Hugo F. Carrer

Ultrastructural changes in the hypothalamic ventromedial nucleus of ovariectomized rats after estrogen treatment.

The changes produced in the hypothalamic ventromedial nucleus (VMN) of ovariectomized rats after administration of 100 microgram estradiol benzoate/kg body weight were studied using light and electron microscopy. Quantitative morphometric studies included number and size of VMN neurons and nuclei, size and density of terminals and synaptic contacts, spine-to-shaft ratio of postsynaptic elements and relative frequency of two types of synaptic vesicles. Evidence was obtained favoring the concept of heterogeneous composition of the VMN: in ovariectomized animals many cells appeared in a state of quiescence, but other neurons showed no major alterations. Estrogen administration to ovariectomized rats produced evidence of metabolic stimulation such as increase in rough surfaced endoplasmic reticulum, condensation of nucleolar material, enlarged Golgi and presence of pleomorphic mitochondria. The number of neurons in the VMN was not modified by estrogen treatment; however, neuron soma and nuclei were larger. In the ventrolateral division of the VMN terminals and synaptic contacts per unit area were increased after estrogen treatment, but synaptic contact length, terminal size and spine-to-shaft ratio were not modified. The possibility that the differences observed may be consequent to changes in synaptic organization of the VMN related to its estrogen-dependent functions is discussed.

Differential effect of oestradiol and astroglia-conditioned media on the growth of hypothalamic neurons from male and female rat brains.

To determine whether soluble products from different CNS regions differ in their ability to support oestrogen‐stimulated neurite growth, hypothalamic neurons from sexually segregated embryos were cultured with astroglia‐conditioned medium (CM) derived from cortex, striatum and mesencephalon, with or without 17‐β‐oestradiol 100u2003n m added to the medium. After 48u2003h in vitro, neurite outgrowth was quantified by morphometric analysis. Astroglia‐CM from mesencephalon (a target for the axons of hypothalamic neurons) induced the greatest axogenic response in males and in this case only a neuritogenic effect could be demonstrated for oestradiol. On the other hand, astroglia‐CM from regions that do not receive projections from ventromedial hypothalamus inhibited axon growth. A sexual difference in the response of hypothalamic neurons to astroglia‐CM and oestradiol was found; growth of neurons from female foetuses was increased by astroglia‐CM from mesencephalon, but no neuritogenic effect could be demonstrated for oestradiol in these cultures. Blot immunobinding demonstrated the presence of receptors for neurotrophic factors in cultures of hypothalamic neurons; Western blot analysis of these cultures demonstrated that oestradiol increased the concentration of trkB and IGF‐I Rβ, whereas trkA was not detected and the concentration of trkC was not modified. These results support the hypothesis that target regions produce some factor(s) that stimulate the growth of axons from projecting neurons and further indicate that in the case of males this effect is modulated by oestradiol, perhaps mediated through the upregulation of trkB and IGF‐I receptors.

Correlation between threshold to induce long-term potentiation in the hippocampus and performance in a shuttle box avoidance response in rats

The relationship between the learning ability of normal rats and the facility to induce long-term potentiation (LTP) in the same animals was investigated. Behavioral performance was measured in a shuttle box avoidance paradigm, using a buzzer as conditioned stimulus. Three days later animals were sacrificed; frequency threshold necessary to induce LTP was determined in transverse hippocampal slices taken from these animals and maintained in vitro. A linear regression analysis on the behavioral and electrophysiological data showed a negative correlation (Spearman rank correlation coefficient rs = -0.705; P less than 0.001) between percent of conditioned responses in the shuttle box and threshold frequency necessary to induce LTP in gyrus dentatus in response to tetanic stimulation of the perforant path. It is concluded that learning ability of normal rats in a shuttle box avoidance paradigm is correlated with hippocampal synaptic plasticity.

Differential threshold for long-term potentiation in the hippocampus of rats with inborn high or low learning capacity

Threshold of stimulation frequency in the perforant path to induce long-term potentiation (LTP) in dentate gyrus was determined in hippocampal slices obtained from two different lines of rats inbred for 30 generations according to their performance in an avoidance escape test in a shuttle box. High-performance (HP) rats were defined as those giving at least 70% conditioned responses (CRs) and low-performance (LP) rats as those giving less than 15% CRs. LTP was defined as a 30% or more increase in the amplitude of the evoked population spike (PS), lasting at least 20 min. Stimulation frequency threshold was determined by stimulating with a train of pulses of 0.5 ms duration during 1 s. The same slice was stimulated with trains of increasing frequency from 5 to 400 Hz, each train separated by an interval of at least 20 min. HP rats showed a lower threshold (13 +/- 4 Hz) than LP rats (92 +/- 42 Hz) for the induction of LTP; there were no differences in the magnitude of LTP. The greater learning ability of HP rats may be related to the plasticity of hippocampal synaptic transmission.

