Carlos A. Beltramino

Detection of conspecific pheromones elicits fos expression in GABA and calcium-binding cells of the rat vomeronasal system–medial extended amygdala

The olfactory accessory system is specialized in the detection of pheromones, being an afferent to medial extended amygdala. In spite of the fact that numerous phenotypes are found in these structures, in the current literature, there are no detailed descriptions about the phenotype of neurons in the vomeronasal system–medial extended amygdale after their activation by pheromonal stimuli. Using immunohistochemistry for fos and dual immunohistochemistry for fos and phenotypes, here we show that females have a greater number of activated neurons by the pheromonal stimulus. Likewise, a great colocalization of fos with GABA, calretinin, and calbindin was observed in the vomeronasal system–medial extended amygdala. These data suggest that in amygdaloid areas, neuronal excitability is controlled by GABAergic neurons that contain different calcium-binding proteins, indicating the important role of inhibitory control on the incoming sensory pheromonal and olfactory inputs controlled and processed by the vomeronasal system.

Timed changes of synaptic zinc, synaptophysin and MAP2 in medial extended amygdala of epileptic animals are suggestive of reactive neuroplasticity.

Repeated seizures induce permanent alterations of the brain in experimental models and patients with intractable temporal lobe epilepsy (TLE), which is a common form of epilepsy in humans. Together with cell loss and gliosis in many brain regions, synaptic reorganization is observed principally in the hippocampus. However, in the amygdala this synaptic reorganization has been not studied. The changes in Zn density, synaptophysin and MAP(2) as markers of reactive synaptogenesis in medial extended amygdala induced by kainic acid (KA) as a model of TLE was studied. Adult male rats (n=6) were perfused at 10 days, 1, 2, 3 and 4 months after KA i.p. injection (9 mg/kg). Controls were injected with saline. The brains were processed by the Timms method to reveal synaptic Zn and analyzed by densitometry. Immunohistochemistry was used to reveal synaptophysin and MAP(2) expression. A two-way ANOVA was used for statistics, with a P<0.05 as a significance limit. Normal dark staining was seen in all medial extended amygdala subdivisions of control animals. At 10 days post KA injection a dramatic loss of staining was observed. A slow but steady recovery of Zn density can be followed in the 4 month period studied. Parallel, from 10 days to 2 months stronger synaptophysin expression could be observed, whereas MAP(2) expression increased from 1 month with peak levels at 3-4 months. The results suggest that a process of sprouting exists in surviving neurons of medial extended amygdala after status epilepticus and that these neurons might be an evidence of a reactive synaptogenesis process.

Alcohol-induced neuronal death in central extended amygdala and pyriform cortex during the postnatal period of the rat.

Mothers who consume alcohol during pregnancy may cause a neurotoxic syndrome defined as fetal alcohol spectrum disorder (FASD) in their offspring. This disorder is characterized by reduction in brain size, cognitive deficits and emotional/social disturbances. These alterations are thought to be caused by an alcohol‐induced increase in apoptosis during neurodevelopment. Little is known about neuroapoptosis in the central extended amygdala and the pyriform cortex, which are key structures in emotional/social behaviors. The goal of this study was to determine the vulnerability of neuroapoptotic alcohol effects in those areas. Rats were administered alcohol (2.5 g/kg s.c. at 0 and 2 h) or saline on postnatal day (PND) 7, 15 and 20. The Amino‐cupric‐silver technique was used to evaluate neurodegeneration and immunohistochemistry to detect activated caspases 3–8 and 9 at 2 h, 4, 6, 8, 12 and 24 h after drug administration. We measured blood alcohol levels each hour, from 2 to 8 h post second administration of alcohol in each of the ages studied. Results showed alcohol induced apoptotic neurodegeneration in the central extended amygdala on PND 7 and 15, and pyriform cortex on PND 7, 15 and 20. These structures showed activation of caspase 3 and 9 but not of caspase 8 suggesting that alcohol‐induced apoptosis could occur by the intrinsic pathway. The pharmacokinetic differences between ages did not associate with the neurodegeneration age dependence. In conclusion, these limbic areas are damaged by alcohol, and each one has their own window of vulnerability during the postnatal period. The possible implications in emotional/social features in FASD are discussed.

