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The bath-to-cell transport, cytosolic concentration, and tubular content of methylmercury (Me203Hg+) and the sulfhydryl-amino acids and sulfhydryl-amino acid derivatives conjugated to Me203Hg+ were studied in the non-perfused S2 segments of the proximal tubule of the rabbit kidney. Active transport of Me203Hg+ was established by a temperature dependent (greater than 100% reduction in bath-to-cell transport, 99% decrease in cytosolic concentration, 63% decline in the tubular contents at 12°C when compared to 37°C). Conjugates of Me203Hg+ showed mixed results, with L-cysteine and L-taurine demonstrating the most significant increase in uptake. Transport of Me203Hg+-L-cysteine was also temperature dependent with a 77% reduction in bath-to-cell transport, 76% decrease in cytosolic concentration, and 86% decline in tubular contents at 12°C when compared to 37°C. A significant decrease in transport was seen with the classic organic anion transport (OAT) inhibitors of PAH (71% and 67%) and probenicid (48% and 38%), as well as, the dicarboxylates, adepate (over 100%) and glutarate (69% and 52%) in both bath-to-cell and cytosolic concentration respectively. The addition of L-methionine to the Me203Hg+-L-cysteine conjugate significantly reduced the bath-to-cell transport by 64% and the cytosolic contents by 47%. The Me203Hg+-L-taurine conjugate also demonstrated temperature dependence (99% reduction and 91% decrease in bath-to-cell and cytosolic concentration respectively, at 12°C when compared to 37°C). Inhibition with PAH was also seen (77% reduction) in bath-to-cell transport and 67% decline in cytosolic concentration giving further evidence to the transport of the Me203Hg+-L-taurine conjugate via OAT. When Me203Hg+ was conjugated to L-methionine a 55% reduction in bath-to-cell transport was seen which was also temperature dependent (59% decrease at 12°C when compared to 37°), although no significant decrease in transport was noted with the addition of PAH. Analysis of the methylmercury conjugates via mass spectrometry demonstrated that L-cysteine, L-taurine, and L-methionine all are binding with methylmercury in the same unknown common configuration (MW=409). These results indicate the mercuric conjugate is gaining entry into the renal epithelial cells via the OAT and the amino acid transport system in the basolateral membrane of the proximal tubule.

O núcleo basolateral anterior da amígdala (BLAa) tem sido extensivamente investigado em estudos de condicionamento pavloviano envolvendo estímulos aversivos físicos. Até o presente momento não há descrição funcional específica do BLAa frente aos estímulos etologicamente relevantes. Neste trabalho, inicialmente revisitamos as conexões aferentes e eferentes do BLAa. Nossos achados confirmam em grande medida relatos anteriores da literatura e mostram que o núcleo integra informações de sistemas relacionados ao alerta emocional (tais como o locus coeruleus, dorsal da rafe e substância inominada). Este também se relaciona intimamente a estruturas ligadas à circuitaria do córtex pré-frontal, como o núcleo acúmbens, o caudo-putamen dorsomedial além dos córtices pré-límbico e cingulado anterior. Além disso estabelece conexões bidirecionais importantes com o córtex insular e com a região para-hipocampal. Testamos ainda o papel específico do BLAa frente à ameaça predatória e vimos que este influencia respostas de medo inato e contextual à ameaça predatória. Primeiramente, sugerimos que o BLAa responde ao estímulo do predador pelos sistemas de controle de alerta emocional, dado que ele recebe aferências de estruturas responsivas à presença do predador, como o locus coeruleus, que estão ligadas ao controle do alerta. Sugerimos também que através de suas projeções para o núcleo acúmbens, a região estudada, possa influenciar as respostas de defesa inata. Ademais as respostas de medo aprendido, ao contexto em que o rato foi exposto ao predador, podem ser afetadas por meio das relações do BLAa com os córtices pré-limbico, cingulado anterior e com a região para-hipocampal. / The anterior basolateral nucleus of the amygdala (BLAa) has been extensively investigated in studies of Pavlovian conditioning involving physical aversive stimuli. To date, there is no specific functional study of the BLAa regarding its functional roles on responses to ethologically relevant threats. In this work, we initially revisited the afferent and efferent connections of the BLAa. Our findings largely confirm previous reports in the literature, and show that the nucleus integrates information from systems related to emotional alertness (such as the locus coeruleus, dorsal raphe and innominate substance), and is also closely related to the prefrontal cortex circuitry, including the nucleus accumbens, the dorsomedial caudoputamen, and the prelimbic and anterior cingulate cortices. It also establishes important bi-directional connections with the insular cortex and parahippocampal region. We also tested the specific role of BLAa in innate and contextual responses to predatory threat. Thus, we have seen that BLAa influences innate and contextual fear responses to predatory menace. Firstly, we suggest that the BLAa responds to the predator\'s stimulus by the emotional arousal systems, given that it receives inputs from alert related structures highly responsive to the predator threat, such as the locus coeruleus. We also suggest that through its projections to the nucleus accumbens, the BLAa may influence innate defensive responses. In addition, we suggested that the BLAa influences contextual fear responses mostly through its relationships with the prelimbic, anterior cingulate and parahippocampal cortices.

The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

Thesis advisor: Jon C. Horvitz / Previous research has yielded conflicting results regarding the involvement of mesolimbic dopamine in Pavlovian and operant tasks. While there is abundant evidence that an operant lever press requires intact dopamine (DA) D1 transmission in the nucleus accumbens (ACB) and in the basolateral amygdala (BLA), there is conflicting evidence regarding the specific brain sites at which DA mediates a Pavlovian approach response. The present study was designed to compare the effects of ACB and BLA D1 receptor-blockade on an operant and Pavlovian task, while minimizing differences in behavioral response topography. Animals were trained on either a Pavlovian cued approach task or an operant cued nosepoke task. In the Pavlovian approach task, a tone signaled a pellet delivery to which animals responded with a head entry into a food compartment. In the operant nosepoke task, animals were trained to emit a nosepoke in response to the same tone, in order to trigger a pellet delivery. Bilateral microinfusions of the D1 antagonist SCH 23390 (0, 1 or 2 microgram/side) into either the ACB or the BLA produced a dose-dependent disruption of the operant nosepoke. In contrast, the Pavlovian cued approach response was unaffected by D1 antagonist microinfusions into either the ACB or the BLA. In addition, infusion of SCH 23390 into either site suppressed general locomotion. The results suggest a dissociation of the anatomical substrates mediating an operant nosepoke and a Pavlovian approach, despite similar response topographies. These findings are consistent with the notion that D1 activity at the ACB and BLA plays a role in the expression of operant responses, but not in the expression of Pavlovian approach responses. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology.

Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.

The present study examined social status and adult neurogenesis in the naked mole rat. These animals live in large colonies with a strict reproductive dominance hierarchy; one female and 1-3 males breed, while other members are subordinate and reproductively suppressed. We examined whether social status affects doublecortin (DCX; a marker for immature neurons) immunoreactivity in the dentate gyrus, piriform cortex (PCx), and basolateral amygdala (BLA) by comparing breeders to subordinates. We also examined subordinates removed from their colony and paired with opposite- or same-sex conspecifics for 6 months. Breeders had reduced DCX immunoreactivity in all areas, with BLA effects confined to females. Effects of housing condition were region-specific, with higher PCx DCX immunoreactivity observed in opposite- than same-sex paired subordinates regardless of gonadal status. The opposite pattern was observed in the BLA. Future work will clarify whether findings are attributable to status differences in stress, behavioural plasticity, or life stage.

