Mapping of endogenous morphine-like compounds in the adult mouse brain: Evidence of their localization in astrocytes and GABAergic cells.

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions.

Glucocorticoid-mediated nycthemeral and photoperiodic regulation of tph2 expression.

In the Syrian hamster dorsal and median raphé nuclei, the tryptophan hydroxylase 2 gene (tph2), which codes the rate-limiting enzyme of serotonin synthesis, displays daily variations in its expression in animals entrained to a long but not to a short photoperiod. The present study aimed to assess the role of glucocorticoids in the nycthemeral and photoperiodic regulation of daily tph2 expression. In hamsters held in long photoperiod from birth, after adrenalectomy and glucocorticoid implants the suppression of glucocorticoid rhythms induced an abolition of the daily variations in tph2-mRNA concentrations, a decrease in the amplitude of body temperature rhythms and an increase in testosterone levels. All these effects were reversed after experimental restoration of a clear daily rhythm in the plasma glucocorticoid concentrations. We conclude that the photoperiod-dependent rhythm of glucocorticoids is the main regulator of tph2 daily expression.

Plasma corticosterone in rats is specifically increased at recovery from propofol anesthesia without concomitant rise of plasma ACTH.

General anesthesia combined with surgery is commonly associated with post-operative stress-response in humans. Effects on the hypothalamic-pituitary-adrenal axis (HPA) during and after anesthesia are correlated with the magnitude of surgery and choice of anesthetics. The aim of our study in rats was to characterize the effects of general anesthesia without any surgery on HPA regulation of corticosterone and adrenocorticotropic hormone (ACTH) secretions. First, to assess whether the acute effects of general anesthesia on corticosterone concentration depend on time of day, rats were anesthetized with propofol at three different Zeitgeber times (ZT6, ZT10, and ZT16; with lights-on and -off at ZT0 and ZT12, respectively). Second, to determine the prolonged effects of general propofol anesthesia on daily corticosterone and ACTH concentrations, rats were anesthetized at ZT16 (4 h after lights-off) and euthanized either 1, 4, 12, 16, 20, or 24 h later. Third, the effects of propofol anesthesia on corticosterone and ACTH secretion were studied in rats instrumented with intracarotid cannulation. This permitted us to examine the individual patterns of corticosterone responses to propofol anesthesia as compared to their respective baseline corticosterone secretion. All of the results obtained showed that general propofol anesthesia, independent of the time-of-day of its administration, induces a significant increase of corticosterone secretion during the early recovery period without effect on ACTH secretion (i.e., no pituitary mediated stress-response).

Daily rhythm of tryptophan hydroxylase-2 messenger ribonucleic acid within raphe neurons is induced by corticoid daily surge and modulated by enhanced locomotor activity.

Tryptophan hydroxylase (TPH, the rate-limiting enzyme of serotonin synthesis) protein and mRNA levels display a circadian expression in the rat dorsal and median raphe. These patterns suggest a rhythmic synthesis of serotonin under the control of the master clock of suprachiasmatic nuclei. In the present study, we examined the involvement of endocrine and behavioral output signals of the master clock upon the Tph2 mRNA levels by quantitative in situ hybridization. In the absence of adrenals, a complete suppression of Tph2 mRNA rhythm was observed in dorsal and median raphe over 24 h. The restoration of corticosterone daily variations in adrenalectomized rats induced a Tph2 mRNA rhythmic pattern de novo, indicating that Tph2 mRNA rhythm is dependent upon daily fluctuations of glucocorticoids. Enhanced voluntary locomotor activity during 6 wk increased the level of Tph2 mRNA in both raphe nuclei of control rats without concomitant increase of corticosterone plasma levels. Moreover, this long-term enhanced locomotor activity was able to restore significant variation of Tph2 mRNA in adrenalectomized rats. In conclusion, both endocrine and behavioral cues can modulate Tph2 expression in dorsal and median raphe. The corticosterone surge acts as a rhythmic cue that induces the rhythmic expression of Tph2 in the raphe neurons. On the other hand, long-term exercise modulates the expression levels of this gene. Thus, the serotonin neurons are a target for both endocrine and behavioral circadian cues, and the serotoninergic input to the suprachiasmatic nuclei might feedback and influence the functioning of the clock itself.

