RESUMEN
In this Letter, we report a low-cost, portable, two-photon excitation fluorescence microscopy imager that uses a fiber-based approach for both femtosecond supercontinuum (SC) generation and light delivery to the optical head. The SC generation is based on a tapered polarization-maintaining photonic crystal fiber that uses pre-chirped femtosecond narrowband pulses to generate a coherent SC spectrum with a bandwidth of approximately 300 nm. Using this approach, high-power, near-transform-limited, wavelength-selectable SC pulses are generated and directly delivered to the imaging optical head. Preliminary testing of this imager on brain slices is presented, demonstrating a high signal-to-noise ratio and sub-cellular imaging capabilities to a depth of approximately 200 µm. These results demonstrate the suitability of the technology for ex vivo and potentially in vivo cellular-level biomedical imaging applications.
Asunto(s)
Luz , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Fibras Ópticas , Fenómenos Ópticos , Diseño de Equipo , Dinámicas no LinealesRESUMEN
Tight coupling of neuronal metabolism to synaptic activity is critical to ensure that the supply of metabolic substrates meets the demands of neuronal signaling. Given the impact of temperature on metabolism, and the wide fluctuations of brain temperature observed during clinical hypothermia, we examined the effect of temperature on neurometabolic coupling. Intrinsic fluorescence signals of the oxidized form of flavin adenine dinucleotide (FAD) and the reduced form of nicotinamide adenine dinucleotide (NADH), and their ratios, were measured to assess neural metabolic state and local field potentials were recorded to measure synaptic activity in the mouse brain. Brain slice preparations were used to remove the potential impacts of blood flow. Tight coupling between metabolic signals and local field potential amplitudes was observed at a range of temperatures below 29 °C. However, above 29 °C, the metabolic and synaptic signatures diverged such that FAD signals were diminished, but local field potentials retained their amplitude. It was also observed that the declines in the FAD signals seen at high temperatures (and hence the decoupling between synaptic and metabolic events) are driven by low FAD availability at high temperatures. These data suggest that neurometabolic coupling, thought to be critical for ensuring the metabolic health of the brain, may show temperature dependence, and is related to temperature-dependent changes in FAD supplies.
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Corteza Auditiva/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Corteza Motora/metabolismo , NAD/metabolismo , Neuronas/metabolismo , Animales , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , TemperaturaRESUMEN
Retinal-based opsins are light-sensitive proteins. The photoisomerization reaction of these proteins has been studied outside cellular environments using ultrashort tailored light pulses1-5. However, how living cell functions can be modulated via opsins by modifying fundamental nonlinear optical properties of light interacting with the retinal chromophore has remained largely unexplored. We report the use of chirped ultrashort near-infrared pulses to modulate light-evoked ionic current from Channelrhodopsin-2 (ChR2) in brain tissue, and consequently the firing pattern of neurons, by manipulating the phase of the spectral components of the light. These results confirm that quantum coherence of the retinal-based protein system, even in a living neuron, can influence its current output, and open up the possibilities of using designer-tailored pulses for controlling molecular dynamics of opsins in living tissue to selectively enhance or suppress neuronal function for adaptive feedback-loop applications in the future.
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Sleep and wakefulness are characterized by distinct states of thalamocortical network oscillations. The complex interplay of ionic conductances within the thalamo-reticular-cortical network give rise to these multiple modes of activity and a rapid transition exists between these modes. To better understand this transition, we constructed a simplified computational model based on physiological recordings and physiologically realistic parameters of a three-neuron network containing a thalamocortical cell, a thalamic reticular neuron, and a corticothalamic cell. The network can assume multiple states of oscillatory activity, resembling sleep, wakefulness, and the transition between these two. We found that during the transition period, but not during other states, thalamic and cortical neurons displayed chaotic dynamics, based on the presence of strange attractors, estimation of positive Lyapunov exponents and the presence of a fractal dimension in the spike trains. These dynamics were quantitatively dependent on certain features of the network, such as the presence of corticothalamic feedback and the strength of inhibition between the thalamic reticular nucleus and thalamocortical neurons. These data suggest that chaotic dynamics facilitate a rapid transition between sleep and wakefulness and produce a series of experimentally testable predictions to further investigate the events occurring during the sleep-wake transition period.
