Am J Physiol Regul Integr Comp Physiol 290: R1175-R1182, 2006;
doi:10.1152/ajpregu.00755.2005
0363-6119/06 $8.00
INVITED REVIEW
CALL FOR PAPERS
Neurohypophyseal Hormones: From Genomics and Physiology to Disease
Remodeling of astrocytes, a prerequisite for synapse turnover in the adult brain? Insights from the oxytocin system of the hypothalamus
Dionysia T. Theodosis,
Andrei Trailin, and
Dominique A. Poulain
Laboratory of Morphofunctional Neurobiology Institut National de la Santé et de la Recherche Médicale U 378; University Victor-Segalen-Bordeaux
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ABSTRACT
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Neurons, including their synapses, are generally ensheathed by fine processes of astrocytes, but this glial coverage can be altered under different physiological conditions that modify neuronal activity. Changes in synaptic connectivity accompany astrocytic transformations so that an increased number of synapses are associated with reduced astrocytic coverage of postsynaptic elements, whereas synaptic numbers are reduced on reestablishment of glial coverage. A system that exemplifies activity-dependent structural synaptic plasticity in the adult brain is the hypothalamo-neurohypophysial system, and in particular, its oxytocin component. Under strong, prolonged activation (parturition, lactation, chronic dehydration), extensive portions of somatic and dendritic surfaces of magnocellular oxytocin neurons are freed of intervening astrocytic processes and become directly juxtaposed. Concurrently, they are contacted by an increased number of inhibitory and excitatory synapses. Once stimulation is over, astrocytic processes again cover oxytocinergic surfaces and synaptic numbers return to baseline levels. Such observations indicate that glial ensheathment of neurons is of consequence to neuronal function, not only directly, for example by modifying synaptic transmission, but indirectly as well, by preparing neuronal surfaces for synapse turnover.
NEURONS, GLIA, AND SYNAPTIC inputs are not static elements because they undergo dynamic transformations. This can take place under normal physiological conditions and highlights the adult nervous system’s remarkable capacity to undergo restructuring to meet particular functional requirements. A change in the morphology of neurons, and especially of afferent inputs controlling their activity, can have important consequences on their respective functions. These modifications are often accompanied by remodeling of adjacent glia, which has further impact on neuronal activity by modifying the immediate extracellular microenvironment, thereby influencing synaptic and volume transmission.
The hypothalamo-neurohypophysial system (HNS), and in particular, its oxytocinergic component, is a familiar model for this kind of morphological neuronal and glial plasticity (57, 76). Oxytocin (OT) neurons accumulate in well-defined regions in the hypothalamus, the supraoptic (SON), and paraventricular (PVN) nuclei, and release OT mainly from axon terminals in the neurohypophysis to act as a neurohormone in vital functions like parturition, lactation, and osmotic regulation. Critical for this systemic secretion is the concomitant exocytotic release of the peptide from somata and dendrites of OT neurons themselves in the SON and PVN. This central release facilitates the synchronous high-frequency activity of OT neurons, which is the basis of the intermittent, bolus, systemic release of the neurohormone required for activation and upregulation of OT receptors in the uterus and mammary gland (66).
Under stimulation, the overall morphology of OT neurons is significantly modified since they progressively hypertrophy, their dendrites shorten and display few branches, whereas their axons enlarge and ramify. These changes occur together with modifications in the morphology of their accompanying glia, represented by astrocytes in the hypothalamus and astrocyte-like pituicytes in the neurohypophysis. In addition, there is a significant remodeling of their afferent inputs, which results in increased numbers of inhibitory and excitatory synapses controlling their activity. The system reverts to its prestimulated morphology when central and peripheral OT release return to low baseline levels. This plasticity has been described in detail in several recent reviews (49, 57, 76, 79, 82), and we will here limit our discussion to those features relevant to synaptic remodeling. Moreover, while such plasticity involves the whole system in all the magnocellular nuclei, we will focus on the SON, where, because of its cellular homogeneity and ready accessibility, most work has been carried out.
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SYNAPSE TURNOVER IN THE MAGNOCELLULAR NUCLEI
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The term "synaptic plasticity" generally connotes long-lasting functional modifications within preexisting synapses, like long-term potentiation or depression. Nevertheless, it has been obvious for at least the last two decades that synaptic transmission can be significantly modified by structural synaptic plasticity as well. The OT system is exemplary in this domain and was one of the first neuronal systems in which we obtained clear evidence of activity-dependent formation and elimination of synapses in the adult brain (86).
In the magnocellular nuclei, the most obvious manifestation of synapse turnover is a changing number of synapses originating from boutons making synaptic contacts onto more than one postsynaptic element in the same plane of section (shared synapses) (86). Visible in the magnocellular nuclei under all conditions, their incidence significantly increases during conditions of enhanced or sustained OT release (parturition, lactation, chronic dehydration, restrain stress), an increase that affects those boutons synapsing onto OT profiles (84). When we applied stereology to evaluate synaptic densities, we found that synaptic remodeling in the OT system is even more extensive than reported originally, because it affects terminals making single, as well as shared synaptic contacts (25, 29). Thus in the SON of rats that have lactated for several days, in spite of neuronal hypertrophy (9, 24, 25, 29, 78), the overall synaptic density is not significantly less than that evaluated in virgin animals (about 40 x 106 synapses/mm3 SON) (25, 29, 81). A clear increase in synaptic densities becomes obvious when they are evaluated in relation to the neuropil (25, 29).
In vivo observations indicated that synapse formation occurs within 24 h of the onset of stimulation (52, 89). From our more recent in vitro work on acute hypothalamic slices containing the SON, we have found that new synapses appear very rapidly indeed. Within 2 h after activation of the system, there is a significantly increased incidence of synaptically coupled OT neurons (43), whereas significantly increased synaptic densities can be detected even after 1 h (Trailin A and Theodosis DT, unpublished observations). These findings thus add further evidence to the contention that mature neurons can form synapses reliably and quickly (see also, Refs. 27 and 58). There is synapse elimination in the OT system as well since the number of synapses return to unstimulated values on arrest of stimulation (52, 83), a phenomenon that also takes place within hours (43).
