One of the shortcomings of publishing in scientific journals is severe space restriction imposed by the publishers. Many journals restrict the number of figures or charge prohibitive fees for each additional figure. In other cases the additional figures are allowed by are banned to "supplemental materials" which are not easily accessible. To enhance our publication list on this site we include additional materials from each study that add valuable information not found in the original papers. These materials come in the form of a figure, video clip or perhaps as an additional experiment or a commentary.
One of the shortcomings of publishing in scientific journals is severe space restriction imposed by the publishers. Many journals restrict the number of figures or charge prohibitive fees for each additional figure. In other cases the additional figures are allowed by are banned to "supplemental materials" which are not easily accessible.
To enhance our publication list on this site we include additional materials from each study that add valuable information not found in the original papers. These materials come in the form of a figure, video clip or perhaps as an additional experiment or a commentary.
Organotypic Slice Cultures for Studies of Postnatal Neurogenesis
|Title:||Organotypic Slice Cultures for Studies of Postnatal Neurogenesis|
|Authors:||Adam J. Mosa, Sabrina Wang, Yao Fang Tan, J. Martin Wojtowicz|
|Abstract:||Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique.
This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological
agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study
the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while
excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier.
For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.
Adult neurogenesis. From circuits to models
|Title:||Adult neurogenesis. From circuits to models|
|Publication:||Behav. Brain Res.227|
|Abstract:||Our understanding of the hippocampus as a memory-encoding device is greatly helped by our knowledge
of neuronal circuits and their plasticity. The trisynaptic hippocampal circuit carrying afferent input from the entorhinal cortex, controlled by a network of inhibitory interneurons and supplemented by modu- latory subcortical inputs forms a platform for multiple forms of synaptic plastic mechanisms. Long-term potentiation of synaptic transmission in its various forms is an outstanding example of hippocampal ability to adapt to past neuronal activity. Adult neurogenesis is a profound plastic mechanism incorpo- rating structural and functional changes that were previously thought to be present only in developing neural systems. These powerful forms of plasticity can mask experimental results by compensating for experimentally induced changes in the neurons or circuits. Circuit lesions have been one of the most common techniques in scienti?c investigations of the hippocampus. Although the effects of such lesions can be quite revealing and ground-breaking, in many cases the results are masked by compensatory mechanisms producing misleading results. This review will highlight such mechanisms and argue that the experimental results, in spite of their shortcomings, can be better understood when viewed in light of our knowledge of the neuronal circuitry, and with guidance by conceptual and computational models. Studies demonstrating a role of neurogenesis in pattern separation and memory interference are a good example of fruitful interaction between modeling and experimental approaches.
Adult hippocampal neurogenesis and memory interference.
|Title:||Adult hippocampal neurogenesis and memory interference.|
|Authors:||Winocur,G., Becker, S., Luu,P., Rosenzweig,S.,Wojtowicz,J.M.|
Rats, subjected to low-dose irradiation that suppressed hippocampal neurogenesis, or a sham treatment, were administered a visual discrimination task under conditions of high, or low interference. Half of the rats engaged in running activity and the other half did not. In the non-runners, there was no effect of irra- diation on learning, or remembering the discrimination response under low interference, but irradiation treatment increased their susceptibility to interference, resulting in loss of memory for the previously learned discrimination. Irradiated rats that engaged in running activity exhibited increased neuronal growth and protection from memory impairment. The results, which show that hippocampal cells gen- erated in adulthood play a role in differentiating between conflicting, context-dependent memories, provide further evidence of the importance of neurogenesis in hippocampus-sensitive memory tasks. The results are consistent with computational models of hippocampal function that specify a central role for neurogenesis in the modulation of interfering influences during learning and memory.
Comments: One problem with publishing is that lots of data never sees the publishing light due to space restrictions imposed by the journals. One such piece of data is included here. Although the differences between the groups are subtle, it is clear that non-irradiated runners (NI/R) light-blue and dark-blue (corresponding to low and high interference, respectively) were the only two groups able to improve during the 5 days of testing. All other groups performed about equally across all days. Such differences cannot be seen in the bar graph.
