Lesson 3.

Neurons, basic structural and functional units of the nervous system

Golgi staining 1873

Golgi's method stains a limited number (1%) of cells at random in their entirety. The mechanism by which this happens is still largely unknown.

Golgi's staining is achieved by impregnating fixed nervous tissue with potassium dichromate and silver nitrate. Stained cells are thus filled by microcrystallization of silver chromate

Golgi stained Pyramidal neuron in the hippocampus https://commons.wikimedia.org/wiki/File:Pyramidal_

hippocampal_neuron_40x.jpg

Golgi Stained Rat brain http://www.redhotsci.com/the-immensity-of-the-brain/

Camillo Golgi versus Santiago Ramón y Cajal

The Nobel Prize in Physiology or Medicine 1906

Santiago Ramón y Cajal Prize share: 1/2

Camillo Golgi Prize share: 1/2

"in recognition of their work on the structure of the nervous system"

Ramón y Cajal even commented that: "What a cruel irony of fate of pair, like Siamese twins united by the shoulders, scientific adversaries of such contrasting character!"

The Reticular Theory

Support builds for Reticular Theory 1857 Leydig Working with insects, grainy substance into which neuronal processes disappear. 1867 Kollicker “All cells are interconnected through the nets of their branched extensions”. 1872 Network of Gerlach Credited as “originator” of the net theory of neuron tissue. “A nerve fibre is here seen to divide and the two branches to communicate with the plexus of nerve fibres that are in connection with two nerve cells”.

Interconnected neurons suggesting a ‘reticular’ structure of interneuronal connections from Gerlach (1872)

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Neuron Theory Begins to Take Form

Support builds for Neuron Doctrine 1852 Waller, Gudden, Forel Neuro degeneration restricted 1886 His Observed nerve processes as direct outgrowths of young neurons. 1886 His “we shall ultimately have to accommodate to the idea that the transmission of a stimulus without direct continuity is possible…”

Ramón y Cajal's illustration of the neuronal morphologies in the auditory cortex

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Reticularist view The Neuron Doctrine Golgi’s View Cajal’s View

The Neuron Doctrine 1889

Viewed the protoplasmic processes (dendrites) as

syncytium, purely nutritive in function

The neuron is the

fundamental structural and functional unit of the nervous system

Neurons are discrete cells which are not continuous with other cells

The neuron is composed of 3 parts – the dendrites, axon and cell body, and

The information flows along the neuron in one direction (from the dendrites to the axon, via the cell body).

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Dynamic polarization and Connectional Specificity

Two Physiological corollaries of the Neuron Doctrine diagramed in the anatomy by Cajal Law of Dynamic polarization Dendrites or cell body (receptive receive inputs and signal axons to deliver output to other cells.

Law of Connectional Specificity Neurons communicate only with certain postsynaptic targets.

These are the cellular basis for modern connectionist approach to the brain and the initial discovery of the Synapse (from Greek “TO CLASP”), the major sites of communication between neurons, where cognition arises

Cajal’ Nobel Prize Speech 1906

“Our observations revealed, in my opinion, the terminal arrangement of the nerve fibres. These fibres, ramifying several times, always proceed towards the neuronal body, or towards the protoplasmic expansions around which arises plexuses or very tightly bound and rich nerve nests….[the] morphological structures, whose form varies according to the nerve centres being studied, confirm that the nerve elements are possess reciprocal relationships in contiguity but not in continuity. It is confirmed also that those more or less intimate contacts are always established, not between the nerve arborizations alone, but between these ramifications on the one hand, and the body and protoplasmic processes on the other.”

The Neuron Doctrine Today: what is true?

Reticularists Neuronists

1. Nerve cells originate developmentally as independent entities.

2. Much (but not all) of the communication between nerve cells occurs at one-way (i.e. polarized) synaptic junctions, where two neurons are in contact with each other, not in continuity.

3. Fused neurons, neurons coupled by gap junctions, nerve cells that multiplex, do not ‘break any rules’.

4. Nerve cells develop from several fused structures, show small areas of cytoplasmic continuity, have several functionally independent units within a single cell, have significant trophic influences upon each other, and even have one part (the cell body) that can survive injury to another (the axon).

