Sleep-Deprivation Regulates α-2 Adrenergic Responses of Rat Hypocretin/Orexin Neurons

We recently demonstrated, in rat brain slices, that the usual excitation by noradrenaline (NA) of hypocretin/orexin (hcrt/orx) neurons was changed to an inhibition following sleep deprivation (SD). Here we describe that in control condition (CC), i.e. following 2 hours of natural sleep in the morning, the α2-adrenergic receptor (α2-AR) agonist, clonidine, had no effect on hcrt/orx neurons, whereas following 2 hours of SD (SDC), it hyperpolarized the neurons by activating G-protein-gated inwardly rectifying potassium (GIRK) channels. Since concentrations of clonidine up to a thousand times (100 µM) higher than those effective in SDC (100 nM), were completely ineffective in CC, a change in the availability of G-proteins is unlikely to explain the difference between the two conditions. To test whether the absence of effect of clonidine in CC could be due to a down-regulation of GIRK channels, we applied baclofen, a GABAB agonist known to also activate GIRK channels, and found that it hyperpolarized hcrt/orx neurons in that condition. Moreover, baclofen occluded the response to clonidine in SDC, indicating that absence of effect of clonidine in CC could not be attributed to down-regulation of GIRK channels. We finally tested whether α2-ARs were still available at the membrane in CC and found that clonidine could reduce calcium currents, indicating that α2-ARs associated with calcium channels remain available in that condition. Taken together, these results suggest that a pool of α2-ARs associated with GIRK channels is normally down-regulated (or desensitized) in hcrt/orx neurons to only become available for their inhibition following sleep deprivation

Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

In a previous study we proposed that the depolarized state of the wake-promoting hypocretin/orexin (hcrt/orx) neurons was independent of synaptic inputs as it persisted in tetrodotoxin and low calcium/high magnesium solutions. Here we show first that these cells are hyperpolarized when external sodium is lowered, suggesting that non-selective cation channels (NSCCs) could be involved. As canonical transient receptor channels (TRPCs) are known to form NSCCs, we looked for TRPCs subunits using single-cell RT-PCR and found that TRPC6 mRNA was detectable in a small minority, TRPC1, TRPC3 and TRPC7 in a majority and TRPC4 and 5 in the vast majority (,90%) of hcrt/orx neurons. Using intracellular applications of TRPC antibodies against subunits known to form NSCCs, we then found that only TRPC5 antibodies elicited an outward current, together with hyperpolarization and inhibition of the cells. These effects were blocked by co-application of a TRPC5 antigen peptide. Voltage-clamp ramps in the presence or absence of TRPC5 antibodies indicated the presence of a current with a reversal potential close to 215 mV. Application of the non-selective TRPC channel blocker, flufenamic acid, had a similar effect, which could be occluded in cells pre-loaded with TRPC5 antibodies. Finally, using the same TRPC5 antibodies we found that most hcrt/orx cells show immunostaining for the TRPC5 subunit. These results suggest that hcrt/orx neurons are endowed with a constitutively active non-selective cation current which depends on TRPC channels containing th

Single-cell RT-PCR of TRPCs in hcrt/orx neurons.

<p>(A) RT-PCR experiment on 500 pg total RNAs isolated from the hypothalamus. Agarose gel electrophoresis showing that mRNAs encoding the TRPC1, 3, 4, 5, 6, and 7 are all detected and generate PCR fragments with sizes of respectively 363, 333, 402, 129, 114 and 337 bp. The hcrt/orx PCR fragment size is 189 bp. MW: molecular weight (100 bp DNA ladder). (B) Single-cell RT-PCR experiment. Agarose gel electrophoresis illustrating the more frequently observed TRPC expression pattern, found in 12 out of 41 hcrt/orx cells. It corresponds to TRPC1, 3, 4, 5 and 7. MW: molecular weight (100 bp DNA ladder). (C) Different TRPC expression patterns identified in the 41 hcrt/orx cells investigated. The asterisk indicates the more frequently observed pattern also illustrated in panel B. (D) Histogram showing the occurrence of the different TRPC subunits in the 41 investigated cells.</p

α₂-ARs activation in the control condition reduces total calcium current in identified hcrt/orx neurons.

<p>(A) Total calcium current measured in response to a voltage step from −90 to 0 mV in a typical hcrt/orx neuron before (control), during (clonidine) and after (wash) bath-application of clonidine at 10 µM. In the insets, the recorded neuron (arrowhead) is shown to be Hcrt/Orx-positive (middle inset) and neurobiotin-positive (right inset). (B) Time-dependent reduction of the total calcium current following transient application of clonidine at 10 µM. Experiments were done in presence of 10 µM NBQX, 50 µM D-APV, 30 µM bicuculline, 1 µM tetrodotoxin, 20 mM TEA, 4 mM 4-AP and 3 mM ClCs in the ACSF.</p

Hyperpolarization of hcrt/orx neurons by substitution of sodium with choline chloride.

<p>(A–B) Tonic firing in presence of a depolarizing step (A) and a low-threshold spike (dot) followed by plateau potential (double arrow) when the depolarizing step is given under DC hyperpolarization (B) is characteristic of hcrt/orx neurons. (C) Substitution of sodium chloride with choline chloride in the bath produces a hyperpolarization and cessation of firing. (D) Voltage-clamp ramps in control (Ctrl) conditions and after choline substitution. The subtraction of ramps shown in the inset suggests the presence of a voltage-dependent cationic current.</p