Pacemaker Neurons Research Articles

Cocaine increases dopaminergic neuron and motor activity via midbrain α1 adrenergic signaling.

Cocaine reinforcement is mediated by increased extracellular dopamine levels in the forebrain. This neurochemical effect was thought to require inhibition of dopamine reuptake, but cocaine is still reinforcing even in the absence of the dopamine transporter. Here, we demonstrate that the rapid elevation in dopamine levels and motor activity elicited by cocaine involves α1 receptor activation within the ventral midbrain. Activation of α1 receptors increases dopaminergic neuron burst firing by decreasing the calcium-activated potassium channel current (SK), as well as elevates dopaminergic neuron pacemaker firing through modulation of both SK and the hyperpolarization-activated cation currents (Ih). Furthermore, we found that cocaine increases both the pacemaker and burst-firing frequency of rat ventral-midbrain dopaminergic neurons through an α1 adrenergic receptor-dependent mechanism within the ventral tegmental area and substantia nigra pars compacta. These results demonstrate the mechanism underlying the critical role of α1 adrenergic receptors in the regulation of dopamine neurotransmission and behavior by cocaine.

Dynamics of self-sustained asynchronous-irregular activity in random networks of spiking neurons with strong synapses.

Random networks of integrate-and-fire neurons with strong current-based synapses can, unlike previously believed, assume stable states of sustained asynchronous and irregular firing, even without external random background or pacemaker neurons. We analyze the mechanisms underlying the emergence, lifetime and irregularity of such self-sustained activity states. We first demonstrate how the competition between the mean and the variance of the synaptic input leads to a non-monotonic firing-rate transfer in the network. Thus, by increasing the synaptic coupling strength, the system can become bistable: In addition to the quiescent state, a second stable fixed-point at moderate firing rates can emerge by a saddle-node bifurcation. Inherently generated fluctuations of the population firing rate around this non-trivial fixed-point can trigger transitions into the quiescent state. Hence, the trade-off between the magnitude of the population-rate fluctuations and the size of the basin of attraction of the non-trivial rate fixed-point determines the onset and the lifetime of self-sustained activity states. During self-sustained activity, individual neuronal activity is moreover highly irregular, switching between long periods of low firing rate to short burst-like states. We show that this is an effect of the strong synaptic weights and the finite time constant of synaptic and neuronal integration, and can actually serve to stabilize the self-sustained state.

Mmp1 processing of the PDF neuropeptide regulates circadian structural plasticity of pacemaker neurons.

In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

Differentially timed extracellular signals synchronize pacemaker neuron clocks.

Author SummaryCircadian molecular clocks are essential for daily cycles in animal behavior and we have a good understanding of how these clocks work in individual pacemaker neurons. However, the accuracy of these individual clocks is meaningless unless they are synchronized with one another. In this study we show that synchronizing the principal pacemaker LNv neurons in Drosophila larvae require two extracellular signals that are received at opposite times of day: namely, the neuropeptide PDF released from LNvs themselves at dawn and glutamate released from dorsal clock neurons at dusk. LNvs perceive both PDF and glutamate via G-protein coupled receptors that increase or decrease intracellular cAMP, respectively. The alternating phases of PDF and glutamate release generate oscillations in intracellular cyclic AMP. In addition to maintaining synchrony between LNvs, this rhythm is also required for molecular clock oscillations in individual larval LNvs. We show that disruption of PDF and glutamate signaling also reduces synchrony in adult LNvs. This impairs the oscillations of clock proteins and flies have delayed onset of sleep. Our data highlight the importance of intercellular signaling in ensuring synchrony between clock neurons within the circadian network. Our findings help extend the conservation of clock properties between Drosophila and mammals beyond clock genes to include clock circuitry.

Daily Dance of Synaptic Partners

The number of synapses and the identities of synaptic partners change over the course of a day in circadian pacemaker neurons.

