DOI: 10.5061/dryad.76hdr7t6z
title: Recovery of the full in vivo firing range in post-lesion surviving DA SN neurons associated with Kv4.3-mediated pacemaker plasticity

description: Dopamine (DA) neurons in the substantia nigra (SN) control several
 essential functions, including the voluntary movement, learning and
 motivated behavior. Healthy DA SN neurons show diverse firing patterns in
 vivo, ranging from slow pacemaker-like activity (1-10 Hz) to transient
 high frequency bursts (&lt;100 Hz), interspersed with pauses that can
 last hundreds of milliseconds. Recent in vivo patch experiments have
 started to reveal the subthreshold mechanisms underlying this
 physiological diversity, but the impact of challenges like cell loss on
 the in vivo activity of adult DA SN neurons, and how these may relate to
 behavioral disturbances, are still largely unknown. We investigated the in
 vivoelectrophysiological properties of surviving SN DA neurons after
 partial unilateral 6-OHDA lesions, a single-hit, non-progressive model of
 neuronal cell loss. We show that mice subjected to this model have an
 initial motor impairment, measured by asymmetrical rotations in the open
 field test, which recovered over time. At 3 weeks post-lesion, when open
 field locomotion was strongly impaired, surviving DA SN neurons showed a
 compressed in vivo dynamic firing range, characterized by a 10-fold
 reduction of in vivo burst firing compared to controls. This in vivo
 phenotype was accompanied by pronounced in vitro pacemaker instability. In
 contrast, in the chronic post-lesion phase (&gt;2 months), where
 turning symmetry in open field locomotion had recovered, surviving SN DA
 neurons displayed the full dynamic range of in vivo firing, including in
 vivo bursting, similar to controls. The normalized in vivo firing pattern
 was associated with a 2-fold acceleration of stable in vitro pacemaking,
 mediated by Kv4.3 potassium channel downregulation. Our findings
 demonstrate the existence of a homeostatic pacemaker plasticity mechanism
 in surviving DA SN neurons after pronounced cell loss.
description: Animals Male and
 female C57Bl/6N mice (Charles River Laboratories) were used for the study.
 The mice were 8 weeks old, group housed and maintained on a 12-hour
 light-dark cycle. All experiments and procedures involving mice were
 approved by the German Regierungspräsidium Darmstadt
 (V54-19c20/15-F40/30). In total, 152 mice were used for this study (see
 table below). ** ** 
 
 <strong>Early
 phase</strong> <strong>Late
 phase</strong> 
 ** ** Mice (#)
 Cells (#) Mice (#)
 Cells (#) <em><strong>In
 vivo</strong></em> <strong>juxta & behavior –
 vehicle</strong> 15 12 –
 medial/ 28 – all 12 10 – medial/
 26 – all <em><strong>In
 vivo</strong></em> <strong>juxta & behavior –
 6-OHDA</strong> 16 9 –
 medial/ 16 – all 10 9 – medial/
 11 – all <em><strong>In
 vitro</strong></em> <strong>– vehicle</strong>
 3 16
 3 12 
 <em><strong>In
 vitro</strong></em> <strong>– 6-OHDA</strong>
 6 37
 5 25 
 <em><strong>In
 vitro</strong></em> <strong>& AmmTx3 –
 vehicle</strong> -
 - 3 15
 
 <em><strong>In vitro</strong></em>
 <strong>& AmmTx3 – 6-OHDA</strong> 
 - - 3
 14 <em><strong>In
 vitro</strong></em> <strong>voltage clamp experiments –
 vehicle</strong> -
 - 3 17
 
 <em><strong>In vitro</strong></em>
 <strong>voltage clamp experiments – 6-OHDA</strong>
 -  
 3 15 
 <strong>Female behavior experiments
 – vehicle, 6-OHDA</strong> -
   10  
 <strong>Female histology – vehicle,
 6-OHDA</strong> -
   8  
 <strong>6-OHDA dose
 response</strong> 12
      
 ** **
 Mice (#) Mice (#)
 <strong>Kv4.3
 Immunochemistry</strong> 8
 8 
 <strong>SN
 quantification – vehicle</strong> 9
 3 
 <strong>SN
 quantification – 6-OHDA</strong> 9
 3 
 <strong>Aggregate/ phospho- α-synuclein
