The voltage-clamp technique is applicable only to spherical cells. In nonspherical cells, such as neurons, the membrane potential is not clamped distal to the voltage-clamp electrode. This means that the current recorded by the voltage-clamp electrode is the sum of the local current and of axial currents from locations experiencing different membrane potentials. Furthermore, voltage-gated currents recorded from a nonspherical cell are, by definition, severely distorted due to the lack of space clamp. Justifications for voltage clamping in nonspherical cells are, first, that the lack of space clamp is not severe in neurons with short dendrites. Second, passive cable theory may be invoked to justify application of voltage clamp to branching neurons, suggesting that the potential decay is sufficiently shallow to allow spatial clamping of the neuron. Here, using numerical simulations, we show that the distortions of voltage-gated K+ and Ca2+ currents are substantial even in neurons with short dendrites. The simulations also demonstrate that passive cable theory cannot be used to justify voltage clamping of neurons due to significant shunting to the reversal potential of the voltage-gated conductance during channel activation. Some of the predictions made by the simulations were verified using somatic and dendritic voltage-clamp experiments in rat somatosensory cortex. Our results demonstrate that voltage-gated K+ and Ca2+ currents recorded from branching neurons are almost always severely distorted.

Constructing physiologically relevant compartmental models of neurones is critical for understanding neuronal activity and function. We recently suggested that measurements from multiple locations along the soma, dendrites and axon are necessary as a data set when using a genetic optimization algorithm to constrain the parameters of a compartmental model of an entire neurone. However, recordings from L5 pyramidal neurones can routinely be performed simultaneously from only two locations. Now we show that a data set recorded from the soma and apical dendrite combined with a parameter peeling procedure is sufficient to constrain a compartmental model for the apical dendrite of L5 pyramidal neurones. The peeling procedure was tested on several compartmental models showing that it avoids local minima in parameter space. Based on the requirements of this analysis procedure, we designed and performed simultaneous whole-cell recordings from the soma and apical dendrite of rat L5 pyramidal neurones. The data set obtained from these recordings allowed constraining a simplified compartmental model for the apical dendrite of L5 pyramidal neurones containing four voltage-gated conductances. In agreement with experimental findings, the optimized model predicts that the conductance density gradients of voltage-gated K + conductances taper rapidly proximal to the soma, while the density gradient of the voltage-gated Na + conductance tapers slowly along the apical dendrite. The model reproduced the back-propagation of the action potential and the modulation of the resting membrane potential along the apical dendrite. Furthermore, the optimized model provided a mechanistic explanation for the back-propagation of the action potential into the apical dendrite and the generation of dendritic Na + spikes.

Voltage-gated potassium channels effectively regulate dendritic excitability in neurones. It has been suggested that in the distal apical dendrite of layer 5B (L5B) neocortical pyramidal neurones, K + conductances participate in active dendritic synaptic integration and control regenerative dendritic potentials. The ionic mechanism for triggering these regenerative potentials has yet to be elucidated. Here we used two-electrode voltage clamp (TEVC) to quantitatively record K + conductance densities of a sustained K + conductance in the soma and apical dendrite of L5B neurones of adult rats. We report that the somatic and proximal dendritic sustained voltage-gated K + conductance density is more than 10-fold larger than previous estimates. The results obtained using TEVC were corroborated using current-clamp experiments in combination with compartmental modelling. Possible error sources, including inaccurate measurement of the passive membrane parameters and unknown axonal and basal dendritic conductance distributions, were shown not to distort the density estimation considerably. The sustained voltage-gated K + conductance density was found to decrease steeply along the apical dendrite. The steep negative K + conductance density gradient along the apical dendrite may help to define a distal, low-threshold region for amplification of distal synaptic input in L5B pyramidal neurones. It has been shown that the apical dendrite of layer 5B (L5B) pyramidal neurones performs several non-linear transformations of synaptic input, most clearly exemplified by large, regenerative Ca 2+ potentials that have been readily recorded from the apical dendrite of L5B neocortical pyramidal neurones (Amitai et al.

There is a considerable gap between investigating the dynamics of single neurons and the computational aspects of neural networks. A growing number of studies have attempted to overcome this gap using the excitation in brain slices elicited by various chemical manipulations of the bath solution. However, there has been no quantitative study on the effects of these manipulations on the cellular and network factors controlling excitability. Using the whole-cell configuration of the patch-clamp technique we recorded the membrane potential from the soma of layer 5 pyramidal neurons in acute brain slices from the somatosensory cortex of young rats at 22°C and 35°C. Using blockers of synaptic transmission, we show distinct changes in cellular properties following modification of the ionic composition of the artificial cerebrospinal fluid (ACSF). Thus both cellular and network changes may contribute to the observed effects of slice excitation solutions on the physiology of single neurons. Furthermore, our data suggest that the difference in the ionic composition of current standard ACSF from that of CSF measured in vivo cause ACSF to depress network activity in acute brain slices. This may affect outcomes of experiments investigating biophysical and physiological properties of neurons in such preparations. Our results strongly advocate the necessity of redesigning experiments routinely carried out in the quiescent acute brain slice preparation.

Recent years have seen increased study of dendritic integration, mostly in acute brain slices. However, due to the low background activity in brain slices the integration of synaptic input in slice preparations may not truly reflect conditions in vivo. To investigate dendritic integration, back-propagation of the action potential (AP) and initiation of the dendritic Ca(2+) spike we simultaneously recorded membrane potential at the soma and apical dendrite of layer 5 (L5) pyramidal neurons in quiescent and excited acute brain slices. After excitation of the brain slice the somatic input resistance decreased and the apparent passive space constant shortened. However, the back-propagating AP and dendritic Ca(2+) spike were robust during increased synaptic activity. The dendritic Ca(2+) spike was suppressed by the ionic composition of the bath solution required for slice excitation, suggesting that Ca(2+) spikes may be smaller in vivo than in the acute slice preparation. The results presented here suggest that, under the conditions of slice excitation examined in this study, the increased membrane conductance induced by activation of voltage-gated channels during back-propagation of the AP and dendritic Ca(2+) spike initiation is sufficiently larger than the membrane conductance at subthreshold potentials to allow these two regenerative dendritic events to remain robust over several levels of synaptic activity in the apical dendrite of L5 pyramidal neurons.