* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Glutamate Receptors Form Hot Spots on Apical Dendrites of
Electrophysiology wikipedia , lookup
Mirror neuron wikipedia , lookup
Neural oscillation wikipedia , lookup
Multielectrode array wikipedia , lookup
Caridoid escape reaction wikipedia , lookup
Central pattern generator wikipedia , lookup
Aging brain wikipedia , lookup
Axon guidance wikipedia , lookup
Neural coding wikipedia , lookup
End-plate potential wikipedia , lookup
Single-unit recording wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Development of the nervous system wikipedia , lookup
Neuromuscular junction wikipedia , lookup
Biological neuron model wikipedia , lookup
Signal transduction wikipedia , lookup
Dendritic spine wikipedia , lookup
Spike-and-wave wikipedia , lookup
Circumventricular organs wikipedia , lookup
Holonomic brain theory wikipedia , lookup
Optogenetics wikipedia , lookup
Neuroanatomy wikipedia , lookup
Neurotransmitter wikipedia , lookup
Nonsynaptic plasticity wikipedia , lookup
Evoked potential wikipedia , lookup
Endocannabinoid system wikipedia , lookup
Long-term depression wikipedia , lookup
Activity-dependent plasticity wikipedia , lookup
Nervous system network models wikipedia , lookup
NMDA receptor wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Synaptogenesis wikipedia , lookup
Synaptic gating wikipedia , lookup
Pre-Bötzinger complex wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
Chemical synapse wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Glutamate receptor wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Apical dendrite wikipedia , lookup
Glutamate Receptors Form Hot Spots on Apical Dendrites of Neocortical Pyramidal Neurons A. FRICK, W. ZIEGLGÄNSBERGER, AND H.-U. DODT Clinical Neuropharmacology, Max-Planck-Institute of Psychiatry, 80804 Munich, Germany Received 29 December 2000; accepted in final form 14 May 2001 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-hydroxy5-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 m) 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, 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: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. INTRODUCTION 1412 0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 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 m) 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 m) 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-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methylD-aspartate (NMDA) receptors and not a recruitment of voltageactivated 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. GLUTAMATE HOT SPOTS ON NEOCORTICAL DENDRITES 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). METHODS 1413 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 m 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 Electrophysiology and data acquisition 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 J Neurophysiol • VOL FIG. 1. Infrared-guided laser photostimulation combined with whole cell patch-clamp recording in the somatosensory cortex of rat brain slices. A: schematic representation of the experimental set-up. B: schematic representation of the recording and stimulation geometry [the diameter of the ultraviolet (UV) spots not to scale]. Montage of photographs of an infrared (IR) videomicroscopy image of a layer V pyramidal neuron. The pial surface of the neocortex is toward the top. A and B: neurons in the brain slice were visualized by using a combination of illumination with infrared light and the gradient contrast system. At the same time, light pulses of a UV laser were directed via a quartz fiber into the microscope and by a dichroic mirror onto the recorded neuron. The intensity of the UV light at the front of the objective was 0.5–2 mW. The microscope could be positioned in x-y-z by remote controls. The laser spot of an optical diameter of 1 m formed by the objective in the specimen plane was made visible before the experiment by a fluorescent paper, and its position was marked on the TV monitor. By positioning the site of the neuron to be stimulated at this point, the laser stimulation could be precisely guided by visual control. C: lateral resolution of the laser stimulation. The data were fitted with a Gaussian function (R ⫽ 0.998, n ⫽ 6). Half-maximal peak amplitude was evoked when the light spot was moved 4.8 m away from the dendrite. Data are normalized to values at 0 m. D: focal photolysis of caged glutamate elicits fast glutamatergic responses that are mediated by N-methyl-Daspartate (NMDA) and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Bath application of the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) (1 M) attenuates glutamatergic responses. The residual response is abolished by addition of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV; 100 M). 