Hyperactivity and Learning Deficits in Transgenic Mice Bearing a Human Mutant Thyroid Hormone β1 Receptor Gene

Resistance to thyroid hormone (RTH) is a human syndrome mapped to the thyroid receptor β (TRβ) gene on chromosome 3, representing a mutation of the ligandbinding domain of the TRβ gene. The syndrome is characterized by reduced tissue responsiveness to thyroid hormone and elevated serum levels of thyroid hormones. A common behavioral phenotype associated with RTH is attention deficit hyperactivity disorder (ADHD). To test the hypothesis that RTH produces attention deficits and/or hyperactivity, transgenic mice expressing a mutant TRβ gene were generated. The present experiment tested RTH transgenic mice from the PV kindred on behavioral tasks relevant to the primary features of ADHD: hyperactivity, sustained attention (vigilance), learning, and impulsivity. Male transgenic mice showed elevated locomotor activity in an open field compared to male wild-type littermate controls. Both male and female transgenic mice exhibited impaired learning of an autoshaping task, compared to wild-type controls. On a vigilance task in an operant chamber, there were no differences between transgenics and controls on the proportion of hits, response latency, or duration of stimulus tolerated. On an operant go/no-go task measuring sustained attention and impulsivity, there were no differences between controls and transgenics. These results indicate that transgenic mice bearing a mutant human TRβ gene demonstrate several behavioral characteristics of ADHD and may serve a valuable heuristic role in elucidating possible candidate genes in converging pathways for other causes of ADHD.     

CA1 Long-Term Potentiation Is Diminished but Present in Hippocampal Slices from α-CaMKII Mutant Mice

Previous work has shown that mice missing the α-isoform of calcium–calmodulin-dependent protein kinase II (α-CaMKII) have a deficiency in CA1 hippocampal long-term potentiation (LTP). Follow-up studies on subsequent generations of these mutant mice in a novel inbred background by our laboratories have shown that whereas a deficiency in CA1 LTP is still present in α-CaMKII mutant mice, it is different both quantitatively and qualitatively from the deficiency first described. Mice of a mixed 129SvOla/SvJ;BALB/c;C57Bl/6 background derived from brother/sister mating of the α-CaMKII mutant line through multiple generations (>10) were produced by use of in vitro fertilization. Although LTP at 60 min post-tetanus was clearly deficient in these (−/−) α-CaMKII mice (42.6%, n=33) compared with (+/+) α-CaMKII control animals (81.7%, n=17), α-CaMKII mutant mice did show a significant level of LTP. The amount of LTP observed in α-CaMKII mutants was normally distributed, blocked by APV (2.7%, n=8), and did not correlate with age. Although this supports a role for α-CaMKII in CA1 LTP, it also suggests that a form of α-CaMKII-independent LTP is present in mice that could be dependent on another kinase, such as the β-isoform of CaMKII. A significant difference in input/output curves was also observed between (−/−) α-CaMKII and (+/+) α-CaMKII animals, suggesting that differences in synaptic transmission may be contributing to the LTP deficit in mutant mice. However, tetani of increasing frequency (50, 100, and 200 Hz) did not reveal a higher threshold for potentiation in (−/−) α-CaMKII mice compared with (+/+) α-CaMKII controls.     

Reduced K+ Channel Inactivation, Spike Broadening, and After-Hyperpolarization in Kvβ1.1-Deficient Mice with Impaired Learning

A-type K⁺ channels are known to regulate neuronal firing, but their role in repetitive firing and learning in mammals is not well characterized. To determine the contribution of the auxiliary K⁺ channel subunit Kvβ1.1 to A-type K⁺ currents and to study the physiological role of A-type K⁺ channels in repetitive firing and learning, we deleted the Kvβ1.1 gene in mice. The loss of Kvβ1.1 resulted in a reduced K⁺ current inactivation in hippocampal CA1 pyramidal neurons. Furthermore, in the mutant neurons, frequency-dependent spike broadening and the slow afterhyperpolarization (sAHP) were reduced. This suggests that Kvβ1.1-dependent A-type K⁺ channels contribute to frequency-dependent spike broadening and may regulate the sAHP by controlling Ca²⁺ influx during action potentials. The Kvβ1.1-deficient mice showed normal synaptic plasticity but were impaired in the learning of a water maze test and in the social transmission of food preference task, indicating that the Kvβ1.1 subunit contributes to certain types of learning and memory.     

Impairment of spatial learning and hippocampal synaptic potentiation in c-kit mutant rats

The c-kit receptor tyrosine kinase encoded by the white-spotting (W) gene is highly expressed in rat hippocampal CA1–CA4 regions. We found an impaired spatial learning and memory in homozygous c-kit (Ws/Ws) mutant rats that have a 12-base deletion in the tyrosine kinase domain of the c-kit gene and a very low kinase activity. Electrophysiological studies in hippocampal slices revealed that the long-term potentiation (LTP) induced by the tetanic stimulation (100 Hz, 1 sec) in the mossy fiber (MF)–CA3 pathway, but not in the Schaffer collaterals/commissural–CA1 pathway, was significantly reduced in c-kit mutants compared with wild-type (+/+) rats. The paired-pulse facilitation (PPF) was measured before the tetanus and after the establishment of the LTP in each slice. The initial PPF in the MF–CA3 pathway positively correlated with the amplitude of the LTP in the wild-type rats but not in the c-kit mutant rats. Furthermore, they failed to show the normal characteristics observed in the MF–CA3 pathway of +/+ rats; that is, the negative correlation between the initial PPF and the changes in PPF measured after the LTP. These findings suggest an involvement of SCF/c-kit signaling in hippocampal synaptic potentiation and spatial learning and memory.     

