Effect of electroacupuncture on the activity of corticotrophin-releasing hormone neurons in the hypothalamus and amygdala in rats exposed to restraint water-immersion stress.

OBJECTIVE: To investigate the effects of electroacupuncture (EA) treatment on gastric mucosal lesions and the activity of corticotrophin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus and the central nucleus of the amygdala (CNA) in a rat model of restraint water-immersion stress (RWIS). METHODS: 24 male Wistar rats were randomly divided into three groups: normal, RWIS, and RWIS+EA (n=8 per group). Rats in the RWIS group and RWIS+EA group received RWIS for 3 hours. For rats in the RWIS+EA group, EA was applied at ST36 in the bilateral hind legs for 30 min before RWIS. Rats in the normal group did not receive stressors or EA treatment. The gastric mucosal lesions of each rat were evaluated by the erosion index (EI) according to the methods of Guth. The activity of CRH neurons in the PVN and CNA was measured by a dual immunohistochemical test for Fos and CRH in the brain sections. RESULTS: RWIS induced serious gastric mucosal lesions. The mean gastric EI was significantly decreased in the RWIS+EA group versus the RWIS group (P=0.005). Stress induced significant activation of CRH neurons in the PVN and CNA compared with the normal group (P<0.001 for both). The mean number of Fos+CRH immunoreactive neurons in the PVN and CNA were both decreased inRWIS+EA versusRWIS groups (P<0.001 and P=0.001). CONCLUSIONS: EA at ST36 can ameliorate RWIS-induced gastric mucosal lesions and suppress the Fos expression of CRH neurons in the PVN and CNA, suggesting a potentially therapeutic role for EA in stress-related gastric disorders.

Altered Neuronal Activity in the Central Nucleus of the Amygdala Induced by Restraint Water-Immersion Stress in Rats.

Restraint water-immersion stress (RWIS), a compound stress model, has been widely used to induce acute gastric ulceration in rats. A wealth of evidence suggests that the central nucleus of the amygdala (CEA) is a focal region for mediating the biological response to stress. Different stressors induce distinct alterations of neuronal activity in the CEA; however, few studies have reported the characteristics of CEA neuronal activity induced by RWIS. Therefore, we explored this issue using immunohistochemistry and in vivo extracellular single-unit recording. Our results showed that RWIS and restraint stress (RS) differentially changed the c-Fos expression and firing properties of neurons in the medial CEA. In addition, RWIS, but not RS, induced the activation of corticotropin-releasing hormone neurons in the CEA. These findings suggested that specific neuronal activation in the CEA is involved in the formation of RWIS-induced gastric ulcers. This study also provides a possible theoretical explanation for the different gastric dysfunctions induced by different stressors.

Ventromedial hypothalamic nucleus in regulation of stress-induced gastric mucosal injury in rats.

Previous studies showed that restraint water-immersion stress (RWIS) increases the expression of Fos protein in the ventromedial hypothalamic nucleus (VMH), indicating the VMH involving in the stress-induced gastric mucosal injury (SGMI). The present study was designed to investigate its possible neuro-regulatory mechanisms in rats receiving either VMH lesions or sham surgery. The model for SGMI was developed by restraint and water (21 ± 1 °C) immersion for 2 h. Gastric mucosal injury index, gastric motility, gastric acid secretion and Fos expression in the hypothalamus and brainstem were examined on the 15th postoperative day in RWIS rats. Gastric mucosal injury in VMH-lesioned rats was obviously aggravated compared to the control. Gastric acidity under RWIS was obviously higher in VMH-lesioned rats than that in sham rats. Meantime, the VMH-lesioned rats exhibited marked increases in the amplitude of gastric motility in the VMH lesions group after RWIS. In VMH-lesioned rats, Fos expression significantly increased in the dorsal motor nucleus of the vagus (DMV), the nucleus of the solitary tract (NTS), the area postrema (AP), the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) in response to RWIS. These results indicate that VMH lesions can aggravate the stress-induced gastric mucosal injury through the VMH-dorsal vagal complex (DVC)-vagal nerve pathway.

Effects of electrical stimulation at different locations in the central nucleus of amygdala on gastric motility and spike activity.

