Flavoprotein Autofluorescence Research Articles

Altered brain state during episodic dystonia in tottering mice decouples primary motor cortex from limb kinematics.

Episodic Ataxia Type 2 (EA2) is a rare neurological disorder caused by a mutation in the CACNA1A gene, encoding the P/Q-type voltage-gated Ca2+ channel important for neurotransmitter release. Patients with this channelopathy exhibit both cerebellar and cerebral pathologies, suggesting the condition affects both regions. The tottering (tg/tg) mouse is the most commonly used EA2 model due to an orthologous mutation in the cacna1a gene. The tg/tg mouse has three prominent behavioral phenotypes: a dramatic episodic dystonia; absence seizures with generalized spike and wave discharges (GSWDs); and mild ataxia. We previously observed a novel brain state, transient low-frequency oscillations (LFOs) in the cerebellum and cerebral cortex under anesthesia. In this study, we examine the relationships among the dystonic attack, GSWDs, and LFOs in the cerebral cortex. Previous studies characterized LFOs in the motor cortex of anesthetized tg/tg mice using flavoprotein autofluorescence imaging testing the hypothesis that LFOs provide a mechanism for the paroxysmal dystonia. We sought to obtain a more direct understanding of motor cortex (M1) activity during the dystonic episodes. Using two-photon Ca2+ imaging to investigate neuronal activity in M1 before, during, and after the dystonic attack, we show that there is not a significant change in the activity of M1 neurons from baseline through the attack. We also conducted simultaneous, multi-electrode recordings to further understand how M1 cellular activity and local field potentials change throughout the progression of the dystonic attack. Neither putative pyramidal nor inhibitory interneuron firing rate changed during the dystonic attack. However, we did observe a near complete loss of GSWDs during the dystonic attack in M1. Finally, using spike triggered averaging to align simultaneously recorded limb kinematics to the peak Ca2+ response, and vice versa, revealed a reduction in the spike triggered average during the dystonic episodes. Both the loss of GSWDs and the reduction in the coupling suggest that, during the dystonic attack, M1 is effectively decoupled from other structures. Overall, these results indicate that the attack is not initiated or controlled in M1, but elsewhere in the motor circuitry. The findings also highlight that LFOs, GSWDs, and dystonic attacks represent three brain states in tg/tg mice.

The role of early visual experience in the development of spatial-frequency preference in the primary visual cortex.

The mature functioning of the primary visual cortex depends on postnatal visual experience, while the orientation/direction preference is established just after eye-opening, independently of visual experience. In this study, we find that visual experience is required for the normal development of spatial-frequency (SF) preference in mouse primary visual cortex. We show that age- and experience-dependent shifts in optimal SFs towards higher frequencies occurred similarly in excitatory neurons and parvalbumin-positive interneurons. We also show that some excitatory and parvalbumin-positive neurons preferentially responded to visual stimuli consisting of very high SFs and posterior directions, and that the preference was established at earlier developmental stages than the SF preference in the standard frequency range. These results suggest that early visual experience is required for the development of SF representation and shed light on the experience-dependent developmental mechanisms underlying visual cortical functions. Early visual experience is crucial for the maturation of visual cortical functions. It has been demonstrated that the orientation and direction preferences in individual neurons of the primary visual cortex are well established immediately after eye-opening. The postnatal development of spatial frequency (SF) tuning and its dependence on visual experience, however, has not been thoroughly quantified. In this study, macroscopic imaging with flavoprotein autofluorescence revealed that the optimal SFs shift towards higher frequency values during normal development in mouse primary visual cortex. This developmental shift was impaired by binocular deprivation during the sensitive period, postnatal 3weeks (PW3) to PW6. Furthermore, two-photon Ca2+ imaging revealed that the developmental shift of the optimal SFs, depending on visual experience, concurrently occurs in excitatory neurons and parvalbumin-positive inhibitory interneurons (PV neurons). In addition, some excitatory and PV neurons exhibited a preference for visual stimuli consisting of particularly high SFs and posterior directions at relatively early developmental stages; this preference was not affected by binocular deprivation. Thus, there may be two distinct developmental mechanisms for the establishment of SF preference depending on the frequency values. After PW3, SF tuning for neurons tuned to standard frequency ranges was sharper in excitatory neurons and slightly broader in PV neurons, leading to considerably attenuated SF tuning in PV neurons compared to excitatory neurons by PW5. Our findings suggest that early visual experience is far more important than orientation/direction selectivity for the development of the neural representation of the diverse SFs.

