Name: determining ultrasonic vocalization preferences in mice using a two-choice playback test
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Este trabalho foi apoiado por um Grant-in-Aid para JSPS Fellows para AA; por Grants-in-Aid para a Investigação Científica em áreas inovadoras para jsps Fellows (No. 4501 e nº 25132712) para TK; e por um subsídio concedido pelo projeto de pesquisa Azabu University. Figura 2 é reproduzido a partir de Kihara, T., Harada, T., &amp; Koshida, fabricação e características de silício nanocristalino emissores de ultra-som induzido termicamente compatível-Wafer N.. Em: sensores e atuadores A: físico, volume 125, Elsevier, p. 426, (2006), com a permissão da Elsevier.

<html> Todos os procedimentos foram aprovados pelo Comitê de Ética da Universidade de Azabu o. Todas as experiências foram realizadas numa câmara à prova de som. 1. Preparação de animal <ol><li> Os machos para gravação <ol><li> Obter camundongos machos sexualmente maduros experientes no acasalamento. </li></ol></li><li> Indivíduos do sexo feminino <ol><li> Obter camundongos fêmeas virgens que foram alojados com 2 a 5 ninhada por gaiola (geralmente 8 - 12 semanas de idade). </li><li> Obter esfregaços vaginais diariamente para determinar a fase do ciclo estral antes do teste, de acordo com McLean 21. Cuidados devem ser tomados para minimizar a estimulação vaginal para evitar pseudogravidez. </li><li> Corar as manchas com Giemsa, e determinar a fase do ciclo estral com base na presença ou ausência de leucócitos, células epiteliais cornificadas e nucleadas células epiteliais, de acordo com Nelson 22. </li></ol></li></ol> 2. Dispositivos (Figura 1) _content &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/53074/53074fig1.jpg" /> Figura 1. Esquema do teste de Box e dispositivos Two-escolha. Os ratos podem acessar salas A e B através das pequenas portas localizadas entre eles ea zona neutra. A caixa-dois e dois emissores escolha de ultra-som são colocados no chão de uma câmara à prova de som. O microfone está suspenso dentro da câmara à prova de som. Abreviatura:. PC, computador pessoal <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol><li> Para Gravação <ol><li> Ligue um microfone condensador de ultra-som a um PC usando um conversor USB A / D. </li><li> Defina o nível de ganho do conversor A / D para &quot;7&quot;. </li><li> Dados acústicos, discos em arquivos de formato de 16 bits com uma taxa de amostragem de 400 kHz usando o software de captura de áudio. </li></ol></li><li> Para Reprodução <ol><li> Conecte um conversor D / A para o PC usandouma interface USB. </li><li> Conecte dois amplificadores para o conversor D / A através de um atenuador. (Este último dispositivo serve para evitar a sobrecarga.) </li><li> Conecte um emissor de ultra-som para cada amplificador. </li></ol></li></ol> 3. USV gravação <ol><li> Coloque um rato macho em uma pequena (17 x 10 x 11 cm) ou (21 x 14 x 13 cm) gaiola de tamanho médio com um buraco de seis centímetros de diâmetro na parede lateral eo buraco coberto com uma tela de arame de 0,5 centímetros. Quando se utiliza uma gaiola de tamanho médio, construir uma partição entre o centro da gaiola para reduzir a área. </li><li> Não use roupas de cama na gaiola para evitar ruídos sussurrantes que podem contaminar as gravações. </li><li> Coloque um microfone ao lado da malha. </li><li> Iniciar o monitoramento vocalização ultra-sônica com a configuração de gravação mostrado na etapa 2.1. </li><li> Coloque um rato fêmea sexualmente madura em qualquer metaestro ou diestro na gaiola com o rato macho. </li><li> USVs recorde para 3-5 min. </li></ol> 4. Test Box <ol><li> Construir caixa de teste usando a placa de acrílico (Figura 1). Nota: A caixa de teste (35 x 20 e 20 x cm de altura) é dividido em três compartimentos: a zona neutra (15 x 20 cm), e quartos A e B (20 x 10 cm, cada). Salas A e B cada um tem um buraco de 4 cm de diâmetro, que é coberta com uma malha de arame de 0,5 centímetros. Os buracos da malha e estão localizados em cada extremidade de salas A e B. Adicione portões (5 x 5 cm cada) entre a zona neutra e salas A e B. </li><li> Para evitar fuga de som isolar cada emissor de ultra-sons, com excepção para o lado voltado para a malha, utilizando placas de borracha, selando-os