Retinal stem cells in the adult mammalian eye.

The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium, indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.

An irreducible knee dislocation: a case report.

An 8-year-old man presented after sustaining an injury during a fall. A closed reduction attempt failed, and after several tests, an open reduction was performed. With posterolateral dislocation of the knee, there can be anterior cruciate ligament, posterior cruciate ligament, and medial collateral ligament disruption. At the 6-month (final) follow-up, the patient had no subjective pain or instability. With this type of injury, the approach can be conservative monitoring or repair of all of the ligaments. Because of the age and activity level of our patient, we opted for repair of the medial collateral ligament initially with the possibility of late anterior cruciate ligament and/or posterior cruciate ligament reconstruction.

Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.

The adult derivatives of the embryonic forebrain germinal zones consist of two morphologically distinct cell layers surrounding the lateral ventricles: the ependyma and the subependyma. Cell cycle analyses have revealed that at least two proliferating populations exist in this region, one that is constitutively proliferating and one that is relatively quiescent and thought to include the endogenous adult neural stem cells. Earlier studies demonstrated that specific dissection of the region surrounding the lateral ventricles was necessary for the in vitro isolation of multipotent, self-renewing neural stem cells. However, in these studies, the ependymal layer was not physically separated from the subependymal layer to identify the specific adult laminar localization of the neural stem cells around the lateral ventricles. To determine which cellular compartment in the adult forebrain contained the neural stem cells, we isolated and cultured the ependyma separately from the subependyma and tested for the presence of neural stem cells using the in vitro neurosphere assay. We demonstrate that the ependymal cells can proliferate in vitro to form sphere-like structures. However, the ependymal cells generating spheres do not have the ability to self-renew (proliferate to form secondary spheres after dissociation) nor to produce neurons, but rather only seem to generate glial fibrillary acidic protein-positive ependymal cells when plated under differentiation conditions in culture. On the other hand, a subpopulation of subependymal cells do possess the self-renewing and multipotential characteristics of neural stem cells. Therefore, the adult forebrain neural stem cell resides within the subependymal compartment.

Intra-amygdala infusion of an end-capped antisense oligodeoxynucleotide to c-fos accelerates amygdala kindling.

The changes produced by repeated stimulation of the brain that result in the kindling effect remain unknown. It is known, however, that activation of immediate early genes including c-fos is a very early event in the development of kindling. Here we show that c-fos expression in the amygdala can be knocked-down by an end-capped antisense oligodeoxynucleotide (but not by vehicle, or control antisense oligodeoxynucleotides) and that this results in an acceleration of kindling. These results suggest that, at least in the amygdala, the expression of c-fos is associated with an attempt by the brain to retard the development of kindling.

Putative roles for the inducible transcription factor c-fos in the central nervous system: studies with antisense oligonucleotides.

Although immediate-early genes such as c-fos are widely believed to play an important role in neuroplasticity, there is limited evidence to support involvement in the initiation of molecular events leading to medium- and long-term changes in brain function following a stimulus. Results using techniques such as transgenic knockout of the gene are often difficult to interpret. Antisense oligonucleotide technology offers an alternative. Infusion of antisense oligonucleotide to modify the expression of c-fos in the brain results in dramatic changes in rotation behaviour in animals challenged with psychostimulant drugs such as amphetamine. Similarly, the knockdown of c-fos expression using antisense oligonucleotides can also alter the rate of amygdala kindling in response to electrical stimulation of the brain. While studies using antisense oligonucleotides to knockdown c-fos expression provide evidence that the expression of c-fos plays an important role in regulating neuronal function, the use of antisense nucleotides has limitations and experiments must be very carefully controlled. Many details of antisense oligonucleotide actions remain unknown.

Antisense Therapeutics in the Central Nervous System : The Induction of c-fos.

Immediate-early genes (IEGs) are members of a class of genes that respond, in many cell types, to a variety of stimuli by rapid, but transient expression (1). Several of these IEGs code for transcription factors and include the widely studied activator protein-1 (AP-1) transcription factor complex believed to be homo- and heterodimeric assemblies of the Fos and Jun families (1-3). IEGs are induced in the central nervous system (CNS) by diverse physiological and pharmacological stimuli, many of which, when presented once or on multiple occasions, can alter the "normal" functioning of the brain in a permanent or semipermanent fashion. Examples of pharmacological stimuli that lead to long-term changes are the highly addictive psychostimulant drugs, amphetamine and cocaine These drugs produce a robust activation of IEGs (e.g., c-fos, jun-B, egr-1) in areas of the brain that are believed to be part of the neural substrates of addiction (4-8) In animal models of epileptogenesis or memory, such as kindling and long-term potentiation (LTP), respectively, electrical stimuli produce activation of IEGs within the brain structures thought to underlie the long-lasting changes associated with these experimental procedures (9-16). IEGs can also be induced by noninvasive stimuli, such as a simple light pulse given to animals in a dark room. The circadian rhythms of animals that are housed in darkened conditions can be shifted by exposing them to a light during then subjective night. Activation of IEGs in such experiments are restricted to the suprachiasmatic nucleus (SCN), which is believed to be the seat of the biological clock (17).

Amygdala kindling and immediate-early genes.

We examined if the first afterdischarge (AD) following a kindling stimulation to the amygdala is a necessary and/or sufficient stimulus for immediate-early gene (IEG) activation and whether activity that results in IEG expression in the amygdala was sufficient to cause activation in the hippocampus. We demonstrate that a single, short duration, AD results in activation of c-fos and egr-1 in the amygdala/piriform following a kindling stimulation to the amygdala. Conversely, considerably longer and perhaps more complex ADs are required for IEG induction in the hippocampus of amygdala kindled animals. These results suggest that a single AD is sufficient to activate c-fos and egr-1 in amygdala/piriform and establishes a putative role for IEGs in amygdala kindling.

