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. 2024 Feb 20;14(1):104.
doi: 10.1038/s41398-024-02815-0.

Multi-level profiling of the Fmr1 KO rat unveils altered behavioral traits along with aberrant glutamatergic function

George Ntoulas  1 , Charalampos Brakatselos  1 , Gerasimos Nakas  1 , Michail-Zois Asprogerakas  1 , Foteini Delis  1 , Leonidas J Leontiadis  2 , George Trompoukis  2 , Costas Papatheodoropoulos  2 , Dimitrios Gkikas  3 , Dimitrios Valakos  3 , Giannis Vatsellas  3 , Panagiotis K Politis  3 , Alexia Polissidis  1   4 , Katerina Antoniou  5
Affiliations

Multi-level profiling of the Fmr1 KO rat unveils altered behavioral traits along with aberrant glutamatergic function

George Ntoulas et al. Transl Psychiatry. .

Abstract

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disabilities and the most prevalent monogenic cause of autism. Although the knockout (KO) of the Fmr1 gene homolog in mice is primarily used for elucidating the neurobiological substrate of FXS, there is limited association of the experimental data with the pathophysiological condition in humans. The use of Fmr1 KO rats offers additional translational validity in this regard. Therefore, we employed a multi-level approach to study the behavioral profile and the glutamatergic and GABAergic neurotransmission status in pathophysiology-associated brain structures of Fmr1 KO rats, including the recordings of evoked and spontaneous field potentials from hippocampal slices, paralleled with next-generation RNA sequencing (RNA-seq). We found that these rats exhibit hyperactivity and cognitive deficits, along with characteristic bidirectional glutamatergic and GABAergic alterations in the prefrontal cortex and the hippocampus. These results are coupled to affected excitability and local inhibitory processes in the hippocampus, along with a specific transcriptional profile, highlighting dysregulated hippocampal network activity in KO rats. Overall, our data provide novel insights concerning the biobehavioral profile of FmR1 KO rats and translationally upscales our understanding on pathophysiology and symptomatology of FXS syndrome.

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Conflict of interest statement

