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β-Noradrenergic receptor activation specifically modulates the generation of sighs in vivo and in vitro.

Viemari JC, Garcia AJ, Doi A, Elsen G, Ramirez JM - Front Neural Circuits (2013)

Bottom Line: By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (I(CAN)) remained unaffected.Moreover, riluzole, which blocks bursting in I(Nap) pacemakers abolished sighs altogether, while flufenamic acid (FFA) which blocks the I(CAN) current did not alter the sigh-increasing effect caused by β-NR.Our results suggest that the selective β-NR action of sighs may result from the modulation of I(Nap) pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response.

View Article: PubMed Central - PubMed

Affiliation: Team P3M, Institut de Neurosciences de la Timone, UMR 7289, CNRS, Aix Marseille Univesité , Marseille, France.

ABSTRACT
The pre-Bötzinger complex (preBötC), an area that is critical for generating breathing (eupnea), gasps and sighs is continuously modulated by catecholamines. These amines and the generation of sighs have also been implicated in the regulation of arousal. Here we studied the catecholaminergic modulation of sighs not only in anesthetized freely breathing mice (in vivo), but also in medullary slice preparations that contain the preBötC and that generate fictive eupneic and sigh rhythms in vitro. We demonstrate that activating β-noradrenergic receptors (β-NR) specifically increases the frequency of sighs, while eupnea remains unaffected both in vitro and in vivo. β-NR activation specifically increased the frequency of intrinsically bursting pacemaker neurons that rely on persistent sodium current (I(Nap)). By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (I(CAN)) remained unaffected. Moreover, riluzole, which blocks bursting in I(Nap) pacemakers abolished sighs altogether, while flufenamic acid (FFA) which blocks the I(CAN) current did not alter the sigh-increasing effect caused by β-NR. Our results suggest that the selective β-NR action of sighs may result from the modulation of I(Nap) pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response. The β-NR action on the preBötC may be an important mechanism in modulating behaviors that are specifically associated with sighs, such as the regulation of the early events leading to the arousal response.

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Related in: MedlinePlus

Fictive sigh activity is observed in the in vitro transverse slice preparation. Anatomical characterization of the transverse slice preparation (P7 mouse): (A) rostral and (B) caudal surface of the same live transverse slice preparation. This representative slice preparation was cut into three sections and Nissl staining was used to characterize the rostral (C,C′) and caudal surface (E,E′), as well as the Center/Middle portion of the slice (D,D′). NK1+ and DAPI+ immunoreactive neurons are depicted in (C′–E′). Note that NK1R staining, which is indicative of the preBötC, is most abundant in the center, but NK1R staining extends also into the rostral and caudal portions of the slice. (F) Schematic of the brainstem slice preparation including the anatomical landmarks of the preBötC and recording sites of integrated VRG activity (∫VRG upper trace) and whole-cell patch clamp recordings (membrane potential, Vm, lower trace). Both traces depict fictive eupneic activity and fictive sigh activity recorded from a slice. Sighs are typically followed by a post-sigh apnea. Note that fictive sigh bursts occurred spontaneously at a slower frequency than fictive respiratory activity. Histograms summarize the significant differences between spontaneous sighs and fictive respiratory activity, in burst amplitude (G), in burst frequency (H) and burst duration (I). Results are expressed as mean ± SE. Asterisk (*) shows significant differences. (*p < 0.05, n = 18).
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Figure 2: Fictive sigh activity is observed in the in vitro transverse slice preparation. Anatomical characterization of the transverse slice preparation (P7 mouse): (A) rostral and (B) caudal surface of the same live transverse slice preparation. This representative slice preparation was cut into three sections and Nissl staining was used to characterize the rostral (C,C′) and caudal surface (E,E′), as well as the Center/Middle portion of the slice (D,D′). NK1+ and DAPI+ immunoreactive neurons are depicted in (C′–E′). Note that NK1R staining, which is indicative of the preBötC, is most abundant in the center, but NK1R staining extends also into the rostral and caudal portions of the slice. (F) Schematic of the brainstem slice preparation including the anatomical landmarks of the preBötC and recording sites of integrated VRG activity (∫VRG upper trace) and whole-cell patch clamp recordings (membrane potential, Vm, lower trace). Both traces depict fictive eupneic activity and fictive sigh activity recorded from a slice. Sighs are typically followed by a post-sigh apnea. Note that fictive sigh bursts occurred spontaneously at a slower frequency than fictive respiratory activity. Histograms summarize the significant differences between spontaneous sighs and fictive respiratory activity, in burst amplitude (G), in burst frequency (H) and burst duration (I). Results are expressed as mean ± SE. Asterisk (*) shows significant differences. (*p < 0.05, n = 18).

