Jack B. Kelly

Biosynthesis of the N-Linked Glycan in Campylobacter jejuni and Addition onto Protein through Block Transfer

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.

Origins of medial geniculate body projections to physiologically defined zones of rat primary auditory cortex.

Medial geniculate body neurons projecting to physiologically identified subregions of rat primary auditory cortex (area 41, Te1) were labeled with horseradish peroxidase in adult rats. The goals were to determine the type(s) of projection neuron and the spatial arrangement of these cells with respect to thalamic subdivisions. Maps of best frequency were made with single neuron or unit cluster extracellular recording at depths of 500-800 microm, which correspond to layers III-IV in Nissl preparations. Tracer injections were made in different cortical isofrequency regions (2, 11, 22, or 38 kHz, respectively). Labeled neurons were plotted on representative sections upon which the architectonic subdivisions were drawn independently. Most of the cells of origin lay in the ventral division in every experiment. Injections at low frequencies labeled bands of neurons laterally in the ventral division; progressively more rostral deposits at higher frequencies labeled bands or clusters more medially in the ventral division, and through most of its caudo-rostral extent. Medial division labeling was variable. Labeled cells were always in the lateral half of the nucleus and were often scattered. There were few labeled cells in the dorsal division. Seven types of thalamocortical neuron were identified: ventral division cells had a tufted branching pattern, while medial division neurons have heterogeneous shapes and sizes and were larger. Dorsal division neurons had a radiate branching pattern. The size range of labeled neurons spanned that of Nissl stained neuronal somata. Area 41 may receive two types of thalamic projection: ventral division input is strongly convergent, highly topographic, spatially focal, and restricted to one type of neuron only, while the medial division projection is more divergent, coarsely topographical, involves multiple cortical areas, and has several varieties of projection neuron. Despite species differences in local circuitry, many facets of thalamocortical organization are conserved in phylogeny.

Neural architecture of the rat medial geniculate body

The rat medial geniculate body was subdivided using Nissl preparations to establish nuclear boundaries, with Golgi-Cox impregnations to identify projection and local circuit neurons, and in fiber stained material to delineate the fiber tracts and their distribution. Three divisions were recognized (ventral, dorsal and medial): the first two had subdivisions. The ventral division had lateral and medial parts. The main cell type had bushy tufted dendrites which, with the afferent axons, formed fibrodendritic laminae oriented from dorso-lateral to ventro-medial; such laminae were not as regular medially, in the ovoid nucleus. The dorsal division contained several nuclei (dorsal superficial, dorsal, deep dorsal, suprageniculate, and ventrolateral) and neurons with radiating or bushy dendrites; the nuclear subdivisions differed in the concentration of one cell type or another, and in packing density. A laminar organization was present only in the dorsal superficial nucleus. Medial division neurons were heterogeneous in size and shape, ranging from tiny cells to magnocellular neurons; the various cell types intermingled. so that no further subdivision could be made. This parcellation scheme was consistent with, and supported by, the findings from plastic embedded or fiber stained material. There were very few small neurons with locally ramifying axons and which could perform an intrinsic role like that of Golgi type II cells. Their rarity was consistent with the small number of such profiles in plastic embedded or Nissl material and the few GABAergic medial geniculate body neurons seen in prior immunocytochemical work. While similar neuronal types and nuclear subdivisions are recognized in the rat and cat, there may be major interspecific differences with regard to interneuronal organization in the auditory thalamus whose functional correlates are unknown.

Projections from the superior olive and lateral lemniscus to tonotopic regions of the rat's inferior colliculus.

