Temporal plasticity of dorsal horn somatosensory neurons after acute and chronic spinal cord hemisection in rat

Unilateral T13 hemisection of the rat spinal cord produces a model of chronic spinal cord injury (SCI) that is characterized by bilateral hyperexcitability of lumbar dorsal horn neurons, and behavioral signs of central pain. While we have demonstrated that responsiveness of multireceptive (MR) dorsal horn neurons is dramatically increased at 28 days after injury, the effects of acute hemisection are unknown and predicted to be different than observed chronically. In the present study, the consequences of T13 hemisection are examined acutely at 45 min in MR neurons both ipsilateral and contralateral to the site of injury, and compared to the same class of cells at 28 days after injury (n=20 cells total per group: 2–3 cells/side of the cord from n=5 animals). Acutely, ipsilateral to the hemisection, both spontaneous and evoked activity of MR neurons were significantly increased, whereas contralaterally, only evoked activity was significantly increased. In animals 28 days after hemisection, spontaneous activity of MR neurons was comparable to intact levels ipsilaterally, and cells exhibited hyperexcitability to evoked stimuli bilaterally. Expansion of cutaneous receptive fields was observed only in hindpaws ipsilateral to the lesion, acutely. These results demonstrate dynamic plasticity in properties of dorsal horn somatosensory neurons after SCI.     

GABA decreases in the spinal cord dorsal horn after peripheral neurectomy

A significant fall in the number of GABA-immunoreactive cells in laminae I–III of the rat spinal cord occured in the somatotopic area of projection of the sciatic nerve after nerve transection. The decrease started at 2 weeks post-neurectomy, and at 4 weeks ipsilateral mean cell numbers were approximately 72% of contralateral control values. Similarly, the concentration of GABA in spinal homogenates was significantly reduced 4 weeks post-neurectomy. These data, together with our recent finding of an increase in spinal GABA during chronic inflammation of the hindlimb, suggest that the level of GABA in the dorsal horn is regulated by the amount of primary afferent input.     

Increased receptive field size of dorsal horn neurons following chronic spinal cord hemisections in cats

The somatotopic organization of the 17 dorsal horn was studied using extracellular recordings in normal cats, and in cats with acute or chronic spinal cord hemisection at T13, sparing the dorsal columns. Based on data concerning recovery of function and collateral sprouting of afferents following hemisections, we predicted that the lesion would result in increases in receptive field size and decreases in the specificity of the somatotopic map. In normal animals, the usual mediolateral, rostrocaudal and dorsoventral somatotopic sequences were found. Following acute hemisections (6 h–5 days), there were changes in spontaneous and evoked activity, but receptive field sizes and somatotopic organization remained unchanged. Following chronic hemisections (88–174 days), proximal hindlimb receptive fields in the lateral dorsal horn ipsilateral to the lesion increased dramatically in size and were significantly larger than similar receptive fields on the contralateral side. The largest of these fields extended from the dorsal midline to the middle of the foot. Receptive field sizes elsewhere in the dorsal horn remained unchanged, as did somatotopic organization in general. These findings indicate that hemisections result in a complex series of changes consisting of an early stage of anatomically generalized changes in excitability and a later stage of highly localized changes in receptive field size. Possible mechanisms for these changes, as well as their relationship to recovery of function, are discussed.     

Red nucleus modulation of somatosensory responses of cat spinal cord dorsal horn neurons

Single unit extracellular recordings were obtained from cat lumbar spinal cord dorsal horn neurons activated by cutaneous inputs. The effect of electrical conditioning stimuli applied to the red nucleus (RN) was predominantly that of inhibition although in some cases excitation was seen. All the neurons that projected to the lateral cervical nucleus were inhibited; none of these were excited. These findings suggest that the RN may exert a dynamic modulatory action on the transmission of cutaneous information during the execution of a motor program.     

Long-term potentiation in spinal nociceptive systems--how acute pain may become chronic

Chronic pain is a major problem since it is difficult to treat and the understanding of the underlying neurobiology is sparse. The mechanisms underpinning the transition of acute into chronic pain remain unclear. However, long-term potentiation (LTP) in spinal nociceptive systems may be one such mechanism. Here, we briefly review the literature regarding LTP in spinal nociceptive systems including our own data on LTP in deep convergent nociceptive neurons. Furthermore, we discuss the role of this phenomenon in understanding the neurobiology of chronic pain and the possible therapeutic implications.     

