Jeffrey L. Barker

Sex differences in expression of serotonin receptors (subtypes 1A and 2A) in rat brain: a possible role of testosterone.

Sexual differences in the expression of messenger RNA and in the binding of serotonin receptors (subtypes 1A and 2A) were studied by in situ hybridization and autoradiography ¿[3H]8-hydroxy-2(di-n-propylamino)tetralin and [3H]ketanserin binding) in the rat brain. Serotonin-1A receptor messenger RNA showed distinct expression patterns for female and male rats. Expression of serotonin-1A receptor messenger RNA was greater in males in subregions of the hypothalamus and amygdala, and less in males in subregions of the hippocampus. No significant differences in the distribution of serotonin-1A receptor binding sites were found between the sexes. Serotonin-2A receptor messenger RNA expression was comparable in males and females in all brain regions except the ventromedial hypothalamic nuclei, where lower levels were seen in females. However, the binding of serotonin-2A receptor measured with [3H]ketanserin was significantly higher in females in all regions of the hippocampus. In a separate study, gonadectomy in males significantly increased serotonin-1A messenger RNA content in the cortex, hypothalamus, hippocampus, amygdala and dorsal raphe, and decreased serotonin-2A messenger RNA in ventromedial hypothalamic nuclei only. Almost all gonadectomy-induced changes were reversed by concomitant administration of testosterone. Our data provide evidence for region-specific sex differences in serotonin receptor subtype 1A and 2A transcription and concentration in the rat brain, and further suggest a modulatory role of testosterone in serotonin (particularly subtype 1A) receptor expression. Gender and gonadal steroid effects on central serotonergic systems may underlie the reported sexual dimorphisms in affective state regulation, response to psychopharmacological agonists or pituitary adrenal activation.

Single channel currents activated by gamma-aminobutyric acid, muscimol, and (-)-pentobarbital in cultured mouse spinal neurons

The patch electrode technique was used to record single channel current pulses in tissue-cultured mouse spinal cord neurons. In agreement with earlier noise studies, channels activated by gamma-aminobutyric acid (GABA), muscimol, and (-)-pentobarbital were found to have equal unit conductances. The kinetics of channel closing were studied by analyzing the distributions of open state lifetimes. Channels activated by (-)- pentobarbital and muscimol had longer mean open times than channels activated by GABA. As a result, the kinetics of (-)-pentobarbital- and muscimol-activated channels could be studied in greater detail. Most observed open state lifetime distributions were not exponential but contained an excess of short duration events. A sum of two exponential functions gave a much better fit than a single exponential function to most observed open state lifetime distributions. A critical comparison of noise analysis with single channel recording shows that the fast process responsible for the rapid closures would be very difficult to detect in a noise experiment. The channel noise is dominated by the slower process, and as a result, the relaxation time of the slower kinetic component derived from single channel studies is close to the mean open state lifetime derived from noise measurements. The observation of a faster process points toward either an additional population of channels or a scheme for the channel closing transition which is not a simple first order process.

Interaction of barbiturates with benzodiazepine receptors in the central nervous system

The interaction of barbiturates with benzodiazepine receptors was studied in extensively washed membrane preparations from rat brain. Sedative/hypnotic and anesthetic barbiturates such as pentobarbital, and convulsant barbiturates such as DMBB, enhanced [3H]diazepam binding in a stereospecific fashion. Freeze-thawing of membranes resulted in a decrease in the potency of barbiturates to enhance [3H]diazepam binding, while the maximum response to barbiturates remained unchanged. Significant differences in both the potency and maximum enhancement of [3H]diazepam binding by pentobarbital was observed among brain regions. The rank order potency of pentobarbital in different brain regions was: cerebellum greater than cortex greater than hippocampus, while the rank order efficacy of pentobarbital in these brain regions was reversed. The effects of a combination of anesthetic and/or convulsant barbiturates on [3H]diazepam binding suggested that these compounds function as partial agonists while a combination of anesthetic or convulsant barbiturates with phenobarbital suggested that latter compound antagonized the actions of both anesthetic and convulsant barbiturates. The convulsant benzodiazepine Ro-5-3663 and inosine were more potent as inhibitors of pentobarbital-enhanced than basal (non-pentobarbital enhanced) [3H]diazepam binding. Solubilization of benzodiazepine receptors with Lubrol-PX resulted in a complete loss of barbiturate enhanced [3H]diazepam binding, and greater than a 75% loss in efficacy in the remaining (insoluble receptor) tissue. These data, coupled with recent observations from this and other laboratories, suggests that the site(s) at which barbiturates act to enhance [3H]diazepam binding to benzodiazepine receptors is distinct from the site at which GABA acts to enhance [3H]diazepam binding. The phenomenon of enhanced benzodiazepine binding by barbiturates may be related to the depressant actions of the barbiturates, that is, their direct effects to increase chloride conductance. Although it is premature to assign a pharmacologic correlate to this neurochemical phenomenon, it appears that this action may be related to the anesthetic effects of the barbiturates. However, the definitive assignment of either the electrophysiologic or pharmacologic sequelae to this neurochemical action will require further investigation.

