Articles in PresS. J Appl Physiol (September 6, 2012). doi:10.1152/japplphysiol.00098.2012
1 2 3 Phrenic Long Term Facilitation Following Acute Intermittent Hypoxia Requires
Spinal ERK Activation but not TrkB Synthesis
4 5 M.S. Hoffman, N.L. Nichols, P.M. Macfarlane, and G.S. Mitchell
6 7 8 9 Department of Comparative Biosciences
University of Wisconsin,
Madison, WI 53706, USA
10 12 13 14 15 16 17 18 19 20 21 22 23 24 Running Title:
Phrenic LTF requires ERK activation
Corresponding Author: Gordon S. Mitchell
Department of Comparative Biosciences
University of Wisconsin
2015 Linden Drive
Madison, WI 53706-1102 USA
Tel: (608) 263-9826
Fax:(608) 263-3926
Email: [email protected] 25 26 27 28 29 30 31 32 Copyright © 2012 by the American Physiological Society.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
11 ABSTRACT
34 Acute intermittent hypoxia (AIH) elicits a form of spinal respiratory plasticity known as
35 phrenic long term facilitation (pLTF). pLTF requires spinal serotonin receptor-2
36 activation, synthesis of new brain-derived neurotrophic factor (BDNF) and activation of
37 its high affinity receptor tyrosine kinase, TrkB. Spinal adenosine 2A receptor activation
38 elicits a distinct pathway to phrenic motor facilitation (pMF); this BDNF synthesis-
39 independent pathway instead requires new synthesis of an immature TrkB isoform.
40 Since hypoxia increases extracellular adenosine levels, we tested the hypothesis that
41 new synthesis of TrkB and BDNF contribute to AIH-induced pLTF. Further, given that
42 signaling mechanisms "downstream" from TrkB are unknown in either mechanism, we
43 tested the hypothesis that pLTF requires MEK/ERK and/or PI3K/Akt activation. In
44 anesthetized Sprague Dawley rats, an intrathecal catheter at C4 was used to deliver
45 drugs near the phrenic motor nucleus. Since pLTF was blocked by spinal injections of
46 small interfering RNAs targeting BDNF mRNA, but not TrkB mRNA, only new BDNF
47 synthesis is required for AIH-induced pLTF. Pretreatment with the MEK inhibitor U0126
48 blocked pLTF, whereas a PI3K inhibitor (PI-828) had no effect. Thus, AIH-induced pLTF
49 requires the MEK/ERK (not PI3K/AKT) signaling pathways. When U0126 was injected
50 post-AIH, pLTF development was halted, but not reversed, suggesting that ERK is
51 critical for the development, but not maintenance of pLTF. Thus, there are clear
52 mechanistic distinctions between AIH-induced pLTF (i.e. BDNF synthesis and
53 MEK/ERK dependent) versus adenosine 2A receptor induced pMF (i.e. TrkB synthesis
54 and PI3K/Akt dependent).
55 56 Key Words: Respiratory plasticity, BDNF, TrkB, ERK, respiratory control
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
33 INTRODUCTION
58 59 The neural system controlling breathing exhibits considerable plasticity in response to
60 environmental and physiological perturbations [13]. One frequently studied model of
61 respiratory plasticity is observed following exposure to acute intermittent hypoxia (AIH).
62 AIH elicits long-lasting facilitation of phrenic motor output, an effect known as phrenic
63 long term facilitation [pLTF; 13]. A hallmark of pLTF is that it requires episodic versus
64 continuous hypoxia [1, 39]. In our working model of pLTF, AIH repeatedly activates
65 medullary raphe serotonergic neurons [42], releasing serotonin in the phrenic motor
66 nucleus [23] where it activates 5-HT2 receptors on or near phrenic motor neurons [23].
67 Indeed, spinal serotonin receptor activation is both necessary [4] and sufficient [30] for
68 pLTF. Episodic serotonin receptor activation initiates new synthesis of brain-derived
69 neurotrophic factor (BDNF), subsequently activating its high affinity receptor, TrkB [2].
70 Signaling pathways downstream from TrkB are unknown.
71 A distinct mechanism of long-lasting phrenic motor facilitation (pMF) results from
72 activation of spinal metabotropic receptors coupled to Gs-proteins, such as the
73 adenosine-2A receptor [A2A; 26] or serotonin-7 receptors [5-HT7; 29]. A2A agonist-
74 induced pMF is independent of serotonin receptor activation and new BDNF synthesis
75 [16]; instead, it requires new synthesis of an immature TrkB isoform believed to auto-
76 activate and signal from within phrenic motor neurons [16, 20, 28]. Because adenosine
77 released into the extracellular space during hypoxia [18] may activate A2A receptors, we
78 wondered whether immature TrkB signaling contributes to AIH-induced pLTF.
79 Once activated, TrkB signals via multiple cascades including Ras/MAPK
80 (mitogen-activated protein kinase), PI3K (phosphatidylinositol 3-kinase) and PLCγ
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
57 [phospholipase-Cγ; 49]. In cultured spinal motor neurons, TrkB receptors signal
82 predominantly via extracellular regulated MAPK (ERK) and/or PI3K/protein kinase B
83 [pAkt; 25]. ERK and PI3K/Akt signaling have been implicated in respiratory neural
84 plasticity. For example, AIH increases phosphorylation and activation of ERK in the
85 ventral cervical spinal cord encompassing the phrenic motor nucleus [54]. Further,
86 repetitive AIH exposure up-regulates ERK and phospho-ERK protein levels within
87 presumptive phrenic motor neurons [47]. On the other hand, spinal A2A receptor
88 activation increases Akt phosphorylation in the ventral cervical spinal cord [16], an effect
89 attributed to increased immature TrkB signaling [16, 53]. Thus, we wondered whether
90 Akt and/or ERK signaling contribute to AIH-induced pLTF.
91 Here we tested the hypotheses that new synthesis of immature TrkB, and TrkB
92 signaling via both ERK and Akt contribute to AIH-induced pLTF. Whereas we confirm
93 that new BDNF synthesis is necessary for AIH-induced pLTF, no evidence was found
94 that new TrkB synthesis plays a role. Further, whereas inhibition of cervical spinal ERK
95 activation blocks AIH-induced pLTF, inhibition of Akt activation has no effect. Finally,
96 whereas ERK activation is necessary for pLTF initiation and development, continued