 Staining</strong> 2
 2 
 <strong>Sum
 = 148</strong>  
 
   
 Stereotactic
 6-OHDA infusion All surgeries were performed under
 general anesthesia in areflexic state. Prior to the induction of
 anesthesia, a premedication of 0.2 mg/kg atropine (atropine-sulfate, Braun
 Melsungen AG, Melsungen) was given as an intraperitoneal (i.p.) injection
 to stabilize circulation. Anesthesia was induced in a plastic chamber,
 which was flooded with 5% Isoflurane (Florene®, AbbVie Deutschland GmbH
 & Co. KG, Ludwigshafen, Germany) in pure oxygen (0.4 l/min). For
 maintenance of anesthesia, isoflurane was delivered through a breathing
 mask with a flow rate of 0.35 l/min and its concentration was regulated to
 1.5-2.2% using an adjustable vaporizer (Uno, Zevenaar, Netherlands). The
 depth of anesthesia was controlled by testing the toe pinch reflex and the
 breathing rate (1-2Hz). Body temperature (36°C) and respiration were
 constantly monitored. Lidocaine/prilocaine ointment (25 mg/g, Emla® creme,
 AstraZeneca GmbH, 22876 Wedel) was applied prior to surgery and after
 suturing of the wound as local anesthetics. Additional analgesia was
 provided by subcutaneous injection of carprofen (4 mg/kg in NaCl,
 Rimadyl®, Pfilzer GmbH, Berlin, Germany) after infusion. Eye lubricant
 (Visidic, Bausch and Lomb, Berlin, Germany) was used to protect eyes from
 desiccation. Desipramine hydrochloride (20 mg/kg, Sigma
 Aldrich) was injected i.p. 20-40 min before intracranial infusions to
 prevent 6-OHDA uptake by noradrenergic neurons. The desipramine solution
 was prepared in sterile, isotonic NaCl solution (B. Braun Melsungen AG,
 Germany) at the day of surgery. The infusion solutions are based on
 sterile artificial cerebrospinal fluid (ACSF, Harvard Apparatus,
 Holliston, MA, USA) with 0.02% L-ascorbic acid (used also as a vehicle
 solution). The 6-OHDA solution (0.2% 6-hydroxydopamine hydrochloride in
 ACSF with 0.02% L-ascorbic acid) was prepared at the day of infusion,
 stored on ice, and shielded from light. During surgery,
 the animals were placed on a heating pad and were fixed in a stereotactic
 frame (Model 1900, Kopf Instruments, Tujunga, USA) with a stereotactic arm
 and a connected three-way digital positioning display. The scalp was
 opened by a longitudinal cut to expose the skull with bregma and lambda on
 display. With a centering scope (Model 1915, Kopf Instruments, Tujunga,
 USA), the bregma-lambda distance was measured and examined for suitable
 anatomy (4.4 ± 0.2 mm distance). Afterwards, the skull was aligned to a
 reference frame with a stereotaxic alignment indicator (Model 1905, Kopf
 Instruments, Tujunga, USA) and the manipulator system was referenced to
 bregma. Using a stereotaxic drill (Model 1911, Kopf
 Instruments, Tujunga, USA) with a 500 µm diameter drill bit, a hole above
 the right striatum was drilled (coordinates: ML: +1.9 mm, AP: +0.5 mm to
 bregma). ACSF or 6-OHDA solution were loaded to a 10 µl NanoFil syringe
 (World Precision Instruments Inc., Sarasota, FL, USA) with a 35G blunt
 needle, which was mounted on a MicroSyringe Pump (UMP3-1, World Precision
 Instruments) and controlled by a SYSMicro4 Controller (World Precision
 Instruments). Using the stereotactic arm, the needle was slowly lowered
 (about 750 µm/min) to a position of -2.2 mm below the brain surface
 (infusion site coordinates: ML: +1.9 mm, AP: +0.5 mm, DV: -2.2 mm to
 bregma). Anatomical references are based on Franklin and Paxinos (2008). A
 volume of 6 µl was infused with a flow rate of 250 nl/min. Once the volume
 was infused, the needle rested for 5 minutes in that position before it
 was slowly moved out of the brain. Directly before and after infusion,
 proper functioning of the syringe system and the needle was checked.
