Articles, Cellular/Molecular

Low-Threshold Ca2+ Current Amplifies Distal Dendritic Signaling in Thalamic Reticular Neurons

Shane R. Crandall, G. Govindaiah and Charles L. Cox
Journal of Neuroscience 17 November 2010, 30 (46) 15419-15429; https://doi.org/10.1523/JNEUROSCI.3636-10.2010

Abstract

The low-threshold transient calcium current (IT) plays a critical role in modulating the firing behavior of thalamic neurons; however, the role of IT in the integration of afferent information within the thalamus is virtually unknown. We have used two-photon laser scanning microscopy coupled with whole-cell recordings to examine calcium dynamics in the neurons of the strategically located thalamic reticular nucleus (TRN). We now report that a single somatic burst discharge evokes large-magnitude calcium responses, via IT, in distal TRN dendrites. The magnitude of the burst-evoked calcium response was larger than those observed in thalamocortical projection neurons under the same conditions. We also demonstrate that direct stimulation of distal TRN dendrites, via focal glutamate application and synaptic activation, can locally activate distal IT, producing a large distal calcium response independent of the soma/proximal dendrites. These findings strongly suggest that distally located IT may function to amplify afferent inputs. Boosting the magnitude ensures integration at the somatic level by compensating for attenuation that would normally occur attributable to passive cable properties. Considering the functional architecture of the TRN, elongated nature of their dendrites, and robust dendritic signaling, these distal dendrites could serve as sites of intense intra-modal/cross-modal integration and/or top-down modulation, leading to focused thalamocortical communication.

Introduction

The GABAergic thalamic reticular nucleus (TRN) plays a central role in modulating information transfer between thalamus and neocortex, as well as within the thalamus (Yingling and Skinner, 1976; Crick, 1984; Steriade et al., 1986, 1993; Crabtree et al., 1998; Crabtree and Isaac, 2002). Centrally located in the thalamocortical circuit, corticothalamic and thalamocortical axons pass through the TRN, providing the primary sources of synaptic inputs to the nucleus (Jones, 1975; Ohara and Lieberman, 1981; Liu and Jones, 1999; Guillery and Harting, 2003). In turn, TRN axons project into the thalamus in which they modify thalamic activities such as neuronal excitability (Cox et al., 1997; Kim et al., 1997) and receptive field properties (Lee et al., 1994a,b). Thus, the TRN is ideally located for modulating thalamocortical communication via neocortical-driven feedforward or thalamic-driven feedback inhibition.

Ultimately, the magnitude of inhibition onto the dorsal thalamus depends critically on the voltage state of the TRN neuron. Like most thalamic neurons, TRN neurons respond to excitatory input by discharging Na+-dependent action potentials (APs) in one of two distinct modes: tonic or burst (Llinás and Jahnsen, 1982; Spreafico et al., 1988; Avanzini et al., 1989; Contreras et al., 1993). Transitioning between burst and tonic mode depends on the presence of the low-threshold transient calcium (Ca2+) current, IT, which is inactivated at relatively depolarized membrane potentials and deinactivated at relatively hyperpolarized membrane potentials (Huguenard and Prince, 1992; Huguenard, 1996). In the deinactivated state, sufficient excitatory input can activate IT, resulting in a large Ca2+ depolarization known as a low-threshold spike (LTS), which in turn generates a high-frequency burst discharge of Na+-dependent action potentials.

Despite understanding the role of IT in generating burst output, how this current is distributed across the neuron and more importantly how it contributes to the integration of afferent information remains speculative. Previous studies have suggested a dendritic distribution of IT in TRN neurons, but these studies have been limited because of technical considerations (Destexhe et al., 1996; Joksovic et al., 2005; Cueni et al., 2008; Kovács et al., 2010). Although this issue has been explored in thalamocortical relay neurons in which there appears to be a heterogeneous distribution of IT (Munsch et al., 1997; Zhou et al., 1997; Destexhe et al., 1998; Williams and Stuart, 2000), any comparison between TRN and thalamocortical neurons is difficult because of their distinct morphologies and burst properties (Huguenard and Prince, 1992).

In this study, we used two-photon laser scanning microscopy (2PLSM) coupled with whole-cell patch recordings to characterize the somatodendritic distribution of IT along individual TRN dendrites. Here we show that a somatic burst discharge produces a transient Ca2+ current along the entire length of TRN dendrites, which increases in magnitude at greater distances from the soma. Importantly, we also show that the direct stimulation of individual distal dendrites can locally activate distal IT, leading to the amplification of afferent input. These finding have important implications for understanding how TRN neurons modulate thalamic transmission, as well as thalamocortical operations.

Materials and Methods

Slice preparation.

Thalamic slices were prepared from young Sprague Dawley rats (postnatal age, 11–23 d) of either sex by using published procedures with minor modifications (Govindaiah and Cox, 2004). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg) and perfused with cold oxygenated slicing solution before being decapitated. Brains were quickly removed and placed in cold (4°C) oxygenated (5% CO2, 95% O2) slicing solution containing the following (in mm): 2.5 KCl, 1.25 NaH2PO4, 10.0 MgSO4, 0.5 CaCl2, 26.0 NaHCO3, 10.0 glucose, and 234.0 sucrose. Thalamic slices (250–300 μm) were cut in the horizontal plane for TRN/ventrobasal (VB) nucleus recordings or the coronal plane for dorsal lateral geniculate nucleus (dLGN) recordings. Slices were immediately transferred to a holding chamber with oxygenated physiological saline (31 ± 1°C) containing the following (in mm): 126.0 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 MgCl2, 2.0 CaCl2, 26.0 NaHCO3, and 10.0 glucose. After 15–20 min, the holding chamber was reduced to room temperature, and slices were further incubated for a minimum of 60 min before recording.

Whole-cell recording procedures.