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Behavioral sensitization following repeated amphetamine (AMPH) exposure is associated with changes in GABA function in the medial prefrontal cortex (mPFC). In rats exposed to AMPH during adolescence compared to adulthood, there are unique patterns of sensitization that may reflect age-dependent differences in drug effects on prefrontal GABAergic function. In the current study, we used a sensitizing regimen of repeated AMPH exposure in adolescent and adult rats to determine if a post-withdrawal AMPH challenge would alter inhibitory transmission in the mPFC in a manner that depends on age of exposure. Male Sprague-Dawley rats were treated with saline or 3mg/kg AMPH (i.p.) during adolescence [postnatal day (P) 27-P45] or adulthood (P85- P103) and were sacrificed either at similar ages in adulthood (â¼P133; experiment 1) or after similar withdrawal times (3-4 weeks; experiment 2). Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in vitro from deep layer pyramidal cells in the mPFC using the whole-cell configuration. We found no effect of AMPH pre-exposure on baseline sIPSC frequency. Subsequent application of AMPH (25µM) produced a stable increase in sIPSC frequency in controls, suggesting that AMPH increases inhibitory tone in the mPFC. However, AMPH failed to increase sIPSCs in adolescent- or adult-exposed rats. In experiment 2, where withdrawal period was kept similar for both exposure groups, AMPH induced a suppression of sIPSC activity in adolescent-exposed rats. These results suggest that sensitizing treatment with AMPH during adolescence or adulthood dampens inhibitory influences on mPFC pyramidal cells, but potentially through different mechanisms.
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Anfetamina/toxicidad , Estimulantes del Sistema Nervioso Central/toxicidad , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/crecimiento & desarrollo , Análisis de Varianza , Animales , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Técnicas de Placa-Clamp , Corteza Prefrontal/fisiopatología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas Sprague-Dawley , Técnicas de Cultivo de TejidosRESUMEN
Fragile X syndrome (FXS) is the most common form of inheritable mental retardation caused by transcriptional silencing of the Fmr1 gene resulting in the absence of fragile X mental retardation protein (FMRP). The role of this protein in neurons is complex and its absence gives rise to diverse alterations in neuronal function leading to neurological disorders including mental retardation, hyperactivity, cognitive impairment, obsessive-compulsive behaviour, seizure activity and autism. FMRP regulates mRNA translation at dendritic spines where synapses are formed, and thus the lack of FMRP can lead to disruptions in synaptic transmission and plasticity. Many of these neurological deficits in FXS probably involve the prefrontal cortex, and in this study, we have focused on modulatory actions of dopamine in the medial prefrontal cortex. Our data indicate that dopamine produces a long-lasting enhancement of evoked inhibitory postsynaptic currents (IPSCs) mediated by D1-type receptors seen in wild-type mice; however, such enhancement is absent in the Fmr1 knock-out (Fmr1 KO) mice. The facilitation of IPSCs produced by direct cAMP stimulation was unaffected in Fmr1 KO, but D1 receptor levels were reduced in these animals. Our results show significant disruption of dopaminergic modulation of synaptic transmission in the Fmr1 KO mice and this alteration in inhibitory activity may provide insight into potential targets for the rescue of deficits associated with FXS.