para a caixa em torno do perímetro de cada malha. </li><li> Lugar de chumbo vedantes em ambos os lados da parede entre os quartos A e B para isolamento de som. </li></ol> 5. Preparação para a reprodução de som <ol><li> Criação de sons de reprodução <ol><li> Captar um segmento de 20 segundos de USV do arquivo gravado usando o software de edição de áudio digital. </li><li> Selecione e captar o fundo nãoise (0,35 seg) do arquivo de segmento 20 seg USV. Crie um arquivo de ruído de fundo 20 segundos, repetindo o segmento de ruído. </li><li> Para reproduzir os dois arquivos de som em simultâneo, exportá-los como um único arquivo de som estéreo, com um arquivo para a &quot;orelha esquerda&quot; e outro arquivo para a &quot;orelha direita.&quot; </li><li> Filtrar os arquivos de som usando como filtro passa-alta com um corte de 40 kHz. </li><li> Reduzir o ruído utilizando a ferramenta de redução de ruído no software de pós-processamento. Selecione e capturar um segmento do arquivo que não contém USVs como o perfil de redução de ruído, e executar a redução do ruído com nível 40. </li></ol></li><li> Calibragem dos Sons Reprodução <ol><li> Certifique-se de que o espaço entre os emissores de ultra-som e microfone é de 10 cm. </li><li> Para calibrar os níveis de pressão sonora de os emissores de ultra-som, realizar o monitoramento de ultra-som usando a configuração descrita no Passo 2.1. Medir o nível de pressão sonora pelo microfone em decibéis (dB). </li><li> Using do atenuador e amplificadores, ajustar o volume dos USVs geradas pelos emissores de ultra-sons para o mesmo nível de pressão do som como os USVs macho gravados no Passo 3. </li><li> Ao usar os dois emissores de ultra-som e um arquivo de som composto por USVs e ruído de fundo, confirmar que os dois emissores de ultra-som apresentar o mesmo nível de pressão sonora utilizando um som de calibração (por exemplo, um tom puro de 75 kHz), antes de realizar o teste comportamental. </li><li> Ao reproduzir um arquivo de som composto por duas correntes de USVs, ajuste os dois níveis de pressão sonora gerada para o mesmo nível, antes do teste. </li></ol></li></ol> 6. Teste Duas escolha <ol><li> Coloque os emissores de ultra-som por trás das malhas da caixa de teste. </li><li> Feche as portas para salas A e B com uma placa de acrílico, e habituar o assunto do sexo feminino para a zona neutra para 30 min. Cobrir a caixa com placa de acrílico para evitar que o rato pode escapar. </li><li> Iniciar a gravação de vídeo usando um CCD câmara montada por cima da caixa. A câmara (não mostrada na Figura 1) abrange a área de todos os três compartimentos. </li><li> Retire a tampa e porta-board e permitir que a fêmea para explorar livremente a caixa de teste, incluindo as zonas de som. </li><li> Uma vez que a fêmea tem investigado as duas malhas, e voltou para a zona neutra, começar a jogar um arquivo de som 20 seg repetidamente para 5 ou 10 min. </li><li> Realizar monitoramento comportamental para 5 ou 10 min. </li><li> Para reduzir pistas olfactivas indesejáveis ​​depositados a partir do objecto anterior, limpar as gaiolas de teste entre os testes com etanol a 70%. </li><li> Mude o local de emissores de ultra-som A e B aleatoriamente para evitar o efeito de viés lado inerente no sentido de quartos A ou B. </li></ol> 7. Análise estatística <ol><li> Usando o software evento de scoring comportamental, analisar os seguintes parâmetros: número total de entradas em cada quarto; duração total de permanência em cada zona intermediária; duração total de permanência em cada zona de som; eduração total de busca de cada malha em frente do emissor de ultra-som. A figura 1 ilustra o que se entende por &quot;zona central&quot; e &quot;zona de som.&quot; Nota: Nos casos em que o rato permanece na linha divisória entre as duas zonas (Figura 1, linha tracejada) determinar os locais pela direcção da cabeça. Quando a cabeça voltada para o lado da malha, ele é marcado como uma estadia na zona de som. </li><li> Para cada parâmetro comportamental, comparar as respostas para as salas A e B utilizando um teste de sinais com posto Wilcoxon, ou -Testes t pareado com nível de significância de 0,05. </li></ol>

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The authors declare that they have no competing interests.

