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Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization

Abstract

Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in 4–7 h (excluding histology) and the awake-patched version requires 1–4 h per day for 2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors.

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Figure 1: UV anchoring method and pipette anchoring test system.
Figure 2: Head-fixed whole-cell patching and animal release system.
Figure 3: Diagram of procedures for anesthetized-patched/wake-up (top) and awake-patched (bottom) whole-cell recording in freely moving rats.
Figure 4: Anesthetized-patched whole-cell recordings in freely moving rats.
Figure 5: Awake-patched whole-cell recordings in freely moving rats.
Figure 6: Spatial tuning of subthreshold and suprathreshold activity in awake-patched hippocampal CA1 neurons in freely moving rats.

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Acknowledgements

We thank J. Macklin, J. Colonell and B. Karsh for advice in designing the pipette monitoring system; M. Kim for assistance with surgery and behavioral training; L. Ramasamy for assistance with the remote UV shutter control system and Ronal Tool Company for fabricating custom-designed components. This work was supported by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

D.L. and A.K.L. designed the experiments; D.L., J.E.O. and A.K.L. designed the custom equipment; D.L. conducted the experiments; G.S. provided the idea of the UV curing–aided pipette anchoring method; and D.L. and A.K.L. wrote the manuscript.

Corresponding authors

Correspondence to Doyun Lee or Albert K Lee.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Head-mounted equipment for original anesthetized-patched/wakeup experiments.

The equipment is similar to the protocol described in the main text except for different headplate and headplate clamps, the use of a fixed C-shaped base (instead of an exchangeable base), and pipette anchoring using a 1 mm (instead of 3 mm) tall sapphire collar, side connector, and side support. Anesthetized-patched experiments are now performed with the 3 mm tall collar and without a side connector or side support.

Supplementary Figure 2 Additional equipment for whole-cell recording in freely moving rats.

(a) Top (left) and front (right) views of headstage/LED assembly. (b) Shape of pipette used for hippocampal CA1 target (lower magnification, left; higher magnification, right). (c) Recording electrode/air tube assembly. (d) Detachable reference electrode (top) for awake patching across multiple sessions over multiple days. Reference electrode inserts into the headplate chamber through a 1 mm diameter capillary mounted on the headplate. (e) Transfer box with the urine absorber for awake-patched experiments. A plate of sintered stainless steel is placed onto a tissue covering the floor of the box (left). A transfer box on the sliding platform (right). (f) Hinged eye cover for rapid uncovering during release of animal from head fixation in awake-patched experiments.

Supplementary Figure 3 UV-induced voltage offset

(a) Each UV illumination pulse (blue rectangle) induced a large transient change in voltage. This voltage offset is partly due to UV-susceptible chemicals in the internal solution. (b) Painting the pipette black completely prevents the UV-induced voltage offset. In (a) and (b), asterisks mark voltage deviations due to 500 ms, 300 pA hyperpolarizing current pulses used to measure series resistance.

Supplementary information

Supplementary Figure 1

Head-mounted equipment for original anesthetized-patched/wakeup experiments. (PDF 12609 kb)

Supplementary Figure 2

Additional equipment for whole-cell recording in freely moving rats. (PDF 10949 kb)

Supplementary Figure 3

UV-induced voltage offset. (PDF 262 kb)

Supplementary Methods: Pipette anchoring tests (PDF 116 kb)

Supplementary Note: Development of an improved dental acrylic anchoring method. (PDF 86 kb)

Pipette movement during pipette stabilization using the previous dental acrylic method

Left and right camera views. Dental acrylic was applied at 00 min 00 sec. “x” indicates the pipette tip position at 00:00. (AVI 1686 kb)

Pipette movement during pipette stabilization using the UV-cured collar-based method

Left and right camera views. Here, four pulses of UV light were applied over a period of 2 min. “x” indicates the pipette tip position at 00:00. (AVI 605 kb)

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Lee, D., Shtengel, G., Osborne, J. et al. Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization. Nat Protoc 9, 2784–2795 (2014). https://doi.org/10.1038/nprot.2014.190

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