Activation of excitatory amino acid inputs to supraoptic neurons. I. Induced increases in dye-coupling in lactating, but not virgin or male rats

Mitral cells of the main and accessory olfactory bulbs have been shown to project monosynaptically to the supraoptic nucleus (SON) via the lateral olfactory tract (LOT) which uses excitatory amino acid transmitters. Data collected during characterization of these projections suggested that synaptic activation of SON neurons via LOT stimulation in slices influenced the incidence of dye-coupling. The present study pursued this suggestion using horizontally cut slices from male, virgin female and lactating rats. Neurons were confirmed to be excited by electrical stimulation of the tract, injected with Lucifer yellow, and synaptically activated for 10 min at 10 Hz (n = 92). Another 94 neurons were similarly confirmed and injected, but received no further stimulation. In an additional 8 slices, injected neurons were antidromically activated for 10 min at 10 Hz. Analyses done on 194 injected neurons from the 3 groups showed that synaptic activation resulted in a significant (P less than 0.01) increase in the incidence of coupling only in tissue from lactating rats. This increase was entirely due to larger numbers of cells being coupled dendrodendritically to the injected cells in the stimulated slices. Antidromic activation did not influence coupling. Increased coupling occurred among both oxytocin and vasopressin cell types. This is the first report of increased coupling resulting from synaptic activation in mammalian CNS. Changes seen only in lactating rats may be related to their altered SON ultrastructural morphology (i.e. dendritic bundling). Strong olfactory and vomeronasal input associated with some maternal behaviors may increase neuronal coupling and enhance hormone release in response to other incoming stimuli (e.g. suckling, dehydration).     

Alterations in supraoptic nucleus ultrastructure of maternally behaving virgin rats

Adult, nulliparous female rats were induced to behave maternally via constant cohousing with rat pups. After exhibiting maternal behaviors for 3 days, the animals were transcardially perfused, the supraoptic nuclei (SON) excised and examined quantitatively by transmission electron microscopy. Relative to virgin controls, the animals behaving maternally were found to have significant increases in: a) mean number of dendrites in large (9-12) dendritic bundles, b) mean area per single dendritic profile, c) area of the dendritic zone occupied by dendritic profiles and d) size of the dendritic zone. No significant changes were observed in the cell body zone or in the number of double synapses in the dendritic region. The observed changes are likely to be at least in part associated with the oxytocin-containing cells of the SON. These observations suggest a role for the SON in promoting maternal behaviors and constitute a novel demonstration of a neural modification in the mammalian central nervous system that appears conjointly with a complex set of behaviors.     

Activation of locus coeruleus by prefrontal cortex is mediated by excitatory amino acid inputs

We examined the role of excitatory amino acids (EAAs) in activation of noradrenergic locus coeruleus (LC) neurons evoked by electrical stimulation of the medial prefrontal cortex (mPFC) in halothane-anesthetized rats. Microinfusion of the specific N-methyl-

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-aspartate antagonist 2-amino-5-phosphonopentanoic acid (AP5, 50 or 100 μM) into the LC significantly suppressed LC responses evoked by mPFC stimulation. Microinfusion of the selective non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 25 or 50 μM) also significantly reduced evoked LC responses. Simultaneous microinfusion of both AP5 and CNQX considerably increased the proportion of LC neurons which exhibited complete suppression of evoked responses (81%), compared to either AP5 or CNQX alone (50% each). These results indicate that LC activation by mPFC stimulation is mediated by both NMDA- and non-NMDA-type EAA channels.     

Oxytocin neurons in the supraoptic nucleus receive excitatory inputs from the bilateral dorsomedial hypothalamic nuclei

To examine whether inputs from the dorsomedial hypothalamic nucleus (DMH) alter the discharge of putative oxytocin (OT) neurons with hypothesis that excitation of DMH neurons would increase the activity of OT neurons, electrical stimulation was applied to the DMH in both sides of the hypothalamus while electrical activity of single OT neurons in the supraoptic nucleus (SON) was recorded in urethane-anesthetized lactating rats. About half of the OT neurons showed orthodromic excitation or inhibition followed by excitation in response to electrical stimulation of the DMH on both sides. Continuous electrical stimulation of the DMH both ipsi- and contralateral to the recording side at 10-50 Hz for 30-60 s increased firing rate in 58% of OT neurons tested. Continuous electrical stimulation of the DMH not only excited spiking activity of single OT neurons but also increased intramammary pressure. The results may suggest that some of the projections from the DMH to the SON are bilateral and possibly contribute to coordinated bilateral activation of OT neurons in the hypothalamus during the milk-ejection reflex.     