The aim of the study was to determine the effects of electrical stimulation of different locations in the central nucleus of amygdala (CNA) on gastric motility and spike activity in dorsal vagal complex. Gastric motility index (GMI) and firing rate (FR) of dorsal vagal complex neurons were measured in adult Wistar rats respectively. Neuronal spikes in dorsal vagal complex (DVC) were recorded extracellularly with single-barrel glass microelectrodes. Each type of responses elicited by electrical stimulation in medial (CEM) and lateral (CEL) subdivisions of CNA were recorded, respectively. GMI was significantly increased after stimulation of CEM (p<0.01), and significantly decreased in response to CEL stimulation (p<0.01). After stimulation of CEM, FR in medial nucleus of the solitary tract (mNST) decreased by 31.6 % (p<0.01) and that in dorsal motor nucleus of the vagus (DMNV) increased by 27.1 % (p<0.01). On the contrary, FR in mNST increased (p<0.01) and that in DMNV decreased in response to CEL stimulation (p<0.05). In conclusions, our findings indicated that different loci of CNA may mediate differential effects on gastric activity via changes in the firing of brainstem neurons controlling gut activity.

Arginine Vasopressin Injected into the Dorsal Motor Nucleus of the Vagus Inhibits Gastric Motility in Rats.

Background. Until now, the effect of arginine vasopressin (AVP) in the DMV on gastric motility and the possible modulating pathway between the DMV and the gastrointestinal system remain poorly understood. Objectives. We aimed to explore the role of AVP in the DMV in regulating gastric motility and the possible central and peripheral pathways. Material and Methods. Firstly, we microinjected different doses of AVP into the DMV and investigated its effects on gastric motility in rats. Then, the possible central and peripheral pathways that regulate gastric motility were also discussed by microinjecting SR49059 (a specific AVP receptor antagonist) into the DMV and intravenous injection of hexamethonium (a specific neuronal nicotinic cholinergic receptor antagonist) before AVP microinjection. Results. Following microinjection of AVP (180 pmol and 18 pmol) into the DMV, the gastric motility (including total amplitude, total duration, and motility index of gastric contraction) was significantly inhibited (P < 0.05). Moreover, the inhibitory effect of AVP (180 pmol) on gastric motility could be blocked completely by both SR49059 (320 pmol) and hexamethonium (8 µmol). Conclusions. It is concluded that AVP inhibits the gastric motility by acting on the specific AVP receptor in the DMV, with the potential involvement of the parasympathetic preganglionic cholinergic fibers.

Microinjection of l-glutamate into the nucleus ambiguus partially inhibits gastric motility through the NMDA receptor - nitric oxide pathway.

We have previously reported that both l-glutamate (l-Glu) and nitric oxide (NO) modulate gastric motility in the nucleus ambiguus (NA). The aim of this study is to explore the potential correlation between the l-Glu and NO. A latex balloon connected to a pressure transducer was inserted into the pylorus through the fundus of anesthetized male Wistar rats to continuously record changes in gastric smooth muscle contractile curves. Pretreatment with the NO-synthase inhibitor N-nitro-l-arginine methylester (l-NAME) did not completely abolish the inhibitory effect of l-Glu on gastric motility, but intravenous injection of the ganglionic blocker hexamethonium bromide (Hb) did. By using a specific N-methyl-d-aspartic acid (NMDA) receptor antagonist, we blocked the inhibitory effect of the NO-donor sodium nitroprusside (SNP) on gastric motility. These results suggest that microinjections of l-Glu into the NA inhibits gastric motility by activating the cholinergic preganglionic neurons, partially through the NMDA receptor - NO pathway.

Surveillance and molecular characterization of Newcastle disease virus in seafowl from coastal areas of China in 2011.

