Neuroetologie a smyslová neurofyziologie Behaviorální neurobiologie Vztah mezi nervovým systémem a chováním je velmi těsný motivation rhythms inhibition sense organs sensory pathway integration motor pathway muscles hormones memory Problémy neurofyziologie se často zkoumají sledováním chování: •Pohyb •Cirkadiánní rytmy •Smyslové schopnosti •Působení drog a farmak •Agresivita •Stárnutí •Paměť a učení... Pohyb ■---------------------------------------------------------------------------------------- —..... .......... Wind velocity control .-^ _>" >* Wind tunnel Strobe lamp Camera Preamplifiers ] Lift scale Strobe control Oscilloscope and camera Cirkadiánní rytmy ..—^-^-------------------- -1^ **»Hfci Ť ■Ty., , 71 Iv *■""" j t/ Smyslové schopnosti electric shutter J*^"- hcat beam yaw torque signal flaue ^ UTOue mel light source light guides stepping motor opaque screen pattern position Stárnutí ™ n * rw ■ ■ Hl b ■ ■ ■ ■ Agresivita Působení drog a farmak Mutace genu pro mateřské chování Sociální deprivace Orientace a navigace živočichu **H *taipdii- Lij Um-" - . il. itrvHm in<> ri-< ri ^ bJH h TÜÜ.".*^*1^ " MMhi U f tau *vJJ t^rt I IHFMV IM*- lit Immmmawtikr íám y^-mmßi +*w wpmimi -h «ft* i»n aaivq i-Ji-i rtu "^hK . If. urtaiLSfirtKwf . 2ľ-!Jahi#RS4tfrh#i i ■ hr ■*■* rtu m Vi figure. The orientation and size of each black bar mark the angle and degree (percentage) of polarization, respectively. The open circle indicates the zenith. The solar meridian (the line from the zenith down to the horizon) and the anti-solar meridian represent the symmetry plane of the celestial E-vector pattern. From Wehner (1994a). g— Ô spS^*^*,*«^* h 270° 300° m (b) Figure 10.10 Maze used for demonstrating the existence of a cognitive map. (a) The rat depletes arms 1-7 of food. It is then removed from the maze for a short while, (b) The maze is rotated and the rat i.s returned. I MIHI .:: ■ ü" * J • UiImMiI ■"■\r.z:~z ■ ■■ uu LĽ *-*. ■ mi ■■ ■ " -m . i -r . r1 (A) Training (R) (R) (L) (L) Test (R) (L) (L) DD DD m o ■# Nŕin» L X- O ' o fc\* Figura 1 Routes around a barrier, (a) Desert ants performed a detour around a 10m long barrier on their homeward journey after visiting a feeder 10 m south of their nest. Returning ants about to enter their nest were taken to the end of an identical barrier on a test ground, (b) Barrier in test orientation: ants recreate the path that, in training, took them to the nest, (c) Barrier rotated through 45". The ants' path is controlled by the barrier, rather than by their sky compass. (Redrawn from [15].) 0) oo 5 6 Figure 10* The rat's behaviour in the tank employed by Morris. The rat's route over six trials is shown. At first (1) the rat takes a while to locate die submerged platform. Over trials (2)-(6), the improvement in performance is clear. Note that, irrespective of where the rat starts off, it finally comes to swim directly to the platform. Insects as a model group for sensory neurophysiology •Extraordinary sensory capabilities •Simple and well described nervous system •Easy accessible NS •Durability of preparates •Some species easily reared in lab •Almost unlimited numbers of animals •No animal rights paperwork yet Behavioral assay precedes neuro- and molecular