help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2007-0048
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zeng, H.
Right arrow Articles by Bergmann, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zeng, H.
Right arrow Articles by Bergmann, J. E.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
Medline Plus Health Information
*Anxiety
*Depression
Molecular Endocrinology 21 (11): 2795-2804
Copyright © 2007 by The Endocrine Society

Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice Display Increased Depression and Anxiety-Like Behavior

Hongkui Zeng, Brian A. Schimpf, Alex D. Rohde, Maria N. Pavlova, Alexander Gragerov and John E. Bergmann

Omeros Corporation, Seattle, Washington 98101

Address all correspondence and requests for reprints to: Hongkui Zeng or John E. Bergmann, Omeros Corp., 1420 Fifth Avenue, Suite 2600, Seattle, Washington 98101. E-mail: hongkuiz{at}alleninstitute.org or jbergmann{at}omeros.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Experimental Animals
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH is a neuropeptide with a variety of hormonal and neurotransmitter/neuromodulator functions. In particular, TRH has pronounced acute antidepressant effects in both humans and animals and has been implicated in the mediation of the effects of other antidepressive therapies. Two G protein-coupled receptors, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified. Here we report the generation and phenotypic characterization of mice deficient in TRH-R1. The TRH-R1 knockout mice have central hypothyroidism and mild hyperglycemia but exhibit normal growth and development and normal body weight and food intake. Behaviorally, the TRH-R1 knockout mice display increased anxiety and depression levels while behaving normally in a number of other aspects examined. These results provide the first direct evidence that the endogenous TRH system is involved in mood regulation, and this function is carried out through TRH-R1-mediated neural pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Experimental Animals
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH WAS THE FIRST hypothalamic hormone to be isolated (reviewed in Ref. 1). It is a tripeptide, pyro-Glu-His-Pro-NH2, that is the central regulator of the hypothalamic-pituitary-thyroid (HPT) axis. TRH is produced in the paraventricular nucleus of hypothalamus and stimulates the synthesis and secretion of TSH from the anterior pituitary, which, in turn, stimulates the synthesis and release of thyroid hormones. Outside of the hypothalamus, TRH is widely distributed in many parts of the brain and exerts a variety of effects as a neuromodulator or neurotransmitter (1). These effects include seizure modulation, autonomic nervous system function, food intake, antinociception, thermoregulation, and activation of arousal, cognition, locomotion, and mood. It has been proposed that the main neurobiological function of TRH is to promote homeostasis (2). TRH is also present in many peripheral tissues, such as gastrointestinal tract, pancreas and reproductive tissues and implicated in diverse physiological functions.

Two mammalian G protein-coupled receptors for TRH, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified (reviewed in Refs. 3 and 4). They share approximately 50% identity at the amino acid level. TRH-R1 is found in mouse, rat, and human and is highly conserved among these species with about 95% identity (5, 6, 7). TRH-R2 is present in mouse and rat but not human (8, 9). Rodent receptors of the two types exhibit similar affinities for TRH and appear to signal via similar transduction pathways primarily mediated by Gq/11 and phospholipase C. There are marked differences between the expressions of the two receptors. TRH-R1 is widely distributed in both central and peripheral tissues, whereas TRH-R2 has limited peripheral distribution and is concentrated in the central nervous system (8, 10, 11). In the brain, the two receptors are also differentially distributed (12, 13, 14). TRH-R1 is highly expressed in the anterior pituitary and the neuroendocrine brain regions, the autonomic nervous system, and the visceral brainstem regions. TRH-R2 is enriched in brain areas that are important for the transmission of somatosensory signals and higher central nervous system functions.

Targeted disruption of the prepro-TRH gene (15) results in tertiary hypothyroidism, with significantly reduced thyroid hormone levels, whereas the prolactin and GH levels are normal. These TRH knockout (KO) mice exhibit elevation of serum TSH level and diminished TSH biological activity. Nonetheless, the mice are viable and fertile and exhibit normal development. The TRH KO mice also exhibit hyperglycemia accompanied by impaired insulin secretion after glucose challenge. In TRH KO mice at birth, the blood level of thyroid hormone is similar to that of wild-type (WT) controls. The staining intensity and number of TSH-immunopositive pituitary cells are not affected by TRH deficiency, showing that neither embryonic nor maternal TRH is required for the normal development or differentiation of pituitary thyrotrophs during the embryonic period. However, the mutant mice exhibit decreased number and intensity of staining of TSH-immunoreactive pituitary cells after postnatal d 10, suggesting that TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs (16, 17). No behavioral characterization of these mice has been reported.

A strain of mice with targeted deletion of the TRH-R1 gene has also been reported (18). These TRH-R1 deletion mice have growth retardation while appearing normal otherwise. The mice exhibit a considerable decrease in serum T3, T4, and prolactin levels but not in serum TSH levels. The numbers of thyrotrophs, somatotrophs, and lactotrophs are not affected by the deletion of the TRH-R1 gene.

It has long been recognized that TRH has antidepressant effect. Intravenous or intrathecal administration of TRH induces rapid (within hours) remission of major depression in humans (19, 20, 21, 22) and decreased immobility in forced swim test in rats (23). In animals, TRH has also been shown to enhance the pharmacological action of antidepressant drugs (24, 25). Electroconvulsive shock (ECS) in rats induces synthesis of TRH in multiple subcortical and frontal cortical regions known in humans to be involved in both depression and sleep, such as hippocampus, amygdala, entorhinal cortex, and pyriform cortex (26). Nocturnal TRH administration produces a rapid and sustained antidepressant effect in patients with bipolar disorders (27).