Depletion of new neurons by image guided irradiation
|Title:||Depletion of new neurons by image guided irradiation|
|Authors:||Tan, Y-F., Rosenzweig, S., Jaffray, D., Wojtowicz, J.M.|
|Publication:||Front. Neurosci. 5|
Ionizing radiation continues to be a relevant tool in both imaging and the treatment of cancer. Experimental uses of focal irradiation have recently been expanded to studies of new neurons in the adult brain. Such studies have shown cognitive deficits following radiation treatment and raised caution as to possible unintentional effects that may occur in humans. Conflicting outcomes of the effects of irradiation on adult neurogenesis suggest that the effects are either transient or permanent. In this study, we used an irradiation apparatus employed in the treatment of human tumors to assess radiation effects on rat neurogenesis. For subjects we used adult male rats (Sprague-Dawley) under anesthesia. The irradiation beam was directed at the hippocampus, a center for learning and memory, and the site of neurogenic activity in adult brain. The irradiation was applied at a dose-rate 0.6 Gy/min for total single-fraction, doses ranging from 0.5 to 10.0 Gy. The animals were returned to home cages and recovered with no sign of any side effects. The neurogenesis was measured either 1 week or 6 weeks after the irradiation. At 1 week, the number of neuronal progenitors was reduced in a dose-dependent manner with the 50% reduction at 0.78 Gy. The doseýresponse curve was well fitted by a double exponential suggesting two processes. Examination of the tissue with quantitative immunohistochemistry revealed a dominant low-dose effect on neuronal progenitors resulting in 80% suppression of neurogenesis. This effect was partially reversible, possibly due to compensatory proliferation of the remaining precursors. At higher doses (>5 Gy) there was additional, nearly complete block of neurogenesis without compensatory proliferation. We conclude that notwithstanding the usefulness of irradiation for experimental purposes, the exposure of human subjects to doses often used in radiotherapy treatment could be damaging and cause cognitive impairments.
Supplementary data for Tan et al., 2011
This figure never got published but it shows an informative set of data demonstrating that targeted irradiation of the hippocampus spares Rostral Migratory Stream (RMS) and olfactory bulb (OB).
- Image (A) shows a saggital section with the subventricular zone (SVZ) on the left and OB on the right. RMS carries neural progenitors from the SVZ to OB where they differentiate into olfactory neurons.
- Images (B-D) are taken in coronal planes as shown in (A) to illustrate the presence of Doublecortin (DCX)-positive progenitors in both control and irradiated (3 Gy) brains. The irradiation did not deplete the progenitors in the SVZ-OB pathway while the hippocampal neurogenesis was almost completely eliminated at this dose (see the published article).Thus, the targeted irradiaiton is both effective and accurate.
Analyzing dendritic growth in a population of immature neurons in the adult dentate gyrus using laminar quantification of disjointed dendrites
|Title:||Analyzing dendritic growth in a population of immature neurons in the adult dentate gyrus using laminar quantification of disjointed dendrites|
|Authors:||Rosenzweig, S. and Wojtowicz, J.M.|
In the dentate gyrus (DG) of the hippocampus, new granule neurons are continuously produced throughout adult life. A prerequisite for the successful synaptic integration of these neurons is the sprouting and extension of dendrites into the molecular layer of the DG. Thus, studies aimed at investigating the developmental stages of adult neurogenesis often use dendritic growth as an important indicator of neuronal health and maturity. Based on the known topography of the DG, characterized by distinct laminar arrangement of granule neurons and their extensions, we have developed a new method for analysis of dendritic growth in immature adult-born granule neurons. The method is comprised of laminar quantification of cell bodies, primary, secondary and tertiary dendrites separately and independently from each other. In contrast to most existing methods, laminar quantification of dendrites does not require the use of exogenous markers and does not involve arbitrary selection of individual neurons. The new method relies on immunohistochemical detection of endogenous markers such as doublecortin to perform a comprehensive analysis of a sub-population of immature neurons. Disjointed, ýorphaný dendrites that often appear in the thin histological sections are taken into account. Using several experimental groups of rats and mice, we demonstrate here the suitable techniques for quantifying neurons and dendrites, and explain how the ratios between the quantified values can be used in a comparative analysis to indicate variations in dendritic growth and complexity.
