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The dendritic processing in cerebellar Purkinje cells (PCs), which integrate synaptic inputs coming from hundreds of thousands granule cells and molecular layer interneurons, is still unclear. Here we have tested a leading hypothesis maintaining that the significant PC output code is represented by burst-pause responses (BPRs), by simulating PC responses in a biophysically detailed model that allowed to systematically explore a broad range of input patterns. BPRs were generated by input bursts and were more prominent in Zebrin positive than Zebrin negative (Z+ and Z−) PCs. Different combinations of parallel fiber and molecular layer interneuron synapses explained type I, II and III responses observed in vivo. BPRs were generated intrinsically by Ca-dependent K channel activation in the somato-dendritic compartment and the pause was reinforced by molecular layer interneuron inhibition. BPRs faithfully reported the duration and intensity of synaptic inputs, such that synaptic conductance tuned the number of spikes and release probability tuned their regularity in the millisecond range. Interestingly, the burst and pause of BPRs depended on the stimulated dendritic zone reflecting the different input conductance and local engagement of voltage-dependent channels. Multiple local inputs combined their actions generating complex spatio-temporal patterns of dendritic activity and BPRs. Thus, local control of intrinsic dendritic mechanisms by synaptic inputs emerges as a fundamental PC property in activity regimens characterized by bursting inputs from granular and molecular layer neurons.
Purkinje cells (PCs) are the final common collector of the whole neuronal activity generated in the granular and molecular layer of the cerebellar cortex (Eccles et al., 1967; Ramón y Cajal, 1995). A complex structural and functional organization allows PCs to elaborate the largest synaptic input of the brain, amounting to over 200,000 synapses in rodents (Korbo et al., 1993). PCs are characterized by active dendrites (Llinás and Sugimori, 1980a,b) endowed with different types of voltage-dependent channels. Several experiments and models have been developed with the aim to explain how PCs integrate synaptic inputs (De Schutter and Bower, 1994a,b; Rapp et al., 1994; Roth and Häusser, 2001; Brunel et al., 2004; Santamaria and Bower, 2005; Steuber et al., 2007; Masoli et al., 2015), but the role of voltage-dependent currents in local computation has not been fully clarified. PCs have recently been proposed to react to input bursts by generating a burst-pause response (BPR), which has been correlated with animal behavior (Cao et al., 2012; Herzfeld et al., 2015). While the intervention of inhibitory molecular layer interneurons (molecular layer interneurons; Barmack and Yakhnitsa, 2008; Bower, 2010; Grasselli et al., 2016) has been proposed to regulate the pause, the role played by dendritic properties in BPR generation remained unclear.
BPRs are thought to reflect a stereotyped PC response to bursting granular layer inputs. The granule cells generate brief spike bursts in vitro (D’Angelo et al., 1995; Nieus et al., 2006) and in vivo (Chadderton et al., 2004; van Beugen et al., 2013; Powell et al., 2015) in response to bursts in the mossy fibers (Rancz et al., 2007). These granule cell bursts are finely regulated by GoC inhibition (Mapelli et al., 2009; Nieus et al., 2014) generating specific patterns in the number and timing of spikes (D’Angelo and De Zeeuw, 2009; Arleo et al., 2010). The granule cell bursts are then conveyed to molecular layer activating PCs and molecular layer interneurons, which in turn generate feed-forward inhibition on PCs (Santamaria et al., 2007; Rieubland et al., 2014; Zhang and Südhof, 2016). Therefore, granule cells and molecular layer interneuron set-up complex spatio-temporal patterns of activity generating PC responses that have been classified as type I, II and III (De Zeeuw et al., 2011) and are differentiated depending on Zebrin positive or negative (Z+ or Z−) PCs in different cerebellar areas (Zhou et al., 2014, 2015).
