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. 2008 May 15;586(Pt 14):3353–3364. doi: 10.1113/jphysiol.2008.155580

High frequency action potential bursts (≥ 100 Hz) in L2/3 and L5B thick tufted neurons in anaesthetized and awake rat primary somatosensory cortex

C P J de Kock 1, B Sakmann 2
PMCID: PMC2538819  PMID: 18483066

Abstract

High frequency (≥ 100 Hz) bursts of action potentials (APs) generated by neocortical neurons are thought to increase information content and, through back-propagation, to influence synaptic integration and efficacy in distal dendritic compartments. It was recently shown in acute slice experiments that intrinsic bursting properties differ between neocortical L2/3 and L5B (thick tufted) neurons. In L2/3 neurons for instance, dendritic APs were brief and generated only one additional AP after the initial somatic AP. In L5B neurons, dendritic plateau potentials facilitated the generation of trains of three or more APs. We recently showed in vivo that spiking frequencies are very different for L2/3 and L5B thick tufted neurons under anaesthesia. Here, we addressed the question whether in vivo the bursting properties are different for these two cell types. We recorded from L2/3 and L5B thick tufted neurons of rat primary somatosensory (barrel) cortex under anaesthetized and awake conditions and found that AP activity is dominated by single APs. In addition, we found that in the anaesthetized animal also bursts of two APs were observed in L2/3 neurons but the relative occurrence of these bursts was low. In L5B thick tufted neurons, bursts consisting of up to six APs were recorded and their relative occurrence was significantly higher. Frequencies within bursts were also significantly higher in L5B thick tufted neurons than in L2/3 neurons. In awake (head-restrained) animals, average spike frequencies of L2/3 and L5B thick tufted neurons were surprisingly similar to spike rates under anaesthesia. However, bursting behaviour in L2/3 neurons was comparable to L5B thick tufted neurons. Thus, the distribution of interspike intervals was changed in L2/3 neurons without affecting the average spiking rate. We observed bursts consisting of up to five APs in both cell types and both probability of bursts and AP frequency within bursts were similar for L2/3 and L5B thick tufted neurons. Our analysis shows that most cortical APs occur as single APs, although a minor fraction of APs in L2/3 and L5B thick tufted neurons are part of high frequency bursts (15%). This AP bursting is dependent on the behavioural state of the animal in a cell-type dependent manner.

Neocortical areas are characterized by a large variety of cell types that differ in morphology, anatomical projections, membrane properties and physiological responses (Connors & Gutnick, 1990; Mountcastle, 1997; Binzegger et al. 2004; Markram et al. 2004; Shepherd et al. 2005; de Kock et al. 2007; Schubert et al. 2007). One of the differences that we recently demonstrated was the level of action potential (AP) spiking in primary somatosensory (barrel) cortex of the in vivo (urethane) anaesthetized animal (de Kock et al. 2007). We found that spontaneous and evoked activity was significantly higher in L5B thick tufted neurons compared to L2/3 neurons. In view of different neuronal coding strategies, however, AP frequency might not be the only relevant parameter. Rather, it was suggested that communication in the form of short bursts could increase information content (Lisman, 1997). In addition, short AP bursts can trigger regenerative calcium activity in dendritic compartments to induce plasticity mechanisms and affect dendritic integration properties during synaptic transmission (Stuart & Sakmann, 1994; Larkum et al. 1999b). In the cortex, AP spiking patterns are highly irregular (Ranck, 1973; Softky & Koch, 1993; Gray & McCormick, 1996) and AP bursts are probably the most extreme example (Connors & Gutnick, 1990). The short AP bursts, particularly of ≥ 100 Hz, are crucial for dendritic calcium electrogenesis in distal compartments of cortical L2/3 and L5B neurons, which in turn determines dendritic plasticity mechanisms (Stuart & Sakmann, 1994; Larkum et al. 1999b, 2007; Stuart et al. 1997). In these studies it was also shown that the two cell types have different intrinsic burst capacity. We therefore addressed the question of whether there are differences in the in vivo burst properties of the relatively silent L2/3 and more active L5B thick tufted neurons, irrespective of their spiking frequency. We recorded from anaesthetized and from awake, head-fixed Wistar rats and show that occurrence of bursts is differentially regulated in L2/3 and L5B thick tufted neurons, depending on the behavioural state of the animal. In general, most APs (> 85%) in L2/3 and L5B thick tufted neurons occur as single APs with interspike intervals of > 10 ms. However, both L2/3 and L5B thick tufted neurons are capable of generating trains consisting of up to five APs at frequencies ≥ 100 Hz under awake conditions.

