The small heat shock protein, Hsp27, promotes axonal regeneration in peripheral neurons; however, an analogous role in the central nervous system has not been described. This study examined the relationship between Hsp27 expression and regeneration in mature retinal ganglion cells (RGCs). Adult rat optic nerves were transected and exposed to peripheral nerve autografts to stimulate regeneration of cut RGC axons. There was a five-fold increase in the Hsp27-positive fraction of RGCs that extended new axons into the graft when compared with those that survived injury but did not regenerate (30% versus 6% respectively, P = 0.001). Hsp27 protein was located throughout somata and neuritic processes, and there was a significant positive correlation between Hsp27 expression and axonal regeneration in injured neurons (R = 0.92, P < 0.0001). These findings are consistent with the growth-associated role of Hsp27 demonstrated in peripheral neurons and suggest that Hsp27 may mediate similar physiological functions in the central nervous system.

Cockayne syndrome manifests a spectrum of neurological dysfunction that includes medically intractable movement disorders. Deep brain stimulation has not been well studied in such rare neurodegenerative conditions. In this case, stimulation of the ventral intermediate nucleus of the thalamus was used to manage severe motor symptoms in a young man with Cockayne syndrome. There was a marked and progressive response to thalamic stimulation within weeks of surgery. These results suggest that patients with Cockayne syndrome should be considered for deep brain stimulation to treat refractory movement disorders.

Double immunostaining for Fos and γ-aminobutyric acid (GABA) was used in a previously established animal model of striatal dysfunction to examine whether GABA-immunoreactive neurons in the globus pallidus (GP) and entopeduncular nucleus (EP) are activated to express Fos immunoreactivity by intraperitoneal injection of amphetamine. Striatal efferent activity was suppressed by intrastriatal infusions of antisense oligodeoxynucleotide targeted to the messenger RNA of the immediate early gene, c-fos. This suppression produced robust rotational behavior and expression of Fos in the ipsilateral GP and EP following amphetamine challenge. The expression of Fos in the ipsilateral GP and EP following amphetamine challenge is not observed in naı̈ve or control antisense-treated animals. Quantitative analysis revealed that a majority of the amphetamine-activated (Fos-immunoreactive) neurons in the GP and EP express GABA. The present results suggest that inhibitory GABAergic projection neurons within these two nuclei are regulated by inhibitory striatal output and suggests that decreased inhibitory striatal output may contribute to the motor dysfunction observed in patients with Huntington's disease.

The application of antisense oligonucleotides is becoming a popular technique with which to study the function of genes in the brain. These compounds can serve as a useful alternative when conventional pharmacological tools lack adequate specificity or are unavailable. Theoretically, the versatility of this technology is enormous. However, as with any genetic methodology, theoretical success is hindered by numerous practical difficulties. In this chapter we address several potential pitfalls that arise when using antisense oligonucleotides. Importantly, these include the biodistribution of the molecules within the region of delivery, as well as their potential transport to one or more projection nuclei. Also, determination of oligonucleotide pharmacokinetics is essential for effective experimental design and assessing the interval of maximal antisense effects. Furthermore, the stability of the oligonucleotides, which can be altered by biochemical modification (i.e., phosphorothioate substitution), may impact on the temporal development and duration of antisense effects. Finally, it may be important to assess the neuronal—glial interactions in a particular region and the effects of antisense oligonucleotides on gene expression in both cell types.

In this report, the authors provide a novel description of a population of gamma-aminobutyricacid-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. J. Comp. Neurol. 416:30-44, 2000. (C) 2000 Wiley-Liss, Inc.

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.

Huntington's disease (HD) is one of a number of neurodegenerative disorders caused by expansion of polyglutamine-encoding CAG repeats within specific genes. Huntingtin, the protein product of the HD gene, is widely expressed in neural and nonneural human and rodent tissue. The function of the wild-type or mutated form of huntingtin is currently unknown. We have observed that relative to naive and male animals, huntingtin protein was significantly increased in the arcuate nucleus of postpartum rats. Using an oligonucleotide probe, in sih and Northern blot hybridization confirmed the expression of hunting-tin mRNA, Quantification of the in situ hybridization signal in the arcuate nucleus revealed an approximate sevenfold increase in the expression of huntingtin mRNA in postpartum, lactating animals compared with naive female or male animals. Emulsion autoradiography and immunohistochemistry revealed that the cells with elevated huntingtin expression had a stellate conformation that morphologically resembled astrocytes. Dual label immunofluorescence immunohistochemistry demonstrated the colocalization of huntingin and glial fibrillary acidic protein in these cells, confirming that they were astrocytes, Astrocytes expressing huntingtin were consistently found in close apposition to neuronal soma, suggesting interactions between these cell types. During the perinatal and postnatal period, the hypothalamus undergoes alterations in metabolic function. Our results support the idea of glia-induced metabolic changes in the hypothalamus, These results provide the first demonstration of naturally occurring changes in the expression of the Huntington's disease gene in the brain and suggest that huntingtin may play an important role in the processes that regulate neuroendocrine function.

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. (C) 1999 IBRO. Published by Elsevier Science Ltd.

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.

Stimuli that activate the cells of the central nervous system (CNS) can have permanent or semi-permanent effects on the functioning of the brain. In many cases the stimuli responsible for this change in brain function also activate transcription factors (TF), some of which are of the immediate-early gene (IEG) family. Stimuli of both physiological and pathophysiological significance have been shown to activate the prototypical IEG, c-fos. Consequently, studies attempting to examine the role of IEGs in the CNS abound. In this chapter we describe studies which have associated IEGs with brain function and demonstrate the emerging role that antisense technology has played in this field and other areas of CNS pharmacology.

Activation of glutamate receptors triggers the release of adenosine, which exerts important inhibitory actions in the brain. Evoked adenosine release is potentiated when either adenosine kinase or adenosine deaminase are inhibited. We studied the effects of concurrent inhibition of adenosine kinase and adenosine deaminase on N-methyl-d-aspartate (NMDA)-evoked formation of extracellular adenosine in slices of rat parietal cortex, to determine if combinations of inhibitors of adenosine kinase and adenosine deaminase can produce supra-additive potentiation of this adenosine formation. Combinations of low concentrations of the adenosine kinase inhibitors 5′-amino-5′-deoxyadenosine (0.2 μM) or 5′-iodotubercidin (0.01 μM) with a low concentration of the adenosine deaminase inhibitor 2′-deoxycoformycin (0.2 μM) produced additive potentiations of NMDA-evoked adenosine release from slices of rat parietal cortex. However, combinations of low concentrations of 5′-amino-5′-deoxyadenosine (0.2 μM) or 5′-iodotubercidin (0.01 μM) with a maximal concentration of 2′-deoxycoformycin (200 μM) produced supra-additive potentiation of NMDA-evoked adenosine release. These findings suggest that such combinations of adenosine kinase inhibitors with adenosine deaminase inhibitors may provide useful strategies for developing therapies to treat disorders associated with excessive NMDA receptor activation, such as seizures, ischemic damage and neurodegenerative diseases.

Sulfur modification of oligodeoxynucleotides produces nuclease resistance but also leads to toxic effects when these compounds are administered in vivo. To assess their potential as viable alternatives to full phosphorothioate derivatives, we have used rotational behavior and immunohistochemistry to investigate the efficacy and longevity of phosphorothioate, end-capped antisense oligodeoxynucleotides in suppression of c-fos and ngfi-a in the striatum of adult rats. Our results suggest that, despite having a limited duration of action, these end-capped, chimeric oligodeoxynucleotides are capable of specifically inhibiting gene expression in vivo and may, therefore, possess broader application potential in chronic suppression models as the reduction of sulfur content is likely to greatly minimize their toxic effects.

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.