Although administration of exogenous TRH or its analog at pharmacological doses helps to identify TRH’s potential functions, further elucidation of the endogenous TRH function and the mediation by its two receptors is impeded by the lack of antagonist, especially subtype-selective antagonist. In this study, we report the generation of a line of TRH-R1 KO mice through retroviral insertional mutagenesis, and the behavioral and physiological characterization of these mice. Our TRH-R1 KO mice have lower thyroid hormone level and increased fasting glucose level, but they are normal in body length and weight without any overt developmental abnormalities. This is consistent with the phenotypes of the TRH KO mice (15) but differs from the previously reported TRH-R1 deletion mice that exhibit postnatal growth retardation (18). The normal growth of our TRH-R1 KO mice allowed a comprehensive behavioral evaluation, which revealed that the KO mice have elevated anxiety and depression levels. No data of mood alterations had been provided in either of the previously reported TRH KO or TRH-R1 deletion mice strains. Therefore, our results provide the first line of evidence that the endogenous TRH system is involved in mood regulation and that this function is carried out through TRH-R1-mediated neural pathways in rodents. The TRH-R1 KO mice reported here can be a good model for the study of the broad biological functions of the TRH system.


   Experimental Animals
TOP
 ABSTRACT
 INTRODUCTION
 Experimental Animals
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals were kept on a constant 12-h light, 12-h dark cycle at all times (with lights on at 0700 h and off at 1900 h) with ad libitum food and water access. All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 Experimental Animals
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of TRH-R1 KO Mice
An embryonic stem (ES) cell library was created using a mutagenic retrovirus (51). From this library, a mutant clone with a viral insertion in the TRH-R1 gene (Fig. 1AGo) was identified, isolated, and used to produce mice. The murine TRH-R1 gene contains five exons and spans about 45 kb. The coding sequence is located in exons 2 and 3 (28). The virus insertion was localized in the first intron by sequencing of the genomic DNA. Because the retroviral vector (Fig. 1BGo) included a splice acceptor, termination codons in all reading frames, a polyadenylation site, a selection marker, and a transcription terminator (29), the insertion is expected to interrupt the transcript before exon 2, resulting in no production of the receptor. Southern blot analysis showed that there were no additional viral insertions anywhere else in the genome (data not shown).


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Generation of TRH-R1 KO Mice

A, Schematic diagram of the retroviral vector insertion in the TRH-R1 gene. Numbers 1–5 indicate exons, with start codon (ATG) in exon 2 and stop codon (TAA) in exon 4. The large triangle indicates the viral insertion. Primers a/b and c/d were used for RT-PCR in C to confirm inactivation of the gene. B, Structure of the retroviral vector with multiple mutagenic features. The retroviral vector contains the virus backbone [including 5'-long terminal repeat (LTR) and 3' LTR], an SV40 splice acceptor (SA)-stop codons (stop)-IRES cassette immediately followed by rtTA, a PGK promoter (Pr)-driven neo selection marker flanked by two loxP sites (L), and a transcriptional terminator sequence (t). pA, Polyadenylation sequence. From this locus, transcription initiated from the endogenous TRH-R1 gene continues through rtTA to form a TRHR1-SA-Stops-IRES-rtTA-polyA hybrid transcript in place of the full-length endogenous TRH-R1 transcript. C, RT-PCR using primers a and b, or c and d, in which a 540- or 550-bp band is expected to be amplified from uninterrupted WT allele. RT+, Reverse transcription reaction with the presence of reverse transcriptase. RT–, Control reverse transcription reaction without reverse transcriptase. M, DNA marker; pit, pituitary.

 
To confirm the null mutation, RT-PCR was used to examine the TRH-R1 transcript from the brains and pituitaries of homozygous mutant (KO) and WT control mice. Using two pairs of primers covering different exons, one pair spanning the retroviral insertion and the other pair downstream of the insertion, PCR products from both pairs were amplified from the WT control but not the KO animal (Fig. 1CGo). To further test the sensitivity of the RT-PCR, the TRH-R1 transcript, assessed using both pairs of primers, and a positive control ß-actin transcript in WT, heterozygous, and KO mice were examined by real-time quantitative PCR (qPCR). ß-Actin transcript levels remain the same in all three types of mice (the ß-actin signal appeared after 23–26 cycles in all three types of samples). The TRH-R1 signal appeared after 26–30 cycles in samples from the WT mice and heterozygous mice. After normalization to the level of ß-actin mRNA, the TRH-R1 mRNA level appeared to be two to three times lower in the heterozygous mice than in the WT mice. In contrast, the TRH-R1 mRNA level from the KO mice was below the lower limit of quantification of the qPCR assay even after 40 cycles. Therefore, the retroviral insertion has blocked TRH-R1 transcription completely or decreased it to an undetectable level.

TRH-R1 KO Mice Display Hypothyroidism and Hyperglycemia but Normal Body Size
The thyroid hormone status of the mutant mice was evaluated after they reached 3 months of age. There was a significant reduction in plasma T4 levels of the KO mice compared with their WT littermates (Fig. 2AGo; WT 4.1 ± 0.5 µg/dl, n = 8; KO 2.3 ± 0.3 µg/dl, n = 9; P = 0.005). There was also a mild but significant increase of fasting glucose levels in the KO mice (Fig. 2AGo; WT 76 ± 8 mg/dl, n = 7; KO 102 ± 8 mg/dl, n = 9; P = 0.043). Plasma cholesterol levels were normal (WT 191 ± 17 mg/dl, n = 8; KO 200 ± 15 mg/dl, n = 9; P = 0.674).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. TRH-R1 KO Mice Have Hypothyroidism and Mild Hyperglycemia but Normal Body Size and Food Intake

A, TRH-R1 KO mice have decreased plasma T4 level (left panel) and increased fasting blood glucose level (right panel). *, P < 0.05; **, P < 0.01. B, TRH-R1 KO mice have normal body weight (left panel) and body length (middle panel) and average 24-h food intake (right panel).

 
The TRH-R1 KO mice we generated were obtained at the expected Mendelian ratio, suggesting that there was no reduction in fetal viability. Furthermore, we did not observe any gross abnormality in their growth rate or general appearance (data not shown). Adult body weight, body length, and daily food intake of the KO mice were not significantly different from those of the WT mice (Fig. 2BGo; WT n = 18; KO n = 18). Their baseline body temperature was also normal (WT 36.2 ± 0.1 C, n = 18; KO 36.0 ± 0.1 C, n = 18; P = 0.240).

TRH-R1 KO Mice Display Normal Behavioral Responses in Locomotor Activity, Thermal Pain, Sensorimotor Gating, and Fear Conditioning
Because TRH-R1 is widely distributed in the central nervous system, we subjected the TRH-R1 KO mice to a series of behavioral tests that examine behavioral changes related to general activity, nociception, anxiety, depression, psychosis, and learning.

Home cage activity levels were monitored under the animals’ normal 24-h light/dark cycle (Fig. 3AGo). No differences were observed between the activity cycles of the TRH-R1 KO mice and the WT mice. As expected for nocturnal animals, the mice were generally more active during the dark phase of the cycle. Overall, there was no significant difference between genotypes in total daytime activity or total nighttime activity (WT n = 18, KO n = 18, repeated-measures ANOVA). However, at several isolated time points, the KO mice showed lower activity levels than the WT mice, indicating that the KO mice may have a mild reduction in activity.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 3. TRH-R1 KO Mice Have Normal Locomotor Activity and Contextual Fear Conditioning

A, Twenty-four-hour home cage activity. Activity levels indicate the number of horizontal photobeam breaks. Data plotted are in 1-h bins. B, Open-field activity. Activity levels indicate the number of horizontal photobeam breaks and are binned every 4 min. C, Contextual fear conditioning. The percentage of time spent freezing during the 5-min context testing period is plotted.

 
To evaluate locomotor activity in a novel environment, WT and KO mice were subjected to an open-field activity test (Fig. 3BGo). The TRH-R1 KO mice showed similar levels of activity compared with the WT mice throughout the 20-min testing period. There was no significant difference between TRH-R1 KO and WT mice in the horizontal activity (F(1,34) = 0.100, P = 0.753, WT n = 18, KO n = 18, repeated-measures ANOVA), as well as in total vertical activity, stereotypy, and number of clockwise or counterclockwise rotations (data not shown).

The mice were further tested in contextual fear conditioning, a measure of emotion-based associative learning and memory. The TRH-R1 KO mice showed an average percentage of time in freezing that is not significantly different from that of the WT mice when placed back into the context (Fig. 3CGo, F(1,16) = 0.052, P = 0.823, WT n = 9, KO n = 9), indicating normal fear response and fear-based memory.

No significant differences were seen between TRH-R1 KO and WT mice in the hot plate test, a measure of thermal pain sensation, nor were differences seen in the prepulse inhibition (PPI) of the acoustic startle response, suggesting that the brain’s sensorimotor gating function was unaffected in these mutants (data not shown).

TRH-R1 KO Mice Show Increased Anxiety
In the elevated plus maze test, which is predictive of the efficacy of known anxiolytics, mice are placed on an elevated platform from which they can see a 50-cm drop. The platform is attached to two arms with opaque walls that give them apparent security. It is also attached to two other arms that are fully exposed but that provide a view of the environment. More anxious animals enter the open arms less frequently and spend less time in those open arms. Overall animal locomotor activity can be measured with the total number of entries into both open and closed arms. As shown in Fig. 4Go, no significant differences were detected between TRH-R1 KO (n = 17) and WT (n = 17) mice in the total number of arm entries (F(1, 32) = 1.577, P = 0.218) confirming the lack of differences in the overall activity of these mutants. In contrast, the TRH-R1 mice exhibited markedly enhanced anxiety as seen by the significantly lower percentage of open arm entries (F(1, 32) = 9.235, P = 0.005) and the total time spent in the open arms (F(1, 32) = 6.051, P = 0.019).


Figure 4
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 4. TRH-R1 KO Mice Exhibit Increased Anxiety in the Elevated Plus Maze Test

Left panel, Total number of entries into both open and closed arms; middle panel, percentage of entries into the open arms compared with total number of arm entries; right panel, the time spent in the open arms during the 5-min testing period. *, P < 0.05; **, P < 0.01.

 
TRH-R1 KO Mice Show Increased Depressive Behavior
Two tests that are predictive of the efficacy of known antidepressants were used to evaluate depressive behavior in the mice. In both cases, decreased escaping behavior (struggling or swimming) and increased immobility indicate a form of behavioral despair under adverse situations. The tail suspension test measures how much time mice suspended by their tails spend immobile rather than continuing to struggle in an attempt to get free. As shown in Fig. 5AGo, TRH-R1 KO mice had significantly longer immobility time than the WT mice (F(1, 33) = 6.610, P = 0.015, WT n = 17, KO n = 18).


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. TRH-R1 KO Mice Exhibit Increased Immobility in Depression Tests

A, Tail suspension test. The amount of time spent immobile during the 6-min testing period is plotted. B, Porsolt forced swim test. The amount of time spent immobile during the 5-min testing period is plotted. *, P < 0.05; **, P < 0.01.

