Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

KEY POINTS: Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. ABSTRACT: Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24 h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

Purinoceptors on neuroglia.

Purinergic transmission is one of the most ancient and widespread extracellular signalling systems. In the brain, purinergic signalling plays a unique role in integrating neuronal and glial cellular circuits, as virtually every type of glial cell possesses receptors to purines and pyrimidines. These receptors, represented by metabotropic P1 adenosine receptors, metabotropic P2Y purinoceptors and ionotropic P2X purinoceptors, control numerous physiological functions of glial cells and are intimately involved in virtually every form of neuropathology. In this essay, we provide an in depth overview of purinoceptor distribution in two types of CNS glia--in astrocytes and oligodendrocytes--and discuss their physiological and pathophysiological roles.

Peripherally applied neuropeptide SF is equally algogenic in wild type and ASIC3-/- mice.

RFa-related peptides play a significant role in the processing of pain in the CNS of mammals. Recently it has been found that, when applied subcutaneously, these peptides elicit a powerful algogenic effect. The question arises whether this peripheral effect can be connected with the ability of RFa-related peptides to decrease the rate of desensitization of acid sensing ionic channels (ASICs) expressed in primary sensory neurons. We have addressed this question by comparing the effects of neuropeptide SF (NPSF), mammalian RFa peptide, in ASIC3-/- and wild-type C57BL/6J mice. Knockout of ASIC3 gene results in the changes in some of the behavioral parameters. However, subcutaneous injections of the NPSF into the n.saphenous innervation area result in a clearly nociceptive behavior in both strains of mice. There is no significant difference in the total time of licking of injected paw in the ASIC3-/- (194+/-22s) and C57BL/6J (227+/-25s) animals. Thus peripheral algogenic effects of NPSF cannot be explained only in terms of their action on the ASIC3 channels and involves some other, still unidentified mechanism.

The beta subunit increases the ginkgolide B sensitivity of inhibitory glycine receptors.

We investigated the effect of ginkgolide B (GB), a component of the extract from the leaves of the Ginkgo biloba tree, on recombinant glycine receptors (GlyRs) expressed in Xenopus oocytes by using voltage-clamp recording. GB (0.01-10 microM) inhibited glycine-induced currents of homo-oligomeric alpha1, alpha2 and alpha 3 GlyRs, with the highest potency being found at the alpha1 GlyR (IC(50) value=0.61+/-0.1 microM). Coexpression of the alpha subunits with the beta subunit resulted in a shift of the IC(50) value of GB to nanomolar values, indicating selectivity of GB for beta subunit containing GlyRs. We also analyzed the mechanism of GB inhibition and the effect of point mutations introduced into the alpha1 subunit. Our results are consistent with a channel blocking effect, since (i) GB inhibited glycine currents non-competitively, and (ii) a point mutation in the pore forming M2 domain reduced GB potency. In conclusion, GB is a potent blocker of beta subunit containing GlyR channels and hence can be used to discriminate homo- from hetero-oligomeric GlyRs. As hetero-oligomeric GlyRs are known to be synaptically localized, GB represents a channel blocker that may be employed to separate extrasynaptic from synaptic glycine currents.

RFa-related peptides are algogenic: evidence in vitro and in vivo.