 Finally, after suture the animal was placed on a heating pad for full
 recovery. Oats, wet food pallets and water were placed inside the cage to
 ease consumption. Behavioral
 testing Open field
 Spontaneous locomotion (track length, wall distance, time in
 center and number of rearings) and rotations of all mice were monitored in
 open field (50 × 50 cm, center 30 × 30 cm; red illumination, 3 lx) for 10
 min in 3 baseline sessions and every 4<sup>th</sup> or
 7<sup>th</sup> day post-infusion of ACSF/6-OHDA till the day
 of <em>in vivo</em> or <em>in vitro</em>
 experiment (e.g. 21<sup>st</sup> or
 &gt;68<sup>th</sup>post-operative day). The open field was
 cleaned before and after each mouse with 0,1% acetic acid in distilled
 water. Using a video tracking system (Viewer II/III, Biobserve)
 spontaneous behavior was recorded and analyzed both online and offline.
 Data was extracted from Viewer as Excel-tables and the final analysis was
 made with custom made Matlab-scripts. Cylinder test
 Forelimb use during explorative activity was explored with
 cylinder test. The test was performed at corresponding termination time
 point (20-21<sup>st</sup> and 64<sup>th</sup>
 post-infusion day). Mice were placed individually in a glass beaker (9 cm
 diameter, 19 cm height) at room light and were video recorded with a
 camera (Logitech HD Webcam C615) for about 5 min. No habituation was
 allowed before video recording. The glass cylinder was cleaned before and
 after every mouse with 0,1% acetic acid in distilled water. Only
 weight-bearing wall contacts made by each and both forelimb on the
 cylinder wall were scored. Wall exploration was expressed in terms of the
 percentage of contralateral to the infusion side (in the 6-OHDA-infused
 mice also impaired forepaw) to all forelimb wall contacts.
 <em>In
 vivo</em> electrophysiology Extracellular
 recording <em>In vivo</em> extracellular
 single-unit activities of SN and VTA neurons were recorded in ACSF-infused
 (vehicle) and 6-OHDA-infused mice, similar procedures were used in other
 studies from our lab (Farassat et al. 2019; Schiemann et al. 2012;
 Subramaniam, Althof, et al. 2014). Briefly, mice were anesthetized
 (isoflurane; induction 4.5-5%, maintenance 1–2% in 0.4 l/min O2) and
 placed into a stereotactic frame. The craniotomies were performed as
 described above to target the lateral SN (AP: -3.08 mm, ML: 1.4 mm) and
 medial SN (AP: -3.08 mm, ML: 0.9 mm). Borosilicate glass electrodes (10–25
 MΩ; Harvard Apparatus, Holliston, MA, USA) were made using a horizontal
 puller (DMZ-Universal Puller, Zeitz, Germany) and filled with 0.5 M NaCl,
 10 mM HEPES (pH 7.4) and 1.5% neurobiotin (Vector Laboratories,
 Burlingame, CA, USA). A micromanipulator (SM-6; Luigs & Neumann,
 Ratingen, Germany) was used to lower the electrodes to the recording site.
