This study was undertaken to examine the effects of two acetylcholinesterase inhibitors (donepezil and physostigmine) on cognitive deficits in mice after repeated administration of the NMDA receptor antagonist phencyclidine (PCP). In the novel object recognition test, PCP (10 mg/kg/day for 10 days)-induced cognitive deficits were significantly improved by subsequent subchronic (14 days) administration of donepezil (1.0 mg/kg/day), but not donepezil (0.1 mg/kg/day). Furthermore, the effect of donepezil (1.0 mg/kg/day) on PCP-induced cognitive deficits was significantly antagonized by co-administration of the selective sigma-1 receptor antagonist NE-100 (1.0 mg/kg/day), suggesting the role of sigma-1 receptors in the active mechanisms of donepezil. In contrast, PCP-induced cognitive deficits were not improved by subsequent subchronic (14 days) administration of physostigmine (0.25 mg/kg/day). Moreover, repeated administration of PCP significantly caused the reduction of sigma-1 receptors in the hippocampus. The present study suggests that agonistic activity of donepezil at sigma-1 receptors plays a role in the active mechanisms of donepezil on PCP-induced cognitive deficits in mice. Therefore, it is likely that donepezil would be potential therapeutic drugs for the treatment of the cognitive deficits in schizophrenia.

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.

Binding of [3H]AMPA was increased above control levels in rat brain membranes that had been incubated with concanavalin A (Con A) or a lectin from Lens culinaris (LC), both of which bind mannose residues. This did not occur with any of six lectins with other specificities. The magnitude of the increased binding varied from 15% in cortex to 70% in hippocampus and decreased significantly between 3 weeks and 6 months of age. Succinylated Con A was without effect and neither Con A nor LC increased binding to solubilized AMPA receptors. Increases in binding were not obtained in membranes purified from HEK293 cell lines expressing homomeric AMPA receptors. This indicates that mannose specific lectins may enhance binding by cross-linking AMPA receptors to each other or to proteins that are specific to brain. Con A has been reported to reduce glutamate receptor desensitization with higher efficacy at kainate than at AMPA receptors; the increase in binding reported here appears to be unrelated to such effects because (1) it was not affected by drugs that block desensitization and (2) [3H]kainate binding was reduced rather than increased by Con A. These observations suggest that AMPA receptor kinetic properties not involving desensitization are influenced by extracellular interactions between the receptors and other transmembrane proteins.

Programmed cell death has been implicated in the loss of neurons that occurs in many neurodegenerative diseases. This has led to an increased interest in the types of stimuli that can initiate neurons to undergo programmed cell death. Previously, we have shown that cross-linking of membrane receptors with the lectin concanavalin A can trigger programmed cell death in neurons [D.H. Cribbs, V.M. Kreng, A.J. Anderson, C.W. Cotman, Cross-linking of Concanavalin A receptors on cortical neurons induces programmed cell death, Neuroscience 75 (1996) 173-185]. Concanavalin A, however, binds to many surface glycoproteins and therefore, it is important to determine whether certain specific receptors can initiate the program. We found that surface immobilized anti-neural cell adhesion molecules (NCAM) monoclonal antibodies provide a good substrate for adhesion and neurite outgrowth for cortical neurons. However, neurons treated directly with soluble anti-NCAM monoclonal antibodies show significant cell death after 24 h and exhibit the morphological and biochemical features indicative of apoptosis, including membrane blebbing, cell shrinkage, condensation of nuclear chromatin and internucleosomal DNA cleavage.

