Characterization of intrinsic membrane properties of vestibulo-spinal neurons through xenopus development

(1)

HAL Id: hal-02413482

https://hal.archives-ouvertes.fr/hal-02413482

Submitted on 16 Dec 2019

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Characterization of intrinsic membrane properties of vestibulo-spinal neurons through xenopus development

A Olechowski-Bessaguet, L Cardoit, M Thoby-Brisson, François Lambert

To cite this version:

A Olechowski-Bessaguet, L Cardoit, M Thoby-Brisson, François Lambert. Characterization of intrinsic membrane properties of vestibulo-spinal neurons through xenopus development. 2019 annual meeting Society for Neurosciences, Oct 2019, Chicago, United States. �hal-02413482�

(2)

Characterization of intrinsic membrane properties of vestibulo-spinal neurons through xenopus development

Olechowski-Bessaguet A., Cardoit L., Thoby-Brisson M., Lambert F.M.

FRM team

M. Thoby-Brisson

October 19 - 23 2019 Chicago, IL

402 . 14

Introduction

Materials and methods

Spinal cord Brainstem

IVth V.

Optic tectum

LVST

RDA dye application

RDA+ LVST neurons IVth

vent.

VIII N.

Patch-clamp recording horizontal slice transversal slice

Conclusion

Rostro-caudal brainstem position

63,16%

26,32%

10,53%

Larval Adult

64,71%

23,52%

11,76%

78%

22%

Adult Rana

Tonic Tonic

Tonic

Phasic

Phasic Phasic

Inter.

IVth vent.

VIIIth N.

Summary table Spatial localization in brainstem Current ramp-evoked spiking threshold

Larval

(st53-56) Adult

(st65-66) Metamorphosis

Proportion

Experiments were performed on post-metamorphosis adult-like (juvenile) stage 65-66 and larval stage 53-56 of south African clawed toad xenopus laevis. Stages were identi- fied according to external body criteria (Nieuwkoop and Faber, 1956).

Briefly, consecutive to anesthesia in a 0.05% MS-222 water solution and after fore- brain removal, the CNS was dissected in cold Ringer’s saline (93.5 mM NaCl, 3 mM KCl, 30 mM NaHCO3, 0.5 mM NaH2PO4, 2.6 mM CaCl2, 1 mM MgCl2, and 11 mM glucose, pH 7.4).

Rhodamine dextran (RDA) crystals were applied in a tiny incision performed at the ventral sur- face of the rostral hemi-cord. CNS in vitro preparation was incubated in circulating ringer’s saline at 16°C for at least 3h to allow retrograde labeling of vestibulospinal neurons.

Animals and Tissu preparation

Fluorescent confocal imaging after recording

Patch-clamp recorded RDA+

LVST neurons were intracellularly filled with biocytin. After recor- dings slices were fixed in PFA 4%

and revealed with Alexa fluor 488 streptavidin. Slices were imaged with a confocal microscope at 10x and 40x to allow a good medio lateral and rostro-caudal localiza- tion of recorded neurons within the entire LVST population.

Proportion of tonic and phasic neurons in xenopus compared to adult Rana

Retrograde labeling of VSN

after 3h

IVth vent.

VIII N.

Comparison of membrane intrinsic properties with vestibulo-ocular neurons, involved in other vestibulo-motor function and commissu- ral vestibular neurons, involved in the push-pull pathways between left and right vestibular nuclei.

Phasic and tonic neurons were found in the LVST at both larval and adult stages. Adult Xenopus exhibited the same proportion of phasic and tonic neurons than in adult Rana, with similar basic membrane properties (Beraneck et al., 2007). However the proportion was inver- sed in larvae with a majority of tonic neurons recorded so far and some variations in basic membrane properties. These results could constitute a first difference between larval and adult stages. Interestin- gly, a third class of neurons, demonstrating intermediate discharge dynamics, was found in larval and adult stages. This last vestibular neuron type, not found in adult terrestrial Rana, could be related to the pure aquatic life style of the Xenopus anuran.

