Diminished Fear Extinction in Adolescents Is Associated With an Altered Somatostatin Interneuron-Mediated Inhibition in the Infralimbic Cortex.

BACKGROUND: Rodents and humans show an attenuation of fear extinction during adolescence, which coincides with the onset of several psychiatric disorders. Although the ethological relevance and the underlying mechanism are largely unknown, the suppression of fear extinction during adolescence is associated with a diminished plasticity in the glutamatergic neurons of the infralimbic medial prefrontal cortex, a brain region critical for fear extinction. Given the putative effect of synaptic inhibition on glutamatergic neuron activity, we studied whether gamma-aminobutyric acidergic neurons in the infralimbic medial prefrontal cortex are involved in the suppression of fear extinction during adolescence. METHODS: We assessed membrane and synaptic properties in parvalbumin-positive interneurons (PVINs) and somatostatin-positive interneurons (SSTINs) in male preadolescent, adolescent, and adult mice. The effect of fear conditioning and extinction on PVIN-pyramidal neuron and SSTIN-pyramidal neuron synapses in male preadolescent, adolescent, and adult mice was evaluated using an optogenetic approach. RESULTS: The development of the membrane excitability of PVINs is delayed and reaches maturity only by adulthood, while the SSTIN membrane properties are developed early and remain stable during development from preadolescence to adulthood. Although the synaptic inhibition mediated by PVINs undergoes a protracted development, it does not exhibit a fear behavior-specific plasticity. However, the synaptic inhibition mediated by SSTINs undergoes an adolescence-specific enhancement, and this increased inhibition is suppressed by fear learning but is not restored by extinction training. This altered plasticity during adolescence overlapped with a reduction in calcium-permeable glutamate receptors in SSTINs. CONCLUSIONS: The adolescence-specific plasticity in the SSTINs might play a role in fear extinction suppression during adolescence in mice.

Lack of experience-dependent intrinsic plasticity in the adolescent infralimbic medial prefrontal cortex.

Fear extinction, an inhibitory learning that suppresses a previously learned fear memory, is diminished during adolescence. Earlier studies have shown that this suppressed fear extinction during adolescence involves an altered glutamatergic plasticity in infralimbic medial prefrontal cortical (IL-mPFC) pyramidal neurons. However, it is unclear whether the excitability of IL-mPFC pyramidal neurons plays a role in this development-dependent suppression of fear extinction. Therefore, we examined whether fear conditioning and extinction affect the active and passive membrane properties of IL-mPFC layer 5 pyramidal neurons in preadolescent, adolescent and adult mice. Both preadolescent and adult mice exhibited a bidirectional modulation of the excitability of IL-mPFC layer 5 pyramidal neurons following fear conditioning and extinction, i.e., fear conditioning reduced membrane excitability, whereas fear extinction reversed this effect. However, the fear conditioning-induced suppression of excitability was not reversed in adolescent mice following fear extinction training. Neither fear conditioning nor extinction affected GABAergic transmission in IL-mPFC layer 5 pyramidal neurons, suggesting that GABAergic transmission did not play a role in experience-dependent modulation of neuronal excitability. Our results suggest that the extinction-specific modulation of excitability is impaired during adolescence.

A Neural Circuit Mechanism for Encoding Aversive Stimuli in the Mesolimbic Dopamine System.

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT.

Infralimbic prefrontal cortex structural and functional connectivity with the limbic forebrain: a combined viral genetic and optogenetic analysis.

