Alison M. Crane

Regional distribution of monoamines in the cerebral cortex and subcortical structures of the rhesus monkey: concentrations and in vivo synthesis rates.

Endogenous monoamine concentrations and turnover rates vary markedly in different regions of neocortex as well as in various subcortical structures of young adult rhesus monkeys. Monoamine levels and synthesis rates in amygdala, hippocampus, neostriatum, thalamus and brain stem are generally similar to comparable measures previously reported in a variety of species. However, extending and confirming the results of an earlier study, cortical monoamines exhibit topographically specific patterns of distribution. Thus, dopamine concentration is highest in the prefrontal and temporal neocortex; it decreases along the fronto-occipital axis and only trace amounts are detectable in the visual cortex. The distribution of norepinephrine is similar to that of dopamine except that the highest concentrations of norepinephrine are found in somatosensory cortex instead of prefrontal cortex. The pattern of distribution of serotonin is more uniform. However, the distribution of its metabolite, 5-hydroxyindoleacetic acid, is complementary to that of dopamine: the concentration is lowest in prefrontal cortex and highest in posterior regions of the telencephalon. Synthesis of catecholamines as measured by DOPA accumulation in monkeys treated with an aromatic amino acid decarboxylase inhibitor, NSD 1015, generally parallels the distribution of the catecholamines while indoleamine synthesis, as measured by 5-HTP accumulation, is similar to the distribution of 5-hydroxyindoleacetic acid. It may be significant that synthesis rates for the catecholamines are especially high in various areas of association cortex.

Measurement of Local Cerebral Blood Flow with [14C]Iodoantipyrine in the Mouse

Local cerebral blood flow was measured in the mouse by means of the [14C]iodoantipyrine method. This method has been previously used in the monkey, dog, cat, and rat, but its application to small mammals such as the mouse requires special attention to potential sources of error. The small size of the mouse brain requires special attention to the rapid removal and freezing of the brain to minimize effects of postmortem diffusion of tracer in the tissue. Because of the relatively low diameter/length ratios of the catheters needed for arterial sampling in small animals, substantial errors can occur in the determination of the time course of the [14C]iodoantipyrine concentration in the arterial blood unless corrections for lag time and dead space washout in the catheter are properly applied. Local cerebral blood flow was measured in seven awake mice with appropriate care to minimize these sources of error. The values were found to vary from 48 ml/100 g/min in the corpus callosum to 198 ml/100 g/min in the inferior colliculus. The results demonstrate that the [14C]iodoantipyrine method can be used to measure local cerebral blood flow in the mouse and that the values in that species are, in general, somewhat higher than those in the rat.

Local Changes in Cerebral Glucose Utilization during Ketamine Anesthesia

Ketamine produces both excitatory and depressant actions in the brain, but there have been conflicting results regarding which structures are affected and the magnitude of the alteration in cerebral metabolism produced. The authors applied the 2-[14C]deoxyglucose method quantitatively to a study of

Adaptation of the quantitative 2-[14C]deoxyglucose method for use in freely moving rats

A procedure for venous and arterial catheterization is described which allows the quantitative 2-[14C]deoxyglucose method to be applied to freely moving animals for behavioral and pharmacological studies. The catheterization method is rapid, minimally invasive, and requires no complicated equipment. Physiological conditions and rates of cerebral glucose utilization in freely moving rats and in restrained rats have been compared. The results demonstrate that local cerebral glucose utilization can readily be measured in freely moving animals engaged in behavioral experiments.

Invalidity of criticisms of the deoxyglucose method based on alleged glucose-6-phosphatase activity in brain.

Abstract: The observations made by Sacks et al. [Neurochem. Rea.8, 661–685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose‐6‐phosphate. predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze‐blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose‐6‐phos‐phate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose‐6‐phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose‐6‐phosphate in brain, that infusion of [12P]glucose‐6‐phosphate results in no labeling of brain, and that the blood‐brain barrier is impermeable to glucose‐6‐phosphate. Glucose‐6‐phosphate cannot, therefore, cross the blood‐brain barrier, and the observation by Sacks and coworkers [J. Appl. Physiol.24, 817–827 (1968); Neuro‐chein. Res.8, 661–685 (1983)J of a positive cerebral arteriovenous difference for [14C]glucose‐6‐phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose‐6‐phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity.

