EMG-activity of the hindlimbs muscles of decerebrate DAT-KO rats

The position of decerebration plane influences various parameters of animals’ hindlimb muscle activity. Rats with lack of dopamine transporter (DAT-KO) possess the decreased functional connectivity of the midbrain dopaminergic regions. We compared the tonic activity and responses to L2 and L6 spinal segment electrical epidural stimulation (ES) of mm. tibialis anterior and gastrocnemius medialis of decerebrated wild-type (WT) and DAT-KO rats. Each group was subdivided into two subgroups based on the damage of the substantia nigra (SN), determined by post mortem histological analysis. It was shown that both subgroups WT and DAT-KO rats with damaged SN exhibit elevated tonic and ES-induced muscle activity compared to subgroups with intact SN. The current strength of ES needed to induce maximal middle response was lower for DAT-KO rats. We conclude that in DAT-KO rats the descending dopaminergic projections from SN influence spinal networks despite the known reduced functional connectivity of dopaminergic midbrain regions. We hypothesized that faster dopamine deactivation after decerebration in DAT-KO rats with damaged SN additionally facilitates their ES-evoked responses. EMG-activity of the hindlimbs muscles of decerebrate DAT-KO rats Lyakhovetskii V. 1, Shkorbatova P. 1, Kalinina D. 2,3, Gorskii O. 1,2,4, Bazhenova E. 1,2, Belskaia A. 2, Merkulyeva N. 1 ✝ , Musienko P. 2,3,5 ✝ 1 Pavlov Institute of Physiology, Russian Academy of Sciences, 199034 St. Petersburg, Russia 2 Institute of Translational Biomedicine, State University Hospital, St. Petersburg State University, 199034 St. Petersburg, Russia 3 Department of Neurobiology, Sirius University of Science and Technology, Federal Territory of Sirius, Russia 4 Life Improvement by Future Technologies Center “LIFT”, Mosсow, Russia 5 Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, Moscow, Russia, [email protected] Number of figures: 2 Number of words: 2932 Short title: EMG of decerebrate DAT-KO rats List of abbreviations: DAT – dopamine transporter DAT-KO – DAT-knockout EMG – electromyographic ES – epidural stimulation GM – m. gastrocnemius medialis MR – middle response L1 – 1 st lumbar spinal segment L2 – 2 nd lumbar spinal segment L6 – 6 th lumbar spinal segment SN – substantia nigra TA – m. tibialis anterior VT13 – 13 th thoracic vertebra VL2 – 2 nd lumbar vertebra WT – wild-type The position of decerebration plane influences various parameters of animals’ hindlimb muscle activity. Rats with lack of dopamine transporter (DAT-KO) possess the decreased functional connectivity of the midbrain dopaminergic regions. We compared the tonic activity and responses to L2 and L6 spinal segment electrical epidural stimulation (ES) of mm. tibialis anterior and gastrocnemius medialis of decerebrated wild-type (WT) and DAT-KO rats. Each group was subdivided into two subgroups based on the damage of the substantia nigra (SN), determined by post mortem histological analysis. It was shown that both subgroups WT and DAT-KO rats with damaged SN exhibit elevated tonic and ES-induced muscle activity compared to subgroups with intact SN. The current strength of ES needed to induce maximal middle response was lower for DAT-KO rats. We conclude that in DAT-KO rats the descending dopaminergic projections from SN influence spinal networks despite the known reduced functional connectivity of dopaminergic midbrain regions. We hypothesized that faster dopamine deactivation after decerebration in DAT-KO rats with damaged SN additionally facilitates their ES-evoked responses.

DAT-KO rats; decerebration; tonic activity; epidural stimulation; substantia nigra.