 
The Porsolt forced swim test measures how long mice exhibit immobility and stop swimming or wall climbing in an attempt to escape the deep water in which they have been placed. In this test also, TRH-R1 KO mice showed significantly longer immobility time (Fig. 5BGo, F(1, 34) = 8.304, P = 0.007, WT n = 18, KO n = 18). Both tests demonstrate increased depressive behavior in TRH-R1 KO mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 Experimental Animals
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study shows that TRH-R1 KO mice display central hypothyroidism with decreased thyroid hormone levels. This is similar to the observations made in the TRH KO mice (15) and previously reported TRH-R1 deletion mice (18) and is consistent with the presence of TRH-R1 but not TRH-R2 in the pituitary (8). These data support the notion that TRH regulates thyroid hormone production through the pituitary TRH-R1. Also consistent with the TRH KO mice (15), the TRH-R1 KO mice reported here exhibit mild hyperglycemia but are otherwise normal in body length, body weight, and daily food intake on a regular chow, suggesting that TRH’s effect on insulin secretion and glucose control may be mediated by TRH-R1 in the pancreas as proposed (15) or in the brain.

The normal body growth in our TRH-R1 KO mice is in sharp contrast with the previously reported TRH-R1 deletion mice, which have pronounced postnatal growth retardation (18). This difference is apparently not due to differences in the inbred mouse strains used in the two studies. Although our TRH-R1 KO mice were only characterized in the 129/sv background, the growth retardation phenotype of the reported TRH-R1 deletion mice was observed in three different backcrossed backgrounds: C57BL/6, 129/sv, and NMRI. We speculate that the difference might arise from the different nature of the gene inactivation methods. In our case, the TRH-R1 gene was disrupted by a retroviral insertion, which terminates gene transcription before the coding sequence. There was no loss of any endogenous genomic sequences. In the previously reported TRH-R1 deletion mice, all coding sequences along with a large genomic region (~36 kb) between exons 2 and 5 were deleted through homologous recombination (18). Because our TRH-R1 KO mice resemble TRH KO mice in this respect, there might be certain elements unrelated to the TRH-R1 protein in the deleted genomic region that are responsible for the growth retardation. It should be noted that the TRH KO mice do exhibit transient growth retardation at the age of 4 wk but later on are able to catch up, so that by 8 wk of age, their body weight is not significantly different from that of the WT mice (15). We have not investigated whether our TRH-R1 KO mice also have transient growth retardation during development. On the other hand, it is reported that some children with short statures were found to have isolated central hypothyroidism (30), in particular, a patient with short stature had compound heterozygotic mutations of both alleles of the TRH-R1 gene (31). In light of this, an alternative possibility is that the viral insertion in our TRH-R1 KO mice might not completely shut down the TRH-R1 gene expression during certain early development periods even though it appears to do so in adult animals.

The normal development of the TRH-R1 KO mice allowed us to conduct a meaningful behavioral characterization to examine TRH-R1’s neuronal function in adult mice. Such studies have not been done with the TRH KO mice and were not appropriate to do with the developmentally abnormal TRH-R1 KO mice previously described. In a comprehensive behavioral test battery, we found that TRH-R1 KO mice display selective impairments in aspects of anxiety and depression; namely, they appear to be more anxious and depressive. This is consistent with the prominent presence of TRH and TRH-R1 in regions of the brain that regulate emotion and mood and the well-recognized fact that exogenous TRH administration has antidepressant effects. In the normal brain, both TRH immunoreactivity and TRH-R1 mRNA are present in the limbic regions, including hippocampus, amygdala, and nucleus accumbens (12, 13, 14, 32, 33, 34, 35, 36, 37). Intravenous or intrathecal administration of TRH induces rapid (within hours) remission of major depression in humans (19, 20, 21, 22). Nocturnal TRH administration produces a rapid and sustained antidepressant effect in patients with bipolar disorders (27). TRH administration decreases immobility in forced swim test in rats (23) and reduces anxiety-like behavior and learning impairment in senescence-accelerated mice (38). In animals, TRH administration also enhances the pharmacological action of antidepressant drugs (24, 25).

Furthermore, TRH production may play a role in the efficacy of ECS therapy, an effective treatment for many patients with major depression who are refractory to antidepressant drug treatments. In rats, ECS induces prolonged increase of synthesis of TRH in multiple subcortical and frontal cortical (limbic forebrain) regions known in humans to be involved in both depression and sleep, such as hippocampus, amygdala, entorhinal cortex, and pyriform cortex (26, 39). Similarly, the TRH system may play a role in the mechanism of action of lithium, an antidepressive and neuroprotective agent that alters TRH and TRH-R1 levels in different brain regions (40). Here, our data extend these observations by demonstrating that the endogenous TRH-R1/TRH system plays a key role in regulating anxiety and depression in mice.

It should be noted that alterations in anxiety- and depression-like behavior in the TRH-R1 KO mice may also be due to the reduced thyroid hormone levels as a result of TRH-R1 gene inactivation. Indeed, there is extensive evidence that thyroid hormones (T3 and T4) themselves also have variable antidepressive effects and can augment the effects of known antidepressants in humans and animals (41, 42, 43, 44, 45). There is considerable evidence to support the potentiation by T3 of the actions of the neurotransmitter serotonin (46). In two lines of thyroid hormone receptor {alpha} (TR{alpha}) mutant mice, a TR{alpha} null mutant line (47) and a functional but thyroid hormone-resistant TR{alpha}1 mutant line (48), both lines of animals show increased anxiety behavior. In the latter case, the anxiety deficiency can be relieved by treatment with high doses of T3 (48). The anxiety- and depression-like behaviors in TRH-R1 KO mice may result from either the direct involvement of TRH/TRH-R1 in the central mood-regulating pathways and/or the indirect consequence of lowered thyroid hormone levels. In either of these scenarios, normalization of the T3 or T4 levels by systemic administration might alleviate these behaviors. To further dissect the mechanism of mood alteration by the TRH/TRH-R1 system, it may be informative to create region-specific conditional TRH-R1 KO mice.

Consistent with the widespread distribution of TRH in neural and nonneural tissues far beyond the scope of the HPT axis, a plethora of physiological functions have been proposed. TRH-R1 is the major receptor for TRH in rodents in both peripheral tissues and central nervous system, whereas in humans it is the only receptor for TRH identified so far. TRH-R2, on the other hand, is restricted to the brain and mostly expressed in brain regions that are important for the transmission of somatosensory signals and higher brain functions. Thus, TRH-R1 KO mice will prove to be a good model to explore the various functions of the endogenous TRH system and the differential mediation of these functions by the two TRH receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 Experimental Animals
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of TRH-R1 KO Mice
TRH-R1 KO mice were produced by retroviral mutagenesis as described previously (49). Briefly, an ES cell library was constructed by infecting 129/Sv ES cells with a retroviral vector (29). The ES cell line used was subcloned from CJ7 (50) to obtain a high-frequency germline clone. Mutations in the TRH-R1 gene were found in the library by PCR analysis of genomic DNA using vector-specific and gene-specific primers. Mutant clones isolated from the library were used for animal production using standard injection methods. Chimeric mice were bred with 129S1/SvImJ mice (which was developed to serve as a control inbred strain for many of the steel-derived ES cell lines including CJ7) to generate heterozygotes in an inbred background. The resulting progeny were genotyped by PCR of tail DNA to identify pups containing a disruption in the TRH-R1 gene.

For phenotypic studies, heterozygous males and females bearing the TRH-R1 mutation backcrossed two generations in 129S1/SvImJ were bred with each other to obtain homozygous TRH-R1 KO mice and WT littermates in 129S1/SvImJ inbred background. In all the experiments described here, the WT littermates were used as controls.

RT-PCR was employed to confirm inactivation of the TRH-R1 gene. Brains and pituitaries were dissected and stored in RNALater (Ambion, Austin, TX) at 4 C until RNA isolation. RNA was isolated from homogenized tissue by phenol extraction and LiCl precipitation using a Totally RNA Kit (Ambion). RNA was treated with DNase I (Ambion) for 1 h at 37 C. Equal amounts of RNA (~100 ng for brain, ~20 ng for pituitary) were used in each sample for reverse transcription reactions using a Super-Script First Strand Synthesis kit (Invitrogen, Carlsbad, CA). Each reaction was run in duplicate with (RT+) or without (RT–) reverse transcriptase to control for possible genomic DNA contamination. cDNA was amplified for 32 cycles (94 C for 1 min, 58 C for 1 min, 72 C for 1 min, and final extension at 72 C for 7 min) with TRH-R1-specific primers (primer a, 5'-ggtttagaggaactgccgctctg-3', and primer b, 5'-ggcagccaacatagccatagaccca-3'; or primer c, 5'-catgttcaataacggcctttacc-3', and primer d, 5'-gggctggagagaaatgagttgaca-3') that were predicted to produce a 550- or 540-bp PCR fragment. The primer binding regions are located in exon 1 (primer a), exon 2 (primers b and c), and exon 3 (primer d) of the TRH-R1 gene, with the inactivating retroviral insertion located in the intron 1.

Real-time qPCR was conducted using cDNA from the above RT reactions with TRH-R1-specific primer pairs (a+b and c+d) as well as a positive primer pair of ß-actin (QuantumRNA ß-actin internal standards from Ambion). The reactions are carried out in a final volume of 25 µl that contains 12.5 µl SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 0.5 µM of each primer, and about 20–50 ng cDNA. Each sample was run in triplicates. The following standard cycle was employed: 95 C for 10 min followed by 40 cycles of 94 C for 15 sec, 56 C for 15 sec, and 72 C for 1 min in a 7900HT Fast Real-Time PCR system (Applied Biosystems). On completion of a real-time qPCR experiment, thermal denaturation profile of the resulting amplicon was determined to make sure that the same specific amplicon is detected in different samples. Difference in number of cycles needed to reach a threshold level of fluorescence with TRH-R1 primers as compared with ß-actin primers ({Delta}Ct, change in threshold cycle) was used as measure of relative TRH-R1 mRNA abundance.

Behavioral Testing
TRH-R1 KO and WT littermate male mice were tested sequentially in a battery of primary screen behavioral assays in the following order: 24-h home cage activity with body weight and food intake, open-field activity, hot plate, tail suspension, PPI, and contextual fear conditioning. The testing order was predetermined, with the less stressful tests conducted before the more stressful ones. Additional mice were used to replicate any near-significant findings. Both sets of mice were also used for the elevated plus maze, forced swim, and blood chemistry tests. All experimental mice were singly housed and were tested at ages of 3 months or later.

Home cage activity test was conducted in MicroMax chambers (Accuscan Instruments, Columbus, OH) that were exterior to the cages. The photobeams provide information of when an animal is moving around in its home cage, therefore monitoring the animal’s circadian pattern of sleep-wake cycles. Animals singly housed in their home cages were placed in the photobeam boxes for 3 d, and activity was recorded continually. Measurements used to assess home cage activity include horizontal activity, total distance traveled, and periods of rest time and active time. Twenty-four-hour activity was averaged across the 3 d. In addition, the animal’s body weight and the amount of food at the top of the cage were weighed before and after the 3-d activity monitoring. Average daily food intake was calculated from the change in the amount of food for each cage over the 3 d.

Open-field activity test was done in VersaMax chambers (Accuscan Instruments) measuring 40 x 40 cm and detected by infrared photobeam breaks. Mice were monitored in the chamber for 20 min. Measurements used to assess locomotor activity included horizontal activity, total distance traveled, vertical activity, rotation, stereotypy, and distance traveled in the center compared with total distance traveled (center to total distance ratio).

