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Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by a reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development–during the integration of post-embryonic born GABAergic neurons into the circuit–produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, likely through distinct mechanisms in the developing and mature nervous system.

The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities—advances vs. delays—engage the molecular clockwork remains incompletely understood. We investigated molecular substrates of photic entrainment of the clock in the SCN by stably entraining hamsters to T cycles (non–24-h light/dark cycles) consisting of a single 1-h light pulse repeated as either a short (23.33-h) or a long (24.67-h) cycle; under these conditions, the light pulse of the short cycle acts as “dawn,” whereas that of the long cycle acts as “dusk.” Analyses of the expression of the photoinducible and rhythmic clock genes Period 1 and 2 ( Per1 and Per2 ) in the SCN revealed fundamental differences under these two entrainment modes. Light at dawn advanced the clock, advancing the onset of the Per1 mRNA rhythm and acutely increasing mRNA transcription, whereas light at dusk delayed the clock, delaying the offset of the Per2 mRNA rhythm and tonically increasing mRNA stability. The results suggest that the underlying molecular mechanisms of circadian entrainment differ with morning (advancing) or evening (delaying) light exposure, and such differences may reflect how entrainment takes place in nocturnal animals under natural conditions.

An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output., Author Summary Animal behavior is profoundly affected by contextual information about the internal state of the organism as well as sensory information about the external environment. A class of signaling molecules known as neuropeptides have been implicated in driving transitions between behavioral states (e.g., from food seeking to satiety and back) but we have only a limited understanding of how neuropeptide signaling modulates neural circuit activity and elicits context-dependent behaviors. Here we identify a novel mechanism by which C. elegans modulate their behavior in response to sensory information about food. We show that dopaminergic regulation of NLP-12, a C. elegans homolog of the mammalian neuropeptide cholecystokinin (CCK), shapes behavioral transitions that are central to food searching. Given the conserved nature of these signaling pathways, our work raises the interesting possibility that dopamine modulation of CCK signaling represents a general mechanism by which nervous systems shape context-dependent behavioral changes.

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.

Prokineticin 2 (PK2) is a putative output molecule from the SCN. PK2 RNA levels are rhythmic in the mouse SCN, with high levels during the day, and PK2 administration suppresses nocturnal locomotor activity in rats. The authors examined the PK2 system in a diurnal rodent, Arvicanthis niloticus, to determine whether PK2 or PK2 receptors differ between diurnal and nocturnal species. The major transcript variant of A. niloticus PK2 ( AnPK2) encodes a 26-residue signal peptide followed by the presumed mature peptide of 81 residues. Within the grass rat signal sequence, polymorphic sequences and amino acid substitutions were observed relative to mouse and laboratory rats, but the hydrophobic core and cleavage site of the signal sequence were preserved. The mature PK2 peptide is identical among A. niloticus, rat, and mouse. AnPK2 mRNA is rhythmically expressed in the SCN, with peak RNAlevels occurring in the morning, preceding peaks of Per1 and Per2 as in mouse SCN. Analysis of prokineticin receptor 2 (PKR2) sequences revealed polymorphisms among the grass rats studied. PKR2 mRNAwas expressed in the SCN and paraventricular nuclei of the thalamus and hypothalamus. While further analysis is necessary, there is no clear evidence indicating that a difference in the PK2 ligand/receptor system accounts for diurnality in this rodent species. These data contribute to a growing body of evidence suggesting that the key to diurnality lies downstream of the SCN in A. niloticus.

SummaryThe circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.