Clinical Neuropharmacology, Max-Planck-Institute of Psychiatry, 80804 Munich, Germany
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ABSTRACT |
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Frick, A.,
W. Zieglgänsberger, and
H.-U. Dodt.
Glutamate Receptors Form Hot Spots on Apical Dendrites of
Neocortical Pyramidal Neurons.
J. Neurophysiol. 86: 1412-1421, 2001.
Apical dendrites of layer V cortical
pyramidal neurons are a major target for glutamatergic synaptic inputs
from cortical and subcortical brain regions. Because innervation from
these regions is somewhat laminar along the dendrites, knowing the
distribution of glutamate receptors on the apical dendrites is of prime
importance for understanding the function of neural circuits in the
neocortex. To examine this issue, we used infrared-guided laser
stimulation combined with whole cell recordings to quantify the spatial
distribution of glutamate receptors along the apical dendrites of layer
V pyramidal neurons. Focally applied (<10 µm) flash photolysis of
caged glutamate on the soma and along the apical dendrite revealed a
highly nonuniform distribution of glutamate responsivity. Up to four
membrane areas (extent 22 µm) of enhanced glutamate responsivity (hot
spots) were detected on the dendrites with the amplitude and integral of glutamate-evoked responses at hot spots being three times larger than responses evoked at neighboring sites. We found no association of
these physiological hot spots with dendritic branch points. It appeared
that the larger responses evoked at hot spots resulted from an increase
in activation of both -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)
receptors and not a recruitment of voltage-activated sodium or calcium
conductances. Stimulation of hot spots did, however, facilitate the
triggering of both Na+ spikes and
Ca2+ spikes, suggesting that hot spots may serve
as dendritic initiation zones for regenerative spikes.
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INTRODUCTION |
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The apical dendrites of layer
V pyramidal neurons of the neocortex are a major receptive zone for
excitatory glutamatergic synaptic input (Peters 1987).
Release of glutamate activates
N-methyl-D-aspartate (NMDA)- and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors at these synapses
(Cauller and Connors 1994
; Jones and Baughman
1988
; Thomson et al. 1993
), and it has been
shown immunocytochemically that these glutamate receptor subtypes are
differentially distributed in the various layers of the neocortex
(Huntley et al. 1994a
; Petralia and Wenthold 1992
). Until recently, however, the specific location and
density of these receptors could not be determined on the surface of
single neocortical neurons (Currie et al. 1994
;
Dodt et al. 1998
).
Although electrophysiological and immunocytochemical studies have shown
that NMDA and AMPA receptors are clustered at spines and often
colocalized (Bekkers and Stevens 1989; Craig et
al. 1994
; Jones and Baughman 1991
; Kornau
et al. 1995
; O'Brien et al. 1998
; Rao
and Craig 1997
), glutamate receptors have also been detected
and recorded at extrasynaptic sites (Clark et al. 1997
; Häusser and Roth 1997
; Rao and
Craig 1997
; Rosenmund et al. 1995
; Spruston et al. 1995
). Dendritic recordings in
hippocampal pyramidal neurons and in cerebellar Purkinje cells have
provided evidence that the functional properties of extrasynaptic and
synaptic glutamate receptors are similar (Häusser and Roth
1997
; Spruston et al. 1995
).
To functionally map glutamate receptor activation on single neurons, we
developed an infrared-guided laser photostimulation technique
(Dodt et al. 1999), combined with whole cell patch-clamp recording, that allowed us to resolve local responses with high temporal and spatial resolution; necessary criteria for mimicking synaptic transmission (Denk 1994
; Dodt et al.
1999
; Katz and Dalva 1994
; Pettit and
Augustine 2000
). The focus of an ultraviolet (UV) laser beam
was visually guided by infrared videomicroscopy to a neuronal structure
in the brain slice, and flashes of UV light caused the release of
glutamate by localized photolysis from the caged compound. The ability
to focally release glutamate (<10 µm) at multiple sites on a single
neuron in brain slices overcomes limitations inherent to other modes of
glutamate application, such as iontophoretic application or electrical
stimulation. Furthermore, and in contrast to binding studies using
labeled ligands, functional receptors can be assigned to a particular
compartment of a neuron.
