Horacio O. de la Iglesia

Autistic-like behaviour in Scn1a +/− mice and rescue by enhanced GABA-mediated neurotransmission

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel NaV1.1 causes Dravet’s syndrome, a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome are poorly understood. Here we report that mice with Scn1a haploinsufficiency exhibit hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive to Scn1a+/− mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of NaV1.1 channels in forebrain interneurons is sufficient to cause these behavioural and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABAA receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravet’s syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for NaV1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravet’s syndrome.

Circadian regulation of Kiss1 neurons: implications for timing the preovulatory gonadotropin-releasing hormone/luteinizing hormone surge.

The preovulatory GnRH/LH surge depends on the presence of estradiol (E(2)) and is gated by a circadian oscillator in the suprachiasmatic nucleus (SCN) that causes the surge to occur within a specific temporal window. Although the mechanisms by which the clock times the LH surge are unclear, evidence suggests that the SCN is linked to GnRH neurons through a multisynaptic pathway that includes neurons in the anteroventral periventricular nucleus (AVPV). Recently, Kiss1 neurons in the AVPV have been implicated in the surge mechanism, suggesting that they may integrate circadian and E(2) signals to generate the LH surge. We tested whether Kiss1 neurons display circadian patterns of regulation in synchrony with the temporal pattern of LH secretion. Mice housed in 14 h light, 10 h dark were ovariectomized, given E(2) capsules (or nothing), and transferred into constant darkness. Two days later, the mice were killed at various times of day and their LH and Kiss1 levels assessed. In E(2)-treated females, LH levels were low except during late subjective day (indicative of an LH surge). Similarly, AVPV Kiss1 expression and c-fos coexpression in Kiss1 neurons showed circadian patterns that peaked coincident with LH. These temporal changes in Kiss1 neurons occurred under steady-state E(2) and constant environmental conditions, suggesting that Kiss1 neurons are regulated by circadian signals. In the absence of E(2), animals displayed no circadian pattern in LH secretion or Kiss1 expression. Collectively, these findings suggest that the LH surge is controlled by AVPV Kiss1 neurons whose activity is gated by SCN signals in an E(2)-dependent manner.

Hormone complement of the Cancer productus sinus gland and pericardial organ: An anatomical and mass spectrometric investigation

In crustaceans, circulating hormones influence many physiological processes. Two neuroendocrine organs, the sinus gland (SG) and the pericardial organ (PO), are the sources of many of these compounds. As a first step in determining the roles played by hemolymph‐borne agents in the crab Cancer productus, we characterized the hormone complement of its SG and PO. We show via transmission electron microscopy that the nerve terminals making up each site possess dense‐core and/or electron‐lucent vesicles, suggesting diverse complements of bioactive molecules for both structures. By using immunohistochemistry, we show that small molecule transmitters, amines and peptides, are among the hormones present in these tissues, with many differentially distributed between the two sites (e.g., serotonin in the PO but not the SG). With several mass spectrometric (MS) methods, we identified many of the peptides responsible for the immunolabeling and surveyed the SG and PO for peptides for which no antibodies exist. By using MS, we characterized 39 known peptides [e.g., β‐pigment‐dispersing hormone (β‐PDH), crustacean cardioactive peptide, and red pigment‐concentrating hormone] and de novo sequenced 23 novel ones (e.g., a new β‐PDH isoform and the first B‐type allatostatins identified from a non‐insect species). Collectively, our results show that diverse and unique complements of hormones, including many previously unknown peptides, are present in the SG and PO of C. productus. Moreover, our study sets the stage for future biochemical and physiological studies of these molecules and ultimately the elucidation of the role(s) they play in hormonal control in C. productus. J. Comp. Neurol. 493:607–626, 2005.

Identification of a population of sleep-active cerebral cortex neurons

The presence of large-amplitude, slow waves in the EEG is a primary characteristic that distinguishes cerebral activity during sleep from that which occurs during wakefulness. Although sleep-active neurons have been identified in other brain areas, neurons that are specifically activated during slow-wave sleep have not previously been described in the cerebral cortex. We have identified a population of cells in the cortex that is activated during sleep in three mammalian species. These cortical neurons are a subset of GABAergic interneurons that express neuronal NOS (nNOS). Because Fos expression in these sleep-active, nNOS-immunoreactive (nNOS-ir) neurons parallels changes in the intensity of slow-wave activity in the EEG, and these neurons are innvervated by neurotransmitter systems previously implicated in sleep/wake control, cortical nNOS-ir neurons may be part of the neurobiological substrate that underlies homeostatic sleep regulation.

