Increased neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet brainstem

We examined the immunohistochemical expression of caspase-3 (CASP3), active caspase-3 and TUNEL in the normal piglet brainstem at 13-14 days of age and evaluated the effects of exposure to 2 vs. 4 days of intermittent hypercapnic hypoxia (IHH) on their expression. Eight nuclei from the level of the caudal medulla were studied. In control piglets, CASP3 was present in approximately 45% of neurons while active caspase-3 and TUNEL were present in approximately 5%, indicating that approximately half the neuronal population of the piglet medulla express caspase-3 in a latent state and that only approximately 5% undergo 'normal' programmed cell death. After 2 days of IHH, CASP3 increased in the nucleus of the solitary tract (NTS), gracile and cuneate nuclei (P<0.05 for all). Active caspase-3 increased in the dorsal motor nucleus of the vagus (DMNV) (P<0.05) but decreased in the lateral reticular nucleus (LRt) (P<0.05), while TUNEL increased in both the DMNV and LRt (P<0.05 for both). After 4 days of IHH, CASP3 remained elevated in the cuneate nucleus (P<0.01) but decreased in the hypoglossal and DMNV (P<0.05) when compared to controls. Active caspase-3 levels were not changed, whereas TUNEL was increased in the DMNV, LRt, and inferior olivary nucleus (P<0.05 for all). These results show that IHH induces neuronal cell death within certain nuclei in the piglet caudal medulla that are functionally important in cardiorespiratory, sleep and arousal control. This could have important implications for clinical conditions including obstructive apnea and prone sleeping as a risk for SIDS.     

Impaired response to hypoxia in the respiratory center is a major cause of neonatal death of the PACAP‐knockout mouse

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a neuropeptide expressed widely in nervous tissues. PACAP‐knockout (^?/?) mice display a sudden infant death syndrome (SIDS)‐like phenotype, although the underlying physiological mechanism to explain this remains unclear. Here, we report on the presence of abnormal respiratory activity in PACAP^?/? mice under hypoxic conditions, which provides a basis for the SIDS‐like phenotype. PACAP^?/? mice display a lowered baseline respiratory activity compared with wild‐type animals, and an abnormal response to hypoxia. More specifically, PACAP^?/? mice at postnatal day 7 showed respiratory arrest in response to hypoxia. In contrast, their response to hypercapnic conditions was the same as that of wild‐type mice. Histological and real‐time PCR analyses indicated that the catecholaminergic system in the medulla oblongata was impaired in PACAP^?/? mice, suggesting that endogenous PACAP affects respiratory centers in the medulla oblongata via its action on the catecholaminergic system. We propose that disruption of this system is involved in the SIDS‐like phenotype of PACAP^?/? mice. Thus, disorders of the catecholaminergic system involved with O₂ sensing could be implicated in underlying neuronal mechanisms responsible for SIDS.     

Correlations between brainstem NMDA receptor changes and active neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet

The role of the N-methyl-D-aspartate (NMDA) receptor in cell death was evaluated in the piglet brainstem after exposure to intermittent hypercapnic hypoxia (IHH). Study groups comprised controls (n=6) and piglets exposed to IHH on 2 (n=6) or 4 (n=5) successive days prior to euthanasia. All piglets had the caudal medulla evaluated at 13-14 days of age using double immunohistochemistry for TUNEL and the NMDA receptor 1 (NR1) subunit. The percent of TUNEL positive neurons amongst NR1 (% TUN in NR1) and non-NR1 neurons (% TUN in non-NR1) was determined in eight nuclei. After 2 days of IHH, %TUN in NR1 was increased in the dorsal motor nucleus of the vagus (DMNV, P=0.007) and the inferior olivary nucleus (ION, P=0.05). After 2 days IHH, %TUN in non-NR1 neurons was increased in the lateral reticular nucleus (LRt, P=0.05), nucleus of the solitary tract (NTS, P=0.004) and gracile nucleus (P=0.05). After 4days IHH, the increase of %TUN in NR1 was sustained in the ION (P=0.05), while %TUN in non-NR1 neurons was sustained in NTS (P=0.04) and LRt (P=0.006). Daily IHH exposure induces neuronal death within NR1 and non-NR1 neurons, but the neuronal phenotype is consistent within affected brainstem nuclei. Involvement of the NMDA receptor tended to occur in nuclei with higher basal NR1 expression, and thus occurred in nuclei relevant to cardiorespiratory function. We speculate that IHH exposures, such as occurs during obstructive apnea or facial entrapment in prone sleeping during infancy, can induce abnormalities of cardiorespiratory control.     

