Submicrosecond pacemaker precision is behaviorally modulated: The gymnotiform electromotor pathway

What are the limits and modulators of neural precision? We address this question in the most regular biological oscillator known, the electric organ command nucleus in the brainstem of wave-type gymnotiform fish. These fish produce an oscillating electric field, the electric organ discharge (EOD), used in electrolocation and communication. We show here that the EOD precision, measured by the coefficient of variation (CV = SD/mean period) is as low as 2 × 10−4 in five species representing three families that range widely in species and individual mean EOD frequencies (70–1,250 Hz). Intracellular recording in the pacemaker nucleus (Pn), which commands the EOD cycle by cycle, revealed that individual Pn neurons of the same species also display an extremely low CV (CV = 6 × 10−4, 0.8 μs SD). Although the EOD CV can remain at its minimum for hours, it varies with novel environmental conditions, during communication, and spontaneously. Spontaneous changes occur as abrupt steps (250 ms), oscillations (3–5 Hz), or slow ramps (10–30 s). Several findings suggest that these changes are under active control and depend on behavioral state: mean EOD frequency and CV can change independently; CV often decreases in response to behavioral stimuli; and lesions of one of the two inputs to the Pn had more influence on CV than lesions of the other input.

Novel Gq alpha isoform is a candidate transducer of rhodopsin signaling in a Drosophila testes-autonomous pacemaker.

DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. We have uncovered three duplicated exons in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA (phospholipase C)-binding, and rhodopsin-binding. We examined the in vivo splicing of dgq in adult flies and find that, in all but the male gonads, only two isoforms are expressed. One, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, we have found a third isoform, dgqC, which is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. We suggest that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that rhodopsin 2 and arrestin 1, two photoreceptor-cell-specific genes, are also expressed in male gonads.

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Altered emotional and locomotor responses in mice deficient in the transcription factor CREM

Various transcription factors act as nuclear effectors of the cAMP-dependent signaling pathway. These are the products of three genes in the mouse, CREB, CRE modulator (CREM), and ATF-1. CREM proteins are thought to play important roles within the hypothalamic–pituitary axis and in the control of rhythmic functions in the pineal gland. We have generated CREM-mutant mice and investigated their response in a variety of behavioral tests. CREM-null mice show a drastic increase in locomotion. In contrast to normal mice, the CREM-deficient mice show equal locomotor activity during the circadian cycle. The anatomy of the hypothalamic suprachiasmatic nuclei, the center of the endogenous pacemaker, is normal in mutant mice. Remarkably, CREM mutant mice also elicit a different emotional state, revealed by a lower anxiety in two different behavioral models, but they preserve the conditioned reactiveness to stress. These results demonstrate the high degree of functional specificity of each cAMP-responsive transcription factor in behavioral control.

Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

We investigated the circadian function of Drosophila dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.

Thyroid hormone receptor β-dependent expression of a potassium conductance in inner hair cells at the onset of hearing

To elucidate the role of thyroid hormone receptors (TRs) α1 and β in the development of hearing, cochlear functions have been investigated in mice lacking TRα1 or TRβ. TRs are ligand-dependent transcription factors expressed in the developing organ of Corti, and loss of TRβ is known to impair hearing in mice and in humans. Here, TRα1-deficient (TRα1−/−) mice are shown to display a normal auditory-evoked brainstem response, indicating that only TRβ, and not TRα1, is essential for hearing. Because cochlear morphology was normal in TRβ−/− mice, we postulated that TRβ regulates functional rather than morphological development of the cochlea. At the onset of hearing, inner hair cells (IHCs) in wild-type mice express a fast-activating potassium conductance, IK,f, that transforms the immature IHC from a regenerative, spiking pacemaker to a high-frequency signal transmitter. Expression of IK,f was significantly retarded in TRβ−/− mice, whereas the development of the endocochlear potential and other cochlear functions, including mechanoelectrical transduction in hair cells, progressed normally. TRα1−/− mice expressed IK,f normally, in accord with their normal auditory-evoked brainstem response. These results establish that the physiological differentiation of IHCs depends on a TRβ-mediated pathway. When defective, this may contribute to deafness in congenital thyroid diseases.