Previous reports have shown that the inbred strains of rat, Lewis (LEW) and Fischer 344 (F344), differ in several behavioural and biochemical indices of mesolimbic dopamine (DA) function. Specifically, these two strains differ in their behavioural and neurochemical response to novel environments, and acute amphetamine or cocaine challenge as well as in their susceptibility to addiction. To investigate if differences in DA D1-like, D2-like, D3 receptors and DA transporter could be correlated with these behavioural differences between strains, a comparative autoradiographic study of DA receptors and transporter within the striatal and accumbal regions was undertaken. We observed strain and region specific differences in binding levels for DA D2-like and D3 receptors and for the DA transporter. Namely, DA transporter levels in the striatum, nucleus accumbens and olfactory tubercle of LEW rats were significantly lower than in F344 rats. DA D3 densities in the shell of the nucleus accumbens and olfactory tubercle of LEW rats were lower than the levels found in the F344 rats. Finally, LEW rats have a lower levels of D2-like receptors in the striatum and the core of the nucleus accumbens compared to F344 rats. These data suggest that differences in DA transporter and DA receptors may in part contribute to differences in DA related behaviour seen between these two strains.

Previous studies have shown that: (1) activation of neurons in the dorsomedial hypothalamus (DMH) of the rat by blockade of local GABAA receptors with bicuculline methiodide (BMI) elicits cardiovascular changes resembling those seen in experimental stress, including marked sympathetically-mediated tachycardia, and (2) inhibition of neurons in the same region by local microinjection of the GABAA receptor agonist muscimol can virtually abolish stress-induced tachycardia. This study examined the possibility that GABAB receptors exist in the neural circuitry of the DMH, and that stimulation of these receptors might suppress the cardiovascular response to local disinhibition with BMI. Microinjection of BMI 10 pmol into the DMH in urethane-anesthetized rats resulted in marked tachycardia with little or no effect on arterial pressure. Simultaneous injection of the GABAB receptor agonist baclofen at doses of 2.5, 5.0 and 10 pmol produced dose-related suppression of BMI induced tachycardia. Coinjection of the GABAB receptor antagonist 2-hydroxysaclofen 100 or 200 pmol had no significant effect on the heart rate response to BMI, but reversed the suppression elicited in the presence of baclofen. These findings indicate that (1) functional GABAB receptors exist in the DMH, and (2) stimulation of these receptors inhibits the tachycardia resulting from blockade of local GABAA receptors.

The effects of chronic ethanol (EtOH) consumption on the central nervous system may be related in part to its action on biological membranes by altering various receptor functions. In the current study, we examined the effects of chronic EtOH (4 day inhalation) on cannabinoid receptors (CB1) labeled with [3H]CP55,940 in synaptic plasma membranes (SPM) isolated from mouse brain. Our results indicate the presence of a high level of CB1 receptors in controls (Bmax=12.0+/-0.3 pmol mg-1 protein) which decreased significantly (-58%) in SPM from mouse brain chronically exposed to EtOH. This effect occurs without any changes in the receptor affinity (Kd=2. 3+/-0.3 nM for control and 2.9+/-0.3 nM for EtOH group, P>0.05). Dissociation kinetic results showed a dissociation rate constant (K-1) of 0.09+/-0.01 min-1 for control and this dissociation rate constant decreased significantly in the chronic EtOH treated mice brain (0.05+/-0.01 min-1, P<0.05). The competition studies with anandamide resulted in a substantial decrease in [3H]CP55,940 binding in both the control and EtOH group, with a decrease (P<0.05) in the Ki values in the SPM of chronic EtOH exposed mice. Hill transformation analysis showed an nH close to one in control (0. 92+/-0.01). This did not change significantly after chronic EtOH (0. 95+/-0.01) administration, which indicates the existence of a single class of receptor for [3H]CP55,940 binding in SPM from control and EtOH treated mice. The observed down-regulation of CB1 receptors by chronic EtOH may indicate the involvement of cannabinoid receptors in EtOH tolerance and dependence.

