J.J. Mulchahey

Effects of trauma-related audiovisual stimulation on cerebrospinal fluid norepinephrine and corticotropin-releasing hormone concentrations in post-traumatic stress disorder

BACKGROUND Although elevated concentrations of both corticotropin-releasing hormone (CRH) and norepinephrine are present in the cerebrospinal fluid (CSF) of patients with post-traumatic stress disorder (PTSD), the effects of exposure to traumatic stimuli on these stress-related hormones in CSF are unknown. METHODS A randomized, within-subject, controlled, cross-over design was used, in which patients with war-related PTSD underwent 6-h continuous lumbar CSF withdrawal on two occasions per patient (6-9 weeks apart). During one session the patients watched a 1-h film containing combat footage (traumatic film) and in the other a 1-h film on how to oil paint (neutral film). At 10-min intervals, we quantified CRH and norepinephrine in CSF, and ACTH and cortisol in plasma, before, during, and after symptom provocation. Subjective anxiety and mood were monitored using 100-mm visual analog scales. Blood pressure and heart rate were obtained every 10min from a left leg monitor. RESULTS Eight of 10 patients completed two CSF withdrawal procedures each. A major drop in mood and increases in anxiety and blood pressure occurred during the traumatic relative to the neutral videotape. CSF norepinephrine rose during the traumatic film relative to the neutral videotape; this rise directly correlated with magnitude of mood drop. In contrast, CSF CRH concentrations declined during the trauma-related audiovisual stimulus, both absolutely and relative to the neutral stimulus; the magnitude of CRH decline correlated with degree of subjective worsening of anxiety level and mood. Plasma cortisol concentrations were lower and ACTH levels similar during the stress compared with the neutral videotape. CONCLUSIONS CSF concentrations of the stress hormones norepinephrine and CRH differentially change after exposure to 1h of trauma-related audiovisual stimulation in chronic, combat-related PTSD. While the CSF norepinephrine increase was postulated, the decline in CSF CRH levels is surprising and could be due to audiovisual stress-induced increased uptake of CSF CRH into brain tissue, increased CRH utilization, increased CRH degradation, or to an acute stress-related inhibition or suppression of CRH secretion.

Corticotropin‐Releasing Hormone (CRH) Expression and Protein Kinase A Mediated CRH Receptor Signalling in an Immortalized Hypothalamic Cell Line

Corticotropin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH‐expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus‐mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH‐like immunoreactivity (CRH‐LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase‐polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule‐associated protein‐2. We verified that the CRH‐LI from IVB cell lysates coeluted with CRH standard via reversed‐phase high‐performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration‐dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho‐cyclic‐AMP response element binding protein (CREB) imunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB‐mediated gene expression as determined via the PathDetect CREB trans‐reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH‐R1‐mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Cerebrospinal fluid and plasma testosterone levels in post-traumatic stress disorder and tobacco dependence

BACKGROUND Little is known about the relationship between endogenous central nervous system (CNS) testosterone and any psychiatric syndrome. The goal of this study was to screen for potential abnormalities in CNS testosterone levels in patients with post-traumatic stress disorder (PTSD) and/or tobacco dependence. METHODS We sampled cerebrospinal fluid (CSF) via a subarachnoid catheter over six hours and determined hourly basal CSF concentrations of testosterone in 11 combat veterans with PTSD and 12 normal volunteers. Smokers were abstinent for 11-17 h. Testosterone in CSF and matching plasma samples was assayed by radioimmunoassay. RESULTS A factor analysis for effects of PTSD status, smoking status and sample time revealed significant effects of PTSD or smoking status, but not time, on CSF testosterone. CSF testosterone levels were lower in individuals with PTSD as compared with normal volunteers. When divided by smoking status, abstinent smokers had mean CSF testosterone levels higher than those of non-smokers. A similar analysis of plasma testosterone revealed no significant effects of any factor on plasma testosterone. CONCLUSIONS These results indicate that CSF testosterone is significantly influenced by PTSD and smoking status. The exposure of the brain to altered levels of testosterone in smokers and patients with PTSD may have pathophysiologic significance in these conditions.

Age-related alterations in emotional behaviors and amygdalar corticotropin-releasing factor (CRF) and CRF-binding protein expression in aged Fischer 344 rats.

