Retinal Electrical Synapse Plasticity and Visual Performance

General Principles of Electrical Synapses and Plasticity

• Synaptic Composition: Nervous systems utilize both chemical and electrical synapses. Chemical synapses typically form amplifying feedforward or feedback connections in neural chains, transmitting signals of the same or opposite signs to define circuit functions. Electrical synapses, formed by gap junctions, generally transmit sign-conserving signals with gains less than $1$.

• Network Coordination: In homologous groups (same neuron type), electrical synapses coordinate activity to facilitate synchrony. In heterologous groups (different neuron types), they coordinate diverse neuron populations and form feedforward circuits allowing bidirectional signal propagation.

• Dynamic Plasticity: Plasticity is not limited to chemical synapses; electrical synapses change their coupling strength based on cellular activity, activity of connected cells, or activation of neurotransmitter and neurohormone receptors. This plasticity allows for the modification of the size of coordinated neuronal groups and can range from subtle changes in spike synchrony to complete circuit reconfiguration.

• Homeostasis and Evaluation: Baseline states of most gap junctions exhibit low phosphorylation, implying relatively low coupling with significant room for enhancement. Understanding the upper limit of physiological regulation is necessary to evaluate the role of electrical synapse plasticity in neural network tuning.

Phosphorylation as the Regulator of Connexin 36 (Cx36) Coupling

• Connexin 36 (Cx36): This protein, or its homologue, forms the majority of electrical synapses in the mammalian central nervous system. Its functional states are primarily determined by phosphorylation.

• Regulatory Sites: Multiple studies have identified four key phosphorylation sites in mouse Cx36:

  • Ser110 (Serine 110): Located in the cytoplasmic loop; targeted by Protein Kinase A (PKA) and Calmodulin-dependent protein kinase II (CaMKII).

  • Thr111 (Threonine 111): Located in the cytoplasmic loop; specifically phosphorylated by CaMKII.

  • Ser293 (Serine 293): Found in the C-terminal tail; corresponds to Ser276 in fish Cx35. It is phosphorylated by PKA and Nitric Oxide-regulated Protein Kinase G (PKG).

  • Ser315 (Serine 315): Located near the tip of the C-terminus; corresponds to Ser298 in fish homologues. It is potently phosphorylated by CaMKII and regulates the binding of PDZ-domain and 14-3-3 proteins.

• Regulatory Mechanism: Phosphorylation at these sites opens the gap junction channels, while dephosphorylation closes them. In many neurons and HeLa expression systems, PKA signaling activates Protein Phosphatase 2A (PP2A), which dephosphorylates Cx36, thereby reducing coupling.

Experimental Development of the Phosphomimetic Cx36-DEDD Mutant

• Phosphomimetic Rationale: Replacing phosphorylatable serine or threonine residues with acidic residues (Aspartic acid - Asp/D, or Glutamic acid - Glu/E) introduces a negative charge to mimic the phosphorylated state.

• Iterative Mutagenesis and Testing:

  • DTD/ETE Mutants: Cx36-S110D, S293D (DTD) and Cx36-S110E, S293E (ETE) were insufficient to significantly open channels or lock them against regulation.

  • Triple Mutants (DED and 3D): Combinations like Cx36-S110D, T111E, S293D (DED) and Cx36-S110D, T111D, S293D (3D) still failed to produce constitutively open channels.

  • S315D Mutation: Mutation of Ser315 alone significantly altered PKA regulation, where both inhibition and activation of PKA increased coupling compared to wild type (WT).

• The DEDD Mutant: The final saturated mutant, Cx36-S110D, T111E, S293D, S315D (abbreviated Cx36-DEDD), was found to be constitutively open. It displayed significantly high coupling in control conditions and resisted dephosphorylation driven by PP2A.

Validation of the Cx36-DEDD Mutant in Expression Systems

• HeLa Cell Tracer Coupling: Using Neurobiotin and scrape loading, Cx36-DEDD showed persistent coupling despite PKA activator (Sp-8-cpt-cAMPS) or inhibitor (Rp-8-cpt-cAMPS) treatments. In control conditions, its diffusion coefficient $k$ was significantly higher than WT ($p < 0.0001$).

• Resistance to Signaling Pathways: While glutamate (100 ̀μM) and glycine (100 ̀μM) stimulation (activating CaMKII) enhanced WT coupling, they had no effect on the already maximally-coupled Cx36-DEDD.

• Interpretation: Functional opening requires heavy phosphorylation; essentially all subunits in a gap junction must be fully phosphorylated for the synapse to be maximally active.

Generation and Anatomy of the Cx36-DEDD Conditional Knockin Mouse

• Targeting Strategy: The saturated mutant Cx36-DEDD was used for gene knock-in via homologous recombination at the second exon of the $(gjd2)$ gene. Initial direct knock-in attempts resulted in non-viable offspring, suggesting constitutive global expression disrupts vital brain function.

