Methylphenidate regulates c-fos and fosB expression in multiple regions of the immature rat brain

Methylphenidate (Ritalin, MPH) is a common psychostimulant used to treat childhood attention-deficit hyperactivity disorder (ADHD). Little is known about the long-term developmental effects on gene expression and behavior, which may occur with extended MPH use. We reported previously that the striatum is a major target of MPH, consistent with human MRI studies. In the present study, we tested the hypothesis that MPH is likely to have widespread effects in extra-striatal regions of the brain. We used the expression of two immediate early genes, c-fos and fosB, as probes to map the response of the immature rat brain to single (1 day) versus repeated (14 days) MPH treatment (2 or 10 mg/kg; s.c.) from postnatal day 25 to 38. Consistent with previous reports, the striatum is a major target of acute MPH action, as indicated by elevated levels of cFOS-immunoreactivity (-ir). Increases in c-fos expression were also seen in the nucleus accumbens, cingulate/frontal cortex and piriform cortex, and Islands of Calleja. FosB expression was elevated only in the striatum following a single stimulation. Chronic MPH treatment (10 mg/kg/day for 14 days) resulted in an attenuation of c-fos expression in the striatum and Islands of Calleja. However, levels of cFOS-ir remained elevated in the nucleus accumbens and frontal cortex. In contrast to the inhibitory effect of repeated MPH exposure on c-fos expression, FOSB-ir was further elevated in the striatum, and an increase was observed in the cingulate/frontal and piriform cortices. Thus, chronic MPH differentially regulated expression of c-fos and fosB in several brain regions. Our data suggest that MPH may exert its stimulant effects at multiple sites in the immature brain, which has implications for long-term treatment in children.     

Hypoxia differentially regulates the expression of neuroglobin and cytoglobin in rat brain

Neuroglobin (Ngb) and Cytoglobin (Cygb) are new members of the globin family and display heterotopic expression patterns. To examine the effect of different hypoxia profiles on expression of Ngb and Cygb in rodent brain, rats were exposed to either sustained hypoxia (SH; 10% O(2)) or intermittent hypoxia (IH; 10% and 21% O(2) alternating every 90 s) for 1, 3, 7 and 14 days, and mRNA and protein expression of Ngb and Cygb were assessed in brain cortex. SH increased Ngb mRNA and protein expression throughout the exposure, while IH only elicited slight increases in Ngb expression at day 1. Neither SH nor IH elicited increases in Cygb expression. Thus, hypoxic stimulus presentation is a major determinant of the regulation of hypoxic sensitive genes such as Ngb. Furthermore, disparities between Ngb and Cygb responses to hypoxia further suggest that these two globins may play divergent roles in brain.     

Scopolamine attenuates haloperidol-induced c-fos expression in the striatum.

Haloperidol increases the expression of Fos, the protein product of the proto-oncogene c-fos, in some parts of the central nervous system. Haloperidol also produces catalepsy in rodents and extrapyramidal side effects in humans, both of which are reduced by muscarinic receptor antagonists. In order to gain insight into the neurochemical and neuroanatomical substrates of haloperidol-induced catalepsy we examined the effects of the muscarinic receptor antagonist scopolamine on haloperidol-induced Fos expression in the striatum, nucleus accumbens and lateral septal nucleus. At a dose that reduced the cataleptic effect of haloperidol, scopolamine decreased the neuroleptic-induced Fos expression in the striatum and lateral septal nucleus but not the nucleus accumbens. These results indicate that haloperidol may increase c-fos expression in medium spiny striatal neurons indirectly by enhancing striatal acetylcholine release. They are also consistent with the hypothesis that neuroleptic-induced increases in striatal c-fos expression are predictive of extrapyramidal side effects produced by these compounds.     

Transient compartmental expression of neurocan in the developing striatum of the rat

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum. J. Neurosci. Res. 51:612–618, 1998. © 1998 Wiley-Liss, Inc.     

Androgenic-anabolic steroids blunt morphine-induced c-fos expression in the rat striatum: possible role of beta-endorphin

Self-administration of large doses of androgenic-anabolic steroids (AAS) in a significant portion of the population suggests that these agents are drugs of abuse. However, acute administration of AAS did not induce striatal immediate-early genes (IEG) expression in male rats, indicating that AAS do not share a common mechanism of action with other drugs of abuse. Surveys have indicated that people who abuse AAS are more likely to self-administer other drugs of abuse than do people who do not take AAS. In the present study, chronic administration of AAS blunted the striatal c-fos response to morphine, indicating that AAS can alter the molecular responses to at least one drug of abuse. Chronic administration of AAS also increased the content of beta-endorphin in the midline thalamus, suggesting a possible mechanism by which AAS may modulate the response to morphine through regulation of thalamo-striatal neurons.     

