Rats that learn to vocalize for food reward emit longer and louder appetitive calls and fewer short aversive calls.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.

Spontaneously hypertensive rats manifest deficits in emotional response to 22-kHz and 50-kHz ultrasonic playback.

Many symptoms used routinely for human psychiatric diagnosis cannot be directly observed in animals which cannot describe their internal states. However, the ultrasonic vocalizations (USV) rodents use to communicate their emotional states can be measured. USV have therefore become a particularly useful tool in brain disease models. Spontaneously hypertensive rats (SHR) are considered an animal model of attention deficit hyperactivity disorder (ADHD) and schizophrenia. However, the specifics of SHR's behavior have not been fully described and there is very little data on their USV. Recently, we developed a communication model, in which Wistar rats are exposed to pre-recorded playbacks of aversive (22-kHz) or appetitive (50-kHz) USV, and their vocal responses depend on the extent of prior fear conditioning (0, 1, 6 or 10 shocks). Here, we investigated SHR's behavior and heart rate (HR) in our communication model, in comparison to Wistar rats employed as controls. In general, SHR emitted typical USV categories, however, they contained more short 22-kHz and less 50-kHz USV overall. Moreover, fewer SHR, in comparison with Wistar rats, emitted long 22-kHz USV after fear conditioning. SHR did not show a 50-kHz playback-induced HR increase, while they showed a profound 22-kHz playback-induced HR decrease. Finally, the number of previously delivered conditioning shocks appeared to have no effect on the investigated vocal, locomotor and HR responses of SHR. The phenomena observed in SHR are potentially attributable to deficits in emotional perception and processing. A lower number of 50-kHz USV emitted by SHR may reflect observations of speech impairments in human patients and further supports the usefulness of SHR to model ADHD and schizophrenia.

Increased Vocalization of Rats in Response to Ultrasonic Playback as a Sign of Hypervigilance Following Fear Conditioning.

We investigated the effects of prior stress on rats' responses to 50-kHz (appetitive) and 22-kHz (aversive) ultrasonic playback. Rats were treated with 0, 1, 6 or 10 shocks (1 s, 1.0 mA each) and were exposed to playbacks the following day. Previous findings were confirmed: (i) rats moved faster during 50-kHz playback and slowed down after 22-kHz playback; (ii) they all approached the speaker, which was more pronounced during and following 50-kHz playback than 22-kHz playback; (iii) 50-kHz playback caused heart rate (HR) increase; 22-kHz playback caused HR decrease; (iv) the rats vocalized more often during and following 50-kHz playback than 22-kHz playback. The previous shock affected the rats such that singly-shocked rats showed lower HR throughout the experiment and a smaller HR response to 50-kHz playback compared to controls and other shocked groups. Interestingly, all pre-shocked rats showed higher locomotor activity during 50-kHz playback and a more significant decrease in activity following 22-kHz playback; they vocalized more often, their ultrasonic vocalizations (USV) were longer and at a higher frequency than those of the control animals. These last two observations could point to hypervigilance, a symptom of post-traumatic stress disorder (PTSD) in human patients. Increased vocalization may be a valuable measure of hypervigilance used for PTSD modeling.

Playback of Alarm and Appetitive Calls Differentially Impacts Vocal, Heart-Rate, and Motor Response in Rats.

Our rudimentary knowledge about rat intraspecific vocal system of information exchange is limited by experimental models of communication. Rats emit 50-kHz ultrasonic vocalizations in appetitive states and 22-kHz ones in aversive states. Both affective states influence heart rate. We propose a behavioral model employing exposure to pre-recorded playbacks in home-cage-like conditions. Fifty-kHz playbacks elicited the most vocalizations (>60 calls per minute, mostly of 50-kHz type), increased heart rate, and locomotor activity. In contrast, 22-kHz playback led to abrupt decrease in heart rate and locomotor activity. Observed effects were more pronounced in singly housed rats compared with the paired housed group; they were stronger when evoked by natural playback than by corresponding artificial tones. Finally, we also observed correlations between the number of vocalizations, heart rate levels, and locomotor activity. The correlations were especially strong in response to 50-kHz playback.

Maternal Immune Activation Induces Neuroinflammation and Cortical Synaptic Deficits in the Adolescent Rat Offspring.

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders.

Prenatal Exposure to Valproic Acid Affects Microglia and Synaptic Ultrastructure in a Brain-Region-Specific Manner in Young-Adult Male Rats: Relevance to Autism Spectrum Disorders.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental conditions categorized as synaptopathies. Environmental risk factors contribute to ASD aetiology. In particular, prenatal exposure to the anti-epileptic drug valproic acid (VPA) may increase the risk of autism. In the present study, we investigated the effect of prenatal exposure to VPA on the synaptic morphology and expression of key synaptic proteins in the hippocampus and cerebral cortex of young-adult male offspring. To characterize the VPA-induced autism model, behavioural outcomes, microglia-related neuroinflammation, and oxidative stress were analysed. Our data showed that prenatal exposure to VPA impaired communication in neonatal rats, reduced their exploratory activity, and led to anxiety-like and repetitive behaviours in the young-adult animals. VPA-induced pathological alterations in the ultrastructures of synapses accompanied by deregulation of key pre- and postsynaptic structural and functional proteins. Moreover, VPA exposure altered the redox status and expression of proinflammatory genes in a brain region-specific manner. The disruption of synaptic structure and plasticity may be the primary insult responsible for autism-related behaviour in the offspring. The vulnerability of specific synaptic proteins to the epigenetic effects of VPA may highlight the potential mechanisms by which prenatal VPA exposure generates behavioural changes.