Membrane properties of deep dorsal horn neurons from neonatal rat spinal cord in vitro

Whole-cell patch-clamp recordings were undertaken to characterize and compare the membrane properties of deep dorsal horn neurons in transverse slices of rat lumbar spinal cord in two age groups, postnatal days (P) 3–6 and 9–16. In both age groups, significant correlations were observed between membrane time constant and cell resistance and between action potential height and its duration at half-maximal amplitude. Cell resistance and action potential half-width values were lower in the P9–16 age group. Neurons were divided into four categories based on their firing properties in response to intracellular current injection: single spike, phasic firing, repetitive firing, and delayed firing. The distribution of neurons within these categories was similar in both age groups which suggests that the firing properties of deep dorsal horn neurons are functionally differentiated at an early postnatal age.     

Modulation of visceral nociceptive responses of rat spinal dorsal horn neurons by sympathectomy

We determined whether sympathectomy modulates visceral nociception under physiological or inflammatory conditions. Recordings of sacral spinal dorsal horn neurons with sustained responses were performed in pentobarbitone-anesthetized rats. Graded colorectal distension (CRD, 20-100 mmHg) was used as a visceral nociceptive stimulus. Inflammation was induced by intracolonic instillation of turpentine (25%). Sympathectomy was produced by administering 6-hydroxydopamine. Inflammation produced an increase in the CRD-evoked responses. The CRD-evoked responses were attenuated following sympathectomy both under control and inflammatory conditions. These changes in the CRD-evoked responses were associated with corresponding changes in spontaneous discharge rate. The convergent input evoked by noxious pinch of the skin was not changed by any of the experimental conditions. The results indicate that sympathectomy attenuates visceral nociceptive responses and spontaneous activity of sacral spinal cord neurons, without effect on convergent cutaneous inputs, both under physiological and inflammatory conditions.     

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.     

Distribution of c-fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve

The distribution of proto-oncogene c-Fos protein-immunoreactive cells in the spinal cord dorsal horn was studied after electrical stimulation at Aα/Aß-fiber intensity of normal and previously injured sciatic nerves in urethane anesthetized rats. No or only occasional Fos protein-like immunoreactive cells were seen after stimulation of the normal uninjured nerve or after nerve transection without stimulation. Electrical nerve stimulation at 3, 12, and 21 days after sciatic nerve transection resulted in substantial increases in the numbers of Fos protein-like immunoreactive cell nuclei in each of Rexed's laminae I–V. Combined demonstration of Fos protein-like immunoreactivity and of glial fibrillary acidic protein-like immunoreactivity (astroglia) or OX-42 immunoreactivity (microglia), indicated that the observed Fos protein-like response was confined to neurons and not to astroglia or microglia. Combined demonstration in the spinal cord of Fos protein-like immunoreactive neurons and neurons labeled retrogradely with Fluoro-Gold from the gracile nucleus showed that some of the Fos protein-like immunoreactive neurons in Rexed's laminae III and IV contributed to the postsynaptic dorsal column pathway. The results indicate that stimulation at Aα/Aß-fiber intensity of a previously injured nerve gives rise to an abnormally increased activation pattern of postsynaptic neurons in the dorsal horn, some of which contribute to the postsynaptic dorsal column pathway.     

Role of substance P in the modulation of C-fiber-evoked responses of spinal dorsal horn neurons

Substance P (SP) as well as excitatory amino acids (EAAs) appear to be released in response to stimulation of primary afferent C-fibers. Activity atN-methyl-d-aspartate (NMDA) receptors is essential for wind-up (the progressive potentiation of C-fiber-evoked responses of single neurons in response to an electrical stimulation), however, the role of SP in wind-up is unclear. To address this, the effects of iontophoretically applied CP-99,994 (a NK-1 receptor antagonist), SP and SP(1–7) (an N-terminal breakdown product of SP), were compared on responses of spinal dorsal horn wide dynamic range (WDR) neurons of the rat. Post-stimulus time histograms (PSTH) were summed over 12 responses to low frequency (0.5 Hz) electrical stimulation of the cutaneous receptive field. Changes in responses of dorsal horn neurons were evaluated by monitoring C-fiber input, wind-up, and the total number of spikes evoked by C-fiber activity in response to the 12 stimuli. The NK-1 receptor antagonist CP-99,994 significantly inhibited the total number of C-spikes and caused a significant reduction in wind-up without changing the C-fiber input, indicating the involvement of NK-1 receptors in wind-up. Application of SP led to an overall increase in the total number of C-fiber evoked responses of dorsal horn neurons and ('-fiber input, however, wind-up, as defined, was significantly decreased following SP. In contrast, substance P(1–7) evoked a long-lasting increase in the total number of C-fiber-related spikes which was initially sustained by a long-lasting increase in the input followed by a longer lasting increase in wind-up, an effect opposite that of CP-99,994. As NMDA activity has been previously shown to be inhibited and then potentiated by SP N-terminal activity over a similar time interval, the present data are consistent with the mediation of wind-up by NMDA and its modulation by SP N-terminal activity. Release of SP in response to noxious stimulation may, therefore, increase primary afferent C-fiber activity (input) whereas an accumulation of SP N-terminal metabolites appears to potentiate wind-up, perhaps via positive modulation of EAA activity.     

