Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Two Types of Auditory Spatial Receptive Fields in Different Parts of the Chicken's Midbrain.

The optic tectum (OT) is an avian midbrain structure involved in the integration of visual and auditory stimuli. Studies in the barn owl, an auditory specialist, have shown that spatial auditory information is topographically represented in the OT. Little is known about how auditory space is represented in the midbrain of birds with generalist hearing, i.e., most of avian species lacking peripheral adaptations such as facial ruffs or asymmetric ears. Thus, we conducted in vivo extracellular recordings of single neurons in the OT and in the external portion of the formatio reticularis lateralis (FRLx), a brain structure located between the inferior colliculus (IC) and the OT, in anaesthetized chickens of either sex. We found that most of the auditory spatial receptive fields (aSRFs) were spatially confined both in azimuth and elevation, divided into two main classes: round aSRFs, mainly present in the OT, and annular aSRFs, with a ring-like shape around the interaural axis, mainly present in the FRLx. Our data further indicate that interaural time difference (ITD) and interaural level difference (ILD) play a role in the formation of both aSRF classes. These results suggest that, unlike mammals and owls which have a congruent representation of visual and auditory space in the OT, generalist birds separate the computation of auditory space in two different midbrain structures. We hypothesize that the FRLx-annular aSRFs define the distance of a sound source from the axis of the lateral visual fovea, whereas the OT-round aSRFs are involved in multimodal integration of the stimulus around the lateral fovea.SIGNIFICANCE STATEMENT Previous studies implied that auditory spatial receptive fields (aSRFs) in the midbrain of generalist birds are only confined along azimuth. Interestingly, we found SRFs s in the chicken to be confined along both azimuth and elevation. Moreover, the auditory receptive fields are arranged in a concentric manner around the overlapping interaural and visual axes. These data suggest that in generalist birds, which mainly rely on vision, the auditory system mainly serves to align auditory stimuli with the visual axis, while auditory specialized birds like the barn owl compute sound sources more precisely and integrate sound positions in the multimodal space map of the optic tectum (OT).

Enteric neuroanatomy and smooth muscle activity in the western diamondback rattlesnake (Crotalus atrox).

BACKGROUND: Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings. RESULTS: A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments. CONCLUSIONS: The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

"Shepherd's crook" neurons drive and synchronize the enhancing and suppressive mechanisms of the midbrain stimulus selection network.

The optic tectum (TeO), or superior colliculus, is a multisensory midbrain center that organizes spatially orienting responses to relevant stimuli. To define the stimulus with the highest priority at each moment, a network of reciprocal connections between the TeO and the isthmi promotes competition between concurrent tectal inputs. In the avian midbrain, the neurons mediating enhancement and suppression of tectal inputs are located in separate isthmic nuclei, facilitating the analysis of the neural processes that mediate competition. A specific subset of radial neurons in the intermediate tectal layers relay retinal inputs to the isthmi, but at present it is unclear whether separate neurons innervate individual nuclei or a single neural type sends a common input to several of them. In this study, we used in vitro neural tracing and cell-filling experiments in chickens to show that single neurons innervate, via axon collaterals, the three nuclei that comprise the isthmotectal network. This demonstrates that the input signals representing the strength of the incoming stimuli are simultaneously relayed to the mechanisms promoting both enhancement and suppression of the input signals. By performing in vivo recordings in anesthetized chicks, we also show that this common input generates synchrony between both antagonistic mechanisms, demonstrating that activity enhancement and suppression are closely coordinated. From a computational point of view, these results suggest that these tectal neurons constitute integrative nodes that combine inputs from different sources to drive in parallel several concurrent neural processes, each performing complementary functions within the network through different firing patterns and connectivity.

Zebrin Expression in the Cerebellum of Two Crocodilian Species.

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII-) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII-) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/- stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals).

Secretagogin Immunoreactivity Reveals Lugaro Cells in the Pigeon Cerebellum.

