Neurocircuit differences between memory traces of persistent hypoactivity and freezing following fear conditioning among the amygdala, hippocampus, and prefrontal cortex.

We aimed to investigate the persistent trace of one traumatic event on neurocircuit controls in rats. Conditioning was reflected by reductions in rates of 'freezing' and 'other-than-freezing' motor activities, between which rats could alternate on delivery of pulsed footshocks of intensity 0.5 mA but not 1.0 mA. At the latter intensity, freezing began to suppress motor activity. The conditional responses evident during both the context and tone sessions persisted when the tests were repeated on post-conditioning days 7 and 8. Thus, difficulties with fear extinction/reduction remained. However, persistence was not evident on post-conditioning days 1 and 2. One day after the 1.0 mA pulsed footshock, ibotenate lesions and corresponding sham surgeries were performed in unilateral and bilateral hemispheres of the amygdala, hippocampus, and prefrontal cortex, as well as three different disconnections (one unilateral and another contralateral lesions out of three regions, a total of nine groups), and were tested on days 7-8. The drastic restoration of freezing following bilateral amygdala lesions was also evident in animals with three types of disconnection; however, this was not the case for hypoactivity. These results imply that a serious experience can drive different neurocircuits that all involve the amygdala, forming persistent concurrent memories of explicit (e.g., 'freezing') or implicit (e.g., 'other-than-freezing' motor activity) emotions, which may exhibit mutual interference.

Early weaning augments the spontaneous release of dopamine in the amygdala but not the prefrontal cortex: an in vivo microdialysis study of male rats.

Our early weaning schedule was associated with the emergence of trait anxiety in male rodents performing an elevated plus maze but not an open-field test. We previously reported that early weaning weakened excitatory neurotransmission to the amygdala from the prefrontal cortex, where the mesocorticolimbic dopaminergic (DAergic) fiber terminates on each. In this study, we investigated DAergic transmission in both these brain regions. The extracellular levels of amygdalar DA in adulthood were two times higher in rats weaned at 16 days compared to those weaned at 30 days in both the home cage and the open-field. This difference in extracellular DA levels was not apparent in the prefrontal cortex. The concurrently measured locomotor and rearing behaviors did not vary according to the weaning period and the probe-implanted region, respectively. These results suggest that the effects of early weaning on DA tone appear to be specific to the amygdala and do not represent ubiquitous upregulation as these changes were not observed in the prefrontal cortex.

The Effects of Housing Density on Social Interactions and Their Correlations with Serotonin in Rodents and Primates.

Population density has been suggested to affect social interactions of individuals, but the underlying neural mechanisms remain unclear. In contrast, neurotransmission of monoamines such as serotonin (5-HT) and dopamine (DA) has been demonstrated to play important roles in social behaviors. Here, we investigated whether housing density affected social interactions of rodents and non-human primates housed in groups, and its correlations with monoamines. Japanese macaques exhibited higher plasma 5-HT, but not DA, concentrations than rhesus macaques. Similarly, C57BL/6 mice exhibited higher plasma and brain tissue 5-HT concentrations than DBA2 mice. Under crowding, C57BL/6 mice and Japanese macaques exhibited more prominent social avoidance with mates than DBA2 mice and rhesus macaques, respectively. Although DBA2 mice and rhesus macaques in crowding exhibited elevated plasma stress hormones, such stress hormone elevations associated with crowding were absent in C57BL/6 mice and Japanese macaques. Administration of parachlorophenylalanine, which inhibits 5-HT synthesis, increased social interactions and stress hormones in C57BL/6 mice under crowding. These results suggest that, animals with hyperserotonemia may exhibit social avoidance as an adaptive behavioral strategy to mitigate stress associated with crowding environments, which may also be relevant to psychiatric disorder such as autism spectrum disorder.

Early weaning influences short-term synaptic plasticity in the medial prefrontal-anterior basolateral amygdala pathway.

Early weaning in rodents reportedly influences behavioral and emotional traits and triggers precocious myelin formation in the anterior basolateral amygdala (aBLA; Ono et al., 2008), where prefrontal efferents terminate. We studied the correlation between behavior and the synaptic properties of the prefrontal-aBLA pathway. Open-field behaviors of adult male rats weaned at either 16 days or 30 days were measured on two consecutive days. On the first day, the rats received a slight footshock that was reportedly insufficient for fear conditioning. Electrophysiological recordings in the prefrontal-aBLA were then performed under urethane anesthesia. Without group differences in the stimulus intensity or the first evoked response, the overall paired-pulse facilitation was significantly lower in the early-weaned group from 25 to 100 ms. At the 25-ms interval, regression values between paired-pulse facilitation and locomotion on the second day were positive/insignificant and negative/significant in early- and control-weaned groups, respectively, and were statistically different between the groups.

