Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

The vulnerability of thalamocortical circuitry to hypoxic-ischemic injury in a mouse model of periventricular leukomalacia

The vulnerability of thalamocortical circuitry to hypoxic-ischemic injury in a mouse model of... Background: Periventricular leukomalacia (PVL) is the leading cause of neurological disabilities including motor and cognitive deficits in premature infants. Periventricular leukomalacia is characterized by damage to the white matter in the immature brain, but the mechanisms by which damage to immature white matter results in widespread deficits of cognitive and motor function are unclear. The thalamocortical system is crucial for human consciousness and cog‑ nitive functions, and impaired development of the cortico‑ thalamic projections in the neonatal period is implicated to contribute importantly to abnormalities of cognitive function in children with PVL. Results: In this study, using a mouse model of PVL, we sought to test the hypothesis that PVL‑ like injury affects the different components of the thalamocortical circuitry that can be defined by vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2), both of which are required for glutamatergic synaptic transmission in the central nervous system. We combined immunocytochemistry and immuno‑ electron microscopy to investigate changes in cortico‑ thalamic synapses which were specifically identified by vGluT1 immunolabeling. We found that a drastic reduction in the density of vGluT1 labeled profiles in the somatosensory thalamus, with a reduction of 72–74 % in ventroposterior ( VP) nucleus and a reduction of 42–82 % in thalamic reticular nucleus (RTN) in the ipsilateral side of PVL mice. We fur‑ ther examined these terminals at the electron microscopic level and revealed onefold–twofold decrease in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. The present study provides anatomical and ultrastructural evidence to elucidate the cellular mechanisms underlying alteration of thalamic circuitry in a mouse model of PVL, and reveals that PVL‑ like injury has a direct impact on the corticothalamic projection system. Conclusions: Our findings provide the first set of evidence showing that the thalamocortical circuitry is affected and vulnerable in PVL mice, supporting a working model in which vGluT1 defined corticothalamic synapses are altered in PVL mice, and vGluT2 defined thalamocortical synapses are associated with such changes, leading to the compro ‑ mised thalamocortical circuitry in the PVL mice. Our study demonstrates that the thalamocortical circuitry is highly vulnerable to hypoxia–ischemia in the PVL model, thus identifying a novel target site in PVL pathology. Keywords: Prematurity, Periventricular leukomalacia, White matter injury, Oligodendrocyte, Thalamocortical circuitry, Cognitive impairment cause of subsequent neurological disability including Background spastic motor deficits (cerebral palsy) and cognitive Periventricular leukomalacia (PVL) is the predominant impairments. Nearly 90  % of the 13–15 million prema- brain pathology in premature infants and is the leading ture infants born worldwide every year survive beyond infancy, in which ≈5–10 % of the survivors develop cere- *Correspondence: wbdeng@ucdavis.edu bral palsy, and 40–50 % develop cognitive and behavioral Department of Biochemistry and Molecular Medicine, School deficits [ 3, 25]. Periventricular leukomalacia is tradition- of Medicine, University of California, Davis, 2425 Stockton Blvd., Room ally classified as a white matter disorder. However, white 653, Sacramento, CA 95817, USA Full list of author information is available at the end of the article © 2016 Liu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. BMC Neurosci (2016) 17:2 Page 2 of 9 matter damage underlying PVL is now recognized as A recent study further confirmed that these vGluT2 the major component of a more generalized injury to synapses also target NG2 cells in layer III-IV and layer the cerebrum that includes neuronal and axonal injury, VI [19] that contains pyramidal cell projections as cor- and is renamed “encephalopathy of prematurity” [25]. ticothalamic inputs to the somatosensory thalamus. Emerging neuropathological and neuroimaging studies In the present study, we sought to test the hypothesis demonstrate that PVL is associated with injury to many that PVL-like injury first affects the vGluT2-containing cortical and subcortical regions, including thalamus, thalamocortical projection system that in turn pro- cerebral cortex, hippocampus and basal ganglia in vari- foundly compromises vGluT1 containing corticotha- able combinations [4, 11, 21, 25]. The widespread effect lamic projection system and thus alters vGluT1 defined of the disorder may account for cognitive impairments corticothalamic synapses in the thalamus. We applied and intellectual deficits in preterm survivors with PVL. vGluT1 immunocytochemistry and electron micros- In particular, white matter injury impacts on axon fib - copy (EM) to analyze the contralateral (control) and ers ascending and descending from cerebral cortex and ipsilateral (injured) side of the brains of PVL mice. We thalamus and the reciprocal thalamo-cortical circuitry found that the density of vGluT1 immunolabeled termi- that is crucial for generating and maintaining awaken- nals was significantly decreased in the somatosensory ing-sleeping cycle of high mammals and thalamocortical thalamus of the ipsilateral injured side. Furthermore, oscillations that are essential for human consciousness at the EM level, these vGluT1 labeled corticothalamic and cognitive function. terminals were drastically altered in their sizes and syn- It has been widely accepted that thalamocortical con- aptic structures. These findings provide the first set of nections are formed perinatally in the mouse, and the evidence showing that the thalamocortical circuitry synaptic remodeling and circuitry refinement start is affected and vulnerable in PVL mice, supporting a around P7 and continue in the second postnatal week working model in which vGluT1 defined corticotha - [1, 7, 20]. The functional thalamocortical circuitry is lamic synapses are selectively altered in PVL mice, and established around P15 [12]. The second postnatal week vGluT2 defined thalamocortical synapses are associated (P7–P15) is critical for establishing thalamic oscillations with such changes, leading to the compromised thalam- that are crucial for the maturation of the thalamocorti- ocortical circuitry in the PVL mice. cal circuitry. Using a mouse model of PVL [23, 24] we investigate whether PVL induction affects the establish - Methods ment of the thalamocortical circuitry and whether syn- Animals aptic connections in the thalamocortical circuitry are C57B/6 mice (The Jackson Laboratories, Bar Harbor, altered following PVL induction. White matter injury ME) at postnatal day 6 (P6) were used to characterize in PVL may affect a significant portion of glutamatergic neuropathological features under different treatments. axon terminals connecting the somatosensory thalamus NG2-DsRed transgenic mice [The Jackson Laboratories, and the cerebral cortex. Defects in these axon terminals stock#: Tg(Cspg4-DsRed.T1)1Akik/J] were used to study can be identified at cellular and ultrastructural level the identity of postsynaptic profiles in cerebral white using specific glutamatergic synaptic markers, such as matter. Animal research was approved by the University vGluT1 and vGluT2, which have been used to define of California-Davis Committee on Animal Research. All specific thalamo-cortical systems [9 , 13]. Gene expres- procedures were carried out by following the guidelines sion and immunocytochemistry studies have confirmed set by the institutional animal care and use committee. that vGluT1 is primarily expressed in layer III and layer V-VI pyramidal cells and vGluT2 is highly expressed in Unilateral carotid ligation (UCL) plus hypoxia thalamic relay cells and this complementary expression Twenty animals underwent a permanent ligation of the is reflected more clearly in the protein expressions that right common carotid artery under ice anesthesia, fol- mainly occur in the axon terminals. It has been previ- lowed by 1-hour recovery on a thermal blanket, main- ously shown that vGluT1 specifically labels the cortico - taining body temperature at 33–34 °C and with a 1-hour thalamic synapses, whereas vGluT2 specifically defines feeding period with the dam. Animals were then placed the thalamocortical synapses [9, 13]. We demonstrated in a sealed chamber infused with nitrogen to maintain that the synaptic contacts between vGluT2-labeled a level of 6.0  % O . Different durations of hypoxia were thalamocortical axon terminals and NG2  +  oligoden- applied. The body temperature of mice was maintained at droglial progenitor cells (OPCs) in the developing white 33–34 °C by leaving animals on thermal blanket through- matter were selectively affected and caused the post - out hypoxia. After a 1-hour period of recovery, mice were synaptic profiles of NG2 progenitor cells to shrink [23]. returned to their dam. Liu et al. BMC Neurosci (2016) 17:2 Page 3 of 9 Immuno‑electron microscopy rectangular region approximately 100  μm were sub- Ten P10 mice that were subjected to UCL/hypoxia at jected to particle analysis. The particle numbers in each P6 were perfused with 4  % paraformaldehyde plus 0.2  % region were obtained, for each case, mean numbers of glutaraldehyde in 0.1  M phosphate buffer saline (PB, profiles  ±  standard deviation/100  μm was compared in and brains were cut coronally with a vibratome (Leica) each pair (ipsilateral and contralateral) in VP or RTN, at 60  μm. Sections were stained for vGluT1 and vGluT2 respectively. antibodies (Millipore, Billerica, MA) using the immun- operoxidase ABC method. A total three cases showing Quantification by electron microscopy dense vGlut1 immunostaining were selected for electron Electron microscope of vGluT1 immunoperoxidase microscopic study. The stained sections were processed labeled terminals or synapses were taken at X10,000. for EM as described previously [9]. The sections from Electron microscope images were obtained from the the ipsilateral (injury) side and the contralateral (control) contralateral and injured side of VP and RTN. In each side were first examined in the light microscope at 10X side, at least 10–15 images were taken. Electron micro- and 20X, the forebrain containing the cerebral cortex and scope images were converted to TIF files and imported to thalamus was identified and photographed, ventroposte - Image J for measuring. We used the “Analyze” function to rior nucleus (VP) and thalamic reticular nucleus (RTN) measure the area (in μm ) and perimeter (in μm) of each regions (approximately 2  ×  0.5  mm) were cut under a vGluT1 labeled terminals. Given the irregular shape of dissecting microscope and glued to blank resin blocks. the labeled profiles and the fact that the area of the pro - Thin sections were cut at 70 nm using an ultramicrotome file represents closely its size, we therefore used the area (Leica Ultracut) and collected on single-slot (2 ×  1  mm) for quantification. At least two labeled terminals were copper grids and stained with uranyl acetate and lead cit- identified from each image and measured. The mean area rate, and examined in a Philips CM120 EM at 80 kV. The size (μm ) ± standard deviation (SD) from the contralat- regions were carefully scanned to exclude the areas con- eral or ipsilateral side was compared. taining fiber tract in the internal capsule, and the orienta - tion of the region was guided by light microscopic images Results taken before EM processing. Much effort was made to Decrease in number of vGluT1 immunolabeled profiles ensure that the regions were taken from the somatosen- in the somatosensory thalamus of PVL mice sory thalamus. Images were taken by a 2  ×  2  K CCD At the light microscopic level, vGluT1 immunolabeled camera (Gatan, Inc., Pleasanton, CA) and processed by profiles appeared to be thin presynaptic terminals and using the software provided by Gatan, Inc (DigitalMi- small boutons densely distributed in the dorsal thalamus crograph). Images were composed using Photoshop CS including VP and RTN of the contralateral side (Fig.  1). (Adobe System, Mountain View, CA). In the ipsilateral side, a drastic decrease in the number of vGluT1 immunolabeled profiles was noted in some Quantification by light microscopy regions of the dorsal thalamic nuclei including VP and vGluT1 immunoperoxidase labeled sections embed- RTN (Fig. 1). Compared to the contralateral side thalamus ded with Araldite were used for the quantitative analy- which contains dense clusters of vGluT1 labeled profiles, sis. For each case (the contralateral and ipsilateral pair), the labeling in the ipsilateral side was more diffuse and at least one section from each side was examined under became less intense in clustering. Instead, some non-spe- light microscope using X20 and X40 objectives. Light cific lightly labeled somata were uniformly distributed on microscopic images (X40) showing dense vGluT1 labeled the background, but they were easily differentiated from boutons in thalamus (VP and RTN) were captured using the labeled terminals by sizes. A quantitative analysis was a Zeiss imaging system with a CCD camera (Zeiss, made to access the change in number of vGluT1 immu- Thornwood, NY, USA) and the images were processed nolabeled profiles in 100 μm area of VP and RTN of the in Photoshop CS to adjust the brightness and contrast ipsilateral side of the somatosensory thalamus (Fig.  2). and convert to gray scale and imported to Image J (NIH, A total of three independent cases were analyzed and Bethesda, MD, USA) as TIF files. The “analyze particles” quantified, and a drastic change in vGluT1 labeling den - function was used to count the labeled profiles. Assum - sity was found in both VP and RTN of the ipsilateral side. ing the diameters of the labeled corticothalamic termi- In VP, in case 1, the density of labeling was decreased nals were from 0.3 to 0.6 μm [17], accordingly, the profile from 7.69 ± 1.61 profiles per 100 μm (contralateral side) 2 2 sizes (areas) in a range between 0.07 and 0.7  μm were to 2.0  ±  1.17 profiles per 100  μm (ipsilateral side) and included in counting. This range would cover the sizes of approximately 74  % or 3.8-fold reduction, p  <  0.005; in most vGluT1 labeled profiles in thalamus. In each sec - case 2, it was decreased from 2.59  ±  0.93 profiles per tion, at least two regions (up to five regions) and each 100  μm (contralateral side) to 0.73  ±  0.47 profiles per Liu et al. BMC Neurosci (2016) 17:2 Page 4 of 9 Fig. 1 Light microscopic images taken from Araldite embedded vibratome sections showing immunolabeling of vGluT1 in the somatosensory thalamus [ventroposterior ( VP) nucleus and thalamic reticular nucleus (RTN)] of ipsilateral (a) and contralateral (b) sides of the brain in a mouse model of PVL. c is a high magnification image taken from VP showing the area lacking vGluT1 labeling in the ipsilateral side. d is a high magnifica‑ tion image taken from VP of the contralateral side showing clusters of vGluT1 labeled profiles among neuropils. e is a much higher power image showing the labeled profiles including presynaptic boutons. f Light microscopic image showing the lack of vGluT1 labeling in the RTN region of the ipsilateral side; note some nonspecifically labeled somata in the region. g Light microscopic image showing vGluT1 labeled small boutons and profiles in the RTN of the contralateral side. h A high power light microscopic image showing clusters of vGluT1 labeled profiles in the RTN 2 2 100  μm (ipsilateral side) approximately 72 % or 3.5-fold (contralateral side) to 0.46  ±  0.15 profiles per 300  μm reduction, p  <  0.0007; in case 3, it was decreased from (ipsilateral side) approximately 82  % or fivefold reduc - 3.44  ±  2.58 profiles per 100  μm (contralateral side) to tion, p < 0.0006 (Fig. 2). Overall, the decreases of vGluT1 0.94 ± 0.59 profiles per 100 μm (ipsilateral side) approx- labeling density in three cases were consistent in VP and imately 72  % or threefold reduction, p  <  0.00216. Con- RTN and the differences between contralateral and the sistent with changes in VP, in RTN, in case 1, the density ipsilateral sides were highly significant. of labeling was decreased from 2.90  ±  1.17 profiles per 100  μm (contralateral side) to 1.63  ±  0.51 profiles per Alteration in ultrastructure of vGluT1 labeled 100  μm (ipsilateral side) approximately 44  % or 1.8- corticothalamic synapses in the somatosensory thalamus fold reduction, p < 0.03; in case 2, it was decreased from of PVL mice 2.24  ±  0.39 profiles per 100  μm (contralateral side) to The drastic reduction in number of vGluT1 labeled ter - 1.26 ± 0.56 profiles per 100 μm (ipsilateral side) approx- minals in the somatosensory thalamus of PVL mice imately 43 % or 1.8-fold reduction, p < 0.0129; in case 3, strongly indicated that these terminals and their synapses it was decreased from 2.64  ±  0.87 profiles per 100  μm may undergo major changes in their ultrastructure in Liu et al. BMC Neurosci (2016) 17:2 Page 5 of 9 terminals in VP and RTN as reported previously [17]: these terminals were about 0.3–0.6 μm in diameter, con- taining few mitochondria, filled with round or oval shape synaptic vesicles and they usually form asymmetrical synapses associated with prominent postsynaptic densi- ties on dendritic profiles. Interestingly, these terminals usually contact small and presumably distal dendrites of relay cells in VP and contact much wide range sizes of dendritic profiles in RTN [15, 17] (Fig.  3). In contrast to vGluT1 labeled terminals in the contralateral side, the labeled terminals in the ipsilateral