97 ERK activation is not necessary for pLTF maintenance.
98 99 METHODS
100 Animals. All experiments were performed on adult (3-6 months, n = 110) male Harlan
101 Sprague Dawley rats (280-450g, colonies 217, 218a; Inc., Indianapolis, IN, USA). In the
102 first experimental series small interfering RNAs were spinally injected targeting BDNF
103 (siBDNF) or TrkB (siTrkB) mRNA to prevent translation and new synthesis of BDNF or
104 TrkB protein, respectively; these injections enabled us to test the hypothesis that AIH-
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
81 105 induced pLTF requires new TrkB synthesis in anesthetized rats. In the second series of
106 experiments,
107 IC50~0.065μM; 10] and PI3K/Akt [PI-828, IC50 ~ 0.098; 15] were used to test their
108 involvement in AIH-induced pLTF. Since pre-treatment with the MEK/ERK inhibitor
109 (U0126), and not the PI3K/Akt inhibitor (PI-828), blocked pLTF, we tested whether
110 continued MEK/ERK activation is necessary for pLTF maintenance by spinally injecting
111 U0126 after the final hypoxic episode in a third series of experiments.
spinal
injections
of
selective
inhibitors
for
MEK/ERK
[U0126,
All procedures were approved by the Institutional Animal Care and Use
113 Committee of the School of Veterinary Medicine, University of Wisconsin, Madison and
114 are in compliance with the policies and regulations outlined by The American
115 Physiological Society.
116 117 Surgical Preparation. Anesthesia was induced with isoflurane (2.5-3.5% in 50% O2,
118 balance N2), and isoflurane was continued during surgical preparations. Once surgical
119 procedures were completed, rats were converted to urethane anesthesia by slow
120 intravenous injections over 15 minutes (1.8g*kg-1.). Adequate anesthetic depth was
121 tested by lack of blood pressure (pressor) or respiratory neural response to toe pinch
122 with a hemostat. After conversion to urethane anesthesia, a continuous infusion (4-6.5
123 ml*kg-1*hr-1) of 6% Hetastarch (artificial colloid composed and dissolved in 0.9% normal
124 saline) and lactated Ringers (1:4 mixture respectively) was implemented to maintain
125 appropriate blood volume, fluid balance and acid-base status. A tracheal cannula was
126 placed in the neck to enable artificial ventilation (Rodent Respirator, model 683, Harvard
127 Apparatus, Holliston, MA; tidal volume = 2.5ml, variable frequency). A rapidly
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
112 responding flow-through carbon dioxide analyzer (Capnogard, Novametrix, Wallingford,
129 CT) was placed on the expired limb of a Y-tube connected to the tracheal cannula to
130 enable measurements of end-tidal carbon dioxide partial pressures (PETCO2). The
131 vagus nerves were cut in the mid-cervical region to prevent entrainment of respiratory
132 neural activity with the ventilator. During ventilation, rats were paralyzed with
133 pancuronium bromide (2.5mg*kg-1). A polyethylene catheter (PE50, Intramedic) was
134 placed in the right femoral artery and blood pressure was monitored with a pressure
135 transducer (Gould, P23ID). A 3-way stop-cock, attached to the arterial catheter, was
136 used to withdraw blood samples (0.2-0.4ml) for blood gas analysis (ABL-500,
137 Radiometer; Copenhagen, Denmark); during an experiment, blood gas determinations
138 were made during baseline conditions, during the first hypoxic episode and at 15, 30, 60
139 minutes post-AIH (additional 90 min in third experimental series). Body temperature was
140 monitored with rectal thermometer (Fischer Scientific) and maintained (37.5±1ºC) with a
141 heated surgical table.
142 Peak integrated phrenic burst amplitude strongly correlates with tidal volume and
143 respiratory muscle activity in spontaneously breathing animals [11]. Therefore we used
144 this index to quantify changes in respiratory neural output. The left phrenic nerve was
145 isolated using a dorsal approach, cut distally, desheathed and placed on bipolar silver
146 electrodes to record respiratory neural activity. Phrenic nerve signals were amplified
147 (100,000x), band-pass filtered (300-10,000 Hz Model 1800, A-M Systems, Carlsborg,
148 WA), rectified and integrated (Paynter filter; time constant, 50ms, CWE Inc., MA-821;
149 Ardmore, PA). The resulting integrated nerve bursts were digitized (8000 Hz) and
150 analyzed using a WINDAQ data acquisition system (DATAQ Instruments, Akron, OH).
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
128 151 Following completion of the surgical preparation and conversion to urethane anesthesia,
152 rats were allowed a minimum of one hour to stabilize before beginning an experimental
153 protocol.
An intrathecal catheter was placed in the cervical region to enable localized
155 siRNA and drug delivery. The spinal column was exposed dorsally, followed by
156 laminectomy at cervical level 2 (C2), where a small incision was made in the dura. A soft
157 silicone catheter (2 French; Access Technologies) was inserted caudally through the
158 incision until the tip was located at approximately C4. The catheter was attached to a
159 50l Hamilton syringe filled with drug (see treatment groups, below) solutions to allow
160 localized drug injections into the cervical spinal region.
161 162 Experimental protocol. At least one hour after conversion to urethane anesthesia,
163 apneic and recruitment thresholds were determined by increasing ventilation and
164 lowering PETCO2 until rhythmic nerve bursts could no longer be detected (apneic
165 threshold). After one minute, the ventilator rate was slowly decreased and/or inspired
166 carbon dioxide was slowly increased until rhythmic nerve bursts resumed (i.e.,
167 recruitment threshold). Baseline conditions were then established by holding PETCO2 ~2
168 mmHg above the recruitment threshold until neural activity had stabilized (≥15 min). An
169 arterial blood sample was then taken to document baseline blood gas levels. Arterial
170 PCO2 was maintained isocapnic (±1.5mmHg) with respect to baseline by manipulating
171 inspired CO2 and/or ventilation rate. Subsets of rats received intrathecal vehicle (siRNA
172 universal buffer or saline/10% dmso) or drug injections (see treatment groups below);
173 injections were made slowly over one min prior to the first hypoxic episode (or
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
154 174 equivalent time for control groups) and shortly after the final hypoxic episode in AIH
175 treated rats.
AIH consisted of three, five min. episodes of isocapnic (±1.5mmHg) hypoxia
177 (10% inspired O2, PaO2=35-45mmHg), separated by five min intervals of baseline
178 oxygen conditions (50% inspired O2, PaO2≥150mmHg). After the third hypoxic episode,
179 rats were returned to baseline inspired oxygen levels and maintained for the duration of
180 an experiment. To test the hypothesis that new spinal TrkB and/or BDNF synthesis are
181 required for AIH-induced pLTF, siRNAs targeting BDNF and TrkB mRNA were injected
182 via an intrathecal catheter over the cervical spinal cord (~C4) to inhibit new BDNF and
183 TrkB protein synthesis in the vicinity of the phrenic motor nucleus.
184 Rats receiving siRNAs were surgically prepared as described above. A pool of
185 siRNAs targeting TrkB mRNA was used to determine the role of newly synthesized TrkB
186 protein in AIH-induced pLTF. TrkB siRNAs were obtained as a pool of four 21-
187 nucleotide duplexes (ON-TARGET plus, Dharmacon, Lafayette, CO gene, NTRK2;
188 GenBank accession number, NM 012731); this same pool has been shown to
189 effectively block new TrkB synthesis in an earlier study from our laboratory using the
190 same experimental preparation [16]. We also used a pool of siRNAs targeting BDNF to
191 verify AIH-induced pLTF required new BDNF synthesis, and to provide an internal
192 positive control for siRNAs; these same BDNF siRNA sequences have previously been
193 shown to prevent new synthesis of spinal BDNF in the same experimental preparation
194 [2]. siRNAs were reconstituted with siRNA Universal Buffer (Dharmacon) and stored at -
195 20°C. Stock TrkB siRNAs (4µl of 5µM solution) were combined with the transfection
196 reagent, Oligofectamine (16µl; Invitrogen, Carlsbad, CA) and RNase-free water (180 µl,
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
176 197 final conc. 100nM) and incubated at room temperature for 20 min. Stock BDNF siRNAs
198 (1.25µM) were also diluted in Oligofectamine and RNase-free prior to injection (final
199 conc. 100nM). The siRNAs were slowly injected over the C4 spinal segment via an
200 intrathecal catheter (2, 10 µl injections separated by 10 min) two hours prior to making
201 baseline measurements and AIH exposures.
