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The Journal of Neuroscience, March 1, 2002, 22(5):1499-1512
Dendritic Calcium Encodes Striatal Neuron Output during
Up-States
Jason N. D.
Kerr and
Dietmar
Plenz
Unit of Neural Network Physiology, Laboratory of Systems
Neuroscience, National Institute of Mental Health, Bethesda, Maryland
20892
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ABSTRACT |
Striatal spiny projection neurons control basal ganglia outputs via
action potential bursts conveyed to the globus pallidus and substantia
nigra. Accordingly, burst activity in these neurons contributes
importantly to basal ganglia function and dysfunction. These bursts are
driven by multiple corticostriatal inputs that depolarize spiny
projection neurons from their resting potential of approximately 
85
mV, which is the down-state, to a subthreshold up-state of 55 mV. To
understand dendritic processing of bursts during up-states, changes in
intracellular calcium concentration ([Ca2+]i) were measured in
striatal spiny projection neurons from cortex-striatum-substantia nigra organotypic cultures grown for 5-6 weeks using somatic
whole-cell patch recording and Fura-2. During up-states,
[Ca2+]i transients at soma and
primary, secondary, and tertiary dendrites were highly correlated with
burst strength (i.e., the number of spontaneous action potentials).
During down-states, the action potentials evoked by somatic current
pulses elicited [Ca2+]i transients in
higher-order dendrites that were also correlated with burst strength.
Evoked bursts during up-states increased dendritic
[Ca2+]i transients supralinearly by
>200% compared with the down-state. In the presence of tetrodotoxin,
burst-like voltage commands failed to elicit
[Ca2+]i transients at higher-order
dendrites. Thus, dendritic [Ca2+]i
transients in spiny projection neurons encode somatic bursts supralinearly during up-states through active propagation of action potentials along dendrites. We suggest that this conveys information about the contribution of a spiny projection neuron to a basal ganglia
output specifically back to the corticostriatal synapses involved in
generating these outputs.
Key words:
action potential backpropagation; forward propagation; calcium; Fura-2; organotypic culture; spiny projection neuron; dendritic processing; up-state; down-state; cortex; striatum; substantia nigra; electrophysiology; imaging
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INTRODUCTION |
Spiny projection neurons in the
striatum play a major role in linking cortical activity to basal
ganglia outputs. Their dendrites receive the majority of basal ganglia
inputs from the cortex and their axons directly innervate basal ganglia
output neurons (for review, see Gerfen and Wilson, 1996
). Action
potential bursts in spiny projection neurons correlate with important
aspects of basal ganglia function, such as movement initiation and
regulation of ongoing movements (DeLong, 1973; Crutcher and DeLong,
1984; Hikosaka et al., 1989; Kimura et al., 1992; Jaeger et al., 1993). Furthermore, changes in spiny projection neuron firing correlate with
behavioral learning that relies on basal ganglia function (Tremblay et
al., 1998; Jog et al., 1999). Similarly, dysfunction of striatal
activity is directly linked to diseases of the basal ganglia (for
review, see Albin et al., 1989). Given the importance of this burst
activity in basal ganglia function, the question arises as to how
information about bursts is processed within these neurons.
Somatic bursts are critical for modification of glutamatergic synapses
in the cortex (Magee and Johnston, 1997; Markram et al., 1997) and are
likely to be important for modification of corticostriatal
glutamatergic synapses as well, which have been shown to be highly
plastic (Lovinger et al., 1993; Calabresi et al., 1994; Akopian et al.,
2000; Reynolds and Wickens, 2000; Kerr and Wickens, 2001). For
corticostriatal synapses to take advantage of information about somatic
bursts in spiny projection neurons, this information must be present in
the dendrites, which are the main targets of corticostriatal inputs.
One mechanism for conveying this information would involve changes in
dendritic [Ca2+]i
attributable to backpropagation of somatic action potentials into dendrites (for review, see Häusser et al., 2000).
Bursts in spiny projection neurons only occur during up-states, which
are signaled by a transition in intracellular membrane potential from
85 mV to a subthreshold range at approximately 55 mV. Up-states
occur in vivo (Wilson and Kawaguchi, 1996; Stern et al.,
1997; Reynolds and Wickens, 2000) and in striatal cultures receiving
cortical inputs (Plenz and Aertsen, 1996; Plenz and Kitai, 1998). They
are blocked by decortication (Wilson et al., 1983) or glutamate
antagonists (Plenz and Kitai, 1998) and therefore indicate critical
periods in dendritic processing that result from multiple
corticostriatal inputs.
The restriction that bursts only occur during up-states in spiny
projection neurons poses several potential problems for backpropagation of somatic action potentials. In cortical pyramidal neurons,
backpropagation occurs at rest in vitro (Stuart and Sakmann,
1994; Stuart et al., 1997b; Häusser et al., 2000) but can fail in
the presence of many synaptic inputs in vivo (Svoboda et
al., 1999). Striatal neurons receive numerous inhibitory inputs from
fast-spiking interneurons during up-states (Plenz and Kitai, 1998; Koos
and Tepper, 1999), which could potentially suppress backpropagation, as
shown in the hippocampus (Kim et al., 1995; Buzsaki et al., 1996;
Tsubokawa and Ross, 1996). Finally, spiny projection neurons express
A-currents (Surmeier et al., 1989; Nisenbaum and Wilson, 1995) that are
active during the up-state and, if present at dendrites, might prevent backpropagation (Hoffman et al., 1997).
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MATERIALS AND METHODS |
Preparation of organotypic cultures. For the
preparation of cortex-striatum-substantia nigra organotypic
cultures (Plenz and Kitai, 1998), coronal sections from rat brains
(Sprague Dawley, Taconic Farms, MD) at postnatal days 0-2 were cut on
a vibroslicer (VT 1000 S; Leica Microsystems Inc., Allendale, NJ).
Slices containing striatum (500 µm thickness) and cortex (350 µm
thickness) were used for dissection of dorsal or dorsolateral cortical
and striatal tissue. For the substantia nigra (including the pars
compacta and pars reticulata), ventrolateral sections from
500-µm-thick mesencephalic slices were taken; medial tissue regions
were avoided. The tissue was submerged in 25 µl of chicken plasma
(Sigma, St. Louis, MO) on a coverslip and 25 µl of bovine thrombin
was added (1000 National Institutes of Health units/0.75 ml;
Sigma). After plasma coagulation, individual cultures were placed in
tubes (Nunc Inc., Naperville, IL) with 800 µl of culture medium
consisting of 50% basal Eagle's medium, 25% HBSS and
25% horse serum, 0.5% glucose, and 0.5 mM
L-glutamine (all from Invitrogen, Grand
Island, NY). Cultures were rotated in a "rollertube" incubator (0.6 rpm; Heraeus, Göttingen, Germany) at 35.5°C in normal
atmosphere. After 3 and 27 d in vitro, 10 µl of mitosis inhibitor was added for 24 hr (4.4 mM cytosine-5-b-arabino-furanosid, 4.4 mM uridine, and 4.4 mM
5-fluorodeoxyuridine; calculated to a final concentration; all from
Sigma). Medium was changed every 3-5 d.