side were much smaller in their sizes and they showed some structural alterations including less defined membranes and con - taining fewer synaptic vesicles and were lack of promi- nent postsynaptic densities (Fig.  3). To quantitatively access the change in ultrastructure of vGluT1 labeled terminals in the ipsilateral side, a total of 15 images each containing at least two labeled terminals from either VP or RTN of the contralateral and ipsilateral side of PVL mice were subjected to quantitative analysis. In the con- tralateral VP, the mean area size of vGluT1 labeled ter- minals was 0.58  ±  0.23  μm , in the ipsilateral side, the mean area size was 0.27 ± 0.09 μm , there was more than twofold decreased in the size of the terminals, the change was highly significant (p  <  0.00011). In the contralateral RTN, the mean area size was 0.39 ± 0.19 μm , in the ipsi- lateral side, the mean size of vGluT1 labeled terminals was 0.24 ± 0.12 μm , there was more than 1.5-fold reduc- tion in the size of these terminals and the change was sig- nificant (p < 0.0291) (Fig.  4). We also observed a change in the length of postsynaptic density of vGluT1 labeled synapses in the ipsilateral side compared to the contralat- eral side. Discussion In the present study, we combined immunocytochem- istry and EM to investigate the changes associated with corticothalamic synapses which were specifically iden - tified by vGluT1 labeling in a mouse model of PVL. We found that a drastic reduction in number of vGluT1 labeled profiles in the somatosensory thalamus (VP: Fig. 2 Quantitative analysis of the density (number of vGluT1 labeled profiles per 100 μm ) of vGluT1 labeled profiles in the contralateral a reduction of 72–74  % or about threefold–fourfold; and ipsilateral side VP (blue color) and RTN (red color) from three cases RTN: a reduction of 42–82  % or 1.8-fold–fivefold) in of PVL mice. In the contralateral side, the density of vGluT1 labeling is the ipsilateral side of PVL mice. We further examined higher in VP than RTN in three cases. In the ipsilateral side, the density these terminals at the EM level and revealed onefold– is decreased significantly in VP (average 3.8‑fold decrease) and RTN twofold shrinkage in the sizes of vGluT1 labeled cor- (onefold–fivefold decrease) compared to the contralateral side, *p < 0.05; **p < 0.01 ticothalamic terminals in VP and RTN. Based on the experimental observations from the present study and our previous study [23] and the recent finding from PVL mice. We further carried out electron microscopic others [19], we proposed a working model to elucidate analysis to resolve this issue. At the EM level, in con- the underlying cellular mechanisms may account for tralateral side, vGluT1 immunolabeled terminals dis- the alteration of thalamic circuitry in the mouse model played typical ultrastructural features of corticothalamic of PVL. Liu et al. BMC Neurosci (2016) 17:2 Page 6 of 9 Fig. 3 Electron micrographs showing typical vGluT1 immunoperoxidase labeled terminals in the contralateral side VP (a) and RTN (c) and ipsilateral side VP (b) and RTN (d). In the contralateral side, vGluT1 labeled terminals (T) contain densely packed vesicles and form clear asymmetrical synapses (indicated by arrows) with postsynaptic dendrites (D) (note the prominent postsynaptic densities associated with the synapses). In ipsilateral side, the labeled terminals have less defined irregular shapes and form less clear synaptic contacts with postsynaptic dendrites (D). Scale bar = 0.5 μm A working model of the vulnerability of corticothalamic on layer IV cells as well [19]. Layer III-IV cells and layer synapses in the PVL mice VI pyramidal cells which express vGluT1 receive TC syn- In this working model  as shown in Fig.  5 , at the normal aptic inputs and send out their axons through white mat- condition, as illustrated in the contralateral side, thalam- ter and form synapses on RTN cells and TC cells. GAB ocortical (TC) relay cells which express vGluT2 [9] (TC Aergic cells in RTN project back to TC cells, serving as vGluT2) in the thalamic nucleus VP send out their axons a negative feedback loop. Note that axon bundles from through white matter, and they also send branches to syn- vGluT1 containing layer VI corticothalamic cells are mye- apse with GABAergic RTN cells. During postnatal devel- linated.In the ipsilateral side of PVL mice, vGluT2 positive opment, these TC cell axons form synaptic contacts on thalamocortical relay cells are profoundly compromised, NG2 cells in the white matter [23], and they also project their terminals that synapse on NG2 cells in the white to other NG2 cells in layer IV and layer VI and synapse matter undergo structural, biochemical and functional Liu et al. BMC Neurosci (2016) 17:2 Page 7 of 9 significant exceptions, do not co-localize in the same synaptic terminals [6, 13]. It has been suggested that vGluT1 is primarily found at synapses characterized by low-release probability and a capacity for long-term potentiation (LTP), whereas vGluT2 is primarily found at synapses characterized by high-release probability and a capacity for long-term depression (LTD) [5]. Our recent study also confirmed previous findings that the majority of thalamocortical terminals contain vGluT2 and the cor- tico-thalamic terminals derived from layer VI pyramidal cells contain vGluT1 [9, 14, 19]. In somatosensory thala- mus, another source of similar terminals to corticotha- lamic synapses is derived from brainstem which mainly cholinergic and monoaminergic, but a large amount of these terminals do not form classical synapses [12, 16], therefore, vGluT1 labeled terminals here should be con- sidered exclusively corticothalamic. In the present study, a quantitative analysis on vGluT1 immunostaining at the light microscopic level revealed a consistent reduc- tion in labeled profiles in the somatosensory thalamus of the ipsilateral side of PVL mice. Although the degree of changes in three cases shows some variabilities, the trend is consistent. Such fluctuation could be related to the brain regions impacted by PVL. The reduction in labeled profiles in PVL mice was further confirmed by Fig. 4 Quantitative analysis of the size (μm ) of vGluT1 immunola‑ EM studies, showing decreased sizes of vGluT1 labeled beled axon terminals in the contralateral and ipsilateral side of VP (a) terminals, which exhibited typical corticothalamic syn- and RTN (b) of PVL mice. In VP, twofold reduction in the size of the aptic features in previous studies [12, 17]. The reduction labeled terminals is observed in the ipsilateral side (*p < 0.00011). In in vGluT1 labeled terminal numbers could result from a RTN, 1.6‑fold reduction in the size of the labeled terminals is found in the ipsilateral side (*p < 0.0291) down-regulation of vGluT1 protein due to the changes in layer VI pyramidal cells and also the alterations in axons and their terminals because of de-myelination. It is obvi- ous that corticothalamic axon terminals were severely changes, and vGluT2 expression is altered, resulting in the damaged, given the drastic ultrastructural changes. This defects in both NG2 progenitor cell function and myelina- target specific alteration can be reliably detected using tion of corticothalamic axons. Thalamocortical synapses vGluT1 labeling. Further investigation to explore the on NG2 cells in layer III-IV and layer VI are also affected, potential changes occur in cortical projection cells and ultimately leading to the change of vGluT1 expression in possible associated changes in myelinated axons will shed layer III-IV and layer VI cells, down-regulation of vGluT1 light on the underlying thalamocortical circuitry which is expression in corticothalamic terminals, and malfunction highly implicated in PVL and other newborn neurologi- of the synaptic transmission. cal injuries or related diseases. vGluT1 as a reliable synaptic marker for corticothalamic Interaction of vGluT1 and vGluT2 in the thalamocortical synapses circuitry in PVL Synaptic plasticity depends, at least in part, on presyn- vGluT1 and vGluT2 are expressed in distinctive neuronal aptic mechanisms that have been associated with the subtypes and involved in different functional systems. probability of neurotransmitter release [22]. The amount vGluT2 is primarily restricted to projections between of glutamate contained within synaptic vesicles and and within subcortical areas, as well as thalamocortical available for release is regulated by vesicular glutamate projections, while vGluT1 is reserved for intercortical transporters (vGluTs) located on the membrane of the and corticothalamic projections [2, 8, 10, 13, 14, 23]. The vesicles. Two of the three known vGluT isoforms, vGluT1 pathology in corticothalamic terminals in PVL suggests and vGluT2, are relatively abundant throughout the cen- that different vGluT proteins may be engaged in specific tral nervous system (CNS), where they are expressed