Perinatal exposure to octabromodiphenyl ether mixture, DE-79, alters the vasopressinergic system in adult rats.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants considered as neurotoxicants and endocrine disruptors with important biological effects ranging from alterations in growth, reproduction, and effects on the hypothalamus-pituitary-adrenal axis. The vasopressinergic (AVPergic) system is a known target for pentaBDEs mixture (DE-71) and the structurally similar chemicals, polychlorinated biphenyls. However, the potential adverse effects of mixtures containing octaBDE compounds, like DE-79, on the AVPergic system are still unknown. The present study aims to examine the effects of perinatal DE-79 exposure on the AVPergic system. Dams were dosed from gestational day 6 to postnatal day 21 at doses of 0 (control), 1.7 (low) or 10.2 (high) mg/kg/day, and male offspring from all doses at 3-months-old were subjected to normosmotic and hyperosmotic challenge. Male offspring where later assessed for alterations in osmoregulation (i.e. serum osmolality and systemic vasopressin release), and both vasopressin immunoreactivity (AVP-IR) and gene expression in the hypothalamic paraventricular and supraoptic nuclei. Additionally, to elucidate a possible mechanism for the effects of DE-79 on the AVPergic system, both neuronal nitric oxide synthase immunoreactivity (nNOS-IR) and mRNA expression were investigated in the same hypothalamic nuclei. The results showed that perinatal DE-79 exposure AVP-IR, mRNA expression and systemic release in adulthood under normosmotic conditions and more evidently under hyperosmotic stimulation. nNOS-IR and mRNA expression were also affected in the same nuclei. Since NO is an AVP regulator, we propose that disturbances in NO could be a mechanism underlying the AVPergic system disruption following perinatal DE-79 exposure leading to osmoregulation deficits.

Safety of the Transcranial Focal Electrical Stimulation via Tripolar Concentric Ring Electrodes for Hippocampal CA3 Subregion Neurons in Rats.

Epilepsy is a neurological disorder that affects approximately one percent of the world population. Noninvasive electrical brain stimulation via tripolar concentric ring electrodes has been proposed as an alternative/complementary therapy for seizure control. Previous results suggest its efficacy attenuating acute seizures in penicillin, pilocarpine-induced status epilepticus, and pentylenetetrazole-induced rat seizure models and its safety for the rat scalp, cortical integrity, and memory formation. In this study, neuronal counting was used to assess possible tissue damage in rats (n = 36) due to the single dose or five doses (given every 24 hours) of stimulation on hippocampal CA3 subregion neurons 24 hours, one week, and one month after the last stimulation dose. Full factorial analysis of variance showed no statistically significant difference in the number of neurons between control and stimulation-treated animals (p = 0.71). Moreover, it showed no statistically significant differences due to the number of stimulation doses (p = 0.71) nor due to the delay after the last stimulation dose (p = 0.96). Obtained results suggest that stimulation at current parameters (50 mA, 200 µs, 300 Hz, biphasic, charge-balanced pulses for 2 minutes) does not induce neuronal damage in the hippocampal CA3 subregion of the brain.

Perinatal exposure to organohalogen pollutants decreases vasopressin content and its mRNA expression in magnocellular neuroendocrine cells activated by osmotic stress in adult rats.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that produce neurotoxicity and neuroendocrine disruption. They affect the vasopressinergic system but their disruptive mechanisms are not well understood. Our group reported that rats perinatally exposed to Aroclor-1254 (A1254) and DE-71 (commercial mixtures of PCBs and PBDEs) decrease somatodendritic vasopressin (AVP) release while increasing plasma AVP responses to osmotic activation, potentially emptying AVP reserves required for body-water balance. The aim of this research was to evaluate the effects of perinatal exposure to A1254 or DE-71 (30mgkg/day) on AVP transcription and protein content in the paraventricular and supraoptic hypothalamic nuclei, of male and female rats, by in situ hybridization and immunohistochemistry. cFOS mRNA expression was evaluated in order to determine neuroendocrine cells activation due to osmotic stimulation. Animal groups were: vehicle (control); exposed to either A1254 or DE-71; both, control and exposed, subjected to osmotic challenge. The results confirmed a physiological increase in AVP-immunoreactivity (AVP-IR) and gene expression in response to osmotic challenge as reported elsewhere. In contrast, the exposed groups did not show this response to osmotic activation, they showed significant reduction in AVP-IR neurons, and AVP mRNA expression as compared to the hyperosmotic controls. cFOS mRNA expression increased in A1254 dehydrated groups, suggesting that the AVP-IR decrease was not due to a lack of the response to the osmotic activation. Therefore, A1254 may interfere with the activation of AVP mRNA transcript levels and protein, causing a central dysfunction of vasopressinergic system.