OR

Neurons

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What are the main characteristics of neurons?

1. Basic structural and functional units of the nervous system

2. Independent cells, making contacts (synapses) with hundreds/thousands of other neurons

1. Excitable cells

2. Secretory cells

3. Quiescent cells

Neurons

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What are the main characteristics of neurons?

Input signals

Output signals

Integration

From: http://fog.ccsf.edu/~rmeckler/mempot2.htm

Conduction

Neurons

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The somatodendritic tree: the neuron’s receptive pole

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1. Primary dendrites (1 to 9) are simple perikaryal extensions

2. They can divide successively to generate a neuron-specific dendritic tree

3. Irregular outline, diameter (decreasing along branching), acute angles, ultrastructural characteristics

4. They present “spines”: ovoid heads bound to the dendrite by peduncle. ‘spiny’ or ‘smooth’

5. Each spine is a synapse

From: http://www.meritnation.com/cbse/class9/textbook-solutions/science/p-s-verma-&-v-k-aggarwal-%28biology%29/tissues/page166-long-answers-qno10/1177_5729_37132/

The somatodendritic tree: the neuron’s receptive pole

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The neurons of the central nervous system present

different dendritic arborizations.

(a) Photomicrographs of neurons in the central

nervous system as observed under the light

microscope. A – Purkinje cell of the cerebellar

cortex; B – pyramidal cell of the hippocampus; C –

soma of a motoneuron of the spinal cord. Golgi (A

and B) and Nissl (C) staining. The Golgi technique is a

silver staining which allows observation of dendrites,

somas and axon emergence. The Nissl staining is a

basophile staining which displays neuronal regions

(soma and primary dendrites) containing Nissl

bodies (parts of the rough endoplasmic reticulum).

(b) Camera lucida drawings of neurons in the central

nervous system of primates, revealed by the Golgi

silver impregnation technique and reconstructed

from serial sections.

The somatodendritic tree: the neuron’s receptive pole

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Pyramidal neuron of the hippocampus

Highly polarized cells

The axon and its collaterals are the neuron’s trasmitter pole

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1. Smooth appearance

2. Narrow from the origin

3. No ribosome, no ER

4. Axon hillock, action potential begins

5. It is divided in collaterals forming right-angles with the main axon

6. Generate sodium-potential and propagate them over considerable length

The axon and its collaterals are the neuron’s trasmitter pole

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Neurons showing complex axonal arborizations. Drawing of a cat reticulospinal neuron has been stained by intracellular injection of peroxidase and drawn in a parasagittal plane obtained from serial sections. The axon (ax, black) gives off numerous collaterals along its rostrocaudal trajectory, making contacts with different neuronal populations (delimited by broken lines). Scale: 7 mm = 1 mm. (b) A rat GABAergic ‘hub’ neuron of the CA3 region of the hippocampus filled with neurobiotin during whole cell recording shows numerous axonal collaterals (blue) that expand inside and outside the hippocampus. In comparison a control GABAergic interneuron shows a restricted axonal arborization (green). Cells bodies and dendrites are in black.

Axons versus dendrites

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http://www.slideshare.net/hatesh101/nervous-tissue2k1

Human nervous system: facts and curiosities

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How many neurons

How many glial cells

Azevedo FA. et al., 2009

Human nervous system: facts and curiosities

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Number of neurons/glial cells, brain size and evolution

Suzana Herculano-Houzel et al., 2009

Compared to rodents, and probably to whales and elephants as well, human brain is built according to the very economical, space-saving scaling rules that apply to other primates; and, among economically built primate brains, it is the largest, hence containing the most neurons.

Brain size is not a reliable indicator of number of neurons across orders. Because of the different cellular scaling rules that apply to rodent and primate brains, primates always concentrate larger numbers of neurons in the brain than rodents of a similar, or even larger, brain size

Numbers of neurons increase faster in the cerebral cortex and cerebellum than in the remaining brain areas (the combined brainstem, diencephalon and basal ganglia.