by functional pathways. How is the corticothalamic pathway specific glutamatergic neuronal populations and, with Liu et al. BMC Neurosci (2016) 17:2 Page 8 of 9 Fig. 5 A working model illustrating the thalamocortical circuitry and the affected targets in the contralateral versus ipsilateral side of the brain in a mouse model of PVL. NG2 cells are represented in the white matter and in the cerebral cortex. The synaptic contacts between vGluT2 labeled ter‑ minals from thalamocortical ( TC) cells (termed “TC vGluT2” cells) are formed in the developing white matter and the cerebral cortex. These synapses are altered in PVL mice, which may in turn affect the myelination of layer VI pyramidal (P) cells (termed “P vGluT1” cells) that may then change vGluT1 expression in their axon terminals in the VP and RTN (indicated by red crosses) affected in PVL mice? We need to consider the thalam - differentiation and migration of these NG2 cells and ocortical circuitry as a whole in order to identify which ultimately affect the myelination of functionally organ - may be altered at PVL condition. Our previous study [23] ized axon bundles in the white matter and cerebral cortex demonstrated that the postsynaptic targets of vGluT2 and result in malfunction of the thalamocortical circuitry synapses were affected in the white matter of the ipsilat - [18]. Furthermore, the impact of PVL injury may also eral side of PVL mice, the shrinkage of the postsynaptic affect the layer VI corticothalamic projecting pyrami - profiles may be derived from NG2-expressing oligoden - dal cells which express vGluT1 and thus down regulate drocyte precursor cells (OPC). A recent study [19] using the protein expression in the corticothalamic terminals elegant transgenic techniques combined electrophysi- and further alter the synaptic transmissions and their ological recording also demonstrated that vGluT2 labeled ultrastructure, the consequence of the impact would be thalamocortical synapses specifically target NG2 cells in the de-synchronization of the whole circuitry. The pro - layer IV and layer VI and other neurons in these layers posed working model presents a new pathway for elu- and provided strong evidence indicating that thalamo- cidating the cellular mechanisms responsible for PVL cortical synapses are vGluT2 specific and are modified injury. vGluT proteins are involved in regulating gluta- by somatosensory afferents [18]. Based on these findings, mate release in presynaptic terminals, the impact of the it is anticipated that the injury in PVL may firstly strike protein defection has a profound effect on glutamater - the thalamocortical relay cells which receive strong sen- gic transmission and thus alters the neuronal circuitry, sory inputs and form synaptic contacts with NG2 cells such as thalamocortical pathway which control the con- in the white matter and other cortical layers during early ciseness level and cognitive functions of the animals. postnatal development (P2–P10). The alteration in syn - Future investigation to further understand the underly- aptic transmission between vGluT2 expressing thalamo- ing pathology of PVL will be crucial for developing new cortical terminals and NG2 cells may compromise the treatments for this devastating human disorder. Liu et al. BMC Neurosci (2016) 17:2 Page 9 of 9 Authors’ contributions 9. Graziano A, Liu XB, Murray KD, Jones EG. Vesicular glutamate transporters XL and YS carried out all the experiments. XL, DEP and WD designed experi‑ define two sets of glutamatergic afferents to the somatosensory thala‑ ments. XL and WD wrote the paper. WD directed the study. All authors read mus and two thalamocortical projections in the mouse. J Comp Neurol. and approved the final manuscript. 2008;507:1258–76. 10. Hackett TA, de la Mothe LA. Regional and laminar distribution of the Author details vesicular glutamate transporter, VGluT2, in the macaque monkey audi‑ Department of Biochemistry and Molecular Medicine, School of Medicine, tory cortex. J Chem Neuroanat. 2009;38:106–16. University of California, Davis, Sacramento, CA 95817, USA. Center for Neu‑ 11. Inder T, Neil J, Kroenke C, Dieni S, Yoder B, Rees S. Investigation of roscience, School of Medicine, University of California, Davis, Sacramento, CA cerebral development and injury in the prematurely born primate 95817, USA. Institute for Pediatric Regenerative Medicine, School of Medi‑ by magnetic resonance imaging and histopathology. Dev Neurosci. cine, University of California, Davis, CA 95817, USA. Medical College, Hubei 2005;27(2–4):100–11. University of Arts and Science, Xiangyang, Hubei, China. Department of Bio‑ 12. Jones EG. The Thalamus. 2nd ed. Cambridge (UK): Cambridge University chemistry and Molecular Medicine, School of Medicine, University of Califor‑ Press; 2007. nia, Davis, 2425 Stockton Blvd., Room 653, Sacramento, CA 95817, USA. 13. Kaneko T, Fujiyama F. Complementary distribution of vesicular glutamate transporters in the central nervous system. Neurosci Res. 2002;42:243–50. Acknowledgements 14. Kaneko T, Fujiyama F, Hioki H. Immunohistochemical localization of The work was supported by Grants from the National Institutes of Health candidates for vesicular glutamate transporters in the rat brain. J Comp (R01NS059043), National Multiple Sclerosis Society, Feldstein Medical Founda‑ Neurol. 2002;444:39–62. tion and Shriners Hospitals for Children to W.D. 15. Liu XB, Honda CN, Jones EG. Distribution of four types of synapse on physiologically identified relay neurons in the ventral posterior thalamic Competing interests nucleus of the cat. J Comp Neurol. 1995;352:69–91. The authors declare that they have no competing interests. 16. Liu XB, Jones EG. The fine structure of serotonin and tyrosine hydroxylase immunoreactive terminals in the ventral posterior thalamic nucleus of cat Received: 15 July 2015 Accepted: 23 December 2015 and monkey. Exp Brain Res. 1991;85(3):507–18. 17. Liu XB, Jones EG. Predominance of corticothalamic synaptic inputs to thalamic reticular nucleus neurons in the rat. J Comp Neurol. 1999;414:67–79. 18. Mangin JM, Gallo V. The curious case of NG2 cells: transient trend or game changer? ASN Neuro. 2011;3:e00052. References 19. Mangin JM, Li P, Scafidi J, Gallo V. Experience ‑ dependent regulation 1. Agmon A, Yang LT, O’Dowd DK, Jones EG. Organized growth of thalamo‑ of NG2 progenitors in the developing barrel cortex. Nat Neurosci. cortical axons from the deep tier of terminations into layer IV of develop‑ 2012;15:1192–4. ing mouse barrel cortex. J Neurosci. 1993;13:5365–82. 20. Mitrofanis J, Guillery RW. New views of the thalamic reticular nucleus in 2. Balaram P, Hackett TA, Kaas JH. VGLUT1 mRNA and protein expression in the adult and the developing brain. Trends Neurosci. 1993;16:240–5. the visual system of prosimian galagos (Otolemur garnetti). Eye Brain. 21. Nagasunder AC, Kinney HC, Blüml S, Tavaré CJ, Rosser T, Gilles FH, Nelson 2011;2011:81–98. MD, Panigrahy A. Abnormal microstructure of the atrophic thalamus in 3. Deng W. Neurobiology of injury to the developing brain. Nat Rev Neurol. preterm survivors with periventricular leukomalacia. AJNR Am J Neurora‑ 2010;6(6):328–36. diol. 2011;32(1):185–91. 4. Deng W, Pleasure J, Pleasure D. Progress in periventricular leukomalacia. 22. Regher W, Stevens C. Physiology of synaptic transmission and short‑term Arch Neurol. 2008;65:1291–5. plasticity. Baltimore: The John Hopkins University Press; 2001. 5. Fremeau RT Jr, Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm‑ 23. Shen Y, Liu XB, Pleasure DE, Deng W. Axon‑ glia synapses are highly Mathisen J, Chaudhry FA, Nicoll RA, Edwards RH. Vesicular glutamate vulnerable to white matter injury in the developing brain. J Neurosci Res. transporters 1 and 2 target to functionally distinct synaptic release sites. 2012;90:105–21. Science. 2004;304:1815–9. 24. Shen Y, Plane JM, Deng W. Mouse models of periventricular leukoma‑ 6. Fujiyama F, Furuta T, Kaneko T. Immunocytochemical localization of lacia. J Vis Exp. 2010;(39). pii: 1951. http://www.jove.com/index/Details. candidates for vesicular glutamate transporters in the rat cerebral cortex. stp?ID=1951, doi: 10.3791/1951. PubMed ID: 20485263. J Comp Neurol. 2001;435:379–87. 25. Volpe JJ. Brain injury in premature infants: a complex amalgam 7. Grant E, Hoerder‑Suabedissen A, Molnar Z. Development of the cortico ‑ of destructive and developmental disturbances. Lancet Neurol. thalamic projections. Front Neurosci. 2012;6:53. 2009;8(1):110–24. 8. Graziano A, Jones EG. Widespread thalamic terminations of fibers arising in the superficial medullary dorsal horn of monkeys and their relation to calbindin immunoreactivity. J Neurosci. 2004;24:248–56. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Neuroscience Springer Journals

The vulnerability of thalamocortical circuitry to hypoxic-ischemic injury in a mouse model of periventricular leukomalacia

Download PDF
9 pages

Loading next page...
