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Debate over the proper management of our relationship with free-roaming cats has escalated based on concerns over impacts on biodiversity and public health, with some calling for their eradication. It is often waged between animalists, primarily focused on the interests of the individual animal, and traditional conservationists, primarily focused on preserving native species and biodiversity. An ethical paradigm that accounts for the interests of all animals and nature to develop a management scheme that promotes interspecies justice is needed. I propose the political theory of animal rights developed in Zoopolis as a fruitful ethical paradigm. Grounded in modern citizenship theory, it defines universal negative rights due to all sentient beings and positive rights due to them based on our relationships to them as citizens, denizens, or foreigners. Application of this political theory in a modern fictional jurisdiction, Zoopoland, is explored to develop a just management strategy for free-roaming cats.

Cats and Conservationists: The Debate Over Who Owns the Outdoors explores the hotly contested debate surrounding outdoor cats, free-living animals, and humans’ role in nature—a debate grounded in conflicting science, ethics, and public policy goals. The authors attempt to sort out the data and values related to this debate and find common ground. However, in so doing, they create several false equivalencies. More helpful to those working on the ground to address outdoor cats would have been a book that, in sorting out the values and data, meaningfully compared the competing arguments.

Cat Wars is a dangerous book that declares war on all free-roaming cats. Filled with hyperbole and exaggerated statistics, the book argues that cats are a danger to humans, birds, and other free-living animals and should be eradicated from the landscape—a devastating, expensive, inhumane, and useless result. This review exposes the flaws in the authors’ analysis and ethical approach and redirects the dialogue toward an ethic that protects all animals. Compassionate conservationism promotes the use of nonlethal management strategies to protect individual birds and other animals while compassionately reducing the number of free-roaming cats.

On 13–14 November 2015, about a hundred wildlife advocates representing governments and international governmental institutions, nongovernment organizations, and academia attended a workshop at The...

In the 2008 article "A Review of Feral Cat Control," Robertson explored the trend developing in the management of so-called "feral" cats away from lethal methods toward the non-lethal method of trap-neuter-return (TNR). The review explored various issues raised by the presence of these unowned, free-roaming cats in our neighborhoods (e.g., zoonotic disease and wildlife predation), stakeholder interests, and management options-all based on then-available information. Missing from the review, however, was an exploration of the shifting ethics underlying TNR's increasing popularity. In this essay, we explore the ethical aspects of community cat management in the U.S. as reflected in the momentum of the "no-kill movement" generally and TNR in particular. We argue that these powerful cultural currents reflect two interrelated ethical theories: (1) a zoocentric ethic that recognizes the intrinsic value of non-human animals beyond any instrumental value to humans, and (2) a virtue ethic that recognizes the legitimacy of "emotional" considerations (e.g., compassion) that rightly accompany decisions about how best to manage community cats.

Author(s): Schaffner, Joan E; Wandesforde-Smith, Geoffrey; Wolf, Peter Joseph; Levy, Julie; Riley, Sophie; Farnworth, Mark James

INTRODUCTION Laparoscopic sleeve Gastrectomy (LSG) has become the most frequently performed bariatric procedure to date. However, LSG is known to worsen preoperative and result in de novo GERD. Preoperative evaluation reveals a high percentage of silent GERD of so far unknown influence on postoperative GERD. METHODS AND PROCEDURES Prospective data of patients undergoing primary LSG between 01/2012 and 12/2015 were evaluated. Preoperative GERD-specific evaluation consisted of validated questionnaires (GERD-HRQL), upper endoscopy, 24h-pH-manometry, and esophagograms. Patients were followed-up with questionnaires every 6 months and gastroscopies after 1 year. Silent GERD was defined as esophagitis grade >B and/or abnormal esophageal acid exposure in absence of symptoms. LSG was performed over a 32F bougie, hiatal hernias >1cm were addressed with posterior hiatoplasty. Excluded were patients with hiatal hernias >4cm, patients with incorrect anatomy (stenosis, fundus too large) and conversion to RYGB for early leaks. RESULTS 222 patients were included. Overall mean follow-up was 3216 months, 3 patients (1%) were lost to follow-up. 124 patients (56%) presented with postoperative GERD symptoms, of which 85 (69%) had de novo symptoms. Of those, 48 (of 85, 56%) had no preoperative GERD and 37 (of 85, 44%) silent GERD (p=0.2). 61 patients (27%) had neither pre- nor postoperative GERD, 17 (8%) had silent preoperative and no postop GERD, and in 18 patients (8%) GERD was cured with LSG. 31 patients (14%) stayed symptomatic. Of 54 patients (24%) with preoperative silent GERD, 37 (of 54, 69%) became symptomatic. Postoperative GERD-symptoms were typical in 116 (of 124, 94%), respiratory in 12 (10%), obstructive in 19 (15%), and pain in 21 patients (17%). CONCLUSION LSG leads to a considerable rate of postoperative GERD. De novo GERD consist of around half of preoperative silent GERD and completely de novo GERD. Most patients with preoperative silent GERD became symptomatic. These data justify a comprehensive preoperative evaluation.

