Basal Retinal Neurons Research Articles

Role of glutamate in coupling between bilaterally paired circadian clocks in Bulla gouldiana

A group of electrically coupled basal retinal neurons (BRN) in the eye of the marine snail Bulla gouldiana generate a circadian rhythm in the frequency of compound action potentials (CAPs). CAPs are conducted to the contralateral retina via the optic nerves and the cerebral commissures to synchronize the rhythms of both eyes. CAPs can induce an excitatory postsynaptic potential (EPSP) in the contralateral BRNs that can lead to action potential generation. The pathway and mechanism of this bilateral coupling signal have not been elucidated, but the evidence suggests monosynaptic connections between the populations of pacemaker cells in both retinae. The study was designed to further characterize the coupling signal and investigate the role of glutamate as a neurotransmitter in this pathway. We found evidence supporting our hypothesis that glutamate, previously identified in BRNs by an immunocytological study, is involved in bilateral coupling. First, a combination of extracellular and intracellular electrophysiological recordings revealed that both electrically and optically evoked CAPs generate excitatory synaptic potentials and action potentials in contralateral BRNs. Application of glutamate also led to increased neuronal activity of individual BRNs both in the intact retina as well when isolated in cell culture. Lastly, glutamate-induced inward currents were characterized in cultured BRNs using perforated-patch recordings. The reversal potential was close to 0 mV, and the currents were sensitive to N-methyl-d-aspartic acid (NMDA) and non-NMDA antagonists. NMDA and AMPA, as well as aspartate, also induced distinct inward currents in BRNs. We conclude that glutamate can be used by BRNs as a transmitter to influence electrical activity in the contralateral pacemaker population. We propose that glutamate is required for synchronizing of the bilaterally paired retinal clocks producing a unified circadian timing signal.

Cellular analysis of a molluscan retinal biological clock.

The eye of the opisthobranch mollusc Bulla gouldiana expresses a circadian rhythm in optic nerve impulse frequency. The circadian rhythm is generated among approximately 100 neurons at the base of the retina referred to as basal retinal neurons. These cells are electrically coupled to one another and fire spontaneous action potentials in synchrony. Basal retinal neurons recorded intracellularly exhibit a circadian rhythm in membrane potential that appears to be driven by a circadian modulation of membrane conductance. Membrane conductance is relatively high during the subjective night and decreases after subjective dawn. Recent experiments in our laboratory indicate that individual basal retinal neurons in culture can express circadian rhythms in membrane conductance. When completely isolated, these cells continue to show circadian conductance changes. These studies provide the first direct demonstration that individual neurons can act as circadian pacemakers. Although the precise details of the mechanism generating the circadian periodicity remain obscure, our research indicates that several transmembrane ionic fluxes are not involved in rhythm generation, but that a transmembrane Ca2+ flux is critical for entrainment. Both transcription and translation appear to play critical roles in generating the circadian cycle.

Bulla gouldiana period Exhibits Unique Regulation at the mRNA and Protein Levels

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.

Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels.

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.

FMRFamide modulates potassium currents in circadian pacemaker neurons of Bulla gouldiana

The peptide FMRFamide (Phe-Met-Arg-Phe-NH 2) is known to modulate the circadian pacemaker found in the eye of the marine snail Bulla gouldiana. In the present study, we investigated the cellular mechanisms underlying this modulation by examining the effects of FMRFamide on the membrane properties of the circadian pacemaker cells, known as basal retinal neurons in this preparation. Bath application of FMRFamide (0.1–1 μM) increased the membrane conductance, and hyperpolarized the membrane potential of these neurons. Next, perforated-patch recordings were used to demonstrate that FMRFamide reversibly increased the outward current amplitude due to an augmentation of a non-inactivating calcium-independent current. Reversal potential of the tail currents and its dependence on extracellular potassium concentration suggested potassium ions as the charge carrier for this current. The peptide-modulated outward current was blocked by 54% after bath application of the potassium channel blocker tetraethylammonium chloride and completely blocked by substituting cesium for intracellular potassium. Voltage dependence, activation kinetics and tail current kinetics of the FMRFamide-modulated current were consistent with values found for the delayed rectifier current. Overall, our data suggest that FMRFamide modulates a delayed rectifier potassium current and at least one other, less voltage-dependent conductance. This provides a mechanistic explanation for FMRFamide’s ability to both shift the phase and attenuate light-induced phase shifts of the circadian pacemaker in B. gouldiana.

A delayed rectifier current is modulated by the circadian pacemaker in Bulla.

