Excitatory and Inhibitory Neurons of the Spinal Cord Superficial Dorsal Horn Diverge in Their Somatosensory Responses and Plasticity in Vivo.

The superficial dorsal horn (SDH) of the spinal cord represents the first site of integration between innocuous and noxious somatosensory stimuli. According to gate control theory, diverse populations of excitatory and inhibitory interneurons within the SDH are activated by distinct sensory afferents, and their interplay determines the net nociceptive output projecting to higher pain centers. Although specific SDH cell types are ill defined, numerous classifications schemes find that excitatory and inhibitory neurons fundamentally differ in their morphology, electrophysiology, neuropeptides, and pain-associated plasticity; yet little is known about how these neurons respond over a range of natural innocuous and noxious stimuli. To address this question, we applied an in vivo imaging approach in male mice where the genetically encoded calcium indicator GCaMP6s was expressed either in vGluT2-positive excitatory or vIAAT-positive inhibitory neurons. We found that inhibitory neurons were markedly more sensitive to innocuous touch than excitatory neurons but still responded dynamically over a wide range of noxious mechanical stimuli. Inhibitory neurons were also less sensitive to thermal stimuli than their excitatory counterparts. In a capsaicin model of acute pain sensitization, the responses of excitatory neurons were significantly potentiated to innocuous and noxious mechanical stimuli, whereas inhibitory neural responses were only depressed to noxious stimuli. These in vivo findings show that excitatory and inhibitory SDH neurons diverge considerably in their somatosensory responses and plasticity, as postulated by gate control theory.SIGNIFICANCE STATEMENT Gate control theory posits that opposing spinal excitatory and inhibitory neurons, differently tuned across somatosensory modalities, determine the net nociceptive output to higher pain centers. Little is known about how natural stimuli activate these two neural populations. This study applied an in vivo calcium imaging approach to genetically target these neurons and contrast their responses over a range of innocuous and noxious mechanical and thermal stimuli. Compared with excitatory neurons, we found that inhibitory neurons are more sensitive to innocuous touch and far less sensitive to thermal stimuli. An acute model of pain also revealed that these subtypes undergo divergent mechanosensory plasticity. Our data provide important and novel insights for gate-control inspired models of pain processing.

Dorsal column and root stimulation at Aß-fiber intensity activate superficial dorsal horn glutamatergic and GABAergic populations.

Neurostimulation therapies are frequently used in patients with chronic pain conditions. They emerged from Gate Control Theory (GCT), which posits that Aß-fiber activation recruits superficial dorsal horn (SDH) inhibitory networks to "close the gate" on nociceptive transmission, resulting in pain relief. However, the efficacy of current therapies is limited, and the underlying circuits remain poorly understood. For example, it remains unknown whether ongoing stimulation of Aß-fibers is sufficient to drive activity in SDH neurons. We used multiphoton microscopy in spinal cords extracted from mice expressing the genetically encoded calcium indicator GCaMP6s in glutamatergic and GABAergic populations; activity levels were inferred from deconvolved calcium signals using CaImAn software. Sustained Aß-fiber stimulation at the dorsal columns or dorsal roots drove robust yet transient activation of both SDH populations. Following the initial increase, activity levels decreased below baseline in glutamatergic neurons and were depressed after stimulation ceased in both populations. Surprisingly, only about half of GABAergic neurons responded to Aß-fiber stimulation. This subset showed elevated activity for the entire duration of stimulation, while non-responders decreased with time. Our findings suggest that Aß-fiber stimulation initially recruits both excitatory and inhibitory populations but has divergent effects on their activity, providing a foundation for understanding the analgesic effects of neurostimulation devices.Perspective: This article used microscopy to characterize the responses of mouse spinal cord cells to stimulation of non-painful nerve fibers. These findings deepen our understanding of how the spinal cord processes information and provide a foundation for improving pain-relieving therapies.

Success with dorsal root entry zone lesioning after a failed trial of spinal cord stimulation in a patient with pain due to brachial plexus avulsion.

Pain caused by brachial plexopathy (BP) represents a challenging clinical problem with few effective therapeutic options. Here, we present a patient with severe, painful BP after a high-impact motor vehicle accident who failed conservative treatments. A trial of cervical spinal cord stimulation was completed using multiple waveforms (tonic, BurstDR, and 10 kHz) over 14 days with only 30% to 40% pain reduction. Subsequently, he underwent dorsal root entry zone lesioning with a significant decrease in his pain 1 year later. Surgical exploration revealed extensive damage and avulsion of his cervical roots that was not observed on a previous brachial plexus magnetic resonance imaging. We discuss the etiology and diagnosis of traumatic BP, possible reasons for the failed spinal cord stimulation trial, and implications for management.

Leptin increases sympathetic nerve activity via induction of its own receptor in the paraventricular nucleus.