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Dopamina/fisiología , Síndrome del Cromosoma X Frágil/fisiopatología , Corteza Prefrontal/fisiopatología , Animales , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Potenciales Postsinápticos Inhibidores , Masculino , Ratones , Ratones Noqueados , Células Piramidales/fisiología , Receptores Dopaminérgicos/fisiología , Transmisión SinápticaRESUMEN
Numerous developmental changes in the nervous system occur during the first several weeks of the rodent lifespan. Therefore, many characteristics of neuronal function described at the cellular level from in vitro slice experiments conducted during this early time period may not generalize to adult ages. We investigated the effect of dopamine (DA) on inhibitory synaptic transmission in superficial layers of the medial prefrontal cortex (PFC) in prepubertal [postnatal age (P; days) 12-20], periadolescent (P30-48), and adult (P70-100) mice. The PFC is associated with higher-level cognitive functions, such as working memory, and is associated with initiation, planning, and execution of actions, as well as motivation and cognition. It is innervated by DA-releasing fibers that arise from the ventral tegmental area. In slices from prepubertal mice, DA produced a biphasic modulation of inhibitory postsynaptic currents (IPSCs) recorded in layer II/III pyramidal neurons. Activation of D2-like receptors leads to an early suppression of the evoked IPSC, which was followed by a longer-lasting facilitation of the IPSC mediated by D1-like DA receptors. In periadolescent mice, the D2 receptor-mediated early suppression was significantly smaller compared with the prepubertal animals and absent in adult animals. Furthermore, we found significant differences in the DA-mediated lasting enhancement of the inhibitory response among the developmental groups. Our findings suggest that behavioral paradigms that elicit dopaminergic release in the PFC differentially modulate inhibition of excitatory pyramidal neuron output in prepuberty compared with periadolescence and adulthood in the superficial layers (II/III) of the cortex.
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Dopamina/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Corteza Prefrontal/fisiología , Factores de Edad , Animales , Cognición/efectos de los fármacos , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Memoria a Corto Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos , Motivación/efectos de los fármacos , Corteza Prefrontal/citología , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistasRESUMEN
The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher-level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)-containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole-cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro-caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5-2000 nm) depolarized nearly all relay neurons tested (> 98%) in a concentration-dependent manner. Voltage-clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)(1) tachykinin receptors, although significantly smaller inward currents were produced by specific NK2 and NK3 receptor agonists. The selective NK(1) receptor antagonist RP67580 attenuated the SP-mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK2 and NK3 receptor antagonists, GR159897 and SB222200, respectively. The SP-mediated response showed voltage characteristics consistent with a K+ conductance, and was attenuated by Cs+, a K+ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.
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Núcleos Talámicos Laterales/fisiología , Neuronas/fisiología , Sustancia P/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Conductividad Eléctrica , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/agonistas , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-2/metabolismo , Receptores de Neuroquinina-3/agonistas , Receptores de Neuroquinina-3/antagonistas & inhibidores , Receptores de Neuroquinina-3/metabolismo , Sinapsis/fisiología , Vías Visuales/efectos de los fármacos , Vías Visuales/fisiologíaRESUMEN
We have recorded the activity of omnipause neurons (OPNs) in the raphe interpositus during so-called staircase saccades produced by prolonged activation of the superior colliculus (SC) by microstimulation. By showing that OPNs cyclically pause during the periodic movements produced by the steady activation function, we reveal the functional relationship of the OPNs within the recurrent brainstem network that produces dynamic, closed-loop, and feedback control of saccades. Despite persistent, steady activation of the SC, the OPNs followed the periodic activity of the brainstem burst generator. This reveals a dominant influence of the oscillating brainstem circuit over descending control from the SC.
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Neuronas/fisiología , Movimientos Sacádicos/fisiología , Colículos Superiores/fisiología , Animales , Tronco Encefálico/citología , Tronco Encefálico/fisiología , Fijación Ocular/fisiología , Macaca mulatta , Microelectrodos , Estimulación Luminosa/métodos , Desempeño Psicomotor/fisiologíaRESUMEN
We present a case of spontaneously occurring irrepressible saccades in an experimental Rhesus monkey. Though eye jerks are sometimes associated with cerebellar disease, central demyelination or brainstem lesions, there is little consensus on their neurological mechanisms. From neurological and anatomical investigation we report that these irrepressible saccades were caused by a discrete cerebrovascular accident that involved the rostral superior colliculus along with its commissure, and with minor invasion of periaqueductal gray and adjacent mesencephalic reticular formation. Other suspected structures, like the raphe interpositus, substantia nigra and the cerebellum, were unaffected.