Elevated plus maze test uses a maze (Med Associates, St. Albans, VT) elevated 50 cm above the floor. A video camera mounted above the maze is used to record the animal’s behavior. To conduct the test, a mouse was placed in the central platform, facing an open arm, and allowed free exploration in the maze for 5 min. Using an automated video tracking software, ANY-maze (Stoelting, Wood Dale, IL), the following criteria were measured: number of entries into open arms, number of entries into closed arms, time spent in open arms, time spent in closed arms, entries into distal portion of open arms, head-dipping over sides of open arms, etc.

Tail suspension test uses an automated tail suspension apparatus (Med Associates). A mouse was taped by the tail to the hook of the apparatus. The load cell amplifier detected the animal’s movements (struggle to escape), and the data were collected over a 6-min testing session. The immobility time (when the animal was not struggling) was measured as the animal’s movement below a preset threshold.

Porsolt forced swim test was conducted by placing a mouse in a clear Plexiglas cylinder (13 cm in diameter x 24 cm in height) containing water (at room temperature) to 10-cm high. The behavior of the mouse was observed for 6 min, and the time spent in mobile (e.g. swimming or attempting to climb the wall) or immobile was recorded using The Observer Mobile with a Psion Workabout (Noldus Information Technology, Wageningen, The Netherlands). Movements necessary to maintain the animal’s head above water were not scored as immobile.

PPI of the acoustic startle response was tested using the SR-Lab System (San Diego Instruments, San Diego, CA). A test session consists of six trial types with a background of 70-dB white noise. The baseline trial used no prepulse and no startle (just 70-dB white noise). The startle trial used a 40-msec, 120-dB noise as the startle stimulus. Four levels (73, 76, 79, or 82 dB) of prepulse trials were also employed in which 20 msec of acoustic stimulus was presented 100 msec before the 120-dB startle stimulus. Six blocks of these six trial types were presented in each block, and the order of trials was randomized. The prepulse stimuli had been determined not to cause a startle response on their own (not shown). The startle response was recorded for 65 msec starting with the onset of the startle stimulus. PPI was calculated as the percentage of each inhibition of the maximum startle response achieved by each of the four prepulses.

Contextual fear conditioning test was conducted in two sessions across 2 d. In the training session, a mouse was placed in the conditioning chamber (Med Associates) and allowed to explore the environment. Two minutes later, a 75- to 80-dB white noise was turned on for 30 sec. A 0.6-mA electric shock was delivered through the metal bar floor for the last 2 sec of the noise period. Two minutes later, the noise and shock were repeated one more time. The mouse was tested 24 h later by placing it in the same chamber for 5 min and scoring the amount of freezing it displayed in the context (the chamber) in which it had been shocked without the noise (context test). Freezing behavior, i.e. complete immobility during each 1-sec bin, was automatically measured using the FreezeFrame system (Actimetrics, Wilmette, IL).

Hot plate test was carried out by placing a mouse on a 55 C hot plate (Accuscan Instruments) and measuring the latency of a hind limb response (shaking or licking) with a maximum cutoff of 30 sec.

Blood Chemistry
Blood was collected via retroorbital eye bleeding or tail clipping. For plasma separation the blood was spun in microtainer tubes (Becton Dickinson, Franklin Lakes, NJ). Plasma was assayed for T4 or cholesterol using a Prochem V biochemistry analyzer (Drew Scientific, Oxford, CT). For T4 measurement, each lot of Prochem V QVETs for T4 had been precalibrated at the factory for three veterinary species (horse, cat, and dog). When assaying mouse plasma, a set of generic (averaged) calibration parameters was programmed in. Blood glucose level was measured using a One-Touch Ultra glucometer.

Statistics
Data were analyzed by Student’s t test or one-way ANOVA using the SPSS program. The criterion for a statistically significant difference was P < 0.05. Data were presented as mean values ± SEM.


   ACKNOWLEDGMENTS
 
We are grateful to John Hohmann for his role in setting up some of the physiological tests.


   FOOTNOTES
 
Present address for H.Z.: Allen Institute for Brain Science, 551 North 34th Street, Seattle, Washington 98103.

This work was supported by private funding and in part by an Small Business Innovative Research Phase I grant from National Institute of Mental Health.

Disclosure Statement: All authors affiliated with Omeros Corp. hereby declare potential conflict of financial interest. H.Z. was previously employed by Omeros Corp. but has no equity interest in Omeros Corp. B.A.S. was previously employed by Nura, Inc. (acquired by Omeros Corp.) but has no equity interest in either Omeros Corp. or Nura, Inc. A.G., M.N.P., A.D.R., and J.E.B. are employed by and have equity interests in Omeros Corp. of less than $10,000.

First Published Online July 31, 2007

Abbreviations: ECS, Electroconvulsive shock; ES, embryonic stem; HPT, hypothalamic-pituitary-thyroid; KO, knockout; PPI, prepulse inhibition; qPCR, quantitative PCR; TR{alpha}, thyroid hormone receptor {alpha}; TRH-R1, TRH receptor 1; WT, wild type.

