Diversity of firing modes of TRN neurons
Current dogma is that a ubiquitous characteristic of thalamic neurons, including TRN neurons, is the ability to produce burst discharge of action potentials. In this study, we systematically examined the action potential discharge properties of TRN neurons relative to their distribution within TRN. Intracellular recordings were obtained from a total of 211 TRN neurons and these cells were subdivided by their action potential discharge characteristics. Of these neurons 170 biocytin-containing cells were recovered and then mapped as to their localization within TRN. To confirm that all four thalamic slices (dorsal to ventral slices, see Methods) do indeed contain TRN, GAD-immunohistochemistry was performed to localize GABA-positive regions within each of the four slices (Fig. 1A). As expected, GAD-immunoreactive neurons were found in all levels of TRN slices, further supporting that electrophysiological recordings obtained in this study were indeed restricted to TRN. Furthermore, all neurons recorded were found within the boundaries of TRN and the morphology of these neurons is consistent with previous findings that most TRN neurons have fusiform-shaped somata with either bipolar or multipolar primary dendrites (Fig. 1B; Spreafico et al. 1988; Cox et al. 1996).
Surprisingly, the majority of TRN neurons (56%: 71/127) obtained from dTRN (1st and 2nd slices) did not produce burst discharge even from relatively hyperpolarized membrane potentials of −80 mV (Fig. 2Aa, Non-burst). In a smaller population of dTRN neurons (35%: 44/127), the apparent burst discharge consisted of an initial action potential followed by multiple small amplitude, long duration depolarizing potentials (Fig. 2Aa, Atypical burst). Only a minor population of neurons in dTRN (9%: 12/127) produced a stereotypical burst discharge consisting of high frequency, multiple large amplitude action potentials similar to that previously described in TRN and other thalamic neurons (Fig. 2Aa, Typical burst). Considering the apparent difference in the transient discharge by the typical burst and atypical burst neurons, we quantified three different characteristics of action potentials in these cells. Namely, we measured the amplitudes of the first three action potentials, the half-width of the first action potential, and the instantaneous firing frequency of the first two action potential intervals (Table 1). The amplitude of the 1st action potential in the burst did not differ between atypical burst and typical burst neurons (P > 0.2, one-way ANOVA with Tukey–Kramer multiple comparisons test); however, the subsequent two action potentials were significantly smaller in the atypical burst neurons (P < 0.01, t test). The half-width of the initial action potential was also significantly shorter in the typical burst neurons compared to the atypical burst and non-burst neurons (Tables 1, P < 0.001, one-way ANOVA with Tukey–Kramer multiple comparisons test). Throughout this study, long duration current pulses (1 s duration) were used to evoke action potential discharge; however, in many studies, shorter duration current steps are used to produce burst discharge. In a subpopulation of neurons, shorter current steps (30–50 ms) were used to evoke burst discharge in order to assure that the differences that we observed in the discharge properties of the TRN neurons were not a result of the longer current steps. As illustrated in Fig. 3A, the duration of current pulses (50 ms versus 1 s) did not alter the discharge properties of the different TRN neuron subtypes (n= 5 for each TRN neuron subtype).
Table 1. Intrinsic properties and action potential (AP) characteristics of TRN neurons
| ||Intrinsic properties||Action potential characteristics|
|Amplitude||Half-width 1st AP (ms)||Frequency|
|Membrane potential (mV)||Input resistance (mΩ)||1st AP (mV)||2nd AP (mV)||3rd AP (mV)||1st interval (Hz)||2nd Interval (Hz)|
|Atypical Burst||−69.9 ± 4.7||330 ± 76||92.5 ± 8.6||65.5 ± 8.0||70.3 ± 7.0||1.29 ± 0.30||226.2 ± 48.3||140.8 ± 39.7|
|(n= 30)||(n= 30)||(n= 23)||(n= 23)||(n= 15)||(n= 21)||(n= 23)||(n= 15)|
|Typical Burst||−70.0 ± 6.8||226 ± 87||88.9 ± 8.2||88.2 ± 7.6||83.7 ± 7.5||0.69 ± 0.23||124.7 ± 46.9||171.5 ± 65.0|
|(n= 61)||(n= 62)||(n= 22)||(n= 22)||(n= 15)||(n= 22)||(n= 22)||(n= 15)|
|Non-burst||−66.5 ± 8.0|| 421 ± 184||92.6 ± 9.3|| || ||1.37 ± 0.72|| || |
|(n= 46)||(n= 60)||(n= 22)|| || ||(n= 22)|| || |
|P < 0.001||P < 0.001||n.s.||P < 0.01||P < 0.01||P < 0.001||P < 0.01||n.s.|
|ANOVA||ANOVA||ANOVA||t test||t test||ANOVA||t test||t test|
