Faculty Bibliography

In order to identify and understand mechanistically the cortical circuitry of sensory information processing estimates are needed of synaptic input fields that drive neurons. From intracellular in vivo recordings one would like to estimate net synaptic conductance time courses for excitation and inhibition, g(E)(t) and g(I)(t), during time-varying stimulus presentations. However, the intrinsic conductance transients associated with neuronal spiking can confound such estimates, and thereby jeopardize functional interpretations. Here, using a conductance-based pyramidal neuron model we illustrate errors in estimates when the influence of spike-generating conductances are not reduced or avoided. A typical estimation procedure involves approximating the current-voltage relation at each time point during repeated stimuli. The repeated presentations are done in a few sets, each with a different steady bias current. From the trial-averaged smoothed membrane potential one estimates total membrane conductance and then dissects out estimates for g(E)(t) and g(I)(t). Simulations show that estimates obtained during phases without spikes are good but those obtained from phases with spiking should be viewed with skeptism. For the simulations, we consider two different synaptic input scenarios, each corresponding to computational network models of orientation tuning in visual cortex. One input scenario mimics a push-pull arrangement for g(E)(t) and g(I)(t) and idealized as specified smooth time courses. The other is taken directly from a large-scale network simulation of stochastically spiking neurons in a slab of cortex with recurrent excitation and inhibition. For both, we show that spike-generating conductances cause serious errors in the estimates of g(E) and g(I). In some phases for the push-pull examples even the polarity of g(I) is mis-estimated, indicating significant increase when g(I) is actually decreased. Our primary message is to be cautious about forming interpretations based on estimates developed during spiking phases

ABSTRACT: BACKGROUND: Molecular genetic studies suggest the dopamine D1 receptor (D1R) may be implicated in attention-deficit/hyperactivity disorder (ADHD). As little is known about the potential motor role of D1R in ADHD, animal models may provide important insights into this issue. Methods: We investigated the effects of a full and selective D1R agonist, SKF-81297 (0.3, 3 and 10 mg/kg), on motor behaviour and expression of the plasticity-associated gene, c-fos, in habituated young adult male Spontaneously Hypertensive Rats (SHR), the most commonly used animal model of ADHD, and Wistar-Kyoto (WKY; the strain from which SHR were derived). Results: SHR rats were more behaviourally active than WKY rats after injection with vehicle. The 0.3 mg/kg dose of SKF-81297 increased motor behaviour (locomotion, sifting, rearing, and sniffing) in both SHR and WKY rats. Total grooming was also stimulated, but only in WKY rats. The same dose increased c-fos mRNA expression in the piriform cortex of both strains. The 3 mg/kg dose increased sifting and sniffing in both strains. Locomotion was also stimulated towards the end of the testing period. The intermediate dose decreased total rearing in both strains, and produced a significant increase in c-fos mRNA in the striatum, nucleus accumbens, olfactory tuberculum, and in the cingulate, agranular insular and piriform cortices. The 10 mg/kg dose of SKF-81297 produced a biphasic effect on locomotion, which was characterized by an initial decrease followed by later stimulation. The latter stimulatory effect was more pronounced in SHR than in WKY rats when compared to their respective vehicle-injected groups. The 10 mg/kg dose also stimulated sifting and sniffing in both strains. Both the 3 and 10 mg/kg doses had no effect on total grooming. The 10 mg/kg dose induced significantly higher levels of c-fos mRNA expression in the nucleus accumbens and adjacent cortical regions (but not striatum) of SHR when compared to WKY rats. CONCLUSION: The present results suggest a potential alteration in D1R neurotransmission within the frontal-striatal circuitry of SHR involved in motor control. These findings extend our understanding of the molecular alterations in SHR, a heuristically useful model of ADHD

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival

One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14.

In a previous study, we identified three cortical areas in human posterior parietal cortex that exhibited topographic responses during memory-guided saccades [visual area 7 (V7), intraparietal sulcus 1 (IPS1), and IPS2], which are candidate homologs of macaque parietal areas such as the lateral intraparietal area and parietal reach region. Here, we show that these areas exhibit sustained delay-period activity, a critical physiological signature of areas in macaque parietal cortex. By varying delay duration, we disambiguated delay-period activity from sensory and motor responses. Mean time courses in the parietal areas were well fit by a linear model comprising three components representing responses to (1) the visual target, (2) the delay period, and (3) the eye movement interval. We estimated the contributions of each component: the response amplitude during the delay period was substantially smaller (<30%) than that elicited by the transient visual target. All three parietal regions showed comparable delay-period response amplitudes, with a trend toward larger responses from V7 to IPS1 and IPS2. Responses to the cue and during the delay period showed clear lateralization with larger responses to trials in which the target was placed in the contralateral visual field, suggesting that both of these components contributed to the topography we measured.

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

The response of a neuron in primary visual cortex (V1) to an optimal stimulus in its classical receptive field (CRF) can be reduced by the presence of an orthogonal mask, a phenomenon known as cross-orientation suppression. The presence of a parallel stimulus outside the CRF can have a similar effect, in this case known as surround suppression. We used a novel stimulus to probe the time course of cross-orientation suppression and found that it is very fast, starting even before the response to optimal excitatory stimuli. However, it occurs with some delay after the offset response, considered to be a measure of the earliest excitatory signals that reach the CRF. We also examined the time course of response to a stimulus presented outside the CRF and found that cross-orientation suppression begins substantially earlier than surround suppression measured in the same cells. Together, these findings suggest that cross-orientation suppression is attributable to either direct feedforward signal paths to V1 neurons or a circuit involving fast local interneurons within V1. Feedback from higher cortical areas is implicated in surround suppression, but our results make this an implausible mechanism for cross-orientation suppression. We conclude that suppression from inside and outside the CRF occur through different mechanisms