P-sort: an open-source software for cerebellar neurophysiology

Algorithms that perform spike sorting depend on waveforms to cluster spikes. However, a cerebellar Purkinje-cell produces two types of spikes; simple and complex spikes. A complex spike coincides with the suppression of generating simple spikes. Here, we recorded neurophysiological data from three species and developed a spike analysis software named P-sort that relies on this statistical property to improve both the detection and the attribution of simple and complex spikes in the cerebellum.

Place fields of single spikes in hippocampus involve Kcnq3 channel-dependent entrainment of complex spike bursts

Hippocampal pyramidal cells encode an animal’s location by single action potentials and complex spike bursts. These elementary signals are believed to play distinct roles in memory consolidation. The timing of single spikes and bursts is determined by intrinsic excitability and theta oscillations (5–10 Hz). Yet contributions of these dynamics to place fields remain elusive due to the lack of methods for specific modification of burst discharge. In mice lacking Kcnq3-containing M-type K+ channels, we find that pyramidal cell bursts are less coordinated by the theta rhythm than in controls during spatial navigation, but not alert immobility. Less modulated bursts are followed by an intact post-burst pause of single spike firing, resulting in a temporal discoordination of network oscillatory and intrinsic excitability. Place fields of single spikes in one- and two-dimensional environments are smaller in the mutant. Optogenetic manipulations of upstream signals reveal that neither medial septal GABA-ergic nor cholinergic inputs alone, but rather their joint activity, is required for entrainment of bursts. Our results suggest that altered representations by bursts and single spikes may contribute to deficits underlying cognitive disabilities associated with KCNQ3-mutations in humans.

Simple and complex spike responses of mouse cerebellar Purkinje neurons to regular trains and omissions of somatosensory stimuli

Cerebellar Purkinje neurons help compute absolute subsecond timing, but how their firing is affected during repetitive sensory stimulation with consistent subsecond intervals remains unaddressed. Here, we investigated how simple and complex spikes of Purkinje cells change during regular application of air puffs (3.3 Hz for ~4 min) to the whisker pad of awake, head-fixed female mice. Complex spike responses fell into two categories: those in which firing rates increased (at ~50 ms) and then fell (complex spike elevated "CxSE" cells), and those in which firing rates decreased (at ~70 ms) and then rose (complex spike reduced "CxSR" cells). Both groups had indistinguishable rates of basal complex (~1.7 Hz) and simple (~75 Hz) spikes, and initially responded to puffs with a well-timed sensory response of a short-latency (~15 ms), transient (4 ms) suppression of simple spikes. CxSE more than CxSR cells, however, also showed a longer-latency increase in simple spike rate, previously shown to reflect motor command signals. With repeated puffs, basal simple spike rates dropped greatly in CxSR but not CxSE cells; complex spike rates remained constant, but their temporal precision rose in CxSR cells and fell in CxSE cells. Also over time, transient simple spike suppression gradually disappeared in CxSE cells, suggesting habituation, but remained stable in CxSR cells, suggesting reliable transmission of sensory stimuli. During stimulus omissions, both categories of cells showed complex spike suppression with different latencies. The data indicate two modes by which Purkinje cells transmit regular repetitive stimuli, distinguishable by their climbing fiber signals.

Complex spike firing adapts to saliency of inputs and engages readiness to act

The cerebellum is involved in cognition next to motor coordination. During complex tasks, climbing fiber input to the cerebellum can deliver seemingly opposite signals, covering both motor and non-motor functions. To elucidate this ambiguity, we hypothesized that climbing fiber activity represents the saliency of inputs leading to action-readiness. We addressed this hypothesis by recording Purkinje cell activity in lateral cerebellum of awake mice learning go/no-go decisions based on entrained saliency of different sensory stimuli. As training progressed, the timing of climbing fiber signals switched in a coordinated fashion with that of Purkinje cell simple spikes towards the moment of occurrence of the salient stimulus that required action. Trial-by-trial analysis indicated that emerging climbing fiber activity is not linked to individual motor responses or rewards per se, but rather reflects the saliency of a particular sensory stimulus that engages a general readiness to act, bridging the non-motor with the motor functions.In briefMice were trained to identify the saliency of different sensory inputs in that they had to learn to ignore a prominent sound cue and respond to a light tactile cue in a Go/No-Go licking task. As the mice learned to discriminate the two inputs and respond to the proper signal, the Purkinje cells in the lateral cerebellum switched their climbing fiber activity (i.e., complex spike activity) towards the moment of occurrence of the salient stimulus that required a response, while concomitantly shifting the phase of their simple spike modulation. Trial-by-trial analysis indicates that the emerging climbing fiber activity is not linked to the occurrence of the motor response or reward per se, but rather reflects the saliency of a particular sensory stimulus engaging a general readiness to act.

A Purkinje cell model that simulates complex spikes

Purkinje cells are the principal neurons of the cerebellar cortex. One of their distinguishing features is that they fire two distinct types of action potential, called simple and complex spikes, which interact with one another. Simple spikes are stereotypical action potentials that are elicited at high, but variable, rates (0 – 100 Hz) and have a consistent waveform. Complex spikes are composed of an initial action potential followed by a burst of lower amplitude spikelets. Complex spikes occur at comparatively low rates (~ 1 Hz) and have a variable waveform. Although they are critical to cerebellar operation a simple model that describes the complex spike waveform is lacking. Here, a novel single-compartment model of Purkinje cell electrodynamics is presented. The simpler version of this model, with two active conductances and a leak channel, can simulate the features typical of complex spikes recorded in vitro. If calcium dynamics are also included, the model can capture the pause in simple spike activity that occurs after complex spike events. Together, these models provide an insight into the mechanisms behind complex spike spikelet generation, waveform variability and their interactions with simple spike activity.

α2δ-2 protein controls structure and function at the cerebellar climbing fiber synapse

Abstractα2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) only express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in a Cacna2d2 knockout mouse. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked excitatory postsynaptic currents (EPSCs). CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wildtype as a result of increased glutamate re-uptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.Significance Statementα2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but co-expression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), whose deletion causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells only expresses this single isoform, we studied excitatory climbing fiber synaptic function onto Purkinje cells in Cacna2d2 knockout mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in Purkinje cells lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.