Indian Journal of Experimental Biology

Vol. 51, September 2013, pp. 685-693

Quick Golgi method: Modified for high clarity and better neuronal anatomy

Nisha Patro1, Kamendra Kumar

1 & Ishan Patro

1,2,*

1School of Studies in Neuroscience, Jiwaji University, Gwalior India, 474 011 2School of Studies in Zoology, Jiwaji University, Gwalior India, 474 011

Received 3 December 2012; revised 20 June 2013

The Golgi methods have long been used to study the neuronal soma, axons, dendritic arborization and spines. The

major concerns of the Golgi method have been its unpredictable nature (inconsistency of impregnation of the stain), time

consumed, tissue hardening and clear background, resulting in several modifications to improve the cellular visualization. In

the present work we describe a modification of the rapid-Golgi method that takes the benefit of perfusion fixation (with

rapid-Golgi solution) then post-fixation in the same fixative for 36 h followed by 36 h impregnation in aqueous AgNO3

followed by vibratomy. This modification is simpler, faster and inexpensive, provides a consistent staining of neurons with

good resolution of neuronal soma, dendritic arborization as well as spines with much reduced formation of silver chromate

crystals and background in just 3 days.

Keywords: Cerebellum, Cerebral cortex, Dendritic spines, Purkinje neurons, Pyramidal neurons, Rapid-Golgi fixative

Camillo Golgi in 1873 discovered that impregnation

of brain slices with silver chromate solution resulted

in staining of a small population of neurons in their

entirety1. This is known as Golgi method and

continues to be extremely useful and very frequently

used even today for studying the neuronal

architecture. Over the years the method has been

variously modified. The classical Golgi method was

modified by Cajal referred as rapid Golgi staining

method2. Cajal extensively used this method and

demonstrated the previously unimagined neuronal

morphology throughout the nervous system, most of

which continues to be accepted. However, Golgi

method’s importance was best felt when Golgi’s so

called artefacts with no physiological relevant

structures on the dendritic surfaces turned out to be

the dendritic spines in the studies of Cajal. He

identified the real value of Golgi method and was first

to describe spines as small thorns that protruded from

the dendrites of cerebellar Purkinje neurons2,3

. Now

the dendritic spines are known as actual centres of

information processing with the ability to regulate

their own protein synthesis and degradation4,5

.

Dendritic spines are proposed as the primary sites of

synaptic plasticity6, their number, shape and size

changes in response to variations in their extracellular

environment7, environmental changes

8-10 and with

evolution11,12

. Most researchers today rely on a two-

step procedure of Golgi staining, chromation

(potassium chromate and/or potassium dichromate

solution) and silver nitrate impregnation which

subsequently allow the formation of silver chromate

crystals visualized as brownish-black structures13

.

The only concern of the Golgi method has been

its unpredictable nature. This has resulted in several

modifications in terms of variation in solution

composition and pH14-21

, use of single sections for

staining22,23

, use of microwaves13,24-26

, changing

embedding media27,28

, use of vibratome29

, coating of

brain blocks with egg yolk13

, application of vacuum30

,

auto metallographic (AMG) enhancement31

and

variation in temperature of the tissue incubating

media21,24-26,32

. Most modifications aimed at decreasing

the time required for the procedure, reduced

precipitation leading to a clearer background and

uniform impregnation and uptake of the Golgi stain in

the nervous tissue. Golgi staining method is useful for

labelling cell profiles in the central nervous system,

but takes several weeks. The long period of fixation

turns the tissue brittle creating problems in sectioning.

The deeply stained blood vessels appearing as

background interfere in deciphering the neuronal

structures. The present communication reports a

simpler, faster and efficient modification of rapid-

——————

*Correspondent author

Telephone: +91 751 2442789

E-mail: ishanpatro@gmail.com

INDIAN J EXP BIOL, SEPTEMBER 2013

686

Golgi staining involving the perfusion of animal with

rapid Golgi fixative and postfixation in the same

fixative leading to a clear background, reduced blood

vessels staining and clear spines and quicker than the

earlier modifications29,33

.

