Valérie S. Fénelon

Oxytocin neuron activation and fos expression: A quantitative immunocytochemical analysis of the effect of lactation, parturition, osmotic and cardiovascular stimulation

As c-fos expression is generally thought to be linked to neuronal activation, we compared Fos immunoreactivity in identified oxytocinergic and vasopressinergic neurons of female rats under various conditions known to elicit particular patterns of electrophysiological and secretory activity in these neurons. In suckled lactating animals, Fos immunoreactivity was visible only in rare oxytocinergic and vasopressinergic neurons of the paraventricular and supraoptic nuclei, even after interruption of suckling for 18-72 h. On the other hand, many Fos-positive cells were visible in the nuclei of parturient rats; they involved about 25% of supraoptic oxytocinergic elements. Even more Fos-positive elements were visible in the nuclei of lactating rats that had also undergone 24 h water deprivation or haemorrhage. This involved about 75% vasopressinergic neurons and 25% oxytocinergic neurons of the supraoptic nucleus. Fos immunoreactivity was particularly conspicuous in oxytocin neurons of the anterior commissural nucleus after haemorrhage. After water deprivation or haemorrhage, Fos-positive oxytocinergic neurons in the supraoptic nucleus were significantly more numerous in virgin rats than in lactating rats. Our observations show that suckling, although a most potent stimulus for oxytocin neuron activation and oxytocin release, is inefficient in inducing Fos synthesis in magnocellular neurons, even after a period of interruption. On the other hand, parturition, water deprivation and haemorrhage were more potent stimuli for both neurosecretory systems. However, under each type of stimulation, only part of the neuronal populations within each nucleus were Fos-positive, suggesting that different stimulus-specific pathways are involved in these regulations. In so far as electrical activity is one possible mechanism for c-fos expression, comparison of the patterns of c-fos activation with the known electrophysiological behaviour of hypothalamic magnocellular neurons suggests that Fos synthesis in these neurons is linked to the number of action potentials generated over a period of time, more than to the pattern of electrical activity, whatever the physiological impact of this pattern. Furthermore, within a group of neurons, the heterogeneity of the response in terms of Fos synthesis may be correlated to the variability of the electrophysiological response within this group.

Sequential developmental acquisition of cotransmitters in identified sensory neurons of the stomatogastric nervous system of the lobsters, Homarus americanus and Homarus gammarus.

We studied the developmental acquisition of three of the cotransmitters found in the gastropyloric receptor (GPR) neurons of the stomatogastric nervous systems of the lobsters Homarus americanus and Homarus gammarus. By using wholemount immunocytochemistry and confocal microscopy, we examined the distribution of serotonin‐like, allatostatin‐like, and FLRFNH2‐like immunoreactivities within the stomatogastric nervous system of embryonic, larval, juvenile, and adult animals. The GPR neurons are peripheral sensory neurons that send proprioceptive information to the stomatogastric and commissural ganglia. In H. americanus, GPR neurons of the adult contain serotonin‐like, allatostatin‐like, and Phe‐Leu‐Arg‐Phe‐amide (FLRFNH2)‐like immunoreactivities. In the stomatogastric ganglion (STG) of the adult H. americanus and H. gammarus, all of the serotonin‐like and allatostatin‐like immunoreactivity colocalizes in neuropil processes that are derived exclusively from ramifications of the GPR neurons. In both species, FLRFNH2‐like immunoreactivity was detected in the STG neuropil by 50% of embryonic development (E50). Allatostatin‐like immunoreactivity was visible first in the STG at approximately E70–E80. In contrast, serotonin staining was not clearly visible until larval stage I (LI) in H. gammarus and until LII or LIII in H. americanus. These data indicate that there is a sequential acquisition of the cotransmitters of the GPR neurons. J. Comp. Neurol. 408:318–334, 1999.

SEQUENTIAL DEVELOPMENTAL ACQUISITION OF NEUROMODULATORY INPUTS TO A CENTRAL PATTERN-GENERATING NETWORK

The activity of the adult stomatogastric ganglion (STG) depends on a large number of aminergic and peptidergic modulatory inputs. Our aim is to understand the role of these modulatory inputs in the development of the central pattern‐generating networks of the STG. Therefore, we analyze the developmental and adult expressions of three neuropeptides in the stomatogastric nervous system of the lobsters Homarus americanus and Homarus gammarus by using wholemount immunocytochemistry and confocal microscopy. In adults, red pigment‐concentrating hormone (RPCH)‐like, proctolin‐like, and a tachykinin‐like immunoreactivity are present in axonal projections to the STG. At 50% of embryonic development (E50), all three peptides stain the commissural ganglia and brain, but only RPCH‐ and proctolin‐like immunoreactivities stain axonal arbors in the STG. Tachykinin‐like immunoreactivity is not apparent in the STG until larval stage II (LII). The RPCH‐immunoreactive projection to the STG consists of two pairs of fibers. One pair stains for RPCH immunoreactivity at E50; the second RPCH‐immunoreactive pair does not stain until about LII. One pair of the RPCH fibers double labels for tachykinin‐like immunoreactivity. The adult complement of neuromodulatory inputs is not fully expressed until close to the developmental time at which major changes in the STG motor patterns occur, suggesting that neuromodulators play a role in the tuning of the central pattern generators during development. J. Comp. Neurol. 408:335–351, 1999.

Central inputs mask multiple adult neural networks within a single embryonic network

It is usually assumed that, after construction of basic network architecture in embryos, immature networks undergo progressive maturation to acquire their adult properties. We examine this assumption in the context of the lobster stomatogastric nervous system. In the lobster, the neuronal population that will form this system is at first orgnanized into a single embryonic network that generates a single rhythmic pattern. The system then splits into different functional adult networks controlled by central descending systems; these adult networks produce multiple motor programmes, distinctively different from the single output of the embryonic network. We show here that the single embryonic network can produce multiple adult-like programmes. This occurs after the embryonic network is silenced by removal of central inputs, then pharmacologically stimulated to restore rhythmicity. Furthermore, restoration of the flow of descending information reversed the adult-like pattern to an embryonic pattern. This indicates that the embryonic network possesses the ability to express adult-like network characteristics, but descending information prevents it from doing so. Functional adult networks may therefore not necessarily be derived from progressive ontogenetic changes in networks themselves, but may result from maturation of descending systems that unmask pre-existing adult networks in an embryonic system.

Ontogenetic alteration in peptidergic expression within a stable neuronal population in lobster stomatogastric nervous system

In the adult lobster, Homarus gammarus, the stomatogastric ganglion (STG) contains two well‐defined motor pattern generating networks that receive numerous modulatory peptidergic inputs from anterior ganglia. We are studying the appearance of extrinsic peptidergic inputs to these networks during ontogenesis. Neuron counts indicate that as early as 20% of development (E20) the STG neuronal population is quantitatively established. By using immunocytochemical detection of 5‐bromo‐2′‐deoxyuridine incorporation, we found no immunopositive cells in the STG by E70. We concluded that the STG neuronal population remains quantitatively stable from mid‐embryonic life until adulthood.

Fos synthesis in identified magnocellular neurons varies with phenotype, stimulus, location in the hypothalamus and reproductive state

The present study compared Fos expression in identified hypothalamic magnocellular neurons in lactating and non-lactating female rats submitted to acute haemorrhage or 24 h of water deprivation, stimuli that induce the release of both oxytocin and vasopressin. Quantitative analysis of preparations doubly immunostained for Fos and either of the neuropeptides revealed that oxytocin and vasopressin neurons synthesise Fos in response to either stimulus but to a different degree, depending on the type of neuron, the type of stimulus, the location of the neurons and the reproductive state of the animal. Thus, in terms of number of cells, haemorrhage was significantly more potent than water deprivation in inducing Fos immunoreactivity in either type of neuron in the supraoptic, paraventricular and anterior commissural nuclei. However, the Fos reaction of vasopressin cells in response to either stimulus was greater than that of oxytocin cells in the supraoptic and paraventricular nuclei, and in the perifornical posterior nucleus and nucleus circularis in response to water deprivation. Moreover, when considering each neuronal population as a whole, it was obvious that Fos synthesis varied in relation to the location of the neurons in the different hypothalamic nuclei, suggesting the existence of functionally distinct neuronal subgroups. Finally, our analyses clearly indicated that Fos synthesis in either type of magnocellular neuron was closely linked to the reproductive state of the animal since after haemorrhage or water deprivation, the number of Fos-positive oxytocin cells in the supraoptic nucleus and Fos-positive vasopressin cells in the paraventricular nucleus was significantly less in lactating than in virgin rats.

Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers.

Summary Bone cancer pain is not always correlated with spinal overexpression of reactive glia markers, in contrast to neuropathic pain. ABSTRACT Bone cancer pain is a common and disruptive symptom in cancer patients. In cancer pain animal models, massive reactive astrogliosis in the dorsal horn of the spinal cord has been reported. Because astrocytes may behave as driving partners for pathological pain, we investigated the temporal development of pain behavior and reactive astrogliosis in a rat bone cancer pain model induced by injecting MRMT‐1 rat mammary gland carcinoma cells into the tibia. Along with the development of bone lesions, a gradual mechanical and thermal allodynia and hyperalgesia as well as a reduced use of the affected limb developed in bone cancer–bearing animals, but not in sham‐treated animals. Dorsal horn Fos expression after nonpainful palpation of the injected limb was also increased in bone cancer–bearing animals. However, at any time during the evolution of tumor, there was no increase in glial fibrillary acidic protein (GFAP) immunoreactivity in the dorsal horn. Further analysis at 21 days after injection of the tumor showed no increase in GFAP and interleukin (IL) 1&bgr; transcripts, number of superficial dorsal horn S100&bgr; protein immunoreactive astrocytes, or immunoreactivity for microglial markers (OX‐42 and Iba‐1). In contrast, all these parameters were increased in the dorsal horn of rats 2 weeks after sciatic nerve ligation. This suggests that in some cases, bone cancer pain may not be correlated with spinal overexpression of reactive glia markers, whereas neuropathic pain is. Glia may thus play different roles in the development and maintenance of chronic pain in these 2 situations.

Phylogenetic, ontogenetic and adult adaptive plasticity of rhythmic neural networks: a common neuromodulatory mechanism?

Neuromodulatory inputs are known to play a major role in the adaptive plasticity of rhythmic neural networks in adult animals. Using the crustacean stomatogastric nervous system, we have investigated the role of modulatory inputs in the development of rhythmic neural networks. We found that the same neuronal population is organised into a single network in the embryo, as opposed to the two networks present in the adult. However, these adult networks pre-exist in the embryo and can be unmasked by specific alterations of the neuromodulatory environment. Similarly, adult networks may switch back to the embryonic phenotype by manipulating neuromodulatory inputs. During development, we found that the early established neuromodulatory population display alteration in expressed neurotransmitter phenotypes, and that although the population of modulatory neurones is established early, with morphology and projection pattern similar to adult ones, their neurotransmitter phenotype may appear gradually. Therefore the abrupt switch from embryonic to adult network expression occurring at metamorphosis may be due to network reconfiguration in response to changes in modulatory input, as found in adult adaptive plasticity. Strikingly, related crustacean species express different motor outputs using the same basic network circuitry, due to species-specific alteration in neuromodulatory substances within homologous projecting neurones. Therefore we propose that alterations within neuromodulatory systems to a given rhythmic neural network displaying the same basic circuitry may account for the generation of different motor outputs throughout development (ontogenetic plasticity), adulthood (adaptive plasticity) and evolution (phylogenetic plasticity).

Maturation of rhythmic neural network: role of central modulatory inputs.

Modulatory systems are well known for their roles in tuning the cellular and synaptic properties in the adult neuronal networks, and play a major role in the control of the flexibility of functional outputs. However far less is known concerning their role in the maturation of neural networks during the development. In this review, using the stomatogastric nervous system of lobster, we will show that the neuromodulatory system exerts a powerful influence on developing neural networks. In the adult the number of both motor target neurons and their modulatory neurons is restricted to tens of identifiable cells. They are therefore well characterized in terms of cellular, synaptic and morphological properties. In the embryo, these target cells and their neuromodulatory population are already present from mid-embryonic life. However, the motor output generated by the system is quite different: while in the embryo all the target neurons are organized into a single network generating unique motor pattern, in the adult this population splits into two distinct networks generating separate patterns. This ontogenetic partitioning does not rely on progressive acquisition of adult properties but rather on a switch between two possible network operations. Indeed, adult networks are present early in the embryonic life but their expression is repressed by central modulatory neurons. Moreover, embryonic networks can be revealed in the adult system again by altering modulatory influences. Therefore, independently of the developmental age, two potential network phenotypes co-exist within the same neuronal architecture: when one is expressed, the other one is hidden and vice versa. These transitions do not necessarily need dramatic changes such as growth/retraction of processes, acquisition of new intra-membrane proteins etc. but rather, as shown by modelling studies, it may simply rely on a subtle tuning of pre-existing intercellular electrical coupling. This in turn suggests that progressive ontogenetic alteration may not take place at the level of the target network but rather at the level of modulatory input neurons.

Removal of GABA within Adult Modulatory Systems Alters Electrical Coupling and Allows Expression of an Embryonic-Like Network

The maturation and operation of neural networks are known to depend on modulatory neurons. However, whether similar mechanisms may control both adult and developmental plasticity remains poorly investigated. To examine this issue, we have used the lobster stomatogastric nervous system (STNS) to investigate the ontogeny and role of GABAergic modulatory neurons projecting to small pattern generating networks. Using immunocytochemistry, we found that modulatory input neurons to the stomatogastric ganglion (STG) express GABA only after metamorphosis, a time that coincides with the developmental switch from a single to multiple pattern generating networks within the STNS. We demonstrate that blocking GABA synthesis with 3-mercapto-propionic acid within the adult modulatory neurons results in the reconfiguration of the distinct STG networks into a single network that generates a unified embryonic-like motor pattern. Using dye-coupling experiments, we also found that gap-junctional coupling is greater in embryos and GABA-deprived adults exhibiting the unified motor pattern compared with control adults. Furthermore, GABA was found to diminish directly the extent and strength of electrical coupling within adult STG networks. Together, these observations suggest the acquisition of a GABAergic phenotype by modulatory neurons after metamorphosis may induce the reconfiguration of the single embryonic network into multiple adult networks by directly decreasing electrical coupling. The findings also suggest that adult neural networks retain the ability to express typical embryonic characteristics, indicating that network ontogeny can be reversed and that changes in electrical coupling during development may allow the segregation of multiple distinct functional networks from a single large embryonic network.