Playback of ultrasonic vocalizations in rats: habituation, response calls, and drug effects

Rats are highly social animals. They have developed a variety of social behaviors including communication via so-called ultrasonic vocalizations (USV). Among these USV, two types can be distinguished in juvenile and adult rats. Appetitive 50-kHz USV are thought to represent a positive affective state, whereas aversive 22-kHz USV are supposed to depict a negative affective state. Playback of positive 50-kHz USV induces an approach behavior in rats as seen by their approach behavior to the sound source. Previous studies have shown that this behavior is only detectable during the first presentation, whereas a quick habituation towards 50-kHz USV results in the rats not approaching the 50-kHz USV playback a second time, even after several days. This habituation phenomenon seems to rely on learning and memory mechanisms. However, its underlying mechanisms have been studied scarcely so far. This dissertation revealed three factors influencing the habituation phenomenon. First, habituation was dependent on stocks. It was only present in Wistar but not Sprague-Dawley rats. Second, habituation could be prevented with treatment of the dopaminergic agonist d-amphetamine before the second 50-kHz USV playback. Third, habituation was state-dependent. It was shown that when the pharmacologically induced internal state changed between the two playbacks, no habituation occurred. Furthermore, the reciprocal nature of USV was investigated in this dissertation. Calls in response towards 50-kHz USV playback had been reported before, but this dissertation is the first to characterize response calls. We showed that response calls towards 50-kHz playback are around frequencies of 30 kHz, have a rather short duration of 0.3 s and hardly any frequency modulation. These parameters resemble aversive 22-kHz calls, which are unlikely to be found in an appetitive paradigm as the 50-kHz USV playback. Feasible functions of these response calls might be a frustrated state due to expectation violation after playback, appeasement calls to pacify the potential play partner indicated by the playback, or they might serve as social contact calls to establish proximity. Taken together, the findings of this dissertation shed light on the reciprocal nature of USV communication indicated by response calls towards 50-kHz USV playback and present possible mechanisms how to overcome the habituation phenomenon. This provides tools to further investigate neurodevelopmental disorders where communication and social behavior is impaired, such as autism spectrum disorder or the Angelman Syndrome, as well as affective disorders

Microglomerular Synaptic Complexes in the Sky-Compass Network of the Honeybee Connect Parallel Pathways from the Anterior Optic Tubercle to the Central Complex

While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body’s (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway

Transmedulla Neurons in the Sky Compass Network of the Honeybee (Apis mellifera) Are a Possible Site of Circadian Input.

Honeybees are known for their ability to use the sun's azimuth and the sky's polarization pattern for spatial orientation. Sky compass orientation in bees has been extensively studied at the behavioral level but our knowledge about the underlying neuronal systems and mechanisms is very limited. Electrophysiological studies in other insect species suggest that neurons of the sky compass system integrate information about the polarization pattern of the sky, its chromatic gradient, and the azimuth of the sun. In order to obtain a stable directional signal throughout the day, circadian changes between the sky polarization pattern and the solar azimuth must be compensated. Likewise, the system must be modulated in a context specific way to compensate for changes in intensity, polarization and chromatic properties of light caused by clouds, vegetation and landscape. The goal of this study was to identify neurons of the sky compass pathway in the honeybee brain and to find potential sites of circadian and neuromodulatory input into this pathway. To this end we first traced the sky compass pathway from the polarization-sensitive dorsal rim area of the compound eye via the medulla and the anterior optic tubercle to the lateral complex using dye injections. Neurons forming this pathway strongly resembled neurons of the sky compass pathway in other insect species. Next we combined tracer injections with immunocytochemistry against the circadian neuropeptide pigment dispersing factor and the neuromodulators serotonin, and γ-aminobutyric acid. We identified neurons, connecting the dorsal rim area of the medulla to the anterior optic tubercle, as a possible site of neuromodulation and interaction with the circadian system. These neurons have conspicuous spines in close proximity to pigment dispersing factor-, serotonin-, and GABA-immunoreactive neurons. Our data therefore show for the first time a potential interaction site between the sky compass pathway and the circadian clock

Antioxidant Treatment with N-acetyl Cysteine Prevents the Development of Cognitive and Social Behavioral Deficits that Result from Perinatal Ketamine Treatment