Beta2 subunit containing acetylcholine receptors mediate nicotine withdrawal deficits in the acquisition of contextual fear conditioning

Acute nicotine enhances contextual fear conditioning, whereas withdrawal from chronic nicotine produces impairments. However, the nicotinic acetylcholine receptors (nAChR) that are involved in nicotine withdrawal deficits in contextual fear conditioning are unknown. The present study used genetic and pharmacological techniques to investigate the nAChR subtype(s) involved in the effects of nicotine withdrawal on contextual fear conditioning. beta2 or alpha 7 nAChR subunit knockout (KO) and corresponding wild-type (WT) mice were withdrawn from 12 days of chronic nicotine treatment (6.3mg/kg/day), and trained with 2 conditioned stimulus (CS; 85 dB white noise)--unconditioned stimulus (US; 0.57 mA footshock) pairings on day 13. On day 14, mice were tested for contextual and cued freezing. beta2 KO mice did not show nicotine withdrawal-related deficits in contextual fear conditioning, in contrast to WT mice and alpha 7 KO mice. A follow-up study investigated if nicotine withdrawal disrupts acquisition or recall of contextual fear conditioning. The high affinity nAChR antagonist dihydro-beta-erythroidine (DH beta E; 3mg/kg) was administered prior to training or testing to precipitate withdrawal in chronic nicotine-treated C57BL/6 mice. Deficits in contextual fear conditioning were observed in chronic nicotine-treated mice when DH beta E was administered prior to training, but not when administered at testing. These results indicate that beta2-containing nAChRs, such as the alpha 4 beta 2 receptor, mediate nicotine withdrawal deficits in contextual fear conditioning. In addition, nicotine withdrawal selectively affects acquisition but not recall or expression of the learned response.     

Protection from premature habituation requires functional mushroom bodies in Drosophila

Diminished responses to stimuli defined as habituation can serve as a gating mechanism for repetitive environmental cues with little predictive value and importance. We demonstrate that wild-type animals diminish their responses to electric shock stimuli with properties characteristic of short- and long-term habituation. We used spatially restricted abrogation of neurotransmission to identify brain areas involved in this behavioral response. We find that the mushroom bodies and, in particular, the α/β lobes appear to guard against habituating prematurely to repetitive electric shock stimuli. In addition to protection from premature habituation, the mushroom bodies are essential for spontaneous recovery and dishabituation. These results reveal a novel modulatory role of the mushroom bodies on responses to repetitive stimuli in agreement with and complementary to their established roles in olfactory learning and memory.     

Transgenic Brain-Derived Neurotrophic Factor Modulates a Developing Cerebellar Inhibitory Synapse

Brain-derived neurotrophic factor (BDNF) has been shown to promote synapse formation and maturation in neurons of many brain regions, including inhibitory synapses. In the cerebellum, the Golgi cell-granule cell GABAergic synaptic responses undergo developmental transition from slow-decaying to fast-decaying kinetics, which parallels a developmental increase of GABA_A receptor α6 subunit expression in the cerebellar granule cells. In culture, BDNF accelerates the expression of GABA_A receptor α6 subunit expression in granule cells. Here we examined synaptic GABA_A response kinetics in BDNF transgenic mice. The mutant mouse, which carries a BDNF transgene driven by a β-actin promoter, overexpresses BDNF (two- to fivefold increase compared with wild types) in all brain regions. Recordings of the spontaneous GABA_A responses indicate that the decay time constant of the GABAergic responses decreases during early postnatal development; this transition is accelerated in the BDNF transgenic mouse. The amplitude of the spontaneous GABA_A responses was also larger in the transgenic mouse than in the wild-type mouse. However, the frequency of the spontaneous GABA_A responses were not different between the two groups. Our results suggest that BDNF may modulate GABAergic synapse maturation in the cerebellum.     

The effects of multiphasic prepulses on automatic and attention-modulated prepulse inhibition

Prepulse inhibition (PPI) is widely viewed as an operational measure of sensorimotor gating. Previous research has shown that sensorimotor gating can occur automatically and also can be influenced by selective attention. The present research investigated the relationship of the transient detection response (TDR) with automatic and attention-modulated PPI using a novel “multiphasic” prepulse stimulus. Experiment 1 compared discrete versus multiphasic prepulse types in a no-task PPI protocol to validate multiphasic prepulses as effective elicitors of automatic sensorimotor gating. Results revealed that the two prepulse types elicited equivalent levels of PPI. Experiment 2 compared the effectiveness of continuous monophasic versus continuous multiphasic prepulses within a task-based PPI protocol using a lead interval of 120 ms. Results revealed a significant attention effect for monophasic prepulses only. However, robust PPI was produced by the multiphasic prepulses independent of attention as well as over time. These results suggest that multiple influences on PPI can be assessed concurrently depending on prepulse parameters designed to active the TDR when used in a PPI protocol capable of assessing the effects of selective attention on prepulse processing.     

