Neuro 3000 Exam 2

transgenic mice

-foreign DNA = injected into mouse germ line (ancestry) by injection of one celled egg or injection of embryonic stem cells into blastocyst before embryo)

-native gene is still present

-addition of new gene

-cannot control where the gene gets integrated in the genome sequence

-usually not a problem but can sometimes affect gene functions

-can be used to express a gene in specific cells to determine the effects of cell or circuits (not applied to whole body)

-can overexpress genes, then purify out to make product

-can be used to "mark" cells (GFP, luciferase, or Lac Z)

-used to be expensive and time consuming to do

uses to TM

-mark specific organelles

-trace axons and dendrites with membrane bound fluorescent reporters

-put in genes measuring neural activity (Ex: detect

ca, voltage, vesicle fusion)

-introduce genes to induce/inhibit neural activity (Ex: channel rhodopsinis, halorhodopsins, DREADDS)

-ablate/kill cells by expressing toxin or toxin receptors

making TM

-transgene = what is added to specific cells

-inject into eggs (new gene can be human or mouse)

-implant in pseudopregnant mouse (she is given hormones to be able to carry eggs to term)

-has babies, then PCR ran (shows which have new gene)

-use those to breed and experiment

knockout/knockin mice

Why:

KO

-inactivate gene (look at function)

-inactivate gene in certain cells

-inactivate at certain time in development (if early activation = lethal)

KI

-can replace with modified gene (determined effect in disease gene)

Construction:

-create targeting construct

-place in ES cells

-select for proper incorporation

-place ES --> blastocyst

-place blastocyst --> pseudopregnant mouse

-breed

general TM strategy

1. use stem cells --> blastocyst

2. construct targeting vector

-contains pieces homologous to the target & new inserted portions

-goes to specific locations

-homologous - new -homologous - HSV-tk

3. transfection

-HSV-tk = lost if done correctly

-new (neo) is like a bubble that gets added in

4. proliferation

-only small number of cells get the new (neo) gene

-positive-negative selection (kill cells that do NOT have new gene to isolate new ES cells)

-selects for yes neo, no HSV-tk

5. inject ES --> blastocyst

-this makes a mosaic (both types of cells)

-implant into pseudomother

6. breeding

-mosaic babies + normal babies = normal or mutated (no mosaic)

-check if successful : do tail snips then run gel & compare

knock out

-disrupt DNA

-can be added at center or end

knock in

-doesn't disrupt

-only adds at end

Cre-Lox system

-KO gene at specific time or in specific place

-tissue specific promoter (Cre Recombinase) + Lox before and after targeted gene = gene cut out between lox for specific spot/time (at the site of Cre)

Place

-aka conditional knockout

-LoxP + Promoter & Cre

-Cre-excised in promoted & not excised in others

Time

-aka inducible knockout

-LoxP + CreER & Promoter

-LoxP in promoted and not others (Cre= inactive) + 4-hydroxytamoxifen (makes Cre active) = Cre-excised in promoted

CRISPR-Cas9

-CRISPRs = clustered regularly interspaced palindromic sequences

-array of identical repeats intercalated with DNA - targeting spacers derived from bacteria and plasmids

-present in bacteria to defend against viruses and plasmids (provides memory or adaptive immunity to organisms previously exposed to)

-RNA generated from spacers and CRISPRs targets Cas9 nuclease to invading DNA (CRISPR loci = transcribed)

-targeted KOs

HOW:

-a cell is transfected with Cas9 and guide RNA (gRNA/crRNA) sequence that matches the target gene

-finds cell that have PAM and sequence

-clips both DNA strands

-KO (normal) : gene is disrupted by small insertions/deletions (NHEJ)

-new sequence (KI) : replace segment with new gene by assisted recombinase (HDR)

mRNA methods of altering endogenous

-RNA intereference or morpholins

-block RNA expression removes functions too

Method 1:

-dsRNA = dicer that cleaves into siRNA(small interfering)

-RISC complex = dicer + siRNA + other protheins

-RISC targets mRNA with a complimentary sequence to siRNA

-RISC degenerates mRNA

Method 2:

-shRNA = folded and pairs with self

protein stage of endogenous altering

-can test the role of a specific protein by blocking functions

-dominant negative can block function even if in just 1/4 subunits

brainbow labeling

-labels different neurons in different colors

-uses TM and Cre-Lox technology to generate animals expressing unique combos of fluorescent markers in indv. cells