Received for publication January 25, 2007. Accepted for publication July 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Experimental Animals
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Nillni EA, Sevarino KA 1999 The biology of pro-thyrotropin-releasing hormone-derived peptides. Endocr Rev 20:599–648[Abstract/Free Full Text]
  2. Gary KA, Sevarino KA, Yarbrough GG, Prange Jr AJ, Winokur A 2003 The thyrotropin-releasing hormone (TRH) hypothesis of homeostatic regulation: implications for TRH-based therapeutics. J Pharmacol Exp Ther 305:410–416[Abstract/Free Full Text]
  3. Gershengorn MC, Osman R 1996 Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev 76:175–191[Abstract/Free Full Text]
  4. Sun Y, Lu X, Gershengorn MC 2003 Thyrotropin-releasing hormone receptors: similarities and differences. J Mol Endocrinol 30:87–97[Abstract]
  5. Straub RE, Frech GC, Joho RH, Gershengorn MC 1990 Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci USA 87:9514–9518[Abstract/Free Full Text]
  6. de la Pena P, Delgado LM, del Camino D, Barros F 1992 Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells. Biochem J 284 (Pt 3):891–899
  7. Matre V, Karlsen HE, Wright MS, Lundell I, Fjeldheim AK, Gabrielsen OS, Larhammar D, Gautvik KM 1993 Molecular cloning of a functional human thyrotropin-releasing hormone receptor. Biochem Biophys Res Commun 195:179–185[CrossRef][Medline]
  8. Cao J, O’Donnell D, Vu H, Payza K, Pou C, Godbout C, Jakob A, Pelletier M, Lembo P, Ahmad S, Walker P 1998 Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem 273:32281–32287[Abstract/Free Full Text]
  9. Itadani H, Nakamura T, Itoh J, Iwaasa H, Kanatani A, Borkowski J, Ihara M, Ohta M 1998 Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem Biophys Res Commun 250:68–71[CrossRef][Medline]
  10. Satoh T, Feng P, Kim UJ, Wilber JF 1993 Identification of thyrotropin-releasing hormone receptor messenger RNA in the rat central nervous system and eye. Brain Res Mol Brain Res 19:175–178[Medline]
  11. Fukusumi S, Ogi K, Onda H, Hinuma S 1995 Distribution of thyrotropin-releasing hormone receptor mRNA in rat peripheral tissues. Regul Pept 57:115–121[CrossRef][Medline]
  12. Calza L, Giardino L, Ceccatelli S, Zanni M, Elde R, Hokfelt T 1992 Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: an in situ hybridization study. Neuroscience 51:891–909[CrossRef][Medline]
  13. Zabavnik J, Arbuthnott G, Eidne KA 1993 Distribution of thyrotrophin-releasing hormone receptor messenger RNA in rat pituitary and brain. Neuroscience 53:877–887[CrossRef][Medline]
  14. Heuer H, Schafer MK, O’Donnell D, Walker P, Bauer K 2000 Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats. J Comp Neurol 428:319–336[CrossRef][Medline]
  15. Yamada M, Saga Y, Shibusawa N, Hirato J, Murakami M, Iwasaki T, Hashimoto K, Taketo MM, Mori M 1997 Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene. Proc Natl Acad Sci USA 94:10862–10867[Abstract/Free Full Text]
  16. Shibusawa N, Yamada M, Hirato J, Monden T, Satoh T, Mori M 2000 Requirement of thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice. Mol Endocrinol 14:137–146[Abstract/Free Full Text]
  17. Yamada M, Satoh T, Mori M 2003 Mice lacking the thyrotropin-releasing hormone gene: what do they tell us? Thyroid 13:1111–1121[CrossRef][Medline]
  18. Rabeler R, Mittag J, Geffers L, Ruther U, Leitges M, Parlow AF, Visser TJ, Bauer K 2004 Generation of thyrotropin-releasing hormone receptor 1-deficient mice as an animal model of central hypothyroidism. Mol Endocrinol 18:1450–1460[Abstract/Free Full Text]
  19. Prange Jr AJ, Lara PP, Wilson IC, Alltop LB, Breese GR 1972 Effects of thyrotropin-releasing hormone in depression. Lancet 2:999–1002[Medline]
  20. Kastin AJ, Ehrensing RH, Schalch DS, Anderson MS 1972 Improvement in mental depression with decreased thyrotropin response after administration of thyrotropin-releasing hormone. Lancet 2:740–742[CrossRef][Medline]
  21. Marangell LB, George MS, Callahan AM, Ketter TA, Pazzaglia PJ, L’Herrou TA, Leverich GS, Post RM 1997 Effects of intrathecal thyrotropin-releasing hormone (protirelin) in refractory depressed patients. Arch Gen Psychiatry 54:214–222[Abstract/Free Full Text]
  22. Callahan AM, Frye MA, Marangell LB, George MS, Ketter TA, L’Herrou T, Post RM 1997 Comparative antidepressant effects of intravenous and intrathecal thyrotropin-releasing hormone: confounding effects of tolerance and implications for therapeutics. Biol Psychiatry 41:264–272[CrossRef][Medline]
  23. Ogawa N, Mizuno S, Mori A, Nukina I, Ota Z, Yamamoto M 1984 Potential anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues. Peptides 5:743–746[CrossRef][Medline]
  24. Reny-Palasse V, Rips R 1985 Potentiation by TRH of the effect of imipramine on the forced-swimming test. Br J Pharmacol 85:463–470[Medline]
  25. Reny-Palasse V, Constans M, Rips R 1989 Potentiation by TRH of the effect of antidepressants in the forced-swimming test, involvement of dopaminergic and opioid systems. Br J Pharmacol 97:197–205[Medline]
  26. Sattin A 1998 A heuristic model of mental depression derived from basic and applied research on thyrotropin-releasing hormone. Thyroid 8:957–962[Medline]
  27. Szuba MP, Amsterdam JD, Fernando 3rd AT, Gary KA, Whybrow PC, Winokur A 2005 Rapid antidepressant response after nocturnal TRH administration in patients with bipolar type I and bipolar type II major depression. J Clin Psychopharmacol 25:325–330[CrossRef][Medline]