Basal retinal neurons of the marine mollusc Bulla gouldiana continue to express a circadian modulation of their membrane conductance for at least two cycles in cell culture. Voltage-dependent currents of these pacemaker cells were recorded using the whole-cell perforated patch-clamp technique to characterize outward currents and investigate their putative circadian modulation. Three components of the outward potassium current were identified. A transient outward current (IA) was activated after depolarization from holding potentials greater than -30 mV, inactivated with a time constant of 50 ms, and partially blocked by 4-aminopyridine (1-5 mM). A Ca(2+)-dependent potassium current (IK(Ca)) was activated by depolarization to potentials more positive than -10 mV and was blocked by removing Ca2+ from the bath or by applying the Ca2+ channel blockers Cd2+ (0.1-0.2 mM) and Ni2+ (1-5 mM). A sustained Ca(2+)-independent current component including the delayed rectifier current (IK) was recorded at potentials positive to -20 mV in the absence of extracellular Na+ and Ca2+ and was partially blocked by tetraethylammonium chloride (TEA, 30mM). Whole-cell currents recorded before and after the projected dawn and normalized to the cell capacitance revealed a circadian modulation of the delayed rectifier current (IK). However, the IA and IK(Ca) currents were not affected by the circadian pacemaker.

Opsin-like immunoreactivity in the circadian pacemaker neurons and photoreceptors of the eye of the opisthobranch mollusc Bulla gouldiana.

Circadian pacemaker cells in the eyes of the opisthobranch mollusc Bulla gouldiana generate a near 24-h rhythm in the frequency of optic nerve impulses. Previous electrophysiological studies suggest that these basal retinal neurons are intrinsically photosensitive and transduce light signals that shift the phase of their pacemaker mechanism. To test whether the pacemaker neurons contain opsin-like proteins, several polyclonal antibodies that recognize opsins of vertebrate photoreceptors have been tested on histological sections of the eye and on the neurons in primary cell culture. The antibodies label both the pacemaker cells and the large distal photoreceptors that surround the lens. Immunoblot analyses of the proteins of the eye have identified a single band at 62+/-4 kDa. These opsin antibodies may label the photopigment used in the entrainment of the circadian pacemaker.

Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni 2+. To test directly if an influx of Ca 2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K +. Calcium levels in the BRNs remained elevated during treatments with 50 mM K + lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca 2+ (approx. 1.7 × 10 −7 M) during depolarization blocked the rise in intracellular Ca 2+, verifying that a Ca 2+ influx is required. The sustained Ca 2+ elevation during depolarization was also prevented with 50 mM Ni 2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni 2+, which does not block phase shifts. The initial rise in [Ca 2+] i in response to 50 mM K + was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca 2+ signal.

Circadian rhythm in membrane conductance expressed in isolated neurons.

Although isolated neurons can generate rhythmic activity, they have not yet been shown to generate rhythms with a period in the circadian range (near 24 hours). The eye of the mollusk Bulla gouldiana expresses a circadian rhythm in optic nerve impulses that is generated by electrically coupled cells known as basal retinal neurons (BRNs). Daily fluctuations in the membrane potential of the BRNs appear to be driven by a rhythm in membrane conductance. Isolated BRNs exhibited spontaneous conductance changes similar to those observed in the intact retina. Membrane conductance was high in the late subjective night and decreased approximately twofold near projected dawn during at least two circadian cycles in culture. The persistence of daily conductance changes in isolated BRNs indicates that individual neurons can function as circadian pacemakers.

Unwinding the Snail's Clock: Cellular Analysis of a Retinal Circadian Pacemaker

The eye of the cloudy bubble snail, Bulla gouldiana, expresses a circadian rhythm in spontaneous optic nerve impulse frequency. The circadian rhythm is generated among a group of approximately 100 neurons (basal retinal neurons - BRNs) at the base of the retina. Each BRN appears to be a competent circadian pacemaker. Using the Bulla retina as a model pacemaker system our laboratory has addressed three central issues of circadian physiology: Synchronization: Entrainment of the ocular rhythm to light cycles is mediated by depolarization of pacemaker neurons with a resultant transmembrane calcium flux. Phase shifts of the rhythm are prevented when calcium entry or membrane depolarization is blocked and light-induced phase shifts can be mimicked by depolarization. Depolarization leads to a persistent increase in intraccllular calcium in pacemaker neurons. Expression: The circadian rhythm in impulse frequency is driven by a rhythm in membrane conductance. Membrane conductance is relatively high during the subjective night and decreases by approximately 50 percent near subjective dawn. The 'clock-controlled' conductances are TEA sensitive and recent experiments reveal a circadian modulation in a sustained calcium-inclependent potassium conductance. Rhythm generation: Protein synthesis and transcription appear to play critical roles in rhythm genesis. Alterations in the rates of transcription and translation profoundly affect the velocity of the circadian pacemaker in a phase-dependent manner. Transmembrane ionic fluxes, while not involved in rhythm generation, play critical roles in pacemaker synchronization and rhythm expression.