Corticotropin-releasing factor (CRF) coordinates the mammalian response to stress. In the amygdala, the CRF system appears to be responsible, at least in part, for the behavioral responses resulting from stress. Associated with amygdalar CRF is a 37 kDa binding protein (CRF-BP) which may also play a role in regulating stressful stimuli. Aging has been shown to be associated with abnormal neuroendocrine stress systems and little is known with regards to how amygdalar stress systems change with aging. In our study, we have assessed levels of amygdalar CRF and CRF-BP mRNA in Fischer 344 rats of 4, 12 or 24 months of age following 14 days of hourly restraint. Prior to sacrifice, rats were also tested for anxiety-like behaviors on the elevated plus maze. After behavioral testing, rats were perfused with 4% paraformaldehyde and the brains were processed for in situ hybridization. Twenty micron sections were hybridized with a CRF as well as a CRF-BP riboprobe. Following hybridization, tissue sections were oppossed to X-ray film and relative amounts of mRNA in the amygdala were quantitated. Levels of CRF mRNA in the amygdala of 12 and 24 month-old rats following chronic restraint were significantly lower relative to rats which were handled for 14 days. There were no significant differences in amygdalar CRF gene expression between stressed and handled 4 month-old rats. At 12 and 24 months of age but not 4 months, there were also significant effects of restraint associated with decreases in amygdalar CRF-BP gene expression. Furthermore, there were reciprocal decreases in anxiety-like behaviors in the 12 and 24 month-old rats which were significant; the changes in anxiety-like behaviors between restrained vs. handled 4 month-old rats were not significantly different. The decreased gene expression of CRF in the amygdala in concert with decreased anxiety-like behaviors following restraint is consistent with the known behavioral effects of exogenously applied intra-amygdalar CRF. The changes in amygdalar CRF-BP observed may be secondary to the known regulatory effects that CRF exhibits on its binding-protein. These studies have relevance to better understanding the molecular basis of aging related changes in neuroendocrine stress systems.

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

RETRACTED: In vitro regulation of corticotropin-releasing hormone

Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.

Regulation of Corticotropin-Releasing Factor-Binding Protein Expression in Amygdalar Neuronal Cultures

Corticotropin‐releasing factor‐binding protein (CRF‐BP) is known to regulate the bioavailability of CRF and may also play a role in stress behaviours. CRF‐BP has been localized in the pituitary as well as central nervous system (CNS) limbic and cortical areas, including the amygdala. The signal transduction pathways which regulate amygdalar CRF‐BP are not well understood. In this report, we have examined the effect of protein kinase A and C activators, CRF, dexamethasone and interleukin‐6 (IL6) on CRF‐BP mRNA and protein expression in dissociated fetal amygdalar cultures. CRF‐BP mRNA levels were determined by Northern analysis following 12 h treatment with the following agents: forskolin (1–30 μM), CRF (1–1000 nM), phorbol‐12‐myristate‐13‐acetate (TPA; 1–50 nM), dexamethasone (1–100 nM) and IL6 (10–500 pM). Significant increases in CRF‐BP mRNA were observed in response to forskolin (30 mM), CRF (100, 1000 nM), IL6 (100, 500 pM), TPA (50 nM) and dexamethasone (100 nM; P<0.05 for all; n=3–6 for all). We extended our observations of CRF‐BP expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with the same agents. Twenty‐four hour treatment with 30 μM forskolin, 1000 nM CRF, 50 nM TPA, 100 pM IL6 or 100 nM dexamethasone significantly increased CRF‐BP expression (P<0.05, n=3 for each treatment). The primary cultures were then transfected with a rat CRF‐BP‐reporter construct containing 3500 base pairs of CRF‐BP 5′ flanking DNA. Treatment with all five agents produced statistically significant increases above control (P<0.05; n=3 for each). The results suggest that CRF‐BP in the amygdala is stimulated by numerous pathways which may play a significant role in promoting behavioural changes.

Regulation of corticotropin-releasing factor messenger RNA by nicotine in an immortalized amygdalar cell line.

Preliminary data suggest that amygdalar corticotropin-releasing factor (CRF) is regulated by nicotinic agonists. We sought to confirm and extend these observations by determining the effects of various concentrations of nicotine on CRF messenger RNA expression in the AR-5 immortalized amygdalar cell line. Nicotine produced concentration- and time-dependent increases in CRF mRNA. This cell line thus confirms that nicotinic agonists stimulate amygdalar CRF and appears to be a useful model for studying molecular factors important in this interaction.

Regulation of corticotropin-releasing hormone in vitro

Studies examining regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Most in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Furthermore, clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. CRH levels in the amygdala in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated the signal transduction mechanisms responsible for CRH expression. These mechanistic studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Clearly much more investigative work in better understanding CRH regulation in vitro is needed.

The effect of feeding on cerebrospinal fluid corticotropin-releasing hormone levels in humans.

Corticotropin-releasing hormone (CRH) is a neuropeptide thought to play a role in appetite regulation. In this report, we used a serial cerebrospinal fluid (CSF) sampling technique to examine the relationship between CSF CRH, plasma ACTH and cortisol and perceptions of hunger and satiety in fasting and sated volunteers. CSF was withdrawn continuously from 11:00 AM to 5:00 PM via an indwelling subarachnoid catheter. Blood was withdrawn every 10 min via an antecubital vein catheter. Fed subjects received a meal at 1:00 PM. Subjects who were fed had lower post-prandial ratings on hunger scales and higher ratings on satiety scales. Fed subjects also had slightly lower levels of CSF CRH after feeding. Furthermore, fed subjects had higher ACTH and cortisol concentrations in the first 3 h; by the fourth h the opposite was true. Our findings do not support the hypothesis that CNS CRH is a central satiety factor in the human. Instead our findings of slightly diminished CSF CRH levels after feeding may be accounted for by the rises in glucocorticoids and their associated negative feedback effects on CNS CRH. Alternatively, our findings could also reflect changes in CRH levels associated with feeding in multiple brain areas and in the spinal cord with the net effect being in the negative direction.