Lugaro cells are inhibitory interneurons found in the upper granular layer of the cerebellar cortex, just below or within the Purkinje cell layer. They are characterized by (1) a fusiform soma oriented in the parasagittal plane, (2) two pairs of dendrites emanating from opposite ends of the soma, (3) innervation from Purkinje cell collaterals, and (4) an axon that projects into the molecular layer akin to granular cell parallel fibers. Lugaro cells have been described in mammals, but not in other vertebrate classes, save one report in teleost fish. Here, we propose the existence of Lugaro cells in the avian cerebellum based on the morphological characteristics and connectivity described above. Immunohistochemical staining against the calcium binding protein secretagogin (SCGN) revealed Lugaro-like cells in the pigeon cerebellum. Some SCGN-labeled cells exhibit fusiform somata and dendrites parallel to the Purkinje cell layer in the parasagittal plane, as well as long axons that project into the molecular layer and travel alongside parallel fibers in the coronal plane. While mammalian Lugaro cells are known to express calretinin, the SCGN-labeled cells in the pigeon do not. SCGN-labeled cells also express glutamic acid decarboxylase, confirming their inhibitory function. Calbindin labeling revealed Purkinje cell terminals surrounding the SCGN-expressing cells. Our results suggest that Lugaro cells are more widespread among vertebrates than previously thought and may be a characteristic of the cerebellum of all vertebrates.

Optic and echo-acoustic flow interact in bats.

Echolocating bats are known to fly and forage in complete darkness, using the echoes of their actively emitted calls to navigate and to detect prey. However, under dim light conditions many bats can also rely on vision. Many flying animals have been shown to navigate by optic flow information and, recently, bats were shown to exploit echo-acoustic flow to navigate through dark habitats. Here, we show for the bat Phyllostomus discolor that, in lighted habitats where self-motion-induced optic flow is strong, optic and echo-acoustic flow interact to guide navigation. Echo-acoustic flow showed a surprisingly strong effect compared with optic flow. We thus demonstrate multimodal interaction between two far-ranging spatial senses, vision and echolocation, available in this combination almost exclusively in bats and toothed whales. Our results highlight the importance of merging information from different sensory systems in a sensory-specialist animal to successfully navigate and hunt under difficult conditions.

The Neural Basis of Dim-Light Vision in Echolocating Bats.

Echolocating bats evolved a sophisticated biosonar imaging system that allows for a life in dim-light habitats. However, especially for far-range operations such as homing, bats can support biosonar by vision. Large eyes and a retina that mainly consists of rods are assumed to be the optical adjustments that enable bats to use visual information at low light levels. In addition to optical mechanisms, many nocturnal animals evolved neural adaptations such as elongated integration times or enlarged spatial sampling areas to further increase the sensitivity of their visual system by temporal or spatial summation of visual information. The neural mechanisms that underlie the visual capabilities of echolocating bats have, however, so far not been investigated. To shed light on spatial and temporal response characteristics of visual neurons in an echolocating bat, Phyllostomus discolor, we recorded extracellular multiunit activity in the retino-recipient superficial layers of the superior colliculus (SC). We discovered that response latencies of these neurons were generally in the mammalian range, whereas neural spatial sampling areas were unusually large compared to those measured in the SC of other mammals. From this we suggest that echolocating bats likely use spatial but not temporal summation of visual input to improve visual performance under dim-light conditions. Furthermore, we hypothesize that bats compensate for the loss of visual spatial precision, which is a byproduct of spatial summation, by integration of spatial information provided by both the visual and the biosonar systems. Given that knowledge about neural adaptations to dim-light vision is mainly based on studies done in non-mammalian species, our novel data provide a valuable contribution to the field and demonstrate the suitability of echolocating bats as a nocturnal animal model to study the neurophysiological aspects of dim-light vision.

Synaptic convergence of afferent inputs in primary infrared-sensitive nucleus (LTTD) neurons of rattlesnakes (Crotalinae) as the origin for sensory contrast enhancement.