Four Newcastle disease virus (NDV) isolates were obtained from 997 fecal and tissue samples were collected in 2011 from seafowl that included seagull, sea duck, and swan from the coastal areas of Guangdong, Jiangsu, and Shandong in China. These isolates (SD1, SD2, GD1, and JS1) were characterized for their pathogenicity according to their mean death time, intracerebral pathogenicity index and intravenous pathogenicity index. Full-length fusion protein genes containing the cleavage site were sequenced, and amino-acid sequences around the cleavage site were deduced. One isolate (SD2) was virulent to poultry as indicated by its mean death time, intracerebral pathogenicity index, and fusion gene cleavage site sequence, which was specific for virulent NDV ((112)R-R-Q-K-R-F(117)). The phylogenic analysis indicated that three of the isolates (SD1, GD1, and JS1) belonged to genotype II and the virulent isolate (SD2) belonged to genotype VIId. These findings suggest that some seafowl NDVs in the coastal areas of China have different virulences and molecular characterizations, and these NDVs have some similarity with vaccine- or poultry-adapted isolates.

[Changes of local field potentials in M1 underlying the specific behavior in rat].

The local field potentials (LFPs) underlying specific behavior were recorded and analyzed in this paper from primary motor cortex (M1) with several medium, such as the self-made single channel micro-electrodes, the system of multi-channels physiological signal acquisition and processing and so on. During the experiment, the specific behavior was divided into four periods according to the changes of the recorded LFPs and the changes of the specific behavior recorded simultaneously in rats. The four periods were named prophase of catching period, planning period, catching period and the completion period, respectively. Then several methods were used for the analysis of the LFPs by MATLAB, such as time domain analysis, power spectral distribution analysis and time-frequency analysis. The results suggested that the LFPs which were caused by different behavior from a large number of movement-related neurons of M1 during the specific behavior in the process of catching play an important part in the "code" guiding role in rats. The results demonstrat that the LFPs of M1 may provide a feasibility to discriminate the motor behavior of forelimb.

Genetic and biological characterizations of a Newcastle disease virus from swine in China.

BACKGROUND: Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics. FINDINGS: Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes. CONCLUSIONS: These above data suggested that the swine virus was NDV and it might be generated from La Sota.

Oxytocin and vasopressin involved in restraint water-immersion stress mediated by oxytocin receptor and vasopressin 1b receptor in rat brain.

AIMS: Vasopressin (AVP) and oxytocin (OT) are considered to be related to gastric functions and the regulation of stress response. The present study was to study the role of vasopressinergic and oxytocinergic neurons during the restraint water-immersion stress. METHODS: Ten male Wistar rats were divided into two groups, control and RWIS for 1h. The brain sections were treated with a dual immunohistochemistry of Fos and oxytocin (OT) or vasopressin (AVP) or OT receptor or AVP 1b receptor (V(1b)R). RESULTS: (1) Fos-immunoreactive (Fos-IR) neurons dramatically increased in the hypothalamic paraventricular nucleus (PVN), the supraoptic nucleus (SON), the nucleus of solitary tract (NTS) and motor nucleus of the vagus (DMV) in the RWIS rats; (2) OT-immunoreactive (OT-IR) neurons were mainly observed in the medial magnocellular part of the PVN and the dorsal portion of the SON, while AVP-immunoreactive (AVP-IR) neurons mainly distributed in the magnocellular part of the PVN and the ventral portion of the SON. In the RWIS rats, Fos-IR neurons were identified in 31% of OT-IR neurons and 40% of AVP-IR neurons in the PVN, while in the SON it represented 28%, 53% respectively; (3) V(1b)R-IR and OTR-IR neurons occupied all portions of the NTS and DMV. In the RWIS rats, more than 10% of OTR-IR and V(1b)R-IR neurons were activated in the DMV, while lower ratio in the NTS. CONCLUSION: RWIS activates both oxytocinergic and vasopressinergic neurons in the PVN and SON, which may project to the NTS or DMV mediating the activity of the neurons by OTR and V(1b)R.

Studies on functional connections between the supraoptic nucleus and the stomach in rats.