-used to distinguish specific cells, trace processes adn connections and highlight cell structures

-can be used on fixed or living tissues

HOW :

-overlapping Lox = variations in colors (blending)

-multiple types of Lox

-Cre hits one or the other Lox set

-doesn't reverse therefore expressed for life

optogenetics

-uses light to directly excite(channelrhodopsins, blue, Na) or inhibit (halorhodopsins, yellow, Cl) specific neurons in living animals (used to turn on/off cells)

-different rhodopsins used for different times and patters of stimulation

-G-protein coupled rhodopsins modulate intracellular signaling (green light)

-can use methods (promoters, virus, injections) to express rhodopsins in specific cells at specific times

-can make cells expressing rhodopsins constitutionally (enzymes) or inducibly (artificially)

-can come about transgenically : before birth ; specific cells

-can come from virus carriers (mature animals)

-Animals used: c. elegans (worm), drosophila (flies), zebrafish, rat, mouse, primate, human retina cells (ex vivo)

-used to study : addiction, learning, reward, fear, neuromodulation of disease symptoms, sleep, depression

polymerase chain reaction (PCR)

-measure and compare small amounts of RNA and DNA

-tiny amount of tissue needed

-can be done in situ (in place)

-used to compare expression of genes in different neurons, different stages of development, mutant animals, or diseased tissues

Process:

-separate DNA strands with heat

-anneal = cool to allow primers to bond

-DNA polymerase extends from 3' ends

-repeats

-3 cycles = target sequece

microscopy

Light (4 kinds)

Fluorescence (very precise)

-epifluorescent = collects light from entire depth of tissue (can come out blurry)

-confocal = collects light from thin section (1 um) of tissues or whole animal (fruit fly) --- can photobleach from long exposure (more clear and combines on computer)

-two photon = deeper, less photobleaching, less background

-total internal reflectance (TIRF) = visualize molecules at surface only in great details

light microscopy

Bright field

phase contrast

dark field

Nomarski

fluorescent microscopy

-neural structure and function

-protein relationships and localizations of specific proteins

-structural changes due to neural activity

-visualize transport down axons

-different fluorophores can be used at the same time (many labels at once)

electron microscopy

-will see several figures through the concourse of synapses in great detail

-light resolution = 200 nm

- EM o.2 nm = radius of glutamate milecule

-samples must be fixed, dried, and chemically treated

-TEM = transmission = through small slice

-SEM = scanning = over the surface

viruses

-can be used to deliver genes to neural cells

-cultured cells, brain slices, brain regions in vivo , etc

-use to test function of a gene, effect of a mutated gene, used when cannot or do not need to make TM or KOM

neural cell cultures

-cell lines (immortalized) with neural properties or dissociated (out of brain) primary cells can be grown under the right conditions

-slice cultures (250 - 400 um) can be used to maintain connectivity (acute for short term electrophysiology ; organotypic for extended studies of synapse formation ; migration)

-can record them and watch synapses

-slice culture = not fixed yet and maintains some connections

-explant culture = chunk of brain

immortal cell lines

ADV:

-easier to use

-homogenous

-fairly well characterized

-can create stable cell lines expressing gene of interest

-can easily put new genes in

DISADV:

-may not have same properties as neurons or primary cells

-may change over time

primary cell culture

ADV:

-relevant cell type, physiology, and circuitry

DISADV:

-heterogenous populations & high variability

stem cells

-pluripotent cells with capacity to self renew

-identified by function and molecular markers

-classified by source (embryonic, adult, induced) and the tissues they generate

-tissues defined = multipotent and can self renew

-progenerator cells (NPC) have more limited capacity to self renew and may be unipotent (only become neurons)

stem cell cultures

-embryonic stem cells = pluripotent, can be induced to form neural progenitors

-neural stem cells = self renew, produce neurons, astrocytes, oligodendrocytes, more present in embryonic than adult

-induced pluripotent stem cells (iPSC): generate cellsfreom patients with neurological disorders

synapse findings

-1897 - Sherrington : used microscope and named the synapse

- 1959 - Furshpan & Potter : electrical synapse : present in many areas and developing neurons found in crayfish

- 1921 - Loewi & Vagusstoff : chemical (ACh) : vagis nerve stimulation = slow heart and hypothesized was done by chemicals