Human brain: facts and curiosities

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Longest axons

The longest axons in the human body are those of the sciatic nerve, which run from the base of the spinal cord to the big toe of each foot. The diameter of axons is also variable. Most individual axons are microscopic in diameter (typically about one micrometer (µm) across). The largest mammalian axons can reach a diameter of up to 20 µm. The squid giant axon, which is specialized to conduct signals very rapidly, is close to 1 millimetre in diameter, the size of a small pencil lead.

http://hermes.mbl.edu/publications/pub_archive/Loligo/squid/neuro1.html

Human brain: facts and curiosities

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Average number of spines

What is the “normal” dendritic spine density? The numbers range from 0.2 to 3.5 spines per 1 μm of dendrite (the latter is a count in human postmortem cortex). Obviously, this number is dependent on age, cell type, and position along the dendrite. However, it is also dependent on the method of measurement. Spiny neurons (pyramidal neurons of the cortex and hippocampus, medium-sized spiny neurons of the striatum, Purkinje cells of the cerebellar cortex) 40,000-100,000 spines 40-60% of their surface

From: https://en.wikipedia.org/wiki/Dendritic_spine

Human brain: facts and curiosities

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The synapse

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Edward George Gray Gray’s Type I: asymmetric, Round shape vesicles Excitatory Dendritic spines Gray’s Type II: symmetric, pleomorphic vesicles Inhibitory Somata - dendritic shaft - axon

From: http://www.oxfordscholarship.com/view/10.1093/acprof:oso/9780195159561.001.1/acprof-9780195159561-chapter-1

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Patterns of Neuronal Connections and Interactions

Of particular interest is the axo-axonic inhibitory synapse on the axon terminal. It is a highly effective way of preventing the axon's action potential from releasing neurotransmitter at the axon terminal. If the terminal does not release its neurotransmitter(s), no message is communicated farther along the neuronal chain, even though an action potential did fire initially, and propagate down the axon. This phenomenon is called presynaptic inhibition.

The synapse

From: http://walkingwithataxia.com/Neuroanatomy.htm

The synapse

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http://blog.nervousencounter.com/wp-content/uploads/2012/07/synapse-EM+cartoon_21.jpg

The synapse

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Ultrastructure of dendritic spines and synapses in the human brain. A and B: Narrow spine necks (asterisks) emanate from the main dendritic shaft (D). The spine heads (S) contain filamentous material (A, B). Some large spines contain cisterns of a spine apparatus (sa, B). Asymmetric excitatory synapses are characterized by thickened postsynaptic densities (arrows A, B). A perforated synapse has an electron-lucent region amidst the postsynaptic density (small arrow, B). The presynaptic axonal boutons (B) of excitatory synapses usually contain round synaptic vesicles. Symmetric inhibitory synapses (arrow, C) typically occur on the dendritic shaft (D) and their presynaptic boutons contain smaller round or ovoid vesicles. Dendrites and axons contain numerous mitochondria (m). Scale bar = 1 μm (A, B) and 0.6 μm (C).

Different types of Neurons

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By the number of processes 1) Unipolar neuron 2) Pseudounipolar neuron 3) Bipolar neuron 4) Multipolar neuron

By the length of Axon 1) Golgi type I neuron 2) Golgi type II neuron

Expression of specific biomarkers 1) Paravalbumin 2) Somatostatin 3) Neuropeptide Y, etc

Physiological classification 1) Fast spiking 2) Nonadapting 3) Intrinsic bursting 4) Irregularly spiking, etc

Different types of Neurons

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http://slideplayer.com/slide/7446965/ https://www.studyblue.com/notes/note/n/lecture-12/deck/3956691

Neurons

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Different types of Neurons

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Basic division of neuronal types

Inhibitory Local Circuit Neurons Inhibitory Interneurons of the Cerebral Cortex

Projecting Neurons Medium-sized Spiny Cells Purkinje Cells

Excitatory

Local Circuit Neurons Spiny Stellate Cells

Projecting Neurons Pyramidal Cells Spinal Motor Neurons

Neuromodulatory Neurons Dopaminergic Neurons of the Substantia Nigra

Different types of Neurons: specific examples

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They control the timing and flow of information in the cerebral cortex

