Experimental procedures were conducted in accordance with the German Animal Welfare Act (last revised in 2014) and European regulations (2010/63/EU). Experiments were approved by the local animal welfare authority (LaGeSo, Berlin), and conformed to local department and international guidelines.
NOTE: In the presented method two models of electrodes are used to record from the hyperdirect cortico-basal ganglia pathway that connects the primary motor cortex (M1) with the subthalamic nucleus (STN) and the substantia nigra pars reticulate (SNr). For epidural electrocorticogram (ECoG) recordings from the M1 custom-made low-impedance Ag/AgCl electrodes are used. The recordings from the STN and SNr are performed with commercially available high-impedance tungsten electrodes.
1. Construction of Epidural Ag/AgCl Epidural Electrodes
2. Affixing the Electrodes to a Stereotaxic Holder
NOTE: To record MUA and LFPs at the same time, use tungsten microwire electrodes with an impedance of 1.5 MΩ. If the focus of the recordings is on high quality recordings of single units, choose microwire electrodes with a higher impedance (>5 MΩ). If the aim of the study is solely directed at LFPs, electrodes with lower impedances can be acceptable. For small structures, for which dorsoventral stereotaxic adjustments are often necessary, use pairs of electrodes with a suitable dorsoventral tip separation (in this case 250 µm). Furthermore, this provides the advantage of a more local reference electrode, if needed. The stereotaxic coordinates are always measured from the lowermost electrode and are calculated in reference to the bregma.
3. Surgery
4. Electrophysiological Mapping and Recordings
NOTE: For this step, a Faraday cage and a multi-channel electrophysiological recording system with recording software capable of online-filtering and online spike-sorting is necessary. Preferably use a system that works with a preamplifier positioned near the head of the animals to keep electrical noise and artifacts to an absolute minimum. Besides the tungsten microwire electrodes, at least one epidural and one reference electrode are necessary to perform recordings of the hyperdirect pathway. It is recommended to insert epidural and reference electrodes pairwise without them touching each other, this helps in case of malfunctioning and allows for different types of referencing in data analysis.
5. End of Experiment
With the herein used recording electrodes, it is possible to sample LFPs from the primary motor cortex, the subthalamic nucleus and the substantia nigra pars reticulata and MUA from the STN and SNr. Initially, LFPs and multi-unit activity are recorded together in a broad-band signal. Thereafter, LFPs and MUAs are separated by bandpass filters (0.05-250 Hz for LFPs and 300-4,000 Hz for MUA).
For the correct targeting of subcortical nuclei, especially of small structures such as the STN, it is advantageous to align the planned stereotaxic coordinates with online-recorded MUA signal. For the electrode trajectory targeting the STN characteristic MUA pattern can be recorded (Figure 2)9,20.
For later steps of the analysis, it is often mandatory to define single units from multi-unit activity by principle component analysis (Figure 4).
In the LFP recordings from the M1 two spontaneously alternating cortical synchronization states can be identified: the Activated State (AS) and the Slow Wave Activity (SWA) state (Figure 3)18,19. While the SWA state is dominated by high-amplitude slow oscillations of around 1 Hz, the AS is characterized by faster oscillations with a lower amplitude (Figure 3).
Figure 1: Set up of the Deep Brain Microwire Electrodes in a Standard Stereotaxic Holder. Note the tip separation between A, the electrode pair for the STN, and B, the electrode pair for the SNr in dorsoventral direction of approx. 200 µm and anterioposterior direction of approx. 2 mm. Please click here to view a larger version of this figure.
Figure 2: Characteristic Multi-unit Activity from a Dorsoventral Electrode Trajectory Targeting the STN. (A) Multi-unit recordings of the ventral posteromedial thalamic nucleus (VPM), the zona incerta (ZI), the subthalamic nucleus (STN) and the substantia nigra pars reticularis (SNr). The VPM exhibits sparse and irregular spaced high amplitude spikes. This pattern of spikes ceases when approaching the ZI. When the electrode enters the STN a typical high-frequency firing pattern with short bursts with medium amplitude can be observed. The SNr can be identified by its high amplitude and regular firing pattern. (B) STN-trajectories superimposed onto images from a rat stereotactic atlas21. Upper part: coronal plane. Lower part: sagittal plane. Note the passing of the electrode tip through VPM and ZI. Please click here to view a larger version of this figure.
