Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

Abstract

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)¹, a critical brain area for energy and glucose homeostasis^2,3. As this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period (CP) for leptin-dependent development of ARC neurocircuits has been proposed⁴. In other brain areas, CP closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh⁵. Here we report that in humans as well as rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed by PNN-like structures. In mice, these neurons are densely-packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both GABAergic and leptin receptor-positive, including a majority of Agrp neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the CP for Agrp neuron maturation and is dependent on input from circulating leptin, as postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the CP. We conclude that neurons crucial to metabolic homeostasis are enmeshed by PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC-ME junction, where metabolically-relevant humoral signals are sensed.

Figure 1.

Wisteria Floribunda agglutinin (WFA)-labeling in the ventromedial ARC forms a “collar” around the ME. Diagrams at top show mid-sagittal view (left) and ventral view (right) of the mouse brain with insets showing the location and orientation of panel images. (a-d) WFA-labeled (red) coronal sections through the Arc, starting just rostral to and progressing through the ME, show a concentration of WFA-labeled cells located in the ARC at its junction with the ME. Note that the very intense staining below the ME does not correspond to labeling around neurons, but to the pia around the ME. (e) Higher magnification image of the boxed region in (c) showing the dense cluster of WFA-labeled ARC cells. (f) High magnification Imaris 3-dimensional rendering of an isolated WFA-labeled cell at the periphery of the dense cluster (arrow in c) reveals that WFA labels the soma and proximal processes of ARC cells. Inset shows the raw image. (g-h) Low (g) and high (h) magnification images of PNNs labeled by WFA in the visual cortex, where they have been extensively studied, for comparison. Note similar PNN pattern between (h) and (f) wrapping the soma and proximal process. (i-j) WFA-labeled wholemounts of the ARC viewed from the 3^rd ventricle wall en-face (i) or the ventral brain surface (j) reveal the distribution of labeled ARC cells forming a “collar” around the ME, which does not contain labeling. From the ventricular surface view (i), the WFA-labeled ARC cells appear as a continuous band along the ventral margin of the ARC. (k) WFA-labeled coronal section from a wild-type mouse sacrificed 2 days after stereotactic unilateral intra-Arc injection of Chondroitinase ABC, an enzyme that digests chondroitin sulfate carbohydrates. (l) Low-power electron micrograph of an ARC section labeled with WFA-DAB shows electron dense DAB deposits surrounding a single ARC neuron (white arrowheads). (m) High-power electron micrograph corresponding to the boxed region in (l) shows WFA-labeling localized to the membrane around the cell soma (white arrowheads) and neurites (white arrows). Note labeling adjacent to an apparent terminal filled with synaptic vesicles (s.v.), as well as the appearance of non-labeled membranes (black arrowheads). (o-p) Confocal images of coronal sections through the ARC stained for other PNN components, including hyaluronic acid using HABP (o, green) and the chondroitin sulfate proteoglycan phosphacan (p, green), show colocalization with WFA (red) in the ARC, providing evidence that ARC WFA-labeling corresponds to PNNs. Scale bars: 100 um (a-d, g, i-k, o-p), 20 um (e), 10 um (f, h), 2 um (l), 500 nm (m). Images in (a-h), (i-j), (k), (l-m), and (o-p) are representative of data from 10, 6, 5, 4, and 3 animals, respectively.

Figure 2.

PNNs enmesh GABAergic, LepRb-positive, Agrp/NPY neurons in the Arc. Diagram at top shows mid-sagittal view of mouse brain with location and orientation of panel images. (a) Dot plots show the proportion of individual neuronal subtypes enmeshed by PNNs. Dots in this and all subsequent dot plots represent data from independent animals (n=3 animals for each neuronal subtype studied). The left plot shows the percentage of all PNN-enmeshed ARC cells that belong to a particular neuronal subtype. The right plot shows the percentage of all ARC Npy-GFP or POMC-GFP cells that are enmeshed by PNNs. Low (b, e, h, k, n) and high (c, f, i, l, o) magnification images of coronal sections stained with WFA (red) and antibodies to GFP (green) (b, h, k), dsRed (green) (e), or SST (green) and Agrp (white) (n) show that most PNN-enmeshed cells are GAD67-GFP-positive (GABAergic), LepRb-positive, and NPY-positive, while few enmeshed cells express POMC or SST. (d, g, j, m) High magnification Imaris 3-dimensional surface rendering of isolated ARC PNN-enmeshed cells belonging to the various neuronal subtypes (corresponding to b, e, h, k, respectively) show PNNs wrapping the soma and proximal processes. Insets show raw images. See corresponding supplementary movies 1 and 3 for (d) and (j), respectively. Scale bars: 50 um (b, e, h, k, n), 20 um (c, f, i, l, o), 10 um (d, g, j, m). Images in (b-d), (e-g), (h-j), (k-m), and (n-o) are representative of data from 3 GAD67-GFP mice, 3 LepRb-Cre;Ai14 mice, 3 NPY-GFP mice, 3 POMC-GFP mice, and 3 C57B/6 mice injected with ICV colchicine, respectively.

