Interplay between Energy Supply and Glutamate Toxicity in the Primary Cortical Culture.

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.

Absence of somatotrophs, lactotrophs, and thyrotrophs in the pituitary of two dwarfs with hypothyroidism: Deficiency of pituitary transcription factor-1?

Most patients with dwarfism due to growth hormone (GH) deficiency have normal pituitary somatotroph morphology and GH response to GH-releasing hormone (GRH), consistent with decreased GRH synthesis, release, or delivery. Primary pituitary hyposecretion of GH may result from adenohypophysial tissue destruction caused by tumors, such as craniopharyngioma. We report a hitherto undescribed form of primary pituitary dwarfism associated with absence of adenohypophysial GH, prolactin (PRL), and thyrotropin (TSH). Two sisters had dwarfism, hypothyroidism, and hypoglycemia. The first child had craniofacial abnormalities and died at age 11/2 months of fluid overload. The second sibling died at age 4 years of pulmonary congestion. At autopsy, both pituitaries were small and acidophils were conspicuously absent. They contained a normal number of corticotrophs with intense adrenocorticotropin (ACTH) immunoreactivity, and gonadotrophs with normal content of α-subunit and ß-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). No GH, PRL, or ß-TSH immunoreactivity was identified. The thyroid glandswere atrophic. Both children had marked pancreatic nesidioblastosis, with an increased number of insulin-containing cells. This clinicopathologic entity appears to represent a familial disorder in which there is defective development of three adenohypophysial cell types: somatotrophs, lactotrophs, and thyrotrophs. A common pituitary transcription factor, Pit-1, has been implicated in the differentiation of these three cell types. The pituitary changes in these two children resemble those described in Snell, Jackson, and Ames dwarf mice, which have recently been shown to be due to abnormalities of the Pit-1 gene. We suggest that this novel human disease is due to deficient or abnormal pituitary transcription factor Pit-1.