RESUBMISSION: Neural processing underlying auditory pattern recognition in an insect brain

Neuroscience aims at understanding how sensory pathways are organised, how stimuli are processed and how features are extracted to elicit an appropriate motor response. Especially in intraspecific communication the recognition of specific stimulus features plays an important role as species-specific olfactory, visual and/or acoustic signals are employed for mate attraction and rivalry behaviour. As sounds are always transient, signalling by repetitive patterns of pulses is central to acoustic communication in many vertebrates and invertebrates. Especially frogs and insects like bush-crickets and crickets use simple sound pulses generated with species-specific temporal patterns for communication. In the auditory pathway of the receiver, these signals require neural filter mechanisms that respond to the temporal structure of sound patterns. Considerable progress has been made in frogs in describing brain neurons with selective tuning to pulse patterns. However, there is still a very limited understanding of neurons underlying temporal processing of sound patterns in crickets although since decades they are a model system for insect hearing. Female crickets are attracted to the species-specific pattern of male calling song. In behavioural experiments we demonstrated the tuning of their phonotactic behaviour to changes in pulse interval, pulse duration and chirp interval. For example their phonotactic behaviour is tuned to the pulse pattern of the male song as pulses with shorter or longer periods are not attractive. We now aim to understand at the level of identified neurons the filter mechanisms in the brain that allow the recognition of the species-specific song pattern.
The ascending auditory interneuron that forwards the pulse pattern of the calling song from the first thoracic ganglion to the brain exhibits no filter properties. Hypothesis for temporal filtering in the brain have been put forward, however, the actual neural mechanisms have not been revealed. In preliminary experiments we identified local brain neurons which form a ring-like auditory neuropil in the protocerebrum matching the axonal arborisations of the ascending interneuron. Some of these neurons showed a selective response to the pulse interval of the calling song and received inhibitory and excitatory synaptic inputs. In other systems the interaction between inhibition and excitation is crucial for selectivity to pulse patterns. Using the same acoustic paradigms as in the behavioural studies we will analyse the activity of auditory brain neurons. We will focus on four questions:
1). Which brain neurons are involved in temporal filtering of acoustic pulse patterns?
2). What are the neural mechanisms underlying temporal filtering?
3). What is the structural and functional organisation of the auditory neuropil?
4). Which projection neurons connect the auditory neuropil to other brain regions?
The project will be based on our experience of recording intracellularly the activity of auditory brain neurons with sharp microelectrodes. The neural responses will be compared with our behavioural data and quantitative analysis will reveal to what degree the activity of single neurons mirrors the behavioural tuning. The pattern of inhibitory and excitatory synaptic activity will provide crucial information on how temporal filtering is achieved within the auditory network. We will manipulate the membrane potential of neurons by intracellular current injection to analyse the nature of the neural filter mechanisms in detail. Finally we will use confocal microscopy to reveal the structural details of these neurons in the brain and we will identify neurons which link the auditory neuropil to other areas of the brain. The analysis of the pattern recognition network in crickets will provide insight to principle mechanisms of temporal filtering at the level of identified neurons and can be a model for temporal selectivity in other systems.

