Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor

doi:10.3390/cells11233889

. 2022 Dec 1;11(23):3889.

doi: 10.3390/cells11233889.

Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor

Joy Zhou^{1

2

3}, Meike E Van der Heijden^{1

2}, Luis E Salazar Leon^{1

2

3}, Tao Lin^{1

2}, Lauren N Miterko^{2

4}, Dominic J Kizek^{1

2}, Ross M Perez^{1

2

4}, Matea Pavešković^{2

3}, Amanda M Brown^{1

2}, Roy V Sillitoe^{1

2

3

4

5}

Affiliations

¹ Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
² Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, 1250 Moursund Street, Suite 1325, Houston, TX 77030, USA.
³ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Program in Development, Disease Models & Therapeutics, Baylor College of Medicine, Houston, TX 77030, USA.
⁵ Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.

PMID: 36497147
PMCID: PMC9740691
DOI: 10.3390/cells11233889

Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor

Joy Zhou et al. Cells. 2022.

. 2022 Dec 1;11(23):3889.

doi: 10.3390/cells11233889.

Authors

Affiliations

¹ Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
² Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, 1250 Moursund Street, Suite 1325, Houston, TX 77030, USA.
³ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Program in Development, Disease Models & Therapeutics, Baylor College of Medicine, Houston, TX 77030, USA.
⁵ Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.

PMID: 36497147
PMCID: PMC9740691
DOI: 10.3390/cells11233889

Abstract

Tremor is the most common movement disorder. Several drugs reduce tremor severity, but no cures are available. Propranolol, a β-adrenergic receptor blocker, is the leading treatment for tremor. However, the in vivo circuit mechanisms by which propranolol decreases tremor remain unclear. Here, we test whether propranolol modulates activity in the cerebellum, a key node in the tremor network. We investigated the effects of propranolol in healthy control mice and Car8^wdl/wdl mice, which exhibit pathophysiological tremor and ataxia due to cerebellar dysfunction. Propranolol reduced physiological tremor in control mice and reduced pathophysiological tremor in Car8^wdl/wdl mice to control levels. Open field and footprinting assays showed that propranolol did not correct ataxia in Car8^wdl/wdl mice. In vivo recordings in awake mice revealed that propranolol modulates the spiking activity of control and Car8^wdl/wdl Purkinje cells. Recordings in cerebellar nuclei neurons, the _targets of Purkinje cells, also revealed altered activity in propranolol-treated control and Car8^wdl/wdl mice. Next, we tested whether propranolol reduces tremor through β₁ and β₂ adrenergic receptors. Propranolol did not change tremor amplitude or cerebellar nuclei activity in β₁ and β₂ null mice or Car8^wdl/wdl mice lacking β₁ and β₂ receptor function. These data show that propranolol can modulate cerebellar circuit activity through β-adrenergic receptors and may contribute to tremor therapeutics.

Keywords: beta-adrenergic receptors; cerebellum; circuitry; electrophysiology; propranolol; tremor.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

**Figure 1**
**Propranolol reduces pathological tremor in *Car8^wdl/wdl* mice and physiological tremor in control mice.** (A) Schematic of the tremor monitor configuration. (B) Representative raw traces of tremor readings recorded from the tremor monitor for control mice at baseline (pink, N = 12), control mice after propranolol treatment (green, N = 12), *Car8^wdl/wdl* mice at baseline (orange, N = 12), and *Car8^wdl/wdl* mice after propranolol treatment (blue, N = 12). Larger vertical deflections indicate stronger tremor power. Scale bar is 50 mV vertical and 500 ms horizontal. (C) Line graph depicting tremor power versus frequency. Color representation for groups is maintained from panel (B). Power indicates the presence and severity level of tremor, with higher power illustrating stronger severity. Frequency indicates the speed of tremor movements. Following propranolol treatment, both control and *Car8^wdl/wdl* mice exhibit reduced tremor power compared to baseline. (D) Bar graph showing quantifications of peak tremor power at baseline and after propranolol treatment in control and *Car8^wdl/wdl* mice. Circle and square points represent each individual subject’s peak tremor power for control and *Car8^wdl/wdl* mice, respectively, with lines connecting each animal’s data before and after treatment. *Car8^wdl/wdl* mice exhibit significantly stronger peak tremor power at baseline compared to control mice. Following treatment with propranolol, both groups show significantly decreased maximum tremor power. * = p < 0.05; **** = p < 0.0001; ns = not significant, p > 0.05. (E) Schematic illustrating the procedure for a non-tremor movement detection analysis within tremor monitor recordings. Scale bar is 1 s. (F) Quantifications of non-tremor movements show no significant difference between control and *Car8^wdl/wdl* mice at baseline, or within groups following propranolol treatment, indicating that tremor severity is not associated with the overall amount of non-tremor movements as captured in the recordings.

