A new experimental model for the investigation of sequential hermaphroditism.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17ß-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

Expression of the inhibitor of apoptosis protein family in multiple sclerosis reveals a potential immunomodulatory role during autoimmune mediated demyelination.

A failure of autoreactive T cells to undergo apoptosis may contribute to the pathogenesis of multiple sclerosis (MS). The role of the inhibitor of apoptosis (IAP) family of anti-apoptotic proteins such as X-linked IAP (XIAP), human inhibitor of apoptosis-1 (HIAP-1), human inhibitor of apoptosis-2 (HIAP-2), neuronal apoptosis inhibitory protein (NAIP) and Survivin in relapsing-remitting, secondary-progressive, primary-progressive or benign forms of MS is unclear. We report here that expression of the IAP family of genes in peripheral blood samples and brain tissues from MS cases support a role for differential regulation of these potent anti-apoptotic proteins in the pathology of MS. XIAP mRNA and protein levels were elevated in peripheral blood mononuclear cells from patients with active disease relative to normal subjects. In patients with active MS, HIAP-1 and HIAP-2 mRNA levels were elevated in resting T cells while NAIP mRNA was increased in whole blood. In post-mortem MS brain tissue, XIAP and HIAP-1 in myelin lesions were co-localized with microglia and T cells, respectively. Only in primary-progressive patients was Survivin expression elevated suggestive of a distinct pathological basis for this subtype of MS. Taken together, these results suggest that patterns of inhibitor of apoptosis expression in immune cells may have value in distinguishing between MS subtypes and offer insight into the mechanisms responsible for their distinct clinical courses.

Neuroprotective properties of cultured neural progenitor cells are associated with the production of sonic hedgehog.

Numerous studies have shown that abnormal motor behavior improves when neural progenitor cells (NPCs) are transplanted into animal models of neurodegeneration. The mechanisms responsible for this improvement are not fully understood. Indirect anatomical evidence suggests that attention of abnormal motor behavior is attributed, at least in part, to the secretion of trophic factors from the transplanted NPCs. However, there is little direct evidence supporting this hypothesis. Here we show that NPCs isolated from the subventricular zone (SVZ) of neonatal mice are highly teratogenic when transplanted into the neural tube of developing chick embryos and are neuroprotective for fetal dopaminergic neurons in culture because they release sonic hedgehog (Shh). In addition, the neuroprotective properties of NPCs can be exploited to promote better long-term survival of transplanted fetal neurons in an animal model of Parkinson's disease. Thus, cultured NPCs isolated from the SVZ can secrete at least one potent mitogen (Shh) that dramatically affects the fate of neighboring cells. This trait may account for some of the improvement in motor behavior often reported in animal models of neurodegeneration after transplantation of cultured NPCs that were isolated from the SVZ.

Striatal phosphodiesterase mRNA and protein levels are reduced in Huntington's disease transgenic mice prior to the onset of motor symptoms.

Inheritance of a single copy of the gene encoding huntingtin (HD) with an expanded polyglutamine-encoding CAG repeat leads to neuronal dysfunction, neurodegeneration and the development of the symptoms of Huntington's disease (HD). We have found that the steady-state mRNA levels of two members of the phosphodiesterase (PDE) multi-gene family decrease over time in the striatum of R6 transgenic HD mice relative to age-matched wild-type littermates. Phosphodiesterase 10A (PDE10A) mRNA and protein levels decline in the striatum of R6/1 and R6/2 HD mice prior to motor symptom development. The rate of reduction in PDE10A protein correlates with the rate of decline of the message and the decrease in PDE10A mRNA and protein is more rapid in R6/2 compared with R6/1 mice. Both PDE10A protein and mRNA, therefore, decline to minimum levels prior to the onset of overt physical symptoms in both strains of transgenic mice. Moreover, protein levels of PDE10A are decreased in the caudate-putamen of grade 3 HD patients compared with age-matched neuropathologically normal controls. Striatal PDE1B mRNA levels also decline in R6/1 and R6/2 HD mice; however, the decrease in striatal PDE10A levels (>60%) was greater than that observed for PDE1B and immediately preceded the onset of motor symptoms. In contrast, PDE4A mRNA levels are relatively low in the striatum and do not differ between age-matched wild-type and transgenic HD mice. This suggests that the regulation of PDE10A and PDE1B, but not PDE4A, mRNA levels is dependent on the relative expression of or number of CAG repeats within the human HD transgene. The loss of phosphodiesterase activity may lead to dysregulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in the striatum, a region of the brain that contributes to the control of movement and cognition.