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254.

Stimulated vasopressin (VP) release from magnocellular neuroendocrine cells in the supraoptic nucleus (SON) of hyperosmotic rats is inhibited by treatment with the industrial polychlorinated biphenyl (PCB) mixture, Aroclor 1254. Because VP responses to hyperosmotic stimulation are regulated by nitric oxide (NO) signaling, we studied NO synthase (NOS) activity in the SON of hyperosmotic rats as potential target of PCB-induced disruption of neuroendocrine processes necessary for osmoregulation. To examine PCB-induced changes in NOS activity under normosmotic and hyperosmotic conditions, male Sprague-Dawley rats were exposed to Aroclor 1254 (30mg/kg/day) in utero and NADPH-diaphorase (NADPH-d) activity was assessed in SON sections at three ages: postnatal day 10, early adult (3-5 months) or late adult (14-16 months). Hyperosmotic treatment increased mean NADPH-d staining density of oil hyperosmotic controls by 19.9% in early adults and 58% in late adulthood vs normosmotic controls. In utero exposure to PCBs reduced hyperosmotic-induced upregulation of NADPH-d activity to control levels in early adults and by 28% in late adults. Basal NADPH-d was reduced in postnatal rats. Rats receiving PCB exposure as early adults orally for 14 days displayed normal responses. Our findings show that developmental but not adult exposure to PCBs significantly reduces NOS responses to hyperosmolality in neuroendocrine cells. Moreover, reduced NADPH-d activity produced by in utero exposure persisted in stimulated late adult rats concomitant with reduced osmoregulatory capacity vs oil controls (375±9 vs 349±5mOsm/L). These findings suggest that developmental PCBs permanently compromise NOS signaling in the activated neuroendocrine hypothalamus with potential osmoregulatory consequences.

Contaminantes ambientales neurotóxicos cercanos a nuestra vida diaria / Neurotoxic enviromental contaminants near our daily life

Chemical substances play an important role in life quality; they are present in household items and consumer products like furniture, cloths, toys, etc. However some of these substances could be dangerous for health and environment. Among the best known are the organohalogens pollutants like the polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). These substances persist in the enviroment, they bioaccumulate and may cause adverse effects in humans health. There is increasing evidence that organohalogens cause neurotoxicity in rats and human brains during development. We have been studying the neurotoxicity of Aroclor 1254 (PCB mixture) and DE-71 (PBDE mixture) in collaboration with Currás-Collazo team (UC-MEXUS/CONACYT grants). We show in this paper the principal results of our research related with molecules that participate in the osmoregulatory system, learning and memory as vasopressin, PACAP and nitric oxide synthase. We exposed pregnant rats to this organohalogens perinatally; the pups were allowed to grow until three months old for an osmotic challenge. Brains were processed for immunfloures-cence, other group was used to evaluate memory with the passive inhibitory avoidance test and Western-blot was done for presynaptic proteins Synapsin I and Synaptophysin. We found a disruption in the content of VP, PACAP and nNOS suggesting that the PCB and PBDE exposure alter the function of hypothalamic neurons that regulates osmosis and water balance. We demonstrated also that PBDE treatment modifies systolic pressure and plasmatic osmolality compared with controls suggesting a cardiovascular alteration caused by PBDEs. We found an alteration in the nNOS activity in Aroclor-1254 treated rats. Memory test and presynaptic proteins expression showed an important reduction in males, suggesting that PCBs alters the expression and activity of nitric oxide and learning and memory. Therefore, due to the neurotoxicity of the organohalogens and its constant contact with humans there is a big concern about the lack of adequate legislation in Mexico and monitoring programs to evaluate the degree of contamination in the population, especially in infants as well as the regions most affected by such contamination.