 
/lp/springer-journals/the-vulnerability-of-thalamocortical-circuitry-to-hypoxic-ischemic-0qJ4XvVsPT

References (29)

Publisher
Springer Journals
Copyright
Copyright © 2016 by Liu et al.
Subject
Biomedicine; Neurosciences; Neurobiology; Animal Models
eISSN
1471-2202
DOI
10.1186/s12868-015-0237-4
pmid
26733225
Publisher site
See Article on Publisher Site

Abstract

Background: Periventricular leukomalacia (PVL) is the leading cause of neurological disabilities including motor and cognitive deficits in premature infants. Periventricular leukomalacia is characterized by damage to the white matter in the immature brain, but the mechanisms by which damage to immature white matter results in widespread deficits of cognitive and motor function are unclear. The thalamocortical system is crucial for human consciousness and cog‑ nitive functions, and impaired development of the cortico‑ thalamic projections in the neonatal period is implicated to contribute importantly to abnormalities of cognitive function in children with PVL. Results: In this study, using a mouse model of PVL, we sought to test the hypothesis that PVL‑ like injury affects the different components of the thalamocortical circuitry that can be defined by vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2), both of which are required for glutamatergic synaptic transmission in the central nervous system. We combined immunocytochemistry and immuno‑ electron microscopy to investigate changes in cortico‑ thalamic synapses which were specifically identified by vGluT1 immunolabeling. We found that a drastic reduction in the density of vGluT1 labeled profiles in the somatosensory thalamus, with a reduction of 72–74 % in ventroposterior ( VP) nucleus and a reduction of 42–82 % in thalamic reticular nucleus (RTN) in the ipsilateral side of PVL mice. We fur‑ ther examined these terminals at the electron microscopic level and revealed onefold–twofold decrease in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. The present study provides anatomical and ultrastructural evidence to elucidate the cellular mechanisms underlying alteration of thalamic circuitry in a mouse model of PVL, and reveals that PVL‑ like injury has a direct impact on the corticothalamic projection system. Conclusions: Our findings provide the first set of evidence showing that the thalamocortical circuitry is affected and vulnerable in PVL mice, supporting a working model in which vGluT1 defined corticothalamic synapses are altered in PVL mice, and vGluT2 defined thalamocortical synapses are associated with such changes, leading to the compro ‑ mised thalamocortical circuitry in the PVL mice. Our study demonstrates that the thalamocortical circuitry is highly vulnerable to hypoxia–ischemia in the PVL model, thus identifying a novel target site in PVL pathology. Keywords: Prematurity, Periventricular leukomalacia, White matter injury, Oligodendrocyte, Thalamocortical circuitry, Cognitive impairment cause of subsequent neurological disability including Background spastic motor deficits (cerebral palsy) and cognitive Periventricular leukomalacia (PVL) is the predominant impairments. Nearly 90  % of the 13–15 million prema- brain pathology in premature infants and is the leading ture infants born worldwide every year survive beyond infancy, in which ≈5–10 % of the survivors develop cere- *Correspondence: wbdeng@ucdavis.edu bral palsy, and 40–50 % develop cognitive and behavioral Department of Biochemistry and Molecular Medicine, School deficits [ 3, 25]. Periventricular leukomalacia is tradition- of Medicine, University of California, Davis, 2425 Stockton Blvd., Room ally classified as a white matter disorder. However, white 653, Sacramento, CA 95817, USA Full list of author information is available at the end of the article © 2016 Liu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. BMC Neurosci (2016) 17:2 Page 2 of 9 matter damage underlying PVL is now recognized as A recent study further confirmed that these vGluT2 the major component of a more generalized injury to synapses also target NG2 cells in layer III-IV and layer the cerebrum that includes neuronal and axonal injury, VI [19] that contains pyramidal cell projections as cor- and is renamed “encephalopathy of prematurity” [25]. ticothalamic inputs to the somatosensory thalamus. Emerging neuropathological and neuroimaging studies In the present study, we sought to test the hypothesis demonstrate that PVL is associated with injury to many that PVL-like injury first affects the vGluT2-containing cortical and subcortical regions, including thalamus, thalamocortical projection system that in turn pro- cerebral cortex, hippocampus and basal ganglia in vari- foundly compromises vGluT1 containing corticotha- able combinations [4, 11, 21, 25]. The widespread effect lamic projection system and thus alters vGluT1 defined of the disorder may account for cognitive impairments corticothalamic synapses in the thalamus. We applied and intellectual deficits in preterm survivors with PVL. vGluT1 immunocytochemistry and electron micros- In particular, white matter injury impacts on axon fib - copy (EM) to analyze the contralateral (control) and ers ascending and descending from cerebral cortex and ipsilateral (injured) side of the brains of PVL mice. We thalamus and the reciprocal thalamo-cortical circuitry found that the density of vGluT1 immunolabeled termi- that is crucial for generating and maintaining awaken- nals was significantly decreased in the somatosensory ing-sleeping cycle of high mammals and thalamocortical thalamus of the ipsilateral injured side. Furthermore, oscillations that are essential for human consciousness at the EM level, these vGluT1 labeled corticothalamic and cognitive function. terminals were drastically altered in their sizes and syn- It has been widely accepted that thalamocortical con- aptic structures. These findings provide the first set of nections are formed perinatally in the mouse, and the evidence showing that the thalamocortical circuitry synaptic remodeling and circuitry refinement start is affected and vulnerable in PVL mice, supporting a around P7 and continue in the second postnatal week working model in which vGluT1 defined corticotha - [1, 7, 20]. The functional thalamocortical circuitry is lamic synapses are selectively altered in PVL mice, and established around P15 [12]. The second postnatal week vGluT2 defined thalamocortical synapses are associated (P7–P15) is critical for establishing thalamic oscillations with such changes, leading to the compromised thalam- that are crucial for the maturation of the thalamocorti- ocortical circuitry in the PVL mice. cal circuitry. Using a mouse model of PVL [23, 24] we investigate whether PVL induction affects the establish - Methods ment of the thalamocortical circuitry and whether syn- Animals aptic connections in the thalamocortical circuitry are C57B/6 mice (The Jackson Laboratories, Bar Harbor, altered following PVL induction. White matter injury ME) at postnatal day 6 (P6) were used to characterize in PVL may affect a significant portion of glutamatergic neuropathological features under different treatments. axon terminals connecting the somatosensory thalamus NG2-DsRed transgenic mice [The Jackson Laboratories, and the cerebral cortex. Defects in these axon terminals stock#: Tg(Cspg4-DsRed.T1)1Akik/J] were used to study can be identified at cellular and ultrastructural level the identity of postsynaptic profiles in cerebral white using specific glutamatergic synaptic markers, such as matter. Animal research was approved by the University vGluT1 and vGluT2, which have been used to define of California-Davis Committee on Animal Research. All specific thalamo-cortical systems [9 , 13]. Gene expres- procedures were carried out by following the guidelines sion and immunocytochemistry studies have confirmed set by the institutional animal care and use committee. that vGluT1 is primarily expressed in layer III and layer V-VI pyramidal cells and vGluT2 is highly expressed in Unilateral carotid ligation (UCL) plus hypoxia thalamic relay cells and this complementary expression Twenty animals underwent a permanent ligation of the is reflected more clearly in the protein expressions that right common carotid artery under ice anesthesia, fol- mainly occur in the axon terminals. It has been previ- lowed by 1-hour recovery on a thermal blanket, main- ously shown that vGluT1 specifically labels the cortico - taining body temperature at 33–34 °C and with a 1-hour thalamic synapses, whereas vGluT2 specifically defines feeding period with the dam. Animals were then placed the thalamocortical synapses [9, 13]. We demonstrated in a sealed chamber infused with nitrogen to maintain that the synaptic contacts between vGluT2-labeled a level of 6.0  % O . Different durations of hypoxia were thalamocortical axon terminals and NG2  +  oligoden- applied. The body temperature of mice was