Cat Wars is a dangerous book that declares war on all free-roaming cats. Filled with hyperbole and exaggerated statistics, the book argues that cats are a danger to humans, birds, and other free-living animals and should be eradicated from the landscape — a devastating, expensive, inhumane, and useless result. This review exposes the flaws in the authors’ analysis and ethical approach and redirects the dialogue toward an ethic that protects all animals. Compassionate conservationism promotes the use of nonlethal management strategies to protect individual birds and other animals while compassionately reducing the number of free-roaming cats.

Although 3α-substituted metabolites of progesterone are well established to interact with GABAAreceptor/Cl−channels, the nature of the interaction(s) remains uncertain. We used patch-clamp recording to study the interaction with GABAAreceptor/Cl−channels expressed by embryonic hippocampal neurons differentiating in culture and nonneuronal cells transfected with GABAAreceptor subunits. Allopregnanolone primarily induced multiphasic current responses in neurons, which were eliminated by bicuculline, an antagonist of GABA at GABAAreceptor/Cl−channels. Similar multiphasic responses blocked by bicuculline were induced by allopregnanollone in nonneuronal cells transfected with α1and γ2subunits, indicating that the steroid activation of GABAAreceptor/Cl−channels occurred independently of GABA. Fluctuation analyses of current responses to allopregnanolone and GABA revealed underlying channel activities with similar estimated unitary properties. However, although both agonists activated Cl−channels with similar estimated short and long burst-length durations, most of those stimulated by the steroid were short, while most of those opened by GABA were long. Allopregnanolone potentiated GABA-evoked Cl−currents in nonneuronal cells transfected with α1and β2or β3subunits, which did not exhibit multiphasic responses to the steroid, indicating another, independent action of the steroid at activated receptors. Pertussis toxin treatment eliminated the low-amplitude current and attenuated the high-amplitude current induced by allopregnanolone in a reversible manner. Mastoparan, which activates G proteins directly, triggered a high-amplitude current after a delay, which was blocked by bicuculline. The results indicate that allopregnanolone interacts with GABAAreceptor/Cl−channels expressed by embryonic hippocampal neurons in multiple ways, some of which are mediated by G proteins.

Embryonic rat hippocampal neurons were grown on patterned silane surface in order to organize synapse formations in a controlled manner. The surface patterns were composed of trimethoxysilylpropyl-diethylenetriamine (DETA) lines separated by tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F) spaces. Pre- and post-synaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Functional synaptic connections were examined by recording simultaneously from pairs of neurons using the whole-cell configuration of the patch-clamp technique. Spontaneous and evoked synaptic currents were recorded in neurons cultured for 2-14 days. The formation of functional connections was accompanied by the appearance of spontaneous synaptic currents (SSCs), which could be detected after approximately 3 days in culture in the absence of evoked synaptic currents (ESCs). ESCs were detected only after approximately 7 days in culture, mostly in the form of unidirectional synaptic connections. Other forms of synaptic connectivity, such as bidirectional and autaptic connections, were also identified. Both transient GABAergic and glutamatergic signals mediated the transmissions between communicating cells. These results demonstrate the combination of various types of synaptic connections forming simple and complex networks in neurons cultured on line (DETA)-space (13F) patterns. Finally, precisely synchronized SSCs were recorded in neuron pairs cultured on pattern indicating the existence of a fast-acting feedback mechanism mediated by pre-synaptic GABA(A) receptors.

The effects of astrocytes on the emergence of synaptic transients and excitable membrane properties in cultured, embryonic, rat ventral spinal neurons were studied with electrical and optical recording techniques. Neurons on astrocytes had significantly longer neurites and an accelerated rate of growth in surface membrane during the initial 24 h in culture compared to neurons on poly-D-lysine (PDL). GABAergic (GABA, gamma-aminobutyric acid) and glutamatergic transients appeared spontaneously in co-cultured neurons by 24 h. GABAergic quanta did not appear in neurons on PDL until 4 days in culture, and glutamatergic transients did not emerge until 7 days in culture. Astrocyte-conditioned medium (ACM) partially mimicked the effects of direct astrocytic contact. GABAergic transients appeared by 2 days, and glutamatergic signals by 4 days in neurons on PDL exposed to ACM. All of the spontaneous, synaptic-like transients were eliminated by tetrodotoxin or Ca2+o-free saline, implicating voltage-dependent cation channels in their generation. Astrocytes immediately and significantly increased the density of voltage-dependent Na+ currents compared to neurons on PDL, but by the end of 24 h, Na+ current densities were identical. Electrophysiological and optical recording revealed comparable densities of high-voltage-activated (HVA) Ca2+ currents on both co-cultured neurons and neurons on PDL throughout the first week. However, neurons on astrocytes had significantly greater contributions of P/Q-type currents and lesser contributions of L-type currents beginning at 24 h and continuing for 7 days. The contribution of N-type current was significantly more in co-cultured neurons only at 24 h. Thus, in vitro, astrocytes help to differentiate specific excitable membrane properties in spinal neurons, along with GABAergic and glutamatergic forms of synaptic transmission.