• Conditional Construct:

  • The construct features wild type Exon 2 followed by an artificial polyadenylation signal and flanked by LoxP sites.

  • Following the second LoxP site is the Cx36-DEDD mutant Exon 2, an Internal Ribosome Entry Sequence (IRES), and the tdTomato coding sequence.

  • Cre recombinase expression deletes the WT exon, allowing the transcript to splice into the DEDD mutant and express both the mutant protein and soluble tdTomato.

• Retinal Implementation: Crossing with Six3-Cre mice released the expression in retinal progenitor cells. This resulted in broad expression across the retina, including photoreceptors, bipolar cells, amacrine cells, and retinal ganglion cells (RGCs).

• Cell Identification: Bright tdTomato signals were observed in cells at the bottom of the inner nuclear layer (INL). These were identified as AII amacrine cells using an adeno-associated virus (AAV) with the synthetic HKamac promoter, which colocalized with tdTomato.

Physiological Effects of Cx36-DEDD on Neuronal Coupling

• Rod-Cone Junctional Conductance ($G_j$):

  • In Six3-Cre/Cx36(DEDD/DEDD) homozygous mice, the mean conductance was $1302 ± 251\,pS$.

  • In Six3-Cre controls, the mean was $375 ± 206\,pS$ ($p = 0.026$).

  • Contextual comparison: This value is similar to the maximum conductance observed with dopamine D2 receptor blockade ($1070\,pS$) and matches the theoretical maximum potential for mouse rod-cone pairs ($1228 ± 120\,pS$).

• AII Amacrine Cell Coupling:

  • Neurobiotin tracer injections in homozygous mice yielded a mean diffusion coefficient $k = 0.00049 ± 0.00015\,\text{cells}^2/\text{sec}$.

  • Heterozygous mice showed $k = 0.00013 ± 0.00004\,\text{cells}^2/\text{sec}$ ($p = 0.0045$).

Impact of Locked Electrical Synapses on Visual Performance

• Optomotor Response (OMR) Assessment: Image-forming visual function was evaluated using drifting vertical stripe patterns at a fixed speed of $12\,\text{degrees/sec}$ and mean luminance of $70\,\text{cd/m}^2$ ($82\,\text{lux}$).

• Photopic Visual Acuity:

  • Measured at $99.7\%$ contrast.

  • Acuity was significantly lower in homozygous Cx36-DEDD mice compared to Six3-Cre controls.

  • Heterozygotes displayed no significant difference in acuity.

• Photopic Contrast Sensitivity:

  • Measured at a spatial frequency of $0.1\,\text{cycle/degree}$.

  • Contrast sensitivity was profoundly reduced in DEDD homozygotes compared to control strains (C57Bl6 and Six3-Cre).

• Gender Analysis: There were no significant differences in visual acuity results between male and female mice across any genotype tested.

Mechanisms and Potential Pathological Applications of Electrical Synapse Plasticity

• Requirement for Uncoupling: Optimal photopic vision requires the reduction of electrical coupling in specific retinal circuits. Maintaining maximal coupling (the "nighttime" state) allows saturated rod signals to compromise cone function.

• Spatial Tuning: The reduction in photopic visual acuity suggests that Cx36 plasticity is necessary for tuning retinal ganglion cell spatial response profiles, likely by regulating inhibitory surrounds formed by interconnected amacrine and ganglion cells.

• Neurological Disorders: Excessive gap junction coupling is observed in pathological states:

  • Retinal Degeneration (rd1 model): Characterized by hyperactivity and oscillations in the AII amacrine-bipolar network, linked to excessive Cx36 phosphorylation.

  • Brain Injury: Traumatic and ischemic injuries can trigger excessive neuronal coupling, contributing to neuronal cell death.

  • Model Utility: The Cx36-DEDD mouse provides a tool to investigate the specific contribution of neuronal coupling to cell loss in neurodegenerative contexts.

Experimental Methodologies and Molecular Tools

• Tracer Diffusion Modeling: Tracer movement was quantified using a linear compartmental diffusion model where the rate-limiting step is defined by coefficient $k$.

• Recordings: Pairs of rods and cones were recorded using perforated-patch clamp configuration with $\beta$-escin ($25\,μM$). Junctional current was evoked by stepping the "driver" cell from $-50$ to $+50\,mV$ in $10\,mV$ increments while holding the "slave" cell at $-35\,mV$.

• Antibodies and Markers:

  • Anti-Cx36 (mouse, MAB3045 or 37-4600).

  • Anti-RFP (rabbit, ab62341) to boost tdTomato.

  • DAPI for nuclear counterstaining (ONL, INL, GCL).

• Genotyping Primers:

  • RL1/RL2: Produces a $431\,bp$ product for DEDD and $397\,bp$ for WT.

  • RL3/RL4: Produces a $290\,bp$ product for DEDD (WT: no product).

  • 63F2/CreR2: Produces a $405\,bp$ product for Six3-Cre transgene.