Figure 3. Cortical Synchronisation States in LFP Recordings from the Primary Motor Cortex during Urethane Anesthesia. (A) Representative 600 s LFP recording of the primary motor cortex. Time periods with high frequency, low amplitude activity corresponding to the Activated State (i) and time periods with a slower rhythm and higher amplitude corresponding to the Slow Wave Activity state (ii) can be differentiated. (B) Corresponding time-frequency plot over an interval of 600 s illustrating the 0-20 Hz relative power of the LFPs presented in (A). Warmer colorings indicate higher relative power. Please click here to view a larger version of this figure.
Figure 4: Sorting of Single Units from STN Multi-unit Activity. (A) Three-dimensional view of unit clusters in feature space after principal component analysis. Each cluster represents a putative single unit. (B) Spike waveforms and spike waveform averages corresponding to the clusters in (A). Please click here to view a larger version of this figure.
Coordinates from Bregma | STN | SNr | M1 | reference 1 | reference 2 |
anterior-posterior | -3.6 | -4.8 | +3.0 | -10.0 | -10.0 |
medial-lateral | +2.5 | +2.5 | +3.0 | +3.0 | -3.0 |
dorsal-ventral | -8.0 | n. a. | n. a. | n. a. | n. a. |
Table 1: Stereotaxic Coordinates for the Recording of the Hyperdirect Cortico-basal Ganglia Pathway. All points are measured from the bregma reference point on the skull in mm; n.a.- not applicable.
Ag/AgCl custom epidural electrodes | Goodfellow GmbH D-61213 Bad Nauheim, Germany info@goodfellow.com |
Product-ID AG005127 for 99.99% silver wire | Ag/AgCl electrodes will allow for better signal quality, but may only be used in acute experiments. Possible replacement: Stainless steel electrodes |
Stereotaxic holder with acrylic block | David Kopf Instruments, 7324 Elmo Street, Tujunga, CA 91042, USA |
Product ID Model 1770 Standard Electrode Holder | Make sure the acrylic block has recesses which suit the electrode setup for the desired target. Acrylic blocks can easily be modified with a file to obtain the desired configuration. Possible replacement: Self-constructed electrode holders |
Tungsten microwire electrodes 1.5 MΩ impedance | Microprobes.com 18247-D Flower Hill Way Gaithersburg, Maryland, 20879 USA |
Product-ID WE3ST31.5A5-250um | The 1.5 MΩ is necessary to record MUA and LFP at the same time. Possible replacement: Microelectrodes of different materials can be used. The electrodes have to be straight, robust and as thin as possible. |
Rat alignment tool | David Kopf Instruments, 7324 Elmo Street, Tujunga, CA 91042, USA |
Product ID Model 944 Rat Alignment Tool | Allows the exact orientation of the brain to match stereotaxic atlases. Possible replacement: Stereotaxic holder with a cannula |
Two-component dental acrylic | Associated Dental Products Ltd. Kemdent Works, Purton, Swindon Wiltshire, SN5 4HT, United Kingdom |
Simplex Rapid Powder Clear 225g, Product code: ACR803; Simplex Rapid Liquid 150ml, Product code: ACR920 | Depending in the electrodes used, superglue might be an easy alternative, if the electrodes are small and lightweight. Possible replacement: Superglue (Cyanacrylate-based) |
Faraday cage | Self-construction | A proper Faraday cage will be the best protection from electromagnetic artifacts, but everything which can be formed into a box shape or applied to a frame and is made of conductive material may help. Possible replacement: Aluminum foil or copper mesh | |
Electrophysiological setup with recording software and online spike-sorting capabilities | OmniPlex® Neural Data Acquisition System Plexon Inc 6500 Greenville Avenue, Suite 700 Dallas, Texas 75206 USA |
Offline sorting software is a potential alternative, multiple scripts and softwares can be found for free in the open source community. |
Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.
Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.
Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.