For recording, individual slices were transferred to a recording chamber that was maintained at room temperature (23 ± 2°C) with oxygenated physiological saline (2.5–3 ml/min). Individual neurons were identified using Dodt contrast optics, and whole-cell recordings were obtained using recording pipettes containing the following (in mm): 117 K-methylsulfonate, 13 KCl, 2.0 MgCl2, 10.0 HEPES, 2.0 Na2-ATP, and 0.4 Na-GTP, pH 7.3 (290 mOsm). Pipettes also contained a Ca2+-sensitive indicator (Fluo-4, 125 μm) and a Ca2+-insensitive indicator (Alexa Fluor 594, 25 μm). The dual indicators allowed for visualization of small distal dendrites and provided a substantially less noisy measurement of Ca2+ changes than the use of a single indicator (Yasuda et al., 2004). The final pipette solution resulted in a junction potential of ∼10 mV and was corrected for in all voltage recordings. During recordings, pipette capacitance was neutralized, and the access resistance was continually monitored.

Pharmacological agents were prepared and stored as recommended and diluted in physiological saline to a final concentration before use. All antagonists were bath applied 10–15 min before subsequent experimental tests. Tetrodotoxin (TTX), ω-conotoxin-MVIIC, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), 6, 7-dinitroquinoxaline-2,3-dione (DNQX), and mibefradil were all purchased from Tocris Bioscience, and all other compounds were from Sigma.

Stimulation of distal dendrites was accomplished by local glutamate application or electrical stimulation. Focal glutamate application was achieved by pressure ejection (0.5–1 mm, 0.75–3 psi, 30–50 ms duration) via a glass pipette (1–3 μm tip diameter). Pipettes also contained Alexa Fluor 594 (12.5 μm), to aid in locating, positioning, and visualizing the ejection site. Only focal ejections with a minimal radial spread (<25 μm) were used for analysis. Line scans were performed at the edge of the stimulated area to limit the amount of dendritic movement caused by pressure ejection. Synaptic stimulation was achieved by placing a bipolar electrode adjacent to TRN within the internal capsule (single stimulus: 40–300 μA, 500 μs duration, 0.1 Hz).

Calcium imaging and analysis.

Ca2+ imaging was performed simultaneously with whole-cell recordings using a custom two-photon laser scanning microscope (Ultima; Prairie Technologies) coupled with a titanium:sapphire laser (MaiTai HP; Spectra Physics). Imaging was accomplished by laser excitation (820 nm) via a high numerical aperture water-immersion objective (60×; Olympus). To allow sufficient filling of dendritic arbor with fluorescent indicators, imaging was initiated 20–30 min after obtaining whole-cell configuration. Once a location was selected for imaging, the fluorescence was averaged over the spatial extent of the structure using line scans (15–20× optical zoom) with a temporal resolution of ∼2.5 ms. On average, five line scans were collected at each location with 10–15 s intervals between scans.

Image acquisition was performed using custom Prairie View Software and analyzed post hoc using NIH ImageJ software. The reported change in fluorescence (ΔG/R) was calculated as the change in fluorescence from baseline of the Ca2+-sensitive indicator (Fluo-4) normalized to the average fluorescence of the Ca2+-insensitive indicator (Alexa Fluor 594): ΔG/r = (GPeak − Go)/RAvg (Sabatini et al., 2002). Baseline fluorescence (Go) was calculated as the average fluorescence of the 200 ms before the stimulus. The peak response was calculated as the combined average of each line scan smoothed with a ∼30 ms running average. Physiological recordings were analyzed using pClamp software (Molecular Devices). Population data are expressed as mean ± SD, and significance was defined as p < 0.05. A paired t test was used for analyses unless otherwise indicated.

Results

Combining whole-cell electrophysiological recordings with 2PLSM, we initially measured dendritic Ca2+ changes in response to a somatic burst discharge in TRN neurons. Individual neurons were loaded via patch pipette with a Ca2+-insensitive indicator (Alexa Fluor 594, 25 μm) to facilitate the localization of small dendrites and a Ca2+-sensitive indicator (Fluo-4, 125 μm) to monitor Ca2+ changes (Fig. 1A). A single burst discharge was evoked by somatic current injection (25–75 ms; 100–400 pA), whereas the initial membrane potential at the soma was held between −80 and −90 mV (Fig. 1B). The resulting change in Ca2+ was monitored simultaneously using a high-resolution line scan and quantified as the change in green fluorescence normalized to the average red fluorescence (ΔG/R) (Fig. 1C,D). Although nonbursting neurons have been reported within the TRN (Contreras et al., 1992; Brunton and Charpak, 1997; Lee et al., 2007), in this study, all recordings were obtained from neurons that produced burst discharge.

Figure 1.
Figure 1.

Measurement of dendritic Ca2+ responses evoked by somatic burst discharge in TRN neurons. A, Stacked 2PLSM image of a TRN neuron filled. The yellow box indicates the dendritic region from which a line scan was performed (75 μm). B, Somatic recording of a burst discharge elicited from a hyperpolarized holding potential by injecting a short depolarizing current pulse. C, Simultaneous line scan of both the calcium-insensitive (Alexa Fluor 594, red) and calcium-sensitive (Fluo-4, green) fluorescent signals. D, The average ΔG/R response obtained from the dendritic region shown in A. The average (dark green line) and SD (light green envelope) were obtained from five consecutive burst discharges.

