Ciglieri, Elisa

Ferrini, Francesco Maria

Salio, Chiara

English

Institutional Research Information System University of Turin

GDNF differentially regulates neuronal excitability in DRGs from normal and diabetic miceCiglieri E., Ferrini F., Salio C.University of Turin, Department of Veterinary Sciences, Largo P. Braccini 2, 10095 Grugliasco (TO) - Italy221.1INTRODUCTIONBoth type 1 and type 2 diabetes are associated with complications impairing a wide range of systems and leading to pathologies such as nephropathy ret inopathy, vasculopathy, cardiomyopathy, 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 ACKOWLEDGMENTSThis work was supported by Progetto Ateneo-Compagnia di San Paolo 2012 (Torino, Italy) to CS.Contact: elisa.ciglieri@unito.itMATERIALS AND METHODSŸ Adult CD-1 male miceŸ Single i.p. injection of Streptozotocin or vehicle in one month old miceŸ Measurement of glycemia at two months of age to assess diabetes ( >300 mg/dl)ElectrophysiologyŸ Recordings were performed in whole-cell configuration, with current and voltage clamp protocolŸ DRGs were incubated at 38°C with collagenase for 1 hour to remove the outern connective layerŸ GDNF (100 ng/µL) was bath-applied for 5 minutesImmunofluorescenceŸ 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 microscopyStatistical analysisPaired 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)mR (Mohm) m 2Area (um )V  (mV)mRheobase (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 ± 2.33Small-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 ± 6.46118.50 ± 24.58797.67 ± 65.64-75.33 ± 2.65Medium-size DIAB (n=8)47.38 ± 3.28170.25 ± 26.96787.85 ± 66.84-78.33 ± 2.527.52 ± 1.86-37.78 ± 2.1546.95 ± 29.9347.52 ± 3.12112.33 ± 18.5914.58  ± 0.320.45 ± 0.05*47.94 ± 9.49*18.54  ± 0.889.95 ± 2.38-40.92 ± 2.539.46 ± 2.7444.85 ± 2.69157.05 ± 20.3815.01  ± 0.130.30 ± 0.0385.70 ± 18.4717.32  ± 0.764.40 ± 1.08**-33.97 ± 1.5727.79 ± 4.8949.15 ± 3.1782.04 ± 19.8914.62  ± 0.140.64 ± 0.0832.50  ± 5.47*20.14  ± 1.2412.44 ± 2.28-32.85 ± 3.109.16 ± 1.9643.23 ± 6.52100.87 ± 17.3614.81  ± 0.280.51 ± 0.1558.78 ± 7.7017.33  ± 0.6825 mV50 msCTRDIAB        Maxriseslope(mV/ms)CTR DIAB0100200300400*  Rheobase(pA/pF)CTR DIAB0102030Threshold(mV)CTR DIAB-60-40-200*Overshoot(mV)CTR DIAB020406080RT20-80%(ms)CTR DIAB0.00.51.01.5Maxdecayslope(mV/ms)CTR DIAB050100150200250DIAB DIAB Overshoot(mV)Resting Hyper020406080CTRCTROvershoot(mV)Hyper020406080 **Resting CTRThreshold(mV)Resting Hyper-80-60-40-200***DIABThreshold(mV)Resting Hyper-80-60-40-200*Latency(ms)Resting Hyper0204060100200300400500***Latency(ms)Resting Hyper0204060100200300400500CTR CTRAPPeaktime(ms)Resting Hyper050100150200200300400500600 *DIABAPPeaktime(ms)Resting Hyper050100150200200300400500600DIABTimemaxdecay(ms)Resting Hyper0510152025*Timemaxdecay(ms)Resting Hyper0510152025HP -60 mV 0.5 s100 mVPre-pulse -100 mV Pre-pulse -40 mV2 nA100 msDIABCTRa)     Total b)     IK a) - b)     IA CTR Small-sizeDIAB Small-sizeV (mV)I(pA)-60 -40 -20 0 +20 +400200040006000* * *** **V (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000V (mV)I(pA)-60 -40 -20 0 +20 +400200040006000* *V (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000 V (mV)I(pA)-60 -40 -20 0 +20 +400200040006000800010000 V (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000 V (mV)I(pA)-60 -40 -20 0 +20 +400200040006000800010000CTR Medium-sizeDIAB Medium-size V (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000V (mV)I(pA)-60 -40 -20 0 +20 +40050010001500* * ******V (mV)I(pA)-60 -40 -20 0 +20 +4005001000150020002500******V (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000V (mV)I(pA)-60 -40 -20 0 +20 +400100020003000CTR Small-sizeDIAB Small-sizeCTR Small-sizeDIAB Small-sizeCTR Small-sizeDIAB Small-sizeCTR Small-sizeDIAB Small-sizeCTR Small-sizeDIAB Small-sizeCTR Medium-sizeDIAB Medium-sizeCTR Medium-sizeDIAB Medium-sizeCTR Medium-sizeDIAB Medium-sizeCTR Medium-sizeDIAB Medium-sizeCTR Medium-sizeDIAB Medium-size  DIAB Small-sizeCTR Small-sizeDIAB Medium-sizeCTR Medium-sizeV (mV)I(pA)-50 -70 -90 -110 -130-800-4000** * * * * **V (mV)I(pA)-50 -70 -90 -110 -130-800-4000* * * * * * ** CTRDIABV (mV)I(pA)-50 -70 -90 -110 -130-800-4000** * * * V (mV)I(pA)-50 -70 -90 -110 -130-800-4000** * * * * * * *   V (mV)I (pA)-50 -70 -90 -110 -130-800-4000*V (mV)I(pA)-50 -70 -90 -110 -130-800-4000* * * * * * * *V (mV)I(pA)-50 -70 -90 -110 -130-800-4000V (mV)I(pA)-50 -70 -90 -110 -130-800-4000* * * * * * * **V (mV)I(pA)-50 -70 -90 -110 -130-800-4000 CTRDIAB  CTRDIABDIAB Small-sizeCTR Small-sizeDIAB Small-sizeCTR Small-sizeDIAB Medium-sizeCTR Medium-sizeDIAB Medium-sizeCTR Medium-size50 mV100 ms 50 mV100 msHP -60 mV200 pA100 ms 200 pA100 msCTR Small-sizeDIAB Small-sizeCTR Medium-sizeDIAB Medium-size+ 2+Na  Free & Ca  blocked - VCEFFECT OF GDNF ON DRG NEURONS25 mV250 msaCSFGDNFRheobase(pA/pF)-100102030aCSF GDNFDIABMax rise slope (mV/ms)0100200300400aCSF GDNFDIABMax rise slope (mV/ms)0100200300400aCSF GDNFCTR   CTRThreshold(mV)-60-40-200aCSF GDNF  DIABThreshold(mV)-60-40-200aCSF GDNF*CTRRheobase(pA/pF)-100102030aCSF GDNFCTRRT20-80%(ms)0.00.51.01.5aCSF GDNFDIABRT20-80%(ms)0.00.51.01.5aCSF GDNF CTR HYPERRheobase(pA/pF)-10010203040aCSF GDNF* DIAB HYPERRheobase(pA/pF)-10010203040aCSF GDNF    CTR HYPERThreshold(mV)-60-40-200aCSF GDNF*DIAB HYPERThreshold(mV)-60-40-200aCSF GDNFCTR HYPERRT20-80%(ms)0.00.51.01.5aCSF GDNFDIAB HYPERRT20-80%(ms)0.00.51.01.5aCSF GDNF*CTR HYPERMaxriseslope(mV/ms)0100200300400aCSF GDNFDIAB HYPERMaxriseslope(mV/ms)0100200300400aCSF GDNF*2 nA100 msHP -60 mV 0.5 s100 mVPre-pulse -100 mV Pre-pulse -40 mVa)     Total b)     IK a) - b)     IAGDNF (CTR)aCSF (CTR)aCSFGDNFSmall-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000*aCSFGDNF  Small-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000***** Medium-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +400500100015002000aCSFGDNFMedium-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +40-5000500100015002000 aCSFGDNFSmall-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +40050010001500***** Small-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +40050010001500* Medium-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +40050010001500Medium-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +40050010001500aCSFGDNFaCSFGDNFaCSFGDNFaCSFGDNF Small-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +40010002000300040005000********* Small-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +40010002000300040005000 Medium-size CTRV (mV)I(pA)-60 -40 -20 0 +20 +40010002000300040005000Medium-size DIABV (mV)I(pA)-60 -40 -20 0 +20 +40010002000300040005000aCSFGDNFaCSFGDNFaCSFGDNFaCSFGDNF Small-size CTRV (mV)I(pA)-50 -70 -90 -110 -130-800-600-400-2000200** * Small-size DIABV (mV)I(pA)-50 -70 -90 -110 -130-800-600-400-2000200 V (mV)Medium-size CTRI(pA)-50 -70 -90 -110 -130-800-600-400-2000200Medium-size DIABV (mV)I (pA)-50 -70 -90 -110 -130-800-600-400-2000200aCSFGDNFaCSFGDNFaCSFGDNFaCSFGDNFSmall-size CTRV (mV)I(pA)-50 -70 -90 -110 -130-800-600-400-2000200*Small-size DIABV (mV)I (pA)-50 -70 -90 -110 -130-800-600-400-2000200Medium-size CTRV (mV)I(pA)-50 -70 -90 -110 -130-800-600-400-2000200Medium-size DIABV (mV)I(pA)-50 -70 -90 -110 -130-800-600-400-2000200aCSFGDNFaCSFGDNFaCSFGDNFaCSFGDNF50 mV100 msHP -60 mVCTRV (mV)I(pA)-50 -70 -90 -110 -130-50050aCSFGDNF***** ** DIABV (mV)I(pA)-50 -70 -90 -110 -130-50050** **aCSFGDNF-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 2until 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 BCD ED. 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, medium-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 onesII. Small-sized neurons were more affected by alterations induced by diabetesIII. 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 KKcurrents (I ) after depolarizing protocols A+VI. DIAB neurons showed increased outward K  currents after hyperpolarazing protocolsFF. 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.GG. 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).H ILVII. GDNF may reduce the excitability of DRG neurons from control mice, especially affecting firing properties in small-size neuronsVIII. GDNF exerted its major effect on small-sized CTR neurons+IX. GDNF seemed to act via multiple mechanisms involving K  currents+ 2+Regular Na  & Ca  - CC+ 2+Regular Na  & Ca  - CC+ 2+Na  Free & Ca  blocked - VCCONCLUSIONS2Ÿ Diabetes alters different parameters in both small (until 490 um ) and 2in medium-to-large (between 490 and 1100 um ) DRG neurons. Diabetes seems to increase cellular excitability mostly in small size neurons.+Ÿ  Diabetes seems to increase K  currents, mostly in small-sized neuronsŸ  Administration of GDNF on control animals has a relevant impact on voltage-sensitive mechanisms, as shown in presence of hyperpolarized conditions. This effect is lost in diabetic animals.Ÿ  Applying depolarizing steps, GDNF acts on both sustained and +transient components of K  currents and this effect is almost lost in diabetic condition.Ÿ GDNF seems to exert its effect mostly on small-sized neurons: applying hyperpolarizing steps in voltage clamp mode, it induces a block of inward currents likely mediated by voltage-dependant potassium channels. The effect is lost in medium-sized neurons.Our data indicate that GDNF acts with multiple mechanisms among which stands out an inhibitory effect on small DRG neurons. In addition, changes of holding potential highlighted an impairment of the voltage-dependent response of small-sized neurons in diabetic conditions. Restoring this inhibitory control in sensory neurons from diabetic patients may represent a novel strategy for mitigating the symptoms of painful diabetic neuropathy. IB4 IB4 IB4 IB4CGRPIB4CGRPIB4CGRPIB4CGRPCGRP CGRP CGRP IB4NF200CGRPNF200CGRPNF200CGRPNF200 NF200

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

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