Las sustancias químicas son importantes en nuestra calidad de vida; éstas están presentes en artículos domésticos y de consumo humano. Algunas son nocivas para la salud y el medio ambiente, como los contaminantes organohalogenados, los bifenilos policlorinados (PCB) y los éteres difenílicos polibrominados (PBDE). Existe evidencia de su neurotoxicidad en las ratas y los humanos sobre todo cuando la exposición es durante el desarrollo. Nuestro grupo se ha interesado en estudiar la neurotoxicicidad de los PCB y PBDE sobre la regulación del equilibrio hidroelectrolítico, el aprendizaje y la memoria, en colaboración con la doctora Currás-Collazo. En este artículo presentamos los hallazgos principales de estos estudios. Expusimos a ratas gestantes a estos organohalogenados y las crías se estudiaron a los tres meses de edad; se sometieron al modelo de estrés osmótico o a la prueba de aprendizaje y memoria (evitación pasiva). Los cerebros se procesaron para inmunofluorescencia para VP, nNOS, PACAP o histoquímica de la NADPH-d, Western-blot para nNOS y las proteínas presinápticas sinapsina I y sinaptofisina. Nuestros resultados mostraron en las ratas tratadas con los PCB y PBDE sometidas a estrés osmótico alteraciones en el contenido de VP, PACAP y NOS y un incremento en la presión sistólica y la osmolaridad plasmática al compararla con controles, sugiriendo que los PBDE alteran la función cardiovascular y osmorregulatoria. La prueba de aprendizaje mostró una disminución significativa de la adquisición y/o consolidación del aprendizaje y memoria en las ratas macho tratadas y alteraciones en la actividad de la NOS, la expresión de la nNOS y las sinapsina y sinaptofisina, lo que sugiere que la exposición perinatal a los PCB altera el aprendizaje y la memoria. Debido a la neurotoxicidad de los organohalogenados y a que estamos expuestos a ellos en nuestra vida diaria existe una gran preocupación por la falta de una legislación adecuada en México y programas de monitoreo para evaluar el grado de contaminación en la población mexicana especialmente en los infantes, así como las regiones más afectadas por dicha contaminación.

Toward a noninvasive automatic seizure control system in rats with transcranial focal stimulations via tripolar concentric ring electrodes.

Epilepsy affects approximately 1% of the world population. Antiepileptic drugs are ineffective in approximately 30% of patients and have side effects. We are developing a noninvasive, or minimally invasive, transcranial focal electrical stimulation system through our novel tripolar concentric ring electrodes to control seizures. In this study, we demonstrate feasibility of an automatic seizure control system in rats with pentylenetetrazole-induced seizures through single and multiple stimulations. These stimulations are automatically triggered by a real-time electrographic seizure activity detector based on a disjunctive combination of detections from a cumulative sum algorithm and a generalized likelihood ratio test. An average seizure onset detection accuracy of 76.14% was obtained for the test set (n = 13). Detection of electrographic seizure activity was accomplished in advance of the early behavioral seizure activity in 76.92% of the cases. Automatically triggered stimulation significantly (p = 0.001) reduced the electrographic seizure activity power in the once stimulated group compared to controls in 70% of the cases. To the best of our knowledge this is the first closed-loop automatic seizure control system based on noninvasive electrical brain stimulation using tripolar concentric ring electrode electrographic seizure activity as feedback.