In the second and third experimental series, an intrathecal catheter was used to
203 pre-treat rats 20 min prior to AIH exposures with a PI3K inhibitor (PI-828, 100µM, 12µl;
204 Tocris Bioscience) or a MEK inhibitor (U0126, 100µM, 12µl; Tocris Bioscience). In the
205 final experimental series, rats received an intrathecal injection of U0126 (100µM, 12µl)
206 immediately (< 5 min) following the final hypoxic episode.
207 208 Data Analysis. Nerve recordings were analyzed using custom software (LabView 6.1,
209 National Instruments, Austin, TX, USA; courtesy of Dr. S. Mahamed). Statistical
210 comparisons between treatment groups for mean arterial pressures, PaCO2 and PaO2
211 were made using a two-way ANOVA with a repeated measures design. Integrated
212 phrenic nerve burst amplitude and burst frequency was averaged over one min bins at
213 each experimental time point (baseline 15, 30, 60 and 90 min). Changes in nerve burst
214 amplitude were normalized as a percentage of baseline. Nerve burst frequency was
215 expressed as an absolute change from baseline (bursts per min.). Comparisons for
216 nerve activity (amplitude & frequency) were made using a two-way ANOVA with a
217 repeated measures design.
218 exposures within groups (data not shown), comparisons were made using two-way
219 ANOVA of phrenic burst amplitude during the fifth minute of hypoxic episodes averaged
Since no differences were detected between hypoxic
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
202 220 from all three episodes.
All individual comparisons were made using the Student-
221 Neuman-Keuls (SNK) post hoc test (Sigma-Stat version 2.03; Jandel Scientific, St.
222 Louis, MO). Differences between groups were considered significant if p<0.05. All
223 values are expressed as mean ± 1 S.E.M.
224 225 226 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
227 243 RESULTS
244 Blood gases and mean arterial pressures.
245 during baseline, hypoxia, and 60 and 90 min post-AIH were similar in all experimental
246 groups. Since, arterial carbon dioxide was actively maintained within 1.5 mmHg of
247 baseline, isocapnia was observed at post-AIH time points (see table 1). During hypoxic
248 episodes, PaO2 decreased (~40mmHg), but returned to baseline levels (PaO2 ≥ 150
249 mmHg) for the duration of experiments. Mean arterial pressure (MAP, mmHg) was
250 similar between treatment groups at baseline conditions (Table 1). During hypoxia, MAP
251 decreased as is usually observed in anesthetized rats; this decrease was similar
252 between treatment groups and MAP returned to near-baseline levels by completion of
253 an experiment (Table 1).
Measurements of arterial PCO2 and PO2
Baseline measurements of frequency and
255 Short term hypoxic phrenic responses.
256 amplitude of peak integrated inspiratory phrenic nerve bursts were similar for all
257 treatment groups; therefore, normalization to baseline measurements was appropriate
258 to quantify pLTF magnitude. In rats receiving vehicle or drug injections prior to AIH,
259 hypoxia elicited a rapid increase in phrenic burst amplitude that was significantly
260 elevated from baseline (vehicle: 110±11%, n=7; siTrkB: 100±11%, n=8; siBDNF:
261 84±9%, n=9; U0126: 118±14%, n=9, PI-828: 129±20%, n=6; ∆ baseline, p<0.001,
262 respectively; Fig. 1). Hypoxia also elicited a rapid increase in phrenic burst amplitude in
263 rats receiving vehicle or drug after AIH that was significantly different from baseline
264 (vehicle: 100±15%, n=8, U0126: 77±11%, n=8, ∆baseline, p<0.001, respectively; Fig. 1).
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
254 265 No between-group differences were detected in phrenic burst amplitude from either pre-
266 AIH (p=0.121) or post-AIH treated rats (p=0.226).
267 pLTF requires new BDNF, but not TrkB synthesis. Typical integrated phrenic
269 neurograms during experimental protocols (series 1) are shown in Figure 2. In vehicle
270 (siRNA buffer, n=7) treated rats, phrenic nerve burst amplitude was increased over
271 baseline at 30 min (23±11%, p=0.015), and remained elevated at least 60 min post-AIH
272 (53±9%, p<0.001; Fig. 3). Phrenic burst amplitude was also significantly greater in
273 vehicle treated rats receiving AIH versus those not exposed to AIH (i.e., time controls,
274 n=5) at 60 min post-AIH (17±11%, p<0.011; Fig. 3), thus confirming pLTF in these rats.
275 Following pre-treatment with siRNAs targeting TrkB mRNA (siTrkB; n=8; two
276 hours before AIH), phrenic amplitude was increased over baseline at 30 min and
277 beyond (30 min: 42±8%, 60 min: 68±9%, p<0.001, respectively), and was not different
278 from control rats receiving only AIH at 60 min (68±9% vs. 53±9%, respectively; p=0.118;
279 Fig. 3). Consistent with our previous report [2], phrenic burst amplitude from siBDNF
280 treated rats (n=9) was significantly decreased at 60 min post-AIH compared to vehicle
281 (17±5% vs. 53±9%, p<0.001) and siTrkB (68±9%, p<0.001) treated rats, but was not
282 different from vehicle time control (AIH) rats at 60 min post-AIH (17±9%, p=0.968; Fig.
