Respiratory deficits in a rat model of Parkinson’s disease

NSC 16158

No. of Pages 11

2 April 2015 Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048 1

Neuroscience xxx (2015) xxx–xxx

2

RESPIRATORY DEFICITS IN A RAT MODEL OF PARKINSON’S DISEASE

3 4 5

M. TUPPY, a B. F. BARNA, b L. ALVES-DOS-SANTOS, a L. R. G. BRITTO, b S. CHIAVEGATTO, a T. S. MOREIRA b AND A. C. TAKAKURA a*

6 7 8 9 10

11

a

Department of Pharmacology, Institute of Biomedical Science, University of Sa˜o Paulo, 05508-000 Sa˜o Paulo, SP, Brazil b

Department of Physiology and Biophysics, Institute of Biomedical Science, University of Sa˜o Paulo, 05508-000 Sa˜o Paulo, SP, Brazil

Abstract—Parkinson’s disease (PD) is a neurodegenerative disease characterized by loss of the dopaminergic nigrostriatal pathway. In addition to deficits in voluntary movement, PD involves a disturbance of breathing regulation. However, the cause and nature of this disturbance are not well understood. Here, we investigated breathing at rest and in response to hypercapnia (7% CO2) or hypoxia (8% O2), as well as neuroanatomical changes in brainstem regions essential for breathing, in a 6-hydroxydopamine (6-OHDA) rat model of PD. Bilateral injections of 6-OHDA (24 lg/ll) into the striatum decreased tyrosine hydroxylase (TH+)neurons in the substantia nigra pars compacta (SNpc), transcription factor phox2b-expressing neurons in the retrotrapezoid nucleus and neurokinin-1 receptors in the ventral respiratory column. In 6-OHDA-lesioned rats, respiratory rate was reduced at rest, leading to a reduction in minute ventilation. These animals also showed a reduction in the tachypneic response to hypercapnia, but not to hypoxia challenge. These results suggest that the degeneration of TH+ neurons in the SNpc leads to impairment of breathing at rest and in hypercapnic conditions. Our data indicate that respiratory deficits in a 6-OHDA rat model of PD are related to downregulation of neural systems involved in respiratory rhythm generation. The present study suggests a new avenue to better understand the respiratory deficits observed in chronic stages of PD. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: Parkinson’s disease, breathing, hypercapnia, hypoxia, brainstem. 12

*Corresponding author. Address: Department of Pharmacology, Institute of Biomedical Science, University of Sa˜o Paulo, 1524, Prof. Lineu Prestes Avenue, 05508-000 Sa˜o Paulo, SP, Brazil. Tel: +5511-3091-7314; fax: +55-11-3091-7285. E-mail address: [email protected] (A. C. Takakura). Abbreviations: 6-OHDA, 6-hydroxydopamine; cVRG, caudal ventral respiratory group; fR, respiratory frequency; LC, locus coeruleus; MV, minute ventilation; PD, Parkinson’s disease; phox2b-ir, phox2b immunoreactivity; preBo¨tC, pre-Bo¨tzinger complex; RTN, retrotrapezoid nucleus; rVRG, rostral ventral respiratory group; SN, substantia nigra; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; TH-ir, TH-immunoreactivity; VRC, ventral respiratory column; VT, tidal volume. http://dx.doi.org/10.1016/j.neuroscience.2015.03.048 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

13

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, characterized by resting tremor, rigidity, bradykinesia and postural instability, as well as non-motor features, including abnormalities of the autonomic nervous system. Although the motor symptoms of PD are considered a pathological hallmark of the disease (Fearnley and Lees, 1991), multiple debilitating symptoms are related to the non-motor aspects of PD (Wolters, 2009). These include sleep disturbances, neuropsychiatric and cognitive deficits, autonomic and sensory dysfunction and breathing instability (Bassetti, 2011; Chaudhuri et al., 2011). Breathing depends on a sophisticated neural network in the lower brainstem that controls respiratory rhythm and pattern generation (Feldman and Ellenberger, 1988; Nogue´s et al., 2002; Feldman and Del Negro, 2006; Feldman et al., 2013). Research efforts are escalating to understand the underlying causes of neuronal respiratory dysfunction, which is symptomatic of many diseases that can occur at almost any time during life (Axelrod et al., 2006; Weese-Mayer et al., 2006, 2008). Breathing deficits, including prolonged and frequent apneas during sleep, are present in a number of neurodegenerative diseases such as PD, multiple system atrophy and amyotrophic lateral sclerosis (Benarroch, 2003, 2007; Benarroch et al., 2003; Schwarzacher et al., 2011). Therefore, it becomes important to evaluate how these conditions can modify breathing activity. The aim of this manuscript was to investigate breathing at rest and in response to hypercapnia (7% CO2) or hypoxia (8% O2), as well as neuroanatomical changes in brainstem regions responsible for the neural control of breathing. We selected a widely used rat model of PD created by bilateral injections of 6-hydroxydopamine (6-OHDA) into the striatum.

