Characterization of Perineuronal Nets (PNNs) in the Paraventricular Nucleus of the Hypothalamus (PVN) and their alteration in neurogenic hypertension
IsmaryBlanco
Ph.D.
1,3✉Emailismary.blanco@vumc.orgSichuChen
B.S.
2ErinYeo2
SamanthaReasonover
M.S.
1MonicaM.Santisteban
Ph.D.
1,2,3✉Emailmonica.santisteban@vumc.org1
A
Vanderbilt University Medical Center2220 Pierce Ave, RRB-53637232NashvilleTN2
A
Vanderbilt University2201 W End Ave37235NashvilleTN3
A
2220 Pierce Ave, RRB-53637232NashvilleTN4
A
615 322 5000
5
615 322 7311
6
615-875-3963
Ismary Blanco, Ph.D.1, Sichu Chen, B.S.2, Erin Yeo2, Samantha Reasonover, M.S.1, Monica M. Santisteban, Ph.D.1,2
Affiliations
1Vanderbilt University Medical Center:
Address: 2220 Pierce Ave, RRB-536, Nashville, TN 37232
Phone number: 615 322 5000
Fax number: 615 343 0126
2Vanderbilt University
Address: 2201 W End Ave, Nashville, TN 37235
Phone number: 615 322 7311
Fax number: 615 322 0073
Ismary Blanco: ORCID ID: 000-0003-4499-1427
Sichu Chen: ORCID ID: 0009-0000-4711-9138
Erin Yeo: ORCID ID: 0009-0009-5241-5981
Samantha Reasonover: ORCID ID: 0009-0003-2211-6899
Monica Santisteban: ORCID ID: 0000-0002-2836-9075
Running title: PNNs in the PVN are altered during hypertension
Word count: 1,739 (including in text citations)
Corresponding Authors:
Names: Monica M. Santisteban and Ismary Blanco
Email: monica.santisteban@vumc.org and ismary.blanco@vumc.org
Address: 2220 Pierce Ave, RRB-536, Nashville, TN 37232
Phone: 615-875-3963
Key words:
Hypertension
Perineuronal nets
Paraventricular nucleus of the hypothalamus
Abstract
Perineuronal nets (PNNs) are key regulators of neuronal excitability, yet whether they are altered during neurogenic hypertension is unknown. Here, we mapped the developmental trajectory of PNNs in the paraventricular nucleus of the hypothalamus (PVN), a crucial nucleus involved in blood pressure regulation, and examined their modulation in neurogenic hypertension. We show that PNNs in PVN follow a developmental pattern, enwrapping 25% of neuronal nitric oxide synthase (nNOS)-expressing neurons, with sex differences observed only in oxytocin (OXT)-enwrapped populations. In the DOCA-salt mouse model of neurogenic hypertension, males, but not females, exhibit an increased number and area of PNNs in the PVN. Given that PNNs modulate neuronal activity, our findings may implicate recruitment of previously “silent” neurons as potential contributors of PVN hyperactivity in hypertension. These results demonstrate that PNN remodeling is associated with neurogenic hypertension.
Introduction
Hypertension affects over one billion people worldwide and more than 45% of U.S. adults (Flack et al., 2024; Kario et al., 2024). Resistant hypertension, commonly neurogenic in origin, affects up to 40% of patients (Flack et al., 2024). The latter, is driven by neurohumoral dysregulation and heightened sympathetic output (Flack et al., 2024; Lamptey et al., 2023; Zheng et al., 2022), largely orchestrated by the paraventricular nucleus of the hypothalamus (PVN) (Basting et al., 2018; Guyenet, 2006). Indeed, PVN overactivation is a hallmark of neurogenic hypertension (Goncharuk et al., 2002; Guyenet, 2006; Xi et al., 2024), reflecting altered excitatory/inhibitory (E/I) balance. Several studies report increased glutamatergic transmission (D.-P. Li et al., 2008; D.-P. Li & Pan, 2006; Ye et al., 2012) and reduced inhibition (Y.-F. Li et al., 2001). However, the cellular and molecular mechanisms underlying PVN overactivation remain incompletely understood.
