Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro.

Pyramidal neurons in the rat hippocampus contain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to which the adrenal steroid corticosterone binds with differential affinity. We have used intracellular recording techniques to examine MR-mediated effects on membrane properties of CA1 pyramidal neurons in hippocampal slices from adrenalectomized rats. Low doses of corticosterone (1 nM) applied by perfusion for 20 min decreased the spike accommodation observed during a depolarizing current pulse (0.5 nA for 500 ms) and the amplitude of the subsequent afterhyperpolarization without affecting other membrane properties tested. The decrease became apparent ca. 15 min after steroid perfusion was started and reached its peak value 10-20 min after the steroid perfusion was terminated. The steroid effect was blocked by the MR antagonist spironolactone and mimicked by the natural MR ligand aldosterone (1 nM). Neurons recorded 30-90 min after termination of aldosterone application still displayed a decreased spike accommodation. However, 30-90 min after corticosterone application, the decrease in spike accommodation/afterhyperpolarization appeared to be reversed. Higher doses of corticosterone (greater than or equal to 30 nM) induced a significant increase in accommodation and amplitude of the afterhyperpolarization, as was previously observed for selective GR ligands. The data indicate that MR and GR activations induce opposite actions on the spike accommodation/afterhyperpolarization of CA1 pyramidal neurons, an important intrinsic mechanism of these neurons to regulate their response to excitatory input. We suggest that occupation of both MR and GR by the endogenous ligand corticosterone will result in an initial MR-mediated enhanced cellular excitability, which is gradually reversed and overridden by a GR-mediated suppression of cellular activity.     

Coordinative mineralocorticoid and glucocorticoid receptor-mediated control of responses to serotonin in rat hippocampus.

In a previous study we showed that selective occupation of the mineralocorticoid receptor (MR) in hippocampal slices from adrenalectomized (ADX) rats attenuates the membrane hyperpolarization and resistance decrease induced in CA1 pyramidal neurons by serotonin (5HT). In the present study we established responses to 5HT in the hippocampal slice when not only MRs but also glucocorticoid receptors (GRs) were occupied, using either a combination of selective MR and GR ligands or different concentrations of the endogenous mixed agonist corticosterone. We observed that the GR agonist RU 28362 blocks the attenuating action of the MR agonist aldosterone on responses to 3, 10 and 30 microM 5HT; RU 28362 by itself did not affect 5HT responses. If a low concentration of the mixed agonist corticosterone (0.5 nM, close to the Kd for the MR) was continuously perfused in vitro, 5HT responses were steadily depressed with a delay of 2 h, while high levels of corticosterone (5 nM, around Kd for GR) only temporarily reduced 5HT responses. Finally, 5HT responses in slices from sham-operated rats (with relatively high plasma corticosterone levels) were similar to the responses obtained in slices from ADX rats. These data suggest that the previously reported MR-mediated attenuation of 5HT responses may be limited to conditions of low adrenocortical activity or pathophysiological conditions where the balance of MR- and GR-mediated effects is disturbed.     

Selective control by corticosterone of serotonin1 receptor capacity in raphe-hippocampal system

The effect of short-term bilateral adrenalectomy (ADX) and corticosterone or aldosterone replacement was investigated on serotonin1 (5-HT1) receptor density in rat brain regions. One hour after ADX the 5-HT1 receptor density was increased in subiculum, molecular layer of the dentate gyrus, substantia nigra, and dorsal raphe nucleus as shown by in vitro autoradiography and computerized densitometric analyses of the film images. In subiculum, dentate gyrus, and dorsal raphe nucleus the 5-HT1 receptor density was restored (decreased) towards the level observed in sham-operated rats after administration of a low dose of corticosterone (CORT) at the time of ADX. CORT replacement decreased the 5-HT1 receptor number also in hippocampal pyramidal cell layer CA1, presubiculum, and other dentate gyrus subregions. The 5-HT1 receptor density was not affected by ADX or CORT replacement therapy in cerebral cortex, central grey, CA3, ventral hippocampus, and median raphe nucleus. Aldosterone administered under the same experimental conditions did not change the 5-HT1 receptor number in any of the hippocampus or raphe regions. The dose of CORT (30 micrograms/100 g body weight, s.c.) gave physiological plasma levels and maintained almost complete occupation of CORT receptors (mineralo-corticoid-like receptors) in hippocampus which was also the case in the sham-operated control animals. CORT replacement did not maintain occupancy of glucocorticoid receptors in hippocampus. When the binding of the selective glucocorticoid agonist, 3H-RU 28362, was taken as 100% at 1 h after ADX, CORT replacement and sham ADX left 75 and 50% of these sites available respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-specific topography of corticosteroid receptor types in rat and hamster brain