283 3). Thus, AIH-induced pLTF requires new BDNF synthesis, but is independent of new
284 TrkB synthesis.
285 286 Spinal inhibition of MEK/ERK activity blocks AIH-induced pLTF. Since AIH-induced
287 pLTF requires TrkB receptor activation [2], we wondered if “downstream” MEK/ERK
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
268 and/or PI3K/Akt activity is necessary for pLTF. Typical integrated phrenic neurograms
289 during experimental series 2 are shown in Fig. 4A-and B. In rats pre-treated with spinal
290 injections of a MEK inhibitor (U0126, n=9), pLTF was significantly attenuated 60 min
291 post-AIH compared to vehicle treated rats (22±7% vs. 53±9%, ,respectively; p=0.004;
292 Fig. 4A and C). Thus, AIH-induced pLTF requires activation of the MEK/ERK pathway.
293 On the other hand, spinal pre-treatment with a selective PI3K/Akt inhibitor (PI-828, n=6)
294 had no effect on pLTF; 60 min post-AIH there were no significant differences between
295 PI-828 and vehicle treated rats (63±10% and 53±9%, respectively; p=0.370; Fig. 4B and
296 C). A subset of rats received vehicle (control: saline, n=5) without AIH; in these rats,
297 phrenic burst amplitude was not different from baseline at any time, and was not
298 different from U0126 treated rats at the 60 min time point (17±11% vs. 22±7%,
299 respectively; p=0.664; Fig. 4). These data demonstrate that AIH-induced pLTF requires
300 MEK/ERK, but not PI3K/Akt activation.
301 302 MEK/ERK activity is necessary for pLTF development. Rats that received spinal U0126
303 injections (100µM) following AIH (< 5min) were monitored 30 additional min (90 min.
304 time point) to provide adequate post-drug data. A subset of rats received vehicle (n=8)
305 post-AIH to serve as a control. Representative phrenic neurograms of experimental
306 series 3 are illustrated in Figure 5. Phrenic burst amplitude in vehicle treated rats
307 increased from baseline at 30 min and beyond (Fig. 6), indicating progressive pLTF. In
308 U0126 post-AIH treated rats (n=8), pLTF was evident, but was significantly attenuated
309 at 30 min versus vehicle treated rats (34±11% vs. 50±10%, p=0.004), 60 min (32±12%
310 vs. 70±13%, p<0.001) and 90 min post-AIH (35±9% vs. 77±15%, respectively p<0.001;
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
288 311 Fig. 6). Phrenic burst amplitude in U0126 post-AIH treated rats was increased from
312 vehicle control rats (no hypoxia, n=8) 60 min post-AIH and beyond (60 min: 32±12% vs.
313 3±8%, p=0.047; 90 min: 35±9% vs. 4±8%, p=0.034; Fig. 6), demonstrating residual
314 pLTF. However, residual pLTF was unchanged between 30 and 90 min post-AIH,
315 suggesting that further development of pLTF was effectively blocked.
To address concerns that spinal MEK/ERK inhibition alone affected phrenic
317 activity, a subset of control rats received U0126, without AIH (n=8). In these rats,
318 phrenic burst amplitude was not different than baseline at any time point (Fig. 6).
319 320 Respiratory Frequency Facilitation. AIH-induced frequency LTF in anesthetized,
321 vagotomized rats is generally small and inconsistent [3]. Phrenic burst frequency
322 increased over baseline in vehicle (10±3 bursts/min, p<0.001) and siTrkB pre-treated
323 rats at 60 min post-AIH (6±2 bursts/min, p=0.016; Fig. 7A). Burst frequency differences
324 were not detected between vehicle and siTrkB treated rats (p=0.129). In rats pre-treated
325 with siBDNF, burst frequency was not increased at 60 min post-AIH (2±3 bursts/min,
326 p=0.66), and was decreased relative to vehicle pre-treated rats (60min: p=0.003). In
327 vehicle time control rats, phrenic burst frequency was not different than baseline at any
328 time point (15min: 1±1, p=0.66; 30min: -1±3, p=0.77, 60min: -2±1, p=0.66 bursts/min),
329 and was significantly less than in vehicle (p<0.001) and siTrkB pre-treated rats
330 (p=0.008) 60 min post-AIH. Burst frequency responses in vehicle time control and
331 siBDNF treated rats did not differ 60 min post-AIH (p=0.127; Fig. 7A).
332 Phrenic burst frequency in rats pretreated with the PI3K inhibitor was increased
333 over baseline at 60 min post-AIH (6±2 bursts/min, p=0.005; Fig. 7B). However, phrenic
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
316 burst frequency following MEK inhibition was not different from baseline at the 60 min
335 time point (2±1 bursts/min, p=0.144), and was significantly decreased from vehicle
336 treated rats 60 min post-AIH (p=0.048; Fig. 7B). In contrast, phrenic burst frequency in
337 rats receiving spinal MEK inhibition post-AIH were increased over baseline at 60 (7±2
338 bursts/min, p<0.001) and 90 min (6±2 bursts/min, p=0.001) (Fig. 7C). In rats receiving
339 vehicle post-AIH, phrenic burst frequency was increased over baseline at 90 min (6±2,
340 p=0.003). Phrenic burst frequency in control rats receiving either vehicle alone or U0126
341 alone were not increased above baseline at any time point (Fig. 7C).
342 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
334 343 344 345 DISCUSSION
346 required for AIH-induced pLTF. These conclusions are supported by findings that spinal
347 siTrkB had no effect, whereas siBDNF blocked pLTF (Fig. 3). The requirement for new
348 BDNF synthesis confirms our earlier report [2]. In both cases the specific siRNA
349 sequences and doses used were validated to have the intended effect when used in this
350 same experimental preparation [2, 16]. On the other hand, tyrosine kinase activity is
351 necessary for both AIH-induced pLTF [2] and A2A receptor induced phrenic motor
352 facilitation [PMF; 16]. However, to date, there are no clearly identified signaling
353 mechanisms downstream from TrkB that underlie pLTF expression and/or development.
354 Here we provide the first direct evidence that activation of the MEK/ERK
355 signaling pathway is necessary for AIH-induced pLTF, whereas no clear role for
356 PI3K/Akt signaling was found. MEK/ERK activity appears to be involved in a distinct
357 phase of pLTF since: 1) pre-treatment with U0126 blocked pLTF (Fig. 3), whereas 2)
358 U0126 administered after AIH halted the development of further pLTF, but did not block
359 initial pLTF expression (Fig. 6).
Here we demonstrate that neither new synthesis of spinal TrkB nor PI3-kinase activity is
361 Distinct mechanisms of phrenic motor facilitation. These results provide evidence that
362 AIH-induced pLTF and pMF induced by Gs protein-coupled metabotropic receptors
363 arise from distinct cellular mechanisms. AIH induced pLTF is induced by activation of
364 Gq protein-coupled metabotropic receptors (i.e., 5-HT2), requires new BDNF synthesis
365 [2], mature TrkB activation and subsequent activation of the MEK/ERK pathway. We
366 refer to this mechanism as the "Q-pathway" to pMF since it is initiated by Gq protein-
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
360 367 coupled receptors [7]. In contrast, the "S-pathway" to pMF is induced by activation of Gq
368 protein-coupled metabotropic receptors [i.e. A2A 5-HT7; 16, 19, 21], requires new
369 synthesis of an immature TrkB isoform, is independent of new BDNF synthesis, and
370 requires downstream signaling via the PI3K/Akt pathway [7]. Future studies are
371 necessary to reveal the respective roles of these distinct mechanisms in different
372 physiological conditions and the nature of interactions between them [43].
