Behavioral analysis reveals hyperactivity and cognitive deficits in Fmr1 KO rats
Increased motor activity in Fmr1 KO rats. are
The open field test has been extensively used for measuring spontaneous and habituated motor activity, including horizontal and vertical responses [11,28]. KO rats showed increased horizontal activity compared to WT rats, both during the first period of spontaneous motor activity recorded (Fig. 1a), as well as the second half, representing habituated motor activity (Fig. 1c). Spontaneous vertical activity was not altered, whereas during the habituated period, Fmr1 KOs demonstrated increased vertical mobility (Fig. 1b, d).
FmR1 KO habituate over time
Open field habituation over consecutive exposures has been used for the evaluation of non-associative learning and memory [12]. Two-way repeated ANOVA measures did not reveal any statistically significant effect. Overall, both Fmr1 KO and WT rats exhibited decreased horizontal activity over the three days of testing and thus, habituated over time (Fig. 1e).
Fmr1 KO rats have impaired recognition memory
The NORT assesses short-term recognition memory, and it is a non-rewarding paradigm based on rodents' spontaneous exploratory behavior [29]. Fmr1 KO rats demonstrated a decrease in the NORT discrimination index indicating a deficit in recognition memory (Fig. 1f).
Fmr1 KO rats exhibited impaired spatial recognition memory
The NOLT is based on the spontaneous exploratory behavior of rodents and is used to assess short-term spatial recognition memory [30]. Fmr1 KO exhibited a lower discrimination index in comparison to their WT counterparts (Fig. 1g), indicating deficits in short-term spatial memory for Fmr1 KO animals.
Region-specific glutamate receptor expression perturbations in Fmr1 KO rats
Next, we examined potential brain alterations associated with cognitive dysfunction in Fmr1 KO rats and first focused on glutamatergic status, including the expression of glutamate receptors (NMDA and AMPA) in the prefrontal cortex and the hippocampus of Fmr1 KO and WT rats. The GluN2A/2B ratio was also assessed since it provides an index of synaptic activity [31–33]. Taken together present results, an opposite status, concerning glutamate protein expression, seems to appear in the PFC versus the hippocampus in the KO compared to WT rats.
Fmr1 KO rats have increased NMDA receptor GluN2A/2B expression in the PFC and decreased NR2B protein expression in the hippocampus.
In the PFC, GluN1 subunit expression was unchanged, whereas increased GluN2A and GluN2B subunit expression was observed in KO rats (Fig. 2a, b, c). The GluN2A/GluN2B ratio was also significantly increased in Fmr1 KO rats (Fig. 2d), due to the prominent increased GluN2A protein expression.
There was no statistically significant difference in GluN1 levels in either the ventral or dorsal parts of the hippocampus between genotypes (Fig. 3a, g). In the dorsal hippocampus, GluN2A expression tended towards a decrease (Fig. 3b), while GluN2B expression was significantly lower in the Fmr1 KO rats (Fig. 3c). The ratio GluN2A/GluN2B was significantly increased for Fmr1 KO rats due to decreased GluN2B protein expression levels (Fig. 3d). The same pattern was observed in the ventral hippocampus, however, the GluN2A/GluN2B ratio was similar between both genotypes (Fig. 3h, i, j).
GluA1 and GluA2 expression is altered in the PFC and hippocampus of Fmr1 KO rats
In the prefrontal cortex, AMPA GluA1 subunit expression was unchanged, whereas GluA2 expression was elevated in Fmr1 KO rats (Fig. 2e, f). In the dorsal hippocampus, GluA1 and GluA2 protein expression levels were significantly lower in the KO rats (Fig. 3e, f). In the ventral hippocampus, GluA1 expression was higher in the Fmr1 KO rats (Fig. 3k), while GluA2 expression was the same between genotypes (Fig. 3i).