Immediate-early gene response to methamphetamine, haloperidol, and quinolinic acid is not impaired in Huntington's disease transgenic mice.

Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are resistant to a variety of toxic insults, including quinolinic acid (QA), kainic acid and 3-nitropropionic acid. The basis for this resistance is currently unknown. To investigate the possibility that the immediate-early gene (IEG) response is defective in symptomatic HD mice leading to a lack of response to these compounds, we examined the expression of c-Fos and Krox 24 after administration of the indirect dopamine agonist methamphetamine, the dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and 10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were resistant to methamphetamine-induced gliosis and QA lesion. There was, however, no difference in the number or distribution of c-Fos-immunoreactive nuclei 2 hr after single injections of methamphetamine or haloperidol among 5- and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly, despite their resistance to QA-induced lesioning and lower basal levels of krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD mice as determined by in situ hybridization and densitometry 2 hr after QA administration. These data demonstrate that the c-Fos and Krox 24 IEG response to dopamine agonists, dopamine antagonists and neurotoxic insult is functional in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be conferred by interactions of mutant huntingtin with proteins or transcriptional processes further along the toxic cascade.

Lack of phencyclidine-induced effects in mice with reduced neuronal nitric oxide synthase.

RATIONALE: Phencyclidine (PCP) is widely used as an animal model of schizophrenia, because in humans it can induce positive and negative symptoms associated with schizophrenia. PCP is an antagonist of N-methyl-D-aspartate receptors, which are associated with the nitric oxide (NO) system. OBJECTIVE AND METHODS: The primary objective was to determine whether neuronal NO synthase (nNOS) is involved in PCP-induced behaviours and neuronal activation, as measured by the expression of c-Fos. After characterizing a PCP mouse model (dose-response study, Experiment 1), we measured PCP-induced effects in mice treated with nNOS antisense oligodeoxynucleotides (AS-ODNs) (Experiment 2), and in nNOS knockout (nNOS-/-) mice (Experiment 3). RESULTS: PCP 5 mg/kg induced the maximum behavioural effects of all doses tested, consisting of hyperlocomotion, stereotyped turning behaviour, without the presence of ataxia. PCP also induced an increase in Fos-like immunoreactivity (Fos-LIR) in the frontal cortex, as well as in the midline limbic (thalamic and hypothalamic nuclei) areas. In the AS-ODN-treated mice, PCP induced less behaviour when compared to water-treated controls. In the nNOS-/- mice, PCP induced less behaviour and a decrease in Fos-LIR in the frontal cortex and midline limbic areas, when compared to wild-type littermate controls. CONCLUSIONS: Our findings suggest that the frontal cortex and midline thalamic brain regions are involved in PCP-induced effects in mice. Furthermore, we show that an intact nNOS system is necessary to obtain PCP-induced effects. This may implicate nNOS as a viable drug target in the treatment of schizophrenia.

Effects of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester on phencyclidine-induced effects in rats.

Phencyclidine (PCP) is widely used as an animal model of schizophrenia. In rats, acute PCP treatment increased locomotor activity and induced stereotyped behaviours consisting of head weaving, turning and backpedalling. PCP had differential regional effects on c-fos expression in rat brain, suggesting different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. To elucidate the role of nitric oxide, an important intracellular messenger, in the mechanism of action of PCP the effects of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were studied in PCP-treated animals. L-NAME potentiated PCP-induced behaviours and c-fos expression in many brain regions. The greatest increases were observed in the frontal, retrosplenial granular cortex, cerebellum, thalamic and subthalamic nuclei. While PCP alone induced low c-fos expression in the entorhinal cortex, with almost no expression in the rostral part of caudate putamen, animals pretreated with L-NAME showed marked activation in these brain areas. These results strongly indicate the involvement of the nitric oxide system in the mechanism of action of PCP.

Blockade of phencyclidine-induced effects by a nitric oxide donor.

