Background
In addition to progressive paralysis and the formation of white matter plaques, multiple sclerosis (MS) is often associated with prominent secondary symptoms [
1]. Sensory alterations, including pain and dysesthesia, are frequently reported in the clinical MS population [
2,
3]. A substantial proportion of those affected (up to 40 %) suffer from pain of central neuropathic origin (CNP) [
4,
5]. An increasing awareness of these issues has developed in parallel with an increased focus on the importance of gray matter alterations in the pathobiology of MS [
6]. Furthermore, a connection between maladaptive plasticity within pain-associated gray matter regions of the brain—such as the primary somatosensory cortex (S1)—and CNP has been established in the literature [
7‐
9].
Several recent studies and reviews have indicated that the disease model, experimental autoimmune encephalomyelitis (EAE), shares multiple pathobiological characteristics with MS beyond the hallmark symptoms of demyelination, paralysis, and frank neurodegeneration [
10]. Wide-spread gray matter synaptopathy, driven by diffuse and persistent neuroinflammation throughout the central nervous system (CNS) is emerging as a critical contributing factor in the loss of function, sensory and cognitive abnormalities [
11], and potentially in pain—which is also now known to feature prominently EAE. These reports provide an experimental foundation for investigations into the connections between these phenomena in diseases like MS/EAE. Specifically, earlier studies by Olechowski et al. [
12‐
14] and others [
15‐
17] established the suitability of the female C57/BL6 mouse model of EAE for the study of the underlying mechanisms of CNP in MS. These studies revealed that mice with EAE develop robust mechanical and thermal allodynia prior to the onset of paralytic symptoms. They also found evidence of hyperexcitability within the dorsal horn of the spinal cord (SC-DH), a form of central sensitization [
12,
18]. While a few previous reports have highlighted the existence of altered neuronal structure and function in the neocortex of animals with EAE [
19‐
21], no study to date has directly examined changes in neuronal activity and structure in higher sensory cortex in connection with altered pain behaviors in the early stages of the disease.
S1 is known to play a critical role in processing “sensory-discriminative” aspects of both painful and non-painful touch. Within S1, the body-centric locations of external stimuli are encoded as a spatially organized “somatotopic map” comprised by distinct regions of cortical activation. The intensity (or perceived intensity) of an external stimulus is encoded as the magnitude of cortical activation (the extent of neuronal spiking activity, within an ensemble) in S1. Painful stimuli, which are generally perceived as being more intense, are associated with a greater magnitude of activation in S1 [
22]. Allodynia, such as in EAE/MS with CNP, involves non-noxious stimuli being perceived as painful—and is thought to involve intense activation (hyperexcitability) in S1 and connected “pain-associated” brain regions [
23‐
25]. Indeed, plasticity and enhanced activation in S1 has been shown to enhance activation in other “pain regions,” such as the anterior cingulate cortex, and to enhance chronic pain states [
7,
23].
In the current study, we quantified synaptic densities and neuronal morphologies in S1 of female C57/BL6 mice with EAE using histological methods. This involved immunostaining for vesicular glutamate transporter (VGLUT1)+ presynaptic excitatory terminals and parvalbumin+ (PV+) inhibitory networks and reflectance-mode confocal microscopy of Golgi-Cox-stained cortical neurons. We also quantified sensory-evoked functional neuronal responses in S1 of EAE mice using in vivo flavoprotein autofluorescence imaging (FAI). FAI has recently been employed in several studies of cortical (S1) responses to noxious and non-noxious peripheral stimuli in rodents under acute urethane-induced anesthesia. This technique measures increases in endogenous green fluorescence, produced by oxidized flavoproteins within the mitochondrial respiratory chain, as a quantitative and non-hemodynamic index of neuronal energy metabolism and activity [
26]. The FAI signal has been shown to exhibit a roughly linear correspondence with local-field potentials and intracellular calcium rises and with stimulus amplitude, frequency, and duration [
27]. These features make FAI an ideal technique for investigating cortical nociceptive responses in EAE and for the assessment of novel antinociceptive treatments.
