The simplest reflexes to the most sublime emotional experiences are all rooted in molecular, genetic and cellular structures in the brain. As one of the newest fields in neuroscience, cellular and molecular neuroscience leverages molecular biology to explore how genes, signaling molecules, and cellular morphology influence the nervous system

This video will present landmark discoveries in the field, address key questions in cellular and molecular neuroscience research, and finally showcase a few prominent methods that can be applied to answer those questions.

Let’s begin by taking a look back at the scientific roots of this relatively young field.

Before there were molecular techniques, anatomists and cell biologists dominated the early history of neuroscience.

However, in the 1930s, an influx of physicists, chemists and theoreticians into the ranks of biologists started the molecular revolution, culminating in Watson and Crick’s discovery of DNA structure and the formalization of the central dogma, which explained how DNA encodes the proteins that act as the functional units of cells. For the first time, neuroscientists were able investigate the role specific genes and proteins played in nervous system function.

For example, in the 1960s, Seymour Benzer was able to connect behavioral changes in fruit flies to specific genetic mutations, identifying key genes expressed in neurons controlling behavior, like locomotion.

In the same time period, Eric Kandel demonstrated that complex processes like learning also had a molecular basis. Using the marine mollusk Aplysia he analyzed neuronal tissue extracts, and implicated the intracellular signaling molecule cyclic AMP in a pathway critical to learning.

Then, in 1980s, members of Shosaku Numa’s lab were the first to clone key molecules in neuron function, like the voltage gated sodium channel. Analyses of the functional domains within these large membrane proteins have since improved our understanding of how the control of ion movements leads to neuron firing.

Another important discovery in the molecular mechanisms behind neuronal communication came in the 1990s, when Thomas Sudhof discovered that calcium ions alter the shape of proteins that anchor neurotransmitter-filled vesicles to the cell membrane, explaining how action potentials lead to the precisely timed release of chemical signals into the synapse.

From neurophysiology to behavior, discoveries in molecular neuroscience have improved our understanding of many neuroscientific disciplines.

In spite of this productive history, there’s still a lot to learn! Let’s review some questions that are being asked in labs today.

One area of research asks which genes influence neuronal function. For example, nucleotide expansions in specific genes are known to be involved in several diseases characterized by steady decline of neuron health, like Huntington’s disease.

Another group of genes under investigation encode transcription factors: proteins that regulate the expression of specific genes. Identifying the DNA targets of nervous system-specific transcription factors can give us a better view of the genetic program that makes us think.

Alternatively, neuroscientists may ask how molecules regulate neuronal behavior. For example they may study the molecular machinery that regulates ion movements across the cell membrane, resulting in the propagation of action potentials. Research here has given us anesthetics like lidocaine, which blocks specific ion channels and halts transmission of pain signals to the brain.

Alternatively, scientists may examine the sites of interaction between neurons, known as the synapse. Here, they may ask what molecules are involved in preserving the integrity of the synapse, and how these connections can be restored in disease states.

Neuronal plasticity is the ability of the nervous system to chemically and physically adapt in response to experience. Some research in this field is focused on experience-dependent morphological changes in elaborate neuronal structures, known as dendritic arbors, that form synapses via highly dynamic structures referred to as dendritic spines. Others are interested in how experience can modify the distribution of neurotransmitter receptors on synaptic membranes, which can strengthen the signaling power of a synapse in a phenomenon known as long term potentiation, or LTP.

As you can see, cellular and molecular neuroscientists approach neurobiology from a microscopic view, but the range of processes they study is expansive.

Now that you’re familiar with some of the exciting questions that are asked in cell and molecular neuroscience labs, let’s look at the tools available to answer them.

First, to investigate the genetic control of neuron function, scientists have some very powerful techniques at their disposal. For example, microarrays can quantify the expression of a huge number of genes simultaneously. A microarray is a surface with tens of thousands of snippets of single stranded DNA “probes” attached to it. Genetic material extracted from a neuron is allowed to bind to the probes, and fluorescent markers are used to visualize precisely which sequences are present in the sample.

Alternatively, real-time or quantitative PCR, utilizes equipment that can indirectly measure the relative quantity of specific mRNA transcripts via a PCR based method. This approach is extremely useful for detecting the expression levels of individual genes.

Last but not least, transgenic technology is an important tool for the investigation of gene function in the nervous system. By this method, researchers can produce animals with portions of their genomes removed, known as knockouts, or with modified genes inserted into their genetic code, known as transgenics. Nervous system tissues from these animals can be analyzed in a variety of ways to determine how changes in gene expression impact cellular function.

To study the molecular control of neuronal signaling, researchers often prefer to study brain tissue outside of the animal, either in small tissue pieces or in dissociated cells grown in vitro called primary neuronal cultures. One advantage of this simplified system is the ability to efficiently manipulate the expression of genes of interest in neuronal signaling by transfection. The results of these genetic manipulations can then be observed by visualization of protein localization, analysis of cell morphology, or electrophysiological recordings.

Lastly, dramatic advances in imaging technology are allowing scientists to study neuronal plasticity in exciting new ways. For example, researchers can use two-photon microscopy to visualize deep layers of living tissues in great detail. After creating a “window” in the skull, this technique can be used to image brain cell morphology before and after animals undergo an experience, allowing researchers to see what learning looks like at the neuronal level.

Imaging tools are also used to investigate molecular components of the nervous system in preserved tissues. Fluorescence microscopy is often used in combination with immunohistochemistry, in which samples are stained with fluorescent antibodies that mark the cellular localization of specific proteins.

Having reviewed both important questions and the tools used to answer them, let’s look at some applications of cell and molecular neuroscience research.

First, by applying microarray techniques to a small population of neurons, researchers can examine the differences in expression of various genes in specific neuronal subtypes. Here, single neurons were isolated from the retina and cellular RNA was extracted, allowing the researchers to profile the expression of mRNAs from cells with different functional and morphological characteristics.

Molecular techniques can also be applied to better understand the nervous system’s response to injury. When dorsal root ganglia, or DRGs, are removed from adult mice for primary neuronal culture, axons are necessarily severed. By transfecting the dissociated DRG neurons with gene silencing constructs, it is possible to test the effect of individual proteins on the regrowth of new axons.

Specialized imaging techniques can be used to study how proteins like neurotransmitter receptors are transported to and from the synaptic membrane. In this experiment, cultured neurons were transfected with a gene encoding a fluorescently tagged membrane protein. Using a confocal microscope, small regions of the membrane were then photobleached, meaning that the fluorophores in that area were destroyed. With the bleaching scheme used here, the progressive recovery of fluorescence, or FRAP, reflects the transport of new fluorescent proteins into the plasma membrane. The rate at which this movement occurs can be quantified and compared over many experimental conditions.

You’ve just watched JoVE’s introduction to cellular and molecular neuroscience. In this video we discussed the historical discoveries that made cellular and molecular neuroscience the field it is today, some key questions in the field, and the techniques used to answer them.

As always, thanks for watching JoVE Science Education!