The projections to physiologically defined tonotopic regions of the central nucleus of the inferior colliculus (ICC) from the adult rats superior olivary complex (SOC) and lateral lemniscus were investigated using retrograde tract tracing methods. Iontophoretic injections of the retrograde tracers, Fluoro-Gold (FG) or horseradish peroxidase (HRP), were made into the ICC through a glass micropipette, which also served as a recording electrode to determine the frequency response at the injection site. Injections were made into frequency-specific regions based on the best responses of neurons to contralaterally presented tones between 2 25 kHz. In the dorsal nucleus of the lateral lemniscus (DNLL) neurons were labeled both ipsilaterally and contralaterally to the injection site with a larger proportion projecting to the contralateral side. The distribution of labeled cells was concentric, with high frequencies represented along the outer margin and low frequencies represented centrally within DNLL. The lateral superior olive (LSO) was labeled bilaterally, with high frequencies represented medially and low frequencies laterally along the nuclear axis. The projection from the medial superior olive (MSO) was ipsilateral, with high frequencies represented ventrally and low frequencies dorsally. The projection from the superior paraolivary nucleus (SPN) was also largely ipsilateral, with high frequencies represented medially and low frequencies laterally. The intermediate and ventral nuclei of the lateral lemniscus (INLL and VNLL) were also labeled ipsilaterally and exhibited a distribution of tracer that depended on the frequency of the injection site: the low frequency projection was banded but the high frequency projection was more evenly distributed.

GABAergic projections from the lateral lemniscus to the inferior colliculus of the rat

The objective of the present study was to provide direct evidence regarding GABAergic projections from the nuclei of the lateral lemniscus to the central nucleus of the inferior colliculus (ICC), and from the ICC to the opposite ICC. Projections of GABAergic neurons in the rat were investigated by a combination of fluorogold (FG) retrograde tracing and GABA immunocytochemistry. FG was first injected into a frequency-defined region (11-13 kHz) in the center of the ICC, and 1-2 weeks was allowed for retrograde transport. Vibratome sections were then cut through the brainstem and stained with GABA antibody. Double-labeling was taken as evidence of GABAergic neurons projecting to the ICC. The results from FG retrograde labeling alone showed that neurons in the dorsal nucleus of the lateral lemniscus (DNLL) bilaterally, in the intermediate and ventral nucleus of the lateral lemniscus (INLL and VNLL) ipsilaterally, and in the ICC contralaterally project to the ICC. GABA immunostaining alone showed substantial numbers of GABA positive neurons in the nuclei of the lateral lemniscus and the inferior colliculus. FG and GABA double-labeled neurons were present in all nuclei of the lateral lemniscus that project to the ICC. The greatest concentration of double-labeled neurons was found bilaterally in the DNLL, suggesting a prominent GABAergic projection from the DNLL to the ICC. The presence of many double-labeled neurons in the ipsilateral INLL and VNLL suggests that there are also GABAergic inputs from the INLL and VNLL to the ICC. No double-labeled neurons were found in the contralateral ICC, which suggests the possibility of a prominent non-GABAergic projection.

Representation of the cochlea in primary auditory cortex of the ferret (Mustela putorius)

In seven barbiturate-anesthetized ferrets, we explored the acoustically sensitive cortex with conventional microelectrode mapping techniques. A tonotopically organized field was found whose orientation was such that high tonal frequencies were represented dorsally, and low frequencies ventrally. Within this field, neurons typically had short (12-20 ms) latent periods to first spikes. In conjunction with extant anatomical evidence on the connectivity of this region, these data suggest that this field represents the ferrets primary auditory cortex.

Calpain-Cleaved Collapsin Response Mediator Protein-3 Induces Neuronal Death after Glutamate Toxicity and Cerebral Ischemia

Collapsin response mediator proteins (CRMPs) mediate growth cone collapse during development, but their roles in adult brains are not clear. Here we report the findings that the full-length CRMP-3 (p63) is a direct target of calpain that cleaves CRMP-3 at the N terminus (+76 amino acid). Interestingly, activated calpain in response to excitotoxicity in vitro and cerebral ischemia in vivo also cleaved CRMP-3, and the cleavage product of CRMP-3 (p54) underwent nuclear translocation during neuronal death. The expression of p54 was colocalized with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei in glutamate-treated cerebellar granule neurons (CGNs) and in ischemic neurons located in the infarct core after focal cerebral ischemia, suggesting that p54 might be involved in neuronal death. Overexpression studies showed that p54, but not p63, caused death of human embryonic kidney cells and CGNs, whereas knock-down CRMP-3 expression by selective small interfering RNA protected neurons against glutamate toxicity. Collectively, these results reveal a novel role of CRMP-3 in that calpain cleavage of CRMP-3 and the subsequent nuclear translocation of the truncated CRMP-3 evokes neuronal death in response to excitotoxicity and cerebral ischemia. Our findings also establish a novel route of how calpain signals neuron death.