We report a high resolution-mapping of functional glutamate receptors,
including areas of high density (hot spots), along the apical dendrite
of neocortical pyramidal neurons in brain slices. The results described
herein cannot distinguish between synaptic and extrasynaptic glutamate
receptors, but, given the significantly greater density of glutamate
receptors at synaptic sites compared with extrasynaptic sites
(Craig et al. 1994; Jones and Baughman
1991
; Kornau et al. 1995
; O'Brien et al.
1998
; Rao and Craig 1997
), our results are more
likely to reflect this distribution of receptor sites. Hot spots of
glutamate receptors might be strategically located on the dendritic
tree and could have an important influence on neural information
processing for these neurons. We show that the generation of
Ca2+ spikes is facilitated by stimulation of
these hot spots, which are, for the most part, not associated with
dendritic branch points (sites that have been hypothesized to be
Ca2+ spike "hot spots") (Llinas and
Hess 1976
; Llinas and Sugimori 1980
). These
findings might contribute to the understanding of the synaptic
connectivity in neocortical circuits (Denk 1994
; Huntley et al. 1994b
; Katz and Dalva
1994
), as well as of the synaptic integration in dendrites
(Johnston et al. 1996
; Magee et al. 1998
;
Stuart et al. 1997
; Yuste and Tank 1996
).
Part of this study has been presented in abstract form (Frick et
al. 1998
).
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METHODS |
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Electrophysiology and data acquisition
Patch-pipette recordings were obtained from the somata of layer
V pyramidal neurons in parasagittal neocortical slices (300 µm thick)
from 15- to 25-day-old male Sprague-Dawley rats, as described
previously (Dodt et al. 1998). Individual neurons were visualized using the newly developed infrared "gradient contrast" (IR-GC) optics (Dodt et al. 1999
). The extracellular
solution consisted of the following (in mM): 125 NaCl, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2
(saturated with 95% O2-5%
CO2, pH 7.4). Patch pipettes (DC resistance 4-7
M
) were filled with the following (in mM): 130 K-gluconate, 5 KCl,
10 HEPES, 0.5 EGTA, 2 Mg-ATP, and 5 glucose, pH 7.2, and in some cases
Neurobiotin (5 mg/ml). Whole cell patch-pipette recordings were made
with a SEC 1 l amplifier (npi Electronics, Tamm, Germany) with
appropriate bridge and capacitance compensation. Series resistances ranged from 8 to 25 M
and were monitored on-line. Recordings were
made at room temperature (22-24°C). Data were low-pass filtered at
0.3-1.5 kHz, digitized at 1-5 kHz (ITC 16 A/D board, Instrutec, New
York), and stored and analyzed using Pulse 8.11 (HEKA, Germany) and
Igor Pro 3 (WaveMetrics). Surface and image plots were constructed using Surfer32 (Golden Software). Neurons included in this study had
resting potentials of
61 ± 3 mV (mean ± SD). Cell input
resistances ranged from 44 to 179 M
, and action potential
amplitudes, measured from resting membrane potential to peak, ranged
from 90 to 116 mV. Video images were taken during the experiments to
document the position of the soma being recorded, of the stimulation
sites on the neuronal surface and of a reference cell, which was filled with Neurobiotin at the end of the experiment. The distance between the
recorded neuron and the reference cell served as a measure of a
potential tissue shrinkage after fixation. After electrophysiological recording, the slices were fixed in 4% paraformaldehyde, subsequently stained using cy3-streptavidin, and stained neurons were imaged with a
fluorescence microscope. Slices were not dehydrated after fixation to
minimize tissue shrinkage and were mounted in an aqueous mounting
medium. If necessary, minimal corrections for the position of the
branch points were made for tissue shrinkage. Each stained cell had a
pyramidal-shaped soma in layer V and an apical dendrite extending to
the pial surface with a terminal tuft, and with the first major branch
point 484 ± 129 µm from the soma (mean ± SD, n = 11). When 500 µM CdCl2 was
used, NaH2PO4 was omitted
in the bath to avoid precipitation. Statistical analysis was performed using unpaired two-tailed t-tests.
