An In Vivo Whole- Cell Patch Clamp Study. Abstract. The inferior olivary nucleus (IO) forms the gateway to the cerebellar cortex and receives feedback information from the cerebellar nuclei (CN), thereby occupying a central position in the olivo- cerebellar loop. Here, we investigated the feedback input from the CN to the IO in vivo in mice using the whole- cell patch- clamp technique. This approach allows us to study how the CN- feedback input is integrated with the activity of olivary neurons, while the olivo- cerebellar system and its connections are intact. Whole Cell Patch Clamp Ppt SlidesWhole Cell Patch Clamp Ppt TemplateOur results show how IO neurons respond to CN stimulation sequentially with: i) a short depolarization (EPSP), ii) a hyperpolarization (IPSP) and iii) a rebound depolarization. The latter two phenomena can also be evoked without the EPSPs. The IPSP is sensitive to a GABAA receptor blocker. The IPSP suppresses suprathreshold and subthreshold activity and is generated mainly by activation of the GABAA receptors. The rebound depolarization re- initiates and temporarily phase locks the subthreshold oscillations. Lack of electrotonical coupling does not affect the IPSP of individual olivary neurons, nor the sensitivity of its GABAA receptors to blockers. The GABAergic feedback input from the CN does not only temporarily block the transmission of signals through the IO, it also isolates neurons from the network by shunting the junction current and re- initiates the temporal pattern after a fixed time point. HTS techniques for patch clamp-based ion channel screening. Answers sought by patch – clamp studies Single. The Interpretation of Current-Clamp Recordings in the Cell. Conventional tight seal whole-cell patch-clamp in voltage-clamp mode was performed. Cell(s), electrodes and headstage amplifier are missing on. The PowerPoint PPT presentation: 'The Patch Clamp Technique' is the property of its. Whole-cell vs Cell-attached patch Current Clamp Components of an action potential Voltage Clamp Conductances of an action potential Channel Structure. Properties of the Nucleo-Olivary Pathway: An In Vivo Whole-Cell. Potential in Reality Patch Clamping Properties of the Action. Cable analysis with the whole. These data suggest that the IO not only functions as a cerebellar controlled gating device, but also operates as a pattern generator for controlling motor timing and/or learning. Citation: Bazzigaluppi P, Ruigrok T, Saisan P, De Zeeuw CI, de Jeu M (2. Properties of the Nucleo- Olivary Pathway: An In Vivo Whole- Cell Patch Clamp Study. PLo. S ONE 7(9). e. Zhang. University of Southern California, United States of America. Received: June 2. Accepted: August 2. Published: September 2. Copyright: . This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The present work has been funded by Zon. Mw Grant 9. 17. 9. M. d. J). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction. The inferior olive (IO) is located in the ventral medulla and gives rise to the climbing fibres (CFs), which constitute one of the two main excitatory inputs to the Purkinje cells (PCs) in the cerebellar cortex. Olivary neurons, which are coupled via dendro- dendritic gap junction (GJ) . PCs send inhibitory fibres to the CN, which contain GABAergic, glycinergic and glutamatergic neurons. Part of the CN neurons projects directly to the IO via an inhibitory, GABAergic, pathway . This olivo- cortico- nuclear projection (Figure 1. A) forms the basis of the modular organization of the cerebellum . Despite our anatomical knowledge on this nucleo- olivary projection, its role in motor control and motor learning is still under debate . The GABAergic feedback inhibition on the IO might serve to gate motor learning in the cerebellar cortex . In vivo CN- evoked IO response. A: experimental set- up: the stimulation electrode is placed in CN, the recording pipette is in the IO. Synapse, closed triangle: excitatory, open triangle: inhibitory. B: control experiment, example of LTO cell responding to CN stimulation, top trace: beginning of the experiment (t = 0); middle trace: after twenty minutes (t = 2. There are no significant changes. C: coronal sections of cerebellum. Left: 1. 6 magnifications, arrow points at the lesion in the Interpositus Nucleus, scale bar: 0. Right: same as left, 4. Abbreviations: CN, Cerebellar Nuclei; MDJ, Meso- diencephalic Junction; PC, Purkinje Cell; IO, Inferior Olive. The only two in vitro studies in which olivary GABAergic IPSPs were observed . Both studies are important because they directly show the actual presence and the activation of GABAA receptors on the membrane of IO neurons. However, both studies did not explore the contribution of this inhibitory feedback action under physiological conditions. For these reasons, we directly activated the CN with a stimulation electrode while performing whole- cell recording from the IO in vivo. Our approach succeeded in evoking inhibitory responses (IPSPs) in olivary neurons and allowed us to explore their relation with the subthreshold oscillatory behaviour of the neurons. Moreover, we pharmacologically block the GABAA receptors in the recorded neuron, showing the direct involvement of GABAA receptors, in line with Devor et al. Ultimately, we replicated the experiments in Connexin. GABAergic IPSPs were generated on the recorded neuron and not in the periphery of the electrotonically coupled network. Results. Cerebellar Control of the Inferior Olive. In vivo whole- cell recordings allow us to monitor both intrinsic suprathreshold and subthreshold activities of olivary neurons as well as responses evoked by CN stimulation (Figure 1). The recorded neurons presented subthreshold profiles in line with the ones previously shown by Khosrovani . Briefly, we focused our analysis on the IO neurons which were presenting either low- threshold oscillations (LTO) or sinusoidal subthreshold oscillations (SSTO). It is still unknown whether these two different subthreshold activities reflect two distinct neuronal populations or whether they are two different oscillating profiles of the same type of neurons. The sinusoidal subthreshold oscillation is an intrinsic property. For all the neurons that were orthodromically activated, we measured the passive membrane properties, which were similar to the ones reported by Khosrovani et al. Input resistance was on average 3. The differences between LTO and SSTO neurons with regards to their subthreshold profile are limited to the frequency, the rhythm and the shape of the oscillations as already described by Khosrovani et al. The orthodromic activation of nucleo- olivary pathway by highfrequency stimulation of the CN gave rise to different sets of inhibitory responses. The activation of the nucleo- olivary pathway resulted in a very specific olivary response pattern (Figure 1. B). Both LTO and SSTO neurons are able to respond to the CN stimulation with EPSPs after a latency of 3. LTO (n = 1. 3) and SSTO (n = 7) neurons, respectively. The EPSP, when present, was occasionally accommodating an action potential. The long- latency IPSPs responses were consistently recorded in both LTO (n = 1. SSTO (n = 7) neurons (8. Table 1). The long- latency IPSP fully suppressed the generation of action potentials as well as the generation of subthreshold activity, including oscillations; however, the responses of LTO and SSTO neurons express different characteristic in this respect. The duration of the membrane hyperpolarization in LTO neurons (5. Moreover, the IPSPs’ peak amplitude of SSTO neurons was significantly bigger than the one of LTO neurons (. The long- latency IPSP responses (figure 2. A and 2. B) were observed with and without the preceding short- latency EPSPs, suggesting that these two responses are evoked independently from each other. We then stimulated two times in a row at different time intervals in four neurons that responded to CN stimulation with both a short- latency EPSPs and a long- latency IPSP in order to elucidate for how long the IPSP can prevent the onset of the EPSP evoked by the second stimulation. The second CN stimulation can elicit a second EPSP only if the interstimulus interval is at least 3. Fig. B: same experiment but in a SSTO cell. Filled arrow: short- latency EPSP, empty arrow: rebound depolarization; empty arrowhead: peak of the IPSP. In order to investigate the contribution of GABA in the observed long- latency hyperpolarizing responses, we added a specific GABAA receptor blocker DNDS to our pipette solution . In order to block GABAA receptors internally, DNDS molecules have to travel from the pipette to distal dendritic sites; a time consuming process of approximately 2. Therefore, this experiment requires a stable recording for at least 2. A subset of the neurons presented in Table 1 was recorded long enough to explore the properties of their responses over a time span of more than 2. To quantify the hyperpolarizing response, we measured the duration, peak amplitude and surface area generated by the hyperpolarizing sag, which were not affected by the dialysis of the cytoplasm with our pipette solution (Table 2, Figure 1. B). Control cells (n = 9) recorded for 2. DNDS- free internal solution showed no significant difference in short- latency EPSPs probability, long- latency IPSPs probability, IPSPs duration, IPSP peak amplitude, IPSP area, rebound probability and amplitude between the beginning and the end of the recordings (Table 2). On the other hand, the presence of DNDS (n = 1. The average probability, duration, surface area and peak amplitude of the IPSPs were lower, but not significantly, than the ones measured with DNDS- free solution, suggesting an immediate action of the blocker on somatic GABAA receptors (unpaired t- test, for SSTO, p = 0. LTO, p = 0. 1. 4, p = 0. Table 2). After 2. DNDS dialysis, the chances of triggering a hyperpolarizing response was significantly reduced (LTO: 8. SSTO: 9. 3. 5 vs 3. V, LTO: . On the other hand the chance of triggering the short latency EPSP was not significantly altered (Table 2) and, the chance to evoke a rebound depolarization were slightly but significantly reduced only in the case of SSTO neurons (9. Yet, the CN stimulations can still evoke a small hyperpolarizing response after 2. DNDS dialysis, (Figure 3, Table 2). In order to exclude the putative limiting blocking effects of 5 m. M DNDS, two experiments have been performed with an high DNDS concentration (1.
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