Speech-language cooperation protocol for the fiberoptic laryngoscopy evaluation of larynx mobility

TEMA: protocolo de avaliação da voz. OBJETIVO: proposição de um protocolo de cooperação fonoaudiológica para avaliação nasofibrolaringoscópica da mobilidade laríngea em doenças da tireóide (PAN), visando a composição de um instrumento objetivo, preciso e consensual para avaliação. MÉTODOS: a primeira versão do protocolo foi elaborada a partir de fundamentação bibliográfica; o PAN foi julgado em duas instâncias pelo método de triangulação por seis juízes em três etapas; foi constituída uma versão piloto do protocolo e aplicada em 11 pacientes; houve novo julgamento de médicos e fonoaudiólogos; a partir da concordância dos juízes, após a aplicação do piloto, foi construída a versão final do PAN. RESULTADOS: o PAN final foi composto por duas partes. A primeira parte considerada o procedimento padrão composta por 4 itens imprescindíveis e que devem necessariamente ser avaliados são: inspiração normal; inspiração forçada; vogal /é/ isolada e sustentada e vogal /i/ aguda isolada e sustentada. A segunda parte considerada de complementação fonoaudiológica é composta pelos itens que são entendidos pelos fonoaudiólogos como fatores informativos ou preditivos para a eficácia da terapia. Esses itens são: vogal /é/ sustentada e fraca; vogal /é/ sustentada e aguda; vogal /é/ sustentada e grave; vogal /é/ curta com ataque vocal brusco. CONCLUSÕES: o PAN, em sua versão final, contribui para a sistematização dos procedimentos de avaliação fundamentados em evidências e concordâncias profissionais. O PAN resulta na descrição de itens a serem solicitados durante a avaliação médica e fonoaudiológica no exame de nasofibrolaringoscopia da alteração da mobilidade laríngea em doenças da tireóide.BACKGROUND: voice protocol. AIM: to propose a protocol for the fiberoptic laryngoscopy evaluation of larynx mobility in thyroid illnesses (PAN), with the intention of having an objective, precise and consensual instrument for this assessment. METHOD: the first version of the protocol was elaborated based on data found in the literature; the protocol was judged twice, using the triangulation method; a pilot version was presented and applied in 11 patients; it was then judged again by doctors and speech-language pathologists; based on the analysis of the judges and after the application of the pilot version, the final version of the PAN was proposed. RESULTS: the final protocol was composed by two parts. The first part, considered a standard procedure, is composed by 4 essential items that necessarily should be evaluated: normal inspiration; forced inspiration; vowel /é/ isolated and sustained; and sharp vowel /i/, isolated and sustained. The second part, considered a speech-language complementation, is composed by items that should be understood as being important for speech-language pathologists as they are informative or predictive of the effectiveness of therapy: vowel /é/ sustained and weak; vowel /é/ sustained and sharp; vowel /é/ sustained and deep; vowel /é/ short with abrupt vocal onset. CONCLUSIONS: the PAN, in its final version, contributes for the systematization of the assessment procedures based on evidence and on the agreement of professionals. The PAN results in the description of items to be obtained during medical and speech-language assessment during the fiberopticlaryngoscopy evaluation of larynx mobility in thyroid illnesses

Fear conditioning- and extinction-induced neuronal plasticity in the mouse amygdala