Since synaptic activity patterns impinging on PCs are not fully known and since monitoring the PC dendritic response is challenging, a first insight into the way PCs respond to their inputs can be obtained using in silico simulations. Here we have faced the issue by exploiting a detailed PC model (Masoli et al., 2015) that was extended with excitatory and inhibitory synapses. This model is auto-rhythmic and its electroresponsiveness has been validated against a large set of biological experiments providing an ideal substrate to explore how intrinsic electroresponsiveness is modulated by synaptic inputs. Simulations with this PC model allowed us to face a set of questions. Does the PC model generate BPR in response to synaptic inputs? Is BPR different in Z+ and Z− PC models? Is BPR affected by the specific location of inputs on dendritic branches, as in the case of ascending axons (aa) vs. parallel fiber inputs (Sims and Hartell, 2005, 2006; Walter et al., 2009)? Is BPR different when inputs are randomly distributed rather than concentrated in limited sub-regions? What is the impact on BPR of the number of spikes and of the intensity of inhibition in the input burst? What are the mechanisms of BPR (intrinsic electroresponsiveness or synaptic inhibition) and what is the role of voltage-dependent ionic channels? Can BPR be modulated by synaptic plasticity? Simulations can provide a coherent mechanistic hypothesis on this broad range of questions that would be otherwise be hard to achieve.
The simulations showed that, for a broad set of activity regimens, PCs generate BPRs modulated by the excitatory/inhibitory balance and by synaptic plasticity. The underlying BPR mechanism reflects modulation of intrinsic pacemaking by Ca/KCa currents generated in the dendritic compartment and transmitted to the soma and axon initial segment (AIS) through the internal resistance. Therefore, BPR emerges as a relevant coding strategy for PCs that could be modulated by spatio-temporal granule cell spike patterns and synaptic inhibition and plasticity, generating the specific outputs to be transmitted to DCN.
The present synaptic PC model is based on the recent PC model of intrinsic electroresponsiveness by Masoli et al. (2015), which in turn derives its morphology from Rapp et al. (1994). The present model updates in several respects the previous active model by De Schutter and Bower (1994a,b) by incorporating a completely new set of ionic channels, by dislocating the action potential generation mechanism in the AIS and axon, by using dynamic synapses and by exploiting a wide set or recent experimental evidences for validation. The model by Masoli et al. (2015) (available on ModelDB), which was built to reproduce PC responses to current injections in vitro and in vivo, is extended here by connecting excitatory and inhibitory synapses on the dendrites and by evaluating a large set of combinations in the input space. As a further update to account for recent in vivo data, the maximum conductance of some ionic channels (soma HCN1 = 0.001 mS/cm2; soma Kv3.4 = 0.0515 mS/cm2; AIS Nav1.6 = 0.8 mS/cm2) was adjusted to raise the average frequency from 35 Hz to 40 Hz in the Z+ PC model (Zhou et al., 2014). The Z− differed Z+ models only for the presence of TRPC channels. TRPC channels are cationic channels generating a tonic depolarizing current, which, once placed in Z− PCs, raised background frequency to around 90 Hz (dendrite TRPC = 4.18e−6 mS/cm2). All simulations were performed at 37° with fixed time step (0.025 ms) and were run using NEURON multisplit (Python 2.7; NEURON 7.5; Hines et al., 2007, 2009) to exploit the eight-core processor of an AMD FX 8350 with 16 GB RAM and an AMD Ryzen 1800x 8 cores/16 threads with 32 GB RAM. The data were analyzed with custom python and MATLAB scripts.
PCs are endowed with hundred thousand spines (O’Brien and Unwin, 2006), each one receiving a single contact from an aa or a parallel fibers (pf) synapse (Walter et al., 2009). While spines may be critical to implement local biochemical processing during synaptic transmission, they have been suggested to generate linear attenuation with little impact on the overall synaptic excitation process (Ly et al., 2016). Therefore, spines were not reconstructed in the model and excitatory synapses were placed directly on the dendrites. The dendrites were divided into three orders of branching [branch I, composed by 105 sections with diameters between 3.5 μm and 9 μm; branch II, composed by 1111 sections with diameters between 1.2 μm and 3.5 μm; branch III, composed by 383 sections with a diameter between 0 μm and 1.2 μm] receiving specific synaptic inputs. Concerning excitatory synapses (Figure (Figure1A),1A), branch II received only pf synapses and branch III received only aa synapses (Santamaria et al., 2007; Lu et al., 2009; Bower, 2010). Branch I should have received a cf input (Kaneko et al., 2011; Zhang et al., 2015) but this was not used here (not shown). Concerning inhibitory synapses (Figure (Figure1A),1A), these were distributed only on branches I and II for a total of 221 sections, since branches III were experimentally reported not to have inhibitory synapses (Lu et al., 2009; Bower, 2010). Inhibitory synapses on branches I and II were made identical, although those on section I may have a different control (He et al., 2015). The inhibitory BC synapses on the soma and AIS (Iwakura et al., 2012; Blot and Barbour, 2014; Kole et al., 2015) were not used here (not shown).
