Exclusive postsynaptic action of hypocretin-orexin on sublayer 6b cortical neurons

Abstract

The hypocretin-orexin (hcrt-orx) neurons are thought to maintain wakefulness because their loss results in narcolepsy. This role may be fulfilled by the excitatory action that the hcrt-orx peptide exerts on multiple brainstem and forebrain systems that, in turn, promote cortical activation. Here, we examined whether hcrt-orx may also exert a postsynaptic excitatory action at the level of the cortex, where hcrt-orx fibers project. However, we found that neurons in layers 2-5 in the primary somatosensory cortex (SSp) were unresponsive to hcrt-orx. We then found that although all neurons tested in sublayer 6a were also unresponsive to hcrt-oxr, all those tested in sublayer 6b were highly sensitive to the peptide. The sublayer selectivity of hcrt-oxr was not restricted to the somatosensory cortex, because it was also found to be present in the primary visual cortex, the motor cortex, and the cingulate cortex. In the SSp, in which the hcrt-oxr effect was investigated further, it was demonstrated to be postsynaptic, to result from an interaction with Hcrtr2-OX2 receptors and to depend on the closure of a potassium conductance. Similar to the selectivity of action in the thalamus, where hcrt-oxr excites the nonspecific thalamocortical projection neurons and not the specific sensory relay neurons, here in the cortex, it excites a specific subset of cortical neurons which, through corticocortical projections, may also be involved in promoting widespread cortical activation.

Figure 1.

Exclusive action of hcrt-orx on cortical neurons of sublayer 6b in the SSp. A, Toluidin blue counterstained cortical slice slab containing two recorded neurons (arrowheads) labeled with neurobiotin in sublayers 6a and 6b (separated by a horizontal band of fibers; ^*) and the responses of which to hcrt-oxr are shown in D and E. B₁, Absence of response to hcrt-oxr of a neuron in layer 2/3. B₂, Summarized results showing that all neurons of layer 2/3 were unresponsive to hcrt-oxr. n represents the total number of cells tested. C₁, Absence of response to hcrt-oxr of a neuron in layer 4/5. C₂, Summarized results showing that most neurons of layer 2/3 were unresponsive to hcrt-oxr (see Results). D₁, Absence of response to hcrt-oxr of a neuron in layer 6a. D₂, Summarized results showing that most neurons of layer 6a were unresponsive to hcrt-oxr (see Results). E₁, Depolarizing response to hcrt-oxr of a neuron in layer 6b. E₂, Summarized results showing that all neurons of layer 6b were depolarized by hcrt-oxr. F₁, F₂, Enlargement of neurons 1 and 2 from A. Scale bar, 15 μm. G, Increase in PSPs in a layer 5 neuron (middle panel) is impeded in the presence of a low calcium-high magnesium ACSF. Calibration: 5 mV, 2 sec. CC, Corpus callosum.

Figure 2.

Mechanism of action of hcrt-oxr on SSp sublayer 6b cortical neurons. A, B, Response to hcrt-oxr in the presence of TTX at 1 μm (A) or in the presence of a modified ACSF containing 0.1 mm Ca²⁺ and 10 mm Mg²⁺ (B). C, Dose-responses to hcrt2-oxrB applied between 10 and 50 nm. D, Comparison of the depolarization induced by hcrt1-oxrA and hcrt2-oxrB (applied at 30 nm).

Figure 3.

Hcrt-oxr blocks a K current. A, Voltage-clamp ramps in absence (1) or presence (2) of hcrt-oxr with K_o at 6.25 mm. B, Voltage-clamp ramps in absence (3) or presence (4) of hcrt-oxr with K_o at 12.0 mm. C, Subtractions of curves from A (1, 2) and B (3, 4). Inset, Reversal potentials for both conditions with respect to Nernst relationship (see Materials and Methods).

Figure 4.

Exclusive action of hcrt-oxr in sublayer 6b in primary visual cortex, primary motor cortex, and cingulate cortex. A, B, Excitatory action of hcrt-oxr on sublayer 6b neurons of the primary visual cortex (A), primary motor cortex (B₁), and cingulate cortex (B₂). Scale bar, 15 μm (in all insets showing infrared images of recorded cells). AQ, Aqueduct; CP, caudate-putamen; DG, dentate gyrus; LS, lateral septum; LV, lateral ventricle; PAG, periacqueductal gray.