Infrared-guided laser photostimulation
Caged glutamate (-CNB-caged L-glutamic acid,
Molecular Probes, Amsterdam) was flash photolyzed by a 354-nm UV laser
beam (Liconix), using 3- to 5-ms shuttered pulses (UniBlitz shutter driver/timer, Rochester, New York) (Callaway and Katz
1993
). The properties of the caged glutamate have been
described elsewhere (Wieboldt et al. 1994
). In short,
the caged glutamate used in this study has a quantum product yield of
0.14, and the half-life of the major component of the photolytic
reaction is in the microsecond range (Wieboldt et al.
1994
). The beam of the laser was directed via a multimode
optical fiber into the epifluorescence port of the microscope (Zeiss
Axioskop), and focused through a ×60/0.9 NA objective lens (Olympus)
to a spot about 1 µm wide (see also Fig.
1). The intensity of the UV light at the
front of the objective was 0.5-2 mW. The microscope was mounted on a
motorized, three-dimensional stage (Luigs and Neumann, Ratingen,
Germany) connected to a personal computer. After establishing a
stable whole cell recording, 1-mM caged glutamate was added to a small
volume (3 ml) of the bath solution, reoxygenated, and recirculated.
Caged glutamate was bath applied for at least 15 min before a visually
guided light spot (using IR-GC optics) was moved in small steps (with
glutamate responses evoked at each step) from the soma to the distal
apical dendrite with a frequency of the flash photolysis of
0.1 Hz. The displacement of the laser beam was performed by moving the microscope. Application of 1-mM caged glutamate itself had no effect on
the neuronal activity of the recorded cells. It has previously been
shown that
-CNB-caged glutamate, at 1 mM concentration, does not
desensitize glutamate receptors in hippocampal neurons, and that caged
glutamate does not inhibit the activation of the glutamate receptors by
50 µM glutamate (Wieboldt et al. 1994
). Likewise,
exposure of cells to the relatively brief periods of uncaging light
used in these experiments (in the absence of caged glutamate) had no
effect on neuronal activity. The depth of the somata and dendrites
stimulated was generally within the first 60 µm from the surface of
the brain slice, where dendrites could still be visualized, but in most
cases even closer to the surface.
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RESULTS |
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After establishing stable whole cell patch-clamp recordings from
the somata of layer V pyramidal neurons in neocortical slices, the
recording chamber was perfused with solution containing 1 mM caged
glutamate (Wieboldt et al. 1994). With apparatus
developed for infrared-guided laser photostimulation (Fig. 1,
A and B), we were able to visually direct a
1-µm spot of UV light onto the surface of the neuron of interest.
Because the uncaging in the z-axis depends on the precise
focus onto the center of the dendrite, the focal points of the IR image
of the neuron and of the UV-light flashes were well aligned (Fig. 1).
The measured spatial variations in the glutamate responsivity were
therefore not due to a variability in the z-axis uncaging of
glutamate. UV-light flashes (duration: 3-5 ms) delivered to the soma
and various sites along the apical dendrite (Fig. 1B)
elicited fast depolarizing deflections of the membrane potential. We
first determined the resolution of the active region by recording
responses evoked as the UV-light spot was moved incrementally away from
the dendrite. In this series of experiments, photolytically evoked
responses decayed with increasing distance. A Gaussian curve was fitted
to the data (R = 0.998, n = 6), and the
accuracy was determined to be within 4.8 µm laterally (half-maximal
peak amplitude, Fig. 1C) and 18 µm in the
z-axis. The value for the depth resolution has been
established previously (Dodt et al. 1999
). Based on
these results, we tested the glutamate responsivity for each neuron by
applying the UV-light flashes to 30-120 different sites (mean: 76 sites) along the apical dendrite at ~5-µm steps. In one case, a
region on the apical dendrite ~640 µm from the soma was evaluated.
