Decreased neurogenesis after cholinergic forebrain lesion in the adult rat

Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival-promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.     

Doublecortin expression in the adult rat telencephalon

Doublecortin (DCX) is a protein required for normal neuronal migration in the developing cerebral cortex, where it is widely expressed in both radially and tangentially migrating neuroblasts. Moreover, it has been observed in the adult rostral migratory stream, which contains the neuronal precursors traveling to the olfactory bulb. We have performed DCX immunocytochemistry in the adult rat brain to identify precisely the neuronal populations expressing this protein. Our observations confirm the presence of DCX immunoreactive cells with the characteristic morphology of migrating neuroblasts in the subventricular zone, rostral migratory stream and the main and accessory olfactory bulbs. We have also found putative migratory cells expressing DCX in regions were no adult neuronal migration has been described, as the corpus callosum, the piriform cortex layer III/endopiriform nucleus and the striatum. Surprisingly, many cells with the phenotype of differentiated neurons were DCX immunoreactive; e.g. certain granule neurons in the hilar border of the granular layer of the dentate gyrus, some neuronal types in the piriform cortex layer II, granule and periglomerular neurons in the main and accessory olfactory bulbs, and isolated cells in the striatum. Almost all DCX immunoreactive cells also express the polysialylated form of neural cell adhesion molecule and have a similar distribution to rat collapsin receptor-mediated protein-4, two molecules involved in neuronal structural plasticity. Given these results, we hypothesize that DCX expression in differentiated neurons could be related to its capacity for microtubule reorganization and that this fact could be linked to axonal outgrowth or synaptogenesis.     

Transient expression of the conserved zinc finger gene INSM1 in progenitors and nascent neurons throughout embryonic and adult neurogenesis

INSM1 is a zinc-finger protein expressed in the developing nervous system and pancreas as well as in medulloblastomas and neuroendocrine tumors. With in situ hybridization combined with immunohistochemistry, we detected INSM1 mRNA in all embryonic to adult neuroproliferative areas examined: embryonic neocortex, ganglionic eminence, midbrain, retina, hindbrain, and spinal cord; autonomic, dorsal root, trigeminal and spiral ganglia; olfactory and vomeronasal organ epithelia; postnatal cerebellum; and juvenile to adult subgranular zone of dentate gyrus, subventricular zone, and rostral migratory stream leading to olfactory bulb. In most of these neurogenic areas, subsets of neuronal progenitors and nascent, but not mature, neurons express INSM1. For example, in developing cerebellum, INSM1 is present in proliferating progenitors of the outer external granule layer (EGL) and in postmitotic cells of the inner EGL, but not in mature granule cell neurons. Also, lining the neural tube from spinal cord to neocortex in mouse as well as human embryos, cells undergoing mitosis apically do not express INSM1. By contrast, nonsurface progenitors located in the basal ventricular and/or subventricular zones express INSM1. Whereas apical progenitors are proliferative and generate one or two additional progenitors, basal progenitors are thought to divide terminally and symmetrically to produce two neurons. The nematode ortholog of INSM1, EGL-46, is expressed during terminal symmetric neurogenic divisions and regulates the termination of proliferation. We propose that, in mice and humans, INSM1 is likewise expressed transiently during terminal neurogenic divisions, from late progenitors to nascent neurons, and particularly during symmetric neuronogenic divisions.     

Chronic inhibition of nitric oxide synthesis enhances both subventricular zone neurogenesis and olfactory learning in adult mice

The ability to generate new neurons during the course of adult life is preserved in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus in the mammalian brain. These two regions constitute specifically regulated neurogenic niches, and provide newborn neurons involved in olfactory and spatial learning, respectively. Nitric oxide (NO) is a negative regulator of neurogenesis in the subventricular zone, whereas its role in the dentate gyrus remains controversial. Using systemic administration of NO synthase (NOS) inhibitors to chronically inhibit NO production, we increased neural precursor proliferation in the subventricular zone as well as neurogenesis in the olfactory bulb, without modifying the number of mitotic cells or the granular cell layer thickness in the dentate gyrus. The same treatment specifically improved olfactory learning performance, whereas spatial learning and memory was unchanged, thus demonstrating that olfactory memory is closely associated with the level of ongoing neurogenesis in the subventricular zone-olfactory bulb. The anatomical specificity of the NOS inhibitor actions was not due to differences in the availability of NO, as demonstrated by immunohistochemical detection of neuronal NOS and S-nitrosylated proteins in both regions. Remarkably, the distinct NO sensitivity might result from a differential expression of epidermal growth factor receptor in precursor cells in both regions, as the proliferative effect of NOS inhibitors in the subventricular zone was restricted to the cells that expressed this receptor.     

