Neurogenesis in the dentate gyrus of adult rat following conditioned fear stimuli

To explore the effects of proBDNF on cell proliferation and differentiation in hippocampal dentate gyrus in Alzheimer' disease (AD) rat model. The AD rat model was established. Alzet osmotic minipumps were connected to right hippocampus of AD rat and filled with proBDNF, sheep antibody to proBDNF or normal sheep serum respectively. Rats received the injection for 14 days at the speed of 0.5 microl/h. 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, ip) was injected twice daily for 14 days. BrdU immunohistochemistry was processed to determine the number of newly generated cells. To examine the phenotype of newly generated cells, immunofluorescent triple labeling was conducted to colocalize BrdU-positive cells with rabbit anti-doublecortin (DCX) or mouse anti-glial fibrillary acid protein (GFAP). proBDNF group had fewer BrdU positive cells in dentate gyrus (P < 0.01), while anti-proBDNF group had more BrdU positive cells (P < 0.01) as compared with control group respectively. Immunofluorescent triple labeling showed that there was no phenotypic difference of BrdU positive cells between each group. proBDNF can suppress the proliferation of hippocampal neuron in dentate gyrus in AD rats while anti-proBDNF has the opposite effect. These findings suggest that promoting the hippocampal neurogenesis by blocking the functions of endogenous proBDNF may be a potential therapeutic strategy for AD.

Conditioned fear or novel environmental stimuli suppress vasopressin (VP) and augment oxytocin (OT) and prolactin (PRL) release in rats. We examined the effects of intracerebroventricular (i.c.v.) injections of adrenoceptor antagonists on these neuroendocrine responses to conditioned fear or novel environmental stimuli in male rats. A beta1 antagonist, metoprolol, blocked the VP but not the OT or PRL response to conditioned fear stimuli, but did not abolish neuroendocrine responses to novel environmental stimuli. A beta2 antagonist, ICI118551, impaired the PRL but not the VP or OT response to fear or novel environmental stimuli. In rats injected with a alpha1 adrenoceptor antagonist, benoxathian, conditioned fear stimuli did not significantly induce the VP, OT or PRL responses. The effects of benoxathian were not due to a general reduction of arousal, since benoxathian did not prevent the VP, OT or PRL response to novel environmental stimuli. These data suggest that beta1 adrenoceptors play a selective role in the VP response to conditioned fear stimuli, as do beta2 adrenoceptors in the prolactin response to conditioned fear and novel environmental stimuli. We conclude that alpha1 adrenoceptors play a facilitative role in VP, OT, PRL responses to conditioned fear stimuli.

Objective To explore the effect of different durations of chronic unpredictable mild stress (CUMS) on behaiviors and neurogenesis of bippocampal dentate gyrus in adult rats.Methods (1)Animals were divided into four groups:control group,rats without stress; M1 group,rats subject to CUMS for 1 week; M2 group,rats subject to CUMS for 2 weeks; and M5 group,rats subject to CUMS for 3 weeks.(2) Rats'depression-like behaviors were observed in opening field test and 1％ sucrose preference test was done before and after the experiment of each group.(3) After bahavior test,rats were sacrificed and the hippocampi were isolated.Neurogenesis in hippocampal dentate gyrus was detected by using immunohistochemistry.Results (1) As compared with control group,total diatance (cm) in l0 min/center distance (cm) in 10 min/rearing counts were decreased in MI group (P ＜ 0.05),M2 group (P ＜ 0.05),and M3 group (P ＜0.05).There was no significant difference in the total distance in 10 min/center distance (cm) in 10 min/rearing counts among the M1,M2 and M3 groups (P ＞ 0.05).As compared with control group,levels of total fluid consumption/sucrose consumption/sucrose preference consumption were decreased in MI group (P ＜ 0.05),M2 group (P ＜ 0.05),and M3 group (P ＜ 0.05).There was no significant difference in levels of total fluid consumption among the M1,M2 and M3 groups (P ＞ 0.05) ; (2)As compared with control group (31.75 ± 8.48),BrdU-labeled cells in the dentate gyrus were decreased in M1 group (22.19 ±6.08) (P＜0.05) and M3 group (19.57 ±5.28) (P＜0.05),but there was no significant difference in the levels of BrdU-labeled cells in the dentate gyrus between M1 group and M3 group (P ＞ 0.05).There was no significant difference in the levels of BrdU-labeled cells in the dentate gyrus between M2 group (29.78 ± 6.78) and control group (P ＞ 0.05).Conclusion Different durations of CUMS could lead to the depressive behaviors,but have different influences on the neurogenesis of hippocampal dentate gyrus in adult rats. Key words: Chronic stress ; Hippocampus ; Depression ; Neurogenesis