Whole-cell patch recordings. For electrophysiological
recording, the cultures were submerged in artificial CSF (ACSF)
containing (in mM): 126 NaCl, 0.3 NaH2PO4, 2.5 KCl, 0.3 KH2PO4, 1.6 CaCl2, 1.0 MgCl2, 0.4 MgSO4, 26.2 NaHCO3, and 11 D-glucose saturated with 95%
O2 and 5% CO2. The
osmolarity of the ACSF was at 300 ± 5 mOsm. Bath temperature was
continuously monitored and maintained at 35.5 ± 0.5°C (TC-20;
NPI, Tamm, Germany). The recording chamber was mounted on an
inverted microscope (IX-70; Olympus Optical, Tokyo, Japan) that had
been placed on a custom-made sliding table allowing for a change in
field of view during the experiment.
Patch pipettes for somatic whole-cell recordings were pulled (1.5 mm
outer diameter, 0.75 inner diameter; P-97; Sutter Instruments, Novato, CA) and fire polished (MF-830; Narishige, Tokyo, Japan). The
intracellular patch solution contained (in mM): 132 K-gluconate, 6 KCl, 8 NaCl, 10 HEPES, 2 Mg-ATP, 0.39 Na-GTP.
The solution was supplemented with 100 µM
Fura-2 (pentapotassium salt; Molecular Probes, Eugene, OR) and 0.2%
Neurobiotin (Vector Laboratories, Burlingame, CA). The pH was adjusted
to 7.2-7.4 with KOH and the final osmolarity of the pipette solution
was at 290 ± 10 mOsm. The open resistance of the
pipettes was 4-6 M
. To reduce degradation of ATP, GTP, and Fura-2,
the intracellular working solution was kept on ice in a darkened room
throughout the experiment before backfilling of electrodes.
Intracellular signals were recorded using an Axopatch 1-D amplifier
with a CV-4 1× head stage (Axon Instruments, Foster City, CA). After
the formation of a Giga-seal, electrode capacitance was compensated for
and serial resistance compensation was switched off. Data were recorded
in current clamp (I-clamp), if not stated otherwise,
preamplified (Cyberamp380; Axon Instruments), digitized at 10 kHz for
voltage and 5 kHz for current, and stored in continuous-stream mode
using the CED 1401plus (Cambridge Electronic Design, Cambridge, UK).
Electrophysiological data analysis was performed in Spike2 (Cambridge
Electronic Design), Origin version 6.0 (Microcal, Southampton, MA), and
Excel (Microsoft, Seattle, WA). All membrane potential values were
corrected for K-gluconate liquid junction potential (Neher, 1992).
Striatal spiny projection neurons were identified by a spherical soma
size of 10-12 µm diameter using Hoffmann modulation contrast optics
(40×). A Fura-2 fluorescent image, taken 5-10 min after break-in, was
used to further identify spiny projection neurons based on the presence
of dendritic spines. Neurons were accepted for recordings if (1) the
resting membrane potential was more negative than 75 mV, (2)
suprathreshold current pulse injection resulted in a ramp-like
depolarization that delayed action potential discharge by several
hundreds of milliseconds, and (3) neurons could fire action potentials
repetitively up to 50 Hz.
For tetrodotoxin (TTX) experiments in voltage clamp
(V-clamp), a command voltage protocol was designed that
mimicked a somatic depolarization-induced action potential burst from a
holding potential of 80 mV stepped to 50 mV for 500 msec and five
action potentials spaced at 100 msec (temporal resolution, 0.01 msec).
The action potential command voltage trajectory was obtained from
averages of 100 action potentials taken from 10 spiny projection neurons.
Fluorescence measurement. Neurons were loaded with the
calcium-sensitive indicator dye Fura-2 (100 µM)
via the patch pipette. After break in, the filling was monitored with a
60× water immersion objective (1.2 numerical aperture; working
distance, 260 µm; Olympus Optical) and the neuron was scanned
for dendrites within the horizontal plane of interest. Usually one to
two primary dendrites and corresponding higher-order dendrites were
analyzed simultaneously. Fluorescence measurements were started 10-15
min after break in (Helmchen et al., 1996).
Dye excitation was achieved with a polychromatic illumination system
coupled to the microscope via a quartz light guide and wavelength
selection via diffraction grating (12 nm bandwidth; T.I.L.L. Photonics,
Munich, Germany). For all experiments, a beam splitter at 400 nm and a
wide long-pass filter at 510 ± 20 nm were used (Omega,
Brattleboro, VT). Fluorescence measurements were made with a
thermoelectrically cooled CCD camera with a 0.5 inch interline
transfer chip and on-chip binning of 4 × 4 (Imago, 640 × 480 pixels; T.I.L.L. Photonics). Images were collected using commercially available software (Tillvision version 3.3.1; T.I.L.L. Photonics) and stored on computer hard-drive.
Definition of regions of interest. An overview
picture (1000 msec exposure; 2 × 2 binning) was taken at
completion of the experiment. Individual regions of interest (ROIs)
were grouped into primary, secondary, and tertiary dendrites, with each
area defined by dendritic branching points. For tertiary dendrites, ROIs were only taken for segments that were clearly in the plane of
focus as well as within the frame of view (average segment length,
10-20 µm). Background was defined as the area adjacent to each ROI
that was located outside the neuron of interest. Calculations for ROIs
and associated changes in
[Ca2+]i were
measured with dual-wavelength imaging (340/380 nm) and expressed as a
ratio or measured with single-wavelength imaging (380 nm) and expressed
as 
F/F.
F/F and ratio calculations. F/F values for
ROIs were calculated for each frame as
(Fi F0)/F0
and expressed as percentage of change, where
F0 indicates the baseline fluorescence
obtained from the average of 10-20 frames during the down-state and
Fi indicates individual fluorescence
measures at frame i. Background correction was calculated
for each image frame and subtracted from both
Fi and
F0. For ratio imaging, pixel
intensities from images taken at 340 nm were divided by pixel
intensities from subsequent images taken at 380 nm after background
subtraction for each ROI. F/Fmax was
taken at the time point at which F/F values reached
maximum and was usually within the same frame between ROIs.
[Ca2+]i
during down-state.
[Ca2+]i
concentrations were calculated according to the following equation:
[Ca2+]i = Keff × [(R Rmin)/(Rmax R)], where
Rmin and Rmax are ratio values obtained during the calibration under 0 [Ca2+] and saturating 39.8 µM
[Ca2+] (theoretical saturation for Fura-2),
respectively, and R indicates ratio values measured during
the recording. All values were corrected for background fluorescence.
