Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.     

The distribution and origin of VIP in the spinal cord of six mammalian species

The distribution of VIP-immunoreactivity was studied in the spinal cord and dorsal root ganglia of 6 mammalian species. Immunoreactive fibres and cell bodies were most apparent in the dorsal horn, dorsolateral funiculus, intermediolateral cell columns and the area around the central canal. The distribution of VIP immunoreactivity was similar in all species studied, mouse, rat, guinea pig, cat, horse and the marmoset monkey. There were fewer VIP fibres in the dorsal horn of cervical and thoracic segments than in lumbosacral segments. Using radioimmunoassay this gradient increase was quantitatively most marked in the sacral spinal cord of the cat. In dorsal root ganglia few nerve cell bodies but numerous fibres were present. A dual origin for VIP in the spinal cord is suggested: (A) Extrinsic, from dorsal root afferent fibres since immunoreactivity was decreased in dorsally rhizotomized animals (cats and rats) and in capsaicin pretreated rats (microinjection of dorsal root ganglia). (B) From local cell bodies intrinsic to the spinal cord which became visible after colchicine pretreatment of rats.     

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.     

可视膜片钳法记录初级传入纤维介导的脊髓背角神经元突触后电流

本文描述了用明胶半包埋法制备带背根脊髓薄片的实验步骤,和在脊髓背角记录由初级传入纤维介导的突触后电流的可视膜片钳法。手术制备一段带背根的脊髓标本,并用20％的明胶包埋在琼脂块上,再用振动切片机切片获得带背根的脊髓薄片。通过红外线可视的引导,在脊髓背角神经元上建立全细胞封接模式。在钳制电压为-70mV条件下,记录自发的和背根刺激引起的兴奋性突触后电流。以传入纤维的传导速度与刺激阈值为指标,可以区分A样纤维与C样纤维兴奋性突触后电流。在钳制电压为0mV条件下,记录自发的和背根刺激引起的抑制性突触后电流。用5μmol／L的士宁或20μmol／L的荷包牡丹碱分离出γ-氨基丁酸能或甘氨酸能的抑制性突触后电流。用可视膜片钳方法可以准确测量脊髓背角神经元的突触后电流,从而研究初级传入突触的传递过程。更重要的是,在红外线可视观察的帮助下,建立膜片钳封接的成功率显著提高,同时也使记录研究脊髓背角深层神经元变得更加容易。本研究为探索初级传入突触传递过程提供了一个有效的方法。     

Specific effects of alpha-D,L-aminoadipic acid on synaptic transmission in frog spinal cord

The sucrose gap technique was employed to investigate both synaptic and amino acid evoked responses from motoneurones or primary afferents of frog spinal cord. alpha-D,L-Aminoadipic acid (alpha-D,L-AAD) selectively antagonized responses to acidic amino acids, especially aspartate. The drug was most effective in antagonizing the polysynaptic components of synaptic potentials evoked by dorsal root or lateral column stimulation but had little effect on their monosynaptic components. The ventral root dorsal root potential which is thought to be mediated by a pathway that does not involve acidic amino acids was insensitive to alpha-D,L-AAD. These data, which were confirmed by intracellular recording from motoneurones, provided further evidence for the role of acidic amino acids in polysynaptic pathways in frog spinal cord.     

Long-range afferents in the rat spinal cord. II. Arborizations that penetrate grey matter.

1. The caudal extent of the collateral arborizations of entering sensory fibres in rat spinal cord was investigated by two methods: bulk labelling of peripheral nerves by injection of horseradish peroxidase conjugated to cholera toxin (B-HRP) and by antidromic stimulation using small currents from microelectrodes in the spinal cord while recording from single units in peripheral nerve or dorsal root. 2. The results show that injection of B-HRP into the sural or sciatic nerve labelled sural afferents in the grey matter three to four segments caudal to their root entry and sciatic nerve fibres were located in S4, the most caudal segment examined, four to six segments caudal to their root entry. 3. Detailed mapping with microelectrode stimulation showed that the parent descending fibres from filaments dissected from the L1 dorsal root coursed more than 20 mm, seven to eight segments caudal to the entry point in the dorsal columns and sent branches into the grey matter. Single units from the sural nerve were also followed caudally into the S2 and S3 spinal cord segments and also issued collateral branches into the grey matter. 4. The present results suggest that there is close agreement in the caudal penetration of long-ranging afferents by using complementary anatomical and electrophysiological methods.     

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.     

Sources of porcine longissimus dorsi muscle (LDM) innervation as revealed by retrograde neuronal tract-tracing

The aim of the present study was to establish the origin of the motor, autonomic and sensory innervation of the L1-L2 segment of the porcine longissimus dorsi muscle (LDM), in order to provide morphological basis for further studies focusing on this neural pathway under experimental conditions, e.g. phototerapy and/or lateral electrical surface stimulation. To reach the goal of the study, multiple injections of the fluorescent neuronal tracer Fast Blue (FB) were made into the LDM region between the spinal processes of the vertebrae L1 and L2. The spinal cord (Th13-S1 segments) as well as the sensory and autonomic ganglia of interest, i.e., dorsal root (DRG) and sympathetic chain ganglia from corresponding spinal cord levels were collected three weeks later. FB-positive (FB+) motoneurons were observed exclusively within the nucleus ventromedialis at L1 and L2 spinal cord level, forming the most ventro-medially arranged cell column within this nucleus. Primary sensory and sympathetic chain neurons were found in appropriate ipsilateral ganglia at Th15-L3 levels. The vast majority of retrogradely traced neurons (virtually all motoneurons, approximately 76% of sensory and 99.4% of sympathetic chain ganglia neurons) was found at the L1 and L2 levels. The morphometric evaluation of FB-labeled DRG neurons showed that the majority of them (approximately 66%) belonged to the class of small-diameter perikarya (10-30 microm in diameter), whereas those of medium size (30-80 microm in diameter) and of large diameter (more than 80 microm) constituted 22.6% and 11.5% of all DRG neurons, respectively. The results of the present study demonstrated that the nerve terminals supplying porcine LDM originated from different levels of the spinal cord, dorsal root and sympathetic chain ganglia. Thus, the study has revealed sources and morphological characteristic of somatic, autonomic and spinal afferent neurons supplying porcine LDM, simultaneously pointing out the characteristic features of their distribution pattern.     

Experiments on the central pattern generator for swimming in amphibian embryos

The central nervous system of paralysed Xenopus laevis embryos can generate a motor output pattern suitable for swimming locomotion. By recording motor root activity in paralysed embryos with transected nervous systems we have shown that: (a) the spinal cord is capable of swimming pattern generation; (b) swimming pattern generator capability in the hindbrain and spinal cord is distributed; (c) caudal hindbrain is necessary for sustained swimming output after discrete stimulation. By recording similarly from embryos whose central nervous system was divided longitudinally into left and right sides, we have shown that: (a) each side can generate rhythmic motor output with cycle periods like those in swimming; (b) during this activity cycle period increases within an episode, and there is the usual rostrocaudal delay found in swimming; (c) this activity is influenced by sensory stimuli in the same way as swimming activity; (d) normal phase coupling of the left and right sides can be established by the ventral commissure in the spinal cord. We conclude that interactions between the antagonistic (left and right) motor systems are not necessary for swimming rhythm generation and present a model for swimming pattern generation where autonomous rhythm generators on each side of the nervous system drive the motoneurons. Alternation is achieved by reciprocal inhibition, and activity is initiated and maintained by tonic excitation from the hindbrain.     

相连四组神经的传入对在体肠系膜下神经节细胞兴奋的作用

运用在体细胞内电位记录法,观察了与肠系膜下神经节（IMG）相连的四组神经对IMG神经元自发电位活动的影响,结果显示扩和结肠或膀胱时,IMG细胞自发电位活动增加;切断或阻滞任一组神经均使IMG细胞电位活动受抑。其中结肠神经和腹下神经分别传导源自结肠尾段和膀胱等盆腔脏器的外周性兴奋,节间神经同时传导源自脊髓的中枢性和结肠的外周笥兴奋,肠系膜下神经节细胞的兴奋不仅源自脊髓,而且来源于结肠和膀胱。定量研究表明后者比前者对神经节细胞兴奋的影响更大。     

The dorsal root reflex in isolated mammalian spinal cord

1. The dorsal root reflex has been investigated in an isolated preparation of adult mammalian spinal cord. 2. Both evoked and spontaneous activity can be recorded from the cord in the dorsal spinal roots. 3. The spontaneous activity has a characteristic pattern of firing in bursts of action potentials. Spontaneous and evoked activity are optimum at temperatures between 25 and 27 degrees C; little activity can be detected above 35 degrees C. 4. The spontaneous dorsal root activity has been shown to be correlated with negative potentials in the dorsal horn of the cord, and intracellular recordings made from primary afferent fibres have shown spontaneous primary afferent depolarizations (PAD) which underlie the generation of the spontaneous dorsal root activity. 5. The evoked dorsal root reflex has been shown to spread up to 16 spinal segments both rostrally and caudally from the stimulated dorsal root, and to the contralateral side of the cord. 6. The spontaneous dorsal root activity in widely separated segments has been shown by cross-correlation analysis to be linked both ipsi- and contra-laterally. 7. The significance of such a widespread system for the generation of PAD is discussed.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents.

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early- and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revealed no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Electrophysiological observations on immature rat nerve cells grown in tissue culture

Intracellular measurements were made on immature cultured rat neurons using single or double barrelled microelectrodes; 361 dorsal root ganglion, DRG, cells, 105 spinal cord cells and 12 cerebellar cells were studied. Membrane potentials recorded were in the range ?25 mV to ?50 mV. A statistically significant increase in membrane potentials with time in culture was found for DRG cells during 32 days in culture. This was not found for spinal cord cells. Voltage-current curves showed a non-linearity in about 50% of DRG and spinal cord cells similar to anomalous rectification. Measurements made in DRG and spinal cord cells at ± 10 nA showed significantly greater average cell input resistance during hyperpolarising pulses than during depolarising pulses. The calculated values of Rm and Cm for DRG and spinal cord cells were similar in magnitude to values given for other cells by other workers. The value for L, the dimensionless electrotonic length, was slightly higher than that calculated for motoneurones by other workers. Differences between these results and those of others working on neurons in vitro are ascribed to the immature nature of the cells studied here.     

Electrical synapses between motoneurons in the spinal cord of the newborn rat

Ventral roots of the newborn rat spinal cord were stimulated while recording intracellularly from motoneurons. In many cells, stimulation subthreshold for an antidromic action potential in the impaled cell produced a small, short-latency depolarization, which was unaffected by membrane polarization. This response (antidromic synaptic potential, a.s.p.) was also seen, in some cells, on stimulating the ventral root of an adjacent segment. Replacement of Ca2+ (2 mM) with Mn2+ (3 mM) or Mg2+ (10 mM) completely abolished orthodromic synaptic potentials, but the a.s.p. persisted. These results strongly suggest that the a.s.p. is produced by an electrical interaction between motoneurons.     

Nerve growth factor-induced stimulation of dorsal root ganglion/spinal cord co-grafts in oculo: enhanced survival and growth of CGRP-immunoreactive sensory neurons

Intraocular co-grafts of rat fetal spinal cord and dorsal root ganglia were used to examine the enhanced survival, growth, and differentiation of sensory neurons by nerve growth factor. E14 lumbar spinal segments were implanted into the anterior eye chamber of capsaicin-pretreated rats. Two weeks later, an E14 dorsal root ganglion was implanted beside the spinal cord graft. Nerve growth factor or vehicle was injected weekly for 4 weeks into the anterior eye chamber. Co-grafts were examined weekly and, at 6 weeks, processed for calcitonin gene-related peptide (CGRP) immunofluorescence. No differences in overall size were determined for the grafts. Co-grafts treated with nerve growth factor contained many more CGRP neurons (19.4 cells/20 microm) that were significantly larger (mean 764 microm2) than neurons from control co-grafts (8.6 cells/20 microm; mean 373 microm2). In co-grafts treated with nerve growth factor, CGRP-immunoreactive fibers were extensive in the dorsal root ganglion, adjacent iris, and spinal cord compared to control co-grafts. A few CGRP-positive motoneurons were observed in the spinal cord, but no differences in number or size of motoneurons were found. The current report demonstrates that spinal cord and dorsal root ganglia can be co-grafted in oculo for long periods of time. Many dorsal root ganglion neurons survive and send peripheral processes into the iris and central processes into the spinal cord under the influence of exogenous nerve growth factor. The intraocular graft paradigm can be of use to further examine the role of neurotrophic factors in regulating or modulating dorsal root ganglion and spinal cord neurons.     

