et al. 1999
). In other words, the first spike of a burst contains most of the information and other spikes add little additional information. Thus the information carried by a single action potential riding an LTS is very similar to that carried by a burst.
Regardless of how much of the vertical cluster of action potentials in
Fig. 3 with prespike intervals 100 ms represents burst mode firing,
it should by now be abundantly clear both that there is clear evidence
of some burst mode firing during awake, alert behavior in thalamic
neurons of these monkeys and that this bursting is related to
IT. This conclusion is at odds with the
historic view that thalamic relay cells fire strictly in tonic mode
during wakefulness and burst mode during sleep (Livingstone and
Hubel 1981
; Steriade et al. 1993
). There is now
growing evidence that bursting can occur intermixed with tonic firing
in the awake rat (Nicolelis et al. 1995
), cat
(Guido and Weyand 1995
), monkey (Ramcharan et al.
2000
), and human (Lenz et al. 1998
;
Radhakrishnan et al. 1999
; Tsoukatos et al.
1997
). Studies of the lightly anesthetized cat indicate that
bursts can carry information roughly equivalent in magnitude to tonic
firing (Reinagel et al. 1999
), but that burst firing
provides better signal detectability while tonic firing provides a more
faithful relay of information. Given the close evolutionary
relationship of monkeys to humans and the close similarities in the
lateral geniculate nucleus between macaque monkeys and humans, these
data strongly suggest that burst firing seen in awake humans
(Lenz et al. 1998
; Radhakrishnan et al.
1999
; Tsoukatos et al. 1997
) is also the result
of activating IT.
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FOOTNOTES |
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Address for reprint requests: J. W. Gnadt, Dept. of Neurobiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5230 (E-mail: JGNADT{at}sunysb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 May 2000; accepted in final form 11 July 2000.
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REFERENCES |
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