The antidepressant phenelzine (PLZ) is an atypical monoamine oxidase inhibitor (MAOI). We have previously demonstrated that EAE is associated with a reduction in CNS tissue concentrations of the monoamine neurotransmitters (NTs) serotonin (5-HT), noradrenaline (NA), and dopamine (DA), as well as gamma-amino butyric acid (GABA) [
28]. PLZ can restore CNS tissue concentrations of all of these NTs when given chronically to mice with EAE [
29]. PLZ therefore combines the features of both an anticonvulsant and an antidepressant—the net effect of which, we predicted, would be a promotion of neuronal inhibition within the CNS. As two recent reviews have speculated that a chronic pro-excitatory/disinhibitory state may exist in the CNS in MS/EAE [
10,
11], and as both pain and neocortical plasticity are thought to be regulated by a precise balance of CNS excitation and inhibition (E-I) [
30,
31], we hypothesized that a disrupted E-I balance might underlie both conditions in EAE. We also hypothesized that restoring this balance, by bolstering CNS inhibition with PLZ, would be an effective approach to treatment for these symptoms of the disease.
Discussion
This study is the first investigation of functional neocortical plasticity along with persistent neuroanatomical and synaptic changes occurring in S1 in the very early stages of the C57/BL6 MOG
35–55 EAE model. Specifically, we find in vivo evidence in early EAE of enhanced intensity and spread of the neuronal activation within S1 that is evoked by vibrotactile stimulation of the fore- or hindlimb. Interestingly, a delay exists between the “pre-symptomatic” and “clinical-onset” time points in the sensitization of responses to forelimb stimulation. This delay mirrors the caudal-to-rostral progression of spinal inflammation and paralysis in EAE [
67] and suggests that ascending sensitization within the SC-DH [
12] may precede (or initiate) sensitization of supraspinal sites, as has been observed in other models of neuropathic pain and allodynia [
8,
23,
24].
In addition to the observed enhancement of functional responses, we find histological evidence of an increased density of excitatory pre-synaptic (VGLUT1+) terminals and post-synaptic contacts (dendritic spines) in cortical layers 2/3 and 4/5 of S1 in early EAE. These changes are indicative of pro-excitatory remodeling of the major feed-forward circuit through S1 [
47], in which layer 4 principal neurons receive thalamocortical inputs [
68] and project vertically to pyramidal neurons of layer 2/3—primarily to the distal/basilar branches. Abundant transcolumnar connections in layer 2/3 mediate the horizontal spread of activation through S1, defining the areal extent of a “functional map” [
53,
69]. Synaptic remodeling along this pathway therefore likely contributes to the intensification and expansion of S1 functional responses in early EAE [
70]. These alterations occur prior to the onset of major paresis and temporally coincide with the appearance of prominent pain behaviors in the disease. Moreover, similar functional and synaptic alterations occurring in S1 have been shown to play a causal role in other neuropathic pain models [
7,
8].
We also find evidence in EAE of an early, although transient, disruption of target-cell innervation by basket-forming PV+ inhibitory interneurons in S1. The central role of PV-mediated fast-spiking inhibition in limiting the extent to which large-scale plastic changes may occur in the neocortex, during both adulthood and the perinatal critical period, is well documented in the literature [
71,
72]. Even a transient loss of PV-mediated perisomatic inhibition in early EAE might therefore have profound and lasting consequences, leading to a dysregulated E-I balance and maladaptive cortical plasticity [
73]. Moreover, we find that PV+ interneurons are affected in EAE by an early-appearing and persistent loss of their associated PNN structures. PNNs serve multiple supportive and protective functions for PV+ neurons, including sequestering cations (i.e., Ca
2+) to support fast-spiking activity, limiting synaptic modifications and alterations of connectivity, and protecting the neurons against chemical insults such as reactive oxygen species (ROS) [
59]. The loss of PNNs may therefore be a key precipitating factor in the aberrant structural and synaptic plasticity we find in both the inhibitory and excitatory circuitry of S1 in early EAE. Loss of PNNs may additionally contribute to the unique susceptibility of PV+ interneurons to degeneration in the later stages of EAE/MS, which has been reported by several groups [
21,
74,
75].
