Matthew S. Miller

Differential patterns of regional c-Fos induction in the rat brain by amphetamine and the novel wakefulness-promoting agent modafinil

We examined the neuronal targets in the rat brain for the novel wakefulness-promoting agent modafinil and for amphetamine using c-Fos immunohistochemistry. Both modafinil and amphetamine induced neuronal expression of c-Fos-like immunoreactivity in the paraventricular nucleus of the hypothalamus, anterior hypothalamus and central nucleus of the amygdala. Modafinil also increased c-Fos-like immunoreactivity in the suprachiasmatic nucleus, while amphetamine had no effect. Brain regions in which amphetamine increased c-Fos-like immunoreactivity, but modafinil had no effect, included frontal cortex, striatum, lateral habenula, supraoptic nucleus and basolateral nucleus of the amygdala. These findings suggest that the mechanism of action of modafinil is different from that of amphetamine and that the neuronal targets for modafinil in the brain include nuclei of the hypothalamus and amygdala.

Brain regional substrates for the actions of the novel wake-promoting agent modafinil in the rat: comparison with amphetamine

Modafinil is a novel wake-promoting compound for which the mechanism and sites of action are unknown. We examined the neural substrates in the brain for the actions of modafinil using 2-deoxyglucose autoradiography and compared the findings to those obtained with amphetamine. Modafinil showed a relatively restricted pattern of changes in brain regional metabolic activity, while amphetamine altered glucose utilization in a wide variety of brain regions. Both modafinil and amphetamine increased glucose utilization in all subregions of the hippocampus (subiculum, CA1-CA3 and dentate gyrus) and in the centrolateral nucleus of the thalamus. Modafinil also increased glucose utilization in the central nucleus of the amygdala, but amphetamine had no effect in this region. Brain structures in which amphetamine increased metabolic rate but modafinil had no effect included regions of the basal ganglia, other nuclei of the thalamus, the frontal cortex, the nucleus accumbens, the ventral tegmental area and the pontine reticular fields. These findings suggest that, while both modafinil and amphetamine promote wakefulness, they act via distinctly different mechanisms. Modafinil appears to act on a specific subset of brain pathways which regulate sleep and wakefulness, whereas amphetamine affects a greater number of cerebral structures involved in the regulation of these behavioral states. Modafinil also lacks the pronounced effects on the extrapyramidal motor system which are characteristic of amphetamine and other psychomotor stimulants, implying that the effects of modafinil are not mediated by the dopamine system and that modafinil may selectively increase wakefulness with fewer side effects.

Insulin-like Growth Factor I Reverses Experimental Diabetic Autonomic Neuropathy

Recent studies have suggested a role for neurotrophic substances in the pathogenesis and treatment of diabetic neuropathy. In this study, the effect of insulin-like growth factor I (IGF-I) on diabetic sympathetic autonomic neuropathy was examined in an experimental streptozotocin-induced diabetic rat model. Two months of IGF-I treatment of chronically diabetic rats with established neuroaxonal dystrophy (the neuropathological hallmark of the disease) involving the superior mesenteric ganglion and ileal mesenteric nerves resulted in nearly complete normalization of the frequency of neuroaxonal dystrophy in both sites without altering the severity of diabetes. Treatment with low-dose insulin (to control for the transient glucose-lowering effects of IGF-I) failed to affect the frequency of ganglionic or mesenteric nerve neuroaxonal dystrophy or the severity of diabetes. The striking improvement in the severity of diabetic autonomic neuropathy shown with IGF-I treatment in these studies and the fidelity of the rat model to findings in diabetic human sympathetic ganglia provide promise for the development of new clinical therapeutic strategies.

IMMUNOLOCALIZATION OF THE MITOGEN-ACTIVATED PROTEIN KINASES P42MAPK AND JNK1, AND THEIR REGULATORY KINASES MEK1 AND MEK4, IN ADULT RAT CENTRAL NERVOUS SYSTEM

Cell survival, death, and stress signals are transduced from the cell surface to the cytoplasm and nucleus via a cascade of phosphorylation events involving the mitogen‐activated protein kinase (MAPK) family. We compared the distribution of p42 mitogen‐activated protein kinase (p42MAPK) and its activator MAPK or ERK kinase (MEK1; involved in transduction of growth and differentiation signals), with c‐Jun N‐terminal kinase (JNK1) and its activator MEK4 (involved in transduction of stress and death signals) in the adult rat central nervous system. All four kinases were present in the cytoplasm, dendrites, and axons of neurons. The presence of p42MAPK and JNK1 in dendrites and axons, as well as in cell bodies, suggests a role for these kinases in phosphorylation and regulation of cytoplasmic targets. A high degree of correspondence was found between the regional distribution of MEK1 and p42MAPK. Immunostaining for MEK1 and p42MAPK was intense in olfactory structures, neocortex, hippocampus, striatum, midline, and interlaminar thalamic nuclei, hypothalamus, brainstem, Purkinje cells, and spinal cord. In addition to neurons, p42MAPK was also present in oligodendrocytes. Whereas MEK4 was ubiquitously distributed, JNK1 was more selective. Immunostaining for MEK4 and JNK1 was intense in the olfactory bulb, lower cortical layers, the cholinergic basal forebrain, most nuclei of the thalamus, medial habenula, and cranial motor nuclei. The distribution of MEK1 and p42MAPK proteins only partially overlapped with that of MEK4 and JNK1. This suggests that the growth /differentiation and death /stress pathways affected by these kinases may not necessarily act to counterbalance each other in response to extracellular stimuli. The differential distribution of these kinases may control the specificity of neuronal function to extracellular signals. J. Comp. Neurol. 398:373–392, 1998.