Primary auditory cortex in the ferret (Mustela putorius): neural response properties and topographic organization

Microelectrode recording studies were made of the crown of the ectosylvian auditory cortex of barbiturate-anesthetized ferrets, using calibrated, sealed acoustic stimulus delivery systems. We confirmed our previous finding using free-field stimuli that this region of the ferrets cerebral cortex contains a tonotopically organized field in which neurons are briskly excited by the onset of tonal stimuli. The vast majority of neurons in this field were narrowly tuned to tone pulse frequency, with Q factors comparable to those described for cortical cells in other species. The distribution of minimum tone thresholds across the frequency representation of this field paralleled the behavioral audiogram in the same species. The majority of neurons received input from both ears, and the natures of the influences exerted by tonal stimuli at the two ears were similar in form to those described for other carnivores. The various binaural cell types had characteristic frequencies that spanned the entire tonal spectrum represented in the cortex. Preliminary mapping studies revealed that there may be a topographic segregation of cells according to their binaural interactions. The basic properties of cells in this region of the ferrets cerebrum are similar to those previously described for the cats primary auditory cortex. The data on the binaural properties of these cells, and the spatial distribution of those cells, provide the first evidence in a species other than the cat for segregated binaural representation in the auditory cortex.

Contribution of AMPA, NMDA, and GABAA Receptors to Temporal Pattern of Postsynaptic Responses in the Inferior Colliculus of the Rat

The central nucleus of the inferior colliculus (ICC) is a major site of synaptic interaction in the central auditory system. To understand how ICC neurons integrate excitatory and inhibitory inputs for processing temporal information, we examined postsynaptic responses of ICC neurons to repetitive stimulation of the lateral lemniscus at 10-100 Hz in rat brain slices. The excitatory synaptic currents mediated by AMPA and NMDA receptors and the inhibitory current mediated by GABAA receptors were pharmacologically isolated and recorded by whole-cell patch-clamp techniques. The response kinetics of AMPA receptor-mediated EPSCs and GABAA receptor-mediated IPSCs were similar and much faster than those of NMDA receptor-mediated EPSCs. AMPA EPSCs could follow each pulse of stimulation at a rate of 10-100 Hz but showed response depression during the course of repetitive stimulation. GABAA IPSCs could also follow stimulus pulses over this frequency range but showed depression at low rates and facilitation at higher rates. NMDA EPSCs showed facilitation and temporal summation in response to repetitive stimulation, which was most pronounced at higher rates of stimulation. GABAA inhibition suppressed activation of NMDA receptors and reduced both the degree of AMPA EPSC depression and the extent of temporal summation of NMDA EPSCs. The results indicate that GABAA receptor-mediated inhibition plays a crucial role in maintaining the balance of excitation and inhibition and in allowing ICC neurons to process temporal information more precisely.

Sound frequency and binaural response properties of single neurons in rat inferior colliculus

Sound frequency and binaural response properties were determined for single neurons in the rats inferior colliculus. Nerve cell responses in the central nucleus of the inferior colliculus were narrowly tuned and had clearly defined characteristic frequencies (CF). The central nucleus was tonotopically organized with low frequencies represented dorsolaterally and high frequencies ventromedially from 0.87 to 45 kHz. Sharpness of tuning, as indicated by Q10, covered a wide range of values for neurons with the same CF, but the maximum Q10 at each frequency increased monotonically with CF. Maximum Q10s were larger than previously reported for auditory cortex at the same CF. Binaural responses were classified as either suppression, summation or mixed. Most of the units encountered exhibited binaural suppression but there were substantial numbers of both summation and mixed responses. Each major binaural response type was distributed broadly across sound frequencies within the rats hearing range. Binaural suppression responses were most numerous at high frequencies and summation responses at low frequencies. The binaural response types, their relative proportions and their distribution by CF were similar for neurons in the central nucleus of inferior colliculus and primary auditory cortex of the albino rat.