Methods

Anaesthetized conditions

Recordings from urethane (1.6–1.7 g kg−1) anaesthetized Wistar rats (P26–42, male/female) were made as previously described (de Kock et al. 2007). In summary, experiments were performed on the primary somatosensory cortex of the left hemisphere (2.5 mm posterior, 5.5 mm lateral to bregma). Whiskers contralateral to the recording site were trimmed to ∼5 mm. Spontaneous APs were measured through continuous recording for 125 s. To record evoked activity, the principle whisker was deflected (20 repetitions) at 3.3 deg in the caudal direction. Only the 0–100 ms time interval after whisker deflection onset was analysed.

Awake conditions

Awake Wistar rats were used (male/female) using slightly older animals (P31–45) to ensure stable mount of the head-post for fixation. Animals were first trained for 3–4 days to become accustomed to the experimenter by daily handling. All experimental procedures were carried out according to the animal welfare guidelines of the Erasmus Medical Center in Rotterdam. The animal was anaesthetized with ketamine–xylazine (10 mg ketamine, 0.5 mg xylazine per 100 g body weight) to surgically attach a metal pole to the skull to enable head fixation (experimental day 0). Depth of anaesthesia was checked by both foot and eyelid reflex and vibrissae movements. The animal's temperature was monitored with a rectal probe and maintained at 37°C by a thermostatically controlled heating pad. Experiments were performed on the left primary somatosensory (barrel) cortex (2.5 mm posterior, 5.5 mm lateral from Bregma). From experimental day 1–3, animals were trained daily to become accustomed to head fixation, which was followed by a food reward. On day 4, juxtasomal recordings were made with 6–8 MΩ patch pipettes. Pipettes were filled with (in mm): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes, pH 7.2 with NaOH, and 20 mg ml−1 biocytin. Bath solution contained 0.9% NaCl. Recordings were made using an Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA) in combination with a Lynx 8 amplifier, band filter settings 300 and 9000 Hz. Data were acquired using the Ntrode Virtual Instrument (custom written software, R. Bruno, Columbia University, New York, USA) for Labview (National Instruments, Austin, TX, USA). After physiological properties were measured, cells were filled with biocytin using current pulses (Pinault, 1996). Animals were then transcardially perfused with 0.1 m phosphate buffered saline (pH 7.2), then 4% paraformaldehyde (PFA), and their brains were removed. The recorded cells were revealed with the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB) using the avidin–biotin–peroxidase method (Horikawa & Armstrong, 1988).

Data analysis

Spontaneous activity during anaesthetized conditions was recorded for 125 s. Evoked activity was quantified in the 100 ms poststimulus time window. During recordings in awake animals, we found that AP spiking frequencies in a subset of neurons was strongly correlated to whisker movement and position (data not shown). For comparison with AP spiking under anaesthetized (quiet) conditions, we therefore only analysed spontaneous activity in awake animals during quiet episodes, when the animals were not moving their whiskers. Since quiet episodes were mixed with episodes of whisker movements, interspike intervals were only calculated for APs within the same quiet episode. Data are represented as means ± s.e.m.

