The major findings of this study were as follows: 1) the combination of [18F]-UCB-H and [18F]-FDG measurements was a feasible approach to depict dynamic changes of structure and function following bilateral vestibular loss in the rat; 2) lesion-induced plasticity was not a uniform process, but involved complex structure-function relationships at various brain levels with different temporal scales; 3) a ‘form follows function’ type of plasticity was found in brainstem and cerebellar networks, which were most closely exposed to loss of vestibular input, while a ‘function follows form’ type of plasticity appeared in thalamic nuclei dedicated to multisensory processing and motor adaptation; 4) locomotor training had a mild effect on dynamic balance after BL, which was paralleled by changes in synaptic density and glucose metabolism in the thalamus.
[18F]-UCB-H/ [18F]-FDG dual tracer PET imaging – a novel method to study neuroplasticity
Advances in non-invasive measurements (such as MRI, PET, NIRS, EEG, MEG) have massively contributed to our understanding of adaptive brain plasticity in vivo following neuronal lesions. While MRI has an excellent spatial resolution, EEG and MEG have a better temporal resolution. MR techniques may provide information on the relation of structural changes in grey and white matter (VBM, DTI) 29–33, and functional activity of brain networks (e.g., rs-fMRI) during behavioural recovery processes after a neuronal lesion 34. However, the cellular mechanisms underpinning adaptive structural changes in grey and white matter are not completely understood yet 1.
Molecular imaging may be complementary in this respect, especially since novel tracers targeting synaptic vesicular protein 2A (SV2A such as [11C]-UCB-J or [18F]-UCB-H) are available to quantify synaptic density as a surrogate for axonal structure 24,35,36, which can be compared to ‘classical’ markers of neuronal activation, such as [18F]-FDG. Recently, it was shown that the [11C]-UCB-J signal was not affected by short-term changes in neuronal activity during an activation task 37,38. On the other hand, short-term dynamics in [18F]-UCB-H binding were depicted in a temporal lobe epilepsy model, which depended on the disease stage 39. In a mouse model of Alzheimer disease, therapeutic effects of a disease-modifying drug could be visualized and quantified by [18F]-UCB-H-PET imaging 40. Clinical studies in patients with epilepsy or dementia syndromes have shown that SV2A tracers can reliably detect a loss of synaptic density, which is associated with neuronal damage 41,42. However, to the best of our knowledge a dual tracer approach using serial [18F]-UCB-H/ [18F]-FDG measurements has not been applied to study time dynamics and spatial distribution of neuroplasticity induced by acute lesions. In this study, we strived to depict changes in synaptic density and regional glucose consumption longitudinally in an established sensory deprivation model in the rat. For this methodological purpose, subgroups with and without training were pooled to increase statistical robustness. We think that this approach is reasonable, because subgroups were comparable for most behavioural parameters (see below) and underwent an identical longitudinal imaging protocol. We were able to show dynamic changes of synaptic density across six consecutive imaging time points within 10 weeks, which appeared in biologically plausible networks in the brainstem, cerebellum, thalamus, multisensory cortex and motor-basal ganglia circuits. Most importantly, rCGM changes were found in similar regions, but with different temporal scales. The direct comparison of time courses and spatial distribution of [18F]-UCB-H and [18F]-FDG signals may potentially allow conclusions to be drawn about the mode of plasticity (‘form follows function’ or ‘function follows form’).