 The single-unit activity of each neuron was recorded for at least 10
 minutes at a sampling rate of 12.5 kHz (for firing pattern analyses), and
 then for another one minute at a sampling rate of 20 kHz (for the fine
 analysis of AP waveforms). Signals were amplified 1000x (ELC-03M; NPI
 electronics, Tamm, Germany), notch- and bandpass-filtered 0.3–5000 Hz
 (single-pole, 6 dB/octave, DPA-2FS, NPI electronics) and recorded on a
 computer with an EPC-10 A/D converter (Heka, Lambrecht, Germany).
 Simultaneously, the signals were displayed on an analog oscilloscope and
 an audio monitor (HAMEG Instruments CombiScope HM1508; AUDIS-03/12M NPI
 electronics). Midbrain DA neurons were initially identified by their broad
 biphasic AP (&gt; 1.2 ms duration) and slow frequency (1–8 Hz)
 (Anthony A. Grace and Bunney 1984; Ungless and Grace 2012). AP duration
 was determined as the interval between the start of initial upward
 component and the minimum of following downward component.
 Juxtacellular labeling of single neurons In order to identify the anatomical location and neurochemical identity of the recorded neurons, they were labeled post-recording with neurobiotin using the juxtacellular <em>in vivo</em> labeling technique (Pinault 1996). Microiontophoretic currents were applied (1–10 nA positive current, 200ms on/off pulse, ELC-03M, NPI Electronics) via the recording electrode in parallel to the monitoring of single-unit activity. Labeling was considered successful, when: the firing pattern of the neuron was modulated during current injection (i.e., increased activity during on-pulse and absence of activity in the off-pulse), the process was stable for at least 20s, and was followed by the recovery of spontaneous activity. This procedure allowed for the exact mapping of the recorded DA neuron within the SN and VTA subnuclei (Franklin and Paxinos 2012) using custom written scripts in Matlab (MathWorks, Natick, MA, USA), combined with neurochemical identification using TH-immunostaining. <em>In vitro</em> electrophysiology Slice preparation Animals were anesthetized by intraperitoneal injection of ketamine (250 mg/kg, Ketaset, Zoetis) and medetomidine-hydrochloride (2.5 mg/kg, Domitor, OrionPharma) prior to intracardial perfusion using ice-cold ACSF consisting of the following (in mM): 50 sucrose, 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 glucose, 6 MgCl2, 0.1 CaCl2 and 2.96 kynurenic acid (Sigma-Aldrich), oxygenated with 95% O2 and 5% CO2. Rostral coronal midbrain slices (bregma: -2.92 mm to -3.16 mm) were sectioned at 250 µm using a vibrating blade microtome (VT1200s, Leica). Slices were incubated for 1 h before recordings in a 37°C bath with oxygenated extracellular solution with extra 1µM AmmTx3, containing the following (in mM): 22.5 sucrose, 125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 glucose, 1.2 MgCl2 and 1.2 CaCl2. In vitro patch-clamp recordings Slices were placed in a heated recording chamber (37°C) that was perfused with oxygenated extracellular solution at 2-4 ml min<sup>−1</sup>. CNQX (20 μM; Biotrend), gabazine (SR95531, 4 μM; Biotrend) DL-AP5 (10 μM; Cayman Chemical) were added to inhibit excitatory and inhibitory synaptic transmission. For voltage clamp recordings, TTX (500nM; Tocris) was added to the extracellular solution. Neurons were visualized using infrared differential interference contrast videomicroscopy with a digital camera (VX55, Till Photonics) connected to an upright microscope (Axioskop 2, FSplus, Zeiss). Patch pipettes were pulled from borosilicate glass (GC150TF-10; Harvard Apparatus LTD) using a temperature-controlled, horizontal pipette puller (DMZ-Universal Puller, Zeitz). Patch pipettes (4-6 MΩ) were filled with a solution containing the following (in mM): 135 KGlu,  5 KCl, 10 HEPES, 0.1 EGTA, 5 MgCl2, 0.075 CaCl2, 5 NaATP, 1 LiGTP, 0.1% neurobiotin, adjusted to a pH of 7.35 with KOH. Recordings were performed using an EPC-10 