14

RESULTS

49

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Animal model of PD: bilateral intra-striatal 6-OHDA selectively destroyed tyrosine hydroxylase (TH)expressing-neurons of the substantia nigra (SN)

50

The 6-OHDA neurotoxic lesion within the nigrostriatal dopaminergic system is one of the most widely used for modeling PD in rodents (McDowell and Chesselet, 2012). In this study, 6-OHDA (6, 12 or 24 lg/ll) was administered into the dorsal striatum of three groups of rats. The size and specificity of the lesion were determined by TH-immunoreactivity (TH-ir) in the SN. Compared to vehicle-infused control rats (906 ± 17

53

51 52

54 55 56 57 58 59 60

NSC 16158

No. of Pages 11

2 April 2015

2 61 62 63 64 65 66 67 68 69 70 71 72

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

neurons), 6-OHDA (6, 12 and 24 lg/ll) dramatically and dose-dependently reduced the number of TH-ir neurons in the SN (722 ± 15, 204 ± 18 and 135 ± 23, respectively, p < 0.001; AP 5.32 to 6.04 mm relative to Bregma, counted in one-in-six series of 40-lm brain sections per rat) (Fig. 1a, b, e). We also analyzed TH-ir in A6 (locus coeruleus: LC) and intra-striatal 6-OHDA did not change the number of TH neurons in A6 compared to control rats (Fig. 1c, d, e). Previous studies have shown that intrastriatal injection of 6-OHDA did not cause degeneration of catecholaminergic neurons in the LC (Miguelez et al., 2011).

6-OHDA selectively destroyed neurons involved in respiratory control

73

To further assess the effect of bilateral intrastriatal 6-OHDA on neurons involved in respiratory control, phox2b immunoreactivity (phox2b-ir) was examined within the retrotrapezoid nucleus (RTN) at 60 days after injection. The number of phox2b-ir nuclei counted in the RTN was unchanged after vehicle or 6 lg of 6-OHDA (one-in-six series of 40-lm brain sections per rat) (Fig. 2e). A massive reduction of phox2b-ir nuclei was found after 12 and 24 lg 6-OHDA (51 ± 3 and 30 ± 4,

75

Fig. 1. Bilateral intrastriatal injections of 6-OHDA destroy TH+ neurons within the substantia nigra (SN). Photomicrographs from a control animal that received bilateral intrastriatal injections of vehicle (left column, panels a, c) or 24 lg of 6-OHDA (right column, panels b, d). Compared to vehicle, 6-OHDA caused an almost complete loss of TH-immunoreactivity at the level of the SN (a, b), but A6 neurons (locus coeruleus) were intact (c, d). (e) Group data. Each column represents the total number of neurons of a given type in five consecutive 40-lm-thick coronal sections, separated by 240 lm. Scale bar in b = 500 lm for panels a, b. Scale bar in d = 200 lm for panels c, d. Abbreviations: SNR, substantia nigra reticulata; SNC, substantia nigra pars compacta; ml, medial lemniscus; VTA, ventral tegmental area; 4V, fourth ventricle. Bar: Barrington’s nucleus. ⁄ p < 0.05 relative to vehicle (sham). Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

74

76 77 78 79 80 81 82 83

NSC 16158

No. of Pages 11

2 April 2015

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

3

Fig. 2. 6-OHDA reduced phox2b in RTN. Photomicrographs from a control animal that received bilateral intrastriatal injections of vehicle (left column, panels a, c) or 24 lg of 6-OHDA (right column, panels b, d). Compared to vehicle, 6-OHDA reduced Phox2b-immunoreactive neurons at the level of RTN (a–d). (e) Group data. Each column represents the total number of neurons of a given type in four consecutive 40-lm-thick coronal sections, separated by 240 lm. Scale bar in b = 200 lm for panels a, b. Scale bar in d = 100 lm for panels c, d. Abbreviations: VII, facial motor nucleus; py, pyramidal tract. ⁄p < 0.05 relative to vehicle (sham).

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104

respectively, vs. vehicle 119 ± 16 neurons, p < 0.05) (Fig. 2a–e). Interestingly, the vast majority of the phox2b-ir nuclei that remained within the RTN region after 6-OHDA (24 lg/ll) belonged to the dorsal cap area of RTN (Fig. 2d) (Mulkey et al., 2004). The phox2b neurons were almost absent in the marginal layer, the thin band of myelin-poor brain parenchyma. The marginal layer that lines the ventral surface of the medulla oblongata contains neurons thought to contribute to central chemoreception (Fig. 2c–d) (Mulkey et al., 2004). The dorsal cap area of the RTN includes many neurons that express phox2b and/or TH (consistent with the abundance of C1 adrenergic neurons and the presence of many presympathetic neurons in the region) (Schreihofer and Guyenet, 1997). 6-OHDA also reduced substance P receptor immunoreactivity (neurokinin 1 receptor: NK1R-ir) in the ventral respiratory column (VRC) (Fig. 3). Vehicle and the 6-lg dose of 6-OHDA produced no detectable lesion in any region of the VRC, but 12 and 24 lg of 6-OHDA caused extensive reductions of NK1R-ir within the

pre-Bo¨tzinger complex (preBo¨tC) (23 ± 0.4 and 24 ± 1.1, respectively vs. vehicle 39.7 ± 1.2%, p < 0.05) and in the rostral ventral respiratory group (rVRG) (31 ± 0.9 and 32.5 ± 0.5, respectively vs. vehicle 37.6 ± 0.6%, p < 0.05) (Fig. 3b, d, f, h, i). No significant change of NK1R-ir was detected within the Bo¨tzinger complex (Bo¨tC) or caudal ventral respiratory group (cVRG) (Fig. 3i). NK1R in dorsal brainstem regions such as the solitary tract and motor nucleus of the vagus appeared normal with no sign of toxin-mediated degradation or receptor internalization (data not shown).

