We were now faced with the situation alluded to above: we had the sequence of a putative protein that had not been never before been seen. Tom Kilduff came to the lab on sabbatical just as the Gautviks departed for home. He was immediately interested in the clone 35 project and was given the task of preparing antibodies to the putative hypocretin protein and subsequently of using the antisera to map out the anatomical distribution of the protein. We decided to take two approaches and raised polyclonal antisera against chemically synthesized peptides corresponding to different regions within the putative protein sequence and to bacterially-expressed, histidine-tagged hypocretin protein. Tom was joined during his year in the lab by two postdoctoral fellows, Chiaki Fukuhara and Christelle Peyron, who participated in antibody generation and application.
In Western blots using these antisera and, as target extracts, bacteria expressing the fusion protein, we observed a single prominent immunoreactive band with a migration of approximately 19kDa with the hyperimmune sera, but not with the preimmune sera. Control extracts contained no immunoreactive targets, demonstrating the specificity of the antisera for the hypocretin.3
Immunohistochemical studies on sections from perfused adult male rats detected prominently granular immunoreactivity within widely spaced, large polymorphic neurons exclusively in the dorsalolateral hypothalamic area, coincident with the in situ hybridization-positive cells.3 This coincident staining and its elimination when the sera were preincubated with their immunogens, together with the Western blot studies, provided strong evidence for the specificity of the antiserum for the hypocretin. A few thousand reactive cell bodies were observed between the fornix and the mammillothalamic tracts, 1 mm lateral to the midline, at the level of the median eminence. The neurons spanned the perifornical nucleus and the magnocellular nucleus of the lateral hypothalamus from the medial hypothalamus across the supra-fornical region at mid-to-posterior hypothalamic levels into the myelinated axons of the retrochiasmic optic radiation.
In addition to the hypothalamic neurons, the antisera detected a prominent network of axons located within the posterior hypothalamus and beyond. Fiber projections were observed in apparent terminal fields within septal nuclei in the basal forebrain, the preoptic area, the paraventricular nucleus of the thalamus, the central gray, and the locus coeruleus. Less prominent fiber projections were observed in apparent terminal fields within the colliculi, the laterodorsal tegmental nucleus, and the nucleus of the solitary tract. A thorough mapping of these extensive projections from a relatively small number of hypocretin-expressing neurons was published late in 1998.5 The neuroanatomy of the hypocretins in detail is discussed in other chapters.
The deposition of the immunoreactivity along axons was clearly grainy. Elena Battenberg and Bloom conducted an electron microscopic examination, which revealed hypocretin immunoreactivity within the lateral hypothalamus on perikaryal rough endoplasmic reticulum, cytoplasmic large granular vesicles, and vesicles within myelinated axons and at presynaptic terminals opposite non-immunoreactive dendrites. Within the relatively dense terminal fields in the periaqueductal gray, immunoreactive boutons consistently made asymmetrical synaptic contacts with small-to-medium-sized dendritic shafts. Boutons contained 2-8 large, intensely immunoreactive granular vesicles in the plane of section, but in heavily reactive boutons a lighter peroxidase reaction was observed between the small agranular vesicles.3 The accumulation of the no-longer-putative hypocretin peptides within dense core vesicles at axon terminals suggested that they might have intercellular signaling activity.
4. ARE THE PEPTIDES NEUROTRANSMITTERS?
The putative structures of the hypocretin peptides, their expression within the dorsolateral hypothalamus and accumulation within vesicles at axon terminals suggested that they might have neurotransmitter activity. To test this hypothesis, we collaborated with Tony van den Pol. We supplied a synthetic peptide corresponding to the amidated form of hcrt2 and Tony applied this to rat hypothalamic neurons that had been cultured for 10 days, and recorded postsynaptic currents under voltage clamp.3 At the peptide evoked a substantial, but reversible, increase in the frequency of postsynaptic currents in 75% of the neurons tested, indicative of an excitatory effect (Tony's later studies suggest that approximately 33% of hypothalamic neurons respond to the hypocretins). The other 25% of the cells showed no response to hcrt2. There was little response by hypothalamic neurons that had been in culture for only 3-5 days, suggesting that a certain degree of synaptic maturity was required for the effect. Hcrt2 elicited no response in cultures of synaptically coupled hippocampal dentate granule neurons, which demonstrated target selectivity and suggested that specific receptors for hcrt2 may exist. The physiology of these peptide neurotransmitters is discussed in detail in the chapter by van den Pol.
5. GOING PUBLIC: A VOTE ON NOMENCLATURE
As we began to write the paper describing our discovery of the peptides via cDNA cloning, their immunohistochemical detection, their presence in dense core vesicles at synapses, and their neuroexcitatory properties on hypothalamic neurons, we realized that we needed a name other than clone 35. There were several possible functions for the peptides, but direct evidence for none. We came up with several non-pejorative possibilities, most of which were variations on syllables abstracted from hypothalamic member of the incretin family. The most straightforward of these possibilities was "hypocretin". However, we were aware that this might be perceived as having negative connotations and, instead, initially settled on "hypoincretin". We submitted our manuscript to Science, referring to the peptides as hypoincretins. We then attended the 1997 Society for Neuroscience Meeting, at which JGS presented a poster describing the sequence of the new protein, the expression of its mRNA exclusively in a small number of neurons in the dorsolateral hypothalamus, the electron microscopic detection of immunoreactive vesicles in presynaptic boutons, and the neuroexcitatory properties of the amidated hcrt2 peptide.6 At the adjacent poster, Cristelle Peyron and Tom Kilduff presented the data detecting hypocretin immunoreactivity in the dorsolateral hypothalamic neurons and immunoreactive fibers through the CNS.7
We were not yet comfortable with the name hypoincretin. Therefore, we posted a ballot listing several of the names under consideration and asked poster attendees to express their preference (Figure 3). The plurality of the votes were cast instead for hypocretin. The people spoke. Thus, this became the first democratically named neurotransmitter. Ironically, one of the names suggested as a write-in by an attendee (Karl Bauer) was "gregortin". While he probably meant this in jest, its adoption would have been prescient since the name Gregor is derived from the Greek gregoros, meaning watchful or alert, and the peptides have since been found to have their main function in maintaining arousal.
We came home from the meeting to find our manuscript, returned by Science unreviewed. We sent a reformatted text using the revised name hypocretin to the PNAS, where it was accepted and published on January 6, 1998. In the paper we noted that the existence of two hcrt peptides that differ in their amino acid sequences might indicate two Hcrt receptor subtypes.3
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