The role of adult hippocampal neurogenesis in reducing interference
|Title:||The role of adult hippocampal neurogenesis in reducing interference|
|Authors:||Luu, P., Sill, O., Gao, L. Becker, S., Wojtowicz,|
Adult neurogenesis in hippocampal dentate gyrus is thought to play an important role in learning and memory. However, the exact contribution of the newly-generated neurons to the hippocampal function remains controversial. Emerging evidence points to specific actions of new neurons in contextual learning, memory interference and pattern separation but no study has specifically addressed a possible interplay between these three factors. In the present study we show that cranial irradiation, resulting in selective reduction of neurogenesis within dentate gyrus, produces significant impairment in the olfactory discrimination task, which involves learning two interfering odor lists occurring several days apart. The impairment was significant only when multimodal contextual cues were used to accompany memory retrieval in the second list. Other aspects of learning such as the ability to learn a single odor list or learning on a spatial reversal task were not affected. This is the first study demonstrating selective contributions by new neurons to memories in conditions of context-dependent proactive interference. These findings are consistent with models invoking pattern separation and context as principal mechanisms in defeating memory interference.
Comments: One of the more difficult aspects of reading a behavioural paper is to visualize a learning task being used in the study. To help a reader along, we attach a short video clip showing how a rat performs an olfactory discrimination task. The animal sniffs at one odour in the pair, decides it is not the right one, and proceeds to the other. After digging out the pellet from under the scented bedding material he waits for another trial. The task was developed and performed in the Smith’s laboratory at Cornell University.
Although the rat is not wearing a maple leaf, the animals for this study came across the border from Toronto, Canada.
Link to video here...
Dynamics of neurogenesis in the dentate gyrus of adult rats
|Title:||Dynamics of neurogenesis in the dentate gyrus of adult rats|
|Authors:||McDonald, H.Y., Wojtowicz, J.M.|
|Publication:||Neurosci. Lett. 385|
Hippocampal neurogenesis declines steadily over the first year of life in the rodent, but the process persists into senescence despite a dramatic drop in the number of neurons it produces. At this point though, the survival and development patterns exhibited by new granule cells in the aging brain remain unclear in relation to patterns observed in the younger brain. The present study was carried out in order to obtain a direct quantitative comparison of hippocampal neurogenesis in juvenile and middle-aged rats with a high degree of temporal resolution, and to compare the survival and differentiation of the new cells over time. 38-day and 12-month old, male Sprague-Dawley rats were injected with 5-bromo-2ý-deoxyuridine (BrdU) in order to label cells dividing in the dentate gyrus over a 24-hr. period, and immunohistochemical labeling was performed in order to record cell production and survival at eight different time points over the following two-month period. Using a marker of neuronally committed precursors and immature neurons (doublecortin; DCX), as well as a marker of mature neurons (calbindin d-28K; CaBP), the extent and timeline of neuronal differentiation, maturation, and migration of the new cells were also characterized. Results indicated that 12-month old rats experienced a nearly 94% reduction in neurogenesis relative to juveniles, due almost entirely to a 92% drop in cell production. A largely preserved course of development and migration in the remaining newborn cells suggests treatments that enhance cell proliferation could be crucial in reversing the age-related decline in neurogenesis.
BrdU assay for neurogenesis in rodents
|Title:||BrdU assay for neurogenesis in rodents|
|Authors:||Wojtowicz,J.M. and Kee,N.|
Neurogenesis within the adult central nervous system is demonstrated using exogenous cell tracer, 5ý-bromo-2ý-deoxyuridine (BrdU), in combination with endogenous neuronal markers. Specific primary antibodies raised against these markers are widely available and their visualization is possible with the use of the fluorescently tagged secondary antibodies. BrdU is a thymidine analogue that incorporates into dividing cells during DNA synthesis. Once incorporated into the new DNA, BrdU will remain in place and be passed down to daughter cells upon division. Typically, BrdU is injected intraperitoneally. Different survival times required by the desired experimental time line will yield data on specific phases of neurogenesis: proliferation, differentiation and maturation. One of the drawbacks of using BrdU is the dependence on stressful injection procedure and uncertain penetration of the targeted cells with uniform concentration of the compound. Thus, for experiments requiring measurements of cell proliferation, Ki-67 can be used as an acceptable alternative. The protocol takes three to five days allowing for sectioning and staining.
Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus
|Title:||Electrophysiological correlates of neural plasticity compensating for ischemia-induced damage in the hippocampus|
|Authors:||Sabrina Wang, Nohjin Kee, Edward Preston, J. Martin Wojtowicz|
|Abstract:||Injury to the brain often results in loss of synapses or cell death in the damaged area. Subsequent to the injury, the areas that are not directly affected often exhibit enhanced neuronal plasticity. Although there are many reports of morphological changes resulting from such plasticity, their functional consequences are poorly understood. In this study we examined electrophysiological changes associated with ischemia-induced neurogenesis in the hippocampus, a brain region that is particularly vulnerable but also exceptionally plastic. Transient global ischemia was induced in Sprague-Dawley rats by occlusion of both carotid arteries and a reduction in blood pressure for 12 min. The procedure resulted in delayed cell death in the CA1 field of the hippocampus while the dentate gyrus (DG) was spared. To assess neurogenesis and synaptic changes in parallel we used both hemispheres from each animal. One side was used for immunohistochemistry and the other for in vitro electrophysiological experiments in brain slices. Synaptic field responses and synaptic plasticity (LTP) in perforant path within the DG were reduced by 50% at 10 days after the ischemic injury but recovered at 35 days. Synaptic responses in non-neurogenic CA1 were abolished in parallel with cell death and did not recover. Gamma irradiation applied focally to the head selectively prevented neurogenesis and the synaptic recovery in the DG. These experiments reveal electrophysiological changes associated with reactive neural plasticity in the hippocampus.|
The effects of running and of inhibiting adult neurogenesis on learning and memory in rats
|Title:||The effects of running and of inhibiting adult neurogenesis on learning and memory in rats|
|Authors:||Wojtowicz J.M., Askew, M.L., Winocur G.,|
|Publication:||European Journal of Neuroscience, 27|
The presence of ongoing adult neurogenesis within the highly plastic hippocampal circuitry poses questions as to the relevance of new neurons to learning and memory. Correlational and causal evidence suggests that some, but not all hippocampal, tasks involve the new neurons. The evidence with regard to spatial learning in the water maze, one of the most commonly used hippocampal tasks, is contradictory. We have addressed this issue using this task and another standard hippocampal task, contextual fear conditioning.. Additionally, we considered control caged rats expressing normal levels of neurogenesis in comparison to rats with either enhanced (running) or reduced (irradiation) levels of neurogenesis. The results indicate that reduced neurogenesis had little effect on spatial learning but severely impaired contextual fear conditioning. It was suggested that compensatory mechanisms within the hippocampus may have contributed selectively to sparing of spatial function. Performance on both tasks was unrelated to running behaviour. The results improve our understanding of the functional role of adult neurogenesis in behaving animals.
Homeostatic regulation of adult hippocampal neurogenesis in aging rats
|Title:||Homeostatic regulation of adult hippocampal neurogenesis in aging rats|
|Authors:||Merkley, C., Jian, C., Mosa, A., Tan, Y. F., Wojtowicz, J. M.|
|Publication:||Front. Neurosci. V8|
|Abstract:||Adult neurogenesis is highly responsive to environmental and physiological factors. The majority of studies todate have examined short-term consequences of enhancing or blocking neurogenesis but long-term changes remain less well understood. Current evidence for age-relatedde clines in neurogenesis warrant further investigation in to these long-term changes. In this report we address the hypothesis that early life experience, such as a period of voluntary running in juvenile rats, can alter properties of adult neurogenesis for the remainder of the animal’s life. The results indicate that the number of proliferating and differentiating neuronal precursors is not altered in runners beyond the initial weeks post-running, suggesting homeostatic regulation of these processes. However, the rate of neuronal maturation and survival during a 4week period after cell division was enhanced up to 11months of age (the end of the study period). This study is the first to show that a transient period of physical activity at a young age promotes changes in neurogenesis that persist over the long-term, which is important for our understanding of the modulation of neurogenesis by exercise with age. Functional integration of adult-born neurons within the hippocampus that resist homeostatic regulation with aging, rather than the absolute number of adult-born neurons, maybe an essential feature of adult neurogenesis that promotes the maintenance of neural plasticity in old age.