105

6-OHDA impaired breathing

116

We measured breathing parameters [tidal volume (VT), respiratory frequency (fR) and minute ventilation (MV)] in unrestrained awake rats. Forty days after bilateral intrastriatal 6-OHDA (12 and 24 lg), the breathing pattern began to transform from eupneic breathing to a decrease in fR in room air (Figs. 4a, b, 5a). That effect was observed from 40 to 60 days after the lesion

117

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

106 107 108 109 110 111 112 113 114 115

118 119 120 121 122 123

NSC 16158

No. of Pages 11

2 April 2015

4

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

Fig. 3. 6-OHDA reduced NK1R in Pre-Bo¨tzinger (PreBotC) and rostral ventral respiratory group (rVRG). Photomicrographs from a control animal that received bilateral intrastriatal injections of vehicle (left column, panels a, c, e, g) or 24 lg of 6-OHDA (right column, panels b, d, f, h). 6-OHDA reduced NK1R-ir at the level of the Pre-Bo¨tzinger complex (PreBotC; b, d) and rostral ventral respiratory group (rVRG; f, h). Each column represents total NK1R-immunoreactive density in four consecutive 40-lm-thick coronal sections, separated by 240 lm. Scale bar in f = 500 lm for panels a, b, e, f and h = 100 lm for panels c, d, g and h. Abbreviations: Amb, nucleus ambiguus; LRN, lateral reticular nucleus. ⁄p < 0.05 relative to vehicle (sham). 124 125 126

(77 ± 7 and 74 ± 3, respectively vs. vehicle: 107 ± 13 breaths/min, p < 0.001) (Figs. 4a, b, 5a). The reduction in fR led to a considerable reduction in MV

(636 ± 69 and 651 ± 85, respectively vs. vehicle: 1032 ± 44 breaths/min, p < 0.001) (Figs. 4d, 5c). There were no changes in resting VT (p > 0.05) (Figs. 4c, 5b).

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

127 128 129

NSC 16158

No. of Pages 11

2 April 2015

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

5

Fig. 4. 6-OHDA reduced breathing at rest and in response to hypercapnia in unanesthetized rats. (a) Typical recording showing airflow at rest and during hypercapnia (7% CO2) at 40 days after bilateral intrastriatal 6-OHDA (24 lg/ll) or vehicle. Changes in (b) respiratory rate (fR), (c) tidal volume (VT) and (d) minute ventilation (MV) at rest and during hypercapnia (7% CO2) at 1 day before and 40, 50 and 60 days after bilateral intrastriatal 6-OHDA (6, 12 or 24 lg/ll) or vehicle. ⁄p < 0.05 relative to vehicle (sham). 130 131 132 133 134 135

Arterial blood gases and pH values of 6-OHDA and control groups were evaluated 61 days after the lesion. There were no significant changes in any parameters (pO2, pCO2, pH and HCO3 ), suggesting that the animals could compensate for their breathing deficits using other physiological mechanisms (Table 1).

6-OHDA reduced breathing responses to central but not peripheral chemoreflex

136

Hypercapnia and hypoxia challenges were performed to assess the role of central or peripheral chemoreceptors in the model of PD. Animals were exposed for 10 min to hypercapnia (7% CO2, 21% O2, balanced with N2 in the

138

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

137

139 140 141

NSC 16158

No. of Pages 11

2 April 2015

6

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

Fig. 5. 6-OHDA did not affect breathing in response to hypoxia in unanesthetized rats. Changes in (a) respiratory rate (fR), (b) tidal volume (VT), and (c) minute ventilation (MV) produced by hypoxia (8% O2) at 1 day before and 40, 50 and 60 days after bilateral intrastriatal 6-OHDA (6, 12 or 24 lg/ll) or vehicle. ⁄p < 0.05 relative to vehicle (sham).

Table 1. Effects of bilateral intrastriatal 6-OHDA on blood gas parameters pH Sham 6 lg 12 lg 24 lg

7.50 ± 0.02 7.50 ± 0.01 7.50 ± 0.02 7.51 ± 0.01

PaCO2 (mmHg)

PaO2 (mmHg)

[HCO3] (mmHg)

35.5 ± 0.9 35.4 ± 1.8 36.4 ± 1.2 31.7 ± 1.7

73.8 ± 1.6 80.2 ± 3.7 78.4 ± 2.9 79.4 ± 2.9

29.5 ± 0.5 27.3 ± 1.5 29.2 ± 0.9 26.1 ± 1.1

Values are expressed as mean ± SEM. HCO3, bicarbonate; PaCO2, partial pressure of CO2; PaO2, partial pressure of O2. n = 8.

142 143 144 145 146

147 148 149 150

inspired air) 1 day before and 40, 50 and 60 days after lesions to evaluate the effect of central chemoreflex activation. In unanesthetized animals, the tachypnea response to hypercapnia was mildly reduced on days 40 and 50 after bilateral intrastriatal 6-OHDA (12 or 24 lg/ ll) (133 ± 0.3 and 129 ± 3, respectively vs. vehicle: 144 ± 4 breaths/min, p < 0.001; Fig. 4a, b). The normal tachypneic response was restored by 60 days. Analysis of the fR excluded sniffing and other behavioral artifacts

that could interfere with the analysis. No changes in VT and MV were observed at any dose of 6-OHDA (Fig. 4a, c, d). The same groups of rats were also exposed to hypoxia for 10 min (8% O2, normocapnic and balanced with N2 in the inspired air) 1 day before and 40, 50 and 60 days after lesions to evaluate the effect of peripheral chemoreflex activation. No significant changes in the ventilatory responses to hypoxia were observed in the 6-OHDA group compared to the control group (p > 0.05) (Fig. 5a–c).

