Peculiarities of the synaptic adaptation of the intermediate neurons of the thoracic segments of the spinal cord by cutaneous,muscle, and visceral afferents

The responses of the interneurons of the thoracic segments of the spinal cord to stimulation of the intercostal and splanchnic nerves were studied on decerebrated and narcotized cats. It was established that the neurons of different layers of the gray matter (according to Rexed) differ substantially in type of afferent inputs. Cells in laminae I–III and IV are activated chiefly by somatic afferents: primarily high-threshold in laminae I–III and low-threshold in lamina IV. The neurons of lamina V and most of the neurons of laminae VII and VIII respond to stimulation of high-threshold somatic afferents (cutaneous fibers of the A group and muscle afferents of groups II and III), as well as visceral afferents of group A, conducting impulses at a rate of 9–35 m/sec. Cells of laminae VII and VIII, monosynaptically activated by muscle afferents of group I, do not respond to stimulation of the visceral afferents. The peculiarities of the "functional" laminar organization of the thoracic segments of the spinal cord are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 6, pp. 563–572, November–December, 1970.     

Distribution of five peptides, three general neuroendocrine markers, and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: an immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I-II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I-III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I-II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Experimental morphological study of primary afferent terminals at the base of the dorsal horn of the cat spinal cord

The distribution and ultrastructure of primary afferent terminals in the gray matter of the cervical and lumbar regions of the cat spinal cord were studied by the experimental degeneration method of Fink and Heimer. Most preterminals of primary afferents were shown to be concentrated in the region of the intermediate nucleus of Cajal (central part of Rexed's laminae VI–VII), in the substantial gelatinosa (laminae II–III), and in the nucleus proprius of the dorsal horn (central and medial parts of lamina IV). Fewer are found in the region of the motor nuclei. The number of degenerating axon terminals in the lateral parts of laminae IV and V differed: 31.5 and 0.4% respectively of all axon terminals. Many terminals of primary afferents in lamina IV contribute to the formation of glomerular structures in which they exist as terminals of S-type forming axo-axonal connections with other terminals. These results are in agreement with electrophysiological data to show that interneurons in different parts of the base of the dorsal horn differ significantly in the relative numbers of synaptic inputs formed by peripheral afferents and descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 406–414, July–August, 1973.     

A monoclonal antibody (SN1) identifies a subpopulation of avian sensory neurons whose distribution is correlated with axial level

We have produced a monoclonal antibody, designated SN1, which binds to the surfaces of a subpopulation of avian sensory neurons, but not to other neurons of the peripheral or central nervous systems. The proportion of SN1(+) neurons in brachial and lumbosacral dorsal root ganglia (DRG), which innervate the wings and legs respectively, is low (30-40%), compared to the proportion (80-90%) in the lower thoracic DRG. SN1 immunoreactive fibers project to laminae I and II of the spinal cord dorsal horn, and are seen in the skin, but not the deeper tissues of older embryos. On the basis of the time of appearance, axial level-dependent distribution, and the central and peripheral projections of SN1(+) neurons, we suggest that they are cutaneous afferents that depend on interaction with peripheral targets to differentiate.     

Synaptic activation of thoracic interneurons by reticulospinal fibers of the lateral funiculus

Synaptic responses of different functional groups of interneurons in segments T10 and T11 to stimulation of the ipsilateral and contralateral medullary reticular formation were investigated in anesthetized cats with only the ipsilateral lateral funiculus remaining intact. Activation of reticulospinal fibers of the lateral funiculus with conduction velocities of 30–100 m/sec was shown to induce short-latency and, in particular, monosynptic EPSPs in all types of cells tested: in interneurons excited by group Ia muscle afferents, in cells activated only by high-threshold cutaneous and muscle afferents (afferents of the flexor reflex), in cells activated mainly by descending systems, and, to a lesser degree, in neurons connected with low-threshold cutaneous afferents. These cell populations are located mainly in the central and lateral parts of Rexed's lamina VII. Most neurons in laminae I–V of the dorsal horn, except six cells located in the superficial layers of the dorsal horn, received no reticulofugal influences. The functional organization of connections of the lateral reticulospinal tract with spinal neurons is discussed and compared with the analogous organization of the medial reticulospinal tract, and also of the "lateral" (cortico- and rubrospinal) descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 150–161, March–April, 1978.     

Neck muscle and trigeminal input to the upper cervical cord and lower medulla of the cat.