Ethanol dependence, arising from chronic ethanol exposure, is associated with neuroadaptations of GABAA receptors, evidenced by alterations in various behaviors, receptor responsiveness and subunit gene expression. The present studies explored the effects of ethanol dependence in female rats for comparison with previous studies in our laboratory using male rats. We found that ethanol dependence resulted in differential effects on GABAA receptor gene expression in female rat cerebral cortex compared to ethanol dependent male rats. Notably, chronic ethanol consumption did not change GABAA receptor alpha 1 subunit peptide levels in ethanol dependent female rat cortex, in contrast to previously observed decreases in alpha 1 subunit expression in ethanol dependent male rat cortex. The effects of ethanol dependence on additional GABAA receptor subunit peptide levels (alpha 4, beta 2/3 and gamma 2) were similar, but not identical, between female and male rat cortex. When directly compared within the experiment, male and female rats had similar baseline bicuculline seizure thresholds and displayed a similar increase in seizure susceptibility during ethanol withdrawal. Ethanol withdrawn female rats were cross tolerant to the anticonvulsant effects of diazepam, similar to the findings in ethanol withdrawn male rats. Ethanol withdrawn female rats showed a dose-dependent enhancement of the anticonvulsant effect of the neuroactive steroid, THDOC (3 alpha,21-dihydroxy-5 alpha-pregnan-20-one) compared to control animals. This finding is similar to previous observations of increased sensitivity to the anticonvulsant effect of 3 alpha,5 alpha-THP (3 alpha-hydroxy-5 alpha-pregnan-20-one) in ethanol withdrawn male and female rats. In addition, low dose administration of THDOC elevated seizure thresholds in ethanol withdrawn female but not male rats, suggesting that ethanol withdrawn female rats were more responsive to the anticonvulsant effects of this neurosteroid than were ethanol withdrawn male rats. These findings show that gender impacts on adaptations in GABAA receptors elicited by ethanol dependence. However, the physiological outcomes of the differential alterations are not clear. Taken together, these studies suggest that additional mechanisms, beyond effects on GABAA receptor gene expression are involved in the mediation of ethanol dependence and withdrawal.

α4βδ GABA(A) receptors (GABARs) have low CNS expression, but their expression is increased by 48h exposure to the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one). THP also increases the efficacy of δ-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at α4β2δ (gaboxadol, 10 μM, and β-alanine, 10 μM-1mM), on surface expression of α4β2δ. To this end, we used an α4 construct tagged with a 3XFLAG (F) epitope or measured expression of native α4 and δ. HEK-293 cells or cultured hippocampal neurons were transfected with α4Fβ2δ and treated 24h later with GABA agonists, THP, GABA plus THP or vehicle (0.01% DMSO) for 0.5 h-48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10 μM) plus THP increased α4β2δ surface expression up to 3-fold after 48h, an effect first seen by 0.5h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced α4β2δ expression is likely via receptor insertion. Surface expression of α4β2δ was decreased by rottlerin (10 μM), suggesting a role for PKC-δ. These results suggest that trafficking of α4β2δ GABARs is regulated by high efficacy states.

Functional and pharmacological studies have suggested that there are muscarinic receptors (mAChRs) on the endothelial cells of major cerebral arteries, while recent immunological studies indicate that there are no mAChRs on the endothelium of brain capillaries. This difference may be because the distribution of mAChR on the endothelium varies with the type of vessel. This paper examines the distribution of mAChR on the vascular endothelium along intraparenchymal blood vessels in the rat brain by immunolabelling and laser confocal microscopy. Sections were immunostained by combinations of an anti-mAChR antibody (M35) with antibodies to endothelial (anti-GLUT1), or to smooth muscle markers (anti-actin). Antibody labellings were detected with fluorescent second antibodies. Most of the penetrating vessels bore mAChR immunolabelling which coincided over almost all the vessel surface with endothelial labelling. The mAChR immunolabelling was less widespread over the endothelium on the medium sized vessels (diameter < 50 microm) and only 50% of these vessels had mAChR staining on the endothelium. There was no mAChR immunostaining on the endothelium of the capillaries. In contrast with the basilar artery, there was no mAChR immunolabelling on the smooth muscle layer of the intracortical vessels. These data indicate that the intensity of mAChR immunolabelling decreases along the vascular tree from large conducting vessels to capillaries.

