In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem.

Sugiyama D, Hur SW, Pickering AE, Kase D, Kim SJ, Kawamata M, Imoto K, Furue H.

J Physiol. 2012 May 1;590(Pt 10):2225-31. PMID: 22371480

 

Objective:   The locus coeruleus (LC) contains a large number of noradrenergic neurons that project throughout the brain and modulate activity in a number of centers that control things such as cardiorespiratory activity, arousal, etc.  In order to examine the cellular mechanisms involved in controlling the adrenergic neurons of the LC, this paper used blind in vivo whole-cell patch-clamp to measure and manipulate action potentials caused by hindlimb pinch.

Methods:    Sprague-Dawley rats were anaesthetized and artificially ventilated by tracheostomy.  Extracellular (EC) recording was done with a tungsten microelectrode lowered in to the LC through a burr hole in the skull.  Rats had a portion of the cerebellum removed in order to expose the brainstem.  Patch electrodes were filled with artificial intracellular solution and lowered through the dorsal surface of the pons in to the LC.  Cells were identified for patch clamping through their cell-attached spontaneous firing pattern and response to toe pinch.  Some patched cells were labeled with neurobiotin for later analysis.

Results:

·         EC recording of cells showed a spontaneous firing rate of ~7.4Hz which transiently increased following a contralateral toe pinch.  These characteristics were similar to what was seen using cell-attached and whole- recordings.  This demonstrates a good membrane seal and intracellular solution.

·         Toe pinch under voltage-clamp did not show fast excitatory post-synaptic currents, but a longer slow inward current.

·         Blockade of potassium channels with cesium allowed excitatory and inhibitory post-synaptic currents (EPSCs and IPSCs, respectively) to be isolated.  Drugs could be applied to the surface of the pons, which would affect the neurotransmission of the LC.  EPSCs were blocked with CNQX, an AMPA-receptor-antagonist, but recovered after wash-out.  IPSCs were reversibly blocked by the GABA-A antagonist, bicuculline.

Conclusions:

·         Whole-cell patch clamp can be performed on brainstem neurons in vivo.

·         This preparation allows for pharmacological investigations of the cellular mechanisms of neurotransmission in an in vivo state.

 

 

-DH