Short-term cocaine self administration alters striatal gene expression

Rats self-administered cocaine or received saline during 3 daily 5 h sessions and were euthanized 1 h after the final session. Quantitative in situ hybridization revealed that cocaine self-administration increased levels of preprodynorphin, but not preproenkephalin, c-fos, or zif/268 mRNAs in a patchy pattern in the dorsal striatum. These data demonstrate that the regulation of preprodynorphin gene expression is dissociable from that of c-fos and zif/268 in dorsal striatum following short-term cocaine self-administration.     

Suppression of post-ischemic-induced Fos protein expression by an antisense oligonucleotide to c-fos mRNA leads to increased tissue damage

Activation of c-fos, an immediate early gene, and the subsequent upregulation of Fos protein expression occur following neural injury, including focal cerebral ischemia (fci). Fos and Jun form a heterodimer known as activator protein 1, which regulates the expression of many late effector genes. To study the downstream effects of c-fos expression following ischemia, we suppressed the translation of c-fos by administering an antisense oligonucleotide (AO) to c-fos mRNA. Eighteen hours prior to fci, male, Long Evans (LE) rats received intraventricular injections of AO, mismatched AO (MS) or artificial cerebrospinal fluid (aCSF). Fci was induced by permanent right middle cerebral artery occlusion. At 24-h post-occlusion, neurological function was assessed, and the animals were sacrificed. The brains were removed and stained with triphenyltetrazolium chloride for infarct volume determination. Fos immunohistochemistry was performed in separate animals to determine the effects of treatment on Fos expression number of Fos positive cells. AO administration reduced the number of cells with fci-induced Fos expression by 75%. No differences in neurological scores existed between any of the groups. AO-treated LE developed larger infarcts (40.1±1.0%, mean±S.D., p<0.001) than MS- or aCSF-treated controls (34.3±1.0%, 34.6±1.0%, respectively). These results suggest that c-fos activation and subsequent Fos protein expression exerts a neuroprotective effect, which is likely via upregulation of neurotrophins, following focal cerebral ischemia. This response, among others, may contribute to brain adaptation to injury that underlies functional recovery after stroke.     

Systematic expression of immediate early genes and intensive astrocyte activation induced by intrastriatal ferrous iron injection

The potential role(s) of transitional metals such as iron have been implicated in neurodegeneration through biochemical processes, particularly oxidative stress. We injected ferrous chloride (FeCl2) and ferric chloride (FeCl3) into the striatonigral system of Sprague-Dawley rats to investigate the biological and toxic effects of ferrous iron in the central nervous system. When FeCl2 was injected into the ventral midbrain, rats showed a characteristic behavior which indicated ipsilateral dopaminergic hyperactivity. FeCl2 injection into the striatum induced a dose-dependent damage, the activation of astrocytes and recruitment of macrophage/microglia at the injected site. Interestingly, the activation of astrocytes was also observed in the anatomically remote areas such as the ipsilateral subthalamic nucleus and pars reticulata of the substantia nigra after 1 week. Expression of immediate early genes (IEGs; c-fos and NGFI-A) was observed in the cortex, thalamic nuclei, subthalamic nucleus, pars reticulata of the substantia nigra, lateral and medial geniculate bodies on the ipsilateral side from 3 to 15 h after FeCl2 injection. Pre-treatment with dimethyl sulfoxide, a hydroxyl radical scavenger, prevented FeCl2-induced expression of IEGs in the thalamic nuclei and geniculate bodies, but not in the cerebral cortex. On the other hand, the effects of FeCl3 were faint and limited on IEGs expression and tissue damage. These results suggest that ferrous iron affects the nervous system vigorously, possibly yielding free radicals such as hydroxyl radicals, and could be one of the important candidates for neurodegenerative diseases under the state in which acclimating systems for iron toxicity are disrupted.     