maintained at droglial progenitor cells (OPCs) in the developing white 33–34 °C by leaving animals on thermal blanket through- matter were selectively affected and caused the post - out hypoxia. After a 1-hour period of recovery, mice were synaptic profiles of NG2 progenitor cells to shrink [23]. returned to their dam. Liu et al. BMC Neurosci (2016) 17:2 Page 3 of 9 Immuno‑electron microscopy rectangular region approximately 100  μm were sub- Ten P10 mice that were subjected to UCL/hypoxia at jected to particle analysis. The particle numbers in each P6 were perfused with 4  % paraformaldehyde plus 0.2  % region were obtained, for each case, mean numbers of glutaraldehyde in 0.1  M phosphate buffer saline (PB, profiles  ±  standard deviation/100  μm was compared in and brains were cut coronally with a vibratome (Leica) each pair (ipsilateral and contralateral) in VP or RTN, at 60  μm. Sections were stained for vGluT1 and vGluT2 respectively. antibodies (Millipore, Billerica, MA) using the immun- operoxidase ABC method. A total three cases showing Quantification by electron microscopy dense vGlut1 immunostaining were selected for electron Electron microscope of vGluT1 immunoperoxidase microscopic study. The stained sections were processed labeled terminals or synapses were taken at X10,000. for EM as described previously [9]. The sections from Electron microscope images were obtained from the the ipsilateral (injury) side and the contralateral (control) contralateral and injured side of VP and RTN. In each side were first examined in the light microscope at 10X side, at least 10–15 images were taken. Electron micro- and 20X, the forebrain containing the cerebral cortex and scope images were converted to TIF files and imported to thalamus was identified and photographed, ventroposte - Image J for measuring. We used the “Analyze” function to rior nucleus (VP) and thalamic reticular nucleus (RTN) measure the area (in μm ) and perimeter (in μm) of each regions (approximately 2  ×  0.5  mm) were cut under a vGluT1 labeled terminals. Given the irregular shape of dissecting microscope and glued to blank resin blocks. the labeled profiles and the fact that the area of the pro - Thin sections were cut at 70 nm using an ultramicrotome file represents closely its size, we therefore used the area (Leica Ultracut) and collected on single-slot (2 ×  1  mm) for quantification. At least two labeled terminals were copper grids and stained with uranyl acetate and lead cit- identified from each image and measured. The mean area rate, and examined in a Philips CM120 EM at 80 kV. The size (μm ) ± standard deviation (SD) from the contralat- regions were carefully scanned to exclude the areas con- eral or ipsilateral side was compared. taining fiber tract in the internal capsule, and the orienta - tion of the region was guided by light microscopic images Results taken before EM processing. Much effort was made to Decrease in number of vGluT1 immunolabeled profiles ensure that the regions were taken from the somatosen- in the somatosensory thalamus of PVL mice sory thalamus. Images were taken by a 2  ×  2  K CCD At the light microscopic level, vGluT1 immunolabeled camera (Gatan, Inc., Pleasanton, CA) and processed by profiles appeared to be thin presynaptic terminals and using the software provided by Gatan, Inc (DigitalMi- small boutons densely distributed in the dorsal thalamus crograph). Images were composed using Photoshop CS including VP and RTN of the contralateral side (Fig.  1). (Adobe System, Mountain View, CA). In the ipsilateral side, a drastic decrease in the number of vGluT1 immunolabeled profiles was noted in some Quantification by light microscopy regions of the dorsal thalamic nuclei including VP and vGluT1 immunoperoxidase labeled sections embed- RTN (Fig. 1). Compared to the contralateral side thalamus ded with Araldite were used for the quantitative analy- which contains dense clusters of vGluT1 labeled profiles, sis. For each case (the contralateral and ipsilateral pair), the labeling in the ipsilateral side was more diffuse and at least one section from each side was examined under became less intense in clustering. Instead, some non-spe- light microscope using X20 and X40 objectives. Light cific lightly labeled somata were uniformly distributed on microscopic images (X40) showing dense vGluT1 labeled the background, but they were easily differentiated from boutons in thalamus (VP and RTN) were captured using the labeled terminals by sizes. A quantitative analysis was a Zeiss imaging system with a CCD camera (Zeiss, made to access the change in number of vGluT1 immu- Thornwood, NY, USA) and the images were processed nolabeled profiles in 100 μm area of VP and RTN of the in Photoshop CS to adjust the brightness and contrast ipsilateral side of the somatosensory thalamus (Fig.  2). and convert to gray scale and imported to Image J (NIH, A total of three independent cases were analyzed and Bethesda, MD, USA) as TIF files. The “analyze particles” quantified, and a drastic change in vGluT1 labeling den - function was used to count the labeled profiles. Assum - sity was found in both VP and RTN of the ipsilateral side. ing the diameters of the labeled corticothalamic termi- In VP, in case 1, the density of labeling was decreased nals were from 0.3 to 0.6 μm [17], accordingly, the profile from 7.69 ± 1.61 profiles per 100 μm (contralateral side) 2 2 sizes (areas) in a range between 0.07 and 0.7  μm were to 2.0  ±  1.17 profiles per 100  μm (ipsilateral side) and included in counting. This range would cover the sizes of approximately 74  % or 3.8-fold reduction, p  <  0.005; in most vGluT1 labeled profiles in thalamus. In each sec - case 2, it was decreased from 2.59  ±  0.93 profiles per tion, at least two regions (up to five regions) and each 100  μm (contralateral side) to 0.73  ±  0.47 profiles per Liu et al. BMC Neurosci (2016) 17:2 Page 4 of 9 Fig. 1 Light microscopic images taken from Araldite embedded vibratome sections showing immunolabeling of vGluT1 in the somatosensory thalamus [ventroposterior ( VP) nucleus and thalamic reticular nucleus (RTN)] of ipsilateral (a) and contralateral (b) sides of the brain in a mouse model of PVL. c is a high magnification image taken from VP showing the area lacking vGluT1 labeling in the ipsilateral side. d is a high magnifica‑ tion image taken from VP of the contralateral side showing clusters of vGluT1 labeled profiles among neuropils. e is a much higher power image showing the labeled profiles including presynaptic boutons. f Light microscopic image showing the lack of vGluT1 labeling in the RTN region of the ipsilateral side; note some nonspecifically labeled somata in the region. g Light microscopic image showing vGluT1 labeled small boutons and profiles in the RTN of the contralateral side. h A high power light microscopic image showing clusters of vGluT1 labeled profiles in the RTN 2 2 100  μm (ipsilateral side) approximately 72 % or 3.5-fold (contralateral side) to 0.46  ±  0.15 profiles per 300  μm reduction, p  <  0.0007; in case 3, it was decreased from (ipsilateral side) approximately 82  % or fivefold reduc - 3.44  ±  2.58 profiles per 100  μm (contralateral side) to tion, p < 0.0006 (Fig. 2). Overall, the decreases of vGluT1 0.94 ± 0.59 profiles per 100 μm (ipsilateral side) approx- labeling density in three cases were consistent in VP and imately 72  % or threefold reduction, p  <  0.00216. Con- RTN and the differences between contralateral and the sistent with changes in VP, in RTN, in case 1, the density ipsilateral sides were highly significant. of labeling was decreased from 2.90  ±  1.17 profiles per 100  μm (contralateral side) to 1.63  ±  0.51 profiles per Alteration in ultrastructure of vGluT1 labeled 100  μm (ipsilateral side) approximately 44  % or 1.8- corticothalamic synapses in the somatosensory thalamus fold reduction, p < 0.03; in case 2, it was decreased from of PVL mice 2.24  ±  0.39 profiles per 100  μm (contralateral side) to The drastic reduction in number of vGluT1 labeled ter - 1.26 ± 0.56 profiles per 100 μm (ipsilateral side) approx- minals in the somatosensory thalamus of PVL mice imately 43 % or 1.8-fold reduction, p < 0.0129; in case 3, strongly indicated that these terminals and their synapses it was decreased from 2.64  ±  0.87 profiles per 100  μm may undergo major changes in their ultrastructure in Liu et al. BMC Neurosci (2016) 17:2 Page 5 of 9 terminals in VP and RTN as reported previously [17]: these terminals were about 0.3–0.6 μm in diameter, con- taining few mitochondria, filled with round or oval shape synaptic vesicles and they usually form asymmetrical synapses associated with prominent postsynaptic densi- ties on dendritic profiles. Interestingly, these terminals usually contact small and presumably distal dendrites of relay cells in VP and contact much wide range sizes of dendritic profiles in RTN [15, 17] (Fig.  3). In contrast to