151

6-OHDA did not affect motor behavior

162

In a separate group of rats, we examined motor behaviors 1 day before and 30 and 60 days after 6, 12 or 24 lg of bilateral intrastriatal 6-OHDA. The imunohistochemistry for TH in the SN of these animals, evaluated at the end of the experiments, revealed the same pattern of lesions found in the animals used in the respiratory studies. Spontaneous locomotion, assessed for 5 min in an open

163

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

152 153 154 155 156 157 158 159 160 161

164 165 166 167 168 169

NSC 16158

No. of Pages 11

2 April 2015

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

177

field, was similar across groups at each time point (F(3,27) = 0.86; p > 0.05; Fig. 6a). Motor coordination and balance, assessed as the latency to fall from an accelerating rotarod, was also similar across groups and times (F(3,27) = 0.02; p > 0.05; Fig. 6b). Additionally, catalepsy in the bar test was similar across groups (F(3,27) = 0.38; p > 0.05) and times (F(2,27) = 1.65; p > 0.05) (data not shown).

178

DISCUSSION

179

187

In the current study, 40–60 days after degeneration of dopaminergic neurons in the SN induced by bilateral intrastriatal 6-OHDA, respiration transformed from eupneic breathing to a severe reduction in fR. We also observed a change in the tachypneic response to central, but not peripheral chemoreflex activation. The changes in breathing were strongly correlated to the loss of neurons in the VRC, namely RTN, preBo¨tC and rVRG.

188

Breathing automaticity

189

Clinicians have observed breathing deficits in patients with PD (Idiaquez et al., 2007; Reyes et al., 2013), but they have focused primarily on the hallmark motor symptoms of the disease. Our results provide direct evidence that an experimental model of PD has considerable changes in fR at rest, which could explain the breathing problems seen in patients with severe PD. A previous study by Budzinska and Andrzejewski reported respiratory deficits in the 6-OHDA rat model of PD (Budzinska and Andrzejewski, 2014). However, our study differs from this previous study in several ways. We used unrestrained animals after bilateral intrastriatal 6-OHDA, while Budzinska and Andrzejewski used anesthetized animals after unilateral intrastriatal 6-OHDA. Caution must be exercised when interpreting experiments under anesthesia because anesthetic may affect centrally-mediated cardiorespiratory responses. Furthermore, a bilateral lesion is more representative of the clinical aspect of severe PD. These crucial differences could explain why we observed an effect on resting breathing, while Budzinska and Andrzejewski did not. To our knowledge, the current study is the first to demonstrate that bilateral destruction of the dopaminergic neurons in the SN changes ventilation in conscious rats. The present data will certainly open new perspectives to better understand breathing impairments in chronic stages of the disease. The 6-OHDA rat model of PD has been extensively studied because it induces destruction of the nigrostriatal pathway, which causes neurological impairment, akinesia, and sensory impairment (Schwarting and Huston, 1996). Despite many studies having shown that patients with PD have impairment of respiratory muscle function (Tzelepis et al., 1988; Solomon and Hixon, 1993; Bunton, 2005; Troche et al., 2010; Hammer and Barlow, 2010), Seccombe and collegues (2011) suggested that the respiratory impairment observed could be associated with a dysfunction of respiratory neurons within the brainstem, in a region called the ventral respiratory column (VRC), since in their

170 171 172 173 174 175 176

180 181 182 183 184 185 186

190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227

7

Fig. 6. 6-OHDA did not affect motor behavior. (a) Spontaneous locomotor activity in a 5-min session of an open field. (b) Motor coordination and balance in an accelerating rotarod (5–37 rpm, 6 cmdiameter) over a period of 5 min. Behaviors were assessed in an independent cohort of rats at 1 day before and 30 and 60 days after bilateral intrastriatal 6-OHDA (6, 12, or 24 lg) or vehicle (sham).

study they did not see any kind of respiratory muscle disfunction (Seccombe et al., 2011). In their study, they observed that respiratory muscle weakness is common, but insufficient to cause restriction, there is an impairment of the response to hypercapnia in 47% of the patients with PD analyzed but not to hypoxia, suggesting an abnormal respiratory control (Seccombe et al., 2011). In the current study, chronic bilateral lesions of the striatum performed with 6-OHDA in adult rats attenuate breathing at rest and in response to activation by hypercapnia. Additionally, 6-OHDA caused a massive reduction in different phenotypes (Phox2b and NK1R) of neurons in the VRC. Furthermore, doses of 6-OHDA that eliminated 74% of the phox2b-expressing neurons in the RTN and 39% of pre-Botzinger NK1R neurons caused a reduction of breathing at rest and during central chemoreflex activation. This could be explained by the fact that Phox2b is a transcription factor present in the RTN neuron that is involved in central chemoreception and NK1R demarks a limited region of the rodent ventrolateral respiratory column as the preBo¨tC, responsible for respiratory rhythm generation (Guyenet and Wang, 2001; Stornetta et al., 2006). Bilateral intrastriatal 6-OHDA was associated with changes in neuronal cytoarchitecture of crucial regions involved in the neural control of breathing, but the underlying mechanisms are not known. It is unclear whether the midbrain dopaminergic neurons project directly or indirectly to VRC. Very few studies have