In vivo and in vitro autoradiography with radiolabeled corticosteroid analogs as well as immunocytochemistry with monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to determine the presence and the topography of two corticosteroid receptor systems (type I and type II) in hamster and rat brains. In the rat, the in vivo autoradiograms clearly revealed the retention by the type I receptor of tracer amount of [3H]corticosterone, primarily in the CA1 and CA2 cell field, dentate gyrus and lateral septum. In the hamster, tracer doses of [3H]cortisol were retained not only in the CA1, CA2, dentate gyrus and lateral septum, but also at high level in the CA3 and CA4 areas. In both species, immunocytochemistry showed the widespread distribution of the type II receptor sites in areas such as the hippocampus, lateral septum, hypothalamus (particularly in the paraventricular nucleus), thalamus and cortex (these results were also reflected in the in vitro autoradiography). Strong cell nuclear glucocorticoid immunoreactivity (type II-IR) was observed in the CA1 and CA2 (as well as CA3 and CA4 in the hamster) pyramidal neurons. In the hippocampus of intact animals, type II-IR was seen in the neuronal cell nuclei. Adrenalectomy caused a depletion of the type II-IR signal from the cell nucleus, which returned 1 h following subcutaneous administration of RU 28362 to adrenalectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.     

Estrogen synthesis in the brain--role in synaptic plasticity and memory

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200-1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus. Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1-10nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.     

Melatonin inhibits outward delayed rectifier potassium currents in hippocampal CA1 pyramidal neuron via intracellular indole-related domains

In the present study, we investigated the effect of melatonin on the outward delayed rectifier potassium currents (IK) in CA1 pyramidal neurons of rat hippocampal slices using patch-clamp technique in whole-cell configuration. In a concentration-dependent manner, melatonin caused a reduction of IK with a half-maximal inhibitory concentration (IC50) of 3.75 mm. The inhibitory effect had rapid onset and was readily reversible. Melatonin shifted steady-state inactivation of IK in hyperpolarizing direction but did not alter its steady-state activation. Neither luzindole, an MT1/MT2 receptor antagonist, nor prazosin, an MT3 receptor antagonist, blocked melatonin-induced current reduction. The results indicate that melatonin-induced IK inhibition was not via activation of its own membrane receptors. 5-Hydroxytryptamine (5-HT), a melatonin precursor and an agonist of serotonin receptors, when it was given in pipette internal solution but not bath solution, produced a similar inhibitory effect to that of melatonin. Moreover, indole, a major component of melatonin, reversibly and dose dependently inhibited IK with an IC50 of 3.44 mm. Present results suggest that melatonin inhibits IK in hippocampal CA1 pyramidal neurons probably through its interaction with the intracellular indole-related domains of potassium channels.     

Deprivation-induced synaptic depression by distinct mechanisms in different layers of mouse visual cortex

Long-term depression (LTD) induced by low-frequency synaptic stimulation (LFS) was originally introduced as a model to probe potential mechanisms of deprivation-induced synaptic depression in visual cortex. In hippocampus, LTD requires activation of postsynaptic NMDA receptors, PKA, and the clathrin-dependent endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It has long been assumed that LTD induced in visual cortical layer 2/3 by LFS of layer 4 uses similar mechanisms. Here we show in mouse visual cortex that this conclusion requires revision. We find that LTD induced in layer 2/3 by LFS is unaffected by inhibitors of PKA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor endocytosis but is reliably blocked by an endocannabinoid CB1 receptor antagonist. Conversely, LFS applied to synapses on layer 4 neurons produces LTD that appears mechanistically identical to that in CA1 and is insensitive to CB1 blockers. Occlusion experiments suggest that both mechanisms contribute to the loss of visual responsiveness after monocular deprivation.     