approaches •Electrophysiology •Immunohistochemistry •Genetics •Transcription factors •Lesions •etc. From: Brembs, B.": (2003) Operant conditioning in invertebrates. Current Opinion in Neurobiology, 13, 710-717. Insects as a model for analysis of reception and signal processing ■>i ft 4c| ■■*! From: Tegoni.M, Campanacci, V., Cambillau, Ch.: (2004) Operant conditioning in invertebrates. TRENDS in Biochemical Sciences Vol.29 No.5 May 2004. •Chemoreception •Mechanoreception •Polarised light •IR detection •Memory Vytvoření podmíněného spoje mezi dvěma podněty je metodou studia procesů paměti. Vytvoření podmíněného spoje mezi dvěma podněty je také metodou studia smyslové percepce. Odměna a trest ■'; :>-- II ■*: v m*+ i m $-.&» »> m.w ^ . lull Ir 111!: Kompasová orientace -magnetický smysl živočichů Zdroj pole B (T) MGP vznikající činností mozku 10-12_10-14 Geomagnetické pole ÍO"5 Nejsilnější permanentní magnety KT1 - 10° Supravodivé elektromagnety ÍO1 - 102 IN Azimut ' a / • • S tit/ l,rpĽS- ňř iiLi^rnfl^ řťívV r^i^f aphlcjj frciditlünalJT nartliput* V \ *,„. ulétle ~h**. \ ! / y jih jHjj;m-ric pťíie AM» nuS""* I*K * *rP ^tttHrApEiirjiJ *.\|LĽit^r andThfllKlIiuüDn JUtgtit of 11ip m?gnfiIfC ffeldc^gltfe i "inpsEi Jnřurmd(Hjn. ítfULU m.i^nelliľ piife phü-.iHnihitk?iiaH fiflUthpolľ m.ipncřh: horizontální složka Northern magnetic pole Southern magnetic pole Fig. 1. Schematic view of the earth and the geomagnetic field. ieo" 90* N 60* N 30'N- 0' 30'S-J 60" S Inclination 9C'S 180" 120'W ■ I 60*W 0° 60*E 120*E iao" 120"W 60" W + I 6o*e i 120" E 90" N 60N -30*N -oe - 30S -60*S 9ďS 180" Fig. 1.3, Inclination or dip of the magnetic field of the earth (epoch 1965); negative signs designate upward inclination. (After Skiles 1985) total intensity á (n"0 47 950 47 930- 47 910 47B90 47 870 - 47 850 f- —r~~" 6:00 1--------r —i--------r 9:00 Denní variace GMP 31 7.89 I 12:00 i------------«-------------1------------1------------1------ 15:00 18:00 time (UTC) -2 O 2 3 3 2 001 23 4 5 -1 0 4 5 2 3 57911 11 10 9 8 7 6 ieo" 90* N 60* N 30'N- 0' 30'S-J 60" S Inclination 9C'S 180" 120'W ■ I 60*W 0° 60*E 120*E iao" 120"W 60" W + I 6o*e i 120" E 90" N 60N -30*N -oe - 30S -60*S 9ďS 180" Fig. 1.3, Inclination or dip of the magnetic field of the earth (epoch 1965); negative signs designate upward inclination. (After Skiles 1985) 180 120* W 60 W 90" N 60*E I I 120" E ■ ■t 180" 90*N 60"N- Total Intensity (in 1000 nT) 30BH- 30' S 60* S - 90* S -60N -30*N 30*S Í-60'S 90"S 60'E 120* E Fig. 1.4. Total intensity of the magnetic field of the earth (epoch 1965) in 1000 nT. (After Skiles 1985) -2 O 2 3 3 2 001 23 4 5 -1 0 4 5 2 3 57911 11 10 9 8 7 6 Měření GMP Insect magnetoreception path quite unknown today Vertebrate brain Insect brain ĺ i y — TW..... % ůfů I «-»I - T '% 'i Nemec, P., H. Burda, et al. (2005). "Towards the neural basis of magnetoreception: a neuroanatomical approach." Naturwissenschaften 92: 151 -157. A) Position behaviour Alignement the body axis with the cardinal geomagnetic axes Resting positions Termites, Flies, Cockroaches, Bees Altman, (1981) •. ** "-Sak ***** TrA ♦*•«♦! w45j ;.