Metabolic mapping of the effects of intravenous methamphetamine administration in freely moving rats

The 2-[14C]deoxyglucose method was used to examine the effects of acute intravenous administration of methamphetamine (0.5–2.5 mg/kg) on rates of local cerebral glucose utilization in freely-moving rats. These effects were correlated with the effects of methamphetamine on locomotor activity assessed simultaneously in the same animals. Methamphetamine administration resulted in widespread dose-dependent increases in glucose utilization within structures of the extrapyramidal motor system. Rates of glucose utilization were positively correlated with locomotor activity in the globus pallidus, substantia nigra reticulata, entopeduncular nucleus, subthalamic nucleus, and the lateral cerebellar cortex. In contrast, within the limbic system alterations in metabolic activity were smaller and more selective. Glucose utilization was increased in the nucleus accumbens at all doses tested, but alterations in glucose utilization in the ventral tegmental area, amygdala, and anterior cingulate were observed only at the highest doses of methamphetamine tested. Significant increases in rates of glucose metabolism were also found in the substantia nigra compacta and in the median and dorsal raphe nuclei. Dopamine and serotonin are depleted in these regions, as well as in the ventral tegmental area where glucose utilization was also increased, following chronic treatment with high doses of methamphetamine. These changes in glucose utilization may be indicative of disturbances in the biochemical processes involved in the neurotoxic effects of methamphetamine.

Local Cerebral Glucose Utilization in Normal Female Rats: Variations during the Estrous Cycle and Comparison with Males:

The quantitative 2-[14C]deoxyglucose autoradiographic method was used to study the fluctuations of energy metabolism in discrete brain regions of female rats during the estrous cycle. A consistent though statistically nonsignificant cyclic variation in average glucose utilization of the brain as a whole was observed. Highest levels of glucose utilization occurred during proestrus and metestrus, whereas lower rates were found during estrus and diestrus. Statistically significant fluctuations were found specifically in the hypothalamus and in some limbic structures. Rates of glucose utilization in the female rat brain were compared with rates in normal male rats. Statistically significant differences between males and females at any stage of the estrous cycle were confined mainly to hypothalamic areas known to be involved in the control of sexual behavior. Glucose utilization in males and females was not significantly different in most other cerebral structures.

Damage to Neurons in Culture Following Medium Change: Role of Glutamine and Extracellular Generation of Glutamate

Abstract— Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N‐methyl‐d‐aspartate (NMDA)‐type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10–20 μM) of 2‐amino‐5‐phos‐phonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 μM) of 2‐amino‐5‐phosphonovaleric acid can protect cultures when added at much later times (30–60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 μrn pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6‐Diazo‐5‐oxo‐l‐norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.

A comparison of local rates of glucose utilization in spinal cord and brain in conscious and nitrous oxide- or pentobarbital-treated rats

Local rates of glucose utilization in the spinal cord and brain were measured with the 2-[14C]deoxyglucose method in conscious and in paralyzed and mechanically ventilated pentobarbital- or 70% nitrous oxide-treated rats. In conscious animal lumbar spinal cord glucose utilization is only 40–50% that of the cerebral cortex and shows little laminar heterogeneity. Pentobarbital reduces and nitrous oxide increases the cerebral glucose utilization of most structures. The effect of paralysis and nitrous oxide analgesia on lumbar spinal cord glucose utilization is quantitatively similar to that produced in brain; 15–25% increases occur in most spinal cord laminae and cerebral structures. In contrast, the 10–20% reduction in spinal cord gray matter metabolism in the paralyzed and pentobarbital-treated animals is considerably less than the 20–50% depression measured in most brain structures. From these data the authors conclude that, relative to that of most cerebral structures, spinal cord metabolism is less sensitive to depression by barbiturates and suggest that differences in the cell populations of these tissues may account partially for this observation.

Cell Damage Associated with Changing the Medium of Mesencephalic Cultures in Serum-Free Medium Is Mediated via N-Methyl-D-Aspartate Receptors

Abstract: Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum‐free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N‐methyl‐D‐aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Hams F‐12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbeccos modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.