This work is funded by NSERC grants to BK and RG. The authors would also like to thank Kehe Xie and Russell Hosain for their Golgi-Cox staining expertise.

All experiments were carried out in accordance with the Canadian Council of Animal Care and approved by the University of Lethbridge, Animal Care Committee.
1. Breeding and Tactile Stimulation
<ol>
	<li>Order pregnant rats from common animal suppliers or breed pups in-house using standard laboratory breeding procedures.</li>
	<li>House all animals in a temperature controlled breeding room (21 &deg;C), maintained on a 12:12 hr light:dark cycle and provide access to food and water ad libitum.</li>
	<li>When pups are born, house female rats individually with their offspring.</li>
	<li>To avoid additional stress related to handling immediately after birth, commence tactile stimulation of pups at P3.</li>
	<li>Tactile stimulation should be carried out three times per day (9:00 AM, 1:00 PM, and 4:00 PM) from P3-P21 and pups should be weaned from their mothers at P21.</li>
	<li>When using a cross litter design, half of the pups from each litter will undergo tactile stimulation with the other half serving as controls. Randomly assign equal numbers of male and female pups to each group. To differentiate pups undergoing tactile stimulation and controls, mark one group of pups with a permanent marker on their hind legs and tail. Marking has to be re-applied every other day.</li>
</ol>
Tactile Stimulation
<ol start="7">
	<li>Remove dams from their home cage and place them in a temporary cage with food and water. Keep the dam and temporary cage in the breeding/housing room.</li>
	<li>Weigh rat pups as a group prior to the morning tactile stimulation session (9:00 AM). For each session, transport rat pups their home cages to a separate testing room for tactile stimulation. Place the home cage on a heating pad that is set to 24 &deg;C.</li>
	<li>Use a stiff board to divide the home cage into two halves. Place rat pups in the tactile stimulation group one half and control pups in the other half. Set a timer to 15 min.</li>
	<li>Using a soft feather-like duster, brush all pups in the tactile stimulation group at the same time for 15 consecutive min. The young rat pups (~P3-P12) ususally huddle together and appear to enter a deep sleep cycle making it very easy to stimulate all pups at once. As the pups age, they become more active, with some pups walking and investigating during the session. The experimenter should continuously move wandering pups into the group to ensure each pup receives equal stimulation.</li>
	<li>Once the 15 min session is complete, transport the pups back to the breeding room and return the mother to the cage.</li>
	<li>Repeat this process three times a day for the 19 days of tactile stimulation. After the final tactile stimulation session at P21, the pups should be weaned from their mothers. Pups should then be housed in cages with 5 or 6 other weaned animals of the same-sex.</li>
	<li>If rats are undergoing no additional testing, they should be left undisturbed, on the normal light:dark cycle with access to food and water ad libitm (aside from regular cage cleaning and handling) until they reach approximately 100 days of age. If undergoing other experimental manipulations, animals should be treated or tested in accordance with those laboratory protocols.</li>
</ol>
2. Sacrifice and Golgi-Cox Staining
<ol>
	<li>At approximately P100, administer an overdose of sodium pentobarbital via I.P. injection to the rats and perfuse intracardially with approximately 100 ml of 0.9% saline.</li>
	<li>Extract the brains when the perfusion is complete. Remove the brains from the skull, cutting the optic nerve underneath with effort to keep the cerebellum intact; researchers may choose to keep or remove the olfactory bulbs. Place extracted brains in an opaque Nalgene bottle with 20 ml of Golgi-Cox solution 14.</li>
	<li>Keep the brains in Golgi-Cox solution for 14 days. After 14 days, replace the Golgi-Cox solution with a 30% sucrose solution. Keep the brains in the sucrose solution for 2-5 days before sectioning.</li>
</ol>
Note: If brains cannot be sectioned within 14 days of transfer to the sucrose solution, the researcher needs to replace the sucrose with a new stock of 30% sucrose. The researcher may replace the sucrose every two weeks for 4 months with no adverse effects on staining.
<ol start="4">
	<li>In order to section the brain, the brain should be blotted dry and fixed to the sectioning stage with cyanocacrylic glue. To prevent tearing or uneven sectioning, the researcher must be cautious and ensure that the entire brain is firmly secured to the stage.</li>
	<li>The vibratome reservoir must be filled with 6% sucrose solution to a level that covers the sectioning blade. Set the vibratome parameters to a speed and amplitude to 5, (the midpoint on both scales). Slice the brain into 200 &mu;m sections and place the sections on a 2% gelatinized microscope slide. Be sure to keep the sections wet during the course of sectioning.</li>
	<li>When all of the sections of interest have been collected, press the sections onto the slides by applying pressure to the slides with moistened bibulous paper. Store the slides in a slide rack in the humidity chamber until they are ready to be stained. They should remain in the humidity chamber for at least 12 hr, but not longer than 4 days. The slides need to be moist and should not be permitted to dry out.</li>
	<li>Prepare the staining regiment before commencing the staining process. Label twelve glass staining dishes (10.7 x 8.5 x 6.8 cm) and process in the following manner:
	<ol>
		<li>Distilled Water - 1 min</li>
		<li>Ammonium Hydroxide - 30 min (in the dark)</li>
		<li>Distilled Water - 1 min</li>
		<li>Kodak Fix for Film - 30 min (in the dark)</li>
		<li>Distilled Water - 1 min</li>
		<li>50% Alcohol - 1 min</li>
		<li>70% Alcohol - 1 min</li>
		<li>95% Alcohol - 1 min</li>
		<li>100% Alcohol - 5 min</li>
		<li>100% Alcohol - 5 min</li>
		<li>Solution (1/3 Chloroform, 1/3 HemoDe, 1/3 100% Alcohol) - 15 min</li>
		<li>HemoDe - 15 min</li>
	</ol>
	</li>
</ol>
* Xylene can be used in replace of HemoDe. In order to ensure consistent staining quality, fresh solutions should be used for each slide rack processed.
<ol start="8">
	<li>Following the last 15 min emersion in HemoDe, coverslip the slides with Permount.</li>
	<li>Allow the slides to air dry before examining with a microscope.</li>
</ol>