Differential modes of structural and functional plasticity following complete vestibular loss
The current study shows three major modes of structure-function coupling following complete vestibular loss, which appeared in different neural networks:
i) In brainstem-cerebellar networks, early-onset decrease in rCGM after BL was followed by reduced synaptic density 3 weeks later in the same brain regions (Fig. 3). A rapid drop in neuronal activity in the vestibular and cochlear nucleus is expected after BL, because these nuclei receive primary afferents from the inner ear and are consequently affected most by loss of signal input 43. Of note, rCGM also decreased in secondary hubs of vestibular and auditory processing (e.g., vestibular cerebellum, colliculus inferior) instantaneously after BL. Damage to inner ear inputs resulted in a delayed loss of synaptic density in the same regions (vestibular nuclei, vestibular cerebellum, colliculus inferior), which slowly increased until 9 weeks post BL. This observation can be interpreted as a persistent structural degeneration in brainstem-cerebellar networks following irreversible loss of bilateral vestibular function. Our data seem to be in partial contrast to a previous in vitro study that showed a loss of synaptic density of about 35% in the medial vestibular nucleus 1 week after unilateral vestibular neurectomy in the cat, which was followed by a synaptic reoccupation from 3 weeks to 5 months after the lesion 44. However, it has to be considered that static behavioural deficits after a unilateral vestibular lesion recover rapidly and completely due to central vestibular compensation, while a bilateral vestibular lesion induces symptoms that are more persistent and can in part not be fully compensated. It seems likely that the intact contralateral vestibular inputs contribute to synaptic reoccupation in unilateral vestibular lesions.
ii) In the thalamus, synaptic density started to increase bilaterally from 1 week after BL, which was succeeded by an rCGM increase 2 weeks later (Fig. 4). The thalamus is a well-known hub for integration of multisensory inputs from the vestibular, the visual, and the somatosensory system, as well as vestibular-motor interaction 45. Various thalamic subnuclei with a functional role in multisensory processing (ventroposterior nuclei) 46, motor control (ventrolateral nuclei) 47, heading direction and spatial orientation (anterior nuclei) 48 receive ascending vestibular information directly from the vestibular nuclei or from vestibular cerebellar nuclei 25,26,49,50. Changes in synaptic density and glucose metabolism in the posterior and lateral parts of the thalamus found in the current study likely represent a process of adaptive plasticity, which contributes to multisensory substitution and recalibration. Accordingly, human neuroimaging studies have documented changes in cortical and subcortical multisensory networks in patients with chronic bilateral vestibular loss, which suggest an increased visual substitution 51,52. From a neuroscientific perspective, it is interesting that synaptic density in the thalamus increases before glucose consumption. It seems that during adaptive neuroplasticity novel synaptic connections are established first, which in turn result in a gain of function and increase of neuronal activity. This example shows that function can follow form, similar to the processes in the developing brain during childhood 1. This poses the question, whether the brain reactivates these developmental strategies in the case of a neuronal lesion. Interestingly, dynamic balance, which can be estimated by the number of body rotations, deteriorated until week 3 post BL and stabilized thereafter. This may be interpreted as a behavioural correlate of adaptive plasticity driven by the thalamus.
iii) In frontal-basal ganglia loops, we found an increase of synaptic density, which was paralleled by a relative rCGM decrease especially after 3 weeks post BL (Fig. 5). Frontal-basal ganglia networks are critically involved in supraspinal locomotor control 28,53. An established concept of basal ganglia control of movement states that a direct pathway via striatal medium spiny neurons (dMSNs) expressing dopamine D1 receptor facilitates movements, while an indirect pathway marked by iMSNs expressing dopamine D2 and adenosine 2a receptors suppresses movement 54-56. The same model seems to apply for locomotor control. Stimulation of dMSNs increases and of iMSNs suppresses locomotion by downstream control of the mesencephalic locomotor region (MLR) 53,57. Indeed, connections between the vestibular system and the basal ganglia have been reported 47. Neuronal projections from the medial vestibular nucleus via the parafascicular nucleus of the thalamus to the dorsolateral putamen were documented by tracing experiments in the rat 58. In another study, neurochemical changes in the striatum were induced by vestibular stimulation 59. Furthermore, vestibular signals may project directly to the MLR and thereby modulate the locomotor pattern 60,61. It is a striking and pathognomonic behavioural feature of bilateral vestibular loss in rats that animals develop a persistent hyperlocomotion pattern 