Upregulation of Group I metabotropic glutamate receptors in neurons and astrocytes in the dorsal horn following spinal cord injury

Of the glutamate receptor types, the metabotropic glutamate receptors (mGluRs) are G proteins coupled and can initiate a number of intracellular pathways leading to hyperexcitability of spinal neurons. In this study, we tested the expression of mGluRs to determine which cell types might contribute to sustained neuronal hyperexcitability in the lumbar enlargement with postoperative day (POD) 7 (early), 14 (late), and 30 (chronic phase) following spinal cord injury (SCI) by unilateral hemisection at T13 in Sprague-Dawley rats. Expression was determined by confocal analyses of immunocytochemical reaction product of neurons (NeuN positive) and astrocytes (GFAP positive) in the dorsal horn on both sides of the L4 segment. Neurons were divided into two sizes: small (<20 microm) and large (>35 microm), for physiological reasons. We report a significant increase of mGluR(1) expression in large and small neurons of the dorsal horn on both sides of the cord in late and chronic phases when compared to control sham groups. Expression of mGluR(2/3) significantly increased in large neurons on the ipsilateral (hemisected) side in the late phase. Expression of mGluR(5) significantly increased in large neurons in early, late, and chronic phases. In addition, mGluR(1) and mGluR(5) expression after hemisection was significantly increased in astrocytes in early, late, and chronic phases; whereas mGluR(2/3) did not display any significant changes. In conclusion, our data demonstrate long-term changes in expression levels of Group I mGluRs (mGluR(1) and mGluR(5)) in both neurons and astrocytes in segments below a unilateral SCI. Thus, permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following SCI.     

Simultaneous recordings of wind-up of paired spinal dorsal horn nociceptive neuron and nociceptive flexion reflex in rats

To study the sensory-motor interaction of spinal processing underlying the neuronal mechanisms of the nociceptive flexion reflex (NFR) and its temporal facilitation, 16 spinal dorsal horn (DH) wide-dynamic-range (WDR) neurons and paired 16 single motor units (SMU) from the gastrocnemius soleus muscle (GS) were simultaneously recorded using extracellular single unit and electromyographic techniques in spinal, halothane-anesthetized rats. The paired DH WDR neuron and GS SMU showed a parallel increase in the firing rate and duration of spike responses to noxious pinch stimuli applied to their common cutaneous receptive field (cRF) on the ipsilateral hind paw skin. Innocuous brush or pressure evoked no, or less, firing in the SMU but evoked a graded increase in spike responses in the simultaneously-recorded WDR neuron. Moreover, both pressure and noxious pinch stimuli evoked a short-lasting after-discharge (for several min) in the WDR neuron but without any after-discharge in the simultaneously-recorded SMU. The paired WDR neuron and SMU also showed a parallel basal response (termed as early and late components according to latency), after-discharge and wind-up of the late response to repetitively applied supra-threshold electrical stimulation (intensity: >1.5 T, duration: 1 ms and frequency: 1 Hz for 15 s). Linear regression and cross-correlation histogram analyses showed that the DH WDR neuron had a significant correlation with the simultaneously-recorded SMU and they were functionally located in the spinally-organized NFR circuitry via polysynaptic connections. Systemic administration of fentanyl, an opioid receptor agonist, resulted in a parallel, naloxone-reversible suppression of both basal late response component and wind-up response in both WDR neuron and SMU paired; however, fentanyl suppressed only the early response of the SMU without any effect on that of the DH WDR neuron. The present results provide new direct evidence showing an essential role of spinal DH WDR neurons in the mediation of spinally-organized NFR as well as its temporal facilitation (wind-up). Based on these data, the spinal DH WDR neuron seems to function as a signal discriminator or frequency encoder of multireceptive primary afferent impulses that may determine excitable level of motor output and the occurrence of a behavioral NFR via polysynaptic connections. Consequently, the spinal WDR neuron-mediated NFR and its temporal facilitation are likely to be modulated by spinal endogenous opioid peptides via opioid receptors on the nociceptive sensory components of the spinally-organized NFR circuitry.     

