Microanatomy

Chris Gale

Otago Psychiatry Registrars

17 Feb 2010

(with thanks to Paul Glue)

Things I did not know at medical school.

●The human brain is plastic.● It changes in depression and the recovery from

depression, anxiety, postpartum...● This is not dependant on medicines – similar

results with CBT.●Neurones are able to regenerate in adults.●Most of the gray matter volume is synapses.●Glial cells can transmit information.●Glial cells are part of the synapse.●NMDA signals microglia to prune synapses from neurones.●

Scale issues.● Cells, synapses.

– Glia,– Neurones

● Networks– Imaging techniques

● Pathways.– Dopamine– Serotonin– Glutamate.

In the beginning (late 1800s): - Santiago Ramon Y Cajal

- Golgi (silver chromate) staining to delineate neuronal shape, local spread

- previously invisible axons and dendrites could be seen in surrounding

tissue

Cerebellar purkinje fiber

a, In Brainbow-2.0, Cre triggers inversion of a DNA segment flanked by loxP sites in opposite orientation. In 50% of cells, inversion should end in an antisense orientation and switch gene expression.

b, HEK cells stably expressing CMV-Brainbow-2.0 produce RFP, and stochastically switch to CFP expression when transfected with Cre.

c, The Brainbow-2.1 construct contains two tandem invertible DNA segments. Inversion (i–iii) and excision (iv, v) recombination events create four expression possibilities.

d, Stable CMV-Brainbow-2.1 transfectants express nuclear GFP (nGFP). Cre recombination triggers expression of YFP, RFP or M-CFP. pA1 and pA2, SV40 and bGH polyadenylation signals. Scale bars, 50 μm. Nature 450, 56-62(1 November 2007)

doi:10.1038/nature06293

Brainbow-2: stochastic recombination using Cre-mediated inversion.

XFP expression in Brainbow transgenic mice.

a, b, Thy1-Brainbow-1.0 and Thy1-Brainbow-1.1 transgenic mice were crossed with CreERT2-expressing animals. Tamoxifen injection led to mosaic XFP expression throughout the brain. a, Brainstem, line H; b, hippocampal mossy fibre axons and their terminals (see insets), line M.

c, In Thy1-Brainbow-2.0 mice, transient recombination with the CreERT2/tamoxifen system triggers expression of M-CFP (peripheral motor axons, line N).

d, In Thy1-Brainbow-2.1 mice, CreERT2-mediated recombination leads to expression of multiple XFPs. Left: oculomotor nerve, line R. Right: hippocampus (dentate gyrus), line Q (labelled neurons and astrocytes).

e, Sagittal brain sections of Thy1-Brainbow-1.0 mice line H crossed with the retina-specific Chx10-Cre driver. Recombination is almost completely restricted to retinal ganglion cells, as shown by label of their axons arborizing in the superior colliculus. The boxed area in the left panel is shown at greater magnification in the right panel

Nature 450, 56-62(1 November 2007)

doi:10.1038/nature06293

Structural organization of astrocyte-neuron networks

(a) Confocal image showing GFP-expressing astrocytes (green) and a biocytin-filled neuron (red). A single astrocyte contacts multiple dendrites of a single neuron and single neurons are associated with multiple astrocytes.

(b) The astrocytic network is structurally organized in non-overlapping domains. Each astrocyte occupies a distinct volume (domain) with very little or no overlap with the volumes (domains) occupied by other astrocytes.

Astrocytic Ca2+ signaling in vivo is organized in functional domains.

(a) Spontaneous Ca2+ oscillations are shown for two different time points t1 (left) and t2 (right). Spontaneous Ca2+ signaling in astrocytes occurs independently from the activity of nearby cells and very limited or no correlation between the activity of different astrocytes is observed

(b) The single-cell responsive map shows that during spontaneous oscillations the functional domains of astrocytic Ca2+ response at t1 and t2 (yellow and blue lines respectively) largely overlap with the anatomical domains (black lines) of the astrocytic network.