Brain Rewarding Stimulation Reduces Extracellular Glutamate Through Glial Modulation in Medial Prefrontal Cortex of Rats.

Growing evidence implicates a critical involvement of prefrontal glial modulation of extracellular glutamate (GLU) in aversive behaviors. However, nothing is known about whether prefrontal glial cells modulate GLU levels in rewarding behaviors. To address this question, we measured GLU efflux in the medial prefrontal cortex (PFC) of rats associated with rewarding behaviors. We used intracranial self-stimulation (ICSS) of the medial forebrain bundle (MFB) as the rewarding behavior. GLU was indirectly measured using microdialysis combined with on-line fluorometric detection of NADH resulting from the reaction of GLU and NAD(+) catalyzed by GLU dehydrogenase with a time resolution of 1 min. ICSS caused a minute-by-minute change of extracellular GLU in the medial PFC, with a slight decrease during the stimulation, followed by an increase afterward. This bidirectional change was tetrodotoxin insensitive and abolished by the gliotoxin fluorocitrate. To confirm and extend the previous studies of aversion-induced increase of extracellular GLU in the medial PFC, we also measured prefrontal GLU efflux associated with an aversive stimulation, immobilization stress. The temporal change in extracellular GLU caused by this stress was markedly different from that observed during ICSS. A rapid increase in GLU was detected during the aversive stimulation, followed by a large increase afterward. This bimodal change was tetrodotoxin insensitive, similar to that detected for ICSS. These findings indicate a bidirectional regulation of extracellular GLU by prefrontal glial cells associated with rat ICSS behavior, and reveal that glial modulation of GLU neurochemistry in the medial PFC contributes to rewarding as well as aversive behaviors in rats.

Enigma homolog 1 promotes myogenic gene expression and differentiation of C2C12 cells.

The Enigma homolog (ENH) gene generates several splicing variants. The initially identified ENH1 possesses one PDZ and three LIM domains, whereas ENH2~4 lack the latter domains. The splicing switch from ENH1 to LIM-less ENHs occurs during development/maturation of skeletal and heart muscles. We examined for the roles of ENH splicing variants in muscle differentiation using C2C12 cells. Cells stably expressing ENH1 exhibited significantly higher MyoD and myogenin mRNA levels before differentiation and after 5 days in low serum-differentiating medium than mock-transfected cells. ENH1-stable transformants also retained the ability to exhibit elongated morphology with well-extended actin fibers following differentiation. In contrast, cells stably expressing ENH3 or ENH4 did not show myotube-like morphology or reorganization of actin fibers following culture in the differentiating medium. Transient overexpression of ENH1 using adenovirus supported the increased expression of muscle marker mRNAs and the formation of well-organized stress fibers, whereas ENH4 overexpression prevented these morphological changes. Furthermore, specific suppression of ENH1 expression by RNAi caused a significant reduction in MyoD mRNA level and blocked the morphological changes. These results suggest that ENH1 with multiple protein-protein interaction modules is essential for differentiation of striated muscles, whereas ectopic expression of LIM-less ENH disrupts normal muscle differentiation.

Functional structure of the intermediate and ventral hippocampo-prefrontal pathway in the prefrontal convergent system.

The hippocampo-prefrontal pathway is a unique projection that connects distant ends of the cerebral cortex. The direct hippocampo-prefrontal projection arises from the ventral to intermediate third of the hippocampus, but not from the dorsal third. It forms a funnel-shaped structure that collects information from the large hippocampal area and projects it to the prefrontal cortex. The anatomical regional differentiation of the projection has not been described. The hippocampal region is differentiated into structural and behavioural roles. For example, it has been shown that the ventral, but not the dorsal, hippocampus reciprocally connects with the amygdala and influences emotional behaviours. These data imply that hippocampal variation along the dorso-ventral axis is contained within the hippocampo-prefrontal pathway. Here, we present electrophysiological studies that demonstrate regional differences in short- but not long-term plasticity in the intermediate/posterior-dorsal and ventral routes of the hippocampo-prefrontal pathway. Furthermore, behavioural studies revealed that each route appears to play a different role in working memory. These results suggest that hippocampal regional information is processed through different routes, with the integration of individual regulatory functions in the prefrontal convergent system.

