Nucleus ambiguus cholinergic neurons activated by acupuncture: Relation to enkephalin

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Nucleus ambiguus cholinergic neurons activated by acupuncture: Relation to enkephalin Zhi-Ling Guo⁎, Min Li, John C. Longhurst Susan-Samueli Center for Integrative Medicine and Department of Medicine, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Acupuncture regulates autonomic function. Our previous studies have shown that

Accepted 4 January 2012

electroacupuncture (EA) at the Jianshi–Neiguan acupoints (P5–P6, underlying the median

Available online 12 January 2012

nerve) inhibits central sympathetic outflow and attenuates excitatory cardiovascular reflexes, in part, through an opioid mechanism. It is unknown if EA at these acupoints

Keywords:

influences the parasympathetic system. Thus, using c-Fos expression, we examined activa-

Acupuncture

tion of nucleus ambiguus (NAmb) neurons by EA, their relation to cholinergic (preganglionic

Nucleus ambiguus

parasympathetic) neurons and those containing enkephalin. To enhance detection of cell

Acetylcholine

bodies containing enkephalin, colchicine (90–100 μg/kg) was administered into the sub-

Enkephalin

arachnoid space of cats 30 h prior to EA or sham-operated controls for EA. Following bilat-

c-Fos

eral barodenervation and cervical vagotomy, either EA for 30 min at P5–P6 acupoints or control stimulation (needle placement at P5–P6 without stimulation) was applied. While perikarya containing enkephalin were observed in some medullary nuclei (e.g., raphé), only enkephalin-containing neuronal processes were found in the NAmb. Compared to controls (n = 4), more c-Fos immunoreactivity, located principally in close proximity to fibers containing enkephalin was noted in the NAmb of EA-treated cats (n = 5; P < 0.01). Moreover, neurons double-labeled with c-Fos and choline acetyltransferase in the NAmb were identified in EA-treated, but not control animals. These data demonstrate for the first time that EA activates preganglionic parasympathetic neurons in the NAmb. Because of their close proximity, these EA-activated neurons likely interact with nerve fibers containing enkephalin. These results suggest that EA at the P5–P6 acupoints has the potential to influence parasympathetic outflow and cardiovascular function, likely through an enkephalinergic mechanism. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Department of Medicine, C240 Medical Science 1, University of California, Irvine, Irvine, CA 92697-4075, USA. Fax: +1 949 824 2200. E-mail address: [email protected] (Z.-L. Guo). Abbreviations: ABC, avidin–biotin–peroxidase complex; ACh, acetylcholine; BP, blood pressure; ChAT, choline acetyltransferase; EA, electroacupuncture; HR, heart rate; NAmb, nucleus ambiguus; P5–P6, Jianshi–Neiguan acupoints; PB, phosphate buffer; PBS, phosphate buffered saline; PBST, phosphate buffered saline containing Triton X-100; PBT, phosphate buffer containing Triton X-100; rVLM, rostral ventrolateral medulla; DAB, 3,3′-diaminobenzidine 0006-8993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.006

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1.

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Introduction

Acupuncture has been used to treat a number of diseases for many years. Specifically, acupuncture at the Jianshi–Neiguan acupoints (P5–P6, pericardial meridian overlying the median nerve) is applied commonly to manage cardiovascular dysfunction (Ho et al., 1999; Longhurst, 2010; Longhurst and Costello, 2011; Richter et al., 1991). The mechanisms underlying its effects on central regulation of cardiovascular function have not been fully explored. A series of studies from our laboratory have demonstrated that by inhibiting sympathetic activity, electrical stimulation of the P5–P6 acupoints can lower elevated blood pressure. In this respect, EA at P5–P6 acupoints attenuates acute hypertensive responses evoked by visceral afferent stimulation during and after its application for up to 90 min (Li et al., 2001; Tjen-A-Looi et al., 2003). Preliminary data suggest that after repetitive application of EA for two months, the reduction in blood pressure in hypertensive patients can be maintained for one month after terminating EA (Li and Longhurst, 2010). These results suggest that EA regulates autonomic nerve function. However, the effect of EA on parasympathetic nerve activity has not been evaluated. The nucleus ambiguus (NAmb), located in the ventrolateral division of the hindbrain, is considered to be an important site of origin of parasympathetic preganglionic vagal motor neurons that ultimately regulate autonomic function through the release of acetylcholine (Wang et al., 2001). Specifically, long vagal preganglionic neurons originating in the NAmb directly project to ganglia located near the heart that through short postganglionic neurons modulate heart rate and, to a lesser extent, coronary vascular tone and ventricular contractility (Agarwal and Calaresu, 1991; Blinder et al., 2005; Wang et al., 2001). Acetylcholine (ACh) is an important neurotransmitter in the NAmb and cholinergic neurons are the principal vagal motor phenotype involved in the fast neurotransmission in this region (Zhang et al., 1993). Thus, detection of the cholinergic neurons is often used to locate parasympathetic preganglionic motor neurons in the NAmb (Batten, 1995; Loewy and Spyer, 1990). There is evidence showing that enkephalin influences NAmb function (Laubie and Schmitt, 1981; Wang et al., 2004). In this respect, application of enkephalin into the NAmb causes bradycardia, suggesting the potential for a functional role for this opioid peptide in this region (Agarwal and Calaresu, 1991). Our studies have demonstrated that EA activates enkephalinergic neurons in several brain areas that regulate sympathetic outflow, including the arcuate nucleus, rostral ventrolateral medulla and raphé nuclei, among others (Guo and Longhurst, 2007; Guo et al., 2004, 2008). However, there is no information on the action of EA on nuclei that regulate parasympathetic function. More specifically, no studies have investigated activation of NAmb neurons by EA, particularly, with respect to cholinergic preganglionic neurons and their relationship to neurons or processes containing enkephalin. Demonstration of NAmb activation by EA would imply that it may be involved in the physiological action by EA on parasympathetic function. Expression of c-Fos has been used widely as a marker of neuronal activation (Guo and Longhurst, 2003, 2007; Guo et al., 2004; Lee and Beitz, 1993; Morgan et al., 1987). Thus, we and others