Anatomic patterns of FOS immunostaining in rat brain following systemic endotoxin administration

To identify brain neurons that participate in the acute phase response, rat brains were examined immunocytochemically for Fos protein following the intravenous administration of bacterial endotoxin (lipopolysaccharide, LIPS). Two to three hours after the injection of LPS, 150 μg/kg body weight, to adult male Long-Evans rats, a consistent anatomic pattern of Fos immunostained cell nuclei is seen. In the brain stem, prominant Fos immunostaining is induced in tyrosine hydroxylase immunoreactive neurons of the caudal ventral-lateral medulla (the A1 cell group), in both tyrosine hydroxylase positive and negative neurons of nu. tractus solitarius, in the parabrachial nu., and in a few neurons of the locus ceruleus. In the hypothalamus, endotoxin induces Fos expression in magnocellular neurons of the paraventricular and supraoptic nuclei and intemuclear cell groups. A higher percentage of oxytocin-immunoreactive cells is double labeled for Fos nuclear immunostaining than vasopressin-immunoreactive cells. A minority of somatostatin immunoreactive periventricutar hypothalamic neurons are Fos positive. Other hypothalamic nuclei that contain endotoxin-induced Fos nuclear immunostaining include the parvocellular neurons of the paraventricular nu., the dorsomedial and arcuate nuclei, the lateral hypothalamus, the dorsal hypothalamic area (zona incerta), and the median nucleus of the preoptic area. LPS induces numerous Fos-positive neurons in regions known to respond to a variety of stressful stimuli; these regions include the preoptic area, bed nucleus of the stria terminalis, lateral septum, and the central and medial nuclei of the amygdala. Moreover, Fos nuclear immunostaining is seen in neurons of circumventricular organs: the organum vasculosum of the lamina terminalis, the subfomical organ, and the area postrema. The maximum intensity of Fos nuclear immunostaining occurs 2–3 h after endotoxin administration and declines thereafter. It is attenuated by pretreatment with indomethacin, 25 mg/kg body weight SC, or dexamethasone, l mg/kg III. These observations are consistent with the participation of a variety of brain neuronal systems in the acute phase response and elucidate the functional neuroanatomy of that response at the cellular level.     

Effect of amphetamine on the expression of the metabotropic glutamate receptor 5 mRNA in developing rat brain

Mechanisms underlying the acute effects of amphetamine (AMP) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) and specific ³H-glutamate binding in the developing rat brain. Each of the postnatal day (P) 4, P21 and P60 rats received one intraperitoneal injection of AMP, 5 mg/kg or saline and were sacrificed one hour later. In situ hybridization analysis revealed that the AMP treatment raised the levels of the mGluR5 mRNA by 9–28% in the neurons of the layer 5 of motor and somatosensory cortices, whereas reduced the levels by 12–28% in the layer 5 of perirhinal cortex and the ventromedial part of caudate-putamen of the 3 ages. In the layer 2/3 neurons of cingular cortex, an 18% higher and 14% and 22% lower than control levels of the mRNA were detected in the P4 and in the P21 and P60 rats injected with AMP. Moreover, the levels of mGluR5 mRNA in the hippocampi and dentate gyri were elevated by AMP to 110–151% of controls in the rats of 3 ages. Reversible ³H-glutamate binding assay showed an increase of 25% and a 12% decrease in the binding levels in the cortices of AMP-treated P4 and P21 rats. The AMP administration also produced a 27% reduction and 62% elevation in the binding of the hippocampi of P4 and P60 rats. The results reveal age- and region-dependent changes in the expression of the glutamate receptors induced by AMP and may indicate differential plastic capability of the neurons to the drug perturbation.     

Compartmentally specific effects of quinpirole on the striatal Fos expression induced by stimulation of D1-dopamine receptors in intact rats

Injections of the full D₁-agonist A-77636 (1.45 mg/kg) were found to induce clear Fos-like immunoreactivity (FLI) in the striatum of neurologically intact rats. Pretreatment with the D₂-like agonist quinpirole (3 mg/kg) potentiated staining in the lateral striatum, but actually decreased the number of immunoreactive cells observed in the medial portion of the rostral striatum. Comparison with adjacent sections processed for the calcium binding protein calbindin, indicated that quinpirole pretreatment specifically suppressed staining in the matrix compartment of the striatum while tending to potentiate it in the striosomes, resulting in an extremely patchy pattern of labeling. These results suggest that exogenous stimulation of D₂-receptors, although not essential for the induction of FLI, may play an important role in the compartmental patterning of neuronal activity within the striatum.     