Monday July 3, 200612:00–13:30Hall 3DPlatform SessionBasic Science I: Molecules and Networks for Epileptogenesis

¹ M. Kokaia (   ¹ Wallenberg Neuroscience Center, Lund University Hospital, Sweden )
Purpose: Neural stem cells in the adult mammalian brain (including humans) continue to produce new functional granule cells in the dentate gyrus subgranular zone and new olfactory bulb neurons in the subventricular zone during an entire life. In the hippocampus, neurogenesis has been proposed to play a role in learning and memory and mood regulation. The new cells develop electrophysiological characteristics and synaptic inputs very similar to those of the rest of the cell population. The purpose of the study was to explore whether tissue environment in an epileptic brain influences properties of afferent synapses formed on newborn granule cells.
Method: Rats were exposed to either a physiological stimulus, i.e., running, or status epilepticus, which gives rise to neuronal death, inflammation, increased network excitability and recurrent spontaneous seizures. Both treatments increase neurogenesis in the dentate gyrus. We labelled newborn cells by GFP-retroviral vector injections right after these treatments to identify the cells and apply whole-cell patch-clamp recordings in live hippocampal slices.
Results: Granule cells formed after running and status epilepticus exhibited similar intrinsic membrane properties. However, new neurons born into the epileptic environment differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. The new granule cells formed after status epilepticus exhibited functional connectivity consistent with reduced synaptic network excitability of the dentate gyrus, i.e., decreased excitatory and increased inhibitory input activity.
Conclusion: We demonstrate for the first time a high degree of plasticity in synaptic inputs to the new neurons, which could mitigate pathological activity in the epileptic brain.     

Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development.

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.     

Regeneration of granule neurons after lesioning of hippocampal dentate gyrus: evaluation using adult mice treated with trimethyltin chloride as a model

The hippocampal dentate gyrus in adult animals is known to contain neural progenitors that proliferate and differentiate into neurons in response to brain injury. Little has been observed, however, on regeneration of the granule cell layer of the dentate gyrus that has been directly injured. Using trimethyltin (TMT)-treated mice as an in vivo model, we evaluated the ability of this layer to regenerate after injury. The administration of TMT induced neuronal death in the dentate gyrus selectively 2 days later, with recovery of granule neurons on day 14 and thereafter. At an early stage (days 2-5) after the damage by TMT treatment, 5-bromo-2'-deoxyuridine (BrdU) incorporation into at least two different types of cells was facilitated in the dentate gyrus: BrdU-positive/neuronal nuclear antigen (NeuN)-negative cells were found predominantly in the subgranular zone and granule cell layer, whereas BrdU-positive/NeuN-positive cells were numerous in the dentate molecular layer and hilus. In addition, expression of proliferating cell nuclear antigen, nestin, NeuroD3, and doublecortin, which are markers for proliferating cells and neural progenitors/neuronal precursors, was extremely enhanced in the dentate gyrus at the early stage after treatment. Double staining revealed that BrdU was colocalized with nestin and doublecortin in the subgranular zone. Behavioral analysis revealed that TMT-induced cognition impairment was ameliorated by day 14 after the treatment. Taken together, our data indicate that the hippocampal dentate gyrus itself is capable of regenerating the neuronal cell layer through rapid enhancement of neurogenesis after injury.     

Suppression of insult-induced neurogenesis in adult rat brain by brain-derived neurotrophic factor

In mammals, including humans, the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus contain neural stem cells, which continue to proliferate even in adulthood and give rise to new neurons. Neurogenesis in these areas is enhanced by brain insults. Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and differentiation during the development of the nervous system. In the adult intact brain, BDNF administration in the lateral ventricle or ventricular zone stimulates neurogenesis in several forebrain areas. Here we show that intrahippocampal transduction of recombinant adeno-associated virus carrying the BDNF gene giving rise to levels of BDNF protein sufficient to induce a functional response inhibits the formation of new dentate granule cells triggered by global forebrain ischemia in rats. Our data indicate that long-term delivery of a neurotrophic factor, which is considered as a novel neuroprotective strategy for human brain diseases, may attenuate intrinsic neuroregenerative responses.     