Figure 3. Non-burst TRN neurons lack IT A, similar burst discharges are produced by both long- and short-duration current pulses. a, long-duration current pulses (1 s duration) evoke characteristic discharges in TRN neuron subtypes similar to that in Fig. 2Ab. In the same neurons, a shorter duration current pulse (50 ms duration) evokes a similar discharge as the one second pulse in each subtype of TRN neuron. B, following the addition of TTX (0.5 μm), many non-burst neurons (right) lack an LTS or any transient depolarization in response to depolarizing current steps. In contrast, typical burst (left) and atypical burst (middle) neurons produced large amplitude low threshold Ca2+ spikes in all neurons tested. The amplitude of the low threshold Ca2+ spikes in atypical burst neurons was consistently greater than in typical burst neurons. All neurons were manually hyperpolarized to membrane potentials of −80 mV (dashed line). Ca, the
Download figure to PowerPoint
As expected, when the membrane potential was held at a relatively depolarized level (> −60 mV), all TRN neurons produced tonic action potential discharge indicating the voltage dependence of the burst discharge, when present (Fig. 2Aa). In contrast to dTRN, the majority of neurons recorded from vTRN slices (3rd and 4th slices) displayed typical burst discharge (82%: 69/84, Fig. 2Ab, Typical burst). The remaining 18% (15/84) of the neurons in vTRN slices were non-burst neurons (Fig. 2Ab, Non-burst); however, it is interesting to note that seven of these cells were located near the dorsal surface of the 3rd slices, further supporting that non-burst neurons are preferentially localized in dTRN (Fig. 2Ba, orange circles). It is important to note that we found all three TRN subtypes in slices from older animals as well (up to postnatal age 28 days, Fig. 2Bb), suggesting that the atypical and non-burst neurons are not simply an immature state of typical burst neurons.
As illustrated in Fig. 2Ba, the distribution of 170 TRN neurons has been mapped: 73 non-burst, 40 atypical burst, and 57 typical burst neurons. The non-burst and atypical burst neurons were almost exclusively localized within the dTRN (slices 1 and 2: Fig. 2Ba).These neurons do not appear to be differentially distributed across the anterior/posterior extent of TRN. In contrast, the vast majority of vTRN neurons showed stereotypical burst discharge (Fig. 2Ba, Typical burst, blue circles). Considering TRN neurons have been differentiated by soma shape and polarity within TRN (Spreafico et al. 1991), we also investigated whether these three TRN neuron subtypes may be differentiated by their morphology. The morphology of biocytin-filled neurons was obtained for 73 TRN neurons: 32 non-burst, 16 atypical burst, and 25 typical burst neurons (e.g. Fig. 1B). In these neurons the soma and dendritic arborizations were well-labelled for further analyses. We analysed the soma area, soma shape (ratio of the major and minor axis), and number of primary dendrites (Table 2). Although these basic morphological characteristics varied within each group distinguished by discharge mode, there were no significant differences in any of these morphological characteristics amongst the three groups (P > 0.1, one-way ANOVA). Thus, the size or shape of TRN neurons was not a prediction as to whether the neuron could produce burst discharge of action potentials.
Table 2. Morphological properties of TRN neurons
| ||Soma area (μm2)||Soma shape (minor/major axis)||No. primary dendrites|
|Non-burst||251 ± 83||0.55 ± 0.10||3.3 ± 1.2|
|(n= 32)||(n= 32)||(n= 32)|
|Atypical Burst||249 ± 76||0.53 ± 0.10||4.0 ± 1.0|
|(n= 16)||(n= 16)||(n= 16)|
|Typical Burst||302 ± 107||0.56 ± 0.14||3.7 ± 1.4|
|(n= 25)||(n= 25)||(n= 25)|
Heterogeneous membrane properties among TRN neurons
We next tested if the passive intrinsic membrane properties differed amongst these three TRN neuron subtypes (Table 1). The resting membrane potential significantly differed between burst and non-burst neuronal subtypes (F2,134= 3.9, P < 0.001, one-way ANOVA). The average membrane potential of non-burst neurons (−66.5 ± 8.0 mV, n= 46) significantly differed from the typical burst neurons (−70.0 ± 6.8 mV, n= 61, P < 0.01, Tukey–Kramer multiple comparison test) but did not significantly differ from atypical burst neurons (−69.9 ± 4.7 mV, n= 30, P > 0.1). The apparent input resistances of neurons also significantly varied across the three neuronal populations (F2,149= 32.8, P < 0.01, one-way ANOVA). Non-burst neurons had an average input resistance of 421 ± 184 MΩ (n= 60) which was significantly different from atypical burst neurons (330 ± 76 MΩ, n= 30) and typical burst neurons (226 ± 87 MΩ, n= 62, P < 0.01, Tukey–Kramer multiple comparison test). Furthermore, the input resistance of atypical burst neurons was significantly higher than that of typical burst neurons (P < 0.01, Tukey–Kramer multiple comparison test).