Our preliminary results suggest some specific rhombomeric clustering of phasic and tonic LVST neurons. At both stages, tonic RDA+ neurons were mostly found lateraly in rhombomere 4 at the entry of the VIIIth nerve. Larval LVST phasic neurons were located more medially in Rh 3 and Rh4 whe- reas adult LVST phasic neurons were distributed from Rh3 to Rh6.

HC/Ut AC

PC

42 Hz

-66 mV -64 mV

Phasic 2°VN

Tonic 2°VN Stimulus

ACnerve

Hz0

10 mV 0.2 s

~15 Hz

0-70 Hz 70

Phase lead

r1 r2 r3 r4 r5 r6 r8 r7

spinal cord LVST

TAN

Preliminary results show that LVST larval and adult neurons (phasic/tonic) differ by the type of AHP, the discharge dynamics (mean and inst. firing freq.), the expression of hyperpolarization-activated conductance (Ih). Furthermore, adult LVST neurons (at least phasic) seem to exhibit a higher amplitude of voltage-gated current (IV) than larval neurons that suggests some ongoing maturation processing during metamorphosis. Rectifying IV curve and presence of Ih current in some of LVST neurons suggest the expression of voltage-dependant K+ conductances that are known to be invovled in filtering properties of vestibular neurons like ID conductance (depending of Kv1.1 channel), previously described to confer band- pass filter characteristic in rana phasic neurons (Beraneck et al., 2007). Therefore it will be interessing to investigate in detail the spectrum of Na+

and K+ conductances involved in the setting of AHP shape and of discharges dynamics. These differences observed in intrinsic membrane pro- perties between larval and adult xenopus could reflect a developmental adaptation in LVST pathway related to the metamorphosis-induced remo- deling of the posturo-locomotor system.

-100

0,5 -60

10 mV 0.1 s

+1.1 nA

Tonic 2°VN Phasic 2°VN

20 mV 5 ms

-65 mV

-69 mV

0 20 40 60 80 100

tonic phasic inter. tonic phasic inter.

tonic phasic inter.

Rh1-3 Rh4 Rh5-6

0/12

12/12

3/5

1/2 1/2 1/4

5/11

3/4

2/5 4/11 1/2 1/2

2/11 0/12 0/5

0/4 0/2

0/2

10mV

In adult terrestrial frog, two groups of 2°OVN were described according to their discharge dynamic and their intrinsic membrane properties ( Beraneck et al., 2007). In response to injection of positive current steps phasic neurons exhibit a high-frequency burst of 1-5 spikes, with monophasic AHP, whereas tonic neurons fire continuously spikes with a biphasic AHP. Such membrane properties tune phasic and tonic 2°OVN in band-pass and low-pass neuronal filters, respectively.

Consequently, phasic neurons could encode motion-related high dyna- mic sensory-motor signals whereas tonic neurons could encode position-related slow dynamic signals in vestibulo-motor behaviors. Nonetheless this characteriza- tion was based on 2°OVN that were not related to a specific vestibular functional pathway.

A Intrinsic membrane properties of phasic neurons: Larval Vs adult xenopus

Larval Adult

Number of neurons

Ih current

Resting membrane potential average

5 /19 11 /17

AHP

Spontaneous discharges post-inhibitory rebound

mean Freq/inst freq Rostro-caudal

brainstem position

- 44,8 - 47,8

Single= 3/5

Biphasic= 2/5 Single= 9/11 Biphasic= 2/11

1/5 3/11

0/5 2/11

0/5 3/11

2/25,6 Hz 2,88/28,5 Hz

± 9,7mV ±4,1mV

lateral rh3 lateral rh3

r1 r2 r3 r4

r6

r8 r7

Spinal cord

transversal rh3 slice transversal rh4 slice N.VIII

-45mV

0,5nA 1s

Undulatory swimming

Axial spinal network Appendicular swimming

Thoraco-lumbar spinal network

Adult (n=1)

Larval (n=7)