A novel dynamic network imaging analysis method reveals aging-related fragmentation of cortical networks in mouse.

Network analysis of large-scale neuroimaging data is a particularly challenging computational problem. Here, we adapt a novel analytical tool, the community dynamic inference method (CommDy), for brain imaging data from young and aged mice. CommDy, which was inspired by social network theory, has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network metrics in the auditory and motor cortices by using flavoprotein autofluorescence imaging in brain slices and in vivo. We observed that auditory cortical networks in slices taken from aged brains were highly fragmented compared to networks observed in young animals. CommDy network metrics were then used to build a random-forests classifier based on NMDA receptor blockade data, which successfully reproduced the aging findings, suggesting that the excitatory cortical connections may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity in the awake motor cortex, suggesting that the findings in the auditory cortex reflect general mechanisms during aging. These data suggest that CommDy provides a new dynamic network analytical tool to study the brain and that aging is associated with fragmentation of intracortical networks.

Distinct patterns of activity in individual cortical neurons and local networks in primary somatosensory cortex of mice evoked by square-wave mechanical limb stimulation.

Artificial forms of mechanical limb stimulation are used within multiple fields of study to determine the level of cortical excitability and to map the trajectory of neuronal recovery from cortical damage or disease. Square-wave mechanical or electrical stimuli are often used in these studies, but a characterization of sensory-evoked response properties to square-waves with distinct fundamental frequencies but overlapping harmonics has not been performed. To distinguish between somatic stimuli, the primary somatosensory cortex must be able to represent distinct stimuli with unique patterns of activity, even if they have overlapping features. Thus, mechanical square-wave stimulation was used in conjunction with regional and cellular imaging to examine regional and cellular response properties evoked by different frequencies of stimulation. Flavoprotein autofluorescence imaging was used to map the somatosensory cortex of anaesthetized C57BL/6 mice, and in vivo two-photon Ca2+ imaging was used to define patterns of neuronal activation during mechanical square-wave stimulation of the contralateral forelimb or hindlimb at various frequencies (3, 10, 100, 200, and 300 Hz). The data revealed that neurons within the limb associated somatosensory cortex responding to various frequencies of square-wave stimuli exhibit stimulus-specific patterns of activity. Subsets of neurons were found to have sensory-evoked activity that is either primarily responsive to single stimulus frequencies or broadly responsive to multiple frequencies of limb stimulation. High frequency stimuli were shown to elicit more population activity, with a greater percentage of the population responding and greater percentage of cells with high amplitude responses. Stimulus-evoked cell-cell correlations within these neuronal networks varied as a function of frequency of stimulation, such that each stimulus elicited a distinct pattern that was more consistent across multiple trials of the same stimulus compared to trials at different frequencies of stimulation. The variation in cortical response to different square-wave stimuli can thus be represented by the population pattern of supra-threshold Ca2+ transients, the magnitude and temporal properties of the evoked activity, and the structure of the stimulus-evoked correlation between neurons.

Carotid body type I cells engage flavoprotein and Pin1 for oxygen sensing.

Carotid body (CB) type I cells sense the blood Po2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN-, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND.

