Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected mice.

Journal of Diabetes and Its Complications xxx (2015) xxx–xxx

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Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected mice Hoang Thi Thanh Nguyen a, 1, Janardhan Prasad Bhattarai a, 1, Soo Joung Park a, Jeong Chae Lee c, Dong Hyu Cho b,⁎, Seong Kyu Han a,⁎⁎ a b c

Department of Oral Physiology, School of Dentistry and Institute of Oral Bioscience, Chonbuk National University, Jeonju, Republic of Korea Department of Obstetrics and Gynecology, Chonbuk National University Hospital and School of Medicine, Jeonju, Republic of Korea Department of Orthodontics, School of Dentistry and Institute of Oral Bioscience, Chonbuk National University, Jeonju, Republic of Korea

a r t i c l e

i n f o

Article history: Received 12 November 2014 received in revised form 6 March 2015 accepted 11 March 2015 Available online xxxx Keywords: Streptozotocin Substantia gelatinosa neuron Patch clamp Diabetes mellitus GABA

a b s t r a c t Peripheral neuropathy is a frequent complication of diabetes mellitus and a common symptom of neuropathic pain, the mechanism of which is complex and involves both peripheral and central components of the sensory system. The lamina II of the medullary dorsal horn, called the substantia gelatinosa (SG), is well known to be a critical site for processing of orofacial nociceptive information. Although there have been a number of studies done on diabetic neuropathy related to the orofacial region, the action of neurotransmitter receptors on SG neurons in the diabetic state is not yet fully understood. Therefore, we used the whole-cell patch clamp technique to investigate this alteration on SG neurons in both streptozotocin (STZ)-induced diabetic mice and offspring from diabetic female mice. STZ (200 mg/kg)-injected mice showed a small decrease in body weight and a significant increase in blood glucose level when compared with their respective control group. However, application of different concentrations of glycine, gamma-aminobutyric acid (GABA) and glutamate on SG neurons from STZ-injected mice did not induce any significant differences in inward currents when compared to their control counterparts. On the other hand, the offspring of diabetic female mice (induced by multiple injections of STZ (40 mg/kg) for 5 consecutive days) led to a significant decrease in both body weight and blood glucose level compared to the control offspring. Glycine and glutamate responses in the SG neurons of the offspring from diabetic female mice were similar to those of control offspring. However, the GABA response in SG neurons of offspring from diabetic female mice was greater than that of control offspring. Furthermore, the GABA-mediated responses in offspring from diabetic and control mice were examined at different concentrations ranging from 3 to 1,000 μM. At each concentration, the GABA-induced mean inward currents in the SG neurons of offspring from diabetic female mice were larger than those of control mice. These results demonstrate that SG neurons in offspring from diabetic mice are more sensitive to GABA compared to control mice, suggesting that GABA sensitivity may alter orofacial pain processing in offspring from diabetic female mice. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc), also called the medullary dorsal horn, is thought to be a critical site for the processing of orofacial nociceptive information because it receives synaptic terminations from primary afferents ⁎ Correspondence to: D. H. Cho, Department of Obstetrics and Gynecology, Chonbuk National University Hospital and School of Medicine, Jeonju, 561-756, Republic of Korea. Tel./fax: + 82 63 250 1497. ⁎⁎ Correspondence to: S. K. Han, Department of Oral Physiology & Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju, 561-756, Korea. Tel.: +82 63 270 4030; fax: +82 63 270 4028. E-mail addresses: [email protected] (D.H. Cho), [email protected] (S.K. Han). 1 The first two authors contributed equally to this work.

including myelinated Aδ- and unmyelinated C-fibers (Light & Perl, 1979; Santos, Rebelo, Derkach, & Safronov, 2007; Sugiura, Lee, & Perl, 1986). A substantial number of studies suggest the co-localization of inhibitory and excitatory neurotransmitter receptors like gamma-aminobutyric acid (GABA), glycine and glutamate in the same SG neuron, showing their potential role in the modulation of nociceptive transmission (Kohno, Kumamoto, Higashi, Shimoji, & Yoshimura, 1999; Price, Cervero, & de Koninck, 2005; Todd, Watt, Spike, & Sieghart, 1996). Peripheral neuropathy is a frequent complication of diabetes mellitus, and a common symptom of this disorder is neuropathic pain. Pain associated with diabetic neuropathy is characterized by spontaneity and increased sensitivity to mechanical and chemical stimuli (Galer, Gianas, & Jensen, 2000; Podwall & Gooch, 2004; Sima,

http://dx.doi.org/10.1016/j.jdiacomp.2015.03.007 1056-8727/$© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Nguyen, H.T.T., et al., Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.007

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H.T.T. Nguyen et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx

2003). It has been suggested that the mechanism of diabetic neuropathic pain is complex and is involved in both peripheral and central components of the sensory system. The primary afferent element related to diabetic neuropathic pain has been studied as a result of hyperactivity of damaged small-diameter C-fibers (Burchiel, Russell, Lee, & Sima, 1985; Chen & Levine, 2001). In addition, hyperactivity of dorsal horn neurons in the spinal cord, a major component for the transmission and modulation of the nociceptive pathway, is considered to play an important role in chronic diabetic neuropathic pain (Chen & Pan, 2002; Morgado & Tavares, 2007; Pertovaara, Wei, Kalmari, & Ruotsalainen, 2001). Dorsal horn neurons in the spinal cord have been shown to receive both inhibitory GABAergic/glycinergic and excitatory glutamatergic synaptic inputs (Pan & Pan, 2004; Yoshimura & Nishi, 1995; Zhang, Li, Chen, & Pan, 2005). Consequently, many previous studies have strongly suggested that nerve injuries alter both the synaptic inputs and receptor functions on dorsal horn neurons (Kawamata & Omote, 1996; Polgar, Hughes, Arham, & Todd, 2005; Wang, Zhang, Chen, & Pan, 2007). In streptozotocin (STZ)-induced diabetic rats, there was a pronounced increase in the extracellular GABA concentration in the ventromedial hypothalamic region (Ohtani, Ohta, & Sugano, 1997). Additionally, modulation of GABAC receptor properties was observed on retinal bipolar cells (Ramsey, Ripps, & Qian, 2007) suggesting the effect of diabetic neuropathy on the central nervous system. Furthermore, symptoms of diabetic neuropathy in the orofacial region have been shown to manifest in many ways, including xerostomia (Meurman et al., 1998), taste impairment (Le Floch et al., 1989), burning mouth syndrome (Moore, Guggenheimer, & Orchard, 2007) and trigeminal sensitivity (Arap, Siqueira, Silva, Teixeira, & Siqueira, 2010). Although there are ample studies on diabetic neuropathy related to the orofacial region, the action of neurotransmitter receptors of SG neurons in the diabetic state has not been fully elucidated. Therefore, in this study, we used the whole-cell patch clamp technique to investigate this alteration on the SG neurons of the Vc in both STZ-induced diabetic mice and in offspring from diabetic female mice. 2. Material and methods 2.1. Animals and experimental models of diabetes The ICR mice were housed under 12-h light, 12-h dark cycles with access to food and water ad libitum. All animal experiments were performed with the approval of the Chonbuk University Animal Welfare and Ethics Committee according to protocol CBU-2014-00041 and CBU-2014-00042. 2.1.1. Diabetic mouse model procedure Diabetes mellitus was induced in 3-week-old ICR mice by a single intraperitoneal (i.p.) injection of STZ (200 mg/kg body weight) (Denroche et al., 2011) dissolved in phosphate buffered saline (PBS). Age-matched controls were injected with the same volume of PBS only. In STZ-injected mice, diabetes mellitus was confirmed 3 days later by examining glucose levels (N 240 mg/dl) in blood drawn from the tail vein. The mice from both groups were then electrophysiologically analyzed on alternating days after the 14th day of injection. 2.1.2. Offspring from diabetic dams Mature male and female (postnatal day (PND) N60) mice were mated. On the 11th day of pregnancy, mice were injected with multiple i.p. injections of STZ (40 mg/kg body weight) dissolved in PBS for 5 consecutive days (Maksimovic-Ivanic, Trajkovic, Miljkovic, Mostarica Stojkovic, & Stosic-Grujicic, 2002). Diabetes was confirmed on the last day of injection. The offspring from these dams were used as one group, and the offspring from pregnant mice injected with PBS

alone were considered as another group. The offspring were then electrophysiologically analyzed on alternating days following the 7th postnatal day.

2.2. Brain slice preparation Brain slice preparation was similar to the method performed in our previous study (Nguyen, Bhattarai, Park, & Han, 2013). Briefly, the ICR mice were decapitated, and their brains were excised quickly and immersed in ice-cold bicarbonate-buffered artificial cerebrospinal fluid (ACSF) with the following chemical composition (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 11 D-glucose, 1.4 NaH2PO4 and 25 NaHCO3 (pH 7.3 ~ 7.4, bubbled with 95% O2 and 5% CO2). The segment containing the trigeminal subnucleus caudalis was dissected, supported with a 4% agar block and glued with cyanoacrylate to the chilled stage of a vibratome (Leica VT1200S, Germany). Coronal slices (150 μm in thickness, obtained 1-2 mm from the obex, the most rostral part of the Vc) were prepared in ice-cold ACSF using the vibratome. The slices were kept in oxygenated ACSF at room temperature for at least 1 hour before electrophysiological recording.

2.3. Electrophysiological procedure and data analysis The slices were transferred into a recording chamber, completely submerged and continuously super fused with carboxygenated ACSF at a rate of 4–5 ml/min. An upright microscope (BX51W1, Olympus, Tokyo, Japan) with Nomarski differential interference contrast optics was used to view the slices. The SG (lamina II) was clearly identified as a translucent band just medial to the spinal trigeminal tract that traveled along the lateral edge of the slice. The patch pipettes were pulled from thin-walled borosilicate glass-capillary tubing (PG52154-4, WPI, Sarasota, USA) on a Flaming/Brown puller (P-97, Sutter Instruments Co., Novato, CA). The pipette solution was passed through a disposable 0.22-μm filter and consisted of the following (in mM): 140 KCl, 1 CaCl2, 1 MgCl2, 10 N-2-hydroxyethylpiperazineN ’-2-ethanesulfonic acid (HEPES), 4 MgATP, 10 EGTA (pH 7.3 with KOH). In this study, a high chloride pipette solution was used to amplify the chloride-mediated conductance. The resistance between the recording electrode filled with pipette solution and the reference electrode in the bath was found to be 4–6 MΩ. After a gigaohm seal was formed with the SG neuron, the cell membrane patch was ruptured by negative pressure, and the whole-cell patch clamp recording was performed using an Axopatch 200B (Axon Instruments, Union City, CA). The changes in membrane potentials and membrane currents were sampled online using a Digidata 1322A interface (Axon Instruments) connected to a desktop PC. The signals were filtered (2 kHz, Bessel filter of Axopatch 200B) before being digitized at a rate of 1 kHz. The whole cell voltage clamp currents were recorded at a holding potential of − 60 mV. The mean holding current change within the control and treated period was calculated as the mean of peak-to-peak amplitudes of individual points within each period. The acquisition and subsequent analysis of the acquired data were performed using Clampex9 software (Axon Instruments, USA). The traces were plotted using Origin7 software (MicroCal Software, Northampton, USA). All recordings were made at room temperature.