<ol>
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The authors declare that we have no competing financial interests.

Owing to the overt plasticity of the developing brain, it is important that experimental procedures involving early experiences are thoroughly reviewed and attempts are made to control for all intervening variables. For this reason, a cross litter design is employed during the tactile stimulation procedure to ensure that pups are receiving similar experiences in all other domains of development. In addition, it is also important that multiple pups from multiple litters are selected for analysis to avoid the possibility that effects result from a bias in a single pup or litter.
Tactile stimulation is believed to mimic the naturally occurring maternal behavior, licking and grooming, which is thought to be beneficial to offspring development. Although prior research has emphasized the first week of life (P0-P7) to be a critical period for licking and grooming 16, the sheer magnitude of change identified following 18 days of tactile stimulation may imply that although sensitive times exist, longer exposure is superior. It is also important to note that pups receiving tactile stimulation in this experimental paradigm also experience licking and grooming by their mothers, hence the tactile stimulation is in addition to normal stimulation administered by the mother. Finally, one must also be cognizant of region-dependent changes. Although tactile stimulation during development increased dendritic complexity in the prefrontal cortex, this does not guarantee that structural changes of this nature will be evident in all brain regions. It is possible that neuronal morphology in other brain regions such as the parietal cortex, would respond in a strikingly different manner to the same experience. However, because the tactile stimulation procedure is easily administered and poses no risk to the offspring or dams, it has the potential to serve as a valuable tool for many research studies aimed at improving developmental outcomes.
With respect to Golgi-Cox staining of brain tissue, the critical steps for successful visualization of neuronal cells are as follows: 1) there must be adequate perfusion of the brain with saline solution. Improper or insufficient perfusion of the brain tissue results in blood vessel artifacts that make it difficult to actually visualize neuronal cells through the maze of blood vessels, while also complicating the ability to photograph stained neurons. 2) Perfused brains should be stored in Golgi-Cox solution and sucrose solution in the dark. Storing the brain tissue in the dark reduces the background staining of the tissue, again increasing the chance of successfully visualizing quality neuronal cells. 3) Brain tissue is stored in sucrose solution following the 14 day storage in Golgi-Cox solution. When the tissue is immersed in the 30% sucrose solution for 2-5 days, brains are more pliable which prevents shattering and tearing of sections when cutting. It is important to prevent brain tissue from remaining in the sucrose solution for increased time periods (unless the sucrose solution is continuously replaced with fresh solution) because prolonged storage in sucrose reduces the staining quality. 4) Finally, once slides have been stained and cover slipped, they should be given adequate time to dry before visualization with a microscope. If slides are not permitted to adequately air dry, the tissue may darken, reducing successful visualization of cortical neurons.
Stable changes in psychological functioning and behavioral responses that occur in response to experiences are believed to be facilitated by reorganization of neuronal morphology and synaptic connectivity 17. As these structural changes provide a measurable resource for experience-dependent plasticity, the use of a reliable staining procedure is important. This vibratome based Golgi-Cox staining procedure provides reliable staining of fine branches and dendritic spines that may not be evident with other protocols such as celloidin-embedding. The distinctiveness of the Golgi-Cox procedure stems from its capacity to only stain a small fraction of neuronal elements (1-10%), which permits tracing of single neurons for long distances. Despite the fact that only a small fraction of neurons are impregnated with the stain, cells that are rendered visible, maintain all features including cell body, dendrites, dendritic spines and axons. Moreover, when the staining procedure is carried out correctly, the stained cells stand out clearly and distinctly against a transparent background because other cortical structures remain unstained and transparent. Owing to the realization that persistent changes in outward functioning must be related to the plasticity of the nervous system, i.e. the capability of neurons to modify their structure and connectivity, the Golgi-Cox staining procedure provides researchers with a reliable technique to visualize and quantify this plasticity.

