Thesis (M.Sc.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2009/06/29). Includes bibliographical references.

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Um dos principais mecanismos de regulação da síntese protéica é a fosforilacão do fator de iniciação eIF2α. O aumento de fosforilação de eIF2α leva ao bloqueio do início de tradução geral da célula e tradução de genes específicos a exemplo do ATF4 (fator de ativação transcricional 4). Há 4 cinases específicas que fosforilam eIF2α, cada uma ativada sob condições distintas de estresse. A cinase GCN2, por exemplo, está presente em abundância no encéfalo, e faz parte do controle alimentar, memória e aprendizado de mamíferos. A proteína IMPACT, preferencialmente expressa no encéfalo, atua como uma inibidora da ativação de GCN2. Nesse contexto, torna-se relevante uma análise sistemática da distribuição de IMPACT no encéfalo e do possível envolvimento dessa proteína em condições fisiológicas e/ou patológicas. Um conjunto de resultados foi observado: (i) A proteína IMPACT é preferencialmente expressa em núcleos encefálicos associados ao ritmo circadiano, como também núcleos importantes na geração de ritmos, como o theta hipocampal; ii) A elevada intensidade e densidade de IMPACT no hipotálamo e hipocampo pode condizer com o envolvimento de IMPACT em mecanismos que envolvam homeostase corporal (função hipotalâmica) e memória (função hipocampal). (iii) Em tecido adulto, poucos grupos neuronais mostraram divergências na expressão de IMPACT entre roedores e primata; (iv) Há evidências de expressão diferencial (em uma mesma espécie) de IMPACT em grupos neuronais específicos (GABAérgicos); (v) Neurônios IMPACT-positivos são resistentes a vários tipos de estresse (leves, intensos, agudos ou crônicos, a exemplo de baixas temperaturas e indução de status epilepticus no modelo de epilepsia por pilocarpina); (vi) A proteína IMPACT é constitutiva, mostrou-se insensível a perturbações in vivo; (vii) IMPACT é detectada a partir do estágio embrionário E16. De acordo com os dados aqui dispostos sobre a expressão de IMPACT em neurônios e a evidência de sua expressão áreas encefálicas e em diversas condições, nós podemos especular sobre sua possível relevância na neurofisiologia. Assim, juntos esses dados sugerem que neurônios IMPACT-positivos tem função importante no encéfalo de mamíferos e podem estar envolvidos na geração e controle de ritmos encefálicos e na homeostase. / The control of protein synthesis plays a major role in several physiological conditions, homeostatic mechanisms and several phenomenon of neuronal plasticity. One of the most important mechanisms in regulation of protein synthesis is the phosphorylation of the initiation factor 2 (eIF2α). Increase in the eIF2α phosphorylation blocks general translation and enhance translation of specific genes such ATF4 (activating transcription factor 4) for example. In response to specific stress stimuli four specific kinases can phosphorylate eIF2α. The GCN2 kinase, for instance, is abundant in the brain, and is involved in feeding behavior control, learning and memory of mammalians. The IMPACT protein, preferentially expressed in the brain, acts as an inhibitor of the activation of GCN2. Therefore, a systematic analysis of the distribution of IMPACT in the brain and its possible involvement in physiological and/or pathological conditions are relevant. The following results were observed: (i) The IMPACT protein is preferentially expressed in brain nuclei involved in circadian rhythms, as well as in important nuclei involved in rhythm generation, such as the hippocampal theta rhythm; (ii) The high density and intensity of IMPACT labeling in the hypothalamus and hippocampus may reflect its involvement in homeostatic mechanisms (hypothalamic function) and memory (hippocampal function). (iii) In adult tissue, only a few neuronal groups showed divergence in IMPACT expression between rodents and primate; (iv) Within a single species there was differential expression of IMPACT in specific neuronal groups (e.g. GABAergic neurons); (v) IMPACT-positive