Dendritic spikes mediate negative synaptic gain control in cerebellar Purkinje cells

Ede A. Rancza,b,1 and Michael Häussera

aWolfson Institute for Biomedical Research and Research Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, United Kingdom; and bDivision of , Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom

Edited* by Rodolfo R. Llinás, New York University Medical Center, New York, NY, and approved November 8, 2010 (received for review June 28, 2010) Dendritic spikes appear to be a ubiquitous feature of dendritic dendritic spikes and axonal AP output in Purkinje cells by using excitability. In cortical pyramidal , dendritic spikes increase simultaneous dendritic and somatic whole-cell recordings. Our the efficacy of distal synapses, providing additional inward current results show that a dendritic spike transiently increases synaptic to enhance axonal (AP) output, thus increasing efficacy by promoting short bursts of somatic APs but dampen AP synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger output over longer timescales. The interplay between these two , but their influence on axonal output is not well effects during sustained parallel fiber input results in a “clamping” understood. We have used simultaneous somatic and dendritic of Purkinje cells output over long timescales and, thus, a flattening patch-clamp recordings to directly assess the impact of dendritic of synaptic gain, in striking contrast to pyramidal cells (9). calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, Results followed by pauses in spiking, which cancelled out the extra spikes in Single Dendritic Spikes Differentially Affect Axonal Output on the burst. As a result, average output firing rates during trains of Different Timescales. We made simultaneous somatic and den- input remained independent of the input strength, thus flattening dritic recordings (average distance 141 ± 11 μm, n = 9, range 102– synaptic gain. We demonstrate that this “clamping” of AP output 194 μm) from Purkinje neurons in rat cerebellar slices. Purkinje by the pause following dendritic spikes is due to activation of high cells were spontaneously active (28), and somatic APs were se-

conductance calcium-dependent potassium channels by dendritic verely attenuated at dendritic recording sites (17, 19). To test the NEUROSCIENCE spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, effect of dendritic spikes on somatic AP output, we evoked ex- thus have differential effects on temporally coded and rate coded citatory postsynaptic potentials (EPSPs) by stimulating parallel information: increasing the impact of transient parallel fiber input, fibers close to the dendritic recording electrode (Fig. 1A). Stim- while depressing synaptic gain for sustained parallel fiber inputs. ulus intensity was carefully adjusted to reach dendritic spike threshold (13), such that dendritic spikes were only triggered in cerebellum | | | synaptic integration some trials at identical stimulus strengths (Fig. 1B). Dendritic spikes triggered a brief burst of somatic APs (2.46 ± 0.18 APs, n C D fi hallmark of active is their ability to produce re- = 9; Fig. 1 and ) at high instantaneous ring rates (maximum fi ± ± generative events known as dendritic spikes (1–8). In pyra- ring rate 367 52 Hz with dendritic spikes vs. 236 26 Hz A n P < E midal neurons, the inward currents associated with dendritic without dendritic spikes, =9, 0.02; Fig. 1 ). However, the burst of APs was followed by a prolonged pause that was not spikes provide a strong local that can boost distal C synaptic inputs and enhance their effect on axonal action potential observed in the response to subthreshold EPSPs (Fig. 1 ). The (AP) output (1–4), particularly during burst generation (5, 6). longest somatic interspike intervals (ISIs) were identical before and after EPSPs, which did not trigger dendritic spikes (44 ± 4.6 Furthermore, dendritic spikes can enhance the precision of axonal ± n P APs in hippocampal pyramidal neurons (7) as well as in neo- ms vs. 44 5.4 ms, respectively, =9, = 0.99). In contrast, EPSPs which triggered dendritic spikes were followed by sub- cortical pyramidal cells in vivo (8). Dendritic spikes thus have stantially longer maximal somatic ISIs (71 ± 11 ms, n =9;P < 0.02 a boosting effect on the output of pyramidal cells, thus enhancing compared with subthreshold EPSPs; Fig. 1E). The effects of the gain of the synaptic input-output (I/O) function (9, 10). In dendritic spikes on somatic firing were independent of the loca- contrast, the effect of dendritic spikes on AP output in Purkinje tion of the synaptic input (recording distance along the dendrite, cells is not well understood. dendritic spikes, origi- range 102–194 μm; r = 0.17; r = −0.12). nally discovered in alligator Purkinje cells (11, 12), can be triggered (evoked spikes) (pause) fi fi The net effect of an input on AP output (stimulus-evoked by strong parallel ber (PF) activation (11, 13) or climbing ber spikes) can be quantified by integrating the poststimulus time activation (14, 15) and are due solely to activation of dendritic histograms (PSTH) and subtracting spontaneous activity (Meth- – voltage-gated calcium channels (13, 16 18), because Purkinje cells ods and refs. 29 and 30). This analysis confirmed that dendritic lack dendritic voltage-gated sodium channels and active back- spikes resulted in an increase in the peak number of somatic APs fl propagation of APs (17, 19). Calcium in ux driven by dendritic triggered by the stimulus (Fig. 1F). On average, EPSPs triggering – spikes has an important role in triggering synaptic plasticity (20 dendritic spikes added significantly more somatic APs immedi- 22) and dendritic release of and neuro- ately following the stimulus than subthreshold EPSPs (2.3 ± 0.3 modulators (13, 23, 24). Dendritic spikes triggered by climbing APs vs. 1.7 ± 0.4 APs, n =9,P < 0.01; Fig. 1F). However, the fiber input have virtually no effect on the somatic complex spike increase in somatic AP output was transient, because after a few waveform, probably due to the large synaptic and intrinsic con- ductances activated during the complex spike (14). However, the functional role of parallel fiber-driven dendritic spikes in regulat- Author contributions: E.A.R. and M.H. designed research; E.A.R. performed research; ing axonal output has not been addressed directly. This distinction E.A.R. analyzed data; and E.A.R. and M.H. wrote the paper. is crucial, because the state of the Purkinje cell dendritic tree is The authors declare no conflict of interest. very different during climbing fiber and parallel fiber excitation *This Direct Submission article had a prearranged editor. (25), and because climbing fiber input occurs only at ≈1 Hz in vivo 1To whom correspondence should be addressed. E-mail: [email protected]. fi (26, 27), whereas parallel ber input occurs continuously at high This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rates. We have therefore directly probed the relationship between 1073/pnas.1008605107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008605107 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 dendrite, 184 µm A dendritic stimulation B EPSP + dendritic spike recording electrode electrode 10 mV somatic subthreshold EPSP recording (no dendritic spike) PC electrode 25 ms

