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We have determined the parameters necessary to fabricate reproducible neuronal patterns which we are using to begin studying fundamental issues in developmental neurobiology. The addition of a beam homogenizer, as well as a new surface preparation, has enabled the routine production of reproducible, high-resolution (2−20 μm) organosilane patterns. The effects of surface preparation and beam dosage were monitored using X-ray photoelectron spectroscopy (XPS) and proof of patterning is provided by high-resolution imaging XPS. We also report the guidance of neuronal adhesion and neurite outgrowth and the creation of reproducibly defined circuits of embryonic (E18−19) rat hippocampal neurons using these patterned surfaces in vitro. We have achieved a >50% rate of pattern formation, and at times the rate approaches 90%. We are using these patterns to address the issue of how geometric pattern cues might be used to affect cell-to-cell communication and we report the preliminary results on the synaptic development of the hippocampal neurons using dual patch-clamp electrophysiology. We monitored neurite outgrowth and the emergence of both spontaneous and evoked synaptic activity for both patterned and unpatterned (control) hippocampal cultures. The results indicate the intriguing possibility that geometry itself may be a modulating or trophic factor for cell development.

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

Eighteen dogs with malignant melanoma of the oral cavity were treated with high-dose per fraction (0-7-21 ) radiation therapy. Eight hundred cGy was administered on days 0,7, and 21 for a total dose of 2,400 cGy in 3 weeks. Of 1 7 dogs evaluated, 9 (53%) had a complete remission and 5 (30%) achieved a partial remission with an overall response rate of 83%. Local failure occurred in 2 of the 9 dogs where a complete response was initially observed. One dog died of intercurrent disease, and one died of metastatic disease without evidence of local recurrence. Five dogs are alive elanomas are the most common oropharyngeal ma-

The 0-7-21 radiation therapy protocol was investigated as a palliative treatment in dogs with advanced malignancies. Twenty-four dogs with a variety of tumor types were irradiated using 800 cGy fractions given on days 0, 7, and 2 1 .Twenty-three dogs were evaluated. Palliative response was assessed using a quality of life instrument developed for veterinary use. This pain score was based on owner response to questions regarding analgesic requirement, activity level, appetite, and degree of lameness in the affected dogs. Seventeen (74%) of the 23 dogs experihen cancer patients present with advanced or disseminated disease, the goal of treatment becomes palliative, rather than curative. Palliative therapy is directed at reducing pain and other clinical signs that detract from a patient's quality of life. In many cancers, pain increases in frequency and severity with disease progression.''2 This is a major source of concern for pet owners. In humans, the incidence of pain is greatest in patients with primary bone tumors and cancers of the oral In 1970, Gillette described radiation therapy as an effective means to alleviate pain and extend "meaningful" survival in domestic anirnal~.~ Despite this, the palliative qualities of radiation have received little attention in the veterinary literature. Radiation therapy has generally been approached with curative intent, although the result is often palliative.6 Dose and fractionation have not varied with the intended outcome. Without the possibility of cure, euthanasia has been considered a more humane option than a radical course of radiation therapy.' In humans, close to 50% of radiation therapy is prescribed with palliative intent.' Fractionation patterns have vaned widely and range from single 800 to 1,000 cGy fractions, to 2,000 cGy given over 5 days, to extended courses of 5,000 cGy given over 5 The aim of palliative radiation is to achieve rapid, effective, and lasting symptomatic relief in as short a course as possible, provided the normal tissue reactions can be kept to a minimum.The benefits of palliative therapy are difficult to assess. Traditional end points, such as survival and tumor control are not appropriate measurements of quality of life. Quality of life instruments have not been previously described for animal patients, but should be developed to evaluate palliative protocols.The 0-7-21 radiation therapy protocol is a hypofractionated regime consisting of 800 cGy fractions given on days 0, 7, and 21. This protocol has shown promise in humans for the palliation of mucosal or metastatic melanoma.10-'2 The purposes of this study were to ( 1) determine if the 0-7-2 1 protocol resulted in palliation in a variety of spontaneous canine tumors; and (2) to evaluate the response to therapy using a quality of life instrument. Materials and MethodsFrom June 1989 to July 1991, 24 dogs presented to the Ontario Veterinary College with advanced malignancies received radiation therapy with palliative intent. Dogs were considered eligible if they had ( I ) advanced local...

Tetanus toxin produces spastic paralysis in situ by blocking inhibitory neurotransmitter release in the spinal cord. Although di-and trisialogangliosides bind tetanus toxin, their role as productive toxin receptors remains unclear. We examined toxin binding and action in spinal cord cell cultures grown in the presence of fumonisin B 1 , an inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for 3 weeks in culture in 20 M fumonisin B 1 develop dendrites, axons, and synaptic terminals similar to untreated neurons, even though thin layer chromatography shows a greater than 90% inhibition of ganglioside synthesis. Absence of tetanus and cholera toxin binding by toxin-horseradish peroxidase conjugates or immunofluorescence further indicates loss of mono-and polysialogangliosides. In contrast to control cultures, tetanus toxin added to fumonisin B 1 -treated cultures does not block potassiumstimulated glycine release, inhibit activity-dependent uptake of FM1-43, or abolish immunoreactivity for vesicle-associated membrane protein, the toxin substrate. Supplementing fumonisin B 1 -treated cultures with mixed brain gangliosides completely restores the ability of tetanus toxin to bind to the neuronal surface and to block neurotransmitter release. These data demonstrate that fumonisin B 1 protects against toxin-induced synaptic blockade and that gangliosides are a necessary component of the receptor mechanism for tetanus toxin.Tetanus toxin (TeNT) 1 blocks the release of inhibitory neurotransmitters in the spinal cord leading to hyperactivity of motor neurons and consequent spastic paralysis (for review, see Ref. 1). The toxin is synthesized as a single polypeptide (M r ϭ 150,000), released by bacterial lysis, and cleaved to form an active dichain toxin with the heavy and light chains linked through one disulfide bond. The carboxyl-terminal half of the heavy chain constitutes the receptor binding domain, the amino-terminal half of the heavy chain is responsible for membrane translocation of the toxin, and the light chain contains the catalytic domain (for review, see Ref.2). Upon entering the synaptic cytosol, the toxin arrests neurotransmitter release by its action as a zinc endopeptidase, which cleaves vesicle-associated membrane protein (VAMP), a synaptic vesicle integral membrane protein believed to be critical for neuroexocytosis (3, 4). Whereas TeNT has been shown to bind to the surface of neurons and to act intracellularly (for reviews, see Refs. 2 and 5-7), its cell surface receptor(s) and mechanism of delivery to the cytosol are not well understood.Since the demonstration of the affinity of TeNT for gangliosides present on the neuronal surface (8), there have been a number of studies (for review, see Ref. 6) characterizing the binding of TeNT to gangliosides in various in vitro and in vivo preparations. Although it is difficult to assign an affinity to this binding (7), there is general agreement that TeNT shows the highest affinity for GT1b and GD1b polysialogangliosides, although not nearly as ...