Burst discharge evokes a robust distal Ca2+ response

Consistent with a previous report (Cueni et al., 2008), somatic burst discharge in TRN neurons reliably produced a dendritic Ca2+ response. To determine whether the magnitude of the response was dependent on dendritic location, we initially compared proximal and distal Ca2+ responses obtained from the same dendrite (Fig. 2A). In 13 of 14 TRN dendrites examined (93%), a somatic burst discharge produced a larger Ca2+ response at the distal location (>150 μm) when compared with the proximal location (15–25 μm) [proximal, 20.0 ± 5.6%; distal, 28.5 ± 7.5%; n = 14 dendrites (D)/12 neurons (N); p < 0.001, paired t test]. There were only two instances in which two dendrites were sampled from the same neuron. In both cases, the pair of dendrites displayed a comparable proximal–distal increase. On average, the distal location had a 143% larger response than the proximal location. To determine whether the magnitude of the Ca2+ response observed in TRN neurons was common to other bursting thalamic cell types, we performed the same experiment on thalamocortical projection neurons of the dLGN and VB nucleus (Fig. 2B,C). For dLGN dendrites, no significant difference was observed between distal (>100 μm) and proximal Ca2+ responses (proximal, 8.0 ± 3.6%; distal, 7.6 ± 3.5%; n = 7D/5N; p = 0.69, paired t test). In contrast, VB dendrites did generate a larger distal Ca2+ response (proximal, 5.7 ± 3.1%; distal, 11.0 ± 3.0%; n = 7D/4N; p < 0.01, paired t test). Although a single burst discharge produced a dendritic Ca2+ response in all three thalamic cell types examined, the magnitude of the TRN responses was considerably larger than those of thalamocortical projection neurons.

Figure 2.
Figure 2.

Burst discharge produces a robust Ca2+ response in distal dendrites of TRN neurons. A, Left, Stacked 2PLSM image of an example TRN neuron filled. The blue box indicates the dendritic region from which a proximal line scan was typically obtained (15–25 μm). The red box indicates the dendritic region from which a distal line scan was typically obtained (>150 μm). Middle, Proximal (blue) and distal (red) Ca2+ responses obtained from a different TRN neuron. Right, Population data collected from 14 dendrites. B, Left, Stacked 2PLSM image of an example dLGN neuron filled. Blue box indicates dendritic region from which a proximal line scan was typically obtained (15–25 μm). The red box indicates the dendritic region from which a distal line scan was typically obtained (>100 μm). Middle, Proximal (blue) and distal (red) Ca2+ responses obtained from a different dLGN neuron. Right, Population data collected from seven dendrites. C, Left, Stacked 2PLSM image of an example VB neuron filled. Boxes represent the same region as in B. Middle, Proximal (blue) and distal (red) Ca2+ responses obtained from a different VB neuron. Right, Population data collected from seven dendrites.

To further characterize the dendritic/Ca2+ relationship in TRN neurons, we proceeded to image 22 new dendrites, but this time, we performed multiple line scans (four to six) along the somatodendritic axis at ∼50 μm intervals (Fig. 3A,B). Of the dendrites examined, 19 (86%) displayed a positive dendritic slope or an increased Ca2+ response with a greater measured distance from soma. In general, TRN dendrites displayed a range of slopes with an average increase of 0.05% ΔG/R per micrometer (maximum, 0.14%/μm; minimum, −0.06%/μm). For most dendrites (68%), the largest Ca2+ response was at a distance >100 μm from soma; however, in a small population of dendrites, the largest response occurred between 50 and 75 μm (27%). Despite some variability, the vast majority of TRN dendrites did display an increasing proximal-to-distal gradient, further supporting the hypothesis that distal locations have larger Ca2+ signals.

Figure 3.
Figure 3.

The TRN dendritic/Ca2+ relationship is independent of dendritic size. A, Top, Stacked 2PLSM image of an example TRN neuron filled. Boxes indicate where line scans were performed. Bottom, Corresponding Ca2+ responses obtained from the neuron above. B, Left, Plot depicts Ca2+ responses obtained along 14 different TRN dendrites as a function of dendritic distance. C, Left, Correlation between TRN dendrite diameter and measured distance from soma. Right, Correlation between the peak Ca2+ response and the measured TRN dendritic diameter. D, Left, Correlation between dendrite diameter and measured distance from soma for both dLGN (blue) and VB neurons (red). Right, Correlation between Ca2+ response and the measured dendritic diameter for both thalamocortical projection neurons. E, Left, Diagram illustrating how data were pooled based on their radial distance from soma (soma, proximal, intermediate, distal I, and distal II). Right, Plot summarizing the average response for the three different thalamic cell types (dLGN: soma, 2.9 ± 2.5%, n = 7N; proximal, 7.9 ± 4.2%, n = 11D/7N; intermediate, 8.6 ± 4.2%, n = 13D/8N; distal I, 8.1 ± 3.2%, n = 9D/6N; VB: soma, 2.4 ± 1.0%, n = 4N; proximal, 5.9 ± 2.9%, n = 8D/4N; intermediate, 10.2 ± 2.7%, n = 8D/4N; distal I, 11.0 ± 3.0%, n = 7D/4N).

Considering the relationship between the Ca2+ amplitude and dendritic distance (r = 0.34, p < 0.01, Pearson's correlation; data not shown), could a change in dendritic diameter account for the change in magnitude? In layer V pyramidal neurons, it has been shown that differences in dendritic diameter (i.e., surface-to-volume ratio) can have a significant influence on the amplitude of the Ca2+ response, with smaller/thinner dendrites producing greater changes (Holthoff et al., 2002). When we examined TRN dendrites, the diameter did become smaller with increasing distance from soma (Fig. 3C) (r = −0.53, p < 0.001, Pearson's correlation). However, the relationship between the peak Ca2+ response and dendritic diameter was weak (r = −0.21, p = 0.05, Pearson's correlation). This suggests that the smaller dendritic size does not completely account for the larger response observed in distal TRN dendrites. More importantly, this also implies that the density of current across the dendritic tree is not evenly distributed (i.e., higher densities at more distal locations).