Materials and Methods Wistar rats (250-300 g) used in this study were

raised in the Animal House of School of Studies in

Neuroscience. The animals were housed in chambers

at 25±2 °C room temperature, approximately 50% RH

and 12:12 h L:D conditions. Pelleted standard rat feed

and water was provided ad libitum. The experimental

protocols were pre-approved by the Institutional

Animal Ethics Committee (501/01/a/CPCSEA). A

total of 27 male rats were used in this study.

Rapid-Golgi staining method

Rapid-Golgi fixative solution (RGF) was prepared

as per Patro et al.29

, viz., 5 g potassium dichromate

(Himedia Laboratories, India), 5 g chloral hydrate

(MERCK, India), 8 mL glutaraldehyde (CDH, India),

6 mL formaldehyde (Qualigens, India), 10 drops of

dimethyl–sulphoxide (Qualigens, India) were mixed

and final volume was made up to 100 mL with

distilled water. Three rats were sacrificed by cervical

dislocation followed by decapitation, their brains were

dissected out and the cerebral cortex and cerebellum

were removed. Approximately 1 cm3 tissue blocks

both from cerebral cortex and cerebellar cortex

(through vermis region) were directly immersed in

Golgi fixative and kept in dark amber bottles,

undisturbed, for 2 days followed by a second change

of Golgi fixative for next 2 days. On the 5th day tissue

blocks were rinsed briefly (2-3 times) with 0.75%

aqueous solution of silver nitrate (Qualigens, India)

and kept submerged in the same solution for 2 days in

dark. The tissue blocks were again rinsed with 0.75%

silver nitrate solution (2-3 times) and kept in the same

solution for 3 days in dark. Subsequently, tissue

pieces were washed properly and carefully in 70%

alcohol to remove precipitates of silver and then the

sections were cut using a Leica Automatic Vibratome

(VT1000S Leica Microsystems, Wetzlar, Germany) at

100 µm thickness. The sections were collected in 70%

alcohol, dehydrated in absolute alcohol (Bengal

Chemicals, India), cleared in xylene (Qualigens,

India) and mounted in DPX (Qualigens, India). The

slides were visualized with Leica DM6000

microscope fitted with a digital camera DFC 310 FX

(Leica Microsystems, Wetzlar, Germany).

Quick Golgi staining method (present modifications)

To validate the benefits of the proposed

modifications in the rapid Golgi method, 12 male

Wistar rats were anaesthetized with ether (Qualigens,

India) vapors and brains were perfusion-fixed

(transcardially) with 200 mL of phosphate buffer

saline (PBS) to washout the blood followed by

100 mL of rapid Golgi fixative29

(P-RGF group)

solution at the rate of 20 mL/min. Another group of

12 rats were perfused only with phosphate buffer

saline (P-PBS group). After perfusion with PBS alone

or PBS followed by rapid-Golgi fixative solution

(P-RGF) the brains were dissected out and the

cerebral cortex and cerebellum were removed.

Approximately 1 cm3 tissue blocks from both P-PBS

and P-RGF group rats were post-fixed in RGF and

impregnated with 0.75% aqueous AgNO3 solution in

the following combinations (n=3): 12 h post-fixation

followed by 24 or 36 h AgNO3 impregnation; 24 h

post-fixation followed by 24 or 36 h AgNO3

impregnation; 36 h post-fixation followed by 12, 24

or 36 h AgNO3 impregnation; 48 h post-fixation

followed by 48 h AgNO3 impregnation. On

completion of the respective duration in AgNO3

solution, tissues were washed properly in 70% alcohol

to remove any precipitates and 100 µm thick sections

were cut using a Leica Automatic Vibratome

(VT1000S Leica Microsystems, Wetzlar, Germany).