Protein associated with Myc (PAM) is involved in spinal nociceptive processing

PAM (protein associated with Myc) is a potent inhibitor of adenylyl cyclases (ACs) which is primarily expressed in neurones. Here we describe that PAM is highly expressed in dorsal horn neurones and motoneuron of the spinal cord, as well as in neurones of dorsal root ganglia in adult rats. PAM mRNA expression is differentially regulated during development in both spinal cord and dorsal root ganglia of rats, being strongest during the major respective synaptogenic periods. In adult rats, PAM expression was up-regulated in the spinal cord after peripheral nociceptive stimulation using zymosan and formalin injection, suggesting a role for PAM in spinal nociceptive processing. Since PAM inhibited Galphas-stimulated AC activity in dorsal root ganglia as well as spinal cord lysates, we hypothesized that PAM may reduce spinal nociceptive processing by inhibition of cAMP-dependent signalling. Accordingly, intrathecal treatment with antisense but not sense oligonucleotides against PAM increased basal and Galphas-stimulated AC activity in the spinal cord and enhanced formalin-induced nociceptive behaviour in adult rats. Taken together our findings demonstrate that PAM is involved in spinal nociceptive processing.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early-and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revaled no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.     

Function of gamma-aminobutyric acid receptor/channel rho 1 subunits in spinal cord

gamma-Aminobutyric acid (GABA) receptor/channel rho 1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel rho 1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel rho 1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel rho 1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel rho 1 subunit mutant mice (rho 1-/-). GABA receptor/channel rho 1 subunit expression in the rho 1-/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho 1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho 1-/- mice. A decreased threshold for mechanical pain in the rho 1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel rho 1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.     

Cell mixing in the spinal cords of mouse chimeras.

With the aim of determining whether there is significant cell mixing during development of the spinal cord, experimental chimeric mice containing two genetically distinct cell populations were produced by aggregating BALB/c or BALB/c x LPT hybrid embryos with C3H/HeN embryos. The BALB/c and LPT hybrid spinal cord cells were distinguished histochemically from the C3H/HeN spinal cord cells by using beta-glucuronidase as an independent cell genotype marker. BALB/c and LPT hybrid cells have high levels of beta-glucuronidase activity, while the C3H/HeN cells have low levels. The spinal cords of the chimeras were dissected out regionally (i.e., cervical, thoracic, and lumbar areas) and were sectioned serially. Each region was then analyzed by scoring large- and medium-sized neuronal cell bodies (greater than or equal to 10 microns) whose genotypes were distinguished by their beta-glucuronidase levels. Observations of seven chimeric mice, with coat colors that varied from one extreme (5% albino) to the other (90% albino), suggest that each chimeric spinal cord is a relatively homogeneous population throughout its length. On average only 4 to 5 percentage point differences were observed when comparing left-right, cranial-caudal, and dorsal-ventral regions within a given chimera. The cell mixing, however, is not total, and regional variations were noted. Maximum left-right differences between different spinal cord levels never exceeded 18 percentage points, while in the entire cord the maximum left-right difference was 11 percentage points. When considering dorsal-ventral differences, 18 and 15 percentage points were observed within the spinal cord levels and the entire cord, respectively. However, when comparisons were made between smaller subregions (e.g., right-dorsal-cervical vs left-ventral-lumbar), larger differences of up to 30 percentage points were observed. In addition, the genotype proportions in the spinal cord were closely correlated with the visually estimated proportions of coat color genotypes.