Name: stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro
Uploaded: 2019-08-28T13:00:00
Description: Watch this Scientific Journal Video about Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro at JoVE.com

SPS was supported by the University of Sydney postgraduate research scholarship.

All experimental protocols described were approved by the University of Sydney Animal Ethics Committee (approved protocol number: 2018/1308).
1. Animals
NOTE: Mice were obtained from the Australian Rodent Centre (ARC; Perth, Australia) and held at the Medical Foundation Building Animal Facility at the University of Sydney.
<ol>
	<li>Maintain the mice on a normal 12 h light/dark cycle with environmental enrichment.</li>
	<li>Use male and female C57...

<ol>
	<li>Amiridis, I. G., Hatzitaki, V., Arabatzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-induced+modifications+of+static+postural+control+in+humans.">Age-induced modifications of static postural control in humans.</a> Neuroscience Letters. 350 (3), 137-140 (2003).</li><li>Iwasaki, S., Yamasoba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dizziness+and+imbalance+in+the+elderly:+age-related+decline+in+the+vestibular+system.">Dizziness and imbalance in the elderly: age-related decline in the vestibular system.</a> Aging and disease. 6 (1), (2015).</li><li>Fujimoto, C., 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=Noisy+galvanic+vestibular+stimulation+induces+a+sustained+improvement+in+body+balance+in+elderly+adults.">Noisy galvanic vestibular stimulation induces a sustained improvement in body balance in elderly adults.</a> Scientific Reports. 6, 37575 (2016).</li><li>Breen, P. P., 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=Peripheral+tactile+sensory+perception+of+older+adults+improved+using+subsensory+electrical+noise+stimulation.">Peripheral tactile sensory perception of older adults improved using subsensory electrical noise stimulation.</a> Medical Engineering &amp; Physics. 38 (8), 822-825 (2016).</li><li>Yamamoto, Y., Struzik, Z. R., Soma, R., Ohashi, K., Kwak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noisy+vestibular+stimulation+improves+autonomic+and+motor+responsiveness+in+central+neurodegenerative+disorders.">Noisy vestibular stimulation improves autonomic and motor responsiveness in central neurodegenerative disorders.</a> Annals of Neurology. 58 (2), 175-181 (2005).</li><li>Soma, R., Nozaki, D., Kwak, S., Yamamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1/f+noise+outperforms+white+noise+in+sensitizing+baroreflex+function+in+the+human+brain.">1/f noise outperforms white noise in sensitizing baroreflex function in the human brain.</a> Physical Review Letters. 91 (7), 078101 (2003).</li><li>Wiesenfeld, K., Moss, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+resonance+and+the+benefits+of+noise:+from+ice+ages+to+crayfish+and+SQUIDs.">Stochastic resonance and the benefits of noise: from ice ages to crayfish and SQUIDs.</a> Nature. 373 (6509), 33-36 (1995).</li><li>Moss, F., Ward, L. 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P., 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=Using+low+levels+of+stochastic+vestibular+stimulation+to+improve+locomotor+stability.">Using low levels of stochastic vestibular stimulation to improve locomotor stability.</a> Frontiers in Systems Neuroscience. 9, 117 (2015).</li><li>Iwasaki, S., 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=Noisy+vestibular+stimulation+increases+gait+speed+in+normals+and+in+bilateral+vestibulopathy.">Noisy vestibular stimulation increases gait speed in normals and in bilateral vestibulopathy.</a> Brain stimulation. 11 (4), 709-715 (2018).</li><li>Serrador, J. M., Deegan, B. M., Geraghty, M. C., Wood, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+vestibular+function+in+the+elderly+with+imperceptible+electrical+stimulation.">Enhancing vestibular function in the elderly with imperceptible electrical stimulation.