have detected neurons in brain regions that respond to prolonged (30 min) somatic nerve, (i.e., acupuncture) stimulation through identification of c-Fos expression (Guo and Longhurst, 2007; Guo et al., 2004; Lee and Beitz, 1993). Furthermore, neuronal structures containing ACh or enkephalin are distributed throughout the NAmb (Batten, 1995; Zamir et al., 1985). Considering this background, the present study evaluated expression of c-Fos in the NAmb of cats during EA stimulation, specifically concentrating on neurons containing choline acetyltransferase (ChAT) and/ or enkephalin. We hypothesized that EA applied at P5–P6 acupoints increases c-Fos expression in the NAmb. Moreover, we proposed that neurons demonstrating c-Fos activity in this area co-localize with cholinergic (hence parasympathetic preganglionic) neurons as well as with neurons containing enkephalin.

2.

Results

2.1.

Blood pressure and heart rate

We observed nerve fibers but no cell bodies containing enkephalin in the NAmb of the cat in four animals, in which the same surgical procedures as described below were performed except for administration of colchicine. Colchicine enhances the content of enkephalin in perikarya by disrupting microtubular transport, as described in Methods. Thus, to more definitively evaluate for the possibility of perikarya containing enkephalin in the NAmb, all other cats were treated with colchicine. EA at P5–P6 was employed following colchicine treatment, and bilateral baroreceptor denervation and cervical vagotomy. Mean arterial BP (MAP) decreased slightly (5–10 mm Hg) in two of five cats during EA stimulation, whereas MAP in three animals was not altered. MAP was unchanged in each of four control animals. No remarkable changes in HR (≤5 bpm) were observed during EA or control stimulation in any vagotomized cat from either group. These observations are similar to those described earlier (Guo et al., 2008). Similarly, we did not notice any significant change in BP and HR in both EA-treated and control cats without colchicine treatment following placement of acupuncture needles at P5–P6 before or after electrical stimulation.

2.2.

c-Fos immunohistochemical staining in NAmb

Fos immunoreactivity was distributed throughout the rostrocaudal extension of the NAmb in both control and EA-treated cats treated and those not treated with colchicine after bilateral baro-denervation and cervical vagotomy. Compared to the control animals, Fos-labeled neurons were found at multiplelevels throughout the NAmb in the EA-treated cats. Following EA stimulation, relatively more c-Fos immunoreactivity was observed in the ventrolateral region, compared to other parts of the NAmb. Photomicrographs in Fig. 1 demonstrate the distribution of Fos-labeled neurons in the NAmb of a cat in sham-operated control and EA-treated groups. Similar patterns of c-Fos distribution appeared in the NAmb in single c-Fos labeled sections and double-stained sections containing c-Fos and ChAT (see details below) in cats treated and those not treated with colchicine. We quantitatively evaluated

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Fig. 1 – Photomicrographs demonstrating Fos-like immunoreactive cells in the nucleus ambiguus (NAmb; level P 10.8) of the cat treated with colchicine. A, B: low-power photomicrographs showing region of the NAmb indicated by boxes in a control and electroacupuncture (EA)-treated cat, respectively. C, D: magnified regions shown within boxes in A and B. Arrows indicate dots representing c-Fos labeled cells.

c-Fos immunoreactivity in the double-labeled sections of cats treated with colchicine. Compared to controls (n = 4), a significant increase in the number of Fos positive neurons was found in the NAmb of cats treated with EA (n= 5; P < 0.01, Table 1).

2.3.

Double-labeling with c-Fos and ChAT in NAmb

Similar to ChAT staining in cats without colchicine treatment in the present study and in previous studies (Batten, 1995; Loewy and Spyer, 1990), cell bodies labeled with ChAT were observed in the NAmb of animals treated with colchicine. Moreover, their density and distribution pattern in the NAmb were similar in both groups. Importantly, there was no difference in distribution or density of neurons stained with ChAT in the NAmb comparing controls to EA-treated cats following colchicine (Table 1), indicating that EA did not

influence the density of cholinergic neurons. As mentioned above, more c-Fos immunoreactivity was detected in the NAmb of EA-treated cats compared to controls. In particular, neurons were co-labeled with ChAT and c-Fos in the NAmb of EA-treated cats, but not in control animals (Fig. 2; Table 1). These double-labeled neurons were identified more frequently in the rostral than in the caudal portion of NAmb, as shown in Fig. 2. Compared to controls (n = 4), neurons double-labeled with ChAT and c-Fos as well as their number relative to c-Fos positive cells and the total population of neurons containing ChAT were increased significantly (all P < 0.05; Table 1) in the NAmb of EA-treated cats (n = 5). Similar to these observations, in the group that did not receive colchicine, neurons doublelabeled with ChAT and c-Fos were found in the NAmb of cats treated with EA (n = 2), but not in controls (n = 2). Photomicrographs in Figs. 3 and 4 (Panels A–C) provide an example of

Table 1 – C-Fos immunoreactivity and co-location with cholinergic neurons in the nucleus ambiguus of cats.