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256

NSC 16158

No. of Pages 11

2 April 2015

8

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

295

sought to identify the efferent projections of neurons in the basal ganglia, thalamus and pedunculopontine tegmental nucleus to the brainstem (Prensa and Parent, 2001), and no study has shown SN-TH neurons projecting to VRC. Other possibility is the fact that patients with PD can present a dysfunctional blood–brain barrier (Kortekaas et al., 2005). If that is the case in our model, a disruption in the blood–brain barrier in the medulla could lead to death of brainstem neurons or loss of their markers. However, additional studies will be required to investigate these mechanisms. Bilateral intrastriatal 6-OHDA reduced the tachypneic response to rising CO2 levels. The tachypneic response to hypercapnia was reestablished by 60 days, although animals still had reduced fR. We interpret this as evidence of compensation. A massive reduction in the phox2b-expressing neurons within the RTN region, which act as chemoreceptors, could induce compensation by activating other central chemoreceptors within 60 days. The LC would be a strong candidate, since it is also involved with central chemoreception and we showed no neuronal loss in the current study (Gargaglioni et al., 2010). However, we cannot exclude the role of other putative chemoreceptors like medullary raphe (Richerson and Bekkers, 2004), nucleus of the solitary tract (Dean et al., 1989) and peripheral chemoreceptors (Overholt et al., 2001). In fact we did not further analyze the immunohistochemistry for those candidates, but their involvement with chemoreception is already known. We believe that the lack of effect of 6-OHDA on hypoxia-induced respiration is due to the brain recruiting a constellation of neural circuits to maintain normal respiration during peripheral chemoreceptor activation. However, despite the critical role of chemoreceptor pathways in regulation of cardiorespiratory function, the identity of neurotransmitters and downstream effectors responsible for peripheral chemoreflex control of autonomic function at the level of the brainstem remains incomplete.

296

Motor behavior

297

Interestingly, although bilateral intrastriatal 6-OHDA caused retrograde neurodegeneration of the nigrostriatal pathway, as determined by a dose-dependent reduction of TH+ cells in the SN at 60 days post-lesion, rats did not display any gross motor deficits, even at the highest dose of 6-OHDA. These findings are partially in accordance with previous studies, which found emotional and cognitive deficits, but not locomotor impairment after 12 lg of bilateral 6-OHDA into the ventrolateral area of the dorsal striatum in rats (Lindner et al., 1999; Tadaiesky et al., 2008). Similarly, bilateral intrastriatal injection of a total of 52.5 lg of 6-OHDA bilaterally in rats did not affect traveling distance or average speed in the open field (Yadav et al., 2014). A growing body of evidence suggests that 6-OHDA can model some of the neuropsychiatric aspects of PD, but not motor aspects (Tadaiesky et al., 2008). Nevertheless, it is possible that deficits would have been apparent in more skilled or fine motor behaviors or in latter time points.

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294

298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

CONCLUSION

316

Our results showed that bilateral intrastriatal 6-OHDA led to a massive degeneration of TH+ neurons in the SN, which was associated with deficits in breathing at rest and during hypercapnic conditions without any gross motor impairment. We suggest that clinical PD also produces a downregulation in the functional organization of neural systems involved in respiratory rhythm generation, leading to dysfunction in the neural control of breathing.

317

EXPERIMENTAL PROCEDURES

326

318 319 320 321 322 323 324 325

Animals

327

Experiments were initiated in 63 three- month-old male Wistar rats and the whole protocol lasted two months. Animal use was in accordance with the guidelines of the Animal Experimentation Ethics Committee of the Institute of Biomedical Science at the University of Sa˜o Paulo (ICB/USP).

328

6-OHDA injection

334

Rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (7 mg/kg) intraperitoneally (i.p.) and placed in a stereotaxic frame (model 900; David Kopf Instruments). Two injections of 6-OHDA hydrochloride (6-OHDA hydrochloride, H4381, Sigma, Saint Louis, MO, USA) (6, 12 and 24 lg/ll in 0.3% ascorbic acid saline solution) were made in each dorsal striatum using a 10 ll Hamilton syringe connected by polyethylene tubing (PE-10) to 30-gauge injection cannulas at the following coordinates relative to Bregma: (1) AP: 0 mm, ±2.7 mm ML, 4.5 mm DV and (2) 0.5 mm AP, ±3.2 mm ML, 4.5 mm DV. The injection volume was 0.5 ll/site. Control animals received the same volume of vehicle (0.3% ascorbic acid saline solution).

335

Measurement of lung ventilation

350

fR (breaths/min) and VT (mL/kg) in conscious, freely moving rats were measured by whole-body plethysmography as described in detail previously (Malan, 1973; Favero et al., 2011). All experiments were performed at room temperature (24–26 °C). Briefly, freely moving rats were kept in a Plexiglas recording chamber (5 L) that was flushed continuously with a mixture of 79% nitrogen and 21% oxygen (unless otherwise required by the protocol) at a rate of 1 L/min. Volume calibration was performed during each ventilation measurement throughout the course of the experiments by injecting a known volume of air (1 ml) inside the chamber. VT was calculated using the formula described by Malan (1973) and used in previous studies (Biancardi et al., 2008; Favero et al., 2011; da Silva et al., 2011). Ventilation was calculated as the product of VT and fR and is presented at ambient barometric pressure and at body temperature, which it is saturated with water vapor. Concentrations of O2 and CO2 in the chamber were monitored online using a fast-response O2/CO2 monitor

351

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

329 330 331 332 333

336 337 338 339 340 341 342 343 344 345 346 347 348 349

352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370

NSC 16158

No. of Pages 11

2 April 2015

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

381

(ADInstruments). The pressure signal was amplified, filtered, recorded and analyzed off-line using Powerlab software (Powerlab 16/30, ML880/P; ADInstruments). Rectal temperature was measured before and at the end of the experiments, and the values were averaged. Measurements of fR and VT were made during the last 2 min before exposure to the stimulus and during the 2-min period at the end of each stimulus, when breathing stabilized. Changes in the fR, VT and minute ventilation (MV; fR  VT; in ml/min/kg) were averaged and expressed as mean ± SEM.

382

Chemoreflex analysis

383

390

Unanesthetized rats were allowed at least 30 min to acclimate to the chamber environment at normoxia and normocapnia (21% O2, 79% N2 and <0.5% CO2) before ventilation was measured. Hypoxia was induced by lowering the O2 concentration in the inspired air to 8% for 10 min. Hypercapnia was induced by titrating CO2 into the respiratory mixture up to 7% (21% O2, 69% N2) for 10 min.

