Joseph Christopher Probst

Chronic infusion of a CRH1 receptor antisense oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-related behavior in socially defeated rats.

We studied the role of central amygdala CRH receptors in behavioral responses to an anxiogenic stimulus. An antisense oligodeoxynucleotide corresponding to the rat CRH1 receptor mRNA was infused chronically into the central amygdaloid nucleus of male rats via osmotic minipumps (0.25 micrograms/0.5 microliters/h). Control groups received infusions of either a scrambled sequence oligodeoxynucleotide or vehicle. On the 4th day of treatment, rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Antisense oligodeoxynucleotide-treated rats spent significantly more time exploring the open arms of the plus-maze than scrambled sequence- and vehicle-treated animals, both of which did not differ from each other. The social discrimination test, on the other hand, revealed no difference in juvenile recognition abilities among the treatment groups. Using in situ hybridization and receptor autoradiography, we were not able to detect clear signals of CRH1 receptor mRNA and CRH binding sites in the central amygdaloid nucleus of either group, confirming the reportedly low expression and density of CRH receptors in this brain area. The present data support the view that CRH receptors in the central nucleus of the amygdala are involved in the mediation and expression of anxiety-related behavior, but simultaneously raise questions as to the mechanisms of antisense oligodeoxynucleotide action.

Corticotropin-releasing hormone receptor (type I) antisense targeting reduces anxiety.

Two brain-derived corticotropin-releasing hormone receptors have been cloned, termed corticotropin-releasing hormone receptors type I and type 2. Antisense oligodeoxynucleotides targeted to the cloned rat and mouse corticotropin-releasing hormone receptors type I messenger RNA reduced the binding of the natural ligand of the corticotropin-releasing hormone receptors type I and also the release of adenocorticotrophic hormone in primary rat anterior pituitary cells and in clonal mouse pituitary cells (AtT-20) by up to 60% in an application time-dependent manner. Studies on intracellular uptake of fluorescence-labelled oligodeoxynucleotides indicated a cytoplasmic accumulation starting within two to four hours after application of oligodeoxynucleotides in vitro. In vivo, antisense oligodeoxynucleotides infused intra-cerebroventricularly reduced binding of radiolabelled corticotropin-releasing hormone receptors in central sites of the rat brain. Anxiety induced by i.c.v. administration of corticotropin-releasing hormone was attenuated by corticotropin-releasing hormone receptors type I antisense treatment as determined in the elevated plus maze and in the novel open field test. The corticotropin-releasing hormone-induced behavioural changes were absent in corticotropin-releasing hormone receptors type I antisense-pretreated animals. These results show that the selected antisense probes used were able to suppress corticotropin-releasing hormone receptors type I function in vitro as well as in vivo and suggest that the development of drugs blocking this specific receptor might lead to a novel class of anxiolytics.

Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide treatment attenuates social defeat-induced anxiety in rats

Summary1. The neuropeptide corticotropin-releasing hormone (CRH) is the main mediator of the neuroendocrine and behavioral response to stress. End-capped phosphorothioate antisense and sense oligodeoxynucleotides (ODN) corresponding to the start coding region of rat CRH mRNA were infused intracerebroventricularly (30 µg/3 μl per injection) three times at 12 hr intervals. Six hours after the last injection rats were subjected to social defeat stress and subsequently tested on the elevated plus maze.2. Socially defeated CRH antisense-treated rats displayed markedly reduced anxiety-related behavior, as they spent significantly more time in the open arms of the plus maze compared to sense ODN- and vehicle-treated animals.3. In controls, social defeat evoked a stress-induced elevation of CRH mRNA and CRH in the hypothalamus and a significant increase in plasma corticotropin (ACTH) levels. These parameters were attenuated in antisense-injected rats.4. Our results suggest that CRH antisense treatment is effectively suppressing the neuroendocrine and behavioral effects of social defeat.

Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide induces anxiolytic effects in rat

ANTISENSE oligodeoxynucleotide complementary to the start coding region of rat corticotropin releasing hormone (CRH) mRNA was intracerebroventricularly infused into rats three times at 12-h intervals. In the shuttle-box avoidance procedure antisense-treated rats showed, within 6 h, significant acceleration and increase in the total number of discriminative avoidance responses compared with controls, treated with the corresponding sense probe or vehicle alone. Following the shuttle-box experiment hypothalamic CRH hybridization signals and immuno-reactivity were reduced, while CRH immunoreactivity in the median eminence remained unchanged. Plasma ACTH and corticosterone were decreased in antisense- treated animals. It is likely that in addition to a selective blockade of CRH translation, antisense treatment may also interrupt secretion of CRH. Antisense targeting of the hypothalamo-hypophysial-adrenal axis may provide new strategies for the neuropharmacology of affective disorders.

