Sheryl S. Smith

Estrogen administration increases neuronal responses to excitatory amino acids as a long-term effect.

Ongoing studies from this laboratory have demonstrated marked potentiating actions of the sex steroid 17 beta-estradiol (E2) on glutamate-induced excitation. In the present study, systemic injection of a physiological dose of E2 was demonstrated to augment significantly excitatory responses of cerebellar Purkinje (Pnj) cells to iontophoretic application of the specific excitatory amino acid (e.a.a.) agonists quisqualate and N-methyl-D-aspartate. Potentiation of e.a.a. responses was observed as rapidly as 5-10 min post E2, and was, in many cases, a persistent, non-decremental effect. These observed long-term actions of E2 may provide one mechanism for the activating effects of the steroid on sensorimotor function and seizure activity.

Progesterone withdrawal decreases latency to and increases duration of electrified prod burial: a possible rat model of PMS anxiety.

The purpose of this study was to determine whether withdrawal from chronic exposure to the female sex steroid progesterone (P) alters response of female rats to an electrified prod using the defensive burying paradigm, considered a rat model of anxiety. Withdrawal from chronic exposure to 500 micrograms P (daily, SC, for four days) resulted in a significant decrease in the latency (77%, P < 0.05) to prod burial and an increase in duration (75%, P < 0.05) of this reflexive response, compared with the behavior of oil-injected controls. These results are consistent with the idea that withdrawal from chronic exposure to P increases behaviors that accompany anxiety. At a lower dose (50 micrograms), withdrawal from chronically administered P produced significant changes in response to this paradigm only when the steroid was given concomitantly with estradiol (2 micrograms, SC, for two days). Prior exposure to indomethacin, which blocks the conversion of P to its metabolite 3 alpha,5 alpha-tetrahydroprogesterone (3-alpha-hydroxy-5-alpha-pregnan-20-one), prevented P withdrawal from altering response in the defensive burying paradigm. This finding suggests that it may be withdrawal from this metabolite, rather than P, which increases behaviors associated with increased anxiety.

Sex steroid effects on extrahypothalamic CNS. I. Estrogen augments neuronal responsiveness to iontophoretically applied glutamate in the cerebellum

The purpose of this study was to test whether 17 beta-estradiol (E2) could alter neuronal activity or responsiveness to iontophoretically applied amino acid neurotransmitters in an area not reported to contain classical E2 receptors. Such a region is the cerebellum, which was selected as a model system for these studies because it has been well characterized electrophysiologically. Extracellular activity of cerebellar Purkinje neurons was recorded from urethane-anesthetized, adult, ovariectomized rats using multibarrel glass micropipets. Spontaneous firing rate and responses of single units to microiontophoretic pulses (10 s pulses every 40 s) of GABA (10-50 nA) or glutamate (GLUT, 3-40 nA) were examined before, during and after iontophoretic (0.25 mM 17 beta-estradiol hemisuccinate) or jugular i.v. (100, 300 or 1000 ng/kg 17 beta-estradiol) administration of E2. Both modes of E2 administration resulted in a significant increase in Purkinje cell excitatory responses to GLUT, independent of the direction of change in spontaneous firing rate. This effect was seen as early as one minute after iontophoretic application of E2 and 10-40 min following i.v. E2. In all cases, recovery to the control level of response was not observed by 2 h following E2 administration. 17 alpha-E2 (300 ng/kg) resulted in a less pronounced, transient increase in GLUT response, while a lower dose (100 ng/kg) did not have any effect. Prior administration of the anti-estrogen tamoxifen did not prevent any of the observed E2 effects. In addition, estrogen-priming did not alter E2-induced potentiation of GLUT responses. In contrast to the effect of E2 on GLUT responsiveness, GABA-mediated inhibition of Purkinje cells was either increased, antagonized or unchanged following E2 application. In summary, this study suggests the hypothesis that circulating levels of E2 may alter neuronal sensitivity to specific neurotransmitter substances within the cerebellar circuitry.