391

Determination of blood gases, pH and lactate

392

One day before the experiments, under i.p. ketamine combined with xylazine anesthesia, a polyethylene tubing (PE-10 connected to PE-50; Clay Adams, Parsippany, NJ, USA) was inserted into the abdominal aorta through the femoral artery. The cannula was tunneled subcutaneously to the animals’ backs to allow measurement while they were freely-moving. On the day of experiments, blood samples (150 ll) were taken via the implanted arterial catheter for immediate analysis of arterial pH, partial pressure of arterial carbon dioxide (PaCO2), partial pressure of arterial oxygen (PaO2), plasma bicarbonate (HCO3 ) and O2 saturation with the use of a blood gas analyzer (IStat, Abbott Laboratory, NJ, USA).

371 372 373 374 375 376 377 378 379 380

384 385 386 387 388 389

393 394 395 396 397 398 399 400 401 402 403 404 405

406

Motor tests

407

A different cohort of rats was used to assess spontaneous locomotor activity in the open-field (OF), motor coordination and balance on a rotarod, and catalepsy in the bar test. All behavioral tests were performed on the same day and occurred 1 day before and 30 and 60 days after vehicle (n = 8), 6 lg (n = 8), 12 lg (n = 8) or 24 lg of 6-OHDA (n = 7).

408 409 410 411 412 413

414

Spontaneous locomotor activity

415

Spontaneous locomotor activity was measured as previously described (Chiavegatto et al., 1998) with minor modifications. The OF apparatus consisted of a white wooden square (100  100  38 cm) with the floor marked off in 36 squares. Tests were performed by placing animals on the center of the apparatus and recording the number of squares entered with all four paws over 5 min. The arena was washed with a 5% ethanol solution after each rat had been tested to eliminate possible bias due to odor clues left by previous subjects.

416 417 418 419 420 421 422 423 424

9

Motor coordination

425

Motor coordination was assessed using an automated 4-lane rotarod unit with a 6-cm diameter rod (Insight, Ribeirao Preto, Brazil). Before each test, rats were trained on the apparatus. Training sessions were performed over two consecutive days before vehicle or 6-OHDA infusion or one day before the day 30 and 60 tests. In the first training session before vehicle or 6OHDA infusion, rats were allowed to stay on the rod (motionless) for 5 min. The following day, they were placed on the rod at minimum speed (5 rpm) for 5 min. For each test performed after vehicle or 6-OHDA infusion, a training session one day before the test consisted of placing the rats on the rod at 5 rpm for 5 min. Tests were performed by placing the animals on the rod on accelerated mode, from 5 to 37 rpm over a period of 5 min. The latency to fall in seconds was recorded automatically by a trip switch under the floor of each lane.

426

Catalepsy

444

Catalepsy was measured by placing the rat in a standing position with both forelimbs on a 9.5-cm high circular bar (1.3 cm diameter). The latency to remove one of the forelimbs from the bar in seconds was recorded in three consecutive trials, and the mean was calculated.

445

Histology

450

Rats were anesthetized (pentobarbital overdose, 60 mg/kg, i.p.) and perfused transcardially, first with heparinized saline (150 ml) and then with 4% phosphate-buffered paraformaldehyde (PFA, 500 ml). Brains were removed and postfixed in PFA for 1–2 days at 4 °C. Coronal sections (40 lm) were cut using a vibrating microtome (Vibratome 1000s) and stored in cryoprotectant solution at 20 °C for up to two weeks until histological processing (Schreihofer and Guyenet, 1997). All histological procedures were done with freefloating sections. Sections were rinsed, blocked and subjected to the immunoperoxidase method with antibody specific for detection of TH, phox2b and the substance P receptor (neurokinin 1 receptor, NK1R) as previously described (Barna et al., 2012).

451

Cell mapping, cell counting and imaging

467

A conventional multifunction microscope (brightfield, darkfield and epifluorescence; Zeiss Axioskop 2 microscope (Oberkochen, Germany) was used for all observations except when indicated. The ImageJ software (public domain program available from the NIH; http://rsb.info.nih.gov/ij/) was used to count the various types of neuronal profiles within a defined area. Section alignment between brains was done relative to a reference section. To align sections around SN level, the most caudal section containing the medial geniculate nucleus was identified in each brain and assigned the level 6.60 mm caudal to Bregma (Bregma 6.60 mm) according to the atlas of Paxinos and

468

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443

446 447 448 449

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

469 470 471 472 473 474 475 476 477 478 479 480

NSC 16158

No. of Pages 11

2 April 2015

10

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx

504

Watson (1998).To align sections around the RTN level, the most caudal section containing an identifiable cluster of facial motor neurons was identified in each brain and assigned the level 11.6 mm caudal to Bregma (Bregma 11.6 mm) according to the atlas of Paxinos and Watson (1998). Levels rostral or caudal to this reference section were determined by adding a distance corresponding to the interval between sections multiplied by the number of intervening sections. It was analyzed four sections to RTN. The same method was also used to identify the Bregma level of the LC, BotC, preBotC, rVRG and cVRG. However, for densitometric analysis of NK1R immunoreactivity the region was defined by an oval 350 lm wide and 500 lm long, the top centered on the ventral edge of the ambiguus or retroambiguus and nine sections of the region of VRC were analyzed. All files were exported to the Canvas 9 software drawing program for final modifications. Photographs were taken with a 12-bit color CCD camera (CoolSnap, Roper Scientific, Tucson, AZ, USA); resolution 1392  1042 pixels. The neuroanatomical nomenclature is after Paxinos and Watson (1998).