The medial prefrontal cortex differentially regulates stress-induced c-fos expression in the forebrain depending on type of stressor

The medial prefrontal cortex (mPFC) plays an important inhibitory role in the hypothalamic-pituitary-adrenal (HPA) axis response. The involvement of the mPFC appears to depend on the type of stressor, preferentially affecting 'psychogenic' stimuli. In this study, we mapped expression of c-fos mRNA to assess the neural circuitry underlying stressor-specific actions of the mPFC on HPA reactivity. Thus, groups of mPFC-lesioned and sham-operated rats were restrained for 20 min or exposed to ether fumes for 2 min. In both cases, the animals were killed at 40 min from the onset of stress. Interestingly, bilateral lesions of the mPFC significantly enhanced c-fos mRNA expression in the hypothalamic paraventricular nucleus of restrained animals, an effect that was paralleled by potentiation of circulating ACTH concentrations in these animals. On the other hand, lesions of the mPFC did not affect neither PVN c-fos mRNA expression nor plasma ACTH concentrations in animals exposed to ether. Lesions of the mPFC also enhanced c-fos activation in the medial amygdala following restraint, but not following ether exposure. Additional regions whose activity was affected by mPFC lesions or stressor differences included the ventrolateral division of the bed nucleus of the stria terminalis, CA3 hippocampus, piriform cortex, and dorsal endopiriform nucleus. Expression of c-fos mRNA was nearly absent in the central amygdala of all stressed animals, regardless of lesion. Furthermore, prefrontal cortex lesions did not change stress-induction levels of c-fos in the CA1 hippocampus, dentate gyrus, anteromedial division of the bed nucleus of the stria terminalis, lateral septum, and claustrum. Taken together, this study indicates that the medial prefrontal cortex differentially regulates cellular activation of specific stress-related brain regions, thus exerting stressor-dependent inhibition of the HPA axis.     

Thyroid hormone regulates TAG-1 expression in the developing rat brain

TAG-1 is a member of the immunoglobulin superfamily of cell adhesion molecules thought to play important roles in neuronal differentiation and the establishment of connectivity during brain development. Because these are processes also affected by hypothyroidism, we studied the effects of thyroid hormone deprivation and administration on TAG-1 expression in the developing rat brain. By in situ hybridization, immunohistochemistry and Western blotting we found that TAG-1 RNA and protein levels are upregulated in the hypothyroid brain. From embryonic day 20 to postnatal day (P) 15, elevated TAG-1 RNA was found in several areas including the cerebral cortex, hippocampus and olfactory bulb. In agreement with this, TAG-1 protein was overexpressed in the major fibre tracts arising from these structures, including the corpus callosum, anterior and hippocampal commissures and lateral olfactory tract. A similar overexpression of TAG-1 by hypothyroidism was detected in the cerebellum, but starting only at P15. In all cases, elevation of TAG-1 RNA and protein expression could be reversed by thyroid hormone treatment. These results show that the deregulation of TAG-1 might contribute to the alterations caused by the lack of thyroid hormone during brain development.     

Transient and compartmental expression of the reeler gene product reelin in the developing rat striatum

Mammalian neostriatum is composed of two neurochemically and neuroanatomically defined compartments, called the patches and matrix. The present study concerns a search for neurochemical molecules involved in formation of the striatal compartments. Using the monoclonal antibody CR-50, we here disclose a transient expression of the reeler gene product Reelin, which is known to play a crucial role in neuronal positioning and axon guidance during corticogenesis, in the developing striatum of rats. Furthermore, Reelin protein is differentially concentrated in the two distinct compartments showing a mosaic-like fashion in the early postnatal period: the compartments of heightened CR-50-immunolabeling correspond to so-called “dopamine islands” (i.e., developing striosomes) visualized by tyrosine hydroxylase (TH)-immunostaining. On the basis of these findings, we hypothesize that Reelin protein may play a role in developmental organization of the striatal compartments.     

Daily methylphenidate administration attenuates c-fos expression in the striatum of prepubertal rats

Methylphenidate (Ritalin) is a psychostimulant drug used to treat children with attention deficit hyperactivity disorder. Despite its widespread and increasing clinical use, little is known about the long-term consequences of drug treatment. We compared the effects of a single injection of methylphenidate with that of long-term methylphenidate injections (one/day; 14 days) on immediate-early gene expression (c-fos) in the striatum of prepubertal male rats. Rats (25 days old) were injected once daily for 14 days with either saline or methylphenidate (1, 2 or 10 mg/kg), or for 13 days with saline followed by one injection of methylphenidate (1, 2 or 10 mg/kg) on day 14, and were sacrificed 2 h post-injection. Methylphenidate dose-dependently increased FOS immunoreactivity in the striatum. A single injection of methylphenidate (2 or 10 mg/kg) on day 14, following saline treatment for 13 days, caused a dramatic elevation in c-fos expression. This effect was significantly attenuated in animals treated chronically with methylphenidate (2 or 10 mg/kg) for the entire 14 days. Our data suggest that repeated methylphenidate treatment, at a clinically relevant dose (2 mg/kg), markedly inhibits immediate-early gene expression in the brain. This is the first demonstration of methylphenidate-induced modification of gene expression in developing rat striatum and may have implications for chronic methylphenidate use in children.     

