In the present study, we combined immunocytochemistry and EM to investigate the changes associated with corticothalamic synapses which were specifically identified 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: a reduction of 72–74 % or about threefold–fourfold; RTN: a reduction of 42–82 % or 1.8-fold–fivefold) in the ipsilateral side of PVL mice. We further examined these terminals at the EM level and revealed onefold–twofold shrinkage in the sizes of vGluT1 labeled corticothalamic terminals in VP and RTN. Based on the experimental observations from the present study and our previous study [23] and the recent finding from others [19], we proposed a working model to elucidate the underlying cellular mechanisms may account for the alteration of thalamic circuitry in the mouse model of PVL.
A working model of the vulnerability of corticothalamic synapses in the PVL mice
In this working model as shown in Fig. 5 , at the normal condition, as illustrated in the contralateral side, thalamocortical (TC) relay cells which express vGluT2 [9] (TC vGluT2) in the thalamic nucleus VP send out their axons through white matter, and they also send branches to synapse with GABAergic RTN cells. During postnatal development, these TC cell axons form synaptic contacts on NG2 cells in the white matter [23], and they also project to other NG2 cells in layer IV and layer VI and synapse on layer IV cells as well [19]. Layer III-IV cells and layer VI pyramidal cells which express vGluT1 receive TC synaptic inputs and send out their axons through white matter and form synapses on RTN cells and TC cells. GAB Aergic cells in RTN project back to TC cells, serving as a negative feedback loop. Note that axon bundles from vGluT1 containing layer VI corticothalamic cells are myelinated.In the ipsilateral side of PVL mice, vGluT2 positive thalamocortical relay cells are profoundly compromised, their terminals that synapse on NG2 cells in the white matter undergo structural, biochemical and functional changes, and vGluT2 expression is altered, resulting in the defects in both NG2 progenitor cell function and myelination of corticothalamic axons. Thalamocortical synapses on NG2 cells in layer III-IV and layer VI are also affected, ultimately leading to the change of vGluT1 expression in layer III-IV and layer VI cells, down-regulation of vGluT1 expression in corticothalamic terminals, and malfunction of the synaptic transmission.
vGluT1 as a reliable synaptic marker for corticothalamic synapses
Synaptic plasticity depends, at least in part, on presynaptic mechanisms that have been associated with the probability of neurotransmitter release [22]. The amount of glutamate contained within synaptic vesicles and available for release is regulated by vesicular glutamate transporters (vGluTs) located on the membrane of the vesicles. Two of the three known vGluT isoforms, vGluT1 and vGluT2, are relatively abundant throughout the central nervous system (CNS), where they are expressed by specific glutamatergic neuronal populations and, with 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 cortico-thalamic terminals derived from layer VI pyramidal cells contain vGluT1 [9, 14, 19]. In somatosensory thalamus, another source of similar terminals to corticothalamic 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 considered exclusively corticothalamic. In the present study, a quantitative analysis on vGluT1 immunostaining at the light microscopic level revealed a consistent reduction 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 EM studies, showing decreased sizes of vGluT1 labeled terminals, which exhibited typical corticothalamic synaptic features in previous studies [12, 17]. The reduction in vGluT1 labeled terminal numbers could result from a 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 obvious that corticothalamic axon terminals were severely damaged, given the drastic ultrastructural changes. This target specific alteration can be reliably detected using vGluT1 labeling. Further investigation to explore the potential changes occur in cortical projection cells and possible associated changes in myelinated axons will shed light on the underlying thalamocortical circuitry which is highly implicated in PVL and other newborn neurological injuries or related diseases.
Interaction of vGluT1 and vGluT2 in the thalamocortical circuitry in PVL
vGluT1 and vGluT2 are expressed in distinctive neuronal subtypes and involved in different functional systems. vGluT2 is primarily restricted to projections between and within subcortical areas, as well as thalamocortical projections, while vGluT1 is reserved for intercortical and corticothalamic projections [2, 8, 10, 13, 14, 23]. The pathology in corticothalamic terminals in PVL suggests that different vGluT proteins may be engaged in specific functional pathways. How is the corticothalamic pathway affected in PVL mice? We need to consider the thalamocortical circuitry as a whole in order to identify which may be altered at PVL condition. Our previous study [23] demonstrated that the postsynaptic targets of vGluT2 synapses were affected in the white matter of the ipsilateral side of PVL mice, the shrinkage of the postsynaptic profiles may be derived from NG2-expressing oligodendrocyte precursor cells (OPC). A recent study [19] using elegant transgenic techniques combined electrophysiological recording also demonstrated that vGluT2 labeled thalamocortical synapses specifically target NG2 cells in layer IV and layer VI and other neurons in these layers and provided strong evidence indicating that thalamocortical synapses are vGluT2 specific and are modified by somatosensory afferents [18]. Based on these findings, it is anticipated that the injury in PVL may firstly strike the thalamocortical relay cells which receive strong sensory inputs and form synaptic contacts with NG2 cells in the white matter and other cortical layers during early postnatal development (P2–P10). The alteration in synaptic transmission between vGluT2 expressing thalamocortical terminals and NG2 cells may compromise the differentiation and migration of these NG2 cells and ultimately affect the myelination of functionally organized axon bundles in the white matter and cerebral cortex and result in malfunction of the thalamocortical circuitry [18]. Furthermore, the impact of PVL injury may also affect the layer VI corticothalamic projecting pyramidal cells which express vGluT1 and thus down regulate the protein expression in the corticothalamic terminals and further alter the synaptic transmissions and their ultrastructure, the consequence of the impact would be the de-synchronization of the whole circuitry. The proposed working model presents a new pathway for elucidating the cellular mechanisms responsible for PVL injury. vGluT proteins are involved in regulating glutamate release in presynaptic terminals, the impact of the protein defection has a profound effect on glutamatergic transmission and thus alters the neuronal circuitry, such as thalamocortical pathway which control the conciseness level and cognitive functions of the animals. Future investigation to further understand the underlying pathology of PVL will be crucial for developing new treatments for this devastating human disorder.