Somatic Voltage Clamp Research Articles

Determination of effective synaptic conductances using somatic voltage clamp.

The interplay between excitatory and inhibitory neurons imparts rich functions of the brain. To understand the synaptic mechanisms underlying neuronal computations, a fundamental approach is to study the dynamics of excitatory and inhibitory synaptic inputs of each neuron. The traditional method of determining input conductance, which has been applied for decades, employs the synaptic current-voltage (I-V) relation obtained via voltage clamp. Due to the space clamp effect, the measured conductance is different from the local conductance on the dendrites. Therefore, the interpretation of the measured conductance remains to be clarified. Using theoretical analysis, electrophysiological experiments, and realistic neuron simulations, here we demonstrate that there does not exist a transform between the local conductance and the conductance measured by the traditional method, due to the neglect of a nonlinear interaction between the clamp current and the synaptic current in the traditional method. Consequently, the conductance determined by the traditional method may not correlate with the local conductance on the dendrites, and its value could be unphysically negative as observed in experiment. To circumvent the challenge of the space clamp effect and elucidate synaptic impact on neuronal information processing, we propose the concept of effective conductance which is proportional to the local conductance on the dendrite and reflects directly the functional influence of synaptic inputs on somatic membrane potential dynamics, and we further develop a framework to determine the effective conductance accurately. Our work suggests re-examination of previous studies involving conductance measurement and provides a reliable approach to assess synaptic influence on neuronal computation.

Thorough GABAergic innervation of the entire axon initial segment revealed by an optogenetic 'laserspritzer'.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

The dynamics of somatic input processing in spinal motoneurons in vivo

Uncovering how motoneurons utilize their voltage-sensitive conductances to systematically respond to a variety of inputs is paramount to understanding synaptic integration. In this study, we examine the input dynamics and frequency-dependent characteristics of active conductances in motoneurons as viewed from the soma in the decerebrate cat. We evaluated the somatic response of the motoneuron by superimposing a voltage sinus sweep (a sine wave in which frequency increases with time, which is often referred to as a zap or chirp) at a subset of membrane holding potentials during discontinuous, single-electrode, somatic voltage-clamp. Results from both experimental and modeling data indicate that ionic conductances can respond to a wide variety of input dynamics. Notably, it appears that there is a divergence between low input conductance type S and high input conductance type FF motoneurons in their response to input frequency. Type S motoneurons generate a larger response to lower frequency input dynamics (compared with their response to higher frequencies), whereas type FF generate a larger response to higher input frequency dynamics. Functionally, these results may indicate that motoneurons on the lower end of the motor pool (i.e., recruited first) may favor steady inputs, whereas motoneurons at the higher end (i.e., recruited later) may favor input transients in producing action potentials.

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

Persistent Currents and Discharge Patterns in Rat Hindlimb Motoneurons

We report here the first direct measurements of persistent inward currents (PICs) in rat hindlimb motoneurons, obtained from ketamine-xylazine anesthetized rats during slow voltage ramps performed by single-electrode somatic voltage clamp. Most motoneurons expressed PICs and current-voltage (I-V) relations often contained a negative-slope region (NSR; 13/19 cells). PICs activated at -52.7 ± 3.89 mV, 9 mV negative to spike threshold. NSR onset was -44.2 ± 4.1 mV. PIC amplitudes were assessed by maximum inward currents measured relative to extrapolated leak current and to NSR-onset current. PIC conductance at potentials just positive to activation was assessed by the relative change in slope conductance (g(in)/g(leak)). PIC amplitudes varied widely; some exceeded 5 and 10 nA relative to current at NSR onset or leak current, respectively. PIC amplitudes did not vary significantly with input conductance, but PIC amplitudes normalized by recruitment current decreased with increasing input conductance. Similarly, g(in)/g(leak) decreased with increasing input conductance. Currents near resting potential on descending limbs of I-V relations were often outward, relative to ascending-limb currents. This residual outward current was correlated with increases in leak conductance on the descending limb and with input conductance. Excluding responses with accommodation, residual outward currents matched differences between recruitment and derecruitment currents, suggesting a role for residual outward current in frequency adaptation. Comparison of potentials for PIC activation and NSR onset with interspike trajectories during discharge demonstrated correspondence between PIC activation and frequency-current (f-I) range boundaries. Contributions of persistent inward and outward currents to motoneuron discharge characteristics are discussed.