Non-burst TRN neurons lack low threshold Ca2+ current
Burst firing mode of thalamic neurons requires activation of the low threshold, transient Ca2+ current, IT (Deschênes et al. 1982; Jahnsen & Llinás, 1984b; Coulter et al. 1989). Considering the dependence of IT activation for burst discharge, we speculate that non-burst neurons may in fact lack IT. Alternatively, IT may be masked or inhibited by other voltage-dependent conductances such as the transient K+ current, IA. We initially examined whether a low threshold, transient calcium-dependent depolarization (low threshold spike: LTS) could be evoked in these different TRN neuron subtypes. From an initial membrane potential of −80 mV, depolarizing current steps (1 s duration; 10–40 pA increments; 15 steps) were applied in normal ACSF and TTX. In normal ACSF, we recorded from 26 non-burst neurons in dTRN slices. In the presence of TTX (0.5 μm), the depolarizing current step did not evoke a transient depolarization in 10 of 26 neurons (Fig. 3B, Non-burst). However, in the remaining 16 neurons a small amplitude, transient depolarizing potential was evoked (e.g. see Fig. 4B, Small depolarization). This depolarization averaged 4.6 ± 3.1 mV (n= 16, range 2–10 mV), and was significantly smaller than a normal LTS evoked in typical burst neurons (P < 0.01, Mann–Whitney test). In control conditions, typical burst neurons recorded from vTRN slices produced clear burst discharge (Fig. 3B, Typical burst). In TTX (0.5 μm), a stereotypical LTS was evoked in all typical burst neurons with an average amplitude of 24.9 ± 5.8 mV (n= 16, Fig. 3B, Typical burst). Atypical burst neurons also produced an LTS in the presence of TTX. The LTS amplitude evoked in atypical burst neurons was significantly greater than those in typical burst neurons (Fig. 3B, Atypical burst; 30.6 ± 4.3 mV, n= 16, P < 0.05, Mann–Whitney). In addition, the shape of the LTS differed between typical burst and atypical burst neurons. The duration of the LTS (measured at 50% peak amplitude) in atypical burst neurons averaged 48.2 ± 11.0 ms (n= 16) and was significantly greater than typical burst neurons (34.1 ± 11.4 ms, n= 16, P < 0.05, Mann–Whitney). In these neurons, the atypical burst neurons had significantly higher input resistances (404 ± 130 MΩ; n= 16) compared to the typical burst neurons (205 ± 83 MΩ; P < 0.01, Mann–Whitney); however, there was not a statistically significant relationship between LTS amplitude and input resistance.
Figure 4. The underlying effect of 4-AP on non-burst neurons At a holding potential of −80 mV, a depolarizing step current injection in the presence of TTX produced either no depolarizing potential (n= 6) or a transient depolarizing potential (n= 5) in non-burst neurons (n= 11). A, in TRN neurons that produced no transient depolarization in TTX, 4-AP (5 mm) produced no unmasking of a transient response in 3 of 6 neurons, but unmasked a small depolarization in the remaining 3 neurons (weak unmasking). B, in non-burst neurons that produced small depolarizing potentials in TTX, 4-AP (5 mm) application increased the small depolarization in all 5 neurons in a reversible manner. In 2 of 5 cells, 4-AP application unmasked a normal appearing low threshold Ca2+ spike. Dashed line indicates −80 mV. The bottom traces (I) indicate depolarizing current steps.
Download figure to PowerPoint
In order to examine if non-burst TRN neurons lack the low threshold, transient calcium current, IT, membrane currents were recorded. Following conditioning hyperpolarization voltage steps (−50 to −125 mV, 5 mV increments, 1 s duration, Fig. 3Ca) a step command to −50 mV was applied to evoke the low threshold transient current, IT, while not activating high threshold Ca2+ currents. In non-burst TRN neurons that produced no LTS in response to current steps in current clamp mode (Fig. 3B, Non-burst), the voltage clamp protocol did not produce any transient inward current in 6 of 7 neurons, suggesting a lack of IT (Fig. 3Cb, Non-burst). In the remaining non-burst neuron, a very small transient current (−87.3 pA) was evoked. In all typical burst neurons tested, a transient inward current was evoked and the peak amplitude of the current averaged −630 ± 378 pA (n= 11; Fig. 3Cb, typical burst). The peak inward current evoked in atypical burst neurons did not significantly differ from typical burst neurons (Fig. 3Cb, atypical burst; −654 ± 228 pA, n= 10, P > 0.1, Mann–Whitney test). In order to verify the transient inward current is indeed a Ca2+ current, we next tested if the general Ca2+ channel blocker Ni2+ attenuated the transient inward current. Bath application of Ni2+ (1.0–2.0 mm) completely attenuated the transient inward current in all typical and atypical burst neurons tested (n= 10, Fig. 3Cb).