Centrally administered oligodeoxynucleotides in rats: occurrence of non-specific effects.

We studied the effects of various intracerebroventricularly administered oligodeoxynucleotides on body temperature, locomotor activity, food intake and water consumption in rats during a 24 h period with a radio-telemetric system. Both complete phosphorothioate oligodeoxynucleotides and end-inverted oligodeoxynucleotides dose-dependently elevated body temperature, suppressed food and fluid intake and inhibited nighttime activity. Apparently these effects do not depend on the nucleotide sequence because antisense and sense arginine vasopressin and oxytocin oligodeoxynucleotides, as well as a missense oligodeoxynucleotide produced comparable changes in the autonomous and behavioral parameters. In control experiments neither contaminants from the chemical synthesis nor endotoxins produced such effects, whereas native DNA from salmon sperm did. Fever and sickness-like behavior in response to missense phosphorothioate oligodeoxynucleotides were accompanied by elevated concentrations of circulating corticosterone and by a marked increase in interleukin 6 mRNA in brain and spleen, indicating that centrally administered oligodeoxynucleotides stimulate the production of pyrogenic inflammatory mediators in both central nervous system and peripheral tissues. Our results indicate that centrally administered oligodeoxynucleotides produce beside their intended sequence-specific effects also transient and sequence-independent effects due to their nucleic acid structure.

Increased stress-induced adrenocorticotropin response after long-term intracerebroventricular treatment of rats with antisense mineralocorticoid receptor oligodeoxynucleotides

Brain corticosteroid receptors, the type 1 mineralocorticoid receptor (MR) and the type 2 glucocorticoid receptor (GR), are involved in the regulation of neuroendocrine and behavioral responses during ongoing and stressful conditions. To further investigate the role of MR in these responses, we treated male Wistar rats intracerebroventricularly (icv) for 1 week with an 18-base end-capped phosphorothioate-protected antisense oligodeoxynucleotide (ODN) directed against MR mRNA (MR-AS). A mixed bases sequence (MR-MB) and vehicle (0.9% saline) served as controls. The ODN solution was administered by continuous infusion (1 microgram/0.5 microliter/h) via an icv cannula connected with polyethylene tubing to a subcutaneously implanted Alzet miniosmotic pump. No nonspecific effects of the ODNs, such as signs of sickness behavior or loss of body weight, were observed at any time during the treatment. The MR-AS treatment produced a 21% decline in hippocampal MR concentrations. Hippocampal GR levels were not affected by the treatment. MR and GR levels after MR-MB treatment were similar to those found after treatment with the vehicle. In situ hybridization experiments using an [35S]-labeled antisense MR probe showed that hippocampal MR mRNA levels were increased in MR-AS-treated rats. No changes were found in basal early morning levels of plasma ACTH and corticosterone which is consistent with the lack of any changes in adrenal and thymus weight. When rats were socially defeated for 10 min by a male and female resident and then placed for 5 min in the elevated plus-maze, no changes in the anxiety-like behavior were observed in MR-AS-treated animals. However, MR-AS-treated rats killed immediately after the behavioral test had markedly higher plasma ACTH, but not corticosterone, levels than the MR-MB and saline controls. In conclusion, down-regulation of the brain MR produces an enhanced responsiveness of ACTH to stressful situations which appears to be accompanied by a reduced sensitivity of the adrenal gland to ACTH.

Molecular and cellular analysis of rP1.B in the rat hypothalamus: in situ hybridization and immunohistochemistry of a new P-domain neuropeptide.

P-domain peptides, a new family of secretory polypeptides, have been identified mainly in the gastroenteropancreatic tract of humans, rodents, and amphibians as well as in amphibian skin. In the present study, with PCR and RNA analysis a transcript has been discovered in rat brain termed rP1.B. The deduced polypeptide consists of a single P-domain and its amino acid sequence matches that of rat intestinal trefoil factor (rITF). Thus far, rP1.B is the only P-domain peptide expressed in neuronal cells of the CNS. Immunostained magnocellular perikarya were visible in the paraventricular, supraoptic and periventricular nuclei. Parvocellular rP1.B neurons were found in the arcuate nucleus. Additionally, specific hybridization signals with radiolabeled transcripts were observed in the same regions. rP1.B in the rat hypothalamus may be involved in the control of hypothalamo-hypophysial functions.

