The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity

Abstract

We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.

Figure 1

The hypocretins are novel, secretin-related peptides. (A) Hypocretin rat/mouse nucleotide (coding region only) and amino acid sequences. Differences in nucleotide sequences are indicated by asterisks below each different base, amino acid differences are indicated above triplets. Tandem basic amino acids (putative sites for proteolytic maturation) are indicated in bold, as is the serine residue most likely to represent the end of the secretion signal. (B) Alignment of Hcrt1 and Hcrt2 with rat secretin. The first nine residues of secretin have been repeated to indicate apparent circular permutation. The identities between the hypocretins and members of the glucagon/vasoactive intestinal polypeptide/secretin family (3) are indicated by asterisks, the Hcrt1/2 consensus residues appear above the alignment. The complete rat and mouse sequences were obtained by standard methods.

Figure 2

Hypocretin accumulates postnatally specifically in the brain. Northern blot, prepared by standard methods with poly(A)⁺ from the indicated tissues of (A) adult rats or (B) whole brains (E14 was whole head) from developing rats of the indicated embryonic (E) or postnatal (P) ages. In each case the blot was stripped and reprobed with a cDNA for the ubiquitously expressed cyclophilin as a loading and RNA integrity control.

Figure 3

Hypocretin is expressed in a unique pattern within the hypothalamus. (A) In situ hybridization, performed on free-floating sections as described (40), detects cells in the posterior hypothalamus. Bar = 250 μm (A and B). (B) Immunohistochemical localization of cells containing the preprohypocretin peptide at a rostral-caudal level of the hypothalamus comparable to the view in A. Positive neurons in A and B appear in three clusters: the perifornical, dorsal and lateral hypothalamic areas. Immunoreactivity was observed at levels 25–27 (see ref. 7), and about plates 28–31 of ref. . (C) Immunoreactive fibers within the arcuate nucleus of the hypothalamus. Abbreviations: f, fornix; 3V, third ventricle. Bar = 50 μm. (D) Schematic drawing of a coronal section of the rat brain (from ref. 8) at the level shown in A and B where most hyponcretin-positive cell bodies are located. Greater than 80% of the mRNA-positive and immunoreactive cell bodies are enclosed by the area defined by the square and extend at least 1 mm rostral-caudal from this coronal plane. The area shown in C is indicated by the arrow, however the plane of section was more caudal than the drawing. (E) Electron micrograph of immunoreactive bouton synapsing on dendritic shaft in the periaqueductal gray of the thalamus. Arrowheads indicate the electron-dense peroxidase staining of large, dense-core vesicles. (Bar = 0.25 μm. Magnification, ×37,800.)

Figure 4

Hypocretin 2 has biological activity. (A) Rat hypothalamus from 18-day embryos was cultured for 10 days in vitro. Synaptically coupled hypothalamic neurons were recorded in voltage clamp with a whole cell pipette (holding potential = −60 mV). This recording is typical of nine of 12 cells examined under these conditions. The frequency of postsynaptic events was greatly increased (up to +400%) by 1 μM Hcrt2 applied to the bath. After washout of the peptide, the frequency of postsynaptic events returned to normal baseline levels. (Inset) Higher resolution of the events are indicated by the dotted line. Both boxes (in A and B) were recorded with an identical delay after Hcrt2 administration. (B) A hypothalamic neuron after 4 days of culture was unresponsive to 1 μM Hcrt2. Pipette solution contained 128 mM KMeSO_4, 27 mM KCl, 0.4 mM EGTA, 1 mM ATP, and 0.5 mM GTP.