vGluT1 labeled terminals in the contralateral side, the labeled terminals in the ipsilateral side were much smaller in their sizes and they showed some structural alterations including less defined membranes and con - taining fewer synaptic vesicles and were lack of promi- nent postsynaptic densities (Fig.  3). To quantitatively access the change in ultrastructure of vGluT1 labeled terminals in the ipsilateral side, a total of 15 images each containing at least two labeled terminals from either VP or RTN of the contralateral and ipsilateral side of PVL mice were subjected to quantitative analysis. In the con- tralateral VP, the mean area size of vGluT1 labeled ter- minals was 0.58  ±  0.23  μm , in the ipsilateral side, the mean area size was 0.27 ± 0.09 μm , there was more than twofold decreased in the size of the terminals, the change was highly significant (p  <  0.00011). In the contralateral RTN, the mean area size was 0.39 ± 0.19 μm , in the ipsi- lateral side, the mean size of vGluT1 labeled terminals was 0.24 ± 0.12 μm , there was more than 1.5-fold reduc- tion in the size of these terminals and the change was sig- nificant (p < 0.0291) (Fig.  4). We also observed a change in the length of postsynaptic density of vGluT1 labeled synapses in the ipsilateral side compared to the contralat- eral side. Discussion In the present study, we combined immunocytochem- istry and EM to investigate the changes associated with corticothalamic synapses which were specifically iden - tified by vGluT1 labeling in a mouse model of PVL. We found that a drastic reduction in number of vGluT1 labeled profiles in the somatosensory thalamus (VP: Fig. 2 Quantitative analysis of the density (number of vGluT1 labeled profiles per 100 μm ) of vGluT1 labeled profiles in the contralateral a reduction of 72–74  % or about threefold–fourfold; and ipsilateral side VP (blue color) and RTN (red color) from three cases RTN: a reduction of 42–82  % or 1.8-fold–fivefold) in of PVL mice. In the contralateral side, the density of vGluT1 labeling is the ipsilateral side of PVL mice. We further examined higher in VP than RTN in three cases. In the ipsilateral side, the density these terminals at the EM level and revealed onefold– is decreased significantly in VP (average 3.8‑fold decrease) and RTN twofold shrinkage in the sizes of vGluT1 labeled cor- (onefold–fivefold decrease) compared to the contralateral side, *p < 0.05; **p < 0.01 ticothalamic terminals in VP and RTN. Based on the experimental observations from the present study and our previous study [23] and the recent finding from PVL mice. We further carried out electron microscopic others [19], we proposed a working model to elucidate analysis to resolve this issue. At the EM level, in con- the underlying cellular mechanisms may account for tralateral side, vGluT1 immunolabeled terminals dis- the alteration of thalamic circuitry in the mouse model played typical ultrastructural features of corticothalamic of PVL. Liu et al. BMC Neurosci (2016) 17:2 Page 6 of 9 Fig. 3 Electron micrographs showing typical vGluT1 immunoperoxidase labeled terminals in the contralateral side VP (a) and RTN (c) and ipsilateral side VP (b) and RTN (d). In the contralateral side, vGluT1 labeled terminals (T) contain densely packed vesicles and form clear asymmetrical synapses (indicated by arrows) with postsynaptic dendrites (D) (note the prominent postsynaptic densities associated with the synapses). In ipsilateral side, the labeled terminals have less defined irregular shapes and form less clear synaptic contacts with postsynaptic dendrites (D). Scale bar = 0.5 μm A working model of the vulnerability of corticothalamic on layer IV cells as well [19]. Layer III-IV cells and layer synapses in the PVL mice VI pyramidal cells which express vGluT1 receive TC syn- In this working model  as shown in Fig.  5 , at the normal aptic inputs and send out their axons through white mat- condition, as illustrated in the contralateral side, thalam- ter and form synapses on RTN cells and TC cells. GAB ocortical (TC) relay cells which express vGluT2 [9] (TC Aergic cells in RTN project back to TC cells, serving as vGluT2) in the thalamic nucleus VP send out their axons a negative feedback loop. Note that axon bundles from through white matter, and they also send branches to syn- vGluT1 containing layer VI corticothalamic cells are mye- apse with GABAergic RTN cells. During postnatal devel- linated.In the ipsilateral side of PVL mice, vGluT2 positive opment, these TC cell axons form synaptic contacts on thalamocortical relay cells are profoundly compromised, NG2 cells in the white matter [23], and they also project their terminals that synapse on NG2 cells in the white to other NG2 cells in layer IV and layer VI and synapse matter undergo structural, biochemical and functional Liu et al. BMC Neurosci (2016) 17:2 Page 7 of 9 significant exceptions, do not co-localize in the same synaptic terminals [6, 13]. It has been suggested that vGluT1 is primarily found at synapses characterized by low-release probability and a capacity for long-term potentiation (LTP), whereas vGluT2 is primarily found at synapses characterized by high-release probability and a capacity for long-term depression (LTD) [5]. Our recent study also confirmed previous findings that the majority of thalamocortical terminals contain vGluT2 and the cor- tico-thalamic terminals derived from layer VI pyramidal cells contain vGluT1 [9, 14, 19]. In somatosensory thala- mus, another source of similar terminals to corticotha- lamic synapses is derived from brainstem which mainly cholinergic and monoaminergic, but a large amount of these terminals do not form classical synapses [12, 16], therefore, vGluT1 labeled terminals here should be con- sidered exclusively corticothalamic. In the present study, a quantitative analysis on vGluT1 immunostaining at the light microscopic level revealed a consistent reduc- tion in labeled profiles in the somatosensory thalamus of the ipsilateral side of PVL mice. Although the degree of changes in three cases shows some variabilities, the trend is consistent. Such fluctuation could be related to the brain regions impacted by PVL. The reduction in labeled profiles in PVL mice was further confirmed by Fig. 4 Quantitative analysis of the size (μm ) of vGluT1 immunola‑ EM studies, showing decreased sizes of vGluT1 labeled beled axon terminals in the contralateral and ipsilateral side of VP (a) terminals, which exhibited typical corticothalamic syn- and RTN (b) of PVL mice. In VP, twofold reduction in the size of the aptic features in previous studies [12, 17]. The reduction labeled terminals is observed in the ipsilateral side (*p < 0.00011). In in vGluT1 labeled terminal numbers could result from a RTN, 1.6‑fold reduction in the size of the labeled terminals is found in the ipsilateral side (*p < 0.0291) down-regulation of vGluT1 protein due to the changes in layer VI pyramidal cells and also the alterations in axons and their terminals because of de-myelination. It is obvi- ous that corticothalamic axon terminals were severely changes, and vGluT2 expression is altered, resulting in the damaged, given the drastic ultrastructural changes. This defects in both NG2 progenitor cell function and myelina- target specific alteration can be reliably detected using tion of corticothalamic axons. Thalamocortical synapses vGluT1 labeling. Further investigation to explore the on NG2 cells in layer III-IV and layer VI are also affected, potential changes occur in cortical projection cells and ultimately leading to the change of vGluT1 expression in possible associated changes in myelinated axons will shed layer III-IV and layer VI cells, down-regulation of vGluT1 light on the underlying thalamocortical circuitry which is expression in corticothalamic terminals, and malfunction highly implicated in PVL and other newborn neurologi- of the synaptic transmission. cal injuries or related diseases. vGluT1 as a reliable synaptic marker for corticothalamic Interaction of vGluT1 and vGluT2 in the thalamocortical synapses circuitry in PVL Synaptic plasticity depends, at least in part, on presyn- vGluT1 and vGluT2 are expressed in distinctive neuronal aptic mechanisms that have been associated with the subtypes and involved in different functional systems. probability of neurotransmitter release [22]. The amount vGluT2 is primarily restricted to projections between of glutamate contained within synaptic vesicles and and within subcortical areas, as well as thalamocortical available for release is regulated by vesicular glutamate projections, while vGluT1 is reserved for intercortical transporters (vGluTs) located on the membrane of the and corticothalamic projections [2, 8, 10, 13, 14, 23]. The vesicles. Two of the three known vGluT isoforms, vGluT1 pathology in corticothalamic terminals in PVL suggests and vGluT2, are relatively abundant throughout the cen- that different vGluT