Effects of MK-801 on D1 dopamine receptor-mediated immediate early gene expression in the dopamine-depleted striatum

Previous work indicates that intrastriatal administration of MK-801 does not completely block D1 agonist-induced gene expression in dopamine-depleted rats. The present study examined the effects of systemic MK-801 on such gene expression. A low dose of MK-801 did not affect induction of c-fos or zif268. A high dose completely blocked induction of c-fos, but only slightly suppressed zif268. The data suggest that NMDA receptor activity may not always be necessary for D1-induced gene expression.     

Preproenkephalin mRNA expression in rat dorsal striatum induced by selective activation of metabotropic glutamate receptor subtype-5

Group I metabotropic glutamate receptors (mGluR1 and mGluR5 subtypes) are positively coupled to phosphoinositide hydrolysis through G-proteins and are densely expressed in medium-sized projection neurons of striatum. Selective activation of Group I mGluRs upregulates preproenkephalin (PPE) mRNA expression in the rat dorsal striatum. This study investigated the role of one subtype of Group I receptors, mGluR5, in the regulation of PPE mRNA expression in the rat dorsal striatum using quantitative in situ hybridization. Unilateral injection of the mGluR5 selective agonist (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG) into the dorsal striatum (caudoputamen) of chronically cannulated rats at doses of 50 and 200 nmol elevated basal levels of PPE mRNA in the injected dorsal striatum. The induction of PPE mRNA was evident at 1 h, remained at 3 h, and returned to normal level 6 h after CHPG injection. Pretreatment with an mGluR5 selective antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) at a dose of 10 mg/kg (i.p.) blocked CHPG-stimulated PPE expression. MPEP also attenuated PPE expression induced by dopamine D(2) receptor blockade with eticlopride (0.5 mg/kg, i.p.). Administration of MPEP alone had no significant effects on basal levels of PPE mRNA in the striatum. The results from the present study demonstrate that glutamatergic tone on mGluR5 possesses the ability to positively regulate PPE gene expression in striatal neurons in vivo. Moreover, activation of mGluR5 participates in the mediation of D(2) antagonist-induced PPE expression.     

Increased expression of zif268 mRNA in rat retrosplenial cortex following administration of phencyclidine

Phencyclidine (PCP) has been shown to cause neurotoxicity in rat retrosplenial cortex following a single administration, although the precise mechanism underlying PCP-induced neurotoxicity is unclear. Using in situ hybridization and immunohistochemistry, we studied the effects of PCP on expression of immediate early gene zif268 mRNA and zif268 protein in the rat brain. High constitutive levels of zif268 mRNA and zif268 immunoreactivity were observed in the brain of control rats. Administration of PCP (12.5, 25 or 50 mg/kg, i.p., 6 h) caused marked induction of zif268 mRNA in the rat retrosplenial cortex, in a dose-dependent manner. However, the basal levels of zif268 mRNA in the other regions of cerebral cortex were decreased by administration of PCP. Emulsion-autoradiographical study suggested that marked expression of zif268 mRNA was observed in the layers III and IV of retrosplenial cortex where the neurotoxicity of PCP was detected. Furthermore, zif268 immunoreactivity in the layer IV of retrosplenial cortex was not changed by administration of PCP (25 mg/kg, i.p., 5 h), but that in the other layers of retrosplenial cortex was reduced by PCP. These results suggest that immediate early gene zif268 may, in part, play a role in the neurotoxicity of NMDA receptor antagonists such as PCP.     

Unilateral dopamine depletion paradoxically enhances amphetamine-induced Fos expression in basal ganglia output structures

The ability of amphetamine to induce expression of the immediate early gene protein, Fos, was examined by immunocytochemistry in animals with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle. Amphetamine induced Fos expression in the globus pallidus (GP) on the intact side of the brain, but this response was greatly attenuated on the dopamine-depleted side. In contrast, amphetamine induced little Fos expression in the entopeduncular nucleus (EPN) and the substantia nigra pars reticulata (SNpr) on the intact side of the brain, but resulted in pronounced expression in these structures on the lesioned side. These findings demonstrate that unilateral dopamine depletion results in a pathophysiological state in which some responses to amphetamine are attenuated while others are paradoxically potentiated. One explanation of these effects is that amphetamine may indirectly activate excitatory inputs to the SNpr and the EPN on both sides of the brain. On the intact side, these effects would be opposed by the simultaneous activation of inhibitory pathways arising in the striatum and the GP, with the result that little Fos expression would be seen. On the dopamine-depleted side, however, engagement of these inhibitory pathways would be attenuated and the unopposed effects of the excitatory inputs mobilized by amphetamine would result in exaggerated Fos synthesis.     

