Electron Microscopy

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The hypocretin system influences various brain functions by modulation of synaptic transmission. Electrophysiological data show that Hcrt can act both post-synaptically, by directly altering the membrane potential of neuronal perikarya, and pre-synaptically, by regulating the release of transmitters, such as GABA and glutamate, from adjacent axon terminals. This pre- and post-synaptic action of Hcrt can occur simultaneously on a particular target,17 but examples have also been provided when Hcrt acts predominantly by altering pre-synaptic dynamics of the target neuron, for example, Hcrt neurons.23 Regardless of the type of action, to appreciate the potential of Hcrt to regulate a putative target system, it is essential that the synaptic organization of Hcrt efferents on a postsynaptic target be revealed. It is important to demonstrate the type, location and extent of synaptic interaction between Hcrt efferents and their postsynaptic targets. Equally critical is the establishment of the hierarchical relationship between Hcrt boutons and other pre-synaptic inputs of a given target neuronal population. With the exception of some elementary observations (see below), the majority of these issues have not yet been addressed and, thus, they represent one of the essential areas that must be resolved in order to provide deeper insight to our understanding of Hcrt's regulatory role in the CNS.

As mentioned earlier, the Hcrt circuitry represents an excitatory system. In accordance with this, Hcrt axon terminals, regardless of the brain region, establish almost exclusively asymmetric synaptic contacts (Fig. 7 and 8). Hcrt-containing axon terminals are relatively large (0.6-1 mm diameter) and contain both large-core and small vesicles (Fig. 7). Hcrt-immunoreactivity in axon terminals is associated with the pool of large-core vesicles. Hcrt axon terminals frequently contain mitochondria, sometimes in unusually large numbers (Fig. 8).

An intriguing aspect of the synaptology of Hcrt efferents is that these peptidergic boutons are more often than not situated proximally on target cells. Thus, for example, Hcrt axon terminals on both the arcuate nucleus NPY cells, which are critical in mediating Hcrt's effect on food intake,39 and on the locus coeruleus noradrenergic neurons, which are likely to be vital in mediating Hcrt's effect on arousal,40,41 establish synapses predominantly on the perikaryal membrane or the membranes of dendrites located very proximally to the the perikaryon (Figs. 7, 8).13,40 This appears to be the case also regarding Hcrt interaction with the medio-septal cholinergic neurons.42 This proximal location of Hcrt boutons suggests a superior hierarchical positioning of Hcrt efferents to influence postsynaptic targets. This synaptic organization, together with the fact that in both the arcuate nucleus and locus coeruleus, almost all of the NPY and noradrenergic cells receive massive Hcrt innervation, helps explain the robust influence of Hcrt on both food intake and arousal. Besides this superior positioning of Hcrt boutons on post-synaptic targets, what further amplifies the influence of Hcrt axon terminals is

Electron Microscope Leptin Receptors

Figure 8. Hypocretin axons in synaptic contact with neuropeptide Y neurons containing leptin receptor in the monkey arcuate nucleus. A-D, Correlated light (A) and electron (B-D) micrographs demonstrating that in the monkey arcuate nucleus, two (arrows labeled 1 and 2 on A, B) black-colored hypocretin (HCRT) axon terminals are in close apposition to light stained neuropeptide Y (NPY)-containing neurons (curved arrows on A) HCRT axon 1 contacts a light NPY perikaryon that also expresses Golgi-associated black leptin receptor (LR) labeling (arrowheads on A-C). C and D are high-power magnifications of the boxed regions on B demonstrating that both HCRT boutons 1 and 2 establish asymmetric synaptic contacts (arrowheads on C, D) on the NPY- and leptin receptor-containing cell body (C) and on the NPY-containing dendrite (D). Note the abundance of mitochondria in the HCRT-containing axon terminal on D. Scale bars: A, 10 |xm; B-D, 1 |xm. (From 13)

Figure 8. Hypocretin axons in synaptic contact with neuropeptide Y neurons containing leptin receptor in the monkey arcuate nucleus. A-D, Correlated light (A) and electron (B-D) micrographs demonstrating that in the monkey arcuate nucleus, two (arrows labeled 1 and 2 on A, B) black-colored hypocretin (HCRT) axon terminals are in close apposition to light stained neuropeptide Y (NPY)-containing neurons (curved arrows on A) HCRT axon 1 contacts a light NPY perikaryon that also expresses Golgi-associated black leptin receptor (LR) labeling (arrowheads on A-C). C and D are high-power magnifications of the boxed regions on B demonstrating that both HCRT boutons 1 and 2 establish asymmetric synaptic contacts (arrowheads on C, D) on the NPY- and leptin receptor-containing cell body (C) and on the NPY-containing dendrite (D). Note the abundance of mitochondria in the HCRT-containing axon terminal on D. Scale bars: A, 10 |xm; B-D, 1 |xm. (From 13)

their extensive interaction with other pre-synaptic boutons. These include interactions with putative excitatory as well as putative inhibitory pre-synaptic terminals,1324 and, they frequently exhibit synaptic-like membrane specializations between them (Fig. 6).

An important, albeit labor-intensive, task will be a detailed characterization of the qualitative and quantitative synaptology of Hcrt efferents with respect to both their postsynaptic and pre-synaptic elements. Given all of the limitations of anatomy, only through a thorough immuno-electron microscopic analysis of entire populations of Hcrt-targeted neurons will it be possible to place into perspective all of the data gathered by electrophysiological, molecular biological and physiological techniques.

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