**Figure 2**
**General activity levels, gross locomotor activity, and gait parameters are unaffected by propranolol.** (A) Representative traces of open field activity patterns over a 30 min recording in control mice at baseline (pink, N = 18) and with propranolol (green, N = 8), and in *Car8^wdl/wdl* mice at baseline (orange, N = 20) and with propranolol (blue, N = 8). Lines indicate the animal’s locomotor trajectory over time. The same color assignment for each group is maintained throughout the remaining figure panels (legend above open field traces). Open field data from control and *Car8^wdl/wdl* mice without propranolol (pink and orange) is newly analyzed from the dataset published in Miterko et al., 2021. (B) Quantifications of open field activity in control and *Car8^wdl/wdl* mice. There is no significant difference in total movement time or number of movement episodes between control and *Car8^wdl/wdl* mice at baseline or with propranolol treatment, both within and across groups. No significant difference was found in ambulatory activity with or without propranolol within groups in control or *Car8^wdl/wdl* mice, or with propranolol between groups. * = p < 0.05; *** = p < 0.001; ns = not significant, p > 0.05. (C) Representative traces of forepaw footprinting assays recorded from control (N = 9) and *Car8^wdl/wdl* mice (N = 9), before and after propranolol. Hindpaw prints are shown in purple for context. The 3 gait parameters assessed are stride (the distance between steps of the same paw), sway (the distance between left and right paw placement), and stance (the hypotenuse of stride and sway). Scale bar is 1 cm. (D) Quantifications of footprinting assays for the forepaws. Lines connect before and after treatment data for each animal. There is no significant difference within or between groups in control and *Car8^wdl/wdl* mice, before and after propranolol, for stride or stance. However, sway distances are significantly increased in *Car8^wdl/wdl* mice compared to control mice between groups, before and after propranolol. There is no significant sway difference within groups after propranolol treatment compared to baseline levels in both control and *Car8^wdl/wdl* mice.

**Figure 3**
**Propranolol modulates cerebellar Purkinje cell firing activity.** (A) Top, schematic of awake in vivo electrophysiology recording setup. Bottom, illustrations of a Purkinje cell (bright pink), and downstream cerebellar nuclei neuron (purple), and the two different types of action potentials Purkinje cells produce—simple spikes and complex spikes. (B) Representative raw electrophysiological traces of Purkinje cell activity in control mice before (pink, N = 8, n = 14) and after propranolol treatment (green, N = 5, n = 13), and *Car8^wdl/wdl* mice before (gold, N = 6, n = 14) and after propranolol treatment (blue, N = 4, n = 12). The line graph shown at the top for each condition represents the mean firing rate in Hz at each point in time for the 5 s spike traces shown below each line graph. Below the 5 s spike traces are magnified views of the spikes within the outlined boxes, spanning 500 ms. The same color assignment for each group is maintained throughout the remaining figure panels (legend above electrophysiology graphs). (C) Quantifications of Purkinje cell simple spike firing activity. Propranolol significantly reduces simple spike firing rate in both control and *Car8^wdl/wdl* Purkinje cells. The mode ISI⁻¹ is significantly decreased in *Car8^wdl/wdl* simple spikes following propranolol treatment but not in controls. Simple spike CV and CV2 measures do not significantly change with propranolol in both control and *Car8^wdl/wdl* mice. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant, p > 0.05. (D) Quantifications of Purkinje cell complex spike firing activity. Propranolol significantly reduces complex spike firing rate in both control and *Car8^wdl/wdl* Purkinje cells. The mode ISI⁻¹ is significantly decreased in both control and *Car8^wdl/wdl* complex spikes following propranolol treatment. Complex spike CV is significantly reduced in *Car8^wdl/wdl* mice only, but not in controls. Complex spike CV2 measures do not significantly change with propranolol in both control and *Car8^wdl/wdl* mice.