Embryonic rat hippocampal neurons were cultured for 1-2 days in serum-free, HEPES-buffered Tyrode's solution. The effects of cortical astrocytes and astrocyte-conditioned saline on neuron survival, membrane surface area and the expression of functional amino acid neurotransmitter receptors were studied. Neurons grown in Tyrode's solution alone survived well for 1 day but deteriorated thereafter both in terms of percent neurons surviving and the amplitudes and densities of GABA-, glycine-, kainate-and NMDA-induced currents. Neurons grown in Tyrode's previously conditioned by astrocytes for 24 h had significantly larger apparent plasma membrane surface area, as indexed by whole-cell membrane capacitance, and larger amplitudes and densities of the amino acid-induced currents after both 1 and 2 days. The survival rate and neurite outgrowth were also greater in the astrocyte-conditioned saline group after 2 days in culture. Similarly, neurons cultured on glass cover-slips facing a confluent monolayer of astrocyte were larger in apparent plasma membrane area and amino acid-induced currents than neurons cultured in Tyrode's alone. Neurons cultured in saline conditioned by astrocytes provide a strategy to study the physiological basis of astrocyte-directed neuronal differentiation in the absence of ambiguities arising from the inclusion of sera and other additives often used in vitro.

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 developmen...

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or on cortical astrocytes, some of which had been pretreated for 24 h with amyloid beta-protein (beta-AP). Amino acid-induced currents were quantified. Membrane capacitance (Cm), as well as the amplitude and density of amino acid-evoked currents recorded in neurons cultured on untreated astrocytes were all statistically greater than those recorded in neurons grown on PDL. However, compared to untreated astrocytes, those treated with beta-AP led to significantly lower values in neurons for Cm and GABA, kainate- and NMDA-induced currents, while glycine-activated current values were not significantly different. Furthermore, beta-AP treatment abolished spontaneous Cac2+ fluctuations in astrocytes, which may account for their impaired ability to promote the expression of functional transmitter receptors in neurons.

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl--filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl- channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with alpha1 and gamma2 GABAA receptor subunits exhibited little baseline current variance when recorded with Cl--filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons.

Embryonic rat hippocampal neurons were cultured in a serum-free defined medium (MEM/N3) either directly on poly-D-lysine (PDL) or on a confluent monolayer of postnatal cortical astrocytes, C6 glioma cells, or Rat2 fibroblasts. Neurons on PDL were grown in MEM/N3 or in MEM/N3 conditioned for 24 h by astrocytes or C6 cells. Membrane capacitance (Cm) and γ-aminobutyric acid (GABA)-, glycine-, kainate-, and N-methyl-D-aspartate (NMDA)-induced currents were quantified using whole-cell patch-clamp recordings. Cm as well as the amplitude and the density of these currents in neurons cultured on astrocytes were significantly greater than those in neurons grown on PDL after 24 and 48 h. C6 cells mimicked astrocytes in promoting Cm and GABA-, glycine-, and NMDA-evoked, but not kainate-evoked, currents. Cm and currents in neurons grown on Rat2 cells were comparable to those in neurons on PDL. Astrocytes maintained in culture for 3 months were noticeably less effective than freshly prepared ones just grown to confluence. Suppression of spontaneous cytoplasmic Ca2+ (Cac2+) elevations in astrocytes by 1,2-bis(2-aminophenoxy) ehane-N, N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) loaded intracellularly blocked the observed modulatory effects. Medium conditioned by either astrocytes or C6 cells mimicked the effects of direct coculture of neurons on these cells in promoting Cm and amino acid-evoked currents. Inclusion of antagonists at GABA and glutamate receptors in coculture experiments blocked the observed effects. Thus, diffusible substances synthesized and/or secreted by astrocytes in a Cac2+-dependent manner can regulate neuronal growth and aminoacid receptor function, and these effects may involve neuronal GABA and glutamate receptors. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 848–864, 1997

Application of 0.1-10 microM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 microM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl- equilibrium potential. These MSCs were blocked by the GABAA receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl- channels activated pharmacologically by applying 1-10 microM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABAA receptor agonist muscimol activated Cl- current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl- channels. The GABAB receptor agonist (-)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca2+. Verapamil and Co2+, which block voltage-dependent calcium channels, completely blocked GABA-induced MSCs independent of their effects on the direct activation of a Cl- current response. The results indicate that GABA can trigger GABAergic Cl(-)-dependent MSCs in a Cao(2+)-dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway.