Mentions: Brainstem transverse slice preparation from CD1 mice (P6–P12) were obtained using a technique described in detail previously (Ramirez et al., 1996). The most important steps are summarized here. All surgical and experimental procedures conformed to guidelines from the French Ministry for Agriculture and Fisheries and were approved by the Institutional Animal Care and Use at the Seattle Children's Research Institute. The mice were anesthetized by hypothermia and decapitated. The isolated brainstem was then placed in ice-cold artificial cerebro-spinal fluid (a-CSF) bubbled with carbogen (95% O2 and 5% CO2). The a-CSF contained (in mM): 128 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, pH of 7.4. The brainstem was then glued to an agar block on the mounting plate of a VT 1000 s (Leica Microsystems, Richmond Hill, ON, Canada) with the rostral end up and the ventral face toward the blade. 100 to 200 μm Hundred to two hundred micrometers serial transversal slices at a 20° angle were then made in a rostral to caudal direction until disapperance of parafacial group and appearance of the inferior olive, nucleus ambiguus, the hypoglossal nucleus, and the opening of the fourth ventricle as also described in the P0 atlas by (Ruangkittisakul et al., 2011). Then, a 550–650 μm thick a rhythmic slice containing the preBötC was made. The approach encompasses the preBötC (Figure 2). In this figure we cut the slice into three parts and stained the slices with NK1/DAPI antibodies (Figures 2C–E′). The boundaries of NK1R+ cells in the ventral respiratory column correspond to ~300 μm-thick sections in total. Note that the preBötC area shows a high concentration of NK1 staining (Figures 2C,C′) as previously described by different groups (Gray et al., 1999; Guyenet et al., 2002; Pagliardini et al., 2003). These slices also contain raphé neurons, Chx10 neurons (Figures 3, 6; Crone et al., 2012) and TH-neurons important for the stabilization of the respiratory rhythm (Viemari et al., 2005; Zanella et al., 2006).


β-Noradrenergic receptor activation specifically modulates the generation of sighs in vivo and in vitro.

Viemari JC, Garcia AJ, Doi A, Elsen G, Ramirez JM - Front Neural Circuits (2013)

Fictive sigh activity is observed in the in vitro transverse slice preparation. Anatomical characterization of the transverse slice preparation (P7 mouse): (A) rostral and (B) caudal surface of the same live transverse slice preparation. This representative slice preparation was cut into three sections and Nissl staining was used to characterize the rostral (C,C′) and caudal surface (E,E′), as well as the Center/Middle portion of the slice (D,D′). NK1+ and DAPI+ immunoreactive neurons are depicted in (C′–E′). Note that NK1R staining, which is indicative of the preBötC, is most abundant in the center, but NK1R staining extends also into the rostral and caudal portions of the slice. (F) Schematic of the brainstem slice preparation including the anatomical landmarks of the preBötC and recording sites of integrated VRG activity (∫VRG upper trace) and whole-cell patch clamp recordings (membrane potential, Vm, lower trace). Both traces depict fictive eupneic activity and fictive sigh activity recorded from a slice. Sighs are typically followed by a post-sigh apnea. Note that fictive sigh bursts occurred spontaneously at a slower frequency than fictive respiratory activity. Histograms summarize the significant differences between spontaneous sighs and fictive respiratory activity, in burst amplitude (G), in burst frequency (H) and burst duration (I). Results are expressed as mean ± SE. Asterisk (*) shows significant differences. (*p < 0.05, n = 18).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824105&req=5