1. Phencyclidine (PCP) is widely used as an animal model of schizophrenia. The aim of this study was to better understand the role of nitric oxide (NO) in the mechanism of action of PCP and to determine whether positive NO modulators may provide a new approach to the treatment of schizophrenia. 2. The effects of the NO donor, sodium nitroprusside (SNP), were studied in PCP-treated rats. Following drug administration, behavioural changes and the expression of c-fos, a metabolic marker of functional pathways in the brain, were simultaneously monitored. 3. Acute PCP (5 mg kg(-1), i. p.) treatment induced a complex behavioural syndrome, consisting of hyperlocomotion, stereotyped behaviours and ataxia. Treatment with SNP (2 - 6 mg kg(-1), i.p.) by itself produced no effect on any behaviour studied but completely abolished PCP-induced behaviour in a dose- and time-dependent manner. 4. PCP had differential regional effects on c-fos expression in rat brain, suggesting regionally different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. Pre-treatment with SNP blocked PCP-induced c-fos expression at doses similar to those that suppress PCP-induced behavioural effects. 5. These results implicate the NO system in the mechanism of action of PCP. The fact that SNP abolished effects of PCP suggests that drugs targeting the glutamate-NO system may represent a novel approach to the treatment of PCP-induced psychosis and schizophrenia.

Selective regional blockade of junB gene expression in the hamster suprachiasmatic nucleus by a tyrosine kinase inhibitor.

The hypothalamic suprachiasmatic nucleus (SCN) functions as a circadian pacemaker regulating a variety of physiological and behavioral rhythms in mammals. Retinal illumination evokes expression of several immediate-early genes, including junB, in the ventral SCN early in the subjective night and throughout the SCN later in the subjective night. junB mRNA and protein are also expressed spontaneously around subjective dawn in nocturnal rodents, but only in the dorsal SCN. We examined the biochemical signaling mechanisms underlying both spontaneous and light-evoked expression of junB mRNA in the SCN of Syrian hamsters. Hamsters were injected (i.p.) before subjective dawn with vehicle or with either tyrphostin or genistein, inhibitors of protein tyrosine kinase, and maintained in the dark for 30 min. They were then exposed to a light pulse or kept in darkness for another 30 min. In situ hybridization studies demonstrated that tyrphostin pretreatment (12 or 24 mg/kg) reduced both spontaneous and light-evoked expression of junB mRNA only in the dorsal, and not the ventral, portion of the SCN. Conversely, genistein had little effect on either spontaneous or light-evoked expression of junB mRNA in any part of the SCN. These results indicate that a protein tyrosine kinase sensitive to tyrphostin but not to genistein is involved in the transduction pathways leading to expression of junB mRNA selectively in the dorsal SCN, independently of circadian phase and independently of the involvement of light.

Identification of a subpopulation of substantia nigra pars compacta gamma-aminobutyric acid neurons that is regulated by basal ganglia activity.

In this report, the authors provide a novel description of a population of gamma-aminobutyric acid-containing neurons in the substantia nigra, pars compacta (SNC). By using metabolic mapping of the immediate-early gene, c-fos, the activation pattern of these cells was characterized with respect to basal ganglia stimulation. Dopaminergic stimulation with d-amphetamine or apomorphine induced Fos expression in the central region of the SNC. However, lesions of the nigrostriatal dopamine pathway significantly reduced d-amphetamine- and apomorphine-induced Fos expression in the ipsilateral and contralateral SNC, respectively. Suppression of stimulant-induced Fos expression in the striatum, using antisense oligodeoxynucleotides, also eliminated Fos expression in the ipsilateral SNC, indicating that striatal efferent projections are involved in the activation of these cells. Double-labeling immunohistochemistry revealed that the Fos-positive cells did not express tyrosine hydroxylase but were immunoreactive for glutamic acid decarboxylase. Retrograde labeling of nigrostriatal neurons, combined with Fos immunofluorescence, revealed that these Fos-positive cells did not project to the striatum. Thus, these neurons do not appear to comprise a nondopaminergic nigrostriatal circuit but likely represent locally-projecting interneurons of the substantia nigra.

Daily rhythm of spontaneous immediate-early gene expression in the rat suprachiasmatic nucleus.

