Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.

Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.     

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked,Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.     

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

SNAP-29-mediated modulation of synaptic transmission in cultured hippocampal neurons

Identifying the molecules that regulate both the recycling of synaptic vesicles and the SNARE components required for fusion is critical for elucidating the molecular mechanisms underlying synaptic plasticity. SNAP-29 was initially isolated as a syntaxin-binding and ubiquitously expressed protein. Previous studies have suggested that SNAP-29 inhibits SNARE complex disassembly, thereby reducing synaptic transmission in cultured superior cervical ganglion neurons in an activity-dependent manner. However, the role of SNAP-29 in regulating synaptic vesicle recycling and short-term plasticity in the central nervous system remains unclear. In the present study, we examined the effect of SNAP-29 on synaptic transmission in cultured hippocampal neurons by dual patch clamp whole-cell recording, FM dye imaging, and immunocytochemistry. Our results demonstrated that exogenous expression of SNAP-29 in presynaptic neurons significantly decreased the efficiency of synaptic transmission after repetitive firing within a few minutes under low and moderate frequency stimulations (0.1 and 1 Hz). In contrast, SNAP-29 did not affect the density of synapses and basal synaptic transmission. Whereas neurotransmitter release was unaffected during intensive stimulation, recovery after synaptic depression was impaired by SNAP-29. Furthermore, knockdown of SNAP-29 expression in neurons by small interfering RNA increased the efficiency of synaptic transmission during repetitive firing. These findings suggest that SNAP-29 acts as a negative modulator for neurotransmitter release, probably by slowing recycling of the SNARE-based fusion machinery and synaptic vesicle turnover.     

Antibodies to synaptophysin interfere with transmitter secretion at neuromuscular synapses.

The involvement of synaptophysin, a synaptic vesicle-specific protein, in transmitter release at neuromuscular synapses was studied by intracellular application of synaptophysin antibodies into presynaptic neurons. Polyclonal antibodies or their Fab fragments were loaded into spinal neurons by injection into one of the early blastomeres of Xenopus embryos 1 day prior to culturing or, alternatively, directly through a whole-cell recording pipette at the soma of cultured neurons. At synapses made by antibody-loaded neurons in culture, the spontaneous synaptic currents showed marked reduction in frequency without significant change in their mean amplitude. The impulse-evoked synaptic currents showed reduced amplitude and increased failure rate. These results suggest that interference with synaptophysin function by antibody binding inhibits transmitter secretion.     

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 μm diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca²⁺ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABA_A and GABA_C receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release ^14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal ²¹. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion ¹¹. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level ¹⁹. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced ^{20, 23, 18, 9}. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.     

Experimental analysis and computational modeling of interburst intervals in spontaneous activity of cortical neuronal culture

Rhythmic bursting is the most striking behavior of cultured cortical networks and may start in the second week after plating. In this study, we focus on the intervals between spontaneously occurring bursts, and compare experimentally recorded values with model simulations. In the models, we use standard neurons and synapses, with physiologically plausible parameters taken from literature. All networks had a random recurrent architecture with sparsely connected neurons. The number of neurons varied between 500 and 5,000. We find that network models with homogeneous synaptic strengths produce asynchronous spiking or stable regular bursts. The latter, however, are in a range not seen in recordings. By increasing the synaptic strength in a (randomly chosen) subset of neurons, our simulations show interburst intervals (IBIs) that agree better with in vitro experiments. In this regime, called weakly synchronized, the models produce irregular network bursts, which are initiated by neurons with relatively stronger synapses. In some noise-driven networks, a subthreshold, deterministic, input is applied to neurons with strong synapses, to mimic pacemaker network drive. We show that models with such "intrinsically active neurons" (pacemaker-driven models) tend to generate IBIs that are determined by the frequency of the fastest pacemaker and do not resemble experimental data. Alternatively, noise-driven models yield realistic IBIs. Generally, we found that large-scale noise-driven neuronal network models required synaptic strengths with a bimodal distribution to reproduce the experimentally observed IBI range. Our results imply that the results obtained from small network models cannot simply be extrapolated to models of more realistic size. Synaptic strengths in large-scale neuronal network simulations need readjustment to a bimodal distribution, whereas small networks do not require such changes.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40–48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40–48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Pathway-specific trafficking of native AMPARs by in vivo experience

An accumulating body of evidence supports the notion that trafficking of AMPA receptors (AMPARs) underlies strengthening of glutamatergic synapses and, in turn, learning and memory in the behaving animal. However, without exception, these experiments have been performed using artificial stimulation protocols, cultured neurons, or viral-overexpression systems that can significantly alter the normal function of AMPARs. Using a single-whisker experience protocol that significantly enhances neuronal responses in vivo, we have targeted neurons in and around the spared whisker column of fosGFP transgenic mice for whole-cell recording. Here we show that in vivo experience induces the pathway-specific strengthening of neocortical excitatory synapses. By assaying AMPARs for rectification and sensitivity to joro spider toxin, we find that in vivo experience induces the delivery of native GluR2-lacking receptors at spared, but not deprived, inputs. These data demonstrate that pathway-specific trafficking of GluR2-lacking AMPARs is a normal feature of synaptic strengthening that underlies experience-dependent plasticity in the behaving animal.     

Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons.