neurons were resistant to several types of stress (weak, strong, acute or chronic), such as low temperature and status epilepticus induced by pilocarpine in a model of epilepsy; (vi) IMPACT is a constitutive protein, insensitive to perturbations in vivo; (vii) The detection of IMPACT starts at E16 embryonic stage. According to these results on the neurons expressing IMPACT and the evidence of its presence in some brain areas, we speculate on its possible relevance in neurophysiology. Thus, these data taken together suggest that IMPACT-positive neurons have important functions in the mammalian brains and may be involved in generation and control of brain rhythms and physiological homeostasis in general. / TEDE / BV UNIFESP: Teses e dissertações

Dès les stades précoces du développement et jusqu’à l’âge adulte, le facteur de transcription TSHZ3 est fortement exprimé dans les neurones pyramidaux (PNs) du cortex. Les PNs de la couche V forment la synapse cortico-striée en contactant les neurones épineux moyens (MSNs) du striatum. A ce niveau, l’expression de TSHZ3 n’est pas retrouvée dans les MSNs mais dans les interneurones cholinergiques (CINs). Des données récentes ont établi un lien entre délétion hétérozygote du gène TSHZ3/Tshz3, troubles du spectre autistique (TSA) et dysfonctionnement de la circuiterie cortico-striée (Caubit et al., Nat Genet 2016). Afin de mieux comprendre le rôle de TSHZ3 dans la circuiterie cortico-striée, nous avons caractérisé deux modèles murins de délétion conditionnelle de Tshz3, ciblant soit les neurones de projection à partir de la période postnatale (souris Tshz3-pnCxKO), soit les neurones cholinergiques à partir de la période embryonnaire (souris Tshz3-ChATCre). Chez les souris Tshz3-pnCxKO, la perte de TSHZ3 entraîne une moindre excitabilité des PNs de la couche V, ainsi qu’une diminution de la probabilité de libération du glutamate par leurs afférences. Nous montrons également une profonde altération du fonctionnement de la synapse cortico-striée. Chez les souris Tshz3-ChATCre, nous montrons que la perte de Tshz3 modifie les propriétés membranaires et de décharge d’une proportion des CINs, qui sont les seuls neurones cholinergiques de l'encéphale exprimant TSHZ3 de façon importante. Ces changements fonctionnels suggèrent que TSHZ3 joue un rôle clé dans le développement des PNs du cortex, de la voie cortico-striée et des CINs, confirmant son implication dans les TSA. / The zinc-finger transcription factor TSHZ3 is highly expressed by cortical projection neurons (PNs) from embryonic stages to adulthood, including layer V pyramidal neurons that project to the striatum. There, TSHZ3 is expressed by cholinergic interneurons (CINs) but not by the main targets of PNs, i.e. the medium spiny neurons. Interestingly, recent evidences link heterozygous TSHZ3/Tshz3 gene deletion to autism spectrum disorder (ASD) and to corticostrial circuitry dysfunction (Caubit et al., Nat Genet 2016). In order to provide further insights on the role of Tshz3 in the corticostriatal circuitry, we have characterized two conditional KO mouse models in which its expression is lost either in projection neurons at early postnatal stage (Tshz3-pnCxKO) or in cholinergic cells beginning at embryonic stage (Tshz3-ChATCre). In Tshz3-pnCxKO mice, we confirmed that Tshz3 expression is lost in glutamatergic PNs without altering their number. Our electrophysiological study revealed that layer V PNs are less excitable and that glutamate release probability from their afferents is decreased. We also found dramatic changes of both corticostriatal synaptic transmission and plasticity. In ChAT-Cre mice, we found that Tshz3 is expressed in the striatum by almost 100% of CINs, while it is little or no expressed in the other cholinergic nuclei of the brain. Interestingly, the loss of Tshz3 impacts the spontaneous firing pattern of a subpopulation of CINs without altering their number. These functional changes suggest that TSHZ3 plays a key role in PNs, corticostriatal pathway and CINs development, supporting its implication in ASD.

