Dr Allen's Research Interests
Within individual SCN neurons, molecular clocks consisting of gene transcription feedback loops generate circadian rhythms. Circadian rhythms in individual SCN neurons manifest as a circadian pattern of action potential firing with a higher frequency during the day. However, not all SCN neurons rhythmically express clock genes or action potentials. In the mammalian SCN, these rhythmic and non-rhythmic neurons are organized into anatomical compartments that differ in clock gene expression patterns, as well as in efferent and afferent neuronal connections. Our recent work has focused on determining the synaptic signaling mechanisms of phenotypically identified neurons in a defined SCN compartment, the calbindin subnucleus (CBsn) of the hamster, which is known to be important for generating circadian locomotor behavior. We recently demonstrated using electrophysiological and immunohistochemical techniques that a population of calbindin-expressing neurons believed to contribute to behavioral circadian rhythms do not have a circadian pattern of action potential firing (
Jobst and Allen, Eur J Neurosci, 2002). Our results reveal that CBsn neurons represent a functionally distinct neuronal subpopulation in which rhythmic action potential output is not necessary for the generation of behavioral circadian rhythmicity. Based on these data, we proposed that intercellular communication between rhythmic SCN neurons and nonrhythmic SCN neurons is essential to produce a circadian output in the intact animal (Jobst and Allen, Eur J Neurosci, 2002).
To follow-up on his work we performed studies to provide a neuroanatomical framework for synaptic and gap junction communication, as well as neuron-to-neuron or glia-to-neuron signaling, within the CBsn. Using neuronal reconstructions of recorded neurons, we demonstrated that CB-expressing neurons have significantly more dorsally oriented dendritic arbors than neurons that do not express calbindin. Using double-label confocal microscopy, we showed that calbindin-expressing neurons contain GABA and GABAA receptor subunits and make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFa), a substance shown to inhibit locomotion, was identified as being present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFa. Lastly, we demonstrated that CB-expressing neurons are coupled to CB-expressing and neurons that do not express CB by gap junctions. These data provide a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN (Jobst and Allen, Neuroscience, in press).
We are continuing to pursue understanding of the functions of neurons within the CBsn. Specific studies that are planned include 1) determining the circadian phase dependence of responses of CBsn neurons to optic nerve stimulation, 2) characterizing the regulation of action potential firing and synaptic transmission by vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating polypeptide (PACAP), 3) examining the responses of CBsn neurons to Transforming Growth Factor alpha (TGFa), and 4) determining whether gastrin-releasing peptide (GRP) or VIP immunoreactive neurons fire action potentials in a circadian manner.
To maintain proper temporal coupling between the circadian clock and the environment, neural systems have evolved that sense changing conditions and communicate the information to the clock. The daily change in light intensity (day/night) is the most important of these signals (zeitgebers). The biological clock receives light intensity information via the retinohypothalamic tract (RHT), a direct glutamatergic projection from retinal ganglion cells to SCN neurons. The RHT is both necessary and sufficient for entrainment of the SCN oscillator to the light-dark cycle. Activation of other afferent neural pathways can modify this light input signal and the behavioral responses to light. Presynaptic modulation of neurotransmitter release may involve inhibiting voltage-gated Ca2+ channels, inhibiting vesicular exocytosis, or activation of K+ channels. However, direct experimental evidence demonstrating the mechanisms of presynaptic inhibition of glutamate release from RHT terminals is lacking and the subject of current work. We are using electrophysiological techniques to describe the cellular mechanisms presynaptic pathways use to modulate RHT activity and the environmental light signal. Specific lines of research include 1) identifying the Ca2+ channel subtypes that regulate the release of glutamate from presynaptic RHT terminals, 2) determining the mechanisms coupling activation of GABAB, S-HT, and neuropeptide Y receptors with the inhibition of glutamate release from presynaptic RHT terminals. Experiments are being performed to determine whether GABAB, S-HT and neuropeptide receptors regulate presynaptic RHT terminals by inhibition of voltage-gated Ca2+ channels or activation of K+ channels, 3) examining whether orphanin-FQ receptors produce presynaptic inhibition of RHT terminals, and 4) investigating whether there are co-operative interactions between presynaptic neurotransmitter receptors mediating glutamate release from RHT terminals.