Acute and repeated administration of cocaine differentially regulates expression of PSA-NCAM-positive neurons in the rat hippocampus

Recent data indicating that addictive substances are able to alter brain plasticity and its morphology inclined us to determine whether acute and chronic cocaine administration could modify the expression of a polysialylated form of the neuronal cell adhesion molecule (PSA-NCAM) in the dentate gyrus of the rat hippocampus. Alterations in the PSA-NCAM expression are known to effect a variety of neuroanatomical rearrangements in the brain structure. Cocaine was administered acutely (15 mg/kg, i.p.) or repeatedly (15 mg/kg, i.p. once a day for five consecutive days). The number of PSA-NCAM immunopositive cells was determined at six time points after cocaine treatment: 6 h and 1, 2, 4, 6, and 10 days (both in acute and repeated treatment). It was found that a single injection of cocaine induced a time-dependent decrease in the number of PSA-NCAM cells in the dentate gyrus. The decrease was observed on day 1 after cocaine treatment and lasted for at least 6 days. In contrast, an increase in the number of PSA-NCAM-positive cells in the dentate gyrus was observed 2 and 4 days after the last dose of repeated cocaine. It is concluded that cocaine can evoke long-lasting changes in the PSA-NCAM protein expression in the dentate gyrus and that the direction of cocaine-induced PSA-NCAM changes depends on the regimen of cocaine administration. It is postulated that cocaine may have impact on hippocampal plasticity and subsequent processes that are controlled by plastic changes in the hippocampal structure.     

Differential c-fos expression in the rhinencephalon and striatum after enhanced sleep-wake states in the cat

In order to delimit the supra-brainstem structures that are activated during the sleep-waking cycle, we have examined c-fos immunoreactivity in four groups of polygraphically recorded cats killed after 3 h of prolonged waking (W), slow-wave sleep (SWS), or paradoxical sleep (PS), following microinjection of muscimol (a gamma-aminobutyric acid, GABA agonist) into the periaqueductal grey matter and adjacent areas [Sastre et al. (1996), Neuroscience, 74, 415-426]. Our results demonstrate that there was a direct relationship between a significant increase in c-fos labelling and the amount of PS in the laterodorsalis tegmenti in the pons, supramamillary nucleus, septum, hippocampus, gyrus cingulate, amygdala, stria terminalis and the accumbens nuclei. Moreover, in all these structures, the number of Fos-like immunoreactive neurons in the PS group was significantly higher (three to 30-fold) than in the SWS and W groups. We suggest that the dense expression of the immediate-early gene c-fos in the rhinencephalon and striatum may be considered as a tonic component of PS at the molecular level and that, during PS, the rhinencephalon and striatum are the main targets of an excitatory system originating in the pons.     

Transient and compartmental expression of the reeler gene product Reelin in the developing rat striatum

Mammalian neostriatum is composed of two neurochemically and neuroanatomically defined compartments, called the patches and matrix. The present study concerns a search for neurochemical molecules involved in formation of the striatal compartments. Using the monoclonal antibody CR-50, we here disclose a transient expression of the reeler gene product Reelin, which is known to play a crucial role in neuronal positioning and axon guidance during corticogenesis, in the developing striatum of rats. Furthermore, Reelin protein is differentially concentrated in the two distinct compartments showing a mosaic-like fashion in the early postnatal period: the compartments of heightened CR-50-immunolabeling correspond to so-called “dopamine islands” (i.e., developing striosomes) visualized by tyrosine hydroxylase (TH)-immunostaining. On the basis of these findings, we hypothesize that Reelin protein may play a role in developmental organization of the striatal compartments.     

Transient c-fos expression accompanies naturally occurring cell death in the developing interhemispheric cortex of the rat.

