In the late nineteenth century, the Spanish anatomist Santiago Ramón y Cajal suggested that memories might be formed by the strengthening of connections between nerve cells:
Cerebral gymnastics are not capable of improving the organization of the brain by increasing the number of cells, because it is known that the nerve cells after the embryonic period have lost the property of proliferation; but it can be admitted as very probable that mental exercise leads to a greater development of the dendritic apparatus and of the system of axonal collaterals in the most utilized cerebral regions. In this way, associations already established among certain groups of cells would be notably reinforced by means of the multiplication of the small terminal branches of the dendritic appendages and axonal collaterals; but, in addition, completely new intercellular connections could be established thanks to the new formation of [axonal] collaterals and dendrites.
Today, that idea, which is now called long-term potentiation (LTP), is the prevailing theory of the cellular basis of memory. LTP was first observed in the mid-1960s by Terje Lømo, a Norwegian neurophysiologist, while investigating the role of the hippocampus in short-term memory. Experimenting on anaesthetized rabbits, Lømo isolated a simple neural circuit from the hippocampus, and observed that stimulation of a region at one end of the pathway resulted in changes in the electrical activity of the region at the other end. Unexpectedly, however, he also noticed that an enhanced response could be elicited in one region if a train of high frequency electrical stimuli was first applied to the other.
Lømo had discovered a mechanism whereby experience-dependent electrical activity increases the efficacy of inter-cellular signalling in the nervous system. In LTP, memories are formed by the strengthening and re-arrangement of synaptic connections. This involves a change in the morphology of the cells involved. Numerous synapses are located on a neuron’s dendritic spines, tiny projections found on the dendrites of the cell. The re-arrangement of synaptic connections during memory formation – and forgetting – involves the formation of new spines and the retraction of old ones. It is this process which results in the strengthening or weakening of synaptic connections.
Underlying these changes in neuronal connectivity is the actin cytoskeleton, a cellular scaffold involved in a wide variety of cell functions. For example, it is along the cytoskeleton that chromosomes are segregated prior to cell division, and along which newly-formed synaptic vesicles, filled with neurotransmitter molecules, are transported to the nerve cell terminal. The cytoskeleton, stained green in the image on the left, is composed of microtubules (made of tubulin molecules), micro- filaments (made of actin molecules) and intermiediate filaments. It is a highly dynamic structure – the length of individual microtubules or microfilaments within the cytoskeleton changes constantly, by the addition or removal of its component molecules at one end or the other (polymerization and depolymerization, respectively).
In an advance online publication in the Proceedings of the National Academy of Sciences, Italian researchers describe experiments in which memory and learning were enhanced by a bacterial toxin which induces polymerization of cytoskeletal molecules.
Carla Fiorentini and her colleagues injected cytotoxic necrotizing factor 1 (CNF1), a toxic protein synthesized by the bacterium Escherichia coli, directly into the cerebral ventricles of mice. CNF1 exerts its effects by activating a group of proteins called Rho GTPases, a family of related proteins that are involved in a wide variety of functions, including regulating the polymerization of the actin cytoskeleton.
The effect of the toxin on the behaviour of the mice was examined in a series of tests. The animals were first trained to associate an electric shock with a specific conditioned stimulus. This ‘fear conditioning’ results the formation of associative emotional memories, in which a brain structure called the amygdala is known to be involved. By associating a particular context with an electric shock, the mice learn to avoid particular situations in which they can expect to receive a shock.
It was found that fear conditioning was improved in mice injected with CNF1, suggesting that the animals’ associative learning had been enhanced. The improved ability of CNF1-treated mice to find a hidden platform in a water maze showed that the toxin was also effective in enhancing the animals’ spatial memory. These improvements in fear conditioning and spatial memory were observed only in those animals treated with CNF1, and not in mice injected with either saline or a recombinant form of the protein in which an amino acid residue essential for its function had been modified. In the mice treated with CNF1, the improved learning was observed up to 28 days after conditioning had taken place.
The results of the behavioural tests led the researchers to investigate the effect of CNF1 in vitro. Slices of tissue from the neocortices of mice were cultured and treated with either CNF1, recombinant CNF1 or saline. Fluorescence microscope imaging revealed an enrichment of the cytoskeleton, as well as morphological changes in the dendritic trees, in the CNF1-treated cells, while electro- physiological studies showed that CNF1 enhanced neurotransmission in the hippocampus. The toxin apparently induced LTP in the hippocampal cells; by activating RhoA and Rac1, CNF1 was inducing the polymerization of actin molecules, thus driving the re-arrangement of cytoskeletal microfilaments and the synaptic modifications thought to be essential for memory formation.
As well as providing evidence for LTP, the findings raise the possibilty that pharmacological agents could be used to enhance the changes in neuronal connectivity associated with memory formation. They also identify cytoskeletal components as a possible target for such drugs. As well as improving cognition in healthy, these drugs could, in theory, be used to alleviate the cognitive impairments exhibited by patients with conditions such as Alzheimer’s Disease.
Diana, G., et al. (2007). Enhancement of learning after activation of cerebral Rho GTPases. Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0610059104