Los contaminantes ambientales bifenilos policlorinados (PCB) y sus efectos sobre el Sistema Nervioso y la salud / The polychlorinated biphenyls (PCBS) environmental pollutants and their effects on the Nervous System and health

Environmental pollution is a world-wide issue which is a matter for concern among the international community. Great industrialized cities are the most polluted and Mexico City is among them. However, pollution affects places which are far away from contaminated urban areas, thus damaging eco-systems. Environmental pollution is responsible for an alarming and increasing list of illnesses in humans, animals and plants. This has generated an international interest in this problem. From the 187 chemical agents considered toxic for living organisms, the Inter-Government Committee for the Negotiation of Persistent Organic Pollutants (Pops) has catalogued 12 as the most hazardous for life. Among them are the so-called polychlorinated biphenyls (PCBs). PCBs are a family of 209 structurally chlorinated compounds made up of chlorine, carbon and hydrogen. These compounds are chemically and thermally stable, insoluble in water, non-flammable, electrically resistant, with low volatility at normal temperatures, and bio-degradable only at high temperatures (1200°C). One of their main disadvantages is that they are subject to a process of bioaccumulation where their concentration increases along the food chain. Their physical properties make them widely used in industry, mainly in the electrical and building areas. Not long after PCBs were manufactured, it was determined that food for human intake such as milk, fish and eggs, to mention just a few, presented higher PCBs concentrations than those allowed by the Organism for US Environmental Protection (0.0005mg/l). It has been demonstrated that PCBs can cause damage to the endocrine, immunologic and Nervous Systems, among others. The underlying mechanism of action of these compounds is through the activation of the aril hydrocarbon receptor (AhR), a ligand-dependent cytosolic transcription factor. PCBs act like ligands and, given their lipophilic properties, enter cells by passive diffusion. Two co-chaperone proteins are bound to AhR to form an oligomer which dissociates when binding to a PCB. After ligand binding, a heterodimer is formed which translocates into the nucleus and links to specific DNA regions; this in turn regulates the transcription velocity of specific genes and produces genetic alterations that modify processes and functions in the cell. PCBs belong in the group of chemicals considered endocrine disruptors. Damage caused by these compounds can be irreversible. In the endocrine system they interfere with the production and regulation of steroid and thyroid hormones, acting as agonists or antagonists of hormone receptors. They impair endocrine metabolic pathways, such as those of thyroid hormones (T3 and T4), and inhibit carrier proteins such as transthyretin. Contaminants that harm the endocrine system also affect the reproductive function and disrupt various aspects of sexuality. In males, PCBs inhibit the synthesis of testosterone, alter masculinity, reduce sperm motility and the capacity of binding and penetrating the ovule, induce changes in the shape of the penis as well as its size, retard or inhibit testicle descent, and can generate testicular cancer. In females, they can cause early menarche (first menstruation), enhanced duration of menstrual bleeding, urogenital malformations, endometriosis, spontaneous abortion, fetal death, premature delivery and low-weight in offspring. Our group, as well as other research groups, has encountered that PCB administration to gestating rats causes an increment in offspring mortality, fetal miscarriages, low bodily weight of the offspring and a reduction in the number of males per litter. The immunological system is sensitive to chemicals such as PCBs which originate an immunological response; they act as immunotoxins that cause thymus atrophy, affect innate immunity, compromise host resistance and immunity mediated by B and T cells, as well as humoral immunity. PCBs and their metabolites are carcinogenic and act as general cancer promoters by enhancing the effects of other substances through the generation of oxygen reactive compounds that can induce DNA oxidative damage. Chronic PCB exposure can cause chromosomal aberrations; these compounds have been related to all types of cancer: mammary gland, liver, biliary tract, gastrointestinal, skin (especially malignant melanomas), lung, pancreas and brain. There is evidence that organisms are more vulnerable to PCB exposure during the early embryonic stages. These compounds can cross the placenta and affect the fetus; when they are present in human milk they keep the offspring under high PCB levels thus altering development. In addition, they can contribute to the interruption of growth and development of brain, organs and tissues. As a result, malfunctions or miscarriage occur. PCBs are involved in the neurodegeneration process since they affect dopaminergic neurons in caudate nucleus, ventral tegmental area and substantia nigra. These compounds disrupt neuronal mechanisms such as vesicular transport and dopamine release which lead to cellular death similar to that described for diseases such as Parkinson's. Perinatal exposure to PCBs is associated with neurodevelopmental deficiencies of infants which consist of dysfunctions at the neuropsychological level such as in verbal learning (syllables, words and concepts), performance functions, changes in attention and psychomotor development. Acute or chronic exposure to PCBs is associated with cephalea, insomnia, nervousness, irritability, depression and anxiety; these symptoms in turn modify behavior. At the neurophysiological level, these contaminants impair excitatory and inhibitory synaptic transmission in the hippocampus, inhibit long-term potentiation and synaptic plasticity, alter some mechanisms of cell signaling (GABAergic pathway), and deteriorate learning and memory. Recently, these compounds have been related to cognitive alterations. Our group demonstrated that the administration of PCB-77 and Aroclor 1254 during gestation inhibits the enzymatic activity of nitric oxide synthase (NOS) in 10-day postnatal pups. These rats presented degenerative morphological neuronal changes such as shrinking, picnosis, loss of neurites, neuronal death and decrease in the number of nitrergic neurons in the paraventricular and supraoptic hypothalamic nuclei. We also reported that in these nuclei a decrease in immunoreactivity to vasopressin and neuronal NOS is observed. The evidence in PCB studies is conclusive. The exposure to these environmental toxins interferes with the functioning of various organs and systems such as the endocrine and Nervous Systems, not only in humans but also in animals. These contaminants pose a risk factor for a wide number of neurodegenerative alterations.

