Making connections in the brain

The human brain has a multitude of functions. As well as organising our thoughts, feelings and actions, it also controls bodily processes like fertility. At puberty, the brain is ultimately responsible for switching fertility on. The decision-making process is very complex – it depends on many factors such as the age of the individual, whether they are well nourished and whether are they going through a stressful time.

Dr Rebecca Campbell from the University of Otago is fascinated by how fertility is switched on and off. She studies a network of cells in the brain that control fertility. Rebecca is especially interested in how these cells, called gonadotropin-releasing hormone (or GnRH) neurons, ‘talk’ to each other and to other cells. She uses microscopes to look closely at the shape of GnRH neurons within mouse brains and to trace the connections between them. Rebecca hopes that her work might eventually lead to treatments for infertility.

Neurons under the microscope

Microscopy is crucial for Rebecca’s work because it’s the best way to understand the shape of the cells she studies. Neurons have a very complex shape, with many long skinny branches (called axons and dendrites). They also form a complicated network through their interactions with each other, and microscopy is the best way of studying these networks.

Rebecca’s microscope of choice is the confocal laser scanning fluorescence microscope. This specialised light microscope uses light from lasers to highlight specific parts of a sample – in Rebecca’s case, the branches of GnRH neurons. Only fluorescent molecules show up under the laser light, which makes them easy to find. Confocal microscopy also allows Rebecca to see an entire cell, even a very long skinny one like a neuron, under the microscope.

Finding GnRH neurons in the mouse brain

One of the challenges of studying GnRH neurons is finding them! The mouse brain contains 75 million interconnected neurons, and only about 1,000 of those are GnRH neurons. To make it possible to find individual GnRH neurons under the microscope, Rebecca uses a family of transgenic mice made by members of her research group. These mice are normal except for one thing: their GnRH neurons contain a protein called green fluorescent protein (GFP), which glows green when illuminated with a particular wavelength of light.

When slices of brain from the transgenic mice are illuminated with lasers under the confocal laser scanning fluorescence microscope, the GFP in the GnRH neurons glows bright green. No other neurons are visible, so the GnRH neurons are easy to find.

How GnRH neurons communicate: solving the puzzle

Rebecca’s work has solved a puzzle about how GnRH neurons work together to control fertility. GnRH neurons are scattered throughout the brain and appear disconnected from each other, so scientists didn’t understand how they could form a network. Rebecca and her team changed all this by showing that GnRH neurons actually have unusually long dendrites – more than 2 mm, so the cells are 100 times longer than they are wide! The long dendrites mean they have lots of connections with each other, despite appearing to be distant from each other.

How did Rebecca’s team see the true length of GnRH dendrites for the first time? In their brain slices from their transgenic mice, they found GnRH neurons (glowing green) and filled them with a small protein called neurobiotin. This is a tricky procedure that requires a steady hand! After the cells were filled, they could see where the neurobiotin was under the microscope because it glowed red under laser light.

Because neurobiotin is small, it spread all around the inside of the cells. It even made it all the way to the end of the neuron’s very long dendrites. This meant that the whole length of the dendrites could be seen glowing red under the microscope. Before Rebecca’s experiments, scientists had only looked at the distribution of GnRH peptide, which doesn’t get into all the cell’s nooks and crannies. This is why the cell shape appeared to be less complex than it was.

Looking at neurons in 3D

One of the goals of Rebecca’s research is to understand what the network of GnRH neurons looks like. This would be an almost impossible task without some way of visualising the network in 3D. Like most microscopes, the confocal microscope only takes 2D images, but these images can be built up into a 3D computer model of a GnRH neuron. Using these 3D models, Rebecca has shown that dendrites from several GnRH neurons come together in intertwined bundles. She and her team are now exploring whether these bundles affect how GnRH neurons control fertility.