Modafinil more effectively induces wakefulness in orexin-null mice than in wild-type littermates

Narcolepsy-cataplexy, a disorder of excessive sleepiness and abnormalities of rapid eye movement (REM) sleep, results from deficiency of the hypothalamic orexin (hypocretin) neuropeptides. Modafinil, an atypical wakefulness-promoting agent with an unknown mechanism of action, is used to treat hypersomnolence in these patients. Fos protein immunohistochemistry has previously demonstrated that orexin neurons are activated after modafinil administration, and it has been hypothesized that the wakefulness-promoting properties of modafinil might therefore be mediated by the neuropeptide. Here we tested this hypothesis by immunohistochemical, electroencephalographic, and behavioral methods using modafinil at doses of 0, 10, 30 and 100 mg/kg i.p. in orexin-/- mice and their wild-type littermates. We found that modafinil produced similar patterns of neuronal activation, as indicated by Fos immunohistochemistry, in both genotypes. Surprisingly, modafinil more effectively increased wakefulness time in orexin-/- mice than in the wild-type mice. This may reflect compensatory facilitation of components of central arousal in the absence of orexin in the null mice. In contrast, the compound did not suppress direct transitions from wakefulness to REM sleep, a sign of narcolepsy-cataplexy in mice. Spectral analysis of the electroencephalogram in awake orexin-/- mice under baseline conditions revealed reduced power in the theta; band frequencies (8-9 Hz), an index of alertness or attention during wakefulness in the rodent. Modafinil administration only partly compensated for this attention deficit in the orexin null mice. We conclude that the presence of orexin is not required for the wakefulness-prolonging action of modafinil, but orexin may mediate some of the alerting effects of the compound.

Preservation of cholinergic activity and prevention of neuron death by CEP-1347/KT-7515 following excitotoxic injury of the nucleus basalis magnocellularis

We have identified a class of small organic molecules, derived from the indolocarbazole K-252a, that promote the survival of cultured neurons. However, many of these indolocarbazoles inhibit protein kinase C and neurotrophin-activated tyrosine kinase receptors. These kinase inhibitory activities may limit the utility of these compounds for neurological disorders. A bis-ethyl-thiomethyl analogue of K-252a, CEP-1347/KT-7515, has been identified that lacks protein kinase C and tyrosine kinase receptor inhibitory activities, yet retains the ability to promote survival of cultured neurons, including cholinergic neurons derived from the basal forebrain. In the present studies, CEP-1347/KT-7515 was assessed for neurotrophic activity on basal forebrain neurons of in vivo rats following excitotoxic insult. Ibotenate infusion into the nucleus basalis magnocellularis reduced levels of choline acetyltransferase activity in the cortex, as well as reduced numbers of choline acetyltransferase-immunoreactive and retrogradely (FluoroGold)-labelled cortically-projecting neurons in the nucleus basalis. Systemically administered CEP-1347/KT-7515 attenuated the loss of cortical choline acetyltransferase activity and the loss of the number of choline acetyltransferase-immunoreactive and retrogradely-labelled FluoroGold neurons in the nucleus basalis. Moreover, CEP-1347/KT-7515 ameliorated the loss of cortical choline acetyltransferase if administration was initiated one day, but not seven days post-lesion. Together, these results demonstrate that CEP-1347/KT-7515 protects damaged cortically-projecting basal forebrain neurons from degeneration. Thus, CEP-1347/KT-7515 may have therapeutic potential in neurodegenerative diseases, such as Alzheimers disease, in which basal forebrain cholinergic neurons degenerate.

Morpholinoalkylindenes as antinociceptive agents: Novel cannabinoid receptor agonists

Abstract Indence analogs of pravadoline exhibited antinociceptive activity in several animal models. The inhibition of prostaglandin (PG) synthesis in mouse brain microsomes was diminished in these pravadoline analogs, but they were potent in inhibiting electrically stimulated contractions in the mouse vas deferens (MVD) preparations. Binding studies with ligand WIN 55212-2 have aided to demonstrate that the morpholinoalkyl-indene binding site is functionally equivalent with cannabinoid binding site. The antinociceptive activity of the indene derivatives appears to be mediated by increased affinity for the cannabinoid receptor.