In vivo transcranial flavoprotein autofluorescence imaging of tonotopic map reorganization in the mouse auditory cortex with impaired auditory periphery

Ototoxic-drug-induced hearing disturbances in the auditory periphery are associated with tonotopic map reorganization and neural activity modulation, as well as changes in neural correlates in the central auditory pathway, including the auditory cortex (AC). Previous studies have reported that peripheral auditory impairment induces AC plasticity that involves changes in the balance of excitatory vs. inhibitory synapses, within existing and newly forming patterns of connectivity. Although we know that such plastic changes modulate sound-evoked neural responses and the organization of tonotopic maps in the primary AC (A1), little is known about the effects of peripheral impairment on other frequency-organized AC subfields, such as the anterior auditory field (AAF) and the secondary auditory cortex (A2). Therefore, to examine ototoxic-drug-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of kanamycin sulfate (1 mg/g body weight) and bumetanide (0.05 mg/g body weight), using in vivo transcranial flavoprotein autofluorescence imaging over a 4-week period. At first, ototoxic treatment gradually reduced responses driven by tone bursts with lower- (≤8 kHz) and middle- (e.g., 16 kHz) range frequencies in all AC subfields. Subsequently, response intensities in the A1 recovered to more than 78% of the pre-drug condition; however, in the AAF and A2, they remained significantly lower and were unchanged over 3 weeks. Furthermore, after drug administration, the best frequency (BF) areas of the lower (4 and 8 kHz) and higher (25 and 32 kHz) ranges in all subfields were reduced and shifted to those of a middle range (centered around 16 kHz) during the 3 weeks following drug administration. Our results also indicated that, compared with A1, BF distributions in the AAF and A2 were sharper around 16 kHz 3 weeks after drug administration. These results indicate that the ototoxic-damage-induced tonotopic map reorganizations that occurred in each of the three AC subfields were similar, but that there were subfield-dependent differences in the extent of response intensities and in the activated areas that were responsive to tone bursts with specific frequencies. Thus, by examining cortical reorganization induced by ototoxic drugs, we may contribute to the understanding of how this reorganization can be caused by peripheral damage.

The Role of Extracellular Matrix Stiffness on Cardiac Metabolic Activity

The cardiac L-type Ca2+ channel (LTCC) can regulate mitochondrial metabolic activity via calcium-independent mechanisms. The sarcomeric network plays an important role in this response. Hypertrophic cardiomyopathy (HCM) occurs due to mutations in sarcomeric proteins. Using murine models of human HCM, we have shown that mutations in sarcomeric proteins are associated with altered LTCC kinetics, impaired structural-functional communication between LTCC and mitochondria, and increased metabolic activity (consistent with the human phenotype). However, the mechanisms by which mutations in sarcomeric proteins lead to alterations in metabolic activity remain unknown. Cardiomyocytes can ‘sense’ extracellular matrix (ECM) mechanics via a process called mechanotransduction. This involves conversion of mechanical stimuli into biochemical events that can alter myocardial function. Since human HCM is characterised by a stiff myocardium, we developed an in vitro model to determine the role of increased ECM stiffness on metabolic activity. Wild-type cardiomyocytes were cultured on hydrogels with stiffnesses mimicking healthy (10 kPa) or HCM (40 kPa) myocardium. Cardiomyocytes on 40 kPa hydrogels exhibited increased stiffness versus 10 kPa (atomic force microscopy; 3.8 ± 0.4 kPa, n = 53 versus 1.5 ± 0.2 kPa, n = 31; p < 0.05). Cardiomyocytes on 40 kPa hydrogels also exhibited a larger increase in metabolic activity in response to activation of LTCC (flavoprotein autofluorescence; 40 kPa: 64.6 ± 4.3% increase, n = 35 versus 10 kPa: 20.3 ± 1.8%, n = 27 p < 0.05), that was attenuated by sarcomeric protein depolymerising agents latrunculin A (F-actin) or colchicine (β-tubulin). We conclude that ECM stiffness may regulate cardiac metabolic activity. Increased ECM stiffness may contribute to increased metabolic activity and development of HCM.

Imaging of Neuronal Activity in Awake Mice by Measurements of Flavoprotein Autofluorescence Corrected for Cerebral Blood Flow.

Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can be used to measure neuronal activity and oxygen metabolism in living brains without expressing fluorescence proteins. It is useful for understanding the mechanism of various brain functions and their abnormalities in age-related brain diseases. However, hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurate assessments of brain function in animal models with cerebral blood vessel dysfunctions and subsequent cerebral blood flow (CBF) alterations. In the present study, we developed a new method to correct FAI signals for hemoglobin-dependent green fluorescence reductions by simultaneous measurements of green fluorescence and intrinsic optical signals. Intrinsic optical imaging enabled evaluations of light absorption and scatters by hemoglobin, which could then be applied to corrections of green fluorescence intensities. Using this method, enhanced flavoprotein autofluorescence by sensory stimuli was successfully detected in the brains of awake mice, despite increases of CBF, and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properly quantified in a resting state and during sensory stimulation by a CO2 inhalation challenge, which modified vascular responses without overtly affecting neuronal activities. The flavoprotein autofluorescence signal data obtained here were in good agreement with the previous findings from a condition with drug-induced blockade of cerebral vasodilation, justifying the current assaying methodology. Application of this technology to studies on animal models of brain diseases with possible changes of CBF, including age-related neurological disorders, would provide better understanding of the mechanisms of neurovascular coupling in pathological circumstances.

Micromagnetic Stimulation of the Mouse Auditory Cortex In Vivo Using an Implantable Solenoid System.

Recent studies have reported that micromagnetic stimulation ( MS), which can activate neurons and neural networks via submillimeter inductors, may address several limitations of conventional magnetic stimulation methods. Previous studies have examined the effects of MS on single neurons, yet little is known about how MS can affect brain tissue including local neural networks. Here, we propose a new, readily available implantable MS system and computationally and experimentally evaluate its validity. We conducted numerical calculations and experiments to evaluate the physical characteristics, including magnetic flux density, temperature, coil impedance, and structural integrity of the flexible board supporting the MS coils. We then compared sound- and MS-driven neural responses in the mouse auditory cortex using flavoprotein autofluorescence imaging. Our system successfully activated neural tissue, and we observed activity propagation in local neural networks on the brain surface beyond restricted activation of single neurons. Examining the relationships between stimulation parameters and response characteristics, we found that stimulation amplitude and pulse width were the two most important parameters to effectively induce neural activity. Our MS device has sufficient potential to drive the brain as an implantable magnetic stimulator for basic neuroscience and clinical applications, although further investigation is required. MS can selectively drive and modulate activity in local neural network even at an in vivo tissue level.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.

Salicylate-induced frequency-map reorganization in four subfields of the mouse auditory cortex

Salicylate is the active ingredient in aspirin, and in high-doses it is used as an experimental tool to induce transient hearing loss, tinnitus, and hyperacusis. These salicylate-induced perceptual disturbances are associated with tonotopic-map reorganization and neural activity modulation, and such neural correlates have been examined in the central auditory pathway, including the auditory cortex (AC). Although previous studies have reported that salicylate induces increases in noise-burst-evoked neural responses and reorganization of tonotopic maps in the primary AC, little is known about the effects of salicylate on other frequency-organized AC subfields such as the anterior auditory, secondary auditory, and dorsomedial fields. Therefore, to examine salicylate-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of sodium salicylate (SS, 200 mg/kg), using flavoprotein auto-fluorescence imaging. SS-treatment gradually reduced responses driven by tone-bursts with lower (≤8 kHz) and higher (≥25 kHz) frequencies over 3 h, whereas evoked responses to tone-bursts within middle-range frequencies (e.g., 12 and 16 kHz) were sustained and unchanged in the four subfields. Additionally, in each of the four subfields, SS-treatment induced similar reorganization of tonotopic maps, and the response areas selectively driven by the middle-range frequencies were profoundly expanded. Our results indicate that the SS-induced tonotopic map reorganizations in each of the four AC subfields were similar, and only the extent of the activated areas responsive to tone-bursts with specific frequencies was subfield-dependent. Thus, we expect that examining cortical reorganization induced by SS may open the possibility of new treatments aimed at altering cortical reorganization into the normative functional organization.