The medial prefrontal cortex is critical for contextual appraisal, executive function, and goal-directed behavior. Additionally, the infralimbic (IL) subregion of the prefrontal cortex has been implicated in stress responding, mood, and fear memory. However, the specific circuit mechanisms that mediate these effects are largely unknown. To date, IL output to the limbic forebrain has been examined largely qualitatively or within a restricted number of sites. To quantify IL presynaptic input to structures throughout the forebrain, we utilized a lentiviral construct expressing synaptophysin-mCherry. Thus, allowing quantification of IL efferents that are specifically synaptic, as opposed to fibers of passage. Additionally, this approach permitted the determination of IL innervation on a sub-structural level within the multiple heterogeneous limbic nuclei. To examine the functional output of the IL, optogenetic activation of IL projections was followed by quantification of neuronal activation throughout the limbic forebrain via fos-related antigen (Fra). Quantification of synaptophysin-mCherry indicated that the IL provides robust synaptic input to a number of regions within the thalamus, hypothalamus, amygdala, and bed nucleus of the stria terminalis, with limited input to the hippocampus and nucleus accumbens. Furthermore, there was high concordance between structural connectivity and functional activation. Interestingly, some regions receiving substantial synaptic input did not exhibit significant increases in Fra-immunoreactivity. Collectively, these studies represent a step toward a comprehensive and quantitative analysis of output circuits. This large-scale efferent quantification or 'projectome' also opens the door for data-driven analyses of the downstream synaptic mechanisms that mediate the integrative aspects of cortico-limbic interactions.

[A short essay on the spirituality of placebo from an (evolutionary) psychiatric perspective]. / Ein kurzes Essay über die Spiritualität von Placebo aus (evolutionär) psychiatrischer Sicht.

Different to spirituality, the placebo-effect is well operationalized. Against this background, an attempt is made to look at a possible phenomenological relationship between the therapeutic effectiveness of spirituality and placebo. Similar context influences as well as the possible common use of a phylogenetically well conserved protective route via the mesolimbic dopaminergic reward system are highlighted. For both phenomena, the clinical effectiveness seems to be ubiquitous, with that of the placebo effect being scientifically much more verified than this currently would be methodologically possible with the "spirituality effect". Both effects share their uncertain predictability and are Janus-headed (e. g., placebo effect vs. nocebo effect). Currently, for both, the placebo and spirituality-oriented approaches, there are attempts underway to maximize their clinical effectiveness by modulating the therapeutic framework and conversational content. The discussion ends with the reflexive question of whether the placebo effect could have in essence "spirituality-light" traits.

Sexual rejection via a vomeronasal receptor-triggered limbic circuit.

Mating drive is balanced by a need to safeguard resources for offspring, yet the neural basis for negative regulation of mating remains poorly understood. In rodents, pheromones critically regulate sexual behavior. Here, we observe suppression of adult female sexual behavior in mice by exocrine gland-secreting peptide 22 (ESP22), a lacrimal protein from juvenile mice. ESP22 activates a dedicated vomeronasal receptor, V2Rp4, and V2Rp4 knockout eliminates ESP22 effects on sexual behavior. Genetic tracing of ESP22-responsive neural circuits reveals a critical limbic system connection that inhibits reproductive behavior. Furthermore, V2Rp4 counteracts a highly related vomeronasal receptor, V2Rp5, that detects the male sex pheromone ESP1. Interestingly, V2Rp4 and V2Rp5 are encoded by adjacent genes, yet couple to distinct circuits and mediate opposing effects on female sexual behavior. Collectively, our study reveals molecular and neural mechanisms underlying pheromone-mediated sexual rejection, and more generally, how inputs are routed through olfactory circuits to evoke specific behaviors.

Postnatal TrkB ablation in corticolimbic interneurons induces social dominance in male mice.

