Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Sciatic nerve transection in neonatal rats induces apoptotic neuronal death in L5 dorsal root ganglion

Transection of a peripheral nerve in neonatal rats induces death of the axotomized neurons which may be due to either necrosis or apoptosis. In the present investigation, neuronal cell death in L5 dorsal root ganglion was evaluated after unilateral sciatic nerve transection in rats at 1, 3, 5, 7 and 10 days age. After 5 days, right (experimental) and left (control) dorsal root ganglia in all groups were removed, fixed, processed and embedded for either light or electron microscopy. Normal nucleoli were counted in paraffin embedded serial sections, and correction factors for split and multiple nucleoli were applied as well as the physical disector. The number of neurons in the right dorsal root ganglia, as compared with the controls, was significantly lower in all groups, and the percentage of the reduction at 1, 3, 5, 7 and 10 days was 32.4, 27.2, 23.8, 22.8 and 21.8% respectively. On the other hand, the results of neuronal counts using the disector method showed 34.0, 25.7, 20.2, 20.0 and 14.2% reduction in the number of neurons at 1, 3, 5, 7 and 10 days, respectively. The microscopic and ultrastructural results indicated that there were typical morphological changes similar to those of apoptosis, including condensed basophilic nuclei, formation of nuclear caps, cell shrinkage and apoptotic body formation. We concluded that there is an increase in apoptosis in dorsal root ganglia following sciatic nerve axotomy with the greatest neuronal loss on postnatal day 1.     

Frataxin deficiency induces Schwann cell inflammation and death

Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.     

A histochemical study of the distribution of choline acetyltransferase and acetylcholinesterase activity in sensory ganglia and nerve roots of the bullfrog

Synopsis Histochemical techniques were employed for the localization of choline acetyltransferase (ChAc; EC 2.3.1.6.), acetylcholinesterase (AChE; EC 3.1.1.7) and cholinesterase (ChE; EC 3.1.1.8) activities in dorsal and ventral roots and dorsal root ganglia of the bullfrog. AChE activity was present in most of the neuronal elements of dorsal root ganglia, in some nerve fibres in the dorsal roots, and in all nerve fibres in ventral roots. ChE activity in dorsal root ganglia and in the dorsal roots was confined to non-neuronal elements. No ChE activity was demonstrable in the ventral roots. ChAc activity was localized in many neurons of the dorsal root ganglia and in some nerve fibres of the dorsal roots; however, none of the ventral root fibres were visibly reactive. Some supportive cells of the dorsal roots and ganglia contained small amounts of ChAc activity. Except for the ventral roots, the histochemical distribution of AChE and ChAc activity was similar. The results of solubility studies indicated that under the histochemical conditions, approximately 50% of the ChAc remained bound to the dorsal roots and ganglia, whereas more than 90% of the ChAc in the ventral roots was soluble. This would account for the lack of reactivity in ventral root fibres. Differences in ChAc solubility are discussed in relation to the interpretation of histochemical data and in relation to the concept of multiple forms of ChAc. The results of this study indicate that at least one-third of the neurons of the dorsal root ganglia contain significant levels of the enzymes involved in both the synthesis and hydrolysis of acetylcholine.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Distribution and changes with age of nitric oxide synthase-immunoreactive nerves of the rat urinary bladder, ureter and in lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing nitric oxide (NO) are either postganglionic parasympathetic nerves, with cell bodies in the major pelvic ganglia, or sensory nerves with cell bodies in the lumbosacral dorsal root ganglia. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive for neuronal nitric oxide synthase (NOS) in the urinary bladder, distal ureter and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made of NOS fibres innervating the dome, body and base of the urinary bladder and distal ureter. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for NOS. The dome and the body regions, in both age groups, contained no NOS-immunoreactive axons. The bladder base and distal ureter in young adults showed sparse to moderate numbers of fibres immunoreactive to NOS within the urothelium and in the subepithelium and muscle coat. In the aged rat there were slight reductions in the densities of NOS-immunoreactive nerves in all three regions. In the lumbosacral dorsal root ganglia, the percentage of NOS-immunoreactive neuronal profiles showed a significant reduction from 4.6 +/- 0.2% in young adult to 2.7 +/- 0.2% (means +/- S.E.M) in aged rats. These findings suggest that the effects of NO on the bladder and distal ureteric musculature and also its expression in dorsal root ganglion neurons are affected in aged rats and that the micturition reflex may be perturbed as a result.     

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.     

Heme oxygenase-2 in primary afferent neurons of the guinea-pig

Sensory ganglia (trigeminal, jugular, nodose, cervical and lumbar dorsal root ganglia) of the guineapig were investigated for the presence of a constitutive carbon monoxide-generating enzyme, heme oxygenase-2 (HO-2). A 36-kDa HO-2-immunoreactive protein was identified by Western blotting in protein extracts from dorsal root ganglia and localized by immunohistochemistry to all neuronal perikarya, including both substance P-positive and substance P-negative neurons, in all ganglia investigated. This ubiquitous distribution points to a general requirement for HO-2 in primary afferent neurons rather than to an association with a specific functionally defined subpopulation. Neither the axons of the sensory neurons nor their peripheral terminals in the skin and around visceral arteries were HO-2 immunoreactive. Explants of dorsal root ganglia with crushes placed on the dorsal roots showed accumulation of the neuropeptide, substance P, at the ganglionic side of the crush, but these axons were non-reactive to HO-2, indicating that there is no substantial transport of HO-2 towards the central ending of these sensory neurons. Collectively, the findings suggest that HO-2 exerts it major effects within the sensory ganglia themselves.     

A spatial and temporal analysis of dorsal root and sympathetic ganglion formation in the avian embryo

The present study explores the formation of the dorsal root and sympathetic ganglia in the trunk of the avian embryo. Particular emphasis was given to the timing of gangliogenesis and the relative positions of the neural crest-derived ganglia with respect to the somites. Neural crest cells and their derivatives were recognized by the HNK-1 antibody. The time at which neural crest cell coalesced to form ganglia was assessed by the state of cellular aggregation. The state of ganglionic differentiation was assessed by the expression of neurofilament proteins and the neural cell adhesion molecule (N-CAM). At the level of the 15th somite, neural crest cells were observed in the rostral half of the somite at stage 15, during active neural crest migration, and occupied the rostral two-thirds of the somite at progressive stages. HNK-1 positive cells appeared to be organized in three to four streams of cells oriented mediolaterally and dorsoventrally. The dorsal root ganglia and sympathetic ganglia were first detectable at stages 20 and 21, respectively. Both ganglionic rudiments were aligned with the rostral portion of the somite. The dorsal root ganglia occupied the rostral two-thirds of each somite, whereas cells in the sympathetic ganglia occupied a region corresponding to approximately one-third of each somite. At the time of condensation of the dorsal root ganglia, abundant neurofilament staining was observed within the ganglia. However, no N-CAM immunoreactivity was detected until three stages later at stage 23. In contrast, the sympathetic ganglia demonstrated both neurofilament and N-CAM immunoreactivity at the time of condensation. The observation that both dorsal root and sympathetic ganglia form in register with the rostral portion of somite suggests that cues localized at these axial levels, perhaps within the rostral somite, may influence the position where neural crest cells condense to form ganglia. In sensory ganglia, N-CAM expression does not correlate with the onset of gangliogenesis, suggesting that molecules other than N-CAM may play an important role in the aggregation of some neuronal populations.     

Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane

The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.     

Diabetes is not a potent inducer of neuronal cell death in mouse sensory ganglia,but it enhances neurite regeneration in vitro

We examined the effects of diabetes on the morphological features and regenerative capabilities of adult mouse nodose ganglia (NG) and dorsal root ganglia (DRG). By light and electron microscopy, no apoptotic cell death was detected in the ganglia obtained from either streptozotocin (STZ)-induced diabetic or normal C57BL/6J mice in vivo. Neurite regeneration from transected nerve terminals of NG and DRG explants in culture at normal (10 mM) and high (30 mM) glucose concentrations was significantly enhanced in the diabetic mice. Chromatolytic changes (i.e. swelling and migration of the nucleus to an eccentric position in the neurons, and a loss of Nissl substance in the neuronal perikarya) and apoptotic cell death (less than one-fifth of the neurons) in the cultured ganglia were present, but neither hyperglycemia in vivo nor high glucose conditions in vitro altered the morphological features of the ganglia or the ratios of apoptotic cells at 3 days in culture. By semiquantitative RT-PCR analysis, the mRNA expressions of ciliary neurotrophic factor (CNTF) in DRG from both mice were down-regulated at 1 day in culture. The expression in diabetic DRG, but not in control DRG, was significantly up-regulated at later stages (3 and 7 days) in culture. In summary, hyperglycemia is unlikely to induce cell death in the sensory ganglia, but enhances the regenerative capability of vagal and spinal sensory nerves in vitro. The up-regulation of CNTF mRNA expression during the culture of diabetic DRG may play a role in the enhanced neurite regeneration.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.     

Distribution of spinal sensory neurons supplying the adrenal gland in the pig

The present study was aimed at disclosing which spinal ganglia contribute to the innervation of the adrenal gland in the pig with special regard to the morphology and intraganglionic distribution of the primary sensory neurons within these ganglia. To this end, the animals were injected with a retrograde fluorescent neuronal tracer Fast Blue into the left adrenal gland during laparotomy. After a survival period of three weeks, labelled spinal sensory neurons were found in the ipsilateral dorsal root ganglia (approximately 99% of all retrogradely marked neurons). However, single adrenal gland-projecting perikarya were observed also in the contralateral dorsal root ganglia from Th3 to L3. The majority of the retrogradely labelled afferent neurons (above 65% of all Fast Blue-positive (FB+) perikarya) were located in two groups of spinal ganglia (at neuromeres Th6-7 and Th12-14, approximately 18% and 47% of neurons, respectively), forming two distinct multiganglionic centres of origin for this neural pathway. The morphometric evaluation of FB+ neurons revealed that the vast majority of them (approximately 81%) belonged to the class of small-sized perikarya (10-30 microm in diameter), while the medium-sized (diameter 30-80 microm) and large neurons comprised only up to 13% and 6.5% of adrenal gland-projecting neurons, respectively. Furthermore, the analysis of the intraganglionic distribution pattern of the retrogradely labelled cells revealed that the highest number of them was located in the medio-caudal domain of the dorsal root ganglia, irrespective of the neuromere studied. Thus, the present study has revealed sources and morphological characteristic of spinal afferent neurons supplying the porcine adrenal gland, simultaneously pointing out to the characteristic features of their inter- and intraganglionic distribution pattern.     

A comparison between antisense P75NTR oligonucleotides and neurotrophic factors in promoting the survival of postnatal sensory neurons in vitro

Summary The 75-kDa low-affinity neurotrophin receptor (p75^NTR) has been shown in previous reports to mediate neuronal cell death in vitro and in vivo under certain circumstances. Antisense oligonucleotides directed against p75^NTR promote the survival of nerve growth factor-deprived dorsal root ganglia sensory neurons in vitro (Barrett, G.; Bartlett, P., Proc. Natl. Acad. Sci. USA 91:6501–6505; 1994) and axotomized dorsal root ganglia sensory neurons in vivo (Cheema, S. S.; Barrett, G. L.; Bartlett, P. F., J. Neurosci. Res. 46:239–245; 1996). In this study we compared the neuroprotective effects of antisense p75^NTR oligonucleotides with two neurotrophic factors, namely nerve growth factor (NGF) and leukemia inhibitory factor, on cultured sensory neurons derived from postnatal day 7 and 14 rat dorsal root ganglia. After 3 d in culture, treatment with the neurotrophic factors had significant survival effects on sensory neuron cultures compared to treatment with basal medium (control). However, after 6 and 9 d in culture these rescue effects were not apparent. In contrast, antisense p75^NTR oligonucleotides rescued significantly higher numbers of dorsal root ganglia sensory neurons after 6 and 9 d in culture than treatment with neurotrophic factors, sense oligonucleotides, and basal medium. Furthermore, antisense p75^NTR oligonucleotides rescued trkA-, B-, and C-expressing neurons, while NGF and leukemia inhibitory factor targeted primarily the trkA-positive neurons. These findings suggest that antisense-based strategies that inhibit gene expression of cytotoxic molecules are more efficient at preventing postnatal sensory neuronal death in vitro than treatment with individual neurotrophic factors.     

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.     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.     

IKAP/Elp1 is required in vivo for neurogenesis and neuronal survival, but not for neural crest migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems.     

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.     

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.