-Katz : motor neuron to muscle

-Eccles : CNS : needed better tools because harder to study

MOST IN BRAIN ARE CHEMICAL

electrical synapse

-evolutionarily old

-present at Gap Junctions

-bi directional

-cells are electrically coupled so they are fast (act almost as one big cell)

-common in mammalian cells, glia, cardiac muscles, smoothe muscles, epithelial cells, and liver cells

-instantly sends PSP to second cell and coordinates timing

-used for highly synchronized behaviors like sleep, breathing, or eating

Formation:

-gap junction channel = makes pores and small ions/molecules can pass

- 1 gap junction = 2 connexons

- 1 connexon = 6 connexin (protein)

-distance between membranes = 3.5 nm

chemical synapse

-unidirectional

-pre and post synaptic neurons

-20-50 nm cleft

-cleft = full of extracellular matrix proteins and they help organize the synapse

-contains : secretory granules (large dense vesicles) , active zones (pre) and post synaptic density (receptors and associated proteins)

CNS synapse

-various sizes and configurations

-Grey's 1 and Grey's 22

-more stimulation = more activation = more signaling

Grey's type I

-asymmetrical membrane thickness

-usually excitatory

-round vesicles

Grey's type II

-symmetrical membrane thickness

-inhibitory

-oval vesicles

neuromuscular junction

-between motor neurons and muscles

-much of what we know about synapses were learned here

-similar to CNS synapse, but easier to study

-fast, large, and reliable

-neuron synapses onto a muscle fiber

Principles of synaptic transmission

-NT synthesis

-load NT to vesicles

-vesicles fuse to terminal

-NT spills into cleft

-bind to receptors

-biochemical/electrical response ellicited

-remove NT from cleft

NT synthesis

-various NT have distinct synthetic paths

-in all cells (amino acids)

-specific enzymes in neurons which synthesize unique NT

Peptide NT process:

-rough ER (precursor) -> Golgo (peptide NT) -> down axon -> stored further from cleft in granule

NT process:

-enzyme travels slow down axon to terminal

-makes NT at terminal

-precursor + enzyme -> NT -> transmitter protein loads in vesicle -> docks near terminal

dual transmitter neurons

-the NT, receptors, and geometry dictte the response

they can:

-co release (at the same time from the same vesicles even)

-release at different Ca levels of excitement

-terminal splits to different regions and releases in different areas

NT release

-opening of voltage gate Ca channels = large influx of Ca

-exocytosis (NT release) = rapid (0.2 ms)

-vesicle fuses to the membrane at the active zone before release

-some vesicles could already be docked and waiting for release signals (reserve pool)

-vesicle membrane = recycled by endocytosis (many ways)

-peptides = not at active zone, slower (50 ms), need more Ca to release, parked further back, held to cytoskeleton

-we know Ca channels are numerous becasue of imaging

chromaffin cell

peptide NTs

-adrenal gland releases

vesicle docking

-tSNARES = target snares = attacked to presynaptic cell

-vSNARES = vesicle snares = attached to vesicle

-vesicle attachment site (VAS) = like a jungle gym and allows vesicles to sit and wait in place (presynaptic grid)

-MANY types of snares on the vesicles

-Ex: synaptotagmin = needed to initiate fusion

NT receptors

-more than 100 different ones

-NT gated or ligand gated : binds and opens channels (Na = EPSP , Cl = IPSP)

-G protein coupled (metabotropic) : slower effects

-nAChR = model for ligan gated

basic receptor structure

-hydrophilic outside

-hydrophobic inside

-multiple subunits

transmitter gated channels

-pore/channel closed until ligad/NT binds

- 4 or 5 subunits that change conformation after binding

-opens within microseconds (very fast)

-not as selective as voltage gated channels

-Ex: AChR gates Na, K, and Ca (EPSP or excitatory usually ) = basically like a cation pore for depolarization

-Ex: Cl gates glycine and GABA too (IPSP) = basically an anion pore for hyperpolarization

-takes summation of EPSP and IPSP

-EPSP = Na/Ca in, K out

-IPSP = Cl in

reversal potentials

-when equally permeable to K and Na

-at 0,0 for ACh receptors

-Ex: -60 mV = more permeable to Na (Na out)

-Ex: +60 mV = more permeable to K (K out)

g protein coupled receptors

-slower acting

-amino acids, amines, and peptideds

-also called metabotropic receptors

-same NT can have different cations depending on type of receptor

-autoreceptors = often G protein (regulation and feeback loops)