Human intestinal trefoil factor is expressed in human hypothalamus and pituitary: evidence for a novel neuropeptide.

Human intestinal trefoil factor, hITF, a secretory polypeptide found mainly in the human gastrointestinal tract, is a member of the newly characterized trefoil factor or P‐domain peptide family representing putative growth factors. Here we describe the identification of this gut peptide in the human brain and pituitary. With reverse transcriptase polymerase chain reaction, we were able to isolate and clone the transcript from human hypothalamus. An antibody generated against a synthetic peptide derived from the carboxyl terminus of hITF was used for immunohistochemical studies of appropriate tissue sections. Neurons expressing hITF were identified in two magnocellular hypothalamic nuclei, the paraventricular and periventricular nuclei. hITF polypeptide was also observed in Herring bodies of the neurohypophysis and in secretory cells of the adenohypophysis. Double immunostaining with anti‐growth hormone antibody showed partial coexistence in a selected subpopulation of adenohypophysial cells. Localization of hITF in the hypothalamo‐neurohypo‐ physial system may suggest a modulatory action on the classical magnocellular nonapeptides vasopressin and oxytocin, and further indicates an adenohypophysial importance of this peptide. It is likely that hITF represents a novel neuropeptide of yet unknown function.—Probst, J. C., Zetzsche, T., Weber, M., Theilemann, P., SkuteUa, T., Landgraf, R., Jirik‐ owski, G. F. Human intestinal trefoil factor is expressed in human hypothalamus and pituitary: evidence for a novel neuropeptide. FASEB J. 10, 1518‐1523 (1996)

Vasopressin Antisense Oligonucleotide Induces Temporary Diabetes Insipidus in Rats

The purpose of this study was to downregulate the transcriptiona message of arginine vasopressin (AVP) by antisense treatment. A complete phosphorothioate antisense oligodesoxynucleotide corresponding to the beginning of the coding region of rat AVP mRNA was constructed and injected into the lateral ventricle of rats. Within 3–6 h animals exhibited a temporary diabetes insipidus, which lasted up to 9 h. Accordingly, vasopressin immunoreactivity in the hypothalamic nuclei was reduced. Our results demonstrate that a specific and reversible inhibition of neuropeptide expression can be accomplished in the intact hypothalamo‐neurohypophysial system by antisense treatment, thus providing a novel tool for studies on stimulus‐secretion coupling in vivo.

Downregulation of brain mineralocorticoid and glucocorticoid receptor by antisense oligodeoxynucleotide treatment fails to alter spatial navigation in rats.

Adult male Brown Norway rats were long-term intracerebroventricularly (i.c.v.) infused with antisense oligodeoxynucleotides (18-mer, double endcapped phosphorothioate protected) targeting either mineralocorticoid or glucocorticoid receptor mRNA, or received the respective mixed bases sequence or vehicle. Mineralocorticoid receptor-mixed bases and glucocorticoid receptor-mixed bases oligodeoxynucleotide infusion (1 microg/0.5 microl/h) over a time period of seven days did not alter hippocampal mineralocorticoid receptor and glucocorticoid receptor binding when compared to vehicle treatment. In contrast, i.c.v. administration of mineralocorticoid receptor, as well as glucocorticoid receptor-antisense over the same time period resulted in a significantly reduced binding of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus [mineralocorticoid receptor-antisense group approx. 72% of mineralocorticoid receptor-mixed bases and vehicle groups (100%); glucocorticoid receptor antisense group approx. 77% of glucocorticoid receptor-mixed bases and vehicle]. The specificity of these antisense effects is indicated by the finding that rats treated with mineralocorticoid receptor-antisense did not show any changes in glucocorticoid receptor and vice versa. Animals treated according to this infusion protocol and tested in the Morris water maze for their spatial navigation abilities failed to show significant differences among the groups. These data indicate that a reduction of hippocampal mineralocorticoid receptor or glucocorticoid receptor binding capacity by 20-30% does not interfere with spatial navigation.