The frequency preference of neurons and synapses in a recurrent oscillatory network.

A variety of neurons and synapses shows a maximal response at a preferred frequency, generally considered to be important in shaping network activity. We are interested in whether all neurons and synapses in a recurrent oscillatory network can have preferred frequencies and, if so, whether these frequencies are the same or correlated, and whether they influence the network activity. We address this question using identified neurons in the pyloric network of the crab Cancer borealis. Previous work has shown that the pyloric pacemaker neurons exhibit membrane potential resonance whose resonance frequency is correlated with the network frequency. The follower lateral pyloric (LP) neuron makes reciprocally inhibitory synapses with the pacemakers. We find that LP shows resonance at a higher frequency than the pacemakers and the network frequency falls between the two. We also find that the reciprocal synapses between the pacemakers and LP have preferred frequencies but at significantly lower values. The preferred frequency of the LP to pacemaker synapse is correlated with the presynaptic preferred frequency, which is most pronounced when the peak voltage of the LP waveform is within the dynamic range of the synaptic activation curve and a shift in the activation curve by the modulatory neuropeptide proctolin shifts the frequency preference. Proctolin also changes the power of the LP neuron resonance without significantly changing the resonance frequency. These results indicate that different neuron types and synapses in a network may have distinct preferred frequencies, which are subject to neuromodulation and may interact to shape network oscillations.

Abstract 618: Sympathetic Nervous System-Mediated Low Frequency Fluctuations in Blood Pressure are Independent of Baroreflex Modulation in Autonomic Failure

Autonomic cardiovascular regulation involves sympathetic rhythms that contribute to blood pressure fluctuations in the low frequency range. It has been suggested that fluctuations in sympathetic vasomotor tone reflect a resonance phenomenon due to interplay between the vasculature and baroreflex. Alternatively, this could reflect an intrinsic rhythm of pacemaker neurons. To dissect the autonomic origin of this cardiovascular rhythm, we studied autonomic failure patients with loss of baroreflex buffering. We hypothesized that if this rhythm originates in pacemaker neurons, it would be present in multiple systems atrophy (MSA) patients in whom residual sympathetic tone is intact but not regulated by the baroreflex, and absent in pure autonomic failure (PAF) patients with postganglionic sympathetic denervation. We studied 28 MSA and 34 PAF patients with severe autonomic impairment and neurogenic orthostatic hypotension. Low-frequency systolic blood pressure variability (LFSBP), an index of sympathetic modulation of vasomotor tone, and baroreflex sensitivity (BRS) were assessed by spectral analysis of continuous blood pressure and heart rate recordings. MSA patients had higher LFSBP during supine rest compared with PAF patients (3.3±0.5 versus 1.5±0.2 mmHg2, respectively; p=0.003), despite similarly low BRS (3.6±0.6 PAF versus 4.3±0.7 msec/mmHg MSA; p=0.380). LFSBP was higher in MSA patients with supine hypertension compared with normotensive patients (4.1±0.6 versus 2.3±0.6 mmHg2; p=0.041), with no differences in PAF patients (1.5±0.2 hypertensive versus 1.6±0.3 mmHg2 normotensive, respectively; p=0.984). These findings suggest that LFSBP is driven by an intrinsic rhythm originating in central sympathetic pathways in MSA patients, independent of baroreflex-mediated blood pressure fluctuations. The precise origin of this rhythm is unclear but may include central pacemaker neurons, spinal cord neurons or loops, or hormonal mechanisms. In addition, LFSBP is higher in MSA patients with sympathetically-mediated hypertension, but low and fixed in PAF patients with sympathetic-independent hypertension. Overall, these findings provide new insight into neural regulatory mechanisms involved in blood pressure control.

