Increased glutamatergic neurotransmission between the retinohypothalamic tract and the suprachiasmatic nucleus of old mice.

Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation, and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.

(Pro)renin receptor signaling in hypothalamic tyrosine hydroxylase neurons is required for obesity-associated glucose metabolic impairment.

Glucose homeostasis is achieved via complex interactions between the endocrine pancreas and other peripheral tissues and glucoregulatory neurocircuits in the brain that remain incompletely defined. Within the brain, neurons in the hypothalamus appear to play a particularly important role. Consistent with this notion, we report evidence that (pro)renin receptor (PRR) signaling within a subset of tyrosine hydroxylase (TH) neurons located in the hypothalamic paraventricular nucleus (PVNTH neurons) is a physiological determinant of the defended blood glucose level. Specifically, we demonstrate that PRR deletion from PVNTH neurons restores normal glucose homeostasis in mice with diet-induced obesity (DIO). Conversely, chemogenetic inhibition of PVNTH neurons mimics the deleterious effect of DIO on glucose. Combined with our finding that PRR activation inhibits PVNTH neurons, these findings suggest that, in mice, (a) PVNTH neurons play a physiological role in glucose homeostasis, (b) PRR activation impairs glucose homeostasis by inhibiting these neurons, and (c) this mechanism plays a causal role in obesity-associated metabolic impairment.

Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

GABA Neurotransmission of the Suprachiasmatic Nucleus Is Modified During Rat Postnatal Development.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the brain structure that controls circadian rhythms in mammals. The SCN is formed by two neuroanatomical regions: the ventral and dorsal. Gamma-aminobutyric acid (GABA) neurotransmission is important for the regulation of circadian rhythms. Excitatory GABA effects have been described in both SCN regions displaying a circadian variation. Moreover, the GABAergic system transfers photic information from the ventral to the dorsal SCN. However, there is almost no knowledge about GABA neurotransmission during the prenatal or postnatal development of the SCN. Here, we used whole-cell patch-clamp recordings to study spontaneous inhibitory postsynaptic currents (IPSCs) in the two SCN regions, at two zeitgeber times (day or night), and at four postnatal (P) ages: P3-5, P7-9, P12-15, and P20-25. The results herein show that the three analyzed parameters of the IPSCs, frequency, amplitude, and decay time, were significantly affected by the postnatal age: mostly, the IPSC frequency increased with age, principally in the ventral SCN in both day and night recordings; similarly, the amplitude of IPSCs augmented with age, especially at night, whereas the IPSC decay time was reduced (it was faster) with postnatal age, mainly during the day. Our findings first reveal that parameters of GABA neurotransmission are modified by postnatal development, implying that synaptic adjustments are required for an appropriate maturation of the GABAergic system in the SCN.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

Toxin Ct1a, from venom of Centruroides tecomanus, modifies the spontaneous firing frequency of neurons in the suprachiasmatic nucleus.

The peptide, denominated Ct1a, is a ß-toxin of 66 amino acids, isolated from venom of the scorpion, Centruroides tecomanus, collected in Colima, Mexico. This toxin was purified using size exclusion, cationic exchange, and reverse phase chromatography. It is the most abundant toxin, representing 1.7% of the soluble venom. Its molecular mass of 7588.9 Da was determined by mass spectrometry. The amino acid sequence was determined by Edman degradation and confirmed by transcriptomic analysis. Since neurons of the suprachiasmatic nucleus (SCN) maintain a spontaneous firing rate (SFR), we evaluated the physiological effects of toxin Ct1a on these neurons. The SFR exhibited a bimodal concentration-dependent response: 100 nM of Ct1a increased the SFR by 223%, whereas 500 nM and 1000 nM reduced it to 42% and 7%, respectively. Control experiments, consisting of recordings of the SFR during a time similar to that used in Ct1a testing, showed stability throughout the trials. Experiments carried out with denatured Ct1a toxin (500 nM) caused no variation in SFR recordings. Action potentials of SCN neurons, before and after Ct1a (100 nM) showed changes in the time constants of depolarization and repolarization phases, amplitude, and half-time. Finally, recordings of hNav1.6 sodium currents indicated that Ct1a shifts the channel activation to a more negative potential and reduces the amplitude of the peak current. These results all demonstrate that toxin Ct1a affects the SFR of SCN neurons by acting upon sodium channels of sub-type 1.6, implicating them in regulation of the SFR of SCN neurons.

Synergistic antidepressant-like effect of capsaicin and citalopram reduces the side effects of citalopram on anxiety and working memory in rats.

RATIONALE: We have previously shown that in rats, capsaicin (Cap) has antidepressant-like properties when assessed using the forced swimming test (FST) and that a sub-threshold dose of amitriptyline potentiates the effects of Cap. However, synergistic antidepressant-like effects of the joint administration of Cap and the selective serotonin reuptake inhibitor citalopram (Cit) have not been reported. OBJECTIVES: To assess whether combined administration of Cap and Cit has synergistic effects in the FST and to determine whether this combination prevents the side effects of Cit. METHODS: Cap, Cit, and the co-administration of both substances were evaluated in a modified version of the FST (30-cm water depth) conducted in rats, as well as in the open field test (OFT), elevated plus maze (EPM), and Morris water maze (MWM). RESULTS: In line with previous studies, independent administration of Cap and Cit displayed antidepressant-like properties in the FST, while the combined injection had synergistic effects. In the OFT, neither treatment caused significant increments in locomotion. In the EPM, the time spent in the closed arms was lower in groups administered either only Cap or a combination of Cap and Cit than in groups treated with Cit alone. In the MWM, both Cap and the joint treatment (Cap and Cit) improved the working memory of rats in comparison with animals treated only with Cit. CONCLUSION: Combined administration of Cap and Cit produces a synergistic antidepressant-like effect in the FST and reduces the detrimental effects of Cit on anxiety and working memory.