**Figure 4**
**Propranolol modulates cerebellar nuclei neuron firing activity.** (A) Top, schematic of awake in vivo electrophysiology recording setup. Illustrations of a Purkinje cell (bright pink), and downstream cerebellar nuclei neuron (purple) are shown. Bottom, schematic of a coronal mouse cerebellar section with the cerebellar nuclei outlined in purple. Recordings were conducted in the interposed nucleus (IN) as shown with the electrode placement. FN = fastigial nucleus, DN = dentate nucleus. (B) Representative raw electrophysiological traces of cerebellar nuclei neuron activity in a control mouse before (pink, N = 6, n = 20) and after propranolol treatment (green N = 6, n = 15), and in a *Car8^wdl/wdl* mouse before (gold, N = 6, n = 15) and after propranolol treatment (blue, N = 6, n =15). The line graph shown at the top for each condition represents the mean firing rate in Hz at each point in time for the 5 s spike traces shown below each line graph. Below the 5 s spike traces are magnified views of the spikes within the outlined boxes, spanning 500 ms. The same color assignment for each group is maintained throughout the remaining figure panels (legend above electrophysiology graphs). (C) Quantifications of cerebellar nuclei neuron firing activity. Propranolol significantly reduces cerebellar nuclei neuron firing rate in both control and *Car8^wdl/wdl* mice. The cerebellar nuclei neuron mode ISI⁻¹ is significantly decreased in *Car8^wdl/wdl* mice following propranolol treatment but not in controls. Cerebellar nuclei neuron CV is significantly reduced in *Car8^wdl/wdl* mice only, but not in controls. Cerebellar nuclei neuron CV2 measures do not significantly change with propranolol in both control and *Car8^wdl/wdl* mice. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001; ns = not significant, p > 0.05. (D) Linear regression models correlating mean cerebellar nuclei neuron firing parameters (firing rate, mode ISI⁻¹, CV, and CV2) and mean average tremor power in each group. Solid black lines indicate linear model fit and dotted black lines indicate 95% confidence intervals. Only the model fit between mode ISI⁻¹ and maximal tremor power was significant (p = 0.0002).

**Figure 5**
**The β₁ and β₂ adrenergic receptor antibody signal is expressed throughout the cerebellar cortex.** (A,E) Paraffin staining of a coronal section cut through lobule VIII of the cerebellar cortex in control mice (N = 8) showing β₁ (A) and β₂ (E) adrenergic receptor antibody staining in brown. Purkinje cell somata are positioned in the Purkinje cell layer (PCL) underneath the molecular layer (ML), and directly below the PCL lies the granular layer (GL) containing granule cells and various classes of interneurons. The β₁ and β₂ signal is expressed throughout all three layers of the cerebellar cortex. Scale bar is 50 μm. (B–B″,F–F″) Free-floating fluorescence double staining of the same coronal view of lobule VIII with β₁ (B) and β₂ (F) antibody signal in green, calbindin expression in Purkinje cells in magenta (B′,F′), and the overlay of β₁ or β₂ and calbindin (B″,F″). Co-localized β₁ or β₂ and calbindin expression appears as a brighter, whitish hue. Dotted outlines in B″ and F″ indicate the areas from which the higher magnification images in (C–C″,G–G″) were taken. Scale bar is 100 μm. (C–C″,G–G″) Higher magnification image of the dotted outlined areas from (B″,F″) showing β₁ (C) and β₂ (G) signal expression in green, calbindin expression in Purkinje cells in magenta (C′,G′), and the overlay of β₁ or β₂ and calbindin (C″,G″). Dotted outlines in (C,G) indicate the area from which the higher magnification images in (D–D″,H–H″) were taken. Scale bar is 50 μm. (D–**D″, H**–H″) Even higher magnification image of the dotted outlined areas from (C,G) showing β₁ (D) and β₂ (H) signal expression in green, calbindin expression in Purkinje cells in magenta (D′,H′), and the overlay of β1 (D″) or β2 (H″) and calbindin. At these higher magnifications, the co-localization of β₁ or β₂ and calbindin expression is more easily appreciated. Scale bar is 25 μm.