Experience-dependent changes in behavior are mediated by long-term functional modifications in brain circuits. To study the underlying mechanisms, our lab is using classical auditory fear conditioning, a simple and robust form of associative learning. In classical fear conditioning, the subject is exposed to a noxious unconditioned stimulus (US), such as a foot-shock, in conjunction with a neutral conditioned stimulus (CS), such as a tone or a light. As a result of the training, the tone acquires aversive properties and when subsequently presented alone, will elicit a fear response. In rodents, such responses include freezing behavior, alterations in autonomic nervous system activity, release of stress hormones, analgesia, and facilitation of reflexes. Subsequently, conditioned fear can be suppressed when the conditioned stimulus is repeatedly presented alone, a phenomenon called fear extinction. It emerges from a large number of studies in animals and humans that the amygdala is a key brain structure mediating fear conditioning. The amygdala consists of several distinct nuclei, including the lateral (LA) and basal (BA) nuclei, and the central nucleus (CEA). In the classical circuit model of fear conditioning, the LA is thought of as the primary site where CS-US associations are formed and stored. The formation of CS-US associations in the LA is mediated by N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at glutamatergic sensory inputs originating from auditory thalamus and cortex. In contrast to the LA, the CEA has been considered to be primarily involved in the behavioral expression of conditioned fear responses. While the mechanisms and the circuitry underlying fear conditioning in the LA have been extensively studied, much less is known about the neuronal substrates underlying fear extinction. The question of how conditioned fear can be inhibited by extinction is attracting increasing interest because of its clinical importance for the therapy of anxiety disorders. The amygdala is also a potential site of extinction-associated plasticity since intra-amygdala blockade of NMDA receptors or the MAPK signaling pathway prevents extinction. In the first part of this thesis, a combination of behavioral, pharmacological and in vivo electrophysiological approaches was used to study the role of distinct amygdala sub-nuclei in fear exinction. Single unit recordings in behaving mice revealed that the BA contains distinct types of neurons that are specifically activated upon fear conditioning or extinction, respectively. During acquisition of extinction, the activity of “fear neurons” gradually declines, while “extinction neurons” increase their activity. Conversely, when extinguished fear responses are recovered by placing the animal in an unsafe environment, “extinction neurons” switch off, while “fear neurons” switch on. Using local micro-iontophoretic injection of the GABAA receptor agonist muscimol, we found that inactivation of the BA completely prevents the acquisition of extinction or context-dependent fear recovery, depending on the injection time point. Finally, we could show that “fear neurons” and “extinction neurons” are differentially connected with the medial prefrontal cortex (mPFC) and the ventral hippocampus (vHC), two brain areas involved in context-dependent extinction. In contrast to previous models suggesting that amygdala neurons are active during states of high fear and inactive during states of low fear, our findings indicate that activity in specific neuronal circuits within the amygdala may cause opposite behavioral outcomes, thus providing a new framework for understanding context-dependent expression and extinction of fear behavior. In the second part of the thesis, I examined how inhibitory circuits in the central nucleus of the amygdala (CEA) contribute to fear conditioning. While many studies have demonstrated that neuronal plasticity in the LA is necessary for fear conditioning, the role of the CEA, which is mainly composed of GABAergic inhibitory neurons, is poorly understood. In the classical circuit model, the CEA has been thought of as a passive relay station conveying LA output to downstream targets in the hypothalamus and in the brain stem. However, recent in vivo pharmacological experiments suggest a more active role for the CEA during fear conditioning. To address the role of CEA inhibitory circuits in fear conditioning, we obtained single unit recordings from neurons located in the lateral (CEl) and medial (CEm) subdivisions of the CEA in behaving mice. We found that CEm output neurons, that control fear behavior via projections to brainstem targets, are under tight inhibitory control from a subpopulation of neurons located in CEl. Fear conditioning induced opposite changes in phasic and tonic inhibition in the CEl to CEm pathway. Targeted pharmacological inactivation of CEl and CEm revealed that whereas plasticity of phasic inhibition is necessary for gating CEm output during fear learning and expression, changes in tonic inhibitory network activity control signal-to-noise ratio and stimulus discrimination. Our results identify CEA inhibitory circuits as a major site of plasticity in fear conditioning, and suggest that regulation of tonic activity of inhibitory circuits may be an important mechanism for controlling sensitivity and specificity in associative learning. Taken together, these findings suggest that the amygdala is not a functionally homogeneous structure. Rather, our results reveal that the BA and the CEA contain specialized and discrete neuronal populations that contribute to distinct aspects of fear conditioning and extinction. Ultimately, elucidating these mechanisms is fundamental for an understanding of memory processes in the brain in general, and should also inform novel therapeutic strategies for psychiatric disorders involving excessive fear responses associated with amygdala hypersensitivity such as post-traumatic stress disorder and other anxiety disorders

Distinct memory engrams in the infralimbic cortex of rats control opposing environmental actions on a learned behavior

Conflicting evidence exists regarding the role of infralimbic cortex (IL) in the environmental control of appetitive behavior. Inhibition of IL, irrespective of its intrinsic neural activity, attenuates not only the ability of environmental cues predictive of reward availability to promote reward seeking, but also the ability of environmental cues predictive of reward omission to suppress this behavior. Here we report that such bidirectional behavioral modulation in rats is mediated by functionally distinct units of neurons (neural ensembles) that are concurrently localized within the same IL brain area but selectively reactive to different environmental cues. Ensemble-specific neural activity is thought to function as a memory engram representing a learned association between environment and behavior. Our findings establish the causal evidence for the concurrent existence of two distinct engrams within a single brain site, each mediating opposing environmental actions on a learned behavior

Firing patterns of ventral hippocampal neurons predict the exploration of anxiogenic locations.