Nocturnal light induces the expression of various immediate-early genes (IEGs) in the suprachiasmatic nucleus (SCN), the primary pacemaker of the circadian system of mammals, and causes phase shifts of behavioral rhythms. In the hamster SCN, some IEGs show both sensitivity to light induction at night and a daily peak of spontaneous expression near dawn in different regions of the nucleus. To investigate whether both patterns of IEG expression are observed in the rat SCN, the authors studied the expression of NGFI-A, junB, c-fos, and fosB near the time of subjective dawn in rats entrained to a light-dark 12:12 cycle and then maintained in constant total darkness for approximately 48 h. They found that there were two independent rhythms of expression for junB and c-fos mRNAs in the SCN: (1) a rhythm of photic sensitivity expressed throughout the night and (2) a spontaneous rhythm of expression triggered around dawn and persisting for at least 2 h into the day. By contrast, fosB and NGFI-A transcripts were expressed only after light exposure at night and did not exhibit significant levels of spontaneous expression in the absence of photic input. These observations demonstrate that the circadian clock gates expression of two independent rhythms related to IEG expression in the rat SCN. The rhythm of sensitivity to nocturnal light exposure is expressed more strongly in the ventral SCN and may be related to photic entrainment. The second rhythm is triggered spontaneously in darkness around subjective dawn and is expressed in more dorsal parts of the SCN.

Motor effects and mapping of cerebral alterations in animal models of Parkinson's and Huntington's diseases.

Changes in stimulant-induced behavioral effects and subcortical c-Fos expression were compared between rodent models of Parkinson's disease (PD) and Huntington's disease (HD). Rats received either a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal dopamine pathway (PD model) or a unilateral infusion of antisense oligodeoxynucleotides targeting c-fos into the striatum (HD model). Dopamine-lesioned animals received intraperitoneal injections of either d-amphetamine (6-OHDAamp group) or apomorphine (6-OHDAapo group), whereas all animals that received antisense infusions received d-amphetamine (ASF group). All groups exhibited robust circling behavior upon stimulant challenge. Changes in subcortical activation, as assessed by the induction of Fos-like immunoreactivity (Fos-LI), were examined in several brain regions. The 6-OHDAamp and ASF groups exhibited robust, ipsiversive circling behavior, with similar changes in Fos-LI in the striatum, entopeduncular nucleus, superior colliculus, and ventromedial thalamus. The 6-OHDAapo group exhibited contraversive rotation and had reciprocal patterns of Fos-LI in these regions. Despite exhibiting the same direction of rotation, the 6-OHDAamp and ASF groups had markedly different patterns of Fos-LI in the globus pallidus and the pontine reticular formation. These results suggest that the globus pallidus may undergo distinct alterations in PD and HD and that the pontine reticular formation is particularly susceptible to changes in mesencephalic dopamine sources.

Differential effects of glutamatergic blockade on circadian and photic regulation of gene expression in the hamster suprachiasmatic nucleus.

Nocturnal light exposure induces immediate-early gene (IEG) expression in the hypothalamic suprachiasmatic nucleus (SCN) and causes phase shifts of activity rhythms in mammals. Some IEGs also show a circadian rhythm of expression in the SCN. While excitatory amino acids (EAAs) are known to be involved in mediating photic regulation of entrainment and gene expression, their involvement in spontaneous rhythms of gene expression has not been studied. We assessed the role of NMDA receptors in the expression of NGFI-A, junB and fosB mRNAs induced by light pulses of different intensities late in the night (Zeitgeber Time [ZT] 18). We also examined the spontaneous expression of junB mRNA near subjective dawn (ZT 0). Both dim (5 lx) and bright (100 lx) light pulses induced similar levels of expression of NGFI-A and junB in the SCN late in the night. fosB mRNA was strongly induced by bright light but was less sensitive to dim light. At ZT 18, dizocilpine (MK-801) (3 mg/kg, i.p. ), a non-competitive NMDA receptor antagonist, almost completely blocked light-evoked expression of IEG mRNAs in the ventral SCN but not in the dorsolateral region at a mid-caudal level using either light intensity. At ZT 0, MK-801 strongly reduced light-evoked expression of junB mRNA in both SCN subdivisions, but inhibited spontaneous expression significantly only in the dorsal region. NMDA receptors appear to play an important role in mediating photic input regulating IEG expression only in the ventral SCN at night. At dawn, however, NMDA receptors are involved in mediating photic effects in both parts of the SCN, as well as being involved in spontaneous activation of junB expression selectively in the dorsal SCN. These findings support the idea that the effects in the dorsolateral SCN of nocturnal light exposure are mediated by different mechanisms than those in other portions of the nucleus.

Synergistic influences of the striatum and the globus pallidus on postural and locomotor control.