2.4. Chemicals Glycine, STZ, glutamate, GABA and the chemicals for ACSF were purchased from Sigma (USA). Stocks of chemicals were made according to their solubility in distilled water. Chemicals were diluted to the desired final concentrations in ACSF immediately before use and were applied via bath application.



2.5. Statistics All values were expressed as the mean ± S.E.M. The student t-test was used to compare the amplitudes of mean inward currents induced by chemicals between mouse groups. Statistical significance was defined as p b 0.05. 3. Results 3.1. Effect of STZ on body weight and blood glucose level in STZ-injected mice Although a significant difference in blood glucose levels was found between control and STZ-injected mice 3 and 10 days after a single STZ injection (200 mg/kg) (Fig. 1B), the body weight of the STZ-injected group was not significantly different when compared to the control group either 3 or 14 days (n = 10, p N 0.05, Fig. 1A). Blood glucose levels in the STZ-injected group were 2.64 and 3.28 times higher than those of control group 3 days and 10 days after injection, respectively (p b 0.001, Fig. 1B). 3.2. Responses induced by glycine, GABA and glutamate in SG neurons of STZ-injected mice To investigate whether there were any changes in the response induced by inhibitory or excitatory neurotransmitters in STZ-injected mice, we compared the responses elicited by glycine (100 μM), GABA (100 μM) and glutamate (30 μM) in SG neuron between control and STZ-injected groups by using a voltage clamp at a holding potential of − 60 mV with a high chloride pipette solution. The influence of the inhibitory neurotransmitters GABA and glycine was first investigated. Fig. 2A shows the traces of glycine-induced inward currents from SG neurons in control and STZ-treated mice. The mean inward current (296 ± 39.8 pA, n = 10) in STZ-treated mice was similar to that of the control group (337 ± 95.1 pA, n = 11, Fig. 2D). Fig. 2B shows the traces of GABA-induced inward currents from SG neurons in control and STZ-treated mice. The mean inward current (88.4 ± 37.6 pA, n = 9) in STZ-treated mice was similar to that of the control group (92.8 ± 46.3 pA, n = 10, Fig. 2E). We then investigated the effect of the excitatory neurotransmitter, glutamate. Fig. 2C shows the traces of glutamate-induced inward currents from SG neurons in control and STZ-treated mice. Although, the mean inward current (11.4 ± 6.9 pA, n = 6) in the STZ-treated mice seemed to be lower when compared to the control group (21.0 ± 8.6 pA, n = 6), the difference was not statistically significant (Fig. 2F, p N 0.05). To clarify the above results, in another set of experiments, GABA, glycine and glutamate were all applied in a concentration-dependent manner. The representative trace showing responses induced by

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different concentrations of GABA (3–1,000 μM) in SG neurons from control and STZ-injected mouse are shown in Fig. 3A. The responses induced by GABA were not significantly different when compared between STZ-injected and vehicle-injected mice. The mean inward currents induced by GABA were 5.5 ± 1.5 pA (3 μM), 15.2 ± 3.9 pA (10 μM), 53.4 ± 15 pA (30 μM), 196.7 ± 60.6 pA (100 μM), 378.9 ± 138 pA (300 μM) and 752.9 ± 189 pA (1,000 μM) in the STZ-injected mice and 7.9 ± 2.7 pA (3 μM), 27.6 ± 10.4 pA (10 μM), 61.8 ± 18.4 pA (30 μM), 144 ± 26.8 pA (100 μM), 331 ± 37.3 pA (300 μM) and 531 ± 87 pA (1,000 μM)) in control group (Fig. 3B, p N 0.05). Similarly, in the case of glycine, the induced inward currents were not statistically significant between the two groups. The mean glycine-induced inward currents in control and STZ-injected mice at different concentrations were as follows: 8.6 ± 2.2 pA (3 μM), 52 ± 16.8 pA (10 μM), 254 ± 57.7 pA (30 μM), 524 ± 61.3 pA (100 μM), 700 ± 139 pA (300 μM), 789 ± 153 pA (1,000 μM) and 11.4 ± 1.43 pA (3 μM), 35.4 ± 4.94 pA (10 μM), 196.4 ± 50 pA (30 μM), 585 ± 86.4 pA (100 μM), 692 ± 78.4 pA (300 μM), 701 ± 103 pA (1,000 μM) (Fig. 3C, p N 0.05). Furthermore, when glutamate was applied at different doses, similar results were seen. The inward currents induced in both groups were not significantly different. The mean inward currents in the STZ-injected mice were as follows: 1.9 ± 0.6 pA (3 μM), 6.6 ± 2.5 pA (10 μM), 19.8 ± 5 pA (30 μM), 122 ± 34.1 pA (100 μM), 284 ± 44.7 pA (300 μM) and 332 ± 64.2 pA (1,000 μM). These values were similar to those of the control group: 1.3 ± 0.2 pA (3 μM), 5.2 ± 1 pA (10 μM), 20.4 ± 3.9 pA (30 μM), 66.2 ± 10 pA (100 μM), 229.1 ± 55.2 pA (300 μM) and 297 ± 57.4 pA (1,000 μM) (Fig. 3D, p N 0.05).