La contaminación ambiental es un grave problema mundial que actualmente preocupa a la comunidad internacional. Las grandes ciudades industrializadas, como la de México, son las más contaminadas. Sin embargo, la contaminación llega hasta zonas alejadas de donde se produce y afecta los ecosistemas. La contaminación es responsable de una alarmante y creciente lista de enfermedades en el hombre, los animales y las plantas. Los bifenilos policlorados (PCB) se catalogaron dentro de los 12 contaminantes orgánicos más tóxicos para los organismos vivos. Sus propiedades físicas hicieron que se usaran ampliamente en la industria. No son biodegradables y se acumulan en el ambiente, se transfieren dentro de la cadena alimenticia y tienden a concentrarse más al final de ésta, por lo que en los alimentos se determinaron concentraciones que sobrepasaban los límites establecidos por el Organismo de Protección del Ambiente de los Estados Unidos. Se demostró que los PCB afectan la función de los sistemas endocrino, inmunológico y nervioso, entre otros. El mecanismo de acción descrito para los PCB, es por medio de la activación del receptor aril hidrocarburo, un factor de transcripción citosólico dependiente de ligando. Los PCB actúan como ligandos y son lipofílicos, por lo que entran a la célula y llegan al núcleo para unirse al ADN, lo cual altera la trascripción de genes específicos y provoca alteraciones genéticas que conducen a cambios en los procesos y funciones celulares. Los PCB interfieren con la producción y regulación de las hormonas esteroides y tiroideas al actuar como antagonistas o agonistas de los receptores hormonales. Afectan la función reproductora y alteran diferentes aspectos de la sexualidad. Como otros grupos de investigación, el nuestro ha observado que la administración de PCB a ratas gestantes causa un incremento de la mortalidad de las crías, pérdida fetal, peso corporal bajo y una reducción en el número de machos por camada. Los PCB actúan como inmunotoxinas que causan la atrofia del timo y afectan la respuesta inmune. Los PCB y sus metabolitos son carcinogénicos debido a la generación de especies reactivas de oxígeno que pueden producir daño oxidativo al ADN, provocar aberraciones cromosómicas y generar cáncer de mama, hígado, tracto biliar, gastrointestinal, cerebral, etc. Los organismos son más vulnerables a la exposición de los PCB durante las etapas tempranas del desarrollo embrionario. Los PCB atraviesan la placenta y llegan al feto, permanecen en la leche materna y mantienen niveles altos en las crías. Los PCB afectan así el desarrollo del Sistema Nervioso, los órganos y los tejidos, y pueden llevar a la pérdida fetal. También se asocian a deficiencias en el neurodesarrollo del niño y a alteraciones neuropsicológicas en la atención, el aprendizaje y el desarrollo psicomotor. La exposición aguda o crónica a los PCB se asocia con cefalea, insomnio, nerviosismo, irritabilidad, depresión y ansiedad. Los PCB participan en el proceso de neurodegeneración al afectar el sistema dopaminérgico. En el nivel neurofisiológico, afectan la transmisión sináptica excitatoria e inhibitoria hipocampal; inhiben la potenciación a largo plazo y la plasticidad sináptica; alteran mecanismos de señalización celular como el GABAérgico, en el aprendizaje y la memoria, y producen alteraciones cognoscitivas. Nuestro grupo demostró que la administración de los PCB durante la gestación inhibe la actividad de la enzima sintasa del óxido nítrico y provoca cambios neuronales morfológicos degenerativos en los núcleos paraventricular y supraóptico hipotalámicos. Las evidencias de los estudios realizados con los PCB son concluyentes en cuanto a que la exposición a estos tóxicos ambientales interfiere con el funcionamiento de diferentes órganos y sistemas y a que son un factor de riesgo para un amplio número de alteraciones neurodegenerativas. Actualmente, las poblaciones están expuestas a concentraciones que exceden los niveles límite tolerables recomendados por la Organización Mundial de la Salud. Nuestro grupo está analizando las alteraciones de estos contaminantes en el nivel neuroendocrino y en algunos aspectos del aprendizaje y la memoria. Dada la relevancia de los efectos de los PCB en la salud y de la falta en México de una valoración de los niveles de los PCB existentes en personas y alimentos, es importante que las instituciones de salud fomenten y apoyen las investigaciones en esta área.

