Intracellular recording in vivo and patchclamp recording on brain slices. Wholecell patch clamp, especially in vivo, has a high implementation barrier, as it requires a lot of expertise automation may promote throughput, standardization and implementation of wholecell patch clamp. Show full abstract 23 microcircuit of the c2 barrel column driving sensory processing for the c2 whisker, and combined in vivo wholecell patchclamp recordings in anesthetised and awake head. Preparation of recording electrode is critical for successful intracellular recording in vivo. In vivo field recordings effectively monitor the mouse. Cellular and molecular events can be investigated using electrophysiological techniques.
Our results, including l6 thalamic and callosal projection neurons, indicate that target specificity is a strong predictor of molecular identity of l5 and l6 longrange projection neurons. In vivo wholecell recording from neurons 25 mm below the brain surface, such as in the hippocampus harvey et al. The robot is relatively inexpensive, and can easily be appended to. To study the molecular and functional properties of prenmdars we will use in vivo combinatory molecular knockdownoptogenetic strategies in addition to complimentary wholecell patch clamp recordings using selective pharmacology and calcium imaging. In vivo imaging with miniscope in hippocampus ca1 youtube. Effects of body temperature on neural activity in the. Whole cell patch clamp an overview sciencedirect topics. Electrophysiology from greek, elektron, amber see the etymology of electron. As a critical technique for dissection of synaptic and cellular mechanisms, wholecell patchclamp recording has become feasible for in vivo preparations including both anaesthetized and awake mammalian brains.
Intrinsic membrane properties determine hippocampal. Should you patch hippocampal neurons in acute brain slices or. C procedures and different recording modes of in vivo patch clamp blind patch. Slice patch clamp technique for analyzing learninginduced. In doing so, we will also examine the contribution of prenmdars to hippocampal synaptic. Thus, we carried out an analysis of human brain slice viability by patch clamp recording of individual. For both patchclamp recordings on slices and field recordings in the intact hippocampus preparation, light was provided by a custommade lightemitting diode led system in which blue 473 nm or orange 593 nm leds were coupled to a polymer light guide. The holder houses both an optical fiber and an electrode enabling simultaneous patchclamp recording and optogenetic activation. This approach, combined with pharmacological manipulations, can be used to infer the relative contribution of inhibitory, excitatory, modulatory, and peptidergic inputs. It involves measurements of voltage changes or electric current or manipulations on a wide variety of scales from single ion.
Hippocampal hub neurons maintain unique functional properties. Orthodromic stimulation of the lateral perforant pathway evoked a excitatory postsynaptic current epsc with a latency of 3. Frontiers correlating anatomy and function with gene. Thus, in vitro patch clamp and in vivo fluorescent tracers can be combined to study the relationship between gene expression of individual neurons and axonal projection with the spatial resolution provided by visually guided patch pipette targeting. Whole cell patch clamp is an important tool for studying the function of specific cell types in circuits. Wholecell patchclamp recording is an important neuroscience. Rnaseq blog in workflow december 20, 2017 3,482 views.
By combining viralmediated in vivo gene delivery with in vitro patchclamp recordings, we found that the inhibitory avoidance task. As seen in some labs, people do hippocampal slices for their patch clamping on. Intracellular recording in vivo and patchclamp recording. In vivo imaging with multiphoton microscopy in developmental biology. In vivo crispra decreases seizures and rescues cognitive. Researchers combine in vivo labeling, patch clamp, and single cell rnaseq to correlate anatomy and function with gene expression in individual neurons. In this paper, recent researches on how acupuncture might modulate electrophysiological responses.
In addition, the patchclamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. We prepared acute hippocampal slices from untrained, iatrained, unpaired, and. In vivo wholecell patchclamp recording in the zebrafish. Wholecell patch clamp recording 1,2 of the electrical activity of neurons in vivo utilizes glass micropipettes to establish electrical and molecular access to the insides of neurons in intact. Automated wholecell patch clamp electrophysiology of. B demonstration of blind patch and twophotonguided patch. Wholecell patchclamp recordings were used to study pairedpulse facilitation ppf of the lateral perforant path input to the dentate gyrus in thin hippocampal slices. These exercises form the basis for research projects that address a specific question about the relationship between striatal. Patch clamp recording from cells in sliced tissues the university of. Patchclamp technique in brain slices springer nature experiments. Current clamp recordings were performed in standard external solution in the presence of dlap5 50.
The first few days of the module will comprise exercises that teach the basic principles and techniques of behavioral testing, in vivo single unit and local field potential recording, fiber photometry, and patch clamp. Safinamide maximally inhibited the veratridineinduced glu and gaba release in hippocampus at 15 mgkg, which reached free brain concentrations of 1. Faculty biomedical graduate studies perelman school of. Pten knockdown in vivo increases excitatory drive onto. Advances in the automation of wholecell patch clamp. General description of in vivo patchclamp technique in vivo patchclamp recording can be performed in both anesthetized and awake animals. To shed light on the mechanisms underlying the effect of safinamide, sodium currents were measured by patchclamp recording in rat cortical neurons. Whole cell patch clamp recordings from morphologically digitimer ds2a duration. The emerging role of in vitro electrophysiological methods in cns. Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patchclamp recording. Evidence is provided that pathological synchronization during kainateinduced rhythmic epileptiform activity in the gamma frequency band in vivo results primarily from synchronous inhibition provided by hippocampal. However, the impact of reductions in pten on synaptic and circuit function remains unexplored. Wholecell patch clamp electrophysiology of neurons is a gold standard technique for highfidelity.