Fig. 1. Single dendritic spikes enhance AP output PF. stim. on short, but not long, timescales. (A) Simultaneous whole-cell recordings were made from the No dendritic spikes Dendritic spikes C PF. stim. D PF. stim. and dendrite of the same Purkinje cell while stim- ulating PFs close to the dendritic recording site. (B) Stimulating PFs at the threshold for dendritic spike generation resulted in subthreshold EPSPs (black) or dendritic spikes (red, same traces as in C and D). (C) Somatic (dotted line) and dendritic (thick line) 20 mV voltage recording during a single parallel fiber 50 ms stimulus (arrow) not triggering a dendritic spike. The raster plot and the PSTH contain 10 trials; bin size is 2 ms. (D) Same as in C, except the synaptic stimulus triggered a dendritic spike. The raster plot shows 10 trials, the PSTH contains 45 trials; bin size is 2 ms. Note the somatic AP burst associated with 0.2 the dendritic spike and the following pause in so- matic firing. (E) Pooled averages of maximum so- matic instantaneous firing rates and maximum somatic ISIs (n = 9). Black bars show the effect of E F G EPSPs without dendritic spikes, red bars show the ** 3 * 3 effect of EPSPs with dendritic spikes, and the blue 400 80 1.5 bar represents the average maximal somatic ISI during spontaneous activity showing no significant 1.0 F spont. ISI 2 2 pauses are present without dendritic spikes. ( )The C D 0.5 PSTHs in and were integrated then normalized 200 40 to trial number and spontaneous firing rate, thus dendritic spikes 1 1 0 no dendritic spikes only showing the stimulus-evoked spikes. (G) Bar

maximal firing rate graphs showing the pooled averages of maximum

stimulus evoked spikes 0 100 200 300 400 sustained added spikes maximum added spikes

stim. evoked pause (ms) number of stimulus added spikes and the number of 0 0 time (ms) 0 0 d-sp. d-sp. d-sp. d-sp. sustained added spikes (in a 100-ms window start- no. d-sp. no. d-sp. no. d-sp. no. d-sp. ing 100 ms after the stimulus, n = 9). *P < 0.02.

