D. van der Kooy

Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain

The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.

Post-herpetic neuralgia: Further post-mortem studies of cases with and without pain

&NA; The pathological features associated with post‐herpetic neuralgia require further study. We report here 5 cases, 3 with severe post‐herpetic neuralgia (PHN) and 2 with no persistent pain. The findings of dorsal horn atrophy and cell, axon and myelin loss with fibrosis in the sensory ganglion were found only in patients with persistent pain. Marked loss of myelin and axons in the nerve and/or sensory root were found in cases with and without pain. Some evidence is presented for a more generalized subacute or chronic inflammatory process which may explain the clinical features of some patients. Further studies will be necessary to fully describe the morbid anatomy of this disorder.

Serotonergic and non-serotonergic projections from the nucleus raphe dorsalis to the caudate-putamen complex in the rat, studied by a combined immunofluorescence and fluorescent retrograde axonal labeling technique

Abstract Immunofluorescence, induced in serotonergic neurons, was combined with retrograde axonal tracing by propidium iodide. Evidence is presented for the existence of serotonergic as well as non-serotonergic projections from the nucleus raphe dorsalis to the caudate-putamen complex.

A serotonin-containing pathway from the area postrema to the parabrachial nucleus in the rat

A combined retrograde tracing-immunofluorescent technique was used to identify the relationships between the cellular population projecting to the parabrachial nucleus and the serotonin-immunoreactive cell population of the area postrema in rats. The retrograde fluorescent tracer True Blue was injected in the parabrachial region and 3 days later the animals were perfused. Serial cryostat sections were processed for serotonin immunofluorescence. Three different groups of labeled cells were identified in the area postrema. First, True Blue-positive cells (up to 250/section) that project to the parabrachial nucleus were observed distributed throughout the area postrema. Second, in the pargyline (a monoamine oxidase inhibitor)-treated animals a large number of serotonergic cells (up to 125/section) was observed distributed throughout the area postrema. There was a tendency to a heavier distribution of serotonin-immunoreactive cells in the dorsal two-thirds of the area postrema. Third, double-labelled cells were also seen. Twenty percent of the True Blue-labelled cells projecting to the parabrachial nucleus were serotonin-immunoreactive. Thirty nine percent of the serotonin-immunoreactive population was retrogradely labelled with True Blue. Thus a new serotonergic pathway from the area postrema to the parabrachial nucleus is described; this pathway may be important in the ascending transmission and modulation of chemical and visceral sensory input.

Post-herpetic neuralgia: post-mortem analysis of a case.

&NA; The morphological and biochemical substrates of the severe pain in post‐herpetic neuralgia (PHN) are unclear. This report is an autopsy study of a 67‐year‐old male with severe PHN during the last 5 years of his life over the right T7‐8 dermatomes. The dorsal horn of the thoracic spinal cord of the affected side was atrophic from T4 to T8, with loss of both myelin and axons. Despite this, only the T8 ganglion was affected by fibrosis and cell loss and only the nerve roots at that level appeared affected. Markers of unmyelinated afferents (substance P), substantia gelatinosa neurons (opiate receptors), glial cells (glial fibrillary acidic protein), and descending spinal projections (dopamine‐&bgr;‐hydroxylase and serotonin) were not different at affected versus non‐affected spinal cord levels. The pain of PHN may result from the uninhibited activity of unmyelinated primary afferents as a result of the loss of myelinated afferent fibers and the possible presence of hypersensitive neurons in the dorsal horn.

Is there a non-dopaminergic nigrostriatal pathway?

Abstract Sixteen per cent of the substantia nigra cell bodies normally labeled from the injection of a fluorescent retrograde tracer in the caudate-putamen complex could still be labeled by the same procedure after multiple intracisternal 6-hydroxydopamine treatments that depleted dopamine levels in the caudate-putamen complex to 1.0% of control. However, the demonstration of glyoxylic-acid-induced catecholamine histofluorescence in tissue from these lesioned rats revealed that many of the surviving retrogradely-labeled substantia nigra cell bodies still contained dopamine. The persistence of some dopamine in the substantia nigra of the lesioned animals was confirmed biochemically. Therefore, retrograde tracing in 6-hydroxydopamine lesioned rats overestimated the extent of the non-dopaminergic nigrostriatal tract. The simultaneous combination of retrograde fluorescent tracing and catecholamine histofluorescence in unlesioned animals revealed that only 5% or less of the substantia nigra cell bodies retrogradelylabeled from the caudate-putamen complex were without catecholamine fluorescence. These apparently non-dopaminergic nigrostriatal cells were located primarily in the ventral tegmental area, substantia nigra pars reticulata and extreme medial edge of the substantia nigra pars compacta.