</a> Scientific Reports. 8 (1), 336 (2018).</li><li>Kim, J., Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+primary+vestibular+neurons+to+galvanic+vestibular+stimulation+(GVS)+in+the+anaesthetised+guinea+pig.">Responses of primary vestibular neurons to galvanic vestibular stimulation (GVS) in the anaesthetised guinea pig.</a> Brain Research Bulletin. 64 (3), 265-271 (2004).</li><li>Flores, A., 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=Stochastic+resonance+in+the+synaptic+transmission+between+hair+cells+and+vestibular+primary+afferents+in+development.">Stochastic resonance in the synaptic transmission between hair cells and vestibular primary afferents in development.</a> Neuroscience. 322, 416-429 (2016).</li><li>Huidobro, N., 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=Brownian+Optogenetic-Noise-Photostimulation+on+the+Brain+Amplifies+Somatosensory-Evoked+Field+Potentials.">Brownian Optogenetic-Noise-Photostimulation on the Brain Amplifies Somatosensory-Evoked Field Potentials.</a> Frontiers in Neuroscience. 11, 464-464 (2017).</li><li>Goldberg, J., Ferna, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+vestibular-nerve+afferents+in+the+squirrel+monkey+to+externally+applied+galvanic+currents.">Responses of vestibular-nerve afferents in the squirrel monkey to externally applied galvanic currents.</a> Brain Research. 252 (1), 156-160 (1982).</li><li>Baird, R., Desmadryl, G., Fernandez, C., Goldberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vestibular+nerve+of+the+chinchilla.+II.+Relation+between+afferent+response+properties+and+peripheral+innervation+patterns+in+the+semicircular+canals.">The vestibular nerve of the chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals.</a> Journal of Neurophysiology. 60 (1), 182-203 (1988).</li><li>Remedios, L., 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=Effects+of+Short-Term+Random+Noise+Electrical+Stimulation+on+Dissociated+Pyramidal+Neurons+from+the+Cerebral+Cortex.">Effects of Short-Term Random Noise Electrical Stimulation on Dissociated Pyramidal Neurons from the Cerebral Cortex.</a> Neuroscience. 404, 371-386 (2019).</li><li>Paxinos, G., Franklin, K. 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> The mouse brain in stereotaxic coordinates. , (2004).</li><li>Camp, A. J., Callister, R. J., Brichta, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+synaptic+transmission+differs+in+mouse+type+A+and+B+medial+vestibular+nucleus+neurons+in+vitro.">Inhibitory synaptic transmission differs in mouse type A and B medial vestibular nucleus neurons in vitro.</a> Journal of Neurophysiology. 95 (5), 3208-3218 (2006).</li><li>Camp, A., 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=Attenuated+glycine+receptor+function+reduces+excitability+of+mouse+medial+vestibular+nucleus+neurons.">Attenuated glycine receptor function reduces excitability of mouse medial vestibular nucleus neurons.</a> Neuroscience. 170 (1), 348-360 (2010).</li><li>Iwasaki, S., 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=Effect+of+Noisy+Galvanic+Vestibular+Stimulation+on+Ocular+Vestibular-Evoked+Myogenic+Potentials+to+Bone-Conducted+Vibration.">Effect of Noisy Galvanic Vestibular Stimulation on Ocular Vestibular-Evoked Myogenic Potentials to Bone-Conducted Vibration.</a> Front in Neurology. 8, 26 (2017).</li><li>Goldberg, J., Smith, C. E., Fernandez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+discharge+regularity+and+responses+to+externally+applied+galvanic+currents+in+vestibular+nerve+afferents+of+the+squirrel+monkey.">Relation between discharge regularity and responses to externally applied galvanic currents in vestibular nerve afferents of the squirrel monkey.</a> Journal of Neurophysiology. 51 (6), 1236-1256 (1984).</li></ol>