Whether leptin acts in the paraventricular nucleus (PVN) to increase sympathetic nerve activity (SNA) is unclear, since PVN leptin receptors (LepR) are sparse. We show in rats that PVN leptin slowly increases SNA to muscle and brown adipose tissue, because it induces the expression of its own receptor and synergizes with local glutamatergic neurons. PVN LepR are not expressed in astroglia and rarely in microglia; instead, glutamatergic neurons express LepR, some of which project to a key presympathetic hub, the rostral ventrolateral medulla (RVLM). In PVN slices from mice expressing GCaMP6, leptin excites glutamatergic neurons. LepR are expressed mainly in thyrotropin-releasing hormone (TRH) neurons, some of which project to the RVLM. Injections of TRH into the RVLM and dorsomedial hypothalamus increase SNA, highlighting these nuclei as likely targets. We suggest that this neuropathway becomes important in obesity, in which elevated leptin maintains the hypothalamic pituitary thyroid axis, despite leptin resistance.

Differential modulation of excitatory and inhibitory populations of superficial dorsal horn neurons in lumbar spinal cord by Aß-fiber electrical stimulation.

Activation of Aß-fibers is fundamental to numerous analgesic therapies, yet its effects on dorsal horn neuronal activity remain unclear. We used multiphoton microscopy of the genetically encoded calcium indicator GCaMP6s to characterize the effects of Aß-fiber electrical stimulation (Aß-ES) on neural activity. Specifically, we quantified somatic responses evoked by C-fiber intensity stimulation before and after a 10-minute train of dorsal root Aß-ES in superficial dorsal horn (SDH) neurons, in mouse lumbar spinal cord. Aß-ES did not alter C-fiber-evoked activity when GCaMP6s was virally expressed in all neurons, in an intact lumbar spinal cord preparation. However, when we restricted the expression of GCaMP6s to excitatory or inhibitory populations, we observed that Aß-ES modestly potentiated evoked activity of excitatory neurons and depressed that of inhibitory neurons. Aß-ES had no significant effects in a slice preparation in either SDH population. A larger proportion of SDH neurons was activated by Aß-ES when delivered at a root rostral or caudal to the segment where the imaging and C-fiber intensity stimulation occurred. Aß-ES effects on excitatory and inhibitory populations depended on the root used. Our findings suggest that Aß-ES differentially modulates lumbar spinal cord SDH populations in a cell type- and input-specific manner. Furthermore, they underscore the importance of the Aß-ES delivery site, suggesting that Aß stimulation at a segment adjacent to where the pain is may improve analgesic efficacy.

Spinal Cord Stimulation: Clinical Efficacy and Potential Mechanisms.

Spinal cord stimulation (SCS) is a minimally invasive therapy used for the treatment of chronic neuropathic pain. SCS is a safe and effective alternative to medications such as opioids, and multiple randomized controlled studies have demonstrated efficacy for difficult-to-treat neuropathic conditions such as failed back surgery syndrome. Conventional SCS is believed mediate pain relief via activation of dorsal column Aß fibers, resulting in variable effects on sensory and pain thresholds, and measurable alterations in higher order cortical processing. Although potentiation of inhibition, as suggested by Wall and Melzack's gate control theory, continues to be the leading explanatory model, other segmental and supraspinal mechanisms have been described. Novel, non-standard, stimulation waveforms such as high-frequency and burst have been shown in some studies to be clinically superior to conventional SCS, however their mechanisms of action remain to be determined. Additional studies are needed, both mechanistic and clinical, to better understand optimal stimulation strategies for different neuropathic conditions, improve patient selection and optimize efficacy.

Minimally invasive procedures for neuropathic pain.

Neuropathic pain is "pain arising as a direct consequence of a lesion or disease affecting the somatosensory system". The prevalence of neuropathic pain ranges from 7 to 11% of the population and minimally invasive procedures have been used to both diagnose and treat neuropathic pain. Diagnostic procedures consist of nerve blocks aimed to isolate the peripheral nerve implicated, whereas therapeutic interventions either modify or destroy nerve function. Procedures that modify how nerves function include epidural steroid injections, peripheral nerve blocks and sympathetic nerve blocks. Neuroablative procedures include radiofrequency ablation, cryoanalgesia and neurectomies. Currently, neuromodulation with peripheral nerve stimulators and spinal cord stimulators are the most evidence-based treatments of neuropathic pain.

Electrical stimulation of low-threshold afferent fibers induces a prolonged synaptic depression in lamina II dorsal horn neurons to high-threshold afferent inputs in mice.

