Effects of Wenyang Zhenshuai Granules on the Expression of Key Mitochondrial Autophagy Proteins in the Doxorubicin-Induced Model of H9c2 Cardiomyocyte Injury.

This study aimed to explore the effects of Wenyang Zhenshuai granules (WZG) on the morphology of cardiomyocytes, cell viability, and the expression of key mitochondrial autophagy proteins in the doxorubicin-induced model of H9c2 cardiomyocyte injury. Cardiomyocytes were cultured for 44 h and divided into 4 groups: intact control, doxorubicin-injured cells (DOX), doxorubicin-injured cells treated with WZG (DOX+WZG), and doxorubicin-injured cells treated with valsartan (DOX+valsartan; reference group). The morphology of cardiomyocytes was analyzed under an inverted microscope; cardiomyocyte survival rate was determined by MTT assay. The expression of the key mitochondrial autophagy proteins (PINK1, parkin, LC3-II, and prohibitin-2) was analyzed by Western blotting. WZG down-regulated the expression of the key mitochondrial autophagy proteins in DOX-injured cells, which may be one of the important mechanisms for regulating ventricular remodeling and cardiomyocyte apoptosis.

Expression of the G72/G30 gene in transgenic mice induces behavioral changes.

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially expressed in transgenic mice compared with wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders.

Application of the ERK signaling pathway inhibitor PD98059 in long-term in vivo experiments.

The aim of this study was to explore methods by which the ERK signaling pathway inhibitor PD98059 (PD) could be used in long-term in vivo experiments. Forty healthy New Zealand rabbits were randomly divided into blank control, model control, PD low-dose, PD high-dose, PD blank, dimethyl sulfoxide (DMSO) control, DMSO blank, and positive control groups. The corresponding treatments were administered to each experimental group over the course of four weeks, after which, total ERK1/2 and ERK5 protein levels, protein phosphorylation, and gene expression were measured in myocardial tissues. Treatment of rabbits with Adriamycin (doxorubicin) resulted in the significant overall differences in ERK1/2 and ERK5 phosphorylation (P < 0.05). Compared with the model control group, changes in phosphorylated ERK1/2 and phosphorylated ERK5 were lowest in the PD high-dose group (P < 0.05). No significant differences in total protein and mRNA levels of myocardial ERK1/2 and ERK5 were detected between the groups after four weeks (P > 0.05). Continuous intravenous injection of PD98059 significantly reduced phosphorylation of ERK1/2 and that of ERK5. In conclusion, Adriamycin-induced myocardiopathy and abnormal ERK signaling might constitute a valuable model foruse in long-term experiments. These methods may provide a theoretical basis for related in vivo studies of long duration.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls.

BACKGROUND AND PURPOSE: Huntington's disease is due to a CAG triplet repeat elongation in the huntingtin gene. Boundaries in CAG numbers have been found between healthy people with and without risk to pass the disorder to the next generation, and between people without, with a mild, or with a fully penetrant phenotype. These data have been generated in western populations and it is not clear whether they are also valid amongst Chinese. METHODS: In order to establish normative data in the huntingtin gene for Chinese people, 966 chromosomes from normal controls were tested. Further, the range of CAG repeats was examined in a cohort from six centres and a total of 368 patients with the disease were included. RESULTS: The CAG triplet repeat range in normal controls was between 9 and 35 (mean 18.9, SD 2.57). Triplets in the range between 26 and 35 were found in 2.5%. In the patient cohort, triplet repeats in the shorter allele were between 8 and 37 (mean 17.7, SD 1.6). In the longer allele, a range between 36 and 120 was found. There was a negative correlation (-0.65, r = 0.42) between age at onset and the number of triplet repeats in the larger allele. The mean age at onset was 38 years, with a range between 2 and 70 years. In 23 patients (6%) a childhood or juvenile onset was noted. CONCLUSION: These data show comparable ranges of huntingtin gene CAG triplet repeats in normal people and in patients with Huntington's disease as in western populations.

NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation.

The hippocampal CA1 region is crucial for converting new memories into long-term memories, a process believed to continue for week(s) after initial learning. By developing an inducible, reversible, and CA1-specific knockout technique, we could switch N-methyl-D-aspartate (NMDA) receptor function off or on in CA1 during the consolidation period. Our data indicate that memory consolidation depends on the reactivation of the NMDA receptor, possibly to reinforce site-specific synaptic modifications to consolidate memory traces. Such a synaptic reinforcement process may also serve as a cellular means by which the new memory is transferred from the hippocampus to the cortex for permanent storage.

Prognostic values of STAT3 and HIF-1α in esophageal squamous cell carcinoma.

OBJECTIVE: To investigate the expressions of signal transduction and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1-alpha (HIF-1α) in esophageal squamous cell carcinoma (ESCC), and their potential roles in the pathology of ESCC. PATIENTS AND METHODS: Tumor tissues and clinical data of 202 ESCC patients treated in our hospital from January 2011 to June 2013 were collected. Expressions of STAT3 and HIF-1α in tumor tissues and normal esophageal tissues were detected by immunohistochemical S-P method. Correlation of STAT3 and HIF-1α with clinicopathological parameters and prognosis of ESCC were analyzed. RESULTS: STAT3 was positively expressed in 82/202 ESCC tissues, with a positive expression rate of 40.59%, and HIF-1α was positively expressed in 142/202 ESCC tissues, with a positive expression rate of 70.30%. Both STAT3 and HIF-1α were highly expressed in ESCC tissues than those normal esophageal tissues, showing statistically significant differences (p<0.05). The expression of STAT3 was positively correlated with that of HIF-1α in ESCC tissues (r=0.401, p<0.05). Overall survival (OS) and disease-free survival (DFS) of ESCC patients with positive STAT3 and HIF-1α expression were markedly worse than those with negative expression (p<0.05). STAT3 and HIF-1α were related to the infiltration depth (T stage) of ESCC (p<0.05). Univariate and multivariate analyses revealed that the expression of STAT3 was associated with OS and DFS (p<0.05) and was an independent prognostic factor for ESCC. CONCLUSIONS: High expressions of STAT3 and HIF-1α are closely related to ESCC. STAT3 is an independent prognostic risk factor for ESCC, and HIF-1α may be a poor prognostic survival factor for ESCC, both of which can be used as indicators to predict the prognosis of ESCC patients.

Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces.

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.

Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice.

We produced CA1-specific NMDA receptor 1 subunit-knockout (CA1-KO) mice to determine the NMDA receptor dependence of nonspatial memory formation and of experience-induced structural plasticity in the CA1 region. CA1-KO mice were profoundly impaired in object recognition, olfactory discrimination and contextual fear memories. Surprisingly, these deficits could be rescued by enriching experience. Using stereological electron microscopy, we found that enrichment induced an increase of the synapse density in the CA1 region in knockouts as well as control littermates. Therefore, our data indicate that CA1 NMDA receptor activity is critical in hippocampus-dependent nonspatial memory, but is not essential for experience-induced synaptic structural changes.

Photoperiod modulates pubertal shifts in behavioral responsiveness to testosterone.

