Whole- cell Patch- clamp Recordings from Morphologically- and Neurochemically- identified Hippocampal Interneurons. Cortical networks are controlled by a small, but diverse set of inhibitory interneurons.
Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole- cell recordings from single or synaptically- coupled pairs of neurons with intracellular labeling, post- hoc morphological and immunocytochemical analysis. Date Published: 9/3.
Issue 9. 1; doi: 1. Keywords: Neuroscience, Issue 9. GABAergic interneurons, synaptic transmission, IPSC, GABA- B receptor. Booker, S. A., Song, J., Vida, I. Whole- cell Patch- clamp Recordings from Morphologically- and Neurochemically- identified Hippocampal Interneurons.
Interneurons comprise a small subset of the neuronal population (1. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits.
This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole- cell patch- clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron- specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post- hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons. Hippocampal neuronal circuits have long been the subject of intense scrutiny, with respect to both anatomy and physiology, due to their essential role in learning and memory as well as spatial navigation in both humans and rodents. Equally, the prominent, but simple laminar organization of the hippocampus makes this region a favored subject of studies addressing structural and functional properties of cortical networks.
The patch clamp technique: Principles and technical considerations.
Voltage clamp - Scholarpedia. The voltage clamp is a technique used to control the voltage across the membrane of a small or isopotential area of a nerve cell by an. He concedes 'I had no theory to explain the early inward current. The patch clamp technique is a specialized version of the voltage clamp.
Voltage Clamp Patch Clamp Applications Evidence that potassium is primarily responsible for the RMP. Neuron membrane is at rest Neuron membrane is at rest Now let’s. Patch clamp techniques for single channel and. The input of the ampli
Hippocampal circuits are comprised of excitatory principal cells (> 8. Interneurons release . These inhibitory mechanisms counterbalance excitation and regulate the excitability of principal cells, and thus their timing and pattern of discharge. However, GABA released from interneurons acts not only on principal cells, but also on the interneurons themselves. Pre and postsynaptic receptors mediate feedback regulation and inhibitory mutual interactions among the various types of interneuron. These inhibitory mechanisms in interneuron networks are believed to be central to the generation and shaping of population activity patterns, in particular oscillations at different frequencies.
Measurement of Cellular Excitability by Whole Cell Patch Clamp. An example of inward current. Extracellular ATP-induced inward current in isolated epithelial cells of. Using whole-cell patch-clamp technique and Fura-2. In zero current-clamp. Animation 4.1: The Patch Clamp Method . Chapter 4; Summary; Animations. Animation 4.1; Animation 4.2; Flashcards and Key Terms.
Whole- cell patch- clamp recording is a well- established method for the examination of intrinsic properties and synaptic interactions of neurons. However, due to the high diversity of interneuron types, investigation of inhibitory interneurons requires rigorous identification of the recorded cells. As hippocampal interneuron types are characterized by distinct morphological features and neurochemical marker expression, combined anatomical and immunocytochemical examination can provide a means to determine precise interneuron identity. In the present paper we describe an experimental approach in which whole- cell patch- clamp recordings from single neurons or synaptically- coupled pairs are combined with intracellular labeling, followed by post- hoc morphological and immunocytochemical analysis, allowing for the characterization of slow GABAB receptor mediated inhibitory effects in identified interneurons.
As an example, we focus on one major type of interneuron, a subset of the so called “basket cells” (BC), which innervates the soma and proximal dendrites of its postsynaptic targets and is characterized by a “fast spiking” (FS) discharge pattern, an axon densely covering the cell body layer, and expression of the calcium- binding protein parvalbumin (PV)1. These interneurons display large postsynaptic inhibitory currents, as well as prominent presynaptic modulation of their synaptic output, in response to GABABR activation. The combination of techniques described here can be applied equally well to investigate intrinsic or synaptic mechanisms in a variety of other identified neuron types. Subscription Required.
Please recommend Jo. VE to your librarian. Ethics Statement: All procedures and animal maintenance were performed in accordance with Institutional guidelines, the German Animal Welfare Act, the European Council Directive 8. EEC regarding the protection of animals, and guidelines from local authorities (Berlin, T- 0. Preparation of Acute- hippocampal Slices.
Take a transgenic rat (1. Venus/YFP protein under the v. GAT promoter, which labels the majority of cortical inhibitory interneurons. Rapidly dissect the brain (< 4. O2/5% CO2) sucrose- based artificial cerebrospinal fluid (sucrose- ACSF, Figure 1. A). Assess the dissected rat brain for Venus/YFP fluorescence with a 5.
