Astrocytic plasticity and patterned oxytocin neuronal activity: dynamic interactions.

Astroglial-neuronal interactions are important in brain functions. However, roles of glial fibrillary acidic protein (GFAP) in this interaction remain unclear in acute physiological processes. We explored this issue using the supraoptic nucleus (SON) in lactating rats. At first, we identified the essential role of astrocytes in the milk-ejection reflex (MER) by disabling astrocytic functions via intracerebroventricular application of l-aminoadipic acid (l-AAA). l-AAA blocked the MER and reduced GFAP levels in the SON. In brain slices, l-AAA suppressed oxytocin (OT) neuronal activity and EPSCs. Suckling reduced GFAP in immunocytochemical images and in Western blots, reductions that were partially reversed after the MER. OT, the dominant hormone mediating the MER, reduced GFAP expression in brain slices. Tetanus toxin suppressed EPSCs but did not influence OT-reduced GFAP. Protease inhibitors did not influence OT-reduced GFAP images but blocked the degradation of GFAP molecules. In the presence of OT, transient 12 mm K(+) exposure, simulating effects of synchronized bursts before the MER, reversed OT-reduced GFAP expression. Consistently, suckling first reduced and then increased the expression of aquaporin 4, astrocytic water channels coupled to K(+) channels. Moreover, GFAP molecules were associated with astrocytic proteins, including aquaporin 4, actin, and glutamine synthetase and serine racemase. GFAP-aquaporin 4 association decreased during initial suckling and increased after the MER, whereas opposite changes occurred between GFAP and actin. MER also decreased the association between GFAP and glutamine synthetase. These results indicate that suckling elicits dynamic glial neuronal interactions in the SON; GFAP plasticity dynamically reflects OT neuronal activity.

Neural mechanisms underlying the milk ejection burst and reflex.

One of the more interesting and complex phenomena involving neurohypophysial hormones is the milk ejection reflex and the events surrounding it. Accordingly, many investigations over the years have taken up the challenge of elucidating its myriad aspects. Much has been learned from in vivo preparations about the sequence of events that so regularly occurs: important priming by maternal behaviours, the intermittent rhythms, gating of bursting, synchrony of the oxytocin (OXT) neuronal bursts emitted intermittently in response to the continuous suckling of the young and the factors that influence the amplitude of the bursts/milk ejections (e.g. number of suckling pups). In vivo electrophysiological studies are constrained by the infeasibility of routinely recording transmembrane events and, therefore, cannot offer detailed membrane and/or synaptic analyses. Recent studies have developed an in vitro model of OXT neuronal bursting that has allowed more mechanistic analyses of these bursts as well as factors involved in their generation and structure. Here we review many of the cellular and molecular mechanisms that have been shown to underlie the milk ejection bursts, as revealed by in vitro analyses.

Interaction of extracellular signal-regulated protein kinase 1/2 with actin cytoskeleton in supraoptic oxytocin neurons and astrocytes: role in burst firing.

Neuronal firing patterns determine the manner of neurosecretion, the underlying mechanisms of which are poorly understood. Using supraoptic nuclei in brain slices from lactating rats, we examined the involvement of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and filamentous actin (F-actin) in burst generation by oxytocin (OT) neurons. Blocking phosphorylation of ERK1/2 (pERK1/2) decreased miniature EPSCs and blocked OT-evoked bursts, as did intracellularly loading an antibody against pERK1/2. OT (10 pM) increased cytosolic pERK1/2 close to the cell membrane within the first 5 min, subsiding by 30 min, whereas OT elicited pERK1/2 nuclear translocation in closely associated supraoptic astrocytes. The increased pERK1/2 was tightly correlated with spatiotemporal actin dynamics. In OT neurons, OT initially increased F-actin, particularly at membrane subcortical areas, and then decreased it after 30 min. Both polymerization and depolymerization of actin cytoskeleton were associated with bursts, but only polymerization facilitated OT-evoked bursts. Blocking ERK1/2 activation blocked OT-evoked actin polymerization, whereas depolymerizing F-actin increased pERK1/2 expression. These changes were further identified in vivo. In intact animals, suckling increased ERK1/2 activation in the cytosol and membrane subcortical area F-actin formation in OT neurons, whereas it increased F-actin concentration in astrocytic somata. Coimmunoprecipitation showed that suckling increased molecular interactions between pERK1/2 and actin. Finally, two different blockers of ERK1/2 kinase injected intracerebroventricularly reduced suckling-evoked milk ejections. This is the first demonstration that OT mediation of suckling-evoked bursts/milk ejections is via interactions between pERK1/2 and actin cytoskeleton.