tens of milliseconds, there was no significant difference between however, the average somatic firing rate decreased from 241 ± 8 the effect of suprathreshold and subthreshold EPSPs (1.6 ± 0.2 Hz to 187 ± 12 Hz (P < 0.005, n = 5; Fig. 2E) during the train. APs vs. 1.5 ± 0.4 APs, respectively, n =9,P = 0.9; Fig. 1G). We Dendritic spikes thus reduced average axonal output. These also estimated the time window for reading out dendritic spike- results indicate that dendritic spikes triggered by trains of parallel related axonal firing changes by comparing the distribution of fiber synaptic input can paradoxically inhibit somatic firing. stimulus evoked spikes with or without dendritic spikes across cells for every 1-ms time bin. This measure was significantly Dendritic Spikes Reduce the Gain of the I/O Function. To characterize different for 73 ms following the stimulus (n = 9 cells, P < 0.05). the effect of dendritic spikes on the I/O function using a direct Dendritic spikes thus produce a short-term increase in somatic AP measure of input strength, we substituted synaptic stimulation output. However, this increase is not reflected in a long-term with dendritic injection of synaptic-like currents. The injected change in output, because the transient increase in spiking is current waveforms approximated the temporal dynamics of PF cancelled out by the succeeding pause in spiking. transmission (34), and an I/O curve was constructed by varying the mean amplitude of the injected current from sweep to sweep Dendritic Spikes Triggered by Synaptic Input Bursts Suppress Somatic over a wide range (Fig. 3 A and D). Low levels of synaptic input Firing. Granule cells, whose form the parallel fibers, can increased the average somatic firing rate in a manner linearly respond to sensory stimulation with bursts of APs in vivo (31, 32). related to the amount of injected current (r = 0.98 ± 0.01, n =5; To determine how dendritic spikes triggered by bursts of parallel Fig. 3 G and H). In contrast, for current injections above the fiber input affect somatic output, we applied 10 PF stimuli at 100 Hz dendritic spike threshold, the average somatic firing rate was while monitoring somatic AP output. Somatic AP firing rate pro- independent of the amount of injected current (r = −0.08 ± 0.18, gressively increased during the train (Fig. 2A), due to the facili- P < 0.005 compared with no dendritic spikes, n = 5; Fig. 3 G and tation of PF transmission and summation of synaptic potentials H), essentially flattening the f/I (firing rate vs. injected current) (33). Sufficiently strong synaptic stimulation triggered dendritic curve. As a result, the average somatic firing rate during den- spikes (Fig. 2B). Strikingly, initiation of dendritic spikes was as- dritic spiking was clamped at 214 ± 23 Hz (n = 5; Fig. 3H) across sociated with a decrease in the average somatic firing rate, despite a wide range of input strengths. the presence of continuous parallel fiber synaptic input (Fig. 2C). Activation of dendritic spikes was also associated with a change To quantify this result, we compared somatic AP output in the in the output firing pattern (compare Fig. 3 B and E). The max- first and last 50 ms of the train, because dendritic spikes were imal and minimal somatic firing rates during the train were dif- usually triggered late in the train. When no dendritic spikes were ferentially affected by dendritic spikes (Fig. 3 C and F). When triggered, the average somatic firing rate between the first and dendritic spikes were triggered, maximal firing rates were higher second half of the train increased from 148 ± 16 Hz to 190 ± 13 Hz but were independent of input strength, indicating that dendritic (P < 0.001; n = 5; Fig. 2D). When dendritic spikes were triggered, spikes cause an all-or-nothing rather than a gradual increase in

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008605107 Rancz and Häusser Downloaded by guest on September 27, 2021 A Control analyzed region antagonist of BK channels (37), the clamping effect of dendritic spikes on somatic output disappeared (Fig. 4 A and B). The re- lationship between dendritic current injection and somatic firing rate remained linear (although with a slightly reduced slope) after the appearance of dendritic spikes (r = 0.78 ± 0.12, n =3,P = 0.2 compared with no dendritic spikes; Fig. 4C). Furthermore, den-

PF stim. 350 inst. firing rate dritic spikes were no longer associated with pauses in somatic

250 spiking in the presence of penitrem A. Accordingly, when dendritic

150 spikes were evoked in the presence of penitrem A, the somatic ISI

50 distribution remained unimodal but shifted to the left (Fig. 4D), fi 20 mV consistent with the continuing increase in average somatic ring rate with increasing injected current. These results show that the B Dendritic spikes 20 ms activation of BK channels by dendritic spikes (resulting in long ******* dendritic AHP) is responsible for the pause in somatic firing, clamping the average somatic firing rate.