La mise en place des circuits neuronaux fonctionnels se construit autour d'une grande diversité cellulaire et nécessite l'accomplissement d'une série d'évènements complexes incluant la prolifération, la migration, la différenciation, le guidage axonal, la reconnaissance cellulaire et la synaptogenèse des progéniteurs neuronaux. Dans le cervelet, les interneurones GABAergiques de la couche moléculaire (IGCM) s‘intègrent au cours des deux premières semaines post-natales et se différencient en deux sous-types cellulaires, les cellules en panier (CP) qui innervent le segment initial de la cellule de Purkinje, cellule principale du cervelet, et les cellules étoilées qui innervent l'arbre dendritique de la cellule Purkinje. Bien que ces deux types cellulaires possèdent des morphologies distinctes et innervent des sous-domaines cellulaires spécifiques, aucun marqueur moléculaire ne permet de les discriminer. Depuis près d'un siècle, la controverse existe concernant leur identité et deux théories s'affrontent. La première suggère que ces deux cellules sont des variantes issues d'un même progéniteur et que les différences morphologiques sont dues à un changement progressif de l'environnement cellulaire alors qu'une autre hypothèse suggère que ces deux cellules proviennent de progéniteurs neuronaux différents. Au cours de ma thèse j'ai étudié l'intégration des IGCM au sein de la couche moléculaire (CM) en caractérisant deux étapes clés de la formation des circuits GABAergiques, la migration et l'innervation de leur cible. En utilisant une combinaison de techniques telles que la microscopie bi-photonique et les greffes in vivo de progéniteurs neuronaux, j'ai mis en évidence que durant la première semaine post-natale, les IGCM quittent leur lieu de naissance pour rejoindre la CM en réalisant une seule étape de migration radiale. De manière intéressante certains IGCM accomplissent une étape de migration supplémentaire inédite tangentiellement à la surface piale pendant la deuxième semaine post-natale. Cette nouvelle phase de migration tangentielle des IGCM se déroule au sein de la couche granulaire externe où résident les cellules granulaires pré-migratoires dont les fibres qui expriment TAG-1 jouent un rôle essentiel en tant que support physique et participent à l'établissement des IGCM en mode “inside-out”. De plus, nos résultats suggèrent que seule une sous-population de type cellule étoilée effectuerait cette étape supplémentaire, montrant ainsi une première divergence dans le processus de maturation des IGCM. Par la suite, je me suis intéressée à l'innervation des cellules de Purkinje par les CP nouvellement différenciées. En utilisant des techniques d'immuno-histochimie, j'ai tout d'abord montré que la Neuropiline-1 (NRP1), un des récepteurs de la Sémaphorine-3A, était exprimé au niveau des terminaisons axonales des CP. Enfin, grâce à l'analyse d'un mutant conditionnel pour NRP1, j'ai pu mettre en évidence qu'en plus de son rôle crucial dans le guidage axonal des CP, NRP1 est également impliquée dans l'innervation spécifique du segment initial axonal des cellules de Purkinje en interagissant avec une molécule d'adhésion cellulaire de la famille L1CAM, la Neurofascine. Ces résultats démontrent pour la première fois un rôle de NRP1 dans la transition entre l'étape de guidage avec celle de la reconnaissance cellulaire par les CP. En conclusion, nos résultats suggèrent fortement que les deux sous-types d'IGMC possèdent un programme génétique spécifique leur permettant de s'intégrer de manière unique au sein de la CM. / The establishment of functional neural circuits is built around a large cell diversity and requires the completion of a series of complexe events including proliferation, migration, differentiation, axon guidance, cell recognition and synaptogenesis of neural precursors. In the cerebellum, molecular layer GABAergic interneurons (MLGI) reach their final location during the first two post-natal weeks and differentiate into two cellular subtypes, the basket cells (BC) that innervate the Purkinje cell initial segment and the stellate cells that innervate the dendritic tree of the Purkinje cell, the principal cell of cerebellar cortex. Although these two cell types have distinct morphologies and innervate specific subcellular domains, no molecular marker allows to discriminate between them. For nearly a century, controversy exists concerning their identity and two theories exist. The first one suggests that these two cell types are variants derived from a single progenitor and that morphological divergence is due to a gradual change in the cellular environment while the other hypothesis suggests that these two cell types come from different progenitors. During my thesis, I studied the integration of the MLGI in the molecular layer (ML) characterizing two key steps in the formation of GABAergic