Isolation of Somatic Na+Currents by Selective Inactivation of Axonal Channels with a Voltage Prepulse

We present a simple and effective method for isolating the somatic Na(+) current recorded under voltage clamp from neurons in brain slices. The principle is to convert the axon from an active compartment capable of generating uncontrolled axonal spikes into a passive structure by selectively inactivating axonal Na(+) channels. Typically, whole-cell currents from intact neurons under somatic voltage clamp contain a mixture of Na(+) current and axial current caused by escaped axonal spikes. We found that a brief prepulse to voltages near spike threshold evokes the axonal spike, which inactivates axonal but not somatic channels. A subsequent voltage step then evokes only somatic Na(+) current from electrotonically proximal sodium channels under good voltage-clamp control. Simulations using a neuron compartmental model support the idea that the prepulse effectively inactivates currents from the axon and isolates well controlled somatic currents. Na(+) currents recorded from cortical pyramidal neurons in slices, using the prepulse, were found to have voltage dependence nearly identical to that of currents recorded from acutely dissociated pyramidal neurons. In addition, studies in dissociated neurons show that the prepulse has no visible effect on the voltage dependence and kinetics of Na(+) currents elicited by the subsequent voltage step, only decreasing the amplitude of the currents by 10-20%. The technique was effective in several neuronal types in brain slices from male and female neonatal rats and mice, including raphé neurons, cortical pyramidal neurons, inferior olivary neurons, and hypoglossal motoneurons.

Staircase Currents in Motoneurons: Insight into the Spatial Arrangement of Calcium Channels in the Dendritic Tree

In spinal motoneurons, activation of dendritically located depolarizing conductances can lead to amplification of synaptic inputs and the production of plateau potentials. Immunohistochemical and computational studies have implicated dendritic CaV1.3 channels in this amplification and suggest that CaV1.3 channels in spinal motoneurons may be organized in clusters in the dendritic tree. Our goal was to provide physiological evidence for the presence of multiple discrete clusters of voltage-gated calcium channels in spinal motoneurons and to explore the spatial arrangement of these clusters in the dendritic tree. We recorded voltage-gated calcium currents from spinal motoneurons in slices of mature mouse spinal cords. We demonstrate that single somatic voltage-clamp steps can elicit multiple inward currents with varying delays to onset, resulting in a current with a "staircase"-like appearance. Recordings from cultured dorsal root ganglion cells at different stages of neurite development provide evidence that these currents arise from the unclamped portions of the dendritic tree. Finally, both voltage- and current-clamp data were used to constrain computer models of a motoneuron. The resultant simulations impose two conditions on the spatial distribution of CaV channels in motoneuron dendrites: one of asymmetry relative to the soma and another of spatial separation between clusters of CaV channels. We propose that this compartmentalization would provide motoneurons with the ability to process multiple sources of input in parallel and integrate this processed information to produce appropriate trains of action potentials for the intended motor behavior.

Dendritic NMDA Receptors Activate Axonal Calcium Channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.

Out of control in the dendrites

Although voltage-clamp recordings remain a favorite method for studying synaptic transmission, the space-clamp problems that are associated with somatic voltage-clamp recordings have never been directly measured. A study by Williams and Mitchell in this issue now measures the experimental errors associated with this technique.

Direct measurement of somatic voltage clamp errors in central neurons

The somatic voltage clamp technique has revolutionized understanding of synaptic physiology and the excitability of neurons. Although computer simulations have indicated that the somatic voltage clamp poorly controls voltage in the dendritic tree of neurons, where the majority of synaptic contacts are made, there has not been an experimental description of the performance of the somatic voltage clamp. Here, we directly quantify errors in the measurement of dendritic synaptic input by the somatic voltage clamp using simultaneous whole-cell recordings from the soma and apical dendrite of rat neocortical pyramidal neurons. The somatic voltage clamp did not control voltage at sites other than the soma and distorted measurement of the amplitude, kinetics, slope conductance and reversal potential of synaptic inputs in a dendritic distance-dependent manner. These errors question the use of the somatic voltage clamp as a quantitative tool in dendritic neurons.

