A novel role for cyclic guanosine 3',5'monophosphate signaling in synaptic plasticity: a selective suppressor of protein kinase A-dependent forms of long-term potentiation.

At excitatory synapses onto hippocampal CA1 pyramidal cells, activation of cyclic AMP-dependent protein kinase and subsequent down-regulation of protein phosphatases has a crucial role in the induction of long-term potentiation by low-frequency patterns of synaptic stimulation. Because the second messenger cyclic guanosine 3',5'monophosphate can regulate the activity of different forms of the cyclic AMP degrading enzyme phosphodiesterase, we examined whether increases in cyclic guanosine 3',5'monophosphate can modulate long-term potentiation induction in the mouse hippocampal CA1 region through effects on cyclic AMP signaling. Using the cyclic guanosine 3',5'monophosphate-specific phosphodiesterase inhibitor zaprinast or the nitric oxide donor S-nitroso-D,L-penicillamine to elevate cyclic guanosine 3',5'monophosphate levels we found that increases in cyclic guanosine 3',5'monophosphate strongly inhibit the induction of long-term potentiation by low-frequency patterns of synaptic stimulation where protein kinase A activation is required for long-term potentiation induction. In contrast, zaprinast and S-nitroso-D,L-penicillamine had no effect on the induction of long-term potentiation by high-frequency patterns of synaptic stimulation that induce long-term potentiation in a protein kinase A-independent manner. Directly activating protein kinase A with the phosphodiesterase-resistant cyclic AMP analog 8-Br-cAMP, blocking all phosphodiesterases with 3-isobutyl-1-methylxanthine, or inhibiting protein phosphatases rescued long-term potentiation induction in zaprinast-treated slices. Together, these results suggest that increases in cyclic guanosine 3',5'monophosphate inhibit long-term potentiation by activating phosphodiesterases that interfere with the protein kinase A-mediated suppression of protein phosphatases needed for long-term potentiation induction. Consistent with the notion that this cyclic guanosine 3',5'monophosphate-mediated inhibitory pathway is recruited by some patterns of synaptic activity, blocking cyclic guanosine 3',5'monophosphate production strongly facilitated the induction of long-term potentiation by long trains of theta-frequency synaptic stimulation. Together, our results indicate that increases in cyclic guanosine 3',5'monophosphate can act as a long-term potentiation suppressor mechanism that selectively constrains the induction of protein kinase A-dependent forms of long-term potentiation induced by low-frequency patterns of synaptic stimulation.

Clozapine does not induce a motor impairment in operant responding for heat reinforcement.

This experiment examined the effects of intraperitoneal (i.p.) clozapine (CLZ) compared to haloperidol (HAL) on operant responding for heat reinforcement in a cold environment (-8 degrees C). Three doses of CLZ (1, 3 and 5 mg/kg) were found to dose-dependently increase responding for heat while lowering core temperature (T(c)) only at the highest dose. Three doses of HAL (0.1, 0.3 and 0.5 mg/kg) dose-dependently decreased operant responding which resulted in a dose-dependent decrease in T(c). The highest dose of CLZ was then tested in two other paradigms: a reinforcement schedule in which heat was available as long as the lever was held down, and a temperature gradient (range 7-45 degrees C) in which access to heat required minimal motor effort. The ad libitum reinforcement schedule still did not provide enough heat to overcome the hypothermic effects of CLZ. However, in the gradient, rats receiving CLZ selected a warmer region of the gradient, and T(c) was higher than that of controls. These data support CLZ's reputation for having minimal motor side effects. Unlike HAL, the hypothermic effects of CLZ appear to be unrelated to effects of the drug on movement.

The influence of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells.

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits.

Thermal preference behavior following clonidine, norepinephrine, isoproterenol, and ephedrine.

