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GJs are specialised, low-resistance intercellular junctions, forming an aqueous pore between two closely apposed membranes and are considered the ultrastructural substrate of direct intercellular coupling. The principle structural components of vertebrate GJs are membrane-spanning proteins called connexins (Cxs). It is believed that each GJ is constructed as follows: six Cxs

aggregate to form a hemi-channel, or connexon, within each of the opposed membranes, and the Cx hemi-channels are connected extracellularly to form the complete intercellular channel (Fig. 1.15A). Assemblies of GJs tend to be clustered together forming GJ plaques (Fig. 1.15B). Cx expression decreases during development along with GJ intercellular communication: however neuronal coupling persists in many brain regions in adults (MacVicar and Dudek 1981; Rozental et al. 2000; Rouach et al. 2002).

A number of Cx proteins have been identified in neurones in the central nervous system including Cx26, 32, 36 and 43 and 47 (Dermietzel and Spray 1993; Simon and Goodenough 1998; Rozental et al. 2000). Although each of these Cx types has been detected in neurones by a variety of techniques, much attention has focused on Cx36. The use of ffeeze-fracture electron microscopy showed that Cx36 was the only neuronal connexin whilst Cx32 and Cx43 are localised to glia and astrocytes (Rash et al. 2000; Rash et al. 2001). In addition, Cx 36 is highly expressed in brain areas where electrical coupling has been functionally demonstrated, including the cortex (Galarreta and Hestrin 1999; Gibson et al. 1999; Tamas et al. 2000), hippocampus (Venance et al. 2000; Bartos et al. 2001), cerebellum (Mann-Metzer and Yarom 1999), and thalamic reticular nucleus (Landisman et al. 2002). Cx36 may predominate in networks of electrotonically coupled inhibitory neurones (Galarreta and Hestrin 2001). This does not exclude the participation of other Cx subtypes in interneuronal electrical communication as electrotonic communication is not restricted to inhibitory neurones and has been observed in excitatory neurones in the CNS, including the hippocampus (MacVicar and Dudek 1981; Nunez et al. 1990; Draguhn et al.

1998) and cortex (Venance et al. 2000). Cx47 is a newly discovered member of the mouse Cx family and consequently has received less attention. However, Cx 47 has been localised to cells within areas of grey matter in the spinal cord and brain, forms functional intercellular junctions when transfected into oocytes or HeLa cells (communication-deficient mammalian cell line) and are gated by transjunctional voltage differences and chemical uncouplers (Teubner et al.

2001).

Direct evidence for the existence of intercellular electrotonic communication in a neuronal population comes from dual electrophysiological recordings where pairs

Extracellular M2 M4 M3 CL Intracellular Connexin M Connexon Intracellular channel

B

Figure 1.15 Schematic structure of Gap Junctions.

(A) The transmembrane topology of a generic connexin polypeptide creates four membrane domains (M1-M4), one cytoplasmic (CL) and two extracellular (E1 and E2) loops, and the N- and C-termini face the cytoplasm. Fourier microscopy shows that M1-M4 are packed helices, creating a single connexin subunit. Six subunits oligomerize into a connexon within the membrane of a single cell, which docks with a counterpart in an adjacent cell to form the intercellular channel. (B) Gap junction plaques are variable numbers of intercellular channels clustered at close appositions of the plasma membranes of two cells, leaving a 3-nm 'intercellular gap' and forming axial channels connecting the cytoplasms of the cells. The shaded planes represent the lipid bilayer.

(from Simon and Goodenough 1998).

of electrically coupled neurones are recorded simultaneously. Interneuronal electrotonic communication is demonstrated when current injection into one neurone of the simultaneously recorded pair will evoke a response in both neurones (Fig. 1.16A) (MacVicar and Dudek 1981; Logan et al. 1996; Galarreta and Hestrin 1999; Beierlein et al. 2000; Landisman et al. 2002). Due to the presumed distant position of the GJs and the passive cable properties of the dentrites electrotonic communication more faithfully transmits slower signals. Therefore, action potentials evoked in one of the simultaneously recorded pair of cells can be transmitted to the other neurone and gives rise to small depolarising potentials (called electrotonic coupling potentials or spikelets) (Fig. 1.16A). As the action potential AHP is much slower than the action potential itself, spikelets are characterised by a small rapid depolarisation followed by a longer and often pronounced spikelet AHP (Fig. 1.16A). Electrotonic coupling is often bi­ directional, that is current steps applied to either neurone will evoke an attenuated voltage response in the coupled neurone (Galarreta and Hestrin 2001; Landisman et al. 2002). Spikeletes and action potentials share a number of common features supporting the idea that spikelets are filtered action potentials. In particular spikelets are all-or-none events with characteristic waveforms including: 1) a rapid rising phase; 2) a rapid falling phase indicating active repolarisation (in contrast to post-synaptic potentials); 3) are often followed by a pronounced AHP (which I will refer to as the spikelet AHP); 4) generation is often dependent on neuronal depolarisation, however the amplitude and duration are relatively insensitive to changes in membrane potential (MacVicar and Dudek 1981; Logan et al. 1996; Gibson et al. 1999; Galarreta and Hestrin 2001).