Relative Location of Inhibitory Synapses and Persistent Inward Currents Determines the Magnitude and Mode of Synaptic Amplification in Motoneurons

In some motoneurons, L-type Ca2+ channels that partly mediate persistent inward currents (PICs) have been estimated to be arranged in 50- to 200-microm-long discrete regions in the dendrites, centered 100 to 400 microm from the soma. As a consequence of this nonuniform distribution, the interaction between synaptic inputs to motoneurons and these channels may vary according to the distribution of the synapses. For instance, >93% of synapses from Renshaw cells have been observed to be located 65 to 470 microm away from the cell body of motoneurons. Our goal was to assess whether Renshaw cell synapses are distributed in a position to more effectively control the activation of the L-type Ca2+ channels. Using compartmental models of motoneurons with L-type Ca2+ channels distributed in 100-microm-long hot spots centered 100 to 400 microm away from the soma, we compared the inhibition generated by four distributions of inhibitory synapses: proximal, distal, uniform, and one based on the location of Renshaw cell synapses on motoneurons. Regardless of whether the synapses were activated tonically or transiently, in the presence of L-type Ca2+ channels, inhibitory synapses distributed according to the Renshaw cell synapse distribution generate the largest inhibitory currents. The effectiveness of a particular distribution of inhibitory synapses in the presence of PICs depends on their ability to deactivate the channels underlying PICs, which is influenced not only by the superposition between synapses and channels, but also by the distance away from the somatic voltage clamp.

Multiple Modes of Amplification of Synaptic Inhibition to Motoneurons by Persistent Inward Currents

The ability of inhibitory synaptic inputs to dampen the excitability of motoneurons is augmented when persistent inward currents (PICs) are activated. This amplification could be due to an increase in the driving potential of inhibitory synapses or the deactivation of the channels underlying PICs. Our goal was to determine which mechanism leads to the amplification of inhibitory inputs by PICs. To reach this goal, we measured inhibitory postsynaptic currents (IPSCs) in decerebrate cats during somatic voltage-clamp steps. These IPSCs were generated by tonic activation of Renshaw cells. The IPSCs exhibited a rapid rise and a slower decay to a plateau level. Activation of PICs always led to an increase in the peak of the IPSC, but the amount of decay after the peak of the IPSC was inversely related to the size of the IPSC. These results were replicated in simulations based on compartmental models of motoneurons incorporating distributions of Renshaw cell synapses based on anatomical observations, and L-type calcium channels distributed as 100-microm-long hot spots centered 100 to 400 microm away from the soma. For smaller IPSCs, amplification by PICs was due to an increase in the driving force of the inhibitory synaptic current. For larger IPSCs, amplification was caused by deactivation of the channels underlying PICs leading to a lesser decay of the IPSCs. As a result of this change in the time course of the IPSC, deactivation of the channels underlying PICs leads to a greater amplification of the total inhibitory synaptic current.

Serotonin Modulates Dendritic Calcium Influx in Commissural Interneurons in the Mouse Spinal Locomotor Network

Commissural interneurons (CINs) help to coordinate left-right alternating bursting activity during fictive locomotion in the neonatal mouse spinal cord. Serotonin (5-HT) plays an active role in the induction of fictive locomotion in the isolated spinal cord, but the cellular targets and mechanisms of its actions are relatively unknown. We investigated the possible role of serotonin in modifying dendritic calcium currents, using a combination of two-photon microscopy and patch-clamp recordings, in identified CINs in the upper lumbar region. Dendritic calcium responses to applied somatic voltage-clamp steps were measured using fluorescent calcium indicator imaging. Serotonin evoked significant reductions in voltage-dependent dendritic calcium influx in about 40% of the dendritic sites studied, with no detectable effect in the remaining sites. We also detected differential effects of serotonin in different dendritic sites of the same neuron; serotonin could decrease voltage-sensitive calcium influx at one site, with no effect at a nearby site. Voltage-clamp studies confirmed that serotonin reduces the voltage-dependent calcium current in CINs. Current-clamp experiments showed that the serotonin-evoked decreases in dendritic calcium influx were coupled with increases in neuronal excitability; we discuss possible mechanisms by which these two seemingly opposing results can be reconciled. This research demonstrates that dendritic calcium currents are targets of serotonin modulation in a group of spinal interneurons that are components of the mouse locomotor network.

Dendritic Excitability of Mouse Frontal Cortex Pyramidal Neurons Is Shaped by the Interaction among HCN, Kir2, and KleakChannels