Calibration was performed at the same temperature as experiments using
a Fura-2 calcium-imaging calibration kit ranging from 0 to 10 mM Ca-EGTA and 50 µM
Fura-2 (Molecular Probes). A Kd
estimated as 185.4 nM for 36°C was used for the
calculation of Keff (Groden et al.,
1991).
On-line detection and measurement of spontaneous up-states.
Initial attempts to identify up-states by fitting membrane potential distributions with Gaussian functions were abandoned because membrane potentials in successive sample points at 2 msec were not independent (membrane time constant) and membrane potentials were rectified at
depolarized (spike threshold) and hyperpolarized (anomalous rectification) levels. Therefore, routines written in assembly language
for speed were designed to scan the intracellular membrane potential
on-line to detect up-state transitions as well as down-state periods.
The detection was based on on-line statistical membrane potential
analysis, where a threshold was calculated at 5× SD during periods of
low spontaneous activity. Positive threshold crossing by the membrane
potential indicated a transition to the up-state, whereas negative
threshold crossing indicated a return to a down-state. Threshold
crossings were discarded if the time from positive threshold crossing
to negative crossing was 
50 msec. On average, cells spent 90% of
their time below threshold.
The amplitude of spontaneous membrane potential fluctuations in the
down-state was analyzed off-line. Using a threshold at 2 mV above
average resting value with successive positive peak detection, average
amplitudes were calculated for peak-aligned spontaneous events.
Down-state imaging (20-40 frames; 20-40 msec duration each; 380 and
340 nm, respectively) was performed when the cell was in a down-state
for at least 1.5 sec. This ensured that
[Ca2+]i transients
from the previous up-state did not bias down-state measurements. After
down-state fluorescence measurements, threshold crossing by a
spontaneous up-state subsequently triggered up-state measurements. On
average, 120-300 consecutive images (20-40 msec duration each; 380 nm
or 380/340 nm) were collected to measure a down-state and subsequent
up-state. To exclude possible additive effects of
[Ca2+]i
transients, up-states that occurred closer than 1.5 sec to a preceding
negative threshold crossing were not included in the analysis.
Measurement of fluorescence change during current injection.
Responses during the down-state were measured at least 1.5 sec after
return from an up-state. One to 10 pulses of depolarizing current
injection (500 msec) were given at increasing steps (0.01 nA minimum;
0.01-0.02 nA step size), resulting in subthreshold or both
subthreshold and suprathreshold responses. Either a sequence of images
was taken for a single current pulse (10 images of 380 and 340 nm for
F0 and 50 images of 380 and 340 nm for
Fi) or a continuous sequence of images
was taken for multiple current pulses (10 images of 380 and 340 nm for
F0 and 150 images of 380 and 340 nm
for Fi). Because of the strong effect
of the A-current after depolarization in spiny projection neurons,
F/Fmax values for subthreshold membrane
depolarizations were usually taken from the last frame during the
current pulse and compared with average F/Fmax values taken before current injection.
Dye concentration, bleaching, resting base line, drug
application. Initial experiments using different concentrations of
Fura-2 revealed that Fura-2 concentrations of >100
µM severely interfered with the internal
calcium dynamics (Neher and Augustine, 1992) and were detrimental
during repetitive action potential firing. A concentration of 100 µM allowed recording for at least 25 min, did
not significantly change action potential width, and allowed single-sweep optical recordings from secondary and tertiary dendrites. Frames taken at the beginning and end of the imaging period near the
isofluoremetric point of Fura-2 (358 nm) were used to monitor dye
bleaching. Data were rejected if dye bleaching exceeded the average
baseline fluctuations during the imaging period. TTX (1 µm; Sigma)
was dissolved to final concentration in gassed recording solution and
bath applied.
Data are expressed as mean ± SEM. For statistical data analysis,
ANOVA with Scheffé's post hoc test was used unless
stated otherwise. Correlation and partial correlation was estimated by linear regression analysis (StatView; SAS Institute, Inc., Cary, NC).
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Figure 1.
Membrane properties, spontaneous activity,
and morphological characteristics of spiny projection neurons recorded
in whole-cell patch configuration. A, Membrane potential
responses to somatic current injections during the down-state are
characterized by anomalous rectification at hyperpolarized potentials
(arrow), early outward rectification with depolarization
(closed arrowhead), ramp-like trajectory toward
threshold (open arrowhead), and delayed action potential
firing (maximal depolarization pulse repeated 3 times, 15 min after
break in). Note fluctuations indicative of irregular spontaneous
synaptic inputs. B, Nonlinear steady-state
I-V relationship (from the time indicated by the
bar in A). C,
Characteristic delay in burst firing by somatic current injections
(open arrowhead). D, Spontaneous,
suprathreshold up-state. Note the fast transition to the
up-state, delay in burst firing onset during the up-state (open
arrowhead), and relatively slow return to the down-state.
E, Bimodal membrane potential distribution that reflects
the relatively fast transition between down- and up-states. Values were
taken from the trace in D at 2 msec time resolution.
F, Fluorescence image 35 min after break in at 380 nm
(100 µM Fura-2; 1 sec exposure; composite from 9 individual images).
Circle, Soma; square, primary dendrites;
triangle, secondary dendrites; diamond,
tertiary dendrites. Note spines on dendrites (inset,
asterisks).
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RESULTS |
Membrane properties and spontaneous up-states of cultured spiny
projection neurons recorded in whole-cell patch configuration
Spiny projection neurons (n = 55) showed a
polarized resting membrane potential of 82.5 ± 0.9 mV and
neurons displayed inward and outward rectification as well as delayed
action potential firing in response to somatic current injections (Fig.
1A-C). Their
spontaneous activity in cortex-striatum-substantia nigra cultures was
characterized by irregular up-states with delayed bursts of action
potentials separated by down-states, as described previously (Fig.
1D) (Plenz and Kitai, 1998). In the present study, up-states lasted on average for 1.28 ± 0.10 sec and occurred
irregularly at an average interval of 11.7 ± 1.4 sec
(n = 10 neurons; 10 up-states per neuron). During
up-states, spiny projection neurons were depolarized by 38.8 ± 1.0 mV to reach threshold (n = 40). In the down-state, the spontaneous membrane potential trajectory was characterized by
numerous depolarizations with an average peak value of 4.3 ± 1.3 mV (Figs. 1A,
2A) (1 sec per neuron;
n = 10 neurons). Peak depolarizations during
down-states were significantly smaller compared with depolarizations
reached during up-states (p < 0.0001). Transitions between up- and down-states were relatively fast, resulting
in bimodal membrane potential distributions (Fig.
1E). Dye-loading with Fura-2 (100 µM) during the recording also allowed for
identification of spiny projection neurons based on their spherical
soma and spiny higher-order dendrites (Fig. 1F).