circuits, migration and innervation of their target. Using a combination of techniques such as two-photon microscopy and in vivo transplantation of neural progenitors, I highlighted that during the first post-natal week, MLGI leave their birthplace to join the ML by performing a single radial migration step. Interestingly, some MLGI perform an unexpected additional migration step tangentially to the pial surface during the second post-natal week. This new phase of MLGI tangential migration takes place in the external granule cell layer where resident pre-migratory granule cells whose fibers expressing TAG-1 play an essential role as physical support and participate in the establishment of MLGI « inside-out » mode. In addition, our results suggest that only a stellate-like subpopulation would perform this extra step, bringing the first indication of an early divergence during MLGI maturation process. Then, I was interested in the innervation of Purkinje cells by newly differentiated BC. Using immunohistochemistry experiments, I first showed that Neuropilin-1 (NRP1), a Semaphorin-3A receptor, was expressed in the BC axon terminals. Finally, through the analysis of a NRP1 conditional mutant, I brought out that, in addition to its critical implication in axon guidance, NRP1 is also involved in the specific innervation of the Purkinje cell axon initial segment by interacting with a cell adhesion molecule belonging to the L1 family, Neurofascin. These results demonstrate for the first time a role of NRP1 in the transition between the guidance and the cell recognition steps by BC. In conclusion, our results strongly support that the two MLGI subtypes have a specific genetic program allowing them to integrate within the ML in a unique manner.

Cette thèse étudie la modulation des circuits neuronaux spinaux impliqués dans les fonctions motrices et leur contrôle corticospinal lors de la locomotion chez l’Homme sain et après accident vasculaire cérébral (AVC). Dans ce contexte, la stratégie utilisée a consisté à moduler les comportements de ces circuits en modifiant soit les volées afférentes d’origine suprasegmentaire ou les volées afférentes d’origine périphérique et d’étudier le retentissement de ces modifications sur le comportement des circuits neuronaux spinaux au cours de différentes tâches motrices dont la marche.Trois grands thèmes émergent de ce projet :« L’influence combinée des entrées corticospinales et de l’inhibition réciproque sur l’activité des motoneurones des fléchisseurs plantaires de la cheville » a été étudiée en station debout et lors de la marche stabilisée. Les résultats ont suggéré que l'interaction entre les motoneurones spinaux, les interneurones inhibiteurs Ia et les volées motrices corticospinales descendantes ainsi que leur contribution relative à l'activité des motoneurones des fléchisseurs plantaires de la cheville dépendent de la tâche motrice. Plus d’interaction entre les entrées descendantes et les interneurones Ia pendant la station debout était présente, probablement pour renforcer l’activation tonique des motoneurones du soléaire.« La modulation liée à la tâche de l’inhibition spinale croisée entre les membres inférieurs chez l’Homme » a été étudiée au niveau des muscles soléaires en position assise, debout et lors de la marche stabilisée. Nos résultats suggèrent que la transmission neurale croisée, via les interneurones commissuraux des groupes I et II, est déprimée par les entrées descendantes bilatérales du cortex moteur ou pendant le mouvement volontaire. La modulation spécifique de l’inhibition croisée du groupe II au cours de la locomotion suggère un contrôle des structures mésencéphaliques monoaminergiques et son rôle dans la coordination des jambes pendant la locomotion. « L’influence de la musique sur les automatismes locomoteurs après un Accident Vasculaire Cérébral » a été étudiée durant la marche stabilisée chez les patients atteints d’AVC. Nos résultats préliminaires suggèrent que l’écoute musicale modulerait les réseaux neuronaux médullaires impliqués dans les automatismes locomoteurs. / This thesis investigates the modulation of spinal neuronal circuits involved in motor function and their corticospinal control during locomotion in healthy humans and after stroke. In this context, the strategy was to modulate the behavior of these circuits by modifying either the afferent volleys from suprasegmental origin or from peripheral origin and to study the impact of these changes on the behavior of spinal neuronal circuits during different motor tasks such as walking.Three major themes emerge from this project:« Combined influence of corticospinal inputs and reciprocal inhibition on ankle plantar flexor motoneuron activity during walking » was investigated during standing and during stabilized walking. The results suggested that the interaction between spinal motoneurons, Ia inhibitory interneurones and motor corticospinal descending volleys and their relative contribution to the activity of plantar flexor motoneurons of the ankle depend on the motor task. More interaction between descending inputs and Ia interneurones during standing was present, presumably to strengthen the tonic activation of the soleus motoneurons.