Alterations of the normal redox state can be found in all stages of schizophrenia, suggesting a key role for oxidative stress in the etiology and maintenance of the disease. Pharmacological blockade of N-methyl-D-aspartic acid (NMDA) receptors can disrupt natural antioxidant defense systems and induce schizophrenia-like behaviors in animals and healthy human subjects. Perinatal administration of the NMDA receptor (NMDAR) antagonist ketamine produces persistent behavioral deficits in adult mice which mimic a range of positive, negative, and cognitive symptoms that characterize schizophrenia. Here we tested whether antioxidant treatment with the glutathione (GSH) precursor N-acetyl-cysteine (NAC) can prevent the development of these behavioral deficits. On postnatal days (PND) 7, 9 and 11, we treated mice with subanesthetic doses (30 mg/kg) of ketamine or saline. Two groups (either ketamine or saline treated) also received NAC throughout development. In adult animals (PND 70–120) we then assessed behavioral alterations in a battery of cognitive and psychomotor tasks. Ketamine-treated animals showed deficits in a task of cognitive flexibility, abnormal patterns of spontaneous alternation, deficits in novel-object recognition, as well as social interaction. Developmental ketamine treatment also induced behavioral stereotypy in response to an acute amphetamine challenge, and it impaired sensorimotor gating, measured as reduced prepulse inhibition (PPI) of the startle response. All of these behavioral abnormalities were either prevented or strongly ameliorated by NAC co-treatment. These results suggest that oxidative stress is a major factor for the development of the ketamine-induced behavioral dysfunctions, and that restoring oxidative balance during the prodromal stage of schizophrenia might be able to ameliorate the development of several major symptoms of the disease

Contingent Social Interaction Does Not Prevent Habituation towards Playback of Pro-Social 50-kHz Calls: Behavioral Responses and Brain Activation Patterns

Rats, which are highly social animals, are known to communicate using ultrasonic vocalizations (USV) in different frequency ranges. Calls around 50 kHz are related to positive affective states and promote social interactions. Our previous work has shown that the playback of natural 50-kHz USV leads to a strong social approach response toward the sound source, which is related to activation in the nucleus accumbens. In male Wistar rats, the behavioral response habituates, that is, becomes weaker or is even absent, when such playback is repeated several days later, an outcome found to be memory-dependent. Here, we asked whether such habituation is due to the lack of a contingent social consequence after playback in the initial test and whether activation of the nucleus accumbens, as measured by c-fos immunohistochemistry, can still be observed in a retest. To this end, groups of young male Wistar rats underwent an initial 50-kHz USV playback test, immediately after which they were either (1) kept temporarily alone, (2) exposed to a same-sex juvenile, or (3) to their own housing group. One week later, they underwent a retest with playback; this time not followed by social consequences but by brain removal for c-fos immunohistochemistry. Consistent with previous reports, behavioral changes evoked by the initial exposure to 50-kHz USV playback included a strong approach response. In the retest, no such response was found, irrespective of whether rats had experienced a contingent social consequence after the initial test or not. At the neural level, no substantial c-fos activation was found in the nucleus accumbens, but unexpected strong activation was detected in the anterior cingulate cortex, with some of it in GABAergic cells. The c-fos patterns did not differ between groups but cell numbers were individually correlated with behavior, i.e., rats that still approached in response to playback in the retest showed more activation. Together, these data do not provide substantial evidence that the lack of a contingent social consequence after 50-kHz USV playback accounts for approach habituation in the retest. Additionally, there is apparently no substantial activation of the nucleus accumbens in the retest, whereas the exploratory findings in the anterior cingulate cortex indicate that this brain area might be involved when individual rats still approach 50-kHz USV playback

Central projections of neurons in the lower unit complex of the anterior optic tubercle (AOTU-LUC).

