Fear-Conditioning to Unpredictable Threats Reveals Sex and Strain Differences in Rat Fear-Potentiated Startle (FPS).

Fear-potentiated startle (FPS) has been widely used to study fear processing in humans and rodents. Human studies showed higher startle amplitudes and exaggerated fear reactivity to unpredictable vs. predictable threats in individuals suffering from post-traumatic stress disorder (PTSD). Although human FPS studies use both sexes, a surprisingly limited number of rodent FPS studies use females. Here we investigate the effects of signal-threat contingency, signal-threat order and threat predictability on FPS in both sexes. We use a classic fear-conditioning protocol (100% contingency of cue and shock pairings, with forward conditioning such that the cue co-terminates with the shock) and compare it to modified fear-conditioning protocols (70% contingency; backward conditioning; or cue and shock un-paired). Although there are no sex differences in the startle amplitudes when corrected for body weight, females consistently demonstrate higher shock reactivity during fear-conditioning. Both sexes and strains demonstrate comparable levels of cued, non-cued, and contextual fear in the classic FPS and FPS following fear-conditioning with 70% contingency or backward order (cue co-starts with shock). However, in the classic FPS, Sprague-Dawley females show reduced proportion between cued fear and cue-elicited vigilant state than males. Lastly, a prominent sex difference is uncovered following unpredictable fear-conditioning (cue and shock un-paired), with Wistar, but not Sprague-Dawley, females showing significantly higher startle overall during the FPS recall, regardless of trial type, and higher contextual fear than males. This striking sex difference in processing unpredictable threats in rodent FPS might help to understand the mechanisms underlying higher incidence of PTSD in women.

Fear-conditioning to unpredictable threats reveals sex differences in rat fear-potentiated startle (FPS).

Fear-potentiated startle (FPS) has been widely used to study fear processing in humans and rodents. Human studies have shown higher startle amplitudes and exaggerated fear reactivity to unpredictable vs. predictable threats in individuals suffering from post-traumatic stress disorder (PTSD). Although human FPS studies often use both sexes, a surprisingly limited number of rodent FPS studies use females. Here we investigate the effects of signal-threat contingency, signal-threat order and threat predictability on FPS in both sexes. We use a classic fear-conditioning protocol (100% contingency of cue and shock pairings, with forward conditioning such that the cue co-terminates with the shock) and compare it to modified fear-conditioning protocols (70% contingency; backward conditioning; or cue and shock unpaired). Although there are no sex differences in the startle amplitudes when corrected for body weight, females demonstrate higher shock reactivity during fear-conditioning. Both sexes demonstrate comparable levels of cued, non-cued, and contextual fear in the classic FPS but females show reduced fear discrimination vs. males. Fear-conditioning with 70% contingency or backward order (cue co-starts with shock) induces similar levels of cued, non-cued, and contextual fear in both sexes but they differ in contextual fear extinction. Lastly, a prominent sex difference is uncovered following unpredictable fear-conditioning protocol (cue and shock un-paired), with females showing significantly higher startle overall during the FPS recall, regardless of trial type, and higher contextual fear than males. This striking sex difference in processing unpredictable threats in rodent FPS might help to understand the mechanisms underlying higher incidence of PTSD in women. Highlights: Male and female rats have comparable startle amplitudes when corrected for body weightFemale rats show higher foot-shock reactivity than males during fear-conditioningFemale rats show reduced fear discrimination vs. males in the classic FPSReversed signal-threat order increases contextual fear in both sexesExposure to unpredictable threats increases startle in general and contextual fear only in females.

Oxytocin excites BNST interneurons and inhibits BNST output neurons to the central amygdala.

The dorsolateral bed nucleus of the stria terminalis (BNSTDL) has high expression of oxytocin (OT) receptors (OTR), which were shown to facilitate cued fear. However, the role of OTR in the modulation of BNSTDL activity remains elusive. BNSTDL contains GABA-ergic neurons classified based on intrinsic membrane properties into three types. Using in vitro patch-clamp recordings in male rats, we demonstrate that OT selectively excites and increases spontaneous firing rate of Type I BNSTDL neurons. As a consequence, OT increases the frequency, but not amplitude, of spontaneous inhibitory post-synaptic currents (sIPSCs) selectively in Type II neurons, an effect abolished by OTR antagonist or tetrodotoxin, and reduces spontaneous firing rate in these neurons. These results suggest an indirect effect of OT in Type II neurons, which is mediated via OT-induced increase in firing of Type I interneurons. As Type II BNSTDL neurons were shown projecting to the central amygdala (CeA), we also recorded from retrogradely labeled BNST→CeA neurons and we show that OT increases the frequency of sIPSC in these Type II BNST→CeA output neurons. In contrast, in Type III neurons, OT reduces the amplitude, but not frequency, of both sIPSCs and evoked IPSCs via a postsynaptic mechanism without changing their intrinsic excitability. We present a model of fine-tuned modulation of BNSTDL activity by OT, which selectively excites BNSTDL interneurons and inhibits Type II BNST→CeA output neurons. These results suggest that OTR in the BNST might facilitate cued fear by inhibiting the BNST→CeA neurons.