-more flexible reaction than gated

-binding = shape change

ACh

-ach on g protein receptors = inhibitory

-ach on NT receptors = excite

NT reuptake and degredation

-NT must be destroyed or removed from cleft to terminate signaling

-must be quick because frequency and pattern encode the information being sent

Examples:

-diffusion from cleft

-reuptake by specific transporters (pre or glial)

-degredation by enzymes

-desensitize = down regulate receptors when too much too often

neuropharmacology

-many chemicals, drugs, and diseases can affect each of the steps

antagonists

-block signals

-Ex: curare, cobra venom, Botox (block NT release at SNARE)

agonists

-increase signals

-EX: nicotine, black widow venom (increases ACh by making Ca enter and excite and then uses all the vesicles stored so that the signals cannot send and you stop breathing)

principles of synaptic integration

-post neuron integrates many signals acting through many receptors

-brain performs billions of neural computations/second

-synaptic integration is a process in which these multiple inputs combine within one neuron and the output is then determined (AP or not)

integration of EPSP

-thousands of channels

-amount that open depend on quantity released

-quantum : number of transmitter molecules in a vesicle (several thousand)

-mini post synaptic potentials produced by spontaneous release of one vesicle "accidentally"

-found when random openings were recorded when AP were blocked

-amplitudes of EPSP = multiple minis combined

quantal analysis of EPSP

-compare mini and evoked potentials to decided how much NT was released

-at NMJ : 200 vescicles (-40mV) per AP (muscle contracts ; must work)

-in CNS : could be as little as one vesicel (0.1 mV) : makes sense because of integration needed for computational functions of neuron with many inputs

-AP star at axon hillock

-most synapses on dendrites, some on soma

temporal summation

many firings in one synapse over times leading to AP

spatial summation

many inputs from many other neurons all at once

glia regulation of synaptic development

-structurally normal, but post synaptically silent (EX: thrombospondins)

-facilitates presynaptic activity and increases the probability of NT release : puts receptors on post synaptic (EX: cholesterol)

-induces formation of functional synapses or converts silent to active by facilitating the insertion of receptors on post synaptic (not identified yet)

dendritic cable properties

-electrically passive cables

-currents will dissipate over distance

-lambda = length constant , where depolarization is 37% of original current

- lambda gives some ideas how far away from hillock depolarization can occur and still get A

-want long lambda for higher chance of depolarization

-depends on internal resistance (Ri) and membrane resistance (Rm)

internal resistance depends on

diameter and electrical properties of cytoplasm (constant in mature neurons)

membrane resistance depends on

synaptic activity and how many ion channels are open

excitable dendrites

-some have Na, Ca, and K channels

-usually dont fire AP

-opening of these can add current which allows the propogation of EPSP (pass further along)

inhibition on dendrites

-Cl channel can open

-shunting inhibition = EPSP + IPSP = no stimulation once soma reached

-all depends on where in environment it occurs

NT systems

-first found = ACh (Loewi)

-cells that produce and release ACh = cholinergic (Dale)

-noradrenergic = NE

-adrenergic = Epinephrine

- GABAergic (GABA)

-glutamatergic (Glu)

-peptidergic (peptides)

-ones that receive = ____ceptive

-cells can make more than 1 NT but not as common

how are molecules classified as NT

-must be synthesized and stored in presynaptic

-must release by pre terminal in response to stimulation on command (Ca dependent)

-when applied experimentally, molecule must produce a response in the post synaptic as occurs in vivo

immunocytochemistry

-make antibodies to specific NT or enzymes (they synthesize NTs)

-rabbit used in lab

1. inject NT or enzyme to provoke immune response (antigen)

2. take out antibody from blood

-use labels to find those specific cells

-top = label = constant

-bottoms = variable = binds to NT

in situ hybridization

-detects RNA (for NT) or mRNA (for enzyme) expression using a specific probe

-labeled with radioactivity or colored proteins

-no antibodies needed

-synthesized complementary strand to RNA strand in neuron and it is labeled when it binds

NT release = difficult to study

-fluid from near axons or cells can be tested for substances after stimulation and chemically analyzed (Loewi and Dale)

-in CNS , many synapses using different NT are in close proximity therefore difficult to stimulate a single population of synapses

-now optogenetics is used

- high Ca and high K characterize NT release in in vintro slices

optogenetics reveiw

-chanelrhodopsins = excite

-halorhodopsins = inhibit

-light signaled channel openings

Loewi's Experiment

-vagus sends ACh to heart and slows it when stimulated

-he stimulated the vagus nerve in heart 1 to get this effect

-then used the same solution and put a second heart in it and it slowed shortly after heart 1

testing for response in postsynaptic

-if you believe a certain NT is released somewhere, you can use a pipette to administer a drug onto the synapse and observe the response

analysis of receptors

-each NT can bind to different subtyes of receptors

-two different NT cannot bind to the same receptor

-neuropharm - use antagonist/agonist to classify types

Cholinergic Receptors (ACh)

Nicotinic:

-NT gated

-agonists = ACh and nicotine

-antagonists = curare

Muscarinic

-G protein coupled

-agonists = ACH and muscarine (muschrooms)

-antagonists = atropine

Glutamatergic Receptors (Glu)

-AMPA, NMDA, and Kianate

-let in differnt ions at different speeds

-ionotropic receptors

noradrenergic and adrenergic

-g protein receptors

-NE has alpha and beta

GABAergic receptors

-A = ligand gated

-B = g protein

analysis of NT receptors

-ligand binding = use labeled ligands to bind to specific receptors to see where located

-ligand can be NT, agonist, or antagonist

-ligand can be toxins, or components of venom (snail, snake, spider)

-possible to find recepors before NT

Molecular analysis:

-cloning of many receptors cDNA

-DNA/RNA sequencing

-diversity of subtypes larger than epected from binding and pharmacology

-ability for cell to respond depends on receptors

NT chemistry

-most are AA, amines, or peptides

Dales principle

-neuron has only one NT

-we know this is wrong now

-peptides break this rule as well

cholinergic neurons

-ACh = NT at NMJ

-made by all motor neurons

-used in many ANS circuits

-thalamus = major relay area

-diffuse

Two major groups in brain:

1. Basal forebrain : learning and memory , widespread in corted, AD affects this

2. Dorsolateral potine tegemental constellation

-excitability of sensory relay neurons

ACh pathway

-Choline + Acetyl CoA + ChAT = ACh

-VAChT loads into vesicles

-released into synapse

-taken up by receptors

-excess gets broken down by AChE into Choline and Acetic Acid

-Choline transporter sends Choline back into presynaptic cell

ChAT

choline acetyltransferase

-makes ACh

-good marker for cholinergic cells

choline transporter

-uptake is the RLS

-Na and Cl dependent transporter protein on the synpatic membrae

-EX: AD uses increasing choline levels as treatment

AChE

acetylcholinesterase

-degrades ACh in the cleft and on membranes

-5000/second

-some nerve gas and pesticides block AChE

-made by non ACh neurons too

VAChT

vesicular ACh transporter

-uses proton gradient

- 2 H for 1 ACh

-uses ATP

-not sure how changes in filling are controlled, perhaps by change in driving force or the nuber of transporters/vesicle

Catecholamines

dopamine, norepinephrine, epinephrine

important brain areas for DA

-does not go down SC

-nigro-striatal projection = motor control (dies in parkinsons)

-mesolimbic projection = reward circuit (VTA releases DA to limbic and cortex)

catecholamine synthesis

-tyrosine + tyrosine hydroxylase (TH) = dopa (done in cytosol at terminal)

-dopa + dopa decarboxylase = dopamine (done in cytosol at temrinal)

-domapine + dopamine beta hydroxylase (DBH) = NE (done inside a vesicle)

-NE + PNMT = E (done in cytosol in terminal)

-TH = rate limiting step = could inhibit end products

-increase Ca = increase TH activity

-increase RNA synthesis o TH occurs when large amounts of TH needed

catecholaminergic neurons

-involved in mood, movement, attention, and autonomic functions

-all contain TH

-not degraded in cleft like ACh

-transported back into terminal by specific Na dependent transporter

-amphetamines and cocaine block this action (no reuptake = more DA available in cleft)

-can be degraded by monoamine oxidase (MAO) or reloaded into vesicles

DA pathway

-DA gets loaded into the cell by VMAT (H dependent)

-either take up by receptors on post synaptic neuron or degraded by MAO in liver or reuptake by DAT (Na dependent)

-either broken down by MAO on mitochondria in the cell or recycled into vesicles

dopaminergic neruons

-large amount of dopa decarboxylase present

-amount of DA made depends on amount of dopa available

-basis of treatment for parkinsons (give LDOPA to avoid RLS of TH)

-LDOPA can cross BBB, dopa cannot

-dopamine would cause periphery symptoms

adrenergic neurons

-DBH present in noradrenergic neurons (vesicles not cytosol)

-adrenergic neurons contain PNMT (in cytosol)

-NE is made in the vesicle, released into cytosol, converted into E and then reloaded for release

-NE and E are used as NT in the brain and hormones in the body (released by adrenal gland)

norepinephrine

-most diffuse NT

-activated by new, non painful stimuli (helps us focus on new things happening)

-major group of neurons in locus coeruleus which project to numerous structure including the cortex, hypothalamus, and the hippocampus

-NE neurons modulate attention, feeding, sleeping, mood, arousal, learning, memory, and metabolism

-may communicate with 200,000 + other neurons

NE pathway

-DA loaded by VMAT (H dependent)

-DBH converts DA to NE

-release into cleft

-either taken in by receptors, degraded by MAO in the liver or transported back into the terminal by NET (Na dependent)

-then either recycled into vesicles or degraded by MAO in mitochondria

epinephrine

-removed from synpase and destroyed like DA and NE

-major groups of adrenergic cells in medulla

-present at low levels as compared to other catecholamines

-present in fewer neurons

-function in CNS = not known

-antibodies to enzmes in pathway used to identify catecholaminergic neruons

-in heart, smooth muscle, and brain

-medulla and thalamus = major relay areas

E pathway

-DA loaded by VMAT (H dependent)

-DBH makes DA into NE

-NE released into cytosol

-PNMT makes NE into E

-E is reloaded by VMAT

-same path as DA and NE from here, however, E does not have its on transporter back into the terminal; it is accepted by other catecholamine transporters

serotonin (5HT) synthesis

tryptophan + tryptophan hydroxylase = 5-hydroxytryptophan (5HTP)

-5HTP + 5HTP decarboxylase = 5HT/serotonin/5-hydroxytryptamine

sertainergic neurons

-few neurons (raphe region of pons and upper brainstem) but has widespread projections

-important in the regualtion of sleep, mood, emotional behavior, aggression

-Raphe Nucelus neurons regulate pain as well

-synthesis regulated by amount of tryptophan in etracellular fluid (comes from blood)

-precursor to melatonin in pinneal too (inc. tryp = inc. sleepy)

serotonin

-very diffuse ; goes everywherre

-starts at raphe nuclei (9 of them)

-Prozac inhibits reuptake (more in synapse)

-SSRI used to treat depression (Prozac may increase function of 5HT-1A receptors)

-ectasy induces 5HT release to produce sensory enhancement and empathy

-long term use may destroy 5HT projections

-short term use may produce tachycardia (inc. heart rate), hyperthermia (too hot) or dehydration

seratinergic path

-5HT loaded into vesicle by VMAT (H dependent)

-released into cleft

-taken in by one of many receptors or transported back to terminal by SERT (Na dependent)

-either degraded by MAO in mitochondria or reloaded into vesicles

GABA synthesis

glutamate + GAD = GABA

-GAD = 1-glutamic acid-1-decarboxylase

-present in most GABAergic neurons

-GAD not present in Glu neurons or glia

-vitamin B6 is required for synthesis

glutamate

-involved in many functions and circuits like learning and memory and motor functions

-in most excitatory neurons

-in half of all synapses in brain

-implicated in ALS

-excitotoxicity by Glu during stroke ; perhaps role in AD

-role in development (neuroplasticity) : requires activity to develop

-involved in learning and memory (no Glu = no learning)

Glutamatergic path

-glutamine (Gln) + glutaminase = Glu

-VGLUT loads into vesicle

-released into cleft

-transported back by EAAT (on glia and neurons)

GABA

-major inhibitory NT

-GABAergic neurons present in many areas of the brain (1/3 of all synapses) and spinal cord

-mostly present in the local circuit interneurons but some can be on projection neurons (purkinje cells)

-not reset in peripheral tissues or nerves

-signaling deficits associated with Huntington's diseas, parkinsons, schizophrenia, and senile dementia

-barbiturates = modulators of GABA receptors (treat epilepsy) ; calms

GABA path

-synthesis : Glu + GAD = GABA

-loaded into vesicles by VIAAT (vesicular inhibitory AA transferase)

-released into cleft

-removed by GAT (high affinity transporters on neurons and glia) if not taken in by receptors

-broken down in mitochondria