A thermal gradient (temperature range 7-45 degrees C) was used to assess ambient temperature (Ta) preferences of rats following treatment with clonidine (25 microg/kg), norepinephrine (NE, 250 microg/kg), isoproterenol (ISO, 50 microg/kg), and ephedrine (EPH, 10 mg/kg). Clonidine produced a preference for a temperature (31.5 degrees C) slightly warmer than that preferred after saline (28.3 degrees C), but this resulted in no significant change in posttest colonic temperature (Tc). NE, ISO and EPH produced a preference for a colder region of the gradient (20-22 degrees C) compared to saline (24.5-28.9 degrees C). Posttest Tc was reduced significantly from 37.7-37.9 degrees C after saline to 37.2 degrees C (NE), 37.3 degrees C (ISO), and 36.8 degrees C (EPH). Thus, given the opportunity to select an environmental temperature, the animals selected a Ta that resulted in significantly lower body temperatures after NE, ISO, and EPH. This suggests that paradoxical thermoregulatory effects of these thermogenic adrenergic agonists are due, at least in part, to a preference for a lower body temperature.

A nitric oxide-independent and beta-adrenergic receptor-sensitive form of metaplasticity limits theta-frequency stimulation-induced LTP in the hippocampal CA1 region.

The induction of long-term potentiation (LTP) and long-term depression (LTD) at excitatory synapses in the hippocampus can be strongly modulated by patterns of synaptic stimulation that otherwise have no direct effect on synaptic strength. Likewise, patterns of synaptic stimulation that induce LTP or LTD not only modify synaptic strength but can also induce lasting changes that regulate how synapses will respond to subsequent trains of stimulation. Collectively known as metaplasticity, these activity-dependent processes that regulate LTP and LTD induction allow the recent history of synaptic activity to influence the induction of activity-dependent changes in synaptic strength and may thus have an important role in information storage during memory formation. To explore the cellular and molecular mechanisms underlying metaplasticity, we investigated the role of metaplasticity in the induction of LTP by theta-frequency (5-Hz) synaptic stimulation in the hippocampal CA1 region. Our results show that brief trains of theta-frequency stimulation not only induce LTP but also activate a process that inhibits the induction of additional LTP at potentiated synapses. Unlike other forms of metaplasticity, the inhibition of LTP induction at potentiated synapses does not appear to arise from activity-dependent changes in NMDA receptor function, does not require nitric oxide signaling, and is strongly modulated by beta-adrenergic receptor activation. Together with previous findings, our results indicate that mechanistically distinct forms of metaplasticity regulate LTP induction and suggest that one way modulatory transmitters may act to regulate synaptic plasticity is by modulating metaplasticity.

Effects of alpha 1-adrenoceptor and Ca2+ channel inhibition on norepinephrine-induced thermoregulatory behavior in the cold.

This experiment examined whether paradoxical temperature-dependent effects of norepinephrine (NE) can be blocked by the alpha 1-adrenoceptor antagonist WB 4101 (WB) and the Ca(2+)-channel blocker nifedipine. An operant lever-pressing task was used to measure the demand for heat in a cold environment. As noted previously, NE alone (250 micrograms/kg) produced a substantial and significant increase in the demand for heat, and yet post-test colonic temperature (Tc) fell. When tested alone, WB and nifedipine also increased the demand for heat, but this was sufficient to maintain Tc. When combined with NE, WB and nifedipine reduced the demand for heat and the fall in Tc such that there were no differences between the effects of the blockers given alone or with NE. These results indicate that paradoxical thermoregulatory effects of NE in the cold can be antagonized effectively by either an alpha 1-adrenoceptor antagonist or a Ca(2+)-channel blocker.

Temperature-dependent effects of ephedrine in the cold.