In dog-bite and other animal-related litigation, disputed damages may involve personal injury to humans caused by animals, or injury to animals caused by other animals or humans. The situations that may give rise to such cases are far-raging. Dogs may injure humans, or may get into a fight with, and injure or kill another dog or cat However, there is a seemingly endless variety of cases where humans harm animals. These cases range from heinous cases of a person deliberately and maliciously killing an animal, to the pet food manufacturer that markets tainted food and kills thousands of animals, to the police officer who shoots a dog without cause, to the veterinarian who negligently treats an animal causing injury or death, to the shelter worker who “accidentally” places a dog on the *40 kill list in the shelter, killing the dog before their owner is able to reclaim him. These are just a few examples of the variety of cases that result in injury or death to a human or animal and for which the wrongdoer may be held liable for damages. The law regarding damages for injury caused by animals to humans, including physical injury, pain and suffering, emotional distress, or death, is no different from any other case in which a human is injured by the tortious conduct of another. However, the law regarding damages for the injury or death to an animal is in flux, controversial, and of specific interest in the area of animal law. Animals are considered to be personal property, and this classification dictates the damage types and awards available. For example, some states limit the value of an animal to the animal’s fair market value. Other states allow for veterinary expenses that exceed the animals’ fair market value depending on whether the animal is injured or killed. Furthermore, some jurisdictions include sentimental or intrinsic value of the animal in the determination of damages, while other jurisdictions may consider the owner’s emotional distress or even punitive damages if the animal is killed by the intentional act of another. Determining the proper damages to be awarded for the injury or death of an animal under the law is important to provide both proper compensation to the owner for the loss of a companion animal and the proper incentive for persons to act with reasonable care to avoid harming animals. The following will discuss the various types of damages that may be awarded for the injury or death to an animal.

Embryonic rat hippocampal neurons were cultured on poly-d-lysine (PDL) or a monolayer of postnatal cortical astrocytes to reveal putative changes in neuronal physiology that involve astrocyte-derived signals during the first 4 d of culture. GABA-induced Cl−current (IGABA) was quantified using outside-out and whole-cell patch-clamp recordings beginning at 30 min, when cells had become adherent. The amplitude and density (current normalized to membrane capacitance) of IGABAin neurons grown on astrocytes became statistically greater than that recorded in neurons grown on PDL after 2 hr in culture (HIC). Although the current density remained unchanged in neurons on astrocytes, that in neurons on PDL decreased and became statistically lower beginning after 2 HIC. The differences in amplitude and density of IGABAin the two groups of neurons were maintained during the 4 d experiment. The upregulation effect of astrocytes on neuronal IGABArequired intimate contact between the neuronal cell body and underlying astrocytes. Suppression of spontaneous Cac2+elevations in astrocytes by bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid that was loaded intracellularly decreased their modulatory effects on IGABA. IGABAin all cells was blocked completely by bicuculline and exhibited virtually identical affinity constants, Hill coefficients, and potentiation by diazepam in the two groups. Outside-out patch recordings revealed identical unitary properties of IGABAin the two groups. More channels per unit of membrane area could explain the astrocyte enhancement of IGABA. The results reveal that cortical astrocytes potentiate IGABAin hippocampal neurons in a contact-dependent manner via a mechanism involving astrocyte Cac2+elevation.

We have developed an in vitro system that combines the use of a defined medium with a chemically defined surface for the differentiation of embryonic rat hippocampal neurons. Cells were grown on silica substrates modified with two chemically distinct molecules: poly- d -lysine and an amine-containing organosilane. Cells were dissociated by mechanical or enzymatic methods and grown in serum-containing versus serum-free medium on these surfaces. Our results demonstrate that optimal survival and growth in serum-free medium occurs on the artificial surfaces. X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces both before and after cell culture. In addition, surface properties such as elemental composition, the initial thickness of the substrate material, and the thickness of material deposited during the course of cell culture were quantified after cell removal. Taken together, the results from the cell culture and surface analysis demonstrate that the media, proteins deposited from the media onto the surface, surface composition, and properties intrinsic to neuronal membranes all interact in a complex fashion to determine whether or not the cells will adhere and survive in culture. In particular, the role of material deposited from the medium onto the culture substratum may be more important than has been previously appreciated. This system allows for the study of neuronal differentiation in a well-defined environment.