In the hilar region of the rat hippocampus, large spontaneous excitatory postsynaptic currents (sEPSCs) mediated by non-NMDA glutamate receptors are present in both excitatory spiny mossy cells and inhibitory aspiny hilar interneurons, making these neurons ideal candidates for a comparative study using the tight seal whole-cell recording technique. Although sEPSCs have similar amplitude distributions, the rise and decay times are significantly slower in spiny versus aspiny neurons. Similar kinetic differences are observed in synaptic currents evoked by mossy fiber stimulation. These results demonstrate a physiological difference between the excitatory drive to excitatory and inhibitory neurons in the hilus that certainly contributes to differences in synaptic strength and that may be applicable to other brain regions. Furthermore, since the development or modification of individual spines or groups of spines may affect synaptic strength, these results may be pivotal in establishing a role for spines in modulating synaptic activity.     

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.     

Acute exposure to low-level CW and GSM-modulated 900 MHz radiofrequency does not affect Ba 2+ currents through voltage-gated calcium channels in rat cortical neurons

We have studied the non-thermal effects of radiofrequency (RF) electromagnetic fields (EMFs) on Ba(2+) currents (I Ba 2+) through voltage-gated calcium channels (VGCC), recorded in primary cultures of rat cortical neurons using the patch-clamp technique. To assess whether low-level acute RF field exposure could modify the amplitude and/or the voltage-dependence of I Ba 2+, Petri dishes containing cultured neurons were exposed for 1-3 periods of 90 s to 900 MHz RF-EMF continuous wave (CW) or amplitude-modulated according to global system mobile communication standard (GSM) during whole-cell recording. The specific absorption rates (SARs) were 2 W/kg for CW and 2 W/kg (time average value) for GSM-modulated signals, respectively. The results obtained indicate that single or multiple acute exposures to either CW or GSM-modulated 900 MHz RF-EMFs do not significantly alter the current amplitude or the current-voltage relationship of I Ba 2+, through VGCC.     

In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons

Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains ('hotspots') and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5-6 h, including 1-2 h of two-photon calcium imaging in combination with stable whole-cell recordings.     

Preparation and maintenance of single-cell micro-island cultures of basal forebrain neurons

Micro-island cultures provide a simplified system for studying the expression of cellular phenotype, excitability, synapse formation and pre- and postsynaptic regulatory mechanisms without the usual problems that arise from complex interactions between large numbers of other cells. The technique relies on the ability to constrain the attachment and growth of either single or small groups of neurons to discrete (20-500 microm) 'islands' of cell-permissive substrate applied over a nonadherent background layer. Constrained in this way, neurons form large numbers of conventional synaptic and/or autaptic contacts that can be easily visualized, making them ideally suited for studying synaptic physiology using electrophysiological and/or high-resolution optical imaging techniques. The protocol described here requires approximately 2 h for preparation of the culture dishes and a further 3-4 h for isolation and plating out the cells. Once established, the cultures can be maintained for prolonged periods (>6 weeks) permitting manipulations to be made to their local environment and the effects on individually identified cells to be repeatedly monitored.     

Release-dependent variations in synaptic latency: a putative code for short- and long-term synaptic dynamics

In the cortex, synaptic latencies display small variations ( approximately 1-2 ms) that are generally considered to be negligible. We show here that the synaptic latency at monosynaptically connected pairs of L5 and CA3 pyramidal neurons is determined by the presynaptic release probability (Pr): synaptic latency being inversely correlated with the amplitude of the postsynaptic current and sensitive to manipulations of Pr. Changes in synaptic latency were also observed when Pr was physiologically regulated in short- and long-term synaptic plasticity. Paired-pulse depression and facilitation were respectively associated with increased and decreased synaptic latencies. Similarly, latencies were prolonged following induction of presynaptic LTD and reduced after LTP induction. We show using the dynamic-clamp technique that the observed covariation in latency and synaptic strength is a synergistic combination that significantly affects postsynaptic spiking. In conclusion, amplitude-related variation in latency represents a putative code for short- and long-term synaptic dynamics in cortical networks.     

海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。     

GDNF acutely potentiates Ca2+ channels and excitatory synaptic transmission in midbrain dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) is best known for its long-term survival effect on dopaminergic neurons in the ventral midbrain. A recent study showed that acute application of GDNF to these neurons suppresses A-type potassium channels and potentiates neuronal excitability. Here we have characterized the acute effects of GDNF on Ca(2+) channels and synaptic transmission. GDNF rapidly and reversibly potentiated the high voltage-activated (HVA) Ca(2+) channel currents in cultured dopaminergic neurons. Analyses of channel kinetics indicate that GDNF decreased the activation time constant, increased the inactivation and deactivation time constants of HVA Ca(2+) channel currents. Ca(2+) imaging experiments demonstrate that GDNF facilitated Ca(2+) influx induced by membrane depolarization. To investigate the physiological consequences of the Ca(2+) channel modulation, we examined the acute effects of GDNF on excitatory synaptic transmission at synapses made by these dopaminergic neurons, which co-release the transmitter glutamate. Within 3 min of application, GDNF increased the amplitude of spontaneous and evoked excitatory autaptic- or multiple-postsynaptic currents. The frequency as well as the amplitude of miniature excitatory postsynaptic currents was also increased. These results reveal, for the first time, an acute effect of GDNF on synaptic transmission and its potential mechanisms, and suggest that an important function of GDNF for midbrain dopaminergic neurons is the acute modulation of transmission and ion channels.     

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.