This experiment examined the effects of intraperitoneal ephedrine (5-25 mg/kg) on operant thermoregulatory behavior of rats in the cold (-8 degrees C), and of 10 mg/kg on metabolic rate at a thermoneutral (22 degrees C) ambient temperature (Ta) and in the cold (5 degrees C). Posttest colonic temperature (Tc) decreased dose-dependently in the behavioral tests, yet the demand for heat varied little with respect to saline except for a significant reduction at the lowest dose tested (i.e., there was no compensation for the reduced Tc induced by ephedrine). Ephedrine had a potent thermogenic effect at a Ta of 22 degrees C, increasing both metabolic rate and Tc. In the cold, ephedrine reduced Tc, but this effect could not be accounted for by a reduction in metabolism, which was not significantly different from saline.

Effects of epinephrine on thermoregulatory behavior in lean and obese Zucker rats in the cold.

This series of experiments examined whether epinephrine (EPI) produces the same thermoregulatory effects in the cold that have been reported for norepinephrine and isoproterenol. Lean and obese Zucker rats were trained to press a lever to activate infrared heat lamps in a cold (-8 degrees C) environment. Operant thermoregulatory behavior increased dose-dependently following EPI (0-100 micrograms/kg), but posttest colonic temperature (Tc) fell. Thermal balance calculations showed a substantial increase in net heat loss, more so in obese than lean animals. EPI is therefore thermolytic--i.e., disrupts thermal balance. A low dose (100 micrograms/kg) of the alpha-antagonist phentolamine produced a marked improvement in operant behavior, Tc, and thermal balance, whereas a comparable dose of the beta-antagonist propranolol had no beneficial effect. Increasing the dose of phentolamine worsened the responses with respect to the 100-micrograms/kg dose. The selective alpha 1-antagonist prazosin ameliorated the decrease in Tc induced by EPI but had little effect on operant behavior or thermal balance; the selective alpha 2-antagonist yohimbine had no effect on any parameter compared to EPI alone. These results suggest that the paradoxical effects of EPI in the cold are mediated by alpha-adrenoceptors, but definitive identification of the subclass of receptor involved cannot be determined.

Temperature-dependent effects of alpha-adrenergic agonists and antagonists in the cold.

This series of experiments examined whether temperature-dependent effects of the alpha-antagonists prazosin and yohimbine compromised their use as blockers of alpha-adrenergic agonist responses in cold-exposed rats. An operant leverpressing task was used to measure the demand for heat in a cold environment. The alpha 1-antagonist prazosin had modest effects, but the alpha 2-antagonist yohimbine was thermolytic in that it dose dependently increased operant responding but decreased posttest colonic temperature (Tc). These potent effects of the alpha 2-antagonist led to tests of the alpha 2-agonist clonidine. Clonidine increased operant responding for heat to an extraordinary degree, resulting in significant increases in posttest Tc. However, clonidine was found to be a hypothermic agent when tested in rats at 5 degrees C but denied the opportunity to increase body temperature by operant lever pressing, suggesting a central effect on the control of thermal balance. Measurement of changes in metabolic rate at 5 and 23 degrees C showed that yohimbine increased metabolism at 23 degrees C but decreased it in the cold. Prazosin had little effect on metabolism or Tc at either temperature. Prazosin inhibited the decrease in Tc induced by norepinephrine (NE), but had little effect on the lever-pressing response. Yohimbine had no significant antagonistic effect on NE-induced changes in lever-pressing behavior or posttest Tc, but neither did the thermolytic effects of yohimbine exacerbate those of NE. These results show that alpha-antagonist interactions with agonists can be complicated by temperature-dependent effects of each.

Effects of beta-adrenoceptor agonists and antagonists on thermoregulation in the cold in lean and obese Zucker rats.

