GDNF differentially regulates neuronal excitability in DRGs from normal and diabetic mice

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GDNF differentially regulates neuronal excitability in DRGs from normal and diabetic mice

Ciglieri E., Ferrini F., Salio C.

University of Turin, Department of Veterinary Sciences, Largo P. Braccini 2, 10095 Grugliasco (TO) - Italy

221.1 INTRODUCTION

Both type 1 and type 2 diabetes are associated with complications

impairing a wide range of systems and leading to pathologies such as

n e p h r o p a t h y r e t i n o p a t h y, v a s c u l o p a t h y, c a r d i o m y o p a t h y,

dermatopathy, and neuropathy. One of the main features of diabetic

neuropathy is pain, which arises as a direct consequence of a lesion

or a disease affecting somatosensory system. Several studies

postulated the involvement of dorsal root ganglia (DRGs) in this

pathology considering both the development of sensory abnormalities

in early diabetic patients and the particular vulnerability of sensory

neurons, due to the lack of the blood-brain barrier and the high

metabolic requirements.

DRGs are a group of pseudo-unipolar cells with two branches acting

as a single axon, a distal and a peripheral process, transducing

information from a variety of specialized peripheral receptors.

Nociceptors, the sensory neurons specialized in the transmission of

nociceptive stimuli, can be recognized for their small size (small-to

medium cells) and the binding of speciﬁc markers such as isolectin B4

(IB4; non-peptidergic nociceptors) or the expression of peptides, such

as the calcitonin gene-related peptide (CGRP; peptidergic

nociceptors). Nociceptors are specialized for encoding mechanical,

thermal or chemical stimuli, but they can also encode more than one

modality (polymodal nociceptors).

Small-diameter nociceptors in DRGs of adult animals require speciﬁc

neurotrophins for the maintenance of their phenotype: peptidergic

neurons are under the control of nerve growth factor (NGF) via trkA

receptor, while non-peptidergic neurons are under the control of glial

cell line-derived neurotrophic factor (GDNF) via GFRα-1/Ret

signaling. Moreover, GDNF is expressed both in a population of DRG

neurons and in DRG satellite cells and plays a role in modulating

nociceptive processing.

We focused our attention on the analysis of primary sensory neurons of

the DRGs in a streptozotocin-induced model of type 1 diabetes by

combining electrophysiological and morphological approaches.

The speciﬁc aims are:

§ to compare physiological parameters of

primary sensory neurons in control and

diabetic mice

§ to analyse the effect of GDNF in control and

diabetic mice

§ to analyse the morphological alterations of

DRGs due to diabetes

ACKOWLEDGMENTS

This work was supported by Progetto Ateneo-Compagnia di San Paolo 2012

(Torino, Italy) to CS.

Contact: elisa.ciglieri@unito.it

MATERIALS AND METHODS

Ÿ

Adult CD-1 male mice

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Single i.p. injection of Streptozotocin or vehicle in one month old mice

Ÿ

Measurement of glycemia at two months of age to assess diabetes (

>300 mg/dl)

Electrophysiology

Ÿ

Recordings were performed in whole-cell conﬁguration, with current and

voltage clamp protocol

Ÿ

DRGs were incubated at 38°C with collagenase for 1 hour to remove the

outern connective layer

Ÿ

GDNF (100 ng/µL) was bath-applied for 5 minutes

Immunoﬂuorescence

Ÿ

Immunoﬂuorescence staining was performed on entire ﬂoating DRGs,

ﬁxed in 4% PAF, and on sections from parafﬁn embedded DRGs

Ÿ

Fluorescent images were obtained using a confocal fuorescence

microscopy

Statistical analysis

Paired data were analyzed by Wilcoxon signed rank test and

indipendent groups were analyzed by Mann-Whitney U test. Differences

were considered signiﬁcant for P<0.05.

EFFECT OF DIABETES ON DRG NEURONS

Electrophysiological parameters of small-size and medium-size control and diabe c mice DRG neurons in whole-cell patch-clamp recording experiment.