Control (n = 4) EA-treated (n = 5)

Fos cells (#)

ChAT cells (#)

ChAT + Fos cells (#)

ChAT ± Fos cells

ChAT ± Fos cells

ChAT cells (%)

Fos cells (%)

4±1 20 ± 1**

30 ± 5 33 ± 5

0±0 7 ± 1*

0±0 23 ± 4*

0±0 35 ± 5*

Means ± SE. Average number (#) of c-Fos positive cells, cholinergic neurons and cells co-localized with both stains in the nucleus ambiguus expressed per section. Also shown are percentages (%) of double-labeled neurons to the number of cholinergic neurons or Fos positive cells. ChAT, choline acetyltransferase; EA, electroacupuncture stimulation. * P < 0.05, ** P < 0.01; EA-treated group vs. control group.

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Fig. 2 – Distribution of cholinergic neurons and c-Fos immunoreactivity in NAmb following EA and in a sham-operated control. Four coronal sections (Berman's atlas) were selected from one animal in each experimental group. Each symbol, ●, Δ or + represents one labeled cell with c-Fos, choline acetyltransferase (ChAT) or c-Fos + ChAT, respectively. AMB, nucleus ambiguus; LRN, lateral reticular nucleus; NTS, nucleus tractus solitarius; PT, pyramidal tract. Levels of sections are consistent with those shown in Berman's atlas [Berman, 1968].

co-localization of Fos-like immunoreactive nuclei with perikarya containing ChAT in the NAmb of an EA-treated cat treated and one not treated with colchicine.

2.4.

Double-labeling with ChAT and enkephalin in NAmb

Consistent with our previous findings (Guo et al., 2004, 2008), perikarya containing enkephalin were found in several

medullary nuclei, including the nucleus raphé obscurus, raphé magnus and raphé pallidus, as well as the rostral ventrolateral medulla in both control and EA-treated cats following application of colchicine. Although a few perikarya containing enkephalin were noted in the area adjacent to the NAmb, no enkephalin-containing cell bodies were found within the NAmb (Fig. 5). Conversely, a robust population of neuronal processes that stained positively for enkephalin

Fig. 3 – Confocal microscopic images of neurons double-stained with choline acetyltransferase (ChAT) and c-Fos in NAmb (level P 12.1) of a cat treated with colchicine and EA. A: low-power photomicrograph; B: magnified region shown within box in A. Arrow indicates a neuron double-labeled with c-Fos and ChAT. B is merged image from C and D. Arrows in C and D respectively indicate a neuron containing ChAT and a c-Fos positive nucleus. Scale bars in A and B–D represent 500 and 50 μm, respectively.

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Fig. 4 – Confocal microscopic images showing neurons double-stained with choline acetyltransferase (ChAT) and c-Fos (Panels A–C) or enkephalin (Panels D–F) in NAmb (level P 12.1) of a cat treated with EA, but not colchicine. C and F are merged images from A and B, and D and E, respectively. Arrows in A–C respectively indicate a neuron containing ChAT, a c-Fos positive nucleus and a neuron double-labeled with c-Fos and ChAT. Arrows in D and E respectively indicate cytoplasm of a neuron stained with ChAT and fibers labeled with enkephalin. In F, arrow 1 shows an example of a ChAT-labeled neuron that is in close apposition to neuronal fibers containing enkephalin indicated by arrow 2. Scale bars in A–C and D–F represent 100 and 200 μm, respectively.

was observed in the NAmb. These processes were in a close apposition to perikarya and fibers containing ChAT. These observations were similar to those in cats without colchicine treatment as shown in Fig. 4 (Panels D–F). There was no significant change in the intensity of neuronal processes containing enkephalin after EA, compared to controls. Fig. 5 provides examples of light and confocal NAmb images that demonstrate the distribution of enkephalin-like immunoactivity and its relationship to neurons labeled with ChAT in an EA-treated cat following administration of colchicine.

2.5. Triple-labeling with c-Fos, ChAT and enkephalin in NAmb As described above, we found similar patterns of distribution of neurons labeled with c-Fos, ChAT + c-Fos, ChAT or enkephalin in the NAmb in double and triple labeled sections of cats treated and that not treated with colchicine. Many c-Fos positive nuclei that did not co-localize with ChAT were surrounded or were in close apposition to fibers labeled with enkephalin in both EA-treated and control groups. However, there were more c-Fos nuclei in EA-treated cats, compared to controls, consistent with our findings in double labeled sections. In addition, neurons double-labeled with c-Fos + ChAT were in close proximity to neuronal processes containing

enkephalin in the NAmb of EA-treated cats but not in controls. Photomicrographs in Fig. 6 show a neuron double-labeled with c-Fos and ChAT in very close apposition to enkephalinergic processes in the NAmb of a cat treated with colchicine following EA stimulation.

3.

Discussion

EA at the P5–P6 acupoints overlying the median nerve is commonly used to manage cardiovascular disease (Ho et al., 1999; Longhurst, 2010; Longhurst and Costello, 2011; Richter et al., 1991). Our studies have demonstrated that stimulation of these acupoints reduces myocardial ischemia and sympathoexcitatory pressor reflexes by modulating sympathetic activity (Li et al., 1998, 2004; Tjen-A-Looi et al., 2004). However, it is unknown if EA at P5–P6 acupoints also influences the parasympathetic nervous system. To explore this possibility, the present study utilized c-Fos expression to examine neuronal responses to EA in the NAmb, the principal site of origin of cardiac preganglionic vagal neurons. In this regard, we demonstrated that some (about one third) of the neurons in the NAmb activated by EA were cholinergic. Moreover, vagal preganglionic neurons activated by EA and other c-Fos positive cells (that could not be identified as preganglionic neurons)

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Fig. 5 – A–C: Photomicrographs demonstrating enkephalin labeling in the medulla oblongata (level P 10.8) of the cat treated with colchicine. A: a low-power photomicrograph. Boxes 1 and 2 in A represent nucleus raphé pallidus and NAmb, correspondingly. B, C: magnified regions shown within boxes 1 and 2 within A, respectively. Arrows in B, C indicate enkephalin-containing perikarya and processes, respectively. D–G: confocal microscopic images showing double-labeling with enkephalin and choline acetyltransferase (ChAT) in the NAmb (level P 11.6) following stimulation with EA. D: low-power photomicrograph; E: magnified region shown within box in D. In E, arrow 1 shows an example of a ChAT-labeled neuron that is in close apposition to neuronal fibers containing enkephalin indicated by arrow 2. E is merged image from F and G. Arrows in F and G respectively indicate cytoplasm of a neuron stained with ChAT and fibers labeled with enkephalin. Scale bars in A, B and C, D and E–G represent 1000, 100, 200 and 50 μm, respectively.

were in close apposition to neuronal processes containing enkephalin. As such, the present study provides anatomical data demonstrating activation of a number of NAmb neurons by EA, many of which could be classified as vagal preganglionic neurons, and which likely are influenced by enkephalin. The results imply that EA likely regulates cardiovascular function through its influence on both sympathetic and parasympathetic autonomic nervous systems. Like our and previous studies by others, colchicine was administered to enhance enkephalin labeling in the NAmb (Ceccatelli et al., 1989; Ciriello and Caverson, 1989; Guo et al., 2004, 2008). Many investigators continue to use colchicine to block microtubular transport of neurotransmitters and improve

staining in histological studies (Porteous et al., 2011; Simmons and Yahr, 2011; Stanic et al., 2011). We and others have suggested that the use of colchicine can be problematic in terms of animal wellness and the potential for non-specific influence on gene expression (Gillen and Briski, 1997; Guo et al., 2004). In the present study, the smallest possible dose of colchicine was employed over a very short period (36–48 h) to minimize nonspecific gene expression and side effects (Guo et al., 2004). Importantly, we also included colchicine in the sham-operated control. In the control cat, procedures were replicated with the only difference that acupuncture needles were not stimulated electrically. Thus, over and above control, we believe that the patterns of any changes in NAmb (e.g., an increase in c-Fos) in

Fig. 6 – Confocal microscopic images showing c-Fos immunoreactivity, enkephalin and choline acetyltransferase (ChAT) in NAmb (level P 10.8) in a cat treated with colchicine and EA. Panels A–C show immunostaining of c-Fos (blue), enkephalin (green) and ChAT (red). Panel D demonstrates merged images from Panels A–C. Arrows in Panel A–D show neurons containing c-Fos, enkephalin, ChAT and c-Fos + ChAT in very close apposition to neural processes stained with enkephalin. Scale bars in A–D represent 50 μm.

