Biomedical subjects

John P Welsh

Publications and source records attributed to John P Welsh.

9 recordsLinked to original sources

PubMed2006

Continuous electrical oscillations emerge from a coupled network: a study of the inferior olive using lentiviral knockdown of connexin36.

Do continuous subthreshold oscillations in membrane potential within an electrically coupled network depend on gap junctional coupling? For the inferior olive (IO), modeling and developmental studies suggested that the answer is yes, although physiological studies of connexin36 knock-out mice lacking electrical coupling suggested that the answer is no. Here we addressed the question differently by using a lentivirus-based vector to express, in the IO of adult rats, a single amino acid mutation of connexin36 that disrupts the intracellular trafficking of wild-type connexin36 and blocks gap junctional coupling. Confocal microscopy of green fluorescence protein-labeled dendrites revealed that the mutant connexin36 prevented wild-type connexin36 from being expressed in dendritic spines of IO neurons. Intracellular recordings from lentivirally transduced IO networks revealed that robust and continuous subthreshold oscillations require gap junctional coupling of IO neuron somata within 40 microm of one another. Topological studies indicated that the minimal coupled network for supporting such oscillations may be confined to the dendritic arbor of a single IO neuron. Occasionally, genetically uncoupled IO neurons showed transient oscillations; however, these were not sustained longer than 3 s and were 69% slower and 71% smaller than the oscillations of normal IO neurons, a finding replicated with carbenoxolone, a pharmacological antagonist of gap junctions. The experiments provided the first direct evidence that gap junctional coupling between neurons, specifically mediated by connexin36, allows a continuous network oscillation to emerge from a population of weak and episodic single-cell oscillators. The findings are discussed in the context of the importance of gap junctions for cerebellar rhythms involved in movement.

Action Potentials↗

PubMed2005

Normal motor learning during pharmacological prevention of Purkinje cell long-term depression.

Systemic delivery of (1R-1-benzo thiophen-5-yl-2[2-diethylamino)-ethoxy] ethanol hydrochloride (T-588) prevented long-term depression (LTD) of the parallel fiber (PF)-Purkinje cell (PC) synapse induced by conjunctive climbing fiber and PF stimulation in vivo. However, similar concentrations of T-588 in the brains of behaving mice and rats affected neither motor learning in the rotorod test nor the learning of motor timing during classical conditioning of the eyeblink reflex. Rats given doses of T-588 that prevented PF-PC LTD were as proficient as controls in learning to adapt the timing of their conditioned eyeblink response to a 150- or 350-ms change in the timing of the paradigm. The experiment indicates that PF-PC LTD under control of the climbing fibers is not required for general motor adaptation or the learning of response timing in two common models of motor learning for which the cerebellum has been implicated. Alternative mechanisms for motor timing and possible functions for LTD in protection from excitotoxicity are discussed.

Animals↗

PubMed2004

Fundamental role of inferior olive connexin 36 in muscle coherence during tremor.

Inferior olive (IO) neurons are electrically coupled by cytosolic pores formed by the neuron-specific connexin 36 (Cx36). Electrical coupling in the IO figures prominently in current views about brain control of movement. However, a role for Cx36 in movement has been questioned and not definitively demonstrated. Previous reports have shown that embryonic deletion of the Cx36 gene resulted in almost complete loss of cytosolic and electrical coupling in the IO without an obvious deficit in movement, possibly due to developmental compensations in ionic conductances that can confound the approach of embryonic gene deletion. We used a replication-incompetent lentiviral vector to stably express a dominant-negative Cx36 mutant in the IO of adult rats. We show that interneuronal cytosolic coupling is severely reduced by the mutant Cx36, without effect on neuron morphology or electrical properties. Multisite electromyography revealed that blocking Cx36 in the IO impaired the coherence of muscle firing during harmaline tremor without affecting its rhythm. The data demonstrate that gap junction coupling within the IO mediated by Cx36 adds 10-20 ms of precision to the fine temporal coordination of muscle firing during movement.

Animals↗

PubMed2003

Independently movable multielectrode array to record multiple fast-spiking neurons in the cerebral cortex during cognition.