The present study was to investigate whether there are functional connections between the hypothalamic supraoptic nucleus (SON) and the stomach, which is the case with the paraventricular nucleus. The rats were divided into four groups. Group I: the neuronal discharge was recorded extracellularly in the NTS, DMV or SON before and after cold physiological saline (4°C) was perfused into the stomach and effused from the duodenum. Group II: the rats were stimulated as for Group I and c-Fos expression in NTS, DMV and SON was examined. Group III: the control to Group II. Group IV: gastric motility was recorded continuously before and after microinjection of L: -Glu into the SON. In Group I, the discharge frequency increased in all the three nuclei, while in Group II, Fos expression in NTS, DMV and SON was, respectively, greater than that of Group III. In Group IV, microinjection of L: -Glu (5 nmol) into SON significantly inhibited gastric motility. These data suggest there are functional connections between SON and stomach.

The role of catecholaminergic neurons in the hypothalamus and medullary visceral zone in response to restraint water-immersion stress in rats.

The activity of catecholaminergic neurons in the hypothalamus and the medullary visceral zone (MVZ) in rats in response to restraint water-immersion stress (RWIS) was measured by use of dual Fos and tyrosine hydroxylase (TH) immunohistochemistry. In RWIS rats Fos immunoreactive (Fos-IR) nuclei dramatically increased in the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the dorsal motor nucleus of the vagus (DMV), the nucleus of the solitary tract (NTS), the area postrema (AP), and the ventrolateral medulla (VLM). A small number of TH-immunoreactive (TH-IR) and Fos/TH double-labeling neurons in the PVN, and their absence from the SON, were observed in both RWIS and nonstressed rats. More TH-IR neurons were observed in the MVZ of RWIS rats than in nonstressed rats. In RWIS and nonstressed rats, the percentage of Fos-IR nuclei in TH-IR neurons was 38.0 and 14.3% in the DMV, 34.4 and 9.7% in the NTS, 18.6 and 4.5% in the AP, and 45.7 and 18.9% in the VLM, respectively. In conclusion, catecholaminergic neurons in the MVZ are involved in the response to RWIS; although the PVN and SON also participate in the response to RWIS, the mechanism is not via catecholaminergic neurons.

A mouse model to study infection against porcine circovirus type 2: viral distribution and lesions in mouse.

BACKGROUND: Little information is known about viral distribution and transmission of porcine circovirus type 2 (PCV2) in species other than swine. It is still a debated topic whether the PCV2 could be infected and caused clinical lesions. Our study is aimed to estimate the susceptibility of Kunming mouse to PCV2. Forty-eight, 6-week-old Kunming mice were randomly divided into four groups. Group A (C1-C12) was inoculated with PK-15 cell culture as a control group. Group B (sPCV1-12) was inoculated orally and intramuscularly with PCV2 (10(6.2)TCID50/ml). Group C (mPCV1-12) was inoculated orally and intramuscularly with PCV2 (10(6.2)TCID50/ml) and a booster inoculation at days 14 and 28 after the first inoculation. Group D (MixPCV1-12) was unvaccinated but released into Group C. Each group was sacrificed at 7, 14, 28, and 42 days post-inoculation, respectively. Necropsy was checked on every mouse. Sera samples were collected for the test of PCV2 specific antibody. Tissues were collected for histopathology study and polymerase chain reaction (PCR). RESULTS: The results showed that viral replication, seroconversion, and microscopic lesions were found in inoculated mice. Continuous existence of PCV2 viruses in lymph nodes have been confirmed by PCR, which took at least seven days for the virus to be transferred into other organs from the primary interface, and the diffusion to thymus had been retarded for seven days. Special PCV2 antibody could be found in PCV2 inoculation mice and was significantly higher than that in the control. Further more, microscopic lesions and the main target of PCV2 focused in the lymph nodes with a characteristic depletion and occasional necrosis of lymphocytes in the cortex and paracortex were found in inoculated mice. CONCLUSIONS: The Kunming mouse could be infected by PCV2 virus and used as a PCV2 infected experimental model.

[Effects of L-glutamate microinjection into the ventral horn of spinal cord gray matter on gastrocnemius contractility in toad].

