Correlation of distinct behaviors to the modified expression of cerebral Shank1,3 and BDNF in two autistic animal models

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Highlights

  • VPA- and PPA-induced autistic rat models present different behavioral performances.

  • VPA induces greater anxiety and cognitive deficits than PPA.

  • Prenatal Shank1,3/BDNF expression differences correlated to autism-like behaviors.

Abstract

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder featuring altered neuronal circuitry and consequently impaired social interactions, restrictive interests plus repetitive stereotypic activities. In the present study, differentiated behaviors of valproic (VPA) and propionic (PPA) acid-mediated autism rats were correlated to cerebral scaffolding proteins (Shank1,3) and BDNF expression variations. Sprague–Dawley offspring that received VPA during pregnancy displayed a notably diminished permanence (−78 %, p < 0.01) in the light chamber of light dark (LD) test, reduced exploratory tasks, i.e. grooming (−90 %) and rearing (−65 %). Moreover, they executed extremely greater climbing intervals (+300 %, p < 0.001) in novel cage (NC) test, plus exhibited an extremely reduced (−331 %) discrimination index in novel object recognition (NOR) test when compared to controls. PPA-treated postnatal days (PND) 12–16 rats also displayed anxiety-like behaviors, although in a less evident manner, as indicated by a moderate time (+55 %; p < 0.05) spent in dark chamber along with notable and moderate decreases in digging (−78 %) plus grooming (−52 %), respectively. Contextually, VPA- more than PPA supplied opposite Shank1,3 expression changes in cerebellum (CB; −62 %; +78 %), dorsomedial prefrontal cortex (DM-PFC; +95 % −76 %), respectively, while resulting extremely upregulated in hippocampus (HIP; +125 % – +155 %). Even BDNF resulted to be substantially and notably diminished in HIP (−85 %) and DM-PFC (−72 %), respectively, of VPA rats while it was only moderately reduced (−35 % to −45 %) in these same areas of PPA rats. The early altered brain-specific expression levels accounting for different behavioral performances may provide useful diagnostic indications and constitute valuable therapeutic strategies for autistic patients.

Introduction

Autism spectrum disorder (ASD) is a brain disorder widely characterized by functional and structural circuitry alterations accounting for impaired social interactions, repetitive stereotypic behaviors along with limited interests [1,2]. Recent works have shown that although ASD has been basically investigated for its social and cognitive impacts [3], there are differing sensory processing and motor behaviors that still require to be investigated in order to establish whether behaviors are due to abnormal molecular properties with respect to their developing peers [4]. Indeed, this disorder seems to be tightly linked to the early structural modifications (especially synaptic terminals) of the growing neurons in the various cerebral communicating stations [5]. As of yet molecular changes of such a neurodevelopmental disorder have not been fully established, despite abnormal cerebral morpho-functional features in autism have been linked with cognitive impairments, sleeping difficulties plus anxiety-like and depression disorders [6]. Interestingly, studies have begun to focus their attention on the molecular nature and temporal periods (pre- and post-natal biological stages) in which neurodevelopmental damages in the various cerebral neuronal fields do occur [7] in an attempt to unravel the type of cross-talking mechanisms operating during the onset of autism.

There is a growing tendency to largely consider the morpho-functional differences in appropriate animal models that are capable of displaying autism-like behaviors. It is the case of valproic acid (VPA), a well-known anticonvulsant used for the treatment of seizures, anxiety, bipolar disorder and epilepsy, which has turned out to be a preferred drug for the induction of ASD in human and autistic-like behaviors in rodents [8] although caution has to be taken due to its teratogenic effects [7,9]. The validity of animal models being induced by this compound is based on the assumption that treatment of fetus with VPA interrupts the early neurodevelopmental processes responsible for the typical abnormal motor and cognitive behaviors in autism [10]. Propionic acid (PPA), is another chemical compound capable of promoting autism-like behavioral impairments. This compound, commonly found in dairy plus refined wheat products, has been identified as an intermediate metabolic product of fatty acids, so that it is capable of inducing behavioral, neuropathological and biochemical alterations consistent with those observed in autism when administered to rats during the first three weeks of post-natal development [11]. Moreover PPA, which is also required for immune and physiological functions, does lead to propionic acidemia, a neurodevelopmental metabolic disorder that resembles autism [12] when given in elevated quantities. Despite synaptogenesis occurs during the first 3 postnatal weeks [13], studies have demonstrated that rats treated with PPA display anxiety-like and social behaviors consistent with ASD [11] thus also suggesting this compound as an effective emerging inductor of autism-like behaviors.

It is known that BDNF, a member of the neurotrophin family of growth factors is tightly related to nerve growth by exerting multiple structural and functional roles in neuronal developmental and plasticity events [14]. This growth factor is considered a key element especially during the early induction of autism as indicated by medication-free ASD children supplied altered levels of BDNF [15]. From previous studies conducted on Angelman and Rett syndromes, both of which belong to the spectrum of neurologic disorders associated with autism, it seems that synaptic growth factors do play a key role on the development and plasticity properties of neuronal circuits [16]. Indeed, rats treated with VPA have supplied contrasting expression levels of BDNF in some critical motor and cognitive brain sites [17,18].