We have investigated the role of the globus pallidus in locomotor and postural control in a previously established animal model of striatal dysfunction. Striatal efferent activity was suppressed by intracerebral infusions of antisense oligodeoxynucleotides targeted to the messenger RNA of the proto-oncogene, c-fos. This suppression produced robust circling behavior and an atypical expression of c-fos in the ipsilateral globus pallidus following psychostimulant challenge. Simultaneous infusions of antisense oligodeoxynucleotides into both the caudate-putamen and the ipsilateral globus pallidus produced an approximate threefold increase in the intensity of rotation elicited by D-amphetamine. Excitotoxic lesioning of the globus pallidus produced marked postural asymmetry and circling behavior upon stimulation. The intensity of this rotational behavior was similar to that produced by dual infusions of antisense oligodeoxynucleotides into the caudate-putamen and the globus pallidus, and was not further potentiated by suppression of striatal c-fos expression. These results demonstrate the importance of the globus pallidus in postural and motor control, and suggest that activation of this nucleus through a reduction in striatopallidal inhibition may function to balance the output activity of the basal ganglia.

Alterations of neuronal activity in the superior colliculus of rotating animals.

We have investigated the relationship between alterations in neuronal activity in the superior colliculus and behavioral responses which occur following disruption of basal ganglia circuitry. These changes were analysed following unilateral suppression of the immediate early genes, c-fos and ngfi-a, in the striatum and/or the globus pallidus. Animals with unilateral suppression of immediate early gene expression in the striatum exhibited robust circling activity, following administration of D-amphetamine, that was directed towards the side of suppression. The intensity of rotation was inversely related to the length of the recovery period following antisense infusion and increased significantly when the globus pallidus was infused simultaneously with the striatum. The difference between ipsiversive (towards the antisense-infused hemisphere) and contraversive rotations was calculated and animals were grouped by number according to their ipsiversive bias: I, <50 turns; II, 50-500 turns; III, 500-1000 turns; IV, >1000 turns. Immunohistochemical localization of Fos was used as an indicator of neuronal activity in the superior colliculus. While group I animals showed diffuse Fos-like immunoreactivity throughout the intermediate layers of the superior colliculus, those animals in groups II-IV showed increasing suppression of Fos-like immunoreactivity in the stratum album intermediale and marked enhancement in the stratum griseum intermediale. Correlation and regression analysis revealed a significant positive relationship between the number of ipsiversive rotations and the number of Fos-positive nuclei in the stratum griseum intermediale of the ipsilateral superior colliculus. These data suggest that the degree of rotation elicited in an animal may depend on reciprocal suppression/stimulation of adjacent intermediate strata of the superior colliculus. This study provides the first demonstration, using Fos immunohistochemistry, of changes in tectal activity produced by alterations in basal ganglia function. These findings support previous electrophysiological studies in this region and suggest that the nigrotectal projection may be an important site of altered basal ganglia output.

Circadian and photic regulation of immediate-early gene expression in the hamster suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus is the site of an endogenous circadian clock synchronized by daily light-dark cycles. At some daily phases, light exposure both shifts the clock and alters the expression of several immediate-early genes in cells of the suprachiasmatic nucleus. We have studied both spontaneous circadian and light-induced expression of several immediate-early gene messenger RNAs and proteins in hamsters in constant darkness or in response to brief light exposure. There was no detectable spontaneous expression of NGFI-A messenger RNA in suprachiasmatic nucleus cells at any circadian phase, but light pulses induced its expression selectively during the subjective night, with highest levels of expression 6 h into the night. We also found that there are two independent rhythms of expression of junB messenger RNA and JunB protein, as well as c-fos messenger RNA and c-Fos protein, in the suprachiasmatic nucleus of hamsters: a rhythm of photic sensitivity expressed throughout the night and a spontaneous rhythm of expression triggered around dawn. Induction of NGFI-A messenger RNA and c-fos messenger RNA and c-Fos protein in response to a light pulse were found throughout the suprachiasmatic nucleus, with the highest levels of expression in the ventrolateral subdivision; however, the spontaneous expression of JunB and c-Fos proteins was confined mainly to the dorsomedial suprachiasmatic nucleus. The temporal and anatomical differences in the expression of these immediate-early genes in the mammalian suprachiasmatic nucleus suggest that their protein products may be involved in different signaling mechanisms mediating either photic entrainment or endogenous oscillations within distinct subpopulations of suprachiasmatic nucleus cells.

Intra-amygdala infusion of an end-capped antisense oligodeoxynucleotide to c-fos accelerates amygdala kindling.

The changes produced by repeated stimulation of the brain that result in the kindling effect remain unknown. It is known, however, that activation of immediate early genes including c-fos is a very early event in the development of kindling. Here we show that c-fos expression in the amygdala can be knocked-down by an end-capped antisense oligodeoxynucleotide (but not by vehicle, or control antisense oligodeoxynucleotides) and that this results in an acceleration of kindling. These results suggest that, at least in the amygdala, the expression of c-fos is associated with an attempt by the brain to retard the development of kindling.

Putative roles for the inducible transcription factor c-fos in the central nervous system: studies with antisense oligonucleotides.

Although immediate-early genes such as c-fos are widely believed to play an important role in neuroplasticity, there is limited evidence to support involvement in the initiation of molecular events leading to medium- and long-term changes in brain function following a stimulus. Results using techniques such as transgenic knockout of the gene are often difficult to interpret. Antisense oligonucleotide technology offers an alternative. Infusion of antisense oligonucleotide to modify the expression of c-fos in the brain results in dramatic changes in rotation behaviour in animals challenged with psychostimulant drugs such as amphetamine. Similarly, the knockdown of c-fos expression using antisense oligonucleotides can also alter the rate of amygdala kindling in response to electrical stimulation of the brain. While studies using antisense oligonucleotides to knockdown c-fos expression provide evidence that the expression of c-fos plays an important role in regulating neuronal function, the use of antisense nucleotides has limitations and experiments must be very carefully controlled. Many details of antisense oligonucleotide actions remain unknown.

The effects of hypoxia-ischemia on expression of c-Fos, c-Jun and Hsp70 in the young rat hippocampus.

The expression of c-Fos, c-Jun and Hsp70 was examined in the hippocampus at 6, 12, 24, 48, 72 h, 4, 7 and 42 days following a combination of unilateral common carotid artery ligation and 60 min of systemic hypoxia (8% oxygen, 92% nitrogen) in 25-day-old male rats. While pyknotic cells were not visible in the hippocampus of control animals, pyknosis was evident in the ipsilateral, but not the contralateral hippocampus, of hypoxic-ischemic animals beginning at 24 h post-hypoxia. Immunohistochemical analysis revealed no c-Fos-, c-Jun- or Hsp70-immunoreactivity (IR) in any control animals. However, at 6 h post-hypoxia, Fos- and Jun-IR was evident throughout the injured ipsilateral hippocampus and later appeared throughout the contralateral hippocampus, which never showed signs of pyknosis. In contrast, Hsp70-IR was first observed at 24 h post-hypoxia and was restricted to the injured ipsilateral hippocampus. Hsp70-IR was not, however, limited to dying neurons. H-I/seizure animals did not express these proteins at any time point. These results suggest that, even in irreversibly injured neurons, Fos, Jun and Hsp70 appear to be involved in the aftermath of ischemia but probably do not play a pivotal role in the outcome of H-I compromised cells. Furthermore, compounded injury (H-I/seizure) appears to block the synthesis these proteins.

Coordinate suppression of striatal ngfi-a and c-fos produces locomotor asymmetry and up-regulation of IEGs in the globus pallidus.

We have studied the effects of inhibition of c-Fos and NGFI-A expression by intrastriatal infusion of end-capped antisense oligodeoxynucleotides to determine if their coordinate expression is conferred independently or through regulatory influences exerted between these two proteins. The previously reported locomotor bias that has been associated with unilateral c-Fos suppression was also investigated in animals receiving antisense oligodeoxynucleotides targeted to ngfi-a to determine if the behavior is specific to alterations in c-Fos expression, or if its cause may be a generalized imbalance of striatal IEGs. We show here that while unilateral suppression of c-Fos has negligible effects on NGFI-A, oligodeoxynucleotides targeted to ngfi-a markedly inhibit both NGFI-A and c-Fos expression. Animals with extensive unilateral reduction of either or both proteins demonstrated robust ipsiversive rotation when challenged with D-amphetamine. Infusions of random oligodeoxynucleotides produced neither a reduction in c-Fos or NGFI-A expression, nor a significant rotational bias following D-amphetamine challenge. Surprisingly, animals with extensive striatal IEG suppression were found to have marked up-regulation of c-Fos and NGFI-A in the ipsilateral globus pallidus, a finding which may ultimately shed light on the mechanism of antisense-induced rotational behavior.