Strategies to promote differentiation of newborn neurons into mature functional cells in Alzheimer brain

Adult neurogenesis occurs in the subgranular zone (SGZ) and subventricular zone (SVZ). New SGZ neurons migrate into the granule cell layer of the dentate gyrus (DG). New SVZ neurons seem to enter the association neocortex and entorhinal cortex besides the olfactory bulb. Alzheimer disease (AD) is characterized by neuron loss in the hippocampus (DG and CA1 field), entorhinal cortex, and association neocortex, which underlies the learning and memory deficits. We hypothesized that, if the AD brain can support neurogenesis, strategies to stimulate the neurogenesis process could have therapeutic value in AD. We reviewed the literature on: (a) the functional significance of adult-born neurons; (b) the occurrence of endogenous neurogenesis in AD; and (c) strategies to stimulate the adult neurogenesis process. We found that: (a) new neurons in the adult DG contribute to memory function; (b) new neurons are generated in the SGZ and SVZ of AD brains, but they fail to differentiate into mature neurons in the target regions; and (c) numerous strategies (Lithium, Glatiramer Acetate, nerve growth factor, environmental enrichment) can enhance adult neurogenesis and promote maturation of newly generated neurons. Such strategies might help to compensate for the loss of neurons and improve the memory function in AD.     

Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb

In the adult rat olfactory bulb, neurons are continually generated from progenitors that reside in the lateral ventricle wall. This study investigates long-term survival and cell death of newly generated cells within the adult olfactory bulb. After injecting rats at 2 months of age with 5-bromodeoxyuridine (BrdU), the newly generated cells were quantified over a period of 19 months. A peak of BrdU-positive cells was reached in the olfactory bulb 1 month after BrdU injection, when all new cells have finished migrating from the ventricle wall. Thereafter, a reduction of BrdU-positive cells to about 50% was observed and it was confirmed by dUTP-nick end-labelling (TUNEL) that progenitors and young neurons undergo programmed cell death. However, cells that survived the first 3 months after BrdU injection persisted for up to 19 months. The majority of the BrdU-positive cells that reach the olfactory bulb differentiate into granule cells, but a small fraction migrate further into the glomerular layer. These newborn cells differentiate more slowly into periglomerular interneurons, with a delay of more than 1 month when compared to the granule cells. The newly generated periglomerular neurons, among them a significant fraction of dopaminergic cells, showed a similar decline in number compared to the granule cell layer and long-term survival for the remaining new neurons of up to 19 months. Rather than replacing old neurons, this data suggests that adult olfactory bulb neurogenesis utilizes the overproduction and turnover of young neurons, which is reminiscent of the cellular dynamics observed during brain development.     

Adult neurogenesis in the four-striped mice （Rhabdomys pumilio）

In this study, we investigated non-captive four-striped mice （Rhabdomys pumilio） for evidence that adult neurogenesis occurs in the adult brain of animal models in natural environment. Ki-67 （a marker for cell proliferation） and doublecortin （a marker for immature neurons） immunos-taining conifrmed that adult neurogenesis occurs in the active sites of subventricular zone of the lateral ventricle with the migratory stream to the olfactory bulb, and the subgranular zone of the dentate gyrus of the hippocampus. No Ki-67 proliferating cells were observed in the striatum substantia nigra, amygdala, cerebral cortex or dorsal vagal complex. Doublecortin-immunore-active cells were observed in the striatum, third ventricle, cerebral cortex, amygdala, olfactory bulb and along the rostral migratory stream but absent in the substantia nigra and dorsal vagal complex. The potential neurogenic sites in the four-striped mouse species could invariably lead to increased neural plasticity.     