Catecholaminergic control of mitogen-activated protein kinase signaling in paraventricular neuroendocrine neurons in vivo and in vitro: a proposed role during glycemic challenges.

Paraventricular hypothalamic (PVH) corticotropin-releasing hormone (CRH) neuroendocrine neurons mount neurosecretory and transcriptional responses to glycemic challenges [intravenous 2-deoxyglucose (2-DG) or insulin]. Although these responses require signals from intact afferents originating from hindbrain CA (catecholaminergic) neurons, the identity of these signals and the mechanisms by which they are transduced by PVH neurons during glycemic challenge remain unclear. Here, we tested whether the prototypical catecholamine, norepinephrine (NE), can reproduce PVH neuroendocrine responses to glycemic challenge. Because these responses include phosphorylation of p44/42 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases 1/2 (ERK1/2)], we also determined whether NE activates ERK1/2 in PVH neurons and, if so, by what mechanism. We show that systemic insulin and 2-DG, and PVH-targeted NE microinjections, rapidly elevated PVH phospho-ERK1/2 levels. NE increased Crh and c-fos expression, together with circulating ACTH/corticosterone. However, because injections also increased c-Fos mRNA in other brain regions, we used hypothalamic slices maintained in vitro to clarify whether NE activates PVH neurons without contribution of inputs from distal regions. In slices, bath-applied NE triggered robust phospho-ERK1/2 immunoreactivity in PVH (including CRH) neurons, which attenuated markedly in the presence of the alpha1 adrenoceptor antagonist, prazosin, or the MAP kinase kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Therefore, at a systems level, local PVH delivery of NE is sufficient to account for hindbrain activation of CRH neuroendocrine neurons during glycemic challenge. At a cellular level, these data provide the first demonstration that MAP kinase signaling cascades (MEK-->ERK) are intracellular transducers of noradrenergic signals in CRH neurons, and implicate this transduction mechanism as an important component of central neuroendocrine responses during glycemic challenge.

Dominant role of betagamma subunits of G-proteins in oxytocin-evoked burst firing.

Pulsatile neuropeptide secretion is associated with burst firing patterns; however, intracellular signaling cascades leading to bursts remain unclear. We explored mechanisms underlying burst firing in oxytocin (OT) neurons in the supraoptic nucleus in brain slices from lactating rats. Application of 10 pm OT for 30 min or progressively rising OT concentrations from 1 to 100 pm induced burst firing in OT neurons in patch-clamp recordings. Burst generation was blocked by OT antagonist and ionotropic glutamate receptor blockers or tetanus toxin. Blocking G-protein activation with suramin or intracellular GDP-beta-S, but not intracellularly administered antibody against the OT-receptor (OTR) C terminus, blocked bursts. Moreover, pretreatment of slices with pertussis toxin, an inhibitor of G(i/o)-proteins, did not block OT-evoked bursts, suggesting that G(i)/G(o) activation is unnecessary for burst generation. Thus, we further examined G alpha(q/11)-associated signaling pathways in OT-evoked bursts. Inhibition of phospholipase C or RhoA/Rho kinase did not block bursts. Activation of G betagamma subunits using myristoylated G betagamma-binding peptide (mSIRK) caused bursts, whereas intracellularly loaded antibody against G beta subunit blocked OT-evoked bursts. Blocking Src family kinase, but not phosphatidylinositol 3-kinase, occluded OT-evoked bursts. Similar to the effects of OT on EPSCs, mSIRK inhibited tonic EPSCs and elicited EPSC clustering. Finally, suckling caused dissociation of OTRs and G beta subunits from G alpha(q/11) subunits shown by coimmunoprecipitation and immunocytochemistry, supporting crucial roles for OTRs and G betagamma subunits in the milk-ejection reflex. We conclude that G betagamma subunits play a dominant role in burst firing evoked by applied OT or by suckling.