Within-compound associations between the context and the conditioned stimulus

Vasopressin (VP) release by the neurohypophysis is suppressed and oxytocin (OT) release is augmented after conditioned or unconditioned fear stimuli(3) and after anxiety-producing novel environmental stimuli (2, 4). The NMDA subtype of glutamate receptor is essential for long-term potentiation of excitatory synaptic transmission in the hippocampus and, thereby, has been postulated to play a crucial role in learning and memory. Behavioural experiments have shown that an NMDA-receptor antagonist injected in the amygdala impairs acquisition but not retention or recall of emotional memory associated with conditioned fear response(1). Thus, the present study aimed at investigating in male rats after overnight water deprivation whether an i.p. administered NMDA-receptor antagonist, MK-801 impairs the VP, OT and prolactin (PRL) responses to emotional stimuli. In the experiments with conditioned fear stimuli, rats were trained with environmental stimuli paired with footshocks and tested with the environmental stimuli on the following day. MK-801 administered 30 min before training impaired the suppressive VP and the augmentative OT and PRL responses to conditioned fear stimuli (Fig. 1A). The antagonist injected after training, however, did not block the hormonal responses (data not shown). MK-801 given before testing stimuli abolished the VP and PRL responses and reduced the OT response to conditioned fear stimuli (Fig. 1B). The blocking action of the drug was not due to a reduced level of emotional arousal, since the VP, OT and PRL responses to unconditioned fear stimuli (Fig. 1C) or novel environmental stimuli (Fig. 1D) were not impaired by the drug.

d-cycloserine facilitates extinction of learned fear: Effects on reacquisition and generalized extinction

Objective To observe neural stem cells proliferation, migration and differentiation in hippocampus in developing rats with status epileptictus. Methods 320 healthy SD rats at age 7, 14, 21, 28 d (P7, P14, P21, P28) were randomly divided into status epilepticus (SE) and normal control group. In each group those rats at the same age were further randomly divided into 1, 7, 14, 21, 28 d five time points after PTZ-induced SE (n=8). New cell proliferation and migration were observed by immunohistochemistry studies in the dentate gyrus. Double labeling with Brdu/NeuN and Brdu/GFAP was performed in the P14 rats. Results Nestin positive cells appeared in the dentate gyrus on 1 d after SE in P7, P14, P21, P28 rats. The number of nestin positive cells gradually increased on 7 d and reached a peak on 14 d, then gradually reduced on 21 d, finally fell to a minimum on 28 d after SE. The numbers of nestin positive cells on 7 d（177.00±3.22，t=16.033）and 14 d （195.00±3.41，t=28.840） were significantly higher in the SE group than the NS group (147.50±2.08，136.50±2.65，both P＜0.05). The smaller age of rats with SE onset, the greater the nestin intensity. But the number of nestin positive cells in the dentate gyrus of normal rats were gradually decreased with increasing age. Nestin positive cells were distributed in subgranular zone of dentate gyrus on 1 d and 7 d after SE, then gradually migrated to the granule cell layer on 14 d with morphological changes. Small part of nestin positive cells were ectopically migrated to the hilus of dentate gyrus in P14, P21, P28 age rats, and were also seen in the CA1，CA3 of hippocampus and cortex with various cell morphology. For differentiation of newly generated cells, most of Brdu positive cells co-expressed NeuN and about 4%—5% cells co-expressed GFAP. Conclusions SE could induce neurogenesis in the hippocampal dentate gyrus area in developing rats which has age-related characteristics. Most new cells migrate from the subgranular zone to the granule cell layer of the dentate gyrus, and a small number of newly generated cells ectopically migrated to the hilus of dentate. The majority of newly generated cells differentiate into neurons, and the others differentiate into glial. Key words: Status epilepticus; Hippocampus; Neurogenesis; Neural stem cells

