Faculty Bibliography

Ziconotide, a potent, selective, reversible blocker of neuronal N-type voltage-sensitive calcium channels, is approved in the United States for the management of severe chronic pain in patients for whom intrathecal therapy is warranted, and who are intolerant or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. In the European Union, ziconotide is indicated for the treatment of severe chronic pain in patients who require intrathecal analgesia. Nonclinical investigations of ziconotide included a comprehensive characterization of its toxicology, incorporating acute and subchronic toxicity studies in rats, dogs, and monkeys; reproductive toxicity assessments in rats and rabbits; and mutagenic, carcinogenic evaluations performed in vivo and in vitro. Additional investigations assessed the potential for cardiotoxicity (rats) and immunogenicity (mice, rats, and guinea pigs), and the presence or absence of intraspinal granuloma formation and local cell proliferation and apoptosis (dogs). The resulting nonclinical toxicology profile was predictive of human adverse events reported in clinical trials and consistent with ziconotide's pharmacological activity. Frequently observed nonclinical behavioral effects included tremoring, shaking, ataxia, and hyperreactivity. Occurrences were generally transient and reversible upon cessation of treatment, and intolerable effects occurred at doses more than 45 times the maximum recommended clinical dose. Ziconotide was not associated with target organ toxicity, teratogenicity, or treatment-related gross or histopathological changes; it displayed no mutagenic or carcinogenic potential and no propensity to induce local cell proliferation or apoptosis. Although guinea pigs developed systemic anaphylaxis, antibodies to ziconotide were not detected in mice, rats, or guinea pigs, indicating low immunogenic potential. No evidence of granuloma formation was observed with intrathecal ziconotide treatment. In summary, the results from these nonclinical safety assessments revealed no significant toxicological risk to humans treated with ziconotide as recommended

Intracellular recording and single-cell labeling were combined to investigate the oculomotor circuitry of the goldfish cerebellar vestibulolateral lobe, consisting of the eminentia granularis (Egr) and caudal lobe. Purkinje cells exhibiting highly conserved vertebrate electrophysiological and morphological properties provide the direct output from the caudal lobe to the vestibular nuclei. Biocytin labeling of the Egr distinguished numerous hindbrain precerebellar sources that could be divided into either putative mechano- or vestibulosensitive nuclei based on cellular location and axon trajectories. Precerebellar neurons in a hindbrain nucleus, called Area II, were electrophysiologically characterized after antidromic activation from the Egr (>50% bilateral) and their morphology analyzed after intracellular biocytin labeling (n = 28). Bipolar spindle-shaped somas ranged widely in size with comparably scaled dendritic arbors exhibiting largely closed field configuration. Area II neurons (85%) projected to the ipsilateral Egr with most (93%) sending a collateral through the cerebellar commissure to the contralateral Egr; however, 15% projected to the contralateral Egr by crossing in the ventral hindbrain. Axon terminals in the vestibular nucleus were the only collaterals within the hindbrain. Every Area II neuron received a disynaptic EPSP after contralateral horizontal canal nerve stimulation and a disynaptic IPSP, preceded by a small EPSP (>50%), after ipsilateral activation. Vestibular synaptic potentials were of varying shape/amplitude, unrelated to neuron location in the nucleus, and thus likely a correlate of somadendritic size. The exceptional separation of eye position and eye velocity signals into two separate hindbrain nuclei represents an ideal model for understanding the precerebellar projection to the vestibulocerebellum

Elucidating the causal role of head and eye movement signaling during cerebellar-dependent oculomotor behavior and plasticity is contingent on knowledge of precerebellar structure and function. To address this question, single-unit extracellular recordings were made from hindbrain Area II neurons that provide a major mossy fiber projection to the goldfish vestibulolateral cerebellum. During spontaneous behavior, Area II neurons exhibited minimal eye position and saccadic sensitivity. Sinusoidal visual and vestibular stimulation over a broad frequency range (0.1-4.0 Hz) demonstrated that firing rate mirrored the amplitude and phase of eye or head velocity, respectively. Table frequencies >1.0 Hz resulted in decreased firing rate relative to eye velocity gain, while phase was unchanged. During visual steps, neuronal discharge paralleled eye velocity latency (approximately 90 ms) and matched both the build-up and the time course of the decay (approximately 19 s) in eye velocity storage. Latency of neuronal discharge to table steps (40 ms) was significantly longer than for eye movement (17 ms), but firing rate rose faster than eye velocity to steady-state levels. The velocity sensitivity of Area II neurons was shown to equal (+/- 10%) the sum of eye- and head-velocity firing rates as has been observed in cerebellar Purkinje cells. These results demonstrate that Area II neuronal firing closely emulates oculomotor performance. Conjoint signaling of head and eye velocity together with the termination pattern of each Area II neuron in the vestibulolateral lobe presents a unique eye-velocity brain stem-cerebellar pathway, eliminating the conceptual requirement of motor error signaling

