Environmental enrichment stimulates progenitor cell proliferation in the amygdala

Enriched environments enhance hippocampal neurogenesis, synaptic efficacy, and learning and memory functions. Recent studies have demonstrated that enriched environments can restore learning behavior and long‐term memory after significant brain atrophy and neural loss. Emotional and anxiety‐related behaviors were also improved by enriched stimuli, but the effect of enriched environments on the amygdala, one of the major emotion‐related structures in the central nervous system, remains largely unknown. In this study, we have focused on the effects of an enriched environment on cell proliferation and differentiation in the murine amygdala. The enriched environment increased bromodeoxyuridine (BrdU)‐positive (newborn) cell numbers in the amygdala, almost all of which, immediately after a 1‐week period of enrichment, expressed the oligodendrocyte progenitor marker Olig2. Furthermore, enriched stimuli significantly suppressed cell death in the amygdala. Some of the BrdU‐positive cells in mice exposed to the enriched environment, but none in animals housed in the standard environment, later differentiated into astrocytes. Our findings, taken together with previous behavioral studies, suggest that progenitor proliferation and differentiation in the amygdala may contribute to the beneficial aspects of environmental enrichment such as anxiolytic effects. © 2009 Wiley‐Liss, Inc.     

Anxiety and cognition in histamine H3 receptor-/- mice

Histamine H(3) receptors (H3Rs) were first characterized as autoreceptors modulating histamine release and synthesis via negative feedback. Acute H3R stimulation or blockade with selective agonists and antagonists suggests a role for H3R in anxiety and cognition. However, little is known about the long-term effects of H3R blockade on brain function. In the current study, mice lacking H3 receptors (H3R(-/-)) were used to investigate the role of H3R-mediated signalling in anxiety and cognition. H3R(-/-) mice showed enhanced spatial learning and memory in the Barnes maze. In addition, H3R(-/-) mice showed reduced measures of anxiety in the elevated plus and zero mazes involving exploratory behaviour and avoidable anxiety-provoking stimuli, but enhanced acoustic startle responses involving unavoidable anxiety-provoking stimuli. These behavioural alterations were associated with higher arginine vasopressin levels in the central and basolateral nuclei of the amygdala. These findings support a role for H3Rs in mediating histamine effects on spatial learning and memory and measures of anxiety.     

Emotional stability, anxiety, and natural killer activity under examination stress.

This study was performed to evaluate the relation between a stable personality trait, a mood state and immune response to an examination stress. A self-reported measure of emotional stability (BFQ-ES scale) was obtained in a sample (n = 39) randomly selected from 277 cadets; this personality trait was also investigated by completing a neuroticism scale (Eysenck personality inventory) and a trait-anxiety scale (STAI). Natural killer (NK) cell activity was measured at baseline, long before the examination time and the examination day. The state-anxiety scale evaluated the response to the stressful stimulus. Taking subjects all together, the academic task did not result in significant modification over baseline in NK cell activity. Subjects were then divided into three groups based on emotional stability and state-anxiety scores: high emotional stability/low anxiety, medium, and low emotional stability/high anxiety. Examination stress induced significant increases in NK cell activity in the high emotional stability/low anxiety group, no effect in the medium group, and significant decreases in the low emotional stability/high anxiety group. The repeated-measure ANOVA revealed a significant interaction of group x period (baseline vs. examination) for both lytic units and percent cytolysis. The results did not change after introducing coffee and smoking habits as covariates. Our findings suggest that the state-anxiety acts in concert with a stable personality trait to modulate NK response in healthy subjects exposed to a psychological naturalistic stress. The relation between anxiety and poor immune control has been already described, whereas the ability of emotional stability to associate with an immunoenhancement has not yet reported. The peculiarity of our population, a very homogeneous and healthy group for life style and habits, can have highlighted the role of emotional stability, and may account for the difference with other studies.     