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REMODELING OF ASTROCYTES ACCOMPANIES SYNAPSE TURNOVER
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In the SON, the majority of glia are astrocytes, and like most other central astrocytes, they are quite heterogeneous, both morphologically (4, 6, 36) and physiologically (36). A large number have a radial morphology, with cell bodies lined up along the base of the nucleus and thick, long, processes oriented ventrodorsally from which arise finer processes. They express both glial fibrillary acidic protein (GFAP) and vimentin (6), are not electrotonically coupled, and possess GABA-A receptors (36). Interestingly, although they have the glutamate transporters, GLT-1 and GLAST (Piet R and Theodosis DT, unpublished observations), they do not appear to respond to glutamate (36). The other major population is stellate (protoplasmic), and they also emit thin processes (6). They are coupled by prominent gap junctions, contain GFAP, and possess glutamate transporters. OT neurons often occur in tightly packed clusters, but they remain separated by fine processes from both types of astrocytes. This kind of astrocytic coverage is widely seen in adult neuronal tissues (20, 30, 32, 93). It characterizes the OT and vasopressin (VP) systems under basal conditions of neurosecretion. In contrast, under stimulated conditions, when the neurons are contacted by an increased number of synapses, glial coverage is significantly reduced, and the surfaces of OT somata and dendrites are directly and extensively juxtaposed. When OT secretion returns to baseline levels (for example, after weaning the young), astrocytic processes reappear between neuronal profiles. Ultrastructural analysis of immunoidentified profiles established that these glial changes, like the synaptic changes, are specific to the OT system (84). While VP neurons display some juxtapositions, their incidence and extent are low and, more importantly, do not vary with changing conditions of neurosecretion (12, 78, 85).
As noted earlier, magnocellular neurons hypertrophy progressively with stimulation, and reduced glial ensheathment of oxytocinergic elements may merely represent failure of astrocytes to establish their initial coverage. However, it is difficult to see how cell hypertrophy can explain reduction of astrocytic coverage of dendrites, which often appear as directly juxtaposed bundles (of 3 to 8 dendrites). Moreover, our recent in vitro experiments (43) revealed that these glial transformations occur very rapidly, within 2 h, when the neurons have not significantly hypertrophied. Finally, as noted earlier, VP neurons do not display increased juxtapositions even under strong, persistent stimuli like lactation or chronic dehydration, yet they significantly hypertrophy under these conditions (12, 24, 29). A more likely explanation is that neuronal juxtapositions are the result of active retraction and elongation of astrocytic processes over neuronal surfaces whose morphology is constantly changing. Application of live imaging techniques to the SON should illustrate this more clearly. In the mouse brain stem, for example, a high degree of motility of astrocytic processes over neurons and synapses was visualized even under normal conditions (32). Application of such techniques to the SON should also make it possible to visualize the synapse turnover that is taking place concurrently on OT surfaces free of astrocytic processes.
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NEUROCHEMICAL IDENTITY OF SYNAPSES UNDERGOING REMODELING
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Like most other central neurons, OT and VP cells receive an abundant GABAergic input (19, 25, 29, 38, 81). Our ultrastructural analyses have estimated that under basal conditions, about 35% of all synapses in the SON are GABAergic, with twice as many on dendrites than on somata (25, 29). Moreover, they have clearly demonstrated that GABAergic inputs undergo remodeling so that in the SON of lactating rats, axosomatic and axodendritic GABAergic synapses represent close to 50% of all synapses. By analyzing ultrathin sections in which both pre- and postsynaptic partners were identified immunocytochemically, we confirmed that the increased inputs were on OT neurons (25, 29, 81). This synaptic remodeling can be reproduced in vitro in acute hypothalamic slices, a preparation that permits more direct pharmacological manipulations and therefore, a finer analysis of the modalities of such plasticity. These ongoing studies, using electron microscopy and patch clamp electrophysiology, have already made it clear that synapse formation is very rapid, occurring within a couple of hours and results in the formation of inhibitory synapses that are functional (78a). In vivo observations indicated that the origin of GABAergic inputs to SON neurons is local, from interneurons in close proximity to the magnocellular nuclei (38, 67, 81), as well as from long distance GABA afferents (37). In slices, synaptic densities of putative GABAergic synapses are closely similar to those evaluated in the SON in vivo, which strongly suggests that most GABA afferents to the SON derive from a local interneuron network.
The other major afferent input to OT and VP neurons uses glutamate as the neurotransmitter (24, 25, 38, 47, 90). Under basal conditions, glutamatergic synapses represent about 22% of all synapses in the rat SON; they are twice as numerous on dendrites than on somata (24, 25). Glutamate inputs undergo morphological remodeling under the same conditions that induce changes in GABAergic inputs, so that at lactation, they represent close to 25% of all synapses in the nucleus. The increase is represented only by glutamatergic synapses impinging on OT somata and dendrites (24, 25). Electrophysiological (7, 39) and morphological (14, 38) observations have indicated that many of these glutamatergic afferents derive from local interneurons in adjacent hypothalamic areas.
Finally, an excitatory, noradrenergic input, which represents 
10% of all synapses on magnocellular neurons (48) undergoes activity-dependent remodeling, as well. Thus in virgin rats, noradrenalin afferents innervate equally OT and VP neurons, whereas in lactating rats, there is a significant increase in the noradrenergic (NA) innervation of OT neurons (48). These afferents have an extrahypothalamic source, namely neurons in the A1 and A2 areas of the ventrolateral medulla and nucleus tractus solitarii, respectively (15).