Manipulating L-Type Calcium Channel Activity Alters Mitochondrial Function and Prevents Hypertrophic Cardiomyopathy in a Troponin I Mutant Mouse Model

Hypertrophic cardiomyopathy (HCM) affects 1:200 of the general population. It is associated with altered metabolic activity and hypercontractile function. Mitochondrial function can be regulated by alterations in L-type Ca2+ channel (LTCC) activity, and the cytoskeletal network plays an important role in this response. We have previously demonstrated that human HCM causing cardiac troponin I mutation Gly203Ser leads to a faster LTCC inactivation rate, impaired functional communication between LTCC and mitochondria, and a “hypermetabolic” mitochondrial state in a mouse model of the mutation (cTnI-G203S). These alterations precede development of the cardiomyopathy. A peptide derived against the LTCC alpha-interacting domain (AID-TAT) slows LTCC inactivation rate and decreases metabolic function in wt cardiomyocytes. Here we examined the efficacy of in vitro and in vivo exposure of cTnI-G203S to AID-TAT on normalising mitochondrial function in response to activation of the channel, assessed as changes in mitochondrial membrane potential (Ψm, JC-1 fluorescence) and mitochondrial oxygen consumption (flavoprotein autofluorescence). Acute in vitro exposure of cTnI-G203S cardiomyocytes to AID-TAT normalises Ψm in response to activation of LTCC with channel agonist BayK(-) (cTnI-G203S+AID-TAT = 4.4±0.4% increase, n = 6 versus cTnI-G203S+ inactive scrambled AID-TAT, AID(S)-TAT = 28.4±2.6% increase, n = 5, p<0.05) and flavoprotein autofluorescence (cTnI-G203S+AID-TAT = 4.0±0.3% increase, n=6 versus cTnI-G203S+AID(S)-TAT = 19.2±1.7% increase, n = 3, p<0.05). In vivo treatment of cTnI-G203S mice with AID-TAT via intraperitoneal injection (10µM tri-weekly for 5wks) also normalised Ψm (cTnI-G203S+AID-TAT = 20.1±2.2% increase, n = 18 versus cTnI-G203S+AID(S)-TAT = 30.3±1.8% increase, n = 18, p<0.05), and flavoprotein autofluorescence (cTnI-G203S+AID-TAT = 17.8±2.1% increase, n = 29 versus cTnI-G203S+AID(S)-TAT = 42.3±5.0% increase, n = 26, p<0.05). Treatment also reversed hypertrophy as evidenced by changes on echocardiography. We conclude that treatment of cTnI-G203S mice with AID-TAT appears to restore mitochondrial function and prevent development of hypertrophic cardiomyopathy.

Identifying the Therapeutic Window for Treatment of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) affects 1:200 of the population, and is associated with altered metabolic activity and hypercontractile function. We have previously demonstrated that the cardiac L-type Ca2+ channel (LTCC) can regulate mitochondrial function due to an association via the cytoskeletal network. We have also shown that human HCM causing mutations in cardiac troponin I or β-myosin heavy chain lead to a faster LTCC inactivation rate, and impaired functional communication between LTCC and mitochondria in mouse models of human disease (cTnI-G203S and αMHC403/+ respectively). This results in a “hypermetabolic” mitochondrial state, which precedes development of HCM. Application of a peptide derived against the LTCC alpha-interacting domain (AID-TAT) slows LTCC inactivation rate and decreases metabolic function in wt cardiomyocytes. We examined the efficacy of in vivo exposure of cTnI-G203S and αMHC403/+ mice to AID-TAT (10 μM 3x/wk, 5wk) on mitochondrial function by assessing alterations in mitochondrial membrane potential (Ψm, JC-1 fluorescence) and mitochondrial metabolic activity (MMA, flavoprotein autofluorescence). Treatment of pre-cardiomyopathic cTnI-G203S mice normalised Ψm (AID-TAT: 20.1 ± 2.2% increase, n = 18 vs AID(S)-TAT: 30.3 ± 1.8% increase, n = 18; p < 0.05), and MMA (AID-TAT: 17.8 ± 2.1% increase, n = 29 vs AID(S)-TAT: 42.3 ± 5.0% increase, n = 26; p < 0.05). AID-TAT also prevented hypertrophy as evidenced by changes on echocardiography. Treatment of post-cardiomyopathic αMHC403/+ mice normalised Ψm (AID-TAT: 20.6 ± 3.4% increase, n = 6 vs AID(S)-TAT: 30.3 ± 1.7% increase, n = 24; p < 0.05), but not MMA (AID-TAT: 29.2 ± 2.8% increase, n = 14 vs AID(S)-TAT: 40.8 ± 6.6% increase, n = 13; p = NS). Treatment did not appear to reverse hypertrophy. We conclude that early-intervention AID-TAT treatment may be more effective in restoring mitochondrial function and preventing development of HCM.