The evoked responses (Fig. 1D) were strongly attenuated when
1 µM 2,3-dihydoxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) was added to the perfusion medium (response amplitude
33 ± 4% of control, mean ± SE, n = 11),
and the residual response was almost completely blocked by adding 100 µM D-2-amino-5-phosphonovaleric acid (D-APV;
7 ± 1%, n = 3). These findings demonstrate that
the responses evoked by glutamate photolysis resulted from a
co-activation of AMPA and NMDA receptors, and that dendritic kainate
receptors play only a minor role in these neurons.
Profile of glutamate responsivity along the apical dendrite
Mapping glutamate receptors along the apical dendrites
(n = 36) revealed a nonuniform distribution of the
receptors (Figs. 2,
3, and 5B). UV flashes were
standardized across experiments for laser intensity and duration by
eliciting responses of 4-9 mV when delivered to the soma. The response
amplitude decreased monotonically as the site of stimulation was moved
along the first 50 µm of the apical dendrite away from the soma (Fig.
2B), which is probably due to a small density of spines in
this region (Kunz et al. 1972). With distances >50 µm
from the soma, however, the membrane of the apical dendrite became
progressively more sensitive with distance, eventually exceeding the
somatic sensitivity (i.e., the same intensity and duration of the UV
flashes evoked bigger responses compared with a somatic stimulation).
The increase in responsivity with distance is most easily explained by
an increase in spine density that occurs beyond the proximal 50 µm of
the dendrite (Kunz et al. 1972
). Up to four restricted
areas of high sensitivity (hot spots) were found at dendritic regions
80-600 µm from the soma (Figs. 2, A and B, 3, and 5B). An electrophysiological hot spot was defined as
having a response to glutamate, under conditions where
voltage-dependent Na+ and
Ca2+ conductances are blocked, that was twofold
or more greater than the response recorded by stimulation at an
adjacent site, 5 µm away. The large responses evoked at hot spots
often triggered TTX-sensitive Na+ spikes (not
shown) and Cd2+-sensitive
Ca2+ spikes (see Enhancement of dendritic
calcium spikes at hot spots). In 34 of 36 neurons, at
least one hot spot was located on the apical dendrite at a distance
between 80 and 250 µm from the soma. The amplitude and integral of
glutamate-evoked responses elicited beyond 250 µm from the soma
decreased with increasing distance from the soma (Fig. 2B).
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To verify that the photostimulation method was reliable and that the glutamate responsivity profile of a single neuron could be measured in a reproducible manner, we repeated the stimulation protocol along the same apical dendrite. In all cases (n = 6), the overall profile of glutamate responsivity was the same, and most notably, the location of the hot spots was identical, suggesting that the hot spots were not due to some experimentally introduced artifact such that might occur with slice drift or temporal changes in dendritic excitability.
The decay of the responses was slowest at the most proximal test sites
and became progressively faster as the stimulation site was moved to
the more distal test sites (R2 = 0.79, linear regression fit, n = 14; Fig. 2C). For
responses induced at 500 µm, the decay time constant () was
~50% of responses evoked at the soma (n = 8; Fig.
2C). We also found a decrease in the half-maximal width of
the responses from proximal to more distal dendritic stimulation sites,
but no significant change in the 20-80% rise time values with
distance from the soma (not shown).
Responsivity profile does not reflect activation of voltage-activated calcium and sodium conductances
To test whether voltage-activated Na+ and Ca2+ conductances might be responsible for the spatial variations in responsivity, we bath applied both TTX (1 µM) and various concentrations of CdCl2 (200-500 µM, n = 16 cells). Blocking these conductances accelerated the time course and decreased the peak amplitude and integral of the responses evoked at the soma and along the dendrite. For subthreshold responses in the presence of TTX and Cd2+, peak amplitude, integral, 20-80% rise time, half-width, and the decay time constant were decreased by 23 ± 2%, 47 ± 3%, 40 ± 2%, 44 ± 2%, and 35 ± 2%, respectively (relative to control; mean ± SE, n = 5 cells). These data show that TTX- and Cd2+-sensitive conductances significantly increased the charge that reached the soma and axon. However, regardless of the degree of blockade of these conductances in the presence of TTX and Cd2+, the overall profile of glutamate responsivity (including the hot spots) persisted (Figs. 2B and 5B), indicating that it was not caused by activation of either Na+ or Ca2+ conductances. These results suggest strongly that the glutamate responsivity profile reflects a nonuniform distribution of glutamate receptors along the apical dendrite of neocortical pyramidal neurons.