« Task-related modulation of crossed spinal inhibition between human lower limbs» has been studied at the soleus muscles in sitting, standing and during stabilized walking. Our results suggest that crossed neural transmission via commissural interneurones of groups I and II, is depressed by bilateral descending inputs of motor cortex during voluntary movement. Specific modulation of the crossed inhibition by group II afferents during locomotion suggests a control from monoaminergic mesencephalic structures and its role in legs coordination during locomotion.« The influence of music on locomotor automatisms after stroke» was investigated during stabilized locomotion in stroke patients. Our preliminary results suggest that music modulate the spinal neuronal networks involved in locomotor automatisms.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are necessary in memory and cognition. They are pentameric and consist of α and β subunits. They are most commonly heteromeric but, can sometimes be homomeric. nAChRs are activated by many ligands including nicotine (exogenous) and acetylcholine (endogenous).nAChRs are located on hippocampal interneurons. The interneurons, although sparse, control the synchronous firing of the pyramidal cells. However, the hippocampal interneuron structure and function is quite diverse and not fully characterized. Therefore, we sought to quantify nAChR subunit mRNA levels using real-time PCR of CA1 hippocampal interneurons.Surprisingly we found that the α3 and β2 mRNA subunits were the highest expressed and highest co-expressed subunits. Additionally, the α4 mRNA subunit was the lowest expressed of the subunits detected. The α4 subunit is one of the most pharmacologically targeted nAChR subunits and is found throughout the rest of the brain at much higher levels than the α3 mRNA subunit. Upon PCR analysis two subpopulations of the α3 and β2 subunits emerged: those that contained 3X more α3 than β2 and those that contained 3X more β2 than α3. Therefore, we hypothesized that two likely α3β2 nAChR stoichiometries are present in hippocampal interneurons. We differentiated their kinetic properties using electrophysiology.Additionally, like the α4 subunit, the α7 subunit is highly targeted in cognitive therapeutics. Since, the α7 subunit is the most characterized nAChR subunit, there are current efforts to develop allosteric modulators of the α7 subunit. The α7 subunit is found at moderate levels within hippocampal interneurons and remains a valid target. Current treatment options for Alzheimer's disease, and other dementias are limited and only mildly effective. Therefore, we sought to characterize the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) on α7.Furthermore, there are no current methods to distinguish the α7 from the α7β2 nAChRs during whole cell electrophysiological recordings. Therefore, we also characterized the PAM-2 effect on α7β2 nAChRs. Our results highlight at least 2 ways PAM-2 can be used to differentiate α7 from the α7β2 during whole-cell recordings.

Les premières semaines postnatales sont essentielles pour le développement de la sensibilité à la douleur et sont associées à une réorganisation structurelle et fonctionnelle des systèmes sensoriels. Les interneurones localisés dans la couche II interne (IIi) du sous noyau caudal du trijumeau (Sp5C), premier relais de l’information tactile et nociceptive orofaciale, sont des éléments clés des circuits responsables de l’allodynie mécanique orofaciale. L’objectif de ce travail de thèse est d’étudier le développement postnatal, à la fois morphologique (en utilisant l’immunohistochimie et l’analyse morphologique tridimensionnelle) et fonctionnel (enregistrements en patch-clamp sur tranches de Sp5C), de ces interneurones. Nous nous sommes d’abord intéressés à une population très particulière d’interneurones de la couche IIi, qui expriment l’isoforme gamma de la protéine kinase C (PKCγ). Au stade le plus précoce de notre étude (3 jours postnataux, P3), les interneurones PKCγ sont présents dans toutes les couches superficielles sauf, précisément, la couche IIi. Ce n’est qu’à P6 que les premiers interneurones PKCγ peuvent être observés dans cette couche. Leur nombre y croît ensuite progressivement jusqu’à P11-15. A cet âge, leur nombre dans la couche IIi est quasiment identique à celui observé aux âges plus tardifs. De plus, nous montrons que cette augmentation du nombre d’interneurones PKCγ dans la couche IIi n’est liée ni à une