Reconstruction of neurons of dorsal horn proper using Golgi-stained serial sections

Three neurons located in the dorsal horn (Lamina III) have been reconstructed using Golgi-stained serial sections. These cells including dendritic and axonal branchings appear to remain within the dorsal horn and to belong to small-sized local interneurons of the dorsal horn proper. The measurements have been made with various criteria: the medio-lateral, dorso-ventral and rostro-caudal extents of the dendritic tree as well as the axonal branchings, total dendritic and axonal length, number of axonal endings and distance from the starting point of the axon at the dendrite to the point where the dendrite joins the soma (S-A distance).     

Quantitative analysis of substance P-immunoreactive boutons on physiologically characterized dorsal horn neurons in the cat lumbar spinal cord

A quantitative analysis of substance P (SP)-immunoreactive (IR) terminals contacting physiologically characterized dorsal horn neurons was performed. Three types of neuron were studied: nociceptive specific (NS) from lamina I (n = 3), wide dynamic range (WDR) from laminae II-IV (n = 3), and nonnociceptive (NN) from lamina IV (n = 3). The nociceptive response of focus was a slow, prolonged depolarization to noxious stimuli, because this response was previously shown to be blocked by selective neurokinin-1 (NK-1) receptor antagonists. Ultrastructural immunocytochemistry was used to quantify the relative number of SP-IR boutons apposed to the intracellularly labeled cell per unit of length (density). Densities of the total population (SP immunoreactive+nonimmunoreactive) of apposed boutons were similar in all three regions (cell body, proximal and distal dendrites) for the three functional types of neuron. NS neurons received a significantly higher density of appositions from SP-IR boutons than NN cells in all three regions. However, compared to WDR cells, NS cells possessed a significantly higher density of appositions from SP-IR boutons only in the cell body and proximal dendrites. WDR cells had a higher density of appositions from SP-IR boutons than NN cells, but only in the proximal and distal dendrites. On average, 33.5% of the SP-IR boutons apposed to the cells displayed a synaptic contact. Finally, 30-45% of the SP-IR boutons apposed to the cells colocalized calcitonin gene-related protein (CGRP) immunoreactivity, indicating their primary sensory origin. The data indicate a direct correlation between the amount of SP-IR input and the nociceptive nature of the cells and suggest that SP acts on NK-1 receptors at a short distance from its release site.     

Localization of glutamate receptors in dorsal horn of rat spinal cord

Immunocytochemical localization of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (NMDA-type: NMDAR1 and NMDAR2A-C; AMPA-type: GluR1-4) was performed on sections of rat dorsal horn. Immunoreactivity for mGluR1 alpha was detected in laminae I-III of the dorsal horn, whilst mGluR2/3 immunoreactivity was detected primarily in lamina III. Immunoreactivity for NMDAR1, GluR1, GluR2, GluR2/3, GluR4 and GluR5/6/7 was strongly localized in neuronal elements of laminae I-III. Immunoreactivity for NMDAR2B was localized in laminae I-III. No mGluR5, NMDAR2A and NMDAR2C immunoreactivity was detected. In addition, immunoreactivity for receptors was found to co-localize with immunoreactivity for glutamate in the dorsal horn. The present results indicate that glutamate receptors are differentially localized in neuronal elements of dorsal horn where receptor-neurotransmitter interaction takes place.     

Release of nociceptin-like substances from the rat spinal cord dorsal horn

Electrophysiological studies have shown that 1-Hz repetitive transcranial magnetic stimulation (rTMS) of the primary motor area (M1) can produce a local decrease in excitability. Functional imaging data suggest that this change may be bilateral. In normal subjects, we measured motor evoked potential (MEP) amplitude at a series of stimulation intensities in the contralateral M1 before and after 15 min of active or sham rTMS at just above the MEP threshold. The slope of the curve relating MEP amplitude and stimulation intensity was decreased in the unstimulated hemisphere by active but not sham rTMS. This demonstrates that rTMS can condition cortical excitability at a distance of one or more synapses and suggest that decreased excitability to TMS is a correlate of decreased blood flow and metabolism.     