-61mV

200ms 20mV

10mV

200ms

0 10 20 30 50 60 70 80

Discharge dynamic

Larval Adult

200ms

20mV

Post-inhibitory rebound

Proportion

2,5 2,0 1,5 1,0

-0,5

0,5nA 1s

Current ramp-evoked spiking threshold

IV curve

Summary table

Larval Adult Number of neurons

Ih current

Resting membrane potential average

12 /19 4 /17

AHP

Spontaneous discharges post-inhibitory rebound Rostro-caudal brainstem position

- 50 - 47,8

Single= 3/12

Biphasic= 9/12 Single= 0/4 Biphasic= 4/4

7/12 0/4

6/12

Mean firing frequency= 15HZ 0/4

3/12 0/4

48 Hz 22,66 Hz

± 8,5 mV ±4,1mV

lateral

rh4 lateral or medial rh3

r1 r2 r3 r4

r6

r8 r7

Spinal cord

Spatial localization in brainstem

N.VIII

transversal rh4 slice

transversal rh3 slice

mean +/-SEM

Larval

Larval (n=2)

Adult (n=2)

Inst Freq (Hz) at +150pA

70 60 50 40 30 20 10

Percentage of neurons (%) 0

spike rank 120

0 20 40 60 80 100

2 3 4 5 6

1 7

-51mV

10mV 100ms

10mV

100ms +200pA

Larval Adult

Number of neurons

Ih current

Resting membrane potential average

2 /19 2 /17

AHP

Spontaneous discharges post-inhibitory rebound

Rostro-caudal brainstem position

- 58±11.3mV Single= 2/2

Biphasic= 0/2 Single= 2/2 Biphasic= 0/2

1/2 1/2

0/2 0/2

14/90Hz 9/25 Hz

lateral

rh4 lateral or medial rh3

Summary table

- 51mV

r1 r2 r3 r4

r6

r8 r7

Spinal cord

Intermediate neurons

N.VIII

transversal rh4 slice

transversal rh3 slice -66mV

+150pA +100pA

Discharge dynamic

Spatial localization in brainstem

biphasic AHP Mean spiking threshold= -32, 5mV

TAN LVST

VIIIth N.

SC obex

Larval

r1 r2 r3 r4

r6

r8 r7

r1 r2 r3 r4 r5 r6 r8 r7

r1 r2 r3 r4

r6

r8 r7

Spinal cord

Adult

120 0 20 40 60 80 100

20 30 40

10 Inst. Freq. (Hz) at +150pA

spike rank 20 40

-20 -40

-80

mean +/-SD

Adult

-51mV

mean +/-SEM

r1 r2 r3 r4 r5 r6 r8 r7

In vertebrates, central vestibular neurons (2°OVN) are subdivided in distinct populations according to their pro- jection and their function (vestibulo-ocular/spinal - commissu- ral...). In amphibian, vestibulospinal neurons (VSN), involved in postural control, are organized into lateral vestibulospinal tract (LVST) and tangential (TAN) nuclei projecting to ipsi- and contralateral spinal cord, respectively (Straka et al., 2001). Here, we focused on the LVST neurons, largest group (and common to other vertebrates lineages) localized mostly in rhombomeres 4, at the VIIIth nerve entrance.

The metamorphosis in Xenopus laevis toad, a purely aquatic anuran, offers a unique opportunity to unravel integrative proper- ties of central vestibular neurons in relation to a specific vestibu- lar sensory reference frame (aquatic vs terrestrial, otolith vs canal) and to a given locomotor system (tail- vs limb based swimming).

This study aims to investigate:

Intrinsic membrane properties exhibited by 2°OVN from identified LVST pathway at both larval and adult-like stages.

Underlying developmental plasticity mechanisms related to the posturo-locomotor system re-modeling.

