Corollary discharge modulation of wind-sensitive interneurons in the singing cricket Stefan Schöneich and Berthold Hedwig Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK (Email:
[email protected] /
[email protected])
Summary
Experimental Design
Eserine Injection Brain
Eserine microinjection in specific brain neuropiles elicits singing motor pattern generation in the ventral nerve cord. After cutting all thoracic nerves the “fictive” singing pattern can be recorded as rhythmic motoneuron activity in the truncated wing nerves (open and closed circles mark the wing-opener and wing-closer motoneuron bursts).
SOG
Cricket CNS T1
vGI8-1a
wing nerve T2
T2-N3A
T3-A2
5 mV 50 ms
A3 A4
vGI8-1a
A5 A6
Wind TAG
Cerci
During fictive singing, we intracellularely recorded wind-sensitive afferents and interneurons in the cercal glomerulus of the terminal ganglion. At the end of the experiments the recorded neurons were labeled with fluorescent markers for subsequent morphological identification according the drawings by Jacobs & Murphey 1987.
Cercal Filiform Afferent
Besides sound, the forewing movements of a singing cricket produce notable air currents that stimulate its highly wind-sensitive cercal mechanoreceptors, which would reliably trigger fast predator avoidance reactions in the resting cricket. The male cricket, however, usually sings continuously over several hours while only external wind stimulation may elicit situational predator avoidance responses ranging from either transient silencing to sudden escape. To investigate how the CNS manages to discriminate between such self-generated and external wind stimulation during singing, we recorded intracellularly the activity of wind-sensitive afferents and interneurons in the terminal ganglion of fictively singing crickets. Our recordings revealed rhythmic corollary discharge inhibition (IPSPs) in the dendrites of the ventral giant interneurons 8-1a and 8-1b, but no indication whatsoever for primary afferent depolarizations (PADs) in the terminal branches of the wind-sensitive cercal afferents in phase with the syllable rhythm of singing motor pattern. In fictively singing crickets, where no selfgenerated wind occurs, postsynaptic corollary discharge inhibition largely reduced the spike responses of those two giant interneurons towards external wind stimulation, when the stimulus coincided with the singing motor activity. Paired intracellular recordings revealed that the identified mesothoracic corollary discharge interneuron (T2-CDI), which pre- and postsynaptically inhibits auditory interneurons in singing crickets (Poulet & Hedwig, Science 311:518-522, 2006), is not responsible for the rhythmic inhibition of the recorded giant interneurons, but receives inhibition (IPSPs) in phase with rhythmic cercal wind stimulation. Reducing the responsiveness of wind-sensitive giant interneurons by postsynaptic corollary discharge inhibition is a suitable mechanism to prevent inadvertent triggering of escape reactions by self-generated wind stimulation during singing while maintaining the sensitivity of the cercal predator avoidance pathway. Furthermore our data indicates that the corollary discharge inhibitions in the auditory and wind-sensitive pathways during singing originate from different corollary discharge interneurons, whereby cercal wind stimulation reduces corollary discharge inhibition in the auditory pathway.
Paired intracellular recordings of the Median Giant Interneuron (vGI8-1a) and the mesothoracic corollary discharge interneuron (T2-CDI)
spontaneous afferent activity 60 Afferent spike amplitude
4 mV 100ms
Afferent
mV
TAG
T2
40
Wing Nerve
Chirps=30
T2-CDI
Relative Activity
Wing Nerve
-100 0 100 200 Time to First Syllable in ms
vGI8-1a (MGI)
afferent response to cercal wind stimulation
right cercal glomerulus
* * * * *
Wing Nerve
0.3 Afferent spikes
Wind
25mV 10mV 100ms
AP/ bin
Afferent
left cercal nerve
Wing Nerve
right cercal nerve
Chirps=100
T2-CDI
Relative Activity
Wing Nerve
Intracellular recordings from the axonal terminals of cercal afferents in the terminal ganglion revealed no evidence for presynaptic corollary discharge inhibition at the synapse in the cercal wind-sensitive pathway. We recorded neither PADs nor was there a reduction of the afferent spike amplitudes during fictive singing.
