Name: visualizing the effects of a positive early experience, tactile stimulation, on dendritic morphology and synaptic connectivity with golgi-cox staining
Uploaded: 2013-09-25T09:00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining at JoVE

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>
	<li>Mychasiuk, R., Ilnystkyy, S., Kovalchuk, O., Kolb, B., Gibb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensity+matters:+Brain,+behaviour,+and+the+epigenome+of+prenatally+stressed+rats.">Intensity matters: Brain, behaviour, and the epigenome of prenatally stressed rats.</a> Neuroscience. 180, 105-110 (2011).</li><li>Muhammad, A., Carroll, C., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+during+development+alters+dendritic+morphology+in+the+nucleus+accumbens+and+prefrontal+cortex.">Stress during development alters dendritic morphology in the nucleus accumbens and prefrontal cortex.</a> Neuroscience. 216, 103-109 (2012).</li><li>Wiesel, T., Hubel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+visual+deprivation+on+morphology+and+physiology+of+cells+in+the+cat's+lateral+geniculate+body.">Effects of visual deprivation on morphology and physiology of cells in the cat's lateral geniculate body.</a> Journal of Neurophysiology. 26 (978), 6 (1963).</li><li>Dwyer, J., McQuown, S., Leslie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+effects+of+nicotine+on+the+developing+brain.">The dynamic effects of nicotine on the developing brain.</a> Pharmacology &amp; Therapeutics. 122, 125-139 (2009).</li><li>Richards, S., Mychasiuk, R., Kolb, B., Gibb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+stimulation+during+development+alters+behaviour+and+neuroanatomical+organization+of+normal+rats.">Tactile stimulation during development alters behaviour and neuroanatomical organization of normal rats.</a> Behavioural Brain Research. 231, 86-91 (2012).</li><li>Schanberg, S., Field, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+deprivation+stress+and+supplemental+stimulation+in+the+rat+pup+and+preterm+neonate.">Sensory deprivation stress and supplemental stimulation in the rat pup and preterm neonate.</a> Child Development. 58 (6), 1431-1447 (1987).</li><li>Caldji, C., Tannenbaum, J., Sharma, S., Francis, D., Plotsky, P., Meaney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+care+during+infancy+regulates+the+development+of+neural+systems+mediating+the+expression+of+fearfulness+in+the+rat.">Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat.</a> Proceedings of the National Academy of Sciences. 95 (9), 5335-5340 (1998).</li><li>Imanaka, A., Morinobu, S., Toki, S., Yamamoto, S., Matsuki, A., Kozuru, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+tactile+stimulation+reverses+the+effect+of+neonatal+isolation+on+open-field+and+anxiety-like+behavior,+and+pain+sensitivity+in+male+and+female+adult+Sprague-Dawley+rats.">Neonatal tactile stimulation reverses the effect of neonatal isolation on open-field and anxiety-like behavior, and pain sensitivity in male and female adult Sprague-Dawley rats.</a> Behavioural Brain Research. 186, 91-97 (2008).</li><li>Gibb, R., Gonzalez, C., Wegenast, W., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+stimulation+promotes+motor+recovery+following+cortical+injury+in+adult+rats.">Tactile stimulation promotes motor recovery following cortical injury in adult rats.</a> Behavioural Brain Research. 214 (1), 102-107 (2010).</li><li>Rodrigues, A., Artneni, N., Abel, C., Zylbersztejn, D., Chazan, R., Viola, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+stimulation+and+maternal+separation+prevent+hippocampal+damage+in+rats+submitted+to+neonatal+hypoxia-ischemia.">Tactile stimulation and maternal separation prevent hippocampal damage in rats submitted to neonatal hypoxia-ischemia.</a> Brain Research. 1002, 94-99 (2004).</li><li>Kolb, B., Gibb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+stimulation+after+frontal+or+parietal+cortical+injury+in+infant+rats+facilitates+functional+recovery+and+produces+synaptic+changes+in+adjacent+cortex.">Tactile stimulation after frontal or parietal cortical injury in infant rats facilitates functional recovery and produces synaptic changes in adjacent cortex.</a> Behavioural Brain Research. 214, 115-120 (2010).</li><li>Muhammad, A., Hossain, S., Pellis, S., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+stimulation+during+development+attenuates+amphetamine+sensitization+and+structurally+reorganizes+prefrontal+cortex+and+striatum+in+a+sex-dependent+manner.">Tactile stimulation during development attenuates amphetamine sensitization and structurally reorganizes prefrontal cortex and striatum in a sex-dependent manner.</a> Behavioral Neuroscience. 125 (2), 161-174 (2011).</li><li>Golgi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulla+struttura+della+sostanza+grigia+dell+cervello.">Sulla struttura della sostanza grigia dell cervello.</a> Gaz Med Lomb. 33, 244-246 .</li><li>Glaser, E. M., van der Loos, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thick+brain+sections+by+obverse-reverse+computer+microscopy:+Application+of+a+new,+high+clarity+Golgi-Nissl+stain.">Analysis of thick brain sections by obverse-reverse computer microscopy: Application of a new, high clarity Golgi-Nissl stain.</a> Journal of Neuroscience Methods. 4, 117-125 (1981).</li><li>Gibb, R., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+vibratome+sectioning+of+Golgi-Cox+stained+whole+rat+brain.">A method for vibratome sectioning of Golgi-Cox stained whole rat brain.</a> Journal of Neuroscience Methods. 79, 1-4 (1998).</li><li>Meaney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+care,+gene+expression,+and+the+transmission+of+individual+differences+in+stress+reactivity+across+generations.">Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations.</a> Annual Review of Neuroscience. 24, 1161-1192 (2001).</li><li>Robinson, T. E., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+associated+with+exposure+to+drugs+of+abuse.">Structural plasticity associated with exposure to drugs of abuse.</a> Neuropharmacology. 47, 33-46 (2004).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Potassium Dichromate</td>
			<td>Fisher</td>
			<td>P188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mercuric Chloride</td>
			<td>Fisher</td>
			<td>M1561</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium Chromate</td>
			<td>Fisher</td>
			<td>P220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Fisher</td>
			<td>A669-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kodak Rapid Fix</td>
			<td>Vistek</td>
			<td>Kodak 146 4016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EtOH-95 &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>&nbsp;</td>
			<td>Can be found at most grocery stores</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S-9378</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HemoDe</td>
			<td>Electron Microscopy Sciences</td>
			<td>23410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>Sp15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>160004-365</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>062011-9</td>
			<td>&nbsp;</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).

Watch this Scientific Journal Video about Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining at JoVE

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 ...

visualizing-effects-positive-early-experience-tactile-stimulation-on

Research

JoVE Journal

Neuroscience

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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 ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

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 ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...