BK Channel Block Shifts the Balance of Inward and Outward Currents Associated with Dendritic Spike. Given that physiological synaptic PF stim. 350 inst. firing rate input can produce changes in membrane conductance that can 250 affect excitability and synaptic integration (6, 38–40), we tested 150 the effect of BK channel block on the I/O function associated 50 with parallel fiber synaptic input. In the presence of penitrem A, onset of d-spikes no dampening of somatic output was observed for synaptic input suprathreshold for dendritic spikes (Fig. 5A), with the in- C D E fi

sm stantaneous ring rate increasing monotonically during the syn- 50 Hz sm0

05ts C

300 5tsal s

50 ms sm0 300 aptic stimulus (cf. Fig. 2 ). Furthermore, there were no pauses

m05 r if fi

5t in somatic AP ring following dendritic spikes. Thus BK chan-

t

s

s

ri

al f nels underlie the inhibitory effect of dendritic spikes on somatic NEUROSCIENCE 200 200 output, regardless of how they are triggered. d-spikes To understand the functional relevance of this clamping mech- control onset of 100 100 fi average firing rate (Hz) anism, we compared the maximal somatic ring rates measured average firing rate (Hz) d-spikes control d-spikes with and without dendritic spikes to the frequency limit for reli- Fig. 2. Multiple synaptically triggered dendritic spikes suppress AP output. able axonal propagation of APs in Purkinje cells. The maximal (A) Somatic (gray) and dendritic (red) voltage traces during 10 PF stimuli sustained firing rate was below the axonal propagation limit (236 delivered at 100 Hz. The instantaneous somatic firing rate (blue) is increasing ± 15 Hz; refs. 41 and 42), both when the input was subthreshold as long as the synaptic input is active. (B) Same as in A except with stronger for dendritic spike generation (225 ± 13 Hz, n = 5) and when synaptic stimulus. The onset of dendritic spikes (*) in the dendritic voltage dendritic spikes were activated (214 ± 23 Hz, n = 5; Fig. 5B). trace (orange line) coincides with a sharp drop in the somatic instantaneous fi C fi However, when BK channels were blocked, the sustained somatic ring rate. ( ) Overlay of instantaneous somatic ring rates with and fi ± n without dendritic spikes. (D) Comparison of the average somatic firing rate ring rate could reach up to 320 Hz (average 293 13 Hz; = 3), during the first and last 50 ms of the stimulus when no dendritic spikes were which would result in failures of AP propagation during sustained triggered, showing a clear increase during the stimulus (five cells). (E) Same spike firing. as in D, except for trials with dendritic spikes (five cells). Purkinje cell axons can propagate short bursts of APs more ef- ficiently than sustained firing (41, 42), with the axonal propagation limit being 438 ± 37 Hz for short bursts (42). The highest burst maximal firing rate (Fig. 3G). In contrast, the minimal firing rate, frequency observed in the absence of dendritic spikes was 380 Hz reflecting the pauses following dendritic spikes, decreased with (average 303 ± 26 Hz; n = 5), below the axonal limit for short increasing input and increasing number of dendritic spikes (Fig. bursts. However, when dendritic spikes were triggered, the fastest 3G). The pause was also prominent in the somatic interspike in- somatic bursts reached an instantaneous firing rate of 461 ± 36 Hz, terval distributions (Fig. 3F): When no dendritic spikes were a value comparable to the axonal propagation limit for short bursts evoked, somatic interspike intervals adopted a unimodal distri- (41, 42). When BK channels were blocked, the fastest bursts bution with the peak corresponding to the average firing rate. reached frequencies of 669 ± 73 Hz (n = 3; Fig. 5C), well above the However, when dendritic spikes were triggered the ISI distribu- frequency limit for axonal propagation. Thus, both for sustained tion showed two distinct peaks, one corresponding to faster than somatic firing and for short somatic AP bursts, dendritic spikes average ISIs (i.e., bursts triggered by the dendritic spikes) and maintain somatic firing below the level at which failures of axonal a second peak corresponding to pauses in somatic firing. These transmission occurs. longer ISI values always followed dendritic calcium spikes (Fig. 3E), consistent with the idea that dendritic spikes promote pauses Evaluating the Downstream Consequences of Dendritic Spikes. We in somatic AP firing (cf. Fig. 1). have shown that dendritic spikes, generated by parallel fiber input, alter the temporal pattern of Purkinje cell output. To determine BK Channel Activation Underlies the Pause Following Dendritic how this altered pattern is relayed to the postsynaptic neurons in Spikes. To probe the mechanism underlying the pause and the the deep cerebellar nuclei (DCN), we fed the AP patterns recor- clamping of the f/I relationship triggered by dendritic spikes, we ded from Purkinje cells into a deterministic model of the Purkinje tested the involvement of high conductance calcium-activated po- cell to DCN synapse (ref. 43 and Fig. S2A). The extra APs trig- tassium (BK) channels, which contribute to the afterhyperpolari- gered by single PF inputs caused a brief increase in the inhibitory zation (AHP) of somatic APs (35) and dendritic spikes (13, 36) in synaptic conductance in the DCN (Fig. S2B). When den- Purkinje cells (Fig. S1). We therefore measured the I/O relation- dritic spikes were triggered, the peak synaptic conductance change ship of Purkinje cells after blocking BK channels. When dendritic was larger (P < 0.01; n = 7) and followed by a larger trough (P < spikes were triggered in the presence of penitrem A, a selective 0.05; n =7;Fig. S2C). The change in AP pattern caused by the