patch-clamp amplifier (Heka electronics) with a sampling rate of 20 kHz and a low-pass filter (Bessel, 5 kHz). For voltage clamp recordings only experiments with uncompensated series resistance &lt;10MO were included in this study and series resistance was electronically compensated 75%. Neurons were held at a holding potential of -40mV to minimize HCN activation. To determine Kv4.3 activation kinetics neurons were hyperpolarized to -80mV for 500ms followed by varying voltage steps from -60mV to -20mV in increments of 5mV for 1s. For inactivation kinetics neurons were hyperpolarized from -120mV to -20mV in increments of 10 mV for 1s followed by a fixed voltage step to -20mV for 1s. For analysis of HCN currents neurons were hyperpolarized from -80mV to -120mV in increments of 20mV for 1s. For analysis, action potential thresholds (mV) were determined at dV<sub>m</sub>/dt &gt; 10 mV/ms. Immunohistochemistry Following <em>in vivo</em> recordings, animals were transcardially perfused, as described previously (Farassat et al. 2019; Schiemann et al. 2012; Subramaniam, Althof, et al. 2014). Fixed brains were sectioned into 60µm (midbrain) or 100µm (forebrain) coronal sections using a vibrating microtome (VT1000S, Leica). <em>In vitro</em> slices were fixed in paraformaldehyde after finishing the experiment. Sections were rinsed in PBS and then incubated (in blocking solution (0.2 M PBS with 10% horse serum, 0.5% Triton X-100, 0.2% BSA). For staining with the polyclonal rabbit alpha-synuclein phospho S129 antibody, an antigen retrieval protocol was applied prior to blocking: sections were incubated for 30 minutes at 80 °C in sodium citrate buffer. Afterwards, the standard histological protocol continued with blocking. Sections were incubated in carrier solution (room temperature, overnight) with the following primary antibodies: polyclonal guinea pig anti-tyrosine hydroxylase (TH; 1:1000; Synaptic Systems), monoclonal mouse anti-TH (1:1000; Synaptic Systems) or polyclonal rabbit anti-TH (1:1000; Synaptic Systems); mouse anti-Kv4.3 (1:1000, Alomone Labs); polyclonal rabbit alpha-synuclein phospho S129 (1:200, Abcam), monoclonal rabbit anti alpha-synuclein aggregate [MJFR-14-6-4-2] (1:1000, Abcam). In sequence, sections were again washed in PBS and incubated (room temperature, overnight) with the following secondary antibodies: goat anti-guinea pig 488, goat anti-rabbit 488, goat anti-mouse 488, goat anti-mouse 568 , goat anti-rabbit 568 (all 1:750; Thermofisher). Streptavidin AlexaFluor-568 and Streptavidin AlexaFluor-488 (both 1:1000; Invitrogen) were used for identifying neurobiotin-filled cells. Sections were then rinsed in PBS and mounted on slides with fluorescent mounting medium (Vectashield, Vector Laboratories). DAB immunocytochemistry For DAB (3,3ʼ-diaminobenzidine) staining procedures, a Vectastain ABC Staining Kit (Vector Laboratories) was used. Coronal sections of midbrain (30 μm) areas were cut and rinsed in PBS (3x10 min). Similar to previous immunolabeling procedures, unspecific antigen binding sites were blocked by incubation of the sections with blocking solution (60 min, room temperature). Subsequently, sections were incubated with primary antibody against TH (rabbit anti TH) overnight, rinsed in PBS (3x10 min), and were incubated with biotinylated secondary antibodies (biotinylated anti-rabbit) for two hours at RT. In parallel, an avidin-biotin complex (ABC) was formed by pre-incubation of avidin (1:1000) with biotinylated HRP (1:1000) in PBS for two hours at room temperature. Sections were rinsed in PBS (3x10 min) prior to incubation with ABC solution (60 min, room temperature). Next, sections were rinsed in PBS (2x10 min) and Tris-buffer (1x10 min). Finally, DAB oxidation was developed by application of 2 % H<sub>2</sub>O<sub>2</sub>, 2 % NiCl<sub>2</sub> and 4 % DAB in Tris-buffer using a DAB Substrate Kit (Vector Laboratories, Burlingame, USA). NiCl<sub>2</sub> enhances