Signaled two-way avoidance learning using electrical stimulation of the inferior colliculus as negative reinforcement: effects of visual and auditory cues as warning stimuli

The inferior colliculus is a primary relay for the processing of auditory information in the brainstem. The inferior colliculus is also part of the so-called brain aversion system as animals learn to switch off the electrical stimulation of this structure. The purpose of the present study was to determine whether associative learning occurs between aversion induced by electrical stimulation of the inferior colliculus and visual and auditory warning stimuli. Rats implanted with electrodes into the central nucleus of the inferior colliculus were placed inside an open-field and thresholds for the escape response to electrical stimulation of the inferior colliculus were determined. The rats were then placed inside a shuttle-box and submitted to a two-way avoidance paradigm. Electrical stimulation of the inferior colliculus at the escape threshold (98.12 ± 6.15 (A, peak-to-peak) was used as negative reinforcement and light or tone as the warning stimulus. Each session consisted of 50 trials and was divided into two segments of 25 trials in order to determine the learning rate of the animals during the sessions. The rats learned to avoid the inferior colliculus stimulation when light was used as the warning stimulus (13.25 ± 0.60 s and 8.63 ± 0.93 s for latencies and 12.5 ± 2.04 and 19.62 ± 1.65 for frequencies in the first and second halves of the sessions, respectively, P<0.01 in both cases). No significant changes in latencies (14.75 ± 1.63 and 12.75 ± 1.44 s) or frequencies of responses (8.75 ± 1.20 and 11.25 ± 1.13) were seen when tone was used as the warning stimulus (P>0.05 in both cases). Taken together, the present results suggest that rats learn to avoid the inferior colliculus stimulation when light is used as the warning stimulus. However, this learning process does not occur when the neutral stimulus used is an acoustic one. Electrical stimulation of the inferior colliculus may disturb the signal transmission of the stimulus to be conditioned from the inferior colliculus to higher brain structures such as amygdala

Cardiorespiratory adaptations induced by aerobic training in middle-aged men: the importance of a decrease in sympathetic stimulation for the contribution of dynamic exercise tachycardia

We investigated the effects of aerobic training on the efferent autonomic control of heart rate (HR) during dynamic exercise in middle-aged men, eight of whom underwent exercise training (T) while the other seven continued their sedentary (S) life style. The training was conducted over 10 months (three 1-h sessions/week on a field track at 70-85% of the peak HR). The contribution of sympathetic and parasympathetic exercise tachycardia was determined in terms of differences in the time constant effects on the HR response obtained using a discontinuous protocol (4-min tests at 25, 50, 100 and 125 watts on a cycle ergometer), and a continuous protocol (25 watts/min until exhaustion) allowed the quantification of the parameters (anaerobic threshold, VO2 AT; peak O2 uptake, VO2 peak; power peak) that reflect oxygen transport. The results obtained for the S and the T groups were: 1) a smaller resting HR in T (66 beats/min) when compared to S (84 beats/min); 2) during exercise, a small increase in the fast tachycardia (D0-10 s) related to vagal withdrawal (P<0.05, only at 25 watts) was observed in T at all powers; at middle and higher powers a significant decrease (P<0.05 at 50, 100 and 125 watts) in the slow tachycardia (D1-4 min) related to a sympathetic-dependent mechanism was observed in T; 3) the VO2 AT (S = 1.06 and T = 1.33 l/min) and VO2 peak (S = 1.97 and T = 2.47 l/min) were higher in T (P<0.05). These results demonstrate that aerobic training can induce significant physiological adaptations in middle-aged men, mainly expressed as a decrease in the sympathetic effects on heart rate associated with an increase in oxygen transport during dynamic exercise.

Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion

The aim of this study was to investigate the possible interactions between the nociceptive system, the sympathetic system and the inflammatory process. Thus, the superior cervical ganglion of rats was submitted to chronic inflammation and Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. The painful stimulus consisted of subcutaneously injected formalin applied to the supra-ocular region. Fos-positive neurons were identified by conventional immunohistochemical techniques, and analyzed from the obex through the cervical levels of the spinal cord. In the caudal sub-nucleus of the spinal trigeminal nuclear complex, the number of Fos-positive neurons was much higher in rats with inflammation of the superior cervical ganglion than in control rats, either sham-operated or with saline applied to the ganglion. There was a highly significant difference in the density of Fos-positive neurons between the inflamed and control groups. No significant difference was found between control groups. These results suggest that the inflammation of the superior cervical ganglion generated an increased responsiveness to painful stimuli, which may have been due to a diminished sympathetic influence upon the sensory peripheral innervation.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Ethological analysis of mother-pup interactions and other behavioral reactions in rats: effects of malnutrition and tactile stimulation of the pups

Mother-pup interaction, as well as other behavioral reactions were studied during the lactation period in 24 litters of Wistar rats and their dams fed either a 16% (control - C; 12 litters) or a 6% (malnourished - M; 12 litters) protein diet. The diets were isocaloric. Throughout lactation there was a 36.4% weight loss of M dams and a 63% body weight deficit in the M pups when compared to control pups. During this period, half of the litters were exposed daily to additional tactile stimulation (CS or MS), while the other half were submitted to normal rearing conditions (CN or MN). The tactile stimulation of pups (handling) consisted of holding the animal in one hand and gently touching the dorsal part of the animal's body with the fingers for 3 min. A special camera and a time-lapse video were used to record litter behavior in their home cages. Starting at 6 p.m. and ending at 6 a.m., on days 3, 6, 12, 15, 18 and 21 of lactation, photos were taken at 4-s intervals. An increase in the frequency (154.88 ± 16.19) and duration (455.86 ± 18.05 min) of suckling was observed throughout the lactation period in all groups compared to birth day (frequency 24.88 ± 2.37 and duration 376.76 ± 21.01 min), but the frequency was higher in the C (84.96 ± 8.52) than in the M group (43.13 ± 4.37); however, the M group (470.2 ± 11.87 min) spent more time suckling as compared with the C group (393.67 ± 13.09 min). The M dams showed a decreased frequency of resting position throughout the lactation period (6.5 ± 2.48) compared to birth day (25.42 ± 7.74). Pups from the C group were more frequently observed separated (73.02 ± 4.38) and interacting (258.99 ± 20.61) more with their mothers than the M pups (separated 66.94 ± 5.5 and interacting 165.72 ± 12.05). Tactile stimulation did not interact with diet condition, showing that the kind of stimulation used in the present study did not lead to recovery from the changes induced by protein malnutrition. The changes in mother-pup interaction produced by protein malnutrition of both may represent retardation in neuromotor development and a higher dependence of the pups on their mothers. These changes may represent an important means of energy saving and heat maintenance in malnourished pups.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

Effects of L-arginine on the diaphragm muscle twitches elicited at different frequencies of nerve stimulation

In rats, the nitric oxide (NO)-synthase pathway is present in skeletal muscle, vascular smooth muscle, and motor nerve terminals. Effects of NO were previously studied in rat neuromuscular preparations receiving low (0.2 Hz) or high (200 Hz) frequencies of stimulation. The latter frequency has always induced tetanic fade. However, in these previous studies we did not determine whether NO facilitates or impairs the neuromuscular transmission in preparations indirectly stimulated at frequencies which facilitate neuromuscular transmission. Thus, the present study was carried out to examine the effects of NO in rat neuromuscular preparations indirectly stimulated at 5 and 50 Hz. The amplitude of muscular contraction observed at the end (B) of a 10-s stimulation was taken as the ratio (R) of that obtained at the start (A) (R = B/A). S-nitroso-N-acetylpenicillamine (200 µM), superoxide dismutase (78 U/ml) and L-arginine (4.7 mM), but not D-arginine (4.7-9.4 mM), produced an increase in R (facilitation of neurotransmission) at 5 Hz. However, reduction in the R value (fade of transmission) was observed at 50 Hz. N G-nitro-L-arginine (8.0 mM) antagonized both the facilitatory and inhibitory effects of L-arginine (4.7 mM). The results suggest that NO may modulate the release of acetylcholine by motor nerve terminals.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

Cholinergic stimulation with pyridostigmine reduces the QTc interval in coronary artery disease

Parasympathetic dysfunction is an independent risk factor in patients with coronary artery disease; thus, cholinergic stimulation is a potential therapeutic measure that may be protective by acting on ventricular repolarization. The purpose of the present study was to determine the effects of pyridostigmine bromide (PYR), a reversible anticholinesterase agent, on the electrocardiographic variables, particularly QTc interval, in patients with stable coronary artery disease. In a randomized double-blind crossover placebo-controlled study, simultaneous 12-lead electrocardiographic tracings were obtained at rest from 10 patients with exercise-induced myocardial ischemia before and 2 h after the oral administration of 45 mg PYR or placebo. PYR increased the RR intervals (pre: 921 ± 27 ms vs post: 1127 ± 37 ms; P<0.01) and, in contrast with placebo, decreased the QTc interval (pre: 401 ± 3 ms vs post: 382 ± 3 ms; P<0.01). No other electrocardiographic variables were modified (PR segment, QT interval, QT and QTc dispersions). Cholinergic stimulation with PYR caused bradycardia and reduced the QTc interval without important side effects in patients with coronary disease. These effects, if confirmed in studies over longer periods of administration, may suggest a cardioprotection by cholinergic stimulation with PYR.

Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

Modulation of hormone secretion by functional electrical stimulation of the intact and incompletely dysfunctional dog pancreas

The purpose of the present study was to modulate the secretion of insulin and glucagon in Beagle dogs by stimulation of nerves innervating the intact and partly dysfunctional pancreas. Three 33-electrode spiral cuffs were implanted on the vagus, splanchnic and pancreatic nerves in each of two animals. Partial dysfunction of the pancreas was induced with alloxan. The nerves were stimulated using rectangular, charge-balanced, biphasic, and constant current pulses (200 µs, 1 mA, 20 Hz, with a 100-µs delay between biphasic phases). Blood samples from the femoral artery were drawn before the experiment, at the beginning of stimulation, after 5 min of stimulation, and 5 min after the end of stimulation. Radioimmunoassay data showed that in the intact pancreas stimulation of the vagal nerve increased insulin (+99.2 µU/ml) and glucagon (+18.7 pg/ml) secretion and decreased C-peptide secretion (-0.15 ng/ml). Splanchnic nerve stimulation increased insulin (+1.7 µU/ml), C-peptide (+0.01 ng/ml), and glucagon (+50 pg/ml) secretion, whereas pancreatic nerve stimulation did not cause a marked change in any of the three hormones. In the partly dysfunctional pancreas, vagus nerve stimulation increased insulin (+15.5 µU/ml), glucagon (+11 pg/ml), and C-peptide (+0.03 ng/ml) secretion. Splanchnic nerve stimulation reduced insulin secretion (-2.5 µU/ml) and increased glucagon (+58.7 pg/ml) and C-peptide (+0.39 ng/ml) secretion, and pancreatic nerve stimulation increased insulin (+0.2 µU/ml), glucagon (+5.2 pg/ml), and C-peptide (+0.08 ng/ml) secretion. It was concluded that vagal nerve stimulation can significantly increase insulin secretion for a prolonged period of time in intact and in partly dysfunctional pancreas.