Fractal-like correlations of the fluctuating inter-spike membrane potential of a Helix aspersa pacemaker neuron

We analyzed the voltage fluctuations of the membrane potential manifested along the inter-spike segment of a pacemaker neuron. Time series of intracellular inter-spike voltage fluctuations were obtained in the current-clamp configuration from the F1 neuron of 12 Helix aspersa specimens. To assess the dynamic or stochastic nature of the voltage fluctuations these series were analyzed by Detrended Fluctuation Analysis (DFA), providing the scaling exponent α. The median α result obtained for the inter-spike segments was 0.971 ([0.963, 0.995] lower and upper quartiles). Our results indicate a critical-like dynamic behavior in the inter-spike membrane potential that, far from being random, shows long-term correlations probably linked to the dynamics of the mechanisms involved in the regulation of the membrane potential, thereby endorsing the occurrence of critical-like phenomena at a single-neuron level.

Processing of sub- and supra-second intervals in the primate brain results from the calibration of neuronal oscillators via sensory, motor, and feedback processes.

The processing of time intervals in the sub- to supra-second range by the brain is critical for the interaction of primates with their surroundings in activities, such as foraging and hunting. For an accurate processing of time intervals by the brain, representation of physical time within neuronal circuits is necessary. I propose that time dimension of the physical surrounding is represented in the brain by different types of neuronal oscillators, generating spikes or spike bursts at regular intervals. The proposed oscillators include the pacemaker neurons, tonic inputs, and synchronized excitation and inhibition of inter-connected neurons. Oscillators, which are built inside various circuits of brain, help to form modular clocks, processing time intervals or other temporal characteristics specific to functions of a circuit. Relative or absolute duration is represented within neuronal oscillators by “neural temporal unit,” defined as the interval between regularly occurring spikes or spike bursts. Oscillator output is processed to produce changes in activities of neurons, named frequency modulator neuron, wired within a separate module, represented by the rate of change in frequency, and frequency of activities, proposed to encode time intervals. Inbuilt oscillators are calibrated by (a) feedback processes, (b) input of time intervals resulting from rhythmic external sensory stimulation, and (c) synchronous effects of feedback processes and evoked sensory activity. A single active clock is proposed per circuit, which is calibrated by one or more mechanisms. Multiple calibration mechanisms, inbuilt oscillators, and the presence of modular connections prevent a complete loss of interval timing functions of the brain.

Temperature-dependent bursting pattern analysis by modified Plant model.

Many electrophysiological properties of neuron including firing rates and rhythmical oscillation change in response to a temperature variation, but the mechanism underlying these correlations remains unverified. In this study, we analyzed various action potential (AP) parameters of bursting pacemaker neurons in the abdominal ganglion of Aplysia juliana to examine whether or not bursting patterns are altered in response to temperature change. Here we found that the inter-burst interval, burst duration, and number of spike during burst decreased as temperature increased. On the other hand, the numbers of bursts per minute and numbers of spikes per minute increased and then decreased, but interspike interval during burst firstly decreased and then increased. We also tested the reproducibility of temperature-dependent changes in bursting patterns and AP parameters. Finally we performed computational simulations of these phenomena by using a modified Plant model composed of equations with temperature-dependent scaling factors to mathematically clarify the temperature-dependent changes of bursting patterns in burst-firing neurons. Taken together, we found that the modified Plant model could trace the ionic mechanism underlying the temperature-dependent change in bursting pattern from experiments with bursting pacemaker neurons in the abdominal ganglia of Aplysia juliana.

The ion transport peptide is a new functional clock neuropeptide in the fruit fly Drosophila melanogaster.

The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide pigment dispersing factor (PDF). Here, we propose a role in the control of behavioral rhythms for the ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant Clk(AR), suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, we knocked down ITP in the two clock cells and found that these flies show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. We conclude that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms.