Environmental enrichment upregulates micro‐RNA‐183 and alters acetylcholinesterase splice variants to reduce anxiety‐like behavior in the little Indian field mouse (Mus booduga)

Environmental enrichment (EE) has an influential role in reducing behavioral reactivity to stress. We previously observed that EE reduces the anxiety‐like behavior in the field mouse Mus booduga accompanied by a reduction in the expression of molecules involved in the stress pathway. In this study, we demonstrate the effect of different housing condition on regulation of micro‐RNA‐183‐SC35‐mediated splicing of acetylcholinesterase (AChE). Adult male M. booduga were captured from an agricultural field and housed under nonenriched standard conditions (SC) for 7 days and considered as directly from the wild (DW). On day 8, individuals were randomly assigned to three groups; DW, SC, and EE. The DW group's anxiety‐like behavior was assessed in the elevated plus maze (EPM) and open field test (OFT). The SC and EE groups were transferred to their respective conditions and housed for another 30 days. The mice housed in EE showed less anxiety‐like behavior on EPM and in OFT compared with DW and SC mice. Interestingly, miR‐183 expression was increased following exposure to EPM in EE mice but not in SC mice. Subsequently, the upregulated miR‐183 expression suppresses the SC35 expression and shifting of splicing from AChE‐S (synaptic) to AChE‐R (read‐through) form, whereas standard housing condition downregulate miR‐183 and induces the splicing of AChE. The upregulated AChE‐R form possibly terminates ACh transmission, which is reflected in the level of anxiety‐like behavior. Overall, the present study suggests that EE effectively regulates the miR‐183 pathway to reduce anxiety‐like behavior. © 2012 Wiley Periodicals, Inc.     

Deep brain stimulation affects conditioned and unconditioned anxiety in different brain areas

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) has proven to be an effective treatment for therapy refractory obsessive–compulsive disorder. Clinical observations show that anxiety symptoms decrease rapidly following DBS. As in clinical studies different regions are targeted, it is of principal interest to understand which brain area is responsible for the anxiolytic effect and whether high-frequency stimulation of different areas differentially affect unconditioned (innate) and conditioned (learned) anxiety. In this study, we examined the effect of stimulation in five brain areas in rats (NAc core and shell, bed nucleus of the stria terminalis (BNST), internal capsule (IC) and the ventral medial caudate nucleus (CAU)). The elevated plus maze was used to test the effect of stimulation on unconditioned anxiety, the Vogel conflict test for conditioned anxiety, and an activity test for general locomotor behaviour. We found different anxiolytic effects of stimulation in the five target areas. Stimulation of the CAU decreased both conditioned and unconditioned anxiety, while stimulation of the IC uniquely reduced conditioned anxiety. Remarkably, neither the accumbens nor the BNST stimulation affected conditioned or unconditioned anxiety. Locomotor activity increased with NAc core stimulation but decreased with the BNST. These findings suggest that (1) DBS may have a differential effect on unconditioned and conditioned anxiety depending on the stimulation area, and that (2) stimulation of the IC exclusively reduces conditioned anxiety. This suggests that the anxiolytic effects of DBS seen in OCD patients may not be induced by stimulation of the NAc, but rather by the IC.     

Effects of stress and corticosterone on activity and plasticity in the amygdala

The basolateral amygdala (BLA) has been repeatedly shown to mediate the effects of stress on memory-related processes. However, the way in which stress influences BLA itself has not been fully explored. We studied the effects of stress and corticosterone (CORT) on activity and plasticity in the BLA in the rat, using the electrophysiological procedure of long-term potentiation (LTP) induction in vivo. Rats were exposed to an acute elevated-platform stress or administered vehicle or 5 mg/kg, 10 mg/kg, or 25 mg/kg of CORT systemically, after which they were anesthetized and prepared for field potential recording in the BLA, in response to stimulation of the entorhinal cortex. The elevated platform stress enhanced baseline responses in BLA and plasma CORT but inhibited amygdalar LTP. Systemic injections of CORT enhanced baseline responses in BLA in a dose-dependent manner but did not influence amygdalar LTP. Posttetanic potentiation (PTP) was similarly reduced in CORT- and vehicle-injected groups, possibly because of an additional stress from the injection, thus implying that PTP and LTP in the amygdala differentially react to stress. These results suggest that the increase in amygdalar baseline activity following the exposure to stress may be mediated by the concomitant increase in plasma CORT. However, the suppression of amygdalar LTP is not a result of elevated levels of CORT, suggesting that activity and plasticity in the amygdala might be mediated by different mechanisms.     