sensitivity and intensity of DAB precipitation product. DAB oxidation was developed for 2 to 5 minutes and was stopped with Tris-buffer once a specific high-contrast signal was detectable. Sections were rinsed in Tris-buffer (3x10 min) and transferred onto gelatin-covered slides, air-dried overnight, and dehydrated in consecutive ascending alcohol concentrations (50 %, 70 %, 90 % and 2x 100 %; 10 min each) followed by dehydration in xylol (2x 100 %; 10 min each). Finally, sections were mounted under glass coverslips with hardening mounting medium (Vectamount, Vector Laboratories, Burlingame, USA). Unbiased stereology measurements For quantification of total cell loss, TH-DAB labeled SN DA neurons were counted using unbiased stereology based on optical dissection (Gundersen 1986). In coronal sections (30 μm), the region of interest was selected based on anatomical landmarks including the medial lemniscus, which separates SN and adjacent VTA. Stereological counting provides unbiased data based on random, systematic sampling using an optical fractionator. This method involves counting of neurons with an optical dissector, a three-dimensional probe placed through a reference space (Gundersen 1986). The optical dissector forms a focal plane with a thin depth of field through the selected sections. Objects in focus of this focal plane are located within the reference section and are counted, while objects outside of the focal plane are not counted. On top of the optical dissector, a counting frame is applied. Counting frames ensure that all neurons have equal probabilities of being selected, regardless of shape, size, orientation, and distribution. To avoid counting capped neurons at the border of a section, an additional guard zone was deployed at the upper and lower borders of each section. DA neurons within the counting frame as well as those crossing the green line (acceptance line) were counted, while DA neurons crossing the red line (rejection line) excluded. Moreover, only neurons with a detectable nucleus in focus within the optical dissector were counted. For quantification of total cell loss, StereoInvestigator software (V5, MicroBrightField, Colchester, USA) was used in combination with BX61 microscope (Olympus, Hamburg, Germany). The region of interest was selected and marked using a low magnification objective lens (2x, NA 0.25, Olympus) and 12-30 serial sections of 30 μm thickness were counted, covering the entire caudo-rostral extent of the SN. To count the number of DA neurons in the area of the SN pars compacta, a high magnification oil-immersion objective lens (100x, NA 1.30, Olympus) (counting frame, 50 × 50 μm; sampling grid, 125 × 100 μm) was used. After counting was finished, the total number of neurons was calculated by the software using the formula described by West et al (West 1993). Optical density measurements Optical density measurements of TH-DAB labeled striatal sections were performed using ImageJ software (http://rsbweb.nih.gov/ij/). Following TH-DAB labeling and TH-immunohistochemistry, images of five coronal sections (100 μm) covering the rostrocaudal axis of the striatum were captured using laser-scanning microscope (Nikon Eclipse90i, Nikon GmbH). Images were gray scale converted and mean gray values of desired striatal areas were encircled and measured. Unspecific mean gray values were measured in a defined cortical area (100x100 pixels) that displayed no specific TH signal due to the absence of DA innervation and were subtracted. The ventral edge of lateral ventricles served as an anatomical landmark to separate dorsal and ventral areas. For all animals, the measurement from the ipsilateral to the infusion side were divided by the contralateral side to calculate the relative optical density of the striatum. Immunohistochemical Kv4.3 channel signal quantification A Nikon Eclipse 90i microscope was used for fluorescent signal detection, excitation