Direct neural current imaging in an intact cerebellum with magnetic resonance imaging

Abstract

The ability to detect neuronal currents with high spatiotemporal resolution using magnetic resonance imaging (MRI) is important for studying human brain function in both health and disease. While significant progress has been made, we still lack evidence showing that it is possible to measure an MR signal time-locked to neuronal currents with a temporal waveform matching concurrently recorded local field potentials (LFPs). Also lacking is evidence that such MR data can be used to image current distribution in active tissue. Since these two results are lacking even in vitro, we obtained these data in an intact isolated whole cerebellum of turtle during slow neuronal activity mediated by metabotropic glutamate receptors using a gradient-echo EPI sequence (TR=100ms) at 4.7T. Our results show that it is possible (1) to reliably detect an MR phase shift time course matching that of the concurrently measured LFP evoked by stimulation of a cerebellar peduncle, (2) to detect the signal in single voxels of 0.1mm(3), (3) to determine the spatial phase map matching the magnetic field distribution predicted by the LFP map, (4) to estimate the distribution of neuronal current in the active tissue from a group-average phase map, and (5) to provide a quantitatively accurate theoretical account of the measured phase shifts. The peak values of the detected MR phase shifts were 0.27-0.37°, corresponding to local magnetic field changes of 0.67-0.93nT (for TE=26ms). Our work provides an empirical basis for future extensions to in vivo imaging of neuronal currents.

Fig. 1

Turtle cerebellum. (A) Schematic illustration of the caudal portion of a turtle brain with an intact cerebellum. (B) Cerebellum detached from the rest of the brain at the level of the cerebellar peduncles. Climbing fibers in the peduncle project to the Purkinje cells (Pc). Mossy fibers in the peduncle project to the granule cells (Grc). Grc axons ascend dorsally and bifurcate to form parallel fibers (pf), which make synaptic contacts with the Pc. Bergman cells (BC), stellate cells (sc), Golgi cells (Gc). Dorsal side up.

Fig. 2

Experimental chamber and preparation. (A) The acrylic chamber used for electrophysiology measurements outside the MR scanner and for the MRI-LFP measurements in the MR scanner. (B) Close-up of the chamber. Stimulating and recording electrodes enter the chamber from below. The cerebellum is placed flat on the acrylic platform and is held in place by an inner ring with 3 nylon monofilaments placed from above. (C) Inversion recovery T2-weighted structural image of the cerebellum in the chamber.

Fig. 3

Ionic current phantom used to estimate temporal standard deviation in the phase data. Cyan-colored areas are filled with 0.9% saline containing 5 mM CuSO₄. The 100 μ_A current was restricted to the glass capillary tube. Dimensions of chamber are same as in the cerebellum study.

Fig. 4

(A) MRI-LFP recording apparatus: The magnetic field of the scanner is active to the right of the thick red line. All non-MR-compatible hardware is placed to the left of the red line. The cerebellum is placed in the cradle/tray and is positioned at the scanner isocenter. A surface coil is placed on top of the chamber. The chamber tray has a faceplate that is bolted to the scanner bore using nylon screws. This keeps the chamber level and parallel to the floor. The numbered arrows show the signal flow in this setup: 1 = SYNC pulse, 2 = stimulus timing marker, 3 = trigger pulse to isolator, 4 = current stimulus (delivered to the stimulating electrodes in Cb), 5 = analog LFP signals (from recording electrodes in tissue to amplifier), 6 = digitized LFP signals (fiberoptic cable from amplifier to usb/laptop), 7 = usb input to laptop, 8 = “per-scan” MRI trigger markers. (B) Closeup of tissue submerged in aCSF placed in the chamber. Surface coil is placed on top of the chamber.

Fig. 5

Laminar extracellular potential profile along the depth of the cerebellum at a single location ipsilateral to the stimulation. (A) Measured LFPs at 100 μm increments. (B) Depth profile at the peak of the LFP. The profile suggests an extracellular current source in the granular layer and an extracellular current sink in the molecular layer and in the Purkinje cell layer. Measurement made using a glass micropipette outside the MRI in a magnetically shielded room.

Fig. 6

Electrophysiological characteristics of the slow local field potential (LFP). (A) LFP 200 μm below the ventral surface of the cerebellum (in the granular layer) elicited by peduncular stimulation in a modified aCSF with 1 mM picrotoxin and 10 mM tetraethyammonium. (B) Isopotential pattern of the LFP in (A) at the peak of the response. Tissue shown dorsal side up.

