Hypocretin Excites Thalamocortical Terminals In Prefrontal Cortex

5.1 Pharmacology and lesion studies

The susceptibility of thalamocortical terminals to the generation of terminal spikes would promote bursting in the projecting thalamic neurons through antidromic transmission. It has been suggested that neurotransmitters released in the terminal field could thus influence the bursting of groups of neurons in a distant brain region. Our recent work suggests that hypocretin released in prefrontal cortex could act in this manner.24

In examining the effects of hypocretin in prefrontal cortex, we found that there was very little direct effect of hypocretin on prefrontal neurons. In prefrontal slice, we recorded from layer V pyramidal neurons by whole cell patch clamp and applied hypocretin in the bath. Hypocretin had little effect on the resting membrane potential or the holding current, but potently increased synaptic release of glutamate onto these cells. This effect was observed in voltage clamp as a large increase in spontaneous excitatory postsynaptic currents (sEPSCs) illustrated in Figure 5. Blocking action potentials with tetrodotoxin (TTX) eliminated the hypocretin-induced increase in sEPSCs. This indirect effect of hypocretin occurred preferentially in medial prefrontal cortex, a region which has been shown to receive a greater density of hypocretin projections than other cortical regions.14

We started to suspect that hypocretin was able to induce terminal spikes in thalamocortical terminals because prior thalamic lesions greatly suppressed this effect, as shown in Figure 6. Furthermore, the hypocretin-elicited increase in sEPSCs could be suppressed by agonists of mu-opioid receptors, such as DAMGO. Mu-opioid agonists are known to be able to suppress thalamocortical25 but not cortico-cortical transmission.26

Ipsilateral Prefrontal Cortex
Figure 6. A prior unilateral thalamic lesion almost eliminates hypocretin-induced sEPSCs in ipsilateral cortex. By contrast, hypocretin-elicited sEPSCs remain robust in the medial prefrontal cortex in the opposite hemisphere (** P < 0.01; * P < 0.05). Adapted from Lambe and Aghajanian, 2003.24

Figure 7. A schematic showing how Gq-coupled hypocretin receptors can induce depolarization to threshold in thalamocortical terminals, possibly through inhibiting a potassium conductance (g K+) or enhancing a cation conductance (g cation). In vivo studies have shown that these terminals are particularly susceptible to the induction of terminal spikes that propagate back down the axon to the distant cell body. G;/Go-coupled mu-opioid receptors would physiologically oppose the depolarization induced by hypocretin.

Figure 7. A schematic showing how Gq-coupled hypocretin receptors can induce depolarization to threshold in thalamocortical terminals, possibly through inhibiting a potassium conductance (g K+) or enhancing a cation conductance (g cation). In vivo studies have shown that these terminals are particularly susceptible to the induction of terminal spikes that propagate back down the axon to the distant cell body. G;/Go-coupled mu-opioid receptors would physiologically oppose the depolarization induced by hypocretin.

Figure 8. A prefrontal layer V pyramidal neuron being filled with Oregon green BAPTA-1 and Alexa 594 through the patch pipette on the left. This combination of dyes makes the neuron appear orange when the red and green detection channels are merged.

3 Spikes

Figure 9. The graph in red at the top shows how bath application of hypocretin changes the ratio of green to red intensity at a single spine. The graph in blue shows these measurements from a neighboring spine that did not show calcium transients during hypocretin. The images shown correspond to the points on the graph marked 1,2, and 3. The last shows the overall calcium increase in the dendrite and all spines with a depolarization-induced burst of spikes (adapted from Lambe and Aghajanian, 2003).24

Figure 10. The above images show (a) thalamic neurons and (b, c) axons filled with the anterograde tracer Phaseolus vulgaris conjegated to the green fluorescent marker Alexa 488. Thalamic axons pass through the cingulum (b), as shown in horizontal slice, on their way to prefrontal cortex (c). Adapted from Lambe and Aghajanian, 2003.24

Our model of this process is shown in Figure 7. Hypocretin receptor 2 is abundant in the midline-intralaminar thalamic neurons which project to prefrontal cortex.17,18 Hypocretin stimulation appears to depolarize thalamocortical terminals to threshold for spiking. This effect can be physiologically opposed by stimulation of the Gi/Go-coupled mu-opioid receptors.

5.2 Two-photon calcium imaging studies

To test this hypothesis more rigorously, we used two-photon imaging to identify synapses at which hypocretin induced glutamate release. First, we filled layer V pyramidal neurons with two dyes - a green calcium indicator, Oregon green BAPTA-1 and a red control dye, Alexa 595. As illustrated in Figure 8, this combination of dyes makes the neuron appear orange in the merged image.

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