Paradoxical Excitatory Impact of SK Channels on Dendritic Excitability.

Dendritic excitability regulates how neurons integrate synaptic inputs and thereby influences neuronal output. As active dendritic events are associated with significant calcium influx they are likely to be modulated by calcium-dependent processes, such as calcium-activated potassium channels. Here we investigate the impact of small conductance calcium-activated potassium channels (SK channels) on dendritic excitability in male and female rat cortical pyramidal neurons in vitro and in vivo Using local applications of the SK channel antagonist apamin in vitro, we show that blocking somatic SK channels enhances action potential output, whereas blocking dendritic SK channels paradoxically reduces the generation of dendritic calcium spikes and associated somatic burst firing. Opposite effects were observed using the SK channel enhancer NS309. The effect of apamin on dendritic SK channels was occluded when R-type calcium channels were blocked, indicating that the inhibitory impact of apamin on dendritic calcium spikes involved R-type calcium channels. Comparable effects were observed in vivo Intracellular application of apamin via the somatic whole-cell recording pipette reduced the medium afterhyperpolarization and increased action potential output during UP states. In contrast, extracellular application of apamin to the cortical surface to block dendritic SK channels shifted the distribution of action potentials within UP states from an initial burst to a more distributed firing pattern, while having no impact on overall action potential firing frequency or UP and DOWN states. These data indicate that somatic and dendritic SK channels have opposite effects on neuronal excitability, with dendritic SK channels counter-intuitively promoting rather than suppressing neuronal output.SIGNIFICANCE STATEMENT Neurons typically receive input from other neurons onto processes called dendrites, and use electrical events such as action potentials for signaling. As electrical events in neurons are usually associated with calcium influx they can be regulated by calcium-dependent processes. One such process is through the activation of calcium-dependent potassium channels, which usually act to reduce action potential signaling. Although this is the case for calcium-dependent potassium channels found at the cell body, we show here that calcium-dependent potassium channels in dendrites of cortical pyramidal neurons counter-intuitively promote rather than suppress action potential output.

Characterisation and functional mapping of surface potentials in the rat dorsal column nuclei.

KEY POINTS: The brainstem dorsal column nuclei (DCN) process sensory information arising from the body before it reaches the brain and becomes conscious. Despite significant investigations into sensory coding in peripheral nerves and the somatosensory cortex, little is known about how sensory information arising from the periphery is represented in the DCN. Following stimulation of hind-limb nerves, we mapped and characterised the evoked electrical signatures across the DCN surface. We show that evoked responses recorded from the DCN surface are highly reproducible and are unique to nerves carrying specific sensory information. ABSTRACT: The brainstem dorsal column nuclei (DCN) play a role in early processing of somatosensory information arising from a variety of functionally distinct peripheral structures, before being transmitted to the cortex via the thalamus. To improve our understanding of how sensory information is represented by the DCN, we characterised and mapped low- (<200 Hz) and high-frequency (550-3300 Hz) components of nerve-evoked DCN surface potentials. DCN surface potentials were evoked by electrical stimulation of the left and right nerves innervating cutaneous structures (sural nerve), or a mix of cutaneous and deep structures (peroneal nerve), in 8-week-old urethane-anaesthetised male Wistar rats. Peroneal nerve-evoked DCN responses demonstrated low-frequency events with significantly longer durations, more high-frequency events and larger magnitudes compared to responses evoked from sural nerve stimulation. Hotspots of low- and high-frequency DCN activity were found ipsilateral to stimulated nerves but were not symmetrically organised. In conclusion, we find that sensory inputs from peripheral nerves evoke unique and characteristic DCN activity patterns that are highly reproducible both within and across animals.

GABAB receptors in neocortical and hippocampal pyramidal neurons are coupled to different potassium channels.