Experiments are based on our experience to record the activity of auditory brain neurons. The head of tethered crickets will be positioned in an Eppendorf tube and the head capsule will be opened to expose the brain, which will be stabilized between a silver platform and a ring. Computer generated acoustic test paradigms with systematic variations in pulse interval, pulse duration and pulse period will be presented. The behavioural tuning of females to these paradigms has already been established in behavioural experiments. Sound patterns will be presented by two audio speakers positioned at 45 deg left and right in front of the crickets. During acoustic stimulation we will record the synaptic and spike activity of brain neurons using sharp glass-microelectrodes. Signals will be amplified with high-impedance DC amplifiers and stored on-line to the hard-disk of a PC. For off-line analysis of the neural activity we will use Spike 2 and our custom-designed software Neurolab. Analysis will focus on spike activity and the timing of excitatory and inhibitory postsynaptic potentials relative to the acoustic stimulus patterns. This will reveal to what degree the tuning of neurons matches the tuning of behaviour. The structure of brain neurons will be revealed by intracellular iontophoretic labelling with fluorescent dyes like Alexa or Lucifer yellow followed by histological processing. Brains will be counterstained with antibodies against e.g. synapsin or bruchpilot to reveal the organisation of the auditory neuropil and the projection areas of neurons in whole mounts using a confocal laser scanning microscope. Neural arborisation patterns will be reconstructed from confocal stacks. The named RA has excellent experience with the dissection, intracellular recording and labelling techniques of brain neurons. Most equipment required for the experiments is available and already set up. The Department runs a colony of Gryllus bimaculatus that will be used for the experiments.

1. Neuroscience community: In 2011 the PI gave the prestigious Florey Lecture of the German Neuroscience Society. For 2012 the PI is invited to conferences on the Central Complex of the Insect Brain and on the Evolution of Acoustic Communication Systems at Janelia Farm. The PI will also speak at the Conference on Crickets organised by Prof. S. Noji (Japan), which has been be rescheduled for March 2012. The PI will use these and future meetings to share research methods, to discuss his research with other scientists and to build up collaborations. Neuroscientists exploring the insect brain will benefit from our specific recording techniques; research on auditory pathways in more complex brains may use the outcome of the project to tests hypothesis on processing of temporal patterns. Textbooks describing auditory processing in insects may need revision.

2. Research collaboration: The PI collaborates with the group of Prof. S. Noji (Tokushima/Japan) and with Dr. H. Horch (Bowdoin/US), which aim to generate transgenic crickets expressing calcium reporters like GCaMP3 in their central nervous system. Once this genetic approach is successful, our analysis of identified auditory neurons will be of major advantage. Calcium reporters will allow studying signal processing with both imaging and neurophysiological techniques at the level of identified neurons and neural networks. By combining the research methods of the different labs to study cricket auditory processing we expect new avenues and strong synergetic effects for the analysis of the system (see statements of Prof. Noji and Prof. Horch).

The PI also has an ongoing collaboration with Prof. B. Webb (Edinburgh), who develops bio-inspired robots designed to perform acoustic orientation. As soon as a proper picture of the pattern recognition circuit in the brain of crickets has emerged, our should allow her to implement more realistic neural networks and to fine tune the control circuits of the robots (see statement of Prof. Webb). Travel costs to Edinburgh will occur.

The PI will explore possible collaboration with Prof. K Shaw who is an expert in the field of the genetic basis of cricket speciation.

3. Engage with the public: Two events in Cambridge are suited to give the public insight to our research: the Science Week and the Conversatione of the Natural History Society. The PI will present either a talk to a wider audience or a cricket trackball system used for behavioural studies together with high speed video recordings from walking crickets. Some minor costs for poster printing and a suitable large video monitor will occur. We also will also use opportunities to present our research in radio or TV programs.

4. Industrial exploitation: The PI contacted the research group of Siemens Audiology in Erlangen/Germany, a leading manufacturer of hearing aids. It is my intention to introduce the findings on cricket auditory processing to the engineers from Siemens. Consequently principles of auditory processing may be incorporated into the electronic circuits of hearing aids. Travel costs for a trip to the Siemens group have been included. (see statement from Siemens Audiology).

5. Capacity and involvement: The RA will be trained in neurophysiological research techniques, and will be strongly involved the management of project objectives, time and resources, and in the publication process. The RA will present data at scientific conferences and contribute to any public science events. An essential part of the training will deal with job and grant applications to secure the next position for the RA. The PI will provide substantial support of these RA activities in form of discussions of research results, proof reading of manuscripts and applications, and with advice on career development. Cambridge University runs an excellent program career development and transferable skills. The RA will be strongly encouraged to attend such seminars.