Number of synapses increased in the rat spinal dorsal horn after sciatic nerve transection: a stereological study

We recently found that the number of synapses in the spinal dorsal horn, as estimated by stereological techniques, increased by 86% after chronic constriction injury of sciatic nerve in rats. In this study, we aimed to reveal whether transection of sciatic nerve was also associated with a plasticity change in the number of synapses. 18 adult SD rats were randomly divided into 3 groups undergoing (i) unilateral sham operation, (ii) unilateral sciatic nerve transection, and (iii) unilateral sciatic nerve transection with postoperative medication (parecoxib) for 3 days, respectively. 28 days postoperation, the L4-6 segment of the spinal cord was removed; paraffin-embedded sections were prepared and stained with Nissl's method and synaptophysin immunohistochemistry. The optical disector (a contemporary stereological technique) was used to estimate the numbers of neurons and synapses in the spinal dorsal horn. Compared to the non-operated side, the axotomy induced a 74.3% increase in the number of synapses per unit length of spinal cord or a 67.4% increase in the ratio between the numbers of synapses and neurons in the middle tissue block from the L4-6 segment on the operated side but not in either the rostral or caudal tissue block. Parecoxib had no effect on the parameters. In conclusion, peripheral nerve injury, model for neuropathic pain, is associated with a synaptic plasticity (numerical increase) in the spinal dorsal horn.     

Spinal dorsal horn neurone targets for nociceptive primary afferents: do single neurone morphological characteristics suggest how nociceptive information is processed at the spinal level

It has become increasingly clear that nociceptive information is signalled by several anatomically distinct populations of primary afferents that target different populations of neurones in the spinal cord. It is probable that these different systems all give rise to the sensation pain and hence, an understanding of their separate roles and the processes that they employ, may offer ways of selectively targeting pain arising from different causes. The review focuses on what is known of the anatomy of neurones in LI-III of the spinal dorsal horn that are implicated in nociception. The dendritic geometry and synaptic input of the large LI neurones that receive input from primary afferents containing substance P that express neurokinin 1 (NK₁) receptors suggests that these neurones may monitor the extent of injury rather than the specific localisation of a discrete noxious stimulus. This population of neurones is also critically involved in hyperalgesia. In contrast neurones in LII with the morphology of stalked cells that receive primary afferent input from glomerular synapses may be more suitable for fine discrimination of the exact location of a noxious event such as a sting or parasite attack. The review focuses as far as possible on precisely defined anatomy in the belief that only by understanding these anatomical relationships will we eventually be able to interpret the complex processes occurring in the dorsal horn. The review attempts to be an accessible guide to a sometimes complex and highly specialised literature in this field.     

Spinal administration of capsazepine inhibits noxious evoked responses of dorsal horn neurons in non-inflamed and carrageenan inflamed rats

Vanilloid VR1 receptors are located in the dorsal horn of the spinal cord. The aim of the present study was to determine the role of spinal vanilloid receptors (VR1) during nociceptive processing in control and inflamed rats. Effects of spinal administration of capsazepine (0.5-30 microM/50 microl), a competitive VR1 antagonist, on innocuous and noxious evoked responses of spinal neurones were studied in halothane anaesthetised rats. Transcutaneous electrical-evoked neuronal responses of spinal neurones were recorded in control and carrageenan (2%, 3 h) inflamed rats. Spinal application of capsazepine did not significantly alter Abeta-fibre evoked responses of neurones, however Adelta-fibre evoked responses were significantly inhibited by capsazepine in both non-inflamed and carrageenan inflamed rats (30 microM: non-inflamed 31+/-8% of control, P<0.01: carrageenan-inflamed 43+/-6% of control, P<0.01). Similarly, the evoked C-fibre mediated post-discharge responses of spinal neurones in non-inflamed and carrageenan inflamed rats were reduced by capsazepine (30 microM: non-inflamed 41+/-14% of control, P<0.01: carrageenan-inflamed 31+/-9% of control, P<0.01). These results demonstrate a role of spinal VR1 receptors during noxious, but not innocuous transmission, at the level of the spinal cord. The degree of effect of capsazepine on evoked neuronal responses was similar in control and inflamed rats, suggesting that the role of spinal VR1 receptors is not altered following short-term peripheral inflammation. Our data suggest that following noxious peripheral stimulation, spinal VR1 receptors are activated, but the endogenous ligands mediating this effect remain to be elucidated.     