Activation of adenosine A2A receptors alters postsynaptic currents and depolarizes neurons of the supraoptic nucleus.

Supraoptic nucleus (SON) neurons secrete oxytocin or vasopressin in response to various physiological stimuli (e.g., lactation/suckling, dehydration). Released near fenestrated capillaries of the neurohypophysis, these peptides enter the blood and travel to peripheral target organs. The pervasive neuromodulator adenosine, acting at A1 receptors, is an important inhibitory regulator of magnocellular neuroendocrine cell activity. Another high-affinity adenosine receptor exists in this system, however. We examined the physiological effects of adenosine A2A receptor activation and determined its localization among various cell types within the SON. In whole cell patch-clamp recordings from rat brain slices, application of the selective adenosine A2A receptor agonist CGS-21680 caused membrane depolarizations in SON neurons, often leading to increased firing activity. Membrane potential changes were persistent (>10 min) and could be blocked by the selective A2A receptor antagonist ZM-241385, or GDP-beta-S, the latter suggesting postsynaptic sites of action. However, +/--alpha-methyl-(4-carboxyphenyl)glycine or TTX also blocked CGS-21680 effects, indicating secondary actions on postsynaptic neurons. In voltage-clamp mode, application of CGS-21680 caused a slight increase (approximately 8%) in high-frequency clusters of excitatory postsynaptic currents. With the use of specific antibodies, adenosine A2A receptors were immunocytochemically localized to both the magnocellular neurons and astrocytes of the SON. Ecto-5'nucleotidase, an enzyme involved in the metabolism of ATP to adenosine, was also localized to astrocytes of the SON. These results demonstrate that adenosine acting at A2A receptors can enhance the excitability of SON neurons and modulate transmitter release from glutamatergic afferents projecting to the nucleus. We suggest that adenosine A2A receptors may function in neuroendocrine regulation through both direct neuronal mechanisms and via actions involving glia.

Mechanisms underlying oxytocin-induced excitation of supraoptic neurons: prostaglandin mediation of actin polymerization.

In nonneuronal tissues, activation of oxytocin receptors (OTRs), like other Galpha(q/11) type G-protein-coupled receptors (Galpha(q/11)/GPCRs), increase prostaglandin (PG) expression. This is not known for the OTRs expressed by central OT neurons. We examined mechanisms underlying OT's effects on supraoptic nucleus (SON) OT and vasopressin (VP) neurons in hypothalamic slices from lactating rats. OT application (10 pM, 10 min) significantly increased firing rates of OT and VP neurons, both of which expressed OTRs. Indomethacin, an inhibitor of PG synthetases, blocked these increases. OTR (but not a V1 receptor) antagonist blocked OT effects without blocking the excitatory effect of PGE2. Tetanus toxin blocked OT effects on fast synaptic inputs and firing activity of SON neurons but not OT-evoked depolarization, suggesting involvement of both pre- and postsynaptic neurons. Indomethacin also blocked the excitatory effects of phenylephrine, another Galpha(q/11)/GPCR activating agent but not those of PGE2, a non-Galpha(q/11)/GPCR activating agent in the SON. OT or phenylephrine, but not glutamate or KCl, enhanced cyclooxygenase 2 expression at cytosolic loci in SON neurons and nearby astrocytes, as revealed by immunocytochemistry. This OT effect was not blocked by TTX. Western blot analyses showed that OT significantly increased cyclooxygenase 2 but not actin expression. OT promoted the formation of filamentous actin (F-actin) networks at membrane subcortical areas of both OT and VP neurons. Indomethacin blocked enhancement of F-actin networks by OT but not by PGE2. These results indicate that PGs serve as a common mediator of Galpha(q/11)/GPCR-activating agents in neuronal function.

Autofeedback effects of progressively rising oxytocin concentrations on supraoptic oxytocin neuronal activity in slices from lactating rats.

Suckling stimuli induce somatodendritic oxytocin (OT) release from supraoptic nucleus (SON) neurons, which raises intranuclear OT concentrations and contributes to the effectiveness of the milk-ejection reflex. To clarify how such changes in OT concentrations modulate the activity of OT neurons, we examined OT effects using whole cell patch-clamp recordings from SON neurons in slices from lactating rats. Progressive increases from extremely low OT concentrations (0.1-10 fM) to high concentrations (0.1-10 nM) induced excitation and subsequent spike frequency reduction (SFR) in OT neurons. Significant effects of OT on firing rates were observed starting at 1 fM, reached peak level from 1 fM to 1 pM before SFR occurred in most neurons. The buildup of OT concentrations progressively promoted depolarization of membrane potential, spike broadening, decreases in spike amplitude, and increases in the rise time of spike afterhyperpolarizations, which were unrelated to firing rate. However, intermittent application of OT (1 fM, 1 pM, and 1 nM, each for 5 min) evoked dose-dependent excitation but not the SFR. Application of 1 pM OT for 40 min simulated the effects of progressively increasing OT concentrations. Vasopressin neurons were also activated by OT but did not show SFR. Consistent with presynaptic loci of OT action, ionotropic glutamate receptor antagonists reduced OT effects on firing rate, whereas bicuculline did not change the excitatory effects. These results suggest that the specific autoregulatory effects of OT, and perhaps other neuropeptides as well, are time and concentration dependent.

Burst firing of oxytocin neurons in male rat hypothalamic slices.

Burst firing and single spike activity play different roles in the modulation of local neuronal circuit activity and neurosecretion. In hypothalamic oxytocin (OT) neurons in vivo, burst firing is associated with pulsatile secretion of OT in the milk ejection reflex, and can be observed in slices from both immature and lactating rats in vitro. Whether OT neurons from male rats also possess burst firing capability is still an open question. To examine this possibility, whole-cell patch clamp recordings were made in supraoptic nucleus OT neurons in brain slices from male rats. In low Ca(2+) medium, the alpha(1)-adrenoceptor agonist, phenylephrine evoked bursts that were highly similar to those from lactating rats in vivo and in vitro: explosive onset, short-duration, quickly reaching peak firing rate and displaying an exponential decay in returning to the pre-burst rate. During bursts, spike durations increased, and spike amplitudes decreased, while riding on an arc of depolarization around peak rate. In comparison to those from lactating rats in vitro, the rising phase of male bursts was more rapid, the decay phase was slower, and the rising phase of the spike after hyperpolarization was faster. No significant differences, however, were seen in burst characteristics that are most important in determining the amount of peptide release: burst amplitudes (the number of spikes in a burst), firing frequency within bursts or peak firing rate. Thus, we conclude that OT neurons in males are capable of burst firing highly similar to that seen in lactating rats.

Adenosine postsynaptically modulates supraoptic neuronal excitability.

Effects of adenosine on the excitability of supraoptic nucleus neurons were investigated in whole cell patch-clamp experiments conducted in horizontal slices of rat hypothalamus. Adenosine (10-100 muM) inhibited all neurons tested by reducing or abolishing spontaneous or evoked discharge. Large hyperpolarizations were seen, averaging -6.08 +/- 0.83 mV below resting membrane potential, and action potential durations were significantly reduced by 134 +/- 41 mus in the presence of 100 muM adenosine. The A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 muM) blocked these effects, whereas the A(1) agonists N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA) mimicked the actions of adenosine. A(2) receptor contributions to excitability were assessed by application of an A(2) agonist, carboxamidoadenosine (CPCA). This resulted in membrane depolarizations (3.56 +/- 0.65 mV) and maintenance of firing. The presence of endogenous adenosine in the slice was revealed by both the application of the adenosine uptake inhibitor dilazep (1-100 muM), which resulted in a strong inhibition of firing activity, and the application of DPCPX, which induced firing in cells silenced by negative current injection. We tested for postsynaptic actions of adenosine by blocking G protein activation via GDP-beta-S infusion into recorded neurons. Under these conditions, the adenosinergic inhibition of firing and reduction of spike duration were blocked, suggesting the effects were mediated by postsynaptic adenosine receptors. That the effects on excitability could be due to direct activation of adenosine A(1) receptors on supraoptic neurons was further explored immunocytochemically via the co-labeling of magnocellular neurons with polyclonal antibodies raised against the A(1) receptors. It is concluded that adenosine, acting at postsynaptic A(1) receptors, exhibits a powerful inhibitory influence on supraoptic magnocellular activity and is an important endogenous regulator of magnocellular neuroendocrine function.