The excitatory and inhibitory synapses were built according to Nieus et al. (2006, 2014) following a modified Tsodyks and Markram formalism (Tsodyks et al., 1998). Model EPSCs and IPSCs were adapted to reproduce unitary synaptic currents recorded from PCs at 37°.
The glutamatergic AMPA receptor-mediated EPSC model was derived from granule cells and the maximum synaptic conductance was balanced to reproduce a single pf EPSC (Barbour, 1993; Isope and Barbour, 2002). Release probability was adapted to account for pair-pulse facilitation (Zhang et al., 2015). The aa synapses were made with identical physiological properties as the pf synapses (Walter et al., 2009). The AMPA synapse parameters were: release probability = 0.13, τREC = 35.1 ms, τfacil = 54 ms, τI = 6 ms, Gmax of 2800 pS, reversal potential = 0 mV. The model as a whole faithfully reproduced the response to random pf stimulations (Dittman et al., 2000). The number of synapses was adjusted to imitate experimental observations of pf bursts that elicited excitatory 250 Hz spike burst in the PC soma (Walter and Khodakhah, 2006).
The gabaergic GABA-A receptor-mediated synaptic mechanism was derived from granule cells and modified by maintaining the alpha1 subunit but deleting the alpha6 subunit (absent in PCs). The value for the fitting were taken from Zhang et al. (2015) to account for in vivo recording from stellate cell (SC) in the range P35–P42 to better match the mature PC. The GABA-A synapse parameters were: release probability = 0.35, τREC = 15 ms, τfacil = 4 ms, τI = 1 ms and a Gmax = 1200 pS, reversal potential = −60 mV. The number of synapses was adjusted to imitate experimental observations of multiple SC that elicited background inhibitory activity at 1.5 Hz and single SC bursts up to 150 Hz (Zhang et al., 2015).
Several stimulation protocols were designed to reproduce the basic patterns used experimentally.
This protocol was constructed to investigate the BPR. The fundamental patterns were designed by combining a pf/aa bursts with a SC burst (De Zeeuw et al., 2011; van Beugen et al., 2013; Valera et al., 2016). In a first design, excitatory synapses were randomly distributed over 100 dendrites, both from aa or pf. This allowed to obtain a 250 Hz PC burst as in Walter et al. (2009). The inhibitory burst was composed by three spikes with a 7 ms interspike interval (ISI). Inhibitory synapses were distributed over 25 dendrites and delayed by 4 ms with respect to the excitatory burst to account or synaptic delays along the afferent neuronal chain (granule cell to SC to PC; see also Ramakrishnan et al., 2016). Variants to this pattern were used to reproduce type I, II and III responses (De Zeeuw et al., 2011; Valera et al., 2016), to evaluate the impact of synaptic inhibition, to selectively activate aa rather than pf synapses, to change the intensity or frequency or duration of the bursts, to modify synaptic parameters like release probability and maximum conductance, to restrict pf/aa activity to specific dendritic sectors.
The model response was recorded either in voltage clamp or in current-clamp at the soma, and the voltage-dependent ionic conductances were modified in some cases to test their impact on BPR generation. The membrane voltage, the calcium concentration, the ionic currents from each model section were recorded and saved in a nested MATLAB file. The model response properties were analyzed using MATLAB routines (MathWorks, Natick, MA, USA) and custom made Python scripts, using data recorded during each simulations. Each simulation data file contained information about the membrane voltage, ionic channels and synaptic currents recorded in specific location throughout the models.
Biophysical analysis of the model was carried out according to standard theory (Jack et al., 1975). The model electrotonic properties were analyzed using NEURON functions yielding the input impedance, Zin, for each model section and the corresponding signal attenuation between e.g., a dendritic sections and the soma, A = Vdend/Vsoma. This allowed to determine the electrotonic distance L = ln(A). This definition coincided with the more classical one (L = anatomical distance/length constant) defined when a neuron can be reduced through the 3/2 power branching rule to an equivalent cylinder. The NEURON definition proves particularly useful since PCs do not follow the 3/2 power branching rule (Hines and Carnevale, 2001; Hines et al., 2007, 2009).