Action potential threshold, amplitude, width, slopes, and
afterhyperpolarization did not significantly differ for both evoked and
spontaneous conditions (Table 1).
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Figure 2.
Spontaneous up-states in spiny projection neurons
correlate with large [Ca2+]i
transients in soma and dendrites. A, Simultaneous,
single-sweep measurement of [Ca2+]i
transients in soma and dendrites (upper traces) and
corresponding spontaneous intracellular membrane potential activity at
the soma (bottom trace). F0
values were taken during a period in the down-state indicated by the
leftmost segments (shaded; 20 frames).
Frames were collected at 340 and 380 nm wavelength (42 msec each),
allowing background corrected calculation of both ratio (120 msec;
dotted lines) and F/F values (380 nm;
120 msec; solid lines). Note the similar time course
of estimated [Ca2+]i transients with both
methods. Measurements were arbitrarily scaled to first soma peak
values, respectively, to facilitate comparison. Up-states that occurred
within 1.5 sec of previous up-states (*) and depolarizations above
threshold for <50 msec (**) were discarded from analysis. Numbers of
action potentials are indicated for each up-state.
Circle, Soma; square, primary dendrites;
triangle, secondary dendrites; diamond,
tertiary dendrites. B, Changes in somatic and
dendritic F/F are small during the down-state when
compared with up-state periods (340 and 380 nm; 62 msec each).
C, Relationship between
F/Fmax values during up-states at higher
temporal resolution (taken from A, bar).
D, Summary of F/Fmax
values for the first four up-states shown in A.
E, Averaged normalized decay from
F/Fmax for each region (3 neurons; 3-4
up-states each). Note the slower time course at soma compared with
dendrites. F, Dendritic
F/Fmax values and the number of somatic
action potentials are linearly correlated during spontaneous up-states
(same neuron as in A). Dashed lines
indicate regression functions for each compartment.
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Table 1.
Spontaneous action potentials during up-states are similar
to action potentials elicited by somatic current injection
(I-clamp, whole-cell patch configuration including
100 µM Fura-2; n = 40 neurons)
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Up-states correlate with large
[Ca2+]i transients in soma
and dendrites
The time course and spatial distribution of
[Ca2+]i transients
during spontaneous up-states in spiny projection neurons was addressed
by simultaneously measuring somatic and dendritic
[Ca2+]i transients
as well as somatic membrane potential during spontaneous activity (Fig.
2A-C).
[Ca2+]i transients
followed the overall time course of the somatic membrane potential
(Fig. 2A) when analyzed using either dual-wavelength comparison (ratiometric, 340/380 nm) or single wavelength (380 nm,
relative percentage change as F/F). Thus, for
reasons of higher time resolution, most experiments were performed
using single-wavelength F/F.
At the single-neuron level, the spatial distribution
of F/F transients during individual up-states showed two
characteristics. First, dendritic F/F transients reached
higher F/Fmax values when compared with
soma (p < 0.001; n = 21), and
second, dendritic F/Fmax values tended
to increase from primary to tertiary dendrites, regardless of the
absolute F/Fmax reached within each
compartment (Fig. 2C,D). These relations in
F/Fmax for soma and dendrites during
up-states were also present at the population level (see Fig.
4A). Across neurons,
F/Fmax at the soma was significantly lower than in dendrites (p < 0.001) and
F/Fmax values increased from primary to
tertiary dendrites (see Fig. 4A) (two to three up-states per neuron; n = 21 neurons). During
down-states, both F/F and ratio values were stable in
soma as well as in dendrites; [Ca2+]i was
estimated to range between 30 and 60 nM in soma
and primary dendrites (Fig. 2B) (n = 4 neurons).
The time course in F/F during the return to the
down-state was relatively uniform and was analyzed by fitting a single
exponential function to the decay from normalized
F/Fmax values (Fig.
2E) (r = 0.92-0.99; three to four
up-states per neuron; n = 3 neurons). Dendritic decay
in F/F revealed a single time constant of 790 ± 10 msec that was not different between dendritic compartments (p = 0.518-0.953). In contrast, the somatic
decay of [Ca2+]i
transients had a time constant of 1100 ± 40 msec, which was significantly slower when compared with dendrites
(p < 0.0001).
In neurons that showed a wide range in the number of spontaneous action
potentials during up-states, a surprisingly high correlation, particularly in dendrites, was revealed when plotting spontaneous action potential number against F/Fmax
(Fig. 2F) (r = 0.97 ± 0.01; two
to eight action potentials; n = 3 neurons). This
correlation was also present at the population level (Fig.
3C) (r = 0.79-0.83; n = 21 neurons; three to four up-states per
neuron). Average slope values in F/Fmax
ranged from 24 ± 3% to 36 ± 4% per 10 action potentials
and were similar in dendrites but significantly lower at the soma (Fig.
3C) (p < 0.001; n = 21 neurons).
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Figure 3.
Dynamic range of
F/Fmax values reveals linear correlation
within and continuity between subthreshold and suprathreshold
up-states. Circle, Soma; square, primary
dendrites; triangle, secondary dendrites;
diamond, tertiary dendrites. A,
Time course of F/F during subthreshold up-states.
Note the increase in F/Fmax from soma to
higher-order dendrites. B,
F/Fmax values in suprathreshold up-states
are correlated with the number of spontaneous somatic action
potentials. Note the similar overall time course of up-states but
different number of action potentials (4 and 2, respectively).
C, F/Fmax values
continuously encode the transition from subthreshold to suprathreshold
up-states. Left, F/Fmax
values are linearly correlated with peak membrane potential
depolarization during subthreshold up-states (open
symbols). Right,
F/Fmax values are linearly correlated
with the number of spontaneous action potentials during suprathreshold
up-states (closed symbols). Center,
Broken vertical lines indicate a population action
potential threshold of 44.7 mV aligned to 0 action potentials. Linear
regression is indicated by dashed lines.
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To summarize, up-states in spiny projection neurons were correlated
with large [Ca2+]i
transients that decayed within 1 sec after reaching maximum. The
corresponding F/Fmax values were highly
correlated with the total number of spontaneous action potentials
during the up-state.
Dendritic [Ca2+]i
continuously encodes subthreshold and suprathreshold activity during
up-states
To discriminate between effects of synaptic activity and
somatic action potential firing on
[Ca2+]i
transients, subthreshold up-states were compared with suprathreshold up-states.
In the absence of action potentials, prominent
[Ca2+]i transients
were visible during up-states in both soma and dendrites (Fig. 3A) and F/Fmax modestly
correlated with membrane potential peak values for all regions (Fig.
3C) (r = 0.49-0.69; n = 10 neurons; one to two up-states per neuron). Within the up-state membrane potential range of 60 to 45 mV,
F/Fmax changed between 0 and 20%,
resulting in an average slope of 4 ± 2% to 10 ± 4%
F/Fmax per 10 mV that was not
significantly different between compartments (Fig. 3C)
(p < 0.52; n = 10 neurons).