Collectively with our previous findings [
12,
28], the multiple functional and synaptic changes in S1 evidenced in this study provide support for the hypothesis that EAE involves a profound, pro-excitatory, shift in the E-I balance of the entire somatosensory CNS, beginning very early in the disease course. This disrupted E-I balance promotes functional and structural plasticity within S1 [
30,
71], leading to amplified cortical responses to peripheral stimuli and likely contributing to pain behaviors (i.e., allodynia) in the disease [
7,
23,
31].
While we are the first group to find an increase in both pre- and post-synaptic glutamatergic markers and a concurrent reduction in perisomatic PV+ immunoreactivity in S1 in early EAE, several other groups have found similar or complementary changes in the EAE/MS brain [
21,
74,
75]. A report by Yang et al. (2014) demonstrated enhanced turnover of dendritic spines and axonal boutons in layer 5 pyramidal neurons within S1 in early MOG
35–55 EAE [
19]. As mentioned, loss of PV+ interneurons in EAE has also been demonstrated by several groups in multiple brain regions, including primary motor cortex [
21,
64,
76]. A single report by Tambolo et al. (2015) also suggested, based on functional magnetic resonance imaging (
fMRI)-blood-oxygen-level-dependent (BOLD) data, that the later stages (30–60 dpi) of the Lewis rat model of EAE involve functional expansion of the vibromechanically evoked S1 forelimb representation [
20]. This study also found dendritic spine loss in layer 2/3 and 4 neurons of S1. While some of the findings and interpretations offered in Tambolo et al. (2015) appear to contrast with our observations, it is worth noting that there are significant methodological differences between the studies. Furthermore, inferences about neural activation based strictly on the
fMRI-BOLD signal may potentially be confounded by hemodynamic changes in the disease state. Nevertheless, much agreement exists between these various reports. Indeed, a substantial body of evidence is emerging that early synaptopathy in EAE and MS brains leads progressively to neuronal hyperexcitability, plasticity, excitotoxicity, and eventual dysfunction and degeneration [
21,
65,
77]. In the majority of these studies, inflammation and circulating pro-inflammatory cytokines have been proposed as the proximal causative factors [
10,
11].
In our examination of the role that inflammation plays in initiating or promoting cortical alterations in EAE, we first examined tissues for infiltrating CD3+ T cells and CD45+ leukocytes. As noted, brain-penetrating T cells were absent from S1 at these early stages in our model. However, intracortical Iba-1-reactive microglia were found to be significantly more abundant in EAE compared to CFA controls, both pre-symptomatically (7 dpi), and in the established disease (21 dpi). Previous groups have suggested multiple contributing roles for reactive microglia in EAE/MS-related synaptopathies [
10,
11]. Microglia are capable of modifying neuronal connectivity through multiple mechanisms, including the secretion of diffusible factors such as matrix metalloproteases (i.e., matrix metalloproteinase (MMP)-2, MMP-9), which digest ECMCs such as PNNs, and are known to be elevated in the brain in EAE/MS [
78]. Reactive microglia also secrete cytokines, such as soluble tumor necrosis factor (sTNF)-α and interleukin (IL)-1β [
79,
80] which have been shown to promote synaptic plasticity and scaling, and neuronal hyperexcitability in EAE [
19]. Microglia are furthermore responsive to many activity-dependent signals, such as extracellular glutamate and adenosine triphosphate (ATP) [
81]. The pro-excitatory state found in early EAE cortex therefore likely acts to promote microglial reactivity in a feed-forward manner.