We quantitatively studied the ontogeny of oculomotor behavior in larval fish as a foundation for studies linking oculomotor structure and function with genetics. Horizontal optokinetic and vestibuloocular reflexes (OKR and VOR, respectively) were measured in three different species (goldfish, zebrafish, and medaka) during the first month after hatching. For all sizes of medaka, and most zebrafish, Bode plots of OKR (0.065-3.0 Hz, +/-10 degrees/s) revealed that eye velocity closely followed stimulus velocity (gain > 0.8) at low frequency but dropped sharply above 1 Hz (gain < 0.3 at 3 Hz). Goldfish showed increased gain proportional to size across frequencies. Linearity testing with steps and sinusoids showed excellent visual performance (gain > 0.8) in medaka almost from hatching; but zebrafish and goldfish exhibited progressive improvement, with only the largest equaling medaka performance. Monocular visual stimulation in zebrafish and goldfish produced gains of 0.5 versus <0.1 for the eye viewing a moving versus stationary stimulus pattern but 0.25 versus <0.1 in medaka. Angular VOR appeared much later than OKR, initially at only high accelerations (>200 degrees /s at 0.5 Hz), first in medaka followed by larger (8.11 mm) zebrafish; but it was virtually nonexistent in goldfish. Velocity storage was not observed except for an eye velocity build-up in the largest medaka. In summary, a robust OKR was achieved shortly after hatching in all three species. In contrast, larval fish seem to be unique among vertebrates tested in their lack of significant angular VOR at stages where active movement is required for feeding and survival

Tritonia pedal ganglion peptides (TPeps) are a trio of pentadecapeptides isolated from the brain of the nudibranch Tritonia diomedea. TPeps have been shown both to increase the beating rate of ciliated cells of Tritonia and to accelerate heart contractions in the mollusc Clione limacina. Here we examine the immunocytochemical distribution of TPeps in the Tritonia central nervous system. We found the brain and buccal ganglia to be rich sources of TPep immunoreactivity. Specific cells in both structures, some of them previously identified, were immunoreactive. Moreover, immunoreactive fibers were seen connecting ganglia and exiting almost all the major nerves. In the brain, we found that the paired, ciliated statocysts apparently receive TPep innervation. In addition, we observed unstained cell bodies in each buccal ganglion with extensive TPep immunoreactive projections surrounding their somata and primary neurites. Similar projections were not observed in the brain. We also compared the TPep immunoreactivity with that of SCP(b) in the buccal ganglia. We observed many neurons and processes that were immunoreactive to both peptides. One neuron that contains both TPep- and SCP(b)-like peptides (B12) has an identified role in the Tritonia feeding network. Together, these findings suggest that TPeps may play an active role in the central nervous system of Tritonia as neurotransmitters modulating orientation, swimming, and feeding.

Microscopic analysis of anatomic relationships within the neural networks of adult and developing tissues often requires sampling large spatial regions of neuronal architecture. To accomplish this, there are two common imaging approaches: (1) image the entire area at once with low spatial resolution; or (2) image small sections at higher magnification/resolution and then join the sections back together by mosaic reconstruction (photomontaging). Low magnification imaging is relatively rapid to perform, resulting in a visualization that encompasses a large field of view with an extended depth of field. However, for fluorescence microscopy, low magnification visualizations are often plagued by poor spatial resolution. High magnification imaging possesses superior spatial resolution, but it produces an image with limited depth of field. When creating a larger field of view, the final image is also fragmented at the boundaries where multiple images are stitched together. Using confocal microscopy as well as features of common image processing programs, we outline a new method to transform individual, spatially contiguous z-series into a montage with a seamless field of view and an extended depth of field. In addition, we show that the manual alignment of images our method requires does not introduce significant errors into the final image. We illustrate our method for visualizing neural networks using tissues from the adult gastropod mollusc, Tritonia diomedea, and the developing zebrafish, Danio rerio.

Approximately 10% of newborns with congenital hypothyroidism are unable to convert iodide into organic iodine. This iodide organification defect has a prevalence of 1 in 40,000 newborns and may be caused by defects in the thyroid peroxidase enzyme (TPO), the hydrogen peroxide-generating system, the TPO substrate thyroglobulin, or inhibitors of TPO. We identified a high incidence of severe hypothyroidism due to a complete iodide organification defect in the youngest generation of five nuclear families belonging to an inbred Amish kindred. Genealogical records permitted us to trace their origin to an ancestral couple 7-8 generations back and to identify an autosomal recessive pattern of inheritance. Initial studies of homozygosity by descent using two polymorphic markers within the TPO gene showed no linkage to the phenotype. In fact, 4 of 15 affected siblings from 2 of the nuclear families were heterozygous, resulting in homozygosity values of 73% and 53% in affected and unaffected family members, respectively. A genome-wide homozygosity screen using DNA pools from affected and unaffected family members localized the defect to a locus close to the TPO gene. Linkage analysis using 4 additional polymorphic markers within the TPO gene reduced the number of homozygous unaffected siblings to zero without altering the percent homozygosity initially found in the affected. Sequencing of the TPO gene revealed 2 missense mutations, E799K and R648Q. TPO 779K was found in both alleles of the 11 affected homozygotes, both mutations were present in each of the 3 affected compound heterozygotes, and there were no TPO mutations in 1 subject with hypothyroidism of different etiology. These results demonstrate the power of the DNA pooling strategy in the localization of a defective gene and the pitfalls of linkage analysis when 2 relatively rare mutations coexist in an inbred population.