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MECHANISMS OF SYNAPSE TURNOVER
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In comparison with developing or regenerating systems, one would expect bouton sprouting and degeneration in neuronal centers where synaptic numbers constantly vary. However, we have no clear evidence of growth cones or degenerating boutons in the magnocellular nuclei. It is possible then that new synapses are formed by enlargement and splitting of active zones in already existing boutons. Postsynaptic densities would follow suit if only because there is a tight association of pre- and postsynaptic membranes, mediated by molecules that span or are anchored to the synaptic cleft (94). Shared synapses may be extensions of preexistent terminals, which induced de novo postsynaptic density formation in a neighboring neuronal element. This scenario can be reversed, especially in view of recent studies in the developing Drosophila neuromuscular junction showing formation of postsynaptic densities before the appearance of presynaptic specializations (64). Adhesion between pre- and postsynaptic sites would help to coordinate growth of the synaptic contact. Further maturation of synapses would involve acquisition of the full complement of pre- and postsynaptic components required for stable synaptic transmission, processes that depend on gene regulation and new protein synthesis. This occurs in the SON, because in our in vitro model, synaptic remodeling can be inhibited if the incubation medium includes the protein synthesis inhibitor anisomycin (Langle SL, Trailin A, and Theodosis DT, unpublished observations).
It is obvious that intervening astrocytic processes must participate in the regulation of synapse formation, stability, and elimination. Thus they cannot be present between pre- and postsynaptic partners if synapse formation is to occur while their reinsertion could contribute to undoing of synaptic contacts (see also Refs. 1, 28, and 32). After synapse elimination, pre- and postsynaptic protein components could simply be redistributed in their respective compartments, without involving degeneration of boutons. These possibilities await experimental evidence. What is certain is that synapse formation in the SON, as elsewhere in the central nervous system, must be accompanied by a coordinated development of pre- and postsynaptic molecular and structural specializations, which requires exchange of information from pre- and postsynaptic partners, as well as adjacent astrocytes. Astrocytic membranes are equipped with numerous signaling or cell recognition molecules [transmitter receptors, transporters, adhesion molecules (93)], so that astrocytes are well suited for all of these purposes.
Finally, one should also consider that synaptic remodeling includes a phase of stabilization for which neuronal activity appears critical. Whereas the establishment of synaptic contacts per se does not depend on the secretion of a particular neurotransmitter (91), the specification of synaptic inputs requires neuronal activity (10). It is now clear that membrane depolarization, elevation of intracellular calcium, and action potential generation can lead to posttranslational modifications of synaptic proteins and regulation of gene activity (59). However, the molecular links between these events and synaptic plasticity are not precisely defined. The most likely possibility is that correlated synaptic activity provides critical information about the appropriateness of synapse connections, influencing synapse stability, and elimination (40). This criterion is certainly met by OT neurons, which fire in a highly coordinated manner during parturition and lactation. Nevertheless, what is unclear is how such neuronal activity is translated into long-term structural changes.
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MOLECULAR FACTORS PERMISSIVE FOR SYNAPTIC PLASTICITY
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In view of the evidence presented above, there can be no doubt that the morphology of the OT system, including its synaptic inputs, is repeatedly modified throughout life. This kind of plasticity connotes dynamic cell-cell and cell-matrix interactions that will bring into play cell surface molecules, extracellular matrix, and cytoskeletal proteins, as well as soluble trophic factors, many of which have been identified in developing systems undergoing synaptogenesis and glial transformations (23, 44). It is not too surprising then that the adult HNS continues to express many of these juvenile molecules. The list includes members of the immunoglobulin superfamily of cell adhesion molecules, such as the neural cell adhesion molecule (NCAM), as well as complex extracellular matrix glycoproteins and several cytoskeletal proteins (76, 82). We will here focus our attention on NCAM whose expression is particularly striking in all portions of the HNS and which we are now sure participates in HNS plasticity.
NCAM, which occurs in several isoforms differing in their proteic and carbohydrate moieties, is thought to intervene in most cell interactions via modulation of cell adhesivity and intracellular signaling (see, for example, Ref. 23). Regulation of NCAM-mediated interactions can be achieved by transcriptional control of the type and amount of NCAM expressed on the cell surface. Further regulatory possibilities are provided by the posttranslational addition of the unique carbohydrate polymer, 
-2–8-linked polysialic acid (PSA), on its extracellular domain (68). The resulting high Mr isoforms (about 220 kDa), known as PSA-NCAM, contain more than 30% PSA. PSA-NCAM is abundant in embryonic tissues, whereas most adult tissues contain NCAM with little PSA. Nevertheless, PSA-NCAM continues to be expressed in the adult HNS (5, 41, 55, 87), as it does in other systems endowed with the capacity for morphological and/or physiological plasticity (5, 71).
Neuronal expression of PSA-NCAM is well documented (68) but in the HNS, it is particularly abundant on astrocytic surfaces, especially in the SON and PVN (77). It is also abundant on the surface of all neurosecretory axons and terminals (41, 50, 62, 77). PSA-NCAM is constitutively expressed in HNS neurons and glia (62), which explains why its expression is not markedly affected by changing conditions of neurosecretion (5, 77, 87). Nevertheless, this does not mean that its expression is of no consequence to plasticity. On the contrary, it is a prerequisite for all its dynamic phases. Thus specific enzymatic removal of PSA from NCAM in the SON in situ inhibited the neuronal, glial, and synaptic remodeling associated with lactation and chronic dehydration; there was no effect if such pertubation was performed once remodeling of the nuclei had taken place (77). Likewise, PSA removal from cell surfaces in the neurohypophysis prevented stimulation-related induction and reversal of axon and glial changes but had no effect once remodeling had occurred (50). We do not know how PSA intervenes to permit morphological changes in the HNS, but a possible mechanism is that large quantities of PSA on cell surfaces attenuates adhesion via physical impedance or charge repulsion (69). Cells could then detach from their neighbors or from the extracellular matrix and be able to undergo changes in their conformation. In addition, PSA-NCAM may intervene by transducing intracellular signals resulting in synapse formation (21).
In the HNS, there is concomitant expression of less sialylated isoforms of NCAM, like the trans-membrane NCAM-140 and NCAM-180 (5, 87). Their role in HNS function and plasticity remains unknown. Since they are generally associated with stability of cell contacts (23), they could intervene to maintain the anatomy of the system once it has undergone remodeling. In view of its action in the hippocampus (70), NCAM-180 may also participate directly in the synaptic plasticity of the OT system, via complex intracellular signaling mechanisms.