<p>(A-E) Neurobiotin (NB) injection into the AOTU-LUC reveals central projection areas. Cartoon in A illustrates injection site. (A-C) Direct volume rendering from projections in the contralateral AOTU-LUC shows three morphologically different types of TuTu1 neuron (TuTu1a, TuTu1b, TuTu1c). (A’-C’) Maximum intensity projections of six (A’) or three (B’, C’) adjacent slices from preparations shown in A-C, combined with anti-synapsin labeling (syn-ir, grey). (A’) TuTu1a neurons connecting only the dorsal areas of the AOTU-LUC. (B’, C’) Two similar types of TuTu1 neuron with ramifications in the ventral and median areas of the AOTU-LUC. These neurons had dense (green arrows) and sparse (white arrows) ramification areas. While TuTu1b neurons ramified densely within their dorsal, and sparsely within their ventral branching areas (B’), the opposite was true for TuTu1c neurons (C’). (D, E) Projections of TuLAL1 neurons from the AOTU-LUC to the medial and lateral bulb. (D) Overview (direct volume rendering) shows course of the axons which run within the AOTU-LAL tract. The tract separates into two fascicles that innervate the lateral or the medial bulb (LBU, MBU), respectively. The cell bodies of TuLAL1 neurons were located medially of the AOTU (asterisk). Also stained are axons of TuTu1 neurons that run in the intertubercle tract (ITT). (E) Direct volume rendering of large synaptic terminals of TuLAL1 neurons within the median and the lateral bulbs. (E’) Maximum intensity projection of three adjacent slices of preparation shown in E combined with anti-synapsin labeling (grey) illustrates the projection areas of TuLAL1 neurons with respect to the central complex. AL, antennal lobe; CBU, central body upper division; CBL, central body lower division; UU, upper unit of AOTU. All views in frontal plane. Scale bars: 30 μm in A-C; 100 μm in D; 50 μm in E.</p

Summary of the main findings.

<p>Frontal schematic diagram of the honeybee brain illustrates the main neuropils and the sky compass pathway. The dorsal rim area of the eye (DRA) is connected to the dorsal rim area of the medulla (MEDRA) through long visual fibers. Transmedulla neurons project from the MEDRA to the lower unit complex of the anterior optic tubercle (LUC). TuLAL1 neurons project from the LUC around the vertical lobe of the mushroom body (VL) to the median and lateral bulb (MBU, LBU). Three Types of TuTu1 neurons project to the contralateral LUC. Stippled grey lines in medulla (ME) indicate hypothetical unpolarized light input pathways. Also shown is immunoreactivity in the medulla to antisera against pigment dispersing factor (PDF) and 5HT (5HT). AL, antennal lobe; CBL, lower division of the central body; CBU, upper division of the central body; LA, lamina; LCA and MCA, lateral and medial calyx of the mushroom body; LO, lobula; PED, pedunculus. Scale bar: 200 μm.</p

Primary antibodies, dilutions and fixatives.

<p>GA, glutaraldehyde; GSA, glutathione-S-transferase; KLH, keyhole limpet hemocyanin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; PA, picric acid; PFA, paraformaldehyde.</p><p>Primary antibodies, dilutions and fixatives.</p

Absence of PDF-, 5HT, and GABA-ir in the lower unit complex of the anterior optic tubercle (AOTU-LUC).

<p>Synapsin-immunoreactivity (syn-ir, magenta) and immunoreactivity to antisera against PDH (PDH-ir), 5HT (5HT-ir) and GABA (GABA-ir), shown in green, in the anterior optic tubercle. (A) The entire AOTU is devoid of PDH-ir. (B) 5HT-ir was found in the upper unit of the anterior optic tubercle (UU), but not in the AOTU-LUC. (C) GABA-ir, green was found in the AOTU-UU, but not in the AOTU-LUC. AL, antennal lobe. All views in frontal plane. All scale bars: 30 μm.</p

Spatial relationship between transmedulla neurons and 5HT-ir fibers.

<p>(A) Confocal image of 5HT-immunoreactive (5HT-ir) neurons (green) and transmedulla neurons labelled by dextran biotin/streptavidin Texas Red (magenta). Both stainings are restricted to the dorsal and medial parts of the medulla. (B, C) Higher magnification/resolution images of the areas indicated in A show close proximity, but no colocalization of the two stainings. (B) The dorsal rim area of the medulla (MEDRA) is devoid of 5HT-ir. (C) Transmedulla neurons overlap with the band of 5HT-ir, but the latter is wider and extends more medially. ME, medulla. All views in frontal plane. Scale bars: 100 μm in A; 30 μm in B, C.</p