+2nA
0
vGI8-1a
-100 0 100 200 Time to First Syllable in ms
TAG
-2nA
current
T2-CDI
25mV 10mV 200ms
vGI8-1a (MGI)
afferent recording after cercal nerve transection
Cercal wind stimulation
Wing Nerve
100mV
Wind
Afferent membrane potential 2mV
Afferent
Relative Activity
Wing Nerve
Wing nerve
Chirps=20 Fictive Singing T2-CDI hyperpolarized
Fictive Singing
-100 0 100 200 Time to First Syllable in ms
-2nA
T2-CDI
Fictive Singing T2-CDI depolarized +2nA
No Singing T2-CDI depolarized +2nA
APs=500
20mV
Giant Interneuron 8-1b
rhythmic inhibition of GI8-1b during singing GI8-1b
8 mV 150ms
4mV
GI8-1b
***
***
Chirps=40
Wing Nerve
***
The identified corollary discharge interneuron (T2-CDI), which had previously been shown to inhibit auditory afferents and interneurons during singing (Poulet & Hedwig 2006), is not responsible for corollary discharge inhibition of the wind-sensitive giant interneuron vGI8-1a during singing.
-100 0 100 200 Time to First Syllable in ms
reduces current injection (+3nA) driven spike generation
1 GI8-1b
Chirps=30
GI8-1b receives postsynaptic corollary discharge inhibition (marked by red asterisks) in the syllable rhythm of fictive singing.
***
0
*
** *
Wing Nerve
Wind
vGI8-1a (MGI)
rhythmic inhibition of GI8-1a during singing
*
*
Chirps=40
Chirps=40
5mV
-20 0 20 40 60 Time to First Syllable in ms
-20 0 20 40 60 Time to First Syllable in ms
-20 0 20 40 60 Time to First Syllable in ms
-20 0 20 40 60 Time to T2-CDI AP in ms
Cycles=100
50Hz
Cycles=30
cercal wind stimulation
20mV 250ms vGI8-1a recording
Chirps=40
T2-CDI T2-CDI recording
3 mV
4mV
*
*
*
2mV 130ms
*
*
**
*
Wing Nerve
Wing Nerve Relative Activity
Wing Nerve -100 0 100 200 Time to First Syllable in ms
Wind
Wind
Wing Nerve
*
20mV vGI8-1a (MGI)
vGI8-1a (MGI)
GI8-1a receives postsynaptic corollary discharge inhibition (marked by red asterisks) in the syllable rhythm of fictive singing.
Chirps=40
*
25Hz -100 0 100 200 Time to First Syllable in ms
vGI8-1a (MGI)
Wing Nerve
*
Relative Activity
* *
Wing Nerve
Median Giant Interneuron (vGI8-1a)
*
The identified mesothoracic corollary discharge interneuron (T2-CDI), which rhythmically inhibits the auditory pathway during singing, receives inhibitory inputs from the wind-sensitive cercal pathway. This indicates that external wind stimulation during singing reduces corollary discharge inhibition of the auditory pathway.
AP/ bin
GI8-1b
4mV
Relative Activity
Wing Nerve
... Wind
vGI8-1a (MGI)
Chirps=40
100mV 1.0 vGI8-1a
5mV 130ms
* ** **
***
*
**
***
Wind
vGI8-1a (MGI)
*
*
relative activity in the wing nerve
-20 0 20 40 Time to Wind Cycle in ms
-20 0 20 40 Time to Wind Cycle in ms
Supported by
APs=464
AP/ bin
****
T2-CDI (-1.5nA)
0
Wing Relative Nerve Activity -100 0 100 200 Time to First Syllable in ms
Wing Nerve
and The Isaac Newton Trust (Cambridge, UK)