LED lamp and 5. 15 emission filter, mounted on a pair of goggles. Remove the frontal third of the cortex and cerebellum; then separate the hemispheres, all with a scalpel.
Remove the dorsal surface of the cortex to provide a flat surface to glue the brain down, as previously described. Cut transverse slices (3. Remove additional regions of rostral cortex, midbrain and brainstem. Transfer each slice to a submerged holding chamber containing sucrose- ACSF, which is carbogenated and warmed to 3.
Do this in order to reactivate metabolic processes and facilitate the resealing of cut neuronal processes. Then transfer to room temperature for storage (Figure 1. C). 2. Fabrication and Filling of Recording Pipettes. Pull patch pipettes from glass capillaries, so that a pipette resistance of 2- 4 M.
Keep the intracellular solution chilled on ice to prevent degradation of its constituents. Fill patch pipettes for identification of postsynaptic currents with a solution containing a physiologically relevant low Cl- concentration (ER(Cl- )= - 6. V; see solution list). For paired recordings to identify the presynaptic receptor mediated responses, fill patch pipettes with intracellular solution with low Ca. Cl- concentration (ER(Cl- )= - 2. V) to improve signal- to- noise of observed IPSCs.
Note that changing Cl- concentration can alter IPSC kinetics. Whole Cell Patch- clamp Recording from FS- INs. Carbogenate the ACSF and feed through the perfusion system to the recording chamber, by means of a peristaltic pump (which also removes ACSF from the recording chamber through a suction line, Figure 2. A). Turn on all equipment on the setup in preparation for recording. Transfer a slice to the recording chamber and hold in place with a platinum ring strung with single fibers of silk. Position the slice so that the stratum (str.) pyramidale of CA1 runs vertically through the field of view, allowing access with 2 pipettes to both the str.
Assume good slice quality if a large number of round, moderately contrasted CA1 pyramidal cells (CA1 PC) can be seen in str. Poor quality slices contain large numbers of highly contrasted, shrunken or swollen cells, with an uneven slice surface. Identify putative FS interneurons under epifluorescence illumination as those expressing Venus/YFP (Figure 2. B), with large multipolar somata in or near the str. Select cells reasonably deep within the slice (5. Lower the pipette to the surface of the slice, slightly offset to the center of the selected neuron. Obtain whole- cell recording configuration as described previously.
Figures 2. D and 2. E. Target a cell: Increase the pressure to 7. Bar and rapidly lower the pipette through the slice to just above the soma of the selected cell (Figure 2. D, top). Approach the cell: Press the pipette against the cell membrane to produce a “dimple” on it (Figure 2. D, top). Perform this step swiftly, in order to prevent biocytin labeling of neighboring cells.
Create a giga- ohm seal: Release the pressure and simultaneously apply a 2. V voltage command to the pipette. A giga- ohm seal (1- 5. G. Once sealed, apply the expected resting membrane potential (typically between - 7. V) as a voltage command. Break through the patch: Once sealed, rupture the membrane patch with a short pulse of negative pressure; thereby achieving the whole- cell configuration (Figure 2.
E, bottom). Compensate whole- cell capacitance and series resistance (RS). Rs is normally 5- 2. M. Abandon cells if membrane potential (VM) on break- through is more depolarized than - 5. V; RS is initially greater than 3. M. FS- INs have relatively depolarized VM (typically - 5.
V), short membrane time- constant (< 2. A depolarizing current injection with a train of action potentials (APs) at frequencies > 1. Hz 1. 1 (Figure 2. F, bottom), which are markedly different from those in CA1 PCs (Figure 2.
F, middle). 4. Extracellular Electrical Stimulation to Evoke GABABR- mediated Responses. To observe synaptically evoked responses, position an extracellular stimulation electrode (a patch pipette filled with 2 M Na. Cl; Resistance: 0. M. Position the electrode 2.
Use single stimuli (1. Further isolate the GABABR- mediated IPSC with application of a GABAAR blocker (gabazine . Paired Recordings of Synaptically Coupled FS- IN and CA1 PCs. Assess GABABR- mediated presynaptic control of inhibitory synaptic transmission with simultaneous recordings, performed between synaptically- coupled IN and PC pairs as described below.
First, establish a whole- cell recording of a presynaptic interneuron (as in section 3) and confirm the FS phenotype (Figure 4. A). Then patch a neighboring CA1 PC (2. If a synaptic connection is present, APs in the IN result in IPSCs in the CA1 PC, held in voltage- clamp (compensate RS to about 8.