The decerebrate animal models are widely used in experimental neurophysiology. While the decerebrate cats are mainly considered as a model for studying locomotion (Grillner, Shik, 1973; Whelan, 1996) the decerebrate rats are used not only for this task (Skinner, GarciaRill, 1984) but also for investigations of spinal sensorimotor network (Shkorbatova et al., 2020), regulation of respiration (Hayashi, 2003), cardiovascular (Hayashi, 2003; Ishii et al., 2020) and urinary system (Yoshiyama et al., 2013). It has been shown that in cats the rostrocaudal position of the decerebration plane changes the parameters of locomotion (e.g., (Grillner, Shik, 1973)). In rats, this position influences spontaneous locomotor activity (Hayashi, 2003; Ishii et al., 2020), the cardiovascular and respiratory characteristics (Hayashi, 2003; Ishii et al., 2020). Previously, in pilot experiments in rats, we had shown that the tonic and induced by electrical epidural stimulation (ES) hindlimb muscle activity depends also on rostrocaudal position of decerebration plane (Shkorbatova et al., 2023): it is increased when substantia nigra (SN) is damaged. One of the advantages of the rodent models is the possibility of its genetic modification. Here, we focused on the rats lacking a dopamine transporter (DAT) due to elimination of the DAT gene. DAT-knockout (DAT-KO) rats show the significant decrease of functional connectivity of midbrain dopaminergic regions (Reinwald et al., 2022). Thus, we hypothesized that the damages of midbrain structures including SN due to different rostrocaudal levels of decerebration may differently affect the DAT-KO rat muscle activity relative to wild type (WT) rats. The aim of this study is the comparison of tonic and ES-induced hindlimb muscle activity of decerebrate WT and DAT-KO rats subgrouped by SN damage.