Both AMPA and NMDA components are elevated at hot spots
Under the conditions where Na+ and
Ca2+ conductances were blocked (Figs.
2B, 4, and
5B), the most pronounced hot
spots were found at a distance of 170 ± 61 µm (mean ± SD;
range: 88-357 µm) from the soma with an extent of 22 ± 10 µm
(range: 9-48 µm; n = 16; Fig. 4A). Figure
4C compares responses evoked at these hot spots, a dendritic
region adjacent to them, and the soma (examples are given in Fig.
4B). Peak amplitudes and integrals of responses evoked at
hot spots were on average three times larger as compared with the soma
and adjacent sites (t-test, P < 0.001).
Responses elicited at the soma had 20-80% rise times of 8 ± 1 ms (mean ± SE) and decay time constants () of 72 ± 5 ms.
For responses induced at hot spots, 20-80% rise times were longer
(11 ± 2 ms) and decay times faster (57 ± 4;
P < 0.01). No significant differences were observed
for the 20-80% rise times between the soma and the adjacent sites and
for the half-maximal width values between the three stimulation sites.
In addition, the decay time constants of responses at hot spots and at
adjacent sites were not significantly different.
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To explore whether the activation of both AMPA and NMDA receptors contribute to the hot spots of glutamate responsivity, we selectively blocked AMPA receptors by applying 1 µM NBQX. NBQX reduced the peak amplitude of responses evoked at hot spots by 72%, and the integral by 48% (n = 8). NBQX had a similar effect on responses evoked at dendritic sites adjacent to hot spots, strongly suggesting that the ratio of AMPA and NMDA receptors does not differ between hot spots and neighboring dendritic regions.
Hot spots and dendritic branch points do not overlap
Previous reports showing intradendritic recordings from cerebellar
Purkinje cells suggested that multiple spike generation zones ("hot
spots") exist in the dendritic tree, probably at or near dendritic
bifurcations (Llinas and Hess 1976; Llinas and Sugimori 1980
). To examine the question of whether hot spots of glutamate responsivity are located at branch points, and therefore could be the basis for spike generation zones at these sites, we
injected Neurobiotin intracellulary to label the dendritic tree and
determined the position of the branch points using fluorescence microscopy (n = 15; Fig. 5). The experiments were
carried out in control solution (Fig. 5B, neurons
12-15) or in the presence of TTX (1 µM) and
Cd2+ (200-500 µM; Fig. 5B,
neurons 1-11). The glutamate responsivity profile along the
somato-dendritic axis of the neurons is shown by color coding the
response amplitudes (Fig. 5B). The dendritic branch points
(small horizontal bars) are superimposed on this activity profile to
compare the position of the hot spots and dendritic branch
points. Although in several cases the branch points occurred within the
range or in close proximity of the hot spots, the majority of the
branch points did not overlap with the hot spots. In addition, in cases
where branch points could be detected with infrared videomicroscopy,
photolytic stimulation at these sites did not result in bigger
responses as compared with adjacent sites. Plotting the number of hot
spots and branch points as a function of the distance from the soma
revealed that most hot spots and branch points were found between 50 and 250 µm from the soma (Fig. 5C). In contrast to branch
points, which were also located along the first 50 µm of the dendrite
and beyond 250 µm from the soma, hot spots were not found in these
regions, providing further evidence against an association of hot spots and dendritic branch points. To demonstrate this statistically, we
divided our data into two groups (branch points and hot spots) and
performed unpaired two-tailed t-tests to determine the
relationship between hot spots and branch points within each group.
