FROM HEARING TO SINGING: SENSORY TO MOTOR INFORMATION PROCESSING IN THE GRASSHOPPER BRAIN Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program "Systems Neuroscience (CSN)" of the Georg-August-University School of Science (GAUSS) submitted by Mit Balvantray Bhavsar from Ahmedabad, India Göttingen 2016 Thesis committee Prof.Dr. Andreas Stumpner Dept. Cellular neurobiology, Georg-August-University Göttingen Prof.Dr. Ralf Heinrich Dept. Cellular neurobiology, Georg-August-University Göttingen Prof.Dr. Hansjörg Scherberger The Neurobiology Laboratory, German Primate Center (DPZ), Göttingen Members of the examination board 1st supervisor and reviewer: Prof.Dr. Andreas Stumpner Dept. Cellular neurobiology, Georg-August-University Göttingen 2nd supervisor and reviewer: Prof.Dr. Ralf Heinrich Dept. Cellular neurobiology, Georg-August-University Göttingen Further members of the examination board Prof.Dr. Hansjörg Scherberger The Neurobiology Laboratory, German Primate Center (DPZ), Göttingen Prof.Dr. Henrik Bringmann Sleep and walking Laboratory, Max plank institute for biophysical chemistry, Göttingen Prof.Dr. Tim Göllisch Dept. of Ophthalmology, University medical center, Göttingen Prof.Dr. Gregor Bucher Dept. of Developmental Biology, Johann-Friedrich-Blumenbach Institute, Göttingen Date of oral examination: 13 May, 2016 I Declaration of academic honesty I herewith declare that the Ph.D. thesis entitled “From hearing to singing: sensory to motor information processing in the grasshopper brain” has been written independently and with no other sources and aids than quoted. Mit Balvantray Bhavsar Göttingen, February 2016 II To my parents…… III Contents Abstract………………………………………………………………………………………XI Chapter 1 General Introduction……………………………………………………………..1 1.1 Communication and its sensory aspects ............................................................................... 2 1.2 Acoustic communication in insects ...................................................................................... 2 1.3 Grasshoppers Chorthippus biguttulus as a model system .................................................... 3 1.4 Neuronal basis of hearing in grasshoppers ........................................................................... 4 1.5 Goal of the project ............................................................................................................... 5 1.6 Terminology to describe a grasshopper song ...................................................................... 7 1.7 Thesis outline ....................................................................................................................... 9 Chapter 2 Multielectrode recordings from auditory neurons in the brain of a small grasshopper .......................................................................................................... 10 Abstract .................................................................................................................................... 11 2.1 Introduction ........................................................................................................................ 12 2.2 Materials and methods ....................................................................................................... 13 2.2.1 Animals………………………………………………………………………....13 2.2.2 Animal preparation…….……………...……………………………………..….13 2.2.3 Multielectrode design and setup………………………………………...………13 2.2.4 Acoustic stimulation…………………………………………………………….14 2.2.5 Marking the recording locations………………………..……….………..…….15 2.2.6 Offline spike sorting….…………………………………..…………………….16 2.2.7 Collision analysis…..…………………………………..……………………….17 2.2.8 Constancy of recording conditions...……………………………...…………….18 2.2.9 Unit identification…………………………………………...………………….18 2.3 Results ................................................................................................................................ 19 2.3.1 Marking the recording locations……………………..……...………………….19 IV 2.3.2 Comparison between copper and tungsten wire recordings……..……………..20 2.3.3 Spike sorting…………………….……………………….……….….………….21 2.3.4 Collision analysis.........................................................................................…....23 2.3.5 Constancy of recording conditions….………………………….……………….24 2.3.6 Auditory units………………………….…………………….………………….25 2.3.7 Intensity response functions and unit identification………..……………..…….26 2.4 Discussion .......................................................................................................................... 30 2.4.1 Productions of multielectrodes...………………………...……..……………….30 2.4.2 Marking the recording locations………………………….…………....……….31 2.4.3 Constancy of recording conditions….………………………..………...………31 2.4.4 Spike sorting and collision analysis……….…………………...….……………32 2.4.5 Unit identification………………………..…..............................................…....33 2.4.6 Multielectrode recordings and song recognition in grasshoppers……..………..35 Chapter 3 Population coding among ascending neurons in the brain of a small grasshopper ........................................................................................................... 37 3.1 Introduction ........................................................................................................................ 38 3.2 Materials and methods ....................................................................................................... 40 3.2.1 Animals……………….………………………………………………………...40 3.2.2 Animal preparation…….……………...………………………………..……….40 3.2.3 Acoustic stimulation…………………………………………….………...…….40 3.2.4 Offline spike sorting and collision analysis...………………….……..…..…….42 3.2.5 Data analysis………….……………………………………………..………….42 3.2.6 Decoding using confusion matrix………………………………….…..……….43 3.3 Results ................................................................................................................................ 46 3.3.1 Unit identification……….………………………………………..…………….46 3.3.2 PCA based classification of ascending neurons…………...…………..…….….57 3.3.3 Summed activity for syllable-pause patterns…...………………….………..….58 3.3.4 Summed activity for syllable-gap patterns.…………….…………….…………67 V 3.3.5 Decoding the stimulus identity from the response………………………..…….70 3.4 Discussion .......................................................................................................................... 77 3.4.1 Problems in unit identification……….…………………….…………..……….77 3.4.2 Population response of ascending neurons…………...………..........……….….78 3.5 Outlook……………………………………………………………………………………81 Chapter 4 Recordings and electrical stimulation of local auditory neurons in the brain of a small grasshopper…………………………………………………………………………85 4.1 Introduction ........................................................................................................................ 83 4.2 Materials and methods ....................................................................................................... 84 4.2.1 Animals………………….………………………………………………..…….84 4.2.2 Animal preparation…….……………...………………………………………...84 4.2.3 Acoustic stimulation……………………………………………….…...……….84 4.2.4 Electrical stimulation…………………….....…………………………..……….86 4.2.5 Marking the recording/stimulation sites...……………………………….…..….86 4.2.6 Offline spike sorting…………….…………………..……………….………….87 4.2.7 Syllable-pause and gap tuning………….……………...…………….………….88 4.3 Results ................................................................................................................................ 88 4.3.1 Latency criteria……….……………………………………….………………...88 4.3.2 LBNs recorded in lateral protocerebrum and their locations...…………..….….90 4.3.3 LBNs recorded in anterior protocerebrum and their locations …………...…….97 4.3.4 LBN showing selectivity……...…..…………….………………...……..…….104 4.3.5 LBNs recorded in central complex and their locations ……….………………107 4.3.6 Electrical stimulation of the auditory neuropile ......………………….…….…113 4.3.7 Female Ch.biguttulus song structure…………………………………...……...114 4.3.8 Differences in singing while stimulating different sites……………………….116 4.3.9 Locations of the stimulation sites……………………. ……………………….117 4.4 Discussion ........................................................................................................................ 118 4.4.1 Recording auditory activity in the brain of a grasshopper…………………….118 VI 4.4.2 LBN recorded in lateral protocerebrum and central complex….….….……….118 4.4.3 LBN showing selective response……….……...…..…………….........………119 4.4.4 Electrical stimulation of auditory neuropile………..…………….........………120 4.5 Outlook ............................................................................................................................. 121 Chapter 5 General Discussion on method .......................................................................... 122 Abstract .................................................................................................................................. 123 5.1 Introduction ...................................................................................................................... 124 5.2 Type of material used for production of multielectrodes………………….…………….125 5.3 Number of neurons that can be recorded using multielectrodes ...................................... 127 5.4 Methods to mark the location of the recording ...............................................................