Electrophysiological Techniques Research Articles

Alfaxalone is an effective anesthetic for the electrophysiological study of anoxia-tolerance mechanisms in western painted turtle pyramidal neurons.

Anoxia in the mammalian brain leads to hyper-excitability and cell death; however, this cascade of events does not occur in the anoxia-tolerant brain of the western painted turtle, Chrysemys picta belli. The painted turtle has become an important anoxia-tolerant model to study brain, heart, and liver function in the absence of oxygen, but being anoxia-tolerant likely means that decapitation alone is not a suitable method of euthanasia. Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. We find that Alfaxalone has no long-term impact on action potential parameters or whole-cell conductance. When acutely applied to naïve tissue, Alfaxalone did lengthen GABAA receptor current decay rates by 1.5-fold. Following whole-animal sedation with Alfaxalone, evoked whole cell GABAA receptor current decay rates displayed an increasing trend with 1 and 2 hours after brain sheet preparation, but showed no significant change after a 3-hour washout period. Therefore, we conclude that Alfaxalone is a suitable anesthetic for same day use in electrophysiological studies in western painted turtle brain tissue.

Analysis of EEG characteristics in Alzheimer’s disease

Alzheimer’s disease (AD) brings a heavy burden to society and families because of the high misdiagnosis rate, prevalence rate, and disability rate. At present, the treatment of AD still faces great challenges. Early intervention can effectively delay the disease progression and improve the burden of symptom care. However, the key to early intervention is accurately identifying and assessing the disease. The examination is difficult to be widely used because of its high invasive cost. There is an urgent need for a simple, convenient, and non-invasive diagnosis of AD. Studies have shown that the brain number of AD patients is slower than that of healthy people, and the complexity of EEG is disturbed. Clinical practice has proved that the specificity and sensitivity of α, β, δ, θ absolute power and α/θ absolute power ratio, especially θ absolute power and α/θ absolute power ratio in the model, are important electrophysiological indexes for AD diagnosis EEG. Non-invasive and economical electrophysiological techniques can characterize and identify the EEG characteristics of AD patients from different angles. They are relatively simple to implement, and patients have a high degree of cooperation. Therefore, EEG, as a diagnostic method of AD, can improve the accuracy of differential diagnosis and early detection of AD and quantify the severity of the disease. It has the advantage of non-invasive convenience and price, which is of great significance for early diagnosis of AD.

Neuron-microelectrode junction induced by an engineered synapse organizer

The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1β and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording.

Neural correlates of recalled sadness, joy, and fear states: a source reconstruction EEG study.

The capacity to understand the others' emotional states, particularly if negative (e.g. sadness or fear), underpins the empathic and social brain. Patients who cannot express their emotional states experience social isolation and loneliness, exacerbating distress. We investigated the feasibility of detecting non-invasive scalp-recorded electrophysiological signals that correspond to recalled emotional states of sadness, fear, and joy for potential classification. The neural activation patterns of 20 healthy and right-handed participants were studied using an electrophysiological technique. Analyses were focused on the N400 component of Event-related potentials (ERPs) recorded during silent recall of subjective emotional states; Standardized weighted Low-resolution Electro-magnetic Tomography (swLORETA) was employed for source reconstruction. The study classified individual patterns of brain activation linked to the recollection of three distinct emotional states into seven regions of interest (ROIs). Statistical analysis (ANOVA) of the individual magnitude values revealed the existence of a common emotional circuit, as well as distinct brain areas that were specifically active during recalled sad, happy and fearful states. In particular, the right temporal and left superior frontal areas were more active for sadness, the left limbic region for fear, and the right orbitofrontal cortex for happy affective states. In conclusion, this study successfully demonstrated the feasibility of detecting scalp-recorded electrophysiological signals corresponding to internal and subjective affective states. These findings contribute to our understanding of the emotional brain, and have potential applications for future BCI classification and identification of emotional states in LIS patients who may be unable to express their emotions, thus helping to alleviate social isolation and sense of loneliness.