The retinal cells generating the circadian small spikes in the Bulla optic nerve.

A circadian rhythm in the frequency of compound action potentials (CAPs) in the optic nerve of the mollusc Bulla gouldiana is believed to be generated by the basal retinal neurons (BRNs) of the eye. Along with the CAPs, which are about 100 microV in amplitude, there are 10- to 40-microV impulses from an undetermined cell type in records from the optic nerve. These impulses, called "small spikes," are generated spontaneously in darkness and show a circadian rhythm in frequency that is about 12 hr out of phase with the CAP rhythm. To enable us to determine the origin of the small spikes, intracellular recordings were made from retinal cells while optic nerve activity was monitored. The cells were identified by their light responses and then injected with the fluorescent dye Lucifer Yellow CH or the tracer biocytin. It was found that the large photoreceptors of the distal retina generated graded depolarizations in response to light, and had axons in the optic nerve, but did not show impulses at the level of the photoreceptor layer. By contrast, the spiking retinal cells of the photoreceptor layer generated depolarizations and impulses in response to light. In addition, the spiking cells were found to be dye-coupled to a series of retinal cells approximately 7 microns in diameter, connected to a single axon in the optic nerve. Impulses from the spiking cells occurred spontaneously and correspond with the small spikes in the optic nerve. The BRNs appear to inhibit the retinal cells that generate the small spikes. Hyperpolarization of the BRNs, through constant-current injection, increased the number of small spikes in the optic nerve. Release from hyperpolarization led to a decrease in small spikes. This could explain how circadian changes in BRN membrane potential might modulate spontaneous firing of the spiking cells, resulting in the circadian rhythm in small-spike frequency.

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.

Monoclonal antibodies recognize localized antigens in the eye and central nervous system of the marine snail Bulla gouldiana.

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.

Localization of neuropeptides in efferent terminals of the eye in the marine snail, Bulla gouldiana.

Like several other opisthobranch molluscs, the marine snail Bulla gouldiana possesses two circadian pacemakers, one in each eye. The two ocular pacemakers are mutually coupled such that: the circadian rhythms of spontaneous electrical activity recorded from the optic nerve are normally synchronous and; if experimentally desynchronized the rhythms will return to the synchronized state. This coupling of the pacemakers is mediated by efferent fibers in the optic nerve, terminating in neuropil adjacent to the basal retinal neurons (BRNs), the putative circadian pacemaker cells. Attempts to identify neurotransmitters in efferent terminals that may be involved in the coupling process have failed. In the present study we demonstrate axons in the optic nerve and axon terminals adjacent to the BRNs that exhibit FMRF-amide- (molluscan cardioexcitatory peptide) and NPY-like (neuropeptide-Y) immunoreactivity. The pattern of immunoreactivity to both antisera is identical. Blocking studies indicate that both antisera are recognizing the same site, most likely the arginine-phenylalanine-amide terminus of FMRF, or an FMRF-like molecule. We conclude that FMRF is a candidate for the chemical mediator involved in the interaction between the two ocular pacemakers in Bulla gouldiana.

The Bulla ocular circadian pacemaker. I. Pacemaker neuron membrane potential controls phase through a calcium-dependent mechanism.

In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker of Bulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that: 1. Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization. 2. Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180 degrees in circadian time to depolarization. 3. Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection. 4. The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts. 5. The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3 X 10(-7) M inhibits light-induced phase shifts without blocking the photic response of the BRNs. The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.

Cellular analysis of ocular circadian pacemaker coupling in Bulla: role of efferent impulses in phase shifting.

The eyes of Bulla gouldiana, a marine snail, contain circadian oscillators that are coupled to each other. Obvious candidates for the coupling signals are the optic nerve compound action potentials (CAPs) that express the circadian rhythm and lead to efferent impulses in the contralateral optic nerve. In the present experiments, the role of the CAPs as coupling signals was evaluated. We found that, following desynchronization of the two ocular oscillators by phase-delaying one eye with manganese, subsequent phase shifts in the initially unshifted ocular rhythm only occurred during the time that efferent optic nerve signals were present. In addition, in the absence of ocular desynchrony, phase shifts of the ocular rhythm could still be effected by activation of the efferent pathway. The influence of efferent impulses on identified retinal cells was also evaluated. No effect of efferent signals on receptor layer cells was detected, while it was found that efferent impulses generated depolarizations in basal retinal neurons (BRNs), the putative circadian oscillator cells. Depolarization of the BRNs has been shown previously to be involved in the light entrainment pathway. Depolarization appears to be similarly involved in the coupling pathway, since membrane depolarizations that mimicked the efferent-induced postsynaptic potentials likewise generated phase shifts of the ocular rhythm.