This experiment examines whether the thermoregulatory ability of obese Zucker rats is comparable to that of lean rats following treatment with beta-adrenoceptor agonists and antagonists in a cold (-8 degrees C) environment. Half-maximal doses of the nonselective beta-adrenoceptor agonist isoproterenol (ISO) produced net thermolytic (heat loss) effects in both obese and lean rats in an operant lever pressing for radiant heat task. ISO increased the demand for heat, but posttest colonic temperature (Tc) decreased. A low dose of propranolol (100 micrograms/kg) normalized thermoregulatory behavior, Tc, and thermal balance when coadministered with ISO. Activation of thermogenesis with the selective beta 3-agonist BRL 35135 (BRL) reduced heat influx by both obese and lean rats at doses between 2 and 10 micrograms/kg, but no dose-response effects were evident within this range. Posttest Tc and thermal balance indicated no thermolytic effects. No evidence was found for a beta 2-component in the BRL response when a supramaximal dose (40 micrograms/kg) was tested with the selective beta 2-antagonist ICI 118551 (1 mg/kg). These data show that, despite a higher baseline demand for heat, the obese Zucker rat responds to the thermogenic effects of BRL and the thermolytic effects of ISO as does the lean rat.

Thermoregulatory responses to beta-adrenergic agonists at low ambient temperatures in the rat.

Dose-response effects on heat production (HP) and dry heat loss (DHL) following injection with the non-selective (beta 1/beta 2) adrenergic agonist isoprenaline (ISO) and the atypical B3 agonist BRL 35135 (BRL) were established at an ambient temperature of 25 degrees C in rats. Subsequently, the effects of HP and DHL of a maximal thermogenic dose of ISO (75 micrograms/kg) and a supramaximal dose of BRL (40 micrograms/kg) were tested at ambient temperatures of 5, 10 and 15 degrees C. In terms of heat production, BRL was no different from saline at 5 degrees C, but its thermogenic activity became increasingly evident as ambient temperature increased. For ISO, HP was lower than, or no different from, saline at 5 and 10 degrees C, respectively, but DHL exceeded HP at both temperatures, and colonic temperature fell significantly; ISO and BRL responses were similar at 15 degrees C. ISO was also capable of producing a decrease in HP at 10 degrees C if the rats were shaven. Substitution for endogenous, sympathetically mediated thermogenesis would explain the attenuation of the BRL and ISO effects at cool ambient temperatures, whereas the hypothermic effects of ISO in the cold appeared to be due to an inappropriate increase in DHL, which was exacerbated at 5 degrees C by a reduction in HP below saline values. The increase in DHL was consistent with beta 2-mediated effects of ISO on peripheral blood flow, but the mechanism responsible for the reduction in HP in the cold is unknown, although reduced vascular thermogenesis has been offered as a putative explanation.

Thermoregulatory effects of beta adrenoceptors: effects of selective agonists and the interaction of antagonists with isoproterenol and BRL-35135 in the cold.

Dose-dependent effects of the selective beta 1 adrenergic antagonist atenolol and the beta 2 antagonist erythro-dl-1-(7-methylinden-4-yloxy)-3-isopropylamino-2-ol were tested on the thermoregulatory responses elicited by half-maximal thermogenic doses of the nonselective beta agonist isoproterenol (ISO) in a cold environment. ISO alone increased operant responding for exogenous heat but decreased body temperature and increased net heat loss. These effects of ISO were blocked by erythro-dl-1-(7-methylinden-4-yloxy)3-isopropylamino-2-ol in a dose-dependent manner, whereas atenolol at the highest dose tested (2 mg/kg) exacerbated the effects of ISO. The beta 3 agonist, (R*,R*-(+/-)-methyl 4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propylphenoxyacetate hydrobromide, reduced operant responding for heat and net heat loss in the cold; these effects were sustained in the presence of both beta 1 and beta 2 antagonists. The beta 2 adrenoceptor agonist, fenoterol, produced a dose-dependent increase in operant behavior but colonic temperature fell and thermal balance reflected the characteristic effects of a thermolytic agent. The beta 1 agonist, prenalterol, had no apparent effect on thermoregulatory behavior, colonic temperature or thermal balance but it blocked the thermolytic effects of fenoterol when both agonists were coadministered. Fenoterol had no significant effect on metabolic rate or colonic temperature when tested in a warm ambient temperature of 23 degrees C but it decreased colonic temperature without a significant effect on metabolic rate at an ambient temperature of 5 degrees C, suggesting an effect on heat loss mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ambient temperature on the paradoxical metabolic responses to norepinephrine.