Antidepressive and anxiolytic activity of selective estrogen receptor modulators in ovariectomized mice submitted to chronic unpredictable stress

Estradiol has antidepressive and anxiolytic actions. However, its therapeutic use is limited by its peripheral effects. Selective estrogen receptor modulators may represent an alternative to estradiol for the treatment of depressive symptoms. Here we report that tamoxifen and raloxifene decrease immobility time in the forced swim test and increases the time spent in open arms in the elevated plus maze in ovariectomized mice submitted to chronic unpredictable stress.     

Anxiety and stress responses in female oxytocin deficient mice

Oxytocin is believed to attenuate the response of the hypothalamic-pituitary-adrenal axis to stress and to be anxiolytic. Stressors with a psychological component evoke both central and peripheral secretion of oxytocin in laboratory rodents. Oxytocin gene deletion mice provide a novel way to understand the role of oxytocin in stress and anxiety-related behaviours. We present our experience with female oxytocin deficient mice that were tested in an elevated plus maze (EPM), a behavioural test of anxiety, or exposed to psychogenic stressors (platform shaker or novel environment). Oxytocin-deficient mice not only displayed more anxiety-related behaviour, but also released more corticosterone after a psychogenic stressor and manifested greater stress-induced hyperthermia compared to wild-type mice. The diurnal variation of corticosterone and the response of corticosterone to corticotropin-releasing factor were not significantly different between genotypes. We also measured Fos-immunoreactive protein, an index of neuronal activation, in the medial amygdala of female mice after EPM testing. The medial amygdala is important for processing of psychogenic stress and anxiety and also contains oxytocin pathways and oxytocin receptors. The expression of Fos in the medial amygdala of mice not exposed to the EPM was not different between genotypes. Following EPM exposure, Fos expression was greater in oxytocin null compared to wild-type mice. Our findings support the hypothesis that central oxytocin is anxiolytic, and attenuates the stress response to psychogenic provocation in female mice.     

Mercurius solubilis attenuates scopolamine-induced memory deficits and enhances the motor coordination in mice

Aim: The present study was designed to investigate the effect of mercurius solubilis (merc sol) on scopolamine induced memory deficits and motor coordination in mice.

Materials and methods: Three different formulations of merc sol (30X, 200M, 1M) were screened for their in vitro antioxidant potential through DPPH (2, 2-diphenyl-1-picrylhydrazyl) and nitric oxide scavenging activity using response surface methodology. Memory impairment was induced by the administration of scopolamine (1mg/kg i.p.) for 3 days to mice and assessment of memory acquisition and retention was done using Morris water maze test, passive avoidance test, elevated plus maze test, light and dark box test, motor coordination was evaluated using rotarod test and inclined plan test. The involvement of ion channels and nitric oxide pathway in the observed effect of merc sol was elucidated by administration of veratrine (0.125 μg/kg, i.p.), A23187 (20 μg/kg, i.p.), L- arginine (40 mg/kg, i.p.), aminoguanidine (50 mg/kg, i.p.) 30 min prior to merc sol. Acute toxicity studies were performed in accordance with the OECD (Organisation for Economic Co-operation and Development) guidelines.

Results: In vitro studies have revealed merc sol 30 X to have maximum free radical and nitric oxide scavenging activity. Administration of merc sol 30 X to mice significantly reduced scopolamine induced memory deficits and motor incoordination in all the performance tasks. The calcium ionophore, A23187 significantly altered the effect of merc sol in mice. No major signs of toxicity were observed.

Conclusion: Merc sol has antiamnesic effect in scopolamine induced deficits and motor coordination in mice.     

Behavioural assays to model cognitive and affective dimensions of depression and anxiety in rats

Animal models have been used extensively to investigate neuropsychiatric disorders, such as depression, and their treatment. However, the aetiology and pathophysiology of many such disorders are largely unknown, which makes validation of animal models particularly challenging. Furthermore, many diagnostic symptoms are difficult to define, operationalize and quantify, especially in experimental animals such as rats. Thus, rather than attempting to model complex human syndromes such as depression in their entirety, it can be more productive to define and model components of the illness that may account for clusters of co-varying symptoms, and that may share common underlying neurobiological mechanisms. In preclinical investigations of the neural regulatory mechanisms linking stress to depression and anxiety disorders, as well as the mechanisms by which chronic treatment with antidepressant drugs may exert their beneficial effects in these conditions, we have employed a number of behavioural tests in rats to model specific cognitive and anxiety-like components of depression and anxiety disorders. In the present study, we review the procedures for conducting four such behavioural assays: the attentional set-shifting test, the elevated-plus maze, the social interaction test and the shock-probe defensive burying test. The purpose is to serve as a guide to the utility and limitations of these tools, and as an aid in optimising their use and productivity.     

