Integration

An Integrated view Nerve Muscle and Movement

Assessment  SA Q totalling 70  Specimen paper  http://biolpc22.york.ac.uk/404

 Practical worth 30 marks, deadline 18 Dec  Submit

1 practical report

To join together… Nerve conduction Synaptic physiology Muscle contraction Mechanics of Motion Axon guidance what could be better than … …fly jumping? with a little help from our genetics friends

Aim  How a fly is built to get away  Key reference  Allen, MJ et al (2006) Making an escape: Development and function of the Drosophila giant fibre system Sem Cell & Devel Biol. 17: 31-41

Genetic tools  EMS-induced mutations  Sequenced genome  UAS GAL4 system  tissue

specific knockouts  tissue specific GFP  tissue may be a few cells

How does a fly jump?

Jump using middle leg

Trimarchi & Schneiderman

max distance jumped (mm) mean ± SE

How far do they go?

30

 Wild type flies go 30 mm

20 10 0

1

2

3

4

5

6

CS female fly #

7

How much work/force?  Work  KE

= ½ m g d = ½ 10-6 x 10 x 0.03 = 150 nJ  Power output = 40 µW or 300 W / kg  at the top end of insect muscle output

 Force  measure

contraction isometrically  peak force 25 x weight of fly

Which muscles?  zap head and record muscle potentials

here given one small and one large stimulus

Summary  thoracic muscles, very energetically demanding

Now onto: what neuromuscular systems does the fly use? (What’s in a fly???)

What’s in a fly? IFM TDT GDN CNS mn foregut

VNC tc femur tibia

tarsus

tc - trochanter mn - motor neuron GDN - Giant descending neuron [= GF] IFM – Indirect flight muscles TDT – tergal depressor of the trochanter [= TTM] VNC - ventral nerve cord

What's in the fly CNS ? brain thoracic ganglion

Plan  start with  muscle  motoneuron  giant

descending interneuron  sensory input

 development

TDT muscle

Koenig & Ikeda, 2005

this end pulls • the wing, • thorax, • stretching the IFMs

 TDT has a double whammy

this end pulls • the leg straight

TDT in section

TDT is…  Striated muscle  Tubular muscle  Fast twitch

Innervation  innervated by 3 motoneurons  1 large – very extensive endings  2 small

Neuromodulation  by octopamine – containing neuron

TDT motoneuron  thoracic nervous system  lateral cell body  dorsal neuropil

Summary  thoracic muscles, very energetically demanding  muscle and motoneuron designed for speed

PSI  Relay between GDN and ?  drives

5 DLM motoneurons  failure

occurs separately

 Amplifier ?

GDN (=GF)

GDN

PSI

TDTmn

GDN → TDTmn synapse  electrical ↑  chemical ▼  ACh

GDN → TDTmn synapse  shakingB2  no

electrical synapses  an innexin mutant  asymmetry in innexins

 shakingB2 and chats2  neither

electrical nor cholinergic synapses

Axonal conduction in GDN  AP with para Na+ channels and K channels  identified

shaker potassium channels  differentiate sh from slo  sh

– voltage activated K channel  slo - Ca activated K channel

Excitation of GDN zap head  Visual

flash light

+benzaldehyde

Fly eye

Visual input to GDN  Cobalt fill of GDN in Musca lobular cells probably electrically coupled to GDN

Mechanosensory input

antennal endings GDN (PDB segment)

Summary  thoracic muscles, very energetically demanding  muscle and motoneuron designed for speed  GDN circuit designed for speed and robustness Now onto: how does the circuit grow?

Development  GDN & TDTmn born during embryogenesis  Connect during pupation

Key steps  GDN neurite outgrowth  Axon pathfinding (larval stages—24 h APF)  Target recognition and initial synapse formation (24–55 h APF)  meet

TDTmn

 bend

 Synapse stabilization and maintenance (55– 100 h APF)  So what are the Molecular regulators of growth

bendless  Giant axon stops and does not bend  Part of ubiqutination system for degrading proteins  This degrades signal saying “go”

Semaphorin-1a  Regulates neurite outgrowth  No

sema-1a GDN axon goes to retina (50%)

 Regulates bend  No

sema-1a GDN axon does not bend (50%)  May be the protein bendless degrades

Target of sema-1a  Plexins ?  Which

signal via Rac, a GTPase

Too much rac

rac blocked

Summary  thoracic muscles, very energetically demanding  muscle and motoneuron designed for speed  GDN circuit designed for speed and robustness  Identification of signalling molecules controlling neuronal growth & synapses

Habituation of jump response

dunce (phosphodiesterase) & rutabaga (adenyl cyclase)

Jumping as a test for disease  Epilepsy

Mutants hyperexcitable followed by paralysis

eas

+/+ eas prior after bang

Flies as genetic models  Parkinsonism, Alzheimer, Fragile X…

 Behaviour, anatomy, physiology, cell biology well known  Screen for modifiers

Summary  thoracic muscles, very energetically demanding  muscle and motoneuron designed for speed  GDN circuit designed for speed and robustness  Identification of signalling molecules controlling neuronal growth & synapses  System for physiological mutant analysis