Freely-moving Animals Research Articles

Circuits regulating pleasure and happiness: the evolution of reward-seeking and misery-fleeing behavioral mechanisms in vertebrates.

The very first free-moving animals in the oceans over 540 million years ago must have been able to obtain food, territory, and shelter, as well as reproduce. Therefore, they would have needed regulatory mechanisms to induce movements enabling achievement of these prerequisites for survival. It can be useful to consider these mechanisms in primitive chordates, which represent our earliest ancestors, to develop hypotheses addressing how these essential parts of human behavior are regulated and relate to more sophisticated behavioral manifestations such as mood. An animal comparable to lampreys was the earliest known vertebrate with a modern forebrain consisting of old and new cortical parts. Lampreys have a separate dorsal pallium, the forerunner of the most recently developed part of the cerebral cortex. In addition, the lamprey extrapyramidal system (EPS), which regulates movement, is modern. However, in lampreys and their putative forerunners, the hagfishes, the striatum, which is the input part of this EPS, probably corresponds to the human centromedial amygdala, which in higher vertebrates is part of a system mediating fear and anxiety. Both animals have well-developed nuclear habenulae, which are involved in several critical behaviors; in lampreys this system regulates the reward system that reinforces appetitive-seeking behavior or the avoidance system that reinforces flight behavior resulting from negative inputs. Lampreys also have a distinct glutamatergic nucleus, the so-called habenula-projection globus pallidus, which receives input from glutamatergic and GABAergic signals and gives output to the lateral habenula. Via this route, this nucleus influences midbrain monoaminergic nuclei and regulates the food acquisition system. These various structures involved in motor regulation in the lampreys may be conserved in humans and include two complementary mechanisms for reward reinforcement and avoidance behaviors. The first system is associated with experiencing pleasure and the second with happiness. The activities of these mechanisms are regulated by a tract running via the habenula to the upper brainstem. Identifying the human correlate of the lamprey habenula-projecting globus pallidus may help in elucidating the mechanism of the antidepressant effects of glutamatergic drugs.

Stridulated soft song by singing insects

The study of low-amplitude or ‘soft’ songs and calls has largely been limited to organisms that produce multiple call types that fall neatly into a bimodal distribution with respect to amplitude. The soft vocalizations of many of these animals, including birds and mammals, have proven to be extremely difficult to collect data on due to difficulty in hearing and recording such songs in the wild, the lack of production of these sounds in captivity, and the difficulty in standardizing measurements of the amplitude produced by free-moving animals. Here I suggest we consistently expand the working definition of soft song to allow for the inclusion of insects and other organisms whose calls do not easily fit into a ‘high-amplitude’ versus ‘low-amplitude’ signal paradigm. For instance, some species of moths produce extremely quiet ultrasonic courtship songs without also producing a high-amplitude song, and field crickets sing courtship songs that contain both relatively loud and quiet elements within the same song. Soft-singing moths and crickets may not only prove more practical to work with, but may also provide answers to heretofore untestable hypotheses about the function and evolution of soft song. • Low-amplitude signals receive little study, especially those produced nonvocally. • Common definitions of soft song only apply to species with loud and quiet songs. • Many moths produce only low-amplitude songs. • Many field crickets produce courtship songs with high- and low-amplitude elements. • The definition of soft song should expand to include these and other organisms.

ATGL-mediated triglyceride turnover and the regulation of mitochondrial capacity in skeletal muscle.

Emerging evidence indicates that skeletal muscle lipid droplets are an important control point for intracellular lipid homeostasis and that regulating fatty acid fluxes from lipid droplets might influence mitochondrial capacity. We used pharmacological blockers of the major triglyceride lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase, to show that a large proportion of the fatty acids that are transported into myotubes are trafficked through the intramyocellular triglyceride pool. We next tested whether increasing lipolysis from intramyocellular lipid droplets could activate transcriptional responses to enhance mitochondrial and fatty acid oxidative capacity. ATGL was overexpressed by adenoviral and adenoassociated viral infection in C2C12 myotubes and the tibialis anterior muscle of C57Bl/6 mice, respectively. ATGL overexpression in C2C12 myotubes increased lipolysis, which was associated with increased peroxisome proliferator-activated receptor (PPAR)-∂ activity, transcriptional upregulation of some PPAR∂ target genes, and enhanced mitochondrial capacity. The transcriptional responses were specific to ATGL actions and not a generalized increase in fatty acid flux in the myotubes. Marked ATGL overexpression (20-fold) induced modest molecular changes in the skeletal muscle of mice, but these effects were not sufficient to alter fatty acid oxidation. Together, these data demonstrate the importance of lipid droplets for myocellular fatty acid trafficking and the capacity to modulate mitochondrial capacity by enhancing lipid droplet lipolysis in vitro; however, this adaptive program is of minor importance when superimposing the normal metabolic stresses encountered in free-moving animals.

