BIPN140 Lecture 8: Synaptic Transmission II

1. Postsynaptic Receptors: Metabotropic & Ionotropic

2. Postsynaptic Responses (Postsynaptic Potentials, PSPs)

3. Neurotransmitters

Su (FA16)

Chemical Synapse: an Overview (Fig. 5.3)

Possible fates for neurotransmitters at the synaptic cleft:

1. Function as ligands for a receptor which directly gates an ion channel (ionotropic)

2. Function as ligands for a receptor which regulates the function of enzymes or ion channels (metabotropic).

3. Degraded by enzymes

4. Sequestered by transporters.

5. Diffuse away

Two Types of Neurotransmitter Receptors (Fig. 5.16)

(1) Membrane proteins (2) Embedded in the postsynaptic membrane(3) Having an extracellular NT binding site

In some synapses, a neurotransmitter binds to a receptor that is metabotropic (G-protein coupled receptors).

In this case, movement of ions through a channel depends on one or more metabolic steps.

Binding of a neurotransmitter to a metabotropic receptor activates a signal transduction pathway in the postsynaptic cell involving a second messenger (e.g. cAMP, Ca2+, etc.)

Compared to ligand-gated channels, the effects of second-messenger systems have a slower onset but last longer (hundreds of milliseconds to minutes), allowing more opportunity for temporal integration.

Amplifying effect: can affect many channels

2nd messengers can diffuse outside the postsynaptic sites.

Metabotropic Receptors (Fig. 5.16)

Ligand-gated ion channels:

(1) having a pore that allows a particular type of ion to flow through (cation or anion) upon activation (binding to NTs).

(2) mediate rapid postsynaptic events. Postsynaptic potentials (PSPs) usually arise within a millisecond or two of an presynaptic AP, and last for only a few tens of milliseconds or less.

(3) can allow either positively charged ion (cation) or negatively charged ion (anion) to go through.

(4) mainly affects PSPs (localized effect)

Ionotropic Receptors (Fig. 5.16)

Postsynaptic Responses: ACh Receptors at the NMJ (Fig. 5.17)

Activation of postsynaptic ionotropic receptors:

(1) NT binding to the receptor opens the channel (ligand-gated ion channel)

(2) Single channel event: patch-clamp recording, “voltage-clamp” mode to measure currents

microscopic current

macroscopic inward current

DepolarizationBy ~ 30 mV(not voltage-clamped)

The Influence of PSP on End Plate Currents (Fig. 5.18)

I = g x V (Ohm’s law)

EPC = gACh x (Vm - Erev)

Vm < Erev => inward currentVm = Erev => no currentVm > Erev => outward current

Net driving force on the ion flowing: the difference between membrane potential and the reversal potential(potential where there is no net current flow)

More negative inside, drawing cation inside the cell => Depolarization effect

More positive inside, pushing cation outside the cell => Hyperpolarization effect

Erev = 0 mV, between ENa

and EK, suggesting that the channel is permeable to both Na+ and K+.

Reversal Potential of a Non-selective Cation Channel

For a neuron that is permeable to multiple types of ions, the equilibrium membrane potential depends on the relative permeability of the ions (GHK equation).

At equilibrium, IK = INa ; IK = gK x (Vm-EK); INa = gNa x (Vm-ENa)

gK x (Vm-EK) = gNa x (Vm-ENa)

Vm = gNa ENa + gK EK

(gNa + gK)

For a channel that is permeable to both Na+ and K+ (non-selective cation channel), the reversal potential (Erev) is a linear sum of the weighted conductance times the respective reversal potential of the permeable ions:

Erev = gNa ENa + gK EK

(gNa + gK)

gNa/gK= (EK – Erev)/(Erev- ENa)

Reversal potential is determined experimentally (in between the equilibrium potentials of the permeable ion species)

ENa= +58 mV

EK= -90 mV

Tug-of-war

Postsynaptic Potentials (PSPs)

For a neuron that has two types of conductance (gm, from leak channels and gACh, e.g. conductance of ACh gated ion channel), the membrane potential is determined by the ratio of these two types of the conductance and the reversal potential of the ligand-gated ion channel.

Vm = gm Erest + gACh Erev

(gm + gACh) Erev= 0 mV

Erest= -60 mV

Tug-of-war

The degree to which the voltage approaches the reversal potential of the ligand-gated ion channel is determined by the relative conductance (gm & gACh).

PSPs generated from an single synapse usually do not reach Erev.

In other words, neurons with small gm require fewer ionotropic receptor to reach the same EPSP.