Migrational analysis of the constitutively proliferating subependyma population in adult mouse forebrain

Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a beta-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically beta-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the down-regulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of beta-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of beta-galactosidase-labeled cells with [3H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.

The dopamine innervation of the visceral cortex mediates the aversive effects of opiates

We have previously reported that opiates acting on peripheral receptors produce aversive effects whereas opiates acting on central brain receptors produce rewarding effects. The neurotransmitter dopamine (DA) has previously been implicated in both opiate reinforcing (positive) and aversive (negative) effects. We, therefore, chose to investigate the effects of disruption of DA systems on these two motivational properties of the opiate, morphine. Moreover, we sought to determine the brain site where dopamine might act as a mediator of these motivational effects. One group of rats received 6-hydroxydopamine (6-OHDA) lesions of the visceral (agranular insular) cortex to destroy dopaminergic innervation to this area. A separate group of animals were pretreated with intraperitoneal (IP) injections of the DA receptor blocker, alpha-flupenthixol (0.8 mg/kg), followed in both groups by 15 mg/kg (IP) morphine. Both 6-OHDA-lesioned and alpha-flupenthixol-pretreated subjects failed to develop the normal aversion to saccharin seen in control groups following conditioned taste aversion training with morphine. In a place conditioning paradigm, the aversive effects produced by low IP injections of morphine (acting on peripheral receptors) were blocked by 6-OHDA lesions of the visceral cortex. However, DA depletion of the visceral cortex did not disrupt the ability of animals to acquire a morphine place preference. Taken together, these results indicate that DA innervation of the visceral cortex mediates the aversive, but not the rewarding, properties of opiates.

Dopamine and opiate receptors: Localization in the striatum and evidence for their axoplasmic transport in the nigrostriatal and striatonigral pathways

The axoplasmic transport of dopamine and opiate receptors in the striatonigral and nigrostriatal pathways was investigated by placing coronal knife cuts through these pathways and examining autoradiographically the accumulation of receptors at the site of the cut. In otherwise normal animals build-up of both receptors was found both rostral and caudal to the cut after a survival time of 24 h. Build-up of both receptors was reduced caudal to the cut by prior 6-hydroxydopamine lesions of the substantia nigra-ventral tegmental area, and reduced rostral to the cut by prior kainic acid lesions of the striatum. In addition, it was found by both autoradiographic and membrane binding methods that kainic acid lesions of the striatum produced a larger reduction of striatal opiate compared with dopamine receptors. The results suggest that opiate and dopamine receptors are axonally transported in fibers of both the nigrostriatal and striatonigral pathways possibly to their respective presynaptic terminals. The differential sensitivity of opiate and dopamine receptors in the striatum to local kainic acid lesions suggests the preferential localization of postsynaptic opiate receptors on dendritic trunks and neuronal somata, whereas the major localization of postsynaptic dopamine receptors may be on striatal dendritic spines, which to some extent appear to survive the lesions.

Spatially localized neuronal cell lineages in the developing mammalian forebrain

The role of cell lineage in the organization of the cerebral cortex and striatum of the developing rat forebrain was analysed using retroviral-mediated gene transfer to mark the progeny of individual progenitors. Injections around the onset of neurogenesis (embryonic day 14) produced neuronal- and glial-specific clones in the striatum and cortex. The majority of the neuronal clones were restricted to either the deep or superficial layers of the cortex and to either the striatal patch or matrix compartments of the striatum. Moreover, modeling the distributions of the neuronal clones in various ways revealed that grouping the clones into deep vs superficial cortical compartments and patch vs matrix striatal compartments best accounted for the clone distributions. These results suggest that at the onset of neurogenesis there is a heterogeneity of neuronal progenitors within the proliferative ventricular zone.