Typical of primate cortex: newer forms and different developmental origin

Molecular Markers: 1) Parvalbumin (PV) 2) Calretinin (CR) 3) Kv3.1 4) Vasoactive intestinal

peptide (VIP) 5) Somatostatin (SST) 6) Cholecystokinin (CCK) 7) Neuronal nitric oxide

synthase (nNOS)

Inhibitory Local Circuit Neurons: interneurons

Different types of Neurons

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The Petilla Interneuron Nomenclature Group Ascoli et al., 2008

A combination of anatomical, molecular and physiological features applied to

GABAergic inhibitory interneurons (15-30%) of the cerebral cortex

Inhibitory Local Circuit Neurons: interneurons

Different types of Neurons

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Inhibitory Local Circuit Neurons: interneurons

The final 49 clusters were assigned an identity based on cell location (Fig. 2) and marker gene expression (Fig. 3). Each type is represented by a color bar with the name and number of core cells representing that type. The violin plots represent distribution of mRNA expression on a linear scale, adjusted for each gene (maximum RPKM on the right), for major known marker genes: Snap25 (pan-neuronal); Gad1 (pan-GABAergic); Vip, Sst and Pvalb (GABAergic); Slc17a7 (pan-glutamatergic); Rorb (mostly L4 and L5a); Foxp2 (L6); Aqp4 (astrocytes); Pdgfra (oligodendrocyte precursor cells, OPCs); Mog (oligodendrocytes); Itgam (microglia); Flt1 (endothelial cells); and Bgn (smooth muscle cells, SMC).

Different types of Neurons

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Typical of striatum: major output from the striatum They receive input from cerebral cortex, thalamus, and dopaminergic neurons

J.P. Vonsattel and M. DiFiglia

Proliferative changes are the predominant morphologic alterations and include marked

increase in spine density, recurving of distal dendritic segments, and

abnormal dendritic growth.

Graded loss of medium spiny neurons in caudate and putamen

Huntington’s Disease: Pathology

Inhibitory Projection Neurons: medium-sized spiny neurons

Different types of Neurons

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Inhibitory Projection Neurons: medium-sized spiny neurons

Molecular Markers: 1. GAD65/67 (glutamate

decarboxylase) 2. DARPP32 (dopamine- and

cAMP-regulated neuronal phosphoprotein)

3. DR1 (dopamine receptor 1) 4. DR2 (dopamine receptor 1)

Different types of Neurons

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Inhibitory Projection Neurons: Purkinje cells Typical of cerebellar cortex: single row, very large, enormous number of spines (80,000) They contact cerebellar nuclei or vestibular nuclei Degenerate in Spinocerebellar ataxia

Red color labels GFAP (glial fibrillary acidic protein) in astrocytes. Green color labels the IP3 (inositol triphosphate) receptor protein, concentrated in cytoplasm of Purkinje cells. Blue color labels DNA, most conspicuously in nuclei of granule cells in the granule cell layer. By Tom Deerinck, NCMIR, 2008

http://www.slideshare.net/drpsdeb/cerebellum-2010 http://www.allposters.com/-sp/Purkinje-Nerve-Cells-In-the-Cerebellum-Posters_i9995633_.htm

Different types of Neurons

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Inhibitory Projection Neurons: Purkinje cells

Molecular Markers: 1. GAD67 (glutamate

decarboxylase) 2. Calbindin (CaBP) 3. Aldolase C 4. cGMP-dependent protein

kinase 5. Inositol 1, 4, 5-triphosphate

receptors (IP3R)

http://www.gensat.org/imagenavigator.jsp?imageID=39202

AldoC-EGFP

https://www.researchgate.net/publication/279300985_Mitigation_of_Cerebellar_Neuropathy_in_Globoid_Cell_Leukodystrophy_Mice_by_AAV-Mediated_Gene_Therapy/figures?lo=1

Different types of Neurons

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Excitatory Local Circuits Neurons: Spiny Stellate cells