Although synaptic delivery of ampatype glutamate receptors ampars contributes to experiencedependent synaptic strengthening, its role in hippocampusdependent learning remains elusive. In vivo wholecell patchclamp recordings from ca1 pyramidal neurons and simultaneous lfp recordings were performed in headfixed, fully awake mice. Wholecell patchclamp recordings in brain slices protocol. Patch clamp recordings, extracellular and intracellular recordings, multicellular calcium imagign, voltage sensitive dye imaging, chloride imaging, multiphoton microscopy, confocal microscopy, immunohistochemistry, neuronal cell culture, transgenic animals, in vitro and in vivo recording techniques, optical recordings, eeg recordings. Parvalbumin interneurons of hippocampus tune population.
There is no clear limitation of recording depth for in vivo patchclamp. In particular, the patchclamp method provides detailed information. This helps to minimize pulsation and increases the systems stability, which is critical for any in vivo. In vivo patchclamp recordings from cells in the lc to target patch electrodes to the dorsal pons we exposed the floor of the fourth ventricle through a posterior occipital craniotomy using gentle suction aspiration to remove the central portion of the cerebellum. In vivo wholecell recording with high success rate.
This approach enables researchers to directly unravel different synaptic components and to understand their underlying roles in particular brain functions. With the development of in vivo patchclamp recording, especially in. Researchers combine in vivo labeling, patch clamp, and. We used in vivo stereotaxic injections of lentivirus expressing a short hairpin rna to knock down pten in. The hippocampus contains neural circuitry that is crucial for higher brain functions, such as learning and memory malenka and nicoll, 1999. M to block nmda, ampakainate, and gaba a receptors, respectively. Contextual learning requires synaptic ampa receptor. In vivo wholecell recording with high success rate in. Combining in vivo patchclamp recording with other techniques, such as twophoton imaging or optogenetics, can provide even clearer functional dissection of the synaptic contributions of. The neurons that form the circuits in the hippocampus require transmembrane cation influx for their depolarization and the generation of action potentials nicholls et al.
With the development of in vivo patchclamp recording, especially in vivo voltageclamp recording, researchers can not only directly measure neuronal activity, such as spiking responses or membrane potential dynamics, but also quantify synaptic inputs from excitatory and inhibitory circuits in living animals. Humans and other mammals have two hippocampi, one in each side of the brain. This is a significant advantage in nonslice protocols, such as invivo recording from intact animals, wholecell patchclamping, lfp recording, and spikesingleunit recording. For my doctoral work, i trained in slice patchclamp and in vivo electrophysiology to study the signal transmission at central auditory synapses. To this end, we performed a series of ex vivo whole cell patch clamp recordings in acute brain slices from adult dlx12e7. Presynaptic nmdar function in the hippocampus pablo lituma. A representative in vivo patchclamp setups for anesthetized, awaking and behaving animals. The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. The patchclamp technique, an electrophysiological technique that has been developed in the late 1970s 1,2, is a primary tool for studying single or multiple ion channel functions in live tissue. Optopatcher and optogenetics tools for electrophysiology. Astrocyte intermediaries of septal cholinergic modulation. In particular, we focused on ca3 pyramidal neurons, because they play a key role in higher network functions lisman, 1999.
Therefore, using in vitro patch clamp, peoples are figuring out the function or. Among the different patch configurations that can be achieved, wholecell patchclamp recordings allow the study of the electrical behavior of a substantial part of the neuron. Automated in vivo patchclamp evaluation of extracellular. Recording electrode is pulled from a borosilicate glass tubing with filament 2. Pfeffer ck and beltramo r 2017 correlating anatomy and function with gene expression in individual neurons by combining in vivo labeling. In the anesthetized state, the animals heart rate and breathing is relatively stable and smooth. All experiments were carried out in strict accordance with institutional, national, and european guidelines for animal experimentation. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes, and demonstrated it in the cortex and hippocampus of. A robust ex vivo experimental platform for molecular. Safinamide differentially modulates in vivo glutamate and.
When the pipette approaches a nearby cell, heartbeatassociated changes become notable in test pulses. Synchronization of kainateinduced epileptic activity via. In vivo field recordings from the cortex and hippocampus were performed using a patch electrode b100507. Automated wholecell patchclamp electrophysiology of. The hippocampus plays a central role in learning and memory. Tissue specific deletion of pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. The slice patch clamp technique is a powerful tool for investigating. To examine how synaptic ach release from septohippocampal axons affects hippocampal neurons, we obtained in vivo patchclamp recordings from hippocampal dentate granule cells in anesthetized mice figure 1a. Wholecell patch clamp recording is a powerful technique for interrogating the cellular response to stimulation of inputs. Automated wholecell patchclamp electrophysiology of neurons in vivo. In most cases, in vivo patchclamp recordings are performed in superficial regions. Phaselocked inhibition, but not excitation, underlies. We injected sgo in the hippocampus in vivo, and 48 h after surgery ex vivo patchclamp recordings from brain slices show a significant reduction in glutamatergic synaptic activity in respect to saline injections.