Teresa Morales

Brainstem prolactin-releasing peptide neurons are sensitive to stress and lactation

Prolactin-releasing peptide (PrRP) was originally thought to participate in the control of adenohypophyseal prolactin secretion, but its predominant expression in a subset of medullary noradrenergic neurons is more in line with roles in interoceptive and/or somatosensory information processing. To better define functional contexts for this peptide system, immuno- and hybridization histochemical methods were used to monitor the capacity of PrRP neurons to display activational responses to lactation, suckling, acute footshock or hypotensive hemorrhage. PrRP mRNA signal was reduced in the medulla of lactating dams, relative to both male and diestrus female controls, with cell counts revealing 42% and 43% reductions in the number of positively hybridized cells in the nucleus of the solitary tract (NTS) and ventrolateral medulla, respectively. Lactating mothers killed after a 90 min suckling episode (following 4 h pup removal) failed to show induced Fos expression in identified medullary PrRP neurons, despite the fact that responsive neurons were detected in other aspects of the caudal NTS. By contrast, acute exposure to hypotensive (25%) hemorrhage or footshock each activated substantial complements of medullary neurons expressing PrRP mRNA. A substantially greater fraction of the total medullary PrRP population exhibited sensitivity to footshock than hemorrhage (71 versus 39%, respectively). These results suggest that medullary PrRP neurons are negatively regulated by (presumably hormonal) changes in lactation, and are not recruited to activation by suckling stimuli. These populations exhibit differential sensitivity to distinct acute stressors, and may participate in the modulation of adaptive neuroendocrine and autonomic responses to each.

Recent findings on neuroprotection against excitotoxicity in the hippocampus of female rats.

Newborn mammals are totally dependent on maternal milk and care for survival. The mother’s brain undergoes different behavioural, physiological and emotional adaptations that make the mother more likely to satisfy the demands of the offspring. Recent reports from our group show that, compared to nulliparous rats, lactation diminishes cell damage induced by excitotoxicity in the dorsal hippocampus of the dam after systemic or i.c. administration of kainic acid (KA) and the resulting motor seizures. Elevated levels of prolactin (PRL), oxytocin, progesterone and glucocorticoids are characteristics of lactation, and the pronounced fluctuation of these hormones occurring in this phase may play a role protecting the hippocampus. Indeed, PRL administration to ovariectomised rats significantly diminishes the deleterious effects of KA in the dorsal hippocampus and reduces the progression of KA‐induced seizures. Thus, lactation is a natural model for neuroprotection because it effectively prevents acute and chronic cell damage of the hippocampus induced by excitotoxicity.

Prolactin reduces the damaging effects of excitotoxicity in the dorsal hippocampus of the female rat independently of ovarian hormones.

We reported previously that lactation prevents the cell damage induced by kainic acid (KA) excitotoxicity in the CA1, CA3, and CA4 areas of the dorsal hippocampus compared to rats in diestrus phase, and hypothesize that pronounced fluctuations of hormones, such as ovarian steroids and prolactin (PRL), have a role in the neuroprotection of the dorsal hippocampus during lactation. PRL is thought to be involved in modulating neural excitability and seizure activity. To investigate actions of prolactin that minimize KA-induced cell damage in the hippocampus, female intact and ovariectomized (OVX) rats were treated for 4 days with a daily dose of 100 microg of prolactin or vehicle. On the third day of prolactin treatment, rats received a systemic dose of 7.5 mg/kg of KA and were sacrificed 48 h later. Immunostaining for Neu-N revealed a significant decrease in cell number in the CA1, CA3 and CA4 areas of intact or OVX, vehicle-treated rats after KA, whereas prolactin treatment prevented cell loss in the CA3 area of intact, and in the CA1, CA3, and CA4 of OVX rats. Fluoro-Jade C staining confirmed these observations. Kainate-induced seizure behavior progressed further in OVX rats, but was attenuated in prolactin-treated rats, both intact and OVX, compared to vehicle-treated rats. These data indicate that prolactin diminishes the damaging actions of excitotoxicity in the kainate model of epilepsy.

Neuroprotective effects of lactation against kainic acid treatment in the dorsal hippocampus of the rat.