Effects of cyclic adenosine monophosphate response element binding protein overexpression in the basolateral amygdala on behavioral models of depression and anxiety.

BACKGROUND: Chronic antidepressant administration increases the cyclic adenosine monophosphate response element binding protein (CREB) in the amygdala, a critical neural substrate involved in the physiologic responses to stress, fear, and anxiety. METHODS: To determine the role of CREB in the amygdala in animal models of depression and anxiety, a viral gene transfer approach was used to selectively express CREB in this region of the rat brain. RESULTS: In the learned helplessness model of depression, induction of CREB in the basolateral amygdala after training decreased the number of escape failures, an antidepressant response. However, expression of CREB before training increased escape failures, and increased immobility in the forced swim test, depressive effects. Expression of CREB in the basolateral amygdala also increased behavioral measures of anxiety in both the open field test and the elevated plus maze, and enhanced cued fear conditioning. CONCLUSIONS: Taken together, these data demonstrate that CREB expression in the basolateral amygdala influences behavior in models of depression, anxiety, and fear. Moreover, in the basolateral amygdala, the temporal expression of CREB in relation to learned helplessness training, determines the qualitative outcome in this animal model of depression.     

Reduced anxiety‐like behavior and central neurochemical change in germ‐free mice

Background There is increasing interest in the gut‐brain axis and the role intestinal microbiota may play in communication between these two systems. Acquisition of intestinal microbiota in the immediate postnatal period has a defining impact on the development and function of the gastrointestinal, immune, neuroendocrine and metabolic systems. For example, the presence of gut microbiota regulates the set point for hypothalamic‐pituitary‐adrenal (HPA) axis activity. Methods We investigated basal behavior of adult germ‐free (GF), Swiss Webster female mice in the elevated plus maze (EPM) and compared this to conventionally reared specific pathogen free (SPF) mice. Additionally, we measured brain mRNA expression of genes implicated in anxiety and stress‐reactivity. Key Results Germ‐free mice, compared to SPF mice, exhibited basal behavior in the EPM that can be interpreted as anxiolytic. Altered GF behavior was accompanied by a decrease in the N‐methyl‐D‐aspartate receptor subunit NR2B mRNA expression in the central amygdala, increased brain‐derived neurotrophic factor expression and decreased serotonin receptor 1A (5HT1A) expression in the dentate granule layer of the hippocampus. Conclusions & Inferences We conclude that the presence or absence of conventional intestinal microbiota influences the development of behavior, and is accompanied by neurochemical changes in the brain.     

Environmental enrichment enhances sensitization to GBR 12935-induced activity and decreases dopamine transporter function in the medial prefrontal cortex

Rats raised in an enriched condition (EC) during development display increased hyperactivity to the effect of acute amphetamine compared to rats raised in an impoverished condition (IC). The present study determined whether environmental enrichment differentially alters the effects of GBR 12935 administration, a selective dopamine transporter (DAT) inhibitor. Acutely, EC rats showed a greater, dose-dependent GBR 12935-induced increase in activity compared to IC rats; however, basal activity for EC rats was lower than for IC rats. After repeated GBR 12935, only EC rats exhibited behavioral sensitization. Kinetic analysis of DAT function in medial prefrontal cortex (mPFC) revealed that the maximal velocity of [3H]dopamine ([3H]DA) uptake in EC rats was less than in IC rats (4.9 +/- 0.6 and 7.7 +/- 0.6 pmol/min/mg, respectively), but not in striatum or nucleus accumbens. Furthermore, GBR 12935-induced inhibition of DAT function, [3H]GBR 12935 binding density and DA content in mPFC, striatum and nucleus accumbens were not different between EC and IC rats. However, dihydroxyphenylacetic acid content in mPFC was lower in EC than IC rats, whereas no differences were found in striatum and nucleus accumbens. These results suggest that EC-induced changes in activity may be due to decreased DAT function and decreased DA metabolism in the mPFC.     