A novel mechanism that may contribute to PVN hyperactivity is alteration in perineuronal nets (PNNs) composition, extracellular matrix structures that regulate neuronal firing (Balmer, 2016; Carulli & Verhaagen, 2021; Frischknecht et al., 2009; Tewari et al., 2018). PNNs are distributed throughout the central nervous system (Lupori et al., 2023), including the hypothalamus (Horii-Hayashi et al., 2017), where they typically enwrap neuronal cell bodies, proximal dendrites, axon initial segments, and synaptic terminals (Balmer, 2016; Cabungcal et al., 2013; Carceller et al., 2020; Carstens et al., 2016; Carulli & Verhaagen, 2021; Celio, 1993; Favuzzi et al., 2017; Fawcett et al., 2019; Frischknecht et al., 2009; Tewari et al., 2018). PNNs are composed primarily of chondroitin sulfate proteoglycans (CSPGs) such as aggrecan, brevican, neurocan, and versican, cross-linked together and anchored to the cell surface (Carulli & Verhaagen, 2021; Deepa et al., 2006; Fawcett et al., 2022; Galtrey et al., 2008). PNN condensation and maturation increase with age, coinciding with the closure of critical periods (Carulli & Verhaagen, 2021; Tewari et al., 2022). Of relevance to neurogenic hypertension, an increase in PNN components and PNN-enwrapped neurons is dependent on increased neuronal activity (Carulli et al., 2007; Dityatev et al., 2007). Moreover, PNNs exhibit diurnal modulation (Harkness et al., 2021) paralleling blood pressure regulation. Additionally, they are dynamically remodeled by activity-dependent processes such as learning and memory (Fawcett et al., 2022; Tewari et al., 2022) as well as by inflammation (Dong et al., 2023), which plays a pivotal role in the pathophysiology of hypertension (Santisteban et al., 2022). Based on this, we investigated which PVN neuronal cell types are enwrapped by PNNs and whether these structures are modulated in a mouse model of neurogenic hypertension.
Results
Perineuronal nets are developmentally regulated in the PVN
PNNs follow a developmental trajectory across brain regions, typically emerging alongside circuit maturation and critical period closure (Carulli & Verhaagen, 2021; Tewari et al., 2022). To assess this pattern in the PVN, we examined PNN across developmental time points using Wisteria Floribunda Agglutinin (WFA) staining, an established marker for PNNs (Härtig et al., 2022). WFA + staining was absent at postnatal day 6 (P6) but appeared diffusely by P14 (Fig. 1a), suggesting that PNN components begin condensing during this period. Quantification of WFA area (WFA + area/total area; Fig. 1b) and total WFA intensity (including both condensed and diffuse labeling; Fig. 1c) revealed a developmental increase in PNN components from P6 to P14 in both sexes.
PNNs form dense, net-like structures around somata and proximal dendrites (Balmer, 2016; Cabungcal et al., 2013; Carceller et al., 2020; Carstens et al., 2016; Carulli & Verhaagen, 2021; Celio, 1993; Favuzzi et al., 2017; Fawcett et al., 2019; Frischknecht et al., 2009; Tewari et al., 2018). At P14, WFA + labeling was mostly diffuse, in contrast to the compact PNNs seen at 3 and 25 months (Fig. 1d), indicating PNN maturation by 3 months. This developmental trajectory aligns with findings in other brain regions in mice (Gogolla et al., 2009; Mirzadeh et al., 2019; Pizzorusso et al., 2002) and in humans (Rogers et al., 2018). Interestingly, unlike cortical and hippocampal PNNs that often extend over dendrites, PVN PNNs showed limited dendritic coverage (Fig. 1d), similar to other hypothalamic areas (Alonge et al., 2020). While age-related PNN changes occur in the cortex (Brewton et al., 2016; Karetko-Sysa et al., 2014) and hippocampus (Lehner et al., 2024), we observed no significant differences in PNN area or intensity between 3 and 25 months male mice within the PVN (Fig. 1e,f).
PNNs surround distinct neuronal subpopulations in the PVN
A
Given the PVN’s complex neuronal makeup, we generated a stereological map to identify neuronal subtypes enwrapped by PNNs. Several subtypes exhibited PNN enwrapped neurons (Fig. 2a). Sex differences were only observed for oxytocin (OXT)-expressing neurons, with females exhibiting a higher number of total WFA + neurons and %WFA + neurons than males (Fig. 2b). The number of PNN-enwrapped neurons did not significantly differ between females and males either in absolute number (Fig. 2c) or when normalized to PVN area (Fig. 2d).DOCA-salt sensitive hypertension increases the number of PNN-enwrapped neurons in the PVN of male mice
We next examined the relationship between neurogenic hypertension and PNN expression in the PVN using the DOCA-salt model (Grobe et al., 2011). Both sexes exhibited a significant increase in the change of blood pressure (Fig. 3a). Next, we quantified PNNs throughout the PVN (Fig. 3b). In males, DOCA-salt increased WFA + area in the PVN relative to sham, a result confirmed by cohort-normalized values (Fig. 3c). This increase corresponded to a higher number of PNNs (Fig. 3d). WFA intensity, however, was unchanged (Fig. 3e). In contrast, female DOCA-salt mice showed no significant changes in WFA + area, PNN count, or WFA intensity (Fig. 3f-h).