The tight balance between synaptic excitation and inhibition (E/I) within neocortical circuits in the mammalian brain is important for complex behavior. Many loss-of-function studies have demonstrated that brain-derived neurotrophic factor (BDNF) and its cognate receptor tropomyosin receptor kinase B (TrkB) are essential for the development of inhibitory GABAergic neurons. However, behavioral consequences of impaired BDNF/TrkB signaling in GABAergic neurons remain unclear, largely due to confounding motor function deficits observed in previous animal models. In this study, we generated conditional knockout mice (TrkB cKO) in which TrkB was ablated from a majority of corticolimbic GABAergic interneurons postnatally. These mice showed intact motor coordination and movement, but exhibited enhanced dominance over other mice in a group-housed setting. In addition, immature fast-spiking GABAergic neurons of TrkB cKO mice resulted in an E/I imbalance in layer 5 microcircuits within the medial prefrontal cortex (mPFC), a key region regulating social dominance. Restoring the E/I imbalance via optogenetic modulation in the mPFC of TrkB cKO mice normalized their social dominance behavior. Taken together, our results provide strong evidence for a role of BDNF/TrkB signaling in inhibitory synaptic modulation and social dominance behavior in mice.

Altering gain of the infralimbic-to-accumbens shell circuit alters economically dissociable decision-making algorithms.

The nucleus accumbens shell (NAcSh) is involved in reward valuation. Excitatory projections from infralimbic cortex (IL) to NAcSh undergo synaptic remodeling in rodent models of addiction and enable the extinction of disadvantageous behaviors. However, how the strength of synaptic transmission of the IL-NAcSh circuit affects decision-making information processing and reward valuation remains unknown, particularly because these processes can conflict within a given trial and particularly given recent data suggesting that decisions arise from separable information-processing algorithms. The approach of many neuromodulation studies is to disrupt information flow during on-going behaviors; however, this limits the interpretation of endogenous encoding of computational processes. Furthermore, many studies are limited by the use of simple behavioral tests of value which are unable to dissociate neurally distinct decision-making algorithms. We optogenetically altered the strength of synaptic transmission between glutamatergic IL-NAcSh projections in mice trained on a neuroeconomic task capable of separating multiple valuation processes. We found that induction of long-term depression in these synapses produced lasting changes in foraging processes without disrupting deliberative processes. Mice displayed inflated reevaluations to stay when deciding whether to abandon continued reward-seeking investments but displayed no changes during initial commitment decisions. We also developed an ensemble-level measure of circuit-specific plasticity that revealed individual differences in foraging valuation tendencies. Our results demonstrate that alterations in projection-specific synaptic strength between the IL and the NAcSh are capable of augmenting self-control economic valuations within a particular decision-making modality and suggest that the valuation mechanisms for these multiple decision-making modalities arise from different circuits.

Excitatory connections between the prelimbic and infralimbic medial prefrontal cortex show a role for the prelimbic cortex in fear extinction.

We elucidated the intrinsic circuitry of the medial prefrontal cortex and its role in regulating fear extinction, using neuronal tracing and optogenetic stimulation in vitro and in vivo. We show that pyramidal neurons in layer 5/6 of the prelimbic medial prefrontal cortex project to pyramidal cells in layer 5/6 of the infralimbic cortex. Activation of this connection enhances fear extinction, redefining the role of the prelimbic cortex in extinction learning.

Chemogenetic Excitation of Accumbens-Projecting Infralimbic Cortical Neurons Blocks Toluene-Induced Conditioned Place Preference.