Marked hippocampal changes in response to excitatory amino acid agonists occur during pregnancy (e.g. decreased frequency in spontaneous recurrent seizures in rats with KA lesions of the hippocampus) and lactation (e.g. reduced c-Fos expression in response to N-methyl-d,l-aspartic acid but not to kainic acid). In this study, the possibility that lactation protects against the excitotoxic damage induced by KA in hippocampal areas was explored. We compared cell damage induced 24 h after a single systemic administration of KA (5 or 7.5 mg/kg bw) in regions CA1, CA3, and CA4 of the dorsal hippocampus of rats in the final week of lactation to that in diestrus phase. To determine cellular damage in a rostro-caudal segment of the dorsal hippocampus, we used NISSL and Fluorojade staining, immunohistochemistry for active caspase-3 and TUNEL, and we observed that the KA treatment provoked a significant loss of neurons in diestrus rats, principally in the pyramidal cells of CA1 region. In contrast, in lactating rats, pyramidal neurons from CA1, CA3, and CA4 in the dorsal hippocampus were significantly protected against KA-induced neuronal damage, indicating that lactation may be a natural model of neuroprotection.

Lactation is a natural model of hippocampus neuroprotection against excitotoxicity.

Lactation is a temporary but complex physiological condition in which hormones and neurogenic stimulation from suckling cause maternal brain plasticity. It has been shown that lactation prevents cell damage induced by excitotoxicity in the dorsal hippocampus of the dam after peripheral administration of kainic acid (KA). The aim of this study was to determine whether lactation protects the maternal hippocampus against damage induced by intracerebral application (ICV) of KA and if lactation decreases, or only delays, this damaging effect of KA. Cell damage was assessed by Fluoro-Jade C staining in the hippocampus of virgin and lactating rats 24 or 72 h after ICV KA. Lactation prevented cell damage of the pyramidal layers of the hippocampus (CA1, CA3, and CA4), as compared to virgin rats. The longer period of KA exposure increased the difference in cell damage between these two conditions. The present results confirm that lactation is a natural model for neuroprotection, since it effectively prevents acute and chronic cell damage of the hippocampus induced by exposure to KA.

Suckling-induced activation of neural c-fos expression at lower thoracic rat spinal cord segments.

Suckling stimulation is essential for neuroendocrine and sympathetic reflex activation during lactation. In the present study, the induction of c-fos gene expression was used to identify neuronal populations in the spinal cord activated by acute 5 min suckling or by electrical stimulation of the central stump of the first abdominal mammary nerve in lactating rats previously separated from their litters for 6 or 18 h. In addition, to investigate whether spinal sympathetic preganglionic neurons are activated by suckling, dual immunostaining (Fos and choline acetyltransferase) was performed. Fos was expressed at low levels in continuously suckled and 6 h nonsuckled mothers, but no expression was found after 18 h of nonsuckling. On the other hand, in 6 h nonsuckled rats, significant increments in Fos expression occurred in several regions after acute suckling and after electrical stimulation. Also, the pattern of Fos expression in each spinal laminae was different for the two stimuli, i.e. more intense effects of suckling in deep laminae V-X and more intense effects in laminae I-IV with electrical stimulation. Double-labeling after suckling was found only in sympathetic preganglionic neurons from the intermedio-medial cell column, whereas after electrical stimulation, double label was observed only in neurons from the intermedio-lateral cell column. On the other hand, no effect upon Fos protein expression was observed after suckling and only a minor effect after electrical stimulation of mammary nerve in 18 h nonsuckled rats. These results are consistent with previous findings on the sympathetic reflex regulation of the mammary gland, as well as on the importance of the nonsuckling interval for optimal functioning of lactation.

Microglia modulate respiratory rhythm generation and autoresuscitation.

Inflammation has been linked to the induction of apneas and Sudden Infant Death Syndrome, whereas proinflammatory mediators inhibit breathing when applied peripherally or directly into the CNS. Considering that peripheral inflammation can activate microglia in the CNS and that this cell type can directly release all proinflammatory mediators that modulate breathing, it is likely that microglia can modulate breathing generation. It might do so also in hypoxia, since microglia are sensitive to hypoxia, and peripheral proinflammatory conditions affect gasping generation and autoresuscitation. Here, we tested whether microglial activation or inhibition affected respiratory rhythm generation. By measuring breathing as well as the activity of the respiratory rhythm generator (the preBötzinger complex), we found that several microglial activators or inhibitors, applied intracisternally in vivo or in the recording bath in vitro, affect the generation of the respiratory rhythms both in normoxia and hypoxia. Furthermore, microglial activation with lipopolysaccharide affected the ability of the animals to autoresuscitate after hypoxic conditions, an effect that is blocked when lipopolysaccharide is co‐applied with the microglial inhibitor minocycline. Moreover, we found that the modulation of respiratory rhythm generation induced in vitro by microglial inhibitors was reproduced by microglial depletion. In conclusion, our data show that microglia can modulate respiratory rhythm generation and autoresuscitation. GLIA 2016;64:603–619

Both prolactin (PRL) and a molecular mimic of phosphorylated PRL, S179D-PRL, protect the hippocampus of female rats against excitotoxicity