Considering the similar activation characteristics of the voltage-dependent outward K+ current, IA and IT, previous studies suggest that the lack of burst discharge in dLGN interneurons may result from overlapping activation of these two currents (Pape et al. 1994). We next tested if the transient K+ current, IA, could mask or prevent an LTS in non-burst TRN neurons. In order to test this, recordings were obtained from 11 non-burst TRN neurons that produced either no transient depolarization (n= 6) or a small amplitude transient depolarization (< 5 mV, n= 5) in response to depolarizing current steps (1 s, 10–40 pA increments, 15 steps) in TTX (0.5 μm, Fig. 4). The IA antagonist 4-aminopoyridine (4-AP, 0.05–5 mm) was then bath applied and the subsequent effect on the response to depolarizing current pulses was examined. In six neurons with no transient depolarization, the addition of 4-AP had no effect on the transient depolarizations in three neurons (Fig. 4A, No unmasking). In the remaining three neurons, a small depolarizing potential was unmasked in the presence of 4-AP suggesting that there may be a very small Ca2+-dependant current in a subgroup of non-burst TRN neurons (Fig. 4A, Weak unmasking). In the remaining five neurons, small transient depolarizations (< 5 mV) were evoked by the depolarizing current steps in the presence of TTX (Fig. 4B, Small depolarization). Following the addition of 4-AP, larger amplitude transient depolarizations (range 10–25 mV) were unmasked in all five neurons tested (Fig. 4B, Small depolarization). In two of these cells, a nearly normal looking LTS was unmasked in 4-AP (Fig. 4B, Strong unmasking). In the other three neurons, a larger transient depolarizing potential was unmasked in 4-AP (Fig. 4B, Weak unmasking).
Next, we examined whether the blockade of IA with 4-AP could transform non-burst neurons into burst neurons because the blockade of IA with 4-AP unmasks an apparent LTS in a small population of non-burst neurons. Small depolarizing steps (1 s duration, 10–40 pA increments, 15 steps) were applied to non-burst TRN neurons without TTX in order to evoke action potential discharge (Fig. 5, Non-burst). In these experiments the ionotropic glutamate receptors antagonists DNQX (20 μm) and CPP (10 μm) were included in the bath to attenuate 4-AP induced epileptiform discharges. In seven non-burst neurons held at hyperpolarized membrane potentials (−80 mV), the depolarizing current steps only produced tonic action potential discharge (Fig. 5, Non-burst). Following the addition of 4-AP (0.5 mm), these neurons still produced tonic discharge (Fig. 5, Non-burst; 4-AP), suggesting that the unmasked LTS observed in some non-burst neurons is not sufficient to evoke burst firing. We also examined whether blocking IA in typical burst and atypical burst neurons could affect their burst firing properties. In all five typical burst TRN neurons, the depolarizing current steps produced burst discharge (Fig. 5, Typical burst). Following the addition of 4-AP, the burst discharge of these cells remained. There was a small decrease in the amplitude of the 2nd action potential within the burst (pre: 95.4 ± 7.2 mV, 4-AP: 86.4 ± 8.2 mV, n= 5, P > 0.1, Wilcoxon matched pairs test) but it was not significantly different as with atypical burst neurons. In a similar manner, the addition of 4-AP did not alter the general features of the atypical burst neuron discharge. In control conditions, in response to a depolarizing current step, the atypical burst neurons produced a discharge consisting of an initial short duration action potential followed by a couple of broader transient depolarizations (Fig. 5, Atypical burst). In 4-AP, the discharge had a similar shape as control conditions; however, the last transient depolarization was longer in duration (Fig. 5, Atypical burst inset, arrow). This latter depolarization was produced in 4 of 5 neurons, and was insensitive to TTX, suggesting this response probably results from activation of a high threshold calcium current, such as an L-current.
Figure 5. Blocking IA does not alter action potential discharge properties of non-burst and typical burst TRN neurons Typical burst: in this neuron, the depolarizing current step produces clear burst discharge ring atop an LTS. In the presence of 4-AP (0.5 mm), the burst discharge persists. Atypical burst: in this neuron, the depolarizing current step produces the atypical burst discharge consisting of an initial sharp action potential followed by broader transient depolarizations (see inset). In 4-AP, a similar discharge pattern is observed; however, the last transient depolarization (arrow in inset) has a longer duration. Non-burst: from a membrane potential of −80 mV, depolarizing current steps in control solution produced no burst discharge in this neuron. In the presence of 4-AP, the depolarizing current step still produces only tonic discharge with a shorter latency to the first action potential. Inset scale: 20 mV, 20 ms.
Download figure to PowerPoint