Similar to suprathreshold up-states, average
F/Fmax values during subthreshold
up-states increased toward higher-order dendrites (Fig.
4A).
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Figure 4.
Summary of the dynamic range in
F/Fmax during up-states.
A, Average F/Fmax values
were two to three times higher during suprathreshold up-states
compared with subthreshold up-states. B,
[Ca2+]i transients continuously
encoded the transition from subthreshold to suprathreshold up-states in
soma and dendrites. Values are based on regression functions (mean ± 95% confidence interval) derived from subthreshold (open
circles) and suprathreshold (closed circles)
up-states extrapolated to membrane potential threshold and 0 action
potentials, respectively.
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However, average F/Fmax values during
subthreshold up-states were significantly lower compared with
suprathreshold up-states (Fig. 4A, p < 0.0001), which raised the question of whether
[Ca2+]i transients
show a discontinuity at the transition from subthreshold to
suprathreshold up-states. Therefore, we predicted
F/Fmax values at this transition based
on regression functions. Linear regressions based on subthreshold
up-state membrane potential values or suprathreshold up-states
predicted increasing F/Fmax values from
10 to 20% (soma to higher-order dendrites) at 44.7 ± 1.5 mV
(population action potential threshold) or when extrapolated to 0 action potentials, respectively (Fig. 4B).
Both predictions highly overlapped for each compartment
(p = 0.3-0.84).
Thus,
[Ca2+]i transients
were two to three times smaller during subthreshold up-states compared
with suprathreshold up-states. Nevertheless,
[Ca2+]i transients
continuously encoded excitation in spiny projection neurons despite a
qualitative discontinuity when moving from subthreshold to
suprathreshold up-states.
Action potentials elicit
[Ca2+]i transients in
higher-order dendrites
Continuous encoding of
[Ca2+]i transients
during subthreshold and suprathreshold up-states could simply reflect
the dependency of
[Ca2+]i on
synaptic inputs (Regehr and Tank, 1994). In this view, a rise in
synaptic inputs will increase dendritic
[Ca2+]i as well as
trigger more action potentials, resulting in a correlation between
action potential number and
[Ca2+]i.
Alternatively, action potentials could directly contribute to dendritic
[Ca2+]i by
propagating along dendrites (Häusser et al., 2000). Dendritic [Ca2+]i transients
were therefore measured during repetitive bursts of action potentials
evoked by somatic current injections during the down-state (Fig.
5).
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Figure 5.
Action potentials elicit stereotypical
[Ca2+]i transients in higher-order
dendrites during the down-state. A, Composite
fluorescence image of the spiny projection neuron (Fura-2; 1 sec
exposure at 380 nm; 40 min after break in). Dashed
lines indicate centers of ROIs located within the optical field
of view (dotted box). B, Repetitive burst
discharge by somatic current injection reliably induced
[Ca2+]i transients throughout the
neuron. a-e are same as in A. A simultaneous,
single-sweep measurement of [Ca2+]i
transients in soma and dendrites (upper traces, 380 nm;
42 msec illumination; 120 msec temporal resolution) and
corresponding somatic burst firing (bottom traces) are
shown. C, Onset of
[Ca2+]i transients correlated with the
first action potential (from B, left
bar). Symbols indicate center times for each
frame acquisition. Circle, Soma; diamond,
tertiary dendrites. D, Averaged normalized decay
from F/Fmax for each region (3 neurons;
3-4 up-states each). Note the slower time course at soma
compared with dendrites and delayed action potential firing
(arrow).
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Action potentials from suprathreshold current injection evoked large
[Ca2+]i transients
at soma and primary, secondary, and tertiary dendrites (Fig.
5A,B). At a fixed burst strength (i.e., number of action potentials per 500 msec), these
[Ca2+]i transients
were reliably elicited and did not change over the period of multiple
bursts, resulting in stereotypical transients throughout the neuron
(Fig. 5B). The F/F time course during each burst revealed a fast onset that was correlated with the first action
potential (Fig. 5C). At burst termination, the decay from F/Fmax was fitted by a single
exponential decay (r = 0.97-0.99; soma to tertiary
dendrites; n = 4 neurons). On average, dendritic [Ca2+]i transients
decayed to 37% within 504 ± 12 msec after burst termination,
which was faster compared with the somatic
[Ca2+]i decay
(p < 0.002) and dendritic
[Ca2+]i decay
during up-states (Fig. 5D) (p < 0.0001).
To test whether evoked
[Ca2+]i transients
encode somatic subthreshold membrane potential and burst strength,
current pulses of 500 msec duration at varying amplitudes were applied
during down-states (Fig. 6)
(n = 12 neurons). Subthreshold depolarizations evoked
small but significant
[Ca2+]i transients
at soma (4.82 ± 0.60% F/Fmax;
p < 0.0001) and primary dendrites (3.58 ± 0.46%
F/Fmax; p < 0.01) (Fig.
6B-D). However, they did not elicit
[Ca2+]i transients
at higher-order dendrites (p = 0.100 and
p = 0.255, respectively). Thus,
F/Fmax values during subthreshold
somatic depolarizations were significantly lower in all dendritic
compartments when compared with F/Fmax
values from subthreshold up-states (p < 0.0001), despite covering a similar voltage range for both conditions
(current injection, 50 ± 1 mV; range, 58 to 55 mV; up-state, 55 ± 2 mV; range, 60 to 43 mV). No difference was found for both conditions in somatic
F/Fmax values (p = 0.61).
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Figure 6.
Dendritic [Ca2+]i
transients encode somatic burst firing but not subthreshold membrane
potential during the down-state. A, Fluorescence image
of the spiny projection neuron (Fura-2; 500 msec exposure at 380 nm; 10 min after break in). Dashed lines indicate ROIs located
within the optical field of view (dotted box).
B, Suprathreshold but not subthreshold somatic
depolarizations (500 msec) evoked
[Ca2+]i transients throughout the
dendritic arbor and F/Fmax values varied
with the number of somatic action potentials (numbers).
Note the small increase in F/Fmax at soma
close to threshold (asterisk). C, Dynamic
range of F/Fmax values for subthreshold
and suprathreshold somatic depolarizations. Left,
Dendritic F/Fmax values were not
correlated with subthreshold somatic membrane potential depolarizations
(open symbols; n = 14 neurons; 1-2
depolarizations per neuron). Right,
F/Fmax values linearly correlated with
the number of action potentials in suprathreshold responses
(closed symbols; n = 35 neurons;
1-2 depolarizations per neuron). Center, Dendritic
F/Fmax values revealed a discontinuous
transition (open arrowheads) when moving from
subthreshold to suprathreshold membrane potential responses.