A molecular clock consisting of gene transcription feedback loops generates circadian rhythms in physiological and behavioral processes. The signaling pathways that transmit timing information from the molecular clock to cellular effectors are not known. Given the ubiquity of Ca2+ as a signaling molecule, regulation of the intracellular Ca2+ concentration is proposed to be an important component of both input pathways to and output from the circadian clock. Our central hypothesis is that changes in cytoplasmic and nuclear Ca2+ concentration are a critical step in lights regulation of the circadian clock. We used an innovative combination of fluorescent imaging, cell culture, and electrophysiological recording techniques to study the regulation of Ca2+ in SCN neurons during different portions of the circadian day (Ikeda et al., Neuron, 2003). This demonstration was both a technical and scientific achievement. Technically, we developed methods to monitor both electrical activity and intracellular Ca2+ levels over multiple circadian cycles. Scientifically, we demonstrated that a subpopulation of SCN neurons show a circadian oscillation of the intracellular Ca2+ concentration. The circadian cycle of intracellular free Ca2+ could regulate diverse cellular processes in SCN neurons, including membrane potential, neurotransmitter release, and gene expression. In contrast, the concentration of Ca2+ in the nuclear does not show a circadian oscillation. The cytosolic Ca2+ rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode-array, with a mean advance in phase of 4 hours. Tetrodotoxin blocked MUA, but not Ca2+ rhythms, while ryanodine damped both Ca2+ and MUA rhythms. These results demonstrate cytosolic Ca2+ rhythms regulated by the release of Ca2+ from ryanodine-sensitive stores in SCN neurons.
Future areas of research include:
- Identifying the mechanisms and circadian regulation of the increase of the cytoplasmic Ca2+ concentration produced by NMDA and AMPA receptor activation
- Determining the mechanisms and circadian phase dependence of the increase in the intranuclear Ca2+ concentration produced by NMDA receptor activation
- Examing the role that PACAP plays in regulating changes in the nuclear Ca2+ concentration induced by NMDA and AMPA receptor activation
- Investigating whether the peak of the cytoplasmic Ca2+ rhythm precedes the peak of action potential firing frequency rhythm in SCN neurons. Through this research, we expect to isolate, for the first time, the early steps in the light-signaling pathway.
In mammals, light entrainment of the circadian clock requires input from the retina, which communicates with the SCN via retinohypothalamic tract. This neuronal projection constitutes the sole source of retinal input to the circadian system, and it is formed by the axons of a small subset of retinal ganglion cells (RGCs). Although retinal input is essential for photoentrainment, rod and cone photoreceptors are not required. Instead, recent findings suggest that RGCs that project to the SCN may themselves function as circadian photoreceptors. In striking contrast to their visual counterparts, RGCs projecting to the SCN express a novel pigment, melanopsin, and exhibit intrinsic light sensitivity. In order to understand the process of circadian entrainment, we must first understand how RGCs of the RHT generate and shape the retinal input to the circadian system. Our working model is that RGCs projecting to the circadian system are intrinsically sensitive to light due to a signaling cascade triggered by photoexcited melanopsin that activates a non-selective cation channel that depolarizes the plasma membrane. This depolarization, in turn, activates a suite of voltage-gated conductances that confers unique firing properties on these neurons.
In collaboration with Dr. David Robinson (CROET) and Dr. Lane Brown (NSI), we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN-projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons (
Warren et al., Eur J Neurosci, 2003). First, in response to the injection of a sustained depolarizing current, SCN-projecting cells fired in a transient fashion, in contrast to most RGCs, which fired robust trains of action potentials. Second, in response to light, SCN-projecting RGCs exhibited an intensity-dependent transient depolarization in the absence of rod and cone input. In response to varying light intensities, SCN-projecting RGCs exhibited a graded transient inward current that peaked within 5 seconds and decayed to a plateau. The voltage dependence of the light-activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light-activated current displayed both inward and outward rectification and was unaffected by substitution of extracellular Na+ with choline. In both respects, the intrinsic light-activated current observed in SCN-projecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors (Warren et al., Eur J Neurosci, 2003).
Our current work is:
- Characterizing the signaling properties of hetrologously-expressed melanopsin
- Identifying the intracellular signaling pathway that generates the intrinsic light response
- Determining the molecular identity of ion channels giving rise to the light-activated current.