Functional Pre- and Postsynaptic Changes between the Retinohypothalamic Tract and Suprachiasmatic Nucleus during Rat Postnatal Development.

The suprachiasmatic nucleus (SCN) is the main brain clock in mammals. The SCN synchronizes to the light-dark cycle through the retinohypothalamic tract (RHT). RHT axons release glutamate to activate AMPA-kainate and N-methyl-D-aspartate (NMDA) postsynaptic receptors in ventral SCN neurons. Stimulation of SCN NMDA receptors is necessary for the activation of the signaling cascades that govern the advances and delays of phase. To our knowledge, no research has been performed to analyze the functional synaptic modifications occurring during postnatal development that prepare the circadian system for a proper synchronization to light at adult ages. Here, we studied the pre- and postsynaptic developmental changes between the unmyelinated RHT-SCN connections. Spontaneous NMDA excitatory postsynaptic currents (EPSCs) were greater in amplitude and frequency at postnatal day 34 (P34) than at P8. Similarly, both quantal EPSCs (miniature NMDA and evoked quantal AMPA-kainate) showed a development-dependent increase at analyzed stages, P3-5, P7-9, and P13-18. Moreover, the electrically evoked NMDA and AMPA-kainate components were augmented with age, although the increment was larger for the latter, and the membrane resting potential was more depolarized at early postnatal ages. Finally, the short-term synaptic plasticity was significantly modified during postnatal development as was the estimated number of quanta released and the initial release probability. All of these synaptic modifications in the unmyelinated RHT-SCN synapses suggest that synchronization to light at adult ages requires developmental changes similar to those that occur in myelinated fast communication systems.

Development-Dependent Changes in the NR2 Subtype of the N-Methyl-D-Aspartate Receptor in the Suprachiasmatic Nucleus of the Rat.

The suprachiasmatic nucleus (SCN) is the main brain clock that regulates circadian rhythms in mammals. The SCN synchronizes to the LD cycle through the retinohypothalamic tract (RHT), which projects to ventral SCN neurons via glutamatergic synapses. Released glutamate activates N-methyl-D-aspartate (NMDA) receptors, which play a critical role in the activation of signaling cascades to enable phase shifts. Previous evidence indicates that presynaptic changes during postnatal development consist of an increase in RHT fibers impinging on SCN neurons between postnatal day (P) 1 to 4 and P15. The aim of this study was to evaluate postsynaptic developmental changes in the NR2 subunits that determine the pharmacological and biophysical properties of the neuronal NMDA receptors in the ventral SCN. To identify the expression of NR2 subtypes, we utilized RT-PCR, immunohistochemical fluorescence, and electrophysiological recordings of synaptic activity. We identified development-dependent changes in NR2A, C, and D subtypes in mRNA and protein expression, whereas NR2B protein was equally present at all analyzed postnatal ages. The NR2A antagonist PEAQX (100 nM) reduced the frequency of NMDA excitatory postsynaptic currents (EPSCs) at P8 significantly more than at P34, but the antagonists for NR2B (3 µM Ro 25-6981) and NR2C/D (150 nM PPDA) did not influence NMDA EPSCs differently at the 2 analyzed postnatal ages. Our results point to P8 as the earliest analyzed postnatal age that shows mRNA and protein expression similar to those found at the juvenile stage P34. Taken together, our findings indicate that postsynaptic development-dependent modifications in the NR2 subtypes of the NMDA receptor could be important for the synchronization of ventral SCN neurons to the LD cycle at adult stages.

Capsaicin produces antidepressant-like effects in the forced swimming test and enhances the response of a sub-effective dose of amitriptyline in rats.

Transient receptor potential vanilloid 1 (TRPV1) channels have been implicated in depression and anxiety. The aim of this study was to evaluate the antidepressant-like properties of the TRPV1 agonist capsaicin using the forced swimming test (FST) in rats. Capsaicin (0.001-0.25 mg/kg, i.p.) produced a reduction of immobility in the FST. A maximally effective dose of the tricyclic antidepressant amitriptyline (12 mg/kg) reduced immobility as well. Notably, doses of capsaicin (1 pg/kg, 1 ng/kg, and 0.001 mg/kg) that were ineffective when applied alone produced a significant decrease in immobility when combined with a subthreshold dose of amitriptyline (5 mg/kg). Rats treated with capsaicin (0.01 mg/kg) + amitriptyline (5 mg/kg) displayed less immobility than those treated with a maximally effective dose of amitriptyline. The non-pungent TRPV1 channel agonist palvanil (0.05-0.1 mg/kg, i.p.) also decreased immobility in the FST. Capsaicin (0.05 mg/kg) did not affect general locomotion in the open field test, nor performance in the elevated plus maze, or skeletal muscle contraction strength measured in vitro after the FST (at 0.25 mg/kg). Altogether, our results imply that low doses of capsaicin produce antidepressant-like effects, and enhance the effect of a subthreshold dose of amitriptyline in the FST.