3.3. Effect of STZ injection on body weight and blood glucose level in the offspring of diabetic female mice There are a number of clinical studies suggesting that diabetic neuropathy may develop in the offspring of diabetic mothers (Ornoy et al., 1998; Stenninger, Flink, Eriksson, & Sahlèn, 1998). So, in another set of experiments, the role of inhibitory and excitatory neurotransmitters was examined in the offspring from the control and diabetic mice. Fig. 4A shows the body weights of offspring from diabetic and control mice at PND 3, 6 and 9 days. In the control group, mean body weights gradually increased with age, but not in the offspring of diabetic mice. Fig. 4B shows images of pups from the control and diabetic mice on PND 7. Note that the offspring from diabetic mice failed to gain weight, so their dimensions were smaller than those of the offspring from control mice. The mean blood glucose level (58.8 ± 5.78 mg/dl) in the offspring from diabetic mice was lower than that of the offspring from control mice (109 ± 4.32 mg/dl) at PND 1–2 weeks.

Fig. 1. Bar graphs comparing the mean body weight (A) and blood glucose levels (B) between control and STZ-injected mice at different times

(⁎⁎⁎

p b 0.001).


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Fig. 2. Membrane responses elicited by glycine (100 μM), GABA (100 μM) and glutamate (30 μM) in control and STZ-injected mice. (A), (B), (C) The representative traces show the inward currents induced by glycine, GABA and glutamate in SG neurons from control and diabetic mice. (D), (E), (F) Bar graphs comparing the peak amplitudes by glycine, GABA and glutamate applications between neurons from normal and diabetic mice (p N 0.05).

3.4. Responses induced by glycine, GABA and glutamate in SG neurons in the offspring from diabetic female mice Similarly in the offspring, glycine (100 μM), GABA (100 μM) and glutamate (30 μM) were applied to the SG neurons under the condition of voltage clamp at a holding potential of − 60 mV with a high chloride pipette solution. Fig. 5A shows the traces of glycine-induced inward currents of SG neurons in offspring from control and diabetic mice. The mean inward current (740 ± 122 pA, n = 13) in the offspring from diabetic mice was similar to that of the control group (606 ± 63.1 pA, n = 13, Fig. 5D). Similarly, Fig. 5B shows the traces of GABA-induced inward currents of SG neurons in offspring from control and diabetic mice. The mean inward current (444 ± 43.7 pA, n = 17) in the offspring from diabetic mice was twice as large as that of the control group (210 ± 43.2 pA, n = 21, p b 0.001, Fig. 5E). Additionally, Fig. 5C shows the traces of glutamate-induced inward currents of SG neurons in control and pups from diabetic mice. The mean inward current (43.2 ± 11.8 pA, n = 12) in the offspring from diabetic mice was similar to that of the control group (61.3 ± 18.0 pA, n = 11, Fig. 5F). To confirm the GABA-mediated actions, responses between the two experimental groups were further examined at different concentrations ranging from 3 to 1,000 μM. Fig. 6A shows the representative current traces from SG neurons in offspring from control and diabetic mice, respectively. As we anticipated, GABA-induced inward currents in the offspring from diabetic mice were larger than those of control mice at each GABA concentration (Fig. 6B). The EC50 values for the average GABA-induced current in offspring from

control and diabetic mice were 231 and 142 μM, respectively. The mean inward currents induced by GABA at different concentrations were 0.84 ± 0.47 pA (3 μM), 16.9 ± 4.58 pA (10 μM), 53.4 ± 12.2 pA (30 μM), 161 ± 36.2 pA (100 μM), 290 ± 54.6 pA (300 μM) and 423 ± 70.8 pA (1,000 μM) in control offspring (n = 9) and 17.5 ± 6.60 pA (3 μM), 74.3 ± 20.5 pA (10 μM), 173 ± 38.1 pA (30 μM), 516 ± 69.1 pA (100 μM), 853 ± 114 pA (300 μM) and 1,095 ± 167 pA (1,000 μM) in offspring (n = 10) from diabetic mice (p b 0.05). These results suggest that GABA-binding affinity or GABA receptor expression is increased on SG neurons in the offspring of diabetic mice compared to control offspring. 4. Discussion The first characteristics of the experimental animals that were reported after STZ injections included body weight and blood glucose level. In this study, the body weight increase (29%) in the STZ-injected group, measured 2 weeks after single STZ injection, was slightly less than that of the PBS-injected group (34%) (Fig. 1A). On the other hand, blood glucose levels were 2.64 and 3.28 times higher in the STZ-injected group on the 3rd and 14th day after injection, respectively (Fig. 1B). The mild loss of body weight and elevation of blood glucose in the STZ-injected mice were similar to the results of previous studies (Goss et al., 2002; Martin, Roon, Van Ells, Ganapathy, & Smith, 2004). With regard to the offspring of diabetic mice, they exhibited lower body weight (p b 0.001, Fig. 4A) and blood glucose level compared to their age-matched controls. The lower body weight



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Fig. 3. (A) Representative current traces showing concentration dependent inward currents induced by GABA in SG neurons from control and diabetic mice. (B) A plot showing mean inward currents induced by GABA in SG neurons from control and diabetic mice. No significant difference was found in concentration–response relationship of GABA in control and STZ-injected mice (p N 0.05). (C) Graph showing the mean inward currents induced by glycine in SG neurons from control and diabetic mice. Statistical analysis showed that there was no difference in concentration–response relationship of glycine in control and STZ-injected mice (p N 0.05). (D) The figure illustrates the mean inward currents corresponding with different glutamate concentrations in both groups. Statistical analysis showed no difference between both groups (p N 0.05).