Prolactin (PRL) has many functions in the CNS, including neuroprotection. During lactation, the dorsal hippocampus is protected from excitotoxic kainic acid (KA)-induced cellular damage. We have previously reported that systemic pre-treatment with ovine PRL had similar protective effects in female rats. Here, we asked (1) whether intracerebral human PRL (hPRL) would have the same action, (2) because phosphorylated PRL is high in lactation, whether a mimic of phosphorylated hPRL, human prolactin in which the normally phosphorylated serine at position 179 is replaced with an aspartate (S179D-PRL), had similar activity, and (3) what signaling pathways mediated the protective effect. Female ovariectomized (OVX, 1 month) rats were implanted with micro-osmotic pumps connected to unilateral icv cannulae directed at the right lateral ventricle. The pumps delivered 0.10 ng/h of hPRL, S179D-PRL, a combination of hPRL+S179D-PRL, or saline vehicle for 7 days prior to a systemic dose of 7.5mg/kg of KA. Rats were sacrificed 48 h after KA injection. Immunostaining for neuronal nuclei (Neu-N) revealed a significant KA-induced decrease in cell number in the CA1, CA3, and CA4 hippocampal areas of rats (∼55% of control). Treatment with either hPRL or S179D-PRL or the combination prevented the damaging effect of KA in these hippocampal regions (∼95% of corresponding control), but was not completely effective at preventing early seizure-related behaviors such as staring and wet dog shakes. Analysis of signals generated by hPRL and S179D-PRL showed no activation of signal transducer and activation of transcription 5 (Stat5) or other signaling molecules in the hippocampus, but activation of extracellular-regulated kinase (ERK)1/2 in the amygdala. These results support a central protective effect of both PRL forms and suggest that PRL could be exerting its protective action by indirectly modulating input signals to the hippocampus and thus regulating excitability.

Lactation Reduces Glial Activation Induced by Excitotoxicity in the Rat Hippocampus

Motherhood induces a series of adaptations in the physiology of the female, including an increase of maternal brain plasticity and a reduction of cell damage in the hippocampus caused by kainic acid (KA) excitotoxicity. We analysed the role of lactation in glial activation in the hippocampal fields of virgin and lactating rats after i.c.v. application of 100 ng of KA. Immunohistochemical analysis for glial fibrillary acidic protein (GFAP) and ionised calcium binding adaptor molecule 1 (Iba‐1), which are markers for astrocytes and microglial cell‐surface proteins, respectively, revealed differential cellular responses to KA in lactating and virgin rats. A significant astrocyte and microglial response in hippocampal areas of virgin rats was observed 24 h and 72 h after KA. By contrast, no increase in either GFAP‐ or Iba‐1‐positive cells was observed in response to KA in the hippocampus of lactating rats. Western blot analysis of GFAP showed an initial decrease at 24 h after KA treatment, with an increase at 72 h in the whole hippocampus of virgin but not of lactating rats. The number of GFAP‐positive cells was increased by lactation in the dentate gyrus of the hippocampus but not in CA1 and CA3 areas. The present results indicate that lactating rats exhibit diminished responses of astrocyte and microglial cells in the hippocampus to damage induced by KA, supporting the notion that the maternal hippocampus is resistant to excitotoxic insults.

Estrogen receptors increased expression during hippocampal neuroprotection in lactating rats

Estrogen receptor (ER)-mediated neuroprotection has been demonstrated in both in vitro and in vivo model systems. Two types of estrogen receptors, ERalpha and ERbeta, are the major mediators of the biological functions of estrogens. In the hippocampus, ERbeta is prevalent over ERalpha. Recently, we reported that during the final phase of lactation there is a neuroprotective mechanism in the hippocampus of the adult female rat against neuronal damage induced by systemic kainic acid administration vs. virgin (metestrus) rats. In this study, we assessed differential ER expression and localization in CA1, CA3 and dentate gyrus regions of dorsal hippocampus of metestrus and lactating adult rats at day 19 of lactation, during basal conditions (metestrus and L19, respectively) and 24h after systemic kainate administration. ERs were assessed by western blot and immunohistochemistry. We found a significant increase in the expression of ERs in the hippocampus during lactation as compared with metestrus. ERbeta was significantly increased in the CA1 and CA3 of lactating rats after the kainic acid insult. In addition, we observed a relocalization of ERbeta from the cytoplasm to the nucleus of neuronal cells. Our results suggest that there is a strong correlation between expression of ERs, especially ERbeta, in lactating CA1 and CA3 hippocampus regions in response to kainate administration, and neuroprotection observed during this reproductive period. This may be one of the mechanisms involved in the protection of the maternal brain to ensure offspring survival.