An Exciting New Age for Neuroscience

Neuroscience has undergone a technological revolution in recent years that has allowed us to begin to observe and manipulate neuronal circuits with unprecedented precision. Advances in genetics and molecular biology techniques have led to the introduction of optogenetic tools that allows us to selectively modulate (e.g. channelrhodopsin-2) and monitor (e.g. genetically encoded calcium indicator – GCaMP) specific groups of neurons within intact circuits while new microscopy techniques allow us to see deeper than ever into the living brain with greater spatial and temporal resolution. Super resolution microscopy techniques, including the STED and STORM variants, now allow us to image structures in the brain, such as dendritic spines, with resolution below the diffraction limit and even track the real-time movement of individual molecules within membranes. Our research uses a combination of electrophysiological, optical imaging and computational techniques to study the thin synaptic input receiving structures known as dendrites and to investigate their influence on the computational properties of individual neurons and circuits. Understanding these processes at this level of detail will help us to understand not only how important physiological systems operate but also how these systems break down in disease states.

The laboratory uses a range of techniques including:

  • Whole cell patch clamp recording – including patch clamp recordings from multiple (upto 4) neurons simultaneously.    
  • 2-photon calcium imaging  
  • 2-photon neurotransmitter uncaging  
  • 2-photon fluorescence targeted dendritic patch clamp recording       
  • Optogenetics
  • Immunohistochemistry
  • Computational modelling                                                 

2-photon microscopy

High resolution imaging deep into brain tissue or even the intact brain is beyond the limits of conventional linear single photon microscopy techniques because scattering of light significantly blurs the images. By exploiting the principle of 2-photon excitation originally predicted by Maria Goeppert-Mayer in 1931, in the early 1990’s Winfried Denk and colleagues in the laboratory of Watt W. Webb pioneered the use of 2-photon excitation laser-scanning microscopy. This technique has proven to be revolutionary in the study of neuronal circuits in awake behaving animals and has greatly enhanced our understanding of many critical brain processes. Several key factors are essential in making two photon microscopy the first choice in non-invasive fluorescence imaging in the brain. Firstly, and unlike conventional single photon fluorescence, the process of 2-photon excitation leads to highly localised three dimensional excitation. This occurs because 2-photon excitation requires two photons to arrive near simultaneously (~0.5 fs) at a fluorescent molecule (fluorophore) in order to combine their energies and promote the excitation of the molecule into an excited state (see below). The molecule then follows the normal fluorescence-emission pathway with the notable exception that the emitted photon is ‘bluer’ (i.e. shorter wavelength) than the excitation light used (typically infra-red greater than 800 nm).

In order for 2-photon excitation to occur, however, the excitation photons must be concentrated in space and time since the probability of a co-incident event occuring at ‘normal’ light intensity is very low. Temporal concentration is achieved by the use2-photon excitation of titanium:sapphire lasers that produce extremely brief (femtosecond) pulses of high intensity light. Spatial concentration is achieved by focussing the laser beam through a high numerical aperture (NA) objective lens. The combined effect of spatially and temporally concentrating photons in this way allows 2-photon excitation to occur and ensures that the diffraction limited point-spread function (i.e. the spot in which excitation occurs) of the microscope is extremely small (for high NA lenses the volume is typically less than 1 femtolitre). The ability to use 2-photon excitation to excite fluorescent molecules also means that much longer wavelength light can be used. Many typically fluorophores have peak single photon excitation in the range between 350-600 nm. However, their 2-photon excitation peaks occur at much longer wavelengths in the near infrared and infrared range of 700-1000 nm.  The ability to use these long wavelengths is particularly useful in whole brain imaging as they undergo significantly less scattering in tissue compared to  shorter wavelengths. This allows imaging at depths of greater than 1mm. In rodents this allows us to see into the deep layers of the neocortex of behaving animals.

Several excellent review articles discussing the development and implementation of 2-photon microscopy are available:

Helmchen F. and Denk W. (2005) Deep tissue two-photon microscopy. Nat. Methods 2(12): 932-940.

Svoboda K. and Yasuda R. (2006) Priciples of two-photon excitation microscopy and its applications to neuroscience. Neuron 50(6): 823-829.

Zipfel W.R., Williams R.M. and Webb W.W. (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21(11): 1369-1377.        

2-photon neurotransmitter uncaging     

2-photon neurotransmitter uncaging is a technique used for the activation of synapses with extremely high precision. It relies on the properties of 2-photon excitation, described above, to deliver highly spatially and temporally constrained packets of neurotransmitter to neurons to mimic the actions of neurotransmitter endogenously released at synapses. By using mirror galvanometers the small spot of 2-photon excitation (described above) that is required to cause the photoactivation of caged compounds (e.g. MNI-glutamate) can be rapidly (as fast as 0.1 ms between spots) moved from synapse to synapse. In this way we can study how neurons transform different spatial and temporal sequences of inputs they receive from their many thousands of synaptically connected neighbours into a cellular output (typically action potentials). This technique has contributed to our understanding that dendrites can perform comlicated non-linear input-output transformations that have key physiological roles.