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Fig. 1
Fig. 1. Increased motor activity and impaired recognition and spatial memory for Fmr1 KO rats.
Spontaneous and habituated horizontal and vertical motor activity for Fmr1 WT (n = 12) and KO (n = 12) rats in the open field apparatus. Distance traveled (a) and vertical counts (b) during spontaneous motor activity. Distance traveled (c) and vertical counts (d) during habituated motor activity. Time spent in the center of the open-field apparatus (e). Habituation to Learning (f) of Fmr1 WT and KO rats. Assessment of recognition and spatial short-term memory of Fmr1 WT and KO rats. Discrimination Index (DI) of Fmr1 WT (n = 10) and KO rats (n = 12) for the Novel Object Recognition Task (NORT) (g). Total time spent exploring the two objects during the test phase (T2) of the test (h). Discrimination index (DI) of Fmr1 WT (n = 9) and KO rats (n = 11) for the Novel Object Location Task (NOLT) (i). Total time spent exploring the two objects in the test phase (T2) of the test (j). All results are represented as means ± SEM; *P <��0.05, **P < 0.01, ***P < 0.001.
Fig. 2
Fig. 2. NMDA and AMPA receptors subunits protein expression levels were found elevated in the prefrontal cortex of Fmr1 KO rats.
NMDA receptors subunits protein expression levels in the prefrontal cortex of the Fmr1 WT (n = 6) and KO (n = 6) rats. GluN1 (a), GluN2A (b), GluN2B (c), and ratio of GluN2A/GluN2B (d). AMPA receptors subunits protein expression levels in the prefrontal cortex of the Fmr1 WT (n = 6) and KO (n = 6) rats. GluA1 (e) and GluA2 (f). The optical density (OD) of each band was divided by the corresponding loading marker. Data are presented as relative protein expression to WT rats. Bellow each graph is presented a representative image from each western blot including a band of the protein of interest and the corresponding loading marker band. All results are represented as means ± SEM; *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3. NMDA and AMPA receptor subunit levels were found to be altered in the dorsal and ventral hippocampus of Fmr1 KO rats.
NMDA and AMPA receptors subunits protein expression levels in the dorsal and ventral hippocampus of the Fmr1 WT (n = 6) and KO (n = 6) rats. GluN1(a), GluN2A (b), GluN2B (c), and the ratio of GluN2A/GluN2B (d), GluA1 (e), and GluA2 (f) in the dorsal hippocampus. GluN1 (g), GluN2A (h), GluN2B (i), and the ratio of GluN2A/GluN2B (j), GluA1 (k), and GluA2 (l) in the ventral hippocampus. The optical density (OD) of each band was divided by the corresponding loading marker. Data are presented as relative protein expression of WT rats. Bellow each graph is presented a representative image from each western blot including a band of the protein of interest and the corresponding loading marker band. All results are represented as means ± SEM; *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4. Regionally dependent alterations in glutamatergic and GABAergic neurotransmission activity of Fmr1 KO rats.
Glutamate (Glu), Glutamine (GLN), GABA tissue levels and their cycling rates in the prefrontal cortex, the dorsal, and the ventral hippocampus of Fmr1 KO (n = 8) and WT (n = 8) rats. Glu (a), GLN (b), and GABA (c) along with cycling rate of GLN/Glu (d) and GLN/GABA (e) in the prefrontal cortex. Glu (f), GLN (g) and GABA (h) along with cycling rate of GLN/Glu (i) and GLN/GABA (j) in the dorsal hippocampus. Glu (k), GLN (l) and GABA (m) along with cycling rate of GLN/Glu (n) and GLN/GABA (o) in the ventral hippocampus. All results are represented as means ± SEM; *P < 0.05, **P < 0.01.
Fig. 5
Fig. 5. Alterations in excitability and local inhibitory processes in the hippocampus of the Fmr1 KO rats.
Comparison of synaptic transmission, neuronal excitation, and neuronal excitability in WT and Fmr1 KO in the dorsal (D. Hip) (a–c) and ventral hippocampus (V. Hip) (d–f). Upper graphs in each panel show examples of input–output curves between stimulation current intensity and fEPSP or PS or PS/fEPSP. Synaptic transmission was compared between WT and KO rats by measuring the max fEPSP. Max fEPSP did not significantly differ between the WT and KO rats in either the dorsal (a) (WT n = 15 slices/12 rats and KO n = 19 slices/19 rats) or the ventral hippocampus (d) (WT n = 18 slices/16 rats and KO n = 18 slices/18 rats). Regarding neuronal excitation we found that max PS did not significantly differ between WT and KO rats in either the dorsal (b) (WT n = 42 slices/33 rats and KO n = 46 slices/35 rats) or the ventral hippocampus (e) (WT n = 40 slices/33 rats and KO n = 46 slices/37 rats). Neuronal excitability was compared between WT and KO rats by measuring the ratio PS/fEPSP at max PS value. Max PS/fEPSP significantly increased in KO compared with WT rats in both the dorsal (c) (WT n = 13 slices/11 rats and KO n = 17 slices/16 rats) and the ventral hippocampus (f) (WT n = 15 slices/13 rats and KO n = 16 slices/13 rats). Paired-pulse inhibition increases in the ventral, not dorsal, KO hippocampus. The effectiveness of paired-pulse inhibition was compared between WT and Fmr1 KO rats by measuring the ratio PS2/PS1 recorded at a stimulation strength that produced a half-maximum PS1. PS2/PS1 in the dorsal hippocampus (g) was comparable between WT and KO rats (WT n = 34 slices/29 rats and KO n = 33 slices/27 rats). However, PS2/PS1 was significantly smaller in KO compared with WT ventral hippocampus (h) (WT n = 38 slices/30 rats and KO n = 44 slices/32 rats), suggesting a higher inhibition in the KO vs WT ventral hippocampus. Collective data are shown in the bottom graphs in each panel and they are represented as means ± SEM; *P < 0.05.
Fig. 6
Fig. 6. Comprehensive analysis of gene expression and pathway enrichment in the hippocampus of the Fmr1 KO rats.
Volcano plot displaying the results of differential gene expression analysis. Differentially Expressed Genes (DEGs) are represented as red dots, while non-DEGs are depicted as black dots (a). Histogram presenting the enrichment analysis results from Gene Set Enrichment Analysis (GSEA) performed using the WebGestalt platform (b). The histogram illustrates the enrichment of upregulated and downregulated biological processes among the DEGs. c–e Enrichment plots demonstrating significant enrichment of DEGs in specific biological processes: “Regulation of neuron projection development” (c),“Signal release” (d), and “Synapse organization” (e). Expression heatmap showing gene expression levels of DEGs implicated in the biologic processes mentioned in (d–f). g, h Pathway depictions obtained from the KEGG database: Glutamatergic synapse pathway with upregulated genes outlined in red and labeled in red, and downregulated genes outlined in blue and labeled in blue (g); GABAergic synapse pathway with upregulated genes outlined in red and labeled in red, and downregulated genes outlined in blue and labeled in blue (h). Relative expression levels of the DEGs implicated in both the glutamatergic and GABAergic synapse pathways, allowing for comparative analysis of gene expression changes between the two pathways (i).
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