Here, the results of a representative test showed that female mice can discriminate between artificial male USVs and background noise (Figure 3). The conclusion to be drawn from these results is that the discrimination signal is reflected in the duration of stay in the room and sound zone, and in the duration of searching the mesh in the first 5 min of testing, but not in the second 5 min (Figure 3C, E and F). These data indicate that mice become habituated to the playback sounds, possib...

Muitos animais usam vocalizações para a comunicação intra-específica. No musculus rato Mus, um importante tipo de sinal de comunicação é vocalizações ultra-sônicas (USVs), que têm frequências superiores a 20 kHz. USVs emitidos pelos ratos são considerados um componente de reconhecimento social em masculino-feminino 1-4, fêmea-fêmea 1, 5, e macho-macho 1, 6 interações. USVs também são emitidos por filhotes quando eles são isolados a partir de sua mãe, o que aumenta o seu comportamento de recuperação de cachorro, e, portanto, a sobrevivência dos cachorros 7. Embora muitos relatórios analisados ​​e categorizados USVs rato 8, 9, as respostas comportamentais e mecanismos neurais do animal que recebeu foram documentados menos 10, 11. O último é necessário clarificar o significado biológico das várias características de USVs. Para revelar destes mecanismos, o experimento de reprodução é um método eficiente. Estudos recentes revelaram que a reprodução femininaratos são atraídos para USVs 12, e que eles preferem USVs de homens que são diferentes de seus pais 13, 14. Este artigo explica o ensaio utilizado para avaliar a reprodução preferência USV em ratinhos. Uma caixa de teste de dois escolha foi desenvolvido no qual dois USVs diferentes podem ser reproduzidos simultaneamente em dois compartimentos de uma câmara de ensaio, como se mostra na Figura 1. Este tipo de caixa de teste evita a contaminação do som através da divisão da área de teste em três sub-câmaras , usando paredes de chumbo. Os emissores de ultra-som estão localizados fora cada quarto. Na parede entre os quartos e emissores de ultra-som são buracos cobertos com malha de arame. Ratos podem circular livremente nos três quartos, e mostrar uma &quot;busca da malha&quot; comportamento, como se para responder a USVs reproduzidos pelos emissores de ultra-som. Neste teste, os ratos permanecer por períodos de duração diferente perto de um emissor de som ou de outro. Estes parâmetros podem estar logado para obter uma sensível measu<html> re de preferências de som. Para reproduzir as USVs volta, silício nanocristalino emissores termo-acústicos (ie, &quot;emissor nc-Si&quot;) foram utilizados como em estudos anteriores 15-17. Estes dispositivos são constituídos por um eléctrodo de película fina aquecedor, uma camada de silício nanoporoso, e uma pastilha de silício monocristalino. O ficheiro de som digital é convertido para um sinal analógico e, em seguida, passado através do aquecedor de eléctrodo. O dispositivo converte os sinais térmicos dependentes da voltagem, resultando em pressão sonora significativa com baixa distorção. Este dispositivo é único na medida em que, ao contrário de geradores de som comuns que dependem de vibrações mecânicas, ele pode reproduzir som sem a necessidade de um diafragma. O emissor apresenta um nível de pressão sonora plana a frequências de 20 a 160 kHz (Figura 2), e pode reproduzir USVs murino gravados digitalmente de forma muito precisa em termos de duração, freqüência e nível de pressão sonora de 15, 18, ​​19. ve_content &quot;&gt; Em uma experiência representativa mostrada na Figura 3, C57BL / 6 (B6) fêmeas foram autorizados a escolher entre BALB / c (BALB) USVs macho e ruído de fundo. Além disso, a Figura 4 mostra a escolha das fêmeas B6 e BALB entre playbacks USV simultâneas de um BALB e um macho B6, conforme relatado em um estudo anterior 14. As características dos USVs masculinos diferem entre B6 e BALB estirpes 20. Como mostrado por estes resultados, a atratividade de USVs pode ser avaliada com o presente protocolo, em que os sons são gravados a partir de um indivíduo vivo, acústico analisado, e jogou de volta para outros indivíduos.