í*»*íí^ sje •TS * * S* gíůmagnelic field t*. ^ sä».',- «fT^ K ^É^ compensated magnetic field r** Becker, (1965) Směr vnějšího pole i Expanze Kontrakce i Směr vnějšího pole (a) To brain / Ophthalmic nerve Mandibular nerve (b) Spont.a. 1 / Maxillary nerve Trigeminální ganglium Dolichornix 15000 nT 200 nT 2 5000 nT 3 2mV [_ 50 ms ô 25000 nT 100.000 nT ÖD tí w Singletový stav Tripletový stav excitace fotonem recepce světla Opsin-cis retinal v základním stavu Trans retinal + opsin MP štěpí tripletní stavy a mění pravděpodobnost přeskoků Energie intenzita MP N Polarity vs BH — — mN E B, Local Field Polar Comp a» Inclination Compass Inclination & ■h ------- Vertical Component Reversal '•í. * 1 4ř . roť.. «V k_Mfc '■■r^* Aflh- "s^hbI É* ^^ ^^^(c *. Wtf 'Íf^;,>-.' í * -^Hj. ""^W" * s. " *H£3rľ \! - , ifkj^Ě MEH ■"-• aMlPJ. . %| ^_^___^ ^"**T ^* ■P"^ ■ | "I HPV fc j' rr ift^ Z^ta v*^ flÜ ■ MM T, Ihf I|f Chjiyn IIW Flip ■ Ik In4d clump i \rm dj i lttliJ pbtrhchj ľiLl il Lmrw j*n P4*pY1 h+*l -** i lW-h fHCW «*--ii t* k atw-:*-rmzln.-m uml h-P» rwrirj«- T* do-> un cri üif Apfhrtit I vil lb •..■ tk^m: iJV> HrtrpforfU < Irfa-.j I . .■■■■..-- ..■ .-■.ť..:Jb,.,'rf ■. —| •■ *»H I «1*1 H KH* »Triu n»«.* úfa rill-H N-irikHTi riítrldj AlNIMAi řfAtfi&ATK I 45 S i« 34hlf ppWi iM* i h h 4 I I I I ■ 1 -I h k I ■ I I i Training Rig (Phillips and Sayeed, 1993) a imsn Q. O —I-------■— I ■ M t ■■ES f \ k Magnetoreception in Drosophila (Phillips and Sayeed, 1993) Top Loading Magnetoreception in Drosophila (Phillips and Sayeed, 1993) Top Loading Magnetorecepce potemníka moučného - Tenebrio molitor s © Trénovací tubus Testovací aréna 11 10] •° 4, vj?^ ^^ N v 2 1 w^/* ^k -ŕ 3l \ 10 <3> i luL k- Ji ■n j _g^ n 11 10] 4-i n 11 10] 4-, 12 11 IQ] k Wv \TZ^^—- \ _x-^Í [io[ 0 1 rk /tím _£^ \ v^"" T^ ■• x 2^^^B W/%/ vr \3H Um | g^ 11 4-i \% T, t ■^^ ^^^^ ^^k_ _^E ^^H ^^^ ' ~^M ^^E__^ft^H fZ-T^ž. ' r^_ ■ ^^fi rj 1 ^b^JI ^F 1 ^ ^^^= 'V* / >- Tma 0%RH N = 100 6 = 60° * o ^o-MJi í -S3 J--■■■■i Mfl PP' 1 T« i- r** JlLUiliilINi r Ir ■ 1 i I i II í Et -■■■-. rtu m ipi; Isc-M Hw,:| íŕ*-*« I Wa I "H:o s ■ ■ ■■■■ w m« ...m jnr a) Spontaneous - untrained reaction ■ r_. . .r. irr. L. J -^.r ■ .1 ■ ■ ■■■■ w m« ...m jnr a) Spontaneous - untrained reaction a) Spontaneous -■untrained reaction Before treatment After treatment HI lili B iiiiiiiiiiiiiiiiiiiiiiiiiiiiiV Ih B "■■ B iiiiiiiiiiiiiiiiiiiiiiiiiiiiiV IF iF ^1 (gwalflWWI Amplitude + Time = Integral (Area, Effective amplitude) ■ r_ .fH— irr. L. n i H 'Bi p^p^^^H| r ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 31 After treatment 4 ■ ■ ■ . i .1 ■ ■ ■■■■ w ngf ...m jnr Before treatment i ■ m __&. '- — j ■. ilIÉfiwbl hJ HI H J Area = 82 _L u u i- Mr f ■ 1ipq |tji^i j j-. -. . . - p. liI__RI_ i H i_dr^_ ' -w .__■_—_ _ ■ ■_■'*■ FY ■ ■ - 1 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment Area = 59 ■ .1 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment 1 y . 'SOT I« -A Area = 80 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment \ i I * kfu \ T™ T-" Area = 183 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment Area = 250 ■ .1 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment Area = 317 ■ .1 ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment 4 r' : H ■ . Area = 795 ■ .i ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment 4 r' : H ■ . Area = 981 ■ .i ■ ■ ■■■■ w m« ...m jnr Before treatment i Area = 390 After treatment 4 r' : H ■ . Area = 990 ■ .i ■ ■ ■■■■ w m« ...m jnr Before treatment ^i—* Area = 390 i Wilcoxon pair test ■ r_ .fH— irr. ĺ M ^ ■* l ^b kl -r ■-■ aji__i J -"■ After treatment 4 ■ ■ ■ . Area = 990 ■ .i Frequency o (Q > (D ■0 > 03 ? O 3 3 (D Q. Q. 3 (D (0 (0 Untrained light 26 fr 24 § 22 Í- 20 £ 18 "- 16 14 12 10 8 6 4 2 0 IBefore=53762 IAfter=78691 n=80, Wilcoxon P=n.s. Fxx| Before After ■0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 log Aef Conclusion: Animals did not react spontaneously - no perception ? - no motivation ? Hypothesis: Provided pupae perceive MF, their reaction should be enhanced by negative reinforcement. b) Trained - conditioned reaction Magnetic pulse (10s) Heat pulse (15s) 12 times per night b) Trained - conditioned reaction Movement detector Helmholtz coil MG pulse Heat pulse I"C L IB l_k -W '■ .A * >^_. wt -í■ I 1 .191 . 1II -n ■ JI ' ■ ■ «-•I H ri re ĺ is i_k j- ■■ .a * >^_. j ^11 i .i9i ■ rt _ j j. Conditioned darkness 26 fr 24 c o 3 O" o 22 20 18 LL 16 14 12 10 8 6 4 2 0 IBefore=6535 IAfter=10046 n=87,Wilcoxon / P=0,002** [33 Before After ■0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 log Aef -I:1« ..i ÍT'HRJ &>-'& American cockroach Periplanetta americana J Wf i f / 7 Methods: 2x4 Helmholtz coil system rotating GM F 60°CW i 1 Results:' v\ "... v ■'••, '•••, **» ■■»»... ■iii.J .."■" ....»'" HUM ŕ" ÉÉÉ ."'" ..■' y N=27(1293) Natural Field \u / y North ______ "... "MU, '"■"., ""t, ... .................. 'É. --------- "". "., "'*. N; "». \ \ ■v\ "... v ■'•», '■■■, "t "»»..i ■i.i.J .."■" .......... ŕ" ÉÉÉ .""" .*" ^> y N=27(1293) / Natural Field 11 ii North I - + -I I // y +< .."' ..**• É»«" ::é t^^^Éti ^ifcü; .t^^.t ::^í: "... "MU, '"■"., ""t, ... .................. 'É. --------- "". "., "'*. N; "•. \ i í ! \ •* ■5 •• *1 ___\J/ fe_ ■::»»:::; -w —4f£— e -šttttttxt: "*^ geamagnelic field Eííí.%7 »h- W * S o^ oompereated magnetic lie Id Becker, (1965) .................. N=27(1262) Field Rot. 60° CW Field Rot. 60° CW .................. N=27(1262) s^ ..x. ■'••, '•••, ■.. ■■»»... •ÉÉÉti .."•" lltk»>" HiiM *" ÉÉÉ ."'" É*«' y ÉÉ»»" Quadrimodal orientation Mean vectors distribution Natural Field Field Rot. 60° CW B) Body turns in rotating field **» 13 '«* / Time schedule: Control i 4pm 6pm Oam 6am Dish loading 10pm 1pm 6pm 11.30pm 2.30pm V. J Y 1 picture/min = 270 samples Time schedule: Test GMF60°CW rotation Pulses 5min/5min 4pm 6pm Oam Dish loading 6am 10pm 1pm 6pm 11.30pm 2.30pm V. J Y 1 picture/min = 270 samples í f \*» ^ m*J Z" \. ( ^^ . 1 c y 7 I **» 13 '«* / í" li ■F J r / F f V' i ^%4 ^ '«* IZDOS- OC-01 13:1ü:?l| ^~ ^^T F> ■\^^^^ (* • J A ( i r ^L \ \ ) $7> S Results: 9 8 7 (O I 6 (O F 4 Ä 3 o m 2 GMF 60°CW rotation far test group D test group D control group 10.00 1 10.45 2 11.30 3 12.15 4 13.00 5 13.45 6 14.30 Time and experimental periods (n=62/66) Results: 9 8 7 (O I 6 (O F 4 Ä 3 o m 2 GMF 60°CW rotation far test group D test group D control group y T- T- T- 10.00 1 10.45 2 11.30 3 12.15 4 13.00 5 13.45 6 14.30 Time and experimental periods (n=62/66) Projekt kryptochromy • Metody reverzní genetiky - iRNA • Chirurgie - leze, ablace • Imunochistochemie Central brain Opile Cabe