In vivo optogenetic stimulation of the rodent central nervous system.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

<em>In vivo</em> Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

An accurate and portable eye movement detector for studying sleep in small animals.

Although eye movements are a highly valuable variable in attempts to precisely identify different periods of the sleep-wake cycle, their indirect measurement by electrooculography is not good enough. The present article describes an accurate and portable scleral search coil that allows the detection of tonic and phasic characteristics of eye movements in free-moving animals. Six adult Wistar rats were prepared for chronic recording of electroencephalography, electromyography and eye movements using the scleral search coil technique. We developed a miniature magnetic field generator made with two coils, consisting of 35turns and 15mm diameter of insulated 0.2mm cooper wire, mounted in a frame of carbon fibre. This portable scleral search coil was fixed on the head of the animal, with each magnetic coil parallel to the eye coil and at 5mm from each eye. Eye movements detected by the portable scleral search coil were compared with those measured by a commercial scleral search coil requiring immobilizing the head of the animal. No qualitative differences were found between the two scleral search coil systems in their capabilities to detect eye movements. This innovative portable scleral search coil system is an essential tool to detect slow changes in eye position and miniature rapid eye movements during sleep. The portable scleral search coil is much more suitable for detecting eye movements than any previously available system because of its precision and simplicity, and because it does not require immobilization of the animal's head.

Inside view

inside view

Design of a single-fiber, wavelength-resolved system for monitoring deep tissue oxygenation.

We propose a single-fiber, zero source-detector separation system with wavelength-resolved detection for measuring oxygen saturation in deep brain structures. The system consists of a white light emitting diode (LED) source, optics to couple light into a 240-μm-diameter fiber, a beam splitter to separate the collected from the delivered photons and a spectrometer for detection. Depth resolution is achieved by inserting the fiber, comparable in size to microelectrodes used for electrophysiology, into the tissue of interest. Since most of the diffuse reflected light travels through a small volume at the tip of the fiber, this arrangement allows efficient collection of signal. Fresnel reflections are minimized using polarizers. Monte Carlo simulations across 400-1000 nm indicate that ~0.5% of the incident light can be collected and effectively interrogate a ~0.02 mm(3) volume at the fiber tip. System design, characterization data and phantom experiments using an absorptive dye in scattering media are presented. The simple nature of the instrumentation can potentially lead to a miniaturized system capable of detecting oxygen saturation in deep brain structures in freely-moving animals.

A freely-moving monkey treadmill model

Objective. Motor neuroscience and brain–machine interface (BMI) design is based on examining how the brain controls voluntary movement, typically by recording neural activity and behavior from animal models. Recording technologies used with these animal models have traditionally limited the range of behaviors that can be studied, and thus the generality of science and engineering research. We aim to design a freely-moving animal model using neural and behavioral recording technologies that do not constrain movement. Approach. We have established a freely-moving rhesus monkey model employing technology that transmits neural activity from an intracortical array using a head-mounted device and records behavior through computer vision using markerless motion capture. We demonstrate the flexibility and utility of this new monkey model, including the first recordings from motor cortex while rhesus monkeys walk quadrupedally on a treadmill. Main results. Using this monkey model, we show that multi-unit threshold-crossing neural activity encodes the phase of walking and that the average firing rate of the threshold crossings covaries with the speed of individual steps. On a population level, we find that neural state-space trajectories of walking at different speeds have similar rotational dynamics in some dimensions that evolve at the step rate of walking, yet robustly separate by speed in other state-space dimensions. Significance. Freely-moving animal models may allow neuroscientists to examine a wider range of behaviors and can provide a flexible experimental paradigm for examining the neural mechanisms that underlie movement generation across behaviors and environments. For BMIs, freely-moving animal models have the potential to aid prosthetic design by examining how neural encoding changes with posture, environment and other real-world context changes. Understanding this new realm of behavior in more naturalistic settings is essential for overall progress of basic motor neuroscience and for the successful translation of BMIs to people with paralysis.

