Kevin D. Phelan

The Interstitial System of the Spinal Trigeminal Tract in the Rat: Anatomical Evidence for Morphological and Functional Heterogeneity

Utilizing cyto-, myelo-, and chemoarchitecture as well as connectional criteria, the present study reveals the interstitial system of the spinal trigeminal tract (InSy-SVT) in the rat to be composed of five morphologically and functionally distinct components that are distributed within spatially restricted regions of the lateral medulla. The first component is represented by scattered interstitial cells and neuropil, which extend laterally into SVT from the superficial laminae of the medullary dorsal horn (MDH). The second component, the dorsal paramarginal nucleus (PaMd), consists of a small group of marginal (lamina I)-like neurons and neuropil situated within the dorsolateral part of SVT at the rostral pole of MDH. The third component represents a trigeminal extension of the parvocellular reticular formation (V-Rpc) into the ventromedial aspect of SVT at levels extending from rostral MDH to the caudal part of trigeminal nucleus interpolaris (Vi). The fourth component, the paratrigeminal nucleus (PaV), consists of a large accumulation of neurons and neuropil situated within the dorsal part of SVT throughout the caudal half of Vi. The fifth component is the insular trigeminal-cuneatus lateralis nucleus (iV-Cul), which is a discontinuous collection of neurons and neuropil interspersed among fibers of SVT as well as wedged between it and the spinocerebellar tract. Thalamic projection neurons are located in PaMd and V-Rpc, whereas cerebellar projecting neurons are confined to iV-Cul.

Dual topographic representation of neostriatum in the globus pallidus of rats

Two spatially separate strio-pallidal axonal arborizations were demonstrated autoradiographically. One was restricted to an approximately 100 micrometer thick strio-pallidal border zone of pallidal neuropil and displayed a 2-dimensional topographical relationship with neostriatum. The second exhibited a 3-dimensional neostriatal representation throughout the remainder of the pallidal neuropil. Comparison to the axonal branching pattern of single strio-pallidal neurons suggests that these represent the two pallidal collateral arborizations made by axons of spiny neostriatal neurons.

Protection of neurons and microglia against ethanol in a mouse model of fetal alcohol spectrum disorders by peroxisome proliferator-activated receptor-γ agonists.

Fetal alcohol spectrum disorders (FASD) result from ethanol exposure to the developing fetus and are the most common cause of mental retardation in the United States. These disorders are characterized by a variety of neurodevelopmental and neurodegenerative anomalies which result in significant lifetime disabilities. Thus, novel therapies are required to limit the devastating consequences of FASD. Neuropathology associated with FASD can occur throughout the central nervous system (CNS), but is particularly well characterized in the developing cerebellum. Rodent models of FASD have previously demonstrated that both Purkinje cells and granule cells, which are the two major types of neurons in the cerebellum, are highly susceptible to the toxic effects of ethanol. The current studies demonstrate that ethanol decreases the viability of cultured cerebellar granule cells and microglial cells. Interestingly, microglia have dual functionality in the CNS. They provide trophic and protective support to neurons. However, they may also become pathologically activated and produce inflammatory molecules toxic to parenchymal cells including neurons. The findings in this study demonstrate that the peroxisome proliferator-activated receptor-γ agonists 15-deoxy-Δ12,15 prostaglandin J2 and pioglitazone protect cultured granule cells and microglia from the toxic effects of ethanol. Furthermore, investigations using a newly developed mouse model of FASD and stereological cell counting methods in the cerebellum elucidate that ethanol administration to neonates is toxic to both Purkinje cell neurons as well as microglia, and that in vivo administration of PPAR-γ agonists protects these cells. In composite, these studies suggest that PPAR-γ agonists may be effective in limiting ethanol-induced toxicity to the developing CNS.

Pioglitazone Blocks Ethanol Induction of Microglial Activation and Immune Responses in the Hippocampus, Cerebellum, and Cerebral Cortex in a Mouse Model of Fetal Alcohol Spectrum Disorders