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 Patch-pipette recordings were obtained from the somata of layer V pyramidal neurons in parasagittal neocortical slices (300 m 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 m from the soma (mean ⫾ SD, n ⫽ 11). When 500 M CdCl2 was used, NaH2PO4 was omitted in the bath to avoid precipitation. Statistical analysis was performed using unpaired two-tailed t-tests. 1414 A. FRICK, W. ZIEGLGÄNSBERGER, AND H.-U. DODT evoked at each step) from the soma to the distal apical dendrite with a frequency of the flash photolysis of ⱕ0.1 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 M 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 m from the surface of the brain slice, where dendrites could still be visualized, but in most cases even closer to the surface. RESULTS 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 J Neurophysiol • VOL Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 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-m 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 m laterally (half-maximal peak amplitude, Fig. 1C) and 18 m 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-m steps. In one case, a region on the apical dendrite ⬃640 m from the soma was evaluated. The evoked responses (Fig. 1D) were strongly attenuated when 1 M 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 M 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. FIG. 2. Glutamate receptor mapping. The peak amplitude and decay values of the responses are plotted as a function of the stimulation distance from the soma. A: glutamate responsivity profile for 7 neurons recorded in solution containing TTX. All points are response amplitudes that were normalized to the amplitudes evoked by stimulation at the soma (100%). Ca2⫹ spikes were often evoked at the hot spots. B: summary data of control (●, n ⫽ 14), and in solution containing 1 M TTX ⫹ 200 –500 M Cd2⫹ (䊐, n ⫽ 16). Dendritic response amplitudes are normalized to the amplitude evoked by UV flashes delivered to the soma (mean ⫾ SE). C: the decay kinetics of the glutamate responses became faster as the site of dendritic stimulation was moved farther from the soma (R2 ⫽ 0.79, linear regression fit, n ⫽ 14). All points are mean ⫾ SE. decreased monotonically as the site of stimulation was moved along the first 50 m of the apical dendrite away from the soma (Fig. 2B), which is probably due to a small density of spines in 86 • SEPTEMBER 2001 • www.jn.org GLUTAMATE HOT SPOTS ON NEOCORTICAL DENDRITES 1415 this region (Kunz et al. 1972). With distances ⬎50 m 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 m of the dendrite (Kunz et al. 1972). Up to four restricted areas of high sensitivity (hot spots) were found at dendritic regions 80 – 600 m 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 m 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 m from the soma. The amplitude and integral of glutamate-evoked responses elicited beyond 250 m from the soma decreased with increasing distance from the soma (Fig. 2B). 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 J Neurophysiol • VOL 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 m, 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 voltageactivated 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 M) and various concentrations of CdCl2 (200 –500 M, 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 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 FIG. 3. Dendritic hot spots of glutamate sensitivity in a neocortical pyramidal neuron of layer V. Fluorescent image (A) taken from the neuron following intracellular filling with Neurobiotin and staining with cy-3. Scale bar, 20 m. B: surface plot of responses to focal glutamate photolysis at the soma and along the 1st 400 m of the apical dendrite, showing several dendritic hot spots in this neuron. Note that Ca2⫹ spikes were evoked at the hot spots. Recordings were obtained in solution containing 1 M TTX. 1416 A. FRICK, W. ZIEGLGÄNSBERGER, AND H.-U. DODT 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. 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 M 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 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 m (mean ⫾ SD; range: 88 –357 m) from the soma with an extent of 22 ⫾ 10 m (range: 9 – 48 m; 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 J Neurophysiol • VOL 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 FIG. 4. Properties of responses evoked at hot spots of neurons recorded in solution containing 1 M TTX ⫹ 200 –500 M CdCl2 (n ⫽ 16). A: the most prominent hot spots were measured 170 ⫾ 61 m from the soma with an extent of 22 ⫾ 10 m (mean ⫾ SD). B: example of the significantly larger response evoked at a hot spot compared with the soma or an adjacent region in one neuron. C: comparison of the peak amplitude, integral, 20 – 80% rise time, half-width (duration at 1⁄2 of peak amplitude), and decay values () for responses evoked at hot spots (■), at regions close to the hot spots (s), and at the somata (䊐) in 16 neurons. P ⬍ 0.01 for hot spot/adjacent site (⫹), and for hot spot/soma (*). 