**Figure 6**
**Propranolol does not modulate tremor, gait, or cerebellar nuclei neuron activity in mice lacking β₁ and β₂ adrenergic receptors.** (A) Representative raw traces of tremor readings recorded from the tremor monitor for *β1-AR^−/−;β2-AR^−/−* mice at baseline (vermillion, N = 11), and after propranolol treatment (sky blue, N = 13). The same color assignment for each group is maintained throughout the remaining figure panels (legend below panels (C,D)). Scale bar is 50 mV vertical and 500 ms horizontal. (B) Line graph depicting tremor power versus frequency. Color representation for groups is maintained from panel A. (C) Bar graph showing quantifications of peak tremor power at baseline and after propranolol treatment in *β1-AR^−/−;β2-AR^−/−* mice with lines connecting each animal’s data before and after treatment. There is no significant difference in peak tremor power after *β1-AR^−/−;β2-AR^−/−* mice receive propranolol. * = p < 0.05; **** = p < 0.0001; ns = not significant, p > 0.05. (D) Quantifications of non-tremor movements show no significant difference between *β1-AR^−/−;β2-AR^−/−* mice at baseline or after being treated with propranolol. (E) Representative traces of forepaw footprinting assays recorded from *β1-AR^−/−;β2-AR^−/−* mice before and after propranolol (N = 9). Hindpaw prints are in shown in purple for context. Scale bar is 1 cm. (F) Quantifications of footprinting assays. Lines connect before and after treatment data for each animal. There is no significant difference before and after propranolol for stride, stance, or sway in *β1-AR^−/−;β2-AR^−/−* mice. (G) Representative raw electrophysiological traces of Purkinje cell activity in *β1-AR^−/−;β2-AR^−/−* mice before (N = 5, n = 17) and after propranolol treatment (N = 4, n = 14). The line graph at the top for each condition represents the mean firing rate in Hz at each point in time for the 5 s spike traces shown below each line graph. Below the 5 s spike traces are magnified views of the spikes within the outlined boxes, spanning 500 ms. (H) Quantifications of *β1-AR^−/−;β2-AR^−/−* Purkinje cell simple spike firing activity. Propranolol significantly reduces simple spike firing rate in *β1-AR^−/−;β2-AR^−/−* mice. Mode ISI⁻¹, CV, and CV2 measures are unchanged by propranolol in *β1-AR^−/−;β2-AR^−/−* mice. (I) Quantifications of Purkinje cell complex spike firing patterns before and after propranolol treatment in *β1-AR^−/−;β2-AR^−/−* mice. Propranolol significantly reduces both the firing rate and mode ISI⁻¹ of complex spikes in *β1-AR^−/−;β2-AR^−/−* mice. Inversely, complex spike CV and CV2 measures are increased following propranolol treatment in *β1-AR^−/−;β2-AR^−/−* mice. (J) Representative raw electrophysiological traces of cerebellar nuclei neuron activity in *β1-AR^−/−;β2-AR^−/−* mice before (N = 6, n = 15) and after propranolol treatment (N = 4, n = 14). (K) Quantifications of cerebellar nuclei neuron firing activity. Propranolol has no effect on the firing rate, mode ISI⁻¹, CV, or CV2 measures in *β1-AR^−/−;β2-AR^−/−* cerebellar nuclei neurons.

**Figure 7**
**β₁ and β₂ adrenergic receptor function is required for propranolol to reduce *Car8^wdl/wdl* tremor.** (A) Line graph depicting tremor power versus frequency in *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* mice before (gray, N = 4) and after (indigo, N = 4) propranolol treatment. The same color assignment for each group is maintained throughout the remaining figure panels (legend below panels (B,C)). (B) Quantification of peak tremor power in *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* mice, with lines connecting each subject’s before and after propranolol data points. Propranolol does not significantly reduce tremor in *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* mice. ns = not significant, p > 0.05. (C) Quantifications of non-tremor movements show no significant difference between *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* mice at baseline or after being treated with propranolol. (D) Representative raw electrophysiological traces of cerebellar nuclei neuron activity in *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* mice before (N = 2, n = 6) and after propranolol treatment (N = 2, n = 5). The line graph at the top for each condition represents the mean firing rate in Hz at each point in time for the 5 s spike traces shown below each line graph. Below the 5 s spike traces are magnified views of the spikes within the outlined boxes, spanning 500 ms. (E) Quantifications of *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* cerebellar nuclei neuron activity before and after propranolol. Propranolol has no effect on the firing rate, mode ISI⁻¹, CV, or CV2 in *β1-AR^−/−;β2-AR^−/−;Car8^wdl/wdl* nuclei neurons.