Classically, GABAB receptors are thought to regulate neuronal excitability via G-protein-coupled inwardly rectifying potassium (GIRK) channels. Recent data, however, indicate that GABAB receptors can also activate two-pore domain potassium channels. Here, we investigate which potassium channels are coupled to GABAB receptors in rat neocortical layer 5 and hippocampal CA1 pyramidal neurons. Bath application of the non-specific GIRK channel blocker barium (200 µm) abolished outward currents evoked by GABAB receptors in CA1 pyramidal, but only partially blocked GABAB responses in layer 5 neurons. Layer 5 and CA1 pyramidal neurons also showed differential sensitivity to tertiapin-Q, a specific GIRK channel blocker. Tertiapin-Q partially blocked GABAB responses in CA1 pyramidal neurons, but was ineffective in blocking GABAB responses in neocortical layer 5 neurons. Consistent with the idea that GABAB receptors are coupled to two-pore domain potassium channels, the non-specific blockers quinidine and bupivacaine partially blocked GABAB responses in both layer 5 and CA1 neurons. Finally, we show that lowering external pH, as occurs in hypoxia, blocks the component of GABAB responses mediated by two-pore domain potassium channels in neocortical layer 5 pyramidal neurons, while at the same time revealing a GIRK channel component. These data indicate that GABAB receptors in neocortical layer 5 and hippocampal CA1 pyramidal neurons are coupled to different channels, with this coupling pH dependent on neocortical layer 5 pyramidal neurons. This pH dependency may act to maintain constant levels of GABAB inhibition during hypoxia by enhancing GIRK channel function following a reduction in two-pore domain potassium channel activity.

Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons.

Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels onN-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive.

Different calcium sources control somatic versus dendritic SK channel activation during action potentials.

Small-conductance calcium-activated potassium (SK) channels play an important role in regulating neuronal excitability. While SK channels at the soma have long been known to contribute to the medium afterhyperpolarization (mAHP), recent evidence indicates they also regulate NMDA receptor activation in dendritic spines. Here we investigate the activation of SK channels in spines and dendrites of rat cortical pyramidal neurons during action potentials (APs), and compare this to SK channel activation at the soma. Using confocal calcium imaging, we demonstrate that the inhibition of SK channels with apamin results in a location-dependent increase in calcium influx into dendrites and spines during backpropagating APs (average increase, ~40%). This effect was occluded by block of R-type voltage-dependent calcium channels (VDCCs), but not by inhibition of N- or P/Q-type VDCCs, or block of calcium release from intracellular stores. During these experiments, we noticed that the calcium indicator (Oregon Green BAPTA-1) blocked the mAHP. Subsequent experiments using low concentrations of EGTA (1 mm) produced the same result, suggesting that somatic SK channels are not tightly colocalized with their calcium source. Consistent with this idea, all known subtypes of VDCCs except R-type were calcium sources for the apamin-sensitive mAHP at the soma. We conclude that SK channels in spines and dendrites of cortical pyramidal neurons regulate calcium influx during backpropagating APs in a distance-dependent manner, and are tightly coupled to R-type VDCCs. In contrast, SK channels activated by APs at the soma of these neurons are weakly coupled to a variety of VDCCs.

In vivo whole-cell recording from morphologically identified mouse superior colliculus neurons.

In vivo whole-cell recording, when combined with morphological characterization after biocytin labeling, is a powerful technique to study subthreshold synaptic processing in cell-type-identified neuronal populations. Here, we describe steps for performing whole-cell recordings in the superior colliculus of urethane-anesthetized mice, a major visual processing region in the rodent brain. We detail two types of visual stimulation techniques: full-field light-emitting diode (LED) flashes and visual stimuli shown on monitors. While we focus on superior colliculus, this protocol is applicable to other brain areas.

Characterization of primary visual cortex input to specific cell types in the superior colliculus.

The superior colliculus is a critical brain region involved in processing visual information. It receives visual input directly from the retina, as well as via a projection from primary visual cortex. Here we determine which cell types in the superficial superior colliculus receive visual input from primary visual cortex in mice. Neurons in the superficial layers of the superior colliculus were classified into four groups - Wide-field, narrow-field, horizontal and stellate - based on their morphological and electrophysiological properties. To determine functional connections between V1 and these four different cell types we expressed Channelrhodopsin2 in primary visual cortex and then optically stimulated these axons while recording from different neurons in the superficial superior colliculus using whole-cell patch-clamp recording in vitro. We found that all four cell types in the superficial layers of the superior colliculus received monosynaptic (direct) input from V1. Wide-field neurons were more likely than other cell types to receive primary visual cortex input. Our results provide information on the cell specificity of the primary visual cortex to superior colliculus projection, increasing our understanding of how visual information is processed in the superior colliculus at the single cell level.

