Neurotrophins and hyperalgesia

Nerve growth factor (NGF), a member of the neurotrophin family, is crucial for survival of nociceptive neurons during development. Recently, it has been shown to play an important role in nociceptive function in adults. NGF is up-regulated after inflammatory injury of the skin. Administration of exogenous NGF either systemically or in the skin causes thermal hyperalgesia within minutes. Mast cells are considered important components in the action of NGF, because prior degranulation abolishes the early NGF-induced component of hyperalgesia. Substances degranulated by mast cells include serotonin, histamine, and NGF. Blockade of histamine receptors does not prevent NGF-induced hyperalgesia. The effects of blocking serotonin receptors are complex and cannot be interpretable uniquely as NGF losing its ability to induce hyperalgesia. To determine whether NGF has a direct effect on dorsal root ganglion neurons, we have begun to investigate the acute effects of NGF on capsaicin responses of small-diameter dorsal root ganglion cells in culture. NGF acutely conditions the response to capsaicin, suggesting that NGF may be important in sensitizing the response of sensory neurons to heat (a process that is thought to operate via the capsaicin receptor VR1). We also have found that ligands for the trkB receptor (brain-derived neurotrophic factor and neurotrophin-4/5) acutely sensitize nociceptive afferents and elicit hyperalgesia. Because brain-derived neurotrophic factor is up-regulated in trkA positive cells after inflammatory injury and is transported anterogradely, we consider it to be a potentially important peripheral component involved in neurotrophin-induced hyperalgesia.

Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.

Circuitry for color coding in the primate retina.

Human color vision starts with the signals from three cone photoreceptor types, maximally sensitive to long (L-cone), middle (M-cone), and short (S-cone) wavelengths. Within the retina these signals combine in an antagonistic way to form red-green and blue-yellow spectral opponent pathways. In the classical model this antagonism is thought to arise from the convergence of cone type-specific excitatory and inhibitory inputs to retinal ganglion cells. The circuitry for spectral opponency is now being investigated using an in vitro preparation of the macaque monkey retina. Intracellular recording and staining has shown that blue-ON/yellow-OFF opponent responses arise from a distinctive bistratified ganglion cell type. Surprisingly, this cone opponency appears to arise by dual excitatory cone bipolar cell inputs: an ON bipolar cell that contacts only S-cones and an OFF bipolar cell that contacts L- and M-cones. Red-green spectral opponency has long been linked to the midget ganglion cells, but an underlying mechanism remains unclear. For example, receptive field mapping argues for segregation of L-and M-cone signals to the midget cell center and surround, but horizontal cell interneurons, believed to generate the inhibitory surround, lack opponency and cannot contribute selective L- or M-cone input to the midget cell surround. The solution to this color puzzle no doubt lies in the great diversity of cell types in the primate retina that still await discovery and analysis.

Emergence of order in visual system development.

Neural connections in the adult central nervous system are highly precise. In the visual system, retinal ganglion cells send their axons to target neurons in the lateral geniculate nucleus (LGN) in such a way that axons originating from the two eyes terminate in adjacent but nonoverlapping eye-specific layers. During development, however, inputs from the two eyes are intermixed, and the adult pattern emerges gradually as axons from the two eyes sort out to form the layers. Experiments indicate that the sorting-out process, even though it occurs in utero in higher mammals and always before vision, requires retinal ganglion cell signaling; blocking retinal ganglion cell action potentials with tetrodotoxin prevents the formation of the layers. These action potentials are endogenously generated by the ganglion cells, which fire spontaneously and synchronously with each other, generating "waves" of activity that travel across the retina. Calcium imaging of the retina shows that the ganglion cells undergo correlated calcium bursting to generate the waves and that amacrine cells also participate in the correlated activity patterns. Physiological recordings from LGN neurons in vitro indicate that the quasiperiodic activity generated by the retinal ganglion cells is transmitted across the synapse between ganglion cells to drive target LGN neurons. These observations suggest that (i) a neural circuit within the immature retina is responsible for generating specific spatiotemporal patterns of neural activity; (ii) spontaneous activity generated in the retina is propagated across central synapses; and (iii) even before the photoreceptors are present, nerve cell function is essential for correct wiring of the visual system during early development. Since spontaneously generated activity is known to be present elsewhere in the developing CNS, this process of activity-dependent wiring could be used throughout the nervous system to help refine early sets of neural connections into their highly precise adult patterns.

Multineuronal codes in retinal signaling.

The visual world is presented to the brain through patterns of action potentials in the population of optic nerve fibers. Single-neuron recordings show that each retinal ganglion cell has a spatially restricted receptive field, a limited integration time, and a characteristic spectral sensitivity. Collectively, these response properties define the visual message conveyed by that neuron's action potentials. Since the size of the optic nerve is strictly constrained, one expects the retina to generate a highly efficient representation of the visual scene. By contrast, the receptive fields of nearby ganglion cells often overlap, suggesting great redundancy among the retinal output signals. Recent multineuron recordings may help resolve this paradox. They reveal concerted firing patterns among ganglion cells, in which small groups of nearby neurons fire synchronously with delays of only a few milliseconds. As there are many more such firing patterns than ganglion cells, such a distributed code might allow the retina to compress a large number of distinct visual messages into a small number of optic nerve fibers. This paper will review the evidence for a distributed coding scheme in the retinal output. The performance limits of such codes are analyzed with simple examples, illustrating that they allow a powerful trade-off between spatial and temporal resolution.

Spatial organization of retinal information about the direction of image motion.

The visual stimuli that elicit neural activity differ for different retinal ganglion cells and these cells have been categorized by the visual information that they transmit. If specific visual information is conveyed exclusively or primarily by a particular set of ganglion cells, one might expect the cells to be organized spatially so that their sampling of information from the visual field is complete but not redundant. In other words, the laterally spreading dendrites of the ganglion cells should completely cover the retinal plane without gaps or significant overlap. The first evidence for this sort of arrangement, which has been called a tiling or tessellation, was for the two types of "alpha" ganglion cells in cat retina. Other reports of tiling by ganglion cells have been made subsequently. We have found evidence of a particularly rigorous tiling for the four types of ganglion cells in rabbit retina that convey information about the direction of retinal image motion (the ON-OFF direction-selective cells). Although individual cells in the four groups are morphologically indistinguishable, they are organized as four overlaid tilings, each tiling consisting of like-type cells that respond preferentially to a particular direction of retinal image motion. These observations lend support to the hypothesis that tiling is a general feature of the organization of information outflow from the retina and clearly implicate mechanisms for recognition of like-type cells and establishment of mutually acceptable territories during retinal development.

Radial and tangential dispersion patterns in the mouse retina are cell-class specific.

The retina is derived from a pseudostratified germinal zone in which the relative position of a progenitor cell is believed to determine the position of the progeny aligned in the radial axis. Such a developmental mechanism would ensure that radial arrays of cells which comprise functional units in the mature central nervous system are also clonally related. The present study has tested this hypothesis by using X chromosome-inactivation transgenic mosaic mice. We report that the retina shows a conspicuous distinction for clonally related neuroblasts of different laminar and functional fates: the rod photoreceptor, Müller, and bipolar cells are aligned in the radial axis, whereas the cone photoreceptor, horizontal, amacrine, and ganglion cells are tangentially displaced with respect to them. These results indicate that the dispersion of cell classes across the retinal surface is differentially constrained. Some classes of retinal neuroblast exhibit a significant tangential, as well as radial, component in their dispersion from the germinal zone, whereas others disperse only in the radial dimension. Consequently, the majority of radial columns within the mature retina must be derived from multiple progenitors. Because the cone photoreceptor, horizontal, amacrine, and ganglion cells establish nonrandom matrices in their cellular distributions within the respective retinal layers, tangential dispersion may be the means by which these matrices are constructed.

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Endothelin-B receptor is expressed by neural crest cells in the avian embryo.

Disruptions of the genes encoding endothelin 3 (EDN3) and its receptor endothelin-B receptor (EDNRB) in the mouse result in defects of two neural crest (NC)-derived lineages, the melanocytes, and the enteric nervous system. To assess the mechanisms through which the EDN3/EDNRB signaling pathway can selectively act on these NC derivatives, we have studied the spatiotemporal expression pattern of the EDNRB gene in the avian embryo, a model in which NC development has been extensively studied. For this purpose, we have cloned the quail homologue of the mammalian EDNRB cDNA. EDNRB transcripts are present in NC cells before and during their emigration from the neural tube at all levels of the neuraxis. At later developmental stages, the receptor remains abundantly expressed in the peripheral nervous system including the enteric nervous system. In a previous study, we have shown that EDN3 enhances dramatically the proliferation of NC cells when they are at the pluripotent stage. We propose that the selective effect of EDN3 or EDNRB gene inactivation is due to the fact that both melanocytes and enteric nervous system precursors have to colonize large embryonic areas (skin and bowel) from a relatively small population of precursors that have to expand considerably in number. It is therefore understandable that a deficit in one of the growth-promoting pathways of NC cells has more deleterious effects on long-range migrating cells than on the NC derivatives which develop close to the neural primordium like the sensory and sympathetic ganglia.

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.