in the offspring from diabetic mice can be explained by an active deleterious process due to poor nourishment as previously reported (Steele, 1988; Tran et al., 2008). Furthermore, the lower blood glucose

Fig. 4. (A) Body weight of the offspring from control and diabetic mice (⁎⁎⁎p b 0.001). (B) Images showing the difference in dimensions between control offspring and offspring (PND 7) from diabetic mice.

level in the offspring from the STZ-treated mice can be explained by fetal hyperinsulinemia. In a pregnant subject with hyperglycemia, high glucose can be transferred from the mother to the fetus, causing fetal hyperglycemia, and this effect can lead to fetal hyperinsulinemia (Atkins, Flozak, Ogata, & Simmons, 1994). This condition can increase both metabolic rate and oxygen consumption leading to fetal hypoxemia (Aerts, Holemans, & Van Assche, 1990) and can increase glucose usage, which may lead to hypoglycemia (Aerts et al., 1990; Catalano & Kirwan, 2001). Maternal diabetes mellitus has been known to increase the risk of congenital malformation in offspring. This defect influences many major organ systems in the offspring, such as the cardiovascular, urogenital, gastrointestinal, musculoskeletal systems and the central nervous system in particular (Becerra, Khoury, Cordero, & Erickson, 1990; Kousseff, 1999; Mills, 2010). A remarkable finding of this study is that the SG neurons of the medullary dorsal horn from offspring of diabetic mice showed higher sensitivity to GABA (Fig. 5B and E). GABA, a major inhibitory neurotransmitter in the mammalian central nervous system, is intimately involved in nociception through actions at spinal and supraspinal/supratrigeminal levels (Hammond, 1997; Sawynok, 1987). Both anatomical and pharmacological evidence suggests that the activation of nociceptive afferents is associated with a release of GABA in the medullary or spinal dorsal horn (Difiglia & Aronin, 1990; Kaneko & Hammond, 1997). In addition, the dysfunction of the GABAergic system leads to a clinical expression of central pain, resulting from damage to the central nervous system (Canavero & Bonicalzi, 1998). Systemic or topical administration of GABA agonists and antagonists modulates neuronal responses in the trigeminal sensory nucleus induced by noxious and non-noxious stimulation of the orofacial area (Chiang, Kwan, Hu, & Sessle, 1999; Mitsikostas & Sanchez del Rio, 2001; Takeda, Tanimoto, Ikeda, Kadoi, & Matsumoto, 2004). Changes in the central nervous system due to STZ-induced diabetes have been demonstrated in many previous studies. A microdialysis study in the ventromedial hypothalamus of


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Fig. 5. Membrane current responses mediated by glycine (100 μM), GABA (100 μM) and glutamate (30 μM) in the offspring from control and diabetic mice. Representative traces showing inward currents induced by the presence of glycine (A), GABA (B) and glutamate (C) in neurons from controls and offspring from diabetic mice. Bar graphs comparing the average values of glycine (D), GABA (E) and glutamate-induced responses (F) between controls and offspring from diabetic mice (⁎⁎⁎p b 0.001).

rats after a single STZ injection revealed a negative correlation between extracellular GABA and extracellular concentrations of noradrenaline (Ohtani et al., 1997). Malmberg, O'Connor, Glennon, Cesena, and Calcutt (2006) measured levels of spinal amino-acid neurotransmitters in control and diabetic rats, and the results indicated that the GABA concentration in spinal dialysates increased in diabetic rats when compared with those of the control (Malmberg et al., 2006). In this study, the GABA-induced inward currents in offspring from diabetic mice provide strong evidence that this kind of offspring has a high sensitivity to GABA. In the adult nervous system, the main inhibitory neurotransmitter associated with pain modulation is GABA. However, GABA also carries out an excitatory function in the immature nervous system (CorderoErausquin, Coull, Boudreau, Rolland, & De Koninck, 2005; Obata, Oide, & Tanaka, 1978). Ordinarily, according to pharmacological criteria and the nature of coupling between the receptor and ion channels, GABA receptors are divided into 3 types: GABAA, GABAB and GABAC. GABAA and GABAC receptors are ligand-gated ion channels that contain a GABA binding site directly linked to a chloride channel (Schofield et al., 1987). Alternatively, GABAB receptors are coupled indirectly to potassium or calcium channels via G-proteins (Andrade, Malenka, & Nicoll, 1986). An impaired inhibitory function of GABA due to diabetes is revealed by the

changes in the activity of GABAA receptors, which mainly depends on K + Cl− cotransporter 2 (KCC2) (Coull et al., 2003; Morales-Aza, Chillingworth, Payne, & Donaldson, 2004). In neuropathic pain models, reductions in KCC2 expression lead to an increase in intracellular chloride. Under such conditions, GABA acts as an excitatory rather than an inhibitory neurotransmitter by binding to post-synaptic GABAA receptors (Morgado, Pinto-Ribeiro, & Tavares, 2008). With a defective central nervous system due to diabetic neuropathy, an elevated sensitivity of GABA receptors in the SG area of offspring from diabetic mice may represent increased excitatory synaptic input due to spontaneous hyperactivity of peripheral nerve fibers. In other words, hyperactivity of dorsal horn neurons in the offspring from diabetic mice plays an important role in transmitting orofacial nociceptive information. In this study, we used a high chloride pipette solution to amplify chloride-mediated conductance. The results showed that the inward currents induced by different concentrations of glycine and glutamate in the diabetic state did not have any significant difference when compared with control mice. However, although GABA-mediated responses were created with nearly the same amplitudes in SG neurons from control and STZ-injected mice, the diabetic state produced a significant change in the properties of GABA receptors in the offspring from diabetic mice. This change is in line with previous studies reporting a higher expression of



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of Education, Science and Technology (2014R1A1A2054241) and (2013R1A1A2058356).