BACKGROUND Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol and are the leading cause of mental retardation in the United States. There is currently no effective treatment that targets the causes of these disorders. Thus, novel therapies are critically needed to limit the neurodevelopmental and neurodegenerative pathologies associated with FASD. METHODS A neonatal mouse FASD model was used to examine the role of the neuroimmune system in ethanol (EtOH)-induced neuropathology. Neonatal C57BL/6 mice were treated with EtOH, with or without pioglitazone, on postnatal days 4 through 9, and tissue was harvested 1 day post treatment. Pioglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits anti-inflammatory activity and is neuroprotective. We compared the effects of EtOH with or without pioglitazone on cytokine and chemokine expression and microglial morphology in the hippocampus, cerebellum, and cerebral cortex. RESULTS In EtOH-treated animals compared with controls, cytokines interleukin-1β and tumor necrosis factor-α mRNA levels were increased significantly in the hippocampus, cerebellum, and cerebral cortex. Chemokine CCL2 mRNA was increased significantly in the hippocampus and cerebellum. Pioglitazone effectively blocked the EtOH-induced increase in the cytokines and chemokine in all tissues to the level expressed in handled-only and vehicle-treated control animals. EtOH also produced a change in microglial morphology in all brain regions that was indicative of microglial activation, and pioglitazone blocked this EtOH-induced morphological change. CONCLUSIONS These studies indicate that EtOH activates microglia to a pro-inflammatory stage and also increases the expression of neuroinflammatory cytokines and chemokines in diverse regions of the developing brain. Further, the anti-inflammatory and neuroprotective PPAR-γ agonist pioglitazone blocked these effects. It is proposed that microglial activation and inflammatory molecules expressed as a result of EtOH treatment during brain development contribute to the sequelae associated with FASD. Thus, pioglitazone and anti-inflammatory pharmaceuticals more broadly have potential as novel therapeutics for FASD.

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Heteromeric Canonical Transient Receptor Potential 1 and 4 Channels Play a Critical Role in Epileptiform Burst Firing and Seizure-Induced Neurodegeneration

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

A comparison of the distribution and morphology of thalamic, cerebellar and spinal projection neurons in rat trigeminal nucleus interpolaris.

The retrograde transport of horseradish peroxidase was used to examine and compare the distribution and morphology of thalamic, cerebellar and spinal projecting neurons in rat trigeminal nucleus interpolaris following large injections into their respective targets. The regional distribution of these three populations was evaluated in relation to the six cytoarchitecturally distinct regions which characterize the nucleus. Cerebellar projecting neurons were distributed throughout the rostrocaudal extent of trigeminal nucleus interpolaris, but were infrequently present in its dorsolateral region and in the rostral pole of the nucleus. Thalamic projecting neurons exhibited a distribution pattern that extensively overlapped with that of the trigeminocerebellar neurons: however, they were particularly concentrated in caudal, dorsomedial and rostral, ventrolateral regions of the nucleus. Trigeminospinal projecting neurons exhibited a more restricted distribution within ventral and lateral regions of trigeminal nucleus interpolaris. Although the three populations of projection neurons could not be distinguished solely on the basis of somatic size or shape, distinct regional variations in the distribution and somatodendritic and axonal morphology of these neurons indicated that they arise largely from independent cell populations. However, several regions were identified in which specific cell types were likely to contribute to axonal collaterilization among these pathways. In the ventrolateral magnocellular region of the nucleus, for example, more than half of the large multipolar-shaped neurons were retrogradely labeled after injections into each of the three target sites. The results of the present study indicate that the thalamic, cerebellar and spinal projections of trigeminal nucleus interpolaris arise from a morphologically heterogeneous group of neurons. In addition, regional variations in the distribution and morphology of these neurons provide evidence for the existence of functionally distinct regions that parallel the cytoarchitecturally defined regions of the nucleus. This study also provides indirect evidence for and against collateralization among these three projections within specific regions of the nucleus.

An AnAlysis of the Cyto- and Myeloarchitectonic Organization of Trigeminal Nucleus Interpolaris in the Rat

The cyto- and myeloarchitectonic organization of trigeminal nucleus interpolaris (Vi) was examined in the rat using correlated Nissl- and myelin-stained sections. The caudal boundary of Vi is marked by a spatial overlap with the rostral pole of the medullary dorsal horn (MDH), where there is a dorsal and medial displacement of the substantia gelatinosa (SG, lamina II) layer of MDH. This spatial displacement was further documented using cytochrome-oxidase-reacted sections through the periobex region (POR) of the medulla, where the relatively unstained SG contrasts sharply with the intensely stained Vi neuropil. The rostral boundary of Vi is characterized partly by a distinct overlap with the caudal pole of the dorsomedial region (DM) of trigeminal nucleus oralis (Vo), and partly by a more gradual transition with ventral and lateral regions of Vo. The presence of the distinct MDH-Vi overlap is discussed in terms of its impact on the widespread contention that Vi is involved in the processing of dental pain afferents in the POR. Six separate and distinct regions of rat Vi can be distinguished on the basis of differences in their overall cyto- and myeloarchitecture: (1) a ventrolateral parvocellular region (vlVipc), which occupies the ventrolateral caudal half of Vi; (2) a ventrolateral magnocellular region (vlVimc), which occupies a similar region in the rostral half of the nucleus; (3) a border region (brVi), interposed between the spinal trigeminal tract (SVT) and vlVipc and vlVimc; (4) a dorsolateral region (dlVi), which lies predominantly in the rostral two-thirds of Vi subjacent to the dorsal half of SVT; (5) a dorsal cap region (dcVi), occupying the dorsomedial aspect of the nucleus throughout its entire rostrocaudal extent; and (6) an intermediate region (irVi), which lies immediately ventral to dcVi within the concavity formed by the medial borders of vlVipc and vlVimc. It is proposed that these cyto- and myeloarchitecturally distinct regions of Vi may largely represent functionally distinct regions, based on reported differences in the organization of afferent and efferent projections within the nucleus.

Transforming growth factor-β2 both stimulates and inhibits neurogenesis of rat cerebellar granule cells in culture

Transforming growth factor-beta 2 (TGF beta 2) is expressed in the developing cerebellar cortex during the period of granule cell proliferation and maturation. However, the role of TGF beta 2 in granule cell development is confused by conflicting observations regarding TGF beta 2 control of neurogenesis. To resolve these conflicts and determine the effect of TGF beta 2 on neurogenesis, rat cerebellar granule cell cultures were treated with TGF beta 2 (0.1-100 ng/ml, 24 h) in the presence or absence of exogenous serum. Neuroblast proliferation was quantified by bromodeoxyuridine and [3H]thymidine incorporation. TGF beta 2 stimulated proliferation to 220% of controls in the presence of serum (ED50 = 0.4 ng/ml) based on bromodeoxyuridine labeled granule cell counts. In contrast, in serum free medium, TGF beta 2 inhibited proliferation 75% (ED50 = 0.7 ng/ml). DNA synthesis measured by [3H]thymidine incorporation was increased to 122% in the presence of serum factors, but inhibited 70% in serum free medium, as a result of TGF beta 2 activity. Thus, TGF beta 2 differentially regulates neurogenesis of cerebellar granule cells depending on the presence of exogenous, undefined regulatory factors derived from serum. This suggests that TGF beta 2 activity in cerebellar neurogenesis is complex as it may be modulated by the repertoire of other endogenous regulatory factors in the developing cerebellar cortex.

The spinotrigeminal pathway and its spatial relationship to the origin of trigeminospinal projections in the rat

The anterograde transport of horseradish peroxidase and tritiated amino acids was used to examine the distribution and morphology of spinal afferent fibers terminating in the rat spinal trigeminal complex. The results confirm the existence of a direct, ipsilateral projection from the spinal cord which is distributed exclusively to the deepest layers of the medullary dorsal horn narrow regions subjacent to the spinal trigeminal tract in trigeminal nucleus interpolaris, trigeminal nucleus oralis and the trigeminal main sensory nucleus. Spinal inputs also terminated in the insular trigeminal-cuneatus lateralis nucleus which is a distinct component of the interstitial system of the spinal trigeminal tract. The spinal afferent fibers which terminated in the dorsolateral parts of the spinal trigeminal complex arose from the dorsal column funiculi, while those that terminated in ventral parts of the complex arose from both the dorsal column and lateral funiculi. The tritiated amino acid experiments indicate that at least part of the spinotrigeminal pathway originates from cells located in the cervical spinal dorsal horn. The present findings also document a complex spatial relationship between the spinotrigeminal and trigeminospinal pathways which includes an extensive overlap between spinotrigeminal fibers and spinal projecting neurons in each of the lateralmost regions of the complex. This spatial overlap supports the existence of anatomical substrates which may underlie functional reciprocal loops between the spinal trigeminal complex and cervical spinal cord. Since these regions are primarily concerned with the processing of sensory information from lateral and posterior parts of the face, it follows that the spinotrigeminal pathway may be primarily concerned with the integration of head and neck functions. In addition, the spatial convergence of spinal inputs and the distribution of other trigeminal efferent neurons suggests that part of the spinotrigeminal pathway may be involved in spino-trigemino-thalamic and spino-trigemino-cerebellar pathways in parallel with other spinobulbar pathways in the medulla.