Electrical field stimulation improves bone mineral density in ovariectomized rats

Osteoporosis and its consequent fractures are a great social and medical problem mainly occurring in post-menopausal women. Effective forms of prevention and treatment of osteoporosis associated with lower costs and the least side effects are needed. Electrical fields are able to stimulate osteogenesis in fractures, but little is known about their action on osteoporotic tissue. The aim of the present study was to determine by bone densitometry the effects of electrical stimulation on ovariectomized female Wistar rats. Thirty rats (220 ± 10 g) were divided into three groups: sham surgery (SHAM), bilateral ovariectomy (OVX) and bilateral ovariectomy + electrical stimulation (OVX + ES). The OVX + ES group was submitted to a 20-min session of a low-intensity pulsed electrical field (1.5 MHz, 30 mW/cm²) starting on the 7th day after surgery, five times a week (total = 55 sessions). Global, spine and limb bone mineral density were measured by dual-energy X-ray absorptiometry (DXA Hologic 4500A) before surgery and at the end of protocol (84 days after surgery). Electrical stimulation improved (P < 0.05) global (0.1522 ± 0.002), spine (0.1502 ± 0.003), and limb (0.1294 ± 0.003 g/cm²) bone mineral density compared to OVX group (0.1447 ± 0.001, 0.1393 ± 0.002, and 0.1212 ± 0.001, respectively). The OVX + ES group also showed significantly higher global bone mineral content (9.547 ± 0.114 g) when compared to both SHAM (8.693 ± 0.165 g) and OVX (8.522 ± 0.207 g) groups (P < 0.05). We have demonstrated that electrical fields stimulate osteogenesis in ovariectomized female rats. Their efficacy in osteoporosis remains to be demonstrated.

Influence of gender on psychomotor vigilance task performance by adolescents

During adolescence, the sleep phase delay associated with early school times increases daytime sleepiness and reduces psychomotor performance. Some studies have shown an effect of gender on psychomotor performance in adults and children. Males present faster reaction times (RT) compared with females. The aim of the present study was to evaluate the influence of gender on Palm psychomotor vigilance task (PVT) performance in adolescents. Thirty-four adolescents (19 girls, 13 to 16 years old) attending morning school classes of a public school in Curitiba, PR, Brazil, participated in the study. Sleep patterns were measured using actigraphy and sleepiness data were accessed with the Karolinska Sleepiness Scale (KSS). KSS and PVT measurements were collected at two times in the morning (8:00 and 11:00 h). The data were compared using one-way ANOVA, considering gender as a factor. ANOVA indicated that gender did not affect sleep patterns and subjective somnolence; however, a statistically significant effect of gender was detected for PVT performance. Boys presented faster RT (RT-PVT1: 345.51 ms, F = 6.08, P < 0.05; RT-PVT2: 343.30 ms, F = 6.35, P < 0.05) and fewer lapses (lapses-PVT1: 8.71, F = 4.45, P < 0.05; lapses-PVT2: 7.82, F = 7.06, P < 0.05) compared with girls (RT-PVT1: 402.96; RT-PVT2: 415.70; lapses-PVT1: 16.33; lapses-PVT2: 17.80). These results showed that this effect of gender, already reported in adults and children, is also observed in adolescents. The influence of gender should be taken into account in studies that use Palm PVT to evaluate psychomotor performance in this age range.

Changes in types of muscle fibers induced by transcutaneous electrical stimulation of the diaphragm of rats

The objective of the present study was to assess the effect of transcutaneous electrical diaphragmatic stimulation (TEDS) on different types of diaphragm muscle fibers. Male Wistar rats (8-12 weeks old) were divided into 2 experimental groups (N = 8 in each group): 1) control, 2) animals submitted to TEDS [frequency = 50 Hz; T ON/T OFF (contraction/relaxation time) = 2/2 s; pulse duration = 0.4 ms, intensity = 5 mA with a 1 mA increase every 3 min for 20 min] for 7 days. After completing this treatment period, the I, IIA, IIB, and IID diaphragm muscle fibers were identified using the mATPase technique. Statistical analysis consisted of the normality, homoscedasticity and t-tests (P < 0.05). There was a 19.6% (P < 0.05) reduction in the number of type I fibers and a 49.7% increase (P < 0.05) in type IID fibers in the TEDS group compared with the control group. An important result of the present study was that electrical stimulation with surface electrodes was efficient in altering the distribution of fibers in diaphragm muscle. This therapeutic resource could be used in the treatment of respiratory muscle alterations.