Estimation of artificial neuron parameters that obtain a required distribution of coupled system periods in a hybrid network

Rhythmic neuronal activity is a hallmark of many higher-level functions of the nervous system including the expression of rhythmic motor patterns and generation of behaviors such as cognition, memory, arousal, and sleep. Many rhythmic networks underlying these behaviors consist of one or more pacemaker neurons that are coupled with non-pacemaking neurons, and exhibit reliable stereotyped activity that however displays diverse levels of activity variability between networks – and sometimes those circuits that exhibit high levels of activity variability even depend upon their variability to accomplish some behavioral goal [1,2]. While it is understood that synaptic feedback from non-pacemaking neurons coupled to pacemaker neurons can limit variability of rhythmic activity [3,4], it is not yet understood how specific neuronal dynamics influence variability of the coupled network. Here we develop a theoretical approach to predict how network activity variability – as quantified by the probability distribution of observed periods – changes with altered phase response dynamics of non-pacemaking neurons, and demonstrate this theoretical approach’s predictive efficacy in hybrid networks established in the stomatogastric nervous system (STNS) of Cancer borealis. These hybrid networks consist of one biological pacemaker neuron (Anterior Burster/Pyloric Dilator complex [AB/PD]) and one artificial non-pacemaker neuron (designed to mimic the burst timing of the Lateral Pyloric [LP] neuron), which allows us to vary the phase response dynamics of the latter element. Our simple theoretical approach to predict the effects of network coupling on activity variability is based on three assumptions. First, within the range of variation of AB/PD neuron’ intrinsic period, it is assumed that there is always at least one stable fixed-point in the dynamics of the coupled system. Second, it is assumed that any drifting of the AP/PD neuron’s intrinsic period is significantly slower than the duration of one intrinsic period, so that the coupled system is capable of reaching a steady-state condition. Third, it is assumed that as the period of AB/PD varies, any changes in this neuron’s phase resetting curve (PRC) are negligibly small. The first two assumptions allow us to directly map an AB/PD neuron’s intrinsic period onto the network period in the coupled system. The last assumption allows us to recalculate the map for different intrinsic periods and predict the distribution of periods in the coupled network based upon the known distribution of intrinsic periods of AP/PD neuron. Using previously developed analyses [5] and a linear tS-tR curve for the artificial neuron we are able to qualitatively predict how slope and bias of the artificial neuron’s tS-tR curve affect the distribution of network periods. All experiments are performed using standard intracellular electrophysiological methods for the pyloric network of the STNS, and hybrid networks are established with the dynamic clamp.

Mathematical modeling and analysis of spinal circuits involved in locomotor pattern generation and frequency-dependent left-right coordination

Coordination between left and right neural activities in the spinal cord during locomotion is controlled by commissural interneurons (CINs). Several CIN types have been genetically identified, including the excitatory V3 and excitatory and inhibitory V0 types. Talpalar et al. [1] recently reported that genetic elimination of the V0 CINs caused switching from a normal left-right alternating pattern of motor activity to a left-right synchronized “hopping” pattern. Furthermore, ablation of only the inhibitory V0 neurons (V0D subtype) resulted in a lack of left-right alternation at low locomotor frequencies and maintaining this alternation at high frequencies, whereas selective ablation of the excitatory V0 neurons (V0V subtype) maintained the left–right alternation at low locomotor frequencies and switched the motor output to a left-right synchronized (hopping) pattern at high frequencies. To analyze and explain the above findings, we developed a simplified mathematical model of neural circuits consisted of four pacemaker neurons representing left (LF) and right (RF) flexor and left (LE) and right (RE) extensor half-centers interacting via commissural pathways representing V3, V0D, and V0V CINs (Fig. ​(Fig.1).1). The “locomotor” frequency in the model was controlled by a parameter defining the excitability of neurons (via the leak reversal potentials) and commissural pathways, whose changes represented the corresponding changes induced by changing the concentration of N-methyl-D-aspartate (NMDA) applied to control the locomotor frequency in the isolated rodent spinal cord preparations [1]. Figure 1 Model schematic. The model demonstrated: (1) a typical left-right alternating pattern under control conditions; (2) switching to a synchronized hopping activity at any frequency after removing commissural connections representing both V0 (V0D and V0V) neurons; (3) a synchronized left-right pattern at low frequencies and left-right alternation of activity at high frequencies after removing the commissural connections representing V0D neurons; (4) an alternating left-right pattern at low frequencies with synchronized hopping at high frequencies after removing the commissural connections representing V0V neurons. We used the bifurcation and fast–slow decomposition methods to analyze the behavior of this network in the four above states/regimes and transitions between them. The model was able to reproduce and provide explanation to several important experimental phenomena and generated predictions that can be tested experimentally.