Since the PC was continuously pacing and input bursts were applied randomly with respect to ongoing spike discharge, repeated PC responses to the inputs differed one from each other. This behavior was represented using raster plots and peri-stimulus time histograms (PSTH), that were constructed using MATLAB routines and were subsequently analyzed to extract the average properties of PC responses.
The impact of cortical input patterns, conveyed through pf, granule cell aa and stellate cells (SC), on PC spike firing was investigated using detailed PC models differentiated into Z+ and Z− types (Masoli et al., 2015). These models differ for the expression of depolarizing TRPC-like channels in the terminal dendrites, resulting in higher background SS activity in Z− than Z+ PCs (around 90 Hz vs. 45 Hz). Both models were endowed with excitatory and inhibitory synapses, which were located according to anatomical measurements. This, combined with the filtering properties of dendrites, caused the expected EPSP electrotonic decay from synapses to soma (Roth and Häusser, 2001). The synaptic models were endowed with dynamic mechanisms (Tsodyks and Markram, 1997; Nieus et al., 2006, 2014; Figures 1A,B) allowing to adapt neurotransmission to arbitrary spike patterns (Dittman et al., 2000; Figure Figure1C1C).
A first question was whether the PC model was able to generate BPRs following activation of excitatory and inhibitory synapses (Cao et al., 2012; Herzfeld et al., 2015) and whether BPRs were different between Z+ and Z− PCs. The PC responses to input bursts generated by Granule cells (Rancz et al., 2007; van Beugen et al., 2013; Powell et al., 2015; Wilms and Häusser, 2015; Delvendahl and Hallermann, 2016) were tested in Z+ and Z− PC models in three stereotyped functional cases corresponding to the definitions of type I, type II and type III responses reported in vivo (De Zeeuw et al., 2011). Type I responses were driven by pure Granule cell excitatory inputs (100 synapses), type II responses were driven by both granule cell excitatory inputs (100 synapses) and SCs inhibitory inputs (25 synapses), type III responses were driven by SC inhibitory inputs only (25 synapses; Figure Figure2).2). In all cases, molecular layer interneuron inhibition was delayed by 4 ms to account for delays accumulated along the synaptic chain (Eccles et al., 1967; Ramakrishnan et al., 2016). In this set of simulations, a random distribution of active synapses was used and maintained the same with all the different stimulation patterns (Figure (Figure2A2A).
In Type I responses, a brief pf burst elicited a PC burst terminating in close coincidence with the stimuli (average frequency of 260 Hz) followed by a pause, configuring a typical BPR. The pause between the last spike of the burst and the first spikes when SS activity restarted showed an ISI of 50.46 ± 25.6 ms. Type II responses showed a similar BPR as type I, with the pause showing an average ISI of 48.2 ± 20.4 ms (Steuber et al., 2007). Type III responses showed just the pause, with an ISI of 27.4 ± 3.7 ms. Thus, with this stimulation pattern, BPRs were determined by intrinsic properties of Z+ PCs and were accentuated by molecular layer interneurons.
In the Z− model, a 10-pulses/500 Hz pf burst generated a PC burst like in the Z+ model type I and type II responses. However, the Z− model was almost unable to generate any pauses, in either type I, II or III responses. This difference with the Z+ model was reduced by raising synaptic inhibition from 25 to 100 molecular layer interneuron synapses, which allowed pauses to emerge in the type I and type II responses (see Figure Figure2C).2C). Therefore, the Z− PC model showed reduced ability to generate intrinsic BPR, in which the pauses were markedly dependent on the amount of molecular layer interneuron inhibition. It should be noted that the reduced ability of Z− PC models to generate intrinsic BPRs was likely to be related to the high basal firing rate, since Z− became similar to Z+ BPR when the basal firing rate was equalized with somatic injection of a constant negative current (Figure (Figure2B2B).
Modulation of BPR was investigated in detail in Z+ PC type II responses, in which BPR was the most pronounced. Increasing the strength of inhibition did not change the burst but prolonged the pause. Moreover, responses to aa synapses were differentiated by considering their specific location on distal dendrites. The aa generated weaker BPR than pf probably because of their longer electrotonic distance from soma (see also Figure Figure6A).6A). However, the aa and pf BPR became very similar when aa transmission strength was increase, reproducing the functional equivalence of transmission along these two transmission lines (Sims and Hartell, 2005, 2006; Walter et al., 2009).