Stimulus-seeking in rats is accompanied by increased c­Fos expression in hippocampal CA1 as well as short 22 kHz and flat 50 kHz calls.

We determined CA1 hippocampal field to be involved in self-exposure, a type of novelty­seeking behaviour that has also been associated with short 22 kHz and flat 50 kHz ultrasonic vocalizations (USV) in adult male Long-Evans rats. Rats were habituated for three days to a self-exposure cage with two nose-poke holes. On day four, the animals from the experimental group were allowed to turn the cage light off for 5 s with a nose­poke (test/self­exposure session), while rats from control-yoked group had changing light conditions coupled and identical to the experimental animals. The experimental rats performed more nose-pokes during self-exposure session than animals from the control group. This effect was accompanied by a higher density of c-Fos-positive nuclei in the hippocampal CA1. There were no significant group differences in c-Fos expression in other brain regions analysed. However, possible involvement of several other structures in self-exposure (i.e., CA3, the dentate gyrus, amygdala, prefrontal cortex, and nucleus accumbens) is also discussed, as their correlational activity, reflected by c-Fos immunoactivity, was observed in the experimental rats. During test sessions, there were more nose-pokes accompanied by short 22 kHz calls and 50 kHz calls performed by the rats of the experimental group than of the control group. The CA1 region has previously been associated with novelty; short 22 kHz USV and flat 50 kHz USV could be associated with self-exposure, also they appear to be emitted correlatively.

Glutamate, Glutamine and GABA Levels in Rat Brain Measured Using MRS, HPLC and NMR Methods in Study of Two Models of Autism.

The disorders of the glutamatergic neurotransmission have been associated with pathogenesis of autism. In this study we evaluated the impact of the in vivo and ex vivo test methodology on measurements of levels of neurotransmitter amino acids in hippocampus of rats for valproic acid- (VPA) and thalidomide- (THAL) induced models of autism. The main goal was to compare the changes in concentrations of glutamate (Glu), glutamine (Gln) and GABA between both autistic groups and the control, measured in vivo and ex vivo in homogenates. The rat pups underwent three in vivo tests: ultrasonic vocalization (USV), magnetic resonance spectroscopy (MRS) and unilateral microdialysis of the hippocampus. Analyses of homogenates of rat hippocampus were performed using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis, we performed univariate and multivariate tests. USV test, which is considered in rodents as an indicator of pathology similar to autism, showed decreased USV in VPA and THAL groups. In vivo MRS studies demonstrated increases of Glu content in male rat's hippocampus in VPA and THAL groups, while the microdialysis, which allows examination of the contents in the extracellular space, detected decreases in the basal level of Gln concentrations in VPA and THAL groups. Ex vivo HPLC studies showed that levels of Glu, Gln and GABA significantly increased in male rat's hippocampus in the VPA and THAL groups, while NMR studies showed increased levels of Gln and GABA in the VPA group. Collectively, these results are consistent with the hypothesis suggesting the role of the glutamatergic disturbances on the pathogenesis of autism. For all methods used, the values of measured changes were in the same direction. The orthogonal partial least square discriminant analysis confirmed that both animal models of autism tested here can be used to trace neurochemical changes in the brain.

Cortical Synaptic Transmission and Plasticity in Acute Liver Failure Are Decreased by Presynaptic Events.

Neurological symptoms of acute liver failure (ALF) reflect decreased excitatory transmission, but the status of ALF-affected excitatory synapse has not been characterized in detail. We studied the effects of ALF in mouse on synaptic transmission and plasticity ex vivo and its relation to distribution of (i) synaptic vesicles (sv) and (ii) functional synaptic proteins within the synapse. ALF-competent neurological and biochemical changes were induced in mice with azoxymethane (AOM). Electrophysiological characteristics (long-term potentiation, whole-cell recording) as well as synapse ultrastructure were evaluated in the cerebral cortex. Also, sv were quantified in the presynaptic zone by electron microscopy. Finally, presynaptic proteins in the membrane-enriched (P2) and cytosolic (S2) fractions of cortical homogenates were quantitated by Western blot. Slices derived from symptomatic AOM mice presented a set of electrophysiological correlates of impaired transmitter release including decreased field potentials (FPs), increased paired-pulse facilitation (PPF), and decreased frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs) accompanied by reduction of the spontaneous transmitter release-driving protein, vti1A. Additionally, an increased number of sv per synapse and a decrease of P2 content and/or P2/S2 ratio for sv-associated proteins, i.e. synaptophysin, synaptotagmin, and Munc18-1, were found, in spite of decreased content of the sv-docking protein, syntaxin-1. Slices from AOM-treated asymptomatic mice showed impaired long-term potentiation (LTP) and increased PPF but no changes in transmitter release or presynaptic protein composition. Our findings demonstrate that a decrease of synaptic transmission in symptomatic ALF is associated with inefficient recruitment of sv proteins and/or impaired sv trafficking to transmitter release sites.