5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Parietal-Frontal Pathway Controls Relapse of Fear Memory in a Novel Context

BackgroundFear responses significantly affect daily life and shape our approach to uncertainty. However, the potential resurgence of fear in unfamiliar situations poses a significant challenge to exposure-based therapies for maladaptive fear responses. Nonetheless, how novel contextual stimuli are associated with the relapse of extinguished fear remains unknown. MethodsUsing a context-dependent fear renewal model, the functional circuits and underlying mechanisms of the posterior parietal cortex (PPC) and anterior cingulate cortex (ACC) were investigated using optogenetic, histological, in vivo, and ex vivo electrophysiological and pharmacological techniques. ResultsWe demonstrated that the PPC-to-ACC pathway governs fear relapse in a novel context. We observed enhanced populational calcium activity in the ACC neurons that received projections from the PPC and increased synaptic activity in the basolateral amygdala–projecting PPC-to-ACC neurons upon renewal in a novel context, where excitatory postsynaptic currents amplitudes increased but inhibitory postsynaptic current amplitudes decreased. In addition, we found that parvalbumin–expressing interneurons controlled novel context-dependent fear renewal, which was blocked by the chronic administration of fluoxetine. ConclusionsOur findings highlight the PPC-to-ACC pathway in mediating the relapse of extinguished fear in novel contexts, thereby contributing significant insights into the intricate neural mechanisms that govern fear renewal.

Berger and the Breakthrough: A Centennial Celebration of the Human Electroencephalogram

ABSTRACT A century ago, it was a challenge for neuroscientists to decipher the mysteries of human brain functioning until German psychiatrist Hans Berger discovered what is now one of the most well-known electrophysiological recording techniques to examine brain function, EEG. He is rightly regarded as the Father of The Electroencephalogram (EEG), since he performed the first human electroencephalogram in 1924. Berger attempted to investigate the connection between psychology and physiology and to solve the “psychic energy” enigma. Despite turbulence in his professional life and slow progress in his research, he persevered and succeeded in giving humankind an indispensable technique that is now widely used in clinical and research practice. His publications on EEG provide valuable insight into our current understanding of several of the brain’s responses to physiological and pathological phenomena. In July 2024, it will be 100 years since Berger recorded the first human EEG, and that calls for a celebration among EEG researchers, neuroscientists, psychiatrists, and neurologists. This article presents a brief account of his journey and commemorates Hans Berger’s contributions to the field of neurodiagnostics.

Glutamatergic neurons in the paraventricular nucleus of the hypothalamus participate in the regulation of visceral pain induced by pancreatic cancer in mice.

Visceral pain induced by pancreatic cancer seriously affects patients' quality of life, and there is no effective treatment, because the mechanism of its neural circuit is unknown. Therefore, the aim of this study is to explore the main neural circuit mechanism regulating visceral pain induced by pancreatic cancer in mice. The mouse model of pancreatic cancer visceral pain was established on C57BL/6N mice by pancreatic injection of mPAKPC-luc cells. Abdominal mechanical hyperalgesia and hunch score were performed to assess visceral pain; the pseudorabies virus (PRV) was used to identify the brain regions innervating the pancreas; the c-fos co-labeling method was used to ascertain the types of activated neurons; in vitro electrophysiological patch-clamp technique was used to record the electrophysiological activity of specific neurons; the calcium imaging technique was used to determine the calcium activity of specific neurons; specific neuron destruction and chemogenetics methods were used to explore whether specific neurons were involved in visceral pain induced by pancreatic cancer. The PRV injected into the pancreas was detected in the paraventricular nucleus of the hypothalamus (PVN). Immunofluorescence staining showed that the majority of c-fos were co-labeled with glutamatergic neurons in the PVN. In vitro electrophysiological results showed that the firing frequency of glutamatergic neurons in the PVN was increased. The calcium imaging results showed that the calcium activity of glutamatergic neurons in the PVN was enhanced. Both specific destruction of glutamatergic neurons and chemogenetics inhibition of glutamatergic neurons in the PVN alleviated visceral pain induced by pancreatic cancer. Glutamatergic neurons in the PVN participate in the regulation of visceral pain induced by pancreatic cancer in mice, providing new insights for the discovery of effective targets for the treatment of pancreatic cancer visceral pain.

The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters.

In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.

Accurate diagnosis and effective treatment of abnormal meridians in erectile dysfunction patients based on infrared thermography: an electrophysiological technique study.