Influence of the corticosterone rhythm on photic entrainment of locomotor activity in rats.

The question of involvement of glucocorticoid hormones as temporal signals for the synchronization of the timekeeping system was addressed in rats with different corticosterone status. The authors showed that adrenalectomy had no effects on the synchronization of wheel-running activity rhythms to a steady-state LD 12:12 cycle, regardless of whether it was compensated for by a corticosterone replacement therapy that either reinstated constant plasma concentrations of the hormone or mimicked its natural rhythm. However, after a 12-h phase shift (daylight reversal), the lack of circulating corticosterone induced a significant shortening of the resynchronization rate (less than 3 days vs. 7 days). Normalization required restoration of a rhythmic corticosterone secretion that was synchronized to the new photoperiod. Under constant darkness, the corticosterone rhythm did not show any synchronizing effect, providing evidence that it participates in entrainment of the locomotor activity rhythm through modulation of light effects. It is proposed that, under stable lighting conditions, circulating glucocorticoids contribute to stabilizing activity rhythms by reinforcing resistance of the circadian timing system to variations of the photoperiod. Experimental evidence that serotonergic neurons are involved in relaying their modulatory effects to the clock is also presented.

Selective expression of a persistent tetrodotoxin-resistant Na+ current and NaV1.9 subunit in myenteric sensory neurons.

Voltage-gated Na(+) currents play critical roles in shaping electrogenesis in neurons. Here, we have identified a TTX-resistant Na(+) current (TTX-R I(Na)) in duodenum myenteric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na(+) channel alpha subunits and the biophysical and pharmacological properties of TTX-R I(Na). Whole-cell patch-clamp recording from in situ neurons revealed the presence of a voltage-gated Na(+) current that was highly resistant to TTX (IC(50), approximately 200 microm) and selectively distributed in myenteric sensory neurons but not in interneurons and motor neurons. TTX-R I(Na) activated slowly in response to depolarization and exhibited a threshold for activation at -50 mV. V(1/2) values of activation and steady-state inactivation were -32 and -31 mV in the absence of fluoride, respectively, which, as predicted from the window current, generated persistent currents. TTX-R I(Na) also had prominent ultraslow inactivation, which turns off 50% of the conductance at rest (-60 mV). Substituting CsF for CsCl in the intracellular solution shifted the voltage-dependent parameters of TTX-R I(Na) leftward by approximately 20 mV. Under these conditions, TTX-R I(Na) had voltage-dependent properties similar to those reported previously for NaN/Na(V)1.9 in dorsal root ganglion neurons. Consistent with this, reverse transcription-PCR, single-cell profiling, and immunostaining experiments indicated that Na(V)1.9 transcripts and subunits, but not Na(V)1.8, were expressed in the enteric nervous system and restricted to myenteric sensory neurons. TTX-R I(Na) may play an important role in regulating subthreshold electrogenesis and boosting synaptic stimuli, thereby conferring distinct integrative properties to myenteric sensory neurons.

Corticosterone-dependent driving influence of the suprachiasmatic nucleus on adrenal sensitivity to ACTH.

We investigated the effects of ablation of the suprachiasmatic nucleus (SCN) on corticosterone (CORT) responses to synthetic ACTH given in either the morning or evening. After dexamethasone treatment, evening ACTH injections in intact rats produced a significantly larger increase in plasma CORT compared with morning ones. In rats with SCN lesions, the ACTH-induced CORT secretion was independent of time of day, providing direct evidence for a driving influence of the SCN on the diurnal rhythm of adrenal sensitivity to ACTH. In the absence of dexamethasone treatment, the SCN-lesioned rats were selected for morning-like (ML) or evening-like (EL) basal levels of CORT. Responses to ACTH were not different in ML rats compared with sham-lesioned morning controls. In contrast, EL rats compared with sham-lesioned evening controls showed an approximately 60% decrease in increment of CORT levels within the first 15 min postinjection. These results indicate that the SCN upregulates ACTH sensitivity of the adrenal cortex during the ascending phase of the daily CORT secretion and point to a critical role of glucocorticoids in determining SCN action.