Selective cortical and segmental control of primary afferent depolarization of single muscle afferents in the cat spinal cord

This study was primarily aimed at investigating the selectivity of the cortico-spinal actions exerted on the pathways mediating primary afferent depolarization (PAD) of muscle spindle and tendon organ afferents ending within the intermediate nucleus at the L6-L7 segmental level. To this end we analyzed, in the anesthetized cat, the effects produced by electrical stimulation of sensory nerves and of the cerebral cortex on (a) the intraspinal threshold of pairs of single group I afferent fibers belonging to the same or to different hindlimb muscles and (b) the intraspinal threshold of two collaterals of the same muscle afferent fiber. Afferent fibers were classified in three categories, according to the effects produced by stimulation of segmental nerves and of the cerebral cortex. Twenty-five of 40 fibers (62.5%) were depolarized by stimulation of group I posterior biceps and semitendinosus (PBSt) or tibialis (Tib) fibers, but not by stimulation of the cerebral cortex or of cutaneous and joint nerves, which instead inhibited the PBSt- or Tib-induced PAD (type A PAD pattern, usually seen in Ia fibers). The remaining 15 fibers (37.5%) were all depolarized by stimulation of the PBSt or Tib nerves and the cerebral cortex. Stimulation of cutaneous and joint nerves produced PAD in 10 of those 15 fibers (type B PAD pattern) and inhibited the PBSt- or Tib-induced PAD in the 5 remaining fibers (type C PAD pattern). Fibers with a type B or C PAD pattern are likely to be Ib. Not all sites in the cerebral cortex inhibited with the same effectiveness the segmentally induced PAD of group I fibers with a type A PAD pattern. With the weakest stimulation of the cortical surface, the most effective sites that inhibited the PAD of individual fibers were surrounded by less effective sites, scattered all along the motor cortex (area 4gamma and 6) and sensory cortex (areas 3, 2 and 1), far beyond the area of projection of group I fibers from the hindlimb. With higher strengths of cortical stimulation, the magnitude of the inhibition was also increased, and previously ineffective or weakly effective sites became more effective. Maps obtained when using the weakest cortical stimuli have indicated that the most effective regions that produced PAD of group I fibers with a type B or type C PAD pattern were also scattered throughout the sensory-motor cortex, in the same general area as those that inhibited the PAD of group I afferents with a type A PAD pattern. In eight fibers with a type A PAD pattern it was possible to examine the intraspinal threshold of two collaterals of the same single afferent fiber ending within the intermediate nucleus at the L7 segmental level. In six fibers, stimulation of the PBSt nerve with trains of pulses between 1.5 and 1.86 times threshold (xT) produced a larger PAD in one collateral than in the other. In seven fibers, stimulation of the sensory-motor cortex and of cutaneous nerves produced a larger inhibition of the PBSt-induced PAD in one collateral than in the other. The ratio of the cortically induced inhibition of the PAD elicited in the two collaterals could be modified by changing the strength of cortical and of PBSt stimulation. In three fibers it was possible to inhibit almost completely the background PAD elicited in one collateral while having little or no effect on the PAD in the other collateral. Changes in the intraspinal threshold of pairs of collaterals following electrical stimulation of segmental nerves and of the somato-sensory cortex were examined in three fibers with a type B and two fibers with a type C PAD pattern. In four fibers the PAD elicited by stimulation of cutaneous (4-20xT) and muscle nerves (1.54-3.7xT), or by stimulation of the sensory-motor cortex, was of different magnitude in the two collaterals. In two experiments it was possible to find cortical sites in which weak surface stimulation produced PAD in one collateral only. (ABSTRACT TRUNCATED)     