An increasing body of research has demonstrated that appropriate stimulation of the meridians and acupoints in the human body can play a preventative and therapeutic role in diseases. This study combines the use of infrared thermography with intelligent electrophysiological diagnostic system (iEDS) to accurately diagnose and apply transdermal low-frequency electrical stimulation to treat abnormal meridians in patients with erectile dysfunction (ED). The treatment protocol included 6 treatments (each lasting 30 min and performed twice a week). The International Index of Erectile Function-5 (IIEF-5), Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), and Erection Hardness Scale were used to assess treatment results. A total of 62 patients were included in this study, with 31 patients in the treatment group and 31 patients in the sham therapy group. After six treatments, the treatment group improved significantly in IIEF-5 (15.52 ± 2.06 vs. 18.84 ± 2.67, p < 0.001), PHQ-9 (8.32 ± 6.33 vs. 4.87 ± 4.41, p < 0.001), GAD-7 (5.32 ± 5.08 vs. 2.94 ± 3.31, p = 0.003), and EHS (2.48 (2.00, 3.00) vs. 2.90 (2.00, 3.00), p = 0.007). After six sham treatment sessions, no improvements in any of the scores were reported in the sham therapy group. Following that, this group had an additional six treatments of regular therapy, which resulted in statistically significant improvements in IIEF-5 (16.65 ± 1.96 VS. 19.16 ± 2.40, p < 0.001), PHQ-9 (8.81 ± 6.25 VS. 4.97 ± 4.36, p < 0.001), GAD-7 (5.74 ± 5.18 VS. 3.68 ± 3.42, p < 0.001), and EHS (2.61 (2.00, 3.00) VS. 3.03 (2.00, 4.00), p = 0.003). No adverse events were reported regarding penile discomfort, pain, injury, or deformity. CLINICAL TRIALS: The study protocol is registered in the Clinical Trials Registry with the identification number ChiCTR2300070262.

Research progress on insect visual electrophysiological techniques.

Insect visual electrophysiological techniques are important to study the electrical characteristics of photoreceptor cells and visual neurons in insects, including electroretinography (ERG) and microelectrode intracellular recording (MIR). ERG records the changes of voltage or electric current in the retina of insects in response to different light stimuli, which occurs outside the cell. MIR records the changes in individual photoreceptor cells or visual neurons of an insect exposed to different lights, which occurs inside the cell. Insect visual electrophysiological techniques can explore the mechanism of electrophysiological response of insects' vision to light and reveal their sensitive light spectra and photoreceptor types. This review introduced the basic structure and the principle of ERG and MIR, and summarized their applications in insect researches in the past 20 years, which would provide references for elucidating the mechanism of light perception in insects and the use of insect phototropism to control pests.

Attention to audiovisual speech shapes neural processing through feedback-feedforward loops between different nodes of the speech network.

Selective attention-related top-down modulation plays a significant role in separating relevant speech from irrelevant background speech when vocal attributes separating concurrent speakers are small and continuously evolving. Electrophysiological studies have shown that such top-down modulation enhances neural tracking of attended speech. Yet, the specific cortical regions involved remain unclear due to the limited spatial resolution of most electrophysiological techniques. To overcome such limitations, we collected both electroencephalography (EEG) (high temporal resolution) and functional magnetic resonance imaging (fMRI) (high spatial resolution), while human participants selectively attended to speakers in audiovisual scenes containing overlapping cocktail party speech. To utilise the advantages of the respective techniques, we analysed neural tracking of speech using the EEG data and performed representational dissimilarity-based EEG-fMRI fusion. We observed that attention enhanced neural tracking and modulated EEG correlates throughout the latencies studied. Further, attention-related enhancement of neural tracking fluctuated in predictable temporal profiles. We discuss how such temporal dynamics could arise from a combination of interactions between attention and prediction as well as plastic properties of the auditory cortex. EEG-fMRI fusion revealed attention-related iterative feedforward-feedback loops between hierarchically organised nodes of the ventral auditory object related processing stream. Our findings support models where attention facilitates dynamic neural changes in the auditory cortex, ultimately aiding discrimination of relevant sounds from irrelevant ones while conserving neural resources.

Insula→Amygdala and Insula→Thalamus Pathways Are Involved in Comorbid Chronic Pain and Depression-Like Behavior in Mice.