Rancz and Häusser PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 Fig. 3. Current injection-evoked dendritic spikes suppress ABC A weak input 40 AP output. ( ) Somatic (gray) and dendritic (red) voltage 0.4 30 trace during dendritic injection of synaptic-like current 20 (green). (B) Superimposed somatic (gray) and average den- 0.2

events / bin events 10 dritic (red) traces triggered by the current injection. AP 0 probability (blue) follows the input strength. (C)Histogram spike probability 0 10 mV 12840 of somatic interspike intervals triggered by current injection 20 mV 5 ms interspike interval (ms) D 2.5 nA EFbelow dendritic spike threshold. (D)AsinA but with stron- * ** 20 ms ger current injection triggering dendritic spikes (asterisks). strong input * * * 40 E 0.4 ( ) Superimposed somatic (gray) and average dendritic (red) 30 traces triggered at dendritic spikes. Somatic AP probability 0.2 20 (blue) is strongly reduced following dendritic spikes. (F)

events / bin events 10 Histogram of somatic interspike intervals triggered by cur- 0 spiking probability 0 10 mV G 12840 rent injection above dendritic spike threshold. ( )Average 5 ms interspike interval (ms) (black), maximal (red), and minimal (blue) somatic firing rates and the number of dendritic spikes (green) during the G H current injection is plotted versus the average injected cur- maximum rate clamped frequency (Hz) 400 1.5 300 rent. Note the linear f/I relationship before dendritic spikes 300 fi AP firing rate (Hz) occur and the input independence of somatic ring during 200 average rate 1 200 dendritic spikes. (H) Pooled data from five cells demon-

100 minimum rate strong input strating the linearity of the f/I curve below (weak input) and 0.5 100 above (strong input) dendritic spike threshold and the firing number of 8 rate where dendritic spikes appear ensuing the clamping 4 dendritic spikes 0 input weak fi 0 f/I curve linearity (r) effect. Average values marked with red, lled circles. Dotted 0 0.5 1 1.5 2 lines connect measurements from the same cell (black, open average injected current (nA) - 0.5 circles). (A–G) Data from the same cell.