Results

To examine the bursting behaviour of L2/3 and L5B thick tufted neurons we made juxtasomal recordings of identified cells of primary somatosensory (barrel) cortex of urethane anaesthetized Wistar rats (de Kock et al. 2007). Spontaneous spiking (in the absence of a sensory stimulus) was continuously monitored (for 125 s) during which we found highly irregular spontaneous spiking patterns. Average spontaneous spiking in L2/3 neurons was significantly lower (mean ± s.d., 0.41 ± 0.54 Hz, n = 14) than spontaneous activity in L5B thick tufted neurons (2.70 ± 1.56 Hz, n = 18, Mann–Whitney U test, P < 0.0001) (Fig. 1B, D, G and I, and online supplemental material, Suppl. Fig. 1). The L5B thick tufted neurons were characterized previously as intrinsically bursting cells (Chagnac-Amitai et al. 1990; Schubert et al. 2001). We found that L5B thick tufted neurons frequently spiked with interspike intervals (ISIs) of ≤ 10 ms, which we defined as (high frequency) bursts (Fig. 1Gb). In contrast, ISIs in L2/3 neurons were significantly longer (Fig. 1Bb). To study bursts in response to a sensory stimulus, we re-analysed the data-set from our previous report (de Kock et al. 2007), supplemented with additionally recorded L2/3 and L5B thick tufted neurons. L2/3 evoked responses were very low and we frequently recorded from L2/3 neurons which did not show a single AP in response to repetitive whisker deflections (20 repetitions, n = 18 recordings) (Fig. 1E). Bursts were therefore rarely encountered. In contrast, in L5B thick tufted neurons we did observe bursts which consisted of three or more APs with individual ISIs of ≤ 10 ms (for example Fig. 1J, trial 17 with an evoked burst of 4 APs).

Figure 1. AP bursts are more prevalent in L5B thick tufted neurons compared to L2/3 neurons; single examples.

Figure 1

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A, neurolucida reconstruction of an example L2/3 pyramidal neuron from rat barrel cortex. Ba, continuous juxtasomal recording from L2/3 pyramidal neuron with typical low spontaneous frequency (0.14 Hz). Bb, example of same L2/3 neuron illustrating two consecutive APs at 33 Hz. C, juxtasomal recording illustrating evoked responses during whisker stimulation. White bar shows duration of stimulus. Note that no action potentials were observed. Interstimulus time interval 2 s. D, raster plot with all APs during 125 s of continuous recording in same L2/3 neuron. E, raster plot indicating the lack of APs during 20 consecutive whisker deflections. F, neurolucida reconstruction of an example L5B thick tufted neuron from rat barrel cortex. Ga, continuous juxtasomal recording from L5B thick tufted neuron with relatively high spontaneous AP frequency (2.7 Hz). Gb, example of same L5B thick tufted neuron firing a spontaneous burst (at maximal observed frequency during this particular recording, interspike interval 2.9 ms, 345 Hz). H, L5B thick tufted neuron evoked responses during whisker stimulation (same neuron). White bar shows duration of stimulus. Interstimulus time interval 2 s. I, raster plot demonstrating all APs during 125 s of continuous recording. Bursts of APs at frequencies ≥ 100 Hz are indicated as red dots. J, raster plot demonstrating APs during 20 consecutive whisker deflections. Bursts of APs at frequencies ≥ 100 Hz are indicated as red dots.

Bursts of APs are crucial to induce regenerative calcium activity in distal dendrites and to initiate local calcium influx (Larkum et al. 1999b; Larkum et al. 2007). It was shown that in particular bursts of ≥ 100 Hz were capable of initiating calcium influx in (apical) dendrites. In L2/3, we incidentally observed bursts in the population, under both spontaneous (Fig. 2A) and sensory evoked (Fig. 2B) conditions. In L5B thick tufted neurons, bursts were more common during spontaneous (Fig. 2C) and evoked (Fig. 2D) conditions and could consist of three or more APs. Since the total number of recorded APs was significantly different for these two cell types, we calculated the relative occurrence of these bursts (Fig. 2E and F). During recordings of spontaneous L2/3 activity we found that only 1.62 ± 1.04% of all recorded APs occurred within bursts (Fig. 2E, n = 14) and bursts consisting of three or more APs were not observed. In contrast, in L5B thick tufted neurons, the probability of an AP being part of a burst during spontaneous activity was significantly higher (15.00 ± 4.18%, n = 18, Mann–Whitney U test, P = 0.0004) and we even observed two bursts of six APs with all ISIs ≤ 10 ms. In response to a sensory stimulus, we recorded only one burst in L2/3 (0.48%, n = 18, Fig. 2F) indicating that under anaesthetized conditions regenerative calcium activity in dendritic compartments of L2/3 neurons has limited physiological relevance. In L5B thick tufted neurons we found that in response to a sensory stimulus, APs were more frequently part of bursts (17.20 ± 5.09%, n = 21, Mann–Whitney U test, P = 0.0016 compared to L2/3 evoked bursting) and bursts incidentally consisted of three or four APs (Fig. 2F). No significant difference was found for bursting probability during spontaneous and evoked activity (P = 0.27 for L2/3 and P = 0.64 for L5B thick tufted neurons, respectively). We conclude that under anaesthetized conditions, back-propagation in L5B thick tufted neurons could be involved in dendritic calcium electrogenesis and induction of synaptic plasticity mechanisms.