Analysis and isolation of embryonic mammalian neurons by fluorescence- activated cell sorting

Cells were dissociated from the CNS of the embryonic mouse and rat to produce cell suspensions suitable for analysis and separation on a fluorescence-activated cell sorter (FACS). Cells from the spinal cord of the embryonic mouse were analyzed in the most detail. Cell suspensions generated three major peaks in histograms of forward-angle light scatter. Examination of material isolated from each peak and labeling of cell suspensions with the nonvital and supravital fluorescent dyes propidium iodide, ethidium bromide, and acridine orange demonstrated that the three peaks represented live cells, dead cells, and subcellular fragments. Passage through the cell sorter did not detectably damage live cells, as shown by light microscopy, FACS analysis, and in vitro culture of sorted cells. Neurons and glial cells collected by sorting survived at least 4 weeks in culture. Cell suspensions dissociated from the dorsal root ganglia, hippocampus, hypothalamus, cerebellum, and cerebral cortex of the embryonic mouse and from the spinal cord of the embryonic rat produced similar results. Analysis of samples prepared at different developmental stages showed that viable cells could be recovered from each of these regions throughout the important stages of neurogenesis and early cellular differentiation, but that few viable cells could be recovered from animals beyond late embryonic or early postnatal ages. Quantitative FACS analysis of monoclonal antibody A2B5, tetanus toxin and cholera toxin, and lectins binding to live dissociated cells from the embryonic spinal cord demonstrated that these cells had already developed binding sites for these cell-surface ligands by embryonic day 13. These results demonstrate that a fluorescence-activated cell sorter can be used for quantitative analysis of specific cellular properties, that FACS analysis and sorting can be used to identify and isolate live cells from many regions of the embryonic mammalian CNS during important developmental periods, and that sorted neurons and glial cells can be maintained for weeks in culture.

p11 IS UP-REGULATED IN THE FOREBRAIN OF STRESSED RATS BY GLUCOCORTICOID ACTING VIA TWO SPECIFIC GLUCOCORTICOID RESPONSE ELEMENTS IN THE p11 PROMOTER

Posttraumatic stress disorder (PTSD) is one of the most common psychiatric disorders. Despite the extensive study of the neurobiological correlates of this disorder, the underlying mechanisms of PTSD are still poorly understood. Recently, a study demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, can up-regulate p11, known as S100A10-protein which is down-regulated in patients with depression, (Yao et al., 1999; Huang et al., 2003) a common comorbid disorder in PTSD. These observations led to our hypothesis that traumatic stress may alter expression of p11 mediated through a glucocorticoid receptor. Here, we demonstrate that inescapable tail shock increased both prefrontal cortical p11 mRNA levels and plasma corticosterone levels in rats. We also found that Dex up-regulated p11 expression in SH-SY5Y cells through glucocorticoid response elements (GREs) within the p11 promoter. This response was attenuated by either RU486, a glucocorticoid receptor (GR) antagonist or mutating two of three glucocorticoid response elements (GRE2 and GRE3) in the p11 promoter. Finally, we showed that p11 mRNA levels were increased in postmortem prefrontal cortical tissue (area 46) of patients with PTSD. The data obtained from our work in a rat model of inescapable tail shock, a p11-transfected cell line and postmortem brain tissue from PTSD patients outline a possible mechanism by which p11 is regulated by glucocorticoids elevated by traumatic stress.