Fig. 1
Developmental regulation of PNNs in the PVN. a WFA staining shows no visible PNNs at P6, with progressive appearance at P14 and maturation by 1Mo. b-c Quantification of WFA + area and PNN intensity across developmental timepoints (each data point = average of 3 mice, 7–12 sections per mouse). d Representative images of PNN-enwrapped neurons at P14, 3Mo, and 25Mo. e-f Quantification of WFA + area (%) and fluorescence intensity in 3- vs. 25-month-old naïve male mice (3Mo n = 4; 25Mo n = 5). Data are shown as mean ± SEM; circles represent individual mice (average of 7–12 bilateral PVN sections/mouse).
Fig. 2
PNNs enwrap multiple neuronal cell types in the PVN. a Representative images of PNN-enwrapped neurons within the PVN. b Quantification of the number and percent of PNN-enwrapped neurons per cell type (n = 3/cell type). PV-neurons were not detected within the PVN and are therefore not shown. c Distribution of absolute PNN number across all mice analyzed for neuronal cell type quantification in B (females vs. males: n = 18; 87.1 ± 4.9 vs. 73.6 ± 5.7, p = 0.0798). d PNN number normalized to PVN area in an independent cohort of 3-month-old mice (females vs. males: n = 3; 43.3 ± 1.7 vs. 46.5 ± 3.5, p = 0.4506, unpaired). Data are mean ± SEM; circles represent independent mice (average of 7–12 bilateral PVN sections/mouse). *p = 0.0169, #p = 0.0077.
Fig. 3
DOCA-salt increases PVN PNNs in male but not female mice. a Systolic blood pressure (SBP) after 21 days of DOCA-salt treatment (males: SHAM n = 11, DOCA n = 9, ∆SBP − 5.0 ± 4.1 vs. 20.6 ± 4.3, p = 0.0004, unpaired t-test; females: SHAM n = 12, DOCA n = 13, ∆SBP − 6.7 ± 4.7 vs. 21.0 ± 5.2, p = 0.0024, Mann-Whitney). b Schematic showing PVN sections included in quantification. c-e Quantification in males: c WFA area (%) and normalized WFA area (SHAM n = 10, DOCA n = 9; 4.2 ± 0.3 vs. 5.7 ± 0.5, p = 0.0255; normalized 1.0 ± 0.1 vs. 1.4 ± 0.1, p = 0.0013), d PNN number (35.6 ± 2.0 vs. 44.8 ± 3.9, p = 0.0447), and e WFA intensity (1.0 ± 0.1 vs. 1.1 ± 0.0, p = 0.2677). f-h Quantification in females: f WFA area (%) and normalized WFA area (SHAM n = 7, DOCA n = 8; 6.7 ± 0.7 vs. 6.3 ± 0.5, p = 0.5532; normalized 1.0 ± 0.1 vs. 1.0 ± 0.1, p > 0.9999), g PNN number (71.5 ± 4.4 vs. 64.9 ± 6.0, p = 0.4011), and h WFA intensity (1.0 ± 0.1 vs. 1.0 ± 0.1, p > 0.9999). Data are mean ± SEM; circles represent independent mice (average of 10–15 bilateral PVN sections/mouse).
Conclusion
We found that PNNs in the PVN follow a developmental trajectory similar to other brain regions. The most prevalent neuronal type enwrapped by PNNs was nNOS-expressing neurons, and sex differences were observed only in OXT-enwrapped neurons. Importantly, neurogenic hypertension was associated with a sex-specific increase in both the number and area of PNNs within the PVN of male mice. Despite an increase in blood pressure in female mice, we did not observe changes in number, area, or intensity of PNNs. This suggests that PNNs may be involved in the increase in blood pressure in male mice only. Of note, we did not assess the estrous cycle at the time of euthanasia in female mice. In some brain regions, but not all, PNNs are modulated by the estrous cycle (Nguyen et al., 2025). Thus, it is plausible that differences in PNNs would be observed if we controlled for estrous cycle at the time of euthanasia.