Like that of TRN neurons, thalamocortical dendrites also became smaller with increased distance from soma (Fig. 3D) (dLGN, r = −0.66, p < 0.001; VB, r = −0.73, p < 0.001, Pearson's correlation). However, unlike TRN neurons, VB dendrites had a threefold stronger inverse relationship between peak Ca2+ response and dendritic diameter (r = −0.67, p < 0.001, Pearson's correlation). This argues that the VB proximal/distal differences (Fig. 2C) are primarily attributable to smaller dendritic size and not a larger current density. No significant correlation between peak Ca2+ response and dendritic diameter was found for dLGN dendrites (r = −0.34, p = 0.054, Pearson's correlation). These analyses indicate that the Ca2+ distribution in TRN dendrites are fundamentally different from those observed in thalamocortical projection neurons.

For a more quantitative comparison within and across thalamic cell types, we divided dendritic length into five subgroups based on radial distance from soma: soma (0 μm), proximal (15–25 μm), intermediate (50–75 μm), distal I (100–130 μm), and distal II (>150 μm) (Fig. 3E). For TRN neurons, the average Ca2+ response for each dendritic region was significantly larger than soma (p < 0.001, one-way ANOVA with post hoc Bonferroni's test) (TRN soma, 5.8 ± 1.6% ΔG/R, n = 17N; proximal, 19.5 ± 4.9%, n = 22D/17N; intermediate, 25.2 ± 8.9%, n = 22D/17N; distal I, 26.7 ± 7.0%, n = 22D/17N; distal II, 28.5 ± 7.5%, n = 14D/14N). In addition to the somatic/dendritic difference, the Ca2+ responses taken from both distal I and distal II locations were significantly larger than proximal (p < 0.01, one-way ANOVA with post hoc Bonferroni's test). Compared with thalamocortical neurons, TRN neurons had significantly larger Ca2+ responses than both dLGN and VB neurons (p < 0.001, two-way ANOVA with post hoc Bonferroni's test).

Dendritic Ca2+ responses are dependent on firing mode

In many neurons, action potentials initiated in either the axon or soma can backpropagate into the dendritic arbor (Stuart and Sakmann, 1994; Stuart et al., 1997). In pyramidal neurons of the neocortex and hippocampus, backpropagating action potentials have been shown to produce Ca2+ responses along their dendrites (Jaffe et al., 1992; Markram et al., 1995; Schiller et al., 1995; Magee and Johnston, 1997). To characterize whether the observed Ca2+ responses were possibly evoked by backpropagating action potentials in TRN neurons, we elicited either a single AP or a tonic burst of action potentials (four APs at 100 Hz) using brief somatic current injections (Fig. 4A). To ensure TRN neurons were not in burst mode (i.e., deinactivation of IT), the membrane potential of the soma was adjusted with current to −60 mV. Under these conditions, a single and tonic burst of APs produced a small Ca2+ response at proximal dendritic locations (Fig. 4B) (single AP, 3.2 ± 2.0% ΔG/R, n = 6; tonic burst, 6.1 ± 2.0% ΔG/R, n = 6). At more distal locations, the Ca2+ responses were difficult to detect from baseline (single AP, 2.3 ± 1.0% ΔG/R, n = 5; tonic burst, 2.6 ± 0.9% ΔG/R, n = 5). Overall, the dendritic Ca2+ responses evoked by action potential discharge at −60 mV were significantly smaller than those produced via a burst discharge at −80 mV (Fig. 4C) (p < 0.001, two-way ANOVA). These data strongly suggest that an underlying LTS must be responsible for the dendritic Ca2+ response.

Figure 4.
Figure 4.

Ca2+ responses are dependent on firing mode. A, Action potentials were discharged in one of three ways. Burst discharge (black) was evoked as previously described in Figure 1B. A single (blue) or tonic burst (red) of action potentials were evoked by holding the soma near −60 mV and injecting short (5 ms) depolarizing current pulses. A tonic burst consisted of four pulses delivered at 100 Hz. B, Ca2+ responses for each of the conditions shown in A at different distances obtained from the same dendrite. The responses shown are from the same dendrite. C, Plot summarizing the average response to burst discharge (black), tonic burst (red), and single action potential (blue). For reference, the average response to a burst discharge is shown in black.

Dendritic Ca2+ responses are mediated by IT

To determine the nature of the dendritic Ca2+ response in TRN neurons, we initially tested whether the burst-induced response was dependent on the activation of voltage-gated Na+ channels. Using TTX (0.5 μm) to block voltage-gated Na+ channels, the same somatic current injection revealed a transient, Ca2+-dependent LTS (Fig. 5A). The LTS in turn produced a Ca2+ response along the entire length of the dendrite similar to that generated by a burst discharge (p = 0.84, two-way ANOVA). To further confirm the independence from voltage-gated Na+ channels, distal (>150 μm) responses were measured from the same location before and after TTX application. Under these conditions, the magnitude of the response remained the same (Fig. 5B: 117.4 ± 34.0% of control, n = 15D/15N, p = 0.17, paired t test). These results indicate that, for TRN neurons, the magnitude of the burst-induced dendritic Ca2+ response is independent of both the backpropagation of somatic action potentials and dendritic voltage-gated Na+ channels.

Figure 5.
Figure 5.

TRN dendritic Ca2+ are independent of voltage-gated Na+ channels but are strongly dependent on voltage-gated T-type Ca2+ channels. A, Left, Example of a somatic recording from a TRN neuron in which a depolarizing current step evoked a burst response in control conditions (ACSF) and an LTS after TTX application (red trace). Right, Population graph illustrating the Ca2+ responses recorded at different dendritic locations produced by somatic current injection in control conditions (ACSF, black) and in TTX (red). The Ca2+ responses in TTX: soma, 5.3 ± 0.7%, n = 4N; proximal, 19.2 ± 5.8%, n = 5D/4N; intermediate, 24.1 ± 6.5%, n = 5D/4N; distal I, 27.4 ± 2.0%, n = 5D/4N; distal II, 28.2 ± 1.5%, n = 5D/5N. B, An example of a Ca2+ response obtained from the same location before (black) and after (red) TTX application. C, Top, In TTX, depolarizing current step evokes a LTS (black). Addition of nifedipine (NIF; 10 μm) slightly alters the LTS (blue); however, subsequent addition of MIB (50 μm) strongly attenuates the LTS (green). Bottom, From the same neuron, the control Ca2+ response (TTX, black) is slightly changed in the presence of nifedipine (blue) but is almost completely blocked by mibefradil (green). D, Population data for five different distal dendrites (>150 μm) obtained from different TRN neurons. The Ca2+ responses are standardized to the response obtained in TTX, and the red data points indicate the mean ± SD.