The slides were visualized using Leica DM6000

microscope equipped with Leica DFC 310 FX digital

camera (Leica Microsystems, Wetzlar, Germany) and

the LAS V4.2 multifocus module that automatically

captured a stack of images at different focus positions

across the depth of the neurons. These images were

then combined into a single image all in-focus

neuronal processes of the stack. The spine density of

220 neurons (110 Purkinje and 110 pyramidal

neurons) was analyzed, in both the staining methods,

using an ocular micrometer scale fitted in the

eye piece of a microscope. For each neuron, 3

measurements were made. The data were analysed

by t-test using SigmaStat version 3.5 for Windows.

Values of P≤0.05 were considered significant.

Results To get better results faster than the earlier modified

Golgi methods, perfusion fixation with the rapid

Golgi fixative solution followed by combinations of

various durations of post-fixation and development

with AgNO3 have been tried in the present study. We

then looked for low background, blood vessels

PATRO et al.: QUICK GOLGI METHOD

687

staining and crystallization with better cellular

details like cell processes, spines, etc. (Table 1).

Combination of 12/24 h and 24/36 h post fixation

with rapid-Golgi fixative solution followed by AgNO3

incubation could not develop much of the details. In

P-PBS group no details could be seen (Fig. 1A and C,

respectively), while in P-RGF group, the interneurons

and Bergmann glial fibers were visible with moderate

impregnation to the central part of the tissue blocks

(Fig. 1B and D, respectively). The combination of

36 h post-fixation following perfusion with rapid-

Golgi fixative solution and 36 h development with

AgNO3 produced wonderful visualization of cellular

details. This combination resulted in minimum

background, least appearance of Golgi stained blood

vessels, complete cellular details (cell soma, axon,

dendrites and spines) of Purkinje neurons and

visibility of interneuron connections with main

projection neurons (stellate, basket and Golgi cells

with Purkinje neurons, Table 1; Fig. 1F) when

compared with corresponding P-PBS group (Table 1;

Fig. 1E). Post fixation for 48 h followed by 48 h

development with AgNO3 also produced similar

results with hardly any further improvement in P-RGF

group (Fig. 1H). However, perfusion with PBS alone

did not provide any such details (Fig. 1G). As

compared to 9 days in rapid Golgi staining procedure,

this modification helps getting better results in 3 days.

It is thus proposed that a combination of perfusion

fixation with rapid-Golgi solution followed by 36 h

post-fixation and 36 h AgNO3 may be used, thus by

the end of the 3rd

day the tissue is ready for sectioning

and visualization. The results thus obtained are

better than the rapid-Golgi method with earlier

modification30

that takes 9 days.

Further, the rapid-Golgi stained brain sections29

were compared with the present Quick Golgi stained

brain sections of 36/36 h combination. Tissue sections

of the present Quick Golgi method presented clearer

details and contrasting cell profiles (Fig. 2B, D and F;

3B, D and F) as compared to the rapid-Golgi method

(Fig. 2A, C and E; 3A, C and E). This difference in

visibility of cell profiles among two different methods

was due to reduced (or even no) staining of the blood

vessels in the proposed Quick Golgi method resulting

in a clear background thus enabling study of the

complete cellular details. The rapid-Golgi stained

blood vessels, prominently visible in the brain

sections of animal perfused with PBS only ruled out

the doubt that blood and its constituents are

responsible for the background. Good camera lucida

tracings were possible with prominent dendritic

spines against a clear background in the proposed

Quick Golgi stained sections (Fig. 2H and 3H). In

Table 1—Qualitative demonstration of rapid Golgi staining in both cerebral cortex and cerebellum in perfusion fixed with rapid-Golgi

fixative solution (P-RGF), perfusion with phosphate buffer saline (P-PBS) only and rapid Golgi method

Impregnation to

central part

Interneurons

Bergmann glial fibers Purkinje cells/spines Pyramidal

cells/spines

Background

Combinations P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS P-RGF P-PBS

12 h fixative &

24 h AgNO3

+ - - - + - - - - - - +

12 h fixative &

36 h AgNO3

+ - - - + - - - - - - +

24 h fixative &

24 h AgNO3

++ - + - ++ - - - - - - ++

24 h fixative &

36 h AgNO3

++ - ++ - +++ - - - - - - ++

36 h fixative &

24 h AgNO3

+++ - +++ - +++ + + - - - - ++

36 h fixative &

36h AgNO3

+++ - +++ + +++ ++ +++ + +++ + - ++

36 h fixative &

12 h AgNO3

+++ - +++ - +++ + + - + - - ++

48 h fixative &

48 h AgNO3

+++ - +++ + +++ ++ +++ + +++ + - ++

Rapid-Golgi

method

+++ ++ +++ +++ +++ +++

- = absent; + = few; ++ = moderate; +++ = enormous

INDIAN J EXP BIOL, SEPTEMBER 2013

688

contrast, because of more background, the camera

lucida tracings were difficult and spine details were

not prominently visible in the rapid-Golgi stained

sections (Fig. 2G and 3G).

There was no significant difference in the dendritic

spine densities of both Purkinje (t=1.579, P = 0.116)

and pyramidal neurons (t=1.125, P = 0.262) when

compared between the rapid Golgi and Quick Golgi

Fig. 1—Photomicrographs showing rapid Golgi stained cerebellar sagittal sections of P-PBS and P-RGF group (n=3). [Green arrows

indicate the Golgi stained fibrous astrocytes, blue arrows indicates Purkinje neurons, pink arrows depicts Golgi stained Bergmann glial

fibers. P-RGF: perfusion with rapid-Golgi fixative solution, P-PBS: perfusion with phosphate buffer saline only. Scale bar = 200 µm.]

PATRO et al.: QUICK GOLGI METHOD

689

Fig. 2—Light microscopic images of the Rapid-Golgi (A, C, E) and Quick Golgi stained (B, D, F) cerebellar sections, showing a

contrasting difference in the clarity of information in Quick Golgi stained preparations. [Red arrows represents the Golgi stained blood

vessels. Scale bar =100 µm.]

INDIAN J EXP BIOL, SEPTEMBER 2013

690

Fig. 3—Photomicrographs showing the rapid-Golgi (A, C and E) and Quick Golgi (B, D and F) stained cortical sections. [Red arrows

indicate the Golgi stained blood vessels (BV). Pyramidal neurons with their processes are clearly visible with both the methods, but with

better cellular details in Quick Golgi method due to reduced staining of the blood vessels. Camera Lucida drawings (1000X) of the main

shaft (MS) and primary branch (PB) of apical dendrite showing the better details of dendritic spines with Quick Golgi procedure (H) than

rapid Golgi (G). Scale bar =100 µm.]

PATRO et al.: QUICK GOLGI METHOD

691

methods, suggesting that the present modification

improves in the qualitative visualization of spine

without compromise in quantitative evaluation (Fig. 4).

The proposed Quick Golgi method involves the

following steps:

1 Anesthetize the animal (rat) with ether vapors.

2 Perfuse transcardially with 200 mL of PBS

followed by 100 mL of rapid-Golgi fixative

solution.

3 Dissect out the brain tissue of interest and cut

tissue blocks of 1 cm3 (or smaller) and post-fix in

the rapid-Golgi fixative for 36 h.

4 Rinse briefly with 2-3 changes of 0.75% aqueous

AgNO3, in dark.

5 Immerse tissue blocks in 0.75% aqueous AgNO3

solution for 36 h in dark amber coloured bottle.

6 Remove any precipitate by rinsing with 70%

alcohol and then store in 70% alcohol.

7 Cut vibratome sections of 100 µm thickness and

collect in 70% alcohol.

8 Dehydrate in 90% and absolute alcohol, clear in

xylene and mount in DPX.

Discussion The importance of Cajal’s Golgi method in

visualization and study of dendritic spines has been

well reviewed by Garcia-Lopez3. Rapid-Golgi method

is frequently used to study the morphology of

neurons, glia and dendritic spines in brain sections.