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Vías Nerviosas , Movimientos Sacádicos , Accidente Cerebrovascular/fisiopatología , Colículos Superiores/lesiones , Animales , Mapeo Encefálico , Femenino , Macaca mulatta , Microelectrodos , Núcleos del Rafe/patología , Accidente Cerebrovascular/patología , Colículos Superiores/patologíaRESUMEN
A fundamental technical hurdle in systems neurophysiology has been to record the activity of individual neurons in situ while using microstimulation to activate inputs or outputs. Stimulation artifact at the recording electrode has largely limited the usefulness of combined stimulating and recording to using single stimulation pulses (e.g., orthodromic and antidromic activation) or to presenting brief trains of pulses to look for transient responses (e.g., paired-pulse stimulation). Using an adaptive filter, we have developed an on-line method that allows continuous extracellular isolation of individual neuron spikes during sustained experimental microstimulation. We show that the technique accurately and robustly recovers neural spikes from stimulation-corrupted records. Moreover, we demonstrate that the method should generalize to any recording situation where a stereotyped, triggered transient might obscure a neural event.
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Potenciales de Acción/fisiología , Algoritmos , Artefactos , Estimulación Eléctrica , Movimientos Oculares/fisiología , Neuronas/fisiología , Procesamiento de Señales Asistido por Computador , Animales , Electrodos Implantados , Retroalimentación , Macaca mulatta , Microelectrodos , Lóbulo Parietal/fisiologíaRESUMEN
A fundamental technical hurdle in systems neurophysiology has been to record the activity of individual neurons in situ while using microstimulation to activate inputs or outputs. Stimulation artifact at the recording electrode has largely limited the usefulness of combined stimulating and recording to using single stimulation pulses or to presenting brief trains of pulses to look for transient responses. We have developed an adaptive filter that in real time allows continuous extracellular isolation of individual neural spikes during sustained experimental microstimulation. Using signal detection analysis we now quantifiably demonstrate the reliability of spike recovery from stimulus-corrupted records. Recordings were made from the regular firing of action potentials from the oculomotor or trochlear nuclei of two macaque monkeys while stimulating at a variety of locations. We found that the adaptive filter technique gave a projected error rate of <1 in 10(4) and the detection index, d', was significantly better than two other methods tested: a 4-parameter 'window discriminator' technique and the sample-and-hold technique. This adaptive technique should generalize to any recording situation where a stereotyped, triggered transient might obscure a neural event and will significantly advance our knowledge in areas of electrophysiology where the experimental design requires prolonged microstimulation.
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Potenciales de Acción/fisiología , Animales , Recuento de Células/métodos , Estimulación Eléctrica/métodos , Macaca mulatta , Macaca radiataRESUMEN
Block of calcium-sensitive potassium SK current (gKCa([SK])) by apamin or bicuculline methiodide potentiates burst firing in dopamine neurons in the presence of N-methyl-D-aspartate (NMDA). The purpose of this study was to test the hypothesis that calcium entry through NMDA-gated channels can potentiate gKCa([SK]) in dopamine neurons in the ventral tegmental area. We used microelectrodes to record an outward tail current that was evoked by membrane depolarization under single-electrode voltage-clamp. Using bicuculline methiodide (50 microM) as a reversible inhibitor of gKCa([SK]), we found that NMDA (15 microM) reduced the peak amplitude of the outward tail current by 39%. Contrary to expectations, our results suggest that stimulation of NMDA receptors reduces the calcium-activated potassium gKCa([SK]), an effect that could facilitate NMDA-dependent burst firing.