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Sympathetic Contribution in Somatosensory Mouse fMRI: Revealing "Hidden" Key Structure Activations by Detailing Functional Response Dynamics1Berlin Ultrahigh Field Facility, Max-Delbrück Center for Molecular Medicine (MDC) in the Helmholtz Association, Berlin, Germany, 2Max-Delbrück Center for Molecular Medicine (MDC) in the Helmholtz Association, Berlin, Germany, 3DZHK (German Centre for Cardiovascular Research), Partner Site, Berlin, Germany, 4Experimental and Clinical Research Center, A Joint Cooperation Between the Charite Medical Faculty and the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
Synopsis
Electrostimulation of the paw is considered a basic paradigm to produce a reliable somatosensory response in animal fMRI. Yet, there is disagreement over the murine BOLD response: based on the anesthetic protocol either unilateral or bilateral patterns were reported. We hypothesize sympathetic activity as an origin of bilateral patterns, which is likely to be suppressed in protocols that employ α2-adrenoreceptor agonists. Utilizing finite impulse response (FIR) basis set modeling we present preliminary data that reveals "hidden" activity of brain structures which are known driving forces of sympathetic pathways. Our overall aim is to reveal the underlying mechanisms of this complex response to a simple paradigm and its implications for somatosensory mouse fMRI.
Introduction
Compared to
state-of-the-art murine fMRI using opto- and chemogenetics, somatosensory
paradigms, like electrostimulation of the paw, appear rather simple. Yet, the
resulting neurosignatures and their causing mechanisms are still controversial,
depending on the anesthetic protocol used: under ketamine-xylazine BOLD
patterns are restricted to the contralateral primary somatosensory cortex (S1)
in which the stimulated limb is represented, while heart rate remains stable throughout
the experiment 1. The very same stimulus under isoflurane elicits bilateral
patterns in S1, S2, insular (IC), parietal cortex (PC), and thalamus (TH),
accompanied by substantial surges in heart rate and mean arterial blood
pressure (MABP) 2-4. The underlying pathways that lead to this bilateral
pattern remain barely understood. We hypothesize a contribution of "hidden"
brain structures whose temporal patterns are insufficiently modeled by a single
HRF that spans the entire stimulus train (up to 20 sec). Here we utilize finite
impulse response (FIR) to include the transient activities of those "hidden"
structures and detail the complex functional dynamics of a basic block paradigm.Methods
Animal preparation: 6 male C57BL/6N mice were studied under 1% isoflurane with continuous, invasive monitoring of MABP as previously described 2,5. Subcutaneous electrostimulation of the hindpaw was performed, 4 blocks, 15 sec, 12 Hz, 1mA. One additional mouse was studied using ketamine-xylazine as described elsewhere 1. MR Imaging: High-resolution sagittal T2-weighted imaging was used to position 19 axial slices for T2*-weighted fMRI (GE-EPI, TR/TE/FA = 2500ms/12.0ms/80°, FOV/matrix/resolution = 16x12x11.4mm / 80x60x19/ 200x200x500μm). All images were acquired on a 9.4T Bruker Biospec with a transceive cryogenic quadrature RF surface coil (Bruker, Ettlingen, Germany). Data analysis: FMRI data were motion corrected, smoothed, registered to the Allen mouse brain atlas 6 and statistically analyzed using FSL FEAT. HRF were modeled for the entire stimulus train: 15 sec block, double gamma convolution + temporal derivative. Alternatively, FIR-modeling was applied to with 2.5 sec window length each, spanning 35 sec from stimulus onset; contrasts were created for single windows (2.5sec) and combinations of up to four windows (10 sec), with F-tests for up to three combinations. Corrected cluster significance thresholds of p<0.05 were determined by Z>3.1.Results
Standard analysis for the stimulation paradigm illustrates the known BOLD patterns: mice treated with ketamine-xylazine showed BOLD in contralateral S1. Isoflurane-anesthetized mice exhibited bilateral BOLD signals that highly correlated with surges in heart rate and MABP (Fig. 1). These cardiovascular effects are driven by sympathetic activity, which is controlled by an assembly of brain stem and subcortical nuclei 7,8. Interestingly, no such structures were found to be significant at the applied statistical threshold in single subjects (Fig. 1c) or at group level 2. By applying FIR we found numerous medullary, midbrain, forebrain and thalamic structures that respond to the stimulus with slightly shifted onsets and durations in respect to the stimulus train (Fig. 2). Among these we found members of the a ascending reticular activating system, the paraventricular hypothalamic nucleus (PVH), the nuclei basalis of Meynert (NBM) as well as midline thalamic and hypothalamic nuclei.Discussion & Conclusion
Our results reveal "hidden" activity of brain structures that contribute to form the bilateral patterns observed with murine electrostimulation under isoflurane. A striking contribution to this pattern is caused by sympathetic activity. This was already evident from the cardiovascular response, yet its neural control regions were unveiled for the first time for murine fMRI: structures like the PVH, which is a key player in cardiovascular control 7, or cholinergic tegmental "arousal" areas that project widely throughout the brain 8. The NBM, which innervate large cortical areas were shown to induce broad patterns of cortical vasodilation in response to somatosensory stimuli 9. All these reactions are well documented for somatosensory stimulation of anesthetized animals. This seems to differ for anesthetic protocols that include potent inhibitors of sympathetic activity, like α2-adrenoreceptor agonists 11. Hence, murine fMRI protocols that employ xylazine or medetomidine lack sympathetic outflow and tend to produce unilateral BOLD responses 1,11. While such protocols prevent the potential occurrence of false-positive BOLD effects induced by stimulus-correlated surges in blood pressure 2,4, one should keep in mind the suppressive effects on (nor)adrenergic central and peripheral mechanisms 10. We are currently evaluating the pros and cons in regards to different research questions. In summary, we have demonstrated that block paradigms may elicit multiple processes, which cannot be modeled by a single HRF. Implementation of more sophisticated analyses like FIR permits to identify diverse sets of brain structures based on their common temporal signatures along a block stimulus train. This provides new perspectives to complete the picture of the underlying neural pathways and overall implications.Acknowledgements
No acknowledgement found.References
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