1. Gap Junctions in the SCN YiDing Yu MCB 186 Circadian Biology

2. Autonomous Oscillators Single-cell autonomous oscillators in the SCN (Welsh 1995) Single-cell autonomous oscillators in the SCN (Welsh 1995) Synchronized firing in vivo Synchronized firing in vivo Heterogeneity within the SCN Heterogeneity within the SCN rhythmicity (phase, period) rhythmicity (phase, period) neurotransmitter neurotransmitter environmental response environmental response rhythms controlled rhythms controlled

3. Multiple Phenotypes Rostral  Caudal (A  E) Moore et al. 2002

4. How are single-cell oscillators synchronized in vivo? Gap Junctions Gap Junctions Neurotransmitters Neurotransmitters GABA GABA Vasoactive intestinal polypeptide (VIP) Vasoactive intestinal polypeptide (VIP) Gastrin-releasing peptide (GRP) Gastrin-releasing peptide (GRP) Prokineticin 2 (PK2) Prokineticin 2 (PK2) Maintain Rhythmicity? Maintain Rhythmicity? Aton et al. 2005

5. Neuronal Gap Junctions Spike-for-spike synchronization Spike-for-spike synchronization Connexins expressed in the SCN Connexins expressed in the SCN Connexin-36 found in murine and human neuronal gap junctions Connexin-36 found in murine and human neuronal gap junctions Long et al. 2005 Long et al. 2005 Electrical coupling between 25% of neighboring cells Electrical coupling between 25% of neighboring cells Electrical coupling lost in connexin-36 (Cx36) KO mice Electrical coupling lost in connexin-36 (Cx36) KO mice Soehl et al. 2005

6. Hypothesis A. Gap junctions are not necessary to maintain rhythmicity of individual SCN cells B. Gap junctions are integral to maintaining synchrony between coupled cells Are gap junctions necessary for maintaining rhythmicity among SCN cells? To what extent do gap junctions maintain synchrony between coupled cells?

7. Protocol (in vitro) Experiment 1: Rhythmicity in WT cells Electrode recording in various regions Electrode recording in various regions Dense vs. sparse neuronal population Dense vs. sparse neuronal population Dorsal shell vs. ventral core Dorsal shell vs. ventral core Neurotransmitter output (AVP, VIP, GRP) Neurotransmitter output (AVP, VIP, GRP) Experiment 2: Synchronization in WT cells Electrode recordings to find coupled SCN neurons Electrode recordings to find coupled SCN neurons Apply transmitter receptor antagonists to eliminate fast chemical synaptic transmission Apply transmitter receptor antagonists to eliminate fast chemical synaptic transmission APPLY TREATMENT: Quinine (Cx36 specific) TREATMENT WASHOUT  Restore WT phenotype Positive control: Cx36 KO mice Negative control: Untreated WT mice

8. Predicted Results Experiment 1: Rhythmicity Experiment 1: Rhythmicity No effect on individual cell rhythmicity upon treatment or washout, regardless of SCN region No effect on individual cell rhythmicity upon treatment or washout, regardless of SCN region Experiment 2: Synchronization Experiment 2: Synchronization Gap junction blocker treatment: Loss of synchrony between coupled cells; similar to Cx36 KO Gap junction blocker treatment: Loss of synchrony between coupled cells; similar to Cx36 KO Washout: Restore WT synchrony Washout: Restore WT synchrony Long et al. 2005

9. Implications & Further Studies All cells remain rhythmic in experiment 1: Gap junctions do not function to maintain rhythmicity of oscillator cells All cells remain rhythmic in experiment 1: Gap junctions do not function to maintain rhythmicity of oscillator cells If some cells become arrhythmic: Gap junctions may play a role in the regulation of some SCN cells by other autonomous cell oscillators  Is this prevalent in a particular region of the SCN? If some cells become arrhythmic: Gap junctions may play a role in the regulation of some SCN cells by other autonomous cell oscillators  Is this prevalent in a particular region of the SCN?

10. Implications & Further Studies Coupled WT cells lose synchrony upon treatment (experiment 2): Cx36 gap junctions are integral in maintaining coupling  Does coupling occur predominately in certain areas of the SCN? Coupled WT cells lose synchrony upon treatment (experiment 2): Cx36 gap junctions are integral in maintaining coupling  Does coupling occur predominately in certain areas of the SCN? If cells remain synchronized: Cx36 gap junctions are not necessary in maintaining coupling; Cx36 KO phenotype confounded If cells remain synchronized: Cx36 gap junctions are not necessary in maintaining coupling; Cx36 KO phenotype confounded Possible other connexins involved? (Octanol, halothane) Possible other connexins involved? (Octanol, halothane) Effects of gap junctions between neurons and glia? Glia and glia? Effects of gap junctions between neurons and glia? Glia and glia?