Abuse rates for inhalants among adolescents continue to be high, yet preclinical models for studying mechanisms underlying inhalant abuse remain limited. Our laboratory has previously shown that, in male rats, an acute binge-like exposure to toluene vapor that mimics human solvent abuse modifies the intrinsic excitability of mPFC pyramidal neurons projecting to the NAc. These changes showed region (infralimbic; IL vs prelimbic; PRL), layer (shallow; 2/3 vs deep; 5/6), target (core vs shell), and age (adolescent vs adult) dependent differences (Wayman and Woodward, 2017). To expand these findings using reward-based models that may better mimic human drug abuse, we used whole-cell electrophysiology and drug receptors exclusively activated by designer drugs to examine changes in neuronal function and behavior in rats showing a conditioned place preference (CPP) to toluene. Repeated pairings of adolescent rats to binge concentrations of toluene vapor previously shown to enhance dopamine release in reward-sensitive areas of the brain produced CPP that persisted for 7 but not 30 d. Toluene-induced CPP was associated with increased excitability of IL5/6 mPFC neurons projecting to the core of the NAc and reduced excitability of those projecting to the NAc shell. No changes in PRL-NAc-projecting neurons were found in toluene-CPP rats. Chemogenetic reversal of the toluene-induced decrease in IL5/6-NAc shell neurons blocked the expression of toluene-induced CPP while manipulating IL5/6-NAc core neuron activity had no effect. These data reveal that alterations in selective mPFC-NAc pathways are required for expression of toluene-induced CPP.SIGNIFICANCE STATEMENT Disturbed physiology of pyramidal neurons projecting from the mPFC to the NAc has been shown to have different roles in drug-seeking behaviors for a number of drugs (e.g., methamphetamine, cocaine, ecstasy, alcohol, heroin). Here, we report that rats repeatedly exposed to the volatile organic solvent toluene, a member of the class of abused inhalants often used for intoxicating purposes by adolescents, induces a preference for the drug-paired environment that is accompanied by altered physiology of a specific population of NAc-projecting mPFC neurons. Chemogenetic correction of this deficit before testing prevented expression of drug preference. Overall, these findings highlight the importance of corticolimbic circuitry in mediating the rewarding properties of abused inhalants.

Neuroanatomical Distribution of DEK Protein in Corticolimbic Circuits Associated with Learning and Memory in Adult Male and Female Mice.

DEK, a chromatin-remodeling gene expressed in most human tissues, is known for its role in cancer biology and autoimmune diseases. DEK depletion in vitro reduces cellular proliferation, induces DNA damage subsequently leading to apoptosis, and down-regulates canonical Wnt/ß-catenin signaling, a molecular pathway essential for learning and memory. Despite a recognized role in cancer (non-neuronal) cells, DEK expression and function is not well characterized in the central nervous system. We conducted a gene ontology analysis (ToppGene), using a cancer database to identify genes associated with DEK deficiency, which pinpointed several genes associated with cognitive-related diseases (i.e., Alzheimer's disease, presenile dementia). Based on this information, we examined DEK expression in corticolimbic structures associated with learning and memory in adult male and female mice using immunohistochemistry. DEK was expressed throughout the brain in both sexes, including the medial prefrontal cortex (prelimbic, infralimbic and dorsal peduncular). DEK was also abundant in all amygdalar subdivisions (basolateral, central and medial) and in the hippocampus including the CA1, CA2, CA3, dentate gyrus (DG), ventral subiculum and entorhinal cortex. Of note, compared to males, females had significantly higher DEK immunoreactivity in the CA1, indicating a sex difference in this region. DEK was co-expressed with neuronal and microglial markers in the CA1 and DG, whereas only a small percentage of DEK cells were in apposition to astrocytes in these areas. Given the reported inverse cellular and molecular profiles (e.g., cell survival, Wnt pathway) between cancer and Alzheimer's disease, these findings suggest a potentially important role of DEK in cognition.

Infant avoidance training alters cellular activation patterns in prefronto-limbic circuits during adult avoidance learning: II. Cellular imaging of neurons expressing the activity-regulated cytoskeleton-associated protein (Arc/Arg3.1).

