Carbonic anhydrase activity in primary sensory neurons

Summary Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled:(1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-m thick Vibratome sections; (3) retention of the reaction product in situ during OsO₄ post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions.     

Carbonic anhydrase activity in primary sensory neurons

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.     

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.     

Noise enhances subthreshold oscillations in injured primary sensory neurons

Noise can play a constructive role in the detection of weak signals in various kinds of peripheral receptors and neurons. What the mechanism underlying the effect of noise is remains unclear. Here, the perforated patch-clamp technique was used on isolated cells from chronic compression of the dorsal root ganglion (DRG) model. Our data provided new insight indicating that, under conditions without external signals, noise can enhance subthreshold oscillations, which was observed in a certain type of neurons with high-frequency (20-100 Hz) intrinsic resonance from injured DRG neurons. The occurrence of subthreshold oscillation considerably decreased the threshold potential for generating repetitive firing. The above effects of noise can be abolished by blocking the persistent sodium current (I(Na, P)). Utilizing a mathematical neuron model we further simulated the effect of noise on subthreshold oscillation and firing, and also found that noise can enhance the electrical activity through autonomous stochastic resonance. Accordingly, we propose a new concept of the effects of noise on neural intrinsic activity, which suggests that noise may be an important factor for modulating the excitability of neurons and generation of chronic pain signals.     

Fibronectin stimulates TRPV1 translocation in primary sensory neurons

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin (FN) plays in the dynamic modulation of transient receptor potential family V type 1 receptor (TRPV1) translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind FN. TG neurons cultured upon or treated with FN experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This FN-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on FN demonstrated an increase in capsaicin-mediated Ca²⁺ accumulation relative to neurons cultured on poly- d -lysine. Data presented from these studies indicate that FN stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying FN as a critical component of the ECM capable of sensory neuron sensitization.     

Localization of substance P immunoreactivity in phrenic primary afferent neurons

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to localize the putative neurotransmitter, substance P, in phrenic primary afferent neurons. Fast Blue was injected into the diaphragm and was found to label phrenic primary afferent neurons in sections from the fifth and sixth cervical dorsal root ganglia. The same sections were then treated with antiserum to substance P. A total of 11.4% of labelled phrenic primary afferent neurons contained substance P immunoreactivity. The diameters of the neurons ranged between 17 to 45 microns with a mean size of 29.7 +/- 0.7 microns (N = 81). The results suggest that substance P could be involved in mediating the transmission of sensory information from the diaphragm to the CNS.     

Localization of basic fibroblast growth factor in a subpopulation of rat sensory neurons

Summary The distribution of basic fibroblast growth factor (bFGF)-immunoreactivity (IR) was studied in rat sensory and autonomic ganglia. In postnatal and adult sympathetic superior cervical ganglia and in adult parasympathetic otic ganglia no bFGF-staining was found. Postnatal and adult neural crest-and placode-derived sensory ganglia displayed intensive bFGF-IR in a neuronal subpopulation. This subpopulation was characterized by use of consecutive sections of adult dorsal root ganglia stained with antibodies against substance P, somatostatin, bombesin, and bFGF. Basic FGF was colocalized with the somatostatin/bombesin subpopulation but not with substance P.     

Reduced number of unmyelinated sensory axons in peripherin null mice

Peripherin is a type III intermediate filament (IF) abundantly expressed in developing neurons, but in the adult, it is primarily found in neurons extending to the peripheral nervous system. It has been suggested that peripherin may play a role in axonal elongation and/or cytoskeletal stabilization during development and regeneration. To further clarify the function of peripherin, we generated and characterized mice with a targeted disruption of the peripherin gene. The peripherin null mice were viable, reproduced normally and did not exhibit overt phenotypes. Microscopic analysis revealed no gross morphological defects in the ventral and dorsal roots, spinal cord, retina and gut, but protein analyses showed increased levels of the type IV IF alpha-internexin in ventral roots of peripherin null mice. Whereas the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged in peripherin knockout mice, there was a substantial reduction ( approximately 34%) in the number of L5 unmyelinated sensory fibers that correlated with a decreased binding of the lectin IB4. These results demonstrate a requirement of peripherin for the proper development of a subset of sensory neurons.     

Nerve growth factor regulates central terminals of primary sensory neurons

Transection of peripheral sensory axons results in transganglionic degenerative atrophy of central terminals of the affected primary sensory neurons. Nerve growth factor applied at the central stump of the transected nerve prevents or delays transganglionic degenerative atrophy. It is concluded that, under normal conditions, nerve growth factor taken up by receptors at peripheral sensory nerve endings and transported retrogradely to perikarya in dorsal root ganglia, regulates synthesis of neuroproteins destined for maintenance of central terminals of these neurons. Accordingly, transganglionic degenerative atrophy is the consequence of failure of nerve growth factor to reach perikarya of primary sensory neurons.     

Nervous system of ascidian larvae: Caudal primary sensory neurons

Summary In larvae of Diplosoma macdonaldi one sensory nerve extends along the dorsal midline of the tail and another extends along the ventral midline. Each nerve is composed of 50–70 naked axons lying in a groove in the base of the epidermis, and each projects to the visceral ganglion. The cell bodies of the caudal sensory neurons occur in pairs within the epidermis, and are situated along the courses of the nerves. A single cilium arises from an invagination in the soma of each neuron, passes through the inner cuticular layer of the tunic and enters a tail fin formed by the outer cuticular layer. We propose that these cells are mechanoreceptors. The caudal sensory system is similar in representative species of ten families of ascidians.Abbreviations a axial complex of the tail - ac accessory centriole - ax axon - bb basal body - bl basal lamina - c cilium - cep common epidermal cells - cs ciliary sheath - dcv dense-cored vesicles - dsn dorsal sensory nerve - ec ependymal cells - ep epidermis - gj gap junction - h hemocoel - hc hemocoelic chamber - icl inner cuticular layer of the tunic - m caudal muscle - nc dorsal nerve cord - ncl neurocoel - no notochord - ocl outer cuticular layer of the tunic - sc sensory cell - sn sensory nerve - sv sensory vesicle - vg visceral ganglion - vsn ventral sensory nerve     

Dynamics of the transganglionic movement of horseradish peroxidase in primary sensory neurons

Summary The dynamics of horseradish peroxidase (HRP) transport in primary sensory neurons were studied in rats by demonstration of the reaction product in spinal nerves, spinal ganglia, dorsal roots and in the spinal cord at different survival times after application of the enzyme to the transected sciatic nerve and to the spinal cord. Using tetramethylbenzidine as the chromogen according to Mesulam (1978), transganglionic transport of HRP was shown in both the disto-proximal direction after peripheral application, and proximo-distal direction after central application. Significant differences in staining intensity between the central and peripheral processes of primary sensory neurons were found after all survival times used in this study. After peripheral application the number of labeled axons and the staining intensity were higher in spinal nerves than in dorsal roots; an inverse situation occurred after central application. These differences as well as the time sequences in staining of different parts of primary sensory neurons suggest that HRP applied to a peripheral nerve and to the spinal cord, respectively, enters the perikarya of spinal ganglion cells in any case before continuing its movement in a cellulifugal direction. Lysosomal degradation of the major portion of the applied HRP is supposed. However, in the post-perikaryal portion of a considerable number of neurons HRP-transport still occurs to a varying extent, thus resulting in labeling of nerve endings. In some neurons a post-perikaryal transport could not be detected light microscopically. The transport rates differ: the calculated transport rate of disto-proximal, cellulipetal movement in the fastest transporting neurons was 7.5 mm/h, that of the disto-proximal cellulifugal movement 2.5 to 3 mm/h.This work was partly supported by the Hartmann Müller-Stiftung I want to thank Miss Regula Eichholzer for the technical assistance     

Possible ATP release through lysosomal exocytosis from primary sensory neurons

The adenosine triphosphate (ATP) plays important roles under physiological and pathological conditions such as traumatic brain injury, neuroinflammation and neuropathic pain. In the present study, we set out to study the role of lysosomal vesicles on ATP release from the dorsal root ganglion neurons. We found that the lysosomal vesicles, which contain the quinacrine-positive fluorescence and express the vesicular nucleotide transporter (VNUT), were localized within the soma and growth cone of the cultured dorsal root ganglion neurons. In addition, the number of the quinacrine staining was decreased by application of lysosomal exocytosis activators, and this decrease was suppressed by the metformin and vacuolin-1, which suppressed lysosomal exocytosis. Thus, these findings suggest that ATP release via the lysosomal exocytosis may be one of the pathways for ATP release in response to stimulation.     

Responses of parietal associative neurons to stimulation of primary sensory areas

Responses of 251 neurons in the anterior part of the middle suprasylvian gyrus to stimulation of primary sensory (auditory, visual, somatosensory) areas and also to acoustic, visual, and somatosensory stimuli were studied in acute experiments on cats anesthetized with chloralose (40 mg/kg) and pentobarbital (20 mg/kg). Three groups of neurons were distinguished by their responses to stimulation of the primary sensory areas: those responding by an increased firing rate (117) or by inhibition (35) and those not responding (99). Responses of 193 neurons to stimulation of the peripheral afferent systems were analyzed. Neurons of the parietal associative cortex responded more frequently to cortical stimulation than to peripheral. By the duration of the latent period of their response to cortical stimulation the neurons were divided into three groups: those with short (less than 20 msec), medium (20–30 msec), and long latent periods (over 30 msec). The first group was the largest.Kemerovo State Medical Institute. Translated from Neirofiziologiya, Vol. 4, No. 5, pp. 524–530, September–October, 1972.     

Pituitary adenylate cyclase-activating polypeptide and islet amyloid polypeptide in primary sensory neurons

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, “neurogenic inflammation,” which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic β-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.     

PHR1, a PH domain-containing protein expressed in primary sensory neurons

Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a beta-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.