Synchronization of two electrically coupled inspiratory pacemaker neurons

Synchronization is considered to be a crucial mechanism that maintains respiratory rhythm. For understanding the effect of electrical coupling on the transition of the firing patterns and synchronization, we coupled two inspiratory pacemaker neurons together, and studied various synchronous behaviors between them. We firstly compared the bifurcation diagrams between the coupled neurons and single neuron, and found that the coupled neurons had a more complicated bifurcation mode. By increasing the coupling strength, the regular variation of phase differences was illustrated so that asynchronous and some synchronous states could be observed. These synchronous states were also shown in detail by phase portraits and firing series. In addition, we explored the ranges of different synchronous states, which attributed to different ranges of membrane capacitance and coupling strength.

The effect of pacemaker neurons on epileptiform activity within the CA3 and CA1 regions of the hippocampus (588.4)

This study simulates pacemaker neurons acting on the CA3 and CA1 regions of the hippocampus and their effect on epileptiform activity. Epileptic seizures are produced from an excess of synchronous positive feedback in neural networks. Research by Netoff et al. (2004) showed that ictal bursting is observed in the CA3 region and prolonged seizures are observed in the CA1 region. Models of these regions were created with the software Simulator for Neural Network and Action Potentials (Ziv et al. 1994). Small world networks of 64 neurons were constructed with the appropriate synaptic connections and strengths, simulating the predicted epileptiform activities in voltage vs. time outputs. A network of 32 electrically connected Morris‐Lecar neurons, pacemaker neurons, were connected to randomly selected neurons in each model. Based on differences in action potential profiles produced, the CA3 region modeled prolonged seizing after the addition of the Morris‐Lecar Network causing an excess of synchronous positive feedback, rather than the expected ictal bursting; however, the prolonged seizing of the CA1 region did not change after adding the Morris‐Lecar Network.

Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior

Author SummaryCircadian clocks provide a mechanism for predicting and adapting behavioral and physiological processes to 24-hour rhythms in the environment. In animal nervous systems, cell-autonomous molecular oscillators are coupled via neural networks that control daily patterns of activity. A major neuropeptide synchronizing neural oscillators in the Drosophila clock network is PIGMENT DISPERSING FACTOR (PDF). Here we identify a fork in the processing of the PDF signal in circadian neurons to independently reset the molecular clock and regulate neuronal activity. We show that the cAMP-activated protein kinase A (PKA) in circadian neurons is necessary and sufficient for many PDF-dependent behaviors. In addition, we find that a PDF>PDF receptor>PKA pathway targets the clock component TIMELESS to control molecular oscillators, and that this process may be influenced by rhythmic expression of PKA. We show that this pathway splits at the level of cAMP generation, with PDF and cAMP acutely increasing the activity of clock neurons in a PKA-independent manner. Thus, PDF operates via dual signaling pathways: one via PKA to reset clocks and the other via cAMP to acutely control activity. These results have broad implications given the conserved involvement of neuropeptide signaling in synchronizing clocks in circadian neural networks.

