Sheryl G. Beck

Androgens modulate glucocorticoid receptor mRNA, but not mineralocorticoid receptor mRNA levels, in the rat hippocampus.

Androgen, mineralocorticoid and glucocorticoid receptors (AR, MR and GR, respectively) are ligand‐activated transcription factors that alter gene expression and have a wide variety of effects in the central nervous system. High levels of AR, MR and GR mRNA have been found in the CA1 pyramidal cell region of the rat hippocampus and all 3 of these proteins bind to a similar hormone response element in DNA suggesting the possibility of common receptor function or cross‐talk between these receptors at the level of transcription. To begin to investigate this hypothesis, we examined the regulation of AR, MR and GR mRNA expression in the rat hippocampus following treatment with androgens in combination with gonadectomy and/or adrenalectomy. Three‐month‐old male Sprague‐Dawley rats were either castrated for 3 weeks, castrated and immediately implanted with 2 Silastic capsules filled with the non‐aromatizable androgen, dihydrotestosterone, or left gonadally intact. Four days prior to sacrifice, these animals were either adrenalectomized or sham operated. GR, MR and AR mRNA were measured in the hippocampal subfields using in situ hybridization. In the CA1 region, dihydrotestosterone treatment of castrates decreased GR mRNA levels to 69 percent of levels found in gonadally intact rats and prevented the adrenalectomy‐induced increases in GR mRNA observed in the gonadally intact and castrated animals. No changes in GR mRNA were observed in the CA3 region or dentate gyrus, where AR expression is low or absent. There was no effect of androgen treatment on MR mRNA levels nor did gonadectomy or androgen replacement alter the increases in MR mRNA following adrenalectomy. AR mRNA levels in the CA1 region were unchanged across all treatment groups. In vitro binding studies revealed almost complete nuclear occupancy of hippocampal AR in dihydrotestosterone‐treated castrates. No appreciable in vitro binding of dihydrotestosterone to hippocampal MR or GR (Ki≈1500u2003nM) was observed which suggests that androgen regulation of GR mRNA in the hippocampus is occurring through AR binding. These data demonstrate a functional similarity of androgens and glucocorticoids in the regulation of GR mRNA levels in an area where AR and GR are colocalized. Androgen‐mediated downregulation of GR expression may prove to be an important event in the adaptive responses of CA1 pyramidal cells to hormonal stimuli.

Androgens selectively modulate c-Fos messenger RNA induction in the rat hippocampus following novelty

We have previously shown that androgen receptors are found in high concentrations in hippocampal CA1 pyramidal cells. To begin to explore the possible roles for androgen receptors in this area of the brain, we studied the effects of endogenous and exogenous androgen on the behaviourally induced expression of cellular immediate early gene messenger RNAs. Adult male Fischer 344 rats were either gonadectomized, gonadectomized and given two Silastic capsules of dihydrotestosterone propionate at the time of surgery, or left intact. Three weeks later, animals were placed into a novel open field for 20 min. This behavioural paradigm caused region- and gene-specific increases of c-fos, jun-B, c-jun and zif268 messenger RNA in the hippocampus as determined by semi-quantitative in situ hybridization histochemistry. The removal of circulating androgen by gonadectomy potentiated, whereas dihydrotestosterone treatment of castrates attenuated, the behaviourally induced expression of c-fos messenger RNA in the CA1 region of the hippocampus. No changes in c-fos messenger RNA expression were detected in the CA3 or dentate gyrus regions where androgen receptor levels are low. Androgen status did not affect either the basal or stimulated expression of Jun-B, c-Jun or zif268 messenger RNA in any of the three cellular regions of the hippocampus examined. These results implicate androgen receptors in modulating the active response of hippocampal neurons to a behaviourally relevant stimulus. Since the products of cellular immediate genes can function to alter an array of downstream genes, the modulation of these genes in the hippocampus by gonadal hormones may have important ramifications for hippocampal function.

Corticosteroids regulate 5-HT1A but not 5-HT1B receptor mRNA in rat hippocampus

The role of mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) in the regulation of serotonin receptors has not been clearly delineated. There is no consensus regarding the regulation of 5-HT(1A) receptors, and corticosteroid regulation of 5-HT(1B) mRNA has not been previously studied. We compared the effects of long-term (two week) adrenalectomy (no MR or GR activation) and several hormone replacement protocols designed to stimulate MR selectively (ALDO), MR and GR (HCT), and continuous MR with cyclical GR activation (SHAM adrenalectomy). 5-HT(1A) and 5-HT(1B) mRNAs were measured by in situ hybridization in hippocampus and raphe nuclei. None of the experimental manipulations altered 5-HT(1B) mRNA levels in the hippocampus or dorsal raphe, and also had no effect on 5-HT(1A) mRNA in dorsal or median raphe. However, 5-HT(1A) mRNA levels were regulated in a complex manner in the different subfields of hippocampus. We conclude that both MR and GR play an integrated role in regulating 5-HT(1A) mRNA levels in hippocampus while having no effect on 5-HT(1B) mRNA levels under these conditions.