References

Fig. 6. Concentration–Response relationship in offspring from control and diabetic mice. (A) Representative traces showing the changes in inward currents evoked by various concentrations of GABA in neurons from control and offspring from diabetic mice. (B) The response curve figure illustrates not only the mean inward current changes corresponding with different GABA concentrations, but also the difference between GABA-induced responses in both groups.

GABA neurotransmitter involved in the brain/blood glucose system in the spinal cord of diabetic animals (Beverly, Beverly, & Meguid, 1995; Malmberg et al., 2006; Morgado et al., 2008). The combination of these results with previous ones may explain the relation between spinal GABA level and nerve fiber hyperactivity in diabetic neuropathic pain. In summary, this study provides emerging evidence that the sensitivity of GABA receptors increases in offspring from diabetic mice. This information is important for understanding the mechanisms responsible not only for central sensitization and neuropathic pain, in general, but also the change in orofacial pain processing in offspring from the diabetic mice. These findings also support the concept that an altered fetal hormonal environment during critical periods of development can yield permanent consequences that contribute to the subsequent development of disease. This study analyzed only the short-term consequences of STZ-induced diabetes, so further studies are needed to ascertain the long-term alterations of SG neurons in the diabetic state.

Acknowledgements This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry

Aerts, L., Holemans, K., & Van Assche, F. A. (1990). Maternal diabetes during pregnancy: Consequences for the offspring. Diabetes/Metabolism Reviews, 6(3), 147–167. Andrade, R., Malenka, R. C., & Nicoll, R. A. (1986). A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science, 234(4781), 1261–1265. Arap, A., Siqueira, S. R., Silva, C. B., Teixeira, M. J., & Siqueira, J. T. (2010). Trigeminal pain and quantitative sensory testing in painful peripheral diabetic neuropathy. Archives of Oral Biology, 55(7), 486–493. Atkins, V., Flozak, A. S., Ogata, E. S., & Simmons, R. A. (1994). The effects of severe maternal diabetes on glucose transport in the fetal rat. Endocrinology, 135(1), 409–415. Becerra, J. E., Khoury, M. J., Cordero, J. F., & Erickson, J. D. (1990). Diabetes mellitus during pregnancy and the risks for specific birth defects: A population-based casecontrol study. Pediatrics, 85(1), 1–9. Beverly, J. L., Beverly, M. F., & Meguid, M. M. (1995). Alterations in extracellular GABA in the ventral hypothalamus of rats in response to acute glucoprivation. The American Journal of Physiology, 269(5), R1174–R1178. Burchiel, K. J., Russell, L. C., Lee, R. P., & Sima, A. A. (1985). Spontaneous activity of primary afferent neurons in diabetic BB/Wistar rats. A possible mechanism of chronic diabetic neuropathic pain. Diabetes, 34(11), 1210–1213. Canavero, S., & Bonicalzi, V. (1998). The neurochemistry of central pain: Evidence from clinical studies, hypothesis and therapeutic implications. Pain, 74(2–3), 109–114. Catalano, P. M., & Kirwan, J. P. (2001). Maternal factors that determine neonatal size and body fat. Current Diabetes Reports, 1(1), 71–77. Chen, X., & Levine, J. D. (2001). Hyper-responsivity in a subset of C-fiber nociceptors in a model of painful diabetic neuropathy in the rat. Neuroscience, 102(1), 185–192. Chen, S. R., & Pan, H. L. (2002). Hypersensitivity of spinothalamic tract neurons associated with diabetic neuropathic pain in rats. Journal of Neurophysiology, 87(6), 2726–2733. Chiang, C. Y., Kwan, C. L., Hu, J. W., & Sessle, B. J. (1999). Effects of GABA receptor antagonist on trigeminal caudalis nociceptive neurons in normal and neonatally capsaicin-treated rats. Journal of Neurophysiology, 82(5), 2154–2162. Cordero-Erausquin, M., Coull, J. A., Boudreau, D., Rolland, M., & De Koninck, Y. (2005). Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: Impact of chloride extrusion capacity. The Journal of Neuroscience, 25(42), 9613–9623. Coull, J. A., Boudreau, D., Bachand, K., Prescott, S. A., Nault, F., Sik, A., et al. (2003). Transsynaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature, 424(6951), 938–942. Denroche, H. C., Levi, J., Wideman, R. D., Sequeira, R. M., Huynh, F. K., Covey, S. D., et al. (2011). Leptin therapy reverses hyperglycemia in mice with streptozotocininduced diabetes, independent of hepatic leptin signaling. Diabetes, 60(5), 1414–1423. Difiglia, M., & Aronin, N. (1990). Synaptic interactions between GABAergic neurons and trigeminothalamic cells in the rat trigeminal nucleus caudalis. Synapse, 6(4), 358–363. Galer, B. S., Gianas, A., & Jensen, M. P. (2000). Painful diabetic polyneuropathy: Epidemiology, pain description, and quality of life. Diabetes Research and Clinical Practice, 47(2), 123–128. Goss, J. R., Goins, W. F., Lacomis, D., Mata, M., Glorioso, J. C., & Fink, D. J. (2002). Herpes simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse. Diabetes, 51(7), 2227–2232. Hammond, D. (1997). Inhibitory neurotransmitters and nociception: role of GABA and glycine. The pharmacology of pain. (pp. 361–383). Kaneko, M., & Hammond, D. L. (1997). Role of spinal gamma-aminobutyric acidA receptors in formalin-induced nociception in the rat. The Journal of Pharmacology and Experimental Therapeutics, 282(2), 928–938. Kawamata, M., & Omote, K. (1996). Involvement of increased excitatory amino acids and intracellular Ca2+ concentration in the spinal dorsal horn in an animal model of neuropathic pain. Pain, 68(1), 85–96. Kohno, T., Kumamoto, E., Higashi, H., Shimoji, K., & Yoshimura, M. (1999). Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord. The Journal of Physiology, 518(Pt 3), 803–813. Kousseff, B. G. (1999). Diabetic embryopathy. Current Opinion in Pediatrics, 11(4), 348–352. Le Floch, J. P., Le Lievre, G., Sadoun, J., Perlemuter, L., Peynegre, R., & Hazard, J. (1989). Taste impairment and related factors in type I diabetes mellitus. Diabetes Care, 12(3), 173–178. Light, A. R., & Perl, E. R. (1979). Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers. The Journal of Comparative Neurology, 186(2), 133–150. Maksimovic-Ivanic, D., Trajkovic, V., Miljkovic, D. J., Mostarica Stojkovic, M., & StosicGrujicic, M. (2002). Down-regulation of multiple low dose streptozotocin-induced diabetes by mycophenolate mofetil. Clinical and Experimental Immunology, 129(2), 214–223. Malmberg, A. B., O'Connor, W. T., Glennon, J. C., Cesena, R., & Calcutt, N. A. (2006). Impaired formalin-evoked changes of spinal amino acid levels in diabetic rats. Brain Research, 1115(1), 48–53.