dendritic spike thus improves the signal-to-noise of the post- excitatory, increasing the gain of synaptic input. We demonstrate synaptic conductance change in the downstream DCN neurons. that the mechanism of this paradoxical inhibition caused by den- Similar results were observed when using a more prolonged PF dritic spikes in Purkinje cells involves the activation of dendritic stimulus (10 PF stimuli delivered at 100 Hz; Fig. S2D). Again, the calcium-activated BK-type calcium channels, which balance the in- occurrence of dendritic spikes increased the peak synaptic con- ward current provided by calcium channel activation. These results ductance (P < 0.01; n = 5) and enhanced the period of decreased indicate that the complement of dendritic voltage-gated con- synaptic inhibition following the PF input train (P < 0.05; n =5; ductances determines the functional signature of dendritic spikes. Fig. S2E). These modeling results suggest that the short–term ef- This signature is cell-type specificandmayreflect the opposite po- fect of dendritic spikes on Purkinje cell output (burst followed by larity of neuronal output in pyramidal cells and Purkinje cells. pause) is further amplified by the properties of the Purkinje- DCN synapse. Dendritic Spikes Trigger Pauses in Axonal Output. We demonstrate that in Purkinje cells, dendritic spikes exert a dual role on axonal Discussion output. On short timescales, they enhance AP firing, triggering We have shown that synaptically evoked dendritic spikes in Pur- a brief burst of spikes. This effect is similar to pyramidal cells, kinje cells serve a dual role: They enhance axonal output on brief where dendritic spikes are also associated with enhanced axonal timescales but paradoxically inhibit average axonal firing rates over AP generation (1, 6, 7), often leading to bursts of spikes (5, 6). In longer timescales. This inhibitory effect presents a striking con- contrast, on longer timescales, the dendritic spike leads to a pro- trast to cortical pyramidal cells, where dendritic spikes are purely longed pause in spontaneous firing following the parallel fiber

Fig. 4. The clamping of the input-output re- A C 1.5 E lationship by dendritic spikes requires BK channels strong input + 100 nM penitrem A strong synaptic input + 100nM Penitrem A inst. firing rate (Hz)

d-spikes 300 and maintains axonal output below the propaga- 1 no d-spikes 250 tion limit. (A) Somatic (gray) and dendritic (red) 20 mV 20 mV 2.5 nA recording during dendritic current injection (green). 200 20 ms 0.5 20 ms BK channels were blocked by 100 nM penitrem A. 150 100 Note the lack of dendritic afterhyperpolarization f/I curve linearity (r) and somatic pause following dendritic spikes. The 0 PF. stim. 50 slow AHP of somatic spikes is also reduced. (B)Av- erage somatic firing rate (black) and the number of dendritic spikes (red) during the current injection is BDF G plotted versus the peak of the injected current. 325 no d-spikes 300 700

12 d-spikes 40 d-spikes 600 Note that with the appearance of dendritic spikes, 275 260 8 30 500 the somatic output continues to correlate positively 225 20 220 400 C 4 with the input, although with a reduced gain. ( ) 175 10 spikes / bin 180 300 Pooled values of the linearity of the f/I curve below 125 0 0 200 max. inst. firing (Hz) somatic firing (Hz) 1.20.80.40 1086420