GABAergic synapses and benzodiazepine receptors are not identically distributed in the primate retina

The distribution of benzodiazepine receptors (BZR) was compared to the distribution of gamma-aminobutyric acid (GABA)-ergic synapses in the rhesus monkey retina using monoclonal antibodies against the BZR and polyclonal antisera to glutamate decarboxylase (GAD), the GABA-synthesizing enzyme which labels the presynaptic terminals of the GABAergic synapses. Indirect immunofluorescence including dual fluorochroming for both BZR and GAD indicates that although both were localized to the inner plexiform layer and adjacent cell body layers, their distributions were largely non-overlapping. Thus, in the primate retina, BZRs are not exclusively associated with GABAergic synapses.

Naloxone antagonism of GABA-evoked membrane polarizations in cultured mouse spinal cord neurons.

Pharmacological studies using an in vitro model system were carried out to determine if naloxone, an opiate receptor antagonist, could have effects on neuronal membranes which were unrelated to its action as an opiate receptor antagonist. Intracellular recordings were made from cultured mammalian spinal cord neurons. Putative amino acid neurotransmitters and naloxone were applied by iontophoresis or superfusion. When naloxone was co-iontophoresed with the amino acids a depression of the GABA response resulted. This depression was dose-dependent and reversible. At the lower doses of naloxone tested, the depression was specific since the glycine and glutamate responses were unaffected. At the higher doses of naloxone tested, alterations in the glycine and/or glutamate responses and membrane input resistance were frequently observed. The naloxone depression of the GABA response did not appear to involve opiate receptors since (+)-naloxone, the inactive isomer, equally depressed the GABA response. Analysis of the effect of naloxone on GABA dose-response curves indicates that naloxone acts as a competitive antagonist at the neuronal GABA receptors. Similar results were obtained when naloxone was applied by superfusion. However, high concentrations of naloxone (0.1-1 mM) were required, suggesting that naloxone has a low affinity for the GABA receptor. These data indicate that under some experimental conditions naloxone could not be considered a specific opiate antagonist.

Involvement of Akt in Preconditioning-Induced Tolerance to Ischemia in PC12 Cells

The serine-threonine protein kinase Akt has been identified as an important mediator of cell survival able to counteract apoptotic stimuli. However, hibernation, a model of natural tolerance to cerebral ischemia, is associated with downregulation of Akt. We previously established a model of ischemic tolerance in a PC12 cell line and using this model we now addressed the question whether ischemic tolerance also downregulates Akt in PC12 cells. Kinetic studies showed decreased Akt phosphorylation in tolerized cells. Similarly, phosphorylated levels of three major targets of Akt and well-known proapoptotic factors, the glycogen synthase kinase 3 (GSK-3), a Forkhead family member, FoxO4, and the protein murine double minute 2 (MDM2), all inactivated upon phosphorylation by Akt, were decreased in preconditioned cells. In addition, pharmacological blockade of the phosphoinositide 3-kinase (PI3K)/Akt pathway reduced cell death induced by oxygen and glucose deprivation (OGD) and increased the protective effect of preconditioning (PC). Furthermore, decreasing availability of P-Akt by transfecting PC12 cells with constructs of inactive Akt also resulted in protection against OGD and potentiation of the protective effect of PC. Depending on the environment, GSK-3, FOXO-4, and MDM2 can trigger apoptotic responses or cell cycle arrest, and thus, in a situation of reduced energy, driving the cells into a state of quiescence might be neuroprotective. This work suggests that in the context of tolerance downregulation of Akt is beneficial.

Rapid transport of proteins in the sonic motor system of the toadfish

A pure cholinergic, motor system of a marine fish has been utilized to study the kinetics and characteristics of proteins rapidly transported from the sonic motor nucleus to the musculature enveloping the swim bladder. Following microinjection of [3H]leucine, [3H]lysine, [35S]methionine, or [3H]fucose into the nucleus a wave of radioactivity was observed moving along the sonic motor nerves with an apparent rate of 96-120 mm/day. Analysis of the rapidly transported methionine-labeled protein using SDS gel electrophoresis revealed at least 9 major peaks of activity. Eight of these proteins were found to incorporate fucose, suggesting that most of the rapidly transported material consists of glycoproteins. These results are consistent with the previously suggested hypothesis relating the function of rapid transport to synaptic vesicles and the maintenance of pre-synaptic terminal membranes.

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABAA receptor/Cl− channels in cultured proliferating rat neuroepithelial cells

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.