RFamide (RFa)-related peptides modulate pain processing in the mammalian CNS. The effects of these peptides are generally considered as 'anti-opioid'. They also decrease the rate of desensitization of acid-sensing ionic channels (ASICs), putative nociceptors in dorsal root ganglia neurons [C. Askwith et al. (2000) Neuron, 26, 133-141]. We have tested the role of mollusc-derived peptide, FMRFa (Phe-Met-Arg-Phe-amide) and its synthetic analogues in peripheral nociception. Here we demonstrate that RFa-related peptides powerfully excite the majority of C-fibres in the skin-nerve preparation of rat: 76% of 55 tested fibres with the conduction velocity below 2 m/s responded with long-lasting discharges to the application of peptides (20 microm). When injected subcutaneously in vivo (mice), they initiate nociceptive behaviour. We confirm the data on humans [S. Ugawa et al. (2002) J. Clin. Invest., 110, 1185-1190]: the activation of C-fibres by acid is inhibited by channel blocker of ASICs, amiloride. However, there is no correlation in the sensitivity of C-fibres to RFa peptides, protons and amiloride: 74% of tested RFa-sensitive C-fibres were insensitive to protons and in 67% of cases the response to peptides was insensitive to amiloride. Thus, powerful excitatory/algogenic action of RFa-related peptides cannot be interpreted solely in terms of their interaction with ASICs. The peptides do not activate any conductance in the somatic membrane of dorsal root ganglion neurons of rats and probably affect still unidentified molecular target(s) responsible for nociceptive signalling.

Ginkgolide B preferentially blocks chloride channels formed by heteromeric glycine receptors in hippocampal pyramidal neurons of rat.

It has been found recently that the platelet activating factor antagonist ginkgolide B is a selective use-dependent blocker of glycine-gated chloride channels. GABAA receptor antagonist picrotoxin is known to block alpha homomeric glycine (Gly) receptors, being less effective for heteromeric alpha1/beta glycine receptors. Studying pyramidal hippocampal neurons of rat, we have confirmed that the effect of picrotoxin depends on the age of the animals. Its blocking ability was characterised by IC50=140+/-12 microM and IC50=354+/-43 microM for 7 and 14 days old rats, respectively, indicating at a possibly increased contribution of heteromeric receptors with animals age. We have revealed that the blocking action of ginkgolide B is subjected to a more drastic change in the same range of ages: the IC50 value is decreased from 1.6+/-0.2 microM for 7 days old rats to 0.27+/-0.01 microM for 14 days old rats. When measured on the background of ginkgolide B (1 microM), IC50 for picrotoxin was 92+/-16 microM. Taken together, these findings indicate that ginkgolide B has higher affinity to heteromeric Gly receptor-gated channels than to the homomeric ones.

Post-synaptic N-methyl-d-aspartate signalling in hippocampal neurons of rat: spillover increases the impact of each spike in a short burst discharge.

High-frequency burst discharges in hippocampus typically consist of less than ten spikes fired at frequencies too high to be followed by a post-synaptic neuron. How significant are these numbers for synaptic signalling? We have measured the N-methyl-d-aspartate (NMDA) component of the excitatory post-synaptic current (EPSC(NMDA)) in hippocampal CA1 neurons of rat after burst discharge of variable duration. The synaptic facilitation is accompanied by a slow-down of the EPSC(NMDA) which develops on a spike-to-spike basis. Consequently the charge transferred by the after-burst EPSC(NMDA) is increased with each spike. The phenomenon is most probably due to the spillover-mediated recruitment of extrasynaptic NMDA receptors. In terms of post-synaptic signalling it dramatically increases the impact of each spike in a short burst discharge.

Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape 'superslow' afterburst EPSC in rat hippocampus.

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA receptor-mediated EPSCs (EPSC(NMDA)). Such a time course has been attributed to the cooperation of synapses through glutamate spillover. Imitating a natural pattern of activity, we have applied short bursts (2-7 stimuli) of high-frequency stimulation and observed a spike-to-spike slow-down of the EPSC(NMDA) kinetics, which accompanied synaptic facilitation. It was found that the early component of the EPSC(NMDA) and the burst-induced late component of the EPSC(NMDA) have distinct pharmacological properties. The competitive NMDA antagonist R-(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP), which has higher affinity to NR2A than to NR2B subunits and lowest affinity at NR2D subunits, significantly slowed down the decay rate of the afterburst EPSC while leaving the kinetics of the control current unaffected. In contrast, ifenprodil, a highly selective NR2B antagonist, and [+/-]-cis-1-[phenanthren-2yl-carbonyl]piperazine-2,3-dicarboxylic acid (PPDA), a competitive antagonist that is moderately selective for NR2D subunits, more strongly inhibited the late component of the afterburst EPSC(NMDA). The receptors formed by NR2B and (especially) NR2D subunits are known to have higher agonist sensitivity and much slower deactivation kinetics than NR2A-containing receptors. Furthermore, NR2B is preferentially and NR2D is exclusively located on extrasynaptic membranes. As the density of active synapses increases, the confluence of released glutamate makes EPSC decay much longer by activating more extrasynaptic NR2B- and NR2D-subunit-containing receptors. Long-term potentiation (LTP) induced by successive rounds of burst stimulation is accompanied by a long-term increase in the contribution of extrasynaptic receptors in the afterburst EPSC(NMDA.)

