Electrical and gap junctions

Barry Connors [email protected] My plan: • • Evolution of gap junctions • History of electrical synapses • Physiology, pharmacology, and functions of electrical synapses Electrical synapses are gap junctions that interconnect . So, what are gap junctions? Chemical : Electrical synapse: presynaptic Pre/postsynaptic

postsynaptic Pre/postsynaptic

Pereda, Nature Rev Neurosci 2014 “Gap junction” is a term from electron microscopy; it refers to a special type of intercellular connection membranes of 2 cells A

TJ=tight junction

Cell 1 Cell 2

GJ=gap junction B

Gap junctions joining adjacent hepatocytes (liver cells) in the mouse. A) Transmission EM; 2–3 nm “gap” (arrows) separating plasma membranes. B) Freeze-fracture replica of a gap junction; particles on “protoplasmic” (P) fracture face and pits on the “ectoplasmic” (E) fracture face.

Goodenough & Paul, Cold Spring Harbor Perspect Biol 2009

Gap junctions are ubiquitous

Gap junctions are found in:

• • Smooth muscle • Immature skeletal muscle • Neurons • Endocrine tissues • Epithelia and endothelia of all sorts • Liver, lung, kidney, pancreas • etc…essentially all tissues with the exception of circulating blood cells and sperm

Epithelial cells in the gut Gap junctions are ubiquitous

Common functions of gap junctions include:

• conducting electrical signals • sharing metabolic needs • coordinating cell development • cell adhesion • suppressors of mutation effects; loss of critical enzyme or in one cell might be compensated by its neighbors

Epithelial cells in the gut Gap junctions are basic

Gap junctions are required for bidirectional signaling between oocytes and granulosa cells.

Gap junctions are densely packed channels that span the membranes of two cells

#1

cell membrane #2

Söhl et al., Nature Rev Neurosci 2005 Gap junctions

Atomic force microscopy shows gap junctions face-on Some nomenclature: 6 = 1 (hemichannel) 2 = 1 GJ channel many GJ channels = 1 gap junction

gap junction channel

connexon gap junction

Bosco et al., Physiol Rev, 2011 Structure of one gap junction channel from crystallography:

Maeda et al., Nature 2009 There are about 20 different connexin genes in mammals; some combinations of connexins form functional channels but many combinations don’t work

Harris, Quarterly Review of Biophysics, 2001 Connexons can be hetero- or homomeric; GJ channels can be hetero- or homotypic

White & Paul, Ann Rev Physiol 1999 Life cycle of gap junction Gap junction

hemichannels

• All metazoans (multicellular organisms) have gap junctions • There are two families of gap junction proteins—connexins and pannexins/innexins

Pannexins Connexins (innexins)

Vertebrates + +

Invertebrates - +

The first diverged from invertebrates ~525 M years ago Pannexins and innexins are orthologues, but they have no sequence similarity to connexins

Bosco et al., Physiol Rev, 2011 Innexins and connexins can form either gap junctions or hemichannels

Pannexins form only hemichannels (probably)

Scemes, Spray, Meda, Europ J Physiol, 2008

Is each an individual unit, or is the brain a continuous network of fused neurons?

Santiago Ramón y Cajal (“”) (“reticular theory”)

Shared the in 1906 Sir Bernard Katz Synapses: “soup” or “sparks”?

“There is no evidence that any other mechanism [besides direct electrical transfer] plays even a subsidiary role in synaptic transmission.”

“One day [Eccles] asked me to cut the grass for him. He had an electric lawn mower operated from the 240 V mains. I rapidly cut down one lane and on the first turnabout managed to cut through the mower’s electric cable…” “Eccles was so concerned that he threw the electric mower out and bought a petrol-driven machine instead…” “And I believe that was the precise moment Sir John Eccles, 1946 when I converted Professor Eccles from electrical to chemical transmission.” Is synaptic transmission mediated by chemicals? Definitive evidence: hyperpolarizing inhibition

Extracellular stimulation of inhibitory

IPSP

Brock, Coombs & Eccles, 1952

“It can now be taken as established that transmission across synapses occurs not by the spread of electrical currents, but by the specific chemical substances which impulses cause to be liberated from the presynaptic membrane.” Eccles, 1957 Eccles overstated the case…

Electrical synapses are gap junctions that interconnect neurons

The first electrical synapses observed: crayfish: Furshpan & Potter, 1957 shrimp: Watanabe, 1958 fish: Bennett, 1959

Presynaptic Presynaptic lateral giant Microelectrodes axon (current and voltage)

50 mV Postsynaptic giant motor axon cord

Giant motor synapse Postsynaptic axon

Giant motor synapse of the crayfish 5 ms

Furshpan & Potter, J Neurophysiol, 1959

Electrical synapses: gap juncons provide a high conductance pathway for ionic current to pass from one cell to another

Cell 1 Cell 2

Ionic No synapses: current

Cell 1 Cell 2

Electrical synapse:

Transmembrane ion channels (e.g. K+, Na+, Ca2+, Cl-)

Gap junction channels Most electrical synapses are bidirectional, symmetrical, and fast

30 mV V1 1 I1 gap junction 3 mV V2 2 neurons 50 ms Gap junction channels are permeable to both inorganic ions and small organic molecules Gap junction channels are permeable to small signaling molecules