Figure 2. AP bursts are more common in L5B thick tufted neurons compared to L2/3 neurons; population average.

Figure 2

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A, example of a spontaneous burst in an L2/3 neuron. B, example of an evoked burst in an L2/3 neuron. C, example of a spontaneous burst of an L5B thick tufted neuron. D, example of an evoked burst in an L5B thick tufted neuron. E, occurrence of spontaneous bursts during continuous recordings. Note that in L2/3 neurons only bursts of 2 APs fired at ≥ 100 Hz were observed. In L5B thick tufted neurons, bursts could consist of up to 6 APs. Since the L2/3 example burst from A demonstrated only 1 ISI of ≥ 100 Hz, this burst is considered a doublet. F, occurrence of evoked high frequency bursts (≥ 100 Hz) during whisker stimulation. Note that only one burst of 2 APs was observed in all L2/3 recordings, whereas for L5B thick tufted neurons bursts were more frequently observed. G, AP amplitude adaptation as a function of ISI for spontaneous APs fired by L2/3 neurons. H, as for G but for L5B thick tufted neurons.

During AP bursts, we regularly observed adaptation of consecutive APs resulting in decreased AP amplitude (see for example Fig. 2A–D). Since amplitude adaptation is relevant for both synaptic release characteristics and efficacy of back-propagation, we quantified the ratio of 2nd AP amplitude versus 1st AP amplitude during spontaneous AP spiking. Usually, AP adaptation was found for ISIs ≤ 25 ms and most obvious during trains consisting of more than three APs (see Fig. 2D). To avoid a cumulative effect of adaptation during trains consisting of more than two APs, we only quantified adaptation for the first two APs within trains of APs with ISIs ≤ 25 ms. In L2/3 we found only a few ISIs of ≤ 25 ms, and AP amplitude was only slightly affected in the range of 0–25 ms (Fig. 2G). In contrast, in L5B thick tufted neurons short ISIs were more frequently found and the amplitude of the 2nd AP was significantly affected depending on the interspike interval (Fig. 2H). More specifically, in the ISI range of 2–5 ms the average amplitude of the 2nd AP was only 72.3 ± 18.1% (mean ± s.d.) and in the range of 5–10 ms 83.6 ± 14.5% (Mann–Whitney U test, P < 0.0001 compared to ISIs of 25–50 ms). Thus, adaptation of AP amplitude is relevant during high frequency bursts in L5B thick tufted neurons and this phenomenon could decrease the efficiency of back-propagation to initiate dendritic calcium electrogenesis.

High frequency bursts in hippocampus have been previously described as ‘complex spikes’ that last ∼25 ms and consist of two to six APs occurring at about 200 Hz (Lisman, 1997; Ranck, 1973). We rarely observed this type of burst in cortex, but for dendritic calcium electrogenesis in cortex it is still of importance to know the occurrence of ISIs in the range of the critical frequency of 100 Hz. We therefore plotted first all ISIs (Fig. 3A) and then the ISIs ≤ 25 ms to compare the frequencies within these cortical complex spikes (Fig. 3B). This analysis again shows that during spontaneous activity in L2/3 neurons bursts (ISIs ≤ 10 ms) were rarely observed, but that bursts close to the critical frequency of 100 Hz were more frequently observed (total of 8.52% of ISIs ≤ 25 ms). As expected, in L5B thick tufted neurons we observed ISIs that were frequently ≤ 10 ms and more AP bursts that could be considered complex spikes (total of 16.72% of ISIs ≤ 25 ms). Thus, frequencies within cortical complex spikes were significantly higher for L5B thick tufted neurons compared to L2/3 neurons (Mann–Whitney U test, P < 0.0001). The analysis of sensory evoked ISIs in L2/3 neurons was severely hampered by the very low number of APs that were observed in the 100 ms after whisker deflection. We found only one burst with an ISI of ≤ 10 ms and only five with a value of ≤ 25 ms. In contrast, in L5B thick tufted neurons we consistently found bursts and the short ISIs known to initiate regenerative calcium activity (Fig. 3D).