Kainic acid-induced early genes activation and neuronal death in the medial extended amygdala of rats.

The medial extended amygdala modulates pheromonal perception, influencing emotional and social behavior. As the amygdala is part of neuronal circuits that are very sensitive to excitability, its neurons are targets of seizures in temporal lobe epilepsy. It has been suggested that the hippocampus is strongly involved this pathology. There is less consistent information, however, on the effects of this disease in the amygdala. The effects of status epilepticus on the medial extended amygdala were analyzed by immunohistochemistry for neural stress and by the amino-cupric-silver technique for neuronal death in rats after kainic acid (KA) administration. Sixty adult Wistar male rats were used. Thirty animals received an injection of KA, and 30 were injected with saline. After 2, 4, 12, 24 and 48 h survival the brains were stained for Fos and FosB and for neuronal death. In the present study we show that KA induces Fos and FosB expression in neurons of the medial extended amygdala after 2, 4-48 h, with time courses that are different between them and from control animals. While Fos-IR peaks at 2-4 h post KA and then decreases, FosB-IR increases in the same period reaching its highest expression at 24-48 h. Moreover, KA injection produced massive neuronal death with a peak at 24 h. This neurodegeneration paralleled FosB-IR protein expression. These findings show that KA produces neuronal stress and activation of early genes and neuronal death in the medial extended amygdala, demonstrating the vulnerability of its neurons to the epileptogenic effects of KA.

Effect of sex differences and gonadal hormones on kainic acid-induced neurodegeneration in the bed nucleus of the stria terminalis of the rat

Previously we have demonstrated that medial nucleus of the amygdala, which is part of medial extended amygdala, is damaged by status epilepticus induced by kainic acid (KA) and this neurodegeneration was prevents by estrogen replacement. The medial bed nucleus of stria terminalis (BSTM) also belong to medial extended amygdala and it is uncertain whether the gonadal hormones are protective or not against this neurotoxicity in the BSTM. Here we show that a single i.p. injection of KA (9 mg/kg) induces neurodegeneration in the subnuclei of the BSTM of rats with different degrees of intensity in males and females. A differential neuroprotective effect of the gonadal hormones was also observed. In diestrous rats, massive neuronal death similar to that in the ovariectomized females was detected. BSTM neurons of proestrous rats, like the ovariectomized treated with estrogen, were significantly less affected by the KA. Testosterone produced a mild neuroprotective action, but dihydrotestosterone did not protect. A similar pattern was observed in all male groups. This results show that estrogen protects BSTM neurons from KA neurotoxicity and androgens are partially neuroprotective; and probably this effect of androgens is due to conversion to estrogen.

Binge alcohol-induced alterations in BDNF and GDNF expression in central extended amygdala and pyriform cortex on infant rats.

Mothers who consume alcohol during pregnancy may cause a neurotoxic syndrome termed fetal alcohol spectrum disorder (FASD) in the offspring, which includes cognitive deficits and emotional/social disturbances. These alterations are thought to be caused, at least in part, by alcohol‐induced imbalance in neurotrophic factor levels, which are critically involved in normal neurodevelopment. Our goal was to study whether brain‐derived neurotrophic factor (BDNF) and glial‐derived neurotrophic factor (GDNF) expression were affected by alcohol in central extended amygdala (CEXA) and pyriform cortex (Pyr), structures strongly involved in emotional/social behaviors. Further, we evaluated how these changes could be related to blood and brain alcohol concentrations. Postnatal day (PND) pups at 7, 15 and 20‐days old were administered alcohol (2.5 g/kg s.c. at 0 and 2 h) or saline. Immunohistochemistry was used to detect the expression of BDNF and GDNF at 2, 12 and 24 h after drug administration. Also, gas chromatography was bused to measure blood alcohol levels (BALs) and brain alcohol levels (BrALs) at each hour, from 2 to 8 h after the second alcohol administration. Results showed: (1) alcohol‐induced enhancement of BDNF positive cells on PND 7 and 20, a decrease on PND 15 in the CEXA, and no changes in the Pyr on PND 7 and 20, but a diminished on PND 15; (2) GDNF positive cells rise after alcohol administration for the three ages in the CEXA and Pyr except on PND 15, where there was a decline; and (3) pharmacokinetics analysis demonstrated age‐related differences showing equal BALs on PND 7 and 20 but higher BALs on PND 15. In contrast, BrALs were higher on PND 7 than 15 and 20. Hence, BALs may not be predictive of BrALs in postnatal rats. Furthermore, we did not find a relationship between age in pharmacokinetic differences and neurotrophins response. In conclusion, the CEXA and Pyr are brain structures sensitive to alcohol‐induced imbalance in neurotrophic factors expression; and BALs are not a mirror of BrALs.