A multiple microelectrode carrier system is described that allows each of 16 microelectrodes in a high-density array to be manipulated in the brain independently by remote control during a cognitive task. Descriptions of the carrier system, advancing techniques, and microelectrode design are presented that allow high-fidelity, extracellular recordings of multiple cerebral cortex neurons with high signal-to-noise ratio and day-to-day repeatability. The motivation for the new carrier system was to provide a method to target multi-microelectrode recordings to a distinct population of fast-spiking neurons in the cerebral cortex during a behavioral task to assess their involvement in selective attention. Recent work using intracellular recording in vitro and single-neuron extracellular recordings in vivo has demonstrated that subpopulations of cortical interneurons can be identified on the basis of their action potential waveform and response to sensory input, and that such interneurons play a fundamental role in generating cortical rhythmicity associated with vigilant wakefulness. A new behavioral paradigm is presented, based on Pavlov's and Kamin's classic work on compound conditioning, that permits the electrophysiological patterns of selective attention among neuronal ensembles to be distinguished from those of sensation without attention in a primary sensory cortex. Our approach of multiple, individually guided cerebral cortical recordings in behaving rats during a complex cognitive task is beginning to provide new support for the role of fast cerebral rhythms in selective attention.

Action Potentials↗

PubMed2002

A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability.

Rat connexin 36 (Cx36) was mutated by substituting serine for cysteine at residue 231 (C231S) and the mutant's effect on the subcellular localization of wild-type Cx36 and the intercellular permeability that it confers was determined in human HeLa and rat PC12 cells. Cells transfected with the mutant or wild-type Cx36 cDNA expressed the expected 36 kDa protein and Cx36 immunoreactivity. Co-immunoprecipitation experiments with monkey COS-7 cells transiently transfected with both mutant and wild-type Cx36 cDNAs demonstrated that the mutant protein bound to the wild-type. Double immunofluorescence microscopy of stably transfected HeLa cells demonstrated that mutant Cx36 blocked the transport of the wild-type Cx36 to the cell membrane, primarily by trapping it in the endoplasmic reticulum around the nucleus. Coexpression of the mutant Cx36 with the wild-type protein abolished the ability of the latter to permit dye transfer in both HeLa and PC12 cells. The findings are the first demonstration of a mutation of Cx36 that inhibits wild-type Cx36 function in mammalian cells.

Amino Acid Substitution↗

PubMed2002

Functional significance of climbing-fiber synchrony: a population coding and behavioral analysis.

Population coding and behavioral approaches were taken toward analyzing the functional significance of the climbing-fiber system. Analyses of neuronal interaction using the joint peristimulus time histogram showed that given a low rate of firing, the climbing-fiber system organizes itself to fire synchronously during movement--a feature that bears little or no relationship to the modulation in firing rate during movement or whether olivary neurons respond to a sensory stimulus. Moreover, the climbing-fiber system avoids synchrony during a passive sensory response but actively makes a transition into synchrony as a movement is initiated. Thus, from a functional viewpoint, the active feature of the climbing-fiber system to organize into synchronously firing cell ensembles is uniquely motor. Analyses of behaving rats without an inferior olive revealed that the climbing-fiber system optimizes the timing of skilled movement by reducing reaction time and the interval between repetitive movements by 100 milliseconds. Finally, using classical delay eyeblink conditioning, it was found that the inferior olive is essential for learning about rapid sequences of events but not the same event sequence when given more slowly. It was concluded that the climbing-fiber system exerts its function through synchrony, which provides a 100 ms advantage in movement speed and the ability to learn about events that are rapidly presented in time.

Action Potentials↗

PubMed2002

The serotonin hypothesis of myoclonus from the perspective of neuronal rhythmicity.