Somatic and dendritic GABA(B) receptors regulate neuronal excitability via different mechanisms.

GABA(B) receptors play a key role in regulating neuronal excitability in the brain. Whereas the impact of somatic GABA(B) receptors on neuronal excitability has been studied in some detail, much less is known about the role of dendritic GABA(B) receptors. Here, we investigate the impact of GABA(B) receptor activation on the somato-dendritic excitability of layer 5 pyramidal neurons in the rat barrel cortex. Activation of GABA(B) receptors led to hyperpolarization and a decrease in membrane resistance that was greatest at somatic and proximal dendritic locations. These effects were occluded by low concentrations of barium (100 µM), suggesting that they are mediated by potassium channels. In contrast, activation of dendritic GABA(B) receptors decreased the width of backpropagating action potential (APs) and abolished dendritic calcium electrogenesis, indicating that dendritic GABA(B) receptors regulate excitability, primarily via inhibition of voltage-dependent calcium channels. These distinct actions of somatic and dendritic GABA(B) receptors regulated neuronal output in different ways. Activation of somatic GABA(B) receptors led to a reduction in neuronal output, primarily by increasing the AP rheobase, whereas activation of dendritic GABA(B) receptors blocked burst firing, decreasing AP output in the absence of a significant change in somatic membrane properties. Taken together, our results show that GABA(B) receptors regulate somatic and dendritic excitability of cortical pyramidal neurons via different cellular mechanisms. Somatic GABA(B) receptors activate potassium channels, leading primarily to a subtractive or shunting form of inhibition, whereas dendritic GABA(B) receptors inhibit dendritic calcium electrogenesis, leading to a reduction in bursting firing.

Action potential generation requires a high sodium channel density in the axon initial segment.

The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

Voltage Clamp Errors During Estimation of Concurrent Excitatory and Inhibitory Synaptic Input to Neurons with Dendrites.

The whole-cell voltage clamp technique is commonly used to estimate synaptic conductances. While previous work has shown how these estimates are affected by series resistance and space clamp errors during isolated synaptic events, how voltage clamp errors impact on synaptic conductance estimates during concurrent excitation and inhibition is less clear. This issue is particularly relevant given that many studies now use the whole-cell voltage clamp technique to estimate synaptic conductances in vivo, where both excitation and inhibition are intact. Using both simplistic and morphologically realistic models, we investigate how imperfect voltage clamp conditions distort estimates of excitatory and inhibitory synaptic conductance estimated using the Borg-Graham method during concurrent synaptic input onto dendrites. These simulations demonstrate that dendritically located conductances are underestimated even when dynamic clamp reinjection faithfully reproduces the voltage response at the soma to the actual conductances. Inhibitory conductances are underestimated more than excitatory conductances, leading to errors in the excitatory to inhibitory conductance ratio and negative inhibitory conductance estimates during distal inhibition. Interactions between unclamped dendritic excitatory and inhibitory conductances also introduce correlations when the actual conductances are uncorrelated, as well as distortions in the time course of estimated excitatory and inhibitory conductances. Finally, we show that space clamp errors are exacerbated by the inclusion of dendritic voltage-activated conductances. In summary, we highlight issues with the interpretation of synaptic conductance estimates obtained using somatic whole-cell voltage clamp during concurrent excitatory and inhibitory input to neurons with dendrites.

High synaptic threshold for dendritic NMDA spike generation in human layer 2/3 pyramidal neurons.

Neurons receive synaptic input primarily onto their dendrites. While we know much about the electrical properties of dendrites in rodents, we have only just started to describe their properties in the human brain. Here, we investigate the capacity of human dendrites to generate NMDA-receptor-dependent spikes (NMDA spikes). Using dendritic glutamate iontophoresis, as well as local dendritic synaptic stimulation, we find that human layer 2/3 pyramidal neurons can generate dendritic NMDA spikes. The capacity to evoke NMDA spikes in human neurons, however, was significantly reduced compared with that in rodents. Simulations in morphologically realistic and simplified models indicated that human neurons have a higher synaptic threshold for NMDA spike generation primarily due to the wider diameter of their dendrites. In summary, we find reduced NMDA spike generation in human compared with rodent layer 2/3 pyramidal neurons and provide evidence that this is due to the wider diameter of human dendrites.

Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.

Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 microm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.

Membrane potential changes in dendritic spines during action potentials and synaptic input.

Excitatory input onto many neurons in the brain occurs onto specialized projections called dendritic spines. Despite their potential importance in neuronal function, direct experimental evidence on electrical signaling in dendritic spines is lacking as their small size makes them inaccessible to standard electrophysiological techniques. Here, we investigate electrical signaling in dendritic spines using voltage-sensitive dye imaging in cortical pyramidal neurons during backpropagating action potentials and synaptic input. Backpropagating action potentials were found to fully invade dendritic spines without voltage loss. The voltage change in dendritic spines during synaptic input ranged from a few millivolts up to approximately 20 mV. During hyperpolarization of the membrane potential, the amplitude of the synaptic voltage in spines was increased, consistent with the expected change resulting from the increased driving force. This observation suggests that voltage-activated channels do not significantly boost the voltage response in dendritic spines during synaptic input. Finally, we used simulations of our experimental observations in morphologically realistic models to estimate spine neck resistance. These simulations indicated that spine neck resistance ranges up to approximately 500 Mohms. Spine neck resistances of this magnitude reduce somatic EPSPs by <15%, indicating that the spine neck is unlikely to act as a physical device to significantly modify synaptic strength.

Initiation of simple and complex spikes in cerebellar Purkinje cells.

Cerebellar Purkinje cells produce two distinct forms of action potential output: simple and complex spikes. Simple spikes occur spontaneously or are driven by parallel fibre input, while complex spikes are activated by climbing fibre input. Previous studies indicate that both simple and complex spikes originate in the axon of Purkinje cells, but the precise location where they are initiated is unclear. Here we address where in the axon of cerebellar Purkinje cells simple and complex spikes are generated. Using extracellular recording and voltage-sensitive dye imaging in rat and mouse Purkinje cells, we show that both simple and complex spikes are generated in the proximal axon, 15-20 mum from the soma. Once initiated, simple and complex spikes propagate both down the axon and back into the soma. The speed of backpropagation into the soma was significantly faster for complex compared to simple spikes, presumably due to charging of the somatodendritic membrane capacitance during the climbing fibre synaptic conductance. In conclusion, we show using two independent methods that the final integration site of simple and complex spikes is in the proximal axon of cerebellar Purkinje cells, at a location corresponding to the distal end of the axon initial segment.

Cortical feed-forward networks for binding different streams of sensory information.

Different streams of sensory information are transmitted to the cortex where they are merged into a percept in a process often termed 'binding.' Using recordings from triplets of rat cortical layer 2/3 and layer 5 pyramidal neurons, we show that specific subnetworks within layer 5 receive input from different layer 2/3 subnetworks. This cortical microarchitecture may represent a mechanism that enables the main output of the cortex (layer 5) to bind different features of a sensory stimulus.

Superior colliculus modulates cortical coding of somatosensory information.

The cortex modulates activity in superior colliculus via a direct projection. What is largely unknown is whether (and if so how) the superior colliculus modulates activity in the cortex. Here, we investigate this issue and show that optogenetic activation of superior colliculus changes the input-output relationship of neurons in somatosensory cortex, enhancing responses to low amplitude whisker deflections. While there is no direct pathway from superior colliculus to somatosensory cortex, we found that activation of superior colliculus drives spiking in the posterior medial (POm) nucleus of the thalamus via a powerful monosynaptic pathway. Furthermore, POm neurons receiving input from superior colliculus provide monosynaptic excitatory input to somatosensory cortex. Silencing POm abolished the capacity of superior colliculus to modulate cortical whisker responses. Our findings indicate that the superior colliculus, which plays a key role in attention, modulates sensory processing in somatosensory cortex via a powerful di-synaptic pathway through the thalamus.

Loss of sensory input increases the intrinsic excitability of layer 5 pyramidal neurons in rat barrel cortex.