Electrical stimulation of low-threshold Aß-fibers (Aß-ES) is used clinically to treat neuropathic pain conditions that are refractory to pharmacotherapy. However, it is unclear how Aß-ES modulates synaptic responses to high-threshold afferent inputs (C-, Aδ-fibers) in superficial dorsal horn. Substantia gelatinosa (SG) (lamina II) neurons are important for relaying and modulating converging spinal nociceptive inputs. We recorded C-fiber-evoked excitatory postsynaptic currents (eEPSCs) in spinal cord slices in response to paired-pulse test stimulation (500 µA, 0.1 millisecond, 400 milliseconds apart). We showed that 50-Hz and 1000-Hz, but not 4-Hz, Aß-ES (10 µA, 0.1 millisecond, 5 minutes) induced prolonged inhibition of C-fiber eEPSCs in SG neurons in naive mice. Furthermore, 50-Hz Aß-ES inhibited both monosynaptic and polysynaptic forms of C-fiber eEPSC in naive mice and mice that had undergone spinal nerve ligation (SNL). The paired-pulse ratio (amplitude second eEPSC/first eEPSC) increased only in naive mice after 50-Hz Aß-ES, suggesting that Aß-ES may inhibit SG neurons by different mechanisms under naive and nerve-injured conditions. Finally, 50-Hz Aß-ES inhibited both glutamatergic excitatory and GABAergic inhibitory interneurons, which were identified by fluorescence in vGlut2-Td and glutamic acid decarboxylase-green fluorescent protein transgenic mice after SNL. These findings show that activities in Aß-fibers lead to frequency-dependent depression of synaptic transmission in SG neurons in response to peripheral noxious inputs. However, 50-Hz Aß-ES failed to induce cell-type-selective inhibition in SG neurons. The physiologic implication of this novel form of synaptic depression for pain modulation by Aß-ES warrants further investigation.

Conventional and kilohertz-frequency spinal cord stimulation produces intensity- and frequency-dependent inhibition of mechanical hypersensitivity in a rat model of neuropathic pain.

BACKGROUND: Spinal cord stimulation (SCS) is a useful neuromodulatory technique for treatment of certain neuropathic pain conditions. However, the optimal stimulation parameters remain unclear. METHODS: In rats after L5 spinal nerve ligation, the authors compared the inhibitory effects on mechanical hypersensitivity from bipolar SCS of different intensities (20, 40, and 80% motor threshold) and frequencies (50, 1 kHz, and 10 kHz). The authors then compared the effects of 1 and 50 Hz dorsal column stimulation at high- and low-stimulus intensities on conduction properties of afferent Aα/ß-fibers and spinal wide-dynamic-range neuronal excitability. RESULTS: Three consecutive daily SCS at different frequencies progressively inhibited mechanical hypersensitivity in an intensity-dependent manner. At 80% motor threshold, the ipsilateral paw withdrawal threshold (% preinjury) increased significantly from pre-SCS measures, beginning with the first day of SCS at the frequencies of 1 kHz (50.2 ± 5.7% from 23.9 ± 2.6%, n = 19, mean ± SEM) and 10 kHz (50.8 ± 4.4% from 27.9 ± 2.3%, n = 17), whereas it was significantly increased beginning on the second day in the 50 Hz group (38.9 ± 4.6% from 23.8 ± 2.1%, n = 17). At high intensity, both 1 and 50 Hz dorsal column stimulation reduced Aα/ß-compound action potential size recorded at the sciatic nerve, but only 1 kHz stimulation was partially effective at the lower intensity. The number of actions potentials in C-fiber component of wide-dynamic-range neuronal response to windup-inducing stimulation was significantly decreased after 50 Hz (147.4 ± 23.6 from 228.1 ± 39.0, n = 13), but not 1 kHz (n = 15), dorsal column stimulation. CONCLUSIONS: Kilohertz SCS attenuated mechanical hypersensitivity in a time course and amplitude that differed from conventional 50 Hz SCS, and may involve different peripheral and spinal segmental mechanisms.

Double dissociation between long-term depression and dendritic spine morphology in cerebellar Purkinje cells.

Experiments in hippocampal area CA1 suggest that long-term potentiation could be associated with spine addition and enlargement, and long-term depression (LTD) with spine shrinkage and loss. Is this a general principle of synaptic plasticity? We used two-photon microscopy to measure dendritic spines in rat cerebellar Purkinje cells. Neither local synaptic induction of LTD nor global chemical induction of LTD changed spine number or size. Conversely, a manipulation that evoked persistent dendritic spine retraction did not alter parallel fiber-evoked excitatory postsynaptic currents.

Dynamic imaging of cerebellar Purkinje cells reveals a population of filopodia which cross-link dendrites during early postnatal development.

Two-photon microscopy was used to image dye-loaded filopodia of Purkinje cells in acute rat cerebellar slices. In the process of examining filopodia in Purkinje cells from a period of rapid dendritic growth (P10-21), we observed a small subset of filopodia which appeared to form connections between two dendrites of the same cell, usually between the tips of two adjacent dendrites or the tip of a dendrite and the shaft of another. There were fewer of these 'filopodial bridges' present at P18-21 than at an earlier stage in development (P10-12) and they were absent in mature Purkinje cells. Filopodial bridges do not appear to be an artifact of living brain slice preparation as they may also be seen by dye-loading Purkinje cells in slices prepared from perfusion-fixed brain. They have varied morphologies which are mostly similar to conventional, unattached filopodia. However, when measured over tens of minutes, filopodial bridges were observed to be less motile than conventional filopodia as indicated by a reduced index of expansion. While the functions of these novel structures are unknown it is attractive to speculate that they play an instructive role in Purkinje cell dendritic development.