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 M) and Cd2⫹ (200 –500 M; 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 m from the soma (Fig. 5C). In contrast to branch points, which were also located along the first 50 m of the dendrite and beyond 250 m 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. GLUTAMATE HOT SPOTS ON NEOCORTICAL DENDRITES 1417 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 M). 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 faciliJ Neurophysiol • VOL tatory 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 m 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 m, 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 M 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 m 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 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 FIG. 5. Localization of physiological hot spots with respect to branch points of the apical dendrites (n ⫽ 15). Fluorescent image (A) taken from 2 neurons following intracellular filling with Neurobiotin and staining with cy-3. Scale bar, 20 m. The scaling does not relate to B. B: color-coded plots of response amplitudes to focal glutamate photolysis at the soma and along the 1st 500 m of the apical dendrite, showing several dendritic hot spots in these neurons. Recordings were obtained in control solution (neurons 12–15) or in the presence of TTX (1 M) and Cd2⫹ (100 –500 M; neurons 1–11). Hot spots are colored in red. The positions of the dendritic branch points was determined using fluorescence microscopy of the Neurobiotin-cy3–stained neurons and superimposed on the activity pattern as small horizontal bars. C: statistical distribution of hot spots and dendritic branch points along the dendrite. The normalized numbers of branch points and of hot spots are plotted against the distance from the soma. As not all dendrites could be followed in the infrared image for stimulation up to a distance of 500 m from the soma, the numbers were normalized, by dividing the absolute values by the number of neurons for this dendritic segment (given in parentheses). 1418 A. FRICK, W. ZIEGLGÄNSBERGER, AND H.-U. DODT 2⫹ FIG. 6. Ca spikes evoked by somatic and dendritic depolarization. A: examples of Ca2⫹ spikes evoked at the soma and at a dendritic site in TTX (black line) and in TTX/TEA (gray line) in one neuron. Arrows and triangles indicate apparent threshold and peak amplitude of the responses, respectively. Plot of Ca2⫹ spike amplitude (B), measured from somatic membrane potential to peak, and apparent threshold (C), evoked by somatic and dendritic glutamate photostimulation at various distances from the soma. Ca2⫹ spikes were measured in solution containing 1 M TTX (●, n ⫽ 17), and in solution containing 1 M TTX ⫹ 10 mM TEA (‚, n ⫽ 9). dendrite and, because dendritic and somatic K⫹ currents were reduced by TEA, propagated actively to the soma. DISCUSSION 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 m) within the region of the dendrite under investigation (up to 640 m). 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 J Neurophysiol • VOL 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 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 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 m 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. GLUTAMATE HOT SPOTS ON NEOCORTICAL DENDRITES 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 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 m 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 J Neurophysiol • VOL 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 m). 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? 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. REFERENCES AGHAJANIAN GK AND MAREK GJ. Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Neuropharmacology 36: 589 –599, 1997. AMITAI Y, FRIEDMAN A, CONNORS BW, AND GUTNICK MJ. Regenerative activity in apical dendrites of pyramidal cells in neocortex. Cereb Cortex 3: 26 –38, 1993. BEKKERS JM AND STEVENS CF. NMDA and non-NMDA receptors are colocalized at individual excitatory synapses in cultured rat hippocampus. Nature 341: 230 –233, 1989. BENKE TA, JONES OT, COLLINGRIDGE GL, AND ANGELIDES KJ. N-methyl-Daspartate receptors are clustered and immobilized on dendrites of living cortical neurons. Proc Natl Acad Sci USA 90: 7819 –7823, 1993. CALLAWAY EM AND KATZ LC. Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc Natl Acad Sci USA 90: 7661–7665, 1993. 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 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. 1419 1420 A. FRICK, W. ZIEGLGÄNSBERGER, AND H.-U. DODT J Neurophysiol • VOL MAGEE J, HOFFMAN D, COLBERT C, AND JOHNSTON D. Electrical and calcium signaling in dendrites of hippocampal pyramidal neurons. Annu Rev Physiol 60: 327–346, 1998. MAGEE JC. Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. J Neurosci 18: 7613–7624, 1998. MAGEE JC AND COOK EP. Somatic EPSP amplitude is independent of synapse location in hippocampal pyramidal neurons [see comments]. Nature Neurosci 3: 895–903, 2000. MARKRAM H, HELM PJ, AND SAKMANN B. Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J Physiol (Lond) 485: 1–20, 1995. MARKRAM H, LUEBKE J, FROTSCHER M, ROTH A, AND SAKMANN B. Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex. J Physiol (Lond) 500: 409 – 440, 1997. MARKRAM H AND SAKMANN B. Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels. Proc Natl Acad Sci USA 91: 5207– 5211, 1994. NUSSER Z, LUJAN R, LAUBE G, ROBERTS JDB, MOLNAR E, AND SOMOGYI P. Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 21: 545–559, 1998. O’BRIEN RJ, LAU LF, AND HUGANIR RL. Molecular mechanisms of glutamate receptor clustering at excitatory synapses. Curr Opin Neurobiol 8: 364 –369, 1998. PETERS A. Number of neurons and synapses in primary visual cortex. In: Cerebral Cortex: Further Aspects of Cortical Function, Including Hippocampus, edited by Jones EG and Peters A. New York: Plenum, 1987, p. 267–294. PETRALIA RS AND WENTHOLD RJ. Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain. J Comp Neurol 318: 329 –354, 1992. PETTIT DL AND AUGUSTINE GJ. Distribution of functional glutamate and GABA receptors on hippocampal pyramidal cells and interneurons. J Neurophysiol 84: 28 –38, 2000. RALL W. Core conductor theory and cable properties of neurons. In: Handbook of Physiology. The Nervous System. Cellular Biology of Neurons. Bethesda, MD: Am. Physiol. Soc., 1977, sect. 1, vol. I, p. 39 –97. RAO A AND CRAIG AM. Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 19: 801– 812, 1997. REUVENI I, FRIEDMAN A, AMITAI Y, AND GUTNICK MJ. Stepwise repolarization from Ca2⫹ plateaus in neocortical pyramidal cells: evidence for nonhomogeneous distribution of HVA Ca2⫹ channels in dendrites. J Neurosci 13: 4609 – 4621, 1993. ROSENMUND C, FELTZ A, AND WESTBROOK GL. Synaptic NMDA receptor channels have a low open probability. J Neurosci 15: 2788 –2795, 1995. SCHILLER J, HELMCHEN F, AND SAKMANN B. Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones. J Physiol (Lond) 487: 583– 600, 1995. SCHILLER J, SCHILLER Y, STUART G, AND SAKMANN B. Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal neurons. J Physiol (Lond) 505: 605– 616, 1997. SCHWINDT PC AND CRILL WE. Local and propagated dendritic action potentials evoked by glutamate iontophoresis on rat neocortical pyramidal neurons. J Neurophysiol 77: 2466 –2483, 1997. SEAMANS JK, GORELOVA NA, AND YANG CR. Contributions of voltage-gated Ca2⫹ channels in the proximal versus distal dendrites to synaptic integration in prefrontal cortical neurons. J Neurosci 17: 5936 –5948, 1997. SPRUSTON N, JAFFE DB, WILLIAMS SH, AND JOHNSTON D. Voltage- and spaceclamp errors associated with the measurement of electrotonically remote synaptic events. J Neurophysiol 70: 781– 802, 1993. SPRUSTON N, JONAS P, AND SAKMANN B. Dendritic glutamate receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons. J Physiol (Lond) 482: 325–352, 1995. STAFSTROM CE, SCHWINDT PC, CHUBB MC, AND CRILL WE. Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro. J Neurophysiol 53: 153–170, 1985. STUART G AND SPRUSTON N. Determinants of voltage attenuation in neocortical pyramidal neuron dendrites. J Neurosci 18: 3501–3510, 1998. 86 • SEPTEMBER 2001 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 CARROLL RC, LISSIN DV, VON ZASTROW M, NICOLL RA, AND MALENKA RC. Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures. Nature Neurosci 2: 454 – 460, 1999. CASH S AND YUSTE R. Linear summation of excitatory inputs by CA1 pyramidal neurons. Neuron 22: 383–394, 1999. CAULLER LJ AND CONNORS BW. Synaptic physiology of horizontal afferents to layer I in slices of rat SI neocortex. J Neurosci 14: 751–762, 1994. CLARK BA, FARRANT M, AND CULL-CANDY SG. A direct comparison of the single-channel properties of synaptic and extrasynaptic NMDA receptors. J Neurosci 17: 107–116, 1997. COOK EP AND JOHNSTON D. Voltage-dependent properties of dendrites that eliminate location-dependent variability of synaptic input. J Neurophysiol 81: 535–543, 1999. CRAIG AM, BLACKSTONE CD, HUGANIR RL, AND BANKER G. Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc Natl Acad Sci USA 91: 12373–12377, 1994. CURRIE SN, WANG XF, AND DAW NW. NMDA receptors in layers II and III of rat cerebral cortex. Brain Res 662: 103–108, 1994. DEISZ RA, FORTIN G, AND ZIEGLGÄNSBERGER W. Voltage dependence of excitatory postsynaptic potentials of rat neocortical neurons. J Neurophysiol 65: 371–382, 1991. DENK W. Two-photon scanning photochemical microscopy: mapping ligandgated ion channel distributions. Proc Natl Acad Sci USA 91: 6629 – 6633, 1994. DODT HU, FRICK A, KAMPE K, AND ZIEGLGÄNSBERGER W. NMDA and AMPA receptors on neocortical neurons are differentially distributed. Eur J Neurosci 10: 3351–3357, 1998. DODT H, EDER M, FRICK A, AND ZIEGLGÄNSBERGER W. Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation [published erratum appears in Science 1999 Dec 17;286(5448): 2273]. Science 286: 110 –113, 1999. FRICK A, ZIEGLGÄNSBERGER W, AND DODT HU. Glutamate sensitivity of neocortical pyramidal neurons investigated with infrared-guided laser stimulation. Soc Neurosci Abstr 24: 325, 1998. HÄUSSER M AND ROTH A. Dendritic and somatic glutamate receptor channels in rat cerebellar Purkinje cells. J Physiol (Lond) 501: 77–95, 1997. HOFFMAN DA, MAGEE JC, COLBERT CM, AND JOHNSTON D. K⫹ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons [see comments]. Nature 387: 869 – 875, 1997. HUNTLEY GW, VICKERS JC, JANSSEN W, BROSE N, HEINEMANN SF, AND MORRISON JH. Distribution and synaptic localization of immunocytochemically identified NMDA receptor subunit proteins in sensory-motor and visual cortices of monkey and human. J Neurosci 14: 3603–3619, 1994a. HUNTLEY GW, VICKERS JC, AND MORRISON JH. Cellular and synaptic localization of NMDA and non-NMDA receptor subunits in neocortex: organizational features related to cortical circuitry, function and disease. Trends Neurosci 17: 536 –543, 1994b. JOHNSTON D, MAGEE JC, COLBERT CM, AND CRISTIE BR. Active properties of neuronal dendrites. Annu Rev Neurosci 19: 165–186, 1996. JONES KA AND BAUGHMAN RW. NMDA- and non-NMDA-receptor components of excitatory synaptic potentials recorded from cells in layer V of rat visual cortex. J Neurosci 8: 3522–3534, 1988. JONES KA AND BAUGHMAN RW. Both NMDA and non-NMDA subtypes of glutamate receptors are concentrated at synapses on cerebral cortical neurons in culture. Neuron 7: 593– 603, 1991. KATZ LC AND DALVA MB. Scanning laser photostimulation: a new approach for analyzing brain circuits. J Neurosci Methods 54: 205–218, 1994. KIM HG AND CONNORS BW. Apical dendrites of the neocortex: correlation between sodium- and calcium-dependent spiking and pyramidal cell morphology. J Neurosci 13: 5301–5311, 1993. KORNAU HC, SCHENKER LT, KENNEDY MB, AND SEEBURG PH. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269: 1737–1740, 1995. KUNZ G, KIRSCHE W, WENZEL J, WINKELMANN E, AND NEUMANN H. Quantitative studies on the dendrite spines of pyramidal neurons in the rat sensory cortex. Z Mikrosk Anat Forsch 85: 397– 416, 1972. LLINAS R AND HESS R. Tetrodotoxin-resistant dendritic spikes in avian Purkinje cells. Proc Natl Acad Sci USA 73: 2520 –2523, 1976. LLINAS R AND SUGIMORI M. Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. J Physiol (Lond) 305: 197– 213, 1980. GLUTAMATE HOT SPOTS ON NEOCORTICAL DENDRITES STUART G, SPRUSTON N, SAKMANN B, AND HÄUSSER M. Action potential initiation and backpropagation in neurons of the mammalian CNS. Trends Neurosci 20: 125–131, 1997. TAKUMI Y, RAMIREZ-LEON V, LAAKE P, RINVIK E, AND OTTERSEN OP. Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nature Neurosci 2: 618 – 624, 1999. THOMSON AM, DEUCHARS J, AND WEST DC. Large, deep layer pyramid-pyramid single axon EPSPs in slices of rat motor cortex display paired pulse and frequency-dependent depression, mediated presynaptically and self-facilitation, mediated postsynaptically. J Neurophysiol 70: 2354 –2369, 1993. VOGT K, LUSCHER HR, AND STREIT J. Analysis of synaptic transmission at 1421 single identified boutons on rat spinal neurons in culture. Pflügers Arch 430: 1022–1028, 1995. WIEBOLDT R, GEE KR, NIU L, RAMESH D, CARPENTER BK, AND HESS GP. Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale. Proc Natl Acad Sci USA 91: 8752– 8756, 1994. YUSTE R, GUTNICK MJ, SAAR D, DELANEY KR, AND TANK DW. Ca2⫹ accumulations in dendrites of neocortical pyramidal neurons: an apical band and evidence for two functional compartments. Neuron 13: 23– 43, 1994. YUSTE R AND TANK DW. Dendritic integration in mammalian neurons, a century after Cajal. Neuron 16: 701–716, 1996. Downloaded from http://jn.physiology.org/ by 10.220.33.3 on April 29, 2017 J Neurophysiol • VOL 86 • SEPTEMBER 2001 • www.jn.org