Broken vertical lines indicate a population action
potential threshold of 44.7 mV, which is aligned with 0 action
potentials. Linear regression is indicated by dashed
lines. av, Average F/F for
subthreshold depolarizing current pulses. Circle, Soma;
square, primary dendrites; triangle,
secondary dendrites; diamond, tertiary
dendrites. D, Average
F/Fmax values were up to 10 times higher
in suprathreshold responses compared with subthreshold responses.
E, Discontinuity of F/Fmax
values in encoding the transition from subthreshold to suprathreshold
responses to somatic current injection. Values from regression
functions (mean ± 95% confidence interval) derived from
subthreshold (open circles) and suprathreshold
(closed circles) responses (from C,
extrapolated to threshold and to 0 action potentials, respectively) are
shown.
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In contrast, suprathreshold current injections resulted in
F/Fmax values throughout the
dendritic arbor that were highly correlated with the number of action
potentials across all neurons examined (Fig. 6B,C)
(r = 0.83-0.86; y = a + b × x; n = 26 neurons). This correlation was not improved when using a simple saturation function [r = 0.81-0.84; y = a × (1 e
bx)].
Within the range of 1-20 action potentials per 500 msec duration, F/Fmax increased by
14 ± 1% to 16 ± 2% F/Fmax
per 10 action potentials for all compartments. This slope was
significantly lower when compared with suprathreshold up-states
(p < 0.005, soma and dendrites). Average
F/Fmax values during suprathreshold current injections were relatively high in soma and dendrites (27-33%) and were not significantly different between dendrites (p = 0.22-0.66), but were significantly higher
compared with F/Fmax during subthreshold
depolarizations (p < 0.001) (Fig.
6D).
To understand the dynamic range of
[Ca2+]i transients
at the transition from subthreshold to suprathreshold somatic current injection, F/Fmax values at this
transition were predicted based on regression functions. Based on
membrane potentials during subthreshold down-states, decreasing
F/Fmax values from 7 to 3% (soma to
higher-order dendrites) were predicted at a membrane potential
threshold of 44.7 ± 1.5 mV. In contrast,
F/Fmax values of between 12 and 16%
(soma to higher-order dendrites) were predicted when based on action
potentials during suprathreshold current injections and extrapolated to
0 action potentials (Fig. 6E). Both
predictions were significantly different for each compartment,
respectively (p < 0.001) (Fig.
6E).
To summarize, subthreshold somatic depolarization by
current injection only elicited
[Ca2+]i transients
at the soma, which is markedly different compared with subthreshold
up-states. However, somatic action potentials temporarily raised
intracellular
[Ca2+]i throughout
the dendritic arbor of spiny projection neurons. These
[Ca2+]i transients
were highly correlated with somatic burst strength (i.e., number of
action potentials per 500 msec), which is similar to suprathreshold
up-states.
Somatic action potentials elicit dendritic
[Ca2+]i transients by active
propagation along dendrites during the down-state
Somatic action potentials have been shown to actively
backpropagate and elicit
[Ca2+]i transients
in higher-order dendrites (Stuart and Sakmann, 1994; Stuart et al.,
1997b; Häusser et al., 2000). However, for neurons with
relatively short dendrites, such as striatal spiny projection neurons,
passive propagation might be sufficient, assuming standard cable
properties (Turner, 1984). Alternatively, spiny projection neurons
might be highly shunted at soma and dendrites, which would require the
presence of dendritic sodium channels for action potentials to
propagate and elicit
[Ca2+]i transients
throughout the dendritic tree.
Dendritic [Ca2+]i
transients were first measured in response to somatic suprathreshold
current injections (Fig. 7)
(n = 3). Next, the sodium channel blocker TTX (1 µM) was bath applied and recording was switched
to V-clamp. Voltage commands were applied that forced the
somatic membrane potential through a similar trajectory to bursts
recorded in I-clamp with suprathreshold depolarizing current
(Fig. 7E,F). In the presence of TTX, spontaneous
activity was abolished and somatic current pulses that were
suprathreshold under control conditions increased
[Ca2+]i in the
soma but not in higher-order dendrites (Fig. 7C). After switching to V-clamp, burst-like voltage commands increased
[Ca2+]i at the
soma but failed to elicit
[Ca2+]i transients
at distal dendrites (Fig. 7D,G). No significant difference
was found in somatic
[Ca2+]i between
both conditions (Fig. 7G) (p = 0.883). Dendritic
[Ca2+]i responses
recovered partially during washout of TTX (Fig. 7I) (n = 3).
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Figure 7.
Action potential-evoked dendritic
[Ca2+]i transients are blocked by TTX.
A, Fluorescence image of the spiny projection neuron
(Fura-2; 1 sec exposure at 380 nm; 50 min after break in).
Dashed lines indicate ROIs located within the optical
field of view (dotted box). B,
Simultaneous, single-sweep measurement of
[Ca2+]i transients in soma and
dendrites (upper traces; 380 nm; 42 msec each frame;
60 msec resolution) and corresponding spontaneous intracellular
neuronal activity at the soma (bottom trace). For the
control (I-clamp), Spontaneous up-states and evoked
somatic action potentials were correlated with
[Ca2+]i transients throughout the
neuron. C, TTX (I-clamp): In the presence
of TTX (1 µM), the same somatic current injection
elicited only weak [Ca2+]i transients
at soma and primary dendrites (asterisks).
D, For TTX (V-clamp), a
V-clamp command that mimicked an action potential burst
elicited strong [Ca2+]i transients at
the soma, but failed to elicit [Ca2+]i
transients in higher-order dendrites. E, Enlarged time
view of the V-clamp command applied in D.
F, Overlay of the average evoked action potential
obtained in I-clamp (dotted line) and the
simulated action potential (solid line) used in the
V-clamp command in D. G,
In the presence of TTX, F/Fmax values
were reduced in higher-order dendrites compared with controls but not
in the soma. Dark bars, Control I-clamp
with three neurons (3 trials per neuron, average of 5 action
potentials). Light bars, TTX
V-clamp, same ROIs and neurons as in the control.
H, In the presence of TTX, action potential-evoked
F/Fmax values decayed to 37% within 17 µm from soma (r = 0.96; single exponential fit).
Closed circles, Control I-clamp with
three neurons (5 trials per neuron, same ROIs). Open
circles, TTX V-clamp, same neurons as in the
control. I, Action potential-evoked dendritic
[Ca2+]i responses before (long
dashed line), during (solid line), and after
partial washout of TTX (10 min, dotted line). Each
line is an average of three responses from the same
neuron.
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Additional quantification of the spatial decay in
F/Fmax revealed that in the presence of
TTX, F/Fmax values decayed to 37%
within 17.5 ± 0.5 µm distance from soma (Fig.
7H) (n = 3 neurons). No significant
difference was found for the decay obtained in the presence of TTX
under I-clamp and V-clamp conditions,
respectively (p > 0.52; data not shown). In
contrast, the spatial decay of F/Fmax
was minimal, and in some cases F/Fmax
was even increased under control conditions (Fig.