Estrogen synthesis in the brain—Role in synaptic plasticity and memory

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200–1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus.Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1–10 nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2 h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.     

Presynaptic m1 muscarinic receptors are necessary for mGluR long-term depression in the hippocampus

To investigate the role of M1 muscarininc acetylcholine receptors (m1 receptors) in metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), we produced mouse lines in which deletion of the m1 gene is restricted to the forebrain (FB–m1KO) or hippocampal CA3 pyramidal neurons (CA3–m1KO). Stimulation in FB–m1KO hippocampal slices resulted in excitatory postsynaptic potentials and long-term synaptic plasticity (long-term potentiation and LTD) similar to controls. The mice were deficient in (S)-3,5-dihydroxyphenylglycine hydrate (DHPG)-induced mGluR LTD, which correlated with a presynaptic increase in the release of neurotransmitters. Protein kinase C (PKC) activity, which is downstream from both mGluRs and m1 receptors, was reduced in CA3 but not in CA1. The presynaptic requirement of m1 receptors was confirmed by the lack of DHPG-induced mGluR LTD in the CA1 of slices from CA3–m1KO mice. mGluR LTD was rescued by stimulating PKC activity pharmacologically in CA3–m1KO mice. These data confirm a role for PKC activation in presynaptic induction of mGluR LTD and distinguish between the roles of mGluRs and m1 receptors.     

Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling

The 5-HT_2A serotonin receptor is the most abundant serotonin receptor subtype in the cortex and is predominantly expressed in pyramidal neurons. The 5-HT_2A receptor is a target of several hallucinogens, antipsychotics, anxiolytics, and antidepressants, and it has been associated with several psychiatric disorders, conditions that are also associated with aberrations in dendritic spine morphogenesis. However, the role of 5-HT_2A receptors in regulating dendritic spine morphogenesis in cortical neurons is unknown. Here we show that the 5-HT_2A receptor is present in a subset of spines, in addition to dendritic shafts. It colocalizes with PSD-95 and with multiple PDZ protein-1 (MUPP1) in a subset of dendritic spines of rat cortical pyramidal neurons. MUPP1 is enriched in postsynaptic density (PSD) fractions, is targeted to spines in pyramidal neurons, and enhances the localization of 5-HT_2A receptors to the cell periphery. 5-HT_2A receptor activation by the 5-HT₂ receptor agonist DOI induced a transient increase in dendritic spine size, as well as phosphorylation of p21-activated kinase (PAK) in cultured cortical neurons. PAK is a downstream target of the neuronal Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 that is important for spine remodeling. Kalirin-7 regulates dendritic spine morphogenesis in neurons but its role in neuromodulator signaling has not been investigated. We show that peptide interference that prevents the localization of kalirin-7 to the postsynaptic density disrupts DOI-induced PAK phosphorylation and spine morphogenesis. These results suggest a potential role for serotonin signaling in modulating spine morphology and kalirin-7''s function at cortical synapses.     

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.     

Modulatory role of locus coeruleus and estradiol on the stress response of female rats

The activity of the hypothalamic-pituitary-adrenal axis is modulated by the norepinephrinergic system and, in females, also by the ovarian hormones. We investigated the role of ovarian steroids and the locus coeruleus (LC) on stress-induced corticosterone secretion in female rats. Ovariectomized rats without hormonal replacement (OVX) or treated with estradiol (OVE) or estradiol plus progesterone (OVEP) were subjected to jugular cannulation. Immediately after that, each hormonal treatment group was subjected to LC lesion or sham surgery or no brain surgery. After 24 h, blood samples of all 9 groups were collected before and after ether inhalation. Other four groups (OVX control, sham and lesioned, and OVE) were perfused for glucocorticoid receptor (GR) immunocytochemistry in hippocampal CA1 neurons and paraventricular nucleus (PVN). Estradiol replacement decreased while LC lesions increased stress-induced corticosterone secretion. The effect of LC lesion was potentiated with the removal of ovarian steroids. Since GR expression of lesioned animals decreased in the hippocampus, but not in PVN, we suggest that the effect of LC lesion on corticosterone secretion could be due to a reduction in the efficiency of the negative feedback system in the CA1 neurons. However, this mechanism is not involved in the estradiol modulation on corticosteroid secretion, as no change in GR expression was observed in estradiol-treated animals.     