Corticosterone alters G protein α-subunit levels in the rat hippocampus

Abstract The hypothalamic-pituitary-adrenal axis regulates the synthesis and secretion of corticosteroid hormones. The hippocampus, a component of the limbic system, contains the highest concentration of corticosteroid receptors in the brain and may play an important role in regulating hypothalamic-pituitary-adrenal axis activity and mediating physiological responses to stress. The corticosteroid hormone corticosterone alters the response elicited by activation of several different G protein-linked neurotransmitter receptors in the hippocampus. In the present study we used Western blot and immunohistochemical techniques to determine the effects of chronic adrenalectomy (ADX), low basal (CT) and high (HCT) corticosterone treatments on G s , G i1 and 2 and G o α -subunit levels and intracellular location in the rat hippocampus. CT treatment increased G s α -subunit levels and HCT treatment increased the levels of G s , G i1 and 2 and G o α -subunits when compared to sham as detected on Western blots. No change in the intracellular location of the G protein α -subunits was detected using immunohistochemistry. Based on our results, we conclude that corticosterone alters G protein α -subunit levels in the rat hippocampus without altering their intracellular location. These results provide an important piece of information towards understanding how corticosteroids alter G protein-linked neurotransmitter receptor-mediated responses.

5-Hydroxytryptamine hyperpolarizes CA3 hippocampal pyramidal cells through an increase in potassium conductance.

The firing rate of hippocampal pyramidal cells recorded from the CA3 subfield is inhibited by 5-hydroxytryptamine (5-HT, serotonin) or by electrical stimulation of the ascending serotonergic fibers from the raphe. The mechanism of action of this inhibitory effect produced by 5-HT has not been determined. Intracellular recording techniques in the hippocampal slice preparation were used to measure the effect of 5-HT perfusion on membrane properties of CA3 pyramidal cells. In 15 out of 16 cells tested, 5-HT elicited a pronounced hyperpolarization concomitant with a decrease in membrane resistance. The hyperpolarization was not altered with either potassium chloride or potassium methylsulphate electrodes; the hyperpolarization by 5-HT was not present when electrodes were filled with cesium chloride. The reversal potential of the 5-HT mediated response was determined to be-105.5 mV in 3 mM KCl buffer using single electrode voltage clamp techniques. Based on these results we conclude that the mechanism of action of the 5-HT inhibition of CA3 hippocampal pyramidal cell excitability is due to an increase in potassium conductance.

Corticosteroids Influence the Action Potential Firing Pattern of Hippocampal Subfield CA3 Pyramidal Cells

Corticosteroids regulate gene expression through the activation of mineralocorticoid and glucocorticoid receptors. The hippocampus contains the highest density of mineralocorticoid and glucocorticoid receptors in the central nervous system. The modulation of neuron excitability by corticosteroids in hippocampal subfield CA1 is well documented. However, it is not known whether corticosteroids produce different effects across the various hippocampal subfields. Therefore, we used intracellular recording techniques to examine the actions of chronic corticosteroid treatment (2 weeks) on the electrophysiological properties of rat hippocampal subfield CA3 pyramidal cells. The treatment groups used in this investigation were: adrenalectomy (ADX), selective mineralocorticoid receptor activation with aldosterone (ALD), mineralocorticoid and glucocorticoid receptor activation with high levels of corticosterone (HCT), and SHAM. Corticosteroid treatment altered the percentage of nonburst and burst firing neurons. The percentages of nonbursting cells were 74 and 62% in tissue from ADX and HCT animals compared to 42 and 41% in ALD and SHAM animals, respectively. The corticosteroid-induced effect on the ratio of nonbursting to bursting cells does not appear to be secondary to changes in the cell’s membrane input resistance, resting potential, time constant, action potential, slow-or fast-afterhyperpolarizing potential properties. Based on these results we conclude that corticosteroids are important for maintaining the ratio of nonburst and burst firing pyramidal neurons in subfield CA3. These novel results are distinct from those previously reported for subfield CA1, suggesting that corticosteroids have different effects across hippocampal subfields.