This technique is also widely used and a very useful

tool in neuroanatomy, CNS pathology and in

estimating the functional status of a brain region

under study in terms of neuronal processes and spines

in them8,9,29

. Dendritic spines are the earliest to be

affected in most neurodegenerative disorders like

Alzheimer’s disease (AD), Parkinson’s disease (PD),

schizophrenia and depression34-36

. The changes are

especially related to the number, size, shape and

origin in the neurons. The available software

(computer program) and hardware (microscope and

related software) have made study of spines easy and

allowed detail study of such structures29,37,38

. Even the

biochemist and molecular biologist can benefit from

this method, as it is easy, procedure is simple, and

requires minimal instrumentation.

Cajal in 1887 modified the original Golgi method

introduced in 1873 by Golgi. It is known as rapid-

Golgi method and has been variously modified largely

based on the type of tissue available (live, post-mortal,

preserved, size, species, etc.) and each modification

has led to a different labeling pattern7,33,39

. The

modifications validated and are in use are many but to

list a few: the rapid Golgi procedure, the Golgi-Cox

technique40,41

; the Golgi-Kopsch method42

; the Del

Rio-Hortega method43

and others.

Most of these variations of original Golgi method

are based on the two step procedure that label the cell

profiles; first, the tissue blocks are immersed in the

chromating solution (either potassium chromate

and/or potassium dichromate) and in the next step the

tissue blocks are immersed in silver nitrate that

produces silver chromate crystals, that allows the

labeling of neurons and other cellular inclusions of

the brain. Other variants replace the silver nitrate with

mercuric chloride and so on.

The major issues with the rapid Golgi method

proposed for modifications are: it takes several weeks

to get results due to long fixation time, the tissue

becomes brittle that causes problem in tissue

sectioning32

; stained particles of blood vessels in the

vicinity of stained neurons and glial cells that

interferes in visualizing and capturing images of

stained cells.

In the present modification the issues like time,

elimination of tissue shrinkage, sectioning, staining of

cellular structures like fine branches of neuronal

processes, uniform clear background, reduced staining

of blood vessels, etc. have been addressed. In addition

to an earlier modification including use of a

vibratome for sectioning that needs no embedding, no

expensive cryostats, ease of cutting sections with a

sharp razor blade, no need of cryopreservation and

less tissue damage due to fragmentation29

have been

proposed.

This modification is novel in the sense that for the

first time perfusion of animals with rapid-Golgi

Fig. 4—Spine density of Purkinje and pyramidal neurons in rapid

Golgi method and quick Golgi method

INDIAN J EXP BIOL, SEPTEMBER 2013

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fixative solution instead of the immersion fixation or

perfusion with PBS alone before immersion fixation

has been tried. This promotes uniform penetration

and thus impregnation of the rapid-Golgi fixative

(chromating solution), much clearer background,

reduced fragments of precipitate and crystals, uniform

impregnation of the chromating solution, better and

homogenous formation of silver chromate thus better

staining of the neurons, interneurons and glial cells.

This also has the advantages of the vibratomy29,41

as

well as better impregnation of the Golgi solution at

the body temperature of 37±1 °C32

and enables to get

preparation ready for study in just 3 days.

The present modification not only have made the

process quicker but also is easy even for the

researchers without an exposure to neuroanatomy,

such as biochemists and molecular biologists, who

can benefit from this simple method that needs no

special infrastructure/instrumentations.

Acknowledgement Financial support from Indian Council of Medical

Research, New Delhi is thankfully acknowledged.

Kamendra Kumar is a Department of Biotechnology

(DBT) Senior Research Fellow. Facilities, developed

through the DBT-Human Resource Development

and Bioinformatics Infrastructural facility from

Department of Biotechnology, used in this study are

thankfully acknowledged.

References 1 Golgi C, Sulla struttura della sostanza grigia del cervello,

Gazz Med Ital (Lombardia), 33 (1873) 244.

2 Cajal S R, Estructura de los centros nerviosos de las aves,

I Cerebelo Rev Trim Histol Norm Patol, 1 (1888) 1.

3 García-López P, García-Marín V & Freire M, The discovery

of dendritic spines by Cajal in 1888 and its relevance in the

present neuroscience, Prog Neurobiol, 83 (2007) 110.