Synaptic plasticity dynamics in the hippocampal-prefrontal pathway in vivo.

We examined the effect of long-term potentiation (LTP) on paired-pulse responses across varied stimulus intensities and interstimulus intervals (ISIs), at ascending synapses from the intermediate and ventral hippocampus to the medial prefrontal cortex in urethane-anesthetized rats. LTP significantly shifted the median effective stimulus towards lower intensities in the intermediate route, and increased at 25-ms ISI the paired-pulse response, which was inversely proportional to the stimulus intensity. In the ventral route, the paired-pulse response varied with ISI rather than intensity, and increased at 50-ms and 100-ms ISI after LTP. The intermediate synaptic plasticity significantly exhibited total amplifier dynamics with wide ranges of frequency at lower intensity and intensity at 100-ms ISI in contrast to the ventral one.

Motion classification using epidural electrodes for low-invasive brain-machine interface.

Brain-machine interfaces (BMIs) are expected to be used to assist seriously disabled persons' communications and reintegrate their motor functions. One of the difficult problems to realize practical BMI is how to record neural activity clearly and safely. Conventional invasive methods require electrodes inside the dura mater, and noninvasive methods do not involve surgery but have poor signal quality. Thus a low-invasive method of recording is important for safe and practical BMI. In this study, the authors used epidural electrodes placed between the skull and dura mater to record a rat's neural activity for low-invasive BMI. The signals were analyzed using a short-time Fourier transform, and the power spectra were classified into rat motions by a support vector machine. Classification accuracies were up to 96% in two-class discrimination, including that when the rat stopped, walked, and rested. The feasibility of a low-invasive BMI based on an epidural neural recording was shown in this study.

Specific role of the posterior dorsal hippocampus-prefrontal cortex in short-term working memory.

Working memory in rats involves neural projections from the hippocampus (HP) to the prefrontal cortex (PFC), based on delayed task experiments in a radial-arm maze, in which the time span of working memory is longer than seconds. To determine whether the HP-PFC pathway is involved in short-term (on the order of seconds) working memory function, we lesioned the PFC and/or HP, and measured performance in an operant delayed alternation task. The posterior dorsal (pdHP) and ventral HP (vHP) were assessed separately. The bilateral PFC and bilateral pdHP ibotenate lesions produced significant working memory deficits, but the vHP lesion did not. Unilateral pdHP lesions combined with a PFC lesion in the opposite hemisphere reproduced the effects of bilaterally symmetrical lesions. By contrast, unilateral lesions of the pdHP combined with a PFC lesion in the same hemisphere had no effect on delayed alternation. These results indicate that the pdHP-PFC pathway is essential for working memory on the order of seconds in rats, and suggest that the pdHP and vHP pathways to the PFC play different behavioral roles.

In vivo temporal property of GABAergic neural transmission in collateral feed-forward inhibition system of hippocampal-prefrontal pathway.

Anatomical evidence suggests that rat CA1 hippocampal afferents collaterally innervate excitatory projecting pyramidal neurons and inhibitory interneurons, creating a disynaptic, feed-forward inhibition microcircuit in the medial prefrontal cortex (mPFC). We investigated the temporal relationship between the frequency of paired synaptic transmission and gamma-aminobutyric acid (GABA)ergic receptor-mediated modulation of the microcircuit in vivo under urethane anesthesia. Local perfusions of a GABAa antagonist (-)-bicuculline into the mPFC via microdialysis resulted in a statistically significant disinhibitory effect on intrinsic GABA action, increasing the first and second mPFC responses following hippocampal paired stimulation at interstimulus intervals of 100-200 ms, but not those at 25-50 ms. This (-)-bicuculline-induced disinhibition was compensated by the GABAa agonist muscimol, which itself did not attenuate the intrinsic oscillation of the local field potentials. The perfusion of a sub-minimal concentration of GABAb agonist (R)-baclofen slightly enhanced the synaptic transmission, regardless of the interstimulus interval. In addition to the tonic control by spontaneous fast-spiking GABAergic neurons, it is clear the sequential transmission of the hippocampal-mPFC pathway can phasically drive the collateral feed-forward inhibition system through activation of a GABAa receptor, bringing an active signal filter to the various types of impulse trains that enter the mPFC from the hippocampus in vivo.