The effects of galvanic vestibular stimulation (GVS) on the vestibular system has been highlighted in vivo in humans3,13,23, guinea pigs14, rodents18 and non-human primates24. However, none of these studies have assessed the direct impact of electrical noise on the sensitivity of individual neurons in the vestibular system. Here we demonstrate the first in v...

The vestibular (or balance) system controls our sense of balance by integrating auditory, proprioceptive, somatosensory and visual information. Degradation of the vestibular system has been shown to occur as a function of age and can result in balance deficits1,2. However, therapies targeting the functioning of the vestibular system are scarce.
Galvanic Vestibular Stimulation (GVS) has been shown to improve balance measures, autonomic functioning and other sensory modalities within humans3,4,5,6. These improvements are said to be due to the Stochastic Resonance (SR) phenomenon, which is the increase in the detection of weaker signals in non-linear systems via the application of subthreshold noise7,8. These studies have shown improvements in static9,10 and dynamic11,12 balance, and vestibular output tests such as Ocular Counter Roll (OCR)13. However, many of these studies have used different combinations of stimulus parameters such as white noise9, colored noise13, different stimulus frequency ranges and thresholding techniques. Therefore, optimal stimulus parameters remain unknown and this protocol can assist with determining the most effective parameters. Besides stimulus parameters, the type of stimulus is also important in therapeutic and experimental efficacy. The above work in humans was performed using electrical noise stimuli, whilst much of the in vivo animal work has used mechanical14,15 or optogenetic16 noise stimuli. This protocol will use electrical noise to examine the effects on vestibular nuclei.
Previously, application of GVS to stimulate primary vestibular afferents was been performed in vivo in squirrel monkeys17, chinchillas18, chicken embryos15 and guinea pigs14. However, only two of these studies examined the effect GVS has on the gain of primary vestibular afferents14,15. These experiments were performed in vivo meaning that the precise patterns of stimulation imposed on vestibular nuclei cannot be determined. To our knowledge, only one other study has applied stochastic noise to individual enzymatically dissociated neurons in the central nervous system19. However, no experiments have been performed in the central vestibular nuclei to assess appropriate stimulus parameters and thresholding techniques, making this protocol more precise in determining stimulus effects on individual neurons within the vestibular nuclei.
Here, we describe how to apply sinusoidal and stochastic (electrical) noise directly to individual neurons in the medial vestibular nucleus (MVN), determine neuronal threshold and measure changes in gain/sensitivity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl</td>
			<td>Scharlau</td>
			<td>CA01951000</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-25G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chem-supply</td>
			<td>PA054-500G</td>
			<td>Used for ACSF, sACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>P1847-100G</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187-500MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>MgCl</td>
			<td>Chem-supply</td>
			<td>MA00360500</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Na3-GTP</td>
			<td>Sigma</td>
			<td>G8877-100MG</td>
			<td>Used for K-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chem-supply</td>
			<td>SO02270500</td>
			<td>Use for ACSF and intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4.2H2O</td>
			<td>Ajax</td>
			<td>AJA471-500G</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761-1KG</td>
			<td>Used for ACSF and sACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Chem-supply</td>
			<td>SA030-500G</td>
			<td>Used for sACSF</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>1169567762</td>
			<td>Used for anaesthetising mice</td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Warner instruments</td>
			<td>GC150T-7.5</td>
			<td>1.5mm OD, 1.16mm ID, 7.5cm length</td>
		</tr>
		<tr>
			<td>Data acquisition software</td>
			<td>Axograph</td>
			<td></td>
			<td>Used for electrophysiology and analysis</td>
		</tr>
		<tr>
			<td>Friedmen-Pearson Rongeurs</td>
			<td>World precision instruments</td>
			<td>14089</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td>Used for micropipette</td>
		</tr>
		<tr>
			<td>Multiclamp amplifier</td>
			<td>Axon instruments</td>
			<td>700B</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sper scientific</td>
			<td>860033</td>
			<td>Used for internal solution</td>
		</tr>
		<tr>
			<td>Standard pattern scissors</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>Sutter micromanipulator</td>
			<td>Sutter</td>
			<td>MP-225/M</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>Used for electrophysiology</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Used for slicing brain tissue</td>
		</tr>
	</tbody>
</table>

stochastic noise application for the assessment of medial vestibular nucleus neuron sensitivity in vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed to be due to the stochastic resonance (SR) phenomenon, which is defined as application of a low-level/subthreshold stimulus to a non-linear system to increase detection of weaker signals. However, it is still unknown how SR exhibits its positive effects on human balance. This is one of the first demonstrations of the effects of sinusoidal and stochastic noise on individual neurons. Using whole-cell patch clamp electrophysiology, sinusoidal and stochastic noise can be applied directly to individual neurons in the medial vestibular nucleus (MVN) of C57BL/6 mice. Here we demonstrate how to determine the threshold of MVN neurons in order to ensure the sinusoidal and stochastic stimuli are subthreshold and from this, determine the effects that each type of noise has on MVN neuronal gain. We show that subthreshold sinusoidal and stochastic noise can modulate the sensitivity of individual neurons in the MVN without affecting basal firing rates.

