Tallie Z. Baram

Glucocorticoid Receptor mRNA Ontogeny in the Fetal and Postnatal Rat Forebrain

Glucocorticoid receptor (GR) ontogeny and distribution in postnatal rat brain have been demonstrated, but onset and distribution of GR gene expression during fetal life has not been reported. This study focuses on the distribution of GR-mRNA in the fetal and postnatal rat forebrain, with emphasis on hypothalamic and limbic structures. Time pregnant rats were decapitated at 8:30-9:30 AM on Gestational Days 14 (F14), F16, F17, F18, and F19. Postnatally, rats were sacrificed on Days 1, 4, 6, 10, and 16. Cryostat sections were subjected to in situ hybridization, using a cRNA probe directed to the GR-mRNA. GR-mRNA was detectible in the hippocamposeptal formation as early as F14. By F16, GR gene expression was evident in the hypothalamic paraventricular nucleus (PVN) as well. During late gestation (F17-F19), GR-mRNA was localized also in the thalamus, hippocampus, amygdala, and discrete cortical regions. Postnatally, GR-mRNA abundance was high in the PVN, CA1/CA2 hippocampal field, piriform cortex and dorsal endopiriform nucleus, specific amygdaloid nuclei, and the suprachiasmatic nucleus. In PVN, GR-mRNA was present prior to the onset of CRH gene expression (F17), which may suggest a role for GR in neuronal differentiation.

Ontogeny of corticotropin releasing hormone gene expression in rat hypothalamus--comparison with somatostatin.

Using in situ hybridization histochemistry, corticotropin‐releasing hormone gene expression is first detectable in the parvocellular portion of the rat paraventricular nucleus on the 17th fetal day. The prevalence of messenger RNA for corticotropin releasing hormone decreases perinatally, specifically between the 19th and 21st fetal days. By the 4th postnatal day, CRH gene expression is similar to that of the adult rat. Somatostatin messenger‐RNA is detectable on the 14th fetal day in the periventricular nucleus. No perinatal hiatus in somatostatin gene expression is evident.

Effects of a specific glucocorticoid receptor antagonist on corticotropin releasing hormone gene expression in the paraventricular nucleus of the neonatal rat

Mechanisms controlling the synthesis of corticotropin releasing hormone (CRH) in neonatal rats, and the ontogeny of glucocorticoid (GC) feedback control of hypothalamic CRH remain unknown. Specific issues are whether stress induces up-regulation of CRH gene expression during the first postnatal week, and the role of GC feedback, at the hypothalamic level, in the stress-hyporesponsive period. We studied the ontogeny of the negative feedback regulation of CRH gene expression by GC in the paraventricular nucleus (PVN). We implanted chronic cannulae containing a GC-receptor antagonist, RU 38486, in rats on postnatal days 3 to 13. Three days later, animals were sacrificed, and brains were analyzed for CRH-messenger RNA (CRH-mRNA), using semi-quantitative in situ hybridization. Animals implanted with cholesterol-containing cannulae served to evaluate the stressful effect of implantation on CRH-mRNA abundance. The presence of GC receptor messenger RNA (GR-mRNA) in the PVN of neonatal rats was also determined. RU 38486 did not increase CRH-mRNA abundance during the first postnatal week, despite the presence of GR-mRNA in the PVN. Chronic-implantation stress also failed to increase CRH synthesis. CRH gene expression in the PVN was enhanced in infant rats implanted with RU-38486 on postnatal day 9 or later. Cholesterol implantation on days 9, 10 (but not later), resulted in increased PVN-CRH-mRNA. Thus, CRH-mRNA is up-regulated by chronic blockade of GC receptors only subsequent to the eighth postnatal day. Furthermore, such blockade does not affect the response of CRH-mRNA to chronic stress in the neonatal rat.

ACTH Does Not Control Neonatal Seizures Induced by Administration of Exogenous Corticotropin-Releasing Hormone

Summary ACTH has been used extensively for treatment of massive infantile spasms (MIS) and other intractable seizures. The mechanisms by which ACTH exerts anticonvulsant effects are unknown. ACTH is a neuropeptide with both endocrine and neuromodulatory properties; its efficacy against MIS could derive from intrinsic anticonvulsant properties or from hormonal effects, either directly or through glucocorticoids. We tested ACTH activity against exogenous corticotropin‐releasing hormone (CRH)‐induced seizures in the infant rat model. CRH was administered into the cerebral ventricles of 85 infant rats aged 5–13 days. ACTH was used either 20–60 min before CRH administration or “chronically” (pretreatment with four doses of ACTH every 6 h, before CRH administration). In a separate group of rat pups, we measured plasma corticosterone to ascertain ACTH availability. Administration of CRH, an age‐specific endogenous convulsant, resulted in a prolonged series of seizures after 2‐ to 55‐min latency. There was no difference in latency between controls (9.5 ± 1.2 min) and ACTH‐treated rats (12.4 ± 2.8 min for combined acute and chronic groups). CRH‐induced seizure duration (88.2 ± 9 vs. 74.7 ± 9.4 min) and severity of seizures was also unchanged by ACTH treatment. ACTH reached the circulation and caused significant increase in plasma glucocorticoids. ACTH does not block the convulsant action of exogenous CRH in infant rats. An alternative mechanism for the anticonvulsant effect of ACTH may be suppression of synthesis and secretion of an endogenous convulsant, i.e., CRH.

