Rula Abbud

Use of Fos-related antigens (FRAs) as markers of neuronal activity: FRA changes in dopamine neurons during proestrus, pregnancy and lactation

This manuscript describes the use of staining of Fos-related antigens (FRAs) as markers for changes in neuronal activity. The model system consisted of the tuberoinfundibular dopamine (TIDA) neurons located in the arcuate nucleus of the hypothalamus. Under normal conditions, these neurons are devoid of c-Fos staining even though the neurons are tonically active and can express FRAs. During specific neuroendocrine states the neurons undergo changes in activity, as described by other studies. At times when the activity is relatively high as in pregnancy and during proestrus, approximately 50%-60% of the TIDA neurons expressed FRA immunoreactivity. Moreover changes over the course of proestrus paralleled known shifts in TIDA activity (declining as the day progressed). At times when TIDA activity is suppressed, such as during lactation, FRA staining in TIDA neurons was markedly reduced or absent. Upon removal of the suckling stimulus, FRA staining rose to reach peak expression 12-24 h after pup removal (without coordinate induction of c-Fos). These data suggest that FRA staining can serve as a useful marker of activity in the TIDA neurons which permits not only assessment of stimulated activity but also suppressed function in the neurons. A cautionary note in using this approach along with acquisition of serial blood samples for hormone measurement is that surgical procedures for monitoring plasma hormone levels are associated with strong long-lived induction of FRAs (and c-Fos) in many neurons (including the TIDA neurons) that can confound interpretation of FRA staining.

Do GnRH neurons express the gene for the NMDA receptor

Previous studies have revealed that in several animal models, N-methyl-D,L-Aspartate (NMA) stimulates LH secretion by acting at a suprapituitary site. In addition, NMDA receptor antagonists appear to block GnRH neuronal activation on the afternoon of proestrous as evidenced by the lack of c-Fos expression in the neurons and by the absence of an ovulatory LH surge. However, administration of NMA does not induce c-Fos or c-Jun expression in GnRH neurons. To better understand the effects of NMDA receptor activation on GnRH neuronal function, we examined whether GnRH neurons express the NMDA receptor in male rats, and in female rats during diestrus and proestrus, by performing double label in situ hybridization. An 35S-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) was used to quantify NMDAR1 mRNA and a digoxigenin-labeled cRNA probe for GnRH was used to identify GnRH neurons. The data were quantified and expressed as grains/average cell area. In male and female rats, less than 5% of GnRH neurons expressed grain levels twice the minimum detectable level and were considered double-labeled. However, many non-GnRH neurons in the same areas as GnRH neurons expressed high levels of NMDAR1 mRNA. These results suggest that the effects of NMA on GnRH secretion are unlikely to be mediated solely by the activation of NMDA receptors on GnRH neurons. Given the widespread expression of NMDAR1 mRNA in the hypothalamus, it is possible that the stimulatory effects of NMA on GnRH neurons are indirect through activation of other neurons.

Cortical refractoriness to N-methyl-D,L-aspartic acid (NMA) stimulation in the lactating rat: recovery after pup removal and blockade of progesterone receptors.

We have previously reported that lactating rats, unlike cycling rats, are refractory to N-methyl-D,L-aspartic acid (NMA), but not kainate, in terms of behavioral responses and activation of cFos expression in the neocortex and hippocampus. To study the factors involved in the suppression of cortical activation in lactating rats in response to NMA, we examined the effects of removing either the suckling stimulus and/or progesterone. The degree of cFos expression was used as a marker for cortical activation. Whereas control suckled animals exhibited little or no cFos activation in the piriform cortex in response to NMA, cycling rats showed a high degree of activation. Blockade of the effects of progesterone or removal of the pups for 24 h, resulted in a moderate level of cFos intensity in response to NMA. Total recovery was observed only in animals who had their pups removed for 24 h and the effects of progesterone were blocked. In general, similar results were obtained in the hippocampus except that the total recovery of hippocampal activation took longer than the cortex. Thus, the deficits in cortical activation depend on the presence of both the suckling stimulus and progesterone. However, progesterone alone cannot induce these cortical deficits since pregnant rats showed no deficits in cortical activation in response to NMA when compared to cycling rats. Therefore, the suckling stimulus is required for the inhibition of NMDA-receptor mediated activation of the cortex and hippocampus. The effects of progesterone appear to act synergistically with the effects of suckling.