C (pF)m R (Mohm) m 2 Area (um ) V (mV)m Rheobase (pA/pF) Threshold (mV) Latency (ms) Overshoot (mV) Max rise slope (mV/ms) Time of max rise slope (ms) Rise me 20-80% (ms) Max decay slope (mV/ms) Time of max decay slope (ms)

367.71 ± 23.69*** Small-size CTR (n=11) 28.82 ± 2.83** 358.91 ± 72.66* -59.55 ± 2.33 Small-size DIAB (n=9) 30.22 ± 2.88*** 434.78 ± 103.12* 386.15 ± 21.71*** -54.22 ± 2.29* Medium-size CTR (n=8) 51.25 ± 6.46 118.50 ± 24.58 797.67 ± 65.64 -75.33 ± 2.65 Medium-size DIAB (n=8) 47.38 ± 3.28 170.25 ± 26.96 787.85 ± 66.84 -78.33 ± 2.52 7.52 ± 1.86 -37.78 ± 2.15 46.95 ± 29.93 47.52 ± 3.12 112.33 ± 18.59 14.58 ± 0.32 0.45 ± 0.05* 47.94 ± 9.49* 18.54 ± 0.88 9.95 ± 2.38 -40.92 ± 2.53 9.46 ± 2.74 44.85 ± 2.69 157.05 ± 20.38 15.01 ± 0.13 0.30 ± 0.03 85.70 ± 18.47 17.32 ± 0.76 4.40 ± 1.08** -33.97 ± 1.57 27.79 ± 4.89 49.15 ± 3.17 82.04 ± 19.89 14.62 ± 0.14 0.64 ± 0.08 32.50 ± 5.47* 20.14 ± 1.24 12.44 ± 2.28 -32.85 ± 3.10 9.16 ± 1.96 43.23 ± 6.52 100.87 ± 17.36 14.81 ± 0.28 0.51 ± 0.15 58.78 ± 7.70 17.33 ± 0.68 25 mV 50 ms CTR DIAB M a x ri s e s lo p e (m V /m s ) CTR DIAB 0 100 200 300 400 * R h e o b a s e (p A /p F ) CTR DIAB 0 10 20 30 T h re s h o ld (m V ) CTR DIAB -60 -40 -20 0 * O v e rs h o o t (m V ) CTR DIAB 0 20 40 60 80 R T 2 0 -8 0 % (m s ) CTR DIAB 0.0 0.5 1.0 1.5 M a x d e c a y s lo p e (m V /m s ) CTR DIAB 0 50 100 150 200 250 DIAB DIAB O v e rs h o o t (m V ) Resting Hyper 0 20 40 60 80 CTR CTR O v e rs h o o t (m V ) Hyper 0 20 40 60 80

**

Resting CTR T h re s h o ld (m V ) Resting Hyper -80 -60 -40 -20 0

***

DIAB T h re s h o ld (m V ) Resting Hyper -80 -60 -40 -20 0

*

L a te n c y (m s ) Resting Hyper 0 20 40 60 100 200 300 400 500

***

L a te n c y (m s ) Resting Hyper 0 20 40 60 100 200 300 400 500 CTR CTR A P P e a k ti m e (m s ) Resting Hyper 0 50 100 150 200 200 300 400 500 600

*

DIAB A P P e a k ti m e (m s ) Resting Hyper 0 50 100 150 200 200 300 400 500 600 DIAB T im e m a x d e c a y (m s ) Resting Hyper 0 5 10 15 20 25

*

T im e m a x d e c a y (m s ) Resting Hyper 0 5 10 15 20 25 HP -60 mV 0.5 s 100 mV Pre-pulse -100 mV Pre-pulse -40 mV 2 nA 100 ms DIAB CTR a) Total b) IK a) - b) IA CTR Small-size DIAB Small-size V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 2000 4000 6000 * * *** * * V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 2000 4000 6000 * * V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 2000 4000 6000 8000 10000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 2000 4000 6000 8000 10000 CTR Medium-size DIAB Medium-size V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 * * **** * * V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 2500 ****_** V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 V (mV) I( p A ) -60 -40 -20 0 +20 +40 0 1000 2000 3000 CTR Small-size DIAB Small-size CTR Small-size DIAB Small-size CTR Small-size DIAB Small-size CTR Small-size DIAB Small-size CTR Small-size DIAB Small-size CTR Medium-size DIAB Medium-size CTR Medium-size DIAB Medium-size CTR Medium-size DIAB Medium-size CTR Medium-size DIAB Medium-size CTR Medium-size DIAB Medium-size DIAB Small-size CTR Small-size DIAB Medium-size CTR Medium-size V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * * * * _* * * * V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * * * * * * _* * CTR DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * * * * _* V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 ** * * * * * * * V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * * * * * * * * V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 * * * * _{* * *} ** V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -400 0 CTR DIAB CTR DIAB DIAB Small-size CTR Small-size DIAB Small-size CTR Small-size DIAB Medium-size CTR Medium-size DIAB Medium-size CTR Medium-size 50 mV 100 ms 100 ms _{50 mV} HP -60 mV 200 pA 100 ms 100 ms _{200 pA}