Environmental enrichment during adolescence reverses the effects of prenatal stress on play behaviour and HPA axis reactivity in rats

Prenatal stress (PS) can produce profound and long-lasting perturbations of individual adaptive capacities, which in turn can result in an increased proneness to behavioural disorders. Indeed, in PS rats there is evidence of impaired social play behaviour, disturbances in a variety of circadian rhythms, enhanced anxiety and increased hypothalamic-pituitary-adrenal (HPA) axis reactivity. This study was designed to experimentally investigate the degree of reversibility of PS-induced disturbances of social play and HPA reactivity by assessing the effect of the enrichment of the physical environment on PS rats during periadolescence. PS subjects showed a reduced expression of social play behaviour and a prolonged corticosterone secretion in response to restraint stress, but both these effects were markedly reversed following environmental enrichment. Interestingly, the enrichment procedure increased social behaviour but had no effect on corticosterone secretion in nonstressed animals, indicating a differential impact of the postnatal environment as a function of prenatal background. As a whole, results clearly indicate that rats prenatally exposed to stress can benefit during periadolescence from the modulatory effects of an enriched environment. Moreover, they confirm that PS may well represent a suitable animal model for the design and testing of new therapeutic strategies for behavioural disorders produced by early insults.     

The development of behavioral and endocrine abnormalities in rats after repeated exposure to direct and indirect stress

The present study compared the effects of direct and indirect stress on the behavior and hypothalamic-pituitary-adrenal axis of rats. Animals were placed in a two compartment box. In one compartment the direct stressed rat was subjected to electric foot shocks randomly applied for 10 minutes (0.5 mA of 1 s duration). In the adjacent compartment, the indirect stressed rats witnessed the application of these electric foot shocks. Our data showed substantial behavioral changes in the open field test, but limited effects in the elevated plus maze. The findings suggested that single and repeated stress exposure may have different consequences, that the effects of stress exposure may develop over time and persist for an extended period, and that both direct and indirect stressed rats displayed a hyposensitive HPA axis following acute restraint stress. Overall our observations moderately indicate direct exposure to elicit behavioral changes, and both direct and indirect exposure to stress to result in aberrations within the neuroendocrine system. With additional development our stress models may be considered for studying the complex interrelationship between an external stressor, and the experience of the organism.     

Alzheimer caregiver stress: Basal natural killer cell activity, pituitary-adrenal cortical function, and sympathetic tone

The association between Alzheimer caregiving and natural killer (NK) cell activity and basal plasma levels of adrenocorticotropic hormone (ACTH), cortisol, β-endorphin, prolactin, epinephrine, norepinephrine, and neuropeptide Y was determined in 100 spousal Alzheimer caregivers and 33 age-and gender-comparable control volunteers upon intake into a study of the psychological and physiologic impact of caregiving. The relationship between these physiologic measures and individual characteristics such as age, gender, medical status, severity of stress, severity of depressive symptoms, and caregiver burden was tested. In addition, the association between NK activity and alterations of the neuroendocrine measures was investigated. As compared to controls, the Alzheimer caregivers had similar levels of NK activity and of basal plasma neuroendocrine hormones and sympathetic measures. While older age and male gender status were associated with increased levels of ACTH, neither medical caseness, severity of life stress, nor severity of depressive symptoms was associated with alterations in any of the multiple physiologic domains. Classification of Alzheimer caregiver burden identified caregivers who were mismatched in terms of the amount of care they were required to provide and the amount of respite time received. The mismatched caregivers had significantly higher basal plasma ACTH but no change in other physiological measures, as compared to non-mismatched caregivers. NK activity was negatively correlated with plasma levels of neuropeptide Y but not with any of the other neuroendocrine measures. Based on this cross-sectional evaluation of NK activity and neuroendocrine and sympathetic measures, we conclude that most Alzheimer caregivers do not show evidence of altered basal physiology.     

Modulation of natural killer cell activity by restraint stress during an influenza A/PR8 infection in mice