When we considered the group of hot spots separately (n = 24), we found that there was no bias for their colocalization either
with or without branch points. However, when we considered the group of branch points, there was a strong significant difference between the
number of branch points associated with a hot spot and the number that
was not associated with a hot spot (P < 0.001, n = 147). This analysis confirmed that there was no
particular bias for hot spots to occur at branch points.
Enhancement of dendritic calcium spikes at hot spots
The dendrites of neocortical pyramidal neurons are known to
contain a rich collection of active Ca2+
conductances (Deisz et al. 1991; Kim and Connors
1993
; Markram and Sakmann 1994
; Markram
et al. 1995
; Schiller et al. 1995
; Yuste et al. 1994
) that can support Ca2+ action
potentials (Amitai et al. 1993
; Kim and Connors
1993
; Schiller et al. 1997
; Schwindt and
Crill 1997
; Seamans et al. 1997
). In particular,
the observation that Ca2+ spikes can arise in the
dendrites of several neural cell types has changed our conception of
how a neuron processes synaptic input (for reviews see Johnston
et al. 1996
; Yuste and Tank 1996
). To determine
whether Ca2+ spikes can be initiated anywhere
along the dendrite, or whether glutamate receptor hot spots might
provide a trigger site for the initiation of regenerative
Ca2+ responses, we recorded from neurons in the
presence of TTX (1 µM). We found that Ca2+
spikes were triggered most easily following stimulation of hot spots as
compared with other sites on the neuron (Figs. 2A and 3),
consistent with the hypothesis that hot spots provide a facilitatory role for generation of Ca2+ spikes. To
photolytically evoke Ca2+ spikes at the soma and
multiple dendritic sites, we adjusted the duration or intensity of the
light flashes (n = 17; Fig.
6). At distances >100 µm from the
soma, the amplitude and threshold of the Ca2+
spikes, measured at the soma, decreased as the UV-light spot was moved
farther from the soma (see Fig. 6A for determination of
amplitude and threshold). At ~450 µm, the amplitude of the Ca2+ spikes had declined from 53 mV by
stimulation at the soma to 30 mV (P < 0.01; Fig.
6B), and the threshold from 23 to 13 mV (P < 0.01; Fig. 6C). In the presence of 1 µM TTX and 10 mM
TEA to block both Na+ and
K+ channels, the amplitudes of the photolytically
evoked Ca2+ spikes were large
(n = 9; Fig. 6). Although the amplitude of the
dendritic Ca2+ spikes was large in TEA (on
average 83 mV; Fig. 6B), their apparent threshold was still
quite low (55% at 450 µm compared with soma, P < 0.01, n = 13; Fig. 6C). The similarity
between amplitude, but not threshold, of the photolytically evoked
Ca2+ spikes in TTX/TEA suggests that the spikes
were initiated locally in the dendrite and, because dendritic and
somatic K+ currents were reduced by TEA,
propagated actively to the soma.
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DISCUSSION |
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Glutamate receptors are non-uniformly distributed along apical dendrites of neocortical pyramidal neurons
We have used local photolysis of caged glutamate to map regional
differences in the distribution of glutamate receptors across a wide
range of the main apical dendrites in layer V pyramidal neurons in
neocortical slices. The main findings are that the responsivity to
glutamate is non-uniformly distributed along the apical dendrite, and
that the glutamate receptors form up to four hot spots of glutamate
responsivity (extent 22 µm) within the region of the dendrite under
investigation (up to 640 µm). The evidence showing this non-uniform
distribution pattern of glutamate receptors is twofold: first,
photolytically induced glutamate responses vary as a function of
distance along the dendrite, suggesting that the density of glutamate
receptors differs (see Figs. 2, A and B,
3B, and 5B); second, blocking voltage-activated
sodium and calcium conductances does not change the responsivity
profile obviating the possibility that the differences in response
magnitude are due to a non-uniform distribution of voltage-activated
conductances (see Fig. 2B). These results are consistent
with the results of other studies showing that the distribution of AMPA
receptors are non-uniform with respect to distance along apical
dendrites of neocortical neurons (Dodt et al. 1998) and
hippocampal pyramidal neurons (Magee and Cook 2000
;
Pettit and Augustine 2000
). With the exception of hot
spots, the amplitude and integral of the glutamate-evoked responses
elicited beyond 250 µm from the soma decreased with increasing
distance. This decline could be caused by a decreased membrane area
exposed to glutamate, electrotonic attenuation (Rall
1977
; Spruston et al. 1993
), or higher densities of transient A-type K+ currents (Hoffman
et al. 1997
) or hyperpolarization-activated currents
(Ih) (Magee 1998
;
Stuart and Spruston 1998
) in the distal apical dendrites.
The decay of the responses was slowest at the soma and became
progressively faster as the stimulation site was moved to the more
distal dendrite (see Fig. 2C). Although not investigated here, this might result from an elevated dendritic density of A-type
K+ channels (Hoffman et al. 1997)
or of Ih channels (Magee
1998
; Stuart and Spruston 1998
), a stronger
contribution of AMPA compared with NMDA receptors (Cauller and
Connors 1994
; Dodt et al. 1998
), a lower
membrane resistivity in the distal dendrites (Stuart and Spruston 1998
), or some combination thereof.
Densities of AMPA and NMDA receptors are elevated at hot spots
We found that both AMPA and NMDA receptors were concentrated in
higher numbers at hot spots, and that stimulation at these sites under
conditions where Na+ and
Ca2+ conductances were blocked, resulted in a
threefold greater response compared with adjacent dendritic sites (see
Fig. 4). The kinetics of the responses evoked at hot spots and adjacent
sites were not statistically different and blockade of the AMPA
receptors with NBQX reduced the responses for both sites to the same
degree. Electrophysiological and anatomical studies of cultured rat
visual cortex (Jones and Baughman 1991), hippocampal
neurons (Bekkers and Stevens 1989
; Benke et al.
1993
; Craig et al. 1994
), and spinal cord
(Vogt et al. 1995
), as well as of neocortical neurons in brain slices (Aghajanian and Marek 1997
) suggest that
subtypes of glutamate receptors are colocalized (Bekkers and
Stevens 1989
; Jones and Baughman 1991
) and
clustered (Aghajanian and Marek 1997
; Benke et
al. 1993
; Craig et al. 1994
; Jones and
Baughman 1991
; Vogt et al. 1995
) at synapses. On
the basis of these and other studies, extrasynaptic receptors may make
an insignificant contribution to the profile of glutamate responsivity
reported here.
Hot spots and dendritic branch points are not spatially correlated
Previous studies of Purkinje cells (Llinas and Hess
1976; Llinas and Sugimori 1980
) suggested that
many dendritic sites were capable of initiating
Ca2+ spikes (hot spots), and that these sites
were located probably at or near dendritic bifurcations. Our study of
pyramidal neurons in the neocortex shows that there is no statistical
relationship between hot spots of glutamate responsivity and
morphological identified branch points on the apical dendrites (see
also Fig. 5). One might speculate that the hot spots of glutamate
responsivity described in our study are strategically located, probably
in a layer- and input-specific manner, but not necessarily at branch points. It remains to be shown whether the density of
Ca2+ channels is elevated at bifurcations, and
whether spike initiation zones are located at these sites.
Hot spots of glutamate responsivity are preferential trigger sites for calcium spikes
We demonstrated that when Na+ channels were
blocked (with the addition of TTX), Ca2+ spikes
could be triggered in the dendrite by photolytically released glutamate
(see Figs. 2A, 3B, and 6).
Ca2+ spikes could be evoked at multiple sites
along the dendrite, although most easily at hot spots, and the
amplitude and apparent threshold of the spikes decreased with
increasing distance of the stimulation site from the soma. These
findings support the hypothesis that in neocortical pyramidal neurons
many dendritic sites are capable of initiating
Ca2+ spikes (Schwindt and Crill
1997). In the presence of TTX and TEA, the amplitudes of the
photolytically evoked Ca2+ spikes were large, but
their apparent thresholds were 45% lower compared with the
Ca2+ spikes evoked by photolysis at the soma.