In male mice exposed to the DOCA-salt, circumventricular organs provide increased excitatory input to the PVN, enhancing its activity (Guyenet, 2006). Increased neuronal activity is associated with both the upregulation of PNN components and an increase in PNN-enwrapped neurons (Carulli et al., 2007; Dityatev et al., 2007). Thus, in DOCA-salt males, the early rise in neuronal activity may recruit previously “silent” neurons, enabling them to form PNNs. The emergence of these new PNNs could then stabilize and sustain their heightened activity, contributing to the persistent overactivation of the PVN observed in neurogenic hypertension. Supporting this idea, neurogenic hypertension is associated with increased expression and secretion of corticotropin-releasing hormone (CRH) neurons (Goncharuk et al., 2002) as well as an increase in glutamatergic transmission (D.-P. Li et al., 2008; D.-P. Li & Pan, 2006; Y.-F. Li et al., 2001; Ye et al., 2012). Future studies will focus on identifying which neuronal populations account for the increase in PNN-enwrapped neurons observed after DOCA-salt.
Methods
Animals
A
All procedures were approved by the Institutional Animal Use and Care Committee of Vanderbilt University Medical Center, protocol number M234000-00. C57BL/6J mice (Jax#664), and CRH-Cre mice (Jax#12704) crossed with Ai14-tdTomato reporter mice (Jax#7914) were used. Naïve C57BL/6J mice (P6–90 days, 3–5 months, and 25 months [NIA]) were used for developmental and DOCA-salt studies.Developmental map
A
Except at P6, animals were perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA), post-fixed overnight, and cryoprotected in 30% sucrose. Brains were sectioned at 40 µm using a vibratome (Leica VT1200S).Immunofluorescence
Free-floating sections were permeabilized (PBS/0.5% Triton X-100, 1h), blocked (PBS/0.1% Triton X-100/10% NDS, 1h), and incubated overnight with primary antibodies at 4°C: WFA (1:300, Vector Laboratories FL-1351-2); HuC/D (1:500, ThermoFisher A-21271); Vasopressin (AVP, 1:500, Abcam AB213708); nNOS (1:300, Millipore Sigma AB5380); OXT (1:300, Abcam AB212193); Somatostatin (SST, ThermoFisher PA585759); Tyrosine hydroxylase (TH, ThermoFisher PA5-85167), Parvalbumin (PV, 1:300, Millipore Sigma MAB1572). Sections were then incubated in secondary antibodies (PBS/0.1% Triton X-100/2% NDS) for 2h at room temperature.
DOCA-salt hypertension
Mice were acclimated to tail-cuff plethysmography (Hatteras MC4000) one week prior to surgery. Animals were randomized to sham or DOCA-salt groups; DOCA mice received a subcutaneous 50 mg DOCA pellet (Innovative Research of America, M-121), while shams underwent surgery without implantation. DOCA mice had free access to 0.9% NaCl in the drinking water; controls received water. Systolic blood pressure (SBP) was monitored twice weekly for 21 days. Change in SBP was calculated as final SBP (day 21) minus baseline (pre-surgery average).
Confocal imaging acquisition and quantification
WFA-labeled sections were imaged with a laser scanning microscope (Zeiss LSM 880). Settings were kept constant across all groups (20X, 2µm z-stacks at room temperature; pixel size: 2.37µm; zoom: 0.7; pinhole: 90; digital gain: 1). Bilateral images of the PVN (7–15 per mouse) were analyzed using ImageJ. PNN-positive neurons were identified and counted. PNN intensity was quantified using the manual method by Slaker et al. (Slaker et al., 2016), with SUM-slice projections, background subtraction, and thresholding.
Statistical analysis
Data were analyzed in GraphPad Prism 10. Normality was assessed (Shapiro-Wilk), outliers were removed (Grubbs test), followed by unpaired two-tailed t-tests. p < 0.05 was considered significant.
Statements and Declarations
A
Funding
This research was supported by National Institute of Health K00NS130872 (IB), K22NS123507 (MMS) and by the Burroughs Wellcome Fund Postdoctoral Diversity Enrichment Program (IB).
Competing interests
Authors have no financial interests to disclose.