373 New protein synthesis is critical for phrenic motor plasticity. Translational regulation of
375 new protein synthesis is fundamental to many forms of synaptic plasticity [36, 48, 51].
376 For example, new BDNF synthesis is necessary for AIH-induced pLTF [2], whereas
377 synthesis of an immature TrkB isoform is necessary for the S-pathway [7, 16]. Although
378 the specific trigger for increased translation of BDNF mRNA has not been identified, we
379 confirm here that impaired translation of spinal BDNF mRNA with siRNAs targeting
380 BDNF mRNA blocks pLTF [2].
381 A2A receptor activation constrains pLTF expression [19], suggesting the S-
382 pathway is marginally activated by AIH but, in fact, inhibits the Q-pathway [7]. Recently
383 we demonstrated that other spinal Gs-protein-coupled receptors also elicit pMF, such as
384 spinal 5-HT7 receptor activation [20]. Hence we wondered whether signaling via the S-
385 pathway “downstream” of Gs protein-coupled receptor activation (i.e. immature TrkB)
386 contributes to AIH-induced pLTF. However, the RNAi experiments performed here
387 provide contrary evidence since new TrkB is not necessary for AIH-induced pLTF.
388 Instead the TrkB activation, critical for AIH-induced pLTF, likely results from ligand
389 interactions (i.e. BDNF binding) with the mature TrkB isoform [2].
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
374 390 Spinal MEK/ERK signaling is required for pLTF expression. BDNF activates ERK MAP
392 kinases 1 and 2 (ERK1/2) in multiple neuronal cell types [17, 46, 55], including spinal
393 motor neurons [25]. ERK1/2 activation is required for multiple forms of synaptic plasticity
394 [50], including hippocampal LTP [12] and intermediate-term facilitation of the
395 sensorimotor synapse in Aplysia [37]. Activated (phosphorylated) ERK1/2 may
396 contribute to pLTF by controlling synthesis of “pro-plasticity” proteins via translational
397 regulation [22]. .
398 We propose that AIH triggers new BDNF protein synthesis which, once released
399 from phrenic motor neurons, acts pre- and/or post-synaptically by activating TrkB
400 receptors and, subsequently, ERK1/2. To date, little direct experimental evidence
401 supports involvement of ERK1/2 in AIH-induced pLTF. However, AIH increases ERK1/2
402 phosphorylation in ventral spinal regions that encompass the phrenic motor nucleus
403 [54]. Furthermore, repetitive AIH exposure increases ventral spinal ERK1/2 expression
404 and phosphorylation state [54], an effect localized near synapses onto presumptive
405 phrenic motor neurons [47]. Conversely, episodic spinal A2A receptor activation
406 increases Akt (not ERK) phosphorylation in cervical spinal regions encompassing the
407 phrenic motor nucleus [16]. Thus, indirect evidence supports a role for both kinases in
408 different forms of phrenic motor plasticity [7]. Here we provide the first direct evidence
409 that MEK/ERK (not PI3K/Akt) activation is critical for AIH-induced pLTF.
410 411 Multiple pathways to phrenic motor facilitation. Many lines of evidence are emerging,
412 suggesting that multiple distinct cellular mechanisms give rise to long-lasting phrenic
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
391 motor facilitation [PMF; 7]. For example, pMF results from episodic spinal administration
414 of: 1) serotonin [33], 2) 5-HT2 receptor agonists [31], 3) BDNF [2], 4) A2A receptor
415 agonists [16], 5) 5-HT7 receptor agonists [20], 6) vascular endothelial growth factor [9],
416 and 7) Erythropoetin [8]. We propose that each molecule ultimately activates a limited
417 number of “downstream” signaling pathways that converge on TrkB receptor signaling
418 via either MEK/ERK and/or PI3K/Akt to elicit pMF. The two pathways investigated in this
419 paper (the “Q” and the “S” pathways) elicit pMF via distinct mechanisms characterized
420 by distinct G-protein-coupled metabotropic receptors (Gq versus Gs), their requirements
421 for BDNF versus TrkB synthesis, and MEK/ERK versus PI3K/Akt activation.
422 Since new BDNF synthesis and ERK activation are required, we propose the
423 predominant mechanism of AIH-induced pLTF is the “Q-pathway” to pMF. It is not yet
424 known when and how Gs protein-coupled receptors interact with mechanisms of AIH-
425 induced pLTF, although spinal application of A2A receptor antagonists amplifies AIH-
426 induced pLTF [19, 43]. Thus, activation of the S-pathway to pMF restrains (rather than
427 augments) the Q-pathway via "cross-talk-inhibition."
428 released during AIH activates multiple receptor subtypes, coupled both to Gq (e.g. 5-
429 HT2 receptors) and Gs proteins [e.g. 5-HT7; 6]. Thus, AIH-induced pLTF via 5-HT2
430 receptor activation may normally be limited (constrained) by co-incident activation of Gs
431 protein-coupled serotonin receptors.
On the other hand, serotonin
432 433 Significance. Although our understanding of detailed cellular/synaptic mechanisms
434 giving rise to pLTF remains incomplete, considerable progress has been made in recent
435 years [3, 32, 34, 41]. A detailed understanding of these cellular mechanisms is
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
413 necessary to fully appreciate the biological significance of pLTF. For example, hypoxic
437 episodes simulating apneas (<25 sec) induce phrenic and hypoglossal LTF [35, 44],
438 suggesting that realistic apneas are sufficient to elicit this mechanism. Facilitation of
439 upper airway motor neurons, which preserve upper airway patency (e.g., hypoglossal
440 LTF), may play a compensatory role, stabilizing breathing during sleep [5, 34, 45]. On
441 the other hand, some have suggested that ventilatory LTF de-stabilizes breathing via
442 increased chemoreflex loop-gain [38]. The net balance of stabilizing/destabilizing
443 influences may depend on the specific physiological (or pathological) condition in which
444 LTF is induced.
445 From a clinical perspective, an understanding of cellular/molecular events that
446 underlie pLTF may aid in the development of strategies to “harness” respiratory
447 plasticity for the treatment of ventilatory insufficiency [41]. For instance, defects in
448 serotonin- and/or BDNF-dependent plasticity may contribute to severe respiratory
449 control disorders, including obstructive sleep apnea [27, 52], apnea of prematurity [14],
450 sudden infant death syndrome [13, 24] and respiratory instability in patients with Rett
451 Syndrome [40]. We have only begun to develop an understanding of basic mechanisms
452 underlying pLTF (or pMF) and to appreciate its potential significance in the treatment of
453 important clinical disorders.
454 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
436 455 456 ACKNOWLEDGEMENTS
The authors thank Kalen Nichols for assistance with blood-gas analysis.