Locally applied estrogens potentiate glutamate-evoked excitation of cerebellar Purkinje cells

Ongoing studies in our laboratory have demonstrated that systemically administered sex steroids 17 beta-estradiol (E2) and progesterone (P) alter cerebellar Purkinje cell responses to the amino acid neurotransmitter glutamate (Glu) in the urethane-anesthetized, ovariectomized adult rat. In the present study, we have examined the effects of locally pressure ejected E2 (0.5 microM) on Purkinje cell responsiveness to microiontophoretically applied Glu. The inactive stereoisomer of E2, 17 alpha-E2 (0.5 microM), estradiol benzoate (EB, 0.5 microM), and estrone (E1, 0.5 microM) were also tested (vehicle: 0.01% propylene glycol-saline, pH 7.4). Extracellular activity of single Purkinje neurons was recorded using multibarrel glass micropipets. Spontaneous firing rate and neuronal responses to microiontophoretic pulses (10 s every 40 s at 10-50 nA) of Glu were examined before, during and after continuous local pressure application of the steroids (1-5 psi, 10-15 min). Local E2 administration increased Glu response by 86% within 2-3 min after the onset of steroid application, with no recovery apparent by 30 min after termination of steroid administration. As such, local E2 application mimicked the effect of systemic injection of this steroid. The inactive estrogen isomer, 17 alpha-E2, failed to significantly enhance Glu responsiveness. Both EB and E1, however, significantly potentiated Glu responsiveness in a manner similar to locally applied E2. In addition, EB administration produced long-lasting increases in background discharge, unlike E2, and eventual recovery of Glu responses to control pre-steroid levels. In summary, this study provides a demonstration of local sex steroid actions on neuronal responsiveness in a model extrahypothalamic CNS area. These effects were specific, as the inactive 17 alpha-E2 isomer did not alter neuronal physiology. The results presented here suggest that the neuronal effects of systemic estrogen may be mediated by local actions of E2 or E1.

Locally applied progesterone metabolites alter neuronal responsiveness in the cerebellum.

Ongoing studies in this laboratory have demonstrated that both systemically and locally administered sex steroids 17 beta estradiol (E2) and progesterone (P) alter cerebellar Purkinje cell responses to microiontophoretically applied amino acid neurotransmitters GABA and glutamate (GLUT) in the urethane-anesthetized, ovariectomized adult rat. In the present study, we have examined the effects of several locally pressure ejected P metabolites on Purkinje cell responsiveness to GABA and GLUT: 5 alpha-pregnane-20-one (5 alpha DHP), 5 alpha-pregnane-3 alpha-ol-20-one (3 alpha OH-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (3 beta OH-DHP). GABA-induced inhibition was markedly enhanced immediately after onset of local application of 3 alpha OH-DHP or 5 alpha DHP, unaccompanied by alterations in background discharge. Both metabolites also attenuated excitatory responses to GLUT by 0-3 min after initiation of steroid application. In both cases, recovery to control levels of response was observed 6-9 min after termination of pressure application. These results are similar to those seen after local or systemic injection of P. In contrast, 3 beta OH-DHP did not produce any alteration in Purkinje cell responses to either amino acid. As 5 alpha DHP and 3 alpha OH-DHP can be localized in cerebellar tissue after P administration, the results presented here suggest that the neuronal effects of systemic P may be mediated by local membrane actions of P or its metabolites.

Sex steroid effects on extrahypothalamic CNS. II. Progesterone, alone and in combination with estrogen, modulates cerebellar responses to amino acid neurotransmitters

In a preliminary report we have shown that both intravenous and local application of progesterone (P) are capable of increasing cerebellar Purkinje cell responsiveness to microiontophoretically applied gamma-aminobutyric acid (GABA) and decreasing responsiveness to glutamate (GLUT) in the urethane-anesthetized, ovariectomized adult rat. In the present study we have examined the time course of effects of several doses of P and different combinations of both E2 and P on responses of individual Purkinje cells to GABA and GLUT. Extracellular activity of single Purkinje neurons was recorded using multibarrel glass micropipets. Spontaneous firing rate and responses of neurons to microiontophoretic pulses (10 s pulses every 40 s) of GABA (10-50 nA) and GLUT (3-40 nA) were examined before and after jugular i.v. administration of P or E2/P combinations to ovariectomized rats. In some cases animals received s.c. injections of E2 (2 micrograms) at 24 and 48 h before the day of recording. This injection schedule results in maximal reproductive effects of P. Within 5-15 min after P administration (5,50 or 500 micrograms) to ovariectomized rats, Purkinje cell responses to GLUT were decreased by 87%, and inhibitory responses to GABA were increased by 50%, with no associated change in spontaneous firing rate. In addition, the magnitude of the change in amino acid response was directly proportional to the dose of P. In most cases, complete recovery was observed 20-45 min after P administration. E2 pretreatment did not alter these P-induced effects. Combinations of E2 (300 ng/kg) and P (50 or 500 micrograms) injected simultaneously resulted in effects on GLUT responsiveness which were similar to those seen with P alone, while effects similar to E2 alone were observed with administration of E2 plus P at 5 micrograms. The administration of a protein synthesis inhibitor, anisomycin (30 mg/kg, i.v.), 20 min before the recording session did not prevent any of the above steroid effects. These results indicate that sex steroids can act to alter neuronal responsiveness to putative neurotransmitters in a CNS region not known to contain steroid receptors and that the particular combination of steroids will determine the neuronal response. These findings further suggest that the observed steroid-induced alterations in Purkinje cell responsiveness do not appear to require genomic mechanisms.