Circadian Timing of REM Sleep Is Coupled to an Oscillator within the Dorsomedial Suprachiasmatic Nucleus

Sleep is consistently concentrated to a specific time of the day. Its timing and consolidation depend on the interplay between a homeostatic and a circadian process of sleep regulation [1-3]. Sleep propensity rises as a homeostatic response to increasing wake time, whereas a circadian clock determines the specific time when sleep will probably occur. This two-process regulation of sleep also determines which specific sleep stage will be manifested, and the circadian process governs tightly the manifestation of rapid eye movement sleep (REMS) [1, 4]. The role of the hypothalamic suprachiasmatic nucleus (SCN) in the circadian gating of sleep and wakefulness has been unequivocally established by lesion studies [5], but its role in the timing of specific sleep stages has remained unknown. Using a forced desynchrony paradigm that induces the stable dissociation of the ventrolateral (vl) and dorsomedial (dm) SCN, and a jetlag paradigm that induces desynchronization between these SCN subregions, we show that the SCN can time the occurrence of specific sleep stages. Specifically, the circadian regulation of REMS is associated with clock gene expression within the dmSCN. We provide the first neurophysiological model for the disruption of sleep architecture that may result from temporal challenges such as rotational-shift work and jetlag.

Circadian desynchronization of core body temperature and sleep stages in the rat

Proper functioning of the human circadian timing system is crucial to physical and mental health. Much of what we know about this system is based on experimental protocols that induce the desynchronization of behavioral and physiological rhythms within individual subjects, but the neural (or extraneural) substrates for such desynchronization are unknown. We have developed an animal model of human internal desynchrony in which rats are exposed to artificially short (22-h) light–dark cycles. Under these conditions, locomotor activity, sleep–wake, and slow-wave sleep (SWS) exhibit two rhythms within individual animals, one entrained to the 22-h light–dark cycle and the other free-running with a period >24 h (τ>24 h). Whereas core body temperature showed two rhythms as well, further analysis indicates this variable oscillates more according to the τ>24 h rhythm than to the 22-h rhythm, and that this oscillation is due to an activity-independent circadian regulation. Paradoxical sleep (PS), on the other hand, shows only one free-running rhythm. Our results show that, similarly to humans, (i) circadian rhythms can be internally dissociated in a controlled and predictable manner in the rat and (ii) the circadian rhythms of sleep–wake and SWS can be desynchronized from the rhythms of PS and core body temperature within individual animals. This model now allows for a deeper understanding of the human timekeeping mechanism, for testing potential therapies for circadian dysrhythmias, and for studying the biology of PS and SWS states in a neurologically intact model.

Chronobiology by moonlight

Most studies in chronobiology focus on solar cycles (daily and annual). Moonlight and the lunar cycle received considerably less attention by chronobiologists. An exception are rhythms in intertidal species. Terrestrial ecologists long ago acknowledged the effects of moonlight on predation success, and consequently on predation risk, foraging behaviour and habitat use, while marine biologists have focused more on the behaviour and mainly on reproduction synchronization with relation to the Moon phase. Lately, several studies in different animal taxa addressed the role of moonlight in determining activity and studied the underlying mechanisms. In this paper, we review the ecological and behavioural evidence showing the effect of moonlight on activity, discuss the adaptive value of these changes, and describe possible mechanisms underlying this effect. We will also refer to other sources of night-time light (‘light pollution’) and highlight open questions that demand further studies.

Moonstruck Primates: Owl Monkeys (Aotus) Need Moonlight for Nocturnal Activity in Their Natural Environment

Primates show activity patterns ranging from nocturnality to diurnality, with a few species showing activity both during day and night. Among anthropoids (monkeys, apes and humans), nocturnality is only present in the Central and South American owl monkey genus Aotus. Unlike other tropical Aotus species, the Azaras owl monkeys (A. azarai) of the subtropics have switched their activity pattern from strict nocturnality to one that also includes regular diurnal activity. Harsher climate, food availability, and the lack of predators or diurnal competitors, have all been proposed as factors favoring evolutionary switches in primate activity patterns. However, the observational nature of most field studies has limited an understanding of the mechanisms responsible for this switch in activity patterns. The goal of our study was to evaluate the hypothesis that masking, namely the stimulatory and/or inhibitory/disinhibitory effects of environmental factors on synchronized circadian locomotor activity, is a key determinant of the unusual activity pattern of Azaras owl monkeys. We use continuous long-term (6–18 months) 5-min-binned activity records obtained with actimeter collars fitted to wild owl monkeys (n = 10 individuals) to show that this different pattern results from strong masking of activity by the inhibiting and enhancing effects of ambient luminance and temperature. Conclusive evidence for the direct masking effect of light is provided by data showing that locomotor activity was almost completely inhibited when moonlight was shadowed during three lunar eclipses. Temperature also negatively masked locomotor activity, and this masking was manifested even under optimal light conditions. Our results highlight the importance of the masking of circadian rhythmicity as a determinant of nocturnality in wild owl monkeys and suggest that the stimulatory effects of dim light in nocturnal primates may have been selected as an adaptive response to moonlight. Furthermore, our data indicate that changes in sensitivity to specific environmental stimuli may have been an essential key for evolutionary switches between diurnal and nocturnal habits in primates.

Na(V)1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms.

NaV1.1 is the primary voltage-gated Na+ channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that NaV1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a+/−), which encodes the pore-forming α-subunit of the NaV1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a+/− mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a+/− mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a+/− mice, and this communication is barely detectable in the homozygous gene KO (Scn1a−/−). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of NaV1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na+ channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of NaV1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light–dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN′s subregional organization to both photic input processing and rhythmic output control.