(c) In response to different visual stimuli (stimulus 1, left; stimulus 2, right) distinct groups of astrocytes respond with Ca2+ oscillations. Visual stimuli with, for example, different orientations elicit Ca2+ signaling in specific groups of astrocytes with some cells responding preferentially to one orientation and not to others. Groups of 2 to more than 10 astrocytes can respond to the same visual stimulus.

(d) The single-cell responsive map shows that, during activity-evoked signals, the astrocytic Ca2+ response is organized in functional domains (red and green lines for stim.1 and stim. 2 respectively) that are larger than the structural domains (black lines).

(A) Examples of NMDA-induced (2 s application, NMDA concentrations are shown near the traces) currents in a single astrocyte acutely isolated from the cortical slice and concentration–response curve constructed from six such experiments

(B) Glycine-dependent potentiation of astrocyte NMDA response. The top trace of the left panel shows the 10 μM NMDA-induced current in glycine-free normal extracellular solution; NMDA-induced currents in the presence of different glycine concentrations (30 nM, 1 μM, 10 μM and 30 μM) are displayed below. The concentration–response curve (ΔInorm represents the amplitudes of current increase normalised to the maximal increase at 30 μM glycine) constructed from seven experiments is shown on the right

(C) Synaptic currents mediated by NMDA receptors in astrocytes. Astrocytes in layer II of the cortical slice (obtained from the transgenic mice with green fluorescent protein selectively expressed in astroglia), were identified by their fluorescence and voltage-clamped; electrical stimulation of synaptic inputs was applied to layer IV. Synaptically evoked currents are predominantly sensitive to NMDA antagonist MK801 (10 μM), the residual current is partially blocked by glutamate transported inhibitor dl-TBOA (100 μM). Each point on the time graphs represents the mean ± SEM for five EPSCs; representative examples of EPSCs are shown below.

NMDA receptor-mediated currents in cortical astrocytes.

Glutamate transporter and Na+ homeostasis in glial cells

A) Glutamate triggers [Na+]i elevation in Bergmann glial cells in acute cerebellar slices. Simultaneous recordings of glutamate-induced inward current (which represents the electrogenic effect of Na+/glutamate transporter) and [Na+]i (measured with SBFI indicator), in the presence of 100 μM of selective AMPA/kainate receptors antagonist CNQX and in Na+-free (Na+ substituted by NMDG+) solution. Note that CNQX does not affect [Na+]i elevation, whereas Na+ removal almost completely eliminates both membrane current and [Na+]i transient.

(B) The scheme demonstrating coordinated activity of astroglial sodium-calcium exchanger (NCX) and Na+/glutamate transporter; Na+ extrusion through NCX maintains transmembrane Na+ gradients hence supporting effective operation of glutamate uptake.

Inducible recombination in adult Brainbow1. 0LTPH2-CreERT2 mice leads to combinatorial expression of CFP and YFP in serotonergic neurons.

Tamoxifen (+Tx) (1mg twice daily) was injected for five consecutive days starting at P90.

(A,B) One week later, mice were sacrificed and red, cyan and yellow fluorescence emission was analyzed by confocal microscopy at the level of dorsal raphe nuclei. Stochastic recombination of loxP-flanked fluorescent genes leads to CFP and YFP expression. Depending on the ratio of CFP to YFP expression, each 5-HT neuron shows a distinct color ranging from dark blue (only CFP expression) to yellow (only YFP expression).

(B) Fluorescence labeled neuronal projections are easily detectable.

Front Mol Neurosci. 2009;2:24. doi: 10.3389/neuro.02.024.2009.

Anatomical micro-domains created by glial cells in the CNS tissue.

Freeman Science 2010;330:774-778

Coordination of astrocyte morphological growth and refinement with synapse formation.

(A) Although most neurons are made during embryonic stages, the major waves of synaptogenesis follow and depend on astrocyte production. The timing of astrocyte growth and morphological refinement overlaps significantly with this window of synaptogenesis.

(B) Astrocytes initially extend large, filopodial processes that overlap significantly with neighboring astrocytes; however, by postnatal day 21, astrocytes refine their morphology to occupy unique spatial domains and elaborate fine processes that closely associate with synapses.

(C) A complex interrelation exists between synapses and astrocyte processes. Eph or ephrin signaling can bidirectionally control astrocyte features, as well as spine morphology

The tripartite synapseThe processes of astrocytes are intimately associated with synapses. This association is both structural and functional.

a, Electron micrograph showing a tripartite synapse in the hippocampus. The astrocyte process (blue) ensheaths the perisynaptic area. The axon of the neuron is shown in green, with the dendritic spine in yellow and the postsynaptic density in red and black.

b, Schematic representation of a tripartite synapse. Perisynaptic astrocyte processes contain transporters that take up glutamate (Glu, green circles) that has been released into the synapse and return it to neurons in the form of glutamine (Gln). Glutamate receptors on astrocytes (such as metabotropic glutamate receptors) sense synaptic glutamate release, which in turn induces a rise in Ca2+ concentration in the astrocytes. One of the main functions of glia at the synapse is to maintain ion homeostasis, for example regulating extracellular K+ concentrations and pH.

Eroglu Nature 2010;468:223–231

doi:10.1038/nature09612

Glutamate-induced microglial cell membrane ruffling and chemotaxis in GFP-transgenic mouse spinal cord slices where microglia fluoresce green.

(A) Representative images of four similar experiments showing membrane ruffling before (control), during (glu 5 min) and washout (washout 5 min) of glutamate (1 mm); the cell ruffles are marked by white arrows in the middle panel.

(B) Representative images of chemotaxis induced by glutamate applied by microinjection from the micropipette with the tip at the centre of a circle diameter 25 μm; the green fluorescent intensity becomes stronger within the circle during injection of glutamate, indicating microglia movement towards the source of glutamate.

(C) Time course of changes in fluorescent intensity within a circle like that in (B) for injection of glutamate (1 mm, n = 4) and for bath solution as control (n = 3), indicating the glutamate-induced chemotaxis was not due to mechanical stimulation that might be generated during injection. The values shown in (C) are the relative values. The relative values are fluorescence intensity after application of glutamate was relative to the average fluorescence intensity prior to injection of glutamate

Lui Eur J Neurosci. 2009 Mar;29(6):1108-18.

Glutamate, chemotaxis, cell destruction, microglia.

Dopamine Pathway

Serotonin Pathway

How does this affect classical teaching?

● Neuroplasticity is new.● Are there real change in dopamine and serotonin?

– Neuronal pathways– Psychopharmacology.– But astrocytes (glutamate, NMDA, other) modulate the

synapse.● Microglia → gliosis leads to a model of synaptic pruning that

may explain changes brain volume.● Astrocyte architecture may be driving neural pathways.

Fellin T. Communication between neurons and astrocytes: relevance to the modulation of synaptic and network activity. Journal of Neurochemistry. 2009;108(3):533-544.

Livet J Weissman, TA Kang, H et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 2007; 450(7166): 56-62 doi.org/10.1038/nature06293

Reininghaus U, Craig T, Fisher H, Hutchinson G, Fearon P, Morgan K, et al. Ethnic identity, perceptions of disadvantage, and psychosis: findings from the ÆSOP study. Schizophr Res. 2010 Dec;124(1-3):43-8.

Weber T, Böhm G, Hermann E, Schütz G, Schönig K, Bartsch D. Inducible gene manipulations in serotonergic neurons. Front Mol Neurosci. 2009;2:24. doi: 10.3389/neuro.02.024.2009

Verkhratsky A. Physiology of neuronal-glial networking. Neurochemistry International. 2010 Nov;57(4):332-343.

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