Intrinsically Active and Pacemaker Neurons in Pluripotent Stem Cell-Derived Neuronal Populations

SummaryNeurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

Chapter 3 - Morphological Characterization of Respiratory Neurons in the Pre-Bötzinger Complex

Although the pre-Bötzinger complex (preBötC) was defined as the inspiratory rhythm generator long ago, the functional-anatomical characterization of its neuronal components is still being achieved. Recent advances have identified the expression of molecular markers in the preBötC neurons that, however, are not exclusive to specific respiratory neuron subtypes and have not always been related to specific cell morphologies. Here, we evaluated the morphology and the axonal projections of electrophysiologically defined respiratory neurons in the preBötC using whole-cell recordings and intracellular biocytin labeling. We found that respiratory pacemaker neurons are larger than expiratory neurons and that inspiratory neurons are smaller than pacemaker and expiratory neurons. Other morphological features such as somata shapes or dendritic branching patterns were not found to be significantly different among the preBötC neurons sampled. We also found that both pacemaker and inspiratory nonpacemaker neurons, but not expiratory neurons, show extensive axonal projections to the contralateral preBötC and show signs of electrical coupling. Overall, our data suggest that there are morphological differences between subtypes of preBötC respiratory neurons. It will be important to take such differences in consideration since morphological differences would influence synaptic responses and action potential propagation.

Acute ethanol exposure inhibits silencing of cerebellar Golgi cell firing induced by granule cell axon input

Golgi cells (GoCs) are specialized interneurons that provide inhibitory input to granule cells in the cerebellar cortex. GoCs are pacemaker neurons that spontaneously fire action potentials, triggering spontaneous inhibitory postsynaptic currents in granule cells and also contributing to the generation tonic GABAA receptor-mediated currents in granule cells. In turn, granule cell axons provide feedback glutamatergic input to GoCs. It has been shown that high frequency stimulation of granule cell axons induces a transient pause in GoC firing in a type 2-metabotropic glutamate receptor (mGluR2)-dependent manner. Here, we investigated the effect ethanol on the pause of GoC firing induced by high frequency stimulation of granule cell axons. GoC electrophysiological recordings were performed in parasagittal cerebellar vermis slices from postnatal day 23 to 26 rats. Loose-patch cell-attached recordings revealed that ethanol (40 mM) reversibly decreases the pause duration. An antagonist of mGluR2 reduced the pause duration but did not affect the effect of ethanol. Whole-cell voltage-clamp recordings showed that currents evoked by an mGluR2 agonist were not significantly affected by ethanol. Perforated-patch experiments in which hyperpolarizing and depolarizing currents were injected into GoCs demonstrated that there is an inverse relationship between spontaneous firing and pause duration. Slight inhibition of the Na+/K+ pump mimicked the effect of ethanol on pause duration. In conclusion, ethanol reduces the granule cell axon-mediated feedback mechanism by reducing the input responsiveness of GoCs. This would result in a transient increase of GABAA receptor-mediated inhibition of granule cells, limiting information flow at the input stage of the cerebellar cortex.

Sexual Interactions Influence the Molecular Oscillations in DN1 Pacemaker Neurons in Drosophila melanogaster

Circadian rhythms can synchronize to environmental time cues, such as light, temperature, humidity, and food availability. Previous studies have suggested that these rhythms can also be entrained by social interactions. Here, we used Drosophila melanogaster as a model to study the influence of socio-sexual interactions on the circadian clock in behavior and pacemaker neurons. If two flies of opposite sex were paired and kept in a small space, the daily activity patterns of the two flies were clearly different from the sum of the activity of single male and female flies. Compared with single flies, paired flies were more active in the night and morning, were more active during females’ active phase, and were less active during males’ active phase. These behavioral phenotypes are related to courtship behavior, but not to the circadian clock. Nevertheless, in male-female pairs of flies with clocks at different speeds (wild-type and per S flies), clock protein cycling in the DN1 pacemaker neurons in the male brain were slightly influenced by their partners. These results suggest that sexual interactions between male-female couples can serve as a weak zeitgeber for the DN1 pacemaker neurons, but the effect is not sufficient to alter rhythms of behavioral activity.