A
Author Contribution
IB and MMS designed the study. IB conducted all experiments, analyzed the data, and wrote the manuscript. SC, EY and SR helped with data collections and quantification. MMS provided funding and contributed to manuscript preparation and editing. All authors reviewed the manuscript.
A
Data Availability
Data is provided within the manuscript. Raw or analyzed data during the current study will be available from the corresponding authors on reasonable request.
References
Alonge KM, Mirzadeh Z, Scarlett JM, Logsdon AF, Brown JM, Cabrales E, Chan CK, Kaiyala KJ, Bentsen MA, Banks WA, Guttman M, Wight TN, Morton GJ, Schwartz MW (2020) Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats. Nat Metabolism 2(10):1025–1033. https://doi.org/10.1038/s42255-020-00275-6
Balmer TS (2016) Perineuronal Nets Enhance the Excitability of Fast-Spiking Neurons. Eneuro 3(4). https://doi.org/10.1523/ENEURO.0112-16.2016. ENEURO.0112-16.2016
Basting T, Sriramula S, Epling J, Lazartigues E (2018) Paraventricular Nucleus Over Activation Is A Critical Driver In The Development Of Neurogenic Hypertension. FASEB J 32(S1). https://doi.org/10.1096/fasebj.2018.32.1_supplement.598.5
Brewton DH, Kokash J, Jimenez O, Pena ER, Razak KA (2016) Age-Related Deterioration of Perineuronal Nets in the Primary Auditory Cortex of Mice. Frontiers in Aging Neuroscience, 8. https://doi.org/10.3389/fnagi.2016.00270
Cabungcal J-H, Steullet P, Morishita H, Kraftsik R, Cuenod M, Hensch TK, Do KQ (2013) Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proceedings of the National Academy of Sciences, 110(22), 9130–9135. https://doi.org/10.1073/pnas.1300454110
Carceller H, Guirado R, Ripolles-Campos E, Teruel-Marti V, Nacher J (2020) Perineuronal Nets Regulate the Inhibitory Perisomatic Input onto Parvalbumin Interneurons and γ Activity in the Prefrontal Cortex. J Neurosci 40(26):5008–5018. https://doi.org/10.1523/JNEUROSCI.0291-20.2020
Carstens KE, Phillips ML, Pozzo-Miller L, Weinberg RJ, Dudek SM (2016) Perineuronal Nets Suppress Plasticity of Excitatory Synapses on CA2 Pyramidal Neurons. J Neurosci 36(23):6312–6320. https://doi.org/10.1523/JNEUROSCI.0245-16.2016
Carulli D, Rhodes KE, Fawcett JW (2007) Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum. J Comp Neurol 501(1):83–94. https://doi.org/10.1002/cne.21231
Carulli D, Verhaagen J (2021) An Extracellular Perspective on CNS Maturation: Perineuronal Nets and the Control of Plasticity. Int J Mol Sci 22(5):2434. https://doi.org/10.3390/ijms22052434
Celio MR (1993) Perineuronal nets of extracellular matrix around parvalbumin-containing neurons of the hippocampus. Hippocampus 3(S1):55–60. https://doi.org/10.1002/hipo.1993.4500030709
Deepa SS, Carulli D, Galtrey C, Rhodes K, Fukuda J, Mikami T, Sugahara K, Fawcett JW (2006) Composition of Perineuronal Net Extracellular Matrix in Rat Brain. J Biol Chem 281(26):17789–17800. https://doi.org/10.1074/jbc.M600544200
Dityatev A, Brückner G, Dityateva G, Grosche J, Kleene R, Schachner M (2007) Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets. Dev Neurobiol 67(5):570–588. https://doi.org/10.1002/dneu.20361
Dong Y, Zhao K, Qin X, Du G, Gao L (2023) The mechanisms of perineuronal net abnormalities in contributing aging and neurological diseases. Ageing Res Rev 92:102092. https://doi.org/10.1016/j.arr.2023.102092
Favuzzi E, Marques-Smith A, Deogracias R, Winterflood CM, Sánchez-Aguilera A, Mantoan L, Maeso P, Fernandes C, Ewers H, Rico B (2017) Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican. Neuron 95(3):639–655e10. https://doi.org/10.1016/j.neuron.2017.06.028