A diagram of motor neuron pool of ventral horn of spinal cord gray matter in toad was first delineated. Different concentrations (1, 0.5, 0.1, 0.01 mol/L) of excitatory amino acid L-Glu or physiological saline (0.65% NaCl) were then microinjected into the motor neuron pool in a urethane-anaesthetized toad. The contraction curve of the gastrocnemius was then recorded by the BL-420 Physiological Signal Recording. We took the maximal tension, the duration of rising phase, the velocity of tension variation, and the duration of descending phase as the parameters to study the characteristic of gastrocnemius contractility. It was found that the gastrocnemius contractility of all the 4 groups was tetanus but differed in degree, especially the maximal tension, and velocity of tension variation. In contrast to physiological saline, gastrocnemius contracted by the stimulation of L-Glu, and the contraction parameter showed dose-effect relationships except for the duration of descending phase, which was caused by the combination rate of L-Glu and the receptor.

Effects and mechanisms of L-glutamate microinjected into nucleus ambiguus on gastric motility in rats.

BACKGROUND: L-glutamate (L-GLU) is a major neurotransmitter in the nucleus ambiguus (NA), which can modulate respiration, arterial pressure, heart rate, etc. This study investigated the effects and mechanisms of L-GLU microinjected into NA on gastric motility in rats. METHODS: A latex balloon connected with a pressure transducer was inserted into the pylorus through the forestomach for continuous recording of the gastric motility. The total amplitude, total duration, and motility index of gastric contraction waves within 5 minutes before microinjection and after microinjection were measured. RESULTS: L-GLU (5 nmol, 10 nmol and 20 nmol in 50 nl normal saline (PS) respectively) microinjected into the right NA significantly inhibited gastric motility, while microinjection of physiological saline at the same position and the same volume did not change the gastric motility. The inhibitory effect was blocked by D-2-amino-5-phophonovalerate (D-AP5, 5 nmol, in 50 nl PS), the specific N-methyl-D-aspartic acid (NMDA) receptor antagonist, but was not influenced by 6-cyaon-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) (5 nmol, in 50 nl PS), the non-NMDA ionotropic receptor antagonist. Bilateral subdiaphragmatic vagotomy abolished the inhibitory effect by microinjection of L-GLU into NA. CONCLUSIONS: Microinjection of L-GLU into NA inhibits the gastric motility through specific NMDA receptor activity, not non-NMDA receptor activity, and the efferent pathway is the vagal nerves.

Comparative study of c-Fos expression in rat dorsal vagal complex and nucleus ambiguus induced by different durations of restraint water-immersion stress.

Restraint water-immersion stress (RWIS) of rats induces vagally-mediated gastric dysfunction. The present work explored the effects of different durations of RWIS on neuronal activities of the dorsal vagal complex (DVC) and the nucleus ambiguous (NA) in rats. Male Wistar rats were exposed to RWIS for 0, 30, 60, 120, or 180 min. Then, a c-Fos immunoperoxidase technique was utilized to assess neuronal activation. Resumptively, c-Fos expression in DVC and NA peaked at 60 min of stress, subsequently decreased gradually with increasing durations of RWIS. Interestingly, the most intense c-Fos expression was observed in the dorsal motor nucleus of the vagus (DMV) during the stress, followed by NA, nucleus of solitary tract (NTS) and area postrema (AP). The peak of c-Fos expression in caudal DMV appeared at 120 min of the stress, slower than that in rostral and intermediate DMV. The c-Fos expression in intermediate and caudal NTS was significantly more intense than that in rostral NTS. These results indicate that the neuronal hyperactivity of DMV, NA, NTS and AP, the primary center that control gastric functions, especially DMV and NA, may play an important role in the disorders of gastric motility and secretion induced by RWIS.

c-Fos expression in the supraoptic nucleus is the most intense during different durations of restraint water-immersion stress in the rat.

Restraint water-immersion stress (RWIS) can induce anxiety, hypothermia, and severe vagally-mediated gastric dysfunction. The present work explored the effects of different durations of RWIS on neuronal activities of the forebrain by c-Fos expression in conscious rats exposed to RWIS for 0, 30, 60, 120, or 180 min. The peak of c-Fos induction was distinct for different forebrain regions. The most intense c-Fos induction was always observed in the supraoptic nucleus (SON), and then in the hypothalamic paraventricular nucleus (PVN), posterior cortical amygdaloid nucleus (PCoA), central amygdaloid nucleus (CeA), and medial prefrontal cortex (mPFC). Moreover, body temperature was reduced to the lowest degree after 60 min of RWIS, and the gastric lesions tended to gradually worsen with the prolonging of RWIS duration. These data strongly suggest that these nuclei participate in the organismal response to RWIS to different degrees, and may be involved in the hypothermia and gastric lesions induced by RWIS.

