Innexins in the lobster stomatogastric nervous system: cloning, phylogenetic analysis, developmental changes and expression within adult identified dye and electrically coupled neurons.

Gap junctions play a key role in the operation of neuronal networks by enabling direct electrical and metabolic communication between neurons. Suitable models to investigate their role in network operation and plasticity are invertebrate motor networks, which are built of comparatively few identified neurons, and can be examined throughout development; an excellent example is the lobster stomatogastric nervous system. In invertebrates, gap junctions are formed by proteins that belong to the innexin family. Here, we report the first molecular characterization of two crustacean innexins: the lobster Homarus gammarus innexin 1 (Hg-inx1) and 2 (Hg-inx2). Phylogenetic analysis reveals that innexin gene duplication occurred within the arthropod clade before the separation of insect and crustacean lineages. Using in situ hybridization, we find that each innexin is expressed within the adult and developing lobster stomatogastric nervous system and undergoes a marked down-regulation throughout development within the stomatogastric ganglion (STG). The number of innexin expressing neurons is significantly higher in the embryo than in the adult. By combining in situ hybridization, dye and electrical coupling experiments on identified neurons, we demonstrate that adult neurons that express at least one innexin are dye and electrically coupled with at least one other STG neuron. Finally, two STG neurons display no detectable amount of either innexin mRNAs but may express weak electrical coupling with other STG neurons, suggesting the existence of other forms of innexins. Altogether, we provide evidence that innexins are expressed within small neuronal networks built of dye and electrically coupled neurons and may be developmentally regulated.

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).

Ontogeny of modulatory inputs to motor networks: early established projection and progressive neurotransmitter acquisition.

Modulatory information plays a key role in the expression and the ontogeny of motor networks. Many developmental studies suggest that the acquisition of adult properties by immature networks involves their progressive innervation by modulatory input neurons. Using the stomatogastric nervous system of the European lobster Homarus gammarus, we show that contrary to this assumption, the known population of projection neurons to motor networks, as revealed by retrograde dye migration, is established early in embryonic development. Moreover, these neurons display a large heterogeneity in the chronology of acquisition of their full adult neurotransmitter phenotype. We performed retrograde dye migration to compare the neuronal population projecting to motor networks located in the stomatogastric ganglion in the embryo and adult. We show that this neuronal population is quantitatively established at developmental stage 65%, and each identified projection neuron displays the same axon projection pattern in the adult and the embryo. We then combined retrograde dye migration with FLRFamide-like, histamine, and GABA immunocytochemistry to characterize the chronology of neurotransmitter expression in individual identified projection neurons. We show that this early established population of projection neurons gradually acquires its neurotransmitter phenotype complement. This study indicates that (1) the basic architecture of the known population of projection inputs to a target network is established early in development and (2) ontogenetic plasticity may depend on changes in neurotransmitter phenotype expression within preexisting neurons rather than in the addition of new projection neurons or fibers.

Progesterone regulation of GABAA receptor plasticity in adult rat supraoptic nucleus.

Marked plasticity in GABAA receptor signalling occurs in adult oxytocin neurons of the supraoptic nucleus (SON) through the modulation of GABAA receptor alpha subunits during pregnancy. The present studies were undertaken to examine the potential mechanisms underlying this plasticity. In vivo microdialysis experiments in conscious rats revealed that no significant changes in extracellular GABA concentrations occurred within the SON over the last two days of pregnancy and the time of parturition itself. In situ hybridization studies examined the effects of gonadal steroid manipulation upon the GABAA receptor subunits expressed by SON neurons (alpha1, alpha2, beta2 and gamma2 subunits) and demonstrated that cellular levels of the alpha1 subunit were increased following 8 days oestrogen and progesterone treatment. Estrogen alone or allopregnanolone, the progesterone derivative, had no effect on alpha1 subunit mRNA expression in the SON. Immunocytochemical experiments demonstrated progesterone receptors in many neural populations but not within the SON of late pregnant rats. These studies indicate that alterations in endogenous GABA release within the SON are unlikely to be responsible for the GABAA receptor plasticity exhibited by oxytocin neurons in late pregnancy. Rather, data demonstrate that the fluctuating concentrations of progesterone during pregnancy act indirectly on SON neurons to modulate alpha1 subunit mRNA expression. Together, these experiments provide evidence for the ligand-independent induction of GABAA receptor plasticity in the adult brain by progesterone.

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 preexisting adult networks in an embryonic system.

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 FLRF(NH2)-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 (FLRF(NH2))-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, FLRF(NH2)-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.

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.

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. We then investigated the ontogeny of FLRFamide- and proctolin-like peptides in the stomatogastric nervous system, from their first appearance until adulthood by using whole mount immunocytochemistry. Numerous FLRFamide-like-immunoreactive STG neuropilar ramifications were observable as early as E45 and remain thereafter. From E50 to the first larval stage, one to three STG somata stained, while somatic staining was not observed in larval stage II and subsequent stages. From E50 and thereafter, the STG neuropilar area was immunopositive for proctolin. One to two proctolinergic somata were detected in the STG of the three larval stages but were not seen in embryos, the post-larval stage or in adults. Thus, peptidergic inputs to the STG are present from mid-embryonic life. Moreover, whereas in the adult, STG neurons only contain glutamate or acetylcholine, some neurons transiently express peptidergic phenotypes during development. Although this system expresses an ontogenetic peptidergic plasticity, the STG neurons produce a single stable embryonic-larval motor output (Casasnovas and Meyrand [1995] J. Neurosci. 15:5703-5718).

Development of rhythmic pattern generators.

In contrast to the wealth of knowledge about the organizational rules of adult central pattern generators, far less is known about how these networks are assembled during development. The basic architecture for adult central pattern generators appears early in development but different generators may follow completely different developmental pathways to reach maturity. Recent evidence suggests that neuromodulatory inputs, in addition to their short-term adaptive control of central pattern generator activity, play a crucial role in both the final developmental tuning and the long-term maintenance of adult network function.

Calbindin-D28k mRNA expression in magnocellular hypothalamic neurons of female rats during parturition, lactation and following dehydration.

Recent studies indicate that calcium binding proteins may play a role in determining the electrical firing patterns of the hypothalamic magnocellular oxytocin (OT) and vasopressin (VP) neurons. In this study we have examined the calbindin-D28k mRNA content of magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei and determined whether changes in expression correlate with the specific patterns of electrical activity displayed by these cells under different physiological circumstances. In situ hybridization with [35S]-labelled oligonucleotides revealed a heterogeneous pattern of calbindin-D28k mRNA expression in the SON and magnocellular PVN. Quantitative analysis demonstrated that the number of silver grains/cell in the dorsal half of the SON was approximately 30% higher (P < 0.05) than that of the ventral half of the nucleus. Within the PVN, calbindin-D28k mRNA-expressing neurons were detected in the medial magnocellular division of the PVN but not in magnocellular cells forming the core of the lateral magnocellular division. Dehydration for 24 h did not alter calbindin-D28k mRNA expression in the SON, PVN or cingulate cortex. In parturient and lactating rats, calbindin-D28k mRNA levels were significantly (P < 0.05) reduced in the medial magnocellular division of the PVN compared with virgin animals. No significant differences in calbindin-D28k mRNA expression were observed in either ventral or dorsal halves of the SON, or in the cingulate cortex of these animals. These results provide evidence for the differential expression of calbindin-D28k mRNA by hypothalamic magnocellular neurons and suggest that OT cells may express more calbindin-D28k mRNA than VP neurons. The reduction in calbindin-D28k mRNA expression by putative OT neurons of the PVN at the time of parturition and lactation supports the hypothesis of Li and colleagues (J. Physiol., 488 (1995) 601-608) that calbindin may play a part in determining the electrical firing patterns of magnocellular neurons. However, the absence of any similar decrease in the SON suggests that changes in calbindin-D28k mRNA expression are not essential for OT neurons to exhibit episodic bursting behavior.

Plasticity in GABAA receptor subunit mRNA expression by hypothalamic magnocellular neurons in the adult rat.

The magnocellular hypothalamic neurons exhibit a substantial degree of structural and functional plasticity over the time of pregnancy, parturition, and lactation. This study has used in situ hybridization techniques to examine whether the content of alpha 1, alpha 2, beta 2, gamma 2 GABAA receptor subunit mRNAs expressed by these cells fluctuates over this period. A process of regional, followed by cellular and then topographical, analyses within the supraoptic (SON) and posterior paraventricular (PVN) nuclei revealed that an increase in magnocellular alpha 1 subunit mRNA content occurred during the course of pregnancy up to day 19, after which a decline in expression was detected on the day of parturition. Significant fluctuations of this nature were observed only in the oxytocin neuron-enriched regions of the SON and PVN. The expression of alpha 2, beta 2, and gamma 2 subunit mRNAs in the SON and PVN and of all subunit mRNAs in the cingulate cortex did not change over this period. During lactation, gamma 2 subunit mRNA content within the PVN increased significantly on day 14 of lactation as compared with day 7, and topographical analysis suggested that it involved principally magnocellular vasopressin neurons. These results demonstrate the cell-and subunit-specific regulation of GABAA receptor mRNA expression within the hypothalamic magnocellular system. In particular, they suggest that fluctuations in alpha 1 subunit expression may contribute to the marked variations in electrical activity exhibited by magnocellular oxytocin neurons at the time of parturition. More generally, they provide evidence in support of GABAA receptor plasticity within a physiological context in the adult rat brain.

In vivo regulation of specific GABAA receptor subunit messenger RNAs by increased GABA concentrations in rat brain.

This study has examined whether changes in endogenous GABA concentrations influence GABAA receptor subunit mRNA expression in vivo. Increased GABA concentrations were achieved by treating female rats with gamma-vinyl-GABA (15 mg/100 g), an irreversible inhibitor of the GABA transaminase, daily for three days. High performance liquid chromatography analysis of brain punches from specific brain regions showed that gamma-vinyl-GABA treatment resulted in approximately two-fold increases in brain GABA content. Using in situ hybridization techniques with specific 35S-labelled oligonucleotides, the mRNA expression of the alpha 1, alpha 2, beta 2, beta 3, gamma 1 and/or gamma 2 subunits of the GABAA receptor was quantified in various brain regions including the medial preoptic nucleus, bed nucleus of the stria terminalis, bed nucleus of the anterior commissure, supraoptic and paraventricular nuclei of the hypothalamus, globus pallidus and cingulate cortex. Silver grain density analysis showed that gamma-vinyl-GABA treatment induced a significant 35 and 49% decrease in gamma 1 mRNA expression in the medial preoptic nucleus and the principle encapsulated nucleus of the bed nucleus of the stria terminalis respectively, and a significant 20% decrease in alpha 2 mRNA expression in the cingulate cortex. Expression of alpha 2 and beta 3 in the former areas was unchanged as was alpha 1, beta 2, beta 3 and gamma 2 subunit expression in the cingulate cortex. Elevation of brain GABA levels also resulted in a specific and significant 17% increase in gamma 2 mRNA expression in the supraoptic nucleus. In the globus pallidus, gamma-vinyl-GABA treatment induced a significant 29% increase in alpha 1 mRNA expression combined with 19 and 30% decreases in beta 2 and gamma 2 mRNA expression, respectively. Levels of GABAA receptor subunits expressed in the bed nucleus of the anterior commissure (alpha 2, beta 3, gamma 1) and paraventricular nucleus (alpha 1, alpha 2, beta 2, gamma 2) were not changed by gamma-vinyl-GABA treatment. These results provide in vivo evidence for a region- and subunit-specific regulation of GABAA receptor subunit mRNA levels following the elevation of brain GABA concentrations and suggest that endogenous GABA levels influence GABAA receptor subunit mRNA expression.

Characterisation of GABAA receptor gamma subunit expression by magnocellular neurones in rat hypothalamus.

Gamma-aminobutyric acid (GABA) is known to inhibit the electrical and secretory activity of oxytocin and vasopressin neurones located in the supraoptic and paraventricular nuclei following osmotic, cardiovascular or suckling stimuli. To understand fully the nature of GABA actions on these magnocellular neurones it is important to define the heteropentameric GABAA receptor proteins they express. In the present study, single and dual labelling in situ hybridisation and immunocytochemical experiments were undertaken to define the GABAA receptor gamma subunits expressed by these cells. In situ hybridisation with 35S-labelled antisense oligonucleotides showed that all magnocellular neurones in the supraoptic and paraventricular nuclei of the female rat expressed mRNA encoding the gamma 2 subunit of the GABAA receptor but not the gamma 1 or gamma 3 subunits. Immunocytochemical experiments using a specific polyclonal rabbit antibody directed against the gamma 2 subunit of the GABAA receptor showed that all hypothalamic magnocellular neurones were strongly immunoreactive for gamma 2 subunit protein. Dual in situ hybridisation experiments using the gamma 2 subunit 35 S-labelled oligonucleotide with alkaline phosphatase-labelled antisense oligonucleotides specific for either oxytocin or vasopressin revealed that essentially all oxytocin and vasopressin neurones in both the supraoptic and paraventricular nuclei expressed the gamma 2 subunit of the GABAA receptor. Similarly, sequential double immunoperoxidase staining revealed that all oxytocin and vasopressin neurones in both magnocellular nuclei of the hypothalamus were immunoreactive for the gamma 2 subunit. This study shows that only the gamma 2 subunit of the GABAA receptor gamma subunit family is expressed by hypothalamic oxytocin and vasopressin neurones. In conjunction with our previous results, these findings indicate that individual magnocellular neurones express a complement of alpha 1, alpha 2, beta 2, beta 3 and gamma 2 subunits of the GABAA receptor. The observation of strong gamma 2 subunit expression by neurones known to also express alpha 1 and alpha 2 subunit proteins suggests that these magnocellular cells may express GABAA receptors with both benzodiazepine type-1 and type-2 pharmacology.

Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain.

This study has examined whether circulating estrogens are involved in regulating gamma-aminobutyric acid (GABA)A receptor mRNA expression in regions of the female rat brain known to contain estrogen receptors (ERs). In situ hybridization experiments using 35S-labeled oligonucleotides specific for alpha 2, beta 3, and gamma 1 subunit mRNAs of the GABAA receptor demonstrated that all three mRNAs were abundant in only the medial preoptic nucleus (MPN), where they were expressed by the vast majority of cells, and specific regions of the bed nucleus of the stria terminalis including the principle encapsulated nucleus (PrN-BNST) and bed nucleus of the anterior commissure (BNAC). Estrogen treatment of ovariectomized rats for 7 d resulted in significant 30-60% increases in alpha 2 and gamma 1, but not beta 3, subunit mRNA expression in the MPN and PrN-BNST. Estrogen treatment for 24 hr resulted in levels of mRNA expression intermediate between those of controls and animals treated with estrogen for 7 d. No changes in subunit mRNA expression were detected for any subunit in the BNAC or cingulate cortex. Double-labeling immunocytochemistry experiments using antibodies directed against the alpha 2 subunit of the GABAA receptor and the ER, revealed that 67 +/- 3% of alpha 2 subunit-immunoreactive cells in the MPN also contained ER immunoreactivity. Cells expressing alpha 2 subunits in the PrN-BNST were also found to possess ERs while those in the BNAC and cingulate cortex did not. These findings suggest the possibility that ER-containing cells in the MPN and PrN-BNST express an alpha 2 beta 3 gamma 1 isoform of the GABAA receptor that has its alpha 2 and gamma 1 subunits regulated by circulating estrogen concentrations. Together, our observations indicate that estrogen may regulate GABAA receptor mRNA expression at a transcriptional level and that this is only likely to occur within regions of the rat brain possessing ERs.

Cellular localization and differential distribution of GABAA receptor subunit proteins and messenger RNAs within hypothalamic magnocellular neurons.

The inhibitory neurotransmitter GABA plays an important role in regulating the activity of magnocellular oxytocin and vasopressin neurons located in the supraoptic and paraventricular nuclei through occupancy of GABAA receptors. However, the GABAA receptor is a hetero-oligomeric protein comprised of different subunits and the subunit types expressed in a given receptor complex appear critical for its sensitivity to GABA, benzodiazepines and/or steroids. Thus, in order to understand fully the GABAergic control of oxytocin and vasopressin secretion, definition of the GABAA receptors synthesized by magnocellular neurons in the supraoptic and paraventricular nuclei is required. In the supraoptic nucleus, antibodies directed against the alpha 1, alpha 2 and beta 2/3 subunits of the GABAA receptor revealed similar strong antigen distribution on all magnocellular neurons. Using sequential double-immunoperoxidase staining, immunoreactivity for all three subunits was observed on both oxytocin and vasopressin neurons of the supraoptic nucleus. In contrast, only alpha 2 subunit immunoreactivity was detected on the cell bodies of oxytocin and vasopressin neurons in the paraventricular nucleus. No sex differences were detected. In situ hybridization experiments using 35S-labelled oligonucleotides showed that all supraoptic neurons expressed alpha 1, alpha 2 and beta 2 subunit messenger RNA transcripts while magnocellular neurons in the paraventricular nucleus were only enriched in alpha 2 subunit messenger RNA. Quantitative analysis showed that the expression of alpha 1 and beta 2 subunit messenger RNAs in the paraventricular nucleus was half that observed in the supraoptic nucleus while expression of beta 3 subunit messenger RNA was very low in both nuclei. These results show that all oxytocin and vasopressin neurons located in the supraoptic nucleus synthesize and express alpha 1, alpha 2 and beta 2 subunits of the GABAA receptor while those in the paraventricular nucleus are only immunoreactive for the alpha 2 subunit. These observations suggest, therefore, that at least two pharmacologically distinct GABAA receptor isoforms exist on supraoptic neurons and that these are different to those expressed by paraventricular magnocellular cells. Thus, in addition to providing a definition of the subunits likely to form specific GABAA receptor isoforms on magnocellular neurons, this study gives direct evidence for GABAA receptor heterogeneity between supraoptic and paraventricular neurons, but not between oxytocin and vasopressin cells.

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.

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.

Electrical activity of dorsal horn neurons during the suckling-induced milk ejection reflex in the lactating rat.

In lactating rats, suckling elicits the milk ejection reflex which consists of an intermittent synchronous activation of hypothalamic oxytocin neurons which releases oxytocin into the bloodstream. We here investigated the electrophysiological behaviour of spinal cord neurons linked to mammary innervation in relation to suckling and the suckling-induced milk ejection reflex. Experiments were carried out on 58 urethane-anaesthetized rats, paralysed with gallamine triethiodide and artificially ventilated. Fixation of the spinal cord and laminectomy significantly slowed down the reflex, which occurred in only 27% of the animals. In these rats, 31 dorsal horn neurons at the thoraco-lumbar level were found to be excited by nipple stimulation. During suckling by a litter of at least nine pups, they displayed an irregular pattern of brief bursts of activity (peak firing rate 22.0 ± 3.2 Hz, mean ± SD) correlated to the bouts of suckling of the pups. Seventeen out of 19 cells tested by stimulation of at least 2 adjacent nipples received convergent input from different ipsilateral nipples. Out of 11 cells tested, 8 were also activated by stimulation of a contralateral nipple. Fourteen out of 30 units were recorded through at least one reflex milk ejection. Their firing rate was significantly higher than the firing rate of cells recorded in animals which failed to milk eject (4.4 Hz ± 2.8 versus 1.5 Hz ± 0.7). At the moment of the high frequency discharge of action potentials, occurring in oxytocinergic cells 10 to 15 s before each milk ejection, spinal neurons showed no systematic change in electrical activity. In contrast, the stretch reaction of the pups, which corresponds to an intense period of suckling when milk ejection has started, induced, in 12 cells, a considerable increase in electrical activity. One unit was found to be inhibited by suckling and during the stretch reaction. Ten more units, which were not activated by stimulation of the nipples but responded to stimulation of excitatory receptive fields near the last three pairs of nipples, were recorded through reflex milk ejections: 8 remained silent during reflex milk ejections but 2 were activated when the pups stimulated their excitatory receptive field. We conclude that some dorsal horn neurons, able to respond readily to the suckling movements of pups, appear to receive an ungated input from the nipples. At the time of the activation of oxytocin neurons, they present no particular pattern of activation or inhibition which could account in a simple manner for the intermittence of the high frequency discharge in oxytocinergic cells. However, in so far as these dorsal horn neurons may be part of the milk ejection reflex pathway, their activity, showing convergence and summation of input, and being facilitated in milk ejecting animals, indicates that the reflex does undergo a certain degree of processing at the spinal cord level.