We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36– 48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

During embryogenesis, neuroblasts proliferate within germinal zones, then migrate to their final positions. Although many neurons migrate along radial glial fibers, evidence suggests that environmental factors, as yet unidentified, also influence neuroblast movement. In vivo, nerve growth factor (NGF) and -α-aminobutyric acid (GABA) colocalize near target destinations of migratory neuroblasts. In vitro, embryonic spinal neurons migrate towards NGF and GABA (Behar et al.: J Neurosci 14:29-38, 1994), implying that the molecules may act as chemoattractants in vivo. Here, we have used an in vitro assay of migration to show that migratory responses to these attractants develop along a ventrodorsal gradient that parallels terminal mitosis during cord development, and that GABA stimulates chemokinesis (motility without a gradient) via heterogeneous receptors involving separate signalling pathways. Both GABAA (muscimol) and GABAB (baclofen) agonists mimicked the effects of GABA in stimulating chemokinesis. Muscimol-induced motility was only blocked by GABAA antagonists (bicuculline or picrotoxin), whereas migration to baclofen was blocked by antagonists of both GABAA and GABAB (2-hydroxysaclofen) receptors. Migration to baclofen, but not muscimol, was abolished in the presence of 8-bromo cAMP or pertussis toxin, indicating that the former, but not the latter, attractant may stimulate motility via Gi/Go GTP binding proteins, and that PKA may modulate migratory responses to baclofen. Migration to GABA was partially attenuated by each of the GABA receptor antagonists. These results lead us to conclude that the natural ligand stimulates neuroblast motility via heterogeneous receptors coupled to different signalling mechanisms. © 1995 Wiley-Liss, Inc.1

The United States anti-cruelty laws are criminal laws and comprise the most basic and fundamental legal protection for animals. As the least controversial of the animal protection laws, these laws protect animals from the intentional and gratuitous infliction of pain and suffering at the hands of humans. However, as we will see, most animals in the United States are not covered by the anti-cruelty code. Animals used for food or research and wild animals generally are exempt from the anti-cruelty laws. The anti-cruelty laws target only individual instances of intentional cruelty not institutionalized cruelty. This reflects a serious flaw in our societal view of animals as beings who exist for human use and our acceptance of institutionalized practices that inflict serious pain and suffering upon other species for our benefit.

A constitution, whether written or unwritten, defines the basic rights and obligations as between the government and the individual human citizen, while private law defines the rights and obligations among human persons. Thus, these two sources of law are to protect human interests directly. As a result, animals, if present, almost always are present as objects — the property of their human owners or part of the “common” held for the benefit of humans — rather than subjects — referenced in their own right. To date, these sources of law provide little opportunity for robust protection of animals’ interests, with a few notable exceptions. However, we will see that the manner in which these two sources of law are addressing animals has begun to change. Both the constitutional law and the private law are beginning to recognize the inherent interests of animals as sentient beings, and with time may do more to promote the animals’ own individual interests independent of their owners.

In the United States, local jurisdictions enact laws pursuant to their police powers to protect the public health, safety, and welfare. Animal control laws, laws regulating the ownership of animals by citizens or unowned animals found within the local jurisdiction, are enacted pursuant to this authority and enforced by a local agency typically called “Animal Control.” As is implied by the title, the goal of these laws is to “control” animals. In recent years, some jurisdictions have elected to modify the agency’s title to “Animal Care and Control.” Although the nature of the laws may not be significantly affected by the title change, indicating that the goal of the agency is not only to control animals but to provide for their care may alter the perception of the enforcers and the public towards animals as beings to be cared for and not merely controlled.

Animal welfare laws regulate humans’ use of animals. Humans use animals in a number of different ways, including for research, entertainment or exhibition, hobby, and food. Animal welfare is defined as an animal’s state of well-being, or “maintenance of animals under conditions of space, environment, nutrition, and so forth, consistent with the physiological and social needs of the species” consistent with the Five Freedoms: freedom from (1) thirst, hunger and malnutrition, (2) discomfort, (3) pain, injury, and disease, (4) fear and distress, and (5) freedom to express normal behavior.1 Arguably these laws should set standards beyond the mere prohibition of cruel practices already outlawed by the anti-cruelty laws. However, this may not necessarily be the case, depending upon the requirements of the particular human use being regulated. As we noted, the anti-cruelty laws often exempt certain communities of animals, such as animals used for food or research and/or practices; for example, “accepted agricultural practices.” For these animals, the animal welfare laws provide the only legal protection available.

Thus far, we have studied how animals fit within the existing legal regime. In this final chapter, we will look briefly to the future of animal protection law. What have we learned from a review of the existing law and how might the law be constructed to better protect animals? The focus of this chapter is to initiate a dialogue and to propose a few suggestions and examples of a legal regime that considers animals as subjects.