The ventral hippocampus (vH) plays a crucial role in anxiety-related behaviour and vH neurons increase their firing when animals explore anxiogenic environments. However, if and how such neuronal activity induces or restricts the exploration of an anxiogenic location remains unexplained. Here, we developed a novel behavioural paradigm to motivate rats to explore an anxiogenic area. Male rats ran along an elevated linear maze with protective sidewalls, which were subsequently removed in parts of the track to introduce an anxiogenic location. We recorded neuronal action potentials during task performance and found that vH neurons exhibited remapping of activity, overrepresenting anxiogenic locations. Direction-dependent firing was homogenised by the anxiogenic experience. We further showed that the activity of vH neurons predicted the extent of exploration of the anxiogenic location. Our data suggest that anxiety-related firing does not solely depend on the exploration of anxiogenic environments, but also on intentions to explore them

Distinct ventral hippocampal inhibitory microcircuits regulating anxiety and fear behaviors

In emotion research, anxiety and fear have always been interconnected, sharing overlapping brain structures and neural circuitry. Recent investigations, however, have unveiled parallel long-range projection pathways originating from the ventral hippocampus, shedding light on their distinct roles in anxiety and fear. Yet, the mechanisms governing the emergence of projection-specific activity patterns to mediate different negative emotions remain elusive. Here, we show a division of labor in local GABAergic inhibitory microcircuits of the ventral hippocampus, orchestrating the activity of subpopulations of pyramidal neurons to shape anxiety and fear behaviors in mice. These findings offer a comprehensive insight into how distinct inhibitory microcircuits are dynamically engaged to encode different emotional states

Fear extinction relies on ventral hippocampal safety codes shaped by the amygdala

Extinction memory retrieval is influenced by spatial contextual information that determines responding to conditioned stimuli (CS). However, it is poorly understood whether contextual representations are imbued with emotional values to support memory selection. Here, we performed activity-dependent engram tagging and in vivo single-unit electrophysiological recordings from the ventral hippocampus (vH) while optogenetically manipulating basolateral amygdala (BLA) inputs during the formation of cued fear extinction memory. During fear extinction when CS acquire safety properties, we found that CS-related activity in the vH reactivated during sleep consolidation and was strengthened upon memory retrieval. Moreover, fear extinction memory was facilitated when the extinction context exhibited precise coding of its affective zones. Last, these activity patterns along with the retrieval of the fear extinction memory were dependent on glutamatergic transmission from the BLA during extinction learning. Thus, fear extinction memory relies on the formation of contextual and stimulus safety representations in the vH instructed by the BLA

Fear extinction relies on ventral hippocampal safety codes shaped by the amygdala.

Extinction memory retrieval is influenced by spatial contextual information that determines responding to conditioned stimuli (CS). However, it is poorly understood whether contextual representations are imbued with emotional values to support memory selection. Here, we performed activity-dependent engram tagging and in vivo single-unit electrophysiological recordings from the ventral hippocampus (vH) while optogenetically manipulating basolateral amygdala (BLA) inputs during the formation of cued fear extinction memory. During fear extinction when CS acquire safety properties, we found that CS-related activity in the vH reactivated during sleep consolidation and was strengthened upon memory retrieval. Moreover, fear extinction memory was facilitated when the extinction context exhibited precise coding of its affective zones. Last, these activity patterns along with the retrieval of the fear extinction memory were dependent on glutamatergic transmission from the BLA during extinction learning. Thus, fear extinction memory relies on the formation of contextual and stimulus safety representations in the vH instructed by the BLA

Amygdala circuitry mediating reversible and bidirectional control of anxiety

Anxiety—a sustained state of heightened apprehension in the absence of immediate threat—becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)—achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA—exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA–CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease

Anxiety-related activity of ventral hippocampal interneurons.

Anxiety is an aversive mood reflecting the anticipation of potential threats. The ventral hippocampus (vH) is a key brain region involved in the genesis of anxiety responses. Recent studies have shown that anxiety is mediated by the activation of vH pyramidal neurons targeting various limbic structures. Throughout the cortex, the activity of pyramidal neurons is controlled by GABA-releasing inhibitory interneurons and the GABAergic system represents an important target of anxiolytic drugs. However, how the activity of vH inhibitory interneurons is related to different anxiety behaviours has not been investigated so far. Here, we integratedin vivoelectrophysiology with behavioural phenotyping of distinct anxiety exploration behaviours in rats. We showed that pyramidal neurons and interneurons of the vH are selectively active when animals explore specific compartments of the elevated-plus-maze (EPM), an anxiety task for rodents. Moreover, rats with prior goal-related experience exhibited low-anxiety exploratory behaviour and showed a larger trajectory-related activity of vH interneurons during EPM exploration compared to high anxiety rats. Finally, in low anxiety rats, trajectory-related vH interneurons exhibited opposite activity to pyramidal neurons specifically in the open arms (i.e. more anxiogenic) of the EPM. Our results suggest that vH inhibitory micro-circuits could act as critical elements underlying different anxiety states

Genetic dissection of an amygdala microcircuit that gates conditioned fear

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ^+ neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ^− neurons in CEl. Electrical silencing of PKC-δ^+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called Cel_(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing