Letrozole delays acquisition of water maze task in female BALB/c mice: Possible involvement of anxiety.

Letrozole, an aromatase inhibitor preventing estrogen synthesis from testosterone, is used as an adjuvant therapy in estrogen receptor-positive breast cancer patients. However, like other aromatase inhibitors, it induces many side effects, including impaired cognition. Despite its negative effect in humans, results from animal models are inconsistent and suggest that letrozole can either impair or improve cognition. Here, we studied the effects of chronic letrozole treatment on cognitive behavior of adult female BALB/c mice, a relevant animal model for breast cancer studies, to develop an appropriate animal model aimed at testing therapies to mitigate side effects of letrozole. In Morris water maze, letrozole 0.1 mg/kg impaired reference learning and memory. Interestingly, most of the letrozole 0.1 mg/kg-treated mice were able to learn the new platform position in reversal training and performed similar to control mice in a reversal probe test. Results of the reversal test suggest that letrozole did not completely disrupt spatial navigation, but rather delayed acquisition of spatial information. The delay might be related to increased anxiety as suggested by increased thigmotactic behavior during the reference memory training. The learning impairment was water maze-specific since we did not observe impairment in other spatial tasks such as in Y-maze or object location test. In contrast, the dose of 0.3 mg/kg did not have effect on water maze learning and facilitated locomotor habituation and recognition in novel object recognition test. The current study shows that letrozole dose-dependently modulates behavioral response and that its effects are task-dependent.

Gestational exposures to organophosphorus insecticides: From acute poisoning to developmental neurotoxicity.

For over three-quarters of a century, organophosphorus (OP) insecticides have been ubiquitously used in agricultural, residential, and commercial settings and in public health programs to mitigate insect-borne diseases. Their broad-spectrum insecticidal effectiveness is accounted for by the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that catalyzes acetylcholine (ACh) hydrolysis, in the nervous system of insects. However, because AChE is evolutionarily conserved, OP insecticides are also toxic to mammals, including humans, and acute OP intoxication remains a major public health concern in countries where OP insecticide usage is poorly regulated. Environmental exposures to OP levels that are generally too low to cause marked inhibition of AChE and to trigger acute signs of intoxication, on the other hand, represent an insidious public health issue worldwide. Gestational exposures to OP insecticides are particularly concerning because of the exquisite sensitivity of the developing brain to these insecticides. The present article overviews and discusses: (i) the health effects and therapeutic management of acute OP poisoning during pregnancy, (ii) epidemiological studies examining associations between environmental OP exposures during gestation and health outcomes of offspring, (iii) preclinical evidence that OP insecticides are developmental neurotoxicants, and (iv) potential mechanisms underlying the developmental neurotoxicity of OP insecticides. Understanding how gestational exposures to different levels of OP insecticides affect pregnancy and childhood development is critical to guiding implementation of preventive measures and direct research aimed at identifying effective therapeutic interventions that can limit the negative impact of these exposures on public health.

Learning and memory retention deficits in prepubertal guinea pigs prenatally exposed to low levels of the organophosphorus insecticide malathion.

High doses of malathion, an organophosphorus (OP) insecticide ubiquitously used in agriculture, residential settings, and public health programs worldwide, induce a well-defined toxidrome that results from the inhibition of acetylcholinesterase (AChE). However, prenatal exposures to malathion levels that are below the threshold for AChE inhibition have been associated with increased risks of neurodevelopmental disorders, including autism spectrum disorder with intellectual disability comorbidity. The present study tested the hypothesis that prenatal exposures to a non-AChE-inhibiting dose of malathion are causally related to sex-biased cognitive deficits later in life in a precocial species. To this end, pregnant guinea pigs were injected subcutaneously with malathion (20 mg/kg) or vehicle (peanut oil, 0.5 ml/kg) once daily between approximate gestational days 53 and 63. This malathion dose regimen caused no significant AChE inhibition in the brain or blood of dams and offspring and had no significant effect on the postnatal growth of the offspring. Around postnatal day 30, locomotor activity and habituation, a form of non-associative learning, were comparable between malathion- and peanut oil-exposed offspring. However, in the Morris water maze, malathion-exposed offspring presented significant sex-dependent spatial learning deficits in addition to memory impairments. These results are far-reaching as they indicate that: (i) malathion is a developmental neurotoxicant and (ii) AChE inhibition is not an adequate biomarker to derive safety limits of malathion exposures during gestation. Continued studies are necessary to identify the time and dose dependence of the developmental neurotoxicity of malathion and the mechanisms underlying the detrimental effects of this insecticide in the developing brain.

Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: from clinical findings to preclinical models and potential mechanisms.

Organophosphorus (OP) insecticides are pest-control agents heavily used worldwide. Unfortunately, they are also well known for the toxic effects that they can trigger in humans. Clinical manifestations of an acute exposure of humans to OP insecticides include a well-defined cholinergic crisis that develops as a result of the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh). Prolonged exposures to levels of OP insecticides that are insufficient to trigger signs of acute intoxication, which are hereafter referred to as subacute exposures, have also been associated with neurological deficits. In particular, epidemiological studies have reported statistically significant correlations between prenatal subacute exposures to OP insecticides, including chlorpyrifos, and neurological deficits that range from cognitive impairments to tremors in childhood. The primary objectives of this article are: (i) to address the short- and long-term neurological issues that have been associated with acute and subacute exposures of humans to OP insecticides, especially early in life (ii) to discuss the translational relevance of animal models of developmental exposure to OP insecticides, and (iii) to review mechanisms that are likely to contribute to the developmental neurotoxicity of OP insecticides. Most of the discussion will be focused on chlorpyrifos, the top-selling OP insecticide in the United States and throughout the world. These points are critical for the identification and development of safe and effective interventions to counter and/or prevent the neurotoxic effects of these chemicals in the developing brain. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Spatial learning impairment in prepubertal guinea pigs prenatally exposed to the organophosphorus pesticide chlorpyrifos: Toxicological implications.

Exposure of the developing brain to chlorpyrifos (CPF), an organophosphorus (OP) pesticide used extensively in agriculture worldwide, has been associated with increased prevalence of cognitive deficits in children, particularly boys. The present study was designed to test the hypothesis that cognitive deficits induced by prenatal exposure to sub-acute doses of CPF can be reproduced in precocial small species. To address this hypothesis, pregnant guinea pigs were injected daily with CPF (25mg/kg,s.c.) or vehicle (peanut oil) for 10days starting on presumed gestation day (GD) 53-55. Offspring were born around GD 65, weaned on postnatal day (PND) 20, and subjected to behavioral tests starting around PND 30. On the day of birth, butyrylcholinesterase (BuChE), an OP bioscavenger used as a biomarker of OP exposures, and acetylcholinesterase (AChE), a major molecular target of OP compounds, were significantly inhibited in the blood of CPF-exposed offspring. In their brains, BuChE, but not AChE, was significantly inhibited. Prenatal CPF exposure had no significant effect on locomotor activity or on locomotor habituation, a form of non-associative memory assessed in open fields. Spatial navigation in the Morris water maze (MWM) was found to be sexually dimorphic among guinea pigs, with males outperforming females. Prenatal CPF exposure impaired spatial learning more significantly among male than female guinea pigs and, consequently, reduced the sexual dimorphism of the task. The results presented here, which strongly support the test hypothesis, reveal that the guinea pig is a valuable animal model for preclinical assessment of the developmental neurotoxicity of OP pesticides. These findings are far reaching as they lay the groundwork for future studies aimed at identifying therapeutic interventions to treat and/or prevent the neurotoxic effects of CPF in the developing brain.

Prenatal exposure of guinea pigs to the organophosphorus pesticide chlorpyrifos disrupts the structural and functional integrity of the brain.