Metaplasticity of amygdalar responses to the stress hormone corticosterone

High levels of corticosteroids (as circulate after stress) quickly and reversibly enhance hippocampal glutamatergic transmission via nongenomic actions requiring mineralocorticoid receptors. Subsequently, the hormone slowly and long-lastingly normalizes hippocampal cell function, through nuclear glucocorticoid receptors. Here we describe a rapid mineralocorticoid receptor-dependent enhancement of glutamatergic transmission in basolateral amygdala neurons. Contrary to the hippocampus, this rapid enhancement is long-lasting, potentially allowing an extended window for encoding of emotional aspects during stressful events. Importantly, the long-lasting change in state of amygdala neurons greatly affects the responsiveness to subsequent surges of corticosterone, revealing a quick suppression of glutamatergic transmission, which requires the glucocorticoid receptor. Responses of basolateral amygdala neurons to the stress hormone corticosterone can thus switch from excitatory to inhibitory, depending on the recent stress history of the organism.     

Serotonin receptor subtypes involved in the elevation of serum corticosterone concentration in rats by direct- and indirect-acting serotonin agonists

The serum corticosterone concentration in rats was increased by injection of quipazine, a relatively nonselective serotonin (5-hydroxytryptamine; 5-HT) agonist, or 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT), a serotonin agonist selective for the 5-HT1A subtype of receptor. The quipazine-induced increase in serum corticosterone was antagonized by 17 different serotonin antagonists; of these, MDL 11939, pirenperone, setoperone, mianserin, LY 281067, ketanserin, ritanserin and clozapine have relatively selective affinity for the 5-HT2 subtype of receptor. The 8-OH-DPAT-induced increase in serum corticosterone was not antagonized by metergoline, the most potent antagonist of the quipazine effect, but was antagonized by pindolol or penbutolol, 5-HT1A receptor antagonists. Pindolol did not block the effect of quipazine. The results support earlier evidence that serum corticosterone concentration in rats can be increased by activation of either 5-HT1A or 5-HT2 receptors. Indirect-acting serotonin agonists - fluoxetine, L-5-hydroxytryptophan and p-chloroamphetamine - also increased serum corticosterone concentrations. The increases elicited by those agents, which earlier had been reported not to be blocked by metergoline pretreatment, also were not blocked by pretreatment with pindolol or with the combination of metergoline and pindolol. Thus, an involvement of a specific serotonin receptor subtype in the actions of these indirect agonists has not been established.     

Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus

Kainate (KA) receptor activation depresses stimulus-evoked gamma-aminobutyric acid (GABA-mediated) synaptic transmission onto CA1 pyramidal cells of the hippocampus and simultaneously increases the frequency of spontaneous GABA release through an increase in interneuronal spiking. To determine whether these two effects are independent, we examined the mechanism by which KA receptor activation depresses the stimulus-evoked, inhibitory postsynaptic current (IPSC). Bath application of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)/KA receptor agonist KA in the presence of the AMPA receptor antagonist GYKI 53655 caused a large increase in spontaneous GABA release and a coincident depression of the evoked IPSC. The depressant action on the evoked IPSC was reduced, but not abolished, by the GABA(B) receptor antagonist SCH 50911, suggesting that the KA-induced increase in spontaneous GABA release depresses the evoked IPSC through activation of presynaptic GABA(B) receptors. KA had no resolvable effect on the potassium-induced increase in miniature IPSC frequency, suggesting that KA does not act through a direct effect on the release machinery or presynaptic calcium influx. KA caused a decrease in pyramidal cell input resistance, which was reduced by GABA(A) receptor antagonists. KA also caused a reduction in the size of responses to iontophoretically applied GABA, which was indistinguishable from the SCH 50911-resistant, residual depression of the evoked IPSC. These results suggest that KA receptor activation depresses the evoked IPSC indirectly by increasing interneuronal spiking and GABA release, leading to activation of presynaptic GABA(B) receptors, which depress GABA release, and postsynaptic GABA(A) receptors, which increase passive shunting.