Inhibition of nociceptive responses of spinal dorsal horn neurons by periaqueductal gray stimulation (author's transl)]

In order to analyze analgesic effects of periaqueductal gray stimulation, experiments were carried out in nitrous oxide anesthetized cats. Spinal dorsal horn neurons receiving C-fiber inputs from the posterior tibial and/or superficial peroneal nerves were excited by noxious heat stimulation of the ipsilateral hindpaw. The evoked spike discharges were inhibited by repetitive stimulation of the periaqueductal gray at currents 300--900 microamperemeter. Effective sites for inhibition were distributed throughout the ventral half of the periaqueductal gray bilaterally. A monotonous relationship was found between stimulus intensity (skin temperature) and number of spike discharges; As predicted by Stevens' law, the number of spike discharges psi grew as a power function: psi = K(psi--psi0)beta where psi is the stimulus intensity, psi0 is the effective threshold, and K is a slope constant of the spike discharges versus stimulus intensity curve depending upon neurons. The exponent beta was approximately 1.0. The periaqueductal gray stimulation resulted in a decrease in the slope constant kappa, without alteration either in the threshold constant psi0 or in the exponent beta.     

Effects of electrical stimulation of thalamic nucleus submedius and periaqueductal gray on the visceral nociceptive responses of spinal dorsal horn neurons in the rat

Electrical stimulation of the nucleus submedius (Sm) has been shown to suppress the viscerosomatic reflex (VSR), which is evoked by colorectal distension (CRD). We have examined the effects of focal electrical stimulation (0.3 ms, 50 Hz, 100 microA, 10 s) of the Sm and the periaqueductal gray (PAG) on the excitatory responses evoked by CRD in spinal dorsal horn neurons within the L6-S1 region in the urethane-anesthetized Wistar rats. Extracellular recordings were made from 32 spinal excitatory CRD responses. All of these neurons were convergent neurons with cutaneous receptive fields. The majority of the neurons (27/32) were wide dynamic range (WDR) neurons (responding to noxious and non-noxious cutaneous stimuli) while the remaining five neurons were nociceptive specific (NS) neurons (responding only to noxious cutaneous stimuli). The effects of electrical stimulation applied to 28 sites within the Sm were assessed for spinal neurons. Electrical stimulation in seven sites within the Sm (25%) inhibited the CRD excitatory response of dorsal horn neurons, while in two sites (7%) the same stimulation yielded facilitation. Electrical stimulation in the majority of the sites in the Sm (19/28, 68%) did not affect spinal excitatory CRD responses. On the other hand, electrical stimulation of the PAG clearly inhibited 20 of 22 (90%) CRD excitatory responses. These results suggest that the majority of Sm neurons may suppress VSR activity at a supraspinal reflex center rather than via a descending inhibition of spinal visceral nociceptive transmission, as is the case for the PAG.     

A-fibres sprouting from lamina I into lamina II of spinal dorsal horn after peripheral nerve injury in rats

We have examined the labeling pattern in the spinal dorsal horn by an intra-sciatic nerve injection of cholera toxin B subunit conjugated horseradish peroxidase (HRP) after transection of the posterior cutaneous nerve and inferior gluteal nerve, and found that the cholera toxin B subunit conjugated HRP labeling in lamina I was expanding into lamina II and there was a shrinking gap between lamina I and lamina III. This result suggests that A-fibre sprouting arise after peripheral nerve injury, but mainly from small calibre Adelta-fibres which terminate in lamina I.     

Temporal and spatial distribution of Fos protein in the lumbar spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve

The temporal and spatial expression pattern of Fos protein in spinal dorsal horn neurons was examined by immunohistochemistry in rats with chronic constriction injury (CCI) to the sciatic nerve. In normal animals, a few Fos-immunoreactive (-IR) neurons were detected in the dorsal horn of the lumbar spinal cord. Following induction of CCI, a very large number of Fos-IR neurons appeared in the spinal dorsal horn, but a significant number of Fos-IR neurons were also observed in the contralateral dorsal horn where primary afferents of the injured sciatic nerve rarely project. Sham-operated animals also had a significant number of Fos-IR neurons in the dorsal horn bilaterally. The number of Fos-IR neurons reached its maximal level 1 day following placement of the ligatures (PO 1d). The ratio of the number of Fos-IR neurons in the ipsilateral dorsal horn to the contralateral dorsal horn, however, had its peak level 3 days following CCI (3.1-fold increase compared to the contralateral dorsal horn). The number of Fos-IR neurons in the dorsal horn gradually decreased, but increased again around PO 15d. On PO 30d, the number of Fos-IR neurons decreased and became comparable to that in normal animals. The present results indicate that the induction of Fos-IR neurons in the dorsal horn caused by CCI is biphasic and reaches its maximal level on PO 3d, near the time of hyperalgesia onset.