Initial characterization showed that male TIP39-KO developed normally and were not different from WT on a battery of tests that included evaluation of home cage activity, motor coordination, sensory functions and general health. (Note – female mice were not investigated in this study because of the potential confounds introduced by varying levels of ovarian hormones during the estrus cycle and the failure of ovarian development in female TIP39-KO.) Under standard test conditions, exploratory and anxiety-related behaviors in the hole-board, novel open field, elevated plus-maze and dark–light emergence tests were also largely similar between TIP39-KO and WT. Exceptions included increased risk-assessment (elevated plus-maze) behaviors and defecation (dark–light emergence) observed in TIP39-KO, which provided the suggestion of a modest increase in anxiety-like behavior.
A robust increase in anxiety-like behavior was seen in TIP39-KO in the shock-probe burying test. In this test, TIP39-KO showed more defensive burying following contact with the shock probe. This phenotype was not explained by increased sensitivity to pain because the genotypes did not differ in responsivity to footshock or in several assays of nociception. Therefore, the manifestation of a clear anxiety-like phenotype in the TIP39-KO in the shock-probe burying test can be argued to be because of an inherently greater anxiety-provoking nature of this task than the exploration-based assays in which TIP39-KO were largely normal.
This hypothesis was supported by the results of two experiments in which mice were tested on the elevated plus-maze under conditions designed to amplify the anxiety-provoking nature of the test – high illumination and prior restraint. Both these putative ‘stressors’ uncovered a significant increase in anxiety-like behavior in TIP39-KO. This was unrelated to changes in control measures of locomotor activity, demonstrating the anxioselectivity of the phenotype. Of note, while these stressors were adequate to reveal an anxiety-like phenotype in KO, they did not increase the anxiety-like behavior measured in WT. Although this was our a priori
intent, it may seem surprising in light of previous evidence that similar manipulations heighten anxiety-like responses in rats (Bignante et al. 2008
; Calvo et al. 1998
; Garcia et al. 2005
; Heinrichs et al. 1994
; Hogg 1996
; Martijena et al. 1997
; Mechiel Korte & De Boer 2003
). However, this could reflect a genuine species difference because we chose a 30-min restraint protocol based on literature, suggesting that either a longer period of restraint or repeated restraint is needed to induce significant anxiety-like responses in mice (Bignante et al. 2008
, Chotiwat & Harris 2006
; Dunn & Swiergiel 1999
; Hata et al. 2001
; Hsu et al. 2007
; Schaefer et al. 2000
). Furthermore, while high illumination levels are generally thought to increase elevated plus-maze anxiety-like responses in rats (Griebel et al. 1993
; Hogg 1996
), some investigators observe a threshold effect at relatively low illumination level (Garcia et al. 2005
), and effects on mice are less well documented. A final consideration is that both the non-stressed TIP39-KO and WT showed relatively less anxiety-like behavior than in our initial experiments conducted under similar non-stressed conditions. This is likely because of the use of a different apparatus and test room, which we have subsequently found to produce less anxiety-like behavior regardless of mouse strain.
The abnormal anxiety-like phenotype of TIP39-KO extended to enhanced conditioned fear responses. The mutants exhibited increased freezing to a tone paired with shock during training and subsequently showed more freezing when re-exposed to either the tone or the conditioned context, indicating enhanced acquisition and/or enhanced expression of fear. This phenotype was replicated across two separate experiments. In contrast, fear extinction learning and recall were unaffected by loss of TIP39. The analysis of nociceptive responses we performed produced no indication that abnormal pain processing could account for genotype differences in conditioned fear. This is an important point to emphasize, given the presence of the PTH2-R in the dorsal horn of the spinal cord and primary afferent neurons (Dobolyi et al. 2003a
) and previous observations that percutaneously or intrathecally delivered TIP39 has pro-nociceptive effects (Dobolyi et al. 2002
Collectively, our data point to a specific increase in fear, and stress-related anxiety-like, behavior following loss of TIP39. This finding suggests that TIP39 normally acts to negatively modulate these behaviors. The distribution of TIP39 in the mouse brain is entirely consistent with this role. There is a high density and quite restricted distribution of TIP39-positive fibers and PTH2 receptors in regions known to mediate fear and anxiety, including parts of the amygdala, bed nucleus of the stria terminalis and lateral septum (Dobolyi et al. 2003b
; Faber et al. 2007
). In turn, the areas containing TIP39-positive neurons in the subparafascicular area receive inputs from these regions, suggesting a reciprocal circuit (Dobolyi et al. 2003b
; Wang et al. 2006
). Other neuropeptide systems, including CRH, neuropeptide Y, arginine vasopressin and galanin have been shown to affect rodent anxiety-like behaviors (Belzung et al. 2006
; Griebel 1999
; Karlsson & Holmes 2006
). In general, however, the neurons synthesizing these peptides are more widely distributed than TIP39 neurons, and based on the available data, these peptides also have more varied actions. These observations suggest that TIP39 might play a relatively selective modulatory role in the brain with prominent effects on fear and anxiety.
That said, we cannot exclude the possibility that developmental compensations outside these neural circuits or in the periphery contribute to the phenotypic abnormalities currently seen in mice constitutively lacking TIP39. However, we did not detect a difference in testosterone level (Usdin et al. 2008
), suggesting that gonadal hormone effects are unlikely to contribute to the behavioral differences. Moreover, although the distribution of TIP39 and the PTH2-R suggests potential effects on cardiovascular function, TIP39-KO showed normal baseline cardiac function. Finally, the projection pattern of TIP39 neurons, together with a pharmacological study in which TIP39 addition to hypothalamic cultures and injection into rat-brain-stimulated CRH and ACTH release (Ward et al. 2001
), suggest that TIP39 affects the function of hypothalamic CRH neurons. However, we found that neither basal nor restraint-stress evoked corticosterone or ACTH (not shown) was affected by loss of TIP39. Thus, while it seems probable based on distribution that TIP39 influences hypothalamic and CRH function, this did not appear to be sufficiently robust to produce measurable differences under these conditions, and thus, it seems unlikely that hypothalamo-pituitary-adrenal axis abnormalities caused the observed stress-related behavioral differences in TIP39-KO. Nonetheless, generation of conditional TIP39-KO that circumvent developmental and peripheral issues may be ultimately required to fully elucidate the physiological role of TIP39.
In conclusion, this study has shown that TIP39, a neuropeptide localized in neural circuits subserving emotional processing, plays an important role in the modulation of fear- and anxiety-like behaviors, as demonstrated in a mouse with TIP39 deletion. The anxiety-like phenotype of TIP39-KO, which is selectively manifested under relatively strong provocation, provides a compelling example of the preferential recruitment of neuropeptides as modulators of anxiety under conditions of stress or pathology (Hokfelt et al. 2003
; Holmes et al. 2003
). While further investigation of its behavioral and physiological functions is required, current data suggest the possibility that the TIP39/PTH2-R system could contribute to the pathophysiology of anxiety disorders and be a novel therapeutic target for these and other stress-related neuropsychiatric diseases.