Inhibition of hippocampal LTP by ginkgolide B is mediated by its blocking action on PAF rather than glycine receptors.

Platelet-activating factor (PAF), a biologically active lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphoholine), is identified in different regions of brain, including hippocampus. Specific PAF-activated receptors (PAFRs) are expressed in corresponding brain areas. PAF has been proposed to be a retrograde messenger of long-term potentiation (LTP): the antagonist of PAFRs, ginkgolide B (or BN52021) prevents induction of LTP. Recently it has been found that ginkgolide B is also an efficient blocker of the glycine receptor (GlyR) operated chloride channels (IC(50)=270+/-10 nM in hippocampal pyramidal neurons). The question is as follows: is the alteration of LTP by BN52021 due to the PAF antagonism or to the inhibition of glycine-gated chloride channels? We have studied the effects of ginkgolides B and J on LTP induced in the CA1 area of rat hippocampus. Ginkgolide J which is the weakest blocker of PAFR (IC(50)=54 microM, as compared to IC(50)=2.5 microM for ginkgolide B) inhibits GlyR-operated channels with IC(50)=2.0 microM. This assures a convenient concentration window which allows to inhibit GlyR-operated channels without affecting PAFRs. An amount of 5 microM of ginkgolide J did not prevent the induction of LTP, while ginkgolide B (5 microM) completely inhibited this phenomenon. The effect of ginkgolide B on LTP did not alter considerably if GlyRs were blocked by strychnine (2 microM). Strychnine itself had no significant effect on the induction of LTP. Both ginkgolides and strychnine significantly facilitated short-term potentiation (STP). Our data support a hypothesis according to which ginkgolides affect LTP by inhibiting PAFRs.

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation.

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.

Role for P2X receptors in long-term potentiation.

ATP receptors participate in synaptic transmission and intracellular calcium signaling in the hippocampus by providing a component of the excitatory input to CA1 pyramidal neurons. The activation of P2X purinoreceptors generates calcium influx that does not require cell depolarization, but this response desensitizes at increased rates of stimulation. Here we show that inhibition of P2X receptors dramatically facilitates the induction of long-term potentiation (LTP). High-frequency stimulation (HFS) (1 sec) induced LTP in CA1, whereas brief HFS (0.2 sec) caused only short-term potentiation. However, when P2X receptors were inhibited by PPADS (pyridoxal phosphate-6-azophenyl-2'-4'-disulphonic acid) or desensitized by the nonhydrolyzable ATP analog alpha,beta-methyleneATP, brief HFS reliably induced LTP. Inhibition of P2X receptors had no facilitatory effect on LTP when NMDA receptors were blocked. We hypothesized that P2X receptors affect the threshold for LTP by altering Ca2+-dependent inactivation of NMDA receptors. In isolated pyramidal CA1 neurons and hippocampal slices, activation of P2X receptors did cause inhibition of NMDA receptor-mediated current. We suggest that, by controlling the background calcium and thus the activity of NMDA receptors at low firing frequencies, P2X receptors act as a dynamic low-frequency filter so that weak stimuli do not induce LTP.

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel.

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential -70mV and in the presence of 200 microM glycine IC50 value for the blocking action of BN52021 was 270+/-10nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl- channels by interacting with the pore region and does not compete for the strychnine-binding centre.