Reversal Potential (Figs. 5.18 & 5.19)

Reversal Potential: voltage atwhich EPC reverse direction(the equilibrium potential for theligand-gated ion channel)

EAChR = ~0 mV(the channel is permeable to multiple types of cations, nonselectivecation channel)

EAChR = ~0 mVThe channel is permeable to bothNa+ and K+, therefore, EAChR is between ENa and EK

At rest, EPC is primarily carried by Na+

influx. Why?Vrest = -60 mVENa = +70 mV; EK = -90 mV Net driving force for Na+ : 130 mV Net driving force for K+ : 30mV

Reducing ENa Increasing EK

Na+ and K+ Movements during EPCs and EPPs (Fig. 5.20)

Vm < Erev => inward currentmore negative inside, drawing Na+ inside=> depolarization

Vm = Erev => no current(no effect on membrane potential)

Vm > Erev => outward currentmore positive inside, pushing K+ outside=> hyperpolarization

Net driving force for Na+ : 160 mV Net driving force for K+ : 10 mV

Net driving force for Na+ : 0 mV Net driving force for K+ : 170 mV

Reversal Potentials and Threshold Potentials Determine Postsynaptic Excitation and Inhibition (Fig. 5.21)

Activation of an ionotropic receptor tend to bring the postsynaptic potential towards the reversal potential of the ionotropic receptor (Erev).

Excitatory Postsynaptic Potential (EPSP): Erev > AP threshold (usually ~ -40 mV)

Inhibitory Postsynaptic Potential (IPSP): Erev < AP threshold

The relationship between resting membrane potential and Erev does not impact whether a PSP is excitatory or inhibitory! It does not matter whether the PSP is depolarizing or hyperpolarizing.

ECl = -70 mV

EPSP; depolarizing IPSP; hyperpolarizing IPSP; depolarizing

ECl = -50 mV

Summation of Postsynaptic Potentials (Fig. 5.22)

V = I x R; I = g x (Vm- Erev); g: number of opened channel x conductance per channel

That is, the magnitude of PSP is determined by the size of PSC, i.e. how many postsynaptic channels are activated by NTs.

NMJ synapse is very strong, i.e. lots of postsynaptic AChRs.

Most CNS synapses are not strong, but neurons are usually innervated by thousands of synapses.

PSPs can sum in space and time (PSP duration >> AP duration).

The summation of EPSPs and IPSPs by a postsynaptic neuron permits a neuron to integrate the electrical information provided by all the inhibitory and excitatory synapses acting on it at any moment.

Synapses closer to axon hillock will have a greater impact on AP generation.

Location of Synapses Matters

If the neuron responds to two identical GABAergic inputs at location (1) and (2) with two identical IPSCs, which of the following is true?

A. IPSC at location 1 is more effective in inhibiting action potentials than location 2.

B. IPSC at location 1 is less effective in inhibiting action potentials than location 2.

C. IPSC at location 1 is as effective in inhibiting action potentials as location 2.

D. None of the above. It depends on the threshold of the neuron.

Size of Neurons Matters

If neuron (a) and neuron (b) respond to glutamatergic inputs with EPSCs of identical amplitude, which of the following is true? (Remember Vm= i x Rinput)

A. The EPSC at neuron (a) is more effective in generating action potentials than at neuron (b).

B. The EPSC at neuron (a) is less effective in generating action potentials than at neuron (b).

C. The EPSC at neuron (a) is equally effective in generating action potentials as at neuron (b).

a b

From NT Release to Postsynaptic Excitation or Inhibition (Fig. 5.23)

NT release at all presynaptic terminals on a cell results in receptor binding, which causes the opening or closing of specific ion channels.

The resulting conductance change (g; I = g x V) causes current (I) to flow, which may change the membrane potential ( V = I x R).

The postsynaptic cell integrates all of the EPSPs and IPSPs, resulting in moment-to-moment control of action potential generation (neuronal output, signal that can be propagated to another neuron).

Major Neurotransmitters (Fig 6.1)

Many types of neurotransmitters (>100)

A given neurotransmitter can bind to different types of receptor (a large number of combinations).

Therefore, a given neurotransmitter can excite postsynaptic cells expressing one receptor and inhibit others expressing a different receptor. The effect of a given neurotransmitter is determined by the receptor it activates.

Neurotransmitter receptors can be regulated by other compounds (drugs!). For example, nicotine can activate certain acetylcholine receptors. Curare (arrow poison) inhibit certain acetylcholine receptor to paralyze muscles.

Biogenic Amines

Biogenic amines include

– Dopamine, Norepinephrine, Epinephrine (adrenaline)

– Serotonin

– Histamine

– Affect coordination of body movement, sleep, mood, reward, attention and learning (some also function as hormones).

They are active in the CNS and PNS

Biogenic amines have a central role in a number of nervous system disorders and treatments (Parkinson’s disease, psychiatric disorders, addiction, depression etc.).

Biogenic Amines (Figs. 6.10 & 6.14)

Local synthesis: using amino acids as precursors (catecholamines: tyrosine; Histamine: histidine; Serotonin: tryptophan).

Modern neuropharmacology: development of many drugs affecting the synthesis, receptor binding or catabolism of these neurotransmitters. (Cocaine inhibits dopamine transporter, DAT, thereby increasing DA concentrations in the synaptic cleft. So is amphetamine, which also affects norepinephrine.)

Antidepressants: MAO inhibitors (MAOI) & selective serotonin reuptake inhibitors (SSRI, e.g. Prozac).

Removal: reuptake by transporters. Catabolism of catecholamine NTs requires monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).

Termination of 5-HT by serotonin transporter (SERT)

Found in neurons in the hypothalamus, mediating arousal and attention. Side effect of Benadryl (antihistamine): drowsiness.