The BPRs mechanism was analyzed by tracking transmembrane currents and intracellular axial currents in different cellular compartments using the Z+ PC type I model, in which the intrinsic ability to generate BPRs was evident (Figure (Figure3A).3A). In the PC model, APs arise from the AIS (Masoli et al., 2015) and, not surprisingly, the pause is characterized by a protracted decrease of the AIS transmembrane current (IAIS; Jack et al., 1975). In fact, this makes the pause appearing as an interruption of pacemaking. Pacemaking in PCs is sustained by the persistent Na currents generated mostly in AIS and by Ca currents generated mostly in the dendrites and transmitted to the AIS through the internal resistance (Llinás and Sugimori, 1980a,b), providing two candidate mechanisms for pause generation.
Na currents in the AIS may be inactivated during the burst and then take time to recover, reducing the depolarizing drive. However, there was no remarkable inactivation of Na currents during the bursts that might prevent reactivation of the pacemaker. Therefore, this first mechanism could be excluded.
Ca channel activation and the consequent Ca entry into the dendrites may activate KCa currents overtaking inward currents and generating a protracted repolarizing drive. Actually, the current transmitted from the dendrite to soma (Idend-soma) and from soma to AIS (Isoma-AIS) was reduced during the pause compared to pacemaking regime. Therefore, reduced depolarizing current transmission from the somato-dendritic compartment to AIS appears as the main responsible of the pause.
The ionic nature of the mechanism was confirmed by specific manipulations of the Ca/KCa ionic mechanisms. When either dendritic KCa conductances or Ca conductances were set to zero, the BPR was altered with a marginal reduction of burst duration (from 0% to −16.7%) but a much more dramatic reduction of the pause (from −20% to −80%) (Figure (Figure3B).3B). It should be noted that all Ca channels proved critical for BPR, especially Cav2.1 and Cav3.1 (Cav3.2 switch-off blocked pacemaking; Masoli et al., 2015) as well as all KCa channels, including KCa1.1, KCa2.2, KCa3.1.
These observations show that making BPRs is an intrinsic property of the PC model, which depends on the generation of large KCa currents in the dendrites. In Z− PCs, the mechanism were the same as in Z+ PCs except that TRP channels injected a constant inward current through the dendrites counterbalancing KCa and making the pause more difficult to elicit (not shown).
A central issue in cerebellar physiology is how PCs transform signals coming from granule cells into specific outputs. The spike patterns emitted by granule cells in response to punctuate stimulation consist of short bursts composed of spikes with variable number and ISI (Chadderton et al., 2004; Rancz et al., 2007; D’Angelo and De Zeeuw, 2009). We have therefore used the PC model to simulate the impact on BPR of stereotyped patterns composed by a fixed number of pulses at different frequencies. Concerning burst length, the PC model showed an almost linear input/output relationship at frequencies ranging from 100 Hz to 500 Hz (Figure (Figure4A,4A, left). The pause also showed a slight dependency on the length of the input burst and this eventually caused the burst/pause ratio to reliably report the duration of the input burst (Figure (Figure4A,4A, right). There are two additional noteworthy properties. First, the linearity of burst and burst/pause coding was maintained almost independently from the input frequency. Secondly, the Ranvier nodes in the PC axon filtered the highest frequencies (Masoli et al., 2015) so that the burst was more reliably transmitted at the lower input frequencies (Figure (Figure4A,4A, bottom).
Another modality of granule cell response, largely investigated in the vestibulo-cerebellum, is to generate spike trains in response to prolonged mossy fiber inputs (Arenz et al., 2008). When such stimuli were used, the PC model followed the input frequency (Figure (Figure4B,4B, left), although the frequency dependence was rather weak (as also noted for the BPR in Figure Figure4A).4A). However, there was a steep relationship between the output frequency and the number of active synapses (Figure (Figure4B,4B, right). Therefore, the PC model was more efficient in detecting the intensity of the granule cell input rather than the frequency at individual synapses.