457 458 GRANTS
459 Research support was provided by National Institutes of Health Grants
460 HL080209. M. S. Hoffman was supported by the National Institutes of Health National
461 Research Service Award Predoctoral Fellowship (HL092785).
462 supported by the National Institutes of Health National Research Service Award
463 Postdoctoral Fellowship (T32 HL007654). P. M. MacFarlane was supported by the
464 Francis Families Foundation.
N. L. Nichols was
466 467 DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
468 469 AUTHOR CONTRIBUTIONS
470 Author contributions: M.S.H, N.L.N., P.M.M. and G.S.M. conception and design
471 of research; M.S.H., N.L.N and P.M.M performed experiments; M.S.H., N.L.N. and
472 P.M.M. analyzed data; M.S.H., N.L.N., P.M.M. and G.S.M. interpreted results of
473 experiments; M.S.H. prepared figures; M.S.H. drafted manuscript; M.S.H., N.L.N.,
474 P.M.M. and G.S.M. edited and revised manuscript; M.S.H., N.L.N., P.M.M. and G.S.M.
475 approved final version of manuscript. 476 477 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
465 478 REFERENCES
479 1.
Bach, KB and Mitchell, GS. Hypoxia-induced long-term facilitation of respiratory
activity is serotonin dependent. Respir Physiol 104(2-3): 251-260, 1996.
480 481 482 2.
Baker-Herman TL, Fuller DD, Bavis RW, Zabka AG, Golder FJ, Doperalski
NJ, Johnson RA, Watters JJ, Mitchell GS. BDNF is necessary and sufficient
484 for spinal respiratory plasticity following intermittent hypoxia. Nat Neurosci 7(1):
485 48, 2004.
486 487 3.
Baker-Herman, TL, Mitchell, GS. Determinants of frequency long-term
488 facilitation following acute intermittent hypoxia in vagotomized rats. Respir
489 Physiol Neurobiol 162(1): 8-17, 2008.
490 491 4.
Baker-Herman TL, Mitchell GS. Phrenic long-term facilitation requires spinal
492 serotonin receptor activation and protein synthesis. J Neurosci 22: 6239-6246,
493 2002.
494 495 5.
Behan M, Zabka AG, Mitchell GS. Age and gender effects on serotonin-
496 dependent plasticity in respiratory motor control. Respir Physiol Neurobiol 131(1-
497 2): 65-77, 2002.
498 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
483 499 6.
Bockaert J, Claeysen S, Bécamel C, Dumuis A, Marin P. Neuronal 5-HT
500 metabotropic receptors: fine-tuning of their structure, signaling, and roles in
501 synaptic modulation. Cell Tissue Res 326: 553-572, 2006.
502 503 7.
Dale-Nagle EA, Hoffman MS, MacFarlane PM, Mitchell GS. Multiple pathways
to long-lasting phrenic motor facilitation. Adv Exp Med Biol 669: 225-230, 2010.
504 505 8.
Dale EA, Satriotomo I, Mitchell GS. Cervical spinal erythropoietin induces
507 phrenic motor facilitation via extracellular signal-regulated kinase and Akt
508 signaling. J Neurosci 32(17): 5973-5983, 2012.
509 510 9.
Dale-Nagle EA, Satriotomo I, Mitchell, GS. Spinal vascular endothelial growth
511 factor induces phrenic motor facilitation via extracellular signal-regulated kinase
512 and Akt signaling. J Neurosci 31(21): 7682-7690, 2011.
513 514 10.
Duncia JV, Santella JB 3rd, Higley CA, Pitts WJ, Wityak J, Frietze WE,
515 Rankin FW, Sun JH, Earl RA, Tabaka AC, Teleha CA, Blom KF, Favata MF,
516 Manos EJ, Daulerio AJ, Stradley DA, Horiuchi K, Copeland RA, Scherle PA,
517 Trzaskos JM, Magolda RL, Trainor GL, Wexler RR, Hobbs RW, Olson RE.
518 MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and
519 cyclization products. Bioorg Med Chem Lett 8(20): 2839-2844, 1998.
520 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
506 521 11.
Eldridge FL. Quantification of electrical activity in the phrenic nerve in the study
of ventilatory control. Chest 70(1 Suppl): 154-157, 1976.
522 523 524 12.
English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase
525 cascade in hippocampal long term potentiation. J Biol Chem 272(31): 19103-
526 19106, 1997.
527 13.
Feldman JL, Mitchell GS, Nattie EE. Breathing: rhythmicity, plasticity,
chemosensitivity. Annu Rev Neurosci 26: 239-266, 2003.
529 530 531 14.
Finer NN, Higgins R, Kattwinkel J, Martin RJ. Summary Proceedings From the
Apnea-of-Prematurity Group. Pediatrics 117(3): S47-51, 2006.
532 533 534 15.
Gharbi SI, Zvelebil MJ, Shuttleworth SJ, Hancox T, Saghir N, Timms JF,
535 Waterfield MD. Exploring the specificity of the PI3K family inhibitor LY294002.
536 Biochem J 404(1): 15-21, 2007.
537 538 16.
Golder FJ, Ranganathan L, Satriotomo I, Hoffman M, Lovett-Barr MR,
539 Watters JJ, Baker-Herman TL, Mitchell GS. Spinal adenosine A2a receptor
540 activation elicits long-lasting phrenic motor facilitation. J Neurosci 28: 2033-2042,
541 2008.
542 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
528 543 17.
Gooney M, Lynch MA. Long-term potentiation in the dentate gyrus of the rat
544 hippocampus is accompanied by brain-derived neurotrophic factor-induced
545 activation of TrkB. J Neurochem 77(5): 1198-1207, 2001.
546 547 18.
Gourine AV, Llaudet E, Dale N, Spyer KM. Release of ATP in the ventral
548 medulla during hypoxia in rats: role in hypoxic ventilatory response. J Neurosci
549 25: 1211-1218, 2005.
551 19.
Hoffman MS, Golder FJ, Mahamed S, Mitchell GS. Spinal adenosine 2A
552 receptor inhibition enhances phrenic long term facilitation following acute
553 intermittent hypoxia. J Physiol 588: 255-266, 2010.
554 555 20.
Hoffman MS, Mitchell GS. Spinal 5-HT7 receptor activation induces long-lasting
phrenic motor facilitation. J Physiol 589: 1397-1407, 2011.
556 557 558 21.
Hoffman MS, Mitchell GS. Spinal 5-HT7 receptor activation induces long-lasting
phrenic motor facilitation. J Physiol 589: 1397-1407, 2011.
559 560 561 22.
Kelleher RJ 3rd, Govindarajan A, Tonegawa S. Translational regulatory
mechanisms in persistent forms of synaptic plasticity. Neuron 44(1): 59-73, 2004.
562 563 564 565 23.
Kinkead R, Bach KB, Johnson SM, Hodgeman BA, Mitchell GS. Plasticity in
respiratory motor control: intermittent hypoxia and hypercapnia activate opposing
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
550 566 serotonergic and noradrenergic modulatory systems. Comp Biochem Physiol A
567 Mol Integr Physiol 130(2): 207-218, 2001.
568 569 24.