Inorganic ions: K+, Na+, Cl-, 2+ + - Ca , H , HCO3 Harris, Quart Rev Biophysics, 2001 Gap junction channel permeability depends on connexin composition Pharmacology: gap junctions can be blocked with drugs, but most gap junction antagonists lack specificity and potency

halothane heptanol

enflurane 18-glycyrrhetinic acid (R=H) - carbenoxolone (R=NaOOC(CH2)2CO )

Rozental, Srinivas, Spray, Meth Molec Biol, 2001 Mefloquine (trade name Lariam): the best blocker of neuronal gap junctions (%control) Gapjunction conductance

Cruikshank et al., Proc Natl Acad Sci, 2004

Connectome of the C. elegans CNS (partial): • 302 total neurons • ~5000 chemical synapses • ~600 neuronal gap junctions

Motor

Sensory

Inter- neuron

Chemical synapse

Electrical synapse

White, Southgate, Thompson, Brenner, Phil Trans Roy Soc Lond B, 1986 Establishing the functions of electrical synapses by knocking them out: C. elegans requires electrical synapses to move effectively • gap junctions couple critical motor control neurons • unc-7 and unc-9 are gap junctions genes • gap junction gene deletions leave worms kinked and unable to move forward

Kawano et al., Neuron, 2011 Mammalian electrical synapses: there are ~20 connexin genes; ~half are expressed in the mammalian brain; the large majority of connexins in the brain are in

Astrocytes Ependymal cells Cx36 has been most Neurons consistently implicated in CNS neuronal gap junctions

Neuron-neuron gap junction = electrical synapse

Rash et al., J Neurosci, 2001 Cx35 (fish) and Cx36 (mammals) immunoreactivity occurs near sites of known electrical synapses

Fish brainstem: auditory afferents onto Mauthner cells (Cx35) : AII amacrine cells

Pereda et al., J Neurosci, 2003 Mills et al., J Comp Neurol, 2001 of inhibitory neurons in the mammalian cortex are often connected by gap junctions (as seen with electron microscopy)

Electrical synapse (neuronal gap junction)

Sloper & Powell, Proc Roy Soc Lond, 1978 Microelectrode recordings from cortical neurons

R S

F S

1 0 µ m

Cortical neurons and Brain slice from a mouse microelectrodes for recording The mouse thalamocortical system

Inhibitory neurons: Excitatory neurons:

• Thalamus and cortex are reciprocally connected by feedforward and feedback TRN inhibitory circuits • Inhibitory neurons are VB electrically coupled in mature thalamus cortex and thalamus; most forebrain excitatory neurons are not coupled Electrical synapses between inhibitory neurons in the neocortex, as demonstrated by electrical recordings

30 mV 6 V 1 200 msec V2

V1

I1 V1

V2 V2

I2

Scott Cruikshank, Jaime Mancill Electrical synapses are very good at synchronizing the activity of neurons they interconnect: Synchronous firing of electrically coupled inhibitory of the neocortex

Misha Beierlein, Jay Gibson Electrical synapses are very good at synchronizing the activity of neurons they interconnect: e.g. neurons of the inferior olive (an input to ): • when Cx36 is knocked out, cells can no longer synchronize • when olivary neurons cannot synchronize, coordination of muscle contractions is impaired (“ataxia”) W T

C x 3 6 K O

50 50 mV 5 m V 5 0 0 m s Long et al., J Neurosci, 2002 The forebrain generates a variety of neuronal rhythms, which can be measured with EEG electrodes

High frequency, low amplitude: Gamma/beta, >20 Hz (alert), Alpha 8-13 Hz (drowsy) WakingWaking

Sleep Stage 1

Sleep spindle (~15 Hz) Stage 2

Stage 3 Low frequency, high amplitude: Delta, <4 Hz

Stage 4

RemREM

1 sec Electrical synapses in most mammalian neurons depend on the gap junction Cx36 What happens if you eliminate electrical synapses from the mammalian brain? (knock out the Cx36 gene to find out)

Cx36 KO

• Severe retinal deficits (rod pathways abolished) • EEG abnormalities (gamma frequency rhythms reduced) • Impairment of fine motor control and cerebellar motor learning • Deficits of circadian behavior • Impairment of more “complex” motor learning tasks, object memory, and habituation; spatial learning, fear conditioning, olfactory learning • Impaired cortical inhibition and experience-dependent plasticity • Impaired short-term spatial memory, spatial coding, theta rhythms in

(references available upon request)

Synaptic plasticity: • Chemical and electrical synapses can be modified by neural activity (i.e. LTP and LTD) • Plasticity of chemical and electrical synapses share some similar mechanisms (i.e. regulated trafficking of membrane receptors and channels)

Chemical synapse: Electrical synapse:

Pereda, Nature Rev Neurosci 2014 Development: • Chemical and electrical synapses are regulated by some of the same processes; e.g. a protein called “neurobeachin” is essential for proper development of both

Chemical Electrical synapse synapse

Pereda, Current Biology, 2015 SUMMARY • Electrical synapses are gap junctions that interconnect neurons • Gap junctions are comprised of transcellular ion channels formed from connexin or pannexin/innexin protein subunits • Gap junction channels in species are symmetrical in function; ionic current passes equally well in both directions • Gap junction channels are large enough to allow small organic molecules to permeate • Many drugs can block gap junctions, but all are poorly selective (i.e. they have other effects) • Electrical synapses are found throughout the central nervous systems of all species, connecting particular types of neurons • Electrical synapses allow neurons to share signals (electrical and chemical) and in some cases synchronize their activity