This study examined the effect of photoperiod on pubertal maturation of steroid-dependent reproductive behaviors in male European ferrets (Mustela putorius furo). In the first experiment, levels of neck gripping, mounting, and pelvic thrusting in gonadally intact prepubertal (PRE) ferrets were compared with those of adults that had undergone puberty either while housed in short days (8 hr light/16 hr darkness per day; SD), or after transfer from SD to long days (18 hr light/6 hr darkness per day; LD) at 12 weeks of age. Both LD and SD adults demonstrated significantly greater amounts of neck gripping and mounting than PRE males. In addition, a significantly greater proportion of adults in both SD and LD displayed at least one incidence of the three behaviors compared to PRE ferrets. There were no statistically significant differences in behavior of the gonadally intact LD and SD adults. In the second experiment, dose-response curves for behavioral responses to subcutaneous injections of 0, 0.5, 1.25, 2.5, 5, and 10 mg/kg testosterone propionate (TP) in oil were generated in castrated PRE, SD, and LD males. The lowest dose of TP elicited significantly greater amounts of all three behaviors in LD adults than in PRE ferrets. In addition, levels of mounting and thrusting elicited by the lowest dose of TP were significantly greater in LD adults than in SD adults. These data indicate that pubertal activation of male sexual behavior in male ferrets is accompanied by a pubertal increase in responsiveness to the behavioral effects of testosterone. Furthermore, the degree of behavioral responsiveness of adult ferrets to testosterone is modulated by environmental photoperiod experienced during reproductive maturation.

Involvement of Endogenous Brain-Derived Neurotrophic Factor in Hypothalamic-Pituitary-Adrenal Axis Activity.

Brain-derived neurotrophic factor (BDNF) appears to be highly involved in hypothalamic-pituitary-adrenal (HPA) axis regulation during adulthood, playing an important role in homeostasis maintenance. The present study aimed to determine the involvement of BDNF in HPA axis activity under basal and stress conditions via partial inhibition of this endogenous neurotrophin. Experiments were conducted in rats and mice with two complementary approaches: (i) BDNF knockdown with stereotaxic delivery of BDNF-specific small interfering RNA (siRNA) into the lateral ventricle of adult male rats and (ii) genetically induced knockdown (KD) of BDNF expression specifically in the central nervous system during the first ontogenesis in mice (KD mice). Delivery of siRNA in the rat brain decreased BDNF levels in the hippocampus (-31%) and hypothalamus (-35%) but not in the amygdala, frontal cortex and pituitary. In addition, siRNA induced no change of the basal HPA axis activity. BDNF siRNA rats exhibited decreased BDNF levels and concomitant altered adrenocortoctrophic hormone (ACTH) and corticosterone responses to restraint stress, suggesting the involvement of BDNF in the HPA axis adaptive response to stress. In KD mice, BDNF levels in the hippocampus and hypothalamus were decreased by 20% in heterozygous and by 60% in homozygous animals compared to wild-type littermates. Although, in heterozygous KD mice, no significant change was observed in the basal levels of plasma ACTH and corticosterone, both hormones were significantly increased in homozygous KD mice, demonstrating that robust cerebral BDNF inhibition (60%) is necessary to affect basal HPA axis activity. All of these results in both rats and mice demonstrate the involvement and importance of a robust endogenous pool of BDNF in basal HPA axis regulation and the pivotal function of de novo BDNF synthesis in the establishment of an adapted response to stress.

Brain region-specific regulation of luteinizing hormone-releasing hormone messenger ribonucleic acid in the male ferret: interactions between pubertal maturation and testosterone.

This study examined the regulation of LHRH messenger RNA (mRNA) during pubertal maturation and by testosterone in male ferrets. Prepubertal and postpubertal ferrets were either intact or were castrated and treated with daily injections of oil or 5 mg/kg testosterone propionate for 14 days. In situ hybridization for LHRH mRNA was performed using an 35S-labeled 48-base oligonucleotide complementary to the human LHRH-coding region. Computerized image analysis was performed on cells in the preoptic area, retrochiasmatic area, arcuate nucleus (ARC), and median eminence; cells were classified as labeled if the number of pixels representing silver grains over the cell was 5 or more times the number of background silver grain pixels. Both pubertal maturation of intact males and castration of prepubertal males resulted in an increase in the number of labeled cells in the ARC. These effects were not observed in any of the other three brain regions, suggesting that ARC LHRH-producing neurons are of primary importance in the presumed increase in LHRH release that occurs as a consequence of either pubertal maturation or castration of prepubertal males. Castration of adults did not increase the number of labeled cells in any brain area, but resulted in an increase in silver grains per labeled cell only in the preoptic area. Thus, LHRH mRNA is regulated during puberty primarily in the ARC, and the particular cell group in which LHRH mRNA is most strongly regulated by testosterone changes with pubertal maturation.

NGFI-B, c-fos, and c-jun mRNA expression in mouse brain after acute carbon monoxide intoxication.

The expression of immediate early genes (IEG) has been documented in the brain after various kinds of insults such as ischemia and hypoxia. To determine whether acute carbon monoxide intoxication (ACOI) might trigger IEG expression, adult ddY mice were subjected to carbon monoxide exposure at a rate of 30 mL/min for 35 seconds. The levels of NGFI-B, c-fos, and c-jun mRNA were determined by Northern blot analysis. A time-course study in the cerebral cortex indicated that the induction of NGFI-B, c-fos, and c-jun mRNA started as early as 15 minutes, reached a peak at 30 minutes, and returned to the basal level at 1 hour after the ACOI. In addition, the temporal feature of the induction of these IEG mRNA in the hippocampus was very similar to that in the cerebral cortex. Examination of brain regions at 30 minutes after the ACOI revealed a significant induction of NGFI-B mRNA in the cerebellum, thalamus-hypothalamus, brainstem. as well as in the cortex and hippocampus, but not in the striatum or olfactory bulb. Furthermore, the neuroanatomical distribution of c-fos mRNA at 30 minutes after the ACOI was very similar to that of the NGFI-B mRNA. The widespread distribution of these IEG in the brain, especially in the cerebellum and brainstem, indicates that the major cause for the triggering of IEG expression in the brain by the ACOI might be a diffuse hypoxia. These findings show for the first time the temporal and spatial expression of IEG in the brain after ACOI.

Differential effects of enrichment on learning and memory function in NR2B transgenic mice.

It has been known that environmental enrichment leads to better learning and memory in mice. However, the molecular mechanisms are not known. In this study, we used the 10th-12th of the NR2B transgenic (Tg) lines, in which the NMDA receptor function is enhanced via the NR2B subunit transgene in neurons of the forebrain, to test the hypothesis of the involvement of NMDA receptor function in enrichment-induced better learning and memory. Consistent with our previous results, both larger long-term potentiation (LTP) in the hippocampus and superior learning and memory were observed in naive NR2B Tg mice even after the 10th-12th generation of breeding. After enrichment, wild-type mice exhibited overall improvement in their performances in contextual and cued conditioning, fear extinctions, and novel object recognition tasks. Interestingly, the same enrichment procedures could not further increase the performance of NR2B Tg mice in contextual conditioning, cued conditioning, or fear extinction, thereby indicating that enhanced NMDA receptor function can occlude these enrichment effects. However, we found that in the novel object recognition task enriched NR2B Tg mice exhibited much longer recognition memory (up to 1 week), compared to that (up to 3 days) in naive NR2B Tg mice. Furthermore, our biochemical experiments showed that enrichment significantly increased protein levels of GluR1, NR2B, and NR2A subunits of glutamate receptors in both wild-type and NR2B Tg mice. Therefore, our results suggest an interactive nature of molecular pathways involved in both environmental and genetic NMDA receptor manipulations for enhancing learning and memory.

Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex.