Dendritically placed, voltage-sensitive ion channels are key regulators of neuronal synaptic integration. In several cell types, hyperpolarization/cyclic nucleotide gated (HCN) cation channels figure prominently in dendritic mechanisms controlling the temporal summation of excitatory synaptic events. In prefrontal cortex, the sustained activity of pyramidal neurons in working memory tasks is thought to depend on the temporal summation of dendritic excitatory inputs. Yet we know little about how this is accomplished in these neurons and whether HCN channels play a role. To gain a better understanding of this process, layer V-VI pyramidal neurons in slices of mouse prelimbic and infralimbic cortex were studied. Somatic voltage-clamp experiments revealed the presence of rapidly activating and deactivating cationic currents attributable to HCN1/HCN2 channels. These channels were open at the resting membrane potential and had an apparent half-activation voltage near -90 mV. In the same voltage range, K+ currents attributable to Kir2.2/2.3 and K+-selective leak (Kleak) channels were prominent. Computer simulations grounded in the biophysical measurements suggested a dynamic interaction among Kir2, Kleak, and HCN channel currents in shaping membrane potential and the temporal integration of synaptic potentials. This inference was corroborated by experiment. Blockade of Kir2/Kleak channels caused neurons to depolarize, leading to the deactivation of HCN channels, the initiation of regular spiking (4-5 Hz), and enhanced temporal summation of EPSPs. These studies show that HCN channels are key regulators of synaptic integration in prefrontal pyramidal neurons but that their functional contribution is dependent on a partnership with Kir2 and Kleak channels.

Comparison of the Inhibition of Renshaw Cells During Subthreshold and Suprathreshold Conditions Using Anatomically and Physiologically Realistic Models

Inhibitory synaptic inputs to Renshaw cells are concentrated on the soma and the juxtasomatic dendrites. In the present study, we investigated whether this proximal bias leads to more effective inhibition under different neuronal operating conditions. Using compartmental models based on detailed anatomical measurements of intracellularly stained Renshaw cells, we compared the inhibition produced by glycine/gamma-aminobutyric acid-A (GABA(A)) synapses when distributed with a proximal bias to the inhibition produced when the same synapses were distributed uniformly (i.e., with no regional bias). The comparison was conducted in subthreshold and suprathreshold conditions. The latter were mimicked by voltage clamping the soma to -55 mV. The voltage clamp reduces nonlinear interactions between excitatory and inhibitory synapses. We hypothesized that for electrotonically compact cells such as Renshaw cells, the strength of the inhibition would become much less dependent on synaptic location in suprathreshold conditions. This hypothesis was not confirmed. The inhibition produced when inhibitory inputs were proximally distributed was always stronger than when the same inputs were uniformly distributed. In fact, the relative effectiveness of proximally distributed inhibitory inputs over uniformly distributed synapses was greater in suprathreshold conditions than that in subthreshold conditions. The somatic voltage clamp minimized saturation of inhibitory driving potentials. Because this effect was greatest near the soma, the current produced by more distal synapses suffered a greater loss because of saturation. Conversely, in subthreshold conditions, the effectiveness of proximal synapses was substantially reduced at high levels of background synaptic activity because of saturation. Our results suggest glycine/GABA(A) synapses on Renshaw cells are strategically distributed to block the powerful excitatory drive produced by recurrent collaterals from motoneurons.

Depressed Responses of Facilitatory Synapses

We show that when temporal summation takes place, depression of postsynaptic responses may ensue when the underlying synaptic conductance change is constant or even facilitatory. We term this phenomenon "apparent depression." Such apparent depression is most notable for slow synaptic conductance changes, for high frequency, and when the synapse is located at distal dendritic sites. We show that, when temporal summation ensues, the erroneous estimation of short-term synaptic plasticity arises partially from the conventional measurement of synaptic dynamics at postsynaptic potential peak time. This can be corrected when measuring overlapping synaptic responses at fixed intervals after stimulus time. Somatic voltage clamp also helps to partially correct for the apparent depression, but a good model of the neuron can do even better in providing a more accurate view of the underlying synaptic conductances.

HCN2 and HCN1 Channels Govern the Regularity of Autonomous Pacemaking and Synaptic Resetting in Globus Pallidus Neurons

The globus pallidus (GP) is a critical component of the basal ganglia circuitry controlling motor behavior. Dysregulation of GP activity has been implicated in a number of psychomotor disorders, including Parkinson's disease (PD), in which a cardinal feature of the pathophysiology is an alteration in the pattern and synchrony of discharge in GP neurons. Yet the determinants of this activity in GP neurons are poorly understood. To help fill this gap, electrophysiological, molecular, and computational approaches were used to identify and characterize GABAergic GP neurons in tissue slices from rodents. In vitro, GABAergic GP neurons generate a regular, autonomous, single-spike pacemaker activity. Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels make an important contribution to this process: their blockade with ZD7288 significantly slowed discharge rate and decreased its regularity. HCN currents evoked by somatic voltage clamp had fast and slow components. Single-cell RT-PCR and immunohistochemical approaches revealed robust expression of HCN2 subunits as well as significant levels of HCN1 subunits in GABAergic GP neurons. Transient activation of striatal GABAergic input to GP neurons led to a resetting of rhythmic discharge that was dependent on HCN currents. Simulations suggested that the ability of transient striatal GABAergic input to reset pacemaking was dependent on dendritic HCN2/HCN1 channels. Together, these studies show that HCN channels in GABAergic GP neurons are key determinants of the regularity and rate of pacemaking as well as striatal resetting of this activity, implicating HCN channels in the emergence of synchrony in PD.