Whole-brain circuit dissection in free-moving animals reveals cell-specific mesocorticolimbic networks

The ability to map the functional connectivity of discrete cell types in the intact mammalian brain during behavior is crucial for advancing our understanding of brain function in normal and disease states. We combined designer receptor exclusively activated by designer drug (DREADD) technology and behavioral imaging with μPET and [18F]fluorodeoxyglucose (FDG) to generate whole-brain metabolic maps of cell-specific functional circuits during the awake, freely moving state. We have termed this approach DREADD-assisted metabolic mapping (DREAMM) and documented its ability in rats to map whole-brain functional anatomy. We applied this strategy to evaluating changes in the brain associated with inhibition of prodynorphin-expressing (Pdyn-expressing) and of proenkephalin-expressing (Penk-expressing) medium spiny neurons (MSNs) of the nucleus accumbens shell (NAcSh), which have been implicated in neuropsychiatric disorders. DREAMM revealed discrete behavioral manifestations and concurrent engagement of distinct corticolimbic networks associated with dysregulation of Pdyn and Penk in MSNs of the NAcSh. Furthermore, distinct neuronal networks were recruited in awake versus anesthetized conditions. These data demonstrate that DREAMM is a highly sensitive, molecular, high-resolution quantitative imaging approach.

Controlled Iontophoresis Coupled with Fast-Scan Cyclic Voltammetry/Electrophysiology in Awake, Freely Moving Animals

Simultaneous electrochemical and electrophysiological data were recorded to evaluate the effects of controlled local application of dopaminergic agonists and antagonists in awake rats. Measurements were made with a probe consisting of a carbon-fiber microelectrode fused to three iontophoretic barrels used to introduce the drugs of interest. The probe and the manipulator used to position it in the brain of behaving animals were optimized to improve their performance. The effect of the dopamine autoreceptor on electrically stimulated release was demonstrated. Dopamine inhibited the release of endogenous dopamine whereas raclopride, a D2 antagonist, enhanced it, with similar responses in anesthetized and awake animals. We also examined changes in the firing rate of nucleus accumbens (NAc) neurons in awake animals during and after brief (15 s) iontophoretic ejections of SCH 23390 (D1 receptor antagonist) or raclopride. Changes in response to these antagonists were seen both immediately and on a prolonged time scale. Application of raclopride increased the firing rate in 40% of medium spiny neurons (MSNs), of which half responded immediately. Decreases in firing rate were observed in 46% of MSNs after SCH 23390 application. Only 11% of MSNs responded to both antagonists and one MSN (3%) showed no response to either drug. The same prolonged response in firing rate was seen for electrically stimulated and locally applied dopamine in 75% of MSNs. These results are in agreement with previously reported distributions for dopamine receptor subtypes on MSNs and probe the effects of dopamine on these cell populations.

Hippocampal excitability is increased in aged mice

Aging is known to be associated with a high risk of developing seizure disorders. Currently, the mechanisms underlying this increased seizure susceptibility are not fully understood. Several previous studies have shown a loss of subgroups of GABAergic inhibitory interneurons in the hippocampus of aged rodents, yet the network excitability intrinsic to the aged hippocampus remains to be elucidated. The aim of this study is to examine age-dependent changes of hippocampal network activities in young adult (3–5months), aging (16–18months), and aged (24–28months) mice. We conducted intracranial electroencephalographic (EEG) recordings in free-moving animals and extracellular recordings in hippocampal slices in vitro. EEG recordings revealed frequent spikes in aging and aged mice but only occasionally in young adults. These EEG spikes were suppressed following diazepam administration. Spontaneous field potentials with large amplitudes were frequently observed in hippocampal slices of aged mice but rarely in slices from young adults. These spontaneous field potentials originated from the CA3 area and their generation was dependent upon the excitatory glutamatergic activity. We therefore postulate that hippocampal network excitability is increased in aged mice and that such hyperactivity may be relevant to the increased seizure susceptibility observed in aged subjects.

