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

Abstract

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 −15 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 the TRPC5 subunit and which is responsible for the depolarized and active state of these cells.

TRPM2 Channels Are Required for NMDA-Induced Burst Firing and Contribute to H2O2-Dependent Modulation in Substantia Nigra Pars Reticulata GABAergic Neurons

“…Given the limited specificity of pharmacological agents to study TRP channels, we used an anti-TRPM2 antibody to block TRPM2 channels selectively in these neurons. Anti-TRP channel antibodies have been used successfully to block specific TRP channels in previous work (e.g., Amaral and Pozzo-Miller, 2007; Hecquet et al, 2008; Zhou et al, 2008; Cvetkovic-Lopes et al, 2010). …”

NMDA Receptor-Dependent Synaptic Activation of TRPC Channels in Olfactory Bulb Granule Cells

“…The general deletion of both TRPC1 and TRPC4 could indirectly impede the LLD generation by detrimental effects on the neuronal excitability of MTCs and GCs (Cvetkovic-Lopes et al, 2010). The aforementioned immunohistochemistry with anti-TRPC1 and anti-TRPC4 antibodies yielded no evidence for the presence of any of the two proteins in olfactory receptor neuron axons.…”

Expression of transient receptor potential (TRP) channel mRNAs in the mouse olfactory bulb

“…For example, dopaminergic substantia nigra neurons as a population were found to express mRNA for 5 TRPC channels [32]. Similarly, populations of hypothalamic neurons containing proopiomelanocortin, gonadotropin-releasing hormone or hypocretin/orexin express 5-6 members of the TRPC family [3,26,36]. Expression of other TRP family channels was not examined in these studies, but it is reasonable to speculate that these and other neuronal types may express channels for other TRP families.…”