Social modulation of associative fear learning by pheromone communication

Mice communicate through visual, vocal, and olfactory cues that influence innate, nonassociative behavior. We here report that exposure to a recently fear-conditioned familiar mouse impairs acquisition of conditioned fear and facilitates fear extinction, effects mimicked by both an olfactory chemosignal emitted by a recently fear-conditioned familiar mouse and by the putative stress-related anxiogenic pheromone β-phenylethylamine (β-PEA). Together, these findings suggest social modulation of higher-order cognitive processing through pheromone communication and support the concurrent excitor hypothesis of extinction learning.Social communication in mammals has evolved to facilitate reproductive behavior and for protection against environmental threat and predation. Mice communicate information about imminent danger through vocal (Seyfarth and Cheney 2003), visual (Kavaliers et al. 2001; Langford et al. 2006), and odor or pheromone cues (Rottman and Snowdon 1972), each with profound influences on defensive responding. There is also evidence of social empathy in mice (Langford et al. 2006). Mice will sensitize to pain-inducing stimuli simply by observing a conspecific that is currently experiencing pain. Importantly, sensitization occurs only when the conspecific is familiar with the observer (i.e., sibling or cage mate), a clear example of social modulation of an innate behavior. Müller-Velten (1966) provided the first evidence of a functional alarm chemosignal in mice by showing that animals would avoid a pathway in which the odor of a stressed mouse was present. Subsequent studies have shown effects of mammalian olfactory chemosignals on a variety of defensive behaviors such as analgesia, vigilance, and avoidance (Rottman and Snowdon 1972; Mackay-Sim and Laing 1981; Fanselow 1985; Zalaquett and Thiessen 1991). To date, research on social modulation of behavior has focused primarily on observational learning and innate or nonassociative processes. Two recent studies have demonstrated an influence of fear-related chemosignals on associative learning in humans (Chen et al. 2006; Prehn et al. 2006), evidence that supports the hypothesis that social modulation of behavior extends to higher-order cognitive processing.In the following experiments, we asked whether exposure to a familiar mouse recently fear conditioned or trained for fear extinction would influence associative fear learning in a conspecific. We find that exposure to a recently fear-conditioned mouse impairs acquisition of conditioned fear, while the same experience facilitates the extinction of conditioned fear; effects mimicked by exposure to an olfactory chemosignal emitted from fear-conditioned mice and by the putative anxiogenic pheromone, β-phenylethylamine (β-PEA). Interestingly, we find that exposure to a recently extinction-trained mouse results in an inhibition of fear extinction learning, an effect not related to an olfactory chemosignal emitted by a recently extinguished mouse or by exposure to β-PEA. These data suggest that mice communicate information about their experience, in part through pheromone communication, with different effects on associative learning depending on the valence of the task.     

Angiotensin-(1-7)-induced plasticity changes in the lateral amygdala are mediated by COX-2 and NO

It is known from studies outside the brain that upon binding to its receptor, angiotensin-(1-7) elicits the release of prostanoids and nitric oxide (NO). Cyclooxygenase (COX) is a key enzyme that converts arachidonic acid to prostaglandins. Since there are no data available so far on the role of COX-2 in the amygdala, in a first step we demonstrated that the selective COX-2 inhibitor NS-398 significantly reduced the probability of long-term potentiation (LTP) induction in the lateral nucleus of the amygdala. Similarly, in COX-2^−/− mice, LTP induced by external capsule (EC) stimulation was impaired. Second, we evaluated the action of angiotensin-(1-7) in the amygdala. In wild-type mice, angiotensin-(1-7) increased LTP. This LTP-enhancing effect of Ang-(1-7) was not observed in COX-2^+/− mice. However, in COX-2^−/− mice, Ang-(1-7) caused an enhancement of LTP similar to that in wild-type mice. The NO synthetase inhibitor L-NAME blocked this angiotensin-(1-7)-induced increase in LTP in COX-2^−/− mice. Low-frequency stimulation of external capsule fibers did not cause long-term depression (LTD) in drug-free and angiotensin-(1-7)-treated brain slices in wild-type mice. In contrast, in COX-2^−/− mice, angiotensin-(1-7) caused stable LTD. Increasing NO concentration by the NO-donor SNAP also caused LTD in wild-type mice. Our study shows for the first time that LTP in the amygdala is dependent on COX-2 activity. Moreover, COX-2 is involved in the mediation of angiotensin-(1-7) effects on LTP. Finally, it is recognized that there is a molecular cross-talk between COX-2 and NO that may regulate synaptic plasticity.     

Selective enhancement of fear learning and resistance to extinction in a mouse model of acute early life trauma

Early life stress (ELS) experiences can cause changes in cognitive and affective functioning. This study examined the persistent effects of a single traumatic event in infancy on several adult behavioral outcomes in male and female C57BL/6J mice. Mice received 15 footshocks in infancy and were tested for stress-enhanced fear learning, extinction learning, discrimination and reversal learning, and novel object recognition. Infant trauma potentiated fear learning in adulthood and produced resistance to extinction but did not influence other behaviors, suggesting restricted effects of infant trauma on behaviors reliant on cortico-amygdala circuitry.