8


Martin, P. M., Roon, P., Van Ells, T. K., Ganapathy, V., & Smith, S. B. (2004). Death of retinal neurons in streptozotocin-induced diabetic mice. Investigative Ophthalmology & Visual Science, 45(9), 3330–3336. Meurman, J. H., Collin, H. L., Niskanen, L., Toyry, J., Alakuijala, P., Keinanen, S., et al. (1998). Saliva in non-insulin-dependent diabetic patients and control subjects: The role of the autonomic nervous system. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics, 86(1), 69–76. Mills, J. L. (2010). Malformations in infants of diabetic mothers. Teratology 25:385-94. 1982. Birth Defects Research. Part A, Clinical and Molecular Teratology, 88(10), 769–778. Mitsikostas, D. D., & Sanchez del Rio, M. (2001). Receptor systems mediating c-fos expression within trigeminal nucleus caudalis in animal models of migraine. Brain Research Reviews, 35(1), 20–35. Moore, P. A., Guggenheimer, J., & Orchard, T. (2007). Burning mouth syndrome and peripheral neuropathy in patients with type 1 diabetes mellitus. Journal of Diabetes and its Complications, 21(6), 397–402. Morales-Aza, B. M., Chillingworth, N. L., Payne, J. A., & Donaldson, L. F. (2004). Inflammation alters cation chloride cotransporter expression in sensory neurons. Neurobiology of Disease, 17(1), 62–69. Morgado, C., Pinto-Ribeiro, F., & Tavares, I. (2008). Diabetes affects the expression of GABA and potassium chloride cotransporter in the spinal cord: A study in streptozotocin diabetic rats. Neuroscience Letters, 438(1), 102–106. Morgado, C., & Tavares, I. (2007). C-fos expression at the spinal dorsal horn of streptozotocin-induced diabetic rats. Diabetes/Metabolism Research and Reviews, 23(8), 644–652. Nguyen, T. T. H., Bhattarai, J. P., Park, S. J., & Han, S. K. (2013). Activation of glycine and extrasynaptic GABA(A) receptors by taurine on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis. Neural Plasticity, 2013 (Article ID 740581, 12 pages). Obata, K., Oide, M., & Tanaka, H. (1978). Excitatory and inhibitory actions of GABA and glycine on embryonic chick spinal neurons in culture. Brain Research, 144(1), 179–184. Ohtani, N., Ohta, M., & Sugano, T. (1997). Microdialysis study of modification of hypothalamic neurotransmitters in streptozotocin-diabetic rats. Journal of Neurochemistry, 69(4), 1622–1628. Ornoy, A., Ratzon, N., Greenbaum, C., Peretz, C., Soriano, D., & Dulitzky, M. (1998). Neurobehaviour of school age children born to diabetic mothers. Archives of Disease in Childhood. Fetal and Neonatal Edition, 79(2), F94–F99. Pan, Y. Z., & Pan, H. L. (2004). Primary afferent stimulation differentially potentiates excitatory and inhibitory inputs to spinal lamina II outer and inner neurons. Journal of Neurophysiology, 91(6), 2413–2421. Pertovaara, A., Wei, H., Kalmari, J., & Ruotsalainen, M. (2001). Pain behavior and response properties of spinal dorsal horn neurons following experimental diabetic neuropathy in the rat: Modulation by nitecapone, a COMT inhibitor with antioxidant properties. Experimental Neurology, 167(2), 425–434. Podwall, D., & Gooch, C. (2004). Diabetic neuropathy: Clinical features, etiology, and therapy. Current Neurology and Neuroscience Reports, 4(1), 55–61.

