Experimental Gerontology 63 (2015) 76–80

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A preliminary study on cerebellar acetylcholine-mediated blood pressure regulation in young and old rats Qingfeng Zhu a,1, Peiling Zhou b,1, Shengnan Wang a, Changzheng Zhang a,⁎, Tianmiao Hua b,⁎⁎ a b

School of Life Sciences, Anqing Normal University, Anqing, Anhui 246011, China School of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China

a r t i c l e

i n f o

Article history: Received 23 September 2014 Received in revised form 5 February 2015 Accepted 6 February 2015 Available online 8 February 2015 Section Editor: Christian Humpel Keywords: Acetylcholine Aging Blood pressure Cerebellar cortex

a b s t r a c t Cholinergic innervation of the cerebellar cortex can modulate cerebellar functions. However, the role of acetylcholine (ACh) in cerebellar regulation of blood pressure (BP) has not been examined. Moreover, the effect of aging on this regulation remains unexplored. In this study, carotid arterial pressure was measured in young (2 months) and old (16–20 months) rats after ACh was microinjected into the cerebellar cortex. The results show a concentration-dependent effect of ACh (30, 100, and 300 mM) on mean arterial pressure (MAP), maximal decreased MAP (MDMAP) and reactive time (all Ps b 0.05). However, responses in the old animals were significantly attenuated compared to the young (Ps b 0.05). The results indicate that cerebellar cholinergic innervation exerts depressor effects on BP regulation, and such effects undergo retrogression with aging. Further studies concerning the mechanism are needed to confirm these preliminary findings. © 2015 Elsevier Inc. All rights reserved.

1. Introduction In addition to its role in the execution of fine movements, balance, and motor learning (Schweighofer et al., 2004; Ito, 2012; Heiney et al., 2014), the cerebellum is involved in a multitude of non-motoric functions, including cardiovascular regulation (Nisimaru, 2004; Demirtas-Tatlidede et al., 2011). Despite a great amount of data concerning fastigial pressor responses (Miura and Reis, 1969; Nisimaru, 2004; Rector et al., 2006), cerebellar cortex-mediated blood pressure (BP) modulation has received much less attention. The cerebellar cortex is innervated by cholinergic fibers, which signal through several subtypes of cholinergic receptors (Jaarsma et al., 1997; Schweighofer et al., 2004). This cholinergic innervation is known to modulate neural and behavioral plasticity (Schweighofer et al., 2004; Prestori et al., 2013; Rinaldo and Hansel, 2013), though the effects on BP regulation are still unclear. The cerebellum undergoes significant morphologic and functional alterations with age, including neuronal loss, structural degeneration, and dysregulation of neurotransmitter systems and neuronal firing, which result in many kinds of cerebellum-dependent behavioral dysfunctions (Zhang et al., 2006, 2010; Kennard et al., 2013). Whether ⁎ Correspondence to: C. Zhang, School of Life Sciences, Anqing Normal University, 128 South Linghu Road, Anqing, Anhui 246011, China. ⁎⁎ Correspondence to: T. Hua, School of Life Sciences, Anhui Normal University, 1 East Beijing Road, Wuhu, Anhui 241000, China. E-mail addresses: [email protected] (C. Zhang), [email protected] (T. Hua). 1 Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.exger.2015.02.003 0531-5565/© 2015 Elsevier Inc. All rights reserved.