Exposure to traumatic events early in childhood is associated with the development of psychiatric disorders (Copeland et al. 2018) and deficits in cognitive and affective functioning (Pechtel and Pizzagalli 2011) in adulthood. In humans, early life stress (ELS) is defined as experiencing traumatic events in childhood (Pechtel and Pizzagalli 2011). Rodent models of ELS suggest that acute versus chronic stress may differentially alter systems that regulate the stress response, producing different behavioral outcomes in adulthood (Pryce et al. 2002; Musazzi et al. 2017).Stress-enhanced fear learning (SEFL), a powerful preclinical model of PTSD- and addiction-like behaviors, captures the enduring, maladaptive effects of a single traumatic event on behavior (Rau et al. 2009; Meyer et al. 2013; Radke et al. 2019). In these studies, exposure to 15 footshocks enhances contextual fear conditioning later in life. For infant SEFL, enhanced contextual fear conditioning occurs months after the initial stressful experience and in the absence of memory for the context in which ELS was experienced (Poulos et al. 2014; Quinn et al. 2014). SEFL protocols have been used to model adult or infant trauma in rats (e.g., Rau et al. 2009; Poulos et al. 2014; Quinn et al. 2014) and have been extended to adult mice (Sillivan et al. 2017; Hassien et al. 2020; Pennington et al. 2020). However, to our knowledge, no studies have established the use of acute, infant footshock as a model of ELS in mice.We sought to characterize the effects of acute, infant trauma exposure across several types of learning in adult mice. We tested mice exposed to 15 footshocks on postnatal day (PND) 17 for contextual fear learning, extinction of fear, discrimination and reversal learning, and novel object recognition. Our results suggest that exposure to acute infant trauma enhances fear learning and resistance to extinction in adulthood, but does not alter other types of learning.Male and female C57BL/6J mice were generated from breeding pairs from The Jackson Laboratory. Mice were group-housed (two to four mice/cage) post-weaning and were provided food and water ad libitum, unless otherwise specified. Mice were on a 12:12 light/ dark cycle. Other than trauma exposure, all behavioral tests were conducted during adulthood (PND 60+). Animals were cared for in accordance with the guidelines set by the National Institutes of Health and all procedures were approved by the Institutional Animal Care and Use Committee at Miami University.We first established that exposure to infant footshock produces enhanced contextual fear conditioning in adulthood. Mice were placed in a MED-Associates conditioning chamber (context A) on PND 17 (Fig. 1A; after Quinn et al. 2014). Context A was brightly lit, contained a uniform grid floor, was scented with vanilla (50%), and was cleaned with odorless 5% sodium hydroxide. Mice received either 0 or 15 footshocks (1 mA, 1 sec) during a 60-min session beginning 180 sec following placement in the chamber. Progressive scan video cameras containing visible light filters monitored mice throughout the session. Video Freeze software (Med Associates, Inc.) analyzed the video and data were expressed as percent of time spent freezing during the session. Fear conditioning experiments were powered to detect sex differences with an n of eight per sex. Because differences were not observed when sex was included as a factor in analyses, all reported results represent data from both sexes. Due to computer malfunction, fear conditioning sessions from 14 mice were hand scored according to standard time-sampling procedures (Chowdhury et al. 2005) and data from three mice had to be excluded on extinction session 2.Open in a separate windowFigure 1.Acute infant trauma produces stress-enhanced fear learning in adulthood. (A) Experimental timeline and visual representation of context A and context B. Infant trauma consisted of 15 footshocks or no footshocks on PND 17 in context A. Adult fear conditioning consisted of one footshock (black bars) or no footshock (blue bars) on PND 60 in context B. Extinction was assessed in context B. Memory of the infant trauma was assessed in context A. Stress enhanced fear learning (SEFL) was observed in mice who were exposed to early life stress (15 shocks) and fear conditioning (one shock) for extinction test 1 (B) and extinction test 2 (C). (*) P < 0.05, (**) P < 0.01 versus no/one shock group (Holm–Sidak test). (D) Mice showed little freezing, indicating an absence of fear memory for the infant trauma in context A. Data are means ± SEM.Contextual fear conditioning occurred on PND 60 in a novel context (context B). Context B was dark with a staggered grid floor and cleaned and scented with acetic acid (5%). Baseline freezing during the first 180 sec in this novel context was assessed and used to measure generalization between the stress exposure context (context A) and the novel fear conditioning context (context B). Mice received either 0 or 1 footshocks (1 mA, 1 sec) 180 sec into a 3.5-min session. Thus, there were four groups (infant trauma/adult fear conditioning): no/no shock, n = 14; 15/no shock, n = 15; no/one shock, n = 15; and 15/one shock, n = 18. To test extinction of fear memory, mice were reintroduced to context B for two 8-min sessions, separated by 24 h. Finally, mice were reintroduced to context A for an 8-min retention test of the original context.Mice exposed to 15 footshocks in infancy and conditioned with one footshock as adults exhibited robust SEFL. There were no differences in baseline freezing during the adult fear conditioning session (infant trauma: F_(1,47) = 2.67, P = 0.109, no/no shocks = 0.26 ± 0.15; no/one shock = 2.17 ± 0.64; 15/no shocks = 2.14 ± 0.76; 15/one shock = 7.17 ± 3.25; all data mean ± SEM). For extinction sessions (Fig. 1B,C), a mixed-effects analysis identified main effects of trauma (F_(1,58) = 5.68, P = 0.020), fear conditioning (F_(1,58) = 12.40, P < 0.001), and session (F_(1,55) = 5.25, P = 0.026). There was a significant interaction between fear conditioning and session (F_(1,55) = 7.10, P = 0.010). Two-way ANOVA was next used to examine each test session. For session 1 (Fig. 1B), there were main effects of trauma (F_(1,58) = 5.72, P = 0.020) and fear conditioning (F_(1,58) = 21.10, P < 0.001). The interaction of trauma and fear conditioning did not reach the threshold for significance (F_(1,58) = 2.87, P = 0.096). Follow-up Holm Sidak''s tests revealed that adult fear conditioning produced greater freezing in mice exposed to infant footshock vs. trauma-naïve mice (P = 0.008). For the second extinction test (Fig. 1C), the main effects of trauma (F_(1,55) = 3.72, P = 0.059) and fear conditioning (F_(1,55) = 3.92, P = 0.053) approached the threshold for significance. Follow-up Holm Sidak''s tests revealed that trauma-exposed mice froze more than trauma-naïve mice following adult fear conditioning only (P = 0.042). When tested for memory of the context used for trauma-exposure on PND 17 (context A), mice demonstrated minimal freezing and two-way ANOVA found no significant main effects or interactions (Fs < 1.39) (Fig. 1D). Percent freezing in infancy and in extinction test 1 in adulthood were correlated (r = −0.481, P = 0.043) for the 15/one group alone. Activity bursts in infancy did not correlate with freezing behavior in adulthood. These results indicate that mice exposed to infant trauma who experienced fear conditioning in adulthood exhibited SEFL on the first and second extinction sessions but the memory of the trauma experience was not retained into adulthood.Since PTSD is associated with deficits in fear extinction (Zuj et al. 2016), we next examined extinction learning using 30-min sessions (Fig. 2A). Mice were exposed to acute infant trauma (0 or 15 footshocks) on PND 17. On PND 60, mice were reintroduced to context A for an 8-min test of memory for the original ELS context prior to fear conditioning (no/one shock = 2.41 ± 0.58; 15/one shock = 3.50 ± 0.42; no/three shocks = 1.86 ± 0.43). On PND 61 fear conditioning occurred in context B. Since we observed that trauma-naïve mice displayed very little fear conditioning following one footshock (Fig. 1B,C), mice in this experiment received either one or three footshocks during adult fear conditioning. There were three groups (infant trauma/adult fear conditioning): no/one shock, n = 17; 15/one shock, n = 17; no/three shocks, n = 18. Retention of fear memory was tested for five subsequent days (PND 62–66) in context B during 30-min sessions.Open in a separate windowFigure 2.Extinction of fear learning is impaired following acute infant trauma. (A) Experimental timeline. Infant trauma consisted of 15 footshocks or no footshocks on PND 17 in context A. Adult fear conditioning consisted of one (yellow symbols) or three (blue symbols) footshocks in trauma-naïve mice and one footshock (black symbols) in trauma-exposed mice in context B. Memory of the infant trauma was assessed in context A. Mice received five extinction sessions in context B. (B) On extinction day 1, trauma-exposed mice initially froze at the same level as trauma-naïve mice conditioned with three footshocks but fear persisted longer, demonstrating within-session resistance to extinction. (*) P < 0.05, (**) P < 0.01 15/one shock group versus no/one shock group; (^#) P < 0.05 15/one shock group versus no/three shocks group; (^†) P < 0.05, (^‡) P < 0.01 no/one shock group versus no/three shock group (Holm–Sidak test). (C) Freezing was averaged across the first 8 min of each extinction session. Trauma-exposed mice demonstrated resistance to extinction across sessions. On session 1, both trauma-exposed (15/one shock group) and mice conditioned with three footshocks (no/three shock group) had elevated freezing compared to mice conditioned with one footshock. On session 2, freezing in trauma-exposed mice remained elevated and was greater than in the other two groups. (**) P < 0.01 15/one shock group versus no/one shock group. (^##) P < 0.01 15/one shock group versus no/three shocks group. (^†) P < 0.05 no/one shock group versus no/three shocks group (Holm–Sidak test). Data are means ± SEM.On the first extinction session, there were significant main effects of time (F_(29,1421) = 2.23, P < 0.001) and group (F_(2,49) = 3.54, P = 0.037) and a significant interaction (F_(58,1421) = 1.93, P < 0.001). Holm–Sidak follow-up comparisons revealed that trauma-exposed mice (15/one shock) and mice conditioned with three footshocks during adulthood (no/three shocks) froze more than the no/one shock group during the first 3 min (P < 0.05 for minute 1 and P < 0.01 for minutes 2 and 3) of the first extinction session (Fig. 2B). Trauma-exposed mice continued to freeze more than the no/one shock group during minutes 4–7 (P < 0.01) and minute 11 (P < 0.05). Freezing in the no/3 shocks group was similar to trauma-exposed mice for the first 4 min but diminished sooner, evidenced by a significant difference between these groups during minutes 5, 6, and 9 (P < 0.05). These results suggest that conditioning with three footshocks produces similar levels of fear in trauma-naïve mice as conditioning with one footshock in trauma-exposed mice, but that within-session extinction is delayed in the trauma-exposed group.To examine extinction across the five sessions, we averaged freezing during the first 8 min of each session. We found significant main effects of session (F_(4,196) = 22.92, P < 0.001) and group (F_(2,49) = 6.38, P = 0.003) and a significant interaction (F_(8,196) = 2.75, P = 0.007). Holm–Sidak follow-up comparisons revealed that mice exposed to infant trauma (15/one shock) and mice in the no/three shock group froze more than those in the no/one shock group on extinction session 1 (P < 0.01 and P < 0.05, respectively) (Fig. 2C). On session 2, freezing was greater in trauma-exposed mice versus both other groups (P < 0.01). Percent freezing in infancy did not correlate with freezing behavior in adulthood. Activity bursts in infancy correlated with freezing behavior in adulthood on extinction test 1 (r = −0.558, P = 0.020). These results further suggest that infant trauma produces resistance to extinction.To determine whether the behavioral alterations observed in mice exposed to infant trauma extend to other types of learning, we tested the effects of infant footshock on operant discrimination and reversal learning for food reward and novel object recognition. For discrimination and reversal learning, we used a subset of mice from the first experiment (no shock = 20, 15 shocks = 17) restricted to 85% of free-feeding weight. Mice underwent one habituation day with ten 14-mg grain pellets (Bio Serv) in their home cages. The following day, mice began 15-min training sessions in a standard mouse operant chamber (Med Associates). There were two nose-poke holes and a reward receptacle on one wall of the chamber and a house light and speaker on the opposite wall. The chamber was housed in a sound and light-attenuating box and connected to a computer for data collection (Med-PC V software suite). For all sessions, the house light was off and lights in the nose-poke holes were on. Following a correct response, a 2-sec, 65-dB tone sounded and there was a timeout period of 20 sec following reward delivery during which the lights above the nose-poke holes were off and no rewards could be earned.During the first session, 30 pellets were automatically delivered into the reward receptacle. Next, mice were trained to respond for the food reward on a fixed ratio 1 (FR1) schedule by responding at either nose-poke hole until meeting criterion of 30 responses in 15-min. For discrimination, mice were trained to respond at the active nose-poke hole (100% probability of reward), which was randomly assigned to the left or right side. The contingencies of the active and inactive nose-pokes holes were reversed once criterion was met (≥30 rewards with 85% reinforced responses over two consecutive sessions).Neither sex nor adult fear conditioning affected any measure of discrimination and reversal learning, so all results are reported collapsed across these two factors. Two trauma-exposed females did not acquire the discrimination in 25 sessions and were not advanced to reversal. Data were analyzed using mixed-effects analyses with phase (i.e., acquisition and reversal) as the within-subjects factor. There were no differences between groups in the total number of sessions required to complete discrimination (no shock = 5.70 ± 0.69; 15 shock = 6.82 ± 1.50) or reversal (no shock = 9.40 ± 0.98; 15 shock = 7.73 ± 1.21). Mice made more total reinforced (main effect of phase: F_(1,33) = 11.62, P = 0.002) and total nonreinforced (main effect of phase: F_(1,33) = 42.54, P < 0.001) responses during reversal but there were no effects of infant trauma on behavior (Fig. 3A,B). These results indicate that acute infant trauma does not influence acquisition of operant discrimination learning or behavioral flexibility in adulthood.Open in a separate windowFigure 3.Acute infant trauma does not affect discrimination and reversal learning or novel object recognition. (A,B) Following infant trauma on PND 17 (no footshock, blue, or 15 footshocks, black), adult mice were trained to respond for a food pellet in an operant, spatial discrimination and reversal learning task. Following acquisition of the discrimination (left or right nose-poke hole reinforced 100% of the time) the contingencies of the responses were reversed. Mice made more total reinforced (A) and unreinforced (B) responses during reversal versus acquisition across sessions ([**] P < 0.01, main effect of training phase) but there were no effects of infant trauma exposure. (C) A separate cohort of mice exposed to infant trauma (no or 15 footshocks on PND 17) was tested for novel object recognition by assessing exploration of a novel object 1 and 24 h after exposure to the familiar object. Mice explored the novel object more ([**] P < 0.01, main effect of object, main effect of testing session, and interaction of object × testing session) but there were no effects of infant trauma exposure. Data are means ± SEM.In a new cohort of mice (no shock = 16, 15 shocks = 16), novel object recognition following infant footshock was tested. In adulthood, mice were handled for 1–3 min for two consecutive days. The following day, mice were placed in 20 × 18 × 25-cm apparatus with white floors and patterned walls (Panlab) for a 10-min habituation session to the chamber. Twenty-four hours later, mice were returned to the apparatus now containing two sample objects for 10-min initial exposure to the objects (two identical small plastic caps, 0.8 cm tall with a 2.1-cm diameter) placed in the back right and left corners of the apparatus. One hour later, mice were returned to the apparatus for a 3-min session (1-h test) where one of the sample objects was replaced with a novel object (a 2.5 × 1.2 × 1.5-cm Lego tower). The object replaced was alternated for each mouse. Mice were returned to the same box 24 h later for another 3-min session (24-h test) with the opposite cap replaced with a second novel object (a 3 × 1.5-cm black binder clip). ANY-maze software recorded each session. Time spent interacting with each object was measured by two independent raters and averaged (after Bevins and Besheer 2006).A three-way ANOVA revealed a significant main effect of object (familiar vs. novel; F_(1,60) = 59.84, P < 0.001). There was also a main effect of testing session (1-h vs. 24-h test; F_(1,60) = 7.89, P = 0.007) and an interaction of object × testing session (F_(1,60) = 11.48, P = 0.001) (Fig. 3C,D). Interrater reliability was confirmed using Pearson''s correlation (r = 0.942). These results indicate that acute infant trauma does not influence hippocampal-dependent object recognition memory.Our results demonstrate that an acute traumatic experience during infancy affects some learned behaviors during adulthood. As previously reported in rats (Quinn et al. 2014; Poulos et al. 2014), 15 footshocks on PND 17 increased adult contextual fear conditioning in mice. We also demonstrated for the first time that the infant SEFL protocol produces resistance to extinction (within-session and between-session) and that behavior in a discrimination and reversal learning task and a novel object recognition task are unaffected. These findings establish the use of infant footshock to study SEFL in mice and further support the use of this paradigm as a model of PTSD-like behavior.The effects observed here differ from those commonly observed following chronic ELS manipulations such as limited nesting and bedding (Ivy et al. 2008; Molet et al. 2016) or maternal separation (Nishi et al. 2014). Chronic ELS impairs acquisition of fear conditioning in adult rodents (Kosten et al. 2006; Stevenson et al. 2009; Lesuis et al. 2019) and performance on hippocampal-dependent memory tasks (Rice et al. 2008; Naninck et al. 2015), for example. These behavioral differences suggest that acute and chronic infant stressors alter neural circuits in unique ways that are worthy of further study.Since the tasks used here rely on distinct neural circuits, the current results provide novel insight into how acute infant trauma impacts brain function. The effects of acute ELS were restricted to fear acquisition and extinction, suggesting alterations in cortico-amygdala circuits (Tovote et al. 2015). However, preservation of novel object recognition as well as discrimination and reversal learning suggest that hippocampal and striatal circuits likely remain intact (Cohen and Stackman 2015; Izquierdo et al. 2017). These findings can guide future studies concerning the neural mechanisms of acute ELS effects on behavior.     

Deficits in acquisition and extinction of conditioned responses in mGluR7 knockout mice

Metabotropic glutamate receptor 7 (mGluR7) is expressed in brain regions implicated in emotional learning and working memory, and previous behavioral experiments indicated contributions of mGluR7 to various complex behaviors. In the present study, we investigated the specific effects of mGluR7 deletion on a variety of conditioning paradigms that model crucial neurocognitive and psychopathological behavioral phenomena. Null-mutant mGluR7^−/− mice displayed defects during scheduled appetitive conditioning, acquisition and extinction of appetitive odor conditioning, extinction of response suppression-based conditioned emotional responding (CER), acquisition of discriminative CER, and contextual fear conditioning. mGluR7^−/− animals were slower to acquire the association between a conditioned stimulus and a positive or negative reinforcer, but eventually reached similar performance levels to their wildtype littermates. Notably, extinction learning of conditioned responses was slower in mGluR7^−/− compared to wildtype animals. The observed delays in the acquisition of complicated stimulus associations across conditioning procedures may suggest a critical role for mGluR7 in neurocognitive functions and psychopathology.     

Reduced delayed-rectifier K+ current in the learning mutant rutabaga

In the Drosophila mutant rutabaga, short-term memory is deficient and intracellular cyclic adenosine monophosphate (cAMP) concentration is reduced. We characterized the delayed-rectifier potassium current (IK_DR) in rutabaga as compared with the wild-type. The conventional whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK_DR was smaller in rutabaga (368±11 pA) than in wild-type (541±14 pA) neurons, measured in a Ca²⁺-free solution. IK_DR was clearly activated at ~0 mV in the two genotypes. IK_DR typically reached its peak within 10–20 msec after the start of the pulse (60 mV). There was no difference in inactivation of IK_DR for wild-type (14±3%) and rutabaga (19±3%). After application of 10 mM TEA, in wild-type, IK_DR was reduced by 46±5%, whereas in rutabaga, IK_DR was reduced by 28±3%. Our results suggest that IK_DR is carried by two different types of channels, one which is TEA-sensitive, whereas the other is TEA-insensitive. Apparently, the TEA-sensitive channel is less expressed in rutabaga neurons than in wild-type neurons. Conceivably, altered neuronal excitability in the rutabaga mutant could disrupt the processing of neural signals necessary for learning and memory.     

Overexpression of hAPPswe impairs rewarded alternation and contextual fear conditioning in a transgenic mouse model of Alzheimer's disease

One of the hallmarks of the pathology in Alzheimer's disease is the deposition of amyloid plaques throughout the brain, especially within the hippocampus and amygdala. Transgenic mice that overexpress the Swedish mutation of human amyloid precursor protein (hAPPswe; Tg2576) show age-dependent memory deficits in hippocampus-dependent learning tasks. However, the performance of aged Tg2576 mice in amygdala-dependent learning tasks has not been thoroughly assessed. We trained young (2–4 mo) and old (16–18 mo) Tg2576 and wild-type mice in a T-maze alternation task (hippocampus-dependent) and a Pavlovian fear-conditioning task (amygdala- and hippocampus-dependent). As previously reported, old Tg2576 mice showed impaired acquisition of rewarded alternation; none of these mice reached the criterion of at least five out of six correct responses over three consecutive days. In contrast, old Tg2576 mice showed normal levels of conditional freezing to an auditory conditional stimulus (CS) and acquired a contextual discrimination normally. However, when the salience of the fear-conditioning context was decreased, old (12–14 mo) Tg2576 mice were impaired at acquiring fear to the conditioning context, but not to the tone CS. Histological examination of a subset of the mice verified the existence of amyloid plaques in the cortex, hippocampus, and amygdala of old, but not young, Tg2576 mice. Hence, learning and memory deficits in old Tg2576 mice are limited to hippocampus-dependent tasks, despite widespread amyloid deposition in cortex, hippocampus, and amygdala.     

Enhanced Hippocampal CA1 LTP but Normal Spatial Learning in Inositol 1,4,5-trisphosphate 3-kinase(A)-Deficient Mice

To define the physiological role of IP₃3-kinase(A) in vivo, we have generated a mouse strain with a null mutation of the IP₃3-kinase(A) locus by gene targeting. Homozygous mutant mice were fully viable, fertile, apparently normal, and did not show any morphological anomaly in brain sections. In the mutant brain, the IP₄ level was significantly decreased whereas the IP₃ level did not change, demonstrating a major role of IP₃3-kinase(A) in the generation of IP₄. Nevertheless, no significant difference was detected in the hippocampal neuronal cells of the wild-type and the mutant mice in the kinetics of Ca²⁺ regulation after glutamate stimulation. Electrophysiological analyses carried out in hippocampal slices showed that the mutation significantly enhanced the LTP in the hippocampal CA1 region, but had no effect on the LTP in dentate gyrus (DG). No difference was noted, however, between the mutant and the wild-type mice in the Morris water maze task. Our results indicate that IP₃3-kinase(A) may play an important role in the regulation of LTP in hippocampal CA1 region through the generation of IP₄, but the enhanced LTP in the hippocampal CA1 does not affect spatial learning and memory.     

Improvements in hippocampal-dependent learning and decremental attention in 5-HT(3) receptor overexpressing mice

The 5-HT3 receptor for serotonin is expressed within limbic structures and is known to modulate neurotransmitter release, suggesting that this receptor may influence learning and memory. Perturbations in serotonergic neurotransmission lead to changes in the ability to attend, learn, and remember. To examine the role of 5-HT3 receptors in learning, memory, and attention, 5-HT3 receptor overexpressing (5-HT3-OE) transgenic mice and their wild-type littermates (WT) were tested in Pavlovian contextual and cued fear conditioning, fear extinction, and latent inhibition (LI) paradigms. Prepulse inhibition (PPI) was assessed to reveal changes in sensorimotor gating. Additionally, anxious behaviors, shock sensitivity, and reactions to novel stimuli were evaluated. 5-HT3-OE mice displayed enhanced contextual conditioning, whereas cued conditioning remained the same as that of WT mice. 5-HT3-OE mice did not differ from WT in extinction rates to either the context or cue. LI was enhanced for 5-HT3-OE mice compared to WT. PPI remained unchanged. No differences in sensitivity to footshock or startle were found. However, 5-HT3-OE mice demonstrated heightened exploratory behavior in response to novel environmental stimuli and decreased anxiety as measured in the elevated plus-maze. Results indicate that overexpression of the 5-HT3 receptor in mouse forebrain results in enhanced hippocampal-dependent learning and attention. Enhanced inspective behavior in response to novelty may contribute to the observed improvements in learning, memory, and attention due to 5-HT3 receptor overexpression.     

Automated Measurement of Mouse Freezing Behavior and its Use for Quantitative Trait Locus Analysis of Contextual Fear Conditioning in (BALB/cJ × C57BL/6J)F2 Mice

The most commonly measured mouse behavior in fear conditioning tests is freezing. A technical limitation, particularly for genetic studies, is the method of direct observation used for quantifying this response, with the potential for bias or inconsistencies. We report the use of a computerized method based on latency between photobeam interruption measures as a reliable scoring criterion in mice. The different computer measures obtained during contextual fear conditioning tests showed high correlations with hand-scored freezing; r values ranged from 0.87 to 0.94. Previously reported strain differences between C57BL/6J and DBA/2J in context-dependent fear conditioning were also detected by the computer-based system. In addition, the use of computer-scored freezing of 199 (BALB/cJ×C57BL/6J)F₂ mice enabled us to detect a suggestive gender-dependent chromosomal locus for contextual fear conditioning on distal chromosome 8 by QTL analysis. Automation of freeze scoring would significantly increase efficiency and reliability of this learning and memory test.     

Tripartite Mushroom Body Architecture Revealed by Antigenic Markers

We have explored the organization of the axonal lobes in Drosophila mushroom bodies by using a panel of immunohistochemical markers. These markers consist of antibodies to eight proteins expressed preferentially in the mushroom bodies: DAMB, DCO, DRK, FASII, LEO, OAMB, PKA RII, and RUT. Previous to this work, four axonal lobes, two projecting dorsally (α and α′) and two medially (β and γ), had been described in Drosophila mushroom bodies. However, our analysis of immunohistochemically stained frontal and sagittal sections of the brain revealed three medially projecting lobes. The newly distinguished lobe, which we term β′, lies along the dorsal surface of β, just posterior to γ. In addition to resolving a fifth lobe, our studies revealed that there are specific lobe sets defined by equivalent marker expression levels. These sets are (1) the α and β lobes, (2) the α′ and β′ lobes, and (3) the γ lobe and heel (a lateral projection formed by a hairpin turn of some of the peduncle fibers). All of the markers we have examined are consistent with these three sets. Previous Golgi studies demonstrate that each mushroom body cell projects one axon that branches into a dorsal lobe and a medial lobe, or one unbranched axon that projects medially. Taken together with the lobe sets listed above, we propose that there are three major projection configurations of mushroom body cell axons: (1) one branch in the α and one in the β lobe, (2) one branch in the α′ and one in the β′ lobe, and (3) one unbranched axon projecting to the heel and the γ lobe. The fact that these neuron types exhibit differential expression levels of a number of mushroom body genes suggests that they may have corresponding functional differences. These functions may be conserved in the larvae, as several of these genes were expressed in larval and embryonic mushroom bodies as well. The basic mushroom body structure, including the denritic calyx, peduncle, and lobes, was already visible by the late stages of embryogenesis. With new insights into mushroom body organization, and the characterization of markers for developing mushroom bodies, we are beginning to understand how these structures form and function.