The NMDA receptor (NMDAR) is a heteromer comprised of NR1 and NR2 subunits. Mice that overexpress the NR2B subunit exhibit enhanced hippocampal LTP, prolonged NMDAR currents, and improved memory ( Tang et al., 1999). In the current study, we explored visual cortex plasticity and NMDAR function in NR2B overexpressing transgenic mice. Unlike the hippocampus, in vitro synaptic plasticity of the visual cortex was unaltered by NR2B overexpression. Consistent with the plasticity findings, NMDAR excitatory postsynaptic current (EPSC) durations from layer 2/3 pyramidal cells were similar in wild-type (wt) and transgenic (tg) mice. Furthermore, temporal summation of NMDAR EPSCs to 10, 20, and 40 Hz stimulation did not differ between cells from wt and tg mice. Finally, although in situ studies clearly demonstrate overexpression of NR2B mRNA in visual cortex, we failed to observe a significant elevation in the synaptic expression of NR2B protein. We conclude that the synaptic ratio of NR2B over NR2A in the NMDA receptor complex in the visual cortex is not significantly influenced by the transgene overexpression. These data suggest that mRNA availability is not a limiting factor for the synthesis of NR2B protein in the visual cortex, and support the hypothesis that levels of NR2A, rather than NR2B, normally determine the subunit composition of NMDARs in visual cortex.

Effects of VA-045 on learning and memory deficits in traumatic brain injury (TBI)-induced retrograde and anterograde amnesic mice.

1. No specific regimen has been developed to treat post-traumatic amnesia in man. In the present study, we examined the effects of (+)-eburnamenine-14-carboxylic acid (2-nitroxyethyl) ester (VA-045), a novel derivative of apovincaminic acid, on learning and memory deficits associated with a mild traumatic brain injury (TBI) in mice. 2. Two kinds of amnesia, TBI-induced retrograde amnesia (TRA) and anterograde amnesia (TAA), were produced by means of post- and pre-acquisition head injury, respectively, by a simple weight-drop device. A novel procedure of water-finding task was used to assess learning and memory functions. 3. Both TRA and TAA mice were dramatically impaired in the task performance, with prolonged latencies for finding and drinking in either retention test or retest, indicating that retention was impaired in TRA mice while learning and retention were impaired in TAA mice. 4. VA-045 administered 30 min post-trauma in TRA mice dramatically shortened the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the retention deficit observed in TRA mice. 5. VA-045 administered 30 min post-trauma in TAA mice dramatically attenuated the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the learning and retention deficits observed in TAA mice. 6. Administration of VA-045 30 min pre-trauma in normal mice markedly attenuated the delay of latencies for finding and drinking after trauma in both retention test and retest, which shows that VA-045 significantly prevented learning and retention deficits after TBI. 7. Motor activities were not significantly affected by either the TBI or the chemical treatment at the time of task examination in either experimental model. 8. It is concluded that VA-045 may have potential effects on learning and memory deficits observed in either TBI-induced retrograde or anterograde amnesia.

Differential effects of testosterone, 5 alpha-dihydrotestosterone and oestradiol-17 beta on plasma concentrations of LH in castrated ferrets.

The biological activity of testosterone often depends on the conversion of testosterone within the target cell to an androgenic or oestrogenic metabolite. The purpose of this study was to compare the relative ability of testosterone and two of its metabolites, dihydrotestosterone (DHT) and oestradiol, to suppress LH secretion in castrated male ferrets. Castrated ferrets were treated with five different doses of steroid by implanting various numbers of s.c. silicone elastomer capsules packed with either testosterone, DHT or oestradiol. The lowest dose of oestradiol (0.1 mm capsule length/100 g body weight, mean estimated total release rate of 25 ng/day) significantly suppressed plasma concentrations of LH in castrated ferrets. Higher amounts of DHT (2.5 mm capsule length/100 g body weight, mean estimated total release rate of 88 ng/day) were required for a significant reduction in plasma concentrations of LH. Concentrations of LH were also significantly lowered by testosterone when administered at a 2.5 mm capsule length/100 g body weight; however, estimated total release rate was 312 ng/day from these capsules. The fact that oestradiol was more effective than DHT, and that DHT was more effective than testosterone in inhibiting LH secretion in castrated ferrets, suggests that in gonadally intact ferrets, testosterone may be converted to DHT or oestradiol within target cells that mediate steroid negative feedback on LH secretion.

A concussive-like brain injury model in mice (I): impairment in learning and memory.