Oxytocin Promotes Accurate Fear Discrimination and Adaptive Defensive Behaviors.

The nonapeptide, oxytocin (OT), known for its role in social bonding and attachment formation, has demonstrated anxiolytic properties in animal models and human studies. However, its role in the regulation of fear responses appears more complex, brain site-specific, sex-specific, and dependent on a prior stress history. Studies have shown that OT neurons in the hypothalamus are activated during cued and contextual fear conditioning and during fear recall, highlighting the recruitment of endogenous oxytocin system in fear learning. OT is released into the extended amygdala, which contains the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), both critical for the regulation of fear and anxiety-like behaviors. Behavioral studies report that OT in the CeA reduces contextual fear responses; whereas in the BNST, OT receptor (OTR) neurotransmission facilitates cued fear and reduces fear responses to un-signaled, diffuse threats. These ostensibly contrasting behavioral effects support growing evidence that OT works to promote fear discrimination by reducing contextual fear or fear of diffuse threats, yet strengthening fear responses to imminent and predictable threats. Recent studies from the basolateral nucleus of the amygdala (BLA) support this notion and show that activation of OTR in the BLA facilitates fear discrimination by increasing fear responses to discrete cues. Also, OTR transmission in the CeA has been shown to mediate a switch from passive freezing to active escape behaviors in confrontation with an imminent, yet escapable threat but reduce reactivity to distant threats. Therefore, OT appears to increase the salience of relevant threat-signaling cues yet reduce fear responses to un-signaled, distant, or diffuse threats. Lastly, OTR signaling has been shown to underlie emotional discrimination between conspecifics during time of distress, social transmission of fear, and social buffering of fear. As OT has been shown to enhance salience of both positive and negative social experiences, it can also serve as a warning system against potential threats in social networks. Here, we extend the social salience hypothesis by proposing that OT enhances the salience of relevant environmental cues also in non-social contexts, and as such promotes active defensive behaviors.

Cell Proliferation, Migration, and Neurogenesis in the Adult Brain of the Pulse Type Weakly Electric Fish, Gymnotus omarorum.

Adult neurogenesis, an essential mechanism of brain plasticity, enables brain development along postnatal life, constant addition of new neurons, neuronal turnover, and/or regeneration. It is amply distributed but negatively modulated during development and along evolution. Widespread cell proliferation, high neurogenic, and regenerative capacities are considered characteristics of teleost brains during adulthood. These anamniotes are promising models to depict factors that modulate cell proliferation, migration, and neurogenesis, and might be intervened to promote brain plasticity in mammals. Nevertheless, the migration path of derived cells to their final destination was not studied in various teleosts, including most weakly electric fish. In this group adult brain morphology is attributed to sensory specialization, involving the concerted evolution of peripheral electroreceptors and electric organs, encompassed by the evolution of neural networks involved in electrosensory information processing. In wave type gymnotids adult brain morphology is proposed to result from lifelong region specific cell proliferation and neurogenesis. Consistently, pulse type weakly electric gymnotids and mormyrids show widespread distribution of proliferation zones that persists in adulthood, but their neurogenic potential is still unknown. Here we studied the migration process and differentiation of newborn cells into the neuronal phenotype in the pulse type gymnotid Gymnotus omarorum. Pulse labeling of S-phase cells with 5-Chloro-2'-deoxyuridine thymidine followed by 1 to 180 day survivals evidenced long distance migration of newborn cells from the rostralmost telencephalic ventricle to the olfactory bulb, and between layers of all cerebellar divisions. Shorter migration appeared in the tectum opticum and torus semicircularis. In many brain regions, derived cells expressed early neuronal markers doublecortin (chase: 1-30 days) and HuC/HuD (chase: 7-180 days). Some newborn cells expressed the mature neuronal marker tyrosine hydroxylase in the subpallium (chase: 90 days) and olfactory bulb (chase: 180 days), indicating the acquisition of a mature neuronal phenotype. Long term CldU labeled newborn cells of the granular layer of the corpus cerebelli were also retrogradely labeled "in vivo," suggesting their insertion into the neural networks. These findings evidence the neurogenic capacity of telencephalic, mesencephalic, and rhombencephalic brain proliferation zones in G. omarorum, supporting the phylogenetic conserved feature of adult neurogenesis and its functional significance.

Spatial distribution and cellular composition of adult brain proliferative zones in the teleost, Gymnotus omarorum.

Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones.