Peripheral nervous system (PNS) toxicity is surveyed inconsistently in nonclinical general toxicity studies. These Society of Toxicologic Pathology "best practice" recommendations are designed to ensure consistent, efficient, and effective sampling, processing, and evaluation of PNS tissues for four different situations encountered during nonclinical general toxicity (screening) and dedicated neurotoxicity studies. For toxicity studies where neurotoxicity is unknown or not anticipated (situation 1), PNS evaluation may be limited to one sensorimotor spinal nerve. If somatic PNS neurotoxicity is suspected (situation 2), analysis minimally should include three spinal nerves, multiple dorsal root ganglia, and a trigeminal ganglion. If autonomic PNS neuropathy is suspected (situation 3), parasympathetic and sympathetic ganglia should be assessed. For dedicated neurotoxicity studies where a neurotoxic effect is expected (situation 4), PNS sampling follows the strategy for situations 2 and/or 3, as dictated by functional or other compound/target-specific data. For all situations, bilateral sampling with unilateral processing is acceptable. For situations 1-3, PNS is processed conventionally (immersion in buffered formalin, paraffin embedding, and hematoxylin and eosin staining). For situation 4 (and situations 2 and 3 if resources and timing permit), perfusion fixation with methanol-free fixative is recommended. Where PNS neurotoxicity is suspected or likely, at least one (situations 2 and 3) or two (situation 4) nerve cross sections should be postfixed with glutaraldehyde and osmium before hard plastic resin embedding; soft plastic embedding is not a suitable substitute for hard plastic. Special methods may be used if warranted to further characterize PNS findings. Initial PNS analysis should be informed, not masked ("blinded"). Institutions may adapt these recommendations to fit their specific programmatic requirements but may need to explain in project documentation the rationale for their chosen PNS sampling, processing, and evaluation strategy.

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A characteristic feature of the rat somatosensory neocortex is a discrete topographic representation of the facial whiskers. Afferent fibers projecting to this vibrissae representation were "bulk-labeled" by injecting horseradish peroxidase into the white matter. Terminal arbors with the morphological characteristics of Lorente de No's (1949) "specific" thalamocortical afferents were then reconstructed through serial sections. These terminal arbors, characterized by the discrete organization of their dense plexus in layer IV, have a laminar distribution of boutons that parallels the laminar pattern of terminal degeneration resulting from lesions of the ventral posterior nucleus of the thalamus. The regional distribution of different-sized arbors corresponds to the distribution of vibrissae-related clusters of different sizes. Larger arbors were found in the posteromedial region corresponding to the mystacial vibrissae representation, while smaller arbors were found in the anterolateral region corresponding to the representation of the anterior sinus hairs. Terminal arbors were also reconstructed from sections stained simultaneously to demonstrate the pattern of vibrissae-related clusters. The greatest concentration of boutons on these axons occurred within a single vibrissae-related cluster. Furthermore, when 2 fibers terminated within a single cluster, their terminal arbors appeared to be largely coextensive. The morphology, size, and distribution of these terminal arbors support the hypothesis that the layer IV plexus of a single specific thalamocortical afferent tends to fill a vibrissae-related cluster. Thus, the organization of specific thalamocortical afferents may be responsible for clustered organization within the somatotopic map of the rodent neocortex.

The granule cell-enriched Ca2+/calmodulindependent protein kinase (CaM kinase-Gr) is a recently discovered neuron-specific enzyme. The kinase avidly phosphorylates synapsin I and contains a polyglutamate sequence, which suggests an association with chromatin as well. A possible role in synapsin I phosphorylation and in nuclear Ca2+ signaling was supported by immunochemical and ultrastructural examination of CaM kinase-Gr distribution. CaM kinase-Gr immunoreactivity was present in the molecular and granule cell layers of the rat cerebellum. This pattern corresponded to the occurrence of the enzyme in the granule cell axons and nuclei, respectively. Immunoblots confirmed these findings. Thus, CaM kinase-Gr may mediate and coordinate Ca2+-signaling within different subcellular compartments.Ca2+/calmodulin-dependent protein kinases (CaM kinases) have been implicated in neuronal communication by regulating neurotransmitter biosynthesis, neurotransmitter release, alterations in cytoskeletal components, and possible regulation of gene expression (1-5). A brain-specific CaM kinase has recently been isolated and purified and part of its encoding nucleotide sequence has been cloned (6). This kinase shows certain catalytic and regulatory similarities to CaM kinase II but exhibits a number of unique characteristics, including amino acid sequence, subunit organization, subcellular distribution, and immunohistochemical localization. Because this kinase protein is enriched in the granule cells of the cerebellar cortex, it has been called the granule cell-enriched CaM kinase (CaM kinase-Gr). The enzyme has been purified to homogeneity and consists of two polypeptides with apparent Mr values of 65,000 and 67,000 (6). Partial cDNA sequence data indicate that mouse and human brains contain homologues to the rat enzyme that, nevertheless, exhibit considerable divergence in their nucleotide sequence (7,8).The potential physiological roles of CaM kinase-Gr depend on its substrate specificity and its subcellular availability. This kinase phosphorylates synapsin I on the head and tail domains (6) and may thereby promote neurotransmitter release by analogy to the action of CaM kinase II (9, 10). Moreover, CaM kinase-Gr contains a polyglutamate-rich sequence (6), which characterizes several chromatinassociated proteins (11). Thus, CaM kinase-Gr may regulate different neuronal reactions in different subcellular compartments. The objective of this study was to determine the subcellular compartments in which CaM kinase-Gr occurs.MATERIALS AND METHODS Primary Antiserum. Rabbit antibodies were raised against a f3-galactosidase fusion product of CaM kinase-Gr expressed in and purified from Escherichia coli. The antiserum was affinity-purified by adsorption to mammalian CaM kinase-Gr isolated from rat cerebellum (6). The resulting monospecific antibody preparation was employed throughout this study.Immunoblots. Punches from the molecular layer and granule cell layer of cerebellar vermis of adult rats (0.1 mg of each tissue) were susp...

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A key trait of developmental neurotoxicants is their ability to cause structural lesions in the immature nervous system. Thus, neuropathologic assessment is an essential element of developmental neurotoxicity (DNT) studies that are designed to evaluate chemically-induced risk to neural substrates in young humans. The guidelines for conventional DNT assays have been established by regulatory agencies to provide a flexible scaffold for conducting such studies; recent experience has launched new efforts to update these recommendations. The present document was produced by an ad hoc subcommittee of the Society of Toxicologic Pathology (STP) tasked with examining conventional methods used in DNT neuropathology in order to define the 'best practices' for dealing with the diverse requirements of both national (EPA) and international (OECD) regulatory bodies. Recommendations (including citations for relevant neurobiological and technical references) address all aspects of the DNT neuropathology examination: study design; tissue fixation, collection, processing, and staining; qualitative and quantitative evaluation; statistical analysis; proper control materials; study documentation; and personnel training. If followed, these proposals will allow pathologists to meet the need for a sound risk assessment (balanced to address both regulatory issues and scientific considerations) in this field today while providing direction for the research needed to further refine DNT neuropathology 'best practices' in the future.

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The organization of the whisker representation within the neocortex of the rat is dependent on an intact periphery during development. To further investigate how alterations in this cortical map arise we examined the organization of thalamocortical afferents to the whisker representation in adult animals in which the infraorbital branch of the trigeminal nerve was cut on the day of birth. The disrupted pattern of thalamocortical projections to the vibrissae representation was apparent in the abnormal pattern of the anterograde transport of horseradish peroxidase from the thalamus, as well as in the abnormal pattern of succinate dehydrogenase activity. To determine the morphology of individual thalamocortical axons associated with this disrupted pattern, terminal arbors were “bulk-labeled” by injections of horseradish peroxidase into the white matter beneath the somatosensory cortex. Terminal arbors were identified by their laminar distribution of boutons corresponding to the specific thalamocortical afferent. The medial to lateral extent of these terminal arbors varied dramatically, from 350 to 1500 microns. In addition, terminal arbors innervating the same local area of cortex appeared to have varying degrees of overlap. Thus, the disruption of the neocortical vibrissae representation appears to involve the abnormal arborization of individual thalamocortical afferents. This finding supports the hypothesis that the fine-grain organization of the somatotopic map is dependent on the morphology and organization of individual thalamocortical arbors, which, in turn, are dependent on the periphery during development.