Muscarinic cholinergic receptor-mediated modulation on striatal c-fos mRNA expression induced by levodopa in rat brain

Summary To clarify the interactions between dopamine receptors and muscarinic cholinergic receptors by which neurotransmitters may affect genetic responses, we studied the effects of the muscarinic cholinergic agonist, carbachol, and the muscarinic cholinergic antagonist, trihexyphenidyl, on levodopa-induced c-fos messenger RNA (mRNA) expression in rat striatum. Animals were administered levodopa (levodopa with one-tenth dosage of carbidopa), carbachol or thrihexyphenidyl alone or administered in combination as levodopa (100 mg/kg) + carbachol, or levodopa + trihexyphenidyl given as a single bolus. Levodopa given alone increase the expression of c-fos mRNA. Although carbachol or trihexyphenidyl alone was ineffective in inducing c-fos mRNA, the combination of levodopa and carbachol ( 0.1 mg/kg) significantly suppressed the induction of c-fos mRNA as compared with levodopa given alone. The combined administration of levodopa and trihexyphenidyl showed a trend toward an additive effect on the induction of c-fos mRNA vs levodopa alone. These findings suggest that the muscarinic cholinergic system may modulate the levodopa-induced c-fos mRNA expression which then regulates the expression of other mRNAs.     

Zif268 and Fos-like immunoreactivity in tetanus toxin-induced epilepsy: reciprocal changes in the epileptic focus and the surround

Altered gene expression for a number of molecules has been suggested as one of the underlying mechanisms of epileptogenesis. Changes in expression of the immediate early genes, zif268 and c-fos, were investigated in chronic focal epilepsy induced by tetanus toxin (TT, 20–35 ng) injected in the rat motor cortex. Most rats injected with TT and perfused on postoperative day 5, 7 or 14 had recurrent focal seizures after a latent period of 4–13 days, and showed enhanced Zif268 immunoreactivity in a cluster of neurons at the injection site, as well as reduced Zif268 immunoreactivity in a distinct cortical zone around this cluster. C-fos or Fos-related immunoreactivity was decreased over widespread areas of frontoparietal and piriform cortex in epileptic rats, except for a focus at the injection site which, in most cases, showed increases in Fos-like immunoreactivity. Some epileptic rats showed increased Zif268 immunoreactivity in neurons of the ipsilateral ventral lateral and central lateral thalamic nuclei and increased Zif268 and Fos-like immunoreactivity in the pontine nuclei. Rats perfused before onset of seizures, showed no overt changes other than a slight decrease in Zif268 and Fos-like immunoreactivity at the injection site. The reciprocal changes in Zif268 immunoreactive neurons in the epileptic focus and the immediate surround parallel changes in gene expression for a number of molecules important in epileptogenesis and suggest a state of functional disconnection of the epileptic focus from other cortical areas that may contribute to the development and maintenance of focal epilepsy.     

Differential induction of immediate early gene mRNAs following cryogenic and impact trauma with/without craniotomy in rats

Expression of immediate early gene (IEG) mRNAs following traumatic brain injury in 3 different models—cryogenic injury, impact injury with craniotomy and impact injury without craniotomy—was investigated using in situ hybridization. Cryogenic brain injury resulted in c-fos and c-jun mRNA expression throughout the ipsilateral cortex, piriform cortex and dentate gyrus on the injured side, with peak at 30 min to 1 h post-injury. Impact injury with craniotomy was associated with hybridization signals in the same areas and also in the subcortical white matter or ependyma underlying the impact site at 30 min post-injury. The expression was rather more prolonged than with cryogenic injury. Impact injury without craniotomy induced the expression of both mRNAs throughout the ipsilateral cortex, piriform cortex and dentate gyrus at 30 min post-injury, but this was promptly attenuated by 1 h post-injury, except for bilateral elevation in the dentate gyrus. The present study, thus, demonstrated that regional and temporal expression of IEG mRNAs is influenced by the intensity, quality and manner of application of the insult. Differences in the expression of IEGs may alter the late response gene expression and affect the succeeding events.     

Transient MRI-detected water apparent diffusion coefficient reduction correlates with c-fos mRNA but not hsp70 mRNA induction during focal cerebral ischemia in rats

Cerebral ischemia induces immediate early genes such as c-fos and stress genes such as hsp70. In this study, the spatial relationships between c-fos and hsp70 mRNA expression and changes detectable with diffusion and perfusion magnetic resonance (MR) imaging were examined. The middle cerebral artery (MCA) of young adult rats was occluded for 30 or 60 min. Diffusion MR (D-MR) images were acquired continuously during the ischemic period and dysprosium-contrast perfusion (P-MR) images were acquired at the end of the ischemic period. C-fos and hsp70 mRNA expression were examined with in situ hybridization. The most significant finding of this work was that for both durations of ischemia, c-fos induction was observed in cortical and sub-cortical regions exhibiting a transient reduction in the apparent diffusion coefficient of water (ADC). Transients which occurred on a time scale of 3 min may have been caused by spreading depression. Those occurring on a 10-min time scale may have been caused by an initial reduction in blood flow with occlusion that was followed by an ischemia-induced increase in collateral blood flow. P-MR imaging showed that perfusion in c-fos positive regions was higher than in regions with persistently reduced ADC. Hsp70 induction did not correlate with transient ADC reduction. It was induced in the MCA territory in regions showing persistent ADC changes, with induction being greatest at the periphery of these regions. It was also induced in regions that exhibited both spontaneous reversal of the diffusion changes and decreased perfusion.     

Increased c-fos expression in spinal neurons induced by electrical stimulation of the ureter in the rat

The spinal processing of afferent input from the ureter was examined using an immunocytochemical technique to detect the expression of c-fos, an immediate early gene. Proximal and distal sites in one ureter were electrically stimulated separately or together at intensities that elicited a pseudo-affective response (an increase in arterial pressure). Very few Fos + cells (range: 0.6–6.6 cells/half section were present in the L₁-L₂, L₅-S₂ spinal segments in sham operated control animals; however, following stimulation of the ureter, a significant increase in the numbers of Fos + cells was detected at spinal levels L₁-L₂ (mean 24.5–33.1 cells/half section) and L₆-S₁ (mean 17.4–33.0 cells/half section). In L₆-S₁,the numbers of Fos + cells were significantly greater ipsilateral (mean 25.2 cells/half section) vs. contralateral (12.3 cells/half section) to stimulation; whereas in L₁-L₂, the numbers were similar on both sides of the spinal cord. In L₁-L₂, a greater percentage of Fos + cells was present in superficial medial (MDH, 49.7%) and lateral dorsal hom (LDH, 40.8%); whereas in L₆-S₁, the cells were more numerous in sacral parasympathetic nucleus (SPN, 38.7%) and LDH (25.6%) regions. This distribution of Fos + cells varies in a number of respects from that noted in previous experiments after chemical irritation of the urinary bladder and urethra which activated neurons only in L₆-S₁ and primarily in the DCM and MDH. The results indicate that nociceptive afferent inputs from different areas of the urinary tract are processed in different regions of the spinal cord.     

l-Dopa stimulates c-fos expression in dopamine denervated striatum by combined activation of D-1 and D-2 receptors

Administration of l-dopa to unilaterally 6-hydroxydopamine-lesioned rats, activates the early gene c-fos in the lesioned caudate-putamen. D-1 receptor blockade by SCH 23390, preventedl-dopa-induced Fos-like immunoreactivity in the whole caudate-putamen, while D-2 receptor blockade by raclopride reduced Fos-like immunoreactivity only in the dorso-lateral portion. The results suggest thatl-dopa induces c-fos primarily through an activation of D-1 receptors, while D-2 receptor stimulation plays a facilitatory influence on D-1 mediated c-fos expression.