A1 neurons and excitatory amino acid receptors in rat caudal medulla mediate vagal excitation of supraoptic vasopressin cells

Extracellular recordings from the supraoptic nucleus of the rat established that vasopressinergic neurosecretory cells were excited by stimulation of cervical but not abdominal vagal afferents. This response was absent or significantly attenuated after microinjection of gamma-aminobutyric acid into a region of the caudal medulla known to contain the A1 noradrenaline cell group. Consistent with the possible involvement of the A1 group, vagal stimulation approximately doubled the frequency of proto-oncogene expression in A1 noradrenaline neurons, as indicated by the occurrence of nuclear Fos-like immunoreactivity in tyrosine hydroxylase-positive neurons of the caudal ventrolateral medulla. Finally, A1 region microinjection of either the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced vasopressin cell responses to vagal stimulation. These findings suggest that: (i) the A1 group is an essential component in a pathway which relays facilitatory vagal input of cardiopulmonary origin to neurosecretory vasopressin cells, and (ii) the activation of A1 neurons in this pathway involves both NMDA and non-NMDA excitatory amino acid receptors, an observation consistent with an input to A1 cells which generates 'mixed' excitatory postsynaptic potentials.     

Noradrenergic excitatory inputs to median preoptic neurones in rats.

Extracellular single-unit activity was recorded from 21 median preoptic nucleus (MnPO) neurones, antidromically identified as projecting to the hypothalamic paraventricular nucleus (PVN), in urethane-anaesthetized male rats. Of these identified MnPO neurones, 14 displayed an excitatory response in neuronal excitability following electrical stimulation (5 Hz, 600 microA) of the A1 noradrenergic region of the ventrolateral medulla, while the remaining neurones were unresponsive. The excitatory response of MnPO neurones was blocked by microiontophoretically applied phentolamine, an alpha-adrenoceptor antagonist, but not by timolol, a beta-adrenoceptor antagonist. These results suggest that the A1 region acts to enhance the activity of MnPO neurones projecting to the PVN via an alpha-adrenoceptor mechanism.     

Vasopressin and oxytocin decrease excitatory amino acid release in adult rat supraoptic nucleus

Oxytocin and vasopressin reduce the amplitude of excitatory postsynaptic responses in magnocellular neuroendocrine cells of the supraoptic nucleus (SON). To test whether synaptic glutamate release is modulated by these neuropeptides, we examined the combined effect of vasopressin and oxytocin on depolarization-induced glutamate and aspartate release from acutely dissected rat SON or fronto-parietal cortex punches. Glutamate release was stimulated with 60 mm K+ for 5-10 min and measured using ion exchange chromatography or high-performance liquid chromatography. During depolarization with high K+, extracellular glutamate levels increased, on average, to 204% of control values. In the presence of vasopressin/oxytocin, K+-stimulated glutamate and aspartate release were significantly reduced by 34% and 62%, respectively, in the SON. Treatment with the aminopeptidase inhibitor amastatin did not mimic the effects of exogenous vasopressin/oxytocin on glutamate or aspartate release, suggesting that, under the conditions tested here, amastatin treatment may produce more complex effects. The effects of exogenous neuropeptides are likely mediated by oxytocin and/or vasopressin receptors, as the oxytocin- and V1a-receptor antagonist, Manning Compound (10-100 micro m), partially reversed the effects of vasopressin/oxytocin on SON glutamate release. In contrast, in cortical punches, glutamate release was enhanced by high K+, but vasopressin/oxytocin did not significantly reduce glutamate/aspartate release, consistent with the relatively sparse distribution of vasopressin/oxytocin receptors in fronto-parietal cortex. These findings suggest that locally released oxytocin and vasopressin may autoregulate SON magnocellular neuroendocrine cell activity in part by modulating the release of excitatory amino acids from afferent terminals targeting these cells and/or from other cellular sources.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular analysis of excitatory subthalamic inputs to the pedunculopontine neurons