Cooperativity between hippocampal-prefrontal short-term plasticity through associative long-term potentiation.

The hippocampal-medial prefrontal cortex (mPFC) pathway provides highly convergent input to the mPFC in rats and shows two types of short-term plasticity in terms of paired-pulse facilitation (PPF) of the field potential under urethane anesthesia. We now report that stimulating either the dorsal or ventral subregions of the posterior hippocampus elicited PPF (by about 335 and 120%, respectively) of field potentials recorded in the mPFC at 100 ms interpulse interval. This PPF-like interaction occurred when projections were stimulated in the ventral-dorsal order (by about 200% of the single-pulsed response), but not vice versa. When weak long-term potentiation (LTP) of the dorsal projection was evoked simultaneously with strong LTP of the ventral projection, an associative effect was revealed (about +55%), although the magnitudes of LTP in each projection were not correlated. Even when the impermutable PPF-like facilitation was further enhanced (by about +120%), the enhancement was not correlated with either form of LTP, but exhibited the interaction of changes in the dorsal PPF, rather than in the heterotopic priming effect through the ventral projection. Moreover, this change was correlated with the associated LTP ratio of dorsal to ventral projection LTP (i.e., LTP associativity). Larger increases in LTP associativity correlated with greater impermutable PPF-like facilitation; in addition, there was hardly attenuation of the response to the dorsal projection by subsequent electrolytic lesions of the ventral subregion. These results indicate that the mPFC functionally integrates discrete sources of hippocampal information via cooperativity between short- and long-term plasticity.

Extracellular level of basolateral amygdalar dopamine responding to reversal of appetitive-conditioned discrimination in young and old rats.

Young and old rats, aged 3 and 24 months old, respectively, were conditioned to press a lever under lamp-on conditions for reward acquisition and lamp-off for no reward using a variable interval reinforcement schedule that averaged 15 s; i.e., the minimal requirement was 4 responses/min. Over a 30-day period, young and old groups showed increased response to lamp-on from 22 to 35/min and from 10 to 23/min, respectively, and shortened response to lamp-off after initial training. Response to lamp-on as a percentage of total response to lamp-on and -off (the discrimination ratio) was over 80%. For the next 30 days, reversal learning was imposed to reinforce discrimination of the lamp-off state. Young rats showed a steadily increasing discrimination ratio from 40% to 70%, and old rats from 30% to 60%. In comparison with the initial training, young rats showed a total response increase from 50% to 60%, while old rats showed an approximately 5% decrease without loss of reward-obtaining efficiency. In vivo microdialysis during reversal revealed that young rats had higher dopamine transmission in the basolateral amygdala than old rats. The dopamine level was positively correlated with the number of responses to state of reward in young rats and negatively with the numbers of both NRF and RF responses to lamp-on and -off states in old rats. These results suggest that in reversal discrimination, basolateral amygdalar dopamine efflux correlates with the manner of age-related conditioned response rather than the ability to learn.

Effects of ventral hippocampal long-term potentiation and depression on the gamma-band local field potential in anesthetized rats.

We examined the effect of long-term potentiation or depression (LTP or LTD) on the local field potential, focusing on the gamma-band (40-100 Hz) power, in the ventral hippocampus CA1 of anesthetized rats. LTP and LTD induction in the CA3-CA1 pathway increased the CA1 spontaneous gamma-band power by around 40 and 80-100 Hz, respectively, while neither changed the evoked levels significantly. These results suggest that the ventral CA1 local field potential can maintain bidirectional plasticity in the steady state for the long term. Given the involvement of synaptic plasticity in learning and memory, the gamma-band power change associated with LTP/LTD may relate to ventral hippocampal functions. The LTP increased the spontaneous power at around 40 Hz of the gamma-band frequency in the ventral CA1, and the LTD did the same at 80-100 Hz. The biphasic increase may distribute the subsequent input appropriately to regulate the relevant synaptic history in the ventral CA1 and anatomically related structures in vivo.

In vivo calcium imaging of cerebral cortex in hypoxia-ischemia followed by developmental stage-specific injury in rats.