Corticosteroids Alter the 5-HT1A Receptor-Mediated Response in CA1 Hippocampal Pyramidal Cells

The hippocampus, prolonged excessive corticosterone secretion, and the 5-hydroxytryptamine (5-HT) neurotransmitter system are implicated in the etiology and treatment of psychiatric disorders. Corticosterone regulates CA1 hippocampal physiology and the 5-HT1A receptor-effector pathway; however the effect of chronic stress levels of corticosterone is unknown. Bilateral adrenalectomy (ADX), adrenalectomy with high dose corticosterone replacement (HCT), or surgical sham (SHAM) treatments were for 2 weeks. Standard intracellular recording techniques were used in hippocampal slices to measure active and passive cellular properties and 5-HT1A receptor-mediated responses in CA1 pyramidal cells. The magnitude and half-decay time of the slow after-hyperpolarization (sAHP) were decreased and the membrane time constant (tau) was increased by HCT treatment. The Emax and EC50, but not the slope, of the concentration-response curve for 5-HT activation of the 5-HT1A receptor were reduced in cells recorded from HCT versus SHAM treated rats. The net effect of treatment with stress levels of corticosterone was to increase the excitability of the CA1 hippocampal pyramidal cell through changes in membrane properties and 5-HT1A receptor-mediated response.

Serotonergic suppression of interhemispheric cortical synaptic potentials

The inhibitory effects of 5-hydroxytryptamine (5-HT) on interhemispheric and intracortical synaptic potentials in layer V neurons of the rat medial prefrontal (MFC) cortex were examined. Low concentrations (1-3 microM) of 5-HT selectively attenuated polysynaptic potentials that were similarly evoked by callosal or white matter stimulation. Maximally effective concentrations of 5-HT blocked interhemispheric transmission by 50-90%, as evidenced by an attention of the short latency callosal depolarizing synaptic potential (e-DPSP). These effects of 5-HT were not associated with a change in membrane potential or input resistance. The e-DPSP was characterized as having an N-methyl-D-aspartate (NMDA) and a non-NMDA component; the non-NMDA component was attenuated by 5-HT. Attenuation of the synaptic potential was accompanied by an attenuation of a postsynaptic glutamate potential. Suppression of both the e-DPSP and the glutamate potential was concentration dependent with 10-100 microM being maximally effective. The 5-HT1A/2 antagonist, spiperone, antagonized the effects of 5-HT on synaptic and glutamate potentials. Spiperone (1 microM) shifted the concentration-effect curves for suppression of the e-DPSP and the glutamate potential to the right; however, the Kb for the glutamate potential concentration-effect curve was 10 times that for the e-DPSP curve. The differential antagonist sensitivity of synaptic and glutamate potentials was an indication that serotonin acted on more than one receptor subtype to reduce interhemispheric transmission.

Differential immunohistochemical labeling of Gs, Gi1 and 2, and Go ?-subunits in rat forebrain

The anatomical and morphological distribution of the G proteins Go, Gi1 and 2, and Go α‐subunits in rat forebrain sections was determined using immunohistochemical techniques. Diffuse Go labeling occurred in the neuropil throughout the cortex, superficial layers of the entorhinal cortex, thalamus, several white matter fiber tracts, and hippocampus. Gi1 and 2 immunareactivity was also located in the neuropil but produced a more fibrous pattern. Fibrous labeling of Gi1 and 2 was observed in the cortex, amygdala, hippocampal subfield CA3, and several white matter fiber tracts. Both Go and Gi1 and 2 labeling was present in the pencil fibers within the striatum and lateral geniculate nucleus. Gs labeling, in contrast to Go and Gi1 and 2, was generally cytoplasmic. Cytoplasmic Gs labeling was observed in the thalamus, habenula, dentate, geniculate nucleus, hypothalamus, and hippocampus. Intense Gs labeling was observed in the striatum parenchyma, choroid plexus, and infundibular stem. Based on our results, we conclude that the G proteins Go, Gi1 and 2, and Gs are anatomically distributed differently throughout the brain. The diffuse neuropil labeling of Go, fibrous neuropil labeling of Gi1 and 2, and cytoplasmic labeling of Gs strongly suggests that the G proteins are also differentially distributed morphologically within a neuron. The differential anatomical and cellular location of G proteins in the CNS may contribute to the coupling specificity between neurotransmitter receptors and G proteins.