Although there are many reported and utilized techniques for the examination of negative early experiences on brain maturation, such as perinatal stress 1,2, sensory deprivation 3, and drug toxicity 4, there are very few methodologies employed to examine the effects of positive experiences in this time period. Aside from environmental enrichment, tactile stimulation is one of a few brain enhancing treatments with demonstrated effects 5. Tactile stimulation is a method of sensory stimulation to the skin that mimics the maternal rat behavior, licking and grooming. Its general acceptance as a positive manipulation arises from studies indicating that tactile stimulation improved maturation of premature infants and newborn rats 6. In addition, research in the Meaney laboratory 7 has demonstrated that higher levels of maternal licking and grooming are linked to positive outcomes in offspring. Owing to these positive influences, tactile stimulation rapidly evolved into a remedial strategy aimed at reducing anxiety 8, improving outcomes associated with brain injury 9-11, and attenuating drug sensitization 12. As such, tactile stimulation is a valuable technique for the promotion of positive early experiences, with a proven ability to dramatically reorganize neuronal morphology and synaptic connectivity of the developing brain.
In order to examine and quantify changes in neuronal morphology, it is necessary to visualize intact neurons. The Golgi-Cox staining procedure is a modification of Camillo Golgi's technique published in the late 1800s that provides discrete staining of a small number of complete neurons 13. Although the procedure appears to stain neurons at random and reproducibility is commonly cited as major flaw, the small impregnation rate permits visualization of the entire neuronal element, including cell bodies, dendrites, dendritic spines, and axons. Similarly, the time required to impregnate a given brain with Golgi solution has also been cited as a downfall. However, given that once staining is complete tissue can be preserved indefinitely, and the time between perfusion of the brain and visualization of neurons with a microscope can be completed in less than 21 days, the time period is not unreasonable. In addition, with minor modifications to the protocol, Golgi-Cox staining can be effectively used to impregnate brains of rodents from all age ranges. As changes at the structural level have been linked to persistent modifications to behavioral and psychological functioning, the Golgi-Cox staining technique provides researchers with a valuable tool to measure neuroplasticity.

<table><tbody><tr><td>Potassium Dichromate</td><td>Fisher</td><td>P188</td><td></td></tr><tr><td>Mercuric Chloride</td><td>Fisher</td><td>M1561</td><td></td></tr><tr><td>Potassium Chromate</td><td>Fisher</td><td>P220</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher</td><td>A669-500</td><td></td></tr><tr><td>Kodak Rapid Fix</td><td>Vistek</td><td>Kodak 146 4016</td><td></td></tr><tr><td>EtOH-95 &amp;amp; Anhydrous</td><td>Commercial Alcohols</td><td>No Cat Numbers</td><td>All other Et-OH are dilutions</td></tr><tr><td>Swiffers- Soft Feather-Like dusters</td><td>Safeway</td><td></td><td>Can be found at most grocery stores</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S-9378</td><td></td></tr><tr><td>HemoDe</td><td>Electron Microscopy Sciences</td><td>23410</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>Sp15</td><td></td></tr><tr><td>Slides</td><td>VWR</td><td>160004-365</td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>062011-9</td><td></td></tr></tbody></table>

visualizing the effects of a positive early experience, tactile stimulation, on dendritic morphology and synaptic connectivity with golgi-cox staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