Animals and their treatment by humans appear in our law in a variety of ways. Consider Matthew, a 27-year-old chimp whose case was accepted by the European Court of Human Rights in 2008.1 Animal rights activist and teacher Paula Stibbe wanted to be declared his legal guardian should the sanctuary where he resides close due to bankruptcy. Austria’s Supreme Court upheld the lower court ruling rejecting her request. The problem, under Austrian law, is that only legal “persons” may be appointed a guardian. Since Matthew is deemed a legal “thing” under Austrian law he cannot be appointed a guardian. In contrast, in 2007 when Michael Vick, an American football star, was arrested for dog-fighting and his “pit bull type” dogs were seized in the civil forfeiture action associated with the criminal case, the government appointed a Special Master/Guardian for the dogs.2 Although animals are deemed “things” under state and federal law in the United States, the court held that such status would not prohibit assigning a human to represent the dogs’ interests.3

Single-cell image analysis of rat pinealocytes has been difficult because they do not attach readily to coated or uncoated surfaces and typically adhere in clusters to fibroblast-like cells. In the present report, a new method for the rapid attachment of rat pinealocytes is described. Cells were prepared using papain digestion and density centrifugation and then were placed on coverslips or slides coated with PepTite-2000 TM , a preparation containing the attachment peptide sequence Arg-Gly-Asp. Cells immobilized with this preparation responded to norepinephrine treatment with an increase in cyclic AMP and melatonin production. Single-cell analysis of Fura-2-loaded cells revealed that norepinephrine increase [Ca 2+ ] i . This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.

It is not well understood when during embryonic development the elements of a cell's responsiveness first appear, nor the factors controlling their appearance. A strategy to approach this issue is to determine which aspects of neuronal development are highly stereotyped in presence, timing, or pattern across a variety of cell types, and which are more diversified by cell type, region, or other parameters. We have used a fluorescent potentiometric oxonol dye in conjunction with a digital video imaging system to record the emergence and distribution of specific forms of excitability in dissociated embryonic rat spinal cord cells. We studied the expression of responses to veratridine, a sodium channel activator; muscimol, a GABAA receptor agonist; and kainic acid, an agonist at a class of glutamate receptors. Responses were consistently detectable in a percentage of cells dissociated from the earliest age examined, embryonic day 13, and increased progressively in later ages. Cells were examined from four regions, with cervical-lumbosacral and ventrodorsal distinctions. In the population of cells from each region, functional sodium channels appeared prior to GABAA receptors, which in turn emerged prior to kainate-activated glutamate receptors. This pattern was common to all spinal cord regions and revealed ventrodorsal and rostrocaudal gradients reflecting the known pattern of spinal cord neurogenesis. Analysis of the individual cell responses indicated that the stereotypical pattern of sequential channel development occurs individually on most cells in each region.

At early developmental stages in the rat spinal cord (embryonic day 13), when neuronal progenitors are still proliferating, most differentiating neurons express truncated forms of glutamic acid decarboxylase (GAD) (approximately 25 kDa) which are the products of alternative splicing of the GAD67 gene. These truncated proteins do not appear to synthesize γ-aminobutyric acid (GABA). The amino acid is detected in cells only after alternative splicing of the GAD67 gene generates a full-length, 67 kDa enzymatically active form of GAD. Both the 67 kDa GAD and GABA colocalize and appear diffusely distributed in the cytoplasm of embryonic neurons. GABA does not appear associated with synaptic vesicles until after birth, when its intracellular distribution becomes punctate and it colocalizes with synaptophysin. At this time, it also colocalizes with an immunologically distinct 65 kDa GAD protein encoded by a second GAD gene (GAD65). Expression of different GAD-related proteins with distinct intracellular distributions during development suggests that GABA, the product of these enzymes, may have trophic or metabolic roles during spinal cord differentiation.

At the neuromuscular junction, spontaneous miniature excitatory synaptic currents mediated by acetylcholine are considered elementary, "quantal" transmissions. These miniature conductances can be quantitatively dichotomized into a large-mode class whose mode is the mean of a normal, bell-shaped distribution and a small-mode class whose distribution is skewed to lower values with its mode being a fraction of the large-mode class. The large-mode class constitutes the population of synaptic signals originally utilized to formulate tenets of "quantal" transmission, which have been tacitly adopted in more recent studies of fast transmission at central synapses. Large- and small-mode conductance classes of inhibitory synaptic elementary conductances mediated by GABA have now been recorded in cultured hippocampal neurons (Vautrin J, Schaffner AE, Barker JL, 1991, Neurosci Lett 138:67). Pairs of hippocampal neurons were patch-recorded at optimal signal-to-noise and, using time course analysis, two elementary fluctuations (0.1-0.3 nS and 1-2 nS) were found within synaptic conductances evoked either by presynaptic action potentials or by presynaptic terminal stimulation. These results were interpreted with a simple model that shows how different frequencies of unitary GABA release can generate either small-mode, skew-distributed conductance (0.5-3 kHz) or large-mode, normally-distributed conductances (> or = 10 kHz). Only the latter satisfies the original tenets of the classic quantal theory.

Amplitude and time course of spontaneous γ-aminobutyric acid (GABA)-mediated miniature postsynaptic currents (MPSCs), recorded in cultured embryonic hippocampal neurons in presence of either tetrodotoxin (TTX) or increased external [ Mg 2+ Ca 2+ ] ratio, revealed that they form two classes. The distribution of the most commonly recorded MPSCs was skewed both in terms of peak amplitude and rise-time (skew-MPSCs, mode: 70–120 pS). Another, less frequent class (mode: 1–3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonically attenuated. Bell-MPSCs did not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.

The present work is concerned with the probability of contamination by Listeria monocytogenes in the artisanal manufacture of Swiss Emmental hard cheese made from raw milk. The simulation model follows the evolution of the contaminant flora from raw milk at the farm to milk mixing, storage at the cheese factory and to the cheese manufacturing process.The simulations are based on models of predictive microbiology, namely the exponential growth model of bacteria including the lag-time, a cardinal growth model and a Log-linear model of thermal deactivation of bacteria.The results of the actual simulation indicate that the contamination of milk at the farm is a rare event (P=0.0036), but the mixing of milk at the cheese factory leads to a higher probability of contamination of cheese milk (P=0.07). Elevated bacterial concentrations are mainly due to cases of mastitis involving Listeria monocytogenes. The decline in bacterial counts during cheese manufacture depends on the curing temperature (52-54 degrees C) and varies between 1.5 and 3.2 Log cfu/ml. Freshly manufactured Emmental-cheese made from contaminated raw milk is expected to have only 4.6 cfu of heat injured Listeria monocytogenes /kg cheese mass in the press.Depending on listeria evolution, from the press to the product consumption, consumer exposure has been evaluated and might result in 1 to 10 Listeria monocytogenes per portion of cheese. The bacterial presence could be due to recontamination during packaging, distribution and cheese preparation by the consumer. Based on the presented data and estimations, it is concluded that the consumption of traditionally/artisanal manufactured Swiss Emmental hard cheese presents an extremely low, but existent risk, especially for people with a deficient or diminished immune system.

Embryonic, ventral spinal cord neurons were grown on poly(d-lysine) (PDL) or on a monolayer of type 1 astrocytes. At various times from 6 h to 2 weeks postplating, cells were fluorescently labeled and fixed with 4% paraformaldehyde. The cell surface immunoreaction allowed visualization of neurons in their entirety, namely, cell bodies and various membranous extensions that included lamellipodia, growth cones, axons, and dendrites. Outlines were drawn for individual neurons and their fractal dimension (D) was calculated. Neurons on poly(d-lysine) reached a peak D at 3 days in vitro, 1 day later than neurons on astrocytes (2 days in vitro). The maximum D was greater for cells on poly(d-lysine) when compared with neurons on astrocytes. In a second experiment the maximum D was similar for neurons on both surfaces but neurons on PDL maintained a higher D for a much longer period than neurons on astrocytes. An examination of fluorescent images revealed that neurons on poly(d-lysine) exhibited lamellipodia and large growth cones for several days and these structures were likely responsible for the high D seen in these cells. These structures were rarely observed in neurons plated on astrocytes. Interestingly, D on both surfaces decreased to a similar value at between 1 and 2 weeks in vitro. The trend for D in these cultures, an initial increase to a peak value followed by a decrease to a stable value, is discussed in light of the chemical nature of the two surfaces and synapse formation and stabilization.

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire beta3 cytoplasmic domain induced constitutive LIBS exposure on alphavbeta3 and alphaIIbeta3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on alphavbeta3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the beta3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the alphavbeta3 ectodomain, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes.

Recent studies indicate that GABA acts as a chemoattractant during rat cortical histogenesis. In vivo, GABA localizes in appropriate locations for a chemoattractant, along migratory routes and near target destinations for migrating cortical neurons. In vitro, GABA induces dissociated embryonic cortical neurons to migrate. Here, embryonic rat cortical slices were cultured in the presence or absence of GABA receptor (GABA-R) antagonists to assess GABA's effects on neuronal migration in situ. Gestational day 18 (E18) cortical slices were incubated overnight in bromodeoxyuridine (BrdU)-containing medium to label ventricular zone (vz) cells as they underwent terminal mitosis. The slices were then cultured in BrdU-free medium with or without GABA-R antagonists. In control slices, most BrdU(+) cells were observed in the cortical plate (cp) after 48 h. In contrast, cultures maintained in either saclofen (a GABA(B)-R antagonist) or picrotoxin (a GABA(A/C)-R antagonist) had few BrdU-labeled cp cells. However, the effects of the two antagonists were distinct. In the picrotoxin-treated slices, nearly half of all BrdU(+) cells remained in the vz and subventricular zone (svz), whereas saclofen treatment resulted in an accumulation of BrdU(+) cells in the intermediate zone (iz). Bicuculline, a GABA(A)-R antagonist, did not block, but rather enhanced migration of BrdU(+) cells into the cp. These results provide evidence that picrotoxin-sensitive receptors promote the migration of vz/svz cells into the iz, while saclofen-sensitive receptors signal cells to migrate into the cp. Thus, as cortical cells differentiate, changing receptor expression appears to modulate migratory responses to GABA.

Whole cell patch-clamp recordings using Cl−-filled pipettes revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a monolayer of astrocytes than those cultured on poly-d-lysine. These effects were mimicked by culturing neurons on poly-d-lysine in astrocyte-conditioned medium (ACM). The baseline current and variance decreased immediately in all cells after either local perfusion with saline or exposure to bicuculline, an antagonist of GABA at GABAAreceptor/Cl− channels. Baseline current and variance in all cells reached a nadir at ∼0 mV, the calculated equilibrium potential for Cl−. Perfusion of ACM rapidly induced a sustained current in neurons, which also reversed polarity at ∼0 mV. Bicuculline attenuated or eliminated the ACM-induced current at a concentration that completely blocked micromolar GABA-induced current. Quantitative analyses of spontaneously occurring fluctuations superimposed on the ACM-induced current revealed estimated unitary properties of the underlying channel activity similar to those calculated for GABA's activation of GABAA receptor/Cl−channels. Bicuculline-sensitive synaptic-like transients, which reversed at ∼0 mV, were also detected in neurons cultured in ACM, and these were immediately eliminated along with the negative baseline current and superimposed current fluctuations by perfusion. Furthermore bicuculline-sensitive synaptic-like transients were rapidly and reversibly triggered when ACM was acutely applied. ACM induced an increase in cytoplasmic Ca2+ in cultured embryonic hippocampal neurons that was completely blocked by bicuculline and strychnine. We conclude that astrocytes release diffusible substances, most likely GABA, that persistently activate GABAA receptor/Cl−channels in co-cultured neurons.

Exocytosis is commonly viewed as the only secretory process able to account for quantal forms of fast synaptic transmission. However, the demonstrated variability and composite properties of miniature postsynaptic signals are not easily explained by all-or-none exocytotic discharge of transmitter in solution from inside vesicles. Recent studies of endocrine secretion have shown that hormone release does not coincide with exocytosis due to its trapping in the core matrix of the granule. Thus, we tested whether the synaptic transmitter GABA could also be held in a matrix before being released. Using confocal microscopy and flow cytometry of embryonic rat hippocampal neurons, we found a GABA immunoreaction at the surface of live cell bodies and growth cones that coincided spatially and quantitatively with the binding of tetanus toxin fragment C (TTFC). TTFC binds predominantly at membrane sites containing the trisialoglycosphingolipid GT1b. Using flow cytometry, GT1b-containing liposomes preincubated in 100 nM GABA exhibited the same relationship between GABA and TTFC surface binding as found on neurons and growth cones. Embryonic neurons differentiated in culture expressed initially a tonic, and after 3-5 days, transient, postsynaptic signals mediated by GABA acting at GABA(A) receptor/Cl(-) channels. A stream of saline applied to the neuronal surface rapidly and reversibly suppressed both tonic and transient signals. A brief application of the GABAmimetic isoguvacine immediately transformed both tonic and transient GABAergic signals into tonic and transient isoguvacinergic signals. These results and those in the literature are consistent with an immediately releasable compartment of transmitter accessible from the presynaptic surface.

GABAergic responses of acutely dissociated rat retinal neurons, including both bipolar cells (BCs) and other, morphologically round cells (RCs), were assayed with the fluorescent (FL), voltage-sensitive probe oxonol DiBaC4(5). Using intensified video microscopy and simultaneous recording, GABA responses were identified in one-third of cells in a typical microscope field; of these 85% hyperpolarized (0.05–0.3 log unit FL decreases) while the remainder depolarized (0.05–0.2 log unit FL increases). GABA-sensitive cells were also TACA-sensitive (trans-4-Aminocrotonic acid), and these ligands appeared interchangeable in ability to evoke responses. In RCs, an asymmetric co-responsive pattern was observed between GABA- and muscimol-evoked events. Muscimol-sensitive RCs responded well to GABA, but not all GABA-sensitive RCs responded to muscimol. In GABA-sensitive BCs, muscimol responses were typically weak or absent. Few BCs or RCs responded to CACA (cis-4-Aminocrotonic acid). Bicuculline-resistant GABA responses occurred in ∼80% of GABA-responsive RCs and BCs. Both bicuculline-sensitive (GABAA-like) and bicuculline-insensitive (GABAC-like) responses were resistant to picrotoxin. Although a small minority of GABA-sensitive cells hyperpolarized in response to R(+)baclofen, bicuculline-insensitive responses were not antagonized by 2-hydroxysaclofen, and were abolished in low [Cl−]o. Results suggested (1) that bicuculline-insensitive, Cl−-dependent, GABAC-like responses were broadly distributed and predominant among dissociated rat retinal neurons; (2) that muscimol was a particularly weak agonist for rat retinal BCs; and (3) that oxonol was a sensitive probe for retinal GABA responses.