Gli autori sono grati a Keali Röhm, Louisa Frieß e Heike Menzel per loro assistenza tecnica; a Helga Brünner per la cura degli animali; e a Chistopher S. Ward per la generazione e la distribuzione del disector ottico griglia plugin per il software ImageJ.

<ol>
	<li>Ip, C. W., 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=AAV1/2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment:+a+new+mouse+model+for+Parkinson's+disease.">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathol Commun. 5 (1), 11 (2017).</li><li>Ip, C. W., Beck, S. K., Volkmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytes+reduce+nigrostriatal+deficits+in+the+6-hydroxydopamine+mouse+model+of+Parkinson's+disease.">Lymphocytes reduce nigrostriatal deficits in the 6-hydroxydopamine mouse model of Parkinson's disease.</a> J Neural Transm (Vienna). 122 (12), 1633-1643 (2015).</li><li>West, M. J., Slomianka, L., Gundersen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+thesubdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.">Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator.</a> Anat Rec. 231 (4), 482-497 (1991).</li><li>Schmitz, C., Hof, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereology+in+neuroscience.">Design-based stereology in neuroscience.</a> Neuroscience. 130 (4), 813-831 (2005).</li><li>Tieu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+neurotoxic+animal+models+of+Parkinson's+disease.">A guide to neurotoxic animal models of Parkinson's disease.</a> Cold Spring Harb Perspect Med. 1 (1), e009316 (2011).</li><li>Abercrombie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+nuclear+population+from+microtome+sections.">Estimation of nuclear population from microtome sections.</a> Anat Rec. 94, 239-247 (1946).</li><li>Rose, R. D., Rohrlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+sectioned+cells+via+mathematical+reconstruction.">Counting sectioned cells via mathematical reconstruction.</a> J Comp Neurol. 263 (3), 365-386 (1987).</li><li>Weibel, E. R., Gomez, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+principle+for+counting+tissue+structures+on+random+sections.">A principle for counting tissue structures on random sections.</a> J Appl Physiol. 17, 343-348 (1962).</li><li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereological+methods+for+counting+neurons.">Design-based stereological methods for counting neurons.</a> Prog Brain Res. 135, 43-51 (2002).</li><li>Keuker, J. I., Vollmann-Honsdorf, G. K., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+use+the+optical+fractionator:+an+example+based+on+the+estimation+of+neurons+in+the+hippocampal+CA1+and+CA3+regions+of+tree+shrews.">How to use the optical fractionator: an example based on the estimation of neurons in the hippocampal CA1 and CA3 regions of tree shrews.</a> Brain Res Brain Res Protoc. 7 (3), 211-221 (2001).</li><li>Mouton, P. R., 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=The+effects+of+age+and+lipopolysaccharide+(LPS)-mediated+peripheral+inflammation+on+numbers+of+central+catecholaminergic+neurons.">The effects of age and lipopolysaccharide (LPS)-mediated peripheral inflammation on numbers of central catecholaminergic neurons.</a> Neurobiol Aging. 33 (2), e427-e436 (2012).</li><li>Barreto, G. E., Sun, X., Xu, L., Giffard, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+proliferation+following+stroke+in+the+mouse+depends+on+distance+from+the+infarct.">Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.</a> PLoS One. 6 (11), e27881 (2011).</li><li>Robinson, 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=Microstructural+and+microglial+changes+after+repetitive+mild+traumatic+brain+injury+in+mice.">Microstructural and microglial changes after repetitive mild traumatic brain injury in mice.</a> J Neurosci Res. 95 (4), 1025-1035 (2017).</li><li>Paxinos, G., Franklin, K. <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. , (2001).</li><li>. Grid_Overlay.java Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/graphic-overlay.html">https://imagej.nih.gov/ij/plugins/graphic-overlay.html</a> (2010)</li><li>. cell_counter.jar Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/cell-counter.html">https://imagej.nih.gov/ij/plugins/cell-counter.html</a> (2010)</li><li>Baquet, Z. C., Williams, D., Brody, J., Smeyne, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+model-based+(2D)+and+design-based+(3D)+stereological+methods+for+estimating+cell+number+in+the+substantia+nigra+pars+compacta+(SNpc)+of+the+C57BL/6J+mouse.">A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse.</a> Neuroscience. 161 (4), 1082-1090 (2009).</li><li>Fu, Y., 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=A+cytoarchitectonic+and+chemoarchitectonic+analysis+of+the+dopamine+cell+groups+in+the+substantia+nigra,+ventral+tegmental+area,+and+retrorubral+field+in+the+mouse.">A cytoarchitectonic and chemoarchitectonic analysis of the dopamine cell groups in the substantia nigra, ventral tegmental area, and retrorubral field in the mouse.</a> Brain Struct Funct. 217 (2), 591-612 (2012).</li><li>German, D. C., Manaye, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+dopaminergic+neurons+(nuclei+A8,+A9,+and+A10):+three-dimensional+reconstruction+in+the+rat.">Midbrain dopaminergic neurons (nuclei A8, A9, and A10): three-dimensional reconstruction in the rat.</a> J Comp Neurol. 331 (3), 297-309 (1993).</li><li>Smeyne, R. J., 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=Assessment+of+the+Effects+of+MPTP+and+Paraquat+on+Dopaminergic+Neurons+and+Microglia+in+the+Substantia+Nigra+Pars+Compacta+of+C57BL/6+Mice.">Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL/6 Mice.</a> PLoS One. 11 (10), e0164094 (2016).</li></ol>