We have searched for the possible correlation of naturally occurring cell death with spontaneously enhanced c-fos expression in the developing cerebral cortex of normal Wistar albino rats. During the late prenatal and early postnatal period, cells with irregular contours and intracytoplasmic electron-dense granules (granule-containing cells) were apparent in the interhemispheric cortex, including the anterior cingulate and the retrosplenial cortices. These cells were loosely packed within the cortical layers derived from the cortical plate. Having excluded the possibility that these cells could be phagocytes by immunocytochemical experiments, we propose that they are cells in different phases of a process of autophagic degeneration and death. Images of extreme nuclear pyknosis were also apparent in identical locations. Cells showing immunoreactivity for c-Fos protein appeared in the same cortical areas. The immunoreactive cells were very abundant in the retrosplenial cortex, but were also present in the anterior cingulate cortex. These cells showed markedly irregular contours and large, densely immunoreactive intracytoplasmic inclusions; these images were similar to those of granule-containing cells revealed by conventional stains. The immunoreactivity for c-Fos protein was ephemeral, occurring exclusively during embryonic days 20 and 21, but granule-containing cells were observed for a longer period. The present results provide evidence, albeit indirect, that c-fos expression may occur in certain neural cells at the onset of a process of death by autophagia, and suggest a possible involvement of the proto-oncogene c-fos in certain forms of naturally occurring neuronal death.     

Long-term suppression of methamphetamine-induced c-Fos expression in rat striatum by the injection of c-fos antisense oligodeoxynucleotides absorbed in water-absorbent polymer

The use of water-absorbent polymer (WAP) as a hydrogel carrier for the slow delivery of antisense oligodeoxynucleotides (ODN) in the brain, was recently developed. In this experiment, 15-mer phosphorothioate ODN, complementary to c-fos gene absorbed in WAP, was injected in the rat striatum. The expression of c-Fos-immunoreactivity induced by methamphetamine (6 mg/kg, intraperitoneally) around the injection site was suppressed until 5 days after injection. Using this method, it was observed that unilateral injection with c-fos antisense ODN into the rat striatum caused robust ipsilateral rotations after methamphetamine challenge 4 days post injection. This method is simple, and the biological and behavioral effects of antisense ODN in WAP can be maintained for several days even after a single injection into the brain.     

Excitatory amino acids differentially regulate the expression of GDNF, neurturin, and their receptors in the adult rat striatum

Glial cell line-derived neurotrophic factor (GDNF) family ligands are important regulators of neuronal development and maintenance of the connectivity in the basal ganglia and show neuroprotective activities in several paradigms of brain injury. The mRNAs of two members of this family, GDNF and neurturin, and also their receptors have been detected in the basal ganglia. In the present work, we analyzed the time course changes in the expression of these neurotrophic factors and receptors in the adult rat striatum, induced by quinolinate or kainate excitotoxicity. Our results show that stimulation of NMDA or non-NMDA receptors induced different effects on the mRNA levels analyzed. Expression of GDNF and its preferred receptor, GDNF family receptor-alpha1 (GFRalpha1), was transiently up-regulated by quinolinate and kainate, but with differing intensity and temporal pattern. Immunohistochemical analysis showed that, although GDNF and GFRalpha1 were initially localized in neurons, excitotoxicity induced the expression of these proteins in astrocyte-like cells. Neurturin mRNA levels were only up-regulated after quinolinate injection, whereas quinolinate or kainate injection did not modify GFRalpha2 mRNA. The mRNA for the common receptor, c-Ret, was up-regulated by both agonists with similar temporal pattern but with differing intensity. Immunohistochemical analysis showed that c-Ret protein was located on neurons. These changes in mRNA levels and protein localization of GDNF family components could reflect an endogenous trophic response of striatal cells to different excitotoxic insults.     

Dopamine D1 autoreceptor function: possible expression in developing rat prefrontal cortex and striatum.

Synthesis-modulating dopamine (DA) autoreceptor function was studied in vivo using gamma-butyrolactone (GBL) to block propagation along DA axons. DA synthesis was measured by the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) after inhibition of aromatic L-amino acid decarboxylase. GBL treatment markedly increased DOPA accumulation in both the striatum and prefrontal cortex of developing rats. The selective DA partial D1 agonist SKF-38393 inhibited this GBL-induced rise in DA synthesis in both the striatum and prefrontal cortex of 15- and 22-day-old rats, but not in adults. The effects of SKF-38393 in developing rats were mimicked by the non-catechol D1 partial agonist CY-208-243, and were blocked by the D1 antagonist SCH-23390, suggesting receptor mediation. The mixed D2/D3 agonist quinpirole attenuated DA synthesis in striatum of both two-week-old and adult rats, but failed to inhibit the GBL-induced increase in DA synthesis in the developing prefrontal cortex. These findings suggest that synthesis-modulating D1-like receptor function may emerge transiently in the developing mammalian forebrain. In the adult striatum these functions appear to be subsumed by D2-like receptors, whereas all synthesis-modulating DA receptor function in prefrontal cortex appears to be essentially lost with maturation.