prolifération cellulaire ni à l’arrivée progressive des fibres afférentes nociceptives dans le Sp5C. Nous avons également étudié le développement des interneurones de la couche IIi dans leur ensemble. Ces neurones sont l’objet d’un grand nombre de changements morphologiques, aussi bien au niveau de leur soma (augmentation du volume) que de leurs neurites (augmentation de leur longueur combinée à une diminution de leur nombre et de leurs ramifications). Sur le plan fonctionnel, les neurones de la couche IIi, à la naissance, sont plus dépolarisés, ont une rhéobase plus basse – ils seraient donc plus excitables – et montrent plus fréquemment un profil de décharge avec un seul potentiel d’action, comparés aux mêmes interneurones chez l’adulte.Toutes ces modifications structurelles et fonctionnelles des interneurones de la couche IIi du Sp5C pourraient contribuer au développement de la sensibilité orofaciale. / The first postnatal weeks are pivotal for the development of pain sensitivity and are associated with structural and functional reorganization of sensory systems. Interneurons located in the inner part of lamina II(IIi) of the caudal trigeminal subnucleus (Sp5C), the first central node in orofacial tactile and nociceptive pathways, are key elements in circuits underlying the orofacial mechanical allodynia. The aim of this thesis is to study the morphological (by using immunohistochemistry and tridimensional morphological analysis) and functional (by using whole-cell patch-clamp recordings) postnatal development of these interneurons. First, we looked at a very specific population of lamina IIi interneurons expressing the gamma isoform of the protein kinase C (PKCγ). At the earliest stage of our study (3 postnatal days, P3), PKCγ interneurons are present in all superficial layers but PKCγ interneurons can be observed in lamina IIi only at P6. The number of PKCγ interneurons within this lamina then increases gradually up to P11-15. At this age, the number of PKCγ interneurons in lamina IIi is almost the same as that at later ages. Interestingly, we show that neither cell proliferation nor the gradual projection of nociceptive fibers within the Sp5C accounts for such increase. We also studied the development of the whole population of lamina IIi interneurons. These interneurons undergo a large number of morphological changes, in their soma (increased volume) as well as neurites (concomitant increase in length and decrease in number and branching). Furthermore, according to electrophysiological properties, lamina IIi interneurons, at birth, are more depolarized, have a lower rheobase – suggesting that they are more excitable – and exhibit more frequently a single action potential discharge profile compared with mature ones. All these structural and functional changes of lamina IIi interneurons might contribute to the development of orofacial sensitivity.

Our lives unfold in space and time—we are able to be aware not only of the present instant but also to recollect the past and imagine the future, and our memories tend to be not instantaneous snapshots but rather bear a temporal, sequential dimension. This faculty of time travel allows us to adjust our current actions in light of what we have previously learned and with respect to what we aspire to become. It depends upon faithful records of our personal experiences, termed episodic memory. While over the last century we have learned a great deal about the molecular changes that support this kind of learning, the circuit-level mechanisms with which the brain implements the formation of episodic memory remain to be discovered. Failures of episodic memory can be catastrophic, and unfortunately, such dysfunction is commonplace in a number of human pathologies. In the neuropsychiatric syndrome of schizophrenia, the capacity to form and utilize episodic memory is compromised, a state of affairs that likely contributes to the difficulty people with schizophrenia have adjusting their actions to meet desired goals. Attempts to understand the pathogenesis of schizophrenia’s memory deficits at the molecular level have yielded frustratingly few leads, making circuit-level inquiries a rational next step. Utilizing a genetic mouse model of schizophrenia susceptibility (Df(16)A+/- mice), we have taken a three-pronged approach to the analysis of the circuit mechanisms and missteps of episodic memory. We first developed a behavioral model of episodic memory, a variation on classical ‘trace’ fear conditioning, which involves the formation of an association between an innocuous stimulus (conditioned stimulus, CS) and a temporally separate aversive stimulus (unconditioned stimulus, US). Next, we turned to a region of the brain known to be required for trace fear conditioning and implicated in the pathogenesis of schizophrenia, dorsal CA1 of the hippocampus. Because