Information

Why do neurones use chemical signalling at synaptic junctions?


When a neurone fires, it sends an electrical signal that jumps down the axon via the nodes of Ranvier very rapidly. At a synaptic junction, chemical Brownian diffusion signalling with receptor surface proteins is relatively slow and is often exploited by venoms and susceptible to toxins (on the plus side it's the reason a lot of medical drugs work). It seems flawed for evolution to have selected for this rather than some alternative quicker and more direct electrical interface.

Question. Why are chemical synaptic interfaces used in higher organisms at the synaptic junction?


This is the coolest part. Those synapses are the reason the brain is so complex! Basically you've got the first part right, the neurones are quicker and they transmit messages from one end to the other. The other thing you have to do is analyse and calculate. Signals from multiple neurones feed into a single neurone using a chemical synapse. Similarly the converse is true, a single neurone can feed multiple synapses. So when we for example walk, receptors monitoring our balance can feed this to our conscious and unconscious mind, but I'm grossly simplifying this. What our brain does is take hundreds and thousands of ports of information, with where the chemical synapse is and the type of synapse and the receptors and a million other things just slightly changing the information until it's perfect and then send that information a million ways however it likes.

The most important thing is this allows inhibition. Most of the brain uses inhibitory GABA rather than activating signals. Furthermore, the system uses the delay created. Different transmitters act for different durations and send a different volume of signal. An impulse is all or nothing, it's of a fixed amplitude, however a chemical signal can be fine tuned. Neurones can not only interact to inhibit themselves or other neurones to increase the resolution of a signal etc. Conversely a system of just physical connections allows little regulation, in fact it'd be practically a seizure.

Does that make sense?


There are both chemical and electrical synapses in many organisms. The electrical synapses are called gap junctions.

As you point out, the primary advantage of gap junctions is their speed, and they are commonly used in systems involving defensive reflexes.

However, as AndroidPenguin indicates, chemical synapses allow for greater computational abilities (changing the gain, integrating multiple inputs, etc). Gap junctions are also disadvantageous because they often (but not always) transmit signals in both directions; chemical synapses tend to be more unidirectional (of course, there's always backpropagation).


There are indeed 'gap junctions' which pass current directly from one cell to the next. So what advantages do we get out of chemical synapses that gap junctions do not provide?

  1. Asymmetry. Synapses do not operate in reverse, thus the postsynaptic cell cannot generate currents in the presynaptic terminal (although there are secondary forms of communication which may operate in the reverse direction).
  2. Ion Selectivity. A synapse allows one set of ionic currents to be transformed into any other set. Thus sodium and calcium influx at the presynaptic terminal might translate to chloride influx to make the synapse inhibitory. Furthermore, the ability of the postsynaptic cell to control calcium levels is critical for plasticity mechanisms.
  3. Insulation. Gap junctions increase the conductance of the cell membrane, which can reduce the fidelity of signals as they propagate along the dendrite. In contrast, an inactive synapse might have virtually no effect on the postsynaptic cell--it does not pass currents.
  4. Kinetics. Because chemical synapses generate their own ionic currents, they are also free to modify the speed of those currents (how quickly they rise and fall). This is much more difficult to achieve with gap junctions.

There is a very long list of functionality provided only by synapses which I have not listed here, but I think most of those are things which could, in principle, also be achieved with gap junctions (for example: amplitude control, metabolic responses, short- and long-term plasticity).


Why do neurones use chemical signalling at synaptic junctions? - Biology

A subscription to J o VE is required to view this content. You will only be able to see the first 20 seconds .

The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.

If that doesn't help, please let us know.

Neurons communicate to each other and to other cells mainly through chemical signaling at synapses. These specialized regions are where the axon terminal of the presynaptic cell, the neuron sending the message, meets the postsynaptic cell receiving the message.

The signal consists of neurotransmitter molecules which are stored in the axon terminal within membrane-bound organelles called synaptic vesicles.

When an electrical signal, known as an action potential, occurs in the presynaptic neuron, it triggers these vesicles to fuse to the cell membrane. When the vesicles fuse, they release their neurotransmitter into the synaptic cleft, the narrow space between cells.

The neurotransmitter then diffuses across and binds to its postsynaptic receptors. This binding elicits a response in the postsynaptic cell, which, in this case, is a neuron, and an action potential may be produced. Ultimately, synaptic signaling allows neurons to transmit information to other cells, near and far.

6.7: Synaptic Signaling

Neurons communicate at synapses, or junctions, to excite or inhibit the activity of other neurons or target cells, such as muscles. Synapses may be chemical or electrical.

Most synapses are chemical. That means that an electrical impulse&mdashor action potential&mdashspurs the release of chemical messengers. These chemical messengers are also called neurotransmitters. The neuron sending the signal is called the presynaptic neuron. The neuron receiving the signal is the postsynaptic neuron.

The presynaptic neuron fires an action potential that travels through its axon. The end of the axon, or axon terminal, contains neurotransmitter-filled vesicles. The action potential opens voltage-gated calcium ion channels in the axon terminal membrane. Ca 2+ rapidly enters the presynaptic cell (due to the higher external Ca 2+ concentration), enabling the vesicles to fuse with the terminal membrane and release neurotransmitters.

The space between presynaptic and postsynaptic cells is called the synaptic cleft. Neurotransmitters released from the presynaptic cell rapidly populate the synaptic cleft and bind to receptors on the postsynaptic neuron. The binding of neurotransmitters instigates chemical changes in the postsynaptic neuron, such as opening or closing ion channels. This, in turn, alters the membrane potential of the postsynaptic cell, making it more or less likely to fire an action potential.

To end signaling, neurotransmitters in the synapse are degraded by enzymes, reabsorbed by the presynaptic cell, diffused away, or cleared by glial cells.

Electrical synapses are present in the nervous system of both invertebrates and vertebrates. They are narrower than their chemical counterparts and transfer ions directly between neurons, allowing faster transmission of the signal. However, unlike chemical synapses, electrical synapses cannot amplify or transform presynaptic signals. Electrical synapses syncronize neuron activity, which is favorable for controlling rapid, invariable signals such as the danger escape in squids.

Neurons can send signals to, and receive them from, many other neurons. The integration of numerous inputs received by postsynaptic cells ultimately determines their action potential firing patterns.

Kennedy, Mary B. &ldquoSynaptic Signaling in Learning and Memory.&rdquo Cold Spring Harbor Perspectives in Biology 8, no. 2 (February 2016). [Source]


Paracrine Signaling

Signals that act locally between cells that are close together are called paracrine signals. Paracrine signals move by diffusion through the extracellular matrix. These types of signals usually elicit quick responses that last only a short amount of time. In order to keep the response localized, paracrine ligand molecules are normally quickly degraded by enzymes or removed by neighboring cells. Removing the signals will reestablish the concentration gradient for the signal, allowing them to quickly diffuse through the intracellular space if released again.

One example of paracrine signaling is the transfer of signals across synapses between nerve cells. A nerve cell consists of a cell body, several short, branched extensions called dendrites that receive stimuli, and a long extension called an axon, which transmits signals to other nerve cells or muscle cells. The junction between nerve cells where signal transmission occurs is called a synapse. A synaptic signal is a chemical signal that travels between nerve cells. Signals within the nerve cells are propagated by fast-moving electrical impulses. When these impulses reach the end of the axon, the signal continues on to a dendrite of the next cell by the release of chemical ligands called neurotransmitters by the presynaptic cell (the cell emitting the signal). The neurotransmitters are transported across the very small distances between nerve cells, which are called chemical synapses. The small distance between nerve cells allows the signal to travel quickly this enables an immediate response.

Figure (PageIndex<1>): Synapsis: The distance between the presynaptic cell and the postsynaptic cell&mdashcalled the synaptic gap&mdashis very small and allows for rapid diffusion of the neurotransmitter. Enzymes in the synapatic cleft degrade some types of neurotransmitters to terminate the signal.


Neuromuscular Junction

A very important synapse in animal physiology is called the neuromuscular junction, This is the junction between motor neurons and skeletal muscle, so it is also called the myoneural junction. The following list outlines the sequence of events involved with synaptic transmission at the neuromuscular junction (seen in depth in the muscle chapter).

  1. An action potential is initiated in the motor neuron and spreads down the axon to the synaptic bulb.
  2. The synaptic bulb depolarizes.
  3. Ca ++ gates open in the neurolemma of the synaptic bulb.
  4. Ca ++ influx causes the exocytosis of ACh from the synaptic vesicles.
  5. The ACh diffuses across the synaptic cleft and binds with receptors in the sarcolemma.
  6. The ACh-receptor complex is formed.
  7. The ACh-receptor complex causes sodium gates to open in the sarcolemma.
  8. Na + ions diffuse into the sarcoplasm and the sarcolemma depolarizes. If threshold is reached, a muscle cell action potential will be generated.
  9. The enzyme acetylcholinesterase (ACh-E) is present in the synaptic cleft.
  10. The ACh-E hydrolizes the ACh (choline is reabsorbed by the presynaptic neuron and the acetyl group leaves the cleft but can be reused elsewhere), such that there is only one muscle stimulation for each neuron action potential.

Methods

C. elegans culture and strains

All worms were raised on agar plates with a layer of OP50 Escherichia coli at 21 °C inside an environmental chamber. The worm strains used in this study are listed in Table 1.

Gene expression pattern analyses

To confirm that lgc-46 and acr-14 are expressed in A-MNs, we coexpressed mStrawberry under the control of Punc-4 (A-MN-specific) and GFP under the control of either Plgc-46 or Pacr-14. To confirm that AVA interneurons are cholinergic neurons, we first created an integrated transgenic strain expressing GFP under the control of Punc-17 (ZW798), and then coinjected two plasmids, pNP259 (Pgpa-14::Cre) and wp1392 (Pflp-18::loxP::LacZ::STOP::loxP::mStrawberry) into the ZW798 strain. The use of Pgpa-14 and Pflp-18 in Cre-loxP recombination results in AVA-specific gene expression 30 . The expression patterns of GFP and mStrawberry were examined and imaged with an inverted microscope (TE-2000U, Nikon) equipped with specific fluorescence filters (HQ Texas Red/41004 and HQ FITC/41001, Chroma Technology Corp., Bellows Falls, VT, USA) and a CCD camera (F-View II, Olympus). Pflp-18 and Pgpa-14 were gifts from Alexander Gottschalk lab 30 . The other promoters, including Punc-4 (2.9 kb), Pacr-14 (3.8 kb), Plgc-46 (3.0 kb) and Punc-17(4.1 kb), were cloned using genomic DNA of the Bristol N2 strain as the template and the primers listed in Table 2.

Rescue experiments

lgc-46 and acr-14 mutants were rescued by expressing wild-type LGC-46 and ACR-14 under the control of either the native promoter or the A-MN-specific Punc-4. Full-length lgc-46 (Y71D11A.5) cDNA and acr-14 (T05C12.2) cDNA were cloned from a Bristol N2 cDNA library using the primers listed in Table 2.

RNA interference

Neuron-specific gene knockdown was achieved by coexpressing two plasmids encoding sense and corresponding antisense RNA fragment of a gene under the control of a specific promoter 62 . A-motor neuron-specific gene knockdown was achieved by using Punc-4, whereas AVA-specific gene knockdown through a Cre-LoxP approach using Pflp-18 and Pgpa-14 (ref. 30). The primers used for cloning the cDNA fragments of lgc-46 (585 bp), unc-17 (473 bp), unc-7 (485 bp) and unc-9 (397 bp) are listed in Table 2. One transgenic line of each kind was randomly chosen for electrophysiological and behavioural analyses.

Auxin-induced UNC-7 degradation

A DNA fragment encoding codon-optimized degron 41 was synthesized (GenScript, Piscataway, NJ, USA) and inserted into wild-type genome using the Crispr/Cas9 approach 42 to tag UNC-7 with degron at the C-terminus. The guide RNA sequence (5′-GGATGCGGAACACGGTCAA) targeting the last exon of unc-7 was inserted into pDD162 (Peft-3::Cas9+Empty sgRNA) (Addgene #47549) by site-directed mutagenesis. The resulting plasmid (wp1685) was coinjected with the transgenic marker Pmyo-2::mStrawberry (wp1613) into wild-type worms. Genome-edited worms were identified through PCR screen and DNA sequencing. The Cre-LoxP system was used to achieve AVA-specific degradation of UNC-7. The TIR1::mRuby sequence was amplified from pLZ31 (Peft-3::TIR1::mRuby::unc-54 3′UTR, pCFJ151) (Addgene #71720) and cloned into Pflp-18::loxP::LacZ::STOP::loxP::mStrawberry (wp1392) to generate Pflp-18::loxP::LacZ::STOP::loxP::TIR1::mRuby (wp1693). This plasmid was coinjected with Pgpa-14::Cre (pNP259) and Pflp-18::loxP::LacZ::STOP::loxP::mCherry::SL2::GFP (wp1383) into the UNC-7 degron strain. For auxin-induced UNC-7 degradation, L4 transgenic worms were transferred to NGM plates containing 1 mM auxin, and the adult worms were used for electrophysiological recording on the following day.

C. elegans electrophysiology

All electrophysiological experiments were performed with adult hermaphrodites. An animal was immobilized on a glass coverslip by applying Vetbond Tissue Adhesive (3M Company, St Paul, MN). Application of the glue was generally restricted to the dorsal anterior portion of the animal, allowing the tail to sway freely during the experiment. A short ( ∼ 300 μm) longitudinal incision was made along the glued region. After clearing the viscera by suction through a glass pipette, the cuticle flap was folded back and glued to the coverslip, exposing several ventral body-wall muscle cells, a small number of motor neurons anterior to the vulva, and some neurons in the head. The dissected worm preparation was treated with collagenase A (Roche Applied Science, catalogue number 10103578001, 0.5 mg ml −1 ) for 10–15 s and perfused with the extracellular solution for 5 to 10-fold of bath volume. Borosilicate glass pipettes were used as electrodes for voltage- and current-clamp recordings. Pipette tip resistance for recording from neurons was ∼ 20 MΩ whereas that for recording from body-wall muscle cells was 3–5 MΩ. Classical whole-cell configuration was obtained by applying a negative pressure to the recording pipette. Motor neurons were identified based on their anatomical locations whereas AVA based on GFP fluorescence from the expression of an integrated transgene, which was created using two promoters and the Cre-LoxP approach as described above, and crossed into various mutants. Spontaneous and evoked PSCs at the neuromuscular junction were recorded and analysed as previously described 63 . Voltage- and current-clamp experiments were performed with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) and the Clampex software (version 10, Molecular Devices). Data were filtered at 2 kHz and sampled at 10 kHz. Postsynaptic currents and exogenous neurotransmitter/agonist-induced whole-cell currents were recorded at a holding voltage of −60 mV. Exogenous neurotransmitters and agonists were applied by pressure-ejection through a glass pipette using a Picospritzer III (Parker Hannifin, Hollis, NH) with pressure set at 10 pSi and pulse duration at 30 ms. The extracellular solution contained (in mM) NaCl 140, KCl 5, CaCl2 5, MgCl2 5, dextrose 11 and HEPES 5 (pH 7.2). The pipette solution contained (in mM) 120 KCl, 20 KOH, 5 Tris, 0.25 CaCl2, 4 MgCl2, 36 sucrose, 5 EGTA, and 4 Na2ATP (pH 7.2) except for that used for recording PSC bursts from body-wall muscle cells, which contained (in mM) 6.8 KCl, 113.2 Kgluconate, 20 KOH, 5 Tris, 0.25 CaCl2, 4 MgCl2, 36 sucrose, 5 EGTA and 4 Na2ATP (pH 7.2).

Xenopus oocyte expression

Oocytes were obtained from wild-type South African clawed frogs (Xenopus Express, Brooksville, FL) following a protocol approved by the Institutional Animal Care and Use Committee of UConn Health. Capped cRNA of lgc-46 was synthesized using a T3 mMessage mMachine Kit (Life Technologies, Carlsbad, CA, USA), and injected into defolliculated oocytes ( ∼ 50 nl per oocyte at 1.0 ng nl −1 ) using a Drummond Nanoject II injector (Drummond Scientific, Broomall, PA, USA). Two-electrode whole-cell recordings were performed 4–5 days after cRNA injection using an oocyte clamp amplifier (OC-725C, Warner Instruments, Hamden, CT, USA) and the Clampex software. The holding voltage was −60 mV. Data were filtered at 1 kHz and sampled at 10 kHz. In each experiment, a single oocyte was placed in an oocyte perfusion chamber (AutoMate Scientific, Berkeley, CA, USA) containing ND96 solution (compositions in mM: 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 HEPES, pH 7.2). Candidate agonists were perfused into the oocyte perfusion chamber through a micro-manifold (AutoMate Scientific). An 8-channel perfusion controller (Valvelink8.2, AutoMate Scientific) was used for perfusion with its valves controlled by pClamp.

Ca 2+ imaging

Young adult worms of an integrated strain expressing GCaMP2 in body-wall muscle cells 54 were glued and filleted for imaging spontaneous fluorescence changes of body-wall muscle cells using an electron-multiplying CCD camera (iXonEM+885, Andor Technology, Belfast, Northern Ireland), a FITC filter set (59222, Chroma Technology Corp.), a light source (Lambda XL, Sutter Instrument, Novato, CA, USA), and the NIS-Elements software (Nikon). Images were acquired at 16 frames per second with 10–40 ms of exposure time (no binning) for 3 min. TTL signals from the camera were used to synchronize the recordings of Ca 2+ transients and PSCs.

Optogenetic stimulation

Worms of an integrated strain expressing channelrhodopsin-2 in command interneurons under the control of Pglr-1 (ref. 16) were grown to L1-L2 stage on standard worm culture plates, and then transferred to new plates either with or without (for negative control) all-trans retinal two days before the experiment. The retinal plates were prepared by spotting each plate (60-mm diameter with 10 ml agar) with 200 μl OP50 E. coli containing 2 mM retinal (R2500, Sigma-Aldrich). Photostimulation was applied through a 40X water immersion objective in 2-s pulses at 30-s intervals using a light source (Lambda XL with SmartShutter, Sutter Instrument) and a 470±20 nm excitation filter (59222, Chroma Technology Corp.). The measured light intensity at the specimen position was 6.7 mW mm −2 , which was sufficient to cause maximal evoked peak responses 16 .

Measurements of backward locomotion

Backward locomotion in response to head touch by a platinum wire was assayed with L4-stage worms. The number of dorsoventral tail swings in response to each touch was counted by the experimenter with a stereomicroscope (SMZ-800, Nikon). One investigator (B.C.) prepared the worm strains whereas another (P.L.) performed the locomotion assay without knowing the strain genotypes.

Chemicals

Acetylcholine (AC159170050, ACROS Organics), GABA (AC103280250, ACROS Organics), aspartate (11240, Sigma-Aldrich), ATP (A3377, Sigma-Aldrich), dopamine (H8502, Sigma-Aldrich), glutamate (49621, Sigma-Aldrich), octopamine (O0250, Sigma-Aldrich), serotonin (H