A quantitative analysis of two rat syndromes of myoclonus are presented, modeling myoclonic epilepsy and postanoxic myoclonus. Like the human conditions, both of the models benefit therapeutically from drugs that act on the serotonin system. The rat model of myoclonic epilepsy is associated with a profound loss of serotonin throughout the brain (except in the striatum) and is generated by an oscillator that is synchronized around the midline. The rat model of posthypoxic myoclonus does not demonstrate a significant reduction in serotonin in any location of its brain and is generated by a non-oscillating circuit in the medulla. Although some forms of myoclonic epilepsy may benefit from serotonin drugs because they are caused by a decrease in brain serotonin, our data indicate that posthypoxic myoclonus is not caused by a decrease in the serotonergic innervation of any region of the brain. That the raphe nuclei do not degenerate after global brain ischemia was noted by C. David Marsden in a discussion of the histologic findings of three of his human cases of posthypoxic myoclonus (page 117 of reference 10) and led him to question the hypothesis that posthypoxic myoclonus was due to a loss of serotonin neurons. Our data confirm his observation in the rat, but also indicate that density of serotonin fibers and terminals throughout the brain is not reduced by the brain ischemia that produces posthypoxic myoclonus. It remains to be determined whether the physiologic responsiveness of serotonin neurons is altered by global brain ischemia and whether changes in serotonin release or serotonin receptor properties are associated with posthypoxic myoclonus. The stability of the serotonin system in posthypoxic myoclonic rats is remarkable when one considers the wide range of disorders that is produced by the prolonged brain ischemia. The inability of the most severely posthypoxic myoclonic rats to perform 7-Hz tongue protrusions indicates substantial physiologic disruption of brainstem motor function. Moreover, the posthypoxic myoclonic rat suffers from ataxia, seizures, retrograde amnesia, and impaired ability to learn. The wide spectrum of these deficits is sharply constrasted by its apparently intact serotonin system. We have identified the inferior olive as a locus that may generate the rhythmic components of tremor and myoclonus in syndromes that are truly associated with a dramatic loss of brainstem serotonin. Serotonin acts within the inferior olive to constrain its rhythmic firing. Without intraolivary serotonin, olivary neurons are predisposed to oscillate continuously, providing a substrate upon which sustained rhythmic spiking may be superimposed. It is clear that such unconstrained rhythmicity produces synchronized whole-body tremor at 10 Hz (33, 41-43). The effects of serotonin to suppress olivocerebellar rhythmicity are mediated by postsynaptic 5-HT2 receptors that reduce the magnitude of the low-threshold calcium conductance, IT. It is notable that dysregulation of this conductance has been associated with hyper-rhythmic states in the thalamus underlying cognitive disorders ranging from depression to tinnitus (49), indicating a common mechanism underlying a variety of neurologic conditions. The identification of a specific brainstem locus (inferior olive), serotonin receptor 5-HT2, and ionic current IT involved in a form of rhythmic myoclonus may provide multiple clues toward which future pharmacotherapies can be directed.

Amnesia, Retrograde↗

PubMed2002

Why do Purkinje cells die so easily after global brain ischemia? Aldolase C, EAAT4, and the cerebellar contribution to posthypoxic myoclonus.