Effects of nucleus ambiguus and dorsal motor nuclei of vagus on gastric H(+) and HCO(3)(-) secretion in rats.

AIM: To determine the effects of electrical stimulation of nucleus ambiguus (NA) and dorsal motor nuclei of vagus (DMV) on gastric acid and bicarbonate secretion in rats. METHODS: NA and DMV in rats were electrically stimulated. Pylorus ligation or esophagus perfusion was used to collect the gastric secretion. The titratable H(+) quantum, H(+) concentration, HCO(3)(-) secretion quantum were measured. RESULTS: Electrical stimulation of NA had no effects on the volume of gastric juice, titratable acidity and acid concentration, but elicited a pronounced increase in the total bicarbonate. However, electrical stimulation of DMV significantly increased the titratable acidity, the volume of gastric juice and the acid concentration. Similarly, electrical stimulation of either NA or DMV decreased the respiratory frequency and sinus bradycardia. CONCLUSION: NA in rats can not control the secretion of gastric acid but the secretion of bicarbonate in gastric juice, while DMV controls the secretion of gastric acid.

[Studies on effects and mechanism of water immersion restraint stress on bile secretion in rats].

AIM: To probe into the operation mechanism of stress, through the studies on the effects of bile secretion in rats at the condition of water immersion restraint. METHODS: The animals were divided into six groups (n=8): Group A: restraint alone under room temperature + saline; Group B: water immersion restraint + saline; Group C: restraint alone under room temperature + Atropine; Group D: water immersion restraint + Atropine; Group E: restraint alone under room temperature + Phentolamine; F group: water immersion restraint + Phentolamine. RESULTS: Compared with group A, the capacity of bile secretion in group B decreased significantly (P < 0.05), changes of bile increased remarkably (P < 0.01), but there were no significant decreases on the capacity of bile secretion in group C (P > 0.05) compared with A, Group C only decreased appreciably. Compared with group A, the capacity of bile secretion in group E decreased appreciably (P < 0.05). Compared with group B, the capacity of bile secretion in group D decreased significantly (P < 0.05), pH of bile had no significant changes in group D. Compared with group B, the capacity of bile secretion in group F decreased significantly (P < 0.05), pH of bile had no significant changes in group F. Compared with group D, the capacity of bile secretion and pH of bile in group F had no significant changes. CONCLUSION: Water immersion restraint stress inhibited evidently on the capacity of bile secretion, and the capacity of bile secretion in water immersion groups decreased significantly, moreover pH of bile increased greatly. At the condition of restraint alone under room temperature, vagus and sympathetic nerve had no significant effects on the bile secretion, but they played important roles in decreases of bile secretion evidently induced by water immersion restraint stress in rats (P < 0.05).

Comparative studies on the wide frequency band electrocardiogram and vectorcardiogram in pigeon and mouse.

Wide frequency band ECG and vectorcardiogram in anesthetized pigeon and mouse were studied from the standpoint of comparison. The key results were as follows: in pigeon, the direction of the main QRS was inverted in leads II, III and aVF, and upright in lead aVR, which was contrary to that in mouse. The T wave was upright in leads II, III and aVF, but inverted in lead aVR in pigeon, which was the same as that in mouse. In pigeon, there was a large notch on the upstroke of the S wave in lead II without exception, but there was no such notch in the corresponding lead in mouse. The QRS vector loop in the frontal plane lay between -90 and -180 degrees in pigeon, while that of mouse lay between 0 and 90 degrees. The relative power of high frequency range (80-1000 Hz) of the QRS in lead II was approximately 15% in pigeon, but 55% in mouse. The direction of the main QRS was contrary in pigeon and mouse because the subepicardial muscles were depolarized before the subendocardial muscles in pigeon, but the latter were depolarized before the former in mouse. The direction of the T waves was the same in both pigeon and mouse because subepicardial muscles were all repolarized before subendocardial muscles.