Objective To evaluate the effects of sevoflurane on hippocampal neurogenesis in dentate gyrus (DG) of mice of different ages. Methods Ninety-six SPF healthy male C57BL/6 mice, aged 2 weeks, 6 weeks, 9 months and 20 months (24 mice for each age, 12 mice for each group), were divided into 2 groups (n=48 each) using a random number table method: control group (group C) and sevoflurane group (group S). Group S inhaled 3.0% sevoflurane for 2 h once a day for 3 consecutive days, while group C inhaled the mixture of air and O2.Six mice of each age were selected, and 5′-bromo-2′-deoxyuridine (BrdU) 50 mg/kg was intraperitoneally injected immediately before and after inhalation once a day for 3 consecutive days in two groups.Mice were sacrificed at 24 h after the last inhalation (T1), brains were removed and hippocampi isolated for determination of the number of nestin and doublecortin (DCX) positive cells in DG by immunohistochemistry.Mice were sacrificed at 4 weeks after the last inhalation (T2), brains were removed and hippocampi isolated for determination of the number of neuronal nuclei antigen (NeuN)/BrdU and glial fibrillary acid protein (GFAP)/BrdU positive cells by immunofluorescence. Results Compared with group C, the number of nestin and DCX positive cells was significantly reduced at T1, and the number of NeuN/BrdU and GFAP/BrdU positive cells was reduced at T2 in mice of 2 weeks and 20 months old (P 0.05). Conclusion Three percent sevoflurane can inhibit hippocampal neurogenesis in DG of immature and old mice and exerts no influence on hippocampal neurogenesis in DG of juvenile and adult mice. Key words: Anesthetic, inhalation; Age; Neurogenesis; Hippocampus

Conditioned fear stimuli reinstate cocaine-induced conditioned place preference

Lesions in the bed nucleus of the stria terminalis disrupt corticosterone and freezing responses elicited by a contextual but not by a specific cue-conditioned fear stimulus

The effect of a conditioned fear stimulus (CS) on responsiveness to pain was examined in three experiments. In Experiment 1, a CS that signaled shock attenuated freezing in response to shock, with the attenuation occurring several minutes after the shock. Naloxone blocked the effect of the CS. The effect of the CS, including its reversibility by naloxone, was retained over an interval of 90 days. Experiment 2 showed that this effect on freezing is due to associative fear conditioning, rather than blocking of conditioning to context by a novel cue. In Experiment 3, presenting a fear CS just prior to administering a tail-flick (radiant heat) test of nociception increased the tail-flick latencies; that is, the fear CS apparently induced hyperalgesia rather than analgesia. Because this result makes it difficult to interpret the change in freezing seen in the first experiment as reflecting antinociception, it raises questions about how pain might differentially affect different measures of pain responsiveness. A memory hypothesis is advanced to resolve the different effects obtained with the freezing and tail-flick tests.

Studies employing response prevention (RP) are reviewed. Considering assessment difficulties and conflicting findings, it is questionable whether RP actually reduces fear to a conditioned stimulus (CS). This study measured fear after RP via a conditioned emotional response (CER) paradigm. Hypotheses were that fear of an auditory CS (conditioned in an avoidance paradigm) is reduced during RP, and that fear conditioning would occur to aspects of the conditioning environment per se. Also evaluated was the effectiveness of RP when fear had been learned under two different conditions: (a) avoidance or (b) classical defensive conditioning. Seven groups of 10 experimentally naive female rats were run. Animals were initially trained to bar press for food pellets on a variable interval (VI) 2 schedule. Three groups were then avoidance trained in a two-way shuttle box to a criterion of 10 successive avoidances. Immediately following acquisition, one group received RP (blocking) in the shuttle box (Condition A-B). This consisted of placing a door between the two sides of the box and presenting the 85 dB (A) white noise CS for 15 20-sec periods with a variable 1-min interstimulus interval. One group did not receive RP (nonblocked) and was instead immediately returned to its home cage (Condition A-NBHC). The third group was treated as was A-B except the CS was not presented (Condition A-NBSB). Two other groups were trained in a classical defensive paradigm. These animals were matched to A-B animals in terms of number, order, and duration of CSs and USs. Following conditioning, one group received the same treatment as A-B (Condition CD-B), and the other received the same treatment as A-NBHC (Condition CD-NBHC). Two groups served as controls. A backward control (Condition BC-NBHC) was matched to A-NBHC in terms of number, order, and duration of CSs and USs. A sensitization control (Condition SC-NBHC) was matched to A-NBHC in terms of number, order, and duration of CS presentations. Immediately following conditioning trials, control animals received the same treatment as A-NBHC animals. After differential treatments all animals were immediately returned to the lever box in which they had learned to bar press, a VI 2 schedule was reinstated, and the CER was measured. A-B showed significant suppression initially but significantly less than A-NBHC, suggesting that although RP was effective in reducing fear to the CS, some fear remained. Controls showed essentially no suppression and did not differ. A-B did not differ from A-NBSB, suggesting that conditioning of fear did occur to the environment and that this fear was subsequently reduced in A-NBSB. A-B suppressed significantly more than CD-B, suggesting that RP was more effective when fear was learned in a classical as compared to an avoidance paradigm. Theoretical implications and generalizations to implosive therapy are discussed.