(black) and above (red) dendritic spike threshold max. sustained firing (Hz) . d-sp. aver. inj. current (nA) interspike interval (ms) w BK block BK block n D axonal limit axonal limit ( = 3). ( ) The interspike interval histogram is ctrl. no. d-sp.ctrl. ctrl. noctrl. d-sp. w. d-sp. shifted to the left and remains unimodal with the (Monsivais 2005) (Monsivais 2005) appearance of dendritic spikes when BK channels are blocked. (A, B, and D) Data from the same cell. (E) Somatic (gray) and dendritic (red) voltage trace during synaptically triggered dendritic spikes. BK channels were blocked by 100 nM penitrem A. The instantaneous somatic firing rate (blue) keeps increasing when the synaptic input is active despite the presence of dendritic spikes. (F) Pooled average of maximal sustained somatic firing rates during synaptic-like current injections. (G) Pooled average of the maximal somatic instantaneous firing rates. Here again, the BK channel-dependent dampening mechanism invoked by dendritic spikes keeps the instantaneous somatic firing rate below the axonal propagation limit.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008605107 Rancz and Häusser Downloaded by guest on September 27, 2021 synaptic input, an inhibitory effect that cancels out the effect of the the f/I relationship becomes flat, with average firing rate remaining burst of spikes on the average axonal firing rate. Thus, the net effect clamped at ≈220 Hz, independently of the input intensity. This of dendritic spikes on average axonal output rate is neutral. When clamping is in sharp contrast to pyramidal cells, where recruitment trains of synaptic inputs activate multiple dendritic spikes, this in- of dendritic voltage-gated calcium channels during dendritic syn- hibitory effect can summate, leading to the clamping of the output aptic input produces an increased gain of the f/I function (9). The firing rate at a fixed value. Interestingly, spike synchronization (a clamping or saturation of the I/O curve at high levels of synaptic timing code) and spike rate modification (a rate code) have been input can also be observed in I/O curves, but it is shown to relay different information in another motor area, the thought to be due primarily to shunting by synaptic conductances. primary motor cortex of macaque monkeys (44), suggesting the two In contrast, Purkinje cells exhibit an intrinsic mechanism based on coding strategies can coexist and complement each other. The balanced voltage-gated conductances for regulating synaptic gain climbing fiber input in Purkinje cells also triggers dendritic spikes, and limiting dynamic range at high input intensities. This rapid, which have recently been shown to regulate the postcomplex spike real-time mechanism will complement other ways to limit output pause in axonal firing (14). However, the dendritic spikes activated gain, such as feedforward inhibition (30), which is active over by the complex spike are global, and their effect on axonal output is a wider range of input strengths, and retrograde endocannabinoid- weakened by the strong synaptic and intrinsic conductances active dependent suppression of parallel fiber input (13), which provides during the complex spike. In contrast, here we show that dendritic negative feedback over longer timescales. Recent advances in spikes triggered by single parallel fiber stimuli, which produce calcium imaging (4) and whole-cell recording in awake, freely highly localized spikes (13), can still produce a significant effect on behaving animals (60) should eventually help make it possible to axonal spiking, both in terms of burst generation and the sub- determine under which conditions this mechanism is called into sequent pauses. This pausing effect of dendritic spikes, an intrinsic action during behavior. counterpart to feed-forward inhibition, is particularly significant What is the functional purpose of the clamping effect of den- given that they are superimposed on top of the high spontaneous dritic spikes on axonal output? Axonal spiking is energetically firing rate of Purkinje cells (28, 45, 46), and may contribute to the expensive (61; see also ref. 62), and given the high spontaneous pauses in Purkinje cell spiking seen in vivo (45, 47) and following firing rates exhibited by Purkinje cells, this intrinsically imposed synchronous PF stimulation in vitro (48). ceiling on activity may represent an energy-saving measure. Fur- thermore, the close match between the firing rates at which den- Relative Contribution of Voltage-Gated Conductances Driven by dritic spikes clamp somatic output and the maximal frequency

Dendritic Spikes. What is the mechanism driving the enhanced for faithful transmission of spiking (41, 42) suggests that the clamp- NEUROSCIENCE pause following a dendritic spike and the clamping effect produced ing of axonal output may be required to maintain optimal trans- during multiple dendritic spikes? We demonstrate that a selective mission of spikes along the by obviating generation of spikes blocker of BK-type calcium-activated potassium channels, which which cannot be transmitted. strongly reduces the dendritic AHP following a dendritic spike (13, Finally, the temporal dynamics of the axonal spiking pattern 36), can prevent the clamping effect. This result indicates that the associated with dendritic spikes—a burst of APs followed by outward current mediated by BK channels is sufficiently strong to a pause—may also have important consequences for information counteract the net effect of the inward current delivered by acti- transfer at the Purkinje cell to deep cerebellar nuclei (DCN) vation of P-type calcium channels during the dendritic spike. This relay (47) by improving the discriminability of learned patterns in finding is consistent with voltage clamp experiments in isolated Purkinje cells (48) and producing an improved signal-to-noise at Purkinje cell somata showing that the net effect of blocking cal- the synaptic connection with DCN neurons, due to the short- cium channels is to remove an outward current (49), indicating term dynamics of Purkinje cell synapses (63, 64). This effect will that calcium-activated potassium currents predominate over cal- further be amplified by the summation of postsynaptic IPSPs (65) cium currents (at least in the somatic membrane). To most ef- and consequently also the rebound excitability of DCN neurons fectively influence the shape of the dendritic spike, and its (54, 66, 67). In this way, even though parallel fiber-triggered den- afterhyperpolarization, the BK channels must be localized close to dritic spikes experience marked attenuation toward the soma the source of the calcium entry triggered by the dendritic spikes, (13), they can still profoundly influence axonal output and its i.e., in the dendrite; this assumption is consistent with anatomical downstream consequences. (50) and electrophysiological evidence for the dendritic location of BK channels in Purkinje cells (51). Once the threshold for den- Methods dritic spiking is reached, this balancing of inward and outward All procedures were carried out with approval from the UK Home Office. conductances remains effective over a wide range of input Sagittal slices (200–250 μm thick) were prepared from the cerebellum strengths, producing a flat I/O curve. Thus, the relative density of 18–25 d postnatal Sprague–Dawley rats (68). ACSF for slicing and re- and dynamics of activation and inactivation of calcium channels cording contained 125 mM NaCl, 26 mM NaHCO3, 25 mM glucose, 2.5 mM and BK-type channels (and possibly other conductances; refs. 39 KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, and 1 mM MgSO4 (305 mOsm). All ± and 52–57) must be carefully calibrated to produce this robust recordings were carried out at 34 1 °C. Simultaneous somatic and dendritic whole-cell patch clamp recordings were made from Purkinje neurons under balancing effect, which is independent of input strength. The visual control using differential interference-contrast optics (13, 68). Patch balancing of inward and outward currents driven by a dendritic electrodes (soma: 5–6MΩ; dendrite: 6–10 MΩ) were filled with 130 mM