Polgar, E., Hughes, D. I., Arham, A. Z., & Todd, A. J. (2005). Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. The Journal of Neuroscience, 25(28), 6658–6666. Price, T. J., Cervero, F., & de Koninck, Y. (2005). Role of cation-chloride-cotransporters (CCC) in pain and hyperalgesia. Current Topics in Medicinal Chemistry, 5(6), 547–555. Ramsey, D. J., Ripps, H., & Qian, H. (2007). Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons. Experimental Eye Research, 85(3), 413–422. Santos, S. F., Rebelo, S., Derkach, V. A., & Safronov, B. V. (2007). Excitatory interneurons dominate sensory processing in the spinal substantia gelatinosa of rat. The Journal of Physiology, 581(Pt 1), 241–254. Sawynok, J. (1987). GABAergic mechanisms of analgesia: An update. Pharmacology, Biochemistry, and Behavior, 26(2), 463–474. Schofield, P. R., Darlison, M. G., Fujita, N., Burt, D. R., Stephenson, F. A., Rodriguez, H., et al. (1987). Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature, 328(6127), 221–227. Sima, A. A. (2003). New insights into the metabolic and molecular basis for diabetic neuropathy. Cellular and Molecular Life Sciences, 60(11), 2445–2464. Steele, E. J. (1988). Observations on offspring of mice made diabetic with streptozocin. Diabetes, 37(8), 1035–1043. Stenninger, E., Flink, R., Eriksson, B., & Sahlèn, C. (1998). Long-term neurological dysfunction and neonatal hypoglycaemia after diabetic pregnancy. Archives of Disease in Childhood. Fetal and Neonatal Edition, 79(3), F174–F179. Sugiura, Y., Lee, C. L., & Perl, E. R. (1986). Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science, 234(4774), 358–361. Takeda, M., Tanimoto, T., Ikeda, M., Kadoi, J., & Matsumoto, S. (2004). Activaton of GABAB receptor inhibits the excitability of rat small diameter trigeminal root ganglion neurons. Neuroscience, 123(2), 491–505. Todd, A. J., Watt, C., Spike, R. C., & Sieghart, W. (1996). Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord. The Journal of Neuroscience, 16(3), 974–982. Tran, S., Chen, Y. W., Chenier, I., Chan, J. S., Quaggin, S., Hebert, M. J., et al. (2008). Maternal diabetes modulates renal morphogenesis in offspring. Journal of the American Society of Nephrology, 19(5), 943–952. Wang, X. L., Zhang, H. M., Chen, S. R., & Pan, H. L. (2007). Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy. The Journal of Physiology, 579(Pt 3), 849–861. Yoshimura, M., & Nishi, S. (1995). Primary afferent-evoked glycine- and GABAmediated IPSPs in substantia gelatinosa neurones in the rat spinal cord in vitro. The Journal of Physiology, 482(Pt 1), 29–38. Zhang, H. M., Li, D. P., Chen, S. R., & Pan, H. L. (2005). M2, M3, and M4 receptor subtypes contribute to muscarinic potentiation of GABAergic inputs to spinal dorsal horn neurons. The Journal of Pharmacology and Experimental Therapeutics, 313(2), 697–704.


Excitation of marginal and substantia gelatinosa neurons in the primate spinal cord: indications of their place in dorsal horn functional organization.

Functional expression of 5-HT7 receptor on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

Ultrastructural studies on postnatal differentiation of neurons in the substantia gelatinosa of rat cervical spinal cord.

Neonatal capsaicin treatment induces invasion of the substantia gelatinosa by the terminal arborizations of hair follicle afferents in the rat dorsal horn.

Morphine, enkephalin and the substantia gelatinosa.

Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons.

Proton-induced currents in substantia gelatinosa neurons of the rat trigeminal subnucleus caudalis.

DAMGO modulates two-pore domain K(+) channels in the substantia gelatinosa neurons of rat spinal cord.

Diencephalic projections from the superficial and deep laminae of the medullary dorsal horn in the rat.

Cholecystokinin concentrations and peptide immunoreactivity in the intact and deafferented medullary dorsal horn of the rat.

Effects of tramadol on substantia gelatinosa neurons in the rat spinal cord: an in vivo patch-clamp analysis.

The action of baclofen on neurons of the substantia nigra and of the ventral tegmental area.

Dual effect of exogenous nitric oxide on neuronal excitability in rat substantia gelatinosa neurons.

Neurons with two axons in the substantia gelatinosa layer of the spinal trigeminal nucleus of the adult cat.

GABA-like immunoreactivity in type I glomeruli of rat substantia gelatinosa.

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa.

Effects of peripheral afferent stimulation on dorsal horn neurons in the chicken (Gallus domesticus).

Contribution of persistent sodium currents to the excitability of tonic firing substantia gelatinosa neurons of the rat.

Effects of spinal kappa-opioid receptor agonists on the responsiveness of nociceptive superficial dorsal horn neurons.

Dynamic alterations in the cutaneous mechanoreceptive fields of dorsal horn neurons in the rat spinal cord.

Inhibition of temporomandibular joint input to medullary dorsal horn neurons by 5HT3 receptor antagonist in female rats.

Immunocytochemical evidence that GABA and neurotensin exist in different neurons in laminae II and III of rat spinal dorsal horn.

Reactive oxygen species increase neuronal excitability via activation of nonspecific cation channel in rat medullary dorsal horn neurons.

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.