Rate-limitingRate-limiting

Unconventional NTs: Gases (Fig. 6.20)

Unconventional: Unlike most neurotransmitters, these are not stored in vesicles but are instead synthesized as needed (local regulator). Release is regulated by Ca2+.

Mediate inter-neuronal communication: Can be release pre-synaptically or post-synaptically (retrograde signaling)

Once synthesized, they can permeate the plasma membrane to enter and act on nearby cells.

Can activate guanylyl cyclase to generate cGMP or modify protein directly via nitrosylation (addition of a nitrosyl group to select amino acid)

NO synthase is regulated by

Ca2+/Calmodulin

Unconventional NTs: Endocannabinoids (Fig. 6.18, Box 6G)

Endocannabinoids: Anandamide and 2-arachidonylglycerol (2-AG)

Unsaturated fatty acids produced by our nervous system to act on the cannabinoid receptors (target of 9-tetrahydrocannabinol (THC, active component of marijuana).

Agonists (e.g. WIN 55,212-2) and antagonists (e.g. rimonabant) are available to study endocannabinoid receptors (CB1 type in the CNS, metabotropic receptor).

endocannabinoid

endocannabinoid

agonist

antagonist

Unconventional NTs: Endocannabinoids (Fig. 6.19)

Production is stimulated by a second messenger in the postsynaptic neurons (usually Ca2+).

Functions as a retrograde signaling molecule (from postsynaptic neuron to presynaptic neuron).

Likely released via diffusion (membrane permeable). Not stored in SVs.

Terminated by enzyme hydrolysis (fatty acids hydrolase, FAAH).

Best known to inhibit the communication between postsynaptic target cells and their presynaptic inputs.

Reduced IPSC

> 90% reduction

CB1 antagonist: blocks the reduction of IPSCs

Depolarization-induced Suppression of Inhibition(DSI)

In the hippocampus, CB1 is found mainly in the presynaptic terminals of the inhibitory interneurons

Unconventional NTs: Endocannabinoids (Fig. 6.19)

Reduced IPSC

Depolarization-induced Suppression of Inhibition(DSI)

In the hippocampus, CB1 is found mainly in the presynaptic terminals of the inhibitory interneurons

Background: Endogenous cannabinoids, e.g. anandamide (as well as THC from marijuana), diffuse across cell membranes and activate receptors in hippocampal interneurons that depress GABA release. Depolarization of hippocampal pyramidal neurons suppresses GABAergic IPSPs the neurons receive, a phenomenon called “Depolarization-induced Suppression of Inhibition” or DSI. DSI was thought to represent a retrograde effect of the postsynaptic neuron onto presynaptic inhibitory terminals, but the mechanism was unknown.

Experiments: Induce DSI by depolarizing a hippocampal pyramidal neuron with a patch pipette, and record the changes that occur in IPSPs elicited by stimulating interneuron input to the neuron. (1) Test the effects of compounds that block cannabinoid receptors to see if they block DSI. (2) Similarly, test compounds that activate cannabinoid receptors to see if they mimic DSI. (3) Determine if the cannabinoid effect is likely to be presynaptic, e.g. affects mEPSC frequency (how frequent are SVs released by the presynaptic neuron) but not mEPSC amplitude (unitary activation of postsynaptic receptors).

Fig. 1. DSI requires endogenous cannabinoids

eIPSC: evoked inhibitory

postsynaptic current

CB1 antagonist

CB1 agonist: mimics DSI(occlusion)

CB1 antagonist: prevents DSI

Control experiment: DSI after 30 min of recording

Fig. 3. DSI and a CB1 agonist suppress IPSCs by the same mechanism

Paired-Pulse Ratio: the ratio of two PSPs or PSCs evoked in close succession. Changes in PPR are usually interpreted as presynaptic changes, reflecting the differences in the availability of readily releasable SVs (the first pulse, or stimulus, may deplete a lot of SVs)

Markedly reduced the first PSC (i.e. not so many SVs were released due to CB1 antagonist or DSI)

Cd2+: blocks VGCC, thereby blocking SV release (positive control)

mIPSC frequency: miniature IPSC frequency,(reflecting how often a SV is released)

TTX: block voltage gated Na+ channel to block action potentials.

KCl: to depolarize presynaptic terminal to favor SV release

Recovery: due to removal of endogenous ligand (e.g. 2-AG) by a transporter

CB1 agonist

Results: Blockers of cannabinoid receptors blocked DSI expression; agonist of the receptors mimicked DSI. Both the agonists and DSI acted on presynaptic machinery in GABAergic (inhibitory) terminals innervating the pyramidal neuron. Finally, DSI was shown to “spread” locally, affecting IPSP amplitude in nearby neurons, exactly as predicted for a diffusible membrane-permeant regulator like cannabinoids.

Take-home message: These DSI relies on the retrograde (back across the synapse) transfer of cannabinoids which inhibit GABA release from presynaptic terminals, thereby reducing the evoked IPSC seen in the postsynaptic pyramidal neuron. Globally applied cannabinoids, e.g. THC, can be expected to globally diminish inhibitory activity and alter information processing and system output.