Positive and negative feedback learning is essential to optimize behavioral performance. We used the two-way active avoidance (TWA) task as an experimental paradigm for negative feedback learning with the aim to test the hypothesis that neuronal ensembles activate the activity-regulated cytoskeletal (Arc/Arg3.1) protein during different phases of avoidance learning and during retrieval. A variety of studies in humans and other animals revealed that the ability of aversive feedback learning emerges postnatally. Our previous findings demonstrated that rats, which as infants are not capable to learn an active avoidance strategy, show improved avoidance learning as adults. Based on these findings, we further tested the hypothesis that specific neuronal ensembles are "tagged" during infant TWA training and then reactivated during adult re-exposure to the same learning task. Using cellular imaging by immunocytochemical detection of Arc/Arg3.1, we observed that, compared to the untrained control group, (1) only in the dentate gyrus the density of Arc/Arg3.1-expressing neurons was elevated during the acquisition phase of TWA learning, and (2) this increase in Arc/Arg3.1-expressing neurons was not specific for the TWA learning task. With respect to the effects of infant TWA training we found that compared to the naïve non-pretrained group (a) the infant pretraining group displayed a higher density of Arc/Arg3.1-expressing neurons in the anterior cingulate cortex during acquisition on training day 1, and (b) the infant pretraining group displayed elevated density of Arc/Arg3.1-expressing neurons in the dentate gyrus during retrieval on test day 5. Correlation analysis for the acquisition phase revealed for the ACd that the animals which showed the highest number of avoidances and the fastest escape latencies displayed the highest density of Arc/Arg3.1-expressing neurons. Taken together, we are the first to use the synaptic plasticity protein Arc/Arg3.1 to label neuronal ensembles which are involved in different phases of active avoidance learning and whose activity patterns are changing in response to previous learning experience during infancy. Our results indicate (1) that, despite the inability to learn an active avoidance response in infancy, lasting memory traces are formed encoding the subtasks that are learned in infancy (e.g., the association of the CS and UCS, escape strategy), which are encoded in the infant brain by neuronal ensembles, which alter their synaptic connectivity via activation of specific synaptic plasticity proteins such as Arc/Arg3.1 and Egr1, and (2) that during adult training these memories can be retrieved by reactivating these neuronal ensembles and their synaptic circuits and thereby accelerate learning.

Mirror trends of plasticity and stability indicators in primate prefrontal cortex.

Research on plasticity markers in the cerebral cortex has largely focused on their timing of expression and role in shaping circuits during critical and normal periods. By contrast, little attention has been focused on the spatial dimension of plasticity-stability across cortical areas. The rationale for this analysis is based on the systematic variation in cortical structure that parallels functional specialization and raises the possibility of varying levels of plasticity. Here, we investigated in adult rhesus monkeys the expression of markers related to synaptic plasticity or stability in prefrontal limbic and eulaminate areas that vary in laminar structure. Our findings revealed that limbic areas are impoverished in three markers of stability: intracortical myelin, the lectin Wisteria floribunda agglutinin, which labels perineuronal nets, and parvalbumin, which is expressed in a class of strong inhibitory neurons. By contrast, prefrontal limbic areas were enriched in the enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), known to enhance plasticity. Eulaminate areas have more elaborate laminar architecture than limbic areas and showed the opposite trend: they were enriched in markers of stability and had lower expression of the plasticity-related marker CaMKII. The expression of glial fibrillary acidic protein (GFAP), a marker of activated astrocytes, was also higher in limbic areas, suggesting that cellular stress correlates with the rate of circuit reshaping. Elevated markers of plasticity may endow limbic areas with flexibility necessary for learning and memory within an affective context, but may also render them vulnerable to abnormal structural changes, as seen in neurologic and psychiatric diseases.

Limbic-motor integration by neural excitations and inhibitions in the nucleus accumbens.