505

Statistical analysis

506

510

Data were analyzed using one-way ANOVAs, followed by Tukey post hoc tests. Behavioral data were analyzed by a two-way repeated measures ANOVA. All results are presented as mean ± SEM. Differences were considered significant when p < 0.05.

511

Experimental protocol

512

Two injections of 0.5 ll/site of 6-OHDA (6, 12 and 24 lg/ ll) or saline + 0.3% ascorbic acid (n = 5–9/group) were

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503

507 508 509

513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537

done bilaterally in the striatum (caudate-putamen) to induce a retrograde neuronal nigrostriatal injury (reviewed by Blum et al., 2001). One day before and 40, 50 and 60 days after 6-OHDA, animals were individually placed in plethysmography chambers. During the initial period, the chamber was ventilated and moistened with atmospheric air (21% O2). After the exploratory phase, resting ventilation was measured, then animals were subjected to hypercapnia for 10 min. Ventilation was measured at 10 min after the start of hypercapnia. After 10 min, the chamber was ventilated again with atmospheric air for 30 min, then animals were subjected to hypoxia for 10 min. Again, ventilation was measured at 10 min after initiation of hypoxia. The sequence of chemoreflexes tests was randomized. Finally, the chamber was ventilated again with atmospheric air for 30 min. At the end of the protocol, the femoral artery was cannulated to conduct blood gas determinations. Motor tests were performed 1 day before and 30 and 60 days after 6-OHDA in a different group of rats (n = 7–8). At the end of each experiment, the animals were sacrificed and perfused, and the brains were removed for histological analysis. The imunohistochemistry for TH was performed in all animals to reach the size of SN lesion.

AUTHOR CONTRIBUTIONS

538

T.S.M. and A.C.T. designed the experiments; L.A.S. and S.C. collected and analyzed behavioral data; M.T., B.F.B. and A.C.T. collected and M.T. and A.C.T. analyzed non-behavioral data; M.T., T.S.M. and A.C.T. wrote the paper; L.R.G.B. edited and commented on the manuscript. All authors approved the final version of the manuscript.

539

Acknowledgments—We gratefully acknowledge J.F. Brunet (Departement de Biologie, Ecole Normale Superieure, Paris, France) for providing the Phox2b antibody. This research was supported by public funding from Sa˜o Paulo Research Foundation (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).

546

REFERENCES

552

Axelrod FB, Chelimsky GG, Weese-Mayer DE (2006) Pediatric autonomic disorders. Pediatrics 118:309–321. Barna BF, Takakura AC, Moreira TS (2012) Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats. Neuroscience 212:120–130. Bassetti CL (2011) Nonmotor disturbances in Parkinson’s disease. Neurodegener Dis 8:95–108. Benarroch EE (2003) Brainstem in multiple system atrophy: clinicopathological correlations. Cell Mol Neurobiol 23:519–526. Benarroch EE (2007) Brainstem respiratory control: substrates of respiratory failure of multiple system atrophy. Mov Disord 22:155–161. Benarroch EE, Schmeichel AM, Low PA, Parisi JE (2003) Depletion of ventromedullary NK-1 receptor-immunoreactive neurons in multiple system atrophy. Brain 126:2183–2190. Biancardi V, Bı´ cego KC, Almeida MC, Gargaglioni LH (2008) Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflugers Arch 455:1119–1128. Budzinska K, Andrzejewski K (2014) Respiratory activity in the 6hydroxydopamine model of Parkinson’s disease in the rat. Acta Neurobiol Exp (Wars) 74:67–81. Bunton K (2005) Patterns of lung volume use during an extemporaneous speech task in persons with Parkinson disease. J Commun Disord 38:331–348. Chaudhuri KR, Odin P, Antonini A, Martinez-Martin P (2011) Parkinson’s disease: the non-motor issues. Parkinsonism Relat Disord 17:717–723. Chiavegatto S, Nasello AG, Bernardi MM (1998) Histamine and spontaneous motor activity: biphasic changes, receptors involved and participation of the striatal dopamine system. Life Sci 62:1875–1888. da Silva GS, Giusti H, Benedetti M, Dias MB, Gargaglioni LH, Branco LG, Glass ML (2011) Serotonergic neurons in the nucleus raphe obscurus contribute to interaction between central and peripheral ventilatory responses to hypercapnia. Pflugers Arch 462:407–418. Dean JB, Lawing WL, Millhorn DE (1989) CO2 decreases membrane conductance and depolarizes neurons in the nucleus tractus solitarii. Exp Brain Res 76:656–661. Favero MT, Takakura AC, de Paula PM, Colombari E, Menani JV, Moreira TS (2011) Chemosensory control by commissural nucleus of the solitary tract in rats. Respir Physiol Neurobiol 179:227–234. Fearnley JM, Lees AJ (1991) Ageing and Parkinson’s disease: substantia nigra regional selectivity. Brain 114(Pt 5):2283–2301. Feldman JL, Del Negro CA (2006) Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7:232–242.