CTR Small-size DIAB Small-size CTR Medium-size DIAB Medium-size

+

2+

Na Free & Ca blocked - VC

EFFECT OF GDNF ON DRG NEURONS

25 mV 250 ms aCSF GDNF R h e o b a s e (p A /p F ) -10 0 10 20 30 aCSF GDNF DIAB M a x ri s e s lo p e (m V /m s ) 0 100 200 300 400 aCSF GDNF DIAB M a x ri s e s lo p e (m V /m s ) 0 100 200 300 400 aCSF GDNF CTR CTR T h re s h o ld (m V ) -60 -40 -20 0 aCSF GDNF DIAB T h re s h o ld (m V ) -60 -40 -20 0 aCSF GDNF * CTR R h e o b a s e (p A /p F ) -10 0 10 20 30 aCSF GDNF CTR R T 2 0 -8 0 % (m s ) 0.0 0.5 1.0 1.5 aCSF GDNF DIAB R T 2 0 -8 0 % (m s ) 0.0 0.5 1.0 1.5 aCSF GDNF CTR HYPER R h e o b a s e (p A /p F ) -10 0 10 20 30 40 aCSF GDNF

*

DIAB HYPER R h e o b a s e (p A /p F ) -10 0 10 20 30 40 aCSF GDNF CTR HYPER T h re s h o ld (m V ) -60 -40 -20 0 aCSF GDNF

*

DIAB HYPER T h re s h o ld (m V ) -60 -40 -20 0 aCSF GDNF CTR HYPER R T 2 0 -8 0 % (m s ) 0.0 0.5 1.0 1.5 aCSF GDNF DIAB HYPER R T 2 0 -8 0 % (m s ) 0.0 0.5 1.0 1.5 aCSF GDNF

*

CTR HYPER M a x ri s e s lo p e (m V /m s ) 0 100 200 300 400 aCSF GDNF DIAB HYPER M a x ri s e s lo p e (m V /m s ) 0 100 200 300 400 aCSF GDNF

*

2 nA 100 ms HP -60 mV 0.5 s _{100 mV} Pre-pulse -100 mV Pre-pulse -40 mV a) Total b) IK a) - b) IA GDNF (CTR) aCSF (CTR) aCSF GDNF Small-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 * aCSF GDNF Small-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 ***** Medium-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 2000 _aCSF GDNF Medium-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 -500 0 500 1000 1500 2000 _aCSF GDNF Small-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 ** * * * Small-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 * Medium-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 Medium-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 500 1000 1500 aCSF GDNF aCSF GDNF aCSF GDNF aCSF GDNF Small-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 1000 2000 3000 4000 5000 ** * * * * ** * Small-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 1000 2000 3000 4000 5000 Medium-size CTR V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 1000 2000 3000 4000 5000 Medium-size DIAB V (mV) I (p A ) -60 -40 -20 0 +20 +40 0 1000 2000 3000 4000 5000 aCSF GDNF aCSF GDNF aCSF GDNF aCSF GDNF Small-size CTR V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 * * * Small-size DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 V (mV) Medium-size CTR I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 Medium-size DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 aCSF GDNF aCSF GDNF aCSF GDNF aCSF GDNF Small-size CTR V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 * Small-size DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 Medium-size CTR V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 Medium-size DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -800 -600 -400 -200 0 200 aCSF GDNF aCSF GDNF aCSF GDNF aCSF GDNF 50 mV 100 ms HP -60 mV CTR V (mV) I (p A ) -50 -70 -90 -110 -130 -50 0 50 aCSF GDNF ***** ** DIAB V (mV) I (p A ) -50 -70 -90 -110 -130 -50 0 50 ** ** aCSF GDNF

-A. Active and passive membrane properties of neurons from control (CTR) and diabetic (DIAB) mice. The first category, identified/classified as “small-sized”, contained neurons

2 2

until 490 µm , while the second category “medium-sized” contained neurons between 490 µm2 and 1100 µm .B. AP-related parameters were recorded from CTR and DIAB

neurons. The only parameters that differ signiﬁcantly were the threshold, which was more depolarized for DIAB neurons, and the maximum rise slope, which was reduced in DIAB neurons (CTR n=19, DIAB n=17; *P<0.05, Mann-Whitney test). On the left, two AP superimposed to show the different kinetics between CTR and DIAB neurons.