These experiments were designed to examine the influences of restraint stress (RST) on natural killer (NK) activity and to determine its consequences on influenza A/PR8 (A/PR8) viral replication in mice. The data showed that RST delayed the recruitment of NK1.1+ cells into the lung parenchyma during infection. Quantification of MIP-1alpha and MCP-1 gene expression by real-time PCR revealed that RST suppressed the chemokines responsible for NK cell recruitment into the infected tissue. Additionally, RST suppressed the expression of several macrophage-derived cytokines involved in the effector response of NK cells. IL-15, which is the main cytokine involved in NK cell development and homeostasis, and IL-12, which is important for NK cytotoxicity, were both suppressed. As the NK cell response is an important innate response to control viral replication, we hypothesized that the RST-mediated reduction in NK cell numbers and function would enable viral replication to continue unchecked. In fact, there was enhanced viral replication in the lungs of RST animals. Interestingly, expression of the anti-viral type I interferons (IFN-alpha and IFN-beta) was elevated presumably in response to the elevated viral load in the stressed mice. Together, these data show that RST suppressed expression of the cytokine genes involved in the recruitment and activation of NK cells during an experimental influenza viral infections. The consequence of this effect was diminished NK cell function and enhanced viral replication.     

Coping in an intermittent swim stress paradigm compromises natural killer cell activity in rats

The effects of intermittent swim stress and stressor controllability on natural killer cell activity (NKCA) was examined. Significant decreases in splenic NKCA were observed immediately post-stress, but only when the stress was controllable. Although decreased NKCA was also observed in yoked rats subjected to the same stressor, it failed to attain statistical significance. Previous results suggest these effects are not due to corticosterone. The results suggest a cost of coping on the acute, in vitro immune measure of NKCA.     

Differential effect of prenatal stress on the expression of corticotrophin-releasing hormone and its receptors in the hypothalamus and amygdala in male and female rats

The present study examined the effect of prenatal stress in rats from days 13-20 of gestation on anxiogenic behaviour in the elevated plus maze (EPM) together with changes in the gene expression of corticotrophin-releasing hormone (CRH), its receptors, CRHR1 and CRHR2, as well as CRH binding protein (CRH-BP) in the paraventricular nucleus (PVN) and amygdala of their male and female offspring. Both prenatally-stressed (PS) males and females showed heightened anxiety in the EPM. Prenatal stress did not alter the gene expression of CRH or its receptors in the male PVN, although it decreased CRH-BP mRNA, which could augment the activity of free CRH. In the PVN of PS females, there was an increase in the expression of CRH, coupled with a decrease in that of CRHR2 and CRH-BP. These changes are compatible with the greater activation of the hypothalamic pituitary adrenal axis to stress in females. Anxiogenic behaviour of PS rats was associated with a reduction of CRHR2 mRNA and of CRH-BP mRNA in the amygdala of males and an increase in CRH mRNA and decrease in CRHR2 mRNA in females. Two hours after acute stress of exposure to the elevated plus maze in which heightened anxiety was manifested, increases were seen only in the amygdala of females in CRH and CRHR1 signalling, whereas CRHR2 mRNA was reduced in both sexes. The data show that both prenatal stress and acute stress in adulthood have a differential sex-dependent effect on the expression of CRH its receptors and binding protein in the PVN and amygdala of rats.     

Alterations in behavior, steroid hormones and natural killer cell activity in male transgenic TGF alpha mice.

The expression of transforming growth factor alpha (TGF alpha) is widely distributed throughout many normal and neoplastic tissues, but its physiological significance remains unclear. We have utilized male transgenic mice overexpressing the gene encoding human TGF alpha in multiple tissues to further identify those functions which are influenced by this protein. Male TGF alpha mice develop hepatocellular carcinoma at the age of 10-15 months. At the age of 2-3 months these mice, compared to age matched CD-1 controls, spent significantly longer times immobile in Porsolt's swim test, a model of stress and depressive behavior, and exhibiting aggressive behavior in the resident-intruder test. In contrast, the transgenic TGF alpha mice did not differ from the controls in either the plusmaze test of anxiety, or in their voluntary alcohol intake. Significantly, the TGF alpha mice exhibited a 25% lower Natural Killer (NK) cell activity and a four-fold increase in the plasma levels of 17-beta-estradiol (E2) than the controls. No significant changes in plasma testosterone or corticosterone levels were noted. The results indicate that transgenic male mice overexpressing TGF alpha exhibit behaviors characteristic of both an impaired ability to cope with stress and an increased aggressivity. The TGF alpha mice also show reduced NK cell activity and increased plasma estradiol concentrations. The present data suggest that TGF alpha may be important in influencing behavioral, immunological and hormonal systems prior to the onset of tumors. It remains to be determined whether hepatocarcinoma is associated with the direct proliferative and transforming effects of TGF alpha and/or indirect effects mediated through immune, hormonal and behavioral mechanisms.