Sodium Butyrate, a Histone Deacetylase Inhibitor, Exhibits Neuroprotective/Neurogenic Effects in a Rat Model of Neonatal Hypoxia-Ischemia.

Neonatal hypoxic-ischemic (HI) injury still remains an important issue as it is a major cause of neonatal death and neurological dysfunctions. Currently, there are no well-established treatments to reduce brain damage and its long-term sequel in infants. Recently, reported data show that histone deacetylase inhibitors provide neuroprotection in adult stroke models. However, the proof of their relevance in vivo after neonatal HI brain injury remains particularly limited. In the present study, we show neuroprotective/neurogenic effect of sodium butyrate (SB), one of histone deacetylase inhibitors (HDACis), in the dentate gyrus of HI-injured immature rats. Postnatal day 7 (P7) rats underwent left carotid artery ligation followed by 7.6 % O2 exposure for 1 h. SB (300 mg/kg) was administered in a 5-day regime with the first injection given immediately after the onset of HI. The damage of the ipsilateral hemisphere was evaluated by weight deficit. Newly produced cells were labeled with BrdU, at 50 mg/kg, injected twice daily for 3 consecutive days. Subsequent differentiation of the newborn cells was investigated 2 and 4 weeks after the insult by immunohistochemistry using neuronal and glial cell-lineage markers and BrdU incorporation. Finally, we performed several behavioral tests to evaluate functional outcome. In summary, SB led to a remarkable reduction of the brain damage caused by HI. Moreover, the application of this HDACi protected against HI-induced loss of neuroblasts and oligodendrocyte precursor cells, as well as against neuroinflammation. The observed neuroprotective action suggests that SB may serve as a potential candidate for future treatment of HI-evoked injury in neonates.

Carnosine Reduces Oxidative Stress and Reverses Attenuation of Righting and Postural Reflexes in Rats with Thioacetamide-Induced Liver Failure.

Cerebral oxidative stress (OS) contributes to the pathogenesis of hepatic encephalopathy (HE). Existing evidence suggests that systemic administration of L-histidine (His) attenuates OS in brain of HE animal models, but the underlying mechanism is complex and not sufficiently understood. Here we tested the hypothesis that dipeptide carnosine (ß-alanyl-L-histidine, Car) may be neuroprotective in thioacetamide (TAA)-induced liver failure in rats and that, being His metabolite, may mediate the well documented anti-OS activity of His. Amino acids [His or Car (100 mg/kg)] were administrated 2 h before TAA (i.p., 300 mg/kg 3× in 24 h intervals) injection into Sprague-Dawley rats. The animals were thus tested for: (i) brain prefrontal cortex and blood contents of Car and His, (ii) amount of reactive oxygen species (ROS), total antioxidant capacity (TAC), GSSG/GSH ratio and thioredoxin reductase (TRx) activity, and (iii) behavioral changes (several models were used, i.e. tests for reflexes, open field, grip test, Rotarod). Brain level of Car was reduced in TAA rats, and His administration significantly elevated Car levels in control and TAA rats. Car partly attenuated TAA-induced ROS production and reduced GSH/GSSG ratio, whereas the increase of TRx activity in TAA brain was not significantly modulated by Car. Further, Car improved TAA-affected behavioral functions in rats, as was shown by the tests of righting and postural reflexes. Collectively, the results support the hypothesis that (i) Car may be added to the list of neuroprotective compounds of therapeutic potential on HE and that (ii) Car mediates at least a portion of the OS-attenuating activity of His in the setting of TAA-induced liver failure.

Role of cytoskeletal motor proteins in viral infection.

Cytoskeleton, composed of actin filaments, microtubules and intermediate filaments, regulates many processes in the cell, e.g. intracellular transport. Actin and microtubules are polarized structures, along which bidirectional transport of motor proteins occurs: myosins along actin and the dynein/dynactin complex and kinesins along microtubules. Viruses interact with the cytoskeleton and motor proteins at different stages during their replication cycle. When entering and egressing the cell, viruses must penetrate the cortical layer of microfilaments, which usually takes place with the contribution of myosin. In the cytoplasm, retrograde transport involving dynein is used to move viruses to the microtubule organizing center. After replication, kinesins participate in anterograde transport of newly produced virions to the peripheral region, close to the plasma membrane. Some families of viruses have developed alternate routes of intracellular transport. The aim of this study is to describe the interactions between virus and cytoskeletal motor proteins and to determine their role in viral infection according to the current literature data.