BIOLOGICAL MECHANISMS OF ANXIETY: A BRAIN RECEPTOR AS SWITCH FOR ANXIETY
04 April 2000 - Max Planck Society

In response to a stressful stimulus, humans as well as other mammals release from the hypothalamus, a defined brain region, a peptide called corticotropin-releasing hormone or CRH. The structure of this peptide was characterized in 1981 at the Salk Institute in San Diego by Joachim Spiess, now director at the Max Planck Institute for Experimental Medicine, Goettingen, and his colleagues Wylie Vale, Jean Rivier and Catherine Rivier. Hypothalamic CRH is secreted into the venous portal blood vessel system connecting the hypothalamus and the pituitary and reaches with the blood flow the pituitary gland. There, it stimulates the release of another peptide hormone called corticotropin which is transported with the blood stream to the adrenal gland, where it stimulates the release of glucocorticoid hormones such as cortisol in humans and corticosterone in rodents. Glucocorticoids act on numerous organs and tissues, such as the immune system, skeletal system, liver, lungs and brain. In the brain and pituitary, glucocorticoids inhibit the release of CRH and corticotropin and thus stop their own production. This chain from the hypothalamus over the pituitary to the adrenal gland is called hypothalamo-pituitary-adrenal axis or stress axis which plays an important role in the adaptation of the organism to stressful situations. Under pathological conditions generated for example by mood and anxiety disorders, the functioning of the HPA axis may be strongly impaired.

CRH and its two receptor subtypes 1 and 2 are not only present in the hypothalamus and pituitary, but also in other brain regions and outside of the brain. Both CRH receptors 1 and 2 are proteins that span the cell membrane and initiate –when activated- a cascade of signals which may start or modulate neuronal processes. One of these signals is cyclic AMP, another is CREB which activates after phosphorylation as transcription factor several genes in cells.

For the investigation of the function of CRH receptors, peptidic and non-peptidic antagonists have been developed. Scientists of the Department of Molecular Neuroendocrinology of the Max Planck Institute for Experimental Medicine in Goettingen developed the CRH receptor 2-specific antagonist anti-sauvagine-30. In 1999, the Max Planck scientists Jelena Radulovic, Andreas Rühmann, Thomas Liepold and Joachim Spiess performed a series of behavioral experiments with mice and demonstrated that CRH receptor 2 of the lateral septum, a brain region, mediates the increase of anxiety in response to stressful stimuli. This observation was concluded from the fact that the CRH receptor 2-specific antagonist antisauvagine-30 injected into the septum prevented the stress-induced anxiety.

To further investigate the role of CRH in anxiety, so-called knock out mice had to be created, which did not contain functional CRH receptors 1 or 2. Knock out mice lacking CRH receptor 1 were created and investigated in 1998 independently in the laboratories of Florian Holsboer of the Max Planck Institute for Psychiatry, Muenchen, and of Wylie Vale of the Salk Institute, and George Koob of the Scripps Research Institute, both in San Diego. It was found that mice lacking CRH receptor 1 had lower anxiety than mice which normally expressed this receptor. Since CRH receptor 1 mediates in the pituitary the CRH action on the HPA axis, it is possible, at least in part, that impaired axis activity and especially lacking availability of glucocorticoid may be responsible for decreased anxiety. This aspect deserves even more attention, since it was recently reported by the group of Günther Schütz (Cancer Reasearch Center, Heidelberg) that removal of the functional gene expressing glucocorticoid receptor also leads to reduced anxiety.

On this basis, it was very important to create a mouse lacking the CRH receptor 2 and to investigate its behavior. This was achieved by three groups, the group of Joachim Spiess and Michael Rosenfeld (Max Planck Institute for Experimental Medicine, Goettingen; Howard Hughes Institute, University of California, San Diego), the group of Wylie Vale and George Koob (The Salk Institute, Scripps Research Institute, San Diego) and the group of Mary Stenzel-Poore, Vollum Institute, Portland, Oregon), who are independently publishing their results side by side in the April issue of Nature Genetics.

The Max Planck scientists found that the male CRH receptor 2-deficient mouse created by Toshimitsu Kishimoto, Chijen Lin, Farideh Hooshmand, Ola Hermanson and Michael Rosenfeld in San Diego, and behaviorally analyzed by Jelena Radulovic, Marko Radulovic, Christina Schrick and Joachim Spiess in Goettingen, exhibited profound anxious behavior. Increased anxiety was found in three different tests used for the determination of anxious behavior of the mouse. In the elevated plus-maze test in which two arms are open and two closed that means protected by side walls, the mice expressed their anxious behavior by spending much more time in the protected than in the open arms, without showing any changes of motor activity. This finding is surprising, since septal CRH receptor 2 was found to mediate stress-induced anxiety, as described above. To clarify the role of CRH receptor 2 in anxiety, Jelena Radulovic of the Max Planck Institute injected the CRH receptor antagonist anti-sauvagine-30 into the lateral septum or into the ventricle system of normal mice. Whereas the septal injection was without effect, the ventricular injection elicited anxious behavior that could not be differentiated from the behavior of the male CRH receptor 2 knock out mouse. This experiment demonstrated that the anxiety seen in the knock out mouse was not due to neurodevelopmental disturbances, but resulted directly from the absence of CRH receptor 2. Thus, the Max Planck scientists concluded that non-septal CRH receptor 2 mediates anxiolytic behavior whereas septal CRH receptor 2 mediates stress-induced anxiety. The researchers in Goettingen are now investigating whether septal and non-septal CRH receptors 2 are structurally different, or whether they are differentially linked to the cellular signalling cascades – what seems more likely.

When the Max Planck scientists compared the stress response of male and female knockout mice, they found that the activity of the HPA axis was normal, and that the anxious behavior of the male knock out mouse could thus not be explained by changes of the HPA axis responsiveness. The scientists also demonstrated that the increased anxiety of the knock out mouse was not due to increased CRH receptor 1 activity by pharmacological blockade of this receptor which did not reduce the CRH receptor 2-deficient mouse’s anxiety.

The initial investigations of the signal chains activated by CRH receptor 2 provided more surprising results. In the male knock out mouse in comparison to the wild type mouse, the phosphorylation of the transcription factor CREB in response to the plus maze was significantly reduced in brain regions (except for the septum) normally expressing CRH receptor 2. This observation suggested that the phosphorylation of CREB is part of a signal chain probably involved in anxiolysis. This observation is very important because CREB which can be phosphorylated by many pathways and then becomes a gene activator, a transcription factor, has been seen to date as an important player in memory formation. The data provided here suggest also a molecular role in anxiety.

The female knockout mice were, surprisingly, not anxious and exhibited normal CREB phosphorylation. The scientists concluded that female mice use either different developmentally programmed mechanisms to cope with anxiety or, which appears more probable- had successfully adapted to the receptor deficit. The behavior of the female knock out mice in different anxiety tests was independent of the phase of the oestrus cycle they were experiencing.

Taken together, the experiments published in Nature Genetics and previously in The Journal of Neuoscience show the following: The relationship between CRH receptor and anxiety is obviously more complex than anticipated. In the mouse, CRH receptor 2 is predominantly anxiolytic. Only in response to a stressful stimulus, septal CRH receptor 2 mediates anxiety. These discoveries open new avenues of pharmacological strategies for the treatment of anxiety disorders. Instead of blockade of CRH receptor 1 (or the glucocorticoid receptor), it has to be considered to activate non-septal CRH receptor 2. The stress axis is not expected to be affected by this treatment. However, the chemical challenge is significant because it would not be desirable to activate septal and peripheral CRH receptor 2.

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