Figure 2: Fictive sigh activity is observed in the in vitro transverse slice preparation. Anatomical characterization of the transverse slice preparation (P7 mouse): (A) rostral and (B) caudal surface of the same live transverse slice preparation. This representative slice preparation was cut into three sections and Nissl staining was used to characterize the rostral (C,C′) and caudal surface (E,E′), as well as the Center/Middle portion of the slice (D,D′). NK1+ and DAPI+ immunoreactive neurons are depicted in (C′–E′). Note that NK1R staining, which is indicative of the preBötC, is most abundant in the center, but NK1R staining extends also into the rostral and caudal portions of the slice. (F) Schematic of the brainstem slice preparation including the anatomical landmarks of the preBötC and recording sites of integrated VRG activity (∫VRG upper trace) and whole-cell patch clamp recordings (membrane potential, Vm, lower trace). Both traces depict fictive eupneic activity and fictive sigh activity recorded from a slice. Sighs are typically followed by a post-sigh apnea. Note that fictive sigh bursts occurred spontaneously at a slower frequency than fictive respiratory activity. Histograms summarize the significant differences between spontaneous sighs and fictive respiratory activity, in burst amplitude (G), in burst frequency (H) and burst duration (I). Results are expressed as mean ± SE. Asterisk (*) shows significant differences. (*p < 0.05, n = 18).
Mentions: Brainstem transverse slice preparation from CD1 mice (P6–P12) were obtained using a technique described in detail previously (Ramirez et al., 1996). The most important steps are summarized here. All surgical and experimental procedures conformed to guidelines from the French Ministry for Agriculture and Fisheries and were approved by the Institutional Animal Care and Use at the Seattle Children's Research Institute. The mice were anesthetized by hypothermia and decapitated. The isolated brainstem was then placed in ice-cold artificial cerebro-spinal fluid (a-CSF) bubbled with carbogen (95% O2 and 5% CO2). The a-CSF contained (in mM): 128 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, pH of 7.4. The brainstem was then glued to an agar block on the mounting plate of a VT 1000 s (Leica Microsystems, Richmond Hill, ON, Canada) with the rostral end up and the ventral face toward the blade. 100 to 200 μm Hundred to two hundred micrometers serial transversal slices at a 20° angle were then made in a rostral to caudal direction until disapperance of parafacial group and appearance of the inferior olive, nucleus ambiguus, the hypoglossal nucleus, and the opening of the fourth ventricle as also described in the P0 atlas by (Ruangkittisakul et al., 2011). Then, a 550–650 μm thick a rhythmic slice containing the preBötC was made. The approach encompasses the preBötC (Figure 2). In this figure we cut the slice into three parts and stained the slices with NK1/DAPI antibodies (Figures 2C–E′). The boundaries of NK1R+ cells in the ventral respiratory column correspond to ~300 μm-thick sections in total. Note that the preBötC area shows a high concentration of NK1 staining (Figures 2C,C′) as previously described by different groups (Gray et al., 1999; Guyenet et al., 2002; Pagliardini et al., 2003). These slices also contain raphé neurons, Chx10 neurons (Figures 3, 6; Crone et al., 2012) and TH-neurons important for the stabilization of the respiratory rhythm (Viemari et al., 2005; Zanella et al., 2006).

Bottom Line: By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (I(CAN)) remained unaffected.Moreover, riluzole, which blocks bursting in I(Nap) pacemakers abolished sighs altogether, while flufenamic acid (FFA) which blocks the I(CAN) current did not alter the sigh-increasing effect caused by β-NR.Our results suggest that the selective β-NR action of sighs may result from the modulation of I(Nap) pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response.

View Article: PubMed Central - PubMed

Affiliation: Team P3M, Institut de Neurosciences de la Timone, UMR 7289, CNRS, Aix Marseille Univesité , Marseille, France.

ABSTRACT
The pre-Bötzinger complex (preBötC), an area that is critical for generating breathing (eupnea), gasps and sighs is continuously modulated by catecholamines. These amines and the generation of sighs have also been implicated in the regulation of arousal. Here we studied the catecholaminergic modulation of sighs not only in anesthetized freely breathing mice (in vivo), but also in medullary slice preparations that contain the preBötC and that generate fictive eupneic and sigh rhythms in vitro. We demonstrate that activating β-noradrenergic receptors (β-NR) specifically increases the frequency of sighs, while eupnea remains unaffected both in vitro and in vivo. β-NR activation specifically increased the frequency of intrinsically bursting pacemaker neurons that rely on persistent sodium current (I(Nap)). By contrast, all parameters of bursting pacemakers that rely on the non-specific cation current (I(CAN)) remained unaffected. Moreover, riluzole, which blocks bursting in I(Nap) pacemakers abolished sighs altogether, while flufenamic acid (FFA) which blocks the I(CAN) current did not alter the sigh-increasing effect caused by β-NR. Our results suggest that the selective β-NR action of sighs may result from the modulation of I(Nap) pacemaker activity and that disturbances in noradrenergic system may contribute to abnormal arousal response. The β-NR action on the preBötC may be an important mechanism in modulating behaviors that are specifically associated with sighs, such as the regulation of the early events leading to the arousal response.

Show MeSH
Related in: MedlinePlus