<table><tbody><tr><td>Soundproof chamber</td><td>Muromachi Kikai</td><td></td><td></td></tr><tr><td>Small cage</td><td>CLEA Japan</td><td>CL-0113-1</td><td></td></tr><tr><td>Middle cage</td><td>CLEA Japan</td><td>CL-0103-1</td><td></td></tr><tr><td>Ultrasound condenser microphones</td><td>Avisoft Bioacoustics</td><td>CM16/CMPA</td><td></td></tr><tr><td>A/D converter</td><td>Avisoft Bioacoustics</td><td>UltraSoundGate116H</td><td></td></tr><tr><td>Audio software</td><td>Avisoft Bioacoustics</td><td>RECORDER USGH</td><td></td></tr><tr><td>Adobe Audition 3.0 / Audio editing software</td><td>Adobe Systems</td><td>Adobe Audition 3.0</td><td></td></tr><tr><td>Nc-Si emitter</td><td>Original</td><td></td><td>not commercially available but it is planned to be so in near future</td></tr><tr><td>D/A converter</td><td>National Instruments</td><td>NI USB-6251 BNC</td><td></td></tr><tr><td>Attenuator</td><td>Original</td><td></td><td></td></tr><tr><td>Amplifier</td><td>Yamatake</td><td></td><td></td></tr><tr><td>PC</td><td>Windows 7 professional</td><td></td><td>Intel&amp;reg; core i7-2600K CPU @ 3.4GHz, 8GB RAM, 64-bit operating system</td></tr><tr><td>Event recorder Excel-macro / Event-scoring software</td><td>original</td><td></td><td>Programmed by Naoto Akagawa &amp;amp; Takeru Yamamoto</td></tr><tr><td>CCD Camera</td><td></td><td></td><td></td></tr><tr><td>Rubber plates (made of elastomer resin)</td><td>Tokyo bouon</td><td>TI-75BK B4</td><td>Cut them to the proper size http://www.piano-bouon.jp/shopping/?pid=1329272401-447630&amp;amp;ca=6&amp;amp;p=3</td></tr><tr><td>Giemsa&#039;s azur eosin methylene blue solution</td><td>Merck Millipore</td><td>1.09204.0500</td><td></td></tr></tbody></table>

determining ultrasonic vocalization preferences in mice using a two-choice playback test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Os USVs gravado a partir de um ratinho BALB-macho (161 sílabas por 20 seg), bem como o ruído de fundo foram usadas como a reprodução de sons na experiência representativa mostrada na Figura 3. Nesta experiência, 7 ratinhos B6 fêmeas foram usadas às 9 semanas de idade . Para determinar a melhor duração de teste de resposta feminina para reproduzir sons, os parâmetros comportamentais foram analisados ​​separadamente para as primeira e a última de cinco minutos de tempo total de ensaio de 1...

Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Determinando Preferências Ultrasonic vocalização em ratos utilizando um teste de reprodução de dois escolha

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

determining-ultrasonic-vocalization-preferences-mice-using-two-choice

Universidad San Sebastian

Research

JoVE Journal

Behavior

11.1K visualizações.  Azabu University. Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Vídeo: Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Avaliação de vocalizações ultra-som durante Drogas auto-administração em ratos

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Neurociência

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Gravação mouse ultra-som vocalizações para avaliar Comunicação Social

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

Comportamento

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Formação específica de vocalizações ultra-som em ratos idosos e Parkinson

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

Eliciando e Analisando Canções de Vocalização Ultrassônica de Rato Masculino (USV)

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Apresentando Clicker Formação como um enriquecimento cognitivo para Laboratório Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

Habituação e Prepulse Inibição de sobressalto acústico em Roedores

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Um ensaio comportamental para investigar o papel da memória espacial durante uma defesa instintiva em ratos

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

A configuração de Baixo Custo para audiometria comportamental em roedores

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Estudando Comportamentos Defensivos Ativos em Camundongos com Condicionamento de Medo Modificado

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Autor em destaque: Explorando correlatos neurais de comportamentos defensivos na aprendizagem e extinção do medo

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

Condicionamento de medo modificado para induzir comportamentos de voo em camundongos

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

Determinando Preferências Ultrasonic vocalização em ratos utilizando um teste de reprodução de dois escolha

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