This study was designed to test the hypothesis that prenatal exposure of guinea pigs to the organophosphorus (OP) pesticide chlorpyrifos (CPF) disrupts the structural and functional integrity of the brain. Pregnant guinea pigs were injected with chlorpyrifos (25 mg/kg, s.c.) or vehicle (peanut oil) once per day for 10 consecutive days, starting approximately on the 50th day of gestation. Cognitive behavior of female offspring was examined starting at 40-45 post-natal days (PND) using the Morris water maze (MWM), and brain structural integrity was analyzed at PND 70 using magnetic resonance imaging (MRI) methods, including T2-weighted anatomical scans and diffusion kurtosis imaging (DKI). The offspring of exposed mothers had significantly decreased body weight and brain volume, particularly in the frontal regions of the brain including the striatum. Furthermore, the offspring demonstrated significant spatial learning deficits in MWM recall compared to the vehicle group. Diffusion measures revealed reduced white matter integrity within the striatum and amygdala that correlated with spatial learning performance. These findings reveal the lasting effect of prenatal exposure to CPF as well as the danger of mother to child transmission of CPF in the environment.

Delayed hippocampal effects from a single exposure of prepubertal guinea pigs to sub-lethal dose of chlorpyrifos: a magnetic resonance imaging and spectroscopy study.

This study was designed to test the hypothesis that in vivo magnetic resonance imaging (MRI) and spectroscopy (MRS) can detect in adulthood the neurotoxic effects of a single exposure of prepubertal guinea pigs to the organophosphorus pesticide chlorpyrifos. Twelve female guinea pigs were given either a single dose of chlorpyrifos (0.6×LD50 or 300mg/kg, sc) or peanut oil (vehicle; 0.5ml/kg, sc) at 35-40 days of age. One year after the exposure, the animals were tested in the Morris water maze. Three days after the end of the behavioral testing, the metabolic and structural integrity of the brain of the animals was examined by means of MRI/MRS. In the Morris water maze, the chlorpyrifos-exposed guinea pigs showed significant memory deficit. Although no significant anatomical differences were found between the chlorpyrifos-exposed guinea pigs and the control animals by in vivo MRI, the chlorpyrifos-exposed animals showed significant decreases in hippocampal myo-inositol concentration using MRS. The present results indicate that a single sub-lethal exposure of prepubertal guinea pigs to the organophosphorus pesticide chlorpyrifos can lead to long-term memory deficits that are accompanied by significant reductions in the levels of hippocampal myo-inositol.

Effects of the triple monoamine uptake inhibitor DOV 102,677 on alcohol-motivated responding and antidepressant activity in alcohol-preferring (P) rats.

BACKGROUND: Concurrent inhibitors of dopamine, norepinephrine, and serotonin uptake have been proposed as novel antidepressants. Given the high comorbidity between alcoholism and depression, we evaluated the activity of DOV 102,677 (DOV) on alcohol-maintained responding and performance in the forced swim test (FST), a model of antidepressant (AD) activity, using alcohol-preferring (P) rats. METHODS: Following training to lever press for either alcohol (10% v/v) or sucrose (3, 2%, w/v) on a fixed-ratio 4 (FR4) schedule, DOV (1.56 to 50 mg/kg; PO) was given 25 minutes or 24 hours prior to evaluation. The effects of DOV (12.5 to 50 mg/kg; PO) in the FST were evaluated 25 minutes posttreatment. RESULTS: DOV (6.25 to 50 mg/kg) dose-dependently reduced alcohol-maintained responding by 59 to 88% at 25 minutes posttreatment, without significantly altering sucrose responding. The reduction in alcohol responding (44% at 50 mg/kg) was sustained for up to 120 hours after a single dose. Administration of a single dose of DOV (25, 50 mg/kg) 24 hours before testing suppressed alcohol responding for 48 hours by 59 to 62%. DOV (12.5 to 50 mg/kg) also dose-dependently reduced immobility of P rats in the FST. CONCLUSIONS: DOV produces both prolonged and selective reductions of alcohol-motivated behaviors in P rats. The elimination kinetics of DOV suggests that its long duration of action may be due to an active metabolite. DOV also produced robust AD-like effects in P rats. We propose that DOV may be useful in treating comorbid alcoholism and depression in humans.

Galantamine counteracts development of learning impairment in guinea pigs exposed to the organophosphorus poison soman: clinical significance.

Galantamine, a drug used to treat Alzheimer's disease, protects guinea pigs against the acute toxicity and lethality of organophosphorus (OP) compounds, including soman. Here, we tested the hypothesis that a single exposure of guinea pigs to 1xLD50 soman triggers cognitive impairments that can be counteracted by galantamine. Thus, animals were injected intramuscularly with saline (0.5 ml/kg) or galantamine (8 mg/kg) and 30 min later injected subcutaneously with soman (26.3 µg/kg) or saline. Cognitive performance was analyzed in the Morris water maze (MWM) four days or three months after the soman challenge. Fifty percent of the saline-injected animals that were challenged with soman survived with mild-to-moderate signs of acute toxicity that subsided within a few hours. These animals showed no learning impairment and no memory retention deficit, when training in the MWM started four days post-soman challenge. In contrast, animals presented significant learning impairment when testing started three months post-challenge. Though the magnitude of the impairment correlated with the severity of the acute toxicity, animals that presented no or only mild signs of toxicity were also learning impaired. All guinea pigs that were treated with galantamine survived the soman challenge with no signs of acute toxicity and learned the MWM task as control animals, regardless of when testing began. Galantamine also prevented memory extinction in both saline- and soman-challenged animals. In conclusion, learning impairment develops months after a single exposure to 1xLD50 soman, and galantamine prevents both the acute toxicity and the delayed cognitive deficits triggered by this OP poison.

An acute exposure to a sub-lethal dose of soman triggers anxiety-related behavior in guinea pigs: interactions with acute restraint.

In this study, we tested the hypothesis that a single exposure of guinea pigs to sub-lethal doses of soman triggers anxiety-related behavior that is modifiable by acute stress. Prepubertal male guinea pigs were subjected to one of the following treatments: (i) saline (0.5 ml/kg, sc), (ii) soman (0.6x or 0.8xLD50, sc), (iii) saline followed 30 min later by 2-h restraint, or (iv) soman followed 30 min later by 2-h restraint. Behavior of the animals was examined 2 and 3 months later in a large open field and in the elevated plus maze. Animals that had been exposed to restraint stress alone or soman alone showed decreased exploratory activity when tested in the open field with bare floor at light intensity of 20-30 lx. Total distance traveled and distance traveled in the center of the field were shorter for animals that were exposed to either restraint stress or soman than for saline-injected animals. In addition, animals challenged with soman or restraint stress remained immobile for a longer time in the open field than did saline-injected guinea pigs. Performance in the elevated plus maze test revealed that exposure of guinea pigs to soman or restraint stress decreased their number of entries and the time spent in the open arms of the maze (measures of anxiety) and reduced their overall locomotor activity. Soman exposure and restraint stress cancelled out each other's effect on locomotion, while only attenuating one another's effect on anxiety-related behavior. It is concluded that a single exposure to sub-lethal doses of soman triggers long-lasting anxiogenesis and decreased locomotor activity and that acute restraint stress modifies the magnitude of these effects.

Deficits in substance P mRNA levels in the CeA are inversely associated with alcohol-motivated responding.

In the present study, in vitro and in vivo studies were conducted to determine the relationship between innate substance P (SP) levels and alcohol-motivated behavior in alcohol-preferring (P) and nonpreferring (NP) rat lines. In Experiment 1, in situ hybridization and quantitative autoradiography were used to detect and measure SP mRNA levels in discrete brain loci of the P and NP rats. The results indicated significantly lower SP mRNA levels in the central nucleus of the amygdala (CeA) of P compared with those of NP rats. Experiment 2 evaluated the effects of SP, microinfused into the CeA, on alcohol (10%, v/v) and sucrose (2%, w/v) motivated responding in the P rat. The results revealed that, when infused into the CeA (1-8 microg), SP reduced alcohol responding by 48-85% of control levels, with no effects on sucrose responding. Neuroanatomical control infusions (1-8 microg) into the caudate putamen (CPu) also failed to significantly alter alcohol- or sucrose-motivated behaviors. Given the selective reductions on alcohol (compared to sucrose) responding by direct intracranial infusion of SP, the data suggest that deficits in SP signaling within the CeA (an anxiety regulating locus) are inversely associated with alcohol-motivated behaviors. Activation of SP receptors in the CeA may reduce anxiety-like behavior in the P rat and contribute to reductions on alcohol responding. The SP system may be a suitable target for the development of drugs to reduce alcohol-drinking behavior in humans.

Preclinical investigation of the topical administration of phenserine: transdermal flux, cholinesterase inhibition, and cognitive efficacy.

Phenserine (PS) was designed as a selective acetylcholinesterase (AChE) inhibitor, with a tartrate form (PST) for oral administration in mild to moderate Alzheimer's disease (AD). Recent phase 3 trials of PST in Europe indicate that any clinically relevant activity of PST may be limited by its duration of action. Like many oral drugs, bioavailability and plasma concentrations of PST are regulated by hepatic and gastrointestinal first-pass effects. To minimize the kinetic limitations of first-pass metabolism, transdermal formulations of PS and PST (ointment/patch) were developed and characterized in vitro and in vivo. Initial in vitro kinetic characterization of PS or PST formulations used a diffusion cell chamber and skin samples isolated from hairless mice. Liquid paraffin and fatty alcohol/propylene glycol (FAPG) were found to be suitable vehicles for ointment formulation. Addition of a penetration enhancer, 1-[2-(decylthio)ethyl]-azacyclopentane-2-one (HPE-101), improved stratum corneum permeability. Application of the optimal formulation of PS/HPE-101/FAPG to the shaved back of rats resulted in significantly lowered plasma and brain AChE activities and improved cognitive performance in animals with scopolamine-induced cognitive impairment. These results suggest that the transdermal application of AChE inhibitors may represent an effective therapeutic strategy for AD. Particular benefits over oral therapies might include avoiding first-pass metabolic effects and improved dosing compliance.

Calorie restriction mimetics: an emerging research field.

When considering all possible aging interventions evaluated to date, it is clear that calorie restriction (CR) remains the most robust. Studies in numerous species have demonstrated that reduction of calories 30-50% below ad libitum levels of a nutritious diet can increase lifespan, reduce the incidence and delay the onset of age-related diseases, improve stress resistance, and decelerate functional decline. A current major focus of this research area is whether this nutritional intervention is relevant to human aging. Evidence emerging from studies in rhesus monkeys suggests that their response to CR parallels that observed in rodents. To assess CR effects in humans, clinical trials have been initiated. However, even if results from these studies could eventually substantiate CR as an effective pro-longevity strategy for humans, the utility of this intervention would be hampered because of the degree and length of restriction required. As an alternative strategy, new research has focused on the development of 'CR mimetics'. The objective of this strategy is to identify compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. For example, drugs that inhibit glycolysis (2-deoxyglucose), enhance insulin action (metformin), or affect stress signaling pathways (resveratrol), are being assessed as CR mimetics (CRM). Promising results have emerged from initial studies regarding physiological responses which resemble those observed in CR (e.g. reduced body temperature and plasma insulin) as well as protection against neurotoxicity (e.g. enhanced dopamine action and up-regulated neurotrophic factors). Ultimately, lifespan analyses in addition to expanded toxicity studies must be accomplished to fully assess the potential of any CRM. Nonetheless, this strategy clearly offers a very promising and expanding research endeavor.

Transient improvement in cognitive function and synaptic plasticity in rats following cancer chemotherapy.

BACKGROUND: Cancer chemotherapy has been associated with cognitive impairment. Several issues complicate such findings including the patients' health, use of multiple chemotherapeutic agents, and proper assessment of cognition. To control these factors, we conducted cognitive studies in female rats receiving cyclophosphamide or 5-fluorouracil (5FU). METHODS: Young (7 months) female Fischer-344 rats received five injections of cyclophosphamide (100 mg/kg), 5FU (150 mg/kg), or saline i.p. every 4 weeks for a total of 18 weeks. Aged (18 months) female Fischer-344 rats were treated with cyclophosphamide (80 mg/kg i.p.) for 16 weeks. After 8 to 10 weeks of recovery, rats were tested in two maze learning tasks, the Morris water maze and the Stone 14-unit T-maze. Neuronal synaptic function was assessed by examining long-term potentiation (LTP) in hippocampal slices obtained from young cyclophosphamide-treated rats. RESULTS: Despite the toxic effects induced by chemotherapy, cyclophosphamide- and 5FU-treated rats showed significantly better maze performance compared with controls. Following 29 to 42 weeks of recovery from chemotherapy, no significant effects were observed on maze performance. In aged rats, cyclophosphamide treatment for 14 weeks also produced toxicity, but no impairment in Stone maze learning after 16 weeks of recovery. When assessed during cyclophosphamide treatment, evidence of impaired LTP emerged; however, with 8 weeks of recovery following five cyclophosphamide treatments, we observed enhanced LTP. CONCLUSION: Despite toxicity accompanying chemotherapy, no evidence of impaired cognitive performance emerged after recovery. Indeed, following 7 to 9 weeks of recovery, we noted evidence of improved learning and LTP.

Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide in rodent.

Like acetylcholinesterase, butyrylcholinesterase (BChE) inactivates the neurotransmitter acetylcholine (ACh) and is hence a viable therapeutic target in Alzheimer's disease, which is characterized by a cholinergic deficit. Potent, reversible, and brain-targeted BChE inhibitors (cymserine analogs) were developed based on binding domain structures to help elucidate the role of this enzyme in the central nervous system. In rats, cymserine analogs caused long-term inhibition of brain BChE and elevated extracellular ACh levels, without inhibitory effects on acetylcholinesterase. In rat brain slices, selective BChE inhibition augmented long-term potentiation. These compounds also improved the cognitive performance (maze navigation) of aged rats. In cultured human SK-N-SH neuroblastoma cells, intra- and extracellular beta-amyloid precursor protein, and secreted beta-amyloid peptide levels were reduced without affecting cell viability. Treatment of transgenic mice that overexpressed human mutant amyloid precursor protein also resulted in lower beta-amyloid peptide brain levels than controls. Selective, reversible inhibition of brain BChE may represent a treatment for Alzheimer's disease, improving cognition and modulating neuropathological markers of the disease.

Development of calorie restriction mimetics as a prolongevity strategy.

By applying calorie restriction (CR) at 30-50% below ad libitum levels, studies in numerous species have reported increased life span, reduced incidence and delayed onset of age-related diseases, improved stress resistance, and decelerated functional decline. Whether this nutritional intervention is relevant to human aging remains to be determined; however, evidence emerging from CR studies in nonhuman primates suggests that response to CR in primates parallels that observed in rodents. To evaluate CR effects in humans, clinical trials have been initiated. Even if evidence could substantiate CR as an effective antiaging strategy for humans, application of this intervention would be problematic due to the degree and length of restriction required. To meet this challenge for potential application of CR, new research to create "caloric restriction mimetics" has emerged. This strategy focuses on identifying compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR, but without actually restricting caloric intake. Microarray studies show that gene expression profiles of key enzymes in glucose (energy) handling pathways are modified by CR. Drugs that inhibit glycolysis (2-deoxyglucose) or enhance insulin action (metformin) are being assessed as CR mimetics. Promising results have emerged from initial studies regarding physiological responses indicative of CR (reduced body temperature and plasma insulin) as well as protection against neurotoxicity, enhanced dopamine action, and upregulated brain-derived neurotrophic factor. Further life span analyses in addition to expanded toxicity studies must be completed to assess the potential of any CR mimetic, but this strategy now appears to offer a very promising and expanding research field.

The influence of rapid-rate transcranial magnetic stimulation (rTMS) parameters on rTMS effects in Porsolt's forced swimming test.

To assess the similarity of the behavioural effects of the rapid transcranial magnetic stimulation (rTMS) to those produced by other antidepressant treatments, in particular to repeated electroconvulsive shock (ECS), we carried out experiments on Wistar rats. The effects of a standard ECS procedure (9 daily treatments; the current parameters: 150 mA, 50 Hz, 0.5 s) were compared with 18 d treatment with rTMS of the same field intensity of 1.6 T but with different stimulation frequency (20 or 30 Hz) and a different number of sessions (9 or 18). Twenty-four hours after the last treatment the forced swimming test was carried out and the immobility time was recorded. The standard ECT reduced the immobility by 50%, the intensive rTMS (90 or 104 K impulses for the whole period of treatment) caused a significant effect, although smaller than that induced by ECT (reduction by 20-30%). The stimulation at 20 Hz required 18 treatment sessions to produce a significant effect, while only 9 sessions with stimulation at 30 Hz were sufficient to produce a comparable result. This suggests that the effectiveness of rTMS may be augmented by increasing the number or frequency of rTMS impulses.