wavelength of 488 nm for TH-signal and 568 nm for Kv4.3-channel signal. From each animal, 4 midbrain slices covering the caudal, intermedial and rostral regions, were selected and imaged for overview with 4x magnification. Then 60x magnification was used to acquire data from 4 areas within each SN (4 images on the ipsilateral and 4 images on the contralateral to infusion side). All images were acquired using the same laser and camera settings. Images were exported from Nikon NIS-Elements Advanced Research (Version 4.20.03) software as 8-bit TIFF files for quantification. Data was analyzed using custom made Python 3.0 scripts with matplotlib, numpy, scimage and scipy modules. First, TH immunosignals were converted to a binary image via Otsu-thresholding algorithm to detect TH areas bigger than 400 pixels. Then, the resulting binary image was used as a mask for Kv4.3 channel immunosignal detection. For all ROIs surface areas and mean Kv.4.3 channel signal intensity was measured. By applying erosion and dilatation algorithms on the ROIs, membrane and cytoplasm areas were segregated, allowing isolation of Kv4.3 channel immunosignal intensity for these cell compartments. Background Kv4.3 channel immunosignal was quantified in TH-immunosignal areas below the Otsu-threshold. All data were then grouped according to medio-lateral and ipsi-/contralateral position for both vehicle and 6-OHDA group. Graphs and statistical analysis for this data was performed using Python custom made scripts. Statistical Analysis Spike train analyses Spike time-stamps were extracted by thresholding above noise levels with IgorPro 6.02 (WaveMetrics, Lake Oswego, OR, USA). Firing pattern properties such as mean frequency, coefficient of variation (CV) and bursting measures were analyzed using custom scripts in Matlab. In order to estimate burstiness and intra-burst properties, we used the burst detection methods described in Grace & Bunney (A. A. Grace and Bunney 1984; Anthony A. Grace and Bunney 1984; Ungless and Grace 2012). All non-burst related ISIs (excluding all ISIs that followed the Grace and Bunney criteria, as well as all pre- and post-burst ISIs) were used to calculate the single spike firing frequency and single spike coefficient of variation. For analysis of general firing patterns, autocorrelation histograms (ACH) were plotted using custom Matlab scripts. We used established criteria for classification of <em>in vivo</em> firing patterns based on visual inspection of autocorrelograms (Farassat et al. 2019; Schiemann et al. 2012; Subramaniam, Althof, et al. 2014): single spike-oscillatory (≥3 equidistant peaks with decreasing amplitudes), single spike-irregular (&lt;3 peaks, increasing from zero approximating a steady state), bursty-irregular (narrow peak with steep increase at short ISIs) and bursty-oscillatory (narrow peak reflecting fast intraburst ISIs followed by clear trough and repetitive broader peaks). Statistics Categorical data is represented as stacked bar graphs. To investigate the assumption of normal distribution, we performed the single-sample Kolmogorov-Smirnov test. The Mann-Whitney-Test, one-/two-way ANOVA were performed in non-parametric data to determine statistical significance. Categorical parameters, such as ACH-based firing pattern, were analyzed with the Chi-squared test. Statistical significance level was set to <em>p</em> &lt; 0.05. All data values are presented as means ± SEM. Statistical tests were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA), Matlab and Python. The scatter plots are represented with median or mean ± SEM. The resulting <em>p</em>values were compared with Bonferroni-corrected α-level or Tukey <em>post hoc</em> comparison. A value of <em>p</em> ≤ 0.05 was considered to be statistically significant; <em>p</em> ≤ 0.05 = *<em>p</em> ≤ 0.005 = **<em>p</em> ≤ 0.0005 = ***. Graphs were plotted using GraphPad Prism software (9.0c), Matlab and Python.
