By David N. Leff

"Smells are surer than sounds or sights to make your heartstrings crack."

Rudyard Kipling's poem got it right, a century ago. A sniffed fragrance travels from nostril to brain in a fraction of a second, along nerve cells that reach sensory climax at sites in the cerebral cortex bordering on the hippocampus. This convoluted cluster of neurons is thought to be a mission control for emotions, memory and sex drive, among other crucial mental processes.

Homo sapiens has pretty much outgrown excessive reliance on olfaction in pursuit of a mate or a meal, or avoidance of enemies. In subhuman forms of life, however, smells are right up there with sights and sounds. Animals not only perceive odors, to identify friend from foe, but generate them, largely in the form of hyper-fine-tuned, sex-seeking pheromones.

One trillionth of a gram of silkworm pheromone is sufficient to summon a distant male to his wannabe female mate.

Hippocrates is said to have recommended inhaling certain odoriferous substances as a means of terminating an unwanted pregnancy. Since his time, two and one-half millennia ago, understanding of olfaction has of course progressed, but pushing ignorance ahead of it.

Research into sight (though not yet hearing) is light years ahead of unravelling the neurosensory basis of smell at the molecular level. One major question mark has been identifying the nerve receptor that picks up the odorant message in the nasal cavity and forwards it, via a cascade of signaling reactions, to the brain.

"A candidate olfactory receptor was cloned six years ago," observed molecular biologist Joji Otaki, at Columbia University, "but no one ever proved it to be the real receptor. People have been calling it a 'putative' olfactory receptor. Someone had to prove that explicitly."

Otaki is a coauthor of a report in the current issue of Science, dated Jan. 9, 1998, titled: "Functional expression of a mammalian odorant receptor." Its senior author is physiologist Stuart Firestein, of Columbia's department of biological sciences.

"We determined a ligand for that particular protein receptor," Otaki told BioWorld Today. "So now we can call it the olfactory receptor, or better, the odorant receptor."

There are several thousand odorant ligands and nearly a thousand presumed receptors in the rat nasal epithelium. Among the 74 ligands that Firestein and his team sent out to hunt down and bind its still-putative receptor was n-octanal, an eight-carbon, straight-chain aldehyde. No wonder: n-octanal is an ingredient of the perfumering and flavoring industries.

Citrus-Like Scent Sprayed Into Rats' Nostrils

And aldehydes are the key constituents of pheromones, which attract male and female animals to each other in mating season. The Columbia team's star ligand, n-octanal, has a fruity or citrus-like scent, Otaki noted.

In their experiments, n-octanal proved the most attractive receptor-seeking odorant in the nasal mucosa of laboratory rats. In an area just a centimeter square, the rat nasal epithelium contains some 6 million neurons, of which a large number are receptors.

"There are a lot of olfactory receptors," Otaki pointed out, "expressed by one of the largest gene families known in mammals. In a single neuron, only one of these is expressed, but in a way that can recognize a variety of odorants." The team targeted one such receptor, rat I7, in its odorant mating game.

Their search engine consisted of two linked genes, one for the I7 receptor, carried in a recombinant adenovirus vector; the other for green fluorescent protein (GFP). "We injected this into the nasal cavity of rats," he recounted, "and infected their olfactory neurons."

The cells glowed green under the fluorescent microscope, confirming that the infection was going well. This in turn "showed that our receptor-GFP gene hybrid was being separately expressed in the same cell, because these two genes encode messenger RNA connected with internal ribosome binding sites," Otaki explained.

They then passed a panel of 74 odorants over the infected olfactory epithelia, and screened them by measuring voltage potentials across the cell membranes." All 74 ligands responded, as did controls.

"We got a particularly strong response compared with uninfected cells," Otaki recalled, "from one odorant — n-octanal."

He continued: "Now that we know the receptor binds a specific ligand, the next thing we have to do is investigate the receptor-ligand structural relationships. Probably we can develop different ligands, such as agonists or antagonists, to different receptor molecules. We can mutate these molecules, or use different ones to make other viruses, and basically repeat the same experiment. Thus, we can infer the real receptor-binding site, or putative active site, for the ligand."

First Step In Decoding How Brain Sees Smells

"This molecule," Otaki went on, "is a so-called 7-transmembrane-domain receptor. Most of the important biological receptor molecules are included in this receptor type. So studying the olfactory system," he concluded, "may have some kind of general implication for a wide variety of receptor-ligand systems."

As the Science article pointed out, "[i]dentifying the molecular receptive field of an olfactory sensory neuron is a critical first step in understanding how olfactory perception is achieved by higher brain centers."

An accompanying editorial commented, "The availability of a large family of related proteins that may couple to a common intracellular second messenger system, combined with the fragrance chemist's almost unlimited collection of compounds, is a rich vein for future mining. The pharmacologist and the psychophysicists may finally be able to join forces and decipher the code with which the brain determines the identity of simple and complex odorant stimuli." *