Figure 3. L5B thick tufted neurons generate AP bursts at higher frequency compared to L2/3 neurons.

Figure 3

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A, cumulative distribution of all ISIs during continuous recording of spontaneous APs. Only ISIs ≤ 1 s are shown. Note that ISIs are significantly smaller in L5B thick tufted neurons compared to L2/3 neurons. Inset: same data but only ISIs ≤ 100 ms are shown. B, same data as A but plotted as a histogram to reveal that spontaneous bursts in L5B thick tufted neurons occur at higher frequency compared to L2/3 neurons. C, cumulative distribution of all ISIs during recording of stimulus evoked APs. Only a very limited number of bursts was observed in L2/3 recordings. D, same data as C but plotted as a histogram to show distribution of ISIs of spontaneous bursts in L5B thick tufted neurons.

Cortical AP frequencies are under control of neuromodulatory projections from forebrain and brainstem nuclei that are almost exclusively active when the animal is awake (McCormick, 1989; Castro-Alamancos, 2004). We therefore examined the bursting properties of L2/3 and L5B thick tufted neurons when the animals were awake and un-anaesthetized to study the possible physiological relevance of regenerative calcium activity in dendritic compartments during normal animal behaviour. We made juxtasomal recordings in different regions of primary somatosensory cortex (septal and column related neurons were pooled). Recordings were less stable and only a percentage (∼30%) of recorded neurons could be loaded with biocytin. To ensure that unidentified neurons from supragranular layers were in fact L2/3 neurons, we only selected regular spiking neurons that were recorded up to a depth of 700 μm below pia. For five of these experiments, we were able to confirm post hoc that we recorded from L2/3 pyramidal neurons. In L5 both slender and thick tufted neurons are found (Agmon & Connors, 1992; Schubert et al. 2001; Schubert et al. 2006; de Kock et al. 2007) and we therefore only analysed neurons that were post hoc identified as L5B thick tufted neurons. Whisker movements were recorded with an infrared video system (ISCAN, Massachusetts, USA) and only episodes in which the animal was not whisking (quiet) were used for AP frequency analysis (see Methods).

Average spiking frequency of L2/3 neurons in the awake, un-anaesthetized animal was also relatively low (mean ± s.d., 0.26 ± 0.18 Hz, n = 17) compared to AP spiking in L5B thick tufted neurons (4.12 ± 3.22 Hz, n = 5, Mann–Whitney U test, P = 0.0086, data not shown). Surprisingly, the average spiking frequency in the awake animals was not significantly different from anaesthetized conditions (Mann–Whitney U test, P = 0.87 for L2/3 and P = 0.49 for L5B thick tufted neurons, respectively).

We observed two remarkable differences in the awake L2/3 recordings. First, AP bursts were more frequent (14.99 ± 2.85%, n = 17, Fig. 4A and B) and second, bursts consisting of three or more APs occurred more frequently. Whereas these bursts were never observed in the anaesthetized animal, they were incidentally encountered during awake recordings (2.31 ± 1.54%). This indicates that the probability of burst firing is increased in L2/3 neurons without an affect on the average spiking frequency. Furthermore, we conclude that bursting in L2/3 neurons depends on the behavioural state of the animal. In L5B thick tufted neurons, we did not observe a difference in bursting behaviour during awake recordings compared to anaesthetized conditions (Mann–Whitney U test, P = 0.50). Again, bursts consisting of two APs were regularly observed (11.03 ± 4.09%, n = 5) and also bursts of three or more APs (3.46 ± 1.51%, n = 5, Fig. 4A and B).

Figure 4. Occurrence of AP bursts in the awake animal.

Figure 4

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A, left panel, burst consisting of 4 APs observed in an L2/3 neuron in an awake, head-fixed animal. Right panel, burst consisting of 4 APs observed in an L5B thick tufted neuron in an awake, head-fixed animal. B, occurrence of spontaneous bursts during continuous recordings of L2/3 and L5B thick tufted neurons in awake, head-fixed animals. Note that in L2/3 neurons also bursts of 3–6 APs at ≥ 100 Hz were observed which were absent in anaesthetized conditions. C, the relationship between the occurrence of bursts and age of the animal for L2/3 neurons. D, same relationship for L5B thick tufted neurons. Note that in L2/3 bursting probability is increased in awake animals, whereas this relationship is not apparent in L5B thick tufted neurons. E, AP amplitude adaptation as a function of ISI for spontaneous APs fired by L2/3 neurons in awake animals. F, as for E but for L5B thick tufted neurons.