7H).
Thus, in the presence of TTX, burst-like voltage commands only elicit
[Ca2+]i transients
near the soma, but not at higher-order dendrites, suggesting that
dendrites in spiny projection neurons are active. This would allow
somatic action potentials to actively backpropagate into the dendrite.
Somatic action potentials increase dendritic
[Ca2+]i transients supralinearly
during the up-state
Electrophysiological properties and
[Ca2+]i dynamics
of dendrites change considerably in the presence of synaptic activity
when compared with resting conditions (Pare et al., 1998; Svoboda et al., 1999). Therefore, we tested whether somatic action potential bursts also increase dendritic
[Ca2+]i during
up-states, which are driven by synaptic activity.
Previous experiments revealed that somatic current
injections during the down-state elicited action potential firing with a delay of up to several hundreds of milliseconds (compare Figs. 1A, 5C). For a suprathreshold
depolarization pulse of 500 msec, six action potentials were elicited
on average during the last 209 ± 17 msec (n = 10 neurons), equivalent to an average firing frequency of 28.7 ± 2.4 Hz. Correspondingly, a pulse length of 200 msec and pulse amplitude of
0.1 ± 0.02 nA were used to elicit action potentials during
up-states (Fig. 8).
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Figure 8.
Supralinear dendritic
[Ca2+]i transients encode somatic
bursts during up-states. A, Dendritic
[Ca2+]i transients (380 nm; 42 msec
each frame; 60 msec resolution) during up-state alone
(left) or with suprathreshold somatic current injection
(right). Note the absence of
[Ca2+]i transients in higher-order
dendrites, when the depolarizing pulse occurs in the down-state.
B, Detail of the resulting
[Ca2+]i transient in tertiary
dendrites from up-state with somatic current injection. Note
F/Fmax at the end of the action
potential burst (expanded from A,
bar). C, Increasing the number of action
potentials by somatic current injection increases
F/Fmax linearly in tertiary dendrites
during up-states. Spontaneous up-states without somatic current
injection averaged 2.5 ± 0.5 action potentials; spontaneous
up-states with somatic current injection averaged 8.7 ± 0.9 action potentials. D,
F/Fmax is reached after termination of
somatic current injection. Individual
F/Fmax values (filled squares)
are related to the time after up-state onset (tertiary dendrite). A
peristimulus histogram of action potential frequency distribution
(solid line) in relation to both up-state onset and somatic
current injection is shown (shaded box).
E, F/Fmax values from
up-states with injected action potentials were highly similar to
population values reached during spontaneous up-states with identical
numbers of action potentials. Dark bars,
Up-states without somatic current injection and an average of 2.5 ± 0.5 spontaneous action potentials (n = 6 neurons). Medium bars,
F/Fmax from up-states with somatic
current injection that resulted in an average of 8.7 ± 0.9 action
potentials (n = 6 neurons). Light
bars, Up-states without somatic current injection and 8.7 ± 0.9 spontaneous action potentials (n = 21 neurons; population data from regression analysis; *p < 0.0001). F, Supralinear effect of
backpropagating action potentials and synaptic inputs during up-states
summarized for all compartments. Bottom solid line,
Measured F/Fmax for up-states without
current injection and an average of 2.5 action potentials.
Bottom broken line, Expected increase in
F/Fmax from 2.5 to 8.7 action potentials
during the down-state. Middle broken line, Linear sum of
both functions. Top solid line, Measured
F/Fmax during up-states with on average
2.5 spontaneous action potentials and 6.2 additional action potentials
by somatic current injection (*p < 0.0001).
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Depolarizing somatic current pulses were injected 20 msec after
up-state onset in six neurons (Fig. 8A,B). In
addition, up-states without current injection were sampled from each
neuron (total of two to four up-states per neuron), and
F/Fmax values were pooled for both
conditions and all neurons (Fig. 8C,E), which averaged
possible differences in synaptic inputs between both groups. These
experiments revealed that somatic and dendritic [Ca2+]i were not
saturated during spontaneous up-states, but were further increased by
additional action potentials. On average, current injection increased
the number of action potentials per up-state by 6.1 ± 0.8 (from
2.5 ± 0.5 to 8.6 ± 0.9 action potentials), which resulted
in an increase in F/Fmax of 22-28%
(Fig. 8E) (soma to higher-order dendrites;
p < 0.001). Similar to current injection experiments
in the down-state, these increases in
F/Fmax were highly correlated with the
total number of action potentials (Fig. 8C)
(r = 0.79; 0.80; 0.85; 0.92; soma to tertiary dendrite).
[Ca2+]i transients
have been shown to correlate with action potential frequency (Helmchen
et al., 1996); therefore, we tested whether the increased
F/Fmax during up-states with current
injections resulted from transient firing rate increases that were not
reflected in the total number of action potentials. Average firing
frequency (fmean) during
up-states with injected currents ranged from 10 to 30 Hz, whereas
maximal firing rates (fmax)
ranged from 20 to 57 Hz. The time course of average firing rate for all
neurons is given in Figure 8D. We then tested which
of these parameters accounted best for the observed increases in
F/Fmax, using data from tertiary
dendrites. Action potential number correlated most strongly with
F/Fmax (r = 0.92) (Fig.
8C) compared with fmax (r = 0.20) or fmean
(r = 0.26). This strong correlation between the number
of action potentials and F/Fmax was
present even when considering possible interactions with either
fmax or
fmean (r = 0.918;
partial correlation). The strong correlation with the total number of
action potentials, which implies an accumulated increase of
[Ca2+]i during the
burst, was also supported by the finding that
F/Fmax on average occurred after
termination of current injection (Fig. 8D).
Action potentials elicited by injecting current during up-states
increased dendritic F/F through propagation along the
dendrite. This should be in contrast to dendritic F/F
during up-states with similar numbers of spontaneous action
potentials driven by synaptic inputs. However,
F/Fmax values from up-states with
current injections were very similar to
F/Fmax values reached during up-states
with identical numbers of spontaneous action potentials (Figs.
3C, 8E). More specifically,
F/Fmax values were identical in
higher-order dendrites (p = 0.81-0.99) and only
slightly but nonsignificantly different at the soma
(p = 0.21). This similarity suggests that
dendritic [Ca2+]i
during up-states is largely determined by the number of action potentials and not synaptic inputs.
Finally, predicted increases in F/Fmax
systematically underestimated actual
F/Fmax increases during the up-state
(Fig. 8F). Predictions in
F/Fmax for additional action potentials
were obtained from down-state measurements. The predicted increase in
F/Fmax from three to nine action
potentials during the down-state ranged between 8 and 10% (Figs.