4 Halpain S, Spencer K & Graber S, Dynamics and pathology

of dendritic spines, Prog Brain Res, 147 (2005) 29.

5 Melendez-Ferro M, Perez-Costas E & Roberts R C, A new

use for long-term frozen brain tissue: Golgi impregnation,

J Neurosci Meth, 176 (2009) 72.

6 Johansson B B, Brain plasticity in health and disease, Keio

J Med, 53 (2004) 231.

7 Ziv N E, Hebb and the art of spine remodeling, F1000

biology reports, 2 (2010) 69.

8 Veena J, Srikumar B, Mahati K, Bhagya V, Raju T &

Shankaranarayana Rao B, Enriched environment restores

hippocampal cell proliferation and ameliorates cognitive

deficits in chronically stressed rats, J Neurosci Res, 87

(2009) 831.

9 Ramkumar K, Srikumar B, Venkatasubramanian D, Siva R,

Shankaranarayana Rao B S & Raju T R, Reversal of stress-

induced dendritic atrophy in the prefrontal cortex by

intracranial self-stimulation, J Neural Transm, 119

(2012) 533.

10 Srivastava U C, Singh S & Singh D, Seasonal fluctuation in

the neuronal classes of parahippocampal area of P. krameri

(Scopoli, 1769) and E. scolopaceus (Linnaeus, 1758), Cell

Mol Biol (Noisy-le-Grand, France), 58 (2012) OL1768.

11 Srivastava U C, Maurya R C & Chand P, Cyto-architecture

and neuronal types of the dorsomedial cerebral cortex of the

common Indian wall lizard, Hemidactylus flaviviridis, Arch

Ital Biol, 147 (2009) 21.

12 Srivastava U C & Maurya R, Evolution of the cerebral cortex

in amniotes: Anatomical consideration of neuronal types,

Nature at Work: Ongoing Saga of Evolution, (2012) 329.

13 Zhang X, Bearer E L, Perles-Barbacaru A T & Jacobs R E,

Increased anatomical detail by in vitro MR microscopy with

a modified Golgi impregnation method, Magnet Reson Med,

63 (2010) 1391.

14 Van der Loos H, Une combinaison de deux vieilles méthodes

histologiques pour le système nerveux central, Eur Neurol,

132 (1956) 330.

15 Bertram E G & Ihrig H K, Improvement of the Golgi method

by pH control, Stain Technol, 32 (1957) 87.

16 Morest D K & Morest R R, Perfusion-fixation of the brain

with chrome-osmium solutions for the rapid Golgi method,

Am J Anat, 118 (1966) 811.

17 Stensaas L J, Pericytes and perivascular microglial cells in

the basal forebrain of the neonatal rabbit, Cell Tissue Res,

158 (1975) 517.

18 Adams J C, A fast, reliable silver-chromate Golgi method for

perfusion-fixed tissue, Stain Technol, 54 (1979) 225.

19 Grandin T, Demotte O D, Greenough W T & Curtis S E,

Perfusion method for preparing pig brain cortex for Golgi-

Cox impregnation, Stain Technol, 63 (1988) 177.

20 Gonzalez-Burgos I, Tapia-Arizmendi G & Feria-Velasco A,

Golgi method without osmium tetroxide for the study of the

central nervous system, Biotech Histochem, 67 (1992) 288.

21 Angulo A, Merchan J A & Molina M, Golgi-Colonnier

method: correlation of the degree of chromium reduction and

pH change with quality of staining, J Histochem Cytochem,

42 (1994) 393.

22 Landas S & Phillips M I, Staining of human and rat brain

Vibratome sections by a new Golgi method, J Neurosci

Meth, 5 (1982) 147.

23 Gabbott P L, Somogyi J, The 'single' section Golgi-

impregnation procedure: Methodological description,

J Neurosci Meth, 11 (1984) 221.

24 Armstrong E & Parker B, A new Golgi method for adult

human brains, J Neurosci Meth, 17 (1986) 247.