Pitvipers have a specialized sensory system in the upper jaw to detect infrared (IR) radiation. The bilateral pit organs resemble simple pinhole cameras that map IR objects onto the sensory epithelium as blurred representations of the environment. Trigeminal afferents transmit information about changing temperature patterns as neuronal spike discharge in a topographic manner to the hindbrain nucleus of the lateral descending trigeminal tract (LTTD). A presumed, yet so far unknown neuronal connectivity within this central nucleus exerts a synaptic computation that constrains the relatively large receptive field of primary afferent fibers. Here, we used intracellular recordings of LTTD neurons in isolated rattlesnake brains to decipher the spatio-temporal pattern of excitatory and inhibitory responses following electrical stimulation of single and multiple peripheral pit organ-innervating nerve branches. The responses of individual neurons consisted of complex spike sequences that derived from spatially and temporally specific interactions between excitatory and inhibitory synaptic inputs from the same as well as from adjacent peripheral nerve terminal areas. This pattern complies with a central excitation that is flanked by a delayed lateral inhibition, thereby enhancing the contrast of IR sensory input, functionally reminiscent of the computations for contrast enhancement in the peripheral visual system.

Congruent representation of visual and acoustic space in the superior colliculus of the echolocating bat Phyllostomus discolor.

The midbrain superior colliculus (SC) commonly features a retinotopic representation of visual space in its superficial layers, which is congruent with maps formed by multisensory neurons and motor neurons in its deep layers. Information flow between layers is suggested to enable the SC to mediate goal-directed orienting movements. While most mammals strongly rely on vision for orienting, some species such as echolocating bats have developed alternative strategies, which raises the question how sensory maps are organized in these animals. We probed the visual system of the echolocating bat Phyllostomus discolor and found that binocular high acuity vision is frontally oriented and thus aligned with the biosonar system, whereas monocular visual fields cover a large area of peripheral space. For the first time in echolocating bats, we could show that in contrast with other mammals, visual processing is restricted to the superficial layers of the SC. The topographic representation of visual space, however, followed the general mammalian pattern. In addition, we found a clear topographic representation of sound azimuth in the deeper collicular layers, which was congruent with the superficial visual space map and with a previously documented map of orienting movements. Especially for bats navigating at high speed in densely structured environments, it is vitally important to transfer and coordinate spatial information between sensors and motor systems. Here, we demonstrate first evidence for the existence of congruent maps of sensory space in the bat SC that might serve to generate a unified representation of the environment to guide motor actions.

Multimodal integration in the chicken.

In everyday life we constantly perceive and discriminate between a large variety of sensory inputs, the vast majority of which consist of more than one modality. We performed two experiments to investigate whether chickens use the information present in multimodal signals. To test whether audiovisual stimuli are better detected than visual or acoustic stimuli alone, we first measured the detection threshold with a staircase paradigm. We found that chickens were able to detect weaker stimuli using audiovisual stimuli. Next, we tested whether the multimodal nature of a stimulus also increases the discrimination between two stimuli by measuring the smallest difference that the animals could still distinguish from each other. We found that chickens can discriminate smaller differences using audiovisual stimuli in comparison to visual stimuli alone, but not in comparison to acoustic stimuli alone. Thus, even in a relatively unspecialized species such as the chicken, the benefits of multimodal integration are exploited for sensory processing.

Echo-acoustic flow affects flight in bats.

Flying animals need to react fast to rapid changes in their environment. Visually guided animals use optic flow, generated by their movement through structured environments. Nocturnal bats cannot make use of optic flow, but rely mostly on echolocation. Here, we show that bats exploit echo-acoustic flow to negotiate flight through narrow passages. Specifically, bats' flight between lateral structures is significantly affected by the echo-acoustic salience of those structures, independent of their physical distance. This is true even though echolocation, unlike vision, provides explicit distance cues. Moreover, the bats reduced the echolocation sound levels in stronger flow, probably to compensate for the increased summary target strength of the lateral reflectors. However, bats did not reduce flight velocity under stronger echo-acoustic flow. Our results demonstrate that sensory flow is a ubiquitous principle for flight guidance, independent of the fundamentally different peripheral representation of flow across the senses of vision and echolocation.

Local cholinergic interneurons modulate GABAergic inhibition in the chicken optic tectum.