cerebellar acetylcholine (ACh)-mediated BP regulation also undergoes age-related changes is unknown. This study was designed to determine whether ACh signaling in the cerebellar cortex is involved in BP regulation, and if such regulation changes with age. 2. Materials and methods 2.1. Animals Male young-adult (2-months-old, 230–260 g; n = 26) and old (16–20-months-old, 500–600 g; n = 14) Sprague–Dawley rats were used in this study. Animals were individually housed in a temperaturecontrolled (23 ± 1 °C) environment with a 12/12 h light/dark-cycle and ad libitum food and water. Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996) and approved by our university's Animal Care and Use Committee. 2.2. Surgical procedures and microinjections Rats were anesthetized (800 mg urethane and 60 mg α-chloralose/ kg body weight; ip) and underwent cervical surgery with tracheal intubation. Rectal temperature was monitored and body temperature was maintained at 37.0 ± 0.5 °C by means of an electrical heating pad, and supplemental anesthetic was applied when necessary. A catheter (BL2020; Taimeng Sci-Tec Co., Ltd., Chongqing, China) was inserted into the left carotid artery that was filled with normal saline containing

Q. Zhu et al. / Experimental Gerontology 63 (2015) 76–80

heparin (500 IU/mL) and was connected to a signal collecting and processing apparatus (BL-420F; Taimeng Sci-Tec Co., Ltd.) through a BP transducer. Rats were then mounted on a stereotaxic instrument (51503 New Standard Stereotaxic; Stoelting Co., Wood Dale, IL, USA), and a craniotomy was performed under aseptic conditions. A Hamilton syringe needle (0.3 mm inner diameter, 0.5 mm outer diameter) was placed into the granular layer of cerebellar vermian lobule VI (x: − 10.8 to − 11.8; y: 0.0–1.6; z: 1.8–2.0; Paxinos and Watson, 2007). Once the BP was stable, microinjections (0.5 μL/5 s) of ACh (30, 100, and 300 mM; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) or normal saline (0.9% NaCl) were administered at 30-min intervals to avoid interference between administrations, and at sites that were separated by at least 1 mm. It has been shown that a volume of 0.5 μL has an estimated diffusion range of 0.5–1.0 mm in the brain tissue (Zhang et al., 2014), and such dosage of ACh is restricted to the cerebellar cortex, and induces sufficient effects on BP modulation at all the concentrations in both young and old rats. For comparison, an additional group of young-adult rats (n = 10) received microinjections of ACh (100 mM) into lobule VII (x: −13.4; y: 0.0–1.0; z: 3.4), which has cholinergic projections and is involved in cardiovascular control (Nisimaru, 2004; Lan et al., 1995), and the interpositus nucleus (x: −11.5; y: 2.0–2.6; z: 6.0), which has cholinergic innervation but is unrelated to BP modulation (Jaarsma et al., 1997; Nisimaru, 2004).

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2.4. Statistical analysis A Student's t-test and one-way analysis of variance followed by a Fisher's least significant difference post hoc test were employed for statistical analyses. All data are presented as the mean ± standard error, and P b 0.05 was considered as significant. 3. Results 3.1. Microinjection of ACh into cerebellar cortex regulates BP in young rats Before microinjections, 16 BP values were measured from 16 young rats. The MAP ranged from 81.31 to 121.54 mm Hg, with an average of 103.49 ± 9.96 mm Hg, in line with other reports (Sei et al., 2002). The MAP was maintained at 103.20 ± 10.67 mm Hg after microinjection of saline into the cerebellar cortex, whereas a decrease was observed after microinjection of ACh (Fig. 1A). Furthermore, a concentrationdependent effect of ACh microinjection was observed on the MAP (F3,70 = 65.633; P b 0.001), MDMAP (F2,55 = 86.867; P b 0.001), and the reactive time (F2,55 = 139.343; P b 0.001) (Fig. 1B–D). In addition, a BP modulatory effect was also observed from microinjections of ACh into lobule VII (P b 0.01; Fig. 2A,B), but not the interpositus nucleus (Fig. 2C,D), indicating that the ACh-mediated modulatory effect on BP is specific to cardiovascular-related regions. 3.2. Microinjection of ACh into cerebellar cortex regulates BP in old rats

2.3. BP measurements The reactive time (duration required for BP to return to basal values), mean arterial pressure (MAP) during the reactive time, and maximal decreased MAP (MDMAP) in each ACh trial were recorded. As there was no alteration in BP with saline, MAP for this treatment was calculated from a 60-s BP sequence beginning 10 s after saline microinjection. The percentage changes in the MAP and in the MDMAP response were calculated with respect to the basal BP.