Retinoic acid signaling at sites of plasticity in the mature central nervous system

We used transgenic reporter mice to determine whether brain regions that respond to retinoic acid (RA) during development do so in maturity. We focused on two prominent sites of embryonic RA signaling: the dorsal spinal cord and the olfactory bulb. In the mature dorsal spinal cord, expression of a direct repeat 5 RA response element (DR5-RARE) transgene is seen in interneurons in laminae I and II, as well as in ependymal cells around the central canal. In the olfactory bulb, DR5-RARE transgene-expressing neurons are seen in the mature granule cell and periglomerular layers, as well as in cells in the subventricular zone of the forebrain-the established source for newly generated granule and periglomerular neurons. In addition, there are transgene-labeled neurons in a small number of other brain regions. These include the spinal trigeminal nucleus, area postrema, habenula, amygdala, and the cerebral cortex. Thus, a distinct type of RA-mediated gene expression, detected with the DR5-RARE reporter transgene, defines neurons, subependymal, or ependymal cells in discrete locations throughout the neuraxis. Some of these cells--particularly those in the spinal cord and olfactory bulb--are found in central nervous system regions that receive local RA signals early in development, and retain a significant amount of functional or structural plasticity in the adult.     

Nestin promoter/enhancer directs transgene expression to precursors of adult generated periglomerular neurons

The subventricular zone (SVZ) is a major neurogenic region in the adult brain. Cells from the SVZ give rise to two populations of olfactory bulb interneurons: the granule cells and periglomerular (PG) cells. Currently, little is known about the signaling pathways that direct these newly generated neurons to become either granule or PG neurons. In the present study, we used the nestin promoter and enhancer to direct expression of the tetracycline transactivator (tTA). We generated two independent strains of nestin-tTA transgenic animals and crossed founder mice from both lines to mice containing a tetracycline-regulated transgene (mCREB) whose expression served as a marker for the activity of the nestin-tTA transgene. mCREB expression occurred in a subset of proliferating cells in the SVZ and rostral migratory stream in both lines. Surprisingly, in both lines of nestin-tTA mice transgene expression in the olfactory bulb was limited to PG neurons and was absent from granule cells, suggesting that this nestin promoter construct differentiates between the two interneuronal populations. Transgene expression occurred in several subtypes of PG neurons, including those expressing calretinin, calbindin, GAD67, and tyrosine hydroxylase. These results suggest that a unique subset of SVZ precursor cells gives rise to PG, and not granule cells. The ability to express different transgenes within this subpopulation of neuronal precursors provides a powerful system to define the signals regulating the differentiation and survival of adult-generated neurons in the olfactory bulb.     

GFAP-expressing radial glia-like cell bodies are involved in a one-to-one relationship with doublecortin-immunolabeled newborn neurons in the adult dentate gyrus

The present study examined the relationship between radial glial cells and newborn neurons in the adult dentate gyrus using three different methods. Single labeling immunocytochemistry for newly born neurons using doublecortin, as well as double labeling using an additional antibody to glial fibrillary acidic protein (GFAP) to label astrocytes were used at the light microscopic level. Furthermore, doublecortin immunoelectron microscopy was used to examine the ultrastructural relationship between newborn neurons and astrocytes in the adult dentate gyrus. These data showed an intimate one-to-one relationship between GFAP-expressing radial glia-like cell bodies and their non-radial processes that wrap around the basal and lateral sides of newborn neurons to cradle them in the subgranular zone. A similar relationship is observed for the newborn neurons at the base of the granule cell layer, but the cell body of the GFAP-expressing radial glia-like cells is not as intimately associated with the cell body of the newborn neurons at this site. Furthermore, newborn neurons with apical dendritic processes and growth cones in the granule cell layer extend them along radial glial processes. These newborn neurons do not receive axosomatic or axodendritic synapses indicating the absence of basket cell innervation. These data show that GFAP-expressing radial glia-like cells in the dentate gyrus cradle newborn neurons in the subgranular zone and that their radial processes provide a scaffold for neuronal process outgrowth.     