The modeling of human concussive brain injury (CBI) in the laboratory has been challenging. In the present study, we developed an experimental CBI model in mice using a novel weight-drop device. Various injury levels were examined by adjusting the height of the falling weight (diameter 10 mm, length 20 cm, weight 21 g). At a height of 50 cm, the impact resulted in a mortality rate of 46.7% with a skull fracture rate of 28.6%. At a height of 25 cm, however, the impact produced a concussive-like brain injury (CLBI) to the mice without skull fracture. A series of pathophysiological and neurobehavioral responses was evaluated at this injury level. The CLBI mice lost muscle tone and righting reflex response immediately following the trauma and recovered from the latter within a short duration of 1.6 +/- 0.32 min (mean +/- SE). Brain edema formation started at 12 h, reached a maximum at 24 h and recovered 48 h. Typically edema was found in the neocortex, hippocampus, and cerebellum, but not in the brain stem. Deficits in the feeding behaviors lasted for 2 days, accompanied by lower body weight persisting for 5 days. The body weight growth rate for 24 h returned to the control levels by the third day postinjury. Learning and memory were evaluated at the end of 1-3 weeks after the trauma using a water-finding task. At 1 week, exploratory behaviors were slightly inhibited while learning and memory were profoundly impaired. Interestingly, the learning and memory deficits lasted for 2 weeks while recovering to the control levels by 3 weeks. No motor disability was found in the CLBI mice during the 3-week evaluations. These results indicate that the weight-drop impact produced graded injury to the brain, and at the injury level of 25 cm it produced a CLBI in the mice in which the characteristics of transient loss of neurobehavioral responses, short duration of brain edema, and long-lasting learning and memory deficits are similar to those of human CBI.

A concussive-like brain injury model in mice (II): selective neuronal loss in the cortex and hippocampus.

A novel concussive-like brain injury (CLBI) model characterized by transient neurobehavioral depression, short duration of brain edema, and long-lasting memory deficits has been reported in our companion paper. This was achieved by dropping a 21-g weight from a height of 25 cm onto the head of a mouse. In the present study, we examined the histopathological changes in this model. Male ddY mice were subjected to either the trauma or sham injury. Gross pathological examination of the brain 1 h posttrauma did not demonstrate subdural, subarachnoid, intraventricular, periventricular, and intraparenchymatous hemorrhage, focal lesions or contusions. Microscopic examination 24 h posttrauma with Nissl staining (cresyl violet), however, revealed a selective bilateral neuronal cell loss in the cerebral cortex and hippocampus but not in the regions of the thalamus, cerebellum, and brain stem. The characteristics of neuronal cell loss in the cortex suggested that this pathology was related in part, to the head impact dynamics, since the cell loss was noted in the central portion of the supraventricular cerebral cortex (p < 0.001), the site of the weight impact, gradually decreasing peripheral to this site, and disappearing in the areas remote from this locus. In contrast, neuronal cell loss seen in the hippocampus did not suggest that this pathology was directly associated with the impact site. Neuronal cell loss was concentrated in the pyramidal cell layer of CA2 (p < 0.01) and CA3 (p < 0.01), and a lesser degree was noted in the subfields of CA3c (p < 0.05) and the hilar region (p < 0.05) but not in the subfields of CA1 and the dentate gyrus layers. The present study characterized the histopathological change seen in the CLBI model, demonstrating the selective neuronal cell loss following weight-drop concussion in mice.

Stress and corticotropin-releasing factor potentiate center region activity of mice in an open field.

The effects of corticotropin-releasing factor (CRF) and previously published effect of stress on the locomotor activity of mice in different regions of an open field were compared. Intracerebroventricular (ICV) administration of 0.2 microgram CRF, like stress, significantly increased center region activity; this effect was reversed by the benzodiazepine diazepam (DZP) at a dose of DZP having no significant effect alone. A dose of DZP that antagonized CRF-potentiated center region activity did not block amphetamine-stimulated center area activity. These results suggest that CRF may normally be responsible for many behavioral changes during conditions of stress.