Response patterns of the pedunculopontine (PPN) area neurons to electrical stimulation of the subthalamic nucleus (STH) were investigated in anesthetized rats. Intracellular recordings demonstrated that STH stimulation evoked short duration (mean value: 11.6 ms) depolarizing potentials which were identified as excitatory postsynaptic potentials (EPSPs) by intracellular current injection. These potentials were considered monosynaptic because the latency was constant in spite of changes in stimulus intensities. The conduction velocities of STH-PPN area afferents ranged from 1.2 to 8.3 m/s (mean value: 1.6 m/s). Similar results were obtained in rats with chronic unilateral coronal lesion just rostral to STH which eliminated the rostral afferents. In some neurons, EPSPs were followed by IPSPs. PPN area neurons were also antidromically activated by STH stimulation. Some of these neurons could also be antidromically activated by substantia nigra stimulation. We could differentiate PPN area projection neurons into two groups based on their conduction velocity. One group had a conduction velocity of 6.0 m/s and the second one 1.7 m/s. The morphology of 5 HRP-labeled PPN area neurons was analyzed. The somata were fusiform, polygonal and oval in shape, and the size varied from 152 to 1310 micron 2. Two to nine dendrites emerged from the somata and extended either radially or more in one direction (e.g. rostrocaudal). Axons arose, in general, from a proximal dendritic trunk close to the soma except one and branched near the soma. These data indicate a reciprocal connection between STH and PPN area and that STH exerts excitatory influence on PPN area neurons.     

Developmental expression of excitatory amino acid responses in cerebellar Purkinje neurons in culture.

We have examined the developmental expression of excitatory amino acid responses in cerebellar Purkinje neurons using a culture model system, extracellular recording techniques and micropressure application of agonists. In mature cultured Purkinje neurons, glutamate (Glu) and the selective receptor agonists quisqualate (Quis) and kainate (KA) elicited multiphasic responses with both excitatory and inhibitory phases. N-methyl-D-aspartate was ineffective. The agonist responses were characterized by an initial excitatory period, a period of burst activity and an inhibitory period. Glu, Quis and KA differed in the ability to evoke the 3 response components. Quis was the most potent agonist and the most effective in producing burst activity and an inhibitory period. Immature Purkinje neurons without visible dendritic structure also exhibit prolonged multiphasic responses when tested with the 3 agonists. However, response components were generally less robust than in mature neurons. Developmental changes in response properties were observed for all agonists, but the developmental changes in total response duration and the duration of the inhibitory period observed for the Quis response were not observed for the Glu or KA responses. These data can be explained by the presence of multiple excitatory amino acid receptor subtypes in both mature and immature Purkinje neurons.     

Localization of striatal excitatory amino acid binding site subtypes to striatonigral projection neurons

Quantitative autoradiography was used to examine the cellular localization of excitatory amino acid binding sites in the striatum following selective lesion of striatonigral projection neurons. Degeneration of striatonigral neurons was induced unilaterally by injection of the suicide transport toxin, volkensin, into the left substantia nigra. Twelve days following nigral volkensin injection there was a reduction of all excitatory amino acid binding site subtypes in the striatum ipsilateral to the injected nigra. The reduction in (NMDA) binding sites was significantly greater than the loss of -α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), kainate and metabotropic binding. These results indicate that there are NMDA, AMPA, metabotropic and kainate binding sites on striatonigral projection neurons and suggest that the NMDA subtype may be selectively enriched on striatonigral neurons.     

Activation of excitatory amino acid receptors increases intracellular free [3H]arachidonate levels in cultured cerebellar neurons

Addition of glutamate to cultured cerebellar neurons increases both intra- and extracellular content of [3H]arachidonic acid. The increase is time- and concentration-dependent and is antagonized by Mg2+ or by specific NMDA receptor antagonists (such as a-aminoadipate or MK-801). An increase in intracellular content of [3H]arachidonic acid is also induced by kainate and the muscarinic receptor agonist, carbamylcholine. However, as opposed to glutamate, carbamylcholine does not enhance the extracellular levels of [3H]arachidonic acid.     

