Michael Häusser

Action potential initiation and backpropagation in neurons of the mammalian CNS

Most neurons in the mammalian CNS encode and transmit information via action potentials. Knowledge of where these electrical events are initiated and how they propagate within neurons is therefore fundamental to an understanding of neuronal function. While work from the 1950s suggested that action potentials are initiated in the axon, many subsequent investigations have suggested that action potentials can also be initiated in the dendrites. Recently, experiments using simultaneous patch-pipette recordings from different locations on the same neuron have been used to address this issue directly. These studies show that the site of action potential initiation is in the axon, even when synaptic activation is powerful enough to elicit dendritic electrogenesis. Furthermore, these and other studies also show that following initiation, action potentials actively backpropagate into the dendrites of many neuronal types, providing a retrograde signal of neuronal output to the dendritic tree.

Initiation and spread of sodium action potentials in cerebellar purkinje cells

Simultaneous whole-cell recordings were made from the soma and dendrites of cerebellar Purkinje cells in rat brain slices. Sodium action potentials, evoked by either depolarizing current pulses or synaptic stimulation of parallel or climbing fibers, always occurred first at the soma and decreased in amplitude with increasing distance into the dendrites. Simultaneous somatic and axonal recordings showed that these action potentials were initiated in the axon. Outside-out patches excised from the soma and dendrites revealed that the sodium channel current density decreased with distance from the soma. Consistent with this finding, comparable attenuation was observed for evoked action potentials and simulated action potential waveforms when sodium channels were blocked. These results show that sodium action potentials in cerebellar Purkinje cells are initiated in the axon and then spread passively into the dendritic tree.

Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons

The site of action potential initiation in substantia nigra neurons was investigated by using simultaneous somatic and dendritic whole-cell recording in brain slices. In many dopamine neurons, action potentials were observed first at the dendritic recording site. Anatomical reconstruction showed that in these neurons, the axon emerged from the dendrite from which the recording had been made. Action potentials showed little attention in the dendritic tree, which in dopamine neurons was shown to be due to recruitment of dendritic sodium channels and may be related to the dendritic release of dopamine. We conclude that in substantia nigra neurons, the site of action potential initiation, and thus the final site of synaptic integration, is in the axon. As the axon can originate from a dendrite, up to 240 microns away from the soma, synaptic input to the axon-bearing dendrite may be privileged with respect to its ability to influence action potential initiation.

Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo.

Here we describe an approach for making targeted patch-clamp recordings from single neurons in vivo, visualized by two-photon microscopy. A patch electrode is used to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus enabling the neuron to be visualized as a negative image (shadow) and identified on the basis of its somatodendritic structure. The same electrode is then placed on the neuron under visual control to allow formation of a gigaseal (shadowpatching). We demonstrate the reliability and versatility of shadowpatching by performing whole-cell recordings from visually identified neurons in the neocortex and cerebellum of rat and mouse. We also show that the method can be used for targeted in vivo single-cell electroporation of plasmid DNA into identified cell types, leading to stable transgene expression. This approach facilitates the recording, labeling and genetic manipulation of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.

Dendritic and somatic glutamate receptor channels in rat cerebellar Purkinje cells

1 The properties of glutamate receptor (GluR) channels in outside‐out patches from the dendrites and somata of rat cerebellar Purkinje cells in brain slices were studied using fast agonist application techniques. Dendritic patches were isolated 40–130 μm from the soma. 2 Outside‐out patches from both dendrites and somata of Purkinje cells responded to application of glutamate with a current which desensitized rapidly and nearly completely. Currents evoked by glutamate application were blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), were mimicked by l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), and were modulated by cyclothiazide. Kainate produced small, non–desensitizing currents. No currents were observed in response to aspartate application. Responses characteristic of NMDA receptor activation were not observed. These findings indicate that glutamate‐activated currents were mediated by the AMPA subtype of GluR. 3 Deactivation of the GluR channels following 1 ms pulses of glutamate occurred with a time constant of 1.23 ± 0.07 ms in dendritic and 1.12 ± 0.04 ms in somatic patches. Desensitization occurred with a time constant of 5.37 ± 0.26 ms in dendritic and 5.29 ± 0.29 ms in somatic patches. The time constant of recovery from desensitization caused by a 1 ms application of 1 mm glutamate was 36 ms in dendritic patches and 33 ms in somatic patches. 4 Half‐maximal activation of the GluR channels was achieved at a glutamate concentration of 432 μm. Deactivation kinetics were not dependent on the glutamate concentration, while desensitization became slower at lower glutamate concentrations. 5 Pre‐equilibration of patches with low concentrations of glutamate reduced the peak current activated by 1 mm glutamate. The IC50 for this effect was 8.7 μm. Equilibrium desensitization did not affect the kinetics of the current activated by 1 mm glutamate. 6 The current–voltage relationship of the peak current was linear in normal Na+‐rich external solution, with a reversal potential near 0 mV. In Ca2+‐rich external solution, the reversal potentials were −51.4 ± 2.9 and −51.5 ± 2.8 mV for dendritic and somatic patches, respectively, indicating that these glutamate channels have a low permeability to Ca2+ (PCa/PCs= 0.053). 7 The mean single‐channel conductance of the GluR channels measured using non–stationary fluctuation analysis was ∼8 pS in dendritic and somatic patches, and the maximum open probability was at least 0.7 with 5 mm glutamate. 8 GluR channel kinetics in patches excised from the soma of neonatal (postnatal day 4; P4) Purkinje cells, before the development of the dendritic arborization of the Purkinje cell, were similar to those in patches excised from more mature (P12–18) Purkinje cells. 9 Dendritic and somatic GluR channels in Purkinje cells appear to be functionally identical, are AMPA‐subtype receptors containing the GluR‐B subunit, and have rapid kinetics and low permeability to Ca2+. A kinetic model was constructed which faithfully reproduces the gating characteristics of the GluR channels.