Axonal arbors of spiny stellate cells and their topographical relation to barrels and layers. (A) Asymmetric spiny stellate neuron which distributes virtually all dendritic and axonal branches toward its ‘home’ barrel column. (B) Symmetric neuron with a virtual columnar confinement of dendritic and axonal arbors. (C) Symmetric spiny stellate possessing a single direct ‘transbarrel’ collateral. Medial is to the left, lateral to the right. The barrels, as seen with cytochrome oxidase-staining, are shaded gray. Roman numerals mark cortical layers. Upper and lower black line delineates pial surface or layer VI white matter border, respectively. By Staigelr et al., 2004

Typical intracortical target Excitatory synapses, glutamate as neurotransmitter

Different types of Neurons

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Excitatory Projection Neurons: Pyramidal cells Responsible of all cortical output Excitatory synapses, glutamate as neurotransmitter Very large body and dendritic arborization (several mm)

From: http://encyclopedia2.thefreedictionary.com/Layer+V

Different types of Neurons

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Excitatory Projection Neurons: Pyramidal cells

Molecular Markers: 1. Neurogranin/RC3 2. CaMK (calcium/calmodulin-

dependent protein kinase II, CaMKII)

3. Glutamate

http://news.mit.edu/2013/discovering-hippocampal-connections

Different types of Neurons

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Excitatory Projection Neurons: Spinal Motor Neurons They are the motor cells of the ventral horns of the spinal cord Very long axons to innervate various parts of the body Spinal motor neurons use acethylcoline as neurotransmitter Degenerate in lower motor neuron disease

Different types of Neurons

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Neuromodulatory neurons: Dopaminergic Neurons They contain the catecholamine-synthesizing enzyme tyrosine hydroxylase Dopamine is their neurotransmitter They project to cerebral cortex and basal ganglia Responsible for movement regulation Severely and selectively affected by Parkinson’s Disease

TH-GFP mouse Coronal session

Marten P. Smidt & J. Peter H. Burbach, 2007 http://mus.brc.riken.jp/en/mouse_of_month/may_2008_mm

Different types of Neurons

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Neuromodulatory neurons: Dopaminergic Neurons

Molecular Markers: 1. Tyrosine hydroxylase (TH) 2. Dopamine transporter (DAT) 3. Nurr1 4. Foxa2

http://www.frontiersin.org/files/Articles/165063/fncel-09-00389-HTML/image_m/fncel-09-00389-g003.jpg

Different types of Neurons

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Neuromodulatory neurons: Dopaminergic Neurons of the Substantia Nigra

They contain the catecholamine-synthesizing enzyme tyrosine hydroxylase Dopamine is their neurotransmitter They project to cerebral cortex and basal ganglia Responsible for movement regulation Severely and selectively affected by Parkinson’s Disease

From: Ghosh et al., 2012

Complex Brain organization

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Grey matter and white matter

Grey matter is a major component of the central nervous system, consisting of neuronal cell bodies, neuropil (dendrites and myelinated as well as unmyelinated axons), glial cells (astroglia and oligodendrocytes), synapses, and capillaries. Grey matter is distinguished from white matter, in that white matter is composed chiefly of long-range myelinated axon tracts and contains relatively very few cell bodies.

Grey matter volume and cognition in elderly people

Grey matter volume associated with bipolar disorder

Effects of smoking

From: http://www.abclawcenters.com/frequently-asked-questions/can-hie-cause-brain-injury-without-cerebral-palsy/

From: http://thebrain.mcgill.ca/flash/d/d_02/d_02_cl/d_02_cl_vis/d_02_cl_vis.html/

Complex Brain organization

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Grey matter and white matter

Luis de la Torre-Ubieta, Wellcome Image Awards 2015

Summary of today

1. Golgi’s staining to study neuronal cells

2. Reticularist view in comparison to the neuron doctrine

3. Golgi’s and Cajal’s contribution to the modern concept of synapse

4. The basic morphological and functional characteristics of neurons (dendrites, soma, axon) FLASH CARD

5. The differences between dendrites and axons FLASH CARD

6. The synapse: significance, morphology and characterization

7. Different neuronal types of the brain and their implication in disease FLASH CARD

8. Gray matter versus white matter