Mino D. C. Belle

Daily Electrical Silencing in the Mammalian Circadian Clock

Quiet Clock Many physiological processes have circadian rhythms driven by a biological clock in the suprachiasmatic nuclei (SCN) of the brain. Within the SCN, some neurons express the molecular components of the clock and others do not. Exactly how the clock mechanism is coupled to neuronal activity is not precisely understood. Investigation of the electrophysiological properties of SCN neurons by Belle et al. (p. 281) found that, contrary to the conventionally expected rapid firing rate of the cells during the day, clock-containing cells tended not to fire, despite being in an electrically excited state. Modeling and experimental characterization of changes in channel activity revealed unexpected electrophysiological properties of the SCN cells requiring a reassessment of how the circadian clock regulates activity of SCN neurons. Clock-containing neurons in the mouse brain display complex electrophysiology not seen in other brain cells. Neurons in the brain’s suprachiasmatic nuclei (SCNs), which control the timing of daily rhythms, are thought to encode time of day by changing their firing frequency, with high rates during the day and lower rates at night. Some SCN neurons express a key clock gene, period 1 (per1). We found that during the day, neurons containing per1 sustain an electrically excited state and do not fire, whereas non-per1 neurons show the previously reported daily variation in firing activity. Using a combined experimental and theoretical approach, we explain how ionic currents lead to the unusual electrophysiological behaviors of per1 cells, which unlike other mammalian brain cells can survive and function at depolarized states.

Morphology of inhibitory and excitatory interneurons in superficial laminae of the rat dorsal horn

If we are to stand any chance of understanding the circuitry of the superficial dorsal horn, it is imperative that we can identify which classes of interneuron are excitatory and which are inhibitory. Our aim was to test the hypothesis that there is a correlation between the morphology of an interneuron and its postsynaptic action. We used in vitro slice preparations of the rat spinal cord to characterize and label interneurons in laminae I–III with Neurobiotin. Labelled cells (n= 19) were reconstructed in 3D with Neurolucida and classified according to the scheme proposed by Grudt & Perl (2002) . We determined if cells were inhibitory or excitatory by reacting their axon terminals with antibodies to reveal glutamate decrboxylase (for GABAergic cells) or the vesicular glutamate transporter 2 (for glutamatergic cells). All five islet cells retrieved were inhibitory. Of the six vertical (stalked) cells analysed, four were excitatory and, surprisingly, two were inhibitory. It was noted that these inhibitory cells had axonal projections confined to lamina II whereas excitatory vertical cells projected to lamina I and II. Of the remaining neurons, three were radial cells (2 inhibitory, 1 excitatory), two were antennae cells (1 inhibitory, 1 excitatory), one was an inhibitory central cell and the remaining two were unclassifiable excitatory cells. Our hypothesis appears to be correct only for islet cells. Other classes of cells have mixed actions, and in the case of vertical cells, the axonal projection appears to be a more important determinant of postsynaptic action.

Acute Suppressive and Long-Term Phase Modulation Actions of Orexin on the Mammalian Circadian Clock

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brains master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K+ currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPYs inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.

Distinct roles for GABA across multiple timescales in mammalian circadian timekeeping

Significance Each day, over 50 billion synaptic signals, mediated by the neurotransmitter GABA, are sent between neurons in the central circadian pacemaker in the mammalian brain to time and coordinate daily events. Although GABA is the only signaling molecule sent and received by most, if not all of these neurons, its role is not well understood. Past studies have shown paradoxically that GABA can synchronize and desynchronize, as well as excite and inhibit, clock neurons. Through experiments and modeling characterizing the role of GABA in timekeeping, we propose the existence of two types of differentially regulated GABA signaling—fast signaling that regulates neuronal output, and slow signaling that modulates synchrony between neurons—a hypothesis that can explain many previous experimental results. The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by—and GABA-inhibited neurons repelled from—this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function.