We consider a molecule like NCAM permissive for plasticity since its expression is not tightly linked to the activity of HNS neurons and yet, as seen from our enzyme pertubation experiments (50, 77), its expression appears obligatory for plasticity. Nonetheless, other molecular mechanisms can substitute for NCAM, because remodeling does occur in lactating and salt-loaded mice genetically deficient in NCAM (88). Similar phenomena have been described in other neuronal systems in which NCAM is highly expressed and which continue to undergo plasticity in NCAM –/– mutants (22). In a sense, the specific identity of molecules permissive for remodeling may not be important, provided that they activate the same intracellular mechanism. On the other hand, the response of HNS cells would be invariant and independent of the identity of the factor, provided the proper stimulus intervenes. As will be discussed next, one such stimulus is OT itself. In other adult neuronal systems capable of morphological plasticity and expressing PSA-NCAM (5, 21, 33, 54, 72), other inductive factors must intervene.
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OT INDUCES MORPHOLOGICAL PLASTICITY
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If molecules like PSA-NCAM act as priming molecules to provide a permissive environment for synaptic remodeling, then there must be molecules whose expression is particular to the HNS that act as specific stimuli to induce structural transformations, triggering cascades of intracellular events that result in neuron-glial transformations and synapse formation. Such molecules include those that would be expressed at the onset of enhanced activity to bring on the neuronal and glial rearrangements as well as molecules, not necessarily identical to the former, which signal the system to revert to its original condition.
In the OT system, the physiological conditions during which remodeling is most striking are parturition and lactation. As noted earlier, these conditions are characterized not only by more OT released from the neurohypophysis but in increased secretion of the peptide within the hypothalamic nuclei themselves. Intranuclear OT is due primarily to somatodendritic exocytotic release, which can be brought on by action potential firing and which is modulated by calcium release from internal stores (42). Central OT facilitates the activity of the OT system by various means, including enhancing activity of excitatory glutamate inputs (34, 39) and inhibiting, via a presynaptic action, GABA release (18). In fine, it facilitates firing of its own neurons and therefore, neurohypophyseal release into the peripheral circulation. It is therefore not too surprising that intracerebroventricular infusion of the peptide, presumably mimicking this central release, induced neuronal-glial and synaptic changes in the SON similar to those detected under physiological stimulation (80). This particular action of central OT is receptor mediated because it can be mimicked by a close analog, 4-Th-OT, whereas peptides like VP or cholecystokinin have no effect. Nevertheless, OT does not act alone but in synergy with sexual steroids (51).
Ongoing experiments using acute hypothalamic slices show that this effect of OT can be reproduced in the SON in vitro. As in vivo, an OT receptor mechanism appears responsible, because synaptic and glial morphological changes were mimicked by application of the specific OT agonist 4-Thr-7-Gly-OT and were inhibited when OT was applied together with the specific OT receptor antagonist, dOVT (43). Experiments with our in vitro preparation also highlight the participation of sexual steroids since inclusion of 17-
estradiol potentiated the effects of OT. This is in accordance with numerous observations showing that estrogens enhance OT secretion by modulating the electrical activity of OT neurons (35), increasing OT gene expression (13) and central and peripheral release (95, 96). However, some caution is necessary in interpreting our results, because reports of gonadal steroid effects on magnocellular neurons are conflicting (74). Indeed, the site of action of the steroid in OT neurons has not been identified, and we still do not know whether its effect is direct or indirect. Long-term genomic effects are mediated by nuclear estrogen receptors, ER or ER (46, 73). Rat OT neurons lack the former (73, 74) and <10% express the latter (75). In view of the rapidity of the effects of the steroid on the anatomy of the SON (43), it is more probable that estrogens act via a plasmalemmal mechanism (65), mobilizing intracellular calcium and inducing signaling cascades that result in activation of gene expression and ultimately synapse formation. Such a mechanism is not that unlikely when one considers the extremely rapid effects of the steroid on the electrical activity of OT neurons resulting in enhanced OT secretion (35). It is noteworthy that estrogens affect OT gene regulation if treatment is accompanied by a progesterone withdrawal protocol (2), endocrine conditions similar to those occurring at the end of gestation (8).
Although OT, with or without steroids, appears essential for remodeling in the magnocellular nuclei, our experiments fail to tell us the level at which it acts or whether it acts indirectly or directly, via its receptor on OT neurons (3) and/or associated astrocytes (31). There are other candidates for such signaling, and OT may act in a secondary fashion by facilitating their expression.
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OTHER SIGNAL CANDIDATES FOR PLASTICITY
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From our recent work on acute hypothalamic slices it has become obvious that OT can be replaced by other molecules to induce morphological changes. Thus exposure to a combination of metabotropic and ionotropic glutamate receptor antagonists prevented OT/estrogen-mediated increases in the proportion of juxtaposed and synaptically coupled profiles (43). Likewise, such a treatment inhibits the appearance of new GABA synapses (Trailin A and Theodosis DT, unpublished observations). This strongly suggests that glutamate itself may be a more immediate signaling agent than OT. The excitatory amino acid is an excellent candidate in this respect, because it can serve as a bidirectional signal to transfer information between activated neurons and adjacent astrocytes. Glutamate is taken up by astrocytes once released from synapses and can be released by astrocytes in response to physiological increases in intracellular Ca2+ (93). Even small increases in intracellular Ca2+ in astrocytes are sufficient to induce large glutamate-activated currents in neighboring neurons (61). Such a mechanism, alone or in combination with synaptically released glutamate, may serve both to activate the electrical activity of OT neurons and to signal initiation of morphological transformations. In line with this reasoning are data showing that OT cannot induce morphological changes in slices if it is used together with the chelator BAPTA-AM (43).
GABA, on the other hand, appears to inhibit synaptic and astrocytic transformations in the SON. Our preliminary observations indicate that application of the GABA-A receptor antagonist bicuculline to slices, even in the presence of the specific OT receptor antagonist dOVT, can reproduce the effects of OT and results in increases of juxtaposed and synaptically coupled neurons similar to those obtained with OT alone (43). Either GABA acts directly or the bicuculline effect reveals the intervention of other signaling or inducing agents.
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FUNCTIONAL CONSIDERATIONS
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As seen from the observations presented in this review, both inhibitory and excitatory synaptic inputs on OT neurons increase during enhanced neurosecretion. This could reflect a compensatory mechanism of the cells to adjust to their hypertrophy induced by sustained stimulation, a mechanism which would provide a degree of regulation equivalent to that in unstimulated animals. Data showing that the number of synaptic contacts per unit length of postsynaptic membrane in the SON of lactating rats remains stable, as does the size of postsynaptic densities (24, 25), would support such a premise. In addition, electrophysiological data obtained in lactating rats indicated that in spite of increased numbers of GABA release sites per hypertrophied OT neuron, synaptic current densities were no different from those recorded in virgin animals (9).
Nevertheless, an increased number of synapses may play an additional, more direct role in facilitating the particular electrical activity that characterizes OT neurons at parturition and lactation (63). That synaptic remodeling involves only those afferents impinging on OT neurons and that it occurs at every parturition and lactation is in favor of our contention that this kind of synaptic change directly influences such activity. A mechanism for increased inhibition could be through synchronization of GABA release from several terminals arising from one single inhibitory neuron and converging on several cells. This strong inhibition may be necessary to prevent the neurons from being stimulated by factors other than those relevant to lactation and would be a means by which the response of the OT system to stimuli other than suckling is significantly attenuated (26, 45). This kind of filtering action of GABA may also contribute to maintain OT neurons at a level of depolarization that would facilitate their synchronous activation just before milk ejection (53, 92).
On the other hand, an enhanced glutamatergic input probably serves to induce and further potentiate this activity. It is now obvious from many observations that glutamate plays a key role in the control of OT neuron firing. For instance, intracellular recordings from OT neurons in organotypic slice cultures showed that each burst of high-frequency action potentials is the result of a volley of postsynaptic potentials arising from glutamatergic synapses (39) originating from interneurons in adjacent hypothalamic areas (38). Moreover, simultaneous recordings from two OT cells revealed a high degree of synchronization due to synchronization of afferent input originating from these glutamate neurons (34). These in vitro observations are supported by in vivo data showing that the activity of OT neurons is under strong control by glutamate circuits from glutamate neurons within the hypothalamus but outside the SON and PVN (7, 16). What is still not clear are the possible effects that would be exerted by the additional glutamate input provided by newly formed glutamate synapses (24, 25).
To the potential facilitation of excitatory drive from newly formed glutamatergic synapses, one must include one derived from the additional noradrenergic synapses that appear at lactation (48). Noradrenergic afferents provide an excitatory input to OT neurons (17). In addition, there appears to be a central synergistic action of noradrenaline with glutamate (16, 60), an action which would further facilitate excitation of OT firing.
Nevertheless, some caution is necessary when attributing too strong a participation from additional synapses in the control of bursting activity of OT neurons at lactation. As noted earlier, enzymatic removal of PSA in all magnocellular nuclei at late gestation inhibits synaptic and glial remodeling (77), yet parturition and lactation, bursting firing, and reflex milk ejections occur normally (11). One possible explanation is that OT bursting activity, resulting from the activity of the intrahypothalamic burst generator, of which OT and glutamate neurons are a part (34, 39), is already controlled by strong high-frequency discharges and will appear unaffected by any additional glutamate and noradrenalin inputs. Within the pulse generator, glutamate neurons driving OT cells display an intense activity at milk ejection, which would bypass any subtle modulation of synaptic transmission (56), including that provided by a few additional excitatory synapses. It is also possible, however, that the additional synapses, whether inhibitory or excitatory, derive from pools of neurons not involved directly in the control of the pulsatile, coordinated firing of OT neurons but which intervene in fine tuning of their activity outside periods of high-frequency firing.
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ACKNOWLEDGMENTS
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This work was supported, in part, by short-term fellowships from Institut National de la Santé et de la Recherche Médicale (Poste Vert) and the Ministry of Research (to A. Trailin).
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FOOTNOTES
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Address for reprint requests and other correspondence: D. T. Theodosis, Laboratory of Morphofunctional Neurobiology Institut National de la Santé et de la Recherche Médicale U 378; University Victor Segalen-Bordeaux, F33077, Bordeaux, France (e-mail: dionysia.theodosis{at}bordeaux.inserm.fr)
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REFERENCES
|
|---|
- Aldskogius H, Liu L, and Svensson M. Glial responses to synaptic damage and plasticity. J Neurosci Res 58: 33–41, 1999.[CrossRef][ISI][Medline]
- Amico JA, Crowley RS, Insel TR, Thomas A, and O’Keefe JA. Effect of gonadal steroids upon hypothalamic oxytocin expression. Adv Exp Med Biol 395: 23–36, 1995.[Medline]
- Barberis C and Tribollet E. Vasopressin and oxytocin receptors in the central nervous system. Crit Rev Neurobiol 10: 119–154, 1996.[ISI][Medline]
- Bobak JB and Salm AK. Plasticity of astrocytes of the ventral glial limitans subjacent to the supraoptic nucleus. J Comp Neurol 376: 188–197, 1996.[CrossRef][ISI][Medline]
- Bonfanti L, Olive S, Poulain DA, and Theodosis DT. Mapping of the distribution of polysialylated neural cell adhesion molecule throughout the central nervous system of the adult rat: an immunohistochemical study. Neuroscience 49: 419–436, 1992.[CrossRef][ISI][Medline]
- Bonfanti L, Poulain DA, and Theodosis DT. Radial glia-like cells in the supraoptic nucleus of the adult rat. J Neuroendocrinol 5: 1–6, 1993.[CrossRef][ISI][Medline]
- Boudaba C, Schrader LA, and Tasker JG. Physiological evidence for local excitatory synaptic circuits in the rat hypothalamus. J Neurophysiol 77: 3396–3400, 1997.[Abstract/Free Full Text]
- Bridges RS. A quantitative analysis of the roles of dosage, sequence, and duration of estradiol and progesterone exposure in the regulation of maternal behavior in the rat. Endocrinology 114: 930–940, 1984.[Abstract]
- Brussaard AB, Devay P, Leytig-Vermeulen JL, and Kits KS. Changes in properties and neurosteroid regulation of GABAergic synapses in the supraoptic nucleus during the mammalian female reproductive cycle. J Physiol 516: 513–524, 1999.[Abstract/Free Full Text]
- Burrone J, O’Byrne M, and Murphy VN. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420: 414–418, 2002.[CrossRef][Medline]
- Catheline G, Touquet B, Lombard M-C, Poulain DA, and Theodosis DT. A study of the role of neuron-glial remodeling in the oxytocin system at lactation. Neuroscience 137: 309–316, 2006.[CrossRef][ISI][Medline]
- Chapman DB, Theodosis DT, Montagnese C, Poulain DA, and Morris JF. Osmotic stimulation causes structural plasticity of neurone-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus. Neuroscience 17: 679–686, 1986.[CrossRef][ISI][Medline]
- Crowley RS and Amico JA. Gonadal steroid modulation of oxytocin and vasopressin gene expression in the hypothalamus of the osmotically stimulated rat. Endocrinology 133: 2711–2718, 1993.[Abstract]
- Csaki A, Kocsis K, and Halasz B. Localization of putatitive glutamatergic/aspartatergic neurons projecting to the supraoptic nucleus area of the rat hypothalamus. Eur J Neurosci 16: 55–68, 2002.[CrossRef][ISI][Medline]
- Cunningham ET, and Sawchenko PE. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 274: 60–76, 1988.[CrossRef][ISI][Medline]
- Daftary SS, Boudaba C, Szabo K, and Tasker JG. Noradrenergic excitation of magnocellular neurons in the rat hypothalamic paraventricular nucleus via intranuclear glutamatergic circuits. J Neurosci 18: 10619–10628, 1998.[Abstract/Free Full Text]
- Day TA, Ferguson AV, and Renaud LP. Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells. J Physiol 355: 237–249, 1984.[Abstract/Free Full Text]
- De Kock CPJ, Wierda KDB, Bosman LWJ, Min R, Koksma JJ, Mansvelder HD, Verhage M, and Brussaard AB. Somatodendritic secretion in oxytocin neurons is upregulated during the female reproductive cycle. J Neurosci 23: 2726–2734, 2003.[Abstract/Free Full Text]
- Decavel C and Van Den Pol AN. GABA: a dominant neurotransmitter in the hypothalamus. J Comp Neurol 302: 1019–1037, 1990.[CrossRef][ISI][Medline]
- Derouiche A and Frotscher M. Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins. Glia 36: 330–341, 2001.[CrossRef][ISI][Medline]
- Dityatev A, Dityateva G, Sytnyk V, Delling M, Toni N, Nikonenko I, Muller D, and Schachner M. Polysialylated neural cell adhesion molecule promotes remodeling and formation of hippocampal synapses. J Neurosci 24: 9372–9382, 2004.[Abstract/Free Full Text]
- Durbec P and Remer HC. Revisiting the function of PSA-NCAM in the nervous system. Mol Neurobiol 24: 53–64, 2001.[CrossRef][ISI][Medline]
- Edelman GM and Crossin KL. Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem 60: 155–190, 1991.[CrossRef][ISI][Medline]
- El Majdoubi M, Poulain DA, and Theodosis DT. The glutamatergic innervation of oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus and its contribution to lactation-induced synaptic plasticity. Eur J Neurosci 8: 1377–1389, 1996.[CrossRef][ISI][Medline]
- El Majdoubi M, Poulain DA, and Theodosis DT. Lactation-induced plasticity in the supraoptic nucleus augments axodendritic and axosomatic GABAergic and glutamatergic synapses: an ultrastructural analysis using the disector method. Neuroscience 80: 1137–1147, 1997.[CrossRef][ISI][Medline]
- Fenelon VS, Theodosis DT, and Poulain DA. Fos synthesis in identified magnocellular neurons varies with phenotype, stimulus, location in the hypothalamus and reproductive state. Brain Res 662: 165–177, 1994.[CrossRef][ISI][Medline]
- Friedman HV, Resler TB, Garner CC, and Ziv NE. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27: 57–69, 2000.[CrossRef][ISI][Medline]
- Garcia-Segura LM, Luquin S, Parducz A, and Naftolin F. Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia 10: 59–69, 1994.[CrossRef][ISI][Medline]
- Gies U and Theodosis DT. Synaptic plasticity in the rat supraoptic nucleus during lactation involves GABA innervation and oxytocin neurons: a quantitative immunocytochemical analysis. J Neurosci 14: 2861–2869, 1994.[Abstract]
- Grosche J, Matyash V, Möller T, Verkhratsky A, Reichenbach A, and Kettenmann H. Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat Neurosci 2: 139–143, 1999.[CrossRef][ISI][Medline]
- Guenot-Di Scala D and Strosser M-T. Oxytocin receptors on cultured astroglial cells. Kinetic and pharmacological characterization of oxytocin-binding sites on intact hypothalamic and hippocampic cells from foetal rat brain. Biochem J 284: 491–497, 1992.[Medline]
- Hirrlinger J, Hülsmann S, and Kirchhoff F. Astroglial processes show spontaneous motility at active synaptic terminals in situ. Eur J Neurosci 20: 2235–2239, 2004.[CrossRef][ISI][Medline]
- Hoyk ZS, Parducz A, and Theodosis DT. The highly sialylated isoform of the neural cell adhesion molecule is required for estradiol-induced morphological synaptic plasticity in the adult arcuate nucleus. Eur J Neurosci 13: 649–656, 2001.[CrossRef][ISI][Medline]
- Israel JM, Le Masson G, Theodosis DT, and Poulain DA. Glutamate afferent input governs periodicity and synchronisation of bursting electrical activity in oxytocin neurons. Eur J Neurosci 17: 2619–2629, 2003.[CrossRef][ISI][Medline]
- Israel JM and Poulain DA. 17-oestradiol modulates in vitro electrical properties and responses to kainate of oxytocin neurones in lactating rats. J Physiol 524: 457–470, 2000.[Abstract/Free Full Text]
- Israel JM, Schipke CG, Ohlemeyer C, Theodosis DT, and Kettenmann H. GABA-A receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents. Glia 44: 102–110, 2003.[CrossRef][ISI][Medline]
- Jhamandas JH, Raby W, Rogers J, Buijs RM, and Renaud LP. Diagonal band projection towards the hypothalamic supraoptic nucleus: light and electron microscopic observations in the rat. J Comp Neurol 282: 15–23, 1989.[CrossRef][ISI][Medline]
- Jourdain P, Dupouy B, Bonhomme R, Poulain DA, Israel JM, and Theodosis DT. Visualization of local afferent inputs to magnocellular oxytocin neurons in vitro. Eur J Neurosci 11: 1–13, 1999.[Medline]
- Jourdain P, Israel JM, Dupouy B, Oliet SHR, Allard M, Vitiello S, Theodosis DT, and Poulain DA. Evidence for a hypothalamic oxytocin-sensitive pattern-generating network governing oxytocin neurons in vitro. J Neurosci 18: 6641–6649, 1998.[Abstract/Free Full Text]
- Katz LC and Shatz CJ. Synaptic activity and the construction of cortical circuits. Science 274: 1133–1138, 1996.[Abstract/Free Full Text]
- Kiss JZ, Wang C, and Rougon G. Nerve-dependent expression of high polysialic acid neural cell adhesion molecule in neurohypophysial astrocytes of adult rats. Neuroscience 53: 213–222, 1993.[CrossRef][ISI][Medline]
- Landgraf R and Neumann ID. Neuropeptide release within the brain: a dynamic concept of multiple and variable modes of communication. Front Neuroendocrinol 25: 150–176, 2004.[CrossRef][ISI][Medline]
- Langle SL, Poulain DA, and Theodosis DT. Induction of rapid, activity-dependent neuronal-glial remodeling in the adult hypothalamus in vitro. Eur J Neurosci 18: 206–214, 2003.[CrossRef][ISI][Medline]
- Levinson JN and El-Husseini A. Building excitatory and inhibitory synapses: balancing neuroligin partnerships. Neuron 48: 171–174, 2005.[CrossRef][ISI][Medline]
- Lightman SL and Young WS III. Lactation inhibits stress mediated secretion of corticosterone and oxytocin and hypothalamic accumulation of CRF and enkephalin messenger ribonucleic acids. Endocrinology 124: 2358–2364, 1989.[Abstract]
- McDonnell DP and Morris JD. Connections and regulation of the human estrogen receptor. Science 296: 1642–1644, 2002.[Abstract/Free Full Text]
- Meeker RB, Swanson DJ, Greenwood RS, and Hayward JN. Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus. Brain Res 600: 112–122, 1993.[CrossRef][ISI][Medline]
- Michaloudi HC, El Majdoubi M, Poulain DA, Papadopoulos GC, and Theodosis DT. The noradrenergic innervation of identified hypothalamic somata and its contribution to lactation-induced synaptic plasticity. J Neuroendocrinol 9: 17–23, 1997.[CrossRef][ISI][Medline]
- Miyata S and Hatton GI. Activity-related, dynamic neuron-glia interactions in the hypothalamo-neurohypophysial system. Microsc Res Tech 56: 143–157, 2002.[CrossRef][ISI][Medline]
- Monlezun S, Ouali S, Poulain DA, and Theodosis DT. Polysialic acid is required for active phases of morphological plasticity of neurosecretory axons and their glia. Mol Cell Neurosci 29: 516–524, 2005.[CrossRef][ISI][Medline]
- Montagnese C, Poulain DA, and Theodosis DT. Influence of ovarian steroids on the ultrastructural plasticity of the adult supraoptic nucleus induced by central administration of oxytocin. J Neuroendocrinol 2: 225–231, 1990.
- Montagnese C, Pouclain DA, Vincent JD, and Theodosis DT. Structural plasticity in the rat supraoptic nucleus during gestation, post-partum lactation and suckling-induced pseudogestation and lactation. J Endocrinol 115: 97–105, 1987.[Abstract]
- Moos FC. GABA-induced facilitation of the periodic bursting activity of oxytocin neurones in suckled rats. J Physiol 488: 103–114, 1995.[Abstract/Free Full Text]
- Muller D, Wang C, Skibo G, Toni N, Cremer H, Calaora V, Rougon G, and Kiss JZ. PSA-NCAM is required for activity-induced synaptic plasticity. Neuron 17: 413–422, 1996.[CrossRef][ISI][Medline]
- Nothias F, Vernier P, Von Boxberg Y, Mirman S, and Vincent JD. Modulation of NCAM polysialylation is associated with morphofunctionnal modification in the hypothalamo-neurohypophysial system during lactation. Eur J Neurosci 9: 1553–1565, 1997.[CrossRef][ISI][Medline]
- Oliet SHR, Piet R, and Poulain DA. Control of glutamate clearance and synaptic efficacy by glial coverage of neurons. Science 292: 923–926, 2001.[Abstract/Free Full Text]
- Oliet SHR, Piet R, Poulain DA, and Theodosis DT. Glial modulation of synaptic transmission: insights from the hypothalamic supraoptic nucleus. Glia 47: 258–267, 2004.[CrossRef][ISI][Medline]
- Olmos G, Naftolin F, Perez J, Tranque PA, and Garcia-Segura LM. Synaptic remodeling in the rat arcuate nucleus during the estrous cycle. Neuroscience 32: 663–667, 1989.[CrossRef][ISI][Medline]
- Pak DT and Sheng M. Targeted protein degradation and synapse remodeling by an inducible protein kinase. Science 302: 1368–1373, 2003.[Abstract/Free Full Text]
- Parker SL and Crowley WR. Stimulation of oxytocin release in the lactating rat by a central interaction of -1-adrenergic and -amino-3-hydroxy-5-methylisoxazole-4-propionic acid-sensitive excitatory amino acid mechanisms. Endocrinology 133: 2855–2860, 1993.[Abstract]
- Parpura V and Haydon PG. Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc Natl Acad Sci USA 97: 8629–8634, 2000.[Abstract/Free Full Text]
- Pierre K, Bonhomme R, Dupouy B, Poulain DA, and Theodosis DT. The polysialylated neural cell adhesion molecule (PSA-NCAM) reaches cell surfaces of hypothalamic neurons and astrocytes via the constitutive pathway. Neuroscience 103: 133–142, 2001.[CrossRef][ISI][Medline]
- Poulain DA and Wakerley JB. Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin. Neuroscience 7: 773–808, 1982.[CrossRef][ISI][Medline]
- Rasse TM, Focuquet W, Scchmid A, Kittel RJ, Mertel S, Sigrist CB, Schmidt M, Guzman A, Merino C, Qin G, Quentin C, Madeo FF, Heckman M, and Sigrist SJ. Glutamate receptor dynamics organizing synapse formation in vivo. Nat Neurosci 8: 898–905, 2005.[ISI][Medline]
- Revankar CM, Cimino DF, Sklar LA, Arterburn JB, and Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307: 1625–1630, 2005.[Abstract/Free Full Text]
- Richard P, Moos F, and Freund-Mercier MJ. Central effects of oxytocin. Physiol Rev 71: 331–370, 1991.[Free Full Text]
- Roland BL and Sawchenko PE. Local origins of some GABAergic projections to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol 332: 123–143, 1993.[CrossRef][ISI][Medline]
- Rougon G. Structure, metabolism and cell biology of polysialic acids. Eur J Cell Biol 61: 197–207, 1993.[ISI][Medline]
- Rutishauser U and Landmesser LT. Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions. Trends Neurosci 19: 422–427, 1996.[ISI][Medline]
- Schuster T, Krug M, Hassan H, and Schachner M. Increase in proportion of hippocampal spine synapses expressing neural cell adhesion molecule NCAM180 following long-term potentiation. J Neurobiol 37: 359–372, 1998.[CrossRef][ISI][Medline]
- Seki T and Arai Y. Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci Res 17: 265–290, 1993.[CrossRef][ISI][Medline]
- Shen H, Watanabe M, Tomasiewicz H, Rutishauser U, Magnuson T, and Glass JD. Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function. J Neurosci 17: 5221–5229, 1997.[Abstract/Free Full Text]
- Shughrue PJ, Komm B, and Merchenthaler I. The distribution of estrogen receptor-beta mRNA in the rat hypothalamus. Steroids 61: 678–681, 1996.[CrossRef][ISI][Medline]
- Sladek CD and Somponpun SJ. Oestrogen receptor beta: role in neurohypophysial neurons. J Neuroendocrinol 16: 365–371, 2004.[CrossRef][ISI][Medline]
- Somponpun SJ and Sladek CD. Osmotic regulation of estrogen receptor-beta in rat vasopressin and oxytocin neurons. J Neurosci 23: 4261–4269, 2003.[Abstract/Free Full Text]
- Theodosis DT. Oxytocin-secreting neurons: a physiological model of morphological neuronal and glial plasticity in the adult hypothalamus. Front Neuroendocrinol 23: 101–135, 2002.[CrossRef][ISI][Medline]
- Theodosis DT, Bonhomme R, Vitiello S, Rougon G, and Poulain DA. Cell surface expression of polysialic acid on NCAM is a prerequisite for activity-dependent morphological neuronal and glial plasticity. J Neurosci 19: 10228–10236, 1999.[Abstract/Free Full Text]
- Theodosis DT, Chapman DB, Montagnese C, Poulain DA, and Morris JF. Structural plasticity in the hypothalamic supraoptic nucleus at lactation affects oxytocin- but not vasopressin-secreting neurones. Neuroscience 17: 661–678, 1986.[CrossRef][ISI][Medline]
- Theodosis DT, Koksma J-J, Trailin A, Langle SL, Piet R, Lodder JC, Timmerman J, Mansvelder H, Poulain DA, Oliet SHR, and Brussaard AB. Oxytocin and estrogen promote rapid formation of functional GABA synapses in the adult supraoptic nucleus. Mol Cell Neurosci. In press.
- Theodosis DT and Macvicar BA. Neuron-glia interactions in the hypothalamus and pituitary. Trends Neurosci 19: 363–367, 1996.[CrossRef][ISI][Medline]
- Theodosis DT, Montagnese C, Rodriguez F, Vincent JD, and Poulain DA. Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system. Nature 322: 738–740, 1986.[CrossRef][Medline]
- Theodosis DT, Paut L, and Tappcaz ML. Immunocytochemical analysis of the GABAergic innervation of oxytocin- and vasopressin-secreting neurones in the rat supraoptic nucleus. Neuroscience 19: 207–222, 1986.[CrossRef][ISI][Medline]
- Theodosis DT, Piet R, Poulain DA, and Oliet SHR. Neuronal, glial and synaptic remodeling in the adult hypothalamus: functional consequences and role of cell surface and extracellular matrix adhesion molecules. Neurochem Int 45: 491–501, 2004.[CrossRef][ISI][Medline]
- Theodosis DT and Poulain DA. Evidence for structural plasticity in the supraoptic nucleus of the rat hypothalamus in relation to gestation and lactation. Neuroscience 11: 183–193, 1984.[CrossRef][ISI][Medline]
- Theodosis DT and Poulain DA. Oxytocin-secreting neurones: a physiological model for structural plasticity in the adult mammalian brain. Trends Neurosci 10: 426–430, 1987.[CrossRef]
- Theodosis DT and Poulain DA. Neuronal-glial and synaptic plasticity in the adult rat paraventricular nucleus. Brain Res 484: 361–366, 1989.[CrossRef][ISI][Medline]
- Theodosis DT, Poulain DA, and Vincent JD. Possible morphological bases for synchronisation of neuronal firing in the rat supraoptic nucleus during lactation. Neuroscience 6: 919–929, 1981.[CrossRef][ISI][Medline]
- Theodosis DT, Rougon G, and Poulain DA. Retention of embryonic features by an adult neuronal system capable of plasticity: polysialylated N-CAM in the hypothalamo-neurohypophysial system. Proc Natl Acad Sci USA 88: 5494–5498, 1991.[Abstract/Free Full Text]
- Theodosis DT, Schachner M, and Neumann ID. Oxytocin neuron activation in NCAM-deficient mice: anatomical and functional consequences. Eur J Neurosci 20: 3270–3280, 2004.[CrossRef][ISI][Medline]
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ABSTRACT
SYNAPSE TURNOVER IN THE...