It was previously shown that the age of the animal can have a significant effect on the probability of observing high-frequency bursting L2/3 neurons (Brumberg et al. 2000). In this previous study on L2/3 neurons of ferret visual cortex, the authors found that bursting neurons (or chattering cells) were only found after 2 months of age (5%), which increased to 39% in ferrets older than 3 months. Since our recordings from awake Wistar rats were from slightly older animals compared to urethane anaesthetized animals (see Methods), we analysed whether there is a correlation between bursting probability and age of the animal (Fig. 4C and D). For both L2/3 and L5B neurons, we did not find a correlation supporting the hypothesis that the difference in bursting probability that we found for L2/3 neurons when comparing anaesthetized and awake recordings might be due to modulatory projections from forebrain and brainstem projections.

We already described AP amplitude adaptation in L5B thick tufted neurons recorded under anaesthesia (Fig. 2). We repeated this analysis for spontaneous AP spiking during awake recordings and found that also in L2/3 recordings from awake animals, AP amplitude significantly decreased depending on the ISI. In the range of 2–5 ms, AP amplitude of the 2nd AP was decreased to 83.8 ± 12.0% (mean ± s.d., Mann–Whitney U test, P < 0.0001 compared to amplitudes for ISIs of 25–50 ms). In the range of 5–10 ms, AP adaptation was not significant (93.3 ± 10.9%, Mann–Whitney U test, P = 0.0924). For L5B thick tufted neurons, we also found significant adaptation of AP amplitude in awake recordings, both for the range of 2–5 ms and 5–10 ms (88.5 ± 12.8% and 92.5 ± 15.1%, Mann–Whitney U test, P = 0.0023 and P = 0.0159, respectively, compared to the ISI range of 25–50 ms). Surprisingly, we also found that adaptation in L5B thick tufted neurons was significantly smaller in awake recordings compared to anaesthetized conditions (P = 0.0002 and P = 0.0065 for 2–5 ms and 5–10 ms, respectively). This could suggest that modulatory projections from forebrain and brainstem potentially not only determine the relative occurrence of high frequency AP bursts, but could also modulate additional membrane properties of cortical neurons to reduce AP amplitude adaptation.

In Fig. 4B, we showed an increase in the occurrence of AP bursts in L2/3 during awake recordings compared to anaesthetized conditions. This increase was also reflected in the cumulative distribution of all L2/3 ISIs (Fig. 5A), which showed a significant shift towards shorter ISIs (Mann–Whitney U test, P < 0.0001). Also within the cortical complex spikes, frequencies were significantly higher in awake compared to anaesthetized animals (Fig. 5B, Mann–Whitney U test, P < 0.0001). In L5B thick tufted neurons, we also found a shift in the cumulative distribution towards smaller values in the awake animal (Fig. 5C, Mann–Whitney U test, P < 0.0001), although in the range of 0–25 ms, we observed a significantly lower spiking frequency within bursts recorded from awake animals compared to anaesthetized animals (Fig. 5C and D, Mann–Whitney U test, P < 0.0001).

Figure 5. Occurrence of AP bursts depends on attentional state.

Figure 5

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A, cumulative distribution of ISIs during continuous recording of spontaneous activity in L2/3 neurons in urethane anaesthetized and in awake, head-fixed animals. Note that ISIs are significantly smaller in awake conditions compared to anaesthetized conditions. B, same data as A but plotted as a histogram to reveal that spontaneous bursts in awake conditions had higher frequencies compared to anaesthetized conditions. C, cumulative distribution of ISIs of spontaneous APs in L5B thick tufted neurons from urethane anaesthetized and awake, head-fixed animals. D, same data as C but plotted as a histogram to reveal that frequency of bursts is not dependent on behavioural state for L5B thick tufted neurons, but that their relative occurrence is changed.