Dynamic neuronal-glial interactions: an overview 20 years later.

After commenting on some perceived reasons why our review may have been relatively frequently cited, a brief overview is presented that first summarizes what we knew 25 years ago about the dynamic neuronal-astroglial interactions that occur in response to changes in the physiological state of the animal. The brain system in which these dynamic interactions were studied was the magnocellular hypothalamo-neurohypophysial system (mHNS) of the rat. The mHNS developed as and continues to be the model system yielding the most coherent picture of dynamic morphological changes and insights into their functional consequences. Many other brain areas, however, have more recently come under scrutiny in the search for glial-neuronal dynamisms. Outlined next are some of the questions concerning this phenomenon that led to the research efforts immediately following the initial discoveries, along with the answers, both complete and incomplete, obtained to those research questions. The basis for this first wave of follow-up research can be characterized by the phrase "what we knew we didn't know at that time." The final section is an update and brief overview of highlights of both "what we know now" and "what we now know that we don't know" about dynamic neuronal-astroglial interactions in the mHNS.

Milk ejection burst-like electrical activity evoked in supraoptic oxytocin neurons in slices from lactating rats.

To examine the mechanisms underlying milk-ejection bursts of oxytocin (OT) neurons during suckling, both in vivo and in vitro studies were performed on supraoptic OT neurons from lactating rats. The bursts were first recorded extracellularly in anesthetized rats. Burst-related electrical parameters were essentially the same as previous reports except for a trend toward transient decreases in basal firing rates immediately preceding the burst. From putative OT neurons in slices with extracellular recordings, bursts that closely mimicked the in vivo bursts were elicited by phenylephrine, an alpha1-adrenoceptor agonist, in a low-Ca(2+) medium. Moreover, in whole cell patch-clamp recordings, the in vitro bursts were recorded from immunocytochemically identified OT neurons. After a transient decrease in the basal firing rate, the in vitro bursts started with a sudden increase in the firing rate, quickly reaching a peak level, then gradually decaying, and ended with a postburst inhibition. A brief depolarization of the membrane potential and an increase in membrane conductance appeared after the onset of the burst. Spikes during a burst were characterized by a significant increase in the duration and decrease in the amplitude around the peak rate firing. These bursts were significantly different from short-lasting burst firing of vasopressin neurons in membrane potential changes, time to reach peak firing rate, spike amplitude and duration during peak rate firing. Our extensive analysis of these results suggests that the in vitro burst is a useful model for further study of mechanisms underlying milk-ejection bursts of OT neurons in vivo.

Taurine and the control of basal hormone release from rat neurohypophysis.

Pituicytes of pituitary neural lobe are rich in the amino acid taurine, which they release upon hypoosmotic stimulation. As a generally inhibitory amino acid, taurine is thought to activate receptors on neural lobe nerve terminals and exert some control over hormone release. Previous work has shown the presence of glycine and GABA(A) receptors in neural lobe, both of which have affinity for taurine. Using a perifused explant system, we studied the effects of taurine activation of glycine and GABA(A) receptors on basal hormone release. Somewhat surprisingly, taurine induced increases in basal release of both vasopressin and oxytocin. Taurine-induced increases in oxytocin release were blocked by bicuculline, suggesting involvement of GABA(A) receptors. Increases in vasopressin release were not blocked by bicuculline, indicating involvement of receptors other than GABA(A). Although combined bicuculline and strychnine, an antagonist at most glycine receptors, also did not block increased vasopressin release, picrotoxin (a Cl(-) channel blocker) was effective in blocking increases in both vasopressin and oxytocin release. The other receptor(s) involved in taurine actions is postulated to be strychnine-insensitive glycine receptors. Thus, taurine in neural lobe may act via both a GABA(A) receptor and one or more types of glycine receptors to depolarize nerve terminal membranes under basal conditions. Taurine-induced partial depolarization resulting in Na(+) channel inactivation is probably responsible for its previously observed inhibition of stimulated hormone release from neural lobe.

Fractones and other basal laminae in the hypothalamus.

The physiological role of basal laminae (BL) and connective tissue (meninges and their projections) in the adult brain is unknown. We recently described novel forms of BL, termed fractones, in the most neurogenic zone of the adult brain, the subependymal layer (SEL) of the lateral ventricle. Here, we investigated the organization of BL throughout the hypothalamus, using confocal and electron microscopy. New types of BL were identified. First, fractones, similar to those found in the lateral ventricle wall, were regularly arranged along the walls of the third ventricle. Fractones consisted of labyrinthine BL projecting from SEL blood vessels to terminate immediately beneath the ependyma. Numerous processes of astrocytes and of microglial cells directly contacted fractones. Second, another form of BL projection, termed anastomotic BL, was found between capillaries in dense capillary beds. The anastomotic BL enclosed extraparenchymal cells that networked with the perivascular cells coursing in the sheaths of adjacent blood vessels. Vimentin immunoreactivity was often detected in the anastomotic BL. In addition, the anastomotic BL overlying macrophages contained numerous fibrils of collagen. We also found that the BL located at the pial surface formed labyrinthine tube-like structures enclosing numerous fibroblast and astrocyte endfeet, with pouches of collagen fibrils at the interface between the two cell types. We suggest that cytokines and growth factors produced by connective tissue cells might concentrate in BL, where their interactions with extracellular matrix proteins might contribute to their effects on the overlying neural tissue, promoting cytogenesis and morphological changes and participating in neuroendocrine regulation.

Histamine H1-receptor modulation of inter-neuronal coupling among vasopressinergic neurons depends on nitric oxide synthase activation.

Inter-neuronal coupling is a relatively recently documented property of a wide variety of cell groups in the mammalian central nervous system. For many of these groups there is evidence that the coupling can be modulated by synaptic inputs. Incidence of dye coupling among vasopressin (VP) neurons of the rat supraoptic nucleus (SON) has been shown to increase in response to either activation of histamine H(1)-receptors or to increased NO production. Both of these effects involve activation of cGMP-dependent pathways. We tested the hypothesis that activation of H(1)-receptors resulted in downstream activation of NO synthase, which then mediated the H(1)-receptor effects. Putative VP neurons were intracellularly recorded and dye-injected in horizontal slices of hypothalamus, in which monosynaptic connections from the tuberomammillary nucleus (TM) were intact and electrically stimulated. Single-pulse TM stimulation evoked EPSPs and repetitive stimulation resulted in a nearly 3-fold increase in coupling incidence over unstimulated slices. TM-stimulated increased coupling was completely blocked by inhibitors of NO synthase (L-NAME) or of soluble guanylyl cyclase (ODQ or methylene blue), or pyrilamine, suggesting that the H(1)-receptor is not directly linked to guanylyl cyclase. Addition of the NO precursor, L-arginine or the NO donor, SNP, in combination with TM stimulation produced increases in coupling that were not significantly larger than those seen with stimulation alone, supporting the idea that a common pathway was used. We conclude that H(1)-receptors activate NO synthase via G-protein-coupled pathways and that NO working though its receptor, induces the downstream cGMP-dependent processes that result in increased inter-neuronal coupling.

Glial-neuronal interactions in the mammalian brain.

Recognition of the importance of glial cells in nervous system functioning is increasing, specifically regarding the modulation of neural activity. This brief review focuses on some of the morphological and functional interactions that take place between astroglia and neurons. Astrocyte-neuron interactions are of special interest because this glia cell type has intimate and dynamic associations with all parts of neurons, i.e., somata, dendrites, axons, and terminals. Activation of certain receptors on astrocytes produces morphological changes that result in new contacts between neurons, along with physiological and functional changes brought about by the new contacts. In response to activation of other receptors or changes in the extracellular microenvironment, astrocytes release neuroactive substances that directly excite or inhibit nearby neurons and may modulate synaptic transmission. Although some of these glial-neuronal interactions have been known for many years, others have been quite recently revealed, but together they are forming a compelling story of how these two major cell types in the brain carry out the complex tasks that mammalian nervous systems perform.

Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network.

Cytogenesis in adult peripheral organs, and in all organs during development, occurs nearby basal laminae (BL) overlying connective tissue. Paradoxically, cytogenesis in the adult brain occurs primarily in the subependymal layer (SEL), a zone where no particular organization of BL and connective tissue has been described. We have reinvestigated the anatomy of the area considered the most neurogenic in the adult brain, the SEL of the lateral ventricle, in zones adjacent to the caudate putamen, corpus callosum, and lateral septal nucleus. Here, we report structural (confocal microscopy using laminin as a marker) and ultrastructural evidence for highly organized extravascular BL, unique to the SEL. The extravascular BL, termed fractones because of their fractal organization, were regularly arranged along the SEL and consisted of stems terminating in bulbs immediately underneath the ependyma. Fractones contacted local blood vessels by means of their stems. An individual fractone engulfed in its folds numerous processes of astrocytes, ependymocytes, microglial cells, and precursor cell types. The attachment site (base) of stems to blood vessels was extensively folded, overlying large perivascular macrophages that belong to a fibroblast/macrophage network coursing in the perivascular layer and through the meninges. In addition, collagen-1, which is associated with BL and growth factors during developmental morphogenetic inductions, was immunodetected in the SEL and particularly regionalized within fractones. Because macrophages and fibroblasts produce cytokines and growth factors that may concentrate in and exert their effect from the BL, we suggest that the structure described is implicated in adult neurogenesis, gliogenesis, and angiogenesis.

Peripartum interneuronal coupling in the supraoptic nucleus.

In the supraoptic nucleus (SON), the incidence of conducting gap junctions (gjs), as indicated by dye coupling, is low in cycling females, but dramatically elevated in nursing mothers. Functionally, this is consistent with the well-established presence of synchronous milk ejection bursts among oxytocin neurons only in the lactating rat. In situ hybridization data, however, revealed elevated gj mRNA expression on the last day of pregnancy, a time when burst firing by putative oxytocin neurons is absent. Using Lucifer Yellow dye coupling, we determined the incidence of high conductance gjs in SONs of proestrous, immediately prepartum, postpartum non-lactating, lactating day 1, and lactating day 9-10 rats. Results indicate that coupling incidence is high only at times when milk ejection bursts are known to occur, and that the elevated gj mRNA expression seen on the last day of pregnancy does not indicate conducting gjs. It is suggested that gj conductance states, but not gj expression, are modulated by plasma estradiol titers.

Synaptic potentials mediated by alpha 7 nicotinic acetylcholine receptors in supraoptic nucleus.

Brain slice preparations preserving projections from nearby forebrain cholinergic neurons to the supraoptic nucleus (SON) were used to study synaptic potentials mediated by nicotinic acetylcholine receptors (nAChRs) in the hypothalamus. Paired-pulse electrical stimulation in an area anterior to the SON that was rich in cholinergic cells confirmed the monosynaptic nature of the connections to putative oxytocin and vasopressin SON neurons. With ionotropic glutamate and GABA(A) transmission blocked, this stimulation evoked fast, atropine-insensitive EPSPs that were sensitive to nAChR antagonists. Evoked EPSPs were blocked by methyllycaconitine and alpha-bungarotoxin, antagonists that are selective for nAChRs containing the alpha7 subunit, but not by dihydro-beta-erythroidine at concentrations known to antagonize alpha4beta2 nAChRs. Although anatomical evidence exists for postsynaptic alpha4beta2 nAChRs in the SON, these results indicate that postsynaptic alpha7 nAChRs are primarily responsible for the cholinergically mediated EPSPs. Repetitive stimulation suggested partial desensitization of the receptors. With ionotropic glutamate transmission blocked, inhibition of AChE increased spontaneous EPSP frequency and amplitude, suggesting spontaneous ACh release. ACh, nicotine, and choline (a selective alpha7 nAChR agonist) were effective in evoking action potentials and repetitive firing with synaptic transmission blocked by low Ca2+, high Mg2+ medium. These agonists were also effective in evoking the type of phasic bursts characteristic of vasopressin neurons, long thought to be completely dependent on activation of NMDA receptors (NMDARs). Because phasic bursting is Ca2+-dependent, the functional equivalence of alpha7 nAChR and NMDAR activation in this regard is likely attributable to their large Ca2+ fluxing capacities. This is the first demonstration that synaptically released ACh results in fast, alpha7 nAChR-mediated EPSPs in hypothalamic neurons.