Simultaneous recording of hippocampal oxygen and glucose in real time using constant potential amperometry in the freely-moving rat

Amperometric sensors for oxygen and glucose allow for real time recording from the brain in freely-moving animals. These sensors have been used to detect activity- and drug-induced changes in metabolism in a number of brain regions but little attention has been given over to the hippocampus despite its importance in cognition and disease. Sensors for oxygen and glucose were co-implanted into the hippocampus and allowed to record for several days. Baseline recordings show that basal concentrations of hippocampal oxygen and glucose are 100.26±5.76μM and 0.60±0.06mM respectively. Furthermore, stress-induced changes in neural activity have been shown to significantly alter concentrations of both analytes in the hippocampus. Administration of O2 gas to the animals’ snouts results in significant increases in hippocampal oxygen and glucose and administration of N2 gas results in a significant decrease in hippocampal oxygen. Chloral hydrate-induced anaesthesia causes a significant increase in hippocampal oxygen whereas treatment with the carbonic anhydrase inhibitor acetazolamide significantly increases hippocampal oxygen and glucose. These findings provide real time electrochemical data for the hippocampus which has been previously impossible with traditional methods such as microdialysis or ex vivo analysis. As such, these sensors provide a window into hippocampal function which can be used in conjunction with behavioural and pharmacological interventions to further elucidate the functions and mechanisms of action of the hippocampus in normal and disease states.

An implantable wireless neural interface for recording cortical circuit dynamics in moving primates

Objective. Neural interface technology suitable for clinical translation has the potential to significantly impact the lives of amputees, spinal cord injury victims and those living with severe neuromotor disease. Such systems must be chronically safe, durable and effective. Approach. We have designed and implemented a neural interface microsystem, housed in a compact, subcutaneous and hermetically sealed titanium enclosure. The implanted device interfaces the brain with a 510k-approved, 100-element silicon-based microelectrode array via a custom hermetic feedthrough design. Full spectrum neural signals were amplified (0.1 Hz to 7.8 kHz, 200× gain) and multiplexed by a custom application specific integrated circuit, digitized and then packaged for transmission. The neural data (24 Mbps) were transmitted by a wireless data link carried on a frequency-shift-key-modulated signal at 3.2 and 3.8 GHz to a receiver 1 m away by design as a point-to-point communication link for human clinical use. The system was powered by an embedded medical grade rechargeable Li-ion battery for 7 h continuous operation between recharge via an inductive transcutaneous wireless power link at 2 MHz. Main results. Device verification and early validation were performed in both swine and non-human primate freely-moving animal models and showed that the wireless implant was electrically stable, effective in capturing and delivering broadband neural data, and safe for over one year of testing. In addition, we have used the multichannel data from these mobile animal models to demonstrate the ability to decode neural population dynamics associated with motor activity. Significance. We have developed an implanted wireless broadband neural recording device evaluated in non-human primate and swine. The use of this new implantable neural interface technology can provide insight into how to advance human neuroprostheses beyond the present early clinical trials. Further, such tools enable mobile patient use, have the potential for wider diagnosis of neurological conditions and will advance brain research.

Controlled Iontophoresis Coupled with Fast-Scan Cyclic Voltammetry/Electrophysiology in Awake, Freely Moving Animals

Controlled Iontophoresis Coupled with Fast-Scan Cyclic Voltammetry/Electrophysiology in Awake, Freely Moving Animals

Commentary: How Ethanol Short-Circuits the Cerebellum-Actions on Golgi Cells in Freely-Moving Animals

This commentary discusses the important contributions of the article published in this journal by Huang and colleagues, titled, "Acute ethanol exposure increases firing and induces oscillations in cerebellar Golgi cells of freely moving rats." In this manuscript, Huang and colleagues present a number of interesting and important findings. While it has been shown previously that ethanol (EtOH) causes an increase in the firing of cerebellar Golgi cells in brain slice preparations and anesthetized animals, here the authors provide the first evidence that this action of EtOH occurs in vivo in freely moving, unanesthetized animals. These results also enhance our understanding of cerebellar functioning by describing the mechanism by which EtOH essentially de-afferentates (blocks specific inputs to) the cerebellum from the normal processing of sensory signals due to EtOH-induced Golgi neuron excitation, resulting in inhibition of granule cells. Furthermore, the authors characterize the novel observation of EtOH-induced neuronal oscillations, which was not previously observed in other preparations.