Development of the cortical map is experience dependent, with different critical periods in different cortical layers. Previous work in rodent barrel cortex indicates that sensory deprivation leads to changes in synaptic transmission and plasticity in layer 2/3 and 4. Here, we studied the impact of sensory deprivation on the intrinsic properties of layer 5 pyramidal neurons located in rat barrel cortex using simultaneous somatic and dendritic recording. Sensory deprivation was achieved by clipping all the whiskers on one side of the snout. Loss of sensory input did not change somatic active and resting membrane properties, and did not influence dendritic action potential (AP) backpropagation. In contrast, sensory deprivation led to an increase in the percentage of layer 5 pyramidal neurons showing burst firing. This was associated with a reduction in the threshold for generation of dendritic calcium spikes during high-frequency AP trains. Cell-attached recordings were used to assess changes in the properties and expression of dendritic HCN channels. These experiments indicated that sensory deprivation caused a decrease in HCN channel density in distal regions of the apical dendrite. To assess the contribution of HCN down-regulation on the observed increase in dendritic excitability following sensory deprivation, we investigated the impact of blocking HCN channels. Block of HCN channels removed differences in dendritic calcium electrogenesis between control and deprived neurons. In conclusion, these observations indicate that sensory loss leads to increased dendritic excitability of cortical layer 5 pyramidal neurons. Furthermore, they suggest that increased dendritic calcium electrogenesis following sensory deprivation is mediated in part via down-regulation of dendritic HCN channels.

Dendritic mechanisms controlling spike-timing-dependent synaptic plasticity.

The ability of neurons to modulate the strength of their synaptic connections has been shown to depend on the relative timing of pre- and postsynaptic action potentials. This form of synaptic plasticity, called spike-timing-dependent plasticity (STDP), has become an attractive model for learning at the single-cell level. Yet, despite its popularity in experimental and theoretical neuroscience, the influence of dendritic mechanisms in the induction of STDP has been largely overlooked. Several recent studies have investigated how active dendritic properties and synapse location within the dendritic tree influence STDP. These studies suggest the existence of learning rules that depend on firing mode and subcellular input location, adding unanticipated complexity to STDP. Here, we propose a new look at STDP that is focused on processing at the postsynaptic site in the dendrites, rather than on spike-timing at the cell body.

Local versus Global Dendritic Integration.

While many studies indicate that dendrites can perform a range of local computations on their inputs, work from the Harnett lab in this issue of Neuron suggests that the vast majority of active dendritic events are synchronized across the somato-dendritic axis of cortical pyramidal neurons.

Peripheral Nerve Activation Evokes Machine-Learnable Signals in the Dorsal Column Nuclei.

The brainstem dorsal column nuclei (DCN) are essential to inform the brain of tactile and proprioceptive events experienced by the body. However, little is known about how ascending somatosensory information is represented in the DCN. Our objective was to investigate the usefulness of high-frequency (HF) and low-frequency (LF) DCN signal features (SFs) in predicting the nerve from which signals were evoked. We also aimed to explore the robustness of DCN SFs and map their relative information content across the brainstem surface. DCN surface potentials were recorded from urethane-anesthetized Wistar rats during sural and peroneal nerve electrical stimulation. Five salient SFs were extracted from each recording electrode of a seven-electrode array. We used a machine learning approach to quantify and rank information content contained within DCN surface-potential signals following peripheral nerve activation. Machine-learning of SF and electrode position combinations was quantified to determine a hierarchy of information importance for resolving the peripheral origin of nerve activation. A supervised back-propagation artificial neural network (ANN) could predict the nerve from which a response was evoked with up to 96.8 ± 0.8% accuracy. Guided by feature-learnability, we maintained high prediction accuracy after reducing ANN algorithm inputs from 35 (5 SFs from 7 electrodes) to 6 (4 SFs from one electrode and 2 SFs from a second electrode). When the number of input features were reduced, the best performing input combinations included HF and LF features. Feature-learnability also revealed that signals recorded from the same midline electrode can be accurately classified when evoked from bilateral nerve pairs, suggesting DCN surface activity asymmetry. Here we demonstrate a novel method for mapping the information content of signal patterns across the DCN surface and show that DCN SFs are robust across a population. Finally, we also show that the DCN is functionally asymmetrically organized, which challenges our current understanding of somatotopic symmetry across the midline at sub-cortical levels.