Bicuculline induced seizures in infant rats: ontogeny of behavioral and electrocortical phenomena

The effects of bicuculline, a gamma-aminobutyric acid (GABA) antagonist, were investigated in 258 immature rats between the third and 22nd postnatal days. Behavioral and electrocorticographic events were correlated. Bicuculline induced both behavioral and electrographic seizures as early as the third postnatal day, an age when the CD50 for bicuculline was lowest, and therefore the sensitivity to it was the greatest. Bicuculline may thus be a suitable convulsant for epilepsy studies involving rats during the first postnatal week.

Status epilepticus results in reversible neuronal injury in infant rat hippocampus: novel use of a marker.

Despite ready induction of severe limbic status epilepticus by systemic kainic acid (KA) in infant rats, excitotoxic neuronal injury has not been observed. The mechanisms of this resistance of the immature hippocampus to excitotoxicity are unknown. Acid fuchsin stain has been used as a marker of irreversibly injured neurons in the adult brain. We speculated that the dye might map reversibly injured neurons in the infant. Subsequent to KA-induced status epilepticus in 11-day-old rats, acid fuchsin stain was evident in the hippocampal CA3, CA1, dentate gyrus and hilus by 24 h, peaked at 48 h and disappeared by 6 days, without evidence for neuronal loss. Acid fuchsin may be a useful tool for delineating the distribution of reversibly injured immature neurons in experimental seizure paradigms.

Short-interval amygdala kindling in neonatal rats

The kindling paradigm provides a powerful tool for studying the generation, propagation and generalization of seizures. Such reproducible quantitative paradigms are a prerequisite for the experimental study of epilepsy in the developing brain. Kindling has been extensively utilized as a model of limbic seizures in the adult rat; amygdala short-interval kindling has been studied in > or = 15-day-old rats. We applied the short-interval kindling method, i.e., stimulation at every 15 min, to 7-12-day-old rats. Stage-5 behavioral seizures were achieved even in 7-day-old rats; however, the progression of behavioral kindling differed somewhat from that of older rats. Correlation of electrographic discharges and behavioral phenomena was inversely related to age. Reliable progressive amygdala discharges were difficult to assess in most < or = 10-day-old rats. Spontaneous seizures occurred relatively frequently in younger age groups. The amygdala short-interval kindling paradigm is reproducibly and reliably applicable to rats during the 2nd postnatal week. The presence of progressive focal to bilateral-generalized seizures suggests a significant functional maturity of the amygdala-limbic circuitry at this age.

Cerebrospinal fluid corticotropin and cortisol are reduced in infantile spasms

Infantile spasms respond to ACTH, and levels of the hormone in cerebrospinal fluid of untreated infants with this disorder were found to be lower than in age-matched controls. In this study we analyzed cerebrospinal fluid cortisol and ACTH using improved immunoassays in a larger cohort of infants with infantile spasms. Analysis of 20 patients and 15 age-matched controls revealed significantly lower levels of both ACTH and cortisol in the cerebrospinal fluid. These data, combined with the efficacy of ACTH and glucocorticoids for infantile spasms, support an involvement of the brain-adrenal-axis in this disorder.

CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy

A regimen of twice daily metyrapone injections (100 mg/kg), resulted in pharmacological adrenalectomy of pregnant rats and fetuses in utero, i.e. depression of plasma corticosterone and elevation of plasma adrenocorticotropic hormone (ACTH). Toxicity was minimal on days 14-17 of pregnancy, and increased with higher maternal weight and pregnancy progression. Corticotropin releasing hormone (CRH) messenger RNA abundance in the pregnant adults increased significantly within 48 h of metyrapone initiation. No change in CRH gene expression in the paraventricular nucleus of fetuses (days 17-18) was seen, even after 72 h of the regimen. This is compatible with the independence of CRH gene expression of glucocorticoid feedback in the fetal rat.

Rhythmic Coma in Children

We describe a syndrome of rhythmic coma in children that consists of an invariant, nonreactive, diffuse cortical activity of a specific frequency, such as alpha, beta, spindle, or theta, recorded from a comatose child.We report 11 cases of children who were found to be in rhythmic coma during their acute illnesses. Their ages ranged from 2 to 15 years, and their diagnoses included encephalitis, head trauma, seizures, near drowning, brain tumors, stroke, and metabolic derangements. The specific frequency of the electroencephalographic pattern, ie, alpha, beta, spindle, or theta, did not influence the outcome. The clinical outcome appeared to depend on the primary disease process rather than the electroencephalographic finding. The prognosis of alpha-frequency rhythmic coma as well as of rhythmic coma in general was better in children than in adults. The pathophysiology in children may be similar, ie, the interruption of reticulothalamocortical pathways by metabolic or structural abnormalities, but the expression of this deafferentation may be more varied in the developing brain. Thus, we propose the term rhythmic coma as a unified concept for alpha, beta, spindle, and theta coma in children. (J Child Neurol 1990;5;242-247).