Descifrando la fisiopatología de la epilepsia en un modelo animal: el pentilentetrazol induce la activación pero no la muerte de las neuronas de la amígdala extendida medial

Introduccion: Desde mitad del siglo XX se ha apuntado a la importancia de la amigdala en la epilepsia, aunque los mecanismos basicos de esta participacion en su mayoria son aun desconocidos. Esta ignorancia es aun mayor cuando se tienen en cuenta las distintas subdivisiones de la amigdala, especialmente sus partes mediales. En este trabajo evaluamos la participacion de la amigdala extendida medial en un modelo animal de epilepsia, asi como las consecuencias que tiene el epileptogeno en esta estructura cerebral. Material y metodos: Se utilizaron ratas adultas Wistar machos (n = 48); 24 animales recibieron inyecciones intraperitoneales de pentilentetrazol y 24, de salina. Luego de 2, 6, 12 y 24 h de sobrevida, los animales se fijaron, y sus cerebros se cortaron seriadamente y se procesaron para fos (inmunoquimica) y muerte neuronal con la tecnica A-Cu-Ag. Los datos se analizaron con un ANOVA de 2 vias seguido de un test post-hoc (LSD de Fisher). Resultados: Muy poca activacion fos se halla en animales controles. En animales experimentales, fos fue rapidamente inducida en la amigdala extendida medial a las 2 h. Esta activacion fue sostenida hasta las 12 h y retorno a valores basales a las 24 h. Sin embargo, el estado epileptico no produjo muerte neuronal. Conclusiones: Se demuestra asi una participacion de la amigdala extendida medial en mecanismos epilepticos en los cuales subyace un componente inhibitorio. Sin embargo, el estado epileptico inducido no produce muerte neuronal en esta estructura.

[Understanding the pathophysiology of epilepsy in an animal model: pentylenetetrazole induces activation but not death of neurons of the medial extended amygdala].

INTRODUCTION since middle of the 20th century the importance of amygdala in epilepsy it has suggested, although the basic mechanisms of this participation are still unknown. This ignorance increases when the different subdivisions of amygdala are considered, especially the medial amygdala. In this work we assess the involvement of the medial extended amygdala in an animal model of epilepsy and the consequences of its application in this brain structure. MATERIAL AND METHODS forty eight adult Wistar male rats were used, of which 24 of them received i.p. injections of pentylenetetrazole, and 24 (controls) were injected with saline. After 2, 6, 12 and 24 h survival, animals were fixed; the brains were sectioned serially and stained for fos (immunochemistry) and for neuronal death with the A-Cu-Ag technique. Data were analysed using two-way ANOVA followed by the Fisher post hoc test. RESULTS very few or no fos-immunoreactive neurons were seen in control animals. In experimental animals, fos was rapidly induced in structures of medial extended amygdala with peak levels at 2 h. Marked fos immunoreactivity persisted up to 12 h followed by a gradual return to baseline at 24 h. However, status epilepticus did not induced neuronal death. CONCLUSIONS these results show involvement of medial extended amygdala in epileptic mechanisms with an inhibitory component. However, neuronal death is not a consequence of status epilepticus-induced by pentylentetrazole.