The chicken optic tectum (TeO) and its mammalian counterpart, the superior colliculus, are important sensory integration centers. Multimodal information is represented in a topographic map, which plays a role in spatial attention and orientation movements. The TeO is organised in 15 layers with clear input and output regions, and further interconnected with the isthmic nuclei (NI), which modulate the response in a winner-takes-all fashion. While many studies have analysed tectal cell types and their modulation from the isthmic system physiologically, little is known about local network activity and its modulation in the tectum. We have recently shown with voltage-sensitive dye imaging that electrical stimulation of the retinorecipient layers results in a stereotypic response, which is under inhibitory control [S. Weigel & H. Luksch (2012) J. Neurophysiol., 107, 640-648]. Here, we analysed the contribution of acetylcholine (ACh) and the NI to evoked tectal responses using a pharmacological approach in a midbrain slice preparation. Application of the nicotinic ACh receptor (AChR) antagonist curarine increased the tectal response in amplitude, duration and lateral extent. This effect was similar but less pronounced when γ-aminobutyric acid(A) receptors were blocked, indicating interaction of inhibitory and cholinergic neurons. The muscarinic AChR antagonist atropine did not change the response pattern. Removal of the NI, which are thought to be the major source of cholinergic input to the TeO, reduced the response only slightly and did not result in a disinhibition. Based on the data presented here and the neuroanatomical literature of the avian TeO, we propose a model of the underlying local circuitry.

Neurochemistry and circuit organization of the lateral spiriform nucleus of birds: A uniquely nonmammalian direct pathway component of the basal ganglia.

We used diverse methods to characterize the role of avian lateral spiriform nucleus (SpL) in basal ganglia motor function. Connectivity analysis showed that SpL receives input from globus pallidus (GP), and the intrapeduncular nucleus (INP) located ventromedial to GP, whose neurons express numerous striatal markers. SpL-projecting GP neurons were large and aspiny, while SpL-projecting INP neurons were medium sized and spiny. Connectivity analysis further showed that SpL receives inputs from subthalamic nucleus (STN) and substantia nigra pars reticulata (SNr), and that the SNr also receives inputs from GP, INP, and STN. Neurochemical analysis showed that SpL neurons express ENK, GAD, and a variety of pallidal neuron markers, and receive GABAergic terminals, some of which also contain DARPP32, consistent with GP pallidal and INP striatal inputs. Connectivity and neurochemical analysis showed that the SpL input to tectum prominently ends on GABAA receptor-enriched tectobulbar neurons. Behavioral studies showed that lesions of SpL impair visuomotor behaviors involving tracking and pecking moving targets. Our results suggest that SpL modulates brainstem-projecting tectobulbar neurons in a manner comparable to the demonstrated influence of GP internus on motor thalamus and of SNr on tectobulbar neurons in mammals. Given published data in amphibians and reptiles, it seems likely the SpL circuit represents a major direct pathway-type circuit by which the basal ganglia exerts its motor influence in nonmammalian tetrapods. The present studies also show that avian striatum is divided into three spatially segregated territories with differing connectivity, a medial striato-nigral territory, a dorsolateral striato-GP territory, and the ventrolateral INP motor territory.

Spatiotemporal analysis of electrically evoked activity in the chicken optic tectum: a VSDI study.

The midbrain is an important processing area for sensory information in vertebrates. The optic tectum and its mammalian counterpart, the superior colliculus, receive multimodal, topographic information and contain a sensory map that plays a role in spatial attention and orientation movements. Many studies have investigated the tectal circuitry by cytochemistry and by characterization of particular cell types. However, only a few studies have investigated network activation throughout the depth of the tectum. Our study provides the first data on spatiotemporal activity profiles in the depth and width of the avian optic tectum. We used an optical imaging approach with voltage-sensitive dyes to investigate population responses at a high temporal and spatial resolution. With the necessary caution due to cell extension across several layers, we can thus link our findings tentatively with the general layout of the avian optic tectum. Single electrical stimuli in the retinorecipient layers 1-4 evoked a complex optical response pattern with two components: a short, strong transient response and a weaker persistent response that lasted several hundred milliseconds. The response started in layer 5 and spread within this layer before it propagated into deeper layers. This is in line with neuroanatomical and earlier physiological data. Analysis of temporal sequence and pharmacological manipulations revealed that these responses were mainly driven by postsynaptic activation. Thus tectal network responses to patterned input can be studied by voltage-sensitive dye imaging.