  28. Duthie SM, Taylor PL, Eidne KA 1993 Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA. J Mol Endocrinol 11:141–149[Abstract/Free Full Text]
  29. Gatanaris GA 2001 Vectors and methods for the mutagenesis of mammalian genes, United States patent US6228639B1, May 8, 2001
  30. Rose SR 1995 Isolated central hypothyroidism in short stature. Pediatr Res 38:967–973[Medline]
  31. Collu R, Tang J, Castagne J, Lagace G, Masson N, Huot C, Deal C, Delvin E, Faccenda E, Eidne KA, Van Vliet G 1997 A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene. J Clin Endocrinol Metab 82:1561–1565[Abstract/Free Full Text]
  32. Jackson IM, Reichlin S 1974 Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates. Endocrinology 95:854–862[Abstract/Free Full Text]
  33. Winokur A, Utiger RD 1974 Thyrotropin-releasing hormone: regional distribution in rat brain. Science 185:265–267[Abstract/Free Full Text]
  34. Morley JE 1979 Extrahypothalamic thyrotropin releasing hormone (TRH): its distribution and its functions. Life Sci 25:1539–1550[CrossRef][Medline]
  35. Tsuruo Y, Ceccatelli S, Villar MJ, Hokfelt T, Visser TJ, Terenius L, Goldstein M, Brown JC, Buchan A, Walsh J 1988 Coexistence of TRH with other neuroactive substances in the rat central nervous system. J Chem Neuroanat 1:235–253[Medline]
  36. Sharif NA 1989 Quantitative autoradiography of TRH receptors in discrete brain regions of different mammalian species. Ann NY Acad Sci 553:147–175[Medline]
  37. Kubek MJ, Knoblach SM, Sharif NA, Burt DR, Buterbaugh GG, Fuson KS 1993 Thyrotropin-releasing hormone gene expression and receptors are differentially modified in limbic foci by seizures. Ann Neurol 33:70–76[CrossRef][Medline]
  38. Miyamoto M, Hirai K, Heya T, Nagaoka A 1994 Effects of a sustained release formulation of thyrotropin-releasing hormone on behavioral abnormalities in senescence-accelerated mice. Eur J Pharmacol 271:357–366[CrossRef][Medline]
  39. Sattin A, Hill TG, Meyerhoff JL, Norton JA, Kubek MJ 1987 The prolonged increase in thyrotropin-releasing hormone in rat limbic forebrain regions following electroconvulsive shock. Regul Pept 19:13–22[CrossRef][Medline]
  40. Sattin A, Senanayake SS, Pekary AE 2002 Lithium modulates expression of TRH receptors and TRH-related peptides in rat brain. Neuroscience 115:263–273[CrossRef][Medline]
  41. Schechter MD, Chance WT 1979 Non-specificity of "behavioral despair" as an animal model of depression. Eur J Pharmacol 60:139–142[CrossRef][Medline]
  42. Martin P, Brochet D, Soubrie P, Simon P 1985 Triiodothyronine-induced reversal of learned helplessness in rats. Biol Psychiatry 20:1023–1025[CrossRef][Medline]
  43. Massol J, Martin P, Brochet D, Belon JP, Puech AJ 1989 Are antidepressant-like effects of triiodothyronine (T3) in rodents related to an hyperthyroid state? Prog Neuropsychopharmacol Biol Psychiatry 13:749–764[CrossRef][Medline]
  44. Joffe RT, Sokolov ST 2000 Thyroid hormone treatment of primary unipolar depression: a review. Int J Neuropsychopharmacol 3:143–147[CrossRef][Medline]
  45. Lifschytz T, Shalom G, Lerer B, Newman ME 2006 Sex-dependent effects of fluoxetine and triiodothyronine in the forced swim test in rats. Eur Neuropsychopharmacol 16:115–121[CrossRef][Medline]
  46. Lifschytz T, Segman R, Shalom G, Lerer B, Gur E, Golzer T, Newman ME 2006 Basic mechanisms of augmentation of antidepressant effects with thyroid hormone. Curr Drug Targets 7:203–210[CrossRef][Medline]
  47. Wilcoxon JS, Nadolski GJ, Samarut J, Chassande O, Redei EE 2007 Behavioral inhibition and impaired spatial learning and memory in hypothyroid mice lacking thyroid hormone receptor alpha. Behav Brain Res 177:109–116[CrossRef][Medline]
  48. Venero C, Guadano-Ferraz A, Herrero AI, Nordstrom K, Manzano J, de Escobar GM, Bernal J, Vennstrom B 2005 Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor {alpha}1 can be ameliorated by T3 treatment. Genes Dev 19:2152–2163[Abstract/Free Full Text]
  49. Zeng H, Gragerov A, Hohmann JG, Pavlova MN, Schimpf BA, Xu H, Wu LJ, Toyoda H, Zhao MG, Rohde AD, Gragerova G, Onrust R, Bergmann JE, Zhuo M, Gaitanaris GA 2006 Neuromedin U receptor 2-deficient mice display differential responses in sensory perception, stress, and feeding. Mol Cell Biol 26:9352–9363[Abstract/Free Full Text]
  50. Swiatek PJ, Gridley T 1993 Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev 7:2071–2084[Abstract/Free Full Text]
  51. Gragerov A, Horie K, Pavlova M, Madisen L, Zeng H, Gragerova G, Rhode A, Dolka I, Roth P, Ebbert A, Moe S, Novas C, Finn E, Bergmann J, Vassilatis DK, Pavlakis GN, Gaitanaris GA 2007 Large-scale, saturating insertional mutagenesis of the mouse genome. Proc Natl Acad Sci USA 104:14406–14411[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Neurosci.Home page
R. Parmentier, S. Kolbaev, B. P. Klyuch, D. Vandael, J.-S. Lin, O. Selbach, H. L. Haas, and O. A. Sergeeva
Excitation of Histaminergic Tuberomamillary Neurons by Thyrotropin-Releasing Hormone
J. Neurosci., April 8, 2009; 29(14): 4471 - 4483.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zeng, H.
Right arrow Articles by Bergmann, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zeng, H.
Right arrow Articles by Bergmann, J. E.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
Medline Plus Health Information
*Anxiety
*Depression

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals