Reduction in parvalbumin expression not loss of the parvalbumin-expressing GABA interneuron subpopulation in genetic parvalbumin and shank mouse models of autism

Abstract

BackgroundA reduction of the number of parvalbumin (PV)-immunoreactive (PV+) GABAergic interneurons or a decrease in PV immunoreactivity was reported in several mouse models of autism spectrum disorders (ASD). This includes Shank mutant mice, with SHANK being one of the most important gene families mutated in human ASD. Similar findings were obtained in heterozygous (PV+/-) mice for the Pvalb gene, which display a robust ASD-like phenotype. Here, we addressed the question whether the observed reduction in PV immunoreactivity was the result of a decrease in PV expression levels and/or loss of the PV-expressing GABA interneuron subpopulation hereafter called “Pvalb neurons”. The two alternatives have important implications as they likely result in opposing effects on the excitation/inhibition balance, with decreased PV expression resulting in enhanced inhibition, but loss of the Pvalb neuron subpopulation in reduced inhibition.MethodsStereology was used to determine the number of Pvalb neurons in ASD-associated brain regions including the medial prefrontal cortex, somatosensory cortex and striatum of PV-/-, PV+/-, Shank1-/- and Shank3B-/- mice. As a second marker for the identification of Pvalb neurons, we used Vicia Villosa Agglutinin (VVA), a lectin recognizing the specific extracellular matrix enwrapping Pvalb neurons. PV protein and Pvalb mRNA levels were determined quantitatively by Western blot analyses and qRT-PCR, respectively.ResultsOur analyses of total cell numbers in different brain regions indicated that the observed “reduction of PV+ neurons” was in all cases, i.e., in PV+/-, Shank1-/- and Shank3B-/- mice, due to a reduction in Pvalb mRNA and PV protein, without any indication of neuronal cell decrease/loss of Pvalb neurons evidenced by the unaltered numbers of VVA+ neurons.ConclusionsOur findings suggest that the PV system might represent a convergent downstream endpoint for some forms of ASD, with the excitation/inhibition balance shifted towards enhanced inhibition due to the down-regulation of PV being a promising target for future pharmacological interventions. Testing whether approaches aimed at restoring normal PV protein expression levels and/or Pvalb neuron function might reverse ASD-relevant phenotypes in mice appears therefore warranted and may pave the way for novel therapeutic treatment strategies.

Prenatal Valproate Exposure Differentially Affects Parvalbumin-Expressing Neurons and Related Circuits in the Cortex and Striatum of Mice

“…Within the pool of VVA + cells, ∼70% and ∼65% of cells in control mice were also identified as PV + in the striatum ( Figure 2A ) and the cortex ( Figures 3A and 4A ), respectively. Similar values were obtained in previous studies (Ye and Miao, 2013; Filice et al, 2016). The number of PV + cells in the striatum of VPA mice was reduced by ∼15% ( p = 0.0093) compared to controls ( Figure 2A ).…”

“…Therefore, in addition to counting PV + cells via anti-PV immunostaining, we simultaneously quantified the number of Vicia Villosa Agglutinin VVA + cells, i.e., neurons surrounded by PNNs in the same brain regions to obtain an alternative estimate for the number of Pvalb cells. VVA is a lectin that binds to N -acetylgalactosamine residues of PNNs, which specifically surround Pvalb neurons (Hartig et al, 1992; Haunso et al, 2000), as also shown in our previous study (Filice et al, 2016). VVA + cells were considered to serve as a correlate for Pvalb neurons; a typical example of PV and VVA co-localization is shown in Figure 1 .…”

“…Altogether, these results strongly indicated that VPA treatment resulted in decreased striatal PV expression. Reduced PV expression was also shown before in the striatum of PV +/- mice and moreover, in the same region in Shank3B -/- knockout mice (Filice et al, 2016). …”

“…CEs for both m = 1 and m = 0 are provided in Table 1 and are expected to bracket the true CE values of the estimates. In our double-labeling experiments, strictly individual fluorescence images for either PV + or VVA + structures were counted as reported before (Filice et al, 2016) without crosschecking the other channel. This ascertained that a cell (or a small part of a cell resulting from the sectioning) with “weak” staining intensity (i.e., below the predefined threshold) and/or characterized by an “atypical” shape (not easily discernable as cell-like) was considered negative, even if a check of the other channel would have probably identified this cell/cell segment as positive for the second marker.…”

“…Rather, PV expression levels might be low or absent, thus falling under detection threshold, while the Pvalb neuron number is unchanged. The latter was shown by stereological analysis of perineuronal nets (PNNs), which represent an alternative marker for Pvalb neurons or using PV-EGFP mice (Meyer et al, 2002) expressing EGFP in Pvalb neurons independent of endogenous PV expression levels (Filice et al, 2016). Here, we investigated whether Pvalb neuron numbers were altered and/or if PV expression was down-regulated in three ASD-linked brain regions, namely the mPFC, SSC and striatum of VPA male mice.…”