Kinney HC, Richerson GB, Dymecki SM, Darnall RA, Nattie EE. The
570 brainstem and serotonin in the sudden infant death syndrome. Annu Rev Pathol
571 4: 517-550, 2009.
572 25.
Kishino A, Nakayama C. Enhancement of BDNF and activated-ERK
574 immunoreactivity in spinal motor neurons after peripheral administration of
575 BDNF. Brain Res 964(1): 56-66, 2003.
576 577 26.
Klotz KN. Adenosine receptors and their ligands. Naunyn Schmiedebergs Arch
Pharmacol 362(4-5): 382-391, 2000.
578 579 580 27.
Kraiczi H, Hedner J, Dahlof P, Ejnell H, Carlson J. Effect of serotonin uptake
581 inhibition on breathing during sleep and daytime symptoms in obstructive sleep
582 apnea. Sleep 22(1): 61-67, 1999.
583 584 28.
Lee FS, Chao MV. Activation of Trk neurotrophin receptors in the absence of
neurotrophins. Proc Natl Acad Sci U S A 98(6): 3555-3560, 2001.
585 586 587 588 29.
Lovenberg TW, Baron BM, Lecea Lde, Miller JD, Prosser RA, Rea MA, Foye PE,
Racke M, Slone AL, Siegel BW, Danielson PE, Sutcliffe JG, Erlander, MG. A novel
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
573 589 adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the
590 regulation of mammalian circadian rhythms. Neuron 11(3): 449-458, 1993.
591 592 30.
MacFarlane PM, Mitchell GS. Episodic spinal serotonin receptor activation
593 elicits long-lasting phrenic motor facilitation by an NADPH oxidase-dependent
594 mechanism. J Physiol 587(Pt 22): 5469-5481, 2009.
595 31.
MacFarlane PM, Mitchell GS. NADPH oxidase activity is necessary for phrenic
597 motor facilitation induced by 5HT2B receptor activation. FASEB J.
598 22(1_MeetingAbstracts): 1232.7, 2008.
599 600 32.
Macfarlane PM, Wilkerson JE, Lovett-Barr MR, Mitchell GS. Reactive oxygen
601 species and respiratory plasticity following intermittent hypoxia. Respir Physiol
602 Neurobiol 164(1-2): 263-271, 2008.
603 604 33.
MacFarlane PM, Mitchell, GS. Serotonin-induced phrenic long-term facilitation
605 requires reactive oxygen species signaling via the NADPH oxidase complex.
606 Soc Neurosci Abst 520.15, 2007.
607 608 609 610 34.
Mahamed S, Mitchell GS. Is there a link between intermittent hypoxia-induced
respiratory plasticity and obstructive sleep apnoea? Exp Physiol 92: 27-37, 2007.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
596 611 35.
Mahamed S, Mitchell GS. Simulated apneas induce serotonin-dependent
respiratory long-term facilitation in rats. J Physiol 586(8): 2171-2181, 2008.
612 613 614 36.
Martin KC, Barad M, Kandel ER. Local protein synthesis and its role in
synapse-specific plasticity. Curr Opin Neurobiol 10(5): 587-592, 2000.
615 616 617 37.
Martin KC, Michael D, Rose JC, Barad M, Casadio A, Zhu H, Kandel ER.
MAP kinase translocates into the nucleus of the presynaptic cell and is required
619 for long-term facilitation in Aplysia. Neuron 18(6): 899-912, 1997.
620 621 38.
Mateika JH, Narwani G. Intermittent hypoxia and respiratory plasticity in humans
622 and other animals: does exposure to intermittent hypoxia promote or mitigate
623 sleep apnoea? Exp Physiol 94(3): 279-296, 2009.
624 625 39.
Millhorn DE, Eldridge FL, Waldrop TG. Prolonged stimulation of respiration by
endogenous central serotonin. Respir Physiol 42: 171-188, 1980.
626 627 628 40.
Mironov SL, Skorova E, Hartfelt N, Mironova LA, Hasan MT, Kugler S.
629 Remodelling of the respiratory network in a mouse model of Rett syndrome
630 depends on brain-derived neurotrophic factor regulated slow calcium buffering. J
631 Physiol 587(Pt 11): 2473-2485, 2009.
632 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
618 633 41.
Mitchell GS. Respiratory plasticity following intermittent hypoxia: a guide for
634 novel therapeutic approaches to ventilatory control disorders. In: Genetic Basis
635 for Respiratory Control Disorders, edited by Gaultier C. New York: Springer,
636 2007, p. 291-306.
637 638 42.
Morris KF, Shannon R, Lindsey BG. Changes in cat medullary neurone firing
rates and synchrony following induction of respiratory long-term facilitation. J
640 Physiol 532(Pt 2): 483-497, 2001.
641 642 43.
Nichols NL, Dale EA, Mitchell GS. Severe acute intermittent hypoxia elicits
643 phrenic long-term facilitation by a novel adenosine-dependent mechanism. J
644 Appl Physiol 112(10): 1678-1688, 2012.
645 646 44.
Peng YJ, Prabhakar NR. Reactive oxygen species in the plasticity of respiratory
647 behaviour elicited by chronic intermittent hypoxia. J Appl Physiol 94: 2342-2349,
648 2003.
649 650 45.
Pierchala LA, Mohammed AS, Grullon K, Mateika JH, Badr MS. Ventilatory
651 long-term facilitation in non-snoring subjects during NREM sleep. Respir Physiol
652 Neurobiol 160(3): 259-266, 2008.
653 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
639 654 46.
Purcell AL, Sharma SK, Bagnall MW, Sutton MA, Carew TJ. Activation of a
655 tyrosine kinase-MAPK cascade enhances the induction of long-term synaptic
656 facilitation and long-term memory in Aplysia. Neuron 37(3): 473-484, 2003.
657 658 47.
Satriotomo I, Dale EA, Mitchell GS. Thrice weekly intermittent hypoxia
increases expression of key proteins necessary for phrenic long-term facilitation:
660 a possible mechanism of respiratory metaplasticity? FASEB J 21(6): A1292,
661 2007.
662 663 48.
Schuman EM, Dynes JL, Steward O. Synaptic regulation of translation of
dendritic mRNAs. J Neurosci 26(27): 7143-7146, 2006.
664 665 666 49.
Segal RA. Selectivity in neurotrophin signaling: theme and variations. Annu Rev
Neurosci 26: 299-330, 2003.
667 668 669 50.
Sweatt JD. The neuronal MAP kinase cascade: a biochemical signal integration
670 system subserving synaptic plasticity and memory. J Neurochem 76(1): 1-10,
671 2001.
672 673 51.
Tartaglia N, Du J, Tyler WJ, Neale E, Pozzo-Miller L, Lu B. Protein synthesis-
674 dependent and -independent regulation of hippocampal synapses by brain-
675 derived neurotrophic factor. J Biol Chem 276(40): 37585-37593, 2001.
676 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
659 677 52.