Focusing on the distal dendrites (>150 μm), we next determined which type(s) of voltage-gated Ca2+ channels underlie the somatic evoked distal response. While in the presence of TTX (0.5 μm), the addition of the L-type channel blocker nifedipine (1 and 10 μm) reduced the LTS amplitude recorded at the soma (1 μm, ∼6% reduction; 10 μm, ∼10% reduction) but did not alter the magnitude of the distal Ca2+ response (Fig. 5C,D: 1.0 μm, 106 ± 16% of control, n = 4D/4N, p = 0.5; 10 μm, 107 ± 22.1% of control, n = 5D/5N, p = 0.7, paired t test). Subsequent addition of the T-type channel blocker mibefradil (MIB) (40–50 μm) significantly reduced the LTS amplitude recorded at the soma (∼39% reduction) and nearly eliminated the distal Ca2+ response (MIB, 85.5 ± 6.1% reduction, n = 5D/5N, p < 0.01, paired t test). To confirm that the mibefradil block was not affected by nifedipine, we also tested mibefradil alone and observed a similar reduction (MIB alone, 97.5 ± 1.6% reduction, n = 4D/4N, p < 0.001, paired t test). Mibefradil also blocked the Ca2+ responses at all other dendritic locations (compared with control condition, p < 0.001, two-way ANOVA). Similar to nifedipine, the N,P/Q-type Ca2+ channel blocker ω-conotoxin-MVIIC (20–40 nm) did not significantly alter the dendritic Ca2+ response (114 ± 21.2% of control, n = 5D/5N, p = 0.5, paired t test). Together, these results suggest that the LTS/burst-evoked Ca2+ response in TRN dendrites is strongly mediated by the activation of T-type Ca2+ channels.

Distal IT is activated independent of soma/proximal dendrites

Although a distal Ca2+ response was always produced with a somatic-evoked LTS/burst, it remained unclear whether this response was generated by distally located T-type Ca2+ channels or was simply the result of the passive invasion of soma/proximal signals into the dendrites. To address this issue directly, individual distal TRN dendrites were stimulated with brief, focal glutamate application (0.5–1.0 mm; 30–50 ms duration) via pressure ejection (Fig. 6A). In control conditions [artificial CSF (ACSF)] and holding the soma at a hyperpolarized potential (−80 mV), glutamate application to an individual distal dendrite (>125 μm) generated a strong burst discharge recorded at the soma (Fig. 6B). After the addition of TTX (0.5 μm), the same application of glutamate now produced an LTS (Fig. 6B, TTX). No discernable difference was detected between the glutamate-evoked burst/LTS and those generated via somatic current injection (APs per burst: soma current injection, 3.1 ± 2.1 APs; glutamate, 3.2 ± 2.0 APs, n = 9, p = 0.25, paired t test; LTS amplitude: soma current injection, 43.1 ± 5.8 mV; glutamate, 43.9 ± 4.9 mV, n = 9, p = 0.30, paired t test). When the soma was depolarized to inactivate soma/proximal IT (−60 mV), distal glutamate application resulted in a much smaller, EPSP-like depolarization (Fig. 6B: −80 mV, 44.9 ± 4.2 mV; −60 mV, 11.0 ± 4.4 mV; n = 8, p < 0.001, paired t test). These data suggest that distal afferent signals are capable, when the appropriate voltage-dependent conditions are present at the soma, of generating a burst/LTS output from the neuron.

Figure 6.
Figure 6.

Focal glutamate application onto a distal dendrite evokes local Ca2+ current independent of the soma/proximal dendrites. A, Stacked 2PLSM image of a TRN neuron filled and pressure-ejection pipette near a distal dendrite (185 μm). The pipette contained glutamate (0.5–1 mm in ACSF) and Alexa Fluor 594 (12.5 μm), the latter aiding in the placement of the pipette and estimating the spread of glutamate ejection, which is represented by the white-dotted line (∼15–20 μm radius). Line scans were performed near the edge of the stimulated area (yellow box). B, Glutamate application (Glu) to distal dendrite produced a burst discharge recorded at the soma in control conditions (ACSF). In TTX (0.5 μm), glutamate produced an LTS when the cell was initially held at −85 mV, but when the cell was depolarized (−60 mV), the same stimulation produced a small transient depolarization (EPSP-like). C, Left, Somatic response to five separate glutamate applications with different puff intensities (0.75–3 psi, 50 ms duration). Right, Corresponding Ca2+ responses recorded near the glutamate pipette. Two subthreshold (red) and three suprathreshold (black) responses are shown. D, Examples of Ca2+ responses measured at a distal dendrite (175 μm) in the presence of TTX resulting from somatic current injection (CI; black trace) and distal glutamate application. The latter Ca2+ responses depict glutamate-evoked responses when the soma was held at either −80 mV (blue) or −60 mV (red). E, The plot illustrates the amplitude of the glutamate-evoked responses in these two conditions (−85 or −60 mV at soma) relative to the Ca2+ responses evoked by somatic current injection.