Developmental exposure to PCBs alters the activation of the auditory cortex in response to GABAA antagonism

Developmental exposure of rats to polychlorinated biphenyls (PCBs) causes impairments in hearing and in the functioning of peripheral and central auditory structures. Additionally, recent work from our laboratory has demonstrated an increase in audiogenic seizures. The current study aimed to further characterize the effects of PCBs on auditory brain structures by investigating whether developmental exposure altered the magnitude of activation in the auditory cortex (AC) in response to electrical stimulation of thalamocortical afferents. Long-Evans female rats were fed cookies containing either 0 or 6mg/kg of an environmental PCB mixture daily from 4 weeks prior to breeding until postnatal day 21. Brain slices containing projections from the thalamus to the AC were collected from adult female offspring and were bathed in artificial cerebrospinal fluid (aCSF) alone, aCSF containing a gamma-aminobutyric acid (GABA) receptor antagonist (200nM SR95531), and aCSF containing an and N-methyl-d-aspartate (NMDA) receptor antagonist (50μM AP5). During each of these drug conditions, electrical stimulations ranging from 25 to 600μA were delivered to the thalamocortical afferents. Activation of the AC was measured using flavoprotein autofluorescence imaging. Although there were no differences seen between treatment groups in the aCSF condition, there were significant increases in the ratio of aCSF/SR95531 activation in slices from PCB-exposed animals compared to control animals. This effect was seen in both the upper and lower layers of the AC. No differences in activation were noted between treatment groups when slices were exposed to AP5. These data suggest that developmental PCB exposure leads to increased sensitivity to antagonism of GABAA receptors in the AC without a change in NMDA-mediated intrinsic excitability.

Neural response differences in the rat primary auditory cortex under anesthesia with ketamine versus the mixture of medetomidine, midazolam and butorphanol

Anesthesia affects central auditory processing. However, it is unclear to what extent the choice of anesthetic agent affects neural responses to sound stimulation. A mixture of three anesthetics (medetomidine, midazolam and butorphanol; MMB) was recently developed as an alternative to ketamine owing to the latter's addictive potential, yet the effect of this combination of anesthetics on neural responses is not known. Here, we compared the spontaneous activity, tuning properties and temporal responses of primary auditory cortical neurons under these two anesthetic conditions, using electrophysiological and flavoprotein autofluorescence imaging methods. Frequency tuning properties were not significantly different between ketamine and MMB anesthesia. However, neural activity under MMB showed decreases in the spontaneous and tone-evoked firing rates in a layer-dependent manner. Moreover, the temporal response patterns were also different between the anesthetics in a layer-dependent manner, which may reflect differences in the anesthetic mechanisms. These results demonstrated how response properties in the primary auditory cortex are affected by the choice of anesthesia.

Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis.

BackgroundChronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear.We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS.MethodsWe used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs.ResultsMice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity—but did not significantly attenuate the loss of PNNs.ConclusionsThese findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0609-4) contains supplementary material, which is available to authorized users.

Characterising the Effects of a Peptide Directed Against the L-Type Ca2+ Channel on Mitochondrial Function in Hypertrophic Cardiomyopathy

Mutations in cardiac troponin I (cTnI) or β-myosin heavy chain (MHC) account for approximately 45% of genotyped families with hypertrophic cardiomyopathy (HCM). HCM is characterised by disorganisation of cytoskeletal proteins and altered energy metabolism. The L-type Ca2+ channel (LTCC) plays a critical role in cardiac excitation and contraction. We have demonstrated that activation of LTCC can also regulate mitochondrial function. HCM mouse models cTnI-G203S and αMHC403/+ exhibit a faster LTCC inactivation rate and altered mitochondrial responses after LTCC activation. This results in a “hypermetabolic” mitochondrial state. We examined the efficacy of in vitro and in vivo exposure of cTnI-G203S and αMHC403/+ mice to a peptide derived against the alpha-interacting domain (AID-TAT) on mitochondrial function by assessing alterations in mitochondrial membrane potential (Ψm, JC-1 fluorescence) and mitochondrial oxygen consumption (flavoprotein autofluorescence). In vitro exposure of cTnI-G203S cardiomyocytes to 10µM AID-TAT attenuated the increase in JC-1 fluorescence in response to activation of LTCC with channel agonist BayK(-) (cTnI-G203S+AID-TAT=4.4±0.36% increase, n=6 versus cTnI-G203S=29.2±1.8% increase, n=15, p<0.05). AID-TAT also attenuated increases in flavoprotein autofluorescence in response to BayK(-) (cTnI-G203S+AID-TAT=4.0±0.4% increase, n=6 versus cTnI-G203S=24.4±6.5% increase, n=8, p<0.05). In vivo treatment of cTnI-G203S mice with IP injection of 10µM AID-TAT attenuated JC-1 fluorescence in response to BayK(-) compared to untreated mice (JC-1: cTnI-G203S+AID-TAT=19.7±3.6% increase, n=6 versus cTnI-G203S=29.2±1.8% increase, n=15, p<0.05). Similar results were observed in αMHC403/+ mice. These data suggest that treatment of cTnI-G203S and αMHC403/+ mice with AID-TAT restores mitochondrial function. Normalising mitochondrial function with AID-TAT may be effective in preventing development of the cardiomyopathy.

Ageing-related changes in GABAergic inhibition in mouse auditory cortex, measured using in vitro flavoprotein autofluorescence imaging.

Ageing is associated with hearing loss and changes in GABAergic signalling in the auditory system. We tested whether GABAergic signalling in an isolated forebrain preparation also showed ageing-related changes. A novel approach was used, whereby population imaging was coupled to quantitative pharmacological sensitivity. Sensitivity to GABAA blockade was inversely associated with age and cortical thickness, but hearing loss did not independently contribute to the change in GABAA ergic sensitivity. Redox states in the auditory cortex of young and aged animals were similar, suggesting that the differences in GABAA ergic sensitivity are unlikely to be due to differences in slice health. To examine ageing-related changes in the earliest stages of auditory cortical processing, population auditory cortical responses to thalamic afferent stimulation were studied in brain slices obtained from young and aged CBA/CAj mice (up to 28 months of age). Cortical responses were measured using flavoprotein autofluorescence imaging, and ageing-related changes in inhibition were assessed by measuring the sensitivity of these responses to blockade of GABAA receptors using bath-applied SR95531. The maximum auditory cortical response to afferent stimulation was not different between young and aged animals under control conditions, but responses to afferent stimulation in aged animals showed a significantly lower sensitivity to GABA blockade with SR95531. Cortical thickness, but not hearing loss, improved the prediction of all imaging variables when combined with age, particularly sensitivity to GABA blockade for the maximum response. To determine if the observed differences between slices from young and aged animals were due to differences in slice health, the redox state in the auditory cortex was assessed by measuring the FAD+/NADH ratio using fluorescence imaging. We found that this ratio is highly sensitive to known redox stressors such as H2 O2 and NaCN; however, no difference was found between young and aged animals. By using a new approach to quantitatively assess pharmacological sensitivity of population-level cortical responses to afferent stimulation, these data demonstrate that auditory cortical inhibition diminishes with ageing. Furthermore, these data establish a significant relationship between cortical thickness and GABAergic sensitivity, which had not previously been observed in an animal model of ageing.

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice(1), we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.