The nucleus accumbens (NAc) has often been described as a "limbic-motor interface," implying that the NAc integrates the value of expected rewards with the motor planning required to obtain them. However, there is little direct evidence that the signaling of individual NAc neurons combines information about predicted reward and behavioral response. We report that cue-evoked neural responses in the NAc form a likely physiological substrate for its limbic-motor integration function. Across task contexts, individual NAc neurons in behaving rats robustly encode the reward-predictive qualities of a cue, as well as the probability of behavioral response to the cue, as coexisting components of the neural signal. In addition, cue-evoked activity encodes spatial and locomotor aspects of the behavioral response, including proximity to a reward-associated target and the latency and speed of approach to the target. Notably, there are important limits to the ability of NAc neurons to integrate motivational information into behavior: in particular, updating of predicted reward value appears to occur on a relatively long timescale, since NAc neurons fail to discriminate between cues with reward associations that change frequently. Overall, these findings suggest that NAc cue-evoked signals, including inhibition of firing (as noted here for the first time), provide a mechanism for linking reward prediction and other motivationally relevant factors, such as spatial proximity, to the probability and vigor of a reward-seeking behavioral response.NEW & NOTEWORTHY The nucleus accumbens (NAc) is thought to link expected rewards and action planning, but evidence for this idea remains sparse. We show that, across contexts, both excitatory and inhibitory cue-evoked activity in the NAc jointly encode reward prediction and probability of behavioral responding to the cue, as well as spatial and locomotor properties of the response. Interestingly, although spatial information in the NAc is updated quickly, fine-grained updating of reward value occurs over a longer timescale.

Two different mirror neuron networks: The sensorimotor (hand) and limbic (face) pathways.

The vast majority of functional studies investigating mirror neurons (MNs) explored their properties in relation to hand actions, and very few investigated how MNs respond to mouth actions or communicative gestures. Since hand and mouth MNs were recorded in two partially overlapping sectors of the ventral precentral cortex of the macaque monkey, there is a general assumption that they share a same neuroanatomical network, with the parietal cortex as a main source of visual information. In the current review, we challenge this perspective and describe the connectivity pattern of mouth MN sector. The mouth MNs F5/opercular region is connected with premotor, parietal areas mostly related to the somatosensory and motor representation of the face/mouth, and with area PrCO, involved in processing gustatory and somatosensory intraoral input. Unlike hand MNs, mouth MNs do not receive their visual input from parietal regions. Such information related to face/communicative behaviors could come from the ventrolateral prefrontal cortex. Further strong connections derive from limbic structures involved in encoding emotional facial expressions and motivational/reward processing. These brain structures include the anterior cingulate cortex, the anterior and mid-dorsal insula, orbitofrontal cortex and the basolateral amygdala. The mirror mechanism is therefore composed and supported by at least two different anatomical pathways: one is concerned with sensorimotor transformation in relation to reaching and hand grasping within the traditional parietal-premotor circuits; the second one is linked to the mouth/face motor control and is connected with limbic structures, involved in communication/emotions and reward processing.

Extinction of Cocaine Seeking Requires a Window of Infralimbic Pyramidal Neuron Activity after Unreinforced Lever Presses.

The infralimbic cortex (IL) mediates extinction learning and the active suppression of cocaine-seeking behavior. However, the precise temporal relationship among IL activity, lever pressing, and extinction learning is unclear. To address this issue, we used activity-guided optogenetics in male Sprague Dawley rats to silence IL pyramidal neurons optically for 20 s immediately after unreinforced lever presses during early extinction training after cocaine self-administration. Optical inhibition of the IL increased active lever pressing during shortened extinction sessions, but did not alter the retention of the extinction learning as assessed in ensuing extinction sessions with no optical inhibition. During subsequent cued reinstatement sessions, rats that had previously received optical inhibition during the extinction sessions showed increased cocaine-seeking behavior. These findings appeared to be specific to inhibition during the post-lever press period because IL inhibition given in a noncontingent, pseudorandom manner during extinction sessions did not produce the same effects. Illumination alone (i.e., with no opsin expression) and food-seeking control experiments also failed to produce the same effects. In another experiment, IL inhibition after lever presses during cued reinstatement sessions increased cocaine seeking during those sessions. Finally, inhibition of the prelimbic cortex immediately after unreinforced lever presses during shortened extinction sessions decreased lever pressing during these sessions, but had no effect on subsequent reinstatement. These results indicate that IL activity immediately after unreinforced lever presses is necessary for normal extinction of cocaine seeking, suggesting that critical encoding of the new contingencies between a lever press and a cocaine reward occurs during that period.SIGNIFICANCE STATEMENT The infralimbic cortex (IL) contributes to the extinction of cocaine-seeking behavior, but the precise relationship among IL activity, lever pressing during extinction, and extinction learning has not been elucidated using traditional methods. Using a closed-loop optogenetic approach, we found that selective inhibition of the IL immediately after unreinforced lever pressing impaired within-session extinction learning and promoted the subsequent cued reinstatement of cocaine seeking. These studies suggest that IL activity immediately after the instrumental response during extinction learning of cocaine seeking encodes information required for such learning and that altering such activity produces long-lasting changes in subsequent measures of cocaine craving/relapse.