Initial recordings can provide information about the effects that sinusoidal and stochastic noise have on basal firing rates of individual MVN neurons and how the stimuli effect the gain of neurons. Figure 2 shows that neither sinusoidal nor stochastic noise change basal firing rates of MVN neurons when compared to control (no noise) recordings. This information is crucial for determining the threshold of the individual neurons. During the application of galvanic vestibul...

Watch this Scientific Journal Video about Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro at JoVE.com

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...

This protocol is one of the first demonstrations of the direct application of electrical noise to individual neurons in tissue slices. In order to observe neuronal responses to electrical stimuli. The main advantage of this technique is, that we're able to assess a specific set of stimulus parameters on individual neurons.This technique allows for the optimization of stimulus parameters to target particular, functional neuronal types. This has implications for the development of better therapeutics for balance disfunction. Before beginning the brainstem extraction, Equilibry s-ACSF with CARPOGEN and cool the solution at minus 80 degrees Celsius for 25 minutes until an ice slurry has formed.After the solution has chilled, use a number 22 rounded razor blade to make a sagittal skin incision in the skull of a three to five week old C-57 black six mouse and use the pointed end of a pair of standard pattern scissors to make a small incision, starting at the lambda, along the sagittal suture. Using a pair of shallow bend Pierson von Jours, carefully reflect away the repaired parietal and exoccipital bones, and bathe the brain in ice cold s-ACSF. Then, isolate the brain stem from the forebrain and its boney encasing using a number 11 straight razor blade to cut down the parietal exoccipital sulcus and at the caudal negula.Mount the isolated brain stem ventral and down on perviously cut trapezoidal polystyrene block, and use a piece of tissue paper to remove any excess fluid from around the dissected tissue. Use cyanoacrylate glue to fix the polystyrene block to the cutting stage with the attached brainstem rostral and down. Use an advanced speed of 0.16 millimeters per second and a vibration amplitude of three millimeters to obtain 200 micrometer transverse slices of the MVN.Then use a plastic trimmed pipette to transfer the tissue slices on to a filter paper disk incarbinginated ACSF at 25 degrees Celsius for at least 30 minutes. For whole-cell patch clamp electrophysiology, first pull micro pipettes with a final resistance that will range from three to five mega Oms when filled with a potassium-based internal solution and placed in the bath. To obtain whole-cell patch clamp recordings from individual neurons in the MVN, transfer a single tissue slice from the incubation chamber to the recording chamber of a standard electrophysiological set up, and use a nylon thread on a U shaped weight to secure the slice.Continuously perfuse the recording chamber with carbonginated ACSF at 25 degrees Celsius at a flow rate of three milliliters per minute and fill a micro pipette with internal solution. Apply a small amount of positive pressure using a pipette to push debris away from the pipette tip. Using a lower power objective, locate the MVN before switching to a high power to locate individual neurons within the MVN.Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip, and use the micro manipulator to move the pipette toward the selected neuron. A small dimple should form on the neuronal membrane. Release the positive pressure, and apply a small amount of negative pressure.Once a one giga Om seal has been achieved, apply gentle short and sharp negative pressure to the pipette holder through the suction port to rupture the membrane and to create a whole-cell configuration. Then obtain whole-cell current clamp recordings according to standard protocols. To apply stochastic and sinusoidal noise to individual medial vestibular nucleus neurons, set the range of amplitudes from three to 24 pico amps to determine the neuronal threshold and firing rate and group the higher and lower stimulus intensities to determine the sensory threshold.Next, calculate the average firing rate over the 10 second period, during which the depolarizing current step will be injected for each individual current level. Use the average firing rate values to generate a firing rate versus current plot. Then perform a linear regression analysis to determine the gradient of the line of best fit to determine the neuronal gain.Neither sinusoidal nor stochastic noise changes basil firing rates of MVN neurons compared to control no noise recordings. In this experiment, the selected noise level of 6 pico amps is sub threshold, as it can be observed that the average firing rate begins to increase from the experimental threshold of 12 pico amps. This threshold was determined objectively by grouping the stimulus levels above and below the experimental threshold.The neuronal gain was evaluated by subjecting the neurons to a suite of depolarizing current steps from zero to 50 pico amps in 10 pico amp increments with and without noise. Indeed, sinusoidal and stochastic noise applied at sub threshold amplitudes of six pico amps can alter the gain of MVN neurons. The established stimulus parameters can be applied to the nerve innovating the individual neurons to provide a more naturalistic stimuli.

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Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...