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either 3H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (14C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 μg ml−1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min−1 (mg cell protein)−1, n= 6–8 for each value. Basal 3H-deoxyglucose and 14C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min−1 (mg cell protein)−1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in 3H-deoxyglucose and 14C-Me-AIB uptakes. Typically, 3H-deoxyglucose and 14C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min−1 (mg cell protein−1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 μg ml−1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in 3H-deoxy-D-glucose and 14C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 μg ml−1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of 3H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)

Daily variation in the electrophysiological activity of mouse medial habenula neurones

Neurones of the suprachiasmatic nucleus (SCN) contain a molecular clock that drives these cells to exhibit daily rhythms in electrical activity. The molecular clock may also be present in another brain structure, the medial habenula, and here we tested whether medial habenula neurones show daily changes in their electrical activity. Using a brain slice preparation in which the medial habenula is isolated from inputs from the SCN, we made recordings from mouse medial habenula neurones and determined that they exhibit daily variation in their electrical properties. By contrast, in mice lacking functional molecular clocks, medial habenula neurones did not show overt daily change in their electrical activity. These studies indicate for the first time that medial habenula neurones exhibit daily changes in electrical activity that require a functional molecular clock, but do not depend on signals from the SCN.

Causes and Consequences of Hyperexcitation in Central Clock Neurons

Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (∼24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.

Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity

Light input from the retina acts on clock neurones in the suprachiasmatic nuclei (SCN) and the intrinsic daily electrical output of these cell autonomous clocks coordinates circadian rhythms in the brain and body. Cells in the lateral habenula express clock genes and anatomical studies indicate that SCN output and retinal pathways terminate in this structure. Using a brain slice preparation isolating the lateral habenula from retinal and SCN inputs, we found mouse lateral habenula neurones exhibit a daily variation in their electrical properties that is dependent on a functional molecular clock. Prokineticin 2, a putative output signal of the SCN, changed lateral habenula neuronal activity through enhancing inhibitory signalling. In response to retinal illumination in vivo, lateral habenula neurones sluggishly altered their electrical activity. These studies indicate that mouse lateral habenula neurones possess intrinsic timekeeping capabilities and show for the first time that they are responsive to extrinsic SCN and retinal signals.

Electrophysiological Effects of Melatonin on Mouse Per1 and non-Per1 Suprachiasmatic Nuclei Neurones In Vitro

The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein‐coupled receptors (MT1 and MT2). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole‐cell patch‐clamp recordings in brain slices prepared from Per1::d2EGFP‐expressing transgenic mice. In current‐clamp mode, bath applied melatonin, at near‐physiological concentrations (1 nm), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non‐Per1::GFP SCN neurones. Melatonin‐induced effects were blocked by the MT1/MT2 antagonist, luzindole (1 μm) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nm) or gabazine (20 μm). In voltage‐clamp recordings, 1 nm melatonin increased the frequency of GABA‐mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones.

Aromatase inhibition abolishes courtship behaviours in the ring dove (Streptopelia risoria) and reduces androgen and progesterone receptors in the hypothalamus and anterior pituitary gland.

The aim of this study was to determine in the ring dove, the effects of aromatase inhibition on the expression of aggressive courtship and nest-soliciting behaviours in relation to the distribution of cells containing immunoreactive androgen (AR) and progesterone (PR) receptor in the hypothalamus and pituitary gland. Isolated sexually experienced ring doves were transferred in opposite sex pairs to individual breeding cages, and then injected with the aromatase inhibitor, fadrozole (four males and four females), or saline vehicle (four males and four females) for 3 days at 12 hourly intervals. Saline-injected control males displayed aggressive courtship behaviours (bow-cooing and hop-charging) and nest-soliciting throughout the study, and control females displayed nest-soliciting. By day 3, fadrozole treatment resulted in the disappearance of all these behaviours and in a decrease or disappearance of AR and PR in the anterior pituitary gland, and in the nucleus preopticus paraventricularis magnocellularis (PPM), nucleus preopticus medialis (POM), nucleus hypothalami lateralis posterioris (PLH), and ventral, lateral and dorsal nucleus tuberalis in the hypothalamus (VTu, LTu, DTu). In the nucleus preopticus anterior (POA), fadrozole treatment decreased AR in both sexes and decreased PR in females but not in males. Cells containing co-localized nuclear AR and PR were found in all hypothalamic areas examined, and in the anterior pituitary gland. Fadrozole is suggested to reduce the local availability of estrogen required indirectly for the induction of AR, and except in cells containing PR in the male POA, for the direct induction of PR. It is suggested that aggressive courtship behaviour is terminated by “cross talk” between aromatase-independent PR and aromatase-dependent AR co-localized in neurons in the POA. Aromatase-independent PR may increase in the male POA in response to visual cues provided by a partner. Aromatase-dependent PR in the POM, and basal hypothalamus may play a role in the facilitatory effect of progesterone on estrogen-induced nest-orientated behaviours. (Mol Cell Biochem 276: 193–204, 2005)