spike appears to be a distinctive signature of Purkinje cells, be- methanesulfonic acid, 7 mM KCl, 2 mM Na2ATP, 2 mM MgATP, 0.5 mM cause in pyramidal cells, inward currents appear to predominate Na2GTP, 0.05 EGTA and 0.4 wt/wt% biocytin at pH 7.30 with KOH (285 and pausing is not observed. mOsm). Recordings were made by using a Multiclamp 700A amplifier (Mo- lecular Devices). PF inputs were stimulated (10–90 V, 0.1–0.2 ms) using ACSF- Consequences for Cerebellar Function. Our results are consistent filled patch pipettes placed under visual control ≈100 μm directly beneath with two modes of integration of parallel fiber synaptic input in the dendritic recording site. EPSPs were mimicked by injection of current cerebellar Purkinje cells. At low input strengths, the relationship waveforms shaped like EPSCs, consisting of a double-exponential function t t between maximal firing frequency and parallel fiber input is linear with rise = 0.6 ms and decay = 6 ms. To mimic physiological PF trains, the dynamics of the EPSC amplitudes were calculated based on a modified fa- (29, 33, 58), enabling the simplest possible encoding strategy for fi cilitation-depression model (ref. 34 and Dataset S1). Different input ampli- parallel ber input strength. Dendritic properties may contribute tudes were randomized from sweep to sweep. Recordings were low-pass to, but are not required for this linearity, because the f/I curve of filtered at 8 kHz and sampled at 20–50 kHz by using an Instrutech ITC18 Purkinje cells remains linear after the removal of the dendrites DAC-board controlled by Axograph, and were analyzed by using Igor Pro. To (59). Above threshold for generation of dendritic spikes, however, determine the net spike output in response to synaptic input with or with-

Rancz and Häusser PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 out dendritic spikes, poststimulus time histograms (PSTH) were computed Sigma or Tocris. Linearity was assessed by using Pearson’s correlation co- fi fi – and integrated. To account for spontaneous ring, a linear tto200 600 ms efficient (Pr), significance by Student’s t test, and all data are given as av- prior the stimulus was extrapolated over the entire trial duration and sub- erage ± SEM. tracted from the integral to yield the spontaneous activity corrected cu- “ ” mulative spike count, which we called stimulus evoked spikes (29, 30). For ACKNOWLEDGMENTS. We thank Jenny Davie, Wolfgang Mittmann, and the synaptic conductance modeling, we used the deterministic model of ref. Arnd Roth for helpful discussions. This work was supported by grants from 43 implemented in Igor Pro. Methanesulfonic acid was obtained from Fluka, the Gatsby Charitable Foundation, the Wellcome Trust, and the European penitrem A from Alomone Labs; all other chemicals were obtained from Commission. E.A.R. holds a Sir Henry Wellcome Postdoctoral Fellowship.

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