A digital wireless system for closed-loop inhibition of nociceptive signals

Neurostimulation of the spinal cord or brain has been used to inhibit nociceptive signals in pain management applications. Nevertheless, most of the current neurostimulation models are based on open-loop system designs. There is a lack of closed-loop systems for neurostimulation in research with small freely-moving animals and in future clinical applications. Based on our previously developed analog wireless system for closed-loop neurostimulation, a digital wireless system with real-time feedback between recorder and stimulator modules has been developed to achieve multi-channel communication. The wireless system includes a wearable recording module, a wearable stimulation module and a transceiver connected to a computer for real-time and off-line data processing, display and storage. To validate our system, wide dynamic range neurons in the spinal cord dorsal horn have been recorded from anesthetized rats in response to graded mechanical stimuli (brush, pressure and pinch) applied in the hind paw. The identified nociceptive signals were used to automatically trigger electrical stimulation at the periaqueductal gray in real time to inhibit their own activities by the closed-loop design. Our digital wireless closed-loop system has provided a simplified and efficient method for further study of pain processing in freely-moving animals and potential clinical application in patients.

Using GFP Video to Track 3D Movement and Conditional Gene Expression in Free-Moving Flies

BackgroundIn vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.ResultsWe report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.ConclusionsFluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.

In vivo characterisation of a Nafion®-modified Pt electrode for real-time nitric oxide monitoring in brain extracellular fluid

A Nafion®-modified Pt sensor developed for real-time neurochemical monitoring in freely-moving animals has now been characterised in vivo for the detection of nitric oxide (NO). Experiments were undertaken to test sensitivity, selectivity and stability. In control experiments, local administration of aCSF resulted in a decrease in signal (42 ± 12 pA, n = 9), while systemic administration of saline caused a transient increase in signal (22 ± 3 pA, n = 9) from baseline levels. Local MD perfusions of known concentrations of NO (1 mM) and L-arginine (720 μM) caused significant increases in current (4 ± 1 nA (n = 7) and 271 ± 37 pA (n = 5) respectively) while systemic administration of the non-selective NOS inhibitor L-NAME produced a significant decrease in current (91 ± 19 pA, n = 4) compared to baseline levels. Confirmation that the sensor remained selective was achieved by injection of ascorbic acid (2 g kg−1, i.p). The oxidation current showed no variation over the selected time interval of 60 min, indicating no deterioration of the polymer membrane. Finally, after an initial decrease in sensitivity (ca. 50%) within the first 24 h of implantation the Nafion®-modified Pt sensor showed excellent stability under physiological conditions over extended periods (8 days) validating its use for continuous chronic in vivo recordings.

The role of cortical and hypothalamic histamine‐3 receptors in the modulation of central histamine neurotransmission: an in vivo electrophysiology and microdialysis study

The current study aimed to investigate the effect of histamine-3 (H(3)) receptors, expressed in the tuberomammillary nucleus (TMN) of the hypothalamus and in the prefrontal cortex (PFC), on histamine neurotransmission in the rat brain. The firing activity of histamine neurons in the TMN was measured using in vivo extracellular single-unit electrophysiology, under propofol anesthesia. Extracellular histamine levels were determined using the dual (PFC and TMN) probe microdialysis, in freely-moving animals. Histamine levels in dialysates were determined using high-performance liquid chromatography (HPLC) and fluorescence detection. It was found that systemic administration of the selective H(3)-agonist, immepip, decreases, and the reverse H(3) /H(4)-agonist, thioperamide, increases the firing activity of histamine neurons in the TMN and the release of histamine in TMN and PFC. Local perfusion of immepip into the TMN increased, and thioperamide decreased, histamine levels in the TMN but not in the PFC. Local perfusion of immepip into the PFC, however, decreased extracellular histamine levels in both TMN and PFC. It can be concluded that brain H(3) receptors, and especially those expressed in the PFC, play an important role in the autoregulation of histamine neurotransmission. It is possible that H(3) receptors in the PFC are expressed on pyramidal neurons projecting to the TMN, and activation of these receptors diminishes glutamate excitatory input from PFC to the TMN. As the brain histamine system has a role in pathophysiology of psychotic, affective, cognitive, sleep and eating disorders, H(3) receptors are potential targets for future CNS medications.