6C, 8F). The linear sum of
F/Fmax for spontaneous up-states
without current injection plus predicted F/Fmax values resulted in final
F/Fmax values of 25-37% (soma, primary
to tertiary dendrites). In contrast, actual
F/Fmax values measured during the
up-state were >10% F/Fmax higher
than predicted (Fig. 8F). This supralinearity, when
expressed as a ratio between measured increase in the up-state and
predicted increase from the down-state, was highly significant and
reached >200% for all compartments (p < 0.0001; two-tailed t test).
To summarize, dendritic
[Ca2+]i transients
supralinearly encode somatic bursting in spiny projection neurons
during up-states, which suggests an interaction between synaptic inputs
and action potentials propagating along dendrites.
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DISCUSSION |
The main result from this study shows that during spontaneous
up-states, information about somatic action potentials is present at
higher-order dendrites. This information is likely to be communicated to dendrites through active backpropagation (Stuart and Sakmann, 1994;
Stuart et al., 1997b; Häusser et al., 2000). The supralinear [Ca2+]i signal
represents a relatively simple code that contains information about a
spiny projection neuron's contribution to basal ganglia output. This
interaction between synaptic inputs, backpropagating action potentials,
and corresponding dendritic
[Ca2+]i shows
three characteristics. First, the dynamic range of dendritic [Ca2+]i during
up-states is balanced, allowing for continuous encoding of both
synaptic inputs and somatic burst strength. During subthreshold up-states, the somatic peak membrane potential predicted
[Ca2+]i transients
with relatively low corresponding F/Fmax
values (<20%) throughout the dendritic tree. Bursts during up-states further correlated with additional increases of
F/Fmax by up to 40%, a dynamic range
increase of 200%. Thus, synaptic inputs that depolarize spiny
projection neurons into up-states do not saturate
[Ca2+]i. This was
also confirmed by the increase of F/Fmax
by additional action potentials through somatic current injection
during up-states. Second, propagating action potentials rather than
synaptic inputs dominate dendritic
[Ca2+]i transients
during up-states. This interpretation is supported by the similarity of
F/Fmax between up-states with additional action potentials and spontaneous up-states with the same final number
of action potentials. Finally, propagating action potentials increased
dendritic [Ca2+]i
supralinearly during up-states, because down-state measurements of
evoked dendritic
[Ca2+]i transients
significantly underestimated up-state measurements.
Potentially, action potentials could be initiated in dendrites and
propagate forward to the soma (Häusser et al., 2000). However,
our results on subthreshold current injection show that the calcium
signal decays within 17 µm from the soma, suggesting a corresponding
fast decrement in membrane potential over space (Figs. 6, 7). If the
spike initiation zone is located far in higher-order dendrites,
eliciting spikes by somatic current injections is difficult, unless the
spike-initiation zone has a low threshold. The existence of
subthreshold up-states, however, makes this possibility unlikely. Therefore, we conclude that the spike-initiation zone in spiny projection neurons is probably located close to the soma.
The present study used organotypic cultures containing cortex,
striatum, and substantia nigra grown for 5-6 weeks. This in vitro model possesses several features that allow for the study of
dendritic processing in mature striatal spiny projection neurons. First, spiny projection neurons can be studied during up-states and
down-states with corticostriatal and nigrostriatal inputs present
(Plenz and Kitai, 1998), although these inputs might be markedly
reduced in overall numbers given the limited size of tissue taken for
culturing. Second, the relatively thin cross section of the culture
allows for the study of dendritic processing in higher-order dendrites
without averaging and at relatively low concentrations of Fura-2.
Propagation of action potentials in dendrites of spiny
projection neurons
The failure to elicit dendritic
[Ca2+]i transients
in the presence of TTX using action potential trajectories as
V-clamp commands implies that dendritic sodium channels are
necessary for action potential propagation in spiny projection neurons.
This finding extends previous ideas about nonlinear dendritic
properties in these neurons (Wilson, 1995). The normalized spatial
decay of [Ca2+]i
transients with distance from soma is close to the lengths of primary
dendrites, suggesting that without regenerative sodium channels,
higher-order dendrites in spiny projection neurons would be decoupled
from somatic firing. Similar findings have been described for cortical
neurons (Schiller et al., 1995) in which subsequent dendritic
recordings showed the existence of action potential backpropagation
(Stuart et al., 1997a).
The finding that action potential backpropagation in spiny
projection neurons is actively maintained is consistent with other reports that show action potential backpropagation to be highly regulated (Stuart et al., 1997b). Action potential backpropagation is
frequency dependent in hippocampal neurons (Callaway and Ross, 1995;
Spruston et al., 1995; Larkum et al., 1999), is regulated by dendritic
A-currents (Hoffman et al., 1997), depends on dendritic morphology
(Vetter et al., 2001), and can be suppressed by inhibitory inputs (Kim
et al., 1995; Buzsaki et al., 1996; Tsubokawa and Ross, 1996). The
present study, by showing the existence of backpropagation during
up-states, now allows additional investigation into the modulation of
backpropagation under conditions similar to those in
vivo.
Supralinear [Ca2+]i transients
during up-states
The supralinear increase in dendritic
[Ca2+]i during
up-states was highly correlated with the number of action potentials
and less with average firing or peak firing frequency during the
up-state. This finding is supported by the decay time constant of 500 msec for dendritic calcium signals in our study. Such a time constant would favor the accumulation of the dendritic calcium signal over the
relatively short duration of the up-state.
In cortical neurons, the coincidence of a backpropagating action
potential with a subthreshold synaptic event gives rise to a
[Ca2+]i transient
that is up to 200% larger than the sum of either event alone, which
links somatic spikes with activated dendritic synapses (Denk et al.,
1995; Yuste and Denk, 1995; Magee and Johnston, 1997; Markram et al.,
1997; Koester and Sakmann, 1998; Schiller et al., 1998; Stuart and
Häusser, 2001). In the present study, the magnitude in
supralinearity was similar, but the source of this supralinearity is
currently unclear in spiny projection neurons. In cortical pyramidal
neurons, calcium, in addition to entering through voltage-gated calcium
channels (VGCCs), also enters through NMDA channels, particularly when
synaptic inputs coincide with backpropagating action potentials
(Koester and Sakmann, 1998; Schiller et al., 1998). In addition,
activation of IP3 and ryanodine receptors
supralinearly increases dendritic
[Ca2+]i by
releasing calcium from internal stores (Nakamura et al., 1999,
2000).
NMDA channels most likely contribute to dendritic
[Ca2+]i in spiny
projection neurons. NMDA receptors have been immunohistochemically localized along their dendrites (Bernard and Bolam, 1998; Gracy et al.,
1999), local NMDA application depolarizes spiny projection neurons
(Cepeda and Levine, 1998; Cepeda et al., 1998), NMDA antagonists reduce
evoked depolarizations (Kita, 1996), and calcium entry through NMDA
receptors triggers immediate early gene expression (Konradi et al.,
1996
TOP
ABSTRACT
INTRODUCTION