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats. J. Neurophysiol. 80: 3356-3360, 1998. Glutamate is a major fast transmitter between primary afferent fibers and dorsal horn neurons in the spinal cord. Recent evidence indicates that ATP acts as another fast transmitter at the rat cervical spinal cord and is proposed to serve as a transmitter for nociception and pain. Sensory synaptic transmission between dorsal root afferent fibers and neurons in the superficial dorsal horn of the lumbar spinal cord were examined by whole cell patch-clamp recording techniques. Experiments were designed to test if ATP could serve as a transmitter at the lumbar spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were completely abolished after the blockade of both glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl--aspartate receptors. No residual current was detected, indicating that glutamate but not ATP is a fast transmitter at the dorsal horn of the lumbar spinal cord. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a selective P2x receptor antagonist, produced an inhibitory modulatory effect on fast EPSCs and altered responses to paired-pulse stimulation, suggesting the involvement of a presynaptic mechanism. Intrathecal administration of PPADS did not produce any antinociceptive effect in two different types of behavioral nociceptive tests. The present results suggest that ATP P2x2 receptors modulate excitatory synaptic transmission in the superficial dorsal horn of the lumbar spinal cord by a presynaptic mechanism, and such a mechanism does not play an important role in behavioral responses to noxious heating. The involvement of other P2x subtype receptors, which is are less sensitive to PPADS, in acute nociceptive modulation and persistent pain remains to be investigated.     

NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau1, 24 vs 42 msec; tau2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco-lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12-T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.     

Increased response to sural nerve input in the dorsal horn following chronic spinal cord hemisection

Monosynaptic input from sural nerve afferents to dorsal horn neurons was mapped bilaterally using electrical stimulation in normal cats and cats with spinal cord hemisections. Animals hemisected 6 h-5 days previously did not differ significantly from normals and the sides of the cord did not differ in either group. In animals hemisected 88-182 days previously there were significantly more sites responsive to sural nerve input ipsilateral to the hemisection, than contralateral to it.     

Identified hair follicle afferent boutons in the spinal cord of the cat are enriched with L-glutamate-like immunoreactivity

Hair follicle afferent boutons in the spinal dorsal horn of the cat were examined for L-glutamate enrichment. Two hair follicle afferent axons were labelled intra-axonally with horseradish peroxidase, and post-embedding immunogold reactions of L-glutamate were performed on thin sections containing horseradish peroxidase-labelled boutons. Quantitative analysis showed that hair follicle boutons were associated with immunogold reactions for L-glutamate which were almost twice as dense as average values for dorsal horn tissue. Further analysis revealed that hair afferent boutons displayed 2.3-times the average gold particle density associated with post-synaptic dendrites and 3-times the average immunogold density for L-glutamate of structures which were known to be immunoreactive for GABA. This enrichment of L-glutamate in identified hair afferent terminals supports the idea that the amino acid is a neurotransmitter of hair follicle primary afferent axons.     

Nociceptive integration in the rat spinal cord: role of non-linear membrane properties of deep dorsal horn neurons

Deep dorsal horn neurons (DHNs) involved in nociception can relay long-lasting inputs and generate prolonged afterdischarges believed to enhance the transfer of nociceptive responses to the brain. We addressed the role of neuronal membrane properties in shaping these responses, by recording lamina V DHNs in a slice preparation of the rat cervical spinal cord. Of 256 neurons, 102 produced accelerating discharges in response to depolarizing current pulses, whereas the other neurons showed spike frequency adaptation. Two mechanisms mediated the firing acceleration: a slow inactivation of a K+ current expressed upon activation of the neuron from hyperpolarized holding potentials, and the expression of a regenerative plateau potential activating around resting membrane potential. The increase in firing frequency was much stronger when sustained by the plateau potential (71 DHNs, 28%). A few neurons produced adaptation and both types of acceleration, in different membrane potential domains, showing that the firing pattern of a deep DHN is not a rigid characteristic. Plateau potentials could be elicited by stimulation of nociceptive primary afferent fibres. The bistability associated with plateau potentials permitted afterdischarges. Because plateau potentials had slow activation kinetics and were voltage-dependent, the neurons had non-linear input-output relationships in both the amplitude and time domains. Nociceptive primary afferent stimulation elicited intense and prolonged responses in plateau-generating DHNs, while brief bursts of spikes were evoked otherwise. These results indicate that in a population of deep DHNs, intense firing and prolonged afterdischarges in response to nociceptive stimulation depend on non-linear intrinsic membrane properties.     

Dynorphin mRNA expression in dorsal horn neurons after traumatic spinal cord injury: temporal and spatial analysis using in situ hybridization

Dynorphin, an endogenous opioid, may contribute to secondary nervous tissue damage following spinal cord injury. The temporal and spatial distribution of preprodynorphin (PPD) mRNA expression in the injured rat spinal cord was examined by in situ hybridization. Rats were subjected to traumatic spinal cord injury at the T13 spinal segment using the weight-drop method. Motor function of these rats was evaluated by their ability to maintain their position on an inclined plane. Two double-labeling experiments revealed that increased PPD mRNA and dynorphin peptide expression were found exclusively in dorsal horn neurons. Neurons exhibiting an increase in the level of PPD mRNA were concentrated in the superficial laminae and the neck of dorsal horn within several spinal segments from the epicenter of the injury at 24 and 48 h after injury. A number of neurons showing increased PPD mRNA were found in gray matter adjacent to the injury areas. Segments caudal to the injury site exhibited a long-lasting elevation of PPD mRNA in neurons, compared to the rostral segments. The number of neurons expressing PPD mRNA in each rat was significantly positively correlated with its motor dysfunction. These findings suggest that increased expression of dynorphin mRNA and peptide in dorsal horn neurons occurs after traumatic spinal cord injury. This also supports the hypothesis that the dynorphin has a pathological role in secondary tissue damage and neurological dysfunction after spinal cord injury.     

Steroid anaesthesia (Althesin - CT 1341) and dorsal horn cell activities in feline spinal cord

The effects of steroid anaesthesia (Althesin -CT 1341) on dorsal horn cells (laminae 4 and 5) were studied by extracellular recordings in the spinal cat. I.v. administration of Althesin (0.2 ml/kg) strongly depressed the spontaneous and evoked activities of both types of cells. No differences were found between cells activated by noxious or innocuous stimuli. These results emphasize the fact that the transmission of afferent messages is depressed by various anaesthetics at the level of the first synapses in the CNS. The depressive effects on lamina 5 cells could explain in part the analgesic effects of Althesin.     

Effects of caudal traction of the spinal cord on evoked spinal cord potentials in the cat

This study attempts clarify the mechanism of neurological deficits in tethered cord syndrome using evoked spinal cord potentials (ESCPs). ESCPs in response to both sciatic nerve (SN-ESCP) and spinal cord stimulation (SC-DESCP) were recorded from the dorsal epidural space. With a fixed degree of caudal traction on the spinal cord in ten cats for 2-4 hours, ESCPs were increased in amplitude in the N1 and N2 deflections of the SC-DESCPs to 158% and 154% at L5 and decreased to 91% and 76% after transient augmentation at L3. On the other hand, the amplitude in the N1 deflection of the SN-ESCPs at L3 and L5 was decreased to 40% and 68%. These findings suggest that not only the force but also the duration of traction influence the degree of the spinal cord dysfunction. When the spinal cords of 17 cats received compression with traction and without traction, the SN-ESCPs of the former became positive earlier than that of the latter. The extent of the recovery in amplitude of both SC-DESCPs and SN-ESCPs propagated over compression site was far limited in the former than in the latter. These results would indicate that the spinal cord subjected to traction is vulnerable to compression.     

Slow intrinsic optical signals in the rat spinal dorsal horn in slice

Tetanic stimulation of high-threshold primary afferent fibers in the dorsal root was found to elicit intrinsic optical signals (IOSs) in transverse slices of 11- to 20-day-old rat spinal cords. The IOS, lasting for 30 s or longer, was most prominent in the lamina II of the dorsal horn. Treatment with a Na+-K+-2Cl- co-transport blocker, furosemide, abolished the IOS, suggesting that the origin of the IOS is the cellular swelling due to an activity-dependent rise in extracellular K+. Substance P antagonist spantide, glutamate antagonists 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, and the mu-opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin suppressed IOSs. Thus, IOSs represent at least in part the slow excitatory response that is known to be generated in dorsal horn neurons after tetanic activation of unmyelinated afferent fibers.