Fawcett JW, Fyhn M, Jendelova P, Kwok JCF, Ruzicka J, Sorg BA (2022) The extracellular matrix and perineuronal nets in memory. Mol Psychiatry 27(8):3192–3203. https://doi.org/10.1038/s41380-022-01634-3
Fawcett JW, Oohashi T, Pizzorusso T (2019) The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. Nat Rev Neurosci 20(8):451–465. https://doi.org/10.1038/s41583-019-0196-3
Flack JM, Buhnerkempe MG, Moore KT (2024) Resistant Hypertension: Disease Burden and Emerging Treatment Options. Curr Hypertens Rep 26(5):183–199. https://doi.org/10.1007/s11906-023-01282-0
Frischknecht R, Heine M, Perrais D, Seidenbecher CI, Choquet D, Gundelfinger ED (2009) Brain extracellular matrix affects AMPA receptor lateral mobility and short-term synaptic plasticity. Nat Neurosci 12(7):897–904. https://doi.org/10.1038/nn.2338
Galtrey CM, Kwok JCF, Carulli D, Rhodes KE, Fawcett JW (2008) Distribution and synthesis of extracellular matrix proteoglycans, hyaluronan, link proteins and tenascin-R in the rat spinal cord. Eur J Neurosci 27(6):1373–1390. https://doi.org/10.1111/j.1460-9568.2008.06108.x
Gogolla N, Caroni P, Lüthi A, Herry C (2009) Perineuronal Nets Protect Fear Memories from Erasure. Science 325(5945):1258–1261. https://doi.org/10.1126/science.1174146
Goncharuk VD, Van Heerikhuize J, Swaab DF, Buijs RM (2002) Paraventricular nucleus of the human hypothalamus in primary hypertension: Activation of corticotropin-releasing hormone neurons. J Comp Neurol 443(4):321–331. https://doi.org/10.1002/cne.10124
Grobe JL, Buehrer BA, Hilzendeger AM, Liu X, Davis DR, Xu D, Sigmund CD (2011) Angiotensinergic Signaling in the Brain Mediates Metabolic Effects of Deoxycorticosterone (DOCA)-Salt in C57 Mice. Hypertension 57(3):600–607. https://doi.org/10.1161/HYPERTENSIONAHA.110.165829
Guyenet PG (2006) The sympathetic control of blood pressure. Nat Rev Neurosci 7(5):335–346. https://doi.org/10.1038/nrn1902
Harkness JH, Gonzalez AE, Bushana PN, Jorgensen ET, Hegarty DM, Nardo D, Prochiantz AA, Wisor A, Aicher JP, Brown SA, T. E., Sorg BA (2021) Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex. Brain Struct Function 226(4):1135–1153. https://doi.org/10.1007/s00429-021-02229-4
Härtig W, Meinicke A, Michalski D, Schob S, Jäger C (2022) Update on Perineuronal Net Staining With Wisteria floribunda Agglutinin (WFA). Front Integr Nuerosci 16:851988. https://doi.org/10.3389/fnint.2022.851988
Horii-Hayashi N, Sasagawa T, Nishi M (2017) Insights from extracellular matrix studies in the hypothalamus: Structural variations of perineuronal nets and discovering a new perifornical area of the anterior hypothalamus. Anat Sci Int 92(1):18–24. https://doi.org/10.1007/s12565-016-0375-5
Karetko-Sysa M, Skangiel-Kramska J, Nowicka D (2014) Aging somatosensory cortex displays increased density of WFA-binding perineuronal nets associated with GAD-negative neurons. Neuroscience 277:734–746. https://doi.org/10.1016/j.neuroscience.2014.07.049
Kario K, Okura A, Hoshide S, Mogi M (2024) The WHO Global report 2023 on hypertension warning the emerging hypertension burden in globe and its treatment strategy. Hypertens Res 47(5):1099–1102. https://doi.org/10.1038/s41440-024-01622-w
Lamptey RNL, Sun C, Layek B, Singh J (2023) Neurogenic Hypertension, the Blood–Brain Barrier, and the Potential Role of Targeted Nanotherapeutics. Int J Mol Sci 24(3):2213. https://doi.org/10.3390/ijms24032213
Lehner A, Hoffmann L, Rampp S, Coras R, Paulsen F, Frischknecht R, Hamer H, Walther K, Brandner S, Hofer W, Pieper T, Reisch L, Bien CG, Blumcke I (2024) Age-dependent increase of perineuronal nets in the human hippocampus and precocious aging in epilepsy. Epilepsia Open 9(4):1372–1381. https://doi.org/10.1002/epi4.12963