The response of striatal neuropeptide Y and cholinergic neurons to excitatory amino acid agonists

The effect of excitatory amino acid antagonists on antagonist on neuropeptide Y (NPY) and cholinergic neurons in the striatum of the rat was studied by means of NPY immunocytochemistry, DFP histochemistry for acetylcholinesterase (AChE), and biochemical determinations of choline acetyltransferase (ChAT). Intrastriatal infusion of drugs revealed that striatal neurons containing NPY are more sensitive than cholinergic neurons to the neurotoxic actions of kainic acid (KA), quinolinic acid (QA) and L-glutamic acid (GA); all 3 compounds produced a marked loss of NPY neurons, but only a moderate decrease in the number of AChE neurons or ChAT activity. Co-injection experiments showed that the neurotoxicity of QA and GA, but not that of KA, can be antagonized by the specific N-methyl-D-aspartate (NMDA) receptor antagonist 3-((+/-)-2-(carboxypiperazine-4-yl))-propyl-1-phosphonic acid (CPP). Destruction of the glutamatergic corticostriatal projection by cerebral decortication protected striatal NPY and cholinergic neurons against KA neurotoxicity. These results indicate that striatal NPY and cholinergic neurons receive prominent cortical amino acid afferents, and that the neurotoxic effect of QA and GA on these neurons is mediated through NMDA receptors.     

Delayed anoxic depolarizations in hippocampal neurons of mice lacking the excitatory amino acid carrier 1.

Hypoxia leads to a rapid increase in vesicular release of glutamate. In addition, hypoxic glutamate release might be caused by reversed operation of neuronal glutamate transporters. An increase in extracellular glutamate concentration might be an important factor in generating anoxic depolarizations (AD) and subsequent neuronal damage. To study the AD and the vesicular release in hippocampal slices from CD1 wild-type mice and mice in which the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) had been knocked out, the authors performed recordings of field potentials and patch clamp recordings of CA1 pyramidal cells. Latency to anoxic depolarizations was enhanced in EAAC1-/- mice, whereas the hypoxia-induced increase in miniature excitatory postsynaptic current frequency occurred with similarly short latencies and to a similar extent in control and mutated animals. Additional block of glial glutamate uptake with TBOA (dl-threo-beta-benzyloxyaspartate), a nontransportable and potent inhibitor, dramatically reduced the latency to onset of AD and abolished the difference between wild-type mice and EAAC1-/- mice. The authors conclude that the neuronal glutamate transporter greatly influences the latency to generation of AD. Because ADs are not prevented in EAAC1-deficient mice, vesicular release mechanisms also seem to be involved. They become prominent when glial glutamate transport is blocked.     

Dye coupling among supraoptic nucleus neurons without dendritic damage: Differential incidence in nursing mother and virgin rats

To assess the possibility that dye coupling among neurons in hypothalamic slices might require dendrotomy, as has been suggested for neocortical neurons, dye coupling was studied in horizontally cut slices containing the supraoptic nucleus (SON). Since the dendrites of SON neurons project toward the pial surface, dendritic damage due to slicing can be avoided in the horizontal plane. Intracellular injections of Lucifer Yellow into individual SON neurons in slices from male, virgin female and lactating, mother rats yielded the following results. When dendrotomy occurred there was a significantly lower incidence of dye coupling than was observed when dendrites were intact. Higher order coupling (3 or more cells dye coupled after a single injection) was only seen among neurons without dendrotomy. Independently of dendritic damage, incidence of dye coupling in nursing mothers was reliably greater than for virgins, confirming previous results from coronal slices. The results of this study indicate that dendrotomy is not an inducer of dye coupling in SON neurons. Taken together with other recent findings, these data suggest that a reinterpretation of the effects of dendrotomy on cortical cell dye coupling may be in order.     

Activation of locus coeruleus neurons by nucleus paragigantocellularis or noxious sensory stimulation is mediated by intracoerulear excitatory amino acid neurotransmission

The nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla, is one of two major afferents to the nucleus locus coeruleus (LC). Electrical stimulation of PGi exerts a robust, predominantly excitatory influence on LC neurons that is blocked by intracerebroventricular (i.c.v.) administration of the broad spectrum excitatory amino acid (EAA) antagonists kynurenic acid (KYN) or gamma-D-glutamylglycine (DGG), but not by the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoate (AP7). I.c.v. injection of KYN or DGG also blocked activation of LC neurons evoked by noxious somatosensory stimuli. These results indicate that activation of LC neurons by PGi and noxious stimuli may be mediated by an EAA acting at a non-NMDA receptor in LC. In the present study, microiontophoretic techniques were used to determine the sensitivity of LC neurons in vivo to the selective EAA receptor agonists kainate (KA), NMDA and quisqualate (QUIS). Microinfusion and microiontophoresis were also used to determine whether direct application of KYN, the preferential non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) or the selective NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5) onto LC neurons blocked excitation elicited by stimulation of PGi or the sciatic nerve. The results demonstrated that individual LC neurons were robustly activated by direct application of KA, NMDA and QUIS. Iontophoretically applied KYN reduced or completely antagonized responses evoked by all 3 agonists. In contrast, iontophoretically applied AP5 strongly attenuated NMDA-evoked excitation, while KA-and QUIS-evoked responses were not affected by this agent. Furthermore, direct application of KYN or the specific non-NMDA receptor antagonist, CNQX, onto LC neurons substantially attenuated or completely blocked synaptic activation produced by PGi or sciatic nerve stimulation in nearly every LC neuron tested. Microinfusion of the selective NMDA receptor antagonist AP5 had no effect on sciatic nerve-evoked responses. These results confirm our hypothesis that activation of LC neurons from PGi is mediated by an EAA operating primarily at a non-NMDA receptor subtype on LC neurons. Furthermore, these findings provide additional support for the hypothesis that this pathway mediates at least some sensory-evoked responses of LC neurons.     

Activation of polyphosphoinositide metabolism as a signal-transducing system coupled to excitatory amino acid receptors in astroglial cells

Excitatory amino acids (EAA) are known to induce an increase in the breakdown of polyphosphoinositides (PI) in brain slices and in dispersed cultures of neurons. We have now used astroglia cultured from newborn rat cerebra to demonstrate that glutamate provokes, in [3H]inositol-labeled cells, an accumulation of inositol phosphates in a time- and concentration-dependent manner. The ED50 value for glutamate was 40 microM. Quisqualate, ibotenate, and kainate were also active, with their relative potencies in the order of quisqualate greater than ibotenate much greater than kainate. No effect was detected with N-methyl-D-aspartate and quinolinic acid in the absence of Mg2+. The nonselective glutamate receptor antagonist gamma-D-glutamylglycine fully inhibited glutamate agonist-induced PI breakdown. A brief pretreatment of the astroglial cells with phorbol esters negated these effects of EAA receptor agonists, suggesting a feedback role for protein kinase C in phospholipase C action. Glutamate also elevated cytosolic free Ca2+ in Fura-2-loaded astroglial cells, as assessed by digital fluorescence imaging microscopy. Since a close metabolic partnership is known to exist between neurons and glia, these findings may have important functional consequences for neural cells in vivo.     

Cholinergic stimulation in the lateral septal area activates anterior hypothalamic area neurons via excitatory amino acid receptors in rats

We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these AHA angiotensin II-sensitive neurons are enhanced in spontaneously hypertensive rats. It is suggested that there exist neuronal projections from the lateral septal area (LSV) to the AHA in rats. In this study, we examined whether neurons in the LSV are involved in activation of AHA angiotensin II-sensitive neurons. Male Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Microinjection of carbachol into the LSV caused an increase in firing rate of AHA angiotensin II-sensitive neurons. The carbachol-induced increase of firing rate of AHA angiotensin II-sensitive neurons was inhibited by pressure application of the excitatory amino acid receptor antagonist kynurenate but not by the AT1 receptor antagonist losartan onto the same neurons. Microinjection of carbachol into the LSV also increased the firing rate of AHA ACh-sensitive neurons, and the carbachol-induced increase of firing rate of ACh-sensitive neurons was again abolished by pressure application of kynurenate but not by the muscarinic receptor antagonist scopolamine onto the same neurons. Microinjection of the muscarinic receptor antagonist 4-DAMP into the LSV did not affect the firing rate of AHA angiotensin II-sensitive neurons. These findings indicate that neurons in the LSV are involved in activation of AHA angiotensin II-sensitive neurons. It seems likely that the carbachol-induced activation of AHA angiotensin II-sensitive neurons is mainly mediated via excitatory amino acid receptors at AHA neurons.