When this staining procedure has been followed appropriately, consistent and uniform staining of dendrites and spines is generated. See Figure 1 for a representation of Golgi-Cox neuronal staining. This procedure produces staining that is comparable to the newly developed in vitro methods and may permit visualization of dendritic fields that are uninterruptable following tissue embedding. The vibratome based technique for Golgi-Cox staining has been found to produce more widespread staining of terminal branches and pyramidal neurons, than staining with either celloidin-embedded and cryostat-sectioned tissue, 15. Furthermore, because this method of staining impregnates the entire brain, all sections can be analyzed either immediately or in the future. As the location of morphological change is it is not always obvious when generating an original hypothesis, this process allows for the exploration of additional brain regions and prevents disposal of tissue that may be useful in the future.
This staining method can be used for reliable staining of any aged rat brains (P0-old age). However, when staining young brains (&lt;1 gram), the brain should only remain in the Golgi-Cox solution for 6 days, rather than the 14 days recommended for adult brains; all other procedures are held constant. The main difficulty that may be encountered during this Golgi-Cox staining process is the inability to produce high-quality staining of central brain regions, including the thalamus. As the entire brain is impregnated at the same time, central regions may not receive ideal concentrations of stain. Despite this finding, the Golgi-Cox technique produces exceptional staining of subcortical regions such as the hippocampus and striatum. A final limitation of the procedure arises from incomplete removal of blood from the brain during the perfusion process. Blood may produce artifacts in the brain tissue that makes it difficult to photograph and trace the neurons.
Golgi-Cox staining techniques provide a reliable measure of dendritic morphology and neuroanatomy that is useful for an extensive range of studies designed to examine changes at the neuronal level. When examining neuronal structure in the prefrontal cortex following tactile stimulation in the early-postnatal period, Golgi-Cox staining revealed dramatic increases in dendritic complexity. Anatomical changes related to early tactile stimulation can be demonstrated visually by comparing spine density of pyramidal neurons from a control rat to pyramidal neurons from a rat that underwent tactile stimulation (see Figure 1). In addition, parameters such as dendritic arborization, dendritic length, and spine density can be calculated and statistically analyzed to generate continuous data that can be compared across animals. To generate reliable results, analysis should be carried out on a minimum of three animals per treatment group and approximately five neurons per hemisphere should be randomly selected for each brain region under analysis. Figure 2 demonstrates the dendritic complexity (spines/&mu;m x &mu;m of dendrite) of animals that received tactile stimulation and animals that did not. In both the apical and basilar fields of the neurons analyzed from the mPFC (Cg3), tactile stimulation resulted in proliferation of neuronal parameters.
<img alt="Figure 1" src="/files/ftp_upload/50694/50694fig1.jpg" /> 
Figure 1. A. Representative photograph of a Golgi-Cox stained pyramidal neuron from layer III of area Cg3 in a rat that received tactile stimulation during development. B. Representative magnified (1,000X) photograph of dendritic spines on the terminal dendrites in the basilar field of Cg3; the lighter image on the left is for a rat in the control group and the darker image on the right is from a rat who received tactile stimulation.
<img alt="Figure 2" src="/files/ftp_upload/50694/50694fig2.jpg" /> 
Figure 2. Illustrative representation of the average apical and basilar dendritic complexity (spines/&mu;m x &mu;m) for neurons in the Cg3 area from adult rats that either received tactile stimulation during development or did not (*p &lt;0.01).

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Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

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18.0K Views.  University of Lethbridge.  Dit artikel beschrijft de procedures voor de tactiele stimulatie van pups rat en de daaropvolgende Golgi-Cox kleuring van neuronale morfologie. Tactiele stimulatie is een positieve ervaring die wordt toegediend in de perinatale periode door strelen pups met een huishouden stofdoek. Golgi-cox kleuring is een betrouwbare procedure waardoor de visualisatie van hele neuronen. 

Video: Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Developmental Biology

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

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