🚀 go-pugleaf

Hello neurobiologists and other interested: here I will give you a glance 
of what we are doing in the University of Trondheim. Please give me 
comments or questions!


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Description of the master-project: "Functional organization of the 
antennal lobe of Heliothis virescens (Lepidoptera: Noctuidae).
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THEORY
Pheromones is odors that modify the behaviour of individuals within one 
species, and there are several different kinds depending on how they work. 
In this project, it's all about sex peromones of the moth H.virescens (tobacco 
budworm moth). The male can detect the females sex pheromones, whitch is 
produced in her abdominal gland. Chemical communication is common in many 
insect groups. In moths, sex pheromones is thorougly investigated because 
of their importance in the moths mating behaviour. 
The North-American moth H.virescens, is one of about 80 species in a 
large group of moths: Heliothine. Many of the species eat cultivated 
plants (e.g. tobacco and sunflower) as larvaes, and this means great damage 
to crops, and farmers may suffer economically. This also goes for 
H.virescens. Its sex pheromones is a mixture of six components: 
cis-11-hexadecenal and cis-9-tetradecenal is produced in largest amount 
(16:1), and in this mixture they will lead to mating behavior in the 
male. These components are called the major components. Minor components 
are also aldehydes, and produced in much smaller scale. Behavioral studies 
have showed that they are important for full imitation of the natural blend.

Until now there has been found receptorneurons for 11-16 and 9-14 
aldehyde, and a acetate produced of a different species (for isolation 
of species!). These are located in, and in the basis of the sensilla 
hairs on the antenna. There are sensilla trichodea type 1, whitch are long 
and upstanding hairs in caracteristical rows on the antenna. Receptorcells 
here detect mainly pheromones, and it is only the male that has got 
these. Both sexes has got sensilla trichodea type 2 and sensilla 
basiconica, and these detect mainly host odors, but also pheromones in 
the male.

Thanks to electrophysiological studies, there has been found that every 
pheromonecomponent activates one type of receptorcells that leads the 
information further to the antennal lobe (labelled line system). The 
neurons are making synapses with interneurons in distinct structures called 
glomeruli. In males, there has been shown that pheromonereceptors leads 
to certain areas of the antennal lobe, called a macro glomerular complex 
(MGC). In H.virescens, the MGC contain four glomeruli put together 
in a unit. Something like this has not been found in females. In 
related species it has been found that different pheromone 
receptors leads to different parts of MGC (subunits). 

PURPOSE OF THE PROJECT
1.	Is the different subunits of MGC funcionally different? Are the 
receptorcells leading to different parts of MGC?
2:	Does it exist receptorneurons for other components (minor) than 
those found until now? By using another method, many more receptorneurons 
can be investigated than by using "old" methods.

HOW TO DO THIS
In this project we will use H.virescens males that is sent to us as pupae 
from Ciba-Gigy, Basel, Switzerland. These are hatched in 24 degrees C 
with a diurnal cycle 16h light and 8h darkness. The insects are fed with 
honey in water. For experimental use, the insects is placed in a plast 
cylinder and the head made unmoveable with wax. Just the antennas, that 
can't be moved by the animal, is sticking up. A chloridized silver wire is 
put into the abdomen of the animal and in contact with a metalrod. A clip 
on the rod hold the plastic cylinder. A moist piece of 
paper is put behind the insect to prevent it from moving and 
dehydrating. Then the rod is placed in a Leitz micromanipulator.

Now the tip of a sensilla trichodea type 1 can be cut by to glassknives. 
A glass electrode is put over the cut hair. This electrode is moved by 
another micromanipulator, and is filled with 0.5 M cobalt-lycine. The 
solution is made by mixing cobolt-chloride with L-lycine in destilled 
water. This is steered for nine hours, and then the pH is adjusted to 
7,3-7,4 with 1M HCl.

The receptorcell we now hopefully have contact with, is to be indentified 
by stimulating it with several components. These are in beforehand 
soluted in hexane and put on filterpaper (10 micrograms) and this is put 
in a pipette. To stimulate the neuron, the pipette is in contact with a 
pulsegenerator that make small puffs of clean air go through the 
pipette. From the pipette, the odor is puffed into a glass cylinder 
through whitch air with a speed of 25 ml/sec flows. Then the airstream over 
the antennas are continous, as in "real life". When (or IF) the 
receptorneuron is identified, the pulsegenerator sends pheromone 
puffs lasting 50 ms over the antenna every other second in one hour.

After one hour, the insect is released from the silver wire and placed in 
a petridish in the refridgerator at 4 degrees C for 48 hours. Then it's 
decapitated and the head put in a saltsolution with ammonina sulfid (38%) 
for 10 min. Then the brain is washed and put in fix overnight. The brain 
is then dissicated with eye lobes, rehydrated and silver intensified 
according to Timms procedure.

When this is finished, the brain is dehydrated, cleared in 
methylsalicylat and photographed. Then put into molten plastic and sliced 
in 10 micrometer thick slices. The slices is coloured in 1:1 mix of azure 
and methylene blue, and reconstructed using camera lucida.

PROBLEMS
1.	I can't find any more receptorcells than for cis-11-hexadecenal 
and a few for a acetate ("11-16-acetate"). This is very frustrating! 
Where should I look for them? 
2.      We don't know for sure how the neurons are stained, but it is clear 
that the dye can't go into the cells. That would kill them. It is 
believed that only the most active receptorneurons are stained, so you 
have to stimulate the neuron to get a successfull filling. There may be 
1-3 receptorneurons in a sensilla, and in theory all of them can be tuned 
to the same compound. Then a maximum of three neurons will be stained 
with dye. But in most all of my successfull fillings there are more than 
one or three neurons that are stained!!!! When I am looking for a neuron 
with nice and clear spikes and response, I go through a number of sensilla 
hairs and test them before I try to fill one. Each test takes only 1/2 -
1 minutes, so this can hardly be the reason why many neunrons are stained. 


Thank you
           Anne Karin Amundgaard 
	   Zoological department
           UniT
           Norway