25 Berbel P J, Chromation at low temperatures improves

impregnation of neurons in Golgi-aldehyde methods,

J Neurosci Meth, 17 (1986) 255.

26 Marani E, Guldemond J M, Adriolo P J, Boon M E & Kok L

P, The microwave Rio-Hortega technique: A 24 hour

method, Histochem J, 19 (1987) 658.

27 Blackstad T W, Osen K K & Mugnaini E, Pyramidal

neurones of the dorsal cochlear nucleus: A Golgi and

computer reconstruction study in cat, Neuroscience, 13

(1984) 827.

28 Kolodziejczyk E, Serrant P & Fernandez-Graf M R, A

simple rapid method to slice biological specimens: an

PATRO et al.: QUICK GOLGI METHOD

693

application for non-embedded and embedded Golgi-stained

tissue, J Neurosci Meth, 31 (1990) 183.

29 Patro N, Shrivastava M, Tripathi S & Patro I K, S100beta

upregulation: A possible mechanism of deltamethrin toxicity

and motor coordination deficits, Neurotoxicol Teratol, 31

(2009) 169.

30 Friedland D R, Los J G & Ryugo D K, A modified Golgi

staining protocol for use in the human brain stem and

cerebellum, J Neurosci Meth, 150 (2006) 90.

31 Orlowski D & Bjarkam C R, Autometallographic

enhancement of the Golgi-Cox staining enables high

resolution visualization of dendrites and spines, Histochem

Cell Biol, 132 (2009) 369.

32 Ranjan A & Mallick B N, A modified method for consistent

and reliable Golgi-cox staining in significantly reduced time,

Front Neurol, 1 (2010) 157.

33 Shankaranarayana Rao B S & Raju T R, The Golgi

techniques for staining neurons, in Brain and behavior,

edited by Raju T R, Kutty B M, Sathyaprabha T N &

Shankaranarayana Rao B S (National Institute of Mental

Health and Neurosciences, Bangalore) 2004, 108.

34 Fiala J C, Spacek J & Harris K M, Dendritic spine pathology:

cause or consequence of neurological disorders? Brain Res

Rev, 39 (2002) 29.

35 Hill J, Hashimoto T & Lewis D, Molecular mechanisms

contributing to dendritic spine alterations in the prefrontal

cortex of subjects with schizophrenia, Mol Psychiatr, 11

(2006) 557.

36 Baloyannis S J, Costa V, Mauroudis I, Psaroulis D,

Manolides S L & Manolides L S, Dendritic and spinal

pathology in the acoustic cortex in Alzheimer's disease:

Morphological and morphometric estimation by Golgi

technique and electron microscopy, Acta Oto-Laryngol, 127

(2007) 351.

37 García-López P, García-Marín V & Freire M, Three-

dimensional reconstruction and quantitative study of a

pyramidal cell of a Cajal histological preparation, J Neurosci,

26 (2006) 11249.

38 Perez-Costas E, Melendez-Ferro M & Roberts R C,

Microscopy techniques and the study of synapses, in Modern

research and educational topics in microscopy, edited by

Medez-Vilas A & Diaz J (Badajoz, Spain, Formatex) 2007,

164.

39 Millhouse O E, The Golgi methods, in Neuroanatomical

tract-tracing methods 1, edited by Heimer L & Robards M J

(Plenum Press, New York) 1981, 311.

40 Glaser E M & Van der Loos H, Analysis of thick brain

sections by obverse-reverse computer microscopy:

Application of a new, high clarity Golgi-Nissl stain,

J Neurosci Meth, 4 (1981) 117.

41 Gibb R & Kolb B, A method for vibratome sectioning of

Golgi-Cox stained whole rat brain, J Neurosci Meth, 79

(1998) 1.

42 Fox C A, Ubeda-Purkiss M, Ihrig H K & Biagioli D, Zinc

chromate modification of the Golgi technic, Biotech

Histochem, 26 (1951) 109.

43 del Rio Hortega P, Contribucion al conocimiento citologico

de los turmores del nervio y quiasma optico, Memor Soc

Espanola Hist Nat, 14 (1928) 7.