The origin of the complex spike in cerebellar Purkinje cells

Activation of the climbing fiber input powerfully excites cerebellar Purkinje cells via hundreds of widespread dendritic synapses, triggering dendritic spikes as well as a characteristic high-frequency burst of somatic spikes known as the complex spike. To investigate the relationship between dendritic spikes and the spikelets within the somatic complex spike, and to evaluate the importance of the dendritic distribution of climbing fiber synapses, we made simultaneous somatic and dendritic patch-clamp recordings from Purkinje cells in cerebellar slices. Injection of large climbing fiber-like synaptic conductances at the soma using dynamic clamp was sufficient to reproduce the complex spike, independently of dendritic spikes, indicating that neither a dendritic synaptic distribution nor dendritic spikes are required. Furthermore, we found that dendritic spikes are not directly linked to spikelets in the complex spike, and that each dendritic spike is associated with only 0.24 ± 0.09 extra somatic spikelets. Rather, we demonstrate that dendritic spikes regulate the pause in firing that follows the complex spike. Finally, using dual somatic and axonal recording, we show that all spikelets in the complex spike are axonally generated. Thus, complex spike generation proceeds relatively independently of dendritic spikes, reflecting the dual functional role of climbing fiber input: triggering plasticity at dendritic synapses and generating a distinct output signal in the axon. The encoding of dendritic spiking by the post-complex spike pause provides a novel computational function for dendritic spikes, which could serve to link these two roles at the level of the target neurons in the deep cerebellar nuclei.

Dendritic patch-clamp recording.

The patch-clamp technique allows investigation of the electrical excitability of neurons and the functional properties and densities of ion channels. Most patch-clamp recordings from neurons have been made from the soma, the largest structure of individual neurons, while their dendrites, which form the majority of the surface area and receive most of the synaptic input, have been relatively neglected. This protocol describes techniques for recording from the dendrites of neurons in brain slices under direct visual control. Although the basic technique is similar to that used for somatic patching, we describe refinements and optimizations of slice quality, microscope optics, setup stability and electrode approach that are required for maximizing the success rate for dendritic recordings. Using this approach, all configurations of the patch-clamp technique (cell-attached, inside-out, whole-cell, outside-out and perforated patch) can be achieved, even for relatively distal dendrites, and simultaneous multiple-electrode dendritic recordings are also possible. The protocol—from the beginning of slice preparation to the end of the first successful recording—can be completed in 3 h.Note: In the version of this article initially published online: P. 1235, left column, last line: Quotation marks were misplaced. The sentence should begin: “Although some early, ‘laborious’ efforts... P. 1236, right column, last four lines, and p. 1238, first text line: References were inserted in the wrong place and misspelled. The sentences should read: “Alternative principal anions are gluconate and methanesulfonate; note that all internal solutions are associated with washout of intracellular factors and some may also have pharmacological effects45 (also see Kaczorowski, C.C., Disterhoft, J.F. &Spruston, N. Soc. Neurosci. Abst. 31, 737.17, 2005). A fluorescent dye (e.g., 1–25 μM Alexa 594) can be included…” P. 1238, first line under EQUIPMENT SETUP: “Recording” was omitted. The sentence should read: “An illustration of a typical setup used for dendritic patch-clamp recording is shown in Figure 1.” P. 1239, Table 1, first item in right column: Mispunctuated. The sentence should read: “Cut sagittally, as parallel to the midline of the cerebellum as possible, on either side of the cerebellar vermis.” P. 1240, last paragraph in Step 11: Text was misplaced. The sentences should read: “It is possible, however, to follow a dendrite deep into the slice from the soma to a distal, more superficial location. Even if the dendrite seems to disappear at points it is possible to spot the same dendrite again at a more distal location. P. 1246, Table 2, last item in right column: Punctuation was misplaced. The sentence should read: “Minimize slice swelling (see above) and rig vibration, and make manipulators as smooth and stable as possible.” These errors have been corrected in all versions of the article.

Linking Synaptic Plasticity and Spike Output at Excitatory and Inhibitory Synapses onto Cerebellar Purkinje Cells

Understanding the relationship between synaptic plasticity and neuronal output is essential if we are to understand how plasticity is encoded in neural circuits. In the cerebellar cortex, motor learning is thought to be implemented by long-term depression (LTD) of excitatory parallel fiber (PF) to Purkinje cell synapses triggered by climbing fiber (CF) input. However, theories of motor learning generally neglect the contribution of plasticity of inhibitory inputs to Purkinje cells. Here we describe how CF-induced plasticity of both excitatory and inhibitory inputs is reflected in Purkinje cell spike output. We show that coactivation of the CF with PF input and interneuron input leads not only to LTD of PF synapses but also to comparable, “balanced” LTD of evoked inhibitory inputs. These two forms of plasticity have opposite effects on the spike output of Purkinje cells, with the number and timing of spikes sensitively reflecting the degree of plasticity. We used dynamic clamp to evaluate plasticity-induced changes in spike responses to sequences of excitation and feedforward inhibition of varied relative and absolute amplitude. Balanced LTD of both excitatory and inhibitory components decreased the net spike output of Purkinje cells only for inputs with small inhibitory components, whereas for inputs with a larger proportion of feedforward inhibition CF-triggered LTD resulted in an increase in the net spike output. Thus, the net effect of CF-triggered plasticity on Purkinje cell output depends on the balance of excitation and feedforward inhibition and can paradoxically increase cerebellar output, contrary to current theories of cerebellar motor learning.