El Receptor ORL-1 y su péptido endógeno, La nociceptina/orfanina FQ: nuevos miembros de la familia de los opioides / The opoid receptor like-1 and its endogenous peptide nociceptin/orphanin FQ: new members of the opioid family

A la clonación de receptores opioides d, µ y ? siguió la identificación y secuenciación de otro miembro de la familia opioide, el receptor ORL-1 (por sus siglas en ingles, opioid receptor like-1). No obstante, su gran homología con receptores µ, d y ?, los ligandos opioides endógenos, como la ß-endorfina, la dinorfina A y las encefalinas, no presentan gran afinidad por este receptor. Después del descubrimiento del ORL-1, se aisló su péptido endógeno; éste tiene gran afinidad (nanomolar) para su receptor. Dos grupos de investigación independientes reportaron con este péptido: el grupo de Meunier lo llamó nociceptina y el grupo de Reinscheid, orfanina FQ. El ORL-1 y la nociceptina/orfanina FQ (N/OFQ) tienen una distribución anatómica amplia en el sistema nervioso, principalmente en los núcleos olfatorios, corteza cerebral, hipocampo, región septal, ganglios basales, tálamo, hipotálamo, puente, bulbo, pedúnculos cerebrales, médula espinal y retina, lo que evidencia su participación en diversas funciones cerebrales. La administración de la N/OFQ puede producir hiperalgesia, alodinia, efectos antiopioides o analgesia. Estos efectos dependen básicamente de la dosis y de la vía de administración. Además, alteraciones en la locomoción, en el comportamiento exploratorio motivacional, en la ansiedad, memoria, alimentación y modulación neuroendocrina. La N/OFQ se deriva de un precursor de alto peso molecular, la prepronociceptina (PPNOC), el cual contiene una sola copia de la secuencia de aminoácidos de la N/OFQ. El gen de la PPNOC está conservado en cinco especies animales estudiadas (rata, ratón, porcino, bovino y humano) y se expresa predominantemente en hipotálamo mesencéfalo, núcleos del lemnisco lateral, oliva superior, núcleo trigeminal espinal y médula espinal. Debido al reciente descubrimiento de este péptido y su receptor, además de su amplia distribución en el sistema nervioso resulta obvio que su participación en la fisiología y sus probables implicaciones farmacológicas todavía no están completas. La presencia de la N/OFQ en especies de los primeros niveles de la escala filogenética como son los moluscos Helix aspersa, H. pomatia y Cepaea nemoralis, donde se demostró también que la administración de N/OFQ tiene un efecto pronociceptivo similar a la hiperalgesia de los mamíferos, sugieren que este nuevo sistema opioide tiene un origen filogenético temprano y una continuidad funcional durante el curso de la evolución