Azimuthal sound localization in the chicken.

Sound localization is crucial for the survival and reproduction of animals, including non-auditory specialist animals such as the majority of avian species. The chicken (Gallus gallus) is a well-suited representative of a non-auditory specialist bird and several aspects of its auditory system have been well studied in the last decades. We conducted a behavioral experiment where 3 roosters performed a sound localization task with broad-band noise, using a 2-alternative forced choice paradigm. We determined the minimum audible angle (MAA) as measure for localization acuity. In general, our results compare to previous MAA measurements with hens in Go/NoGo tasks. The chicken has high localization acuity compared to other auditory generalist bird species tested so far. We found that chickens were better at localizing broadband noise with long duration (1 s; MAA = 16°) compared to brief duration (0.1 s; MAA = 26°). Moreover, the interaural difference in time of arrival and level (ITD and ILD, respectively) at these MAAs are comparable to what measured in other non-auditory specialist bird species, indicating that they might be sufficiently broad to be informative for azimuthal sound localization.

In vivo closed-loop control of a locust's leg using nerve stimulation.

Activity of an innervated tissue can be modulated based on an acquired biomarker through feedback loops. How to convert this biomarker into a meaningful stimulation pattern is still a topic of intensive research. In this article, we present a simple closed-loop mechanism to control the mean angle of a locust's leg in real time by modulating the frequency of the stimulation on its extensor motor nerve. The nerve is interfaced with a custom-designed cuff electrode and the feedback loop is implemented online with a proportional control algorithm, which runs solely on a microcontroller without the need of an external computer. The results show that the system can be controlled with a single-input, single-output feedback loop. The model described in this article can serve as a primer for young researchers to learn about neural control in biological systems before applying these concepts in advanced systems. We expect that the approach can be advanced to achieve control over more complex movements by increasing the number of recorded biomarkers and selective stimulation units.

Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum.

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.

Recurrent antitopographic inhibition mediates competitive stimulus selection in an attention network.

Topographically organized neurons represent multiple stimuli within complex visual scenes and compete for subsequent processing in higher visual centers. The underlying neural mechanisms of this process have long been elusive. We investigate an experimentally constrained model of a midbrain structure: the optic tectum and the reciprocally connected nucleus isthmi. We show that a recurrent antitopographic inhibition mediates the competitive stimulus selection between distant sensory inputs in this visual pathway. This recurrent antitopographic inhibition is fundamentally different from surround inhibition in that it projects on all locations of its input layer, except to the locus from which it receives input. At a larger scale, the model shows how a focal top-down input from a forebrain region, the arcopallial gaze field, biases the competitive stimulus selection via the combined activation of a local excitation and the recurrent antitopographic inhibition. Our findings reveal circuit mechanisms of competitive stimulus selection and should motivate a search for anatomical implementations of these mechanisms in a range of vertebrate attentional systems.

Chronic Multi-Electrode Electromyography in Snakes.

Knowledge about body motion kinematics and underlying muscle contraction dynamics usually derives from electromyographic (EMG) recordings. However, acquisition of such signals in snakes is challenging because electrodes either attached to or implanted beneath the skin may unintentionally be removed by force or friction caused from undulatory motion, thus severely impeding chronic EMG recordings. Here, we present a reliable method for stable subdermal implantation of up to eight bipolar electrodes above the target muscles. The mechanical stability of the inserted electrodes and the overnight coverage of the snake body with a "sleeping bag" ensured the recording of reliable and robust chronic EMG activity. The utility of the technique was verified by daily acquisition of high signal-to-noise activity from all target sites over four consecutive days during stimulus-evoked postural reactions in Amazon tree boas and Western diamondback rattlesnakes. The successful demonstration of the chronic recording suggests that this technique can improve acute experiments by enabling the collection of larger data sets from single individuals.