description: # Recovery of the full in vivo firing range in post-lesion surviving DA SN
 neurons associated with Kv4.3-mediated pacemaker plasticity ## GENERAL
 INFORMATION Date of data collection: 2021–2024 ## OVERVIEW This dataset
 provides raw and processed electrophysiological recordings and
 immunohistochemistry data supporting the results of the study
 investigating intrinsic plasticity of surviving substantia nigra (SN)
 dopamine neurons following partial 6-OHDA lesion. The focus lies on the
 temporal evolution of firing adaptations and Kv4.3-mediated homeostatic
 plasticity. ## FILE STRUCTURE AND CONTENT ``` /physiology_data.zip/ │ ├──
 /in_vitro/ │ ├── /6OHDA_early/ │ │ ├── CChyp.zip │ │ ├── contCC.zip │ │
 └── onCell.zip │ ├── /6OHDA_late/ │ │ ├── CChyp.zip │ │ ├── contCC.zip │ │
 └── onCell.zip │ ├── /6OHDA_late_AmmTx3/ │ │ ├── CChyp.zip │ │ ├──
 contCC.zip │ │ └── onCell.zip │ ├── /6OHDA_late_voltageClamp/ │ │ ├──
 Activation.zip │ │ ├── HCN.zip │ │ └── Inactivation.zip │ ├──
 /vehicle_early/ │ │ ├── CChyp.zip │ │ ├── contCC.zip │ │ └── onCell.zip │
 ├── /vehicle_late/ │ │ ├── CChyp.zip │ │ ├── contCC.zip │ │ └── onCell.zip
 │ ├── /vehicle_late_AmmTx3/ │ │ ├── CChyp.zip │ │ ├── contCC.zip │ │ └──
 onCell.zip │ └── /vehicle_late_voltageClamp/ │ ├── Activation.zip │ ├──
 HCN.zip │ └── Inactivation.zip │ └── /in_vivo/ ├──
 in_vivo_6OHDA_earlyphase.txt ├── in_vivo_6OHDA_latephase.txt ├──
 in_vivo_Vehicle_earlyphase.txt └── in_vivo_Vehicle_latephase.txt ``` ##
 FILE DESCRIPTION ### In Vivo Electrophysiology, in_vivo *
 **in_vivo_6OHDA_earlyphase.txt, in_vivo_6OHDA_latephase.txt,
 in_vivo_Vehicle_earlyphase.txt, in_vivo_Vehicle_latephase.txt** –
 Inter-spike intervals from extracellular recordings in vivo. ### In Vitro
 Electrophysiology, in_vitro * Each subfolder contains zip files with the
 in vitro recording (.zip of .mat files) for the corresponding experiment
 (e.g., CC clamp for the early and late phase, CC clamp under AmmTx3 toxin,
 as well as voltage clamp for either the 6-OHDA or vehicle group). ##
 METHODOLOGICAL SUMMARY * **Model**: Unilateral partial intrastriatal
 injection of 6-hydroxydopamine (6-OHDA; 2 µg/µL, 6 µL). * **Timepoints**:
 * *Early Phase*: 3 weeks post-lesion * *Late Phase*: ≥60 days post-lesion
 * **Electrophysiology**: Whole-cell and on-cell patch-clamp recordings in
 acute midbrain slices, and juxtacellular in vivo recordings under
 isoflurane. * **Voltage Clamp**: Protocols for HCN and Kv4.3 current
 activation/inactivation (see scripts). * **Imaging**: Immunofluorescence
 quantification of Kv4.3 expression in SN DA neurons. ## SOFTWARE AND
 DEPENDENCIES * MATLAB R2022a+ ## FILE FORMATS * `.mat`: MATLAB data files
 * `.txt`: ASCII table format (comma/tab delimited) ## DATA TABLE
 DESCRIPTIONS ### in vitro files (applicable for all .mat-files within all
 zip-files) * `Trace_`: table * first column: vector of recording time (in
 seconds) * second column: membrane potential trace (in V) ### in vivo
 files (applicable for all .txt-files in the folder in vivo) * 1-column
 ASCII table * Each value represents the inter-spike interval (in seconds)
 * Unit: s