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this study were likely to be due to stimulation of acupuncture rather than non-specific responses to colchicine. C-Fos, an immediate early gene, is expressed rapidly after the onset of cellular stimulation. Mapping c-Fos expression is widely used to identify neuronal activation in the brain following stimulation of peripheral sensory nerves (Morgan et al., 1987). In this regard, we and other investigators have identified a number of brain regions that are activated during acupuncture by visualizing Fos-like immunoreactivity (Guo and Longhurst, 2007; Guo et al., 2004, 2008; Lee and Beitz, 1993). Bilateral cervical vagotomy and barodenervation were performed to eliminate possible indirect activation of NAmb neurons induced by input from vagal nerves and baroreceptors resulting from changes in blood pressure. In addition, care was also taken to minimize c-Fos expression in the NAmb caused by other non-specific stimuli, such as, anesthesia, surgical procedures, and administration of the smallest possible dose of colchicines as mentioned above (Guo and Longhurst, 2007; Guo et al., 2004). Thus, compared to the sham-operated controls conducted without electrical stimulation, the increase in c-Fos expression in the NAmb in the EA-treated cat was exclusively related to EA stimulation at the P5–P6 acupoints rather than other non-specific stimuli, for example surgery and simulation of baroreflexes. Concern might be raised about accuracy of cell counting in our analysis due to potentially variable sizes of c-Fos nuclei and ChAT-labeled neurons. More accurate cell counting might be obtained with stereological methods to correct the size of cells. However, as noted by Saper (1996), stereological methods of correction are not necessary when immunoreactive c-Fos protein is counted in animals that have received a physiological stimulus when comparing Fos expression in cells of unstimulated animals, since the size of c-Fos containing cells does not contribute to systematic bias and because any error in estimation of Fos counting resulting from a difference in size typically is much smaller than the biological variation. Consistent with this caveat, we found substantially similar sizes of c-Fos nuclei in control and experimental animals as shown in Fig. 1. Saper also noted that stereological methods are not crucial to accurately determining the percentage of a population of cells that are double-labeled. Rather, estimating the percentage of cells expressing c-Fos without analysis of cell size is acceptable. Clearly both conditions apply to our study of c-Fos labeling in conjunction with immunohistochemistry to visualize double labeled cells. As such, the counting methods used in the present study to identify co-localization of c-Fos with one or two neural substances and which has been used by ourselves and others are accurate and can be justified (Chan and Sawchenko, 1994; Guo and Longhurst, 2003; Guo et al., 2008). The present study showed that BP was decreased slightly (5–10 mm Hg) during EA stimulation in two of the five cats. In both cats, relatively more c-Fos expression in the NAmb was found, compared to the three cats that did not show a change in BP following EA stimulation. There was no change in HR (~5 bpm) during EA stimulation. Bilateral cervical vagotomy in the present study eliminated vagal sensory inputs to the brain, but also limited evaluation of the efferent vagal action from the NAmb on cardiovascular function during EA stimulation. Studies from other laboratories have shown

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that depressor responses can be evoked by stimulation of myelinated somatic afferents (Johansson, 1962; Koizumi et al., 1970). As such, a slight decrease in blood pressure was not an unexpected finding. It is unlikely, however, that the small decreases in blood pressure during EA contributed to c-Fos expression in the NAmb since the barodenervation eliminated any secondary baroreflex influence. The NAmb is an important source of vagal preganglionic neurons that modulates autonomic function of many visceral organs, including the heart. In fact, vagal innervation of the heart in the cat originates significantly from the ventrolateral region of the NAmb (Hsieh et al., 1998; McAllen and Spyer, 1976). Thus, excitation of the NAmb decreases HR (Agarwal and Calaresu, 1991; Wang et al., 2001). Although no previous studies have examined the role of the NAmb during acupuncture stimulation, using an anatomical approach, the present study demonstrated an increase in c-Fos expression in this region of cats following EA stimulation at P5–P6, two acupoints on the forelimb that frequently are used to treat cardiovascular disorders. In this study, bilateral vagotomy was conducted in animals to eliminate activation of NAmb neurons by nonspecific stimulation of vagal nerves during EA. Thus, data from this study suggest that there is a strong potential for EA to influence cardiovascular function though an action on preganglionic parasympathetic outflow to the heart. Neurons in the NAmb synthesize a variety of neurotransmitters and neuropeptides (Agarwal and Calaresu, 1991; Wang et al., 2001). ACh is the primary neurotransmitter that excites vagal neurons influencing the heart and other visceral organs (Jordan, 2008; Loewy and Spyer, 1990; Wang et al., 2001). The present study showed that neurons co-labeled with c-Fos and ChAT in the NAmb were present only in EAtreated animals, indicating that some NAmb neurons activated by EA (at the P5–P6 acupoints) are preganglionic vagal neurons. However, many other neurons expressing c-Fos did not contain ChAT, indicating that EA also has the capability of influencing the activity of interneurons in the NAmb that contain neurotransmitters other than acetylcholine. EA's central actions have been tied closely to the opioid system. In this regard, our previous studies have shown that enkephalins and endorphins regulate sympathetic outflow during EA and that EA activates enkephalinergic neurons in multiple brain regions, including the rVLM and raphé nuclei in the medulla and the arcuate in the hypothalamus (Guo and Longhurst, 2007; Guo et al., 2004, 2008). Unlike an earlier study showing cell bodies labeled with a met-enkephalinarg-gly-leu (MERGL) in the NAmb of rats (Fallon and Leslie, 1986), in the present study, only fibers but not perikarya containing met- and leu-enkephalin were observed within this nucleus of cats after treatment with colchicine, used to accentuate cell body expression of enkephalin in other medullary nuclei (Guo et al., 2004, 2008). MERGL is not the same enkephalin peptide we detected in cats in this study. Different animal species also might contribute to different results of staining for enkephalin. In addition, it might have been possible to detect cell bodies containing enkephalin in the NAmb by increasing the dose of colchicine and/or prolonging the survival time of animals after administration of colchicine. However, adverse effects of colchicine as we described previously prevented us from taking those approaches in this

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study (Guo and Longhurst, 2007; Guo et al., 2004). The present study demonstrated that enkephalinergic processes in the NAmb were abundant and were in close proximity to vagal preganglionic as well as interneurons neurons activated by acupuncture. These data do not support the working hypothesis that perikarya containing enkephalin in the NAmb would be activated by EA. Similar to our results, others have identified NAmb enkephalinergic terminals that monosynaptically contact vagal preganglionic neurons including those supplying the heart (Blinder et al., 2005; Milner et al., 1995). Since administration of enkephalin in the NAmb, at least in pharmacological concentrations, causes a naloxone-reversible bradycardia (Agarwal and Calaresu, 1991; Laubie and Schmitt, 1981; Wang et al., 2004), this peptide has the potential to contribute to EA's action on parasympathetic activity in the NAmb. However, further testing of this hypothesis is warranted. In summary, the present study provides the first evidence showing that EA activates NAmb neurons during stimulation of P5–P6 acupoints. Some of activated cells in this area are vagal preganglionic neurons (cholinergic) and appear to have the potential to interact with closely located neuronal processes containing enkephalin. These results imply that EA may influence the NAmb to regulate cardiovascular function, at least in part, through an opioid mechanism.