Discussion

Our results show that in the anaesthetized animal, high frequency bursts (at least 2 APs occurring at ≥ 100 Hz) are observed regularly in L5B thick tufted neurons. Under spontaneous and sensory evoked conditions, 12% and 14% of all APs were part of bursts, respectively. In L2/3, APs were only rarely observed in bursts both under spontaneous and evoked conditions (∼1%). Considering the very low average spontaneous frequency of L2/3 neurons (∼0.5 Hz), this indicates that bursts and therefore regenerative calcium activity in dendritic compartments are rare events under anaesthesia. In the awake animal, however, we found that bursting behaviour in L2/3 neurons was significantly increased (15% of APs in bursts). Surprisingly, the increased occurrence of bursts in awake animals was not reflected in the average spontaneous activity in L2/3 neurons. Furthermore, the increased probability of bursts correlated only with behavioural state in L2/3 neurons. In L5B thick tufted neurons, the probability of bursts was not increased in the awake animal (14% of APs in bursts). Thus, bursting behaviour in cortical cell types can be very different depending on the behavioural state of the animal (see also (Gray & Viana Di Prisco, 1997) and is regulated in a cell-type specific way.

Physiological consequences of bursts

It has been shown previously, that in vivo spiking patterns of cortical neurons are highly irregular and frequently demonstrate bursts of APs (Hubel & Wiesel, 1965; Connors & Gutnick, 1990; Baranyi et al. 1993; Bair et al. 1994; Gray & McCormick, 1996; Brumberg et al. 2000). One potential physiological function is to increase the information content within AP patterns (Cattaneo et al. 1981; Otto et al. 1991; Lisman, 1997). In these experiments, it was suggested that ‘place fields’ in hippocampus and visual maps in visual cortex were more sharply delineated when only bursts were considered in comparison to maps in which single APs were also included.

Bursts are also essential for regenerative calcium activity in dendritic compartments (Larkum et al. 1999a, 2007; Williams & Stuart, 1999, 2000). The back-propagation of somatic APs depends on a critical frequency of about 100 Hz in cortical L2/3 and L5B thick tufted neurons (Larkum et al. 1999a, 2007). Although we recorded exclusively somatic APs in our experiments, the regular occurrence of somatic bursts of ≥ 100 Hz makes it very likely that under these conditions bursts are back-propagated into the dendrites to initiate calcium electrogenesis. Back-propagation under awake conditions was actually predicted previously from in vivo anaesthetized experiments (Waters & Helmchen, 2004) and also directly demonstrated (Buzsaki & Kandel, 1998; Bereshpolova et al. 2007). Back-propagation leads to local calcium influx through voltage-gated calcium channels, which in turn initiates synaptic plasticity mechanisms (Williams & Stuart, 1999). One of the best documented plasticity mechanisms involving back-propagating APs is spike timing-dependent plasticity (STDP) (Kampa et al. 2007). In this form of synaptic plasticity, the exact timing of presynaptic input relative to postsynaptic depolarization determines whether the synapse is potentiated or depressed and STDP therefore depends on sufficient depolarization of dendritic compartments through back-propagating APs. Interestingly, in L2/3 neurons AP bursts are required to induce long-term changes of synaptic currents (Nevian & Sakmann, 2006).

A third factor for the significance of AP bursts within a network is the reliability of information transfer during a burst of APs. Specifically, synaptic transmission of bursts depends on the release properties of the synaptic terminals and hence the release properties of individual synapses eventually determine the output of the network. For instance, strong depressing synapses filter out high frequency components (low-pass filters), whereas strong facilitating synapses favour transmission of high frequency components (high-pass filter) (Izhikevich et al. 2003). There are also reports that demonstrate that a single synapse can combine properties to perform as a band-pass filter (Glantz & Nudelman, 1988; Izhikevich et al. 2003). In turn, adaptation of AP amplitude during high frequency bursts may contribute to synaptic release dynamics and/or back-propagation. A strongly adapted second AP within a burst thus may be less relevant than would be expected from a second AP. AP adaptation can be clearly observed from previous studies (Mason & Larkman, 1990; Williams & Stuart, 1999; Brumberg et al. 2000), although usually not quantified.