Before microinjections, 14 BP values were obtained from 14 old rats. The MAP ranged from 80.71 to 132.97 mm Hg, with an average of 113.38 ± 15.56 mm Hg. The MAP was maintained at 110.33 ± 9.34 mm Hg after saline was microinjected into the cerebellar cortex, but showed decreases after ACh microinjections (Fig. 3A). A concentration-dependent effect of ACh microinjection was also observed on the MAP (F3,57 = 21.319; P b 0.01), MDMAP (F2,44 = 31.866; P b 0.001), and the reactive time (F2,44 = 58.984; P b 0.001) (Fig. 3B–D).

Fig. 1. Blood pressure changes after microinjection of acetylcholine (ACh) into the cerebellar lobule VI of young rats. (A) Representative blood pressure recordings; gray line = raw arterial pressure, black line = mean arterial pressure (MAP); arrows indicate time of injection. (B) MAP, (C) Maximal decreased MAP (MDMAP), and (D) reactive time (duration required for BP to return to basal values) in response to ACh stimulations. Note, as there was no decrease in blood pressure with saline, this group was not included in statistical analyses in panels C and D. Numbers in parentheses denote the number of animals measured in each group; **P b 0.01.

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Fig. 2. Blood pressure changes after microinjection of acetylcholine (ACh) into cerebellar lobule VII and the interpositus nucleus in young rats. (A) Representative blood pressure recordings and (B) average mean arterial pressure (MAP) after ACh microinjection into lobule VII. (C) Representative blood pressure recordings and (D) average MAP after ACh microinjection into the interpositus nucleus. Numbers in parentheses denote the number of animals measured in each group. Gray lines = raw arterial pressure, black lines = MAP; arrows indicate time of injection; **P b 0.01; and n.s. = not significant.

3.3. Effect of age on cerebellar ACh-mediated BP regulation Compared with the young group, the old rats showed 8.72% higher BPs before drug administration. However, the effects of ACh

microinjection in old rats were less robust than those observed in the young group. There were significant differences between the old and young groups in rates of ACh-mediated MAP decrease, MDMAP increase, and in total reactive time at all doses tested (all Ps b 0.05) (Table 1).

Fig. 3. Blood pressure changes after microinjection of acetylcholine (ACh) into the cerebellar cortex of old rats. (A) Representative blood pressure recordings; gray line = raw arterial pressure, black line = mean arterial pressure (MAP); arrows indicate time of injection. (B) MAP, (C) Maximal decreased MAP (MDMAP), and (D) reactive time (duration required for BP to return to basal values) in response to ACh stimulations. Note, as there was no decrease in the blood pressure with saline, this group was not included in statistical analyses in panels C and D. Numbers in parentheses denote the number of animals measured in each group; *P b 0.05 and **P b 0.01.

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Table 1 Blood pressure modulation due to acetylcholine (ACh) in cerebellar cortex. Measure

MAP decrease (%) MDMAP (%) Reactive time (s)

30 mM ACh

100 mM ACh

300 mM ACh

Young

Old

Young

Old

Young

Old

17.48 ± 8.22 15.51 ± 2.82 47.61 ± 16.09

12.31 ± 5.27⁎ 10.85 ± 2.93⁎⁎ 34.56 ± 10.61⁎⁎

29.06 ± 8.70 33.98 ± 10.58 102.72 ± 20.80

19.46 ± 13.51⁎⁎ 22.54 ± 6.99⁎⁎ 75.79 ± 20.19⁎⁎

50.16 ± 11.35 51.78 ± 8.74 186.06 ± 35.46

28.71 ± 7.95⁎⁎ 34.06 ± 12.68⁎⁎ 135.96 ± 41.76⁎⁎

Abbreviations: MAP, mean arterial pressure; MDMAP, maximal decreased MAP. Note: MDMAP (%) represents the percentage change in MDMAP response with respect to the basal BP. Data are expressed as mean ± standard error. ⁎ P b 0.05. ⁎⁎ P b 0.01 vs. young rat.