The mechanisms by which diabetes impairs cognitive function are not well-established. In the present study, we determined the electrophysiological and biochemical nature of disturbances in the mechanism of long-term potentiation (LTP) in diabetic rats. As previously reported, the administration of streptozotocin (STZ) was found to reduce the magnitude of LTP in the CA1 region of the hippocampus, while the same treatment did not interact with the capacity of the hippocampus to generate long-term depression induced by low-frequency stimulation. In addition, STZ treatment did not modify the component of excitatory postsynaptic potentials mediated by activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, suggesting that NMDA receptor function remained intact in STZ-treated slices. At the biochemical level, the capacity of calcium to increase [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (3H-AMPA) binding to glutamate/AMPA receptors in rat brain tissue sections was markedly affected in most regions of the hippocampus of STZ-treated rats. Moreover, changes in 3H-AMPA binding properties elicited by both exogenous phospholipase A2 and melittin, a potent activator of endogenous phospholipases, were also altered in synaptoneurosomes from diabetic rats. Taken together, the present data suggest that the loss of LTP maintenance in STZ-treated rats is more likely the result of disruption of calcium-dependent processes that are suspected to modulate postsynaptic AMPA receptors during synaptic potentiation. Understanding the biochemical factors participating in the impairment of AMPA receptor modulation might provide important clues revealing the very basis of memory deficits in diabetes.

Using patch-clamp techniques we studied several aspects of intracellular GABA(A) and glycine Cl- current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl- current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 microM PMA reduced the amplitude of the GABA(A) and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 microM okadaic acid, a phosphatase inhibitor, induced potentiation in both currents. The amplitude of GABA(A) and glycine currents recorded with 1 mM internal CaCl2 and 10 mM EGTA (10 nM free Ca2+) decayed by less than 30% of control. Increasing the CaCl2 concentration to 10 mM (34 microM free Ca2+) induced a transient potentiation of the GABA(A) current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca2+ concentration. Activation of G proteins with internal FAl4- induced an inhibition of the GABA(A) current, but potentiated the amplitude of the strychnine-sensitive Cl- current. These results indicate that GABA(A) and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca2+. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.

The neuropeptides angiotensin II (AngII) and oxytocin (OT) play important but opposing roles in the regulation of sodium appetite in the rat, AngII as a stimulatory peptide and OT as an inhibitory peptide. Adrenal steroids increase the density of AngII receptors in brain following in vivo administration, although the neuroanatomical and subtype specificity have not been thoroughly examined. Furthermore, previous studies demonstrate that adrenalectomy (ADX) leads to a reduction in OT receptors, although regions associated with sodium appetite remain to be examined. In the present study, quantitative receptor autoradiography was used to locate regions where perturbations in circulating adrenal steroids affect the density of oxytocin receptors and the angiotensin receptor subtypes AT1 and AT2. The results show that ADX results in a small, but significant decrease in AT1 expression in the paraventricular nucleus of the hypothalamus, subfornical organ, and the area postrema. That this effect is reversed by either aldosterone or low-dose corticosterone replacement suggests that occupancy of the mineralocorticoid receptor is responsible for the steroid effect. No changes were observed in AT2 or OT receptors in nuclei associated with sodium appetite, indicating that perturbations in adrenal steroids did not affect these receptors in brain regions implicated in the control of salt appetite.

Expression of TrkB receptors were studied in the cerebral cortex of normal rats and young rats with hypoxic/ischemic injury. TrkB expressing cells were present in the piriform cortex at birth and increased in number with age, and were finally present in the entire cerebral cortex. Density of TrkB cells reached adult levels at P30. They were morphologically regarded as pyramidal neurons and interneurons. Hypoxic/ischemic injury induced a tentative increase of full-length TrkB receptors. A novel appearance of TrkB expressing neurons and enhanced immunostaining on both cell soma and dendrites were observed in the peri-infarct areas and increased number of TrkB expressing neurons were detected in the contralateral cortex after carotid artery ligation. This increase was no longer evident after 48 h of hypoxia. Double immunostaining using antiserum against GFA or OX-42 revealed no co-localization of TrkB receptors and these molecules, while there were only slight co-localization of TrkB and calbindin-D28k molecules. The altered levels in responses to injury indicate that TrkB may play a crucial role in the early protective mechanism of the neurons with hypoxic/ischemic injury through ligands BDNF and/or NT-4/5.