Patch-clamp recordings of RDA+ LVST neurons were performed in whole-cell configuration on 350µm transversal or horizontal brainstem slices continuously perfused with oxygenated Ringer’s solution at 18-20°C. Patch electrodes were filled with intracellular solution (in mM= 115 K-gluconate,2 MgCl2, 2 EGTA, 10 HEPES, 2 MgATP, 0,2 NaGTP and 0,01% bio- cytin) . Different protocols in current clamp (-200 to +300pA current step serie and current ramp) and voltage clamp configura- tion (Ih, IV curve) were applied to measure discharge dynamics and basic membrane properties of RDA+ LVST neurons .

To be continued...

VN

VN VN from Straka et al., 2001

from Beraneck et al., 2007

x4 x4 x40

VIIIth N.

IVth V.

LVST

x40

Biocytin RDA merged

IV th vent.

x10

1-5 spikes firing (inst. freq xxHz)at both stages, larval phasic neurons exhibit both mono- and biphasic AHP whereas adult phasic neurons exhibit only monophasic AHP Discharge dynamic

20mV

-57mV

biphasic AHP monophasic AHP

Larval

-45mV

Adult

200ms

monophasic AHP

+150pA

+200pA

+150pA

-41mV

r1 r2 r3 r4 r5 r6 r8 r7

B Intrinsic membrane properties of tonic neurons: Larval Vs adult xenopus

biphasic AHP monophasic AHP

Percentage of neurons (%) 70 60 50 40 30 20 10

0 Larval stage

(n=19) Adult stage (n=17)

I (nA)

V(mV) mean +/-SD

Instantaneous Frequency

Mean firing frequency (Hz) 40

0 +50 +100 +150 +200 +250 +300 Positive current steps injections (pA)

firing inst. freq.

C Intrinsic membrane properties of intermediate neurons: Larval Vs adult xenopus

Discharge dynamic

0 10 20 30 50 60

Mean firing frequency (Hz) 40

0 +50 +100 +150 +200 +250 +300 Positive current steps injections (pA)

Firing freq.

monophasic AHP

Instantaneous Frequency

Larval stage

(n=19) Adult stage (n=17)

Phasic neurons

Tonic neurons

Adult

Larval

Proportion IV curve Presence of Ih current

0,5

-100 -80

Adult (n=7)

Larval (n=3)

Percentage of neurons (%)

2,5 2,0 1,5 1,0

40 20

-20 -0,5 -40

-60 70

60 50 40 30 20 10

0 Larval stage

(n=19) Adult stage (n=17)

mean +/-SEM

0 +50 +100 +150 +200 +250 +300 0

3 2 1 4 5

firing inst. freq.

mean +/-SD I (nA)

V(mV)

Mean firing frequency (Hz)

Positive current steps injections (pA)

Adult (n=11)

Larval (n=5)

Adult (n=4)

Larval (n=12) Larval (n=12)

Adult (n=4)

Mean spiking threshold= -29,6mV Mean spiking threshold= -34,9mV

Phasic neurons were more encountered in adult than in larval LVST neurons. At depolarized voltages, adult phasic neurons exhibit higher amplitude of vol- tage-gated current (IV curve) than larval phasic neurons. Ih current was present at both larval and adult stages.

Tonic neurons were more encountered in larval than in adult LVST neurons. Larval tonic neurons are characterized by a higher instantaneous discharge fre- quency than adult tonic neurons.

Firing threshold

Adult tonic neurons exhibit only monophasic AHP whereas larval tonic neurons exhibit both mono and biphasic AHP and some of them exhibit post-inhibi- tory rebound.

Larval and adult intermediate neurons are characterized by an absence of continuous discharge in response to positive current steps, like in phasic neurons, but are able to fire more than 5 spikes at a high instantaneous frequency. At both stages, intermediate neurons exhibit only monophasic AHP.

+150pA

r1 r2 r3 r4 r5 r6 r8 r7

LVST

TAN

from Straka et al., 2001

r1 r2 r3 r4 r5 r6 r8 r7

Electrical stimulation of VIIIth nerve branchs, with trains of current pulses that occur with instantaneous frequencies following a sinus temporal waveform, will allow investigating neuronal filtering pro- perties of LVST RDA+ neurons.