When examining the glutamate-evoked Ca2+ response, there was a clear relationship between the response magnitude and the success/failure of generating a somatic LTS (Fig. 6C). When a subthreshold application of glutamate was ejected, it produced a small depolarization at the soma with little or no distal Ca2+ response (Fig. 6C, red traces); however, a large dendritic response was always associated with a suprathreshold application of glutamate, which in turn always produced an LTS at the soma (Fig. 6C, black traces). More importantly, regardless of whether the somatic membrane potential was held at a hyperpolarized (−80 mV) or depolarized (−60 mV) level, suprathreshold glutamate application always produced a robust Ca2+ response in the distal dendrites (Fig. 6D). Compared with the Ca2+ response elicited by a somatic current injection, the glutamate-evoked response was prolonged. The amplitude of the glutamate-evoked response at −80 mV (24.0 ± 7.0% ΔG/R) was larger than both the somatic-evoked (19.8 ± 9.1% ΔG/R, p < 0.05, paired t test) and the glutamate-evoked (18.3 ± 6.3% ΔG/R, n = 12, p < 0.001, paired t test) response at −60 mV (Fig. 6E). No significant difference was found between the somatic-evoked and glutamate-evoked response at −60 mV (p = 0.34, paired t test). These results indicate that focal glutamate application to a distal dendrite is sufficient to activate a large distal Ca2+ response independent of soma/proximal dendrites.

Considering that glutamate stimulation could activate Ca2+-permeable NMDA receptors, we tested the antagonist CPP on the glutamate-evoked distal Ca2+ response. Bath application of CPP (10–20 μm) produced a small but statistically significant attenuation of the glutamate-evoked Ca2+ response (Fig. 7A) (glutamate–control, 36.9 ± 6.6% ΔG/R; glutamate–TTX + CPP, 31.6 ± 5.8% ΔG/R; n = 8, p < 0.05, paired t test). Importantly, the glutamate-evoked Ca2+ response in the presence of CPP did not differ from the response evoked by somatic current injection (30.2 ± 8.2% ΔG/R; n = 8, p = 0.50, paired t test). In all eight neurons tested, CPP also reduced the prolonged Ca2+ response observed with glutamate application. The subsequent application of the AMPA receptor antagonist DNQX (20–40 μm) eliminated the glutamate-evoked Ca2+ response (CPP + DNQX, 5.0 ± 5.8% ΔG/R; n = 3). Neither CPP nor CPP + DNQX altered the dendritic Ca2+ responses evoked by somatic current injection (p = 0.92, n = 7, paired t test and p = 0.93, n = 6, paired t test, respectively).

Figure 7.
Figure 7.

Characterization of glutamate-evoked distal Ca2+ signals. Ai, Experimental design consisted of applying glutamate (Glu) to an individual distal TRN dendrite while holding the soma at −80 mV in a bath solution that contained TTX (0.5 μm). Aii, Glutamate-evoked response recorded at the soma before (Control) and after CPP or CPP + DNQX application. Aiii, Left, In the same cell as Aii, the corresponding glutamate-evoked Ca2+ response imaged at a distal location. Right, For reference, an LTS Ca2+ response evoked by a somatic current injection (CI) is shown. Aiv, Average amplitude of the Ca2+ response for each of the four conditions tested are plotted. Bi, Experimental design consisted of applying glutamate (Glu) to an individual distal TRN dendrite while holding the soma at either −80 or −60 mV in a bath solution that contained TTX (0.5 μm). Bii, Glutamate-evoked response recorded at the soma while holding at −80 mV before (Control) and after mibefradil (MIB; 50 μm) application. Biii, Control trace (black) is the response to distal glutamate application in presence of TTX (left, −80 mV; right, −60 mV). After the addition of mibefradil, the subsequent Ca2+ responses to glutamate application are significantly reduced. Peak Ca2+ responses for this experiment were measured within the gray box. Biv, Average amplitude of the Ca2+ response for each of the four conditions tested are plotted.

We next tested the contribution of T-type Ca2+ channels on the glutamate-evoked Ca2+ response (Fig. 7B). In mibefradil (50 μm), the amplitude of the glutamate-evoked dendritic Ca2+ responses were significantly reduced regardless of the somatic membrane potential (at −80 mV: control, 25.0 ± 6.6% ΔG/R, n = 5; with MIB, 6.3 ± 2.6% ΔG/R, n = 5, p < 0.01, paired t test; at −60 mV: control, 19.5 ± 5.9% ΔG/R, n = 4; with MIB, 7.7 ± 4.8% ΔG/R, n = 4, p < 0.01, paired t test). These findings suggest that distal glutamate application to an individual TRN dendrite is sufficient to activate local T-type Ca2+ channels. Considering that mibefradil reduced both the distal Ca2+ response and corresponding somatic depolarization (58 and 12% reduction at −80 and −60 mV, respectively), it is likely that IT is amplifying afferent input.

Synaptic activation of distal IT

To probe the functional relevance of distal IT amplification, we stimulated local excitatory synaptic afferents in place of glutamate application. For these experiments, the stimulating electrode was positioned lateral to the TRN within the internal capsule so to activate both corticothalamic and thalamocortical fibers (Golshani et al., 2001; Zhang and Jones, 2004). Using a single stimulus (40–300 μA, 500 μs, 0.1 Hz), burst discharge was generated in our somatic recording when the neuron was held at −80 mV, along with a corresponding large distal Ca2+ response (Fig. 8A). The magnitude of the synaptically evoked distal Ca2+ response did not differ from the response evoked by somatic current injection (at −80 mV: synaptic, 27.6 ± 4.0% ΔG/R; soma current injection, 23.7 ± 6.9% ΔG/R, n = 4, p = 0.23, paired t test). Subsequent application of CPP (20 μm) did not significantly alter the amplitude of the synaptically evoked Ca2+ response (with CPP, 23.1 ± 10.7% ΔG/R, n = 4, p = 0.28, paired t test); however, addition of DNQX (40 μm) eliminated the response (CPP + DNQX, 2.0 ± 0.4% ΔG/R, n = 4, p < 0.001, paired t test). When the soma was depolarized to −60 mV, the same stimulus generated one to two action potentials (Fig. 8B). Under these conditions, the amplitude of the synaptically evoked Ca2+ response displayed a considerable degree of variability (45.9% coefficient of variation, n = 4); however, the large responses generated (21.1 ± 8.3% ΔG/R, n = 4) were not statistically different from the responses generated by a somatic induced LTS (23.7 ± 6.9% ΔG/R, p = 0.48, paired t test). Subsequent bath application of CPP (20 μm) did not alter the amplitude of the Ca2+ response (with CPP, 17.2 ± 12.7% ΔG/R, n = 4, p = 0.36, paired t test), whereas DNQX eliminated the response (1.5 ± 0.5% ΔG/R, n = 4, p < 0.02, paired t test).