553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

540 541 542 543 544 545

547 548 549 550 551

NSC 16158

No. of Pages 11

2 April 2015

M. Tuppy et al. / Neuroscience xxx (2015) xxx–xxx 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 702 703 704

Feldman JL, Ellenberger HH (1988) Central coordination of respiratory and cardiovascular control in mammals. Annu Rev Physiol 50:593–606. Feldman JL, Del Negro CA, Gray PA (2013) Understanding the rhythm of breathing: so near, yet so far. Annu Rev Physiol 75:423–452. Gargaglioni LH, Hartzler LK, Putnam RW (2010) The locus coeruleus and central chemosensitivity. Respir Physiol Neurobiol 173:264–273. Guyenet PG, Wang H (2001) Pre-Bo¨tzinger neurons with preinspiratory discharges ‘‘in vivo’’ express NK1 receptors in the rat. J Neurophysiol 86:438–446. Hammer MJ, Barlow SM (2010) Laryngeal somatosensory deficits in Parkinson’s disease: implications for speech respiratory and phonatory control. Exp Brain Res 201:401–409. Idiaquez J, Benarroch EE, Rosales H, Milla P, Rı´ os L (2007) Autonomic and cognitive dysfunction in Parkinson’s disease. Clin Auton Res 17:93–98. Kortekaas R, Leenders KL, van Oostrom JC, Vaalburg W, Bart J, Willemsen AT, Hendrikse NH (2005) Blood–brain barrier dysfunction in parkinsonian midbrain in vivo. Ann Neurol 57:176–179. Lindner MD, Cain CK, Plone MA, Frydel BR, Blaney TJ, Emerich DF, Hoane MR (1999) Incomplete nigrostriatal dopaminergic cell loss and partial reductions in striatal dopamine produce akinesia, rigidity, tremor and cognitive deficits in middle-aged rats. Behav Brain Res 102:1–16. Malan A (1973) Ventilation measured by body plethysmography in hibernating mammals and in poikilotherms. Respir Physiol 17:32–44. McDowell K, Chesselet MF (2012) Animal models of the non-motor features of Parkinson’s disease. Neurobiol Dis 46:597–606. Miguelez C, Grandoso L, Ugedo L (2011) Locus coeruleus and dorsal raphe neuron activity and response to acute antidepressant administration in a rat model of Parkinson’s disease. Int J Neuropsychopharmacol 14:187–200. Mulkey DK, Stornetta RL, Weston MC, Simmons JR, Parker A, Bayliss DA, Guyenet PG (2004) Respiratory control by ventral surface chemoreceptor neurons in rats. Nat Neurosci 7:1360–1369. Nogue´s MA, Roncoroni AJ, Benarroch E (2002) Breathing control in neurological diseases. Clin Auton Res 12:440–449. Overholt JL, Summers BA, Ficker E, Prabhakar NR (2001) CO2/ HCO3- modulates K+ and Ca2+ currents in glomus cells of the carotid body. Adv Exp Med Biol 499:61–66. Prensa L, Parent A (2001) The nigrostriatal pathway in the rat: a single-axon study of the relationship between dorsal and ventral tier nigral neurons and the striosome/matrix striatal compartments. J Neurosci 21:7247–7260. Reyes A, Ziman M, Nosaka K (2013) Respiratory muscle training for respiratory deficits in neurodegenerative disorders: a systematic review. Chest 143:1386–1394.

11

Richerson GB, Bekkers JM (2004) Learning to take a deep breath– with BDNF. Nat Med 10:25–26. Schreihofer AM, Guyenet PG (1997) Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J Comp Neurol 387:524–536. Schwarting RK, Huston JP (1996) The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol 50:275–331. Schwarzacher SW, Ru¨b U, Deller T (2011) Neuroanatomical characteristics of the human pre-Bo¨tzinger complex and its involvement in neurodegenerative brainstem diseases. Brain 134:24–35. Seccombe LM, Giddings HL, Rogers PG, Corbett AJ, Hayes MW, Peters MJ, Veitch EM (2011) Abnormal ventilatory control in Parkinson’s disease–further evidence for non-motor dysfunction. Respir Physiol Neurobiol 179:300–304. Solomon NP, Hixon TJ (1993) Speech breathing in Parkinson’s disease. J Speech Hear Res 36:294–310. Stornetta RL, Moreira TS, Takakura AC, Kang BJ, Chang DA, West GH, Brunet JF, Mulkey DK, Bayliss DA, Guyenet PG (2006) Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J Neurosci 26:10305–10314. Tadaiesky MT, Dombrowski PA, Figueiredo CP, Cargnin-Ferreira E, Da Cunha C, Takahashi RN (2008) Emotional, cognitive and neurochemical alterations in a premotor stage model of Parkinson’s disease. Neuroscience 156:830–840. Troche MS, Okun MS, Rosenbek JC, Musson N, Fernandez HH, Rodriguez R, Romrell J, Pitts T, Wheeler-Hegland KM, Sapienza CM (2010) Aspiration and swallowing in Parkinson disease and rehabilitation with EMST: a randomized trial. Neurology 75:1912–1919. Tzelepis GE, McCool FD, Friedman JH, Hoppin FG (1988) Respiratory muscle dysfunction in Parkinson’s disease. Am Rev Respir Dis 138:266–271. Weese-Mayer DE, Lieske SP, Boothby CM, Kenny AS, Bennett HL, Silvestri JM, Ramirez JM (2006) Autonomic nervous system dysregulation: breathing and heart rate perturbation during wakefulness in young girls with Rett syndrome. Pediatr Res 60:443–449. Weese-Mayer DE, Lieske SP, Boothby CM, Kenny AS, Bennett HL, Ramirez JM (2008) Autonomic dysregulation in young girls with Rett syndrome during nighttime in-home recordings. Pediatr Pulmonol 43:1045–1060. Wolters ECh (2009) Non-motor extranigral signs and symptoms in Parkinson’s disease. Parkinsonism Relat Disord 15(Suppl. 3):S6–S12. Yadav AP, Fuentes R, Zhang H, Vinholo T, Wang CH, Freire MA, Nicolelis MA (2014) Chronic spinal cord electrical stimulation protects against 6-hydroxydopamine lesions. Sci Rep 4:3839.

(Accepted 19 March 2015) (Available online xxxx)

Please cite this article in press as: Tuppy M et al. Respiratory deficits in a rat model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/ 10.1016/j.neuroscience.2015.03.048

652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701