Cortical layer‐specific modulation of neuronal activity after sensory deprivation due to spinal cord injury
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Evaluation Summary:
Zaforas et al. conducted a high-quality study on a very complex topic, using advanced layer-specific neuronal recording techniques. Their findings might be especially interesting for pre-clinical and clinical researchers as well as clinicians in the field of SCI-related sensory pathologies such as neuropathic pain. However, methodological limitations prevent clear mechanistic insight into the underlying causes of their effects.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Cortical areas have the capacity of large‐scale reorganization following sensory deafferentation. However, it remains unclear whether this phenomenon is a unique process that homogeneously affects the entire deprived cortical region or whether it is susceptible to changes depending on neuronal networks across distinct cortical layers. Here, we studied how the local circuitry within each layer of the deafferented cortex forms the basis for neuroplastic changes after immediate thoracic spinal cord injury (SCI) in anaesthetized rats. In vivo electrophysiological recordings from deafferented hindlimb somatosensory cortex showed that SCI induces layer‐specific changes mediating evoked and spontaneous activity. In supragranular layer 2/3, SCI increased gamma oscillations and the ability of these neurons to initiate up‐states during spontaneous activity, suggesting an altered corticocortical network and/or intrinsic properties that may serve to maintain the excitability of the cortical column after deafferentation. On the other hand, SCI enhanced the infragranular layers’ ability to integrate evoked sensory inputs leading to increased and faster neuronal responses. Delayed evoked response onsets were also observed in layer 5/6, suggesting alterations in thalamocortical connectivity. Altogether, our data indicate that SCI immediately modifies the local circuitry within the deafferented cortex allowing supragranular layers to better integrate spontaneous corticocortical information, thus modifying column excitability, and infragranular layers to better integrate evoked sensory inputs to preserve subcortical outputs. These layer‐specific neuronal changes may guide the long‐term alterations in neuronal excitability and plasticity associated with the rearrangements of somatosensory networks and the appearance of central sensory pathologies usually associated with spinal cord injury.
Key points
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Sensory stimulation of forelimb produces cortical evoked responses in the somatosensory hindlimb cortex in a layer‐dependent manner.
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Spinal cord injury favours the input statistics of corticocortical connections between intact and deafferented cortices.
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After spinal cord injury supragranular layers exhibit better integration of spontaneous corticocortical information while infragranular layers exhibit better integration of evoked sensory stimulation.
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Cortical reorganization is a layer‐specific phenomenon.
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Evaluation Summary:
Zaforas et al. conducted a high-quality study on a very complex topic, using advanced layer-specific neuronal recording techniques. Their findings might be especially interesting for pre-clinical and clinical researchers as well as clinicians in the field of SCI-related sensory pathologies such as neuropathic pain. However, methodological limitations prevent clear mechanistic insight into the underlying causes of their effects.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The study addresses the question of what happens in each cortical layer of the severed limb's primary somatosensory cortical representation when the spinal cord is transected. The assessment is made with 32 iridium wire electrodes in a single vertical array at 50um intervals in the hind-limb area. Spontaneous activity is monitored under urethane anaesthesia before and after transection. Responses to electrical stimulation of the forepaw are assessed by field responses.
The study finds that electrically stimulating the forelimb evokes responses in the hindlimb area almost immediately after transection where none existed before. This is a good demonstration of an effect that has been known for some time. A large amount of the literature on cortical layer specific effects of sensory deprivation is missing as …
Reviewer #1 (Public Review):
The study addresses the question of what happens in each cortical layer of the severed limb's primary somatosensory cortical representation when the spinal cord is transected. The assessment is made with 32 iridium wire electrodes in a single vertical array at 50um intervals in the hind-limb area. Spontaneous activity is monitored under urethane anaesthesia before and after transection. Responses to electrical stimulation of the forepaw are assessed by field responses.
The study finds that electrically stimulating the forelimb evokes responses in the hindlimb area almost immediately after transection where none existed before. This is a good demonstration of an effect that has been known for some time. A large amount of the literature on cortical layer specific effects of sensory deprivation is missing as are references to the seminal studies of Calford and Tweedale. In particular one might consult Calford and Tweedale 1988, 1991 in flying fox (bats) and 1991 in macaque monkey cortex. Jacob et al 2017 regarding layer specific effects of sensory deprivation may also be of particular relevance given the changes in layer 5 versus layer 2/3 responses. The incremental advance in knowledge comes from a description of the effects by cortical layer. The infra granular layer responses increase in size and, perhaps counterintuitively, increase in latency while the supragranular layers show changes in spontaneous activity.
In general, the value of the research is lessened by using electrical rather than natural stimulation, thereby rendering the stimuli saturating, hyper-synchronous and ultimately unrealistic. Electrical stimuli will synchronously stimulate all afferents including myelinated and unmyelinated fibres synchronously, a situation that normally does not arise unless you electrocute yourself, which is obviously very painful. Any surround inhibition that might have been present will be overridden by this level of wide field high intensity stimulation. It would have been useful to know how individual digit receptive fields appeared in hindlimb cortex for example and whether they maintained some level of somatotopy. Tactile stimuli will be more relevant to the human SCI that I believe the authors are trying to understand.
It is also disappointing that the electrodes were not implanted chronically before the recording session as 40 minutes is unlikely to give sufficient time for recovery of the cortex to insertion of the electrodes. The lower levels of responsively in superficial layers in particular are probably due to spreading depression brought about by the 32 iridium wire electrode. A different result might have resulted from implanting the electrode chronically and starting recording several weeks after the damage and inflammation had abated. It is also not clear that the observations on gamma in the urethane anaesthetised state are relatable to the situation in awake animals where gamma waves usually occur.
