Polysialic Acid Acute Depletion Induces Structural Plasticity in Interneurons and Impairs the Excitation/Inhibition Balance in Medial Prefrontal Cortex Organotypic Cultures

Abstract

The structure and function of the medial prefrontal cortex (mPFC) is affected in several neuropsychiatric disorders, including schizophrenia and major depression. Recent studies suggest that imbalances between excitatory and inhibitory activity (E/I) may be responsible for this cortical dysfunction and therefore, may underlie the core symptoms of these diseases. This E/I imbalance seems to be correlated with alterations in the plasticity of interneurons but there is still scarce information on the mechanisms that may link these phenomena. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate, because it modulates the neuronal plasticity of interneurons and its expression is altered in schizophrenia and major depression. To address this question, we have developed an in vitro model using mPFC organotypic cultures of transgenic mice displaying fluorescent spiny interneurons. After enzymatic depletion of PSA, the spine density of interneurons, the number of synaptic puncta surrounding pyramidal neuron somata and the E/I ratio were strongly affected. These results point to the polysialylation of NCAM as an important factor in the maintenance of E/I balance and the structural plasticity of interneurons. This may be particularly relevant for better understanding the etiology of schizophrenia and major depression.

Keywords: E/I balance; PSA-NCAM; mPFC cultures; major depression; neuronal structural plasticity; schizophrenia.

Figure 1

Photographs showing the microscopic architecture and the pattern of polysialylated form of the neural cell adhesion molecule (PSA-NCAM) expression in organotypic cultures of the medial prefrontal cortex (mPFC). (A) Panoramic view of a mPFC slice after 14 days in vitro. (B,C) Microphotographs showing that the layered organization of neurons in the mPFC is preserved after 14 days in vitro. (B) Nissl stained slice. (C) Slice immunostained for NeuN, a marker of mature neurons, (D) PSA-NCAM expression is observed in neuronal somata, neurites and neuropil in control slices (D1), but it is completely absent in Endo-N treated-slices (D2). (E) Glutamic acid decarboxylase (GAD)-enhanced green fluorescent protein (EGFP) expressing interneurons in control slices do not co-express PSA-NCAM. Scale bar: 10 μm for (A) 25 μm for (B) and (C) 100 μm for (D1,D2), 20 μm for (E1–E3).

Figure 2

Confocal microscopy images showing the expression of PSA-NCAM (A1,B1,C1,D1), a marker for pyramidal neurons (CamKII-α; A2) and several markers for interneurons: calbindin (CB; B2) calretinin (CR; C2) and parvalbumin (PV; D2). Note the expression of PSA-NCAM in the soma of interneurons expressing CB and CR (B3,C3, arrowheads) but not in the soma of pyramidal neurons (A3, arrowhead) or interneurons expressing PV (D3, arrowhead). In the neuropil, PSA-NCAM expression can be found in perisomatic boutons surrounding pyramidal neurons (A3, asterisks) and also in dendrites and puncta expressing markers for interneurons (B3,C3,D3). Scale bar: 20 μm.

Figure 3

Confocal analysis of GAD-GFP, vesicular GABA transporter (VGAT), vesicular glutamate transporter 1 (VGLUT1) and synaptophysin (SYN) expression in the neuropil of mPFC organotypic cultures. (A) Graph representing neuropil puncta density (number of puncta/300 μm²) in control (purple bar) and Endo-N treated cultures (turquoise bar) for the different markers shown in (C). Note that statistically significant changes (unpaired Student’s t-test) were found in the density of VGAT, VGLUT1 and SYN expressing puncta. (B) Graph showing statistically significant changes (unpaired Student’s t-test) in the Excitation/Inhibition ratio (VGLUT1/VGAT neuropil puncta density) after Endo-N treatment. (C) Confocal images (single confocal planes) comparing the expression of the different proteins analyzed between control and Endo-N treated cultures. Scale bar 5 μm. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 4

Confocal analysis of GAD-GFP, VGAT, PV, VGLUT1 and SYN expression in the perisomatic region of pyramidal neurons. (A) Graph representing statistically significant changes (unpaired Student’s t-test) in perisomatic puncta density (number of puncta/μm of soma perimeter) after Endo-N treatment for the all the markers shown in (C). (B) Graph representing the Excitation/Inhibition ratio (VGLUT1/VGAT perisomatic puncta density) between control (purple bar) and Endo-N treated cultures (turquoise bar). No statistically significant differences were found after unpaired Student’s t-test. (C) Confocal images (single confocal planes) comparing the density of the puncta expressing the different proteins between control and Endo-N treated cultures. Scale bar 5 μm. **p-value < 0.01; ***p-value < 0.001.

Figure 5

Confocal analysis of dendritic spine density in GAD-EGFP expressing interneurons of mPFC organotypic cultures. (A) Graph comparing the dendritic spine density (spines/μm of dendrite length) of GAD-EGFP expressing interneurons from mPFC slices treated with Endo-N with those from slices treated with vehicle solution (control). 0–60, 60–120 and 120–180 indicate dendrite segments of 60 μm located in 0–60, 60–120 and 120–180 μm segments from the interneuron soma, respectively. Asterisk in bars indicate statistically significant differences in unpaired Student t-test: *p < 0.05, **p < 0.01. (B–D′) Confocal microscopic images of GAD-EGFP expressing interneurons in mPFC organotypic cultures treated with vehicle solution (control; B–D) or Endo-N (B′–D′). Note the slight increase in the density of spines after Endo-N treatment in the 0–60 μm segment (A,B) but the marked decrease in this density in the 120–180 μm segment (A,C). Images (D,D′) are 10X enlargements of the spines marked with red arrowheads in (C,C′), respectively. Scale bar: 5 μm for (B,B′,C,C′); 50 μm for (D,D′).