Distribution and quantification of NMDA R1 mRNA and protein in the piglet brainstem and effects of intermittent hypercapnic hypoxia (IHH)

The first aim of this study was to determine the cellular distribution of NR1 in the piglet brainstem. Documentation of NR1 mRNA was by non-radioactive in situ hybridisation (non-RISH) and of NR1 protein by immunohistochemistry. We found that all neurons expressed mRNA but not all had NR1 protein. Application of non-RISH has permitted us, for the first time, to document the cellular localization of NR1 mRNA showing that it was present in the cytoplasm and nucleolus of motor neurons but dispersed throughout the cellular compartments of sensory neurons. NR1 protein on the other hand, was localized to the cytoplasm only. The second aim of this study was to quantify the distribution of NR1 mRNA and protein. Using image analysis software, we used optical density (OD) to quantify the non-RISH signal for mRNA and cellular counting for protein (expressed as % positive). Results show that NR1 expression is greater in motor than sensory nuclei; for mRNA: 0.46+/-0.04 vs. 0.31+/-0.02 OD units (P<0.001), for protein: 75.9+/-3.1 vs. 58.4+/-2.5% positive (P<0.001). The third aim of this study was to determine the effects of intermittent hypercapnic hypoxia (IHH) on NR1 expression. Chronic IHH exposure caused differential changes in mRNA and protein expression. NR1 mRNA expression increased while the number of neurons expressing NR1 protein showed no change. This finding suggests that NMDA pathways are activated by exposure to IHH.     

Intermittent hypercapnic hypoxia effects on the nicotinic acetylcholine receptors in the developing piglet hippocampus and brainstem

This study investigated the effects of acute (1 day) vs repeated (4 days) exposure to intermittent hypercapnic hypoxia (IHH) on the immunohistochemical expression of α2, α3, α5, α7, α9 and β2 nicotinic acetylcholine receptor (nAChR) subunits in the developing piglet hippocampus and brainstem medulla, and how prior nicotine exposure alters the response to acute IHH. Five piglet groups included: 1 day IHH (1D IHH, n = 9), 4 days IHH (4D IHH, n = 8), controls exposed only to air cycles for 1 day (1D Air, n = 6) or 4 days (4D Air, n = 5), and pre-exposed to nicotine for 13 days prior to 1 day IHH (Nic + 1D IHH, n = 7). The exposure period alternated 6 min of HH (8%O₂, 7%CO₂, balance N₂) and 6 min of air over 48 min, while controls were switched from air-to-air. Results showed that: 1. repeated IHH induces more changes in nAChR subunit expression than acute IHH in both the hippocampus and brainstem medulla, 2. In the hippocampus, α2 and β2 changed the most (increased) following IHH and the CA3, CA2 and DG were mostly affected. In the brainstem medulla, α2, α5, α9 and β2 were changed (decreased) in most nuclei with the hypoglossal and nucleus of the solitary tract being mostly affected. 3. Pre-exposure to nicotine enhanced the changes in the hippocampus but dampened those in the brainstem medulla. These findings indicate that the nAChRs (predominantly with the α2/β2 complex) are affected by IHH in critical hippocampal and brainstem nuclei during early brain development, and that pre-exposure to nicotine alters the pattern of susceptibility to IHH.     

Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus

We have recently used Fos expression in adult rats to map neuronal populations activated in the brainstem and hypothalamus during the acute ventilatory response to moderate hypoxia (O(2) 11%). Although present at birth, this response evolves postnatally. The present investigation aimed at a better understanding of these maturational processes by delineating structures that might functionally develop after birth. The developmental pattern Fos expression evoked by hypoxia was analysed in rats aged from 0 to 26 postnatal days. The numbers of Fos positive neurons markedly increased with the age in the medullary areas related to respiratory control during the 2 first postnatal weeks. Thereafter, the response plateaued in the nucleus tractus solitarius and attenuated in the ventral medulla. In the upper brainstem (parabrachial area, central grey) and the hypothalamus (posterior and dorsomedial nuclei, ventral zone), Fos response to hypoxia was absent or weak at birth and increased until late development. The significance of the development of evoked Fos expression in these rostral sites is discussed together with their possible contribution to the maturation of O(2)-sensitive chemoreflex pathways.     