Neuronal integrative analysis of the "Dumbbell" model for passive neurons.

We analyze the so called "Dumbbell" model of Jackson (J. Neurophysiol. 69 (1993) pp. 464) for a single neuron consisting of a patch-clamped cell body attached to dendritic cable of finite length terminating in an oblique derivative ("natural termination") boundary condition representing a dendritic swelling or a natural ending sealed by a continuous surface of the cell membrane. The model is solved analytically via the Green's function method. Large and small time asymptotic behavior of the membrane potential is developed when there is a somatic voltage-clamp imposed. We discuss the difference in the voltage distribution if a sealed-end (Neumann) termination is used instead of the natural termination boundary condition. If the access resistance is large the differences between the potentials corresponding to the two boundary conditions are small at the soma, but can vary significantly near the dendritic termination. This discrepancy is amplified at the soma if there is a synaptic stimulus introduced between the soma and dendritic tip.

Inhibition of backpropagating action potentials in mitral cell secondary dendrites.

The mammalian olfactory bulb is a geometrically organized signal-processing array that utilizes lateral inhibitory circuits to transform spatially patterned inputs. A major part of the lateral circuitry consists of extensively radiating secondary dendrites of mitral cells. These dendrites are bidirectional cables: they convey granule cell inhibitory input to the mitral soma, and they conduct backpropagating action potentials that trigger glutamate release at dendrodendritic synapses. This study examined how mitral cell firing is affected by inhibitory inputs at different distances along the secondary dendrite and what happens to backpropagating action potentials when they encounter inhibition. These are key questions for understanding the range and spatial dependence of lateral signaling between mitral cells. Backpropagating action potentials were monitored in vitro by simultaneous somatic and dendritic whole cell recording from individual mitral cells in rat olfactory bulb slices, and inhibition was applied focally to dendrites by laser flash photolysis of caged GABA (2.5-microm spot). Photolysis was calibrated to activate conductances similar in magnitude to GABA(A)-mediated inhibition from granule cell spines. Under somatic voltage-clamp with CsCl dialysis, uncaging GABA onto the soma, axon initial segment, primary and secondary dendrites evoked bicuculline-sensitive currents (up to -1.4 nA at -60 mV; reversal at approximatety 0 mV). The currents exhibited a patchy distribution along the axon and dendrites. In current-clamp recordings, repetitive firing driven by somatic current injection was blocked by uncaging GABA on the secondary dendrite approximately 140 microm from the soma, and the blocking distance decreased with increasing current. In the secondary dendrites, backpropagated action potentials were measured 93-152 microm from the soma, where they were attenuated by a factor of 0.75 +/- 0.07 (mean +/- SD) and slightly broadened (1.19 +/- 0.10), independent of activity (35-107 Hz). Uncaging GABA on the distal dendrite had little effect on somatic spikes but attenuated backpropagating action potentials by a factor of 0.68 +/- 0.15 (0.45-0.60 microJ flash with 1-mM caged GABA); attenuation was localized to a zone of width 16.3 +/- 4.2 microm around the point of GABA release. These results reveal the contrasting actions of inhibition at different locations along the dendrite: proximal inhibition blocks firing by shunting somatic current, whereas distal inhibition can impose spatial patterns of dendrodendritic transmission by locally attenuating backpropagating action potentials. The secondary dendrites are designed with a high safety factor for backpropagation, to facilitate reliable transmission of the outgoing spike-coded data stream, in parallel with the integration of inhibitory inputs.

Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings.

1. Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective I(h) blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. 2. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (C(m)), specific membrane resistance (R(m)) and intracellular resistivity (R(i)) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: C(m), 0.77 +/- 0.17 microF cm(-2) (mean +/- S.D.; range, 0.64-1.00 microF cm(-2)), R(m), 122 +/- 18 kOmega cm(2) (98-141 kOmega cm(2)) and R(i), 115 +/- 20 Omega cm (93-142 Omega cm). 3. The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. 4. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. 5. A model of I(h) was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. 6. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients such as synaptic potentials and action potentials are heavily filtered.