Figure 3.

PNN formation in the ARC occurs during the lactation and periweaning period, corresponding with the maturation of Agrp neurons. (a-c) Confocal images of coronal sections stained with WFA (red), Agrp (green), and dapi (blue) from postnatal wild-type mice at age P10 (a), P21 (b), and P30 (c). PNN staining intensity and ARC Agrp fiber density increase in parallel over this time period. (d) Dot plot shows correlated increase in WFA intensity and Agrp fiber density in the ARC from P10 to P30, as well as at P90. Dots (WFA intensity in red, Agrp density in black) represent values from independent animals (n=4 (P10); 5 (P21); 3 (P30); and 3 (P90)), and horizontal bars represent the mean. WFA intensity is represented by the average over all voxels in the ARC region of interest, with range 0–255. Agrp fiber density is measured as the volume of Agrp+ voxels divided by the total volume of the ARC region of interest. Scale bar: 100 um (a-c). Images in (a), (b), and (c) are representative of data from 4, 5, and 3 animals, respectively.

Figure 4.

Leptin-deficient ob/ob mice have impaired PNN formation during postnatal development that can be rescued by leptin administration during the critical period. (a-d) Confocal images of ARC sections from ob/ob (b,d) and ob/+ (a,c) control littermates at P15 (a,b) and P30 (c,d), stained with WFA (red) and Agrp (green), show reduced WFA labeling and apparent disruption of PNN architecture in the ARC. Arrows in (a,b) indicate the ARC region where the earliest PNN formation is seen at P15 in ob/+ mice, but not in ob/ob littermates. Images in (a), (b), (c), and (d) are representative of data from 2, 3, 5, and 5 animals, respectively. (e-f) Confocal images of ARC sections from ob/ob pups that received daily i.p. injections of leptin (f) or vehicle (e) from P10 to P30 before being euthanized for analysis with WFA (red) and Agrp (green). Leptin administration during this critical period appeared to restore WFA labeling intensity and PNN architecture. Insets in (c-f) show higher magnification of the ventromedial ARC region indicated by the arrowhead, revealing an increase in Agrp expression within neuronal soma in leptin deficiency. Images in (e) and (f) are representative of data from 2 and 3 animals, respectively. (g-h) Dot plots show normalized intensity values for WFA in the ARC of P15, P21, and P30 ob/+ (filled circles) and ob/ob (open circles) mice (g), or P30 ob/ob mice treated from P10 onward with daily i.p. leptin (red open circle) or vehicle (black open circle) injection (h). Values were normalized to the mean WFA intensity of the control groups (ob/+ or ob/ob-veh). Dots represent values from independent animals (n= 2 ob/+ and 3 ob/ob (P15); 5 ob/+ and 5 ob/ob (P21); 5 ob/+ and 5 ob/ob (P30); 2 ob/ob-veh and 3 ob/ob-lep (P30 rescue)). Horizontal bars represent the mean. There was a consistent decrease in ARC PNN intensity across multiple postnatal ages in ob/ob mice compared to their ob/+ littermates, which appeared to be restored in ob/ob mice at P30 by leptin administration during the critical period (*P21 ob/ob 0.89±0.01 vs. ob/+ 1.00±0.02, two-tailed t-test p=0.0005, t=5.545, df=8, 95% CI of difference: −0.151 to −0.062; P30 ob/ob 0.87±0.01 vs. ob/+ 1.00±0.01, two-tailed t-test p=0.0001, t=8.975, df=8, 95% CI of difference: −0.168 to −0.099). Scale bars: 100 um (a-f), 20 um (insets in c-f).