The comorbidity of chronic pain and depression poses tremendous challenges for the treatment of either one because they exacerbate each other with unknown mechanisms. As the posterior insular cortex (PIC) integrates multiple somatosensory and emotional information and is implicated in either chronic pain or depression, we hypothesize that the PIC and its projections may contribute to the pathophysiology of comorbid chronic pain and depression. We show that PIC neurons were readily activated by mechanical, thermal, aversive, and stressful and appetitive stimulation in naive and neuropathic pain male mice subjected to spared nerve injury (SNI). Optogenetic activation of PIC neurons induced hyperalgesia and conditioned place aversion in naive mice, whereas inhibition of these neurons led to analgesia, conditioned place preference (CPP), and antidepressant effect in both naive and SNI mice. Combining neuronal tracing, optogenetics, and electrophysiological techniques, we found that the monosynaptic glutamatergic projections from the PIC to the basolateral amygdala (BLA) and the ventromedial nucleus (VM) of the thalamus mimicked PIC neurons in pain modulation in naive mice; in SNI mice, both projections were enhanced accompanied by hyperactivity of PIC, BLA, and VM neurons and inhibition of these projections led to analgesia, CPP, and antidepressant-like effect. The present study suggests that potentiation of the PIC→BLA and PIC→VM projections may be important pathophysiological bases for hyperalgesia and depression-like behavior in neuropathic pain and reversing the potentiation may be a promising therapeutic strategy for comorbid chronic pain and depression.

History of clinical electrophysiology in diagnosing and treatment monitoring of high ventricular vulnerability

The paper presents the history of hope from 1980-1995 to predict the risk of sudden arrhythmic death using electrophysiologic techniques in individual patients. Even if this prediction seems possible in selected highly risk cohorts, many more patients will die in ventricular arrhythmia without fulfilling the criteria. Ultimately, high risk of sudden cardiac death can be predicted in selected patient groups, but not in the majority of patients at risk. It is ahistory of dashed hope.

Nicotinic Receptors in Human Chromaffin Cells: Characterization, Functional and Physical Interactions between Subtypes and Regulation.

This review summarizes our research on nicotinic acetylcholine receptors in human chromaffin cells. Limited research has been conducted in this field on human tissue, primarily due to the difficulties associated with obtaining human cells. Receptor subtypes were characterized here using molecular biology and electrophysiological patch-clamp techniques. However, the most significant aspect of this study refers to the cross-talk between the two main subtypes identified in these cells, the α7- and α3β4* subtypes, aiming to avoid their desensitization. The article also reviews other aspects, including the regulation of their expression, function or physical interaction by choline, Ca2+, and tyrosine and serine/threonine phosphatases. Additionally, the influence of sex on their expression is also discussed.

Moving toward elucidating alternative motor pathway structures post-stroke: the value of spinal cord neuroimaging.

Stroke results in varying levels of motor and sensory disability that have been linked to the neurodegeneration and neuroinflammation that occur in the infarct and peri-infarct regions within the brain. Specifically, previous research has identified a key role of the corticospinal tract in motor dysfunction and motor recovery post-stroke. Of note, neuroimaging studies have utilized magnetic resonance imaging (MRI) of the brain to describe the timeline of neurodegeneration of the corticospinal tract in tandem with motor function following a stroke. However, research has suggested that alternate motor pathways may also underlie disease progression and the degree of functional recovery post-stroke. Here, we assert that expanding neuroimaging techniques beyond the brain could expand our knowledge of alternate motor pathway structure post-stroke. In the present work, we will highlight findings that suggest that alternate motor pathways contribute to post-stroke motor dysfunction and recovery, such as the reticulospinal and rubrospinal tract. Then we review imaging and electrophysiological techniques that evaluate alternate motor pathways in populations of stroke and other neurodegenerative disorders. We will then outline and describe spinal cord neuroimaging techniques being used in other neurodegenerative disorders that may provide insight into alternate motor pathways post-stroke.