I wonder whether the conceptualisation of the study as a sensory deprivation is entirely accurate. The spinal cord transection would presumably have cut axons from cortical cells in the cortico-spinal tract. What would the effect be on these neurones in the cortex? Since they consist some of the larger cortical neurones they may contribute more to the field potentials being analysed than some of the other cells.
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Reviewer #2 (Public Review):
The authors seek to demonstrate that plastic changes already occurred early after complete spinal cord transection somatosensory cortex and these were different between deep and superficial layers in deeply anaesthetized adult rats. They suggest that these changes are a consequence of sensory deprivation.
Strengths:
The study for the first time applied multielectrode array technology to explore the effects of spinal cord transection on the circuitry within the somatosensory cortical region associated with the deprived body region (hind limb). In addition, the activity in this region that was evoked by stimulation of a remote body region (fore limb) was explored.It is well demonstrated that the method can be used in vivo and over a long time period, and it can be anticipated that in combination with a …
Reviewer #2 (Public Review):
The authors seek to demonstrate that plastic changes already occurred early after complete spinal cord transection somatosensory cortex and these were different between deep and superficial layers in deeply anaesthetized adult rats. They suggest that these changes are a consequence of sensory deprivation.
Strengths:
The study for the first time applied multielectrode array technology to explore the effects of spinal cord transection on the circuitry within the somatosensory cortical region associated with the deprived body region (hind limb). In addition, the activity in this region that was evoked by stimulation of a remote body region (fore limb) was explored.It is well demonstrated that the method can be used in vivo and over a long time period, and it can be anticipated that in combination with a mechanistic approach MEA can develop into a routine technique for in vivo brain research in rodent models.
With their approach the authors find corresponding changes that already have been reported for other sensory systems.
Weaknesses:
The MEA technology allows assessment of network activity, however, the connectivity of the somatosensory cortex is highly complex and different populations of local excitatory and inhibitory interneurons collaborate to generate output signals of superficial (L3/3) and deep (L5) Purkinje neurons. MEA recordings as such do not sufficiently allow to unequivocally identify the nature of the neurons from which the electrodes pick up signals and therefore, a major limitation of the approach is the lack of a mechanism leading to the observed changes. For example, a reduction of inhibitory inputs and an increase of excitatory inputs could result in a similar increase of activity in the network.An attempt has been made to elucidate the role of thalamic afferents, however, other brain areas projecting to the somatosensory cortex have not sufficiently been considered.
The study does not clearly assess how the reported changes develop. The activity occurring during the transection injury itself is not recorded but due to the excitatory barrage LTP like synaptic processes may occur in addition to the loss of sensory input
The authors managed to support their aim showing differential changes occurring in different cortical layers after spinal cord transfection. However, it is not entirely clarified whether this is due to deprivation of sensory input or to plastic changes occurring as a consequence of strong excitation ascending pathways by the transection injury.
The utility of the MEA approach can be of great interest and use for researchers exploring cortical circuits. In contrast to the methodical advance offered by the study, the presented data are purely descriptive and do not fully justify the proposed conclusions.
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Reviewer #3 (Public Review):
By means of in vivo electrophysiology within the primary somatosensory hindlimb cortex in a thoracic SCI rat model, the authors tried to identify injury-induced, sensory deprivation-related, and cortical layer-specific changes in neuronal excitability and network plasticity in terms of recorded spontaneous and evoked activity.
Spinal cord transection, peripheral stimulation, electrode placement, and electrophysiological recordings have been performed according to widely established approaches and protocols and appropriate statistical tests were used for the analysis. A strength of the methodological approach is the small-scale assessment of functional cortical reorganization in the somatosensory cortex after SCI, addressing the different functional properties and connections of the distinct cortical layers …
Reviewer #3 (Public Review):
By means of in vivo electrophysiology within the primary somatosensory hindlimb cortex in a thoracic SCI rat model, the authors tried to identify injury-induced, sensory deprivation-related, and cortical layer-specific changes in neuronal excitability and network plasticity in terms of recorded spontaneous and evoked activity.
Spinal cord transection, peripheral stimulation, electrode placement, and electrophysiological recordings have been performed according to widely established approaches and protocols and appropriate statistical tests were used for the analysis. A strength of the methodological approach is the small-scale assessment of functional cortical reorganization in the somatosensory cortex after SCI, addressing the different functional properties and connections of the distinct cortical layers before and after the injury. Moreover, cortical reorganization has so far mostly been studied in a period ranging from days to months after SCI. Investigating changes already acutely after the injury in this study might allow to predict long-term cortical reorganization and serve as a biomarker for functional recovery or associated sensory pathologies. Another major strength of the study is the consideration and analysis of thalamocortical connections when investigating layer-specific changes in neuronal network properties after sensory deprivation. The results are detailed and very nicely illustrated and presented in the figures.
One weakness of the study might be the missing data in a sub-acute or chronic phase after SCI which might shed light on the absent cortical changes in a subset of the animals. Such data could actually help to understand the truly different findings in the distinct subsets of animals immediately after the injury and therefore improve the generalizability of the results and conclusions.
By observing different neuronal and network properties of each layer being responsible for the specific changes in spontaneous and evoked activity in the somatosensory cortex post-SCI in their results, the authors were able to confirm the hypotheses. Their findings shed first light on layer-specific changes in somatosensory cortex activity induced by sensory deprivation upon complete thoracic spinal cord transection and mediated by corticocortical and thalamocortical connections as well as local hindlimb cortex networks. The presented methods and data might well serve as a fundament for future preclinical research on cortical reorganization already at the acute stage after SCI and the findings might serve as biomarkers of long-term alterations in neuronal network plasticity potentially being involved in the development of sensory pathologies after SCI such as neuropathic pain.
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