Similar findings were obtained by Schwindt and Crill
(1997)
, who applied glutamate iontophoretically at sites of the
apical dendrite to evoke Ca2+ spikes. In
agreement with several previous reports (Reuveni et al.
1993
; Schwindt and Crill 1997
; Stafstrom
et al. 1985
; Yuste et al. 1994
), we conclude
that dendritic and somatic K+ channels normally
prevent the active propagation of large-amplitude Ca2+ spikes from the site of initiation to the soma.
Mechanism underlying the hot spots of glutamate responsivity
What is the mechanism of the hot spots of glutamate
responsivity? There are several possibilities: dendritic branch points, increase in agonist-affinity or single-channel conductance, elevated glutamate receptor number or density per synapse, or increases in the
density of spines. Branch points seem unlikely to be responsible. If
this was the case, we should see an association of the dendritic branch
points with the physiological hot spots, but our statistical analysis
has indicated that this does not occur. Increases in affinity or
single-channel conductance also seem unlikely to account for hot spots.
Dendritic recordings in hippocampal pyramidal neurons and in cerebellar
Purkinje cells have provided evidence that the functional properties of
dendritic glutamate receptors, at least along the first part of the
dendrite, are similar (Häusser and Roth 1997;
Spruston et al. 1995
). Therefore it seems most likely that the hot spots (~22 µm in extent) described in our study arise from either higher densities of synapses or more receptors per synapse
in these regions. It has been shown that Schaffer collateral synapses
have a wide range of surface areas, and that the number of glutamate
receptors at these synapses is directly related to the area
(Nusser et al. 1998
; Takumi et al. 1999
).
Thus it is possible that glutamate uncaging at larger synapses would
open more glutamate receptors, and a cluster of those synapses with a
larger area could explain an elevated dendritic glutamate sensitivity of a membrane area the size of a hot spot (extent ~22 µm). Unitary excitatory postsynaptic potentials evoked between layer V pyramidal neurons have been found to be variable in size (e.g., Markram et
al. 1997
; Thomson et al. 1993
), but both pre-
and postsynaptic mechanisms could account for this observation. It is
difficult to compare these data with our own study, because the spatial resolution of the laser photostimulation system is unlikely to activate
single but rather several postsynaptic sites, and presysnaptic mechanisms only play a minor or no role. Either of the two mechanisms (cluster of particularly strong synapses or increase in density of
synapses) should correlate with an elevated dendritic glutamate sensitivity of a hot spot. Ninety-four percent of the cells
investigated had at least one hot spot in layer IV, and hot spots may
be a correlate of functional synaptic connections; it remains to be established which connections form these hot spots.
In conclusion, hot spots may provide a mechanism for weighting
dendritic inputs and/or triggering local Na+- and
Ca2+-dependent action potentials, either of which
could increase the effectiveness of synapses by compensating for
electrotonic attenuation. Infrared-guided laser photostimulation may
also be used to clarify how changes in glutamate receptor density and
active properties of the dendrites interact to allow
location-independent transmission of synaptic responses to the soma
(Cook and Johnston 1999; Magee and Cook
2000
), or linear summation of synaptic responses activated at
different dendritic locations (Cash and Yuste 1999
).
Other relevant issues related to the distribution and clustering of glutamate receptors concern synaptic plasticity or synaptic
connectivity; for example, what are the implications of hot spots for
the events that underlie synaptic remodeling during associative
synaptic modification (Carroll et al. 1999
), and how
might changes in receptor density correlate to specific inputs from the
thalamus or cortical areas?
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ACKNOWLEDGMENTS |
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We thank D. Johnston, N. Spruston, and M. Yeckel for discussions and comments on the manuscript.
This work was supported by a grant from Sonderforschungsbereich (SFB) 391 to H.-U. Dodt and W. Zieglgänsberger.
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FOOTNOTES |
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Present address and address for reprint requests: A. Frick, Div. of Neuroscience, S 700, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail: andreas{at}ltp.neusc.bcm.tmc.edu).
Received 29 December 2000; accepted in final form 14 May 2001.
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REFERENCES |
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