Veasey SC. Serotonin. Culprit or promising therapy for obstructive sleep apnea?
Am J Respir Crit Care Med 163(5): 1045-1047, 2001.
678 679 680 53.
Wiese S, Jablonka S, Holtmann B, Orel N, Rajagopal R, Chao MV, Sendtner
681 M. Adenosine receptor A2A-R contributes to motoneuron survival by
682 transactivating the tyrosine kinase receptor TrkB. Proc Natl Acad Sci U S A
683 104(43): 17210-17215, 2007.
685 54.
Wilkerson JE, Mitchell GS. Daily intermittent hypoxia augments spinal BDNF
686 levels, ERK phosphorylation and respiratory long-term facilitation. Exp Neurol
687 217(1): 116-123, 2009.
688 689 55.
Ying SW, Futter M, Rosenblum K, Webber MJ, Hunt SP, Bliss TV, Bramham
690 CR. Brain-derived neurotrophic factor induces long-term potentiation in intact
691 adult hippocampus: requirement for ERK activation coupled to CREB and
692 upregulation of Arc synthesis. J Neurosci 22(5): 1532-1540, 2002.
693 694 695 696 697 698 699 700 701 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
684 702 TABLE and FIGURE LEGENDS
703 Table 1: Measurements of PaCO2, PaO2 and mean arterial pressure (MAP) during
704 baseline, hypoxia and 60 min post-hypoxia. Values are means ± S.E.M. MAP, mean
705 arterial pressure. *different than baseline; †different than vehicle(+AIH); ‡different than
706 vehicle(-AIH); p<0.05.
707 Fig.1: Phrenic hypoxic response during episodes of hypoxia. Change in phrenic
709 burst amplitudes during five min of hypoxic exposures (average of 3) from rats receiving
710 spinal injections prior to AIH or vehicle (siBuffer, n=7), siTrkB (n = 8), siBDNF (n = 9),
711 MEK inhibitor (U0126, n=9), or PI3-K inhibitor (PI-828, n=6) and rats receiving post-AIH
712 injections of vehicle (n=8) and U0126 (n=8). Phrenic burst amplitudes increased from
713 baseline in all groups, although no between group differences were detected. Values
714 are means ± 1 S.E.M. #different from baseline; p<0.05.
715 716 Fig. 2: Representative phrenic neurograms depicting experimental protocols in
717 rats treated prior to AIH. A: intrathecal vehicle (RNAi buffer) prior (~2hrs) to AIH. B:
718 intrathecal siTrkB prior to AIH. C: intrathecal siBDNF prior to AIH. D: time control
719 receiving intrathecal vehicle without AIH. Pretreatment with siTrkB did not inhibit pLTF
720 compared to vehicle rats exposed to AIH. siBDNF inhibited pLTF vs. vehicle, time
721 control experiments.
722 723 Fig. 3: Small interfering TrkB RNA does not block BDNF synthesis-dependent
724 pLTF. Comparisons were made for integrated phrenic amplitudes (∫Phr) from rats
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
708 725 spinally injected with small interfering TrkB RNA (■, siTrkB; n=8) versus BDNF RNA (,
726 siBDNF; n=9). ∫Phr from siTrkB treated rats were not different from vehicle treated rats
727 (RNAi buffer, □; n=7,) 60 min post-AIH, whereas siBDNF significantly inhibited pLTF 60
728 minutes post-AIH. A group of rats treated with vehicle did not receive AIH (i.e. time
729 controls, n=5; Δ). ∫Phr in vehicle control rats were not different than baseline at any time
730 point. Values are means ± S.E.M. #different from vehicle control; † different from
731 siBDNF; p<0.05
733 Fig. 4: Spinal MEK inhibition blocks phrenic LTF. A: Representative phrenic
734 neurogram after intrathecal MEK inhibitor (U0126) prior to AIH. B: Representative
735 phrenic neurogram after intrathecal PI3-K inhibitor (PI-828) prior to AIH. C:
736 Comparisons were made for integrated phrenic burst amplitudes (∫Phr) from spinally
737 treated rats, and expressed as a percentage change from baseline. ∫Phr from rats
738 pretreated (20 min prior) with a MEK inhibitor (, U0126; n=9) were decreased
739 compared to vehicle (□, n=7) at 60 min post-AIH. In rats pretreated with PI3K inhibitor
740 (■, PI-828; n=6) ∫Phr increased over baseline at 60 min post-AIH and were not different
741 than vehicle (□) treatment. Hence MEK activity, not PI3K, is required for pLTF. Values
742 are means ± 1 S.E.M.
743 p<0.05.
#different from vehicle control; †different from MEK inhibitor;
744 745 Fig. 5: Representative phrenic neurograms depicting experimental protocols in
746 rats treated post-AIH. Spinal injection post-AIH (<5min) of vehicle (12μl) (A), vehicle
747 control (no AIH) (B), MEK inhibitor (UO126, 12μl) (C) and UO126 control (no AIH) (D).
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
732 748 Post-AIH UO126 attenuated pLTF at 60 and 90 min compared to vehicle rats exposed
749 to AIH.
750 Fig. 6: Spinal MEK inhibition post-AIH attenuates pLTF expression. Comparisons
752 were made for integrated phrenic burst amplitudes (∫Phr) from spinally treated rats, and
753 expressed as a percentage change from baseline. ∫Phr from rats treated with vehicle
754 (■,n=8) immediately following AIH (<5min) exhibited significant pLTF up to 90 min post-
755 AIH. To test the hypothesis that pLTF maintanence required MEK activity, rats were
756 given a spinal injection of a selective inhibitor (, U0126, 100µM, n=8) immediately
757 following AIH. U0126 attenuated ∫Phr 60 min post-AIH and beyond, suggesting
758 continued MEK/ERK activity is required to maintain full pLTF expression. Control rats
759 reciving either vehicle (□, n=8) or U0126 (n=8) without AIH exhibited facilitation. Values
760 are means±S.E.M. *different from MEK inhibitor; #different from vehicle control;
761 †different from MEK inhibitor control; p<0.05.
762 763 Fig. 7: Changes in phrenic burst frequency (frequency LTF). Changes in phrenic
764 nerve burst frequency from baseline (bursts/min) in rats receiving spinal injection, pre-
765 AIH, of (A) vehicle (sibuffer), siTrkB, siBDNF and vehicle time control; (B) includes
766 comparisons from spinal MEK inhibitor (U0126) and PI3K inhibitor (PI-828) and (C)
767 shows comparisons from groups receiving post-AIH injections of vehicle, U0126, and
768 time control treatments (vehicle, U0126). Values are means±S.E.M. #different than
769 baseline; ‡different than siBDNF; †different than vehicle (pre-AIH treatment); different
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
751 770 than MEK inhibitor (pre- AIH); $different than vehicle control (post-AIH); & different than
771 MEK inhibitor control (post-AIH); p<0.05.
772 773 774 Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
1
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
1
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
1
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
1
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017