Figure 8.
Figure 8.

Characterization of synaptic-evoked distal Ca2+ signals. Ai, Experimental design consisted of stimulating the internal capsule using a single stimulus (Stim) while holding TRN neurons the soma at −80 in a control ACSF bath solution. Aii, Somatic recording of a synaptically evoked burst discharge while holding at −80 mV. Aiii, Left, Distal Ca2+ response evoked by synaptic stimulation of the internal capsule (black). For reference, a control (red) response to a somatic current injection (CI) is also shown. Right, Resulting synaptically evoked Ca2+ responses after the bath application of CPP (green) and CPP + DNQX (blue). Aiv, Average amplitude of the Ca2+ response for each of the four conditions tested are plotted. Bi, Experimental design consisted of stimulating the internal capsule using a single stimulus (Stim) while holding TRN neurons the soma at −60 mV in a control ACSF bath solution. Bii, Somatic recording of a synaptically evoked burst discharge while holding at −60 mV. Biii, Left, A single Ca2+ response (black) evoked by synaptic stimulation of the internal capsule. Subsequent Ca2+ responses after bath application of CPP (green) and CPP + DNQX (blue). Biv, Average amplitude of the Ca2+ response for each of the four conditions tested are plotted.

Discussion

The results of the present study indicate that a somatic burst discharge or activation of an LTS can evoke a robust Ca2+ response in TRN dendrites mediated by IT. Ca2+ influx was observed along the entire length of the dendrite, with the largest response typically occurring at more distal locations (>100 μm). Although all thalamic neurons tested produced a change in dendritic Ca2+ in response to burst discharge, the magnitude within TRN dendrites was significantly larger than those observed in thalamocortical relay neurons. In addition, we show that distally located IT can be activated independently from the soma/proximal dendrites, therefore possibly serving to boost local afferent inputs onto TRN neurons. The activation of distal IT, by either focal glutamate application or synaptic activation, required activation of AMPA but not NMDA glutamate receptors. Considering that IT kinetics and amplitude are temperature dependent (Coulter et al., 1989; Takahashi et al., 1991), performing these experiments at room temperature have likely altered the typical physiological response observed at body temperature but may have also provided a more accurate measurement of the peak change (Markram et al., 1995). Overall, these findings provide the first physiological evidence of the somatodendritic distribution of IT in TRN neurons, as well as insight regarding the potential functional role of IT in synaptic integration within reticular neurons.

Somatodendritic distribution of IT in TRN neurons

It is well documented that activation of IT and the resulting LTS are responsible for generating a burst discharge of action potentials in TRN and thalamocortical relay neurons (Llinás and Jahnsen, 1982; Deschênes et al., 1984; Jahnsen and Llinás, 1984; Domich et al., 1986; Avanzini et al., 1989; Coulter et al., 1989; Crunelli et al., 1989; Hernández-Cruz and Pape, 1989; Suzuki and Rogawski, 1989; Huguenard and Prince, 1992). However, data regarding the dendritic distribution of IT in the thalamus have been limited to studies focusing on thalamocortical relay neurons (Munsch et al., 1997; Zhou et al., 1997; Destexhe et al., 1998; Williams and Stuart, 2000). For the TRN neuron, any accurate physiological quantification of the distribution of IT has been lacking. The results of the present study indicate a heterogeneous dendritic distribution of IT in TRN neurons, results that are consistent with a previous computational study (Destexhe et al., 1996). Although an accurate quantification of T-type channel density is difficult to extrapolate, the weak correlation between Ca2+ response amplitude and dendritic diameter implies an increased density at more distal locations (Fig. 3C). This interpretation corresponds well with a recent anatomical study that found a higher density of T-type Ca2+ channels in small-diameter TRN dendrites, likely representing distal dendrites (Kovács et al., 2010).

State-dependent amplification

Our findings also support the general hypothesis that IT may serve to amplify afferent input as suggested in different neuronal types (Magee et al., 1995; Gillessen and Alzheimer, 1997; Williams et al., 1997; Urban et al., 1998; Watanabe et al., 1998; Williams and Stuart, 2000; Goldberg et al., 2004). Considering the voltage dependence of IT, namely being activated from relatively hyperpolarized potentials, a non-uniform membrane potential along the somatodendritic axis could have significant influence on dendritic excitability, synaptic amplification, and ultimately action potential output. For example, if the somatodendritic axis is uniformly hyperpolarized, amplified distal input would be uninterrupted and actively propagated toward the soma, ultimately producing a burst discharge. Conversely, if the soma/proximal dendrites were depolarized relative to the distal dendrites, distal signals would still be amplified considering activation of IT, resulting in a larger afferent EPSP at the soma level but not producing a burst discharge. In the later scenario, synapses located further from the soma would still have an ability to contribute to somatic output given sufficient activation of distal IT. Finally, different neuromodulators thought to depolarize TRN via actions at distal dendrites (e.g., cholecystokinin, glutamate via metabotropic receptors) could inactivate IT and thereby dampen afferent inputs by attenuating the amplification by IT (Cox et al., 1995; Sohal et al., 1998). Thus, at the dendritic level, IT could serve to regulate the spatial integrative properties of the TRN neuron and interactions between distinct dendritic compartments (i.e., proximal/soma and distal).