Intracellular injection of low molecular weight dyes such a Biocytin, Neurobiotin or Lucifer yellow (molecular weights: 372.5, 322.8 and 457.2 respectively), into a single neurone can result in the spread of dye-into other neurones (MacVicar and Dudek 1981; Nunez et al. 1990; O'Donnell and Grace 1993; Valiante et al. 1995; Mann-Metzer and Yarom 1999). This effect is referred to as dye-coupling and a correlation between the occurrence of spikelets and dye-coupling in neuronal populations is also taken as an indication of the presence of electronic coupling via GJs (Nunez et al. 1990; Valiante et al. 1995; Mann-Metzer and Yarom 1999). Interestingly, not all populations of neurones that have directly

A Electrotonic coupling between NRT neurones in vitro Neurone 1 /

r

B

Figure 1.16 Electrotonic coupling and fast prepotentials in NRT neurons.

Figure 1.16 Electrotonic coupling and fast prepotentials in NRTneurones.

(A) A pair of simultaneously recorded NRT neurones in rat (age: 14-21 days) thalamic slices. (Neurone 1, left: the applied d.c. current steps, ± 200 pA, are shown below the voltage responses. Neurone 2, right: the applied d.c. current steps, ± 200 pA, are shown below the voltage responses). Bidirectional electrotonic coupling was observed in this pair of neurones. Application of d.c. current steps in neurone 1 evoked a voltage response in both neurones although attenuated in neurone 2 (neurone 1 and neurone 2, left). The converse was also observed, where application of the current steps in neurone 2 evoked voltage responses in both neurones, but this time attenuated in neurone 1 (neurone 1 and 2, right). Note action potential firing in either neurone was associated with the appearance of spikelets in the other neurone, (from Landisman et al. 2002). (B) Fast prepotentials (FPPs) in an adult cat NRT neurone recorded intracellularly in vivo. FPPs evoked by application of depolarising current steps of increasing amplitude (0.6, 0.8, 0.9 and 1nA) from a hyperpolarised membrane (-90mV) potential are shown. Each trace is offset in order to clearly see the voltage response to each current step. Note that depolarising pulses of increasing amplitude triggered increasing numbers of all-or-none FPPs (1). From a more depolarised membrane potential (-85mV), FPPs could lead to the generation of action potentials indicating their ability to modify the output of NRT neurones. Current steps of increasing amplitude (0.2, 0.4, 0.6, 0.8 and 0.9nA) were applied. Note that with the smallest current pulse (lower trace) FPPs only are evoked, but FPPs leading to the generation of action potentials were evoked with larger current pulses (2). Neuronal activity was recorded under urethane anesthesia, (from Contreras et al. 1993).

been demonstrated to communicate electrotonically display dye-coupling (Logan et al. 1996; Galarreta and Hestrin 2001; Landisman et al. 2002). This may be due to methodological constraints or through the type of Cx involved, as it is thought that inhibitory intemeurones that are associated with Cx36 do not demonstrate dye-coupling (Galarreta and Hestrin 2001; Landisman et al. 2002). In addition GJ communication in various systems may be sensitive to a number of factors. In various systems, GJs are modulated by transjunctional voltage, intracellular acidification and a number of pharmacological agents. Agents known to reduce coupling include: 1) lipophiles such as arachidonic acid, octanol, halothane, oleic acid and carbenoxolone; 2) acidifiers such as weak acids like lactate or agents that disrupt oxidative phosphorylation like dinitrophenol or nigeriacin; 3) antibodies or peptides targeted towards a particular region of the connexin protein; and 4) other molecules such as glycyrrhetinic acids (Rozental etal. 2001).