proteins may be engaged in specific tral nervous system (CNS), where they are expressed by functional pathways. How is the corticothalamic pathway specific glutamatergic neuronal populations and, with Liu et al. BMC Neurosci (2016) 17:2 Page 8 of 9 Fig. 5 A working model illustrating the thalamocortical circuitry and the affected targets in the contralateral versus ipsilateral side of the brain in a mouse model of PVL. NG2 cells are represented in the white matter and in the cerebral cortex. The synaptic contacts between vGluT2 labeled ter‑ minals from thalamocortical ( TC) cells (termed “TC vGluT2” cells) are formed in the developing white matter and the cerebral cortex. These synapses are altered in PVL mice, which may in turn affect the myelination of layer VI pyramidal (P) cells (termed “P vGluT1” cells) that may then change vGluT1 expression in their axon terminals in the VP and RTN (indicated by red crosses) affected in PVL mice? We need to consider the thalam - differentiation and migration of these NG2 cells and ocortical circuitry as a whole in order to identify which ultimately affect the myelination of functionally organ - may be altered at PVL condition. Our previous study [23] ized axon bundles in the white matter and cerebral cortex demonstrated that the postsynaptic targets of vGluT2 and result in malfunction of the thalamocortical circuitry synapses were affected in the white matter of the ipsilat - [18]. Furthermore, the impact of PVL injury may also eral side of PVL mice, the shrinkage of the postsynaptic affect the layer VI corticothalamic projecting pyrami - profiles may be derived from NG2-expressing oligoden - dal cells which express vGluT1 and thus down regulate drocyte precursor cells (OPC). A recent study [19] using the protein expression in the corticothalamic terminals elegant transgenic techniques combined electrophysi- and further alter the synaptic transmissions and their ological recording also demonstrated that vGluT2 labeled ultrastructure, the consequence of the impact would be thalamocortical synapses specifically target NG2 cells in the de-synchronization of the whole circuitry. The pro - layer IV and layer VI and other neurons in these layers posed working model presents a new pathway for elu- and provided strong evidence indicating that thalamo- cidating the cellular mechanisms responsible for PVL cortical synapses are vGluT2 specific and are modified injury. vGluT proteins are involved in regulating gluta- by somatosensory afferents [18]. Based on these findings, mate release in presynaptic terminals, the impact of the it is anticipated that the injury in PVL may firstly strike protein defection has a profound effect on glutamater - the thalamocortical relay cells which receive strong sen- gic transmission and thus alters the neuronal circuitry, sory inputs and form synaptic contacts with NG2 cells such as thalamocortical pathway which control the con- in the white matter and other cortical layers during early ciseness level and cognitive functions of the animals. postnatal development (P2–P10). The alteration in syn - Future investigation to further understand the underly- aptic transmission between vGluT2 expressing thalamo- ing pathology of PVL will be crucial for developing new cortical terminals and NG2 cells may compromise the treatments for this devastating human disorder. Liu et al. BMC Neurosci (2016) 17:2 Page 9 of 9 Authors’ contributions 9. Graziano A, Liu XB, Murray KD, Jones EG. Vesicular glutamate transporters XL and YS carried out all the experiments. XL, DEP and WD designed experi‑ define two sets of glutamatergic afferents to the somatosensory thala‑ ments. XL and WD wrote the paper. WD directed the study. All authors read mus and two thalamocortical projections in the mouse. J Comp Neurol. and approved the final manuscript. 2008;507:1258–76. 10. Hackett TA, de la Mothe LA. Regional and laminar distribution of the Author details vesicular glutamate transporter, VGluT2, in the macaque monkey audi‑ Department of Biochemistry and Molecular Medicine, School of Medicine, tory cortex. J Chem Neuroanat. 2009;38:106–16. University of California, Davis, Sacramento, CA 95817, USA. Center for Neu‑ 11. Inder T, Neil J, Kroenke C, Dieni S, Yoder B, Rees S. Investigation of roscience, School of Medicine, University of California, Davis, Sacramento, CA cerebral development and injury in the prematurely born primate 95817, USA. Institute for Pediatric Regenerative Medicine, School of Medi‑ by magnetic resonance imaging and histopathology. Dev Neurosci. cine, University of California, Davis, CA 95817, USA. Medical College, Hubei 2005;27(2–4):100–11. University of Arts and Science, Xiangyang, Hubei, China. Department of Bio‑ 12. Jones EG. The Thalamus. 2nd ed. Cambridge (UK): Cambridge University chemistry and Molecular Medicine, School of Medicine, University of Califor‑ Press; 2007. nia, Davis, 2425 Stockton Blvd., Room 653, Sacramento, CA 95817, USA. 13. Kaneko T, Fujiyama F. Complementary distribution of vesicular glutamate transporters in the central nervous system. Neurosci Res. 2002;42:243–50. Acknowledgements 14. Kaneko T, Fujiyama F, Hioki H. Immunohistochemical localization of The work was supported by Grants from the National Institutes of Health candidates for vesicular glutamate transporters in the rat brain. J Comp (R01NS059043), National Multiple Sclerosis Society, Feldstein Medical Founda‑ Neurol. 2002;444:39–62. tion and Shriners Hospitals for Children to W.D. 15. Liu XB, Honda CN, Jones EG. Distribution of four types of synapse on physiologically identified relay neurons in the ventral posterior thalamic Competing interests nucleus of the cat. J Comp Neurol. 1995;352:69–91. The authors declare that they have no competing interests. 16. Liu XB, Jones EG. The fine structure of serotonin and tyrosine hydroxylase immunoreactive terminals in the ventral posterior thalamic nucleus of cat Received: 15 July 2015 Accepted: 23 December 2015 and monkey. Exp Brain Res. 1991;85(3):507–18. 17. Liu XB, Jones EG. Predominance of corticothalamic synaptic inputs to thalamic reticular nucleus neurons in the rat. J Comp Neurol. 1999;414:67–79. 18. Mangin JM, Gallo V. The curious case of NG2 cells: transient trend or game changer? ASN Neuro. 2011;3:e00052. References 19. Mangin JM, Li P, Scafidi J, Gallo V. Experience ‑ dependent regulation 1. Agmon A, Yang LT, O’Dowd DK, Jones EG. Organized growth of thalamo‑ of NG2 progenitors in the developing barrel cortex. Nat Neurosci. cortical axons from the deep tier of terminations into layer IV of develop‑ 2012;15:1192–4. ing mouse barrel cortex. J Neurosci. 1993;13:5365–82. 20. Mitrofanis J, Guillery RW. New views of the thalamic reticular nucleus in 2. Balaram P, Hackett TA, Kaas JH. VGLUT1 mRNA and protein expression in the adult and the developing brain. Trends Neurosci. 1993;16:240–5. the visual system of prosimian galagos (Otolemur garnetti). Eye Brain. 21. Nagasunder AC, Kinney HC, Blüml S, Tavaré CJ, Rosser T, Gilles FH, Nelson 2011;2011:81–98. MD, Panigrahy A. Abnormal microstructure of the atrophic thalamus in 3. Deng W. Neurobiology of injury to the developing brain. Nat Rev Neurol. preterm survivors with periventricular leukomalacia. AJNR Am J Neurora‑ 2010;6(6):328–36. diol. 2011;32(1):185–91. 4. Deng W, Pleasure J, Pleasure D. Progress in periventricular leukomalacia. 22. Regher W, Stevens C. Physiology of synaptic transmission and short‑term Arch Neurol. 2008;65:1291–5. plasticity. Baltimore: The John Hopkins University Press; 2001. 5. Fremeau RT Jr, Kam K, Qureshi T, Johnson J, Copenhagen DR, Storm‑ 23. Shen Y, Liu XB, Pleasure DE, Deng W. Axon‑ glia synapses are highly Mathisen J, Chaudhry FA, Nicoll RA, Edwards RH. Vesicular glutamate vulnerable to white matter injury in the developing brain. J Neurosci Res. transporters 1 and 2 target to functionally distinct synaptic release sites. 2012;90:105–21. Science. 2004;304:1815–9. 24. Shen Y, Plane JM, Deng W. Mouse models of periventricular leukoma‑ 6. Fujiyama F, Furuta T, Kaneko T. Immunocytochemical localization of lacia. J Vis Exp. 2010;(39). pii: 1951. http://www.jove.com/index/Details. candidates for vesicular glutamate transporters in the rat cerebral cortex. stp?ID=1951, doi: 10.3791/1951. PubMed ID: 20485263. J Comp Neurol. 2001;435:379–87. 25. Volpe JJ. Brain injury in premature infants: a complex amalgam 7. Grant E, Hoerder‑Suabedissen A, Molnar Z. Development of the cortico ‑ of destructive and developmental disturbances. Lancet Neurol. thalamic projections. Front Neurosci. 2012;6:53. 2009;8(1):110–24. 8. Graziano A, Jones EG. Widespread thalamic terminations of fibers arising in the superficial medullary dorsal horn of monkeys and their relation to calbindin immunoreactivity. J Neurosci. 2004;24:248–56. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

Journal

BMC Neuroscience – Springer Journals

Published: Jan 5, 2016

There are no references for this article.