4.

Experimental procedure

4.1.

Surgical preparation

The minimum possible numbers of adult cats (n = 9, 3–4 kg) of both sexes were used to obtain reproducible and statistically significant results. All procedures were carried out in accordance with the US Society for Neuroscience and the National Institutes of Health guidelines. Surgical and experimental protocols of this study were approved by the animal use and care committee at the University of California, Irvine. Throughout the study, steps were taken to minimize discomfort and suffering of the animals. Sterile surgical procedures were conducted for administration of colchicine in the surgical operating room of the vivarium at the University of California, Irvine. Cats were preanesthetized with ketamine (25 mg/kg, im) and valium (5 mg/ kg, im) and anesthesia was maintained with isoflurane (1–2%, inhalation). The head was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) and flexed approximately 30° forward in the frame. A one-inch midline incision was made from the external occipital protuberance located at the base of the skull. After exposing the foramen magnum near the brainstem, a 27× 1.25-gauge hypodermic needle attached to a 1.0 ml syringe was inserted into the subarachnoid space through the atlanto-occipital membrane overlying the fourth ventricle. We injected colchicine (90–100 μg/kg, Sigma, St. Louis, MO, USA) in 0.08–0.13 ml of solution (3000 μg/ml) dissolved in 0.9% normal saline. The dose of colchicine used in the present study was determined based on our and other previous studies (Ciriello and Caverson, 1989; Guo et al., 2004). Following administration of colchicine, the incision was closed and the cats were allowed to recover. After the 22–24 h post-operative period following administration of colchicine, cats were re-anesthetized with ketamine

(40–50 mg/kg, im) and α-chloralose (50–60 mg/kg, iv). Supplemental α-chloralose (5–10 mg/kg, iv) was applied to maintain an adequate depth of anesthesia as judged by stability of respiration, blood pressure and heart rate and the lack of a withdrawal response to toe pinch. All animals were ventilated artificially through a cuffed endotracheal tube after incubation. A femoral artery and vein were cannulated for measuring BP (Statham P 23 ID, Oxnard, CA, USA) and administrating drugs and fluids, respectively. HR was derived from the arterial pressure pulse with a biotach (Gould Instrument, Cleveland, OH, USA). Arterial blood gases and pH were monitored with a blood gas analyzer (Radiometer, Inc., Model ABL-3, Westlake, OH, USA). They were maintained within normal limits (PO2, 100–150 mm Hg; PCO2, 28–35 mm Hg; pH, 7.35–7.45) by adjusting the tidal volume and/or ventilatory rate, enriching the inspired O2 supply and administration of 1 M NaHCO3. Body temperature was kept at 36–38 °C by a water heating pad and a heat lamp. Cardiovascular hemodynamic changes lead to secondary baro- and cardiopulmonary reflex responses, which can alter c-Fos expression in the brain (Guo and Longhurst, 2003; Potts et al., 1997). To control for input from this secondary activation of neural pathways resulting from EA stimulation at the P5–P6 acupoints (Johansson, 1962; Koizumi et al., 1970; Lee and Beitz, 1993; Li et al., 1998), bilateral sino-aortic denervation and cervical vagotomy were conducted. We isolated and transected the carotid sinus nerves and cervical vagus near the internal and common carotid artery, respectively. Subsequently, the carotid bifurcations were stripped of adventitial tissue and painted with 10% phenol (Sigma; St. Louis, MO). Barodenervation was verified by the absence of a normal decrease in HR in response to increased arterial BP induced by intravenous administration of phenylephrine (10 μg/kg, Gensia Sicor Pharmaceuticals, Irvine, CA, USA). Similar to our previous studies (Guo et al., 2004, 2008), cats were stabilized for 4 h after surgical preparation. Approximately 28–30 h after administration of colchicine, pairs of stainless steel, 32 ga acupuncture needles were inserted bilaterally at the P5–P6 acupoints, overlying the median nerves. The needles then were connected to a constant current stimulator with a stimulus isolation unit and stimulator (Grass, model S88, W. Warwick, RI, USA). The P5–P6 acupoints on both forelimbs of small animals are analogous to those in humans (Hua, 1994).

4.2.

Experimental protocols

Cats were divided randomly into a sham-operated control group (n = 4) and an EA-treated group (n = 5). P5–P6 acupoints are located 1.5–2.0 and 2.5–3.0 cm above the wrist over the median nerve between the ligaments of the flexor carpi radialis and the palmaris longus. The correct location of acupuncture needles in these acupoints was confirmed by observing moderate, repeated paw flexion induced by low frequency EA (0.5 ms pulses, 2 Hz, 1–4 mA) in each forelimb. Subsequently, gallamine triethiodide (4 mg/kg) was administered intravenously in both groups to prevent muscle movement during stimulation of somatic nerves. Our previous studies have demonstrated that low frequency EA applied for 30 min activates neurons in several brain nuclei (e.g., medullary raphé

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and rostral ventrolateral medulla), and attenuates reflex sympathetic responses in anesthetized cats (Guo et al., 2008; Moazzami et al., 2010). Thus, in the present study, EA was applied bilaterally at P5–P6 for 30 min. Each set of electrodes was stimulated separately so that current did not flow from one location to the contralateral side. In control cats, acupuncture needles were placed in P5–P6 acupoints for 30 min without electrical stimulation. This form of stimulation in controls does not modulate sympathetic reflexes and serves as an adequate control for EA (Tjen-A-Looi et al., 2004). In a separate group, EA and or sham EA were conducted in four cats that had not received colchicine (two in each subgroup).

4.3.

Immunohistochemical staining

4.3.1.

Tissue preparation

As described previously (Guo and Longhurst, 2003; Guo et al., 2004), 90 min after termination of EA stimulation or control procedures, deep anesthesia was induced by another larger dose of α-chloralose (100 mg/kg, iv). The animal then was perfused transcardially with 0.9% saline and cold 4% paraformaldehyde in phosphate buffer (PB, pH 7.2). The medulla oblongata was removed and stored in 4% paraformaldehyde for 2 h and subsequently in 30% sucrose for 48 h to prevent ice crystallization. Coronal sections of the brain (30 μm) were collected on a cryostat microtome (Leica CM1850 Heidelberger Strasse, Nussloch, Germany) and placed serially in cold cryoprotectant solution (Chan and Sawchenko, 1994). Brain sections were used for performing immunohistochemical labels as described below, or were stained with Nissl to reveal the cellular architecture (Guo and Longhurst, 2003). In this study, freefloating sections were used for labeling.

4.3.2.

C-Fos immunohistochemical staining

c-Fos protein was stained using the avidin–biotin–peroxidase complex (ABC) method (Guo and Longhurst, 2003). Briefly, after rinsing three times (10 min each) with 0.1 M PB (pH 7.2) containing 0.3% Triton X-100 (PBT), brain sections were placed in 0.5% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. The sections then were placed in 1% normal goat serum (Vector ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 20 min. They were incubated with a primary polyclonal rabbit anti-Fos antibody (Ab-5; 1:20,000 dilution, Oncogene research product, Calbiochem, #PC38, San Diego, CA, USA) at 4 °C for 48 h. This antibody was raised against amino acids 4–17 of human Fos protein and stained the 55 kDa c-Fos protein (manufacturer's technical information). Subsequently, sections were washed in 0.1 M PBT three times and were incubated with biotinylated goat anti-rabbit IgG (Vector Kit, 1:200) for 60 min. Following three rinses in 0.1 M PBT, brain tissue was placed in ABC solution (Vector Kit, 1:50) for 30 min. Sections were washed twice, each for 10 min in 0.1 M PB and were incubated in a solution containing hydrogen peroxide and 3,3′-diaminobenzidine (DAB; Vector laboratory) for 5–8 min. DAB is reduced by hydrogen peroxide in the ABC complex and is deposited in brain tissue as a brown reaction product. The DAB reaction was terminated by rinsing sections in distilled water. Sections were mounted on slides in 0.1 M PB. Slides were allowed to airdry, cleared in alcohol and xylene baths and covered by glass

33

slips with Permount (Fisher Scientific, Fair Lawn, New Jersey, USA). The c-Fos immunoreactivity was visualized as darkbrown staining. In immunohistochemical control studies, all c-Fos staining was abolished when 1 ml of the diluted primary antibody was preincubated with 5 μg of the immunizing peptide corresponding to amino acids 4–17 of human c-Fos (SGFNADYEASSSRC, Oncogene Research Produc, Calbiochem, #PP10). In addition, no labeling was detected when the primary antibody was omitted.

4.3.3. Double-fluorescent immunohistochemical labeling for ChAT + c-Fos or enkephalin After rising three times (10 min each) with phosphate buffered saline containing 0.3% Triton X-100 (PBST, pH= 7.4), brain sections were placed in 1% normal donkey serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) for 1 h and incubated with primary antibodies at 4 °C for 48 h. PBST solution containing the two primary antibodies included a goat anti-ChAT (1:250, Chemicon International, Inc. #AB144P, Temecula, CA, USA) and either a rabbit polyclonal anti-Fos antibody (1:2000 dilution, Oncogene Research Product) or a mouse anti-met- and anti-leu-enkephalin antibody (1:400, Chemicon International, Inc. #MAB350). The goat anti-ChAT antibody was prepared against human placental enzyme. The mouse enkephalin antibody displays about 40% cross-reactivity with Cterminal extended met-enkephalin hexapetides and 7% crossreactivity with the extended heptapeptide (-Arg-Phe-OH) but does not recognize other endogenous peptides. There is no cross-reactivity with beta-endorphin or dynorphin. This antiserum was prepared against leu-enkephalin conjugated to bovine serum albumin (manufacturer's technical information). Sections then were incubated with fluorescein-conjugated donkey anti-goat antibodies and rhodamine-conjugated donkey antirabbit antibodies or anti-mouse antibodies (all 1:100; Jackson Immunoresearch Laboratories, Inc.) in PBST at 4 °C for 24 h. These secondary antibodies raised in the donkey are made for multiple labels. They have minimal cross-reactivity to other nonspecific species (Catalog, specializing in second antibodies, Jackson Immunoresearch Laboratories, Inc., 2010). After washing with phosphate buffered saline (PBS, pH= 7.4) for 30 min (10 min× 3 times), sections were mounted on slides and air dried. The slides were covered with glass slips using mounting medium (Vector Laboratories). Staining in the medulla oblongata produced a pattern of ChAT immunoreactivity identical to the pattern described in previous studies (Batten, 1995; Loewy and Spyer, 1990). In addition, immunohistochemical control studies were performed by omission of the primary or secondary antibodies and by preabsorption with excess metand leu-enkephalin peptide (both 10 μg/ml; Biochem Peninsula Labs. #0537500 and #ZN233, San Carlos, CA) or a synthetic peptide corresponding to amino acids 4–17 of human c-Fos, as mentioned above. No labeling was detected under these conditions.