Electrophysiological (EEG) Correlates of Reward Effects on Early Sensory Perception in Humans

Existing research suggests that reward system and sensory perception networks function in concert and that activation in one may influence the other; however, the dynamics of these influences remains poorly understood. There is general agreement regarding the existence of an interaction between bottom-up and top-down signals in perception and attentional processing. While it’s not absolutely sure what stages of perception are influenced by cognition, it has been assumed that cognitive input influences later categorization stages of visual processing, and that earlier stages are solely involved in the pure bottom up extraction of basic features of sensory signals. Several recent experiments have challenged this idea by showing that top-down modulation by cognition may extend to early visual stages of perception. Recent Electrophysiological studies have begun to probe the neural correlates of the interaction between attention/reward, perception and cognitive control in humans. . We posit that selection of the value of our choices and actions from multiple alternatives may cause suppression of the sensory representations of unselected, low value stimuli while the selected, high value stimuli are enhanced. The present project has been proposed to study the dynamics of this selective process where effects of various reward categories on attention and early sensory perception are tracked using behavioral and electrophysiological (EEG) techniques. Methods from neuroscience, signal processing, psychophysics and EEG tools will be used in the project.

Adjunct Methods for Alzheimer's Disease Detection: A Review of Auditory Evoked Potentials.

The auditory afferent pathway as a clinical marker of Alzheimer's disease (AD) has sparked interest in investigating the relationship between age-related hearing loss (ARHL) and AD. Given the earlier onset of ARHL compared to cognitive impairment caused by AD, there is a growing emphasis on early diagnosis and intervention to postpone or prevent the progression from ARHL to AD. In this context, auditory evoked potentials (AEPs) have emerged as a widely used objective auditory electrophysiological technique for both the clinical diagnosis and animal experimentation in ARHL due to their non-invasive and repeatable nature. This review focuses on the application of AEPs in AD detection and the auditory nerve system corresponding to different latencies of AEPs. Our objective was to establish AEPs as a systematic and non-invasive adjunct method for enhancing the diagnostic accuracy of AD. The success of AEPs in the early detection and prediction of AD in research settings underscores the need for further clinical application and study.

Covert cortical processing: a diagnosis in search of a definition.

Historically, clinical evaluation of unresponsive patients following brain injury has relied principally on serial behavioral examination to search for emerging signs of consciousness and track recovery. Advances in neuroimaging and electrophysiologic techniques now enable clinicians to peer into residual brain functions even in the absence of overt behavioral signs. These advances have expanded clinicians' ability to sub-stratify behaviorally unresponsive and seemingly unaware patients following brain injury by querying and classifying covert brain activity made evident through active or passive neuroimaging or electrophysiologic techniques, including functional MRI, electroencephalography (EEG), transcranial magnetic stimulation-EEG, and positron emission tomography. Clinical research has thus reciprocally influenced clinical practice, giving rise to new diagnostic categories including cognitive-motor dissociation (i.e. 'covert consciousness') and covert cortical processing (CCP). While covert consciousness has received extensive attention and study, CCP is relatively less understood. We describe that CCP is an emerging and clinically relevant state of consciousness marked by the presence of intact association cortex responses to environmental stimuli in the absence of behavioral evidence of stimulus processing. CCP is not a monotonic state but rather encapsulates a spectrum of possible association cortex responses from rudimentary to complex and to a range of possible stimuli. In constructing a roadmap for this evolving field, we emphasize that efforts to inform clinicians, philosophers, and researchers of this condition are crucial. Along with strategies to sensitize diagnostic criteria and disorders of consciousness nosology to these vital discoveries, democratizing access to the resources necessary for clinical identification of CCP is an emerging clinical and ethical imperative.

Long-term, high-resolution in vivo calcium imaging in pigeons

Invivo 2-photon calcium imaging has led to fundamental advances in our understanding of sensory circuits in mammalian species. In contrast, few studies have exploited this methodology in birds, with investigators primarily relying on histological and electrophysiological techniques. Here, we report the development of invivo 2-photon calcium imaging in awake pigeons. We show that the genetically encoded calcium indicator GCaMP6s, delivered by the adeno-associated virus rAAV2/7, allows high-quality, stable, and long-term imaging of neuronal populations at single-cell and single-dendrite resolution in the pigeon forebrain. We demonstrate the utility of our setup by investigating the processing of colors in the visual Wulst, the avian homolog of the visual cortex. We report that neurons in the Wulst are color selective and display diverse response profiles to light of different wavelengths. This technology provides a powerful tool to decipher the operating principles that underlie sensory encoding in birds.