Figure 11. Spines in apposition to labeled thalamic boutons were significantly more likely to show calcium transients in response to hypocretin (adapted from Lambe and Aghajanian, 2003).24

This combination of dyes allows us to image postsynaptic calcium transients at single dendritic spines. Under baseline conditions, the dendrite and spines appear orange, but they can turn yellow (indicative of an increase in the green to red ratio) when an influx of calcium increases the brightness of the green calcium indicator. For example, Figure 9 shows an NMDA-mediated calcium transient turning a single spine yellow, and then a depolarization-mediated action potential turning the entire branch yellow.

We found that hypocretin induced glutamate release onto a small percentage of spines on branches of the apical dendrites of prefrontal layer V pyramidal neurons.24 These spines tended to be on higher-order branches located in midlayer V or in layer I of medial prefrontal cortex. Although the apical dendritic location of the responding spines would be congruent with excitation of direct thalamic input, these findings do not identify which glutamatergic projections are excited by hypocretin.24

To further test our hypothesis that hypocretin selectively excites thalamocortical synapses within prefrontal cortex, we labeled thalamic projections to prefrontal cortex by electroporating the thalamus in vivo with the anterograde tracer Phaseolus vulgaris conjugated to a green fluorescent marker, Alexa 488. Several days later, slices were made to examine thalamocortical labeling, as illustrated in Figure 10.

After filling layer V pyramidal neurons with the combination of dyes described before, we found scattered appositions in the x-y plane between green-labeled thalamic boutons and orange-labeled dendritic spines.24 Just as only a fraction of spines responded to hypocretin in our previous experiments, apparent appositions between labeled boutons and spines were infrequent and occurred primarily on higher order branches of the apical dendrites in layers V and I. We imaged these spines to observe the effects of hypocretin, as shown in Figure 11.

Hypocretin induced calcium transients at 7 out of 8 spines appearing to be in apposition to labeled thalamic boutons but only at 2 out of 26 control spines that were in focus but not in apparent apposition to a labeled bouton.24 This dramatic difference showed that spines with thalamic appositions were a significantly different population (Chi-squared test, P < 0.0001). These experiments provide strong convergent evidence that hypocretin excites thalamocortical synapses in prefrontal cortex.

Figure 12. A schematic showing thalamic inputs to a prefrontal layer V pyramidal neuron can be excited different neurotransmitters: hypocretin excites through hypocretin receptor 2, serotonin through 5-HTM receptors (although unclear whether these receptors are pre- or post-synaptic), norepinephrine through a1 receptors, and acetylcholine through the high-affinity a4p2 nicotinic receptors. This synapse can also be inhibited by enkephalin through mu-opioid receptors, glutamate through group 2/3 metabotropic glutamate (mGlu) receptors, and adenosine through A1 adenosine receptors.

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