In addition to characterizing cortical functional and synaptic changes in early EAE, we also demonstrated a novel antinociceptive effect of PLZ treatment in the disease. Chronic treatment with PLZ from 7 dpi, when early cortical and behavioral alterations are already established, fully normalized mechanical withdrawal thresholds in EAE mice at clinical onset. Significantly, we also demonstrated that PLZ treatment normalizes S1 functional responses in EAE at onset. Furthermore, PLZ treatment attenuated S1 structural and synaptic abnormalities–normalizing dendritic spine densities at clinical onset and attenuating VGLUT1 reactivity in the established disease (21 dpi). Notably, this result highlights the possibility that, given the proper intervention, disease-related synaptopathies may be reversible. PLZ restores CNS levels of GABA in EAE through the inhibition of GABA-T by its active metabolite phenylethylidenehydrazine (PEH) and restores monoamine levels by the irreversible inhibition of MAO-A and B [
29]. PLZ has previously been shown to enhance functional intracortical GABA release [
82‐
84]. The enhancement of the GABA-AR-mediated [
55,
56] surround-inhibitory FAI signal we find in PLZ-treated EAE mice supports this proposed mechanism of action. Other groups have also suggested that PLZ may attenuate excessive cortical glutamate release by affecting glutamate-glutamine (neuron-astrocyte) shuttling and conversion [
85,
86]. Defective astrocytic reuptake and metabolism has been suggested to promote excessive synaptic glutamate and CNS hyperexcitability in EAE/MS [
10‐
12]. While PLZ treatment in EAE did not rescue disrupted PNNs, it significantly reduced Iba-1+ cells within S1. Just as excitatory signaling can promote microglial reactivity, inhibitory signaling through G protein-coupled receptors, such as GABA-BRs [
87] and adrenergic receptors [
88], can reduce microglial motility and reactivity. Enhancement of GABAergic/monoaminergic neurotransmission and the concomitant reduction of excitatory signaling may therefore be the means by which PLZ treatment reduces cortical microgliosis in EAE. This synergistic neuroglial action likely aids in the restoration of normal constraints on plasticity within the somatosensory CNS and contributes to the normalization of pain behaviors in EAE. PLZ treatment does not induce a generalized analgesic or sedative effect, as it produced no significant changes in basal mechanical sensitivity or motor function in control (CFA) animals. PLZ also did not affect evoked S1 functional responses in control (CFA) animals.
Although the current experiments did not involve direct manipulation of the sensory cortex in a way that might conclusively establish an immediate causal link between altered S1 structure/function and altered pain behaviors in EAE, the complete dissociation of responses to PLZ treatment in non-disease controls and EAE animals supports the hypothesis that maladaptive cortical plasticity and hyperexcitability within S1 directly contributes to pain in the disease.
Abbreviations
5-HT, 5-hydroxytryptamine (serotonin); ANOVA, analysis of variance; ATP, adenosine triphosphate; BOLD, blood-oxygen-level dependent (signal); CD, cluster of differentiation; CFA, complete Freund’s adjuvant; CNP, chronic neuropathic pain; CNS, central nervous system; DH, dorsal horn; dpi, days post-inoculation; EAE, experimental autoimmune encephalomyelitis; ECMC, extracellular matrix component; E-I, excitatory-inhibitory; FA/FAI, flavoprotein autofluorescence (imaging); FL, forelimb; GABA, gamma-aminobutyric acid; DA, dopamine; GABA-T, GABA-transaminase; HL, hindlimb; Iba, ionized calcium-binding adapter; IHC, immunohistochemistry; fMRI, functional magnetic resonance imaging; IL, interleukin; IP, intraperitoneal; MAO, monoamine oxidase; MAOI, monoamine oxidase inhibitor; MMP, matrix metalloproteinase; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; NA, noradrenaline; NT, neurotransmitter; PB, phosphate buffer; PBS, phosphate-buffered saline; PEH, phenylethylidenehydrazine; PFA, paraformaldehyde; PLZ, phenelzine; PNN, peri-neuronal net; PT, pertussis toxin; PV, parvalbumin; ROI, region of interest; S.C., subcutaneous; S1, primary somatosensory cortex; SC, spinal cord; SNK, Student-Newmnn-Keuls; sTNF, soluble tumor necrosis factor; VEH, vehicle; VF/VFH, Von Frey hair; VGLUT, vesicular glutamate transporter; WFA, Wisteria floribunda agglutinin
Acknowledgements
The authors would also like to acknowledge the technical assistance provided by Bin Dong and Kasia Zubkow from the University of Alberta, as well as Dr. Majid Mohajerani of the University of Lethbridge.