Distribution of cholecystokinin-like immunoreactivity in the rat main olfactory bulb

The anatomical localization of cholecystokinin-like immunoreactivity (CCK-I) within the rat main olfactory bulb was analyzed by using the peroxidase-antiperoxidase immunocytochemical technique. Neurons or neuronal processes containing CCK-I were localized within all laminae of the olfactory bulb except the olfactory nerve fiber layer. A large population of CCK-I neurons, with morphology, size, and distribution corresponding to that of the middle and external tufted cells, was observed within a zone extending from the deep periglomerular region through the superficial one-half to one-third of the external plexiform layer. A smaller number of immunoreactive perikarya were found in the deep external plexiform layer, the glomerular layer, and rarely within the inner plexiform layer. These CCK-I neurons appeared to correspond to internal tufted cells, periglomerular cells, and deep short-axon cells, respectively. Dense CCK-I staining of fibers and terminals was present within the internal plexiform layer and, less densely, within the neuropil of the granule cell layer. In addition, terminal-like CCK-I was localized within layer 1A of the anterior olfactory nucleus, the olfactory tubercle, and the most rostral piriform cortex. This observation provides corroboration for the identification of the principal CCK-I neuron in the rat olfactory bulb as the centrally projecting middle tufted cell. The present results, demonstrating the localization of CCK-I to both local circuit and projection neurons of the olfactory bulb and to terminal-like puncta in the internal plexiform and granule cell layers, suggest that CCK may be significantly involved in olfactory processing at several levels.     

NMDA receptor antagonist treatment induces a long-lasting increase in the number of proliferating cells, PSA-NCAM-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus

During adulthood, neural precursors located in the subgranular zone of the dentate gyrus continue to proliferate, leading to the generation of new granule neurons. These recently generated cells transiently express the polysialylated form of the neural cell adhesion molecule, PSA-NCAM, and are supported by radial glia-like cells that are likely to play a role in neuronal migration and differentiation, or even act as their precursors. Previous reports indicate that treatment with NMDA receptor antagonists stimulates adult neurogenesis in the dentate gyrus, and because of the potential therapeutic value of this approach, we were interested in further characterizing the consequences of pharmacologically modulating this process. We treated adult rats with the competitive NMDA receptor antagonist, CGP43487, and examined cell proliferation, PSA-NCAM expression, and changes in the radial glia cell population in the subgranular zone at different time points. In addition, we sought to determine if this treatment led to changes in cell death or gliotic reactions. The number of proliferating cells in the subgranular region of the dentate gyrus was increased significantly 2 days after treatment and it remained elevated 7 days postinjection. PSA-NCAM-immunoreactive granule cells and nestin-expressing radial glia-like cells also increased in number 7 days after the treatment. In contrast, we did not observe any change in granule cell death, and we were unable to detect any microglial or astroglial reaction during the first 7 days after treatment. Thus, NMDA receptor antagonist treatment serves as a valuable tool to increase neurogenesis in the adult hippocampus without undesirable collateral deleterious effects.     

Caspase-mediated death of newly formed neurons in the adult rat dentate gyrus following status epilepticus

A large proportion of cells that proliferate in the adult dentate gyrus under normal conditions or in response to brain insults exhibit only short-term survival. Here, we sought to determine which cell death pathways are involved in the degeneration of newly formed neurons in the rat dentate gyrus following 2 h of electrically induced status epilepticus. We investigated the role of three families of cysteine proteases, caspases, calpains, and cathepsins, which can all participate in apoptotic cell death. Status epilepticus increased the number of bromodeoxyuridine (BrdU)-positive proliferated cells in the subgranular zone of the dentate gyrus. At the time of maximum cell proliferation, immunohistochemical analyses revealed protein expression of active caspase-cleaved poly (ADP-ribose) polymerase (PARP) in approximately 66% of the BrdU-positive cells, while none of them expressed cathepsin B or the 150-kDa calpain-produced fodrin breakdown product. To evaluate the importance of cysteine proteases in regulating survival of the newly formed neurons, we administered intracerebroventricular infusions of a caspase inhibitor cocktail (zVAD-fmk, zDEVD-fmk and zLEHD-fmk) over a 2-week period, sufficient to allow for neuronal differentiation, starting 1 week after the epileptic insult. Increased numbers of cells double-labelled with BrdU and neuron-specific nuclear protein (NeuN) marker were detected in the subgranular zone and granule cell layer of the caspase inhibitor-treated rats. Our data indicate that caspase-mediated cell death pathways are active in progenitor cell progeny generated by status epilepticus and compromise survival during neuronal differentiation.     

Distribution of neuropeptidelike immunoreactivities in the guinea pig olfactory bulb

The distribution of neuropeptidelike immunoreactivities in the adult guinea pig olfactory bulb was studied immunohistochemically with antisera raised against neurotensin (NT), substance P (SP), methionine-enkephalin-Arg6-Gly7-Leu8 (ENK), somatostatin (SOM), neuropeptide Y (NPY), and cholecystokinin-8 (CCK). In the main olfactory bulb, NT-like immunoreactive (NT-IR) neurons were found among periglomerular cells. In addition, a few periglomerular cells showed ENK-like immunoreactivity. Granule cells displaying SP- or ENK-like immunoreactivities and short axon cells with SOM- or NPY-like immunoreactivities were observed in the deeper half of the granule cell layer. SOM-IR short axon cells were also seen in the external plexiform layer. Dense NT- or NPY-IR fibers were distributed in superficial lamina of the granule cell layer, and sparse SP- or CCK-IR fibers were found in the glomerular layer. In the accessory olfactory bulb, some mitral, periglomerular, and granule cells showed NT-like immunoreactivity. SP- or ENK-IR granule cells were also observed. These results are discussed in relation to laminar organization of the olfactory bulb. The most characteristic features of peptide distribution in guinea pigs, as compared with that of rats in previous studies, were the relative abundance of NT-IR structures and the lack of SP- and CCK-IR juxtaglomerular and tufted cells.     

Neuronal replacement in the injured olfactory bulb

The adult forebrain subventricular zone contains neural stem cells that produce neurons destined for the olfactory bulb, where interneuron populations turnover throughout life. Forebrain injuries can stimulate production of these cells, and re-direct migrating precursors from the olfactory system to areas of damage, where their region-appropriate differentiation and long-term functional integration remain a matter for debate. Paradoxically, little is known about the ability of these progenitors to replace olfactory neurons lost to injury. Their innate capacity to generate bulb neurons may give them an advantage in this regard, and using injections of N-methyl-d-aspartate to kill mature olfactory bulb neurons, combined with bromodeoxyuridine labeling to monitor the fate of adult-born cells, we investigated the potential for injury-induced neurogenesis in this system. Widespread degeneration of bulb neurons did not affect the rate of cell proliferation in the subventricular zone, or cause neuroblasts to divert from their normal migratory route. However migration was slowed by the injury, leading to the accumulation and differentiation of neuroblasts as NeuN+ cells in the rostral migratory stream within 2 weeks of their birth. Despite this, a subset of new neurons successfully invaded the damaged bulb tissue, where they expressed neuronal markers including NeuN, calretinin, GABA, and tyrosine hydroxylase, with some surviving here for as long as 6 months. To test for functional integration of cells born post-injury, we also performed smaller NMDA lesions in restricted portions of the bulb granule cell layer and observed adult-born NeuN+ cells in these areas within 5 weeks, and BrdU+ cells that expressed the immediate-early gene c-fos following odor stimulation. These data suggest that the normal neurogenic capacity of the adult subventricular zone can be adapted to replace subsets of olfactory neurons lost to injury.     

Increased cell proliferation and granule cell number in the dentate gyrus of protein repair-deficient mice

Recent studies have demonstrated that mice lacking protein L-isoaspartate (D-aspartate) O-methyltransferase (Pcmt1-/- mice) have alterations in the insulin-like growth factor-I (IGF-I) and insulin receptor pathways within the hippocampal formation as well as other brain regions. However, the cellular localization of these changes and whether the alterations might be associated with an increase in cell number within proliferative regions, such as the dentate gyrus, were unknown. In this study, stereological methods were used to demonstrate that these mice have an increased number of granule cells in the granule cell layer and hilus of the dentate gyrus. The higher number of granule cells was accompanied by a greater number of cells undergoing mitosis in the dentate gyrus, suggesting that an increase in neuronal cell proliferation occurs in this neurogenic zone of adult Pcmt1-/- mice. In support of this, increased doublecortin labeling of immature neurons was detected in the subgranular zone of the dentate gyrus. In addition, double immunofluorescence studies demonstrated that phosphorylated IGF-I/insulin receptors in the subgranular zone were localized on immature neurons, suggesting that the increased activation of one or both of these receptors in Pcmt1-/- mice could contribute to the growth and survival of these cells. We propose that deficits in the repair of isoaspartyl protein damage leads to alterations in metabolic and growth-receptor pathways, and that this model may be particularly relevant for studies of neurogenesis that is stimulated by cellular damage.