C. W. I. ha servito su comitati scientifici per Merz Pharmaceuticals, LLC e TEVA; ha ricevuto finanziamenti per viaggi da Ipsen, Merz Pharmaceuticals, LLC e Allergan, Inc.; e ha ricevuto onorari altoparlante da Merz, TEVA e Allergan, Inc. di fuori del lavoro presentato. J. V. ha lavorato come consulente per Boston Scientific, Medtronic e AbbVie e ha ricevuto onorari da Medtronic, Boston Scientific, AbbVie, Bial, Allergan e GlobalKinetics di fuori del lavoro presentato.

Stereology inizia con lavorazione del tessuto. Il taglio seriale del tessuto SN deve essere eseguito con attenzione per evitare la perdita delle sezioni durante l'analisi STEREOLOGICHE. Inoltre, un passo essenziale consiste nel contrassegnare un emisfero al fine di distinguere la destra dalla sinistra SN durante l'esecuzione di ansia. Immissione di un piccolo foro nella parte superiore del tronco cerebrale generato i risultati migliori nello studio presentato. Inoltre, dal lavorare con le esigenze di metodo frazionatore ...

La quantificazione del numero di cellulare di un neurone è fondamentale nella ricerca pre-clinica la malattia del Parkinson per determinare il livello della neurodegenerazione nel nigra di substantia (SN)1,2. La valutazione imparziale stereologiche del numero delle cellule in una regione di interesse è considerata il gold standard3,4,5.
Prima dell'avvento di stereology imparziale, il numero dei neuroni nelle sezioni è stato valutato mediante la manipolazione delle cellule contate profili per correggere le probabilità variabile che neuroni entrano in vista in una sezione. Uno dei metodi più comunemente usati era la correzione dell'emocromo quantificati descritto da Abercrombie6. Questo metodo ha tentato di prendere in considerazione che le cellule possono essere quantificate in più di una volta se frammenti della stessa cella si trovano nelle sezioni sottili adiacenti. Di conseguenza, Abercrombie e altri autori generato equazioni che ha richiesto le ipotesi circa la forma, dimensione e orientamento delle cellule contate7,8. Tuttavia, questi presupposti erano solitamente non realizzati e quindi portati a errori sistematici e divergenza dal numero effettivo della cella (cioè, polarizzazione). Inoltre, il bias non potrebbe essere ridotto di campionamento supplementare3.
Per la stima stereologiche di numeri di cellulare usando il frazionatore ottico, principi matematici sono applicati per valutare direttamente i numeri di cellulare direttamente in un volume definito, 3-dimensionale. Il vantaggio di questo metodo è che non comporta ipotesi circa la forma, dimensione e orientamento delle cellule contate. Così, i numeri di cellulare stimato sono più vicini ai valori true e avvicinarsi man mano che aumenta la dimensione del campione (cioè, imparziali)3. Perché molte regole devono essere seguite quando usando stereology per mantenere il metodo imparziale, sono stati sviluppati sistemi di ready-to-use commerciale stereology (per la revisione, Vedi Schmitz e Hof, 2005,4). Sistemi specializzati stereology implementano metodi stereologiche basati su progettazione con aprioristicamente definite sonde e sistemi di campionamento per le valutazioni stereologiche che portano all'indipendenza dalla forma, dimensione, distribuzione spaziale e orientamento della cellule per essere analizzati4,9. Tuttavia, i sistemi di stereology commercialmente disponibili sono costosi; Questo potrebbe limitare implementazione nella ricerca di nuove.
Lo scopo di questo studio era quello di sviluppare una tecnica utilizzabile per la stima stereologiche basati su progettazione della conta delle cellule dopaminergiche nel topo SN, impiegando il metodo ottico frazionatore e utilizzando attrezzature di microscopia standard (cioè, luce microscopio, microscopio standard software e un motorizzato x, y, z fase). Per questo, è presentata una guida passo passo su come elaborare del tessuto di cervello di topo e come stimare i numeri di cell SN utilizzando stereology imparziali basati su progettazione. Inoltre, sono disponibili modelli per il calcolo dei numeri di cellulare stimato e coefficienti di errore (CE).
Il metodo qui descritto non è limitato all'analisi dello SN, ma può essere adattato per l'uso in altre regioni anatomicamente definite del cervello di topo o ratto. Per esempio, stereology imparziale è stato utilizzato per stimare i numeri di cellulare di un neurone l' ippocampo10 e il locus coeruleus11. Inoltre, tipi cellulari diversi da neuroni, astrociti12 e microglia13, possono essere valutati come bene. Pertanto, questo metodo può essere utile agli scienziati che intendono attuare stereology imparziale nella loro ricerca, ma non sono disposti a spendere un sacco di soldi per l'acquisto di un sistema di ansia.