4. Discussion Cholinergic projections into the cerebellar cortex, though at a lower density in comparison to other brain regions, originate predominantly from the lower brainstem (Lan et al., 1995; Schweighofer et al., 2004), and may be accompanied by mossy fibers that use ACh as a cotransmitter (Jaarsma et al., 1997). The cerebellar cortex also shows expression of acetyltransferase and several types of cholinergic receptors, with particularly high expression in the mossy fibers, Purkinje cells, and granular cells (Lan et al., 1995; Schweighofer et al., 2004). It has been shown in previous investigations that ACh can excite cerebellar neurons (Schweighofer et al., 2004), and it has been suggested that ACh input contributes to cerebellar control of movement (Schweighofer et al., 2004). The current study indicates that cholinergic signaling in the cerebellar cortex influences BP regulation and, more importantly, that this regulation is modulated by age. The present study shows that microinjection of ACh into the cerebellar cortex induces a dose-dependent decrease in BP, which is reversible and reproducible, indicating that ACh exerts a substance-specific role on BP modulation via cerebellar cortex activity. Cholinergic effects occur by activation of ligand-gated nicotinic and G-protein-coupled muscarinic ACh receptors. Four different alpha and two beta nicotinic subunits are expressed in the cerebellar Purkinje and granule cells, as well as in the mossy fibers (Jaarsma et al., 1997; Schweighofer et al., 2004), and at least two subtypes of muscarinic receptors (M2 and M3) have been found, principally in Purkinje and granular cells (Takayasu et al., 2003; Rinaldo and Hansel, 2013). These receptors are involved in modulating synaptic plasticity; for example, α7-nicotinic ACh receptors in the mossy fiber terminals and granule cell dendrites contribute to regulating the balance of long-term depression/potentiation in the cerebellar cortex (Prestori et al., 2013). Furthermore, muscarinic receptor activation increases the firing frequency of granule cells and the frequency of excitatory postsynaptic currents in Purkinje cells (Takayasu et al., 2003), which contributes to long-term potentiation at parallel fiber– Purkinje cell synapses (Rinaldo and Hansel, 2013), and cerebellumrelated behavioral adaptation (Prestori et al., 2013). In this study, we find that ACh microinjected into lobule VI induces dose-dependent decreases in BP. This effect might be specific to lobule VI as the modulatory effect was also observed with microinjections of ACh into another cardiovascular-related area (lobule VII), in contrast, microinjections into the interpositus nucleus, which contains cholinergic innervation that does not regulate BP (Jaarsma et al., 1997; Nisimaru, 2004), had no effect. However, the precise pathway for lobule VI involvement in cardiovascular control remains largely unclear. As far as the present study is concerned, this lobule is innervated by vagal afferents and acetylcholinesterase (AChE)-positive fibers (Perrin and Crousillat, 1985; Lan et al., 1995; Jaarsma et al., 1997), and contains several types of ACh receptors (Jaarsma et al., 1997; De Filippi et al., 2005). Remarkably, the principal output of this lobule is to the fastigial nucleus (Ito, 2012), which is involved in the baroreceptor reflex (Nisimaru, 2004). Therefore, we speculate that cholinergic signaling in lobule VI likely influences cardiovascular control through the fastigial nucleus and baroreceptor reflex involvement. Consequently, any degeneration

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A preliminary study on cerebellar acetylcholine-mediated blood pressure regulation in young and old rats.

Cholinergic innervation of the cerebellar cortex can modulate cerebellar functions. However, the role of acetylcholine (ACh) in cerebellar regulation ...
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