C. AP related parameters were recorded from CTR (top) and DIAB (bottom) neurons. For each neurons, the same protocols were repeated in aCSF and hyperpolarized (i.e. -85

mV) conditions, to analyze voltage-dependent differences. Interestingly, only CTR neurons showed signiﬁcant differences in hyperpolarized condition in parameters as threshold, latency, overshoot, AP peak time and time of max decay (CTR n=19, DIAB n=17, *P<0.05, **P<0.01, ***P<0.001, Wilcoxon test).

A

B

C

D

E

D. Top Representative traces of the total Kv current (left), sustained (centre) and transient Kv currents (right). Bottom The mean density of total, sustained and transient Kv

currents was signiﬁcantly higher in DIAB small-sized neurons than in CTR small-sized one, while there was no difference between CTR and DIAB neurons in the medium-sized class. This difference was particularly high measuring the current at the middle point (yellow triangle) (CTR small-sized n=9, sized n=5;DIAB small-sized n=10, medium-sized n=5; *P<0.05, **P<0.01, Mann-Whiteney test). E. Top Representative traces of the current elicited by the hyperpolarizing protocols from -50 mV to -130 mV (500 ms, Δ=20 mV). Currents were measure at the start (orange circle), the middle (yellow triangle) and at the end (blue square). Bottom The mean density currents in DIAB neurons were signiﬁcantly greater than in CTR neurons, mostly due to the effect in small-sized cells (CTR n=15, DIAB n=15; *P<0,05, Mann-Whitney test).

I. Small-sized neurons showed different membrane properties than

medium-sized ones

II. Small-sized neurons were more affected by alterations induced by diabetes

III. Diabetes could affect voltage-sensitive mechanisms

+

IV. Diabetes induced changes in K currents in small-sized neurons

+ +

V. DIAB neurons showed higher sustained K currents (I ) and transient K

_K

currents (I ) after depolarizing protocols

_A

+

VI. DIAB neurons showed increased outward K currents after hyperpolarazing

protocols

F

F. On the left, superimposed APs before and after GDNF. AP-related parameters were recorded from CTR (top)and DIAB (bottom) neurons, before and after administration of

GDNF. The only parameter that differ signiﬁcantly was the threshold in DIAB neurons, which was more hyperpolarized after GDNF administration (CTR n=19, DIAB n=17;

*P<0.05, Wilcoxon test). ). Interestingly, previously described differences between CTR (n=19) and DIAB (n=17) neurons in threshold and kinetics were abolished in presence of GDNF.

G

G. AP-related parameters were recorded from CTR (top)and DIAB (bottom) neurons, before and after administration of GDNF in hyperpolarizied condition (i.e. -85 mV). In

comparison to resting condition, hyperpolarization had a major effect on neurons before GDNF treatment and, mostly, on CTR neurons. The effect recorded in DIAB neurons were lost after reconsidering the samples based on their sizes (CTR n=19, DIAB n=17; *P<0.05, Wilcoxon test).

H. Top Representative traces of the total Kv current (left), sustained (centre) and transient Kv currents (right). The mean density of sustained and transient Kv currents of CTR

small-sized neurons was significantly different after GDNF administration, with a decrease of the sustained component and an increase of the transient component. Contrariwise, the total Kv current did not vary significantly. This effect was not measured in small-sized DIAB neurons or in medium-sized both CTR and DIAB (CTR small-sized n=9, medium-sized n=5;DIAB small-sized n=10, medium-sized n=5; *p<0.05, Wilcoxon test). I. The mean density current was significantly increase after GDNF administration, but only in small-sized CTR neurons and at high negative voltages (CTR small-sized n=9, medium-sized n=5;DIAB small-sized n=10, medium-sized n=5; *p<0.05, **p<0.01, Wilcoxon test). L. Hyperpolarization-activated inward current is calculated as the difference between the steady state currents at the end (blue square) and at the start (orange circle) of the traces. A significant difference was noticed at high negative voltages, both in CTR and DIAB animals. GDNF seemed to block the inward currents (CTR small-sized n=9, medium-sized n=5;DIAB small-sized n=10, medium-sized n=5; *p<0.05, **p<0.01, Wilcoxon test).