2.1. Animals. All procedures were performed under the guidelines established by the European Community Council (Directive 2010/63/EU of 22 September 2010), and animal protocols were approved by the Ethics Committee of St. Petersburg State University, St. Petersburg, Russia (approval number 131-03-6). Dopamine transporter knockout (DAT-KO, 4 males, 4 females, n = 8) and wild-type (WT, 8 males, 3 females, n = 11) rats 6–18 months old were used in the experiments.DAT-KO rats were generated by SAGE Laboratories using zinc-finger nucleases technology for the elimination of the DAT gene (Geurts et al., 2009). Breeding and genotyping of DAT-KO and WT rats were performed as described previously (Shkorbatova et al., 2023; Kalinina et al., 2023). Rats were kept numbering 3 animals in a cage, in standard laboratory conditions (12 h light/dark cycle, 21 0 C and 50–70% humidity), food and water were provided ad libitum . 2.2. Surgical procedures. Surgical procedures and histological control were described in detail in (Shkorbatova et al., 2023). Briefly, animals were anesthetized with a mixture of isoflurane on oxygen. The bipolar myographic electrodes consisting of a pair of Teflon insulated stainless steel wires (AS632, Cooner Wire, Chats worth, CA, USA) 0.15 mm in diameter with 0.5 mm long uninsulated sections were implanted bilaterally in hindlimb muscles, mm. tibialis anterior (TA) and gastrocnemius medialis (GM). The carotid arteries were ligated, then the rat’s head was fixed in stereotaxis and the part of brain rostral to the superior colliculi was cut off by a blade fixed in stereotaxic holder at an angle of 65–90° to achieve different rostrocaudal level of decerebration. Laminectomy of vertebrae VT13–VL2 was performed to provide access to the L1–L6 spinal cord segments. After all surgical procedures were finished the anesthesia was discontinued. 2.3. Electrophysiological study. Tonic activity was recorded during 5-10 s before the beginning of ES when the rat was motionless. ES of the dorsal surface of the spinal cord of the rat placed in the custom stereotaxic frame was performed by rectangular biphasic pulses monopolarly (Model 2100, AM Systems, Sequim, WA, USA) using a silver ball electrode with a diameter of 0.5 mm. An indifferent electrode made of a 21G needle was placed under the abdominal skin. The muscle potentials evoked by ES (frequency 1 Hz, pulse duration 0.2–0.3 ms, current strength 10–370 μA, step 10 μA) of the lumbar spinal segments were recorded (10 responses for each current strength). Based on post mortem anatomical control of the segmental position of the stimulation sites the muscle responses obtained during ES stimulation over the L2 and L6 spinal segments were selected for further analysis. Signals from EMG electrodes were amplified (Model 1700, AM Systems, Sequim, WA, USA) and digitized at a sampling rate of 20 kHz (E-502, LCard, Moscow, Russia). To analyze the tonic activity EMG responses were rectified and averaged. To analyze the evoked potentials, we used the peak-to-peak amplitude of the TA and GM middle response (MR) (Lavrov et al., 2008) component, which was distinguished on the basis of its latency (6-12 ms). For each muscle we determined the current strength, I max, at which the maximal MR amplitude was obtained. To compare TA and GM activity of different animals we took their MR amplitude at current strength 70 μA or at I max if it was less than 70 μA. At these currents, MR was pronounced in most animals. EMG processing was performed in the MATLAB R2016b application software package for technical calculation problems (Mathworks, Natick, MA, USA) using custom scripts. 2.4. Histological analysis . After the end of the experiment, the animals were deeply anesthetized with a mixture of isoflurane (5%) on oxygen, then transcardial perfusion fixation with 4% paraformaldehyde was performed. The exact position of ES points relative to spinal cord segments and the rostrocaudal level of decerebration were determined. The positions of SN were marked bilaterally on parasagittal brain sections stained by Nissl method, the presence of SN damage was assessed ( Fig. 1A ). 2.5. Statistical analysis. Statistical significance was assessed using unpaired Mann–Whitney test at the p < 0.05 level (the subject was the animal hindlimb) using Prism 9.0 statistical data processing software (GraphPadSoftware, La Jolla, CA, USA). 3. Results Post mortem histological analysis revealed that SN was damaged in 7/11 WT and 4/8 DAT-KO animals ( Fig. 1B, C ). Further analysis was performed in subgroups of animals depending on the presence of SN damage. For both groups, tonic activity of TA and GM was higher in subgroups with damaged SN (significantly for TA of WT and DAT-KO rats as well for GM of WT rats) ( Fig. 2A ). During ES of L2 segment, the MR obtained at current strength lower than 80 μA were also similar between WT and DAT-KO rats: they were higher in subgroups with damaged SN (significantly for TA of DAT-KO rats as well for GM of WT and DAT-KO rats) ( Fig. 2B ). The current strength, I max, at which the maximal MR amplitude was obtained, was lower for DAT-KO relative to WT rats (significantly for TA of subgroups with damaged SN). For GM of WT rats, I max was significantly lower for the subgroup with damaged SN relative to the subgroup with intact SN ( Fig. 2C ). During ES of L6 segment, the MR obtained at current strength lower than 80 μA was significantly higher in the DAT-KO subgroup with damaged SN relative to DAT-KO subgroup with intact SN and to WT subgroup with damaged SN for both muscles studied ( Fig. 2D ). For GM, the current strength, I max, at which the maximal MR amplitude was obtained, was lower for DAT-KO relative to WT rats (significantly for subgroups with damaged SN) ( Fig. 2E ).

Intravenous infusion of apomorphine, a mixed D1/D2 agonist, depresses the monosynaptic spinal reflexes in spinal paralyzed cats (Carp, Anderson, 1982) and rats (Gajendiran et al., 1996). Local iontophoretic application of D1-D5 agonists depresses the dorsal horn group II field potential in anesthetized paralyzed cats (Skoog, Noga, 1995). It is known that oxydopamine lesions of rat’s SN lead to elevated TA and GM tonic activity (Double, Crocker, 1993) and increased movement-induced reflex activity of these muscles (Wolfarth et al., 1996). Thus, it can be assumed that SN damage decreases dopamine concentration not only in the nigrostriatal system but also in descending dopaminergic pathways (Ryczko, Dubuc, 2017) and hence facilitates muscle activity. The observed in WT rats phenomena, that are an increase of tonic muscle activity, amplitude of GM MR obtained during ES of L2, and decrease of GM I max in subgroup with damaged SN relative to intact SN subgroup, all may be explained by the partial destruction of the descending dopaminergic pathways due to such position of the decerebrated plane. The similar increase of tonic muscle activity and more pronounced increase of MR amplitude of both muscles studied obtained during ES not only L2 but also L6 in subgroup of DAT-KO rats with damaged SN allows us to propose that dopaminergic pathways in DAT-KO rats preserve some functionality despite the lack of DAT. We hypothesize that in DAT-KO rats, dopamine, located predominantly in the synaptic cleft due to their lower capacity of vesicular dopamine pool (Leo et al., 2018) and absence of dopamine reuptake, is inactivated faster than in WT rats during the time elapsed from decerebration to the start of the experiment. Similarly to DAT-KO mice possessing decreased density of dopamine receptors in the brain (Efimova et al., 2016) DAT-KO rats, presumably, possess decreased density of dopamine receptors also in the spinal cord. Taken together, faster dopamine deactivation and reduced density of dopamine receptors may cause significant reduction of TA I max obtained during ES of L2 and GM I max obtained during ES of L6 in DAT-KO rats with damaged SN relative to WT with damaged SN. To conclude SN damage facilitates tonic and ES-induced muscle activity of decerebrated DAT-KO rats similarly to WT animals. The degree of this facilitation is slightly higher than in WT animals due to peculiarities of functioning of the descending dopaminergic pathway in the condition of lack of DAT. Fig.1. The position of the decerebrate plane relative to the substantia nigra (SN) for wild-type (WT) and dopamine transporter knockout (DAT-KO) rats. A. Examples of parasagittal brain slices at mediolateral level 2-2,5 with damaged and intact SN. B. Position of the decerebrate plane of all examined rats in the midbrain diagram with the projections of some midbrain nuclei contours (green - WT, orange - DAT-KO). SC — anterior colliculi, IC — interior colliculi, RN — red nucleus, MB — mammillary bodies, Hb — habenula, Pn — cerebellar peduncles, VTA — ventral tegmental area C. Drawings of parasagittal slices of the midbrain of individual rats after decerebration, SN is marked in black. Fig.2. EMG-activity of mm. tibialis anterior (TA) and gastrocnemius medialis (GM) of decerebrated wild-type (WT) and dopamine transporter knockout (DAT-KO) rats. A. Tonic activity. B. Maximal evoked by epidural stimulation (ES) of L2 middle response (MR) at current strength lower 80 μA. C. Current strength of maximal MR evoked by ES of L2. D. Maximal evoked by ES of L6 middle response (MR) at current strength lower 80 μA. E. Current strength of maximal MR evoked by ES of L6. Mean ± SD. * - p < 0.05, ** - p < 0.01, *** - p < 0.001.

We thank the Saint Petersburg State University Vivarium of the Research Park of the Saint Petersburg State University for the breeding of mutant rats. Funding This work is supported by Saint-Petersburg State University research project IDs: 125022102790-5 (O.G., E.B.), 129659216 (breeding of the mutant rats), State funding of the Pavlov Institute of Physiology, Russian Academy of Sciences (№124020100105-6, for data analysis) and Ministry of Science and Higher Education of the Russian Feferation (Agreement 075-10-2025-017, P.M.). Breeding and support of rats was performed by the Resource Center Vivarium of St. Petersburg State University, St. Petersburg, Russia Author’s contribution Idea of the study and planning of the experiment (V.L., P.M., N.M.), data collection (P.S., O.G., E.B., D.K., V.L., N.M., Z.F.), data processing (P.S., V.L.), manuscript writing and editing (P.S., V.L., O.G., N.M., D.K., P.M.), supervision (P.E.M., N.M.), funding acquisition (P.E.M., N.M.). Conflicts of Interest: The authors declare no conflict of interest. Data Availability Statement: The raw data used in this study are available upon request from the corresponding author.

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Authors Metrics & Citations Metrics Article Usage 163views 92downloads Citations Download citation Vsevolod Lyakhovetskii, Polina Shkorbatova, Daria Kalinina, et al. EMG-activity of the hindlimbs muscles of decerebrate DAT-KO rats. Authorea. 07 November 2025. DOI: https://doi.org/10.22541/au.176253510.02491855/v1 DOI: https://doi.org/10.22541/au.176253510.02491855/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.