Altered Luteinizing Hormone and Prolactin Responses to Excitatory Amino Acids during Lactation

We have used excitatory amino acids as tools to elucidate changes in hypothalamic function associated with lactation, focusing on the regulation of luteinizing hormone (LH) and prolactin secretion. In these studies, we have compared the responsiveness to NMA (N-methyl-D,L-aspartate), an agonist for the N-methyl-D-aspartate (NMDA) receptor, with that of kainate, an agonist for another type of glutamate receptor, the kainate receptor. To address the issue of the permeability of the blood-brain barrier to either NMA or kainate, systemic and central administration of the drugs were compared. Four injections of either drug were administered at 10-min intervals to cycling or lactating rats suckling 8 pups. All of these treatment significantly stimulated LH secretion in cycling rats. However, neither systemic injections of NMA (40 mg/kg) or kainate (2.5-3.5 mg/kg), nor third-ventricular administration of NMA (2 micrograms/2 microliters) or kainate (0.2-0.3 micrograms/2 microliters) stimulated LH secretion during lactation. In contrast, LH responses to NMA were observed in lactating animals suckling 2 pups. These data demonstrate that the intensity of the suckling stimulus determines the degree of gonadotropin-releasing hormone (GnRH) neuronal inhibition during lactation. Recovery of the LH response to NMA in animals suckling 8 pups was not observed after treatment with RU 486 to block the effects of progesterone. Thus, the elevated levels of progesterone during lactation do not appear to play a role in inhibiting GnRH neuronal responsiveness. Removal of the 8-pup suckling stimulus for 24 h also did not restore the LH response to NMA. However, treatment with RU 486 and removal of the suckling stimulus for 24 h did restore LH responses to NMA, suggesting that progesterone may play a role in prolonging the recovery of GnRH neuronal responsiveness. The prolactin responses to NMA and kainate changed with the reproductive state of the animal and the site of administration. Central injections of either drug stimulated prolactin release in both cycling and lactating animals. In contrast, whereas systemic administration of NMA stimulated prolactin secretion in cycling animals, kainate had no effect. In the lactating animals, systemic administration of either drug inhibited prolactin secretion. Thus, the difference in the prolactin responses to systemic administration of the drugs may not only be due to a difference in the distribution of kainate and NMDA receptors but also to the steady state level of activity of the prolactin-releasing and -inhibiting factors which is determined by the reproductive state of the animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Lactation-induced deficits in NMDA receptor-mediated cortical and hippocampal activation: changes in NMDA receptor gene expression and brainstem activation

During lactation, there is an inhibition of cortical and hippocampal activation in response to N-methyl-D,L-aspartate (NMA), but not kainate, as assessed by induction of c-Fos expression. To study whether changes in NMDA receptor function may account for this inhibition, NMDA receptor subunit (NMDAR1) mRNA levels were measured by both Northern analysis and in situ hybridization. Analysis of NMDAR1 gene expression by Northern blot analysis did not reveal significant differences between cycling and lactating rats. Using in situ hybridization, NMDAR1 mRNA levels in several cortical and hippocampal areas appeared to be smaller in lactating rats, compared to cycling rats, although these differences reached significance only in the fronto-parietal cortex and piriform cortex. These subtle changes in NMDAR1 receptor subunit gene expression during lactation are not likely to account for the global lack of neuronal activation in response to NMA. However, it is possible that there may be changes in other NMDA receptor subunits that could account for the deficits in NMDA receptor activation. We also examined the activation state of afferent pathways in the brainstem that provide excitatory input to the cortex and hippocampus. During lactation, NMA induced c-Fos expression in similar areas of the brainstem as during the cycle, except in the locus coeruleus and dorsal raphe, where c-Fos expression was significantly less than that observed during the cycle. In contrast, no differences in the pattern of c-Fos expression in the brainstem in response to kainate were observed between cycling and lactating rats. The lack of NMA-induced activation of the locus coeruleus and dorsal raphe may contribute to the lack of cortical activation during lactation.

Lactation inhibits hippocampal and cortical activation of cFos expression by nivida but not kainate receptor agonists

Lactation in the rat activates afferent neuronal pathways that cause marked changes in hormone secretion and maternal behavior. Previously, we used excitatory amino acids (EAAs) to challenge the neuroendocrine axis of the lactating rat and showed altered hypothalamic responsiveness to EAAs. In conducting those studies, we noted that the typical hyperactive behavior associated with NMA (N-methyl-d,l-aspartate) treatment was completely absent in lactating animals. In these studies we have examined the effects of lactation on cortical responsiveness to EAAs by using cFos expression as a marker of neuronal activation. Lactation inhibited hippocampal and cortical cFos induction in response to NMA, which was consistent with the absence of behavioral responses. However, NMA responsiveness was observed in other areas of the brain. Recovery of cortical activation in response to NMA was not observed until 24 h after removal of the suckling stimulus. In contrast, treatment with kainate (an agonist for a different type of glutamate receptor) induced similar patterns of cFos expression and behavioral responses (wet-dog shakes) in cycling and lactating rats. These data demonstrate that lactation, a physiological condition, can inhibit cortical activation that is mediated by NMDA but not kainate receptors. In so doing, lactation represents a novel model system in which to study mechanisms of NMDA receptor inactivation and subsequent consequences on hippocampal and cortical function.