To quantify AP waveform adaptation (amplitude and spike width, f.i.) it is crucial though that extracellular recordings are obtained from single units, which is achieved by the juxtasomal approach (Pinault, 1996; Joshi & Hawken, 2006; de Kock et al. 2007). With other standard extracellular recording electrodes (microwire or tetrode), significantly adapted APs within bursts might be mistaken for events from additional units (Fee et al. 1996; Harris et al. 2000) leading to false negatives and an underestimation of burst occurrence. In our single unit recordings we show that the amplitude of a second AP within a high-frequency burst can decrease to 75–85% (depending on the behavioural state of the animal). Most likely it reflects residual inactivated sodium channels during the second AP. Since inactivation of sodium (and calcium) channels on the dendrite can effectively block back-propagation (Williams & Stuart, 1999; Bean, 2007), AP adaptation during high frequency bursts is highly relevant when considering back-propagation and dendritic calcium electrogenesis. In summary, bursting is a critical factor for several network characteristics (e.g. information transfer, induction of synaptic plasticity and network output) and is likely to drive animal behaviour.

Vice versa, we found that bursting behaviour in L2/3 neurons, but not L5B thick tufted neurons, strongly correlated with the behavioural state of the animal. In the anaesthetized animal, bursts were virtually absent in L2/3 neurons but were more frequently (∼15%) observed in the awake animal. Surprisingly, in L5B thick tufted neurons, we did not observe a correlation between bursting probability and behavioural state indicating a cell-type specific effect.

A change in AP patterns during different states of alertness could be due to a decreased state of excitation during urethane anaesthesia (Potez & Larkum, 2008). In this recent study, it was shown that dendritic excitability of L5B (thick tufted) neurons is reduced under urethane anaesthesia. Although this might explain the differences we observed for L2/3 bursting properties, we would also have expected to observe this effect of urethane on in vivo L5B bursting properties. An alternative explanation could therefore be that bursting is regulated on a cell-type specific level by modulatory projections (e.g. noradrenaline, acetylcholine, serotonin and histamine) originating from brainstem (McCormick, 1989; Castro-Alamancos, 2004). For instance, it was shown that acetylcholine is able to regulate GABAergic and glutamatergic synaptic transmission in (auditory) cortex, and acetylcholine could therefore shift the balance between excitation and inhibition (Metherate & Ashe, 1993; Metherate & Ashe, 1995).

The modulatory nuclei are preferentially active during arousal or awake states and project to both thalamus and cortex (McCormick & Prince, 1985; McCormick, 1989; Xiang et al. 1998). In thalamus, burst spiking is inhibited during arousal rather than facilitated (Hirsch et al. 1983; Weyand et al. 2001) and can therefore not explain the increased burst firing we observed in cortical L2/3 neurons. Rather, we propose that a direct action of modulatory projections on specific cell types in cortex regulates bursting behaviour during different attentional states. This was previously demonstrated for distinct interneuron cell types in rat visual cortex (Xiang et al. 1998). In this previous study, it was shown that LTS interneurons in layer V of rat visual cortex were activated via nicotinic receptors, whereas FS interneurons were inhibited via muscarinic receptors (Xiang et al. 1998). Thus even within the same layer, modulatory projections can have opposite effects on spiking. In support of the idea that bursting behaviour is regulated, it was shown that regular spiking cells could switch into a bursting mode after prolonged current injection or during sensory stimulation (Kang & Kayano, 1994; Brumberg et al. 2000). Thus, the switch in bursting behaviour we observed in L2/3 neurons might be directly associated with increased excitation during awake compared to anaesthetized states.

Acknowledgments

We thank Michael Brecht and Arthur Houweling for assistance during initial awake experiments, Radoslav Enchev and Bas Koekkoek for analysis software, Marlies Kaiser (MPI), Ellen Stier (MPI), Elize Haasdijk (ErasmusMC) and Erica Goedknegt (ErasmusMC) for histology, Rolf Rödel and Karl Schmidt for technical assistance, Prof Mayank Mehta and Prof David Colquhoun for comments on a previous version of this manuscript, and Sebastiano Bellanca and Thomas Mock for Neurolucida reconstructions. This work was supported by the ErasmusMC, the Max-Planck Society and a VENI grant (Netherlands Organization for Scientific Research – NWO) to C.d.K.

Supplementary material

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