We used Pavlovian counterconditioning in rats to identify the neural mechanisms for appetitive-aversive motivational interactions. In Stage I, rats were trained on conditioned stimulus (CS)-food (unconditioned stimulus [US]) pairings. In Stage II, this appetitive CS was transformed into a fear CS via pairings with footshock. The development of fear responses was retarded in rats that had received Stage I appetitive training. This counterconditioning was associated with increased levels of phosphorylated mitogen activated protein kinase immunoreactivity (pMAPK-IR) in several brain regions, including midline thalamus, rostral agranular insular cortex (RAIC), lateral amygdala, and nucleus accumbens core and shell, but decreased expression in the ventrolateral quadrant of the midbrain periaqueductal gray. These brain regions showing differential pMAPK-IR have previously been identified as part of the fear prediction error circuit. We then examined the causal role of RAIC MAPK in fear learning and showed that Stage II fear learning was prevented by RAIC infusions of the MEK inhibitor PD098059 (0.5 µg/hemisphere). Taken together, these results show that there are opponent interactions between the appetitive and aversive motivational systems during fear learning and that the transformation of a reward CS into a fear CS is linked to heightened activity in the fear prediction error circuit.

Learning safe versus dangerous cues is crucial for survival. During development, parents can influence fear learning by buffering their children's stress response and increasing exploration of potentially aversive stimuli. Rodent findings suggest that these behavioral effects are mediated through parental presence modulation of the amygdala and medial prefrontal cortex (mPFC). Here, we investigated whether similar parental modulation of amygdala and mPFC during fear learning occurs in humans. Using a within-subjects design, behavioral (final N=48, 6-17 years, mean=11.61, SD=2.84, 60% females/40% males) and neuroimaging data (final N=39, 6-17 years, mean=12.03, SD=2.98, 59% females/41% males) were acquired during a classical fear conditioning task, which included a CS+ followed by an aversive noise (US; 75% reinforcement rate) and a CS-. Conditioning occurred once in physical contact with the participant's parent and once alone (order counterbalanced). Region of interest analyses examined the unconditioned stress response by BOLD activation to the US (vs. implicit baseline) and learning by activation to the CS+ (vs. CS-). Results showed that during US presentation, parental presence reduced the centromedial amygdala activity, suggesting buffering of the unconditioned stress response. In response to learned stimuli, parental presence reduced mPFC activity to the CS+ (relative to the CS-), although this result did not survive multiple comparisons' correction. These preliminary findings indicate that parents modulate amygdala and mPFC activity during exposure to unconditioned and conditioned fear stimuli, potentially providing insight into the neural mechanisms by which parents act as a social buffer during fear learning. RESEARCH HIGHLIGHTS: This study used a within-participant experimental design to investigate how parental presence (vs. absence) affects youth's neural responses in a classical fear conditioning task. Parental presence reduced the youth's centromedial amygdala activation to the unconditioned stimulus (US), suggesting parental buffering of the neural unconditioned response (UR). Parental presence reduced the youth's mPFC activation to a conditioned threat cue (CS+) compared to a safety cue (CS-), suggesting possible parental modulation of fear learning.

Although the conditioned cardiac fear response is an important index of psychophysiological fear processing, underlying neural mechanisms remain unclear. N = 22 participants underwent differential fear conditioning and extinction with face pictures as conditioned stimuli (CS) and loud noise bursts as aversive unconditioned stimulus (US) on Day 1 and a recall test 1 day later. We assessed ERPs, evoked heart period (HP), and time-lagged within-subject correlations of single-trial EEG amplitude and HP as index for corticocardiac coupling in response to the CS. Fear-conditioned stimuli (CS+) triggered cardiac deceleration during fear acquisition and recall. Meanwhile, only during Day 1 acquisition, CS+ evoked larger late positivities in the ERP than CS-. Most importantly, during Day 2 recall, stimulus-evoked single-trial EEG responses in the time window between 250 and 500 ms predicted the magnitude of cardiac fear responses 2 to 5 s later. This marker of corticocardiac coupling selectively emerged in response to not previously extinguished CS+ but was absent in response to CS- or previously extinguished CS+. The present results provide first evidence that fear conditioning and extinction modulate functional corticocardiac coupling in humans. Underlying mechanisms may involve subcortical structures enhancing corticocardiac transmission to facilitate processing of consolidated conditioned fear.