Region-specific dysregulation of excitatory and inhibitory neurotransmission in Fmr1 KO rats
We next assessed potential regionally distinct alterations in glutamatergic and GABAergic neurotransmitter activity of Fmr1 KO rats.
Decreased glutamate levels and elevated cycling rate were observed in the PFC (Fig. 4a, d). However, a trend for decreased GABA levels was observed in KO rats.
In the hippocampus, both glutamate and glutamine levels were increased, but the glutamate cycling rate was unchanged (Fig. 4f, g, i, k, l, n). GABA levels were elevated in the dorsal and ventral hippocampus (Fig. 4h, m).
Overall, these neurochemical findings indicate region-specific differential perturbations of neurotransmitter activity consisted with alterations in glutamate receptor expression levels.
Loss of excitation/inhibition balance in the hippocampus of Fmr1 KO rats
Glutamatergic and GABAergic functions are tightly linked to synaptic transmission and the balance between excitation and inhibition, with the hippocampus being a key brain region for assessing such processes [34].
Excitatory synaptic transmission was not different between the two genotypes
Synaptic transmission and neuronal excitation, respectively, were unchanged in both the dorsal and ventral hippocampus of Fmr1 KO rats (Fig. 5a, d).
Maximum neuronal excitation was not different between the two genotypes
Neuronal excitation was compared between WT and Fmr1 KO rats by measuring the maximum PS that was not different between the two genotypes in both dorsal or ventral hippocampus (Fig. 5b, e).
Neuronal excitability was higher in the Fmr1 KO rats
Neuronal excitability was subsequently assessed by measuring the ratio PS/fEPSP at the maximum PS value. A significant increase in neuronal excitability was observed in Fmr1 KO rats in both the dorsal and the ventral hippocampus (Fig. 5c, f).
Paired-pulse inhibition was higher in the ventral hippocampus of FmR1 KO rats
The effectiveness of paired-pulse inhibition was evaluated by measuring the ratio PS2/PS1 recorded at a stimulation strength that produced a half-maximum PS1.
PS2/PS1 was unchanged in the dorsal hippocampus of Fmr1 KO rats, however, we found that the ratio PS2/PS1 was significantly lower in the ventral hippocampus of Fmr1 KO as compared to WT rats (Fig. 5g, h).
Transcriptomic analysis: RNA-seq analysis
Our last objective was to employ RNA sequencing (RNA-Seq) in the hippocampus to gain a deeper insight into pathological alterations at the transcription level. The RNA sequencing analysis yielded compelling results, identifying a total of 838 genes that were differentially expressed (Fig. 6a). To further explore the functional implications of these gene expression changes, we performed Gene Set Enrichment Analysis (GSEA) on the identified gene set. This analysis revealed significant enrichments in gene ontologies related to various biological processes (Fig. 6b). Among the downregulated gene ontologies, synapse organization, regulation of neuron projection development, and signal release, were significantly affected in Fmr1 KO rats (Fig. 6b, c, d, e, f). A gene ontology associated with anion transport was also dysregulated in the Fmr1 KO rats, indicating potential disruptions in ion homeostasis and signaling mechanisms (Fig. 6b).
We next performed pathway analysis using the KEGG database. Key pathways involved in neurotransmission and neuronal signaling were significantly dysregulated; specifically, the glutamatergic/GABAergic synapse pathway (Fig. 6g, h). Noteworthy genes, such as Cacna1c, encoding a voltage-gated calcium channel subunit, showed upregulation, suggesting impaired glutamatergic and GABAergic synapse function (Fig. 6g, h). Conversely, the downregulation of Shank3, involved in synaptic scaffolding and neurotransmitter receptor clustering, indicated disruptions in postsynaptic density organization and synapse function (Fig. 6g). Finally, the downregulation of the Gng13 gene suggests possible alterations in G protein signaling within the glutamatergic synapse, GABA receptor signaling, and synaptic inhibition (Fig. 6g, h).