Li D-P, Pan H-L (2006) Plasticity of GABAergic control of hypothalamic presympathetic neurons in hypertension. Am J Physiol Heart Circ Physiol 290(3):H1110–H1119. https://doi.org/10.1152/ajpheart.00788.2005
Li D-P, Yang Q, Pan H-M, Pan H-L (2008) Plasticity of pre- and postsynaptic GABAB receptor function in the paraventricular nucleus in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 295(2):H807–H815. https://doi.org/10.1152/ajpheart.00259.2008
Li Y-F, Mayhan WG, Patel KP (2001) NMDA-mediated increase in renal sympathetic nerve discharge within the PVN: Role of nitric oxide. Am J Physiol Heart Circ Physiol 281(6):H2328–H2336. https://doi.org/10.1152/ajpheart.2001.281.6.H2328
Lupori L, Totaro V, Cornuti S, Ciampi L, Carrara F, Grilli E, Viglione A, Tozzi F, Putignano E, Mazziotti R, Amato G, Gennaro C, Tognini P, Pizzorusso T (2023) A comprehensive atlas of perineuronal net distribution and colocalization with parvalbumin in the adult mouse brain. Cell Rep 42(7):112788. https://doi.org/10.1016/j.celrep.2023.112788
Mirzadeh Z, Alonge KM, Cabrales E, Herranz-Pérez V, Scarlett JM, Brown JM, Hassouna R, Matsen ME, Nguyen HT, Garcia-Verdugo JM, Zeltser LM, Schwartz MW (2019) Perineuronal net formation during the critical period for neuronal maturation in the hypothalamic arcuate nucleus. Nat Metabolism 1(2):212–221. https://doi.org/10.1038/s42255-018-0029-0
Nguyen R, Rahyab R, Deshpande A, Legge E, Almeida J, Herz SM, Zylko AL, Damaj MI, Lasek AW (2025) Estrogenic regulation of perineuronal nets in the mouse insular cortex and hippocampus. Neuropharmacology 279:110641. https://doi.org/10.1016/j.neuropharm.2025.110641
Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L (2002) Reactivation of Ocular Dominance Plasticity in the Adult Visual Cortex. Science 298(5596):1248–1251. https://doi.org/10.1126/science.1072699
Rogers SL, Rankin-Gee E, Risbud RM, Porter BE, Marsh ED (2018) Normal Development of the Perineuronal Net in Humans; In Patients with and without Epilepsy. Neuroscience 384:350–360. https://doi.org/10.1016/j.neuroscience.2018.05.039
Santisteban MM, Schaeffer S, Anfray A, Faraco G, Lopez DB, Wang G, Sobanko M, Sciortino R, Racchumi G, Waisman A, Park L, Anrather J, Iadecola C (2022) Meningeal IL-17 producing T cells mediate cognitive impairment in salt-sensitive hypertension [Preprint]. Neuroscience. https://doi.org/10.1101/2022.09.05.506398
Slaker ML, Harkness JH, Sorg BA (2016) A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity. IBRO Rep 1:54–60. https://doi.org/10.1016/j.ibror.2016.10.001
Tewari BP, Chaunsali L, Campbell SL, Patel DC, Goode AE, Sontheimer H (2018) Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy. Nat Commun 9(1):4724. https://doi.org/10.1038/s41467-018-07113-0
Tewari BP, Chaunsali L, Prim CE, Sontheimer H (2022) A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system. Front Cell Neurosci 16:1022754. https://doi.org/10.3389/fncel.2022.1022754
Xi H, Li X, Zhou Y, Sun Y (2024) The Regulatory Effect of the Paraventricular Nucleus on Hypertension. Neuroendocrinology 114(1):1–13. https://doi.org/10.1159/000533691
Ye Z-Y, Li D-P, Byun HS, Li L, Pan H-L (2012) NKCC1 Upregulation Disrupts Chloride Homeostasis in the Hypothalamus and Increases Neuronal Activity-Sympathetic Drive in Hypertension. J Neurosci 32(25):8560–8568. https://doi.org/10.1523/JNEUROSCI.1346-12.2012
Zheng H, Katsurada K, Nandi S, Li Y, Patel KP (2022) A Critical Role for the Paraventricular Nucleus of the Hypothalamus in the Regulation of the Volume Reflex in Normal and Various Cardiovascular Disease States. Curr Hypertens Rep 24(7):235–246. https://doi.org/10.1007/s11906-022-01187-4