<table><tbody><tr><td>Paxinos mouse atlas</td><td></td><td></td><td>The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &amp;amp; created by Paul Halasz</td></tr><tr><td>brain matrix slicer mouse</td><td>Zivic Instruments</td><td>BSMAS 001-1</td><td></td></tr><tr><td>paraformaldehyde</td><td>Merck</td><td>1040051000</td><td></td></tr><tr><td>sucrose /D(+) Saccharose</td><td>Roth</td><td>4621.1</td><td></td></tr><tr><td>isopentane</td><td>Roth</td><td>3927.1</td><td></td></tr><tr><td>glycerol</td><td>Merck</td><td>1040931000</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-1L</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;phosphate buffered saline ingredients:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Sigma Aldrich</td><td>31434-1KG-R</td><td></td></tr><tr><td>potassium dihydrogen phosphate</td><td>Merck</td><td>1048731000</td><td></td></tr><tr><td>di-sodium hydrogen phosphate dihydrate</td><td>Merck</td><td>1065801000</td><td></td></tr><tr><td>potassium chloride</td><td>Merck</td><td>1049360500</td><td></td></tr><tr><td>normal goat serum</td><td>Dako</td><td>X0907</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A4503-100G</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ml</td><td>detergent</td></tr><tr><td>3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td><td>Kem En Tec</td><td>4170</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;/ Hydrogen peroxide 30%</td><td>Merck</td><td>1072090250</td><td></td></tr><tr><td>avidin/biotin reagent</td><td>Thermo Scientific</td><td>32050</td><td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td></tr><tr><td>rabbit anti mouse tyrosine hydroxylase antibody</td><td>abcam</td><td>Ab112</td><td>1:1000</td></tr><tr><td>biotinylated goat-anti-rabbit IgG H+L</td><td>vector laboratories</td><td>BA-1000</td><td>1:100</td></tr><tr><td>StereoInvestigator version 11.07</td><td>MBF</td><td></td><td></td></tr><tr><td>BX53 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Visiview</td><td>Visitron Systems GmbH</td><td>3.3.0.2</td><td></td></tr><tr><td>Axiophot2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td>Version 4.7</td><td></td></tr><tr><td>Tissue-TEK OCT</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>dry ice</td><td></td><td></td><td></td></tr><tr><td>grid overlay plugin</td><td>Wayne Rasband</td><td></td><td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td></tr><tr><td>cell counter plugin</td><td>Kurt de Vos</td><td></td><td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&amp;nbsp;</td></tr><tr><td>optical disector macro</td><td>Christopher Ward</td><td></td><td></td></tr><tr><td>C57Bl/6N male mice</td><td>Charles River, Germany</td><td></td><td></td></tr><tr><td>SuperFrost Plus coated object slides</td><td>Langenbrinck, Germany</td><td></td><td></td></tr><tr><td>25G needle Microlance 3</td><td>BD</td><td>300400</td><td></td></tr><tr><td>REGLO Analog Infusion pump</td><td>Ismatec</td><td>ISM 829</td><td></td></tr><tr><td>StereoInvestigator system</td><td></td><td></td><td>StereoInvestigator version 11.07</td></tr><tr><td>BX53 microscope</td><td></td><td></td><td>BX53 microscope</td></tr><tr><td>self-assorted stereology</td><td></td><td></td><td>Visiview</td></tr><tr><td>Axiophot2</td><td></td><td></td><td>Axiophot2</td></tr></tbody></table>

stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

Nella ricerca pre-clinica la malattia del Parkinson, analisi del tratto nigrostriatal, compresa la quantificazione di perdita di neuroni dopaminergici nel nigra di substantia, è essenziale. Per stimare il numero di neuroni dopaminergici totale, imparziale stereology utilizzando il metodo ottico frazionatore è attualmente considerato il gold standard. Poiché la teoria dietro il metodo ottico frazionatore è complessa e stereology è difficile da raggiungere senza attrezzature specializzate, esistono vari sistemi commercialmente disponibili stereology completi che includono il software necessario, puramente per cella contando i motivi. Dal momento che l'acquisto di un setup stereology specializzato non è sempre fattibile, per molte ragioni, questo rapporto descrive un metodo per la stima stereologiche del cellulare di un neurone dopaminergico conteggi mediante microscopia standard attrezzature, compreso un microscopio ottico, un tavola di oggetto (x, y, z aereo) motorizzata con software di imaging e un computer per l'analisi. È dato una spiegazione dettagliata su come eseguire stereologiche quantificazione mediante il metodo ottico frazionatore, e file di pre-programmati per il calcolo dei conteggi delle cellule stimato vengono forniti. Per valutare la precisione di questo metodo, è stato effettuato un confronto con i dati ottenuti da un apparato di stereology commercialmente disponibili. Numeri paragonabili delle cellule sono stati trovati usando questo protocollo e il dispositivo stereology, dimostrando così la precisione di questo protocollo per stereology imparziali.

Usando il metodo presentato, il numero stimato di TH + neuroni dopaminergici in SN ha variato fra cellule 7.363 e 7.987 a destra e, nella parte sinistra SN, tra le celle di 7.446 e 7.904. Così, il numero medio dei neuroni dopaminergici (± SEM) era 7.647 ± 83 cellule per il diritto SN e 7.675 ± 66 per la sinistra SN. La CE calcolata per ciascun animale è stato inferiore a 0,08 (gamma: 0,073-0,079) (Figura 7). Per accertare la comparabilità di questo meto...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Questo lavoro presenta un protocollo passo-passo per la valutazione imparziale stereologiche dei numeri di un neurone delle cellule dopaminergiche nel nigra di substantia del mouse utilizzando attrezzature di microscopia standard (cioè, un microscopio ottico, una tabella di oggetto motorizzato (x, y, z aereo) e software di pubblico dominio per analisi digitale delle immagini.

Stereologiche stima del numero di neuroni dopaminergici nel Nigra di Substantia del Mouse utilizzando il frazionatore ottica e microscopia Standard attrezzature

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

stereological-estimation-dopaminergic-neuron-number-mouse-substantia

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.9K Views.  University Hospital of Würzburg. Questo lavoro presenta un protocollo passo-passo per la valutazione imparziale stereologiche dei numeri di un neurone delle cellule dopaminergiche nel nigra di substantia del mouse utilizzando attrezzature di microscopia standard (cioè, un microscopio ottico, una tabella di oggetto motorizzato (x, y, z aereo) e software di pubblico dominio per analisi digitale delle immagini.

Video: Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Profiling completa del regolamento dopamina nel substantia nigra e ventrale tegmentale Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Ricerca

Neuroscienze

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulazioni ambientali del numero di Mesencefalo dopamina neuroni in topi adulti

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

La tecnica del frazionatore ottico per stimare i numeri di cella in un modello di ratto della terapia elettroconvulsiva

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identificazione della dopamina D1-alfa del ricevitore all'interno del roditore Nucleus Accumbens da un innovativo RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

Quantificare la distribuzione eterogenea di una proteina sinaptica nel cervello del Mouse mediante immunofluorescenza

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

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 useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Stima stereologica della lunghezza della fibra colinergica nel Nucleus Basalis di Meynert del cervello del topo

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 ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Studio pre-formato Fibril indotta accumulazione di z-sinucleina in primario embrionale Midbrain Dopamine Neuroni

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Determinazione semi-quantitativa della densità neuronale dopaminergica nella Substantia Nigra dei modelli di roditori utilizzando l'analisi automatizzata delle immagini

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

Studio dell'accumulo di alfa-sinucleina nei neuroni della dopamina embrionale primaria di topo

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...