62,63. Behavioural data in our rat model exactly resembled this known phenomenon (Fig. 6). It has been suggested that loss of vestibular input to the striatum may hamper the balance of dMSN and iMSN in favor of the direct pathway, thus activating locomotion 47. From a theoretical point of view, increased locomotion following bilateral vestibular loss could be due to an upregulation of dMSNs or a downregulation of iMSNs. Changes of D2 receptor expression in the striatum were not identified following BL in the rat 64. However, patients with chronic bilateral vestibular loss had a reduced D2/D3 receptor availability in the striatum bilaterally, which also correlated to their handicap 65. How can the findings of the current [18F]-UCB-H/ [18F]-FDG tracer study be interpreted in this context? It is striking that frontal-basal ganglia hubs showed a relatively reduced rCGM throughout the experiment. Although [18F]-FDG data cannot inform about the underlying circuit, in view of the behavioural signature of hyperlocomotion it seems likely that missing vestibular input reduced the locomotor control from the frontal cortex via the indirect basal ganglia pathway activating the direct basal ganglia pathway. The significant increase of synaptic density in the frontal association cortex and striatum could be seen either as an adaptive response to this functional imbalance of direct-indirect pathways, or as a secondary maladaptive effect due to the overactive direct pathway. As the hyperlocomotion does persist after irreversible vestibular loss, it seems that the synaptic plasticity in the basal ganglia is not sufficient to compensate for this behavioural dysregulation.
Impact of locomotor training on adaptive brain plasticity following bilateral vestibular loss
Multimodal physical training is the mainstay of therapy in patients with bilateral vestibular loss 23,66. The applied principles are based on neurophysiological considerations and tailored to training of the vestibular-ocular reflex (habituation training), the interplay of multisensory sources to control posture (sensory pertubation training), and dynamic balance (locomotor training) 67,68. While the effectiveness of these exercises is highly evidence based 69, their mode of action on cerebral function and structure has been completely neglected so far. Cortical reorganization by a motor training is well documented for patients with stroke 70,71. From this literature, we have learned that new training methods based on basic neuroscientific principles such as motor imagery 72,73 or action activation 74 may add to the therapeutic success. In the current study, we aimed to start with a comparably simple training program based on the voluntary use of running wheels installed in the cages, to understand how this intervention may influence behaviour, brain structure and function after bilateral vestibular loss in the rat. This selection was motivated by the fact that active locomotion stimulates multisensory feedback and trains motor control. Furthermore, a beneficial effect of voluntary running wheel training has been shown in various neurological disease models 75,76. In the current study, we found an effect of running wheel training on dynamic balance, as measured by the number of body rotations, with a peak 1-3 weeks post BL, while other markers of locomotor behaviour such as velocity or movement duration remained unchanged (Fig. 6). Accordingly, transient and subtle changes of synaptic density and regional glucose metabolism were found in the thalamus (Fig. 5). Given the role of the thalamus for multisensory adaptation and postural control, it is reasonable that the therapeutic effect of running wheel training was mediated by this brain region. Alterations of [18F]-UCB-H/ [18F]-FDG binding in brainstem-cerebellar circuits and frontal-basal ganglia circuits were not affected by training after BL. The finding of a training effect on adaptive plasticity and balance control in BL is promising, because it gives first evidence for a potential neurobiological correlate of vestibular rehabilitation. However, the rather mild and transient effects documented in this study show that a stereotyped training such as running wheel locomotion likely is not sufficient to induce persistent and functionally relevant effects on balance and locomotor control in a three-dimensional space. Future studies might test the effect of more elaborated training protocols (e.g., visual cueing, vibration feedback, more complex and varying movements) on both adaptive brain plasticity and behavioural recovery in vestibular animal models 77,78.
In conclusion, the current study shows the potential of a [18F]-UCB-H/[18F]-FDG dual tracer application to investigate structural and functional plasticity mechanisms after a neuronal injury and the effect of treatment on this process. Lesion-induced neuroplasticity is a complex process with different modes of structure-function coupling, which arise in various neural networks and at different temporal courses. An improved understanding of these processes could help to tailor interventions to the optimal time window and to target specific networks by therapeutic strategies at different times.