Experiments on chloralose-anaesthetized cats have shown that low-threshold neck muscle afferents project to laminae IV and V in the dorsal horn of the upper cervical cord, to lamina VI including the region which encompasses the central cervical nucleus, as well as to extensive regions of the ventral horn. At posterior medullary levels projections also exist to laminae IV, V, and VI of the spinal nucleus of V (although those to lamina IV are circumscribed), to the deep layers and lateral margin of the cuneate nucleus, and to the inferior olive. These projections are both from low- and high-threshold afferents. Evidence of a functional relationship between the trigeminal and neck muscle afferent system was found both in the upper cervical cord and lower medulla. About 40% of units in both regions receive a convergent input and when convergence could not be demonstrated, prior stimulation of one modality in some instances affected the responsiveness of the unit to the other modality. A motor role was found for some trigeminal afferent projections to the upper cervical cord. Trigeminal afferents consistently activated antidromically identified motoneurons of splenius, biventer cervicis, and complexus.     

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle,as revealed by the retrograde transport of the cholera toxin B subunit

Summary Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L₃–L₆. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.     

Transganglionic transport of the lectin Soybean agglutinin (Glycine max) following injection into the sciatic nerve of the adult rat

The lectin soybean agglutinin (SBA) from Glycine max binds to small-sized dorsal root ganglion cells and their central terminals in the superficial dorsal horn of the spinal cord. Here we investigated the ability of SBA and SBA conjugated to horseradish peroxidase(SBA-HRP) to trace thin calibre afferents into the spinal cord from a peripheral nerve. Following injection into the sciatic nerve, labelled cells in the dorsal root ganglion were predominantly small-sized but some medium-sized cells were also labelled. Colocalisation studies of transported SBA with various neuronal markers showed that all neurons that transported SBA-HRP showed SBA binding, indicating high uptake specificity for the conjugate. 15% were immunoreactive for RT97 indicating that some axons were myelinated, and 54% also expressed binding sites for isolectin B4 from Griffonia simplicifolia, a selective marker for a subpopulation of unmyelinated afferents. With regard to neurotransmitter content, 43% of the SBA cells contained calcitonin gene-related peptide, 33% contained substance P and 2.5% somatostatin. In addition, 3% contained carbonic anhydrase. Centrally, injection of SBA in the sciatic nerve resulted in labelled terminals in somatotopically appropriate regions of laminae I–II of the dorsal horn, and in the gracile nucleus. A few neurons in the dorsal horn were labelled indicating that some transneuronal transport of SBA had occurred. The results show that SBA can be used as a transganglionic tracer to label fine calibre primary afferents that project to laminae I–II of the spinal cord and the gracile nucleus. It appears to label more afferents than isolectin B4, including also a subpopulation of myelinated afferents.     

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.     

Connections of the corticospinal and rubrospinal tracts with neurons of the cervical spinal cord in cats

The distribution of focal potentials over the cross section of the 7th cervical segment of the spinal cord was studied during stimulation of the pyramids, the red nucleus, and a peripheral nerve (ulnar) in adult cats anesthetized with chloralose and Nembutal. The earliest focal potentials in the fasciculus dorsolateralis were recorded 1.4–1.5 msec after stimulation of the pyramids and 0.8–0.9 msec after stimulation of the red nucleus. These times correspond to maximal condution velocities of 56–68 and 105–124 m/sec respectively. The earliest post-synaptic activity in response to pyramidal stimulation was found in the lateral areas of laminae V and VI, and in response to stimulation of the red nucleus in laminae VI and VII in Rexed's classification. The pyramidal wave also evoked considerable postsynaptic activity in medial areas of the dorsal horn. In response to stimulation of peripheral afferents activity was evoked in neurons in the central and medial parts of laminae V and VI. It is postulated on the basis of these results that corticospinal and rubrospinal fibers may be connected monosynaptically with specialized interneurons, free from peripheral influences, in the lateral areas of laminae V and VII respectively; in the lateral part of lamina VI convergence of both types of influences on the same cells is possible. Interaction between descending and afferent influences possibly takes place on more medially located neurons.A.A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 2, pp. 158–167, March–April, 1972.     

Hot receptors in the brain

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X₃. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.     

Cortical Presynaptic Control of Dorsal Horn C–Afferents in the Rat

Lamina 5 sensorimotor cortex pyramidal neurons project to the spinal cord, participating in the modulation of several modalities of information transmission. A well-studied mechanism by which the corticospinal projection modulates sensory information is primary afferent depolarization, which has been characterized in fast muscular and cutaneous, but not in slow-conducting nociceptive skin afferents. Here we investigated whether the inhibition of nociceptive sensory information, produced by activation of the sensorimotor cortex, involves a direct presynaptic modulation of C primary afferents. In anaesthetized male Wistar rats, we analyzed the effects of sensorimotor cortex activation on post tetanic potentiation (PTP) and the paired pulse ratio (PPR) of dorsal horn field potentials evoked by C–fiber stimulation in the sural (SU) and sciatic (SC) nerves. We also explored the time course of the excitability changes in nociceptive afferents produced by cortical stimulation. We observed that the development of PTP was completely blocked when C-fiber tetanic stimulation was paired with cortex stimulation. In addition, sensorimotor cortex activation by topical administration of bicuculline (BIC) produced a reduction in the amplitude of C–fiber responses, as well as an increase in the PPR. Furthermore, increases in the intraspinal excitability of slow-conducting fiber terminals, produced by sensorimotor cortex stimulation, were indicative of primary afferent depolarization. Topical administration of BIC in the spinal cord blocked the inhibition of C–fiber neuronal responses produced by cortical stimulation. Dorsal horn neurons responding to sensorimotor cortex stimulation also exhibited a peripheral receptive field and responded to stimulation of fast cutaneous myelinated fibers. Our results suggest that corticospinal inhibition of nociceptive responses is due in part to a modulation of the excitability of primary C–fibers by means of GABAergic inhibitory interneurons.     

Neurotrophin-3 promotes the survival of a limited subpopulation of cutaneous sensory neurons

In the chick embryo, exogenous neurotrophin-3 (NT3) is sufficient to promote the differentiation of proprioceptive afferents even in the absence of limb muscle targets. To determine if NT3 can promote the differentiation of this phenotype in afferents with cutaneous targets, we analyzed the effects of chronic NT3 on cutaneous and muscle sensory neurons that express trkC, a receptor for NT3. In normal embryos, retrograde labeling and immunohistochemistry showed that about 75% of large-diameter muscle afferents express trkC, whereas only about 7% of large-diameter cutaneous afferents express this protein. After chronic treatment with NT3 during the cell death period, both populations of trkC(+) neurons were increased approximately twofold. Because this treatment is known to block cell death in sensory neurons, these results indicate that NT3 can promote the survival of both proprioceptive afferents and cutaneous afferents. To examine the phenotype of the cutaneous afferents rescued by NT3, we analyzed their projections and connections using transganglionic labeling and electrophysiological recording. The results indicate that exogenous NT3 neither altered the pattern of spinal projections nor caused cutaneous afferents to form monosynaptic connections with motor neurons. These results demonstrate that selective cell death does not contribute to the modality-specific pattern of spinal innervation and suggest that proprioceptive afferents may innervate muscle selectively.     

神经激肽-1(NK-1)受体介导福尔马林诱发的大鼠脊髓c-fos表达

本文用Ｆｏｓ作为背角伤害性反应神经元活动的一个标志物,结合免疫细胞化学和神经药理学方法,观察了速激肽受体拮抗剂对福尔马林诱发的脊髓ｃ－ｆｏｓ原癌基因表达的影响。一侧大鼠后肢跖部皮下注射福尔马林,仅在同侧脊髓背角有ｃ－ｆｏｓ表达。Ｆｏｓ阳性神经元最密集分布于Ｉ层和Ⅱ层背侧的内侧部,中等量分布于Ⅳ层和Ｖ型,少量定位于Ⅱ层腹侧部、Ⅲ、Ⅵ和Ⅹ层。若预先在一侧大鼠后肢跖部皮下注射福尔马林前,鞘内给予神经激肽     

异丙酚抑制炎性痛大鼠脊髓NOS神经元的c -fos表达

用福尔马林致痛模型、c fos基因免疫组织化学法和NADPH d组织化学技术 ,研究大鼠脊髓结构对福尔马林痛刺激的反应及异丙酚在其调节过程中的影响。结果表明 ,福尔马林痛刺激后 ,刺激侧脊髓背角出现大量Fos免疫样阳性神经元 ,其中部分为FLI/NOS双标记神经元 ;痛刺激之前或之后给予异丙酚 ,背角各层FLI神经元和FLI/NOS双标记神经元的数量均显著减少 (P <0 0 5或P <0 0 1) ;单纯腹腔注射异丙酚或生理盐水 ,脊髓未见或偶见FLI神经元。上述结果提示 :异丙酚的抗伤害作用可能与其抑制了脊髓内NOS阳性神经元的活性有关     

Intraganglionic AAV6 Results in Efficient and Long-Term Gene Transfer to Peripheral Sensory Nervous System in Adult Rats

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10⁹ viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.