The scaffolding multiple ankyrin repeated domains proteins (Shank) are other factors specifically located at the synaptic terminals controlling structure maturation plus functions of neural circuits and consequently behavioral performances via their binding to neuronal receptors and/or actin cytoskeleton [19]. Shank1 proteins are distributed in the body of spines while Shank3 are mostly concentrated in the postsynaptic density of excitatory synapses thus attributing the former protein with activity-dependent plasticity regulatory events of spines whereas the latter is instead responsible for synaptic structural and neurotransmission properties [20]. Both proteins have proven to be important factors for investigating on neuropsychiatric disorders including schizophrenia and bipolar disorders [21]. Their roles have been extrapolated from Shank1 mutant mice demonstrating impaired performances during mnemonic tasks, along with vocal plus olfactory communication deficits and increased self-grooming behaviors [22,23]. Similarly, Shank3 deficiency have pointed to evident ASD-related behavioral abnormalities including robust repetitive activities and social interaction difficulties [24].

At date, relatively few works have compared the different behavioral performances of autistic rat models induced via pharmacological treatments and precisely by either VPA or PPA, thereby providing a twofold intention of this current study. At the behavioral level, it was our intention to establish if the two compounds evoked differing behavioral performances and namely permanence in light and/or dark chambers, latency to dark chamber, number of transitions between chambers plus time spent in risk assessment chamber, grooming, rearing, exploration bouts and novel object recognition test. To this aim, 29 postnatal days (PND) Sprague-Dawley rats of both experimental models (VPA and PPA) were evaluated in the following behavioral sessions: Light-Dark (LD) test; Novel Cage (NC) test and Novel Object Recognition (NOR) test. Subsequently, the expression levels of BDNF and Shank1,3 in specific motor, cognitive and mnemonic brain regions such as amygdala (AMY) hippocampus (HIP), hypothalamus (HTH), cerebellum (CB), dorsomedial prefrontal cortex (DM-PFC), plus ventrolateral prefrontal cortex (VL-PFC) were determined in order to establish if the type of pharmacological inductions applied for the two autistic models played important role(s) on the various autistic-like behaviors. It is known that the lack of fully active signaling pathways have been suggested to play a major role during the execution of autism-like behaviors and it could very well be that the above scaffolding and neurotrophin proteins are regulating such behavioral performances. Furthermore, it was interesting to determine if the pre- and/or post-natal treatment with VPA and PPA, which were correlated to the above brain specific molecular expression variations accounted for neuronal developmental insults and consequently increased anxiety-like behaviors together with decreased social interaction [11,25], which may constitute valuable alternative therapeutic strategies for this disorder.

Section snippets

Animals

Adult female and male Sprague Dawley rats (Envigo Laboratories, Udine, Italy; n = 24) weighing 200−300 g were housed in standard cages in groups of five per cage, under controlled environmental conditions [23 ± 2 °C; night-day cycle; 12/12 h (on 08:30)] with ad libitum access to food and water. Animal maintenance and experimental procedures were carried out in compliance with ethical provisions for Care and Use of Laboratory Animals reported in the legislative law n° 26 (04-03-2014) and

Effects of VPA and PPA on anxiety and cognitive events

Rats induced with either VPA or PPA showed evident behavioral differences [F(2,22) = 3.45; p < 0.05] with respect to both CTRLs as well as to the two chemical compounds. As for rats induced with the former compound, they showed a notably (p < 0.01) reduced amount of time (-78 %) in the light chamber with respect to CTRL whereas an extremely (p < 0.001) greater amount of time (+85 %) was spent in the dark chamber (Fig. 2A). The preference for this chamber was supported by a notably reduced

Discussion

The overall distinct behavioral performances of our two experimental autistic models resulted to tightly coincide with the altered expression levels of Shank1,3 and BDNF in limbic sites thereby strongly corroborating scaffolding and neurotrophin proteins as key elements altering social interactions in autism. In particular, the greater behavioral difficulties in VPA-induced autistic rats were strongly linked to the early temporal inducing periods accounting for brain morphological and

Conclusions

This first approach of comparing two different experimental models of autism suggested that VPA-induced rats displayed a far greater number of evident behavioral differences together with greater variations in the expression of Shank1,3 and BDNF with respect to PPA-induced rats, despite both models have proven to be useful to study autism. Such a feature was recently corroborated by a member of the SNARE protein complex, i.e. SNAP- 25 protein involved with the trafficking of the scaffolding

CRediT authorship contribution statement

Raffaella Alò: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Ilaria Olivito: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Gilda Fazzari: Investigation, Formal analysis, Software. Merylin Zizza: Investigation, Formal analysis, Software. Anna Di Vito: Data curation. Ennio Avolio: Investigation, Formal analysis, Software. Maurizio Mandalà: Investigation, Formal analysis, Software.

Declaration of Competing Interest

All authors declare that have no biomedical financial interests or conflict of interest in this study.

Acknowledgments

The authors thank the Italian University Research Ministry (MIUR) for having supplied financial support to handle this work.

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