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain.

Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway.

The effects of amphetamine exposure on juvenile rats on the neuronal morphology of the limbic system at prepubertal, pubertal and postpubertal ages.

Amphetamines (AMPH) are psychostimulants widely used for therapy as well as for recreational purposes. Previous results of our group showed that AMPH exposure in pregnant rats induces physiological and behavioral changes in the offspring at prepubertal and postpubertal ages. In addition, several reports have shown that AMPH are capable of modifying the morphology of neurons in some regions of the limbic system. These modifications can cause some psychiatric conditions. However, it is still unclear if there are changes to behavioral and morphological levels when low doses of AMPH are administered at a juvenile age. The aim of this study was to assess the effect of AMPH administration (1mg/kg) in Sprague-Dawley rats (postnatal day, PD21-PD35) on locomotor activity in a novel environment and compare the neuronal morphology of limbic system areas at three different ages: prepubertal (PD 36), pubertal (PD50) and postpubertal (PD 62). We found that AMPH altered locomotor activity in the prepubertal group, but did not have an effect on the other two age groups. The Golgi-Cox staining method was used to describe the neural morphology of five limbic regions: (Layers 3 and 5) the medial prefrontal cortex (mPFC), the dorsal and ventral hippocampus, the nucleus accumbens and the amygdala, showing that AMPH induced changes at pubertal ages in arborization and spine density of these neurons, but interestingly these changes did not persist at postpubertal ages. Our findings suggest that even early-life AMPH exposure does not induce long-term behavioral and morphological changes, however it causes alterations at pubertal ages in the limbic system networks, a stage of life strongly associated with the development of substance abuse behaviors.

Specific Targeting of the Basolateral Amygdala to Projectionally Defined Pyramidal Neurons in Prelimbic and Infralimbic Cortex.

Adjacent prelimbic (PL) and infralimbic (IL) regions in the medial prefrontal cortex have distinct roles in emotional learning. A complete mechanistic understanding underlying this dichotomy remains unclear. Here we explored targeting of specific PL and IL neurons by the basolateral amygdala (BLA), a limbic structure pivotal in pain and fear processing. In mice, we used retrograde labeling, brain-slice recordings, and adenoviral optogenetics to dissect connectivity of ascending BLA input onto PL and IL neurons projecting to the periaqueductal gray (PAG) or the amygdala. We found differential targeting of BLA projections to PL and IL cortex. Activating BLA projections evoked excitatory and inhibitory responses in cortico-PAG (CP) neurons in layer 5 (L5) of both PL and IL cortex. However, all inhibitory responses were polysynaptic and monosynaptic BLA input was stronger to CP neurons in IL cortex. Conversely, the BLA preferentially targeted corticoamygdalar (CA) neurons in layer 2 (L2) of PL over IL cortex. We also reveal that BLA input is projection specific by showing preferential targeting of L5 CP neurons over neighboring L3/5 CA neurons in IL cortex. We conclude by showing that BLA input is laminar-specific by producing stronger excitatory responses CA neurons in L3/5 compared with L2 in IL cortex. Collectively, this study reveals differential targeting of the BLA to PL and IL cortex, which depends both on laminar location and projection target of cortical neurons. Overall, our findings should have important implications for understanding the processing of pain and fear input by the PL and IL cortex.