network coordination and plasticity in dorsal hippocampal CA1 relies heavily on the activity of soma-targeting, parvalbumin-positive interneurons (PV+ INs), we hypothesized that they may be mediators of the associations built during trace fear conditioning. We therefore sought to characterize their activity during temporal association learning in both wild-type (WT) and Df(16)A+/- mice using two-photon calcium imaging. We simultaneously recorded local field potentials in the contralateral dorsal hippocampus to pair the discrete transformations captured through imaging with information about more global states of hippocampal activity. Finally, we manipulated the activity PV+ INs during various epochs of freely-moving trace fear conditioning to test hypotheses regarding their necessity for trace fear conditioning in healthy and schizophrenia-susceptible mice. We found that Df(16)A+/- mice have severe deficits in trace fear conditioning when compared to mice that do not carry their defining mutation. Calcium imaging of PV+, peri-somatic boutons in dorsal CA1 over the course of trace fear conditioning revealed a marked increase in the number of detected boutons that initiate activity during the presentation of the CS and that sustain their activity across the time gap preceding delivery of the US. This shift in activity was notably absent in recordings from Df(16)A+/- mice. Consistent with the observations of others, analysis of local field potentials indicated that successful learning was associated with modulation of amplitude and theta-phase relation in mid- and fast-gamma frequency oscillations. This modulation was compromised in Df(16)A+/- mice. Finally, we found that inhibition of PV+ INs during encoding in Df(16)A+/- mice restores their response to the CS to near-WT levels of fear expression. Our results support the thesis that temporary downregulation of PV+ IN activity during encoding is essential for the formation of complex, hippocampus-dependent associations including temporal association memory. We suggest that this transient disinhibition may serve to allow for the generation of new pyramidal cell ensembles to represent the associated stimuli. The heightened, sustained inhibition observed during post-learning trials in the calcium imaging experiments is consistent with a transition of the PV+ INs into a role of stabilizing the fledgling memory trace during consolidation. Our results also support the hypothesis that in our model of schizophrenia susceptibility, impairments in learning complex associations may be due to the inability of PV+ INs to modulate their activity appropriately over the changing phases of memory formation. We identify PV+ INs as a promising therapeutic target for schizophrenia as we were able to restore behavior of the susceptible mice during our assay of temporal association memory. Further studies combining pharmacogenetic or optogenetic manipulations with calcium imaging and LFP recording could shed light on the mechanisms of these shifts in network plasticity and may help to identify new approaches to the treatment of the debilitating cognitive deficits associated with schizophrenia.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc) underlies the rewarding properties of ethanol (EtOH) and nicotine (NIC). Although the dogma is that EtOH enhancement of DA neural activity contributes to enhancement of DA transmission, DA neurons are not sensitive to rewarding levels of EtOH. However, VTA GABA neurons are sensitive to low-dose EtOH. We have shown previously that EtOH modulation of DA release in the NAc is mediated by α6-containing nicotinic receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and α6*-nAChRs may be a molecular target for low-dose EtOH. Thus, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on VTA GABAergic input to CINs and DA release in the NAc. Using DIO channel rhodopsin-2 (ChR2) viral injections into the VTA of VGAT Cre mice, we found that VTA GABA neurons send an inhibitory projection to CINs, replicating what has been demonstrated by others. This study investigated the acute and chronic effects of EtOH at this synapse. We demonstrate that EtOH markedly enhances CIN firing rate and that these effects are blocked by the α6-conotoxin MII (α-Ctx MII), knockout of accumbal α6*-nAChRs with α6-shRNA, and atypical GABA receptor antagonists. This study also investigated plasticity at this synapse. We demonstrate that a low frequency stimulation (LFS; 1 Hz, 240 pulses) causes inhibitory long-term depression at this synapse (CIN-iLTD) which is also blocked by α-Ctx MII, α6-shRNA, and atypical GABA receptor antagonists. We also show that CIN-iLTD is blocked in EtOH-dependent mice. Taken together, these findings suggest that EtOH affects the VTA GABAergic projection to CINs via α6*-nAChRs and that atypical GABA receptors also play a role.