4.3.4. Triple-fluorescent immunohistochemical labeling of c-Fos, enkephalin and ChAT The staining procedures were similar to those used for doublefluorescent immunohistochemical labeling described above. Briefly, after treating with PBST and 1% normal donkey serum, brain sections were incubated with the three primary antibodies, i.e., a goat anti-ChAT, a mouse anti-enkephalin

34

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and a rabbit anti-c-Fos antibody (1:1000 dilution) for 48 h at 4 °C. Sections then were incubated with rhodamineconjugated anti-goat, fluorescein-conjugated anti-mouse and coumarin-conjugated anti-rabbit antibodies (all 1:100; Jackson Immunoresearch Laboratories, Inc.) at 4 °C for 24 h. The sections were mounted on slides and covered by glass slips with mounting medium. No staining was detected when the corresponding primary or secondary antibody was omitted during immunohistochemical control studies.

4.4.

Data analysis

Brain sections were scanned and examined with a light and fluorescent microscope (Nikon, E400, Melville, NY, USA). Three epi-fluorescence filters (B-2A, G-2A, or UV-2A) equipped in a fluorescent microscope were used to identify single stains appearing as green (fluorescein), red (rhodamine) or blue (coumarin) in brain sections. Two or three single fluorescent images were captured with a Spot digital camera (RT color v3.0, Spot Diagnostic Instruments, Inc., Sterling Heights, MI, USA) from the same site of the brain section. The images were merged to identify double- or triple-labeled markers using the software provided with the Spot digital camera (Guo and Longhurst, 2006, 2007; Guo et al., 2005). As shown in Figs. 3–5, c-Fos labeling appeared as round dots approximately 7–12 μm in diameter, which at 40x magnification were obviously distinguishable from background staining. Cholinergic neurons were demonstrated as perikarya labeled with ChAT (30–40 μm). Co-localization of c-Fos and ChAT was identified if a c-Fos nucleus was surrounded by cytoplasm stained with ChAT in the same neuron. The co-labeled cells were further confirmed with a laser scanning confocal microscope as described below. In every animal, two sections (not adjacent) were selected for each of four representative planes of the medulla oblongata, which closely matched the standard stereotaxic planes of Berman's atlas (P 14.7, P 13.5, P 12.1, P 10.8; Berman, 1968; Fig. 2). The numbers of single-, or doublelabeled cells in the same single section were counted bilaterally in each animal. The average number of labeled neurons in the four representative levels taken within the rostro-caudal extension of the NAmb (Fig. 2) was obtained by dividing the total number of neurons by eight, representing the number of sections used for cell counting (Guo and Longhurst, 2003; Guo et al., 2004). To confirm co-localization of two or three labels in the same neuron, selected sections that had been used for cell counting with a fluorescent microscope were evaluated further with a laser scanning confocal microscope (Zeiss LSM 710, Meta system, Thornwood, NY, USA). This apparatus was equipped with HeNe and Argon lasers and allowed operation of multiple channels. Lasers of 488 and 543 nm wavelengths were used to excite fluorescein (green) and rhodamine (red), respectively. A 790 nm laser was applied for two-photon excitation of coumarin (blue). Each confocal section analyzed was limited to 0.5 μm thickness in the Z-plane. Digital images of the labels were captured and analyzed with software (Zeiss LSM) provided with this microscope. Images in two or three colors in the same plane were merged to reveal the relationship between two or three labels (Figs. 3, 5 and 6). Single-, double- and triple-labeled neurons were evaluated.

4.4.1.

Statistical analysis

All statistical analyses were conducted with statistical software (SigmaStat, Version 3.0, Jandel Scientific Software, San Rafael, CA, USA). The Kolmogorov–Smirnoff test was used to determine if data were normally distributed. Comparisons between two groups were analyzed with the Student's t-test or Mann–Whitney Rank Sum Test. Values were considered to be significantly different when P < 0.05. Data are expressed as means ± SE.

Acknowledgments This study was supported by the National Heart, Lung, and Blood Institute Grant, HL-072125 and HL-63313, the Larry K. Dodge and Susan-Samueli Endowed Chairs (JC Longhurst).

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