Four experiments were conducted to examine the effect of ambient temperature (Ta) on norepinephrine (NE)-induced metabolism. NE increased oxygen consumption at ambient temperatures above and at acclimation temperature and decreased oxygen consumption at ambient temperatures below acclimation temperature in both nonacclimated (25 degrees C) and cold-acclimated (5 degrees C) rats. Varying the Ta between -5 degrees and 25 degrees C at a fixed dose of NE (250 micrograms/kg, IP) resulted in a temperature-dependent decrease in metabolism in nonacclimated rats as a function of Ta. A similar effect was demonstrated in cold-acclimated rats tested at -15, 5, and 25 degrees C. Varying the dose of NE between 100 and 1,000 micrograms/kg at a Ta of 25 degrees C resulted in a maximal thermogenic response at a dose of 250 micrograms/kg with diminished responsiveness at higher and lower doses. At 5 degrees C, NE inhibited metabolism maximally at a dose of 250 micrograms/kg. Propranolol, a nonspecific beta-antagonist, attenuated the hypometabolic effect of NE in the cold, while the alpha 2-antagonist yohimbine completely blocked this effect. These results suggest that the metabolic suppressive effect of NE may be mediated by the presynaptic alpha 2-receptor and that beta-adrenoceptors may also contribute to this effect.

Potentiation of thermoregulatory responses to isoproterenol by beta-adrenergic antagonists.

The thermoregulatory effects of isothermogenic doses of isoproterenol (Iso) and a novel beta-agonist (BRL 35135) were tested in rats at 22 degrees C and in rats trained to bar press for radiant heat at -8 degrees C. BRL 35135 produced hyperthermia at 22 degrees C and reduced operant responding for heat at -8 degrees C, whereas Iso reduced body temperature and increased operant responding. In both situations, the negative effects of Iso on thermal balance were abolished by propranolol at doses that did not inhibit heat production. In anesthetized rats, propranolol potentiated the Iso-induced rise in brown adipose tissue and colonic temperature. The potentiation was more marked with the beta 2-selective antagonist ICI 118,551, whereas treatment with the beta 1-selective antagonist atenolol resulted in a profound Iso-induced reduction in temperature. The two selective antagonists also produced divergent responses in operant behavior in Iso-treated rats at -8 degrees C. These experiments demonstrate the extent to which responses to a nonselective agonist can be manipulated using appropriately low doses of selective antagonists and indicate that the effects of Iso on thermal balance are due to its beta 2 activity.

Effect of conventional (mixed beta 1/beta 2) and novel (beta 3) adrenergic agonists on thermoregulatory behavior.

The effects of submaximal and maximal thermogenic doses of isoproterenol (ISO) on operant thermoregulatory responses in a cold (-8 degrees C) environment were tested in lean (+/?) Zucker rats trained to barpress for radiant heat. Contrary to expectations, ISO rats pressed for twice as much exogenous heat as controls, but showed a smaller rise in colonic temperature. Conversely, a beta 3-selective adrenergic agonist (RO40-2148) decreased the requirement for exogenous heat and produced larger rises in colonic temperature. RO40-2148 and another beta 3-agonist (ICI D7114) produced similar responses in obese (fa/fa) Zucker rats, but tests with ISO were terminated because it caused profound, and lethal hypothermia. The hypothermic effects of ISO on colonic temperature were also observed in Sprague-Dawley rats at room temperature (22 degrees C), whereas RO40-2148 produced hyperthermia. These results provide behavioral evidence for the high thermogenic selectivity of these novel adrenergic agonists and support the existence of an atypical beta 3-adrenoceptor. The hypothermic effects of ISO are presumed to be due to actions on beta 1- and/or beta 2-adrenoceptors.

Paradoxical effects of exogenous norepinephrine on cold-induced thermogenesis in the rat.

The effect of norepinephrine (NE, 250 microgram/kg IP) on thermoregulatory behavior of rats in a cold environment was tested. Norepinephrine produced an increase in operant responding for heat reward but a decrease in core temperature. Since animals compensate behaviorally for alterations in autonomic thermoregulation, this suggested that NE might inhibit cold-induced thermogenesis, an effect contrary to the expected action of this agent. A second experiment showed that NE increased oxygen consumption when rats were tested at 25 degrees C as expected, but decreased oxygen consumption when tested at 5 degrees C. The beta-adrenoceptor antagonist propranolol decreased oxygen consumption both at 25 and at 5 degrees C as expected. These results suggest that the thermogenic effect of NE is highly dependent on ambient temperature.

Respiratory gaseous exchange in the Zucker rat.

Measurements were made of the oxygen consumption and carbon dioxide production of lean and obese Zucker rats ranging from 4 to 400 days of age. As expected, O2 consumption per animal increased with age and was higher in obese than in lean rats. However, O2 consumption was lower in obese animals when expressed per unit of metabolic body mass. Age per se did not influence the respiratory quotient (R), but obese animals had consistently higher Rs than lean animals. These results confirm and extend previous findings described in the literature.

Food restriction normalizes somatic growth and diabetes in adrenalectomized ob/ob mice.

Carcass composition was determined in intact and adrenalectomized (ADX) ob/ob mice after 6 wk of food restriction from weaning (21 days). Diets (2.6 or 3.2 g/day) led to reduced weight gain in intact ob/ob mice, but fat deposition was still greater, and lean growth was less than isocalorically fed lean mice. When diet was combined with ADX, obese mice fed 3.2 g/day had rates of fat-free body growth equal to ad libitum-fed lean mice but still gained more weight and deposited more fat. With 2.6 g/day, however, body weight gain, fat deposition, fat-free body growth, and protein deposition were all similar to intact lean mice receiving the same diet. Neither diet nor diet combined with ADX improved the defective thermoregulation of obese mice tested at 23 degrees C, 8 degrees C, or in response to food deprivation or food ingestion. Together, diet and adrenalectomy reduced blood glucose and insulin levels in ob/ob mice, and with the 2.6 g/day diet, the values of each were the same as littermate lean controls. The present results indicate that adrenal glucocorticoids are necessary for maintaining elevated fat deposition at the expense of lean body growth in dieted ob/ob mice, whereas hyperphagia, defective thermoregulation, energy efficiency, and diabetes are still expressed after surgery.

Thermoregulation during pentobarbital and ketamine anesthesia in rats.

1.) Core temperature, tail temperature, metabolic heat production, and evaporative heat loss were measured in rats exposed to various ambient temperature conditions. 2.) Control rats increased heat production in the cold and heat loss in a warm environment, thus maintaining a relatively constant core temperature. 3.) Pentobarbital anesthesia reduced the thermoregulatory responses and caused core temperature to vary considerably with ambient temperature. Ketamine anesthesia resulted in minor thermoregulatory deficits. 4.) It is concluded that ketamine can be used in thermal physiological studies that require an anesthetised preparation, although it is not completely devoid of inhibitory effects on thermoregulatory responses.

A reevaluation of the role of the lateral hypothalamus in behavioral temperature regulation.

Rats exposed to the cold (-9 degrees C) were trained to depress a lever to obtain pulses of infrared heat. Half of the animals received bilateral electrolytic lesions of the lateral hypothalamus and half were sham operated. Lesioned and control animals performed similarly postoperatively and responded appropriately to changes in reward duration and intensity. Thus, lateral hypothalamic lesions do not impair behavioral thermoregulation. Despite normal behavioral heat intake, body temperature of the lesioned rats fell significantly during the sessions, which can be attributed to an impairment in metabolic heat production due to the lesions.