Prenatal amphetamine exposure: Ovulation, sexual behavior and hypothalamic monoamine content in rats

Previous observations have pointed out that amphetamine treatment during pregnancy produces behavioral and neurochemical changes in the male offspring. The present study was undertaken in order to determine whether those findings could be extended to sexual behavior, reproductive function and hypothalamic monoamine metabolism in female rats. It was found that offspring of amphetamine treated rats have greater sensitivity to estrogen and estrogen-progesterone for the induction of sexual receptivity. 5-HT content in medial hypothalamus of estrogen-progesterone treated rats was decreased with respect to controls. On the other hand the content of 5-HIAA was not different; noradrenaline levels in hypothalamus were also normal. Sex cycle duration and ovulatory phenomena were not affected.

Effect of CNS depressants and stimulants on latency for the appearance of copulatory response in the female rat

Diethyl ether anesthesia, sodium hexobarbital (20 mg/kg), diphenylhydantoin (50 mg/kg), strychnine (1 mg/kg) and picrotoxin (1-0.25 mg/kg) effectively induced the copulatory response (lordotic behavior) in estradiol benzoate treated ovariectomized rats although no progesterone was given. As none of the tested compounds were effective in replacing progesterone in adrenalectomized animals, adrenal secretion is likely to be implicated in the lordosis activating effect of these compounds. The lordosis response appeared faster after the CNS stimulants than after treatment with the CNS depressants. The influence of diethylether anesthesia, strychnine (0.5 mg/kg) or picrotoxin (0.25 mg/kg) on the latency for the appearance of the lordosis response after IV injection of isopregnenone was studied in estradiol benzoate treated ovariectomized females. A 10 min ether anesthesia delayed the onset of the lordosis response in adrenal intact as well as adrenalectomized animals. Anesthesia given after receptivity had been fully established suppressed the responses for a short period (10-30 min) after the narcosis. The delay of the appearance of the first lordosis response after IV injection of isopregnone exceeded this period. Strychnine but not picrotoxin significantly shortened the latency to the onset of the female copulatory response. It is concluded that the lordotic activating action of progesterone or steroids with progesteronelike ability released from an endogenous source or given IV is influenced by compounds which exert a depressant or stimulant effect on neuronal activity. The total response obtained is not changed but the appearance of the response is prolonged by CNS depressants and shortened by certain CNS stimulants.

Body Sodium Overload Modulates the Firing Rate and Fos Immunoreactivity of Serotonergic Cells of Dorsal Raphe Nucleus

In order to determine whether serotonergic (5HT) dorsal raphe nucleus (DRN) cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT) plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO), animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the “in vivo” electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an “in vivo model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, possibly to modulate sodium and water intake/excretion and avoid extracellular volume expansion.

Sexual Differentiation of the Brain: Genetic, Hormonal and Trophic Factors

The current hypothesis to explain the sexual dimorphism of structure and function in the brain of vertebrates maintains that these differences are produced by the interaction of genetic mechanisms and gonadal hormones. In this chapter we summarize the evidence from our laboratory, as well as other laboratories analyzing the mechanisms that control sexually differentiated growth of axons in hypothalamic neurons in vitro.

Sex differences in depolarizing actions of GABAA receptor activation in rat embryonic hypothalamic neurons

GABAA receptor activation exerts trophic actions in immature neurons through depolarization of resting membrane potential. The switch to its classical hyperpolarizing role is developmentally regulated. Previous results suggest that a hormonally biased sex difference exists at the onset of the switch in hypothalamic neurons. The aim of this work was to evaluate sex differences in GABAA receptor function of hypothalamic neurons before brain masculinization by gonadal hormones. Hypothalamic cells were obtained from embryonic day 16 male and female rat foetuses, 2 days before the peak of testosterone production by the foetal testis, and grown in vitro for 9 days. Whole‐cell and perforated patch‐clamp recordings were carried out in order to measure several electrophysiological parameters. Our results show that there are more male than female neurons responding with depolarization to muscimol. Additionally, among cells with depolarizing responses, males have higher and longer lasting responses than females. These results highlight the relevance of differences in neural cell sex irrespective of exposure to sex hormones.