Isolation and identification of endogenous RFamide‐related peptides 1 and 3 in the mouse hypothalamus

Although the RFamide‐related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C‐terminus of RFRP‐1 and RFRP‐3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF‐NH2 and SHFPSLPQRF‐NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild‐type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP‐immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.     

Oestrogen receptor-alpha and -beta immunoreactivity in gonadotropin-releasing hormone neurones after ovariectomy and chronic exposure to oestradiol

Oestradiol exerts negative- and positive-feedback actions on luteinizing hormone (LH) secretion by modulating gonadotropin-releasing hormone (GnRH) release. Furthermore, a chronic increase in circulating oestradiol in either young ovariectomized (OVX) rats, or in middle-aged persistent oestrous (PE) rats, causes a gradual attenuation of LH surges until the positive-feedback action of oestradiol disappears. Based on these findings, and on the equivocal evidence regarding a direct action of oestradiol on GnRH neurones, we tested the hypothesis that chronic oestradiol abolishes LH surges by decreasing the proportion of GnRH neurones containing oestrogen receptor (ER)alpha or beta. Regularly cycling rats were ovariectomized, and half immediately received oestradiol. Three days, or 2 or 4 weeks later, rats were perfused at 18.00 h, and GnRH was colocalized with ERalpha or ERbeta by immunocytochemistry. ERbeta was expressed in 76% of GnRH neurones, whereas virtually no GnRH cells were immunopositive for ERalpha. The proportion of GnRH cells expressing ERalpha or beta in OVX rats was not altered by oestradiol or time after OVX, and this was the case regardless of their medial to lateral, or rostral to caudal location. The results indicate that the mechanisms for the positive-feedback action of oestradiol, and the loss of LH surges in OVX rats after chronic oestradiol, are not mediated by changes in the proportion of oestrogen-receptor containing GnRH neurones.     

Neonatal Bisphenol A exposure alters sexually dimorphic gene expression in the postnatal rat hypothalamus

Developmental exposure to Bisphenol A (BPA), a component of polycarbonate and epoxy resins, has been purported to adversely impact reproductive function in female rodents. Because neonatal life is a critical window for the sexual dimorphic organization of the hypothalamic-pituitary-gonadal (HPG) axis, interference with this process could underlie compromised adult reproductive physiology. The goal of the present study was to determine if neonatal BPA exposure interferes with sex specific gene expression of estrogen receptor alpha (ERα), ER beta (ERβ) and kisspeptin (Kiss1) in the anterior and mediobasal hypothalamus. Long Evans (LE) neonatal rats were exposed to vehicle, 10 μg estradiol benzoate (EB), 50 mg/kg BPA or 50 μg/kg BPA by subcutaneous injection daily from postnatal day 0 (PND 0) to PND 2. Gene expression was assessed by in situ hybridization on PNDs 4 and 10. Within the anterior hypothalamus ERα expression was augmented by BPA in PND 4 females, then fell to male-typical levels by PND 10. ERβ expression was not altered by BPA on PND 4, but significantly decreased or eliminated in both sexes by PND 10. Kiss1 expression was diminished by BPA in the anterior hypothalamus, especially in females. There were no significant impacts of BPA in the mediobasal hypothalamus. Collectively, BPA effects did not mirror those of EB. The results show that neonatal hypothalamic ER and Kiss1 expression is sensitive to BPA exposure. This disruption may alter sexually dimorphic hypothalamic organization and underlie adult reproductive deficiencies. Additionally, the discordant effects of EB and BPA indicate that BPA likely disrupts hypothalamic organization by a mechanism other than simply acting as an estrogen mimic.     

Laminar and subcellular heterogeneity of GLAST and GLT‐1 immunoreactivity in the developing postnatal mouse hippocampus

Astrocytes express two sodium‐coupled transporters, glutamate–aspartate transporter (GLAST) and glutamate transporter‐1 (GLT‐1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT‐1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high‐resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP‐positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT‐1 was completed only at postnatal days (P) P20–25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3–5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT‐1 exhibited a laminar expression pattern from P10–15 on, with the highest immunoreactivity in the stratum lacunosum‐moleculare. At the cellular level, GLT‐1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT‐1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT‐1 expression in the developing hippocampus. The clustering of GLT‐1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood–brain barrier during formation of the hippocampal network. J. Comp. Neurol. 522:204–224, 2014. © 2013 Wiley Periodicals, Inc.     