Thesis advisor: Gorica D. Petrovich / Environmental cues that are neutral in respect to hunger and feeding can come to predict food through Pavlovian appetitive conditioning. These learned cues can drive food seeking and eating independent of physiological hunger leading to overeating and obesity. However, the food outcome, and thus the value of the cues, can change due to environmental alterations. A change in the values of learned cues requires altering behavioral responses to accurately reflect the cue’s new outcome. This behavioral flexibility is necessary to respond appropriately to changes in the environment and, as such, is an adaptive trait. The aim of this dissertation was to determine critical neural mechanisms specifically within the basolateral amygdala (BLA) and also with its interactions with the medial prefrontal cortex (mPFC) during behavioral flexibility when outcomes of learned appetitive cues change using the appetitive reversal learning paradigm. The main focus was on the BLA (Chapter 2) and its connection with the mPFC (Chapters 3 and 4) since both of these areas are critical in appetitive cue learning and valuation and subsequent behavioral modifications. The first study in this dissertation examined if separate neuronal ensembles within the BLA respond to different learned cues, a cue that signals food availability and a cue that does not. Additionally, we investigated if these potentially distinct neuronal ensembles are necessary during periods of behavioral flexibility when the value of the specific learned cues are changed during reversal learning. We determined that there are distinct neuronal ensembles within the BLA that respond to different learned cues, and that the cue-specific ensembles are necessary for updating the value of each specific cue (Chapter 2). Next, we examined a projection target of the BLA, the mPFC, to determine if BLA-projecting neurons are activated during learning (Chapter 3). Using retrograde tract tracing combined with Fos detection, we found recruitment of the anterior BLA to prelimbic area of the mPFC across cue-food learning, signifying that the BLA can inform the mPFC of the value of learned cues. Then to establish that communication between the BLA and mPFC is necessary for cue value learning and updating (Chapter 4), we functionally disconnected communication between these regions and examined appetitive learning using discriminative conditioning, reversal learning, and devaluation paradigms. We found impairments in cue value recall and subsequent updating of the cues’ values during reversal learning. Together, these studies indicate the BLA may be important in informing the mPFC of the value of learned cues, and their interaction is critical to optimally guide behavioral responding. The findings from these experiments are valuable for our understanding of the neural mechanisms that motivate eating behavior under the control of learned food cues and to understand the mechanisms necessary for behavioral flexibility when the outcomes of learned cues are changed. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-HT) is heavily implicated in severe anxiety and trauma-related disor-ders, such as panic and post-traumatic stress disorders. Overall, site-specific pharmacolog-ical manipulations show that while 5-HT enhances anxiety-associated/avoidance behaviors in the amygdala, 5-HT inhibits panic-associated escape behaviors in the perifornical hypo-thalamus region (PeFR). Yet, our understanding of how specific serotonergic networks and co-transmitters regulate these conditions, but also other aspects of innate panic (e.g., car-dioexcitation or thermal response that occur during a flight or escape response) or condi-tioned fear behaviors is still elusive. Therefore, utilizing circuit-based gain- and loss-of-function approaches to selectively manipulate amygdala- and PeFR-projecting sero-tonergic systems, we hypothesize that specific serotonergic networks projecting to the amygdala and PeFR respectively enhance conditioned fear responses and attenuate innate panic-associated behaviors and physiological responses. There are two main chapters in this dissertation. In Chapter III, retrograde tracing revealed that the amygdala-projecting neurons from dorsal Raphe (DR) were almost exclusively serotonergic (92-95%) concen-trated in the dorsal/ventral (DRD/DRV) DR, with few non-serotonergic neurons. While selective lesioning of this network with saporin toxin (SAP) facilitated the extinction of conditioned fear behavior, selective optogenetic activation of amygdala-projecting DRD/DRV cell bodies using intersectional genetics reduced extinction of conditioned fear behavior and enhanced anxiety avoidance. In Chapter IV, retrograde tracing showed that the PeFR was innervated by equally selective serotonergic networks concentrated in the lateral wings DR (lwDR) and median Raphe (MR). Contrasting with the results from the amygdala-innervating 5-HT system, lesioning the PeFR-projecting serotonergic network from lwDR/MR was accompanied by reduced extinction of conditioned fear behavior, in-creased anxiety avoidance, and increased CO2-induced panic (elevated escape responses and enhanced cardioexcitation). Conversely, selective activation of lwDR/MR serotonergic terminals in the PeFR decreased anxiety-associated behaviors; inhibited CO2-induced panic, and induced unconditioned and conditioned place preferences. The circuit-based ap-proach data presented here show that amygdala- and PeFR-projecting 5-HT neurons com-prise distinct circuits underlying opposite roles enhancing anxiety/fear responses in the amygdala and dampening fear/panic responses in the PeFR. The identification of distinct circuits controlling anxiety, fear, and panic responses is a fundamental step towards the development of more effective therapies for psychiatric conditions such as anxiety and trauma-related disorders. / 2021-11-04

Indiana University-Purdue University Indianapolis (IUPUI) / Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories.