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Diagnosing mushroom poisoning in dogs can be difficult and often includes identification of suspect mushrooms. Visual identification may be hindered by mastication, oral medications, or poor quality of environmental mushroom samples. Other analytical techniques may thus be necessary to aid in mushroom identification. A 5-y-old neutered male Labrador Retriever dog developed acute onset of vomiting, diarrhea, tremors, seizures, and somnolence. The dog was treated at a veterinary clinic and was briefly stabilized, but died during transport to an emergency clinic. On postmortem examination at the University of Kentucky Veterinary Diagnostic Laboratory, the dog's stomach was full of mushrooms covered with activated charcoal. Mushrooms were damaged, fragmented, and discolored, precluding accurate visual identification. Mushroom pieces were sent to the Department of Plant Pathology at the University of California-Davis for PCR identification; the neurotoxic mushroom Amanita muscaria was identified. A qualitative liquid chromatography-mass spectrometry (LC-MS) method was developed to detect ibotenic acid and muscimol, the toxic compounds present in A. muscaria. Mushrooms, stomach contents, and urine were analyzed by LC-MS; ibotenic acid and muscimol were detected in all samples. Because identification of ingested mushrooms is sometimes necessary to confirm mushroom poisoning, PCR can identify ingested mushrooms when visual identification is unreliable.

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.

Glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotransmitter GABA, has been localized in goldfish retina using a new antiserum. We observed at least six types of GAD-immunoreactive amacrine cells, one of which was large and pyriform (Ab type). In addition, immunoreactive synaptic terminals were located throughout the inner plexiform layer (IPL). Amacrine cells that were GAD-immunoreactive also had high-affinity uptake mechanisms for both 3H-GABA and 3H-muscimol that were detectable autoradiographically. Type Ab pyriform amacrine cells were heavily labeled because of 3H-GABA uptake and were GAD-immunoreactive. Other types of GAD-immunoreactive amacrine cells, including a subpopulation of Ab amacrines, were lightly labeled because of 3H-GABA uptake. Because the same neurons that were GAD-immunoreactive also accumulated 3H-GABA and 3H-muscimol, these three are appropriate markers for GABAergic cells in the goldfish retina. However, the uptake of 3H-muscimol by many non-GAD-immunoreactive cells, detectable at longer autoradiographic exposure times, indicates that this label must be used with caution. Thirty percent of goldfish retinal amacrine cells are GABAergic, and their processes are distributed throughout all levels of the IPL. Few GAD-immunoreactive amacrine cells accumulated 3H-glycine, so the goldfish retina contains distinct populations of glycinergic and GABAergic amacrine cells.

Gamma-aminobutyric acid (GABA) is a candidate as a neurotransmitter in the vertebrate retina. The GABA analogue muscimol has been used to probe the properties of GABA receptors in other parts of the vertebrate central nervous system (CNS). We thus used 3H-muscimol to investigate potential GABA receptors in the retinas of goldfish and chick by means of biochemical assay techniques and light microscopic autoradiography. In both animals 3H-muscimol shows specific and saturable binding with a dissociation constant (KD) of about 10 nM. GABA effectively inhibits 3H-muscimol binding at 50% inhibitory concentration (IC50) of 10(-6) M. The labeling pattern of 7 x 10(-7) M 3H-muscimol shows common features for both species in that amacrine cell bodies are intensely labeled, horizontal cells are much less so, and there is a laminar pattern throughout the inner plexiform layer (IPL). A 1 mM concentration of GABA abolishes 3H-muscimol labeling in the chick retina and throughout much of the goldfish retina except for some label over amacrine cells and the distal two thirds of the IPL. The intense somatic labeling suggests neuronal uptake of 3H-muscimol, and indeed, virtually all 3H-muscimol labeling is abolished with the addition of 0.4 mM ouabain. The uptake pattern of 3H-GABA differs from that of 3H-muscimol and is largely unaffected by the addition of 1 mM muscimol. We conclude that 3H-muscimol binding in retinas can be adequately demonstrated biochemically but that only 3H-muscimol uptake is observed with autoradiography from tissue conventionally processed through Epon. The fact that GABA can inhibit 3H-muscimol uptake whereas the reverse is not the case shows that the transport carriers for muscimol and GABA are different. Finally, the strong degree of 3H-muscimol uptake by retinal neurons raises serious questions about the use of 3H-muscimol as a probe for GABA synaptic receptors in the retina with autoradiography.