Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis.
Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for calcium influx: high-threshold voltage-sensitive calcium channels, NMDA receptors, and an APV-resistant influx consistent with calcium-permeable AMPA or kainate receptors. These pathways vary among different populations of spines and are engaged under different stimulation conditions, with peak calcium concentrations reaching >10 microM. Furthermore, as a result of the biophysical properties of the NMDA receptor, the calcium dynamics of spines are exquisitely sensitive to the temporal coincidence of the input and output of the neuron. Our results confirm that individual spines are chemical compartments that can perform coincidence detection. Finally, we demonstrate that functional studies and optical quantal analysis of single, identified synapses is feasible in mammalian CNS neurons in brain slices. (+info)
Optical detection of synaptically induced glutamate transport in hippocampal slices.
Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABAA, nACh, 5-HT3, P2X, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D, L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate. (+info)
Optic flow selectivity in the anterior superior temporal polysensory area, STPa, of the behaving monkey.
Earlier studies of neurons in the anterior region of the superior temporal polysensory area (STPa) have demonstrated selectivity for visual motion using stimuli contaminated by nonmotion cues, including texture, luminance, and form. The present experiments investigated the motion selectivity of neurons in STPa in the absence of form cues using random dot optic flow displays. The responses of neurons were tested with translation, rotation, radial, and spiral optic flow displays designed to mimic the types of motion that occur during locomotion. Over half of the neurons tested responded significantly to at least one of these displays. On a cell by cell basis, 60% of the neurons tested responded selectively to rotation, radial, and spiral motion, whereas 20% responded selectively to translation motion. The majority of neurons responded maximally to single-component optic flow displays but was also significantly activated by the spiral displays that contained their preferred component. Moreover, there was a bias in the selectivity of the neurons for radial expansion motion. These results suggest that neurons within STPa are contributing to the analysis of optic flow. Furthermore, the preponderance of cells selective for radial expansion provides evidence that this area may be specifically involved in the processing of forward locomotion and/or looming stimuli. Finally, these results provide carefully controlled physiological evidence for an extension and specialization of the motion-processing pathway into the anterior temporal lobe. (+info)
The instantaneous monitoring of polyacrylamide gels during electrophoresis.
The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel. (+info)
Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.
Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize alpha3/alpha5, alpha7, and beta2 nAChR subunits, and the results demonstrate the prevalence of alpha3/alpha5. It is this alpha3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application. (+info)
Reacquisition deficits in prism adaptation after muscimol microinjection into the ventral premotor cortex of monkeys.
A small amount of muscimol (1 microl; concentration, 5 microg/microl) was injected into the ventral and dorsal premotor cortex areas (PMv and PMd, respectively) of monkeys, which then were required to perform a visually guided reaching task. For the task, the monkeys were required to reach for a target soon after it was presented on a screen. While performing the task, the monkeys' eyes were covered with left 10 degrees, right 10 degrees, or no wedge prisms, for a block of 50-100 trials. Without the prisms, the monkeys reached the targets accurately. When the prisms were placed, the monkeys initially misreached the targets because the prisms displaced the visual field. Before the muscimol injection, the monkeys adapted to the prisms in 10-20 trials, judging from the horizontal distance between the target location and the point where the monkey touched the screen. After muscimol injection into the PMv, the monkeys lost the ability to readapt and touched the screen closer to the location of the targets as seen through the prisms. This deficit was observed at selective target locations, only when the targets were shifted contralaterally to the injected hemisphere. When muscimol was injected into the PMd, no such deficits were observed. There were no changes in the reaction and movement times induced by muscimol injections in either area. The results suggest that the PMv plays an important role in motor learning, specifically in recalibrating visual and motor coordinates. (+info)
Cerebellar lesions and prism adaptation in macaque monkeys.
If a laterally displacing prism is placed in front of one eye of a person or monkey with the other eye occluded, they initially will point to one side of a target that is located directly in front of them. Normally, people and monkeys adapt easily to the displaced vision and correct their aim after a few trials. If the prism then is removed, there is a postadaptation shift in which the subject misses the target and points in the opposite direction for a few trials. We tested five Macaque monkeys for their ability to adapt to a laterally displacing prism and to show the expected postadaptation shift. When tested as normals, all five animals showed the typical pattern of adaptation and postadaptation shift. Like human subjects, the monkeys also showed complete interocular transfer of the adaptation but no transfer of the adaptation between the two arms. When preoperative training and testing was complete, we made lesions of various target areas on the cerebellar cortex. A cerebellar lesion that included the dorsal paraflocculus and uvula abolished completely the normal prism adaptation for the arm ipsilateral to the lesion in one of the five monkeys. The other four animals retained the ability to prism-adapt normally and showed the expected postadaptation shift. In the one case in which the lesion abolished prism adaptation, the damage included Crus I and II, paramedian lobule and the dorsal paraflocculus of the cerebellar hemispheres as well as lobule IX, of the vermis. Thus in this case, the lesion included virtually all the cerebellar cortex that receives mossy-fiber visual information relayed via the pontine nuclei from the cerebral cortex. The other four animals had damage to lobule V, the classical anterior lobe arm area and/or vermian lobules VI/VII, the oculomotor region. When tested postoperatively, some of these animals showed a degree of ataxia equivalent to that of the case in which prism adaptation was affected, but prism adaptation and the postadaptation shift remained normal. We conclude that in addition to its role in long-term motor learning and reflex adaptation, the region of the cerebellum that was ablated also may be a critical site for a short-term motor memory. Prism adaptation seems to involve a region of the cerebellum that receives a mossy-fiber visual error signal and probably a corollary discharge of the movement. (+info)
Novel form of spreading acidification and depression in the cerebellar cortex demonstrated by neutral red optical imaging.
A novel form of spreading acidification and depression in the rat cerebellar cortex was imaged in vivo using the pH-sensitive dye, Neutral red. Surface stimulation evoked an initial beam of increased fluorescence (i.e., decreased pH) that spread rostrally and caudally across the folium and into neighboring folia. A transient but marked suppression in the excitability of the parallel fiber-Purkinje cell circuitry accompanied the spread. Characteristics differentiating this phenomenon from the spreading depression of Leao include: high speed of propagation on the surface (average of 450 microm/s), stable extracellular DC potential, no change in blood vessel diameter, and repeatability at short intervals. This propagating acidification constitutes a previously unknown class of neuronal processing in the cerebellar cortex. (+info)