Fig. 7

Neuronal and metabotropic origin of LFP. The LFP response recorded 200 μm from the ventral surface (red) is blocked (green) by a metabotropic glutamate receptor antagonist (LY341495, 100 μM) antagonist and is recovered with washout (blue). Measurement made using a glass micropipette outside the MRI in a magnetically shielded room.

Fig. 8

Correlation of MR phase with current in the ionic current phantom with current pointed into the plane of the page. B_o orientation same as in the animal study.

Fig. 9

Distribution of current dipole moment (nA.m) based on the 250 μV isopotential contour in the map in Fig. 6B. Dipoles were located in the middle layer of the cerebellum in a 0.125 × 0.125 mm grid. At each grid location in the active area, a single current dipole was placed (pointing into plane of the image). The maximal current dipole moment was 0.0156 nA.m corresponding to a peak current dipole moment density of 1 nA.m/mm² assumed in this calculation. The pair of yellow dots shows location of the twisted pair of stimulating electrodes.

Fig. 10

z-component of the predicted local magnetic field ΔB, parallel to the axial B_{o fi}eld, for the distributed current dipole source in the model cerebellum. (A) Total ΔB_z (sum of primary and secondary magnetic fields). Active area is shown (dotted line). (B) Primary magnetic field ΔB_z,primary due to the dipoles. (C) Secondary field ΔB_z,secondary due to the boundary effect. (D) Magnitude of the ΔB_z, ΔB_z,primary, and ΔB_z,secondary along the line in A. In (A–C), the pair of black dots shows the location of the twisted pair of stimulating electrodes.

Fig. 11

(A) Time course of the ΔΦ superimposed on simultaneously measured LFP (green). Examples from 3 animals in response medium. Correlation of LFP with ΔΦ for (i–iii) are 0.64, 0.46, 0.36, averaged over 160 trials and over 37, 49, 17 voxels with | ΔΦ | > 6*SEM in the red time window, respectively. (B) Neural origin of the signals. Both ΔΦ and LFP are abolished by 2 mM kynurenate added to modified aCSF, but recover after the washout. RESPONSE and BLOCK data were processed identically (same time window, sign correction, and 37 voxels in average). Correlation of ΔΦ with the RESPONSE LFP was 0.64 (p = 10⁻²³) and WASH LFP was 0.53 (p = 1.8 × 10⁻¹⁵). Correlation of the BLOCK-ΔΦ with the RESPONSE LFP was −0.04 (p = 0.5).

Fig. 12

Single-voxel detection of the ΔΦ. A(i) and B(i) show superimposed single-voxel ΔΦ time courses in all the voxels in the red and blue ROIs in C(i), respectively. Averaged across the 160 trials. Red and blue time courses in A(i) and in B(i)—average of all the single-voxel time courses in the red and blue regions in C(i). Concurrently recorded LFP shown in green. A(ii) and B(ii) show single-voxel ΔΦ time courses in the red and blue voxels labeled in C(ii). ROI in C(i) was selected from a single animal. In C(i–ii), the solid dots show location of the stimulating electrodes while the open dots show location of the recording electrodes.

Fig. 13

Spatial map of ΔΦ during (A) the RESPONSE condition and during the same time window during (B) the BLOCK. Average phase change maps are over n = 5 animals. Phase changes seen in the RESPONSE are not visible in the BLOCK condition. In both A, B, the solid dots are the stimulating electrodes and the open dots are the recording electrodes, respectively.

Fig. 14

Spatial map of ΔΦ and current density matching the active tissue determined with LFPs. (A) Spatial map of ΔΦ during the peak of the LFP averaged across n = 7 animals. (B) Minimum norm estimate of current dipole moment Q (nA.m in each voxel) with a maximum of 1.013 nA.m/mm² (directed into the page ⊗), based on the ΔΦ map in A. (C) Statistical significance map of the density estimate in (B). Areas shown are (p < 10⁻⁵, log scale), after Bonferroni correction. (D) The density map of the active tissue determined with LFP. B_o is rostral to caudal. In (A–C), the solid dots are the stimulating electrodes (bottom) and the open dots are the recording electrodes (top), respectively. In (D), only the stimulating electrodes are shown (as a single solid dot).