Heterogeneous neuronal activity in the lateral habenula after short‐ and long‐term cocaine self‐administration in rats

Cocaine addiction is thought to be the result of drug‐induced functional changes in a neural network implicated in emotions, learning and cognitive control. Recent studies have implicated the lateral habenula (LHb) in drug‐directed behavior, especially its aversive aspects. Limited cocaine exposure has been shown to alter neuronal activity in the LHb, but the impact of long‐term drug exposure on habenula function has not been determined. Therefore, using c‐fos as a marker, we here examined neuronal activity in LHb in rats that self‐administered cocaine for either 10 or 60 days. Both the density of labeled cells and the cellular labeling intensity were measured in the lateral (LHbL) and medial (LHbM) parts of LHb. After 10 days of cocaine self‐administration, both the density and intensity of c‐fos‐positive cells were significantly increased in LHbL, but not LHbM, while after 60 days, an increased density (but not intensity) of labeled neurons in both LHbL and LHbM was observed. Most c‐fos‐labeled neurons were glutamatergic. In addition, we found increased GAD65 expression after 10 but not 60 days of cocaine self‐administration in the rostral mesencephalic tegmental nucleus. These data shed light on the complex temporal dynamics by which cocaine self‐administration alters activity in LHb circuitry, which may play an important role in the descent to compulsive drug use as a result of prolonged cocaine‐taking experience.     

Timing of prenatal exposure to trauma and altered placental expressions of hypothalamic‐pituitary‐adrenal axis genes and genes driving neurodevelopment

Prenatal maternal stress increases the risk for negative developmental outcomes in offspring; however, the underlying biological mechanisms remain largely unexplored. In the present study, alterations in placental gene expression associated with maternal stress were examined to clarify the potential underlying epi/genetic mechanisms. Expression levels of 40 selected genes involved in regulating foetal hypothalamic‐pituitary‐adrenal axis and neurodevelopment were profiled in placental tissues collected from a birth cohort established around the time of Superstorm Sandy. Objective prenatal traumatic stress was defined as whether mothers were exposed to Superstorm Sandy during pregnancy. Among the 275 mother‐infant dyads, 181 dyads were delivered before Superstorm Sandy (ie, Control), 66 dyads were exposed to Superstorm Sandy during the first trimester (ie, Early Exposure) and 28 were exposed to Superstorm Sandy during the second or third trimester (ie, Mid‐Late Exposure). Across all trimesters, expression of HSD11B2, MAOA, ZNF507 and DYRK1A was down‐regulated among those exposed to Superstorm Sandy during pregnancy. Furthermore, trimester‐specific differences were also observed: exposure during early gestation was associated with down‐regulation of HSD11B1 and MAOB and up‐regulation of CRHBP; exposure during mid‐late gestation was associated with up‐regulation of SRD5A3. The findings of the present study suggest that placental gene expression may be altered in response to traumatic stress exposure during pregnancy, and the susceptibility of these genes is dependent on the time of the exposure during pregnancy. Further studies should aim to clarify the biological mechanisms that underlie trimester‐specific exposure by evaluating the differential impact on offspring neurodevelopment later in childhood.     

Similarities in the neural signature for the processing of behaviorally categorized and uncategorized speech sounds

Recent human behavioral studies have shown semantic and/or lexical processing for stimuli presented below the auditory perception threshold. Here, we investigated electroencephalographic responses to words, pseudo‐words and complex sounds, in conditions where phonological and lexical categorizations were behaviorally successful (categorized stimuli) or unsuccessful (uncategorized stimuli). Data showed a greater decrease in low‐beta power at left‐hemisphere temporal electrodes for categorized non‐lexical sounds (complex sounds and pseudo‐words) than for categorized lexical sounds (words), consistent with the signature of a failure in lexical access. Similar differences between lexical and non‐lexical sounds were observed for uncategorized stimuli, although these stimuli did not yield evoked potentials or theta activity. The results of the present study suggest that behaviorally uncategorized stimuli were processed at the lexical level, and provide evidence of the neural bases of the results observed in previous behavioral studies investigating auditory perception in the absence of stimulus awareness.