Application of N-methyl-D-aspartate (NMDA) to the supraoptic nucleus of the hypothalamus (SON) generates clustered firing that may be important in hormone release. However, synaptically evoked EPSPs recorded from SON neurons exhibit varying contributions from NMDA receptors. We used the high resolution of single-channel recording to examine the receptor and ion channel properties of NMDA receptors expressed by SON neurons in 'punch' culture. Biocytin introduced into individual neurons during patch clamp recording revealed large (32.1+/-3.3 micron), oblong somas and bipolar extensions typical of magnocellular neuroendocrine cells (MNCs). Rapid application of NMDA (100-300 microM) in the presence of 10 microM glycine to outside-out macropatches resulted in openings with an average conductance of 46. 9 pS and reversal potential of +3.9 mV. Increasing glycine from 0.03 to 30 microM increased the apparent frequency, duration and occurrence of overlapping NMDA-elicited openings. NMDA responses were inhibited by Mg2+ in a voltage-dependent manner and by the NMDA-site antagonist, D-(-)-2-amino-5-phosphonovaleric acid (D-APV). Application of saturating NMDA or glycine alone with the glycine-site antagonist, 5,7-dichlorokynurenate (DCK) or with D-APV, respectively, did not result in agonist-induced openings. NR1 immunoreactivity was observed in large neurons (>25 micron) with MNC-like morphology. These single-channel and immunocytochemical data confirm the presence of functional NR1-containing NMDA receptors in MNCs.

Recently, [125I]S(-)5-OH-PIPAT (5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)amino-tetralin) was reported to be a selective radioiodinated ligand for dopamine D2-like receptors. This ligand displayed a high binding affinity (Kd = 0.3-0.4 nM) and an agonist binding profile to dopamine D2 and D3 receptors expressed in HEK293 cells and dopamine D4 receptors expressed in CHO cells. Herein, a series of studies to characterize D3 receptors in native tissues is presented. Based on studies of the distribution of receptor mRNA, D3, but not D2, receptors are present in the rat cerebellum. Quantitative autoradiographic experiments using [125I]S(-)5-OH-PIPAT to label molecular layers 9 and 10 of rat cerebellum were conducted. Saturation experiments demonstrated that [125I]S(-)5-OH-PIPAT bound with high affinity (Kd = 0.1 nM) to a low density (approximately 3 fmol/mg protein) of sites in molecular layers 9 and 10 of rat cerebellum. Increasing concentrations of Gpp(NH)p, but not ATP, decreased the specific binding of [125I]S(-)5-OH-PIPAT in rat cerebellum slices. In comparison studies, binding of [125I]NCQ298, a dopamine D2/D3 receptor antagonist, with a similar affinity (Kd = 0.2 nM) for D3 receptors as [125]S(-)5-OH-PIPAT, was not sensitive to Gpp(NH)p. Analysis of inhibition by S(-)5-OH-PIPAT of [125I]NCQ298 binding to rat cerebellum resulted in two-site binding with IC50 values of 0.07 nM and 6.0 nM. In the presence of GTP (300 microM), the data best fit a one-site model with an IC50 value of 1.6 nM. Agonists and antagonists inhibited the binding of [125I]S(-)5-OH-PIPAT in the cerebellum with a rank order of potency consistent with an interaction at D3 receptors. These results indicate that [125I]S(-)5-OH-PIPAT binds to D3 receptors in rat cerebellum. Furthermore, [125I]S(-)5-OH-PIPAT binds to GTP sensitive and GTP insensitive states of D3 receptors with distinctive high and low affinity states, respectively.

Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any mu-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. Mu-, delta-, kappa1- and total kappa-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin), [3H]DELTI (D-Ala2 deltorphinI), [3H]CI-977 and [3H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, mu-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from mu-knockout mice there were no detectable mu-receptors in any brain regions and no evidence for mu-receptors derived from another gene. Delta-, kappa1- and total kappa-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in kappa- or delta-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in delta- and kappa-receptors throughout the brains of animals deficient in mu-receptors.

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)7 receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT7 receptors induced by a treatment with agonists (0.1, 1, and 10 muM, 0.5 to 3.5 h). Maximum responses, but not the EC50 values, in the concentration-response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor alpha-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT7 receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.

There is increasing evidence that diabetes may be related to sensory changes in the trigeminal system. Long lasting facial heat hyperalgesia has been described in diabetic rats, but the mechanisms remain to be elucidated. Herein, the contribution of peripheral and central TRPV1 receptors to facial heat hyperalgesia in diabeticrats was investigated. Diabetes was induced in male Wistar rats by streptozotocin (60mg/kg, i.p) and facial heat hyperalgesia was assessed once a week up to four weeks. The role of TRPV1 receptors in the heat hyperalgesia in diabetic rats was evaluated through: 1) the ablation of TRPV1 receptors by resiniferatoxin (RTX) treatment and 2) injection of the TRPV1 antagonist, capsazepine, into the upper lip, trigeminal ganglion or medullary subarachnoid space, at doses that completed prevented the heat hyperalgesia induced by capsaicin in naïve rats. Western blot was used to estimate the changes in TRPV1 expression in diabetic rats. Diabetic rats exhibited facial heat hyperalgesia from the first up to the fourth week after streptozotocin injection, which was prevented by insulin treatment. Ablation of TRPV1-expressing fibers prevented facial hyperalgesia in diabetic rats. Capsazepine injection in all sites resulted in significant reduction of facial heat hyperalgesia in diabetic rats. Diabetic rats exhibited a significant decrease in TRPV1 expression in the trigeminal nerve, increased expression in the trigeminal ganglion and no changes in subnucleus caudalis when compared to normoglycemic ones. In conclusion, our results suggest that facial heat hyperalgesia in diabetic rats is maintained by peripheral and central TRPV1 receptors activation.

The impact of 'binge-like' ethanol exposure on postnatal days (PD) 4-9 was examined on development of gamma-aminobutyric acid type A receptors (GABAAR) during the first month of life in the rat. Whole-cell patch-clamp recordings in acutely isolated medial septum/diagonal band (MS/DB) neurons were used to define effects of rapidly applied ethanol and other allosteric modulators on bicuculline-sensitive GABA currents. Three age groups were examined including 'pups' (PD 4-10), 'juveniles' (PD 11-16) and 'young adults' (PD 25-35). In untreated neurons, maximum responses to GABA and the apparent GABA EC50 increased approximately 2-fold during the first month of life. Potentiation of GABA responses by pentobarbital, midazolam, and loreclezole all increased with age, while Zn2+ inhibition declined. Initial inhibition by ethanol switched to potentiation of GABA responses during this time. In vivo, binge-like ethanol treatment (4.5 g kg-1 day-1 divided into two doses, 2 h apart on PD 4-9) reduced both the GABA maximal response and GABA EC50 measured on PD 11-16. These measures returned to control levels by PD 25-35. After binge-like postnatal ethanol exposure, age-dependent loss of Zn2+ inhibition of GABA responses was increased, while potentiating actions of in vitro ethanol were blocked. GABAAR modulation by other drugs was unaffected. These data suggest that early postnatal ethanol exposure disrupts the expected developmental pattern of GABAAR function in MS/DB neurons, an action that could contribute to neurobehavioral deficits associated with the fetal alcohol syndrome. Whether these changes are due to cellular damage, delayed gene expression or post-translational modification needs to be determined.