The recoding of input into output spike patterns in PCs is thought to depend on how long-term synaptic plasticity modifies PC synaptic responsiveness. While classical pf-PC LTD has postsynaptic expression and simply causes a scale-down of postsynaptic currents, a presynaptic change in release probability (P) would modify neurotransmission dynamics (Tsodyks and Markram, 1997; Nieus et al., 2006). These can shift from the typical short-term facilitation at low P to short-term depression at high P. These changes can therefore differentially affect the PC BPR.
At different P values (range 0.1 and 0.9), the overall response pattern in PC output bursts did not change remarkably. In all cases, the output spikes followed the input spikes quite closely during the burst, then one or two extra spikes were generated and the pause occurred. However, at a closer inspection of raster plots, the precision of action potential emission changed. By increasing P (from P = 0.13 to P = 0.91) the PC model responses were characterized by a greater precision, with decreased variability in burst spike pattern and pause length (see also Figure Figure2C).2C). Increased precision derived from the decreased paired pulse ratio (A2/A1, where A1 and A2 are the amplitudes of the first and second response in a pair). In this way, the first EPSP in the train became more precisely aligned with the input burst leading to a repeatable spike generation independent from the relative phase of background firing activity (see also Figure Figure88 below). Therefore, the highest precision was obtained when the pf-PC synapse expressed presynaptic pf-PC LTP. This suggests that release probability controls fine regulation of spike timing on millisecond scale.
In the case of a postsynaptic change in synaptic conductance G (−30% to 30% with respect to control), the only remarkable effect was a reduction in burst spikes precision when G was reduced, while no appreciable changes were observe in pause precision. The spike precision decrease at low G was likely related to a greater influence of previous spikes on burst initiation. Therefore, in the present conditions, precision could be more effectively tuned by P than G.
The PC shows complex branching (Nedelescu and Abdelhack, 2013) and active electroresponsiveness in the dendrites, so that the specific location of afferent synapses may influence the response pattern. The model was exploited to redirect to specific dendritic sectors (numbered I–IV) the same stimuli that were used before for synapses randomly distributed over the whole dendritic tree (see Figures Figures11–6).
The PC response was different depending on the stimulated sector (Figure (Figure6A).6A). When excitatory pf and inhibitory stimulations were delivered to dendritic sector I or IV, the PC BPRs were similar to those obtained using a random distribution of synapses. However, when excitatory stimulation was delivered to sector II or III, the pause was much longer than usual (over 250 ms in the Z+ model). When aa was substituted to pf stimulation, the responses were similar except for sector I, which showed a longer pause with aa then pf stimulation (the reason of this will be explained below). The difference between sectors was poorly sensitive to the location of inhibition, and BPRs did not change remarkably when inhibition was moved to sectors different from the one that was excited by pf synapses (Figure (Figure6B).6B). Similar response properties were observed in the Z− model, although pauses were shorter (data not shown).
In order to understand the mechanism differentiating responses among dendritic sectors, we compared sector I to sector II, which showed a remarkably different pause lengths. The impact of dendritic structure was considered first (Figure (Figure7A).7A). The local currents generated by synaptic activation (that were identical in the two sectors) caused a stronger depolarization in sector II than sector I. This reflected the different Zin, that was about twice as large for sector II than sector I. Accordingly, this determined different voltage attenuation profiles, so that sector I depolarization started from a higher level and then decayed over a longer distance while approaching the soma. It should be noted that, in these simulations, we used single-synapse stimuli causing small depolarizations from a hyperpolarized membrane potential, so that voltage-dependent currents were not remarkably activated.
Then we considered the activation of voltage-dependent currents in the dendrites (Anwar et al., 2014) by delivering the appropriate multisynaptic stimulation pattern to the PC model in pacemaking regime. The higher synaptic depolarization in sector II than in sector I resulted in a stronger voltage-dependent activation of Ca channels in the former than in the latter (Figure (Figure7B).7B). In sector II, LVA currents amplified the EPSPs leading membrane potential to raise enough to activate also the HVA currents, thus causing a regenerative calcium spike. Consequently, a large raise in intracellular calcium activated the KCa system. This effect eventually influenced the spike-generating mechanisms in the AIS and regulated the pause, that was longer in sector II than sector I.
The non-linear nature of voltage-dependent mechanisms of BPR and their topographical nature bear about two main consequences.
While sector I phenomena remained almost locally confined, the voltage-dependent effects initiated in sector II rapidly spread to neighboring sectors and eventually to the whole dendrite (Figure (Figure8B).8B). Full-blown calcium spikes appeared with some delay in a region close to sector II, involving LVA followed by HVA calcium channel activation causing a large [Ca2+]i raise and eventually KCa activation and the pause. Therefore, also dendritic sectors that are not activated synaptically can generate a local [Ca2+]i increase and take part to control the BPR pause.
The analysis of Figures Figures7,7, ,88 shows that voltage-dependent channels in sector II (but not sector I) cross the activation threshold, so that a local Ca spike is activated bringing about a remarkable KCa channels activation and a long pause. In sector II, Ca spikes and threshold crossing could be prevented by down-tuning the synaptic input. Indeed, when the number of pf synapses was increased progressively from 50 to 100, a sharp threshold in Ca spike activation was observed around 70–80 synapses. Interestingly, with sub-threshold responses the BPR of sector II became almost the same as for sector I (Figure (Figure99).
A last question is how responses generated in different dendritic sectors can integrate to generate BPRs.
When sector I and II were activated together, different behaviors appeared depending on the intensity of dendritic activation. Below Ca spike threshold, the conjoint BPR showed a burst enhancement but the pause was almost the same as in the two sectors alone (Figure (Figure10).10). Conversely, when sector II Ca spike was supra-threshold, BPRs were dominated by the pause (Figure (Figure10).10). The BPR burst became shorter and smaller and the pause longer and deeper than in any one of the two sectors activated alone. This was likely to reflect boosting of KCa current activation due to the conjoint action of the two sectors.
When activation in sector I preceded sector II, or vice versa, the bursts were initially fused together but then separated generating characteristic response profiles (Figure (Figure11).11). When sector II preceded sector I, the unified burst initially decreased and then approached the level of the burst in sector II before separating into two individual bursts. For longer delays, the second burst was reduced along the time course of the BPR pause and finally recovered to sector I burst amplitude. When sector I preceded sector II, the responses behaved similarly. Starting from a reduced conjoint burst, the burst amplitude increased toward that of the burst in sector I. For longer delays, the second burst was reduced along the time course of the BPR pause and finally recovered to sector II burst amplitude.
The present simulations allowed to investigate the foundations of PC responsiveness to synaptic bursts in a way that would not be possible with experiments only. Using the model, we could predetermine the composition of synaptic input patterns and analyze the neuron response mechanisms by independently monitoring several parameters (like membrane potential, ionic currents and calcium concentration) over multiple dendritic compartments. In response to pf and SC inputs, the PC model generated BPRs based on voltage-dependent activation of the dendritic Ca-KCa channel system. BPRs could reliably represent complex granular and molecular layer input patterns and depended on the specific sector of the dendritic tree that was stimulated, on the ionic channel complement, and on the excitatory/inhibitory synaptic pattern. These simulations suggest therefore that PCs exploit their intrinsic electroresponsiveness and the input pattern topography on the dendrites in order to generate a flexible BPR code (Herzfeld et al., 2015) to be relayed to DCN neurons.
This investigation was made possible by the use of an advanced model of the PC (Masoli et al., 2015), which was developed from earlier ones (De Schutter and Bower, 1994a,b; Rapp et al., 1994; Roth and Häusser, 2001; Steuber et al., 2007; Masoli et al., 2015) by including novel electrophysiological features and was validated against a large set of recent experimental data. The model was implemented with ionotropic synapses coming from granule cells (both aa and parallel fibers) and SCs. Additional synaptic mechanisms that may affect the response to specific input patterns (e.g., see Barbour et al., 1994; Takahashi et al., 1995; Tabata et al., 2005; Blot and Barbour, 2014) remain to be assessed.
The pf synaptic transmission was calibrated to match responses evoked by random input patterns (Dittman et al., 2000), ensuring that pf synapses could precisely reproduce the temporal dynamics of short-term synaptic plasticity. The aa were made identical to pf synapses but were located on distal rather than proximal dendrites. A potentiation of the aa (Sims and Hartell, 2005, 2006; Walter et al., 2009) was required to counterbalance the longer electronic distance (L = 1.4 vs. L = 0.4) from soma (Roth and Häusser, 2001), thus ensuring the reported functional equivalence of aa and pf synapses (Walter et al., 2009).
It should be noted that in no case the model generated bistable switching between up-down states (Loewenstein et al., 2005), which may require additional mechanisms or specific modulation of receptors and ionic channels.
Model simulations brought about a number of testable predictions.
These observations suggest that BPRs represents a flexible coding strategy accounting for the timing, duration and intensity of input granule cell spike patterns and that can be modulated by the spatiotemporal distribution of the input, by its intensity, by synaptic plasticity and by inhibitory interneuron activity.
Model simulations also helped hypothesizing how PC dendrites process incoming synaptic inputs through BPRs. Dendritic Ca channels, primed by pf or aa EPSPs, would generate a depolarizing current flowing through the dendrites and activate a spike burst in the AIS. The corresponding raise in [Ca]i would activate KCa channels causing a protracted hyperpolarization, interrupting pacemaking and generating the pause. The whole process turned out to be very sensitive to the location of synaptic inputs on the dendrites. Actually, EPSPs amplitude was larger at longer electrotonic distances and this enhanced the threshold crossing for generating local Ca spikes, that could then spread around invading neighboring dendritic regions. Thus, the combined effect of dendritic structure and voltage-dependent ionic channels generated non-linear interactions sculpting the spatio-temporal profile of local [Ca]i and BPRs. It is tempting to speculate that this would eventually extend the plastic and computational properties of PCs beyond the linear perceptron hypothesized previously (Brunel et al., 2004).
A peculiarity of the PC dendrites is the enrichment in Ca channels (Llinás and Sugimori, 1980a,b), which include both HVA (Cav2.1) and multiple LVA (Cav3.1, Cav3.2, Cav3.3) subtypes (Ly et al., 2016). While Cav2.1 (the classical PC P-type channel) was known to regulate KCa1.1 (BK) channel activation and therefore the fast action potential AHP, the role of LVA channels remained uncertain. The present simulations show that LVA Ca channels (including at least cav3.1 and Cav3.3) are critical for BPR, since they are activated during the burst and then play a local but key role in activating the KCa2.2 (SK2) and KCa3.1 (SK4) channels, thereby regulating the pause. The relevant role of LVA calcium channels in EPSP generation by input bursts has recently been reported experimentally (Ly et al., 2016).
It should be noted that, when simulating intrinsic electroresponsiveness with somatic recordings, LVA Ca channels and KCa channels proved either noncritical or subcritical (Masoli et al., 2015). Therefore, in light of the present simulations, the tuning procedures of maximum ionic conductances in neuronal models should be targeted toward features addressing synaptic response pattern (Marasco et al., 2013) rather than just responses generated by somatic current injection (Druckmann et al., 2007, 2008; Masoli et al., 2015, 2017). Since some of these channels are located in the spines (specifically Cav3.1), a future assessment of the impact of spines in the PC model is warranted.
In aggregate, these simulations showed that BPRs could fully exploit dendritic ionic channels (Llinás and Sugimori, 1980a,b) to generate voltage-dependent responses. However, this mechanism did not compromise the linearity of BPR input-output relationships supporting the predictions that PC may work as a linear perceptron (Brunel et al., 2004) and a perfect integrator (Phoka et al., 2010). Simulations supported the concept that BPRs represent a fundamental operating mode of PCs (Walter and Khodakhah, 2006, 2009; Chen et al., 2016). First, BPRs could discriminate among different input patterns coming from the mossy fibers and expanded/recoded in the granular and molecular layer (Marr, 1969). Second, BPRs sensitivity on the millisecond scale would allow PCs to operate as precise temporal devices (Eccles, 1973). Third, BPRs could discriminate among synaptic locations, generating an exquisite sensitivity to the topography of input patterns (Migliore et al., 2008). Finally, BPRs could generate complex combinations and sequences (Santamaria and Bower, 2005). By expressing these properties, BPRs would effectively integrate the spatio-temporal activity patterns generated in the cerebellar cortex into salient engrams, a prediction warranted experimental testing through electrophysiological, imaging and optogenetic recordings.
SM wrote the model, carried out the simulation and took part to manuscript writing and discussion. ED coordinated the work and wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding. This work was supported by the European Union grant Human Brain Project (HBP-29 604102) to ED.