Given that TRN neurons can have multiple primary dendritic branches (Scheibel and Scheibel, 1966; Jones, 1975; Lübke, 1993; Ohara and Havton, 1996), an interesting and potentially important issue is whether or not the voltage state across the somatodendritic axis could differ between individual dendrites of the same TRN neuron. Such dendritic isolation could promote highly localized processing and would allow individual branches to act as independent integrators of different information (Euler et al., 2002; Häusser and Mel, 2003).

Heterogeneity among TRN neurons

Although this study focused on the functional role of dendritic IT in TRN neurons capable of burst discharge, there are studies that have clearly identified a subpopulation of nonbursting TRN neurons (Contreras et al., 1992; Brunton and Charpak, 1997; Lee et al., 2007). For these neurons, the lack of burst discharge seems to result from a minimal amount of IT recorded at the soma. However, the inability to induce a somatic burst discharge does not suggest a complete absences of dendritic IT. It is possible that nonbursting TRN neurons could represent a subpopulation with an extreme proximal–distal difference in their distribution of IT. In theory, maintaining a high density of distal IT would still allow for the amplification of distal afferent input with the lack of soma/proximal IT preventing the neuron from generating a burst response. In fact, a more distal distribution of IT in nonbursting TRN neurons is supported by the fact that in a subset of these neurons a small LTS could be unmasked when a K+-channel blocker was applied (Lee et al., 2007). Although such a somatodendritic distribution remains to be tested, the functional consequence of such a distribution may play an important role in the transfer of modality-specific information as well as thalamocortical operations.

Functional significance of distal IT

Considering the functional architecture of the TRN, elongated nature of TRN dendrites, and robust responses in distal processes, could distal dendrites serve as sites of intense integration within and across different modalities? The TRN is situated at the interface between the thalamus and neocortex and receives collateral innervation from both thalamocortical and corticothalamic projection neurons. As the fibers pass through the TRN, they assemble in several well defined modality-related sectors (Jones, 1975; Montero, 1997; Guillery and Harting, 2003). Furthermore, sectors can be subdivided into slabs that receive afferent information from focal areas of the neocortex and dorsal thalamus or slabs of TRN neurons projecting to specific regions of the dorsal thalamus (Crabtree, 1999).

Because most TRN neurons possess elongated dendrites (Scheibel and Scheibel, 1966; Jones, 1975; Lübke, 1993; Ohara and Havton, 1996), it is likely that neurons near the borders would have some distal dendrites cross into neighboring sectors or slabs. Because the majority of afferent inputs onto distal dendrites arise from corticothalamic neurons (Liu et al., 1995), it is possible that a strong “top-down” modulation of distal TRN dendrites would significantly influence the integrative properties of the TRN by altering the resting voltage-state of the somatodendritic axis. Such top-down modulation could arise from one or a combination of inputs originating from either the primary sensory neocortex (Montero, 2000) or prefrontal cortex (Zikopoulos and Barbas, 2006). Ultimately, the degree of top-down influence onto distal dendrites could dictate the degree in which the TRN gates “bottom-up” information transfer within and/or across different modalities.

Another potential function of distal dendritic IT is that such mechanisms could serve to facilitate intra-TRN communication. Considering that many TRN neurons are coupled by electrical synapses (Landisman et al., 2002; Long et al., 2004; Landisman and Connors, 2005) and the electrical signals through these synapses are subjected to severe attenuation and low-pass filtering (Galarreta and Hestrin, 2001), it is possible that high densities of distal IT could also serve to amplify dendritic electrical synapses among TRN neurons and thereby facilitate intra-TRN communication in a state-dependent manner. It is also intriguing to speculate as to whether IT and electrical synapses could help generate and preserve the endogenous spindle rhythmic activity observed in the isolated TRN in vivo (Steriade et al., 1987). Furthermore, given the possibility of dendrodendritic synapses within cat TRN neurons, distal IT could strongly influence such communication if present in rodents (Deschênes et al., 1985). The fact that we did not observe strong distal Ca2+ responses in relay neurons and they are not electrically coupled in the relatively mature animal (Lee et al., 2010) further supports this possibility.

To date, there are limited reports of in vivo electrophysiological recordings of TRN neurons responding to more than one sensory modality (Sugitani, 1979; Shosaku and Sumitomo, 1983). However, recent studies indicate that TRN activity is subjected to cross-modal modulation (Yu et al., 2009) and may be affected by various attentional demands (McAlonan et al., 2006, 2008; Yu et al., 2009). In conjunction with our results, we speculate that the contribution of distal dendrites may underlie such cross-modal actions in a state-dependent manner. Clearly, the resting state of the dendrites and distribution of IT will have significant influence on thalamocortical operations that requires future investigation.

Footnotes

  • This work was supported by the National Institutes of Health/National Eye Institute (C.L.C.) and a National Institute on Deafness and Other Communication Disorders training grant (S.R.C.). We thank Drs. Yanyan Wang and Kush Paul for helpful comments on this manuscript.

  • Correspondence should be addressed to Charles L. Cox, 2357 Beckman Institute, 405 North Mathews Avenue, Urbana, IL 61801. cox2{at}illinois.edu

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Low-Threshold Ca2+ Current Amplifies Distal Dendritic Signaling in Thalamic Reticular Neurons
Shane R. Crandall, G. Govindaiah, Charles L. Cox
Journal of Neuroscience 17 November 2010, 30 (46) 15419-15429; DOI: 10.1523/JNEUROSCI.3636-10.2010
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