Sex steroids modulate motor-correlated increases in cerebellar discharge.

Adult female rats implanted with a microelectrode drive unit were trained to walk on a computer-controlled treadmill apparatus (10 s on every 20 s for 2 h) during recording of single Purkinje neurons in the paravermal area of the anterior cerebellum. Vigorous increases in the firing rate of individual units were found to be correlated with movement of specific limbs in particular stages of the step cycle during treadmill locomotion. Both spontaneous and motor-evoked discharge of individual Purkinje neurons were monitored before and after s.c. injection of either 17 beta estradiol (E2, 100 ng/kg) or progesterone (P, 50 micrograms). The percent increase in firing rate during locomotion versus rest was determined as a measure of the evoked:spontaneous discharge ratio. Drug-induced changes in this ratio indicate differential effects on the individual parameters, rather than simple excitatory or inhibitory effects. For all neurons tested, E2 augmented the movement-evoked discharge over pre-E2 control levels. The onset for this effect occurred at 15 min post-steroid, with a peak response noted at 30-35 min post-steroid. By 60-90 min, a partial recovery of the evoked:spontaneous ratio was noted, although absolute increases in both parameters were still observed, indicating long-term effects on neuronal activity. These effects were independent of the stage of the estrous cycle. In contrast, P decreased absolute firing rates of Purkinje cells during stationary and locomotor phases. However, the evoked:spontaneous ratio was decreased to an even greater degree. The latency for this effect was 9-12 min, with recovery to control levels of response seen at 30 min post-steroid. This response was typical of cells tested on estrus and diestrus 1. Cells tested on proestrus or diestrus 2, when E2 levels are increasing, were not modulated by P using the above paradigm.

Progesterone enhances inhibitory responses of cerebellar Purkinje cells mediated by the GABAA receptor subtype

Previous studies from this laboratory have indicated that the sex steroid progesterone (P) is able to augment inhibitory responses of cerebellar Purkinje cells to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the urethane anesthetized adult female rat. Because both GABAA and GABAB receptors are localized in this CNS area, agonist/antagonists for these receptors were utilized in the present evaluation in order to delineate the specific receptor subtype involved in P-GABA interactions. Systemically administered P was found to augment GABA-mediated inhibition by an average of 80%. Concomitant application of the GABAA blocker, bicuculline, greatly reduced this modulatory effect of the steroid. In contrast, P administration had no effect on baclofen-induced inhibition. These data suggest that P effects on GABA physiology are mediated predominantly at the GABAA receptor.

GABAB receptor stimulation by baclofen and taurine enhances excitatory amino acid induced phosphatidylinositol turnover in neonatal rat cerebellum

Excitatory amino acid stimulation of phosphatidylinositol (PI) hydrolysis has been associated with development of the CNS. Normally minimally ineffective in stimulating PI hydrolysis in the neonatal rat cerebellum, N-methyl-D-aspartate (NMDA) increased levels of PI hydrolysis 82.3 +/- 5.5% above basal values in the presence of 1 microM baclofen, a gamma-aminobutyric acidB (GABAB) receptor agonist. This effect was observed at day 7 but not in adult cerebellum. The effect of baclofen could be mimicked by low dose GABA and taurine, actions which were blocked by prior application of a specific GABAB antagonist. Therefore, the ability of NMDA to stimulate PI hydrolysis in neonatal cerebellar tissue may be regulated by the degree of GABAB receptor stimulation.

A paradigm for determination of direct drug-induced modulatory alterations in Purkinje cell activity during treadmill locomotion

This paper describes a paradigm employing chronic single unit recording techniques and videotape analysis of treadmill locomotion in order to determine drug-induced modulation of sensorimotor neuronal activity. Animals implanted with a chronic headstage microdrive unit and an indwelling jugular cannula are trained to walk on a treadmill (10 s on, 10 s off). Characteristically, cerebellar Purkinje cells recorded 1-1.5 mm from midline, exhibit increased rates of discharge in phase with movement of specific limbs during a particular stage of the step-swing cycle, as assessed by video analysis of locomotor patterns. Drug-induced alterations in this movement-correlated discharge relative to changes in the spontaneous firing rate can then be determined to assess drug-induced neuromodulatory effects beyond general non-specific excitatory or inhibitory actions.