The parietal area is a part of the cortex that is vulnerable in the rat to hypoxia-ischemia (HI) within the early postnatal period. To investigate the localizing mechanism of this cortical injury, we spatiotemporally detected the cortical intracellular calcium changes, as revealed by a calcium-sensitive fluorescence dye, Rhod 2-AM, during 1h of HI on postnatal days 7-21 in vivo. The calcium level rose to different levels at different cortical points in all animals within the first 20 min. Over the whole cortical area in the camera field, the changes in three groups significantly differed across time at 30 and 60 min, and a chronic increase appeared at days 7-8. After 3h of reperfusion, microtubule-associated protein 2 (MAP-2) immunoreactivity confirmed that parietal injury was more serious at day 7, whereas the imaging of calcium distribution did not segregate the injured and uninjured areas. Our in vivo findings in the whole brain structure indicate that the age-specific vulnerability of the parietal cortex injury is affected indirectly by the chronic increase in the late HI phase in the early postnatal period, suggesting that each cortical area differs postnatally with respect to the development of calcium regulation and signal transduction involving neural cell death and/or survival.

Differences between paired-pulse facilitation and long-term potentiation in the dorsal and ventral hippocampal CA1-prefrontal pathways of rats.

We studied the interaction between paired-pulse facilitation (PPF) and long-term potentiation (LTP) in the hippocampo-prefrontal cortex (PFC, prelimbic area) pathway, stimulating the ventral or posterior dorsal CA1 region (vCA1 or pdCA1). In the vCA1-PFC, the group averaged PPF did not change after the LTP induction, and there was a negative correlation between the post-LTP PPF change and LTP magnitude. In contrast, the post-LTP PPF of the pdCA1-PFC appeared to decrease significantly, and the PPF change was independent of the LTP magnitude. We found that there were at least two mechanisms of PPF regulation following LTP induction in the pathway resulting from extensive CA1 projections into the prelimbic area. The results imply that the CA1-PFC pathway regulates the PFC PPF quantitatively in LTP-dependent and independent manners, which depend on the local properties of the CA1 regions.

Compatibility of bidirectional synaptic plasticity on hippocampo-prefrontal cortex pathway in rats.

The hippocampo-prefrontal cortex pathway reportedly expresses long-term potentiation (LTP) and depression (LTD) in anesthetized rats. We examined whether there were any effects governing the induction of LTD after prior induction of LTP, or vice versa. Induction in sequence of LTP and LTD resulted in significantly stable changes of about 140 and 70% of a common control for 1 h each. The reversed sequence, LTD and LTP, showed a mirror image of about 65 and 135% of control, which were not different from the respective changes in the first sequence (P>0.3 for each). The correlation coefficient between changes was significantly positive in the first sequence and weakly negative in the reverse. These results indicate that this pathway can express compatibility of bidirectional synaptic plasticity while historical changes remain covert.

Effects of hippocampus-induced prefrontal long-term depression on gamma-band local field potential in anesthetized rats.

To determine whether long-term depression (LTD) affects cortical gamma-band local field potential (40-100 Hz), we conducted a LTD induction experiment in the hippocampus-prefrontal cortex (PFC) pathway of an anesthetized rat. The LTD induction increased the spontaneous level of PFC gamma-band power of 70-100 Hz, which was not affected after the long-term potentiation (LTP) induction in our previous experiment. In addition, the LTD induction increased the evoked PFC gamma-band power at 900 ms after hippocampal test stimulation; this latency appeared to differ from that (500-700 ms) observed in our previous LTP experiment. The results indicate that the PFC field potential increases its gamma-band power following both LTP and LTD in the hippocampus-PFC pathway, which is involved in working memory. Particularly, the sustained increase by LTD may reflect a representation of working memory.

Local properties of CA1 region in hippocampo-prefrontal synaptic plasticity in rats.

We studied paired-pulse facilitation and long-term potentiation/depression in anesthetized rats to determine whether the hippocampal CA1 region inhibits local differences in short-term and long-term synaptic plasticity in its projections to the prefrontal cortex. We compared projections with the PFC from the posterior dorsal and ventral hippocampal CA1 regions (pdCA1 and vCA1 respectively). The two pathways displayed similar properties. However, the PPF properties of the pdCA1, projections differed dramatically from those of the pdCA1 projections. The pdCA1 projections showed the opposite of facilitation (i.e. suppression) at 25-50 ms intervals and more pronounced facilitation at 100-400 ms intervals. These results suggest that there are functional differences between these pathways.