The experiments strongly suggested that the reason why Purkinje cells die so easily after global brain ischemia relates to deficiencies in aldolase C and EAAT4 that allow them to survive pathologically intense synaptic input from the inferior olive after the restoration of blood flow. This conclusion is based on: (a) the remarkably tight correspondence between the regional absence of aldolase C and EAAT4 in Purkinje cells and the patterned loss of Purkinje cells after a bout of global brain ischemia; (b) the necessity of the olivocerebellar pathway for the ischemic death of Purkinje cells; and (c) the build-up of pathologically synchronous and high-frequency burst activity within the inferior olive during recovery from ischemia. Indeed, the correspondence between the absence of aldolase C and EAAT4 to sensitivity to ischemia could be demonstrated for zones of Purkinje cells as small as two neurons. A second finding was that Purkinje cells are not uniformly sensitive to transient ischemia, since they die most frequently in zones where aldolase C and EAAT4 are absent. One implication of the experiment is that factors beyond the unique synaptic and membrane properties of Purkinje cells play an important role in determining this neuron's high sensitivity to ischemia. The data strongly imply that two properties of Purkinje cells that make them susceptible to ischemic death are their reduced capability to sequester glutamate and reduced ability to generate energy during anoxia. The patterned death of Purkinje cells is sufficient to induce a form of audiogenic myoclonus, as determined with a neurotoxic dose of ibogaine. Ibogaine-induced myoclonus is recognized behaviorally as a reduced ability to habituate to a startle stimulus and resembles the myoclonic jerk of rats during recovery from a prolonged bout of global brain ischemia. Commonalities of ischemia and ibogaine-induced neurodegeneration are the intricately striped Purkinje cell loss in the posterior lobe and a nearly complete deafferentation of the lateral aspect of the fastigial nucleus from the cerebellar cortex, in particular the dorsolateral protuberance. Thus, the data point strongly to a cerebellar contribution to audiogenic myoclonus. Single-neuron electrophysiology experiments in monkeys have demonstrated that the evoked activity in the deep cerebellar nuclei occurs too late to initiate the startle response (60) and electromyography of the postischemic myoclonus of rats corroborates this view (see Chapter 31) (20). However, the nearly complete loss of GABAergic terminals in the dorsolateral protuberance after Purkinje cell death would be expected to dramatically increase its tonic firing and the background excitation of the brain-stem structures that it innervates. The fastigial nucleus innervates a large number of autonomic and motor structures in the brainstem and diencephalon, including the ventrolateral nucleus of the thalamus and the gigantocellular reticular nucleus in the medulla--structures that have been implicated in human posthypoxic myoclonus (6, 7). We propose that the posthypoxic myoclonic jerk of rats is, at least in part, due to disinhibition of the fastigial nucleus produced by patterned Purkinje cell death in the vermis. The argument is as follows: the loss of GABAergic inhibition in the fastigial nucleus after ischemia leads to diaschisis of the motor thalamus and reticular formation which, in turn, is responsible for enhanced motor excitability and myoclonus. That the audiogenic myoclonus after global brain ischemia in the rat gradually resolves over a period of 2 to 3 weeks is consistent with this view, as restoration of background excitability after CNS damage in rats has been documented to occur within this time-frame (61). Our view brings together the physiologic finding that posthypoxic myoclonus appears to originate in the sensory-motor cortices and/or reticular formation with the consistent anatomical finding of Purkinje cell loss after ischemia, and explains the puzzle of Marsden's unique cases of myoclonus associated with coeliac disease (1). Moreover, our argument is consistent with findings both in rats (62, 63) and humans (64) that damage to the vermis impairs the long-term habituation of the startle reflex. It remains to be determined whether the pathologically enhanced startle responses after vermal damage resemble brain-stem reticular or cortical myoclonus at the electrophysiologic level of analysis. What is the purpose of the regional expression of aldolase C and EAAT4 in Purkinje cells? The close correspondence between the spatial distribution of aldolase C and the parasagittal anatomy of the cerebellum (48) has led to the view that aldolase C may help specify connectivity during development. While the present experiments do not address this issue, they underscore the fact that aldolase plays a fundamental role in metabolism. Because Purkinje cells have a repressed expression of aldolase A (31), whatever role the absence of aldolase C may play during development comes at the price of metabolic frailty later in adulthood. From another point of view, aldolase C and EAAT4 appear to confer upon Purkinje cells the ability to survive their own climbing fiber. Indeed, climbing fibers form a distributed synapse that synchronously releases glutamate (or aspartate) at all levels of the dendritic tree simultaneously (65, 66). Such synchronous activation triggers calcium influx throughout the Purkinje cell dendrites at a magnitude that is unparalleled in the nervous system (12), and, thus, places an extraordinarily high metabolic demand on the Purkinje cell. The apparently reduced level of aldolase in a subpopulation of Purkinje cells provides the condition for energy failure and death during anoxia so long as the climbing fibers are intact or when climbing fiber activation is pharmacologically enhanced under normoxic conditions, such as after ibogaine (53-56). Lastly, the argument that diaschisis produced by patterned cerebellar degeneration leads to thalamo-cortical and reticular hyperexcitability agrees with C. David Marsden and his colleagues' bold demonstration of an inhibitory influence of cerebellar cortex on motor cortex in humans (67). Our anatomic data indicate that the spatially distinct zones of Purkinje cells, which are killed by global brain ischemia, may be the origin of such inhibition.

Amino Acid Transport System X-AG↗

PubMedDate not supplied

Is autism due to brain desynchronization?

The hypothesis is presented that a disruption in brain synchronization contributes to autism by destroying the coherence of brain rhythms and slowing overall cognitive processing speed. Particular focus is on the inferior olive, a precerebellar structure that is reliably disrupted in autism and which normally generates a coherent 5-13 Hz rhythmic output. New electrophysiological data reveal that the continuity of the rhythmical oscillation in membrane potential generated by inferior olive neurons requires the formation of neuronal assemblies by the connexin36 protein that mediates electrical synapses and promotes neuronal synchrony. An experiment with classical eyeblink conditioning is presented to demonstrate that the inferior olive is necessary to learn about sequences of stimuli presented at intervals in the range of 250-500 ms, but not at 700 ms, revealing that a disruption of the inferior olive slows stimulus processing speed on the time scale that is lost in autistic children. A model is presented in which the voltage oscillation generated by populations of electrically synchronized inferior olivary neurons permits the utilization of sequences of stimuli given at, or faster than, 2 per second. It is expected that the disturbance in inferior olive structure in autism disrupts the ability of inferior olive neurons to become electrically synchronized and to generate coherent rhythmic output, thereby impairing the ability to use rapid sequences of cues for the development of normal language skill. Future directions to test the hypothesis are presented.

Animals↗