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References

1. Louis E.D., McCreary M. How Common is Essential Tremor? Update on the Worldwide Prevalence of Essential Tremor. Tremor Other Hyperkinetic Mov. 2021;11:28. doi: 10.5334/tohm.632. - DOI - PMC - PubMed
1. Louis E.D., Machado D.G. Tremor-related quality of life: A comparison of essential tremor vs. Parkinson’s disease patients. Park. Relat. Disord. 2015;21:729–735. doi: 10.1016/j.parkreldis.2015.04.019. - DOI - PMC - PubMed
1. Jiménez-Jiménez F., Alonso-Navarro H., García-Martín E., Álvarez I., Pastor P., Agúndez J. Genomic Markers for Essential Tremor. Pharmaceuticals. 2021;14:516. doi: 10.3390/ph14060516. - DOI - PMC - PubMed
1. Graham J.D.P. STATIC TREMOR IN ANXIETY STATES. J. Neurol. Neurosurg. Psychiatry. 1945;8:57–60. doi: 10.1136/jnnp.8.3-4.57. - DOI - PMC - PubMed
1. Tyrer P.J., Lader M.H. Tremor in Acute and Chronic Anxiety. Arch. Gen. Psychiatry. 1974;31:506–509. doi: 10.1001/archpsyc.1974.01760160056012. - DOI - PubMed

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[1] Louis E.D., McCreary M. How Common is Essential Tremor? Update on the Worldwide Prevalence of Essential Tremor. Tremor Other Hyperkinetic Mov. 2021;11:28. doi: 10.5334/tohm.632. - DOI - PMC - PubMed

[2] Louis E.D., McCreary M. How Common is Essential Tremor? Update on the Worldwide Prevalence of Essential Tremor. Tremor Other Hyperkinetic Mov. 2021;11:28. doi: 10.5334/tohm.632. - DOI - PMC - PubMed

[3] Louis E.D., Machado D.G. Tremor-related quality of life: A comparison of essential tremor vs. Parkinson’s disease patients. Park. Relat. Disord. 2015;21:729–735. doi: 10.1016/j.parkreldis.2015.04.019. - DOI - PMC - PubMed

[4] Louis E.D., Machado D.G. Tremor-related quality of life: A comparison of essential tremor vs. Parkinson’s disease patients. Park. Relat. Disord. 2015;21:729–735. doi: 10.1016/j.parkreldis.2015.04.019. - DOI - PMC - PubMed

[5] Jiménez-Jiménez F., Alonso-Navarro H., García-Martín E., Álvarez I., Pastor P., Agúndez J. Genomic Markers for Essential Tremor. Pharmaceuticals. 2021;14:516. doi: 10.3390/ph14060516. - DOI - PMC - PubMed

[6] Jiménez-Jiménez F., Alonso-Navarro H., García-Martín E., Álvarez I., Pastor P., Agúndez J. Genomic Markers for Essential Tremor. Pharmaceuticals. 2021;14:516. doi: 10.3390/ph14060516. - DOI - PMC - PubMed

[7] Graham J.D.P. STATIC TREMOR IN ANXIETY STATES. J. Neurol. Neurosurg. Psychiatry. 1945;8:57–60. doi: 10.1136/jnnp.8.3-4.57. - DOI - PMC - PubMed

[8] Graham J.D.P. STATIC TREMOR IN ANXIETY STATES. J. Neurol. Neurosurg. Psychiatry. 1945;8:57–60. doi: 10.1136/jnnp.8.3-4.57. - DOI - PMC - PubMed

[9] Tyrer P.J., Lader M.H. Tremor in Acute and Chronic Anxiety. Arch. Gen. Psychiatry. 1974;31:506–509. doi: 10.1001/archpsyc.1974.01760160056012. - DOI - PubMed

[10] Tyrer P.J., Lader M.H. Tremor in Acute and Chronic Anxiety. Arch. Gen. Psychiatry. 1974;31:506–509. doi: 10.1001/archpsyc.1974.01760160056012. - DOI - PubMed

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Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor

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Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor

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