Infusion into the brain of an antisense oligonucleotide to the immediate-early gene c-fos suppresses production of fos and produces a behavioral effect.

While many studies have examined the numerous physiological and pharmacological factors which can induce the expression of c-fos and other immediate-early genes, few have examined the physiological/biochemical consequences of altering their expression pattern. Using antisense oligonucleotides to c-fos, we demonstrate that D-amphetamine-induced c-fos expression can be attenuated in specific brain regions in vivo. This unilateral attenuation of c-fos expression in D-amphetamine-stimulated animals results in a directed rotational behavior. We show that animals rotate only when they express a difference in Fos-like immunoreactivity between hemispheres. The attenuation of Fos-like immunoreactivity by the antisense oligonucleotides appears to be dependent on the c-fos messenger RNA site that these antisense oligonucleotides target and the degree of chemical protection of the oligonucleotide against degradation. The attenuation of Fos-like immunoreactivity and the increase in unilaterally directed rotation are both time- and dose-dependent. These results demonstrate that manipulating immediate-early gene expression by the direct infusion of antisense oligonucleotides in specific brain regions can have behavioral consequences.

The application of antisense oligonucleotide technology to the brain: some pitfalls.

1. Amphetamine-induced c-fos and egr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity. 2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.

Morphological changes and functional recovery following axotomy of a serotonergic cerebrobuccal neurone in the land snail Achatina fulica.

We have examined an identified serotonergic neurone in Achatina fulica and described the normal morphological and physiological characteristics of this cell. Injury-induced changes in this neurone following in vivo recovery are described and compared with in vitro gastropod models of regeneration. Nickel-lysine and biocytin dye-fills of the metacerebral giant (MCG) neurone, together with serotonin-like immunoreactivity, revealed an extensive innervation of the ipsilateral buccal ganglion, much greater than that previously reported. Labelled MCG fibres were seen to ramify throughout the ganglion, providing extensive neuropilar innervation. Serotonin-immunoreactive fibres were seen not only within the neuropile but also within the cell body layer of the buccal ganglia, surrounding many of the cell bodies with varicose fibres. Dye-fills also revealed a minor contralateral buccal innervation not previously described. This view of a predominantly ipsilateral innervation of the buccal ganglia by the MCG was supported by electrophysiological measurements. The ipsilateral buccal follower cell B1 displayed an increase in depolarization in response to repeated trains of action potentials to the MCG, whereas the contralateral B1 showed only a weak depolarization in response to the identical stimuli. Following a crush to the cerebral-buccal connective (CBC), the MCG rapidly regenerated its injured projections, displaying both morphological and physiological recovery within 5-10 days. The original, severed fibres of the MCG were, however, replaced by a multitude of smaller neurites, which persisted for up to 3 months (the longest recovery period examined). Despite this morphological difference between normal and regenerated fibres, the MCG re-established functionally equivalent connections upon B1. In contrast with previous in vitro studies using gastropods, serotonin-like immunoreactivity revealed that severed distal fibres from the MCG rapidly degenerated (2-6 days), resulting in a transient unilateral depletion of serotonin in the buccal ganglia. We suggest that this loss of serotonin in the lesioned ganglion may play a functional role in regeneration, as has been suggested in vitro.

Antisense oligonucleotide eliminates in vivo expression of c-fos in mammalian brain.

Immediate-early genes such as c-fos and NGFI-A are rapidly and transiently expressed in the striatum following amphetamine administration in vivo. Here we show that direct infusion of an antisense oligodeoxynucleotide to c-fos into striatum will reduce amphetamine-induced production of Fos-like immunoreactivity without affecting NGFI-A expression. These results suggest that it is possible to use antisense technology to study the role of immediate-early genes in specific sites in the brain in vivo.

Distribution of catecholamines and of immunoreactivity to substances like vertebrate enzymes for the synthesis of catecholamines within the central nervous system of the snail, Lymnaea stagnalis.

Catecholamines (CAs) were detected histochemically within over 185 cell bodies in the central nervous system (CNS) of juvenile and young adult Lymnaea. This distribution of CA-containing cells in all central ganglia except the pleural ganglia is more widespread than previously described but is consistent with other reports suggesting numerous roles for CAs within the nervous system. This study also describes the distribution of substances which are antigenically similar to four bovine enzymes for catecholamine synthesis, but the distribution patterns showed little or no overlap with each other or with CA. These results suggest the need for caution in the interpretation of such immunohistochemical studies.

Postembryonic development of serotoninlike immunoreactivity in the central nervous system of the snail, Lymnaea stagnalis.

Posthatching growth in the pond snail Lymnaea stagnalis involves approximately a 20-fold increase in the linear dimensions of the ganglia composing the central nervous system. Developmental change within the population of neurons exhibiting serotoninlike immunoreactivity (SLIR) was examined in order to explain this growth in cellular terms. The study indicates that at least two factors contribute to the growth of the nervous system. First, SLIR cells approximately double in number from the 200-250 cells in hatchlings to the complement found in animals approaching sexual maturity. Much of this increase in cell number occurred within identifiable discrete clusters of neurons with different clusters adding cells at different rates and at different times. The number of SLIR cells also increased in more diffuse populations, particularly along the medial aspects of the paired pedal and the right parietal ganglion. No identified cells were added postembryonically. In addition to the increases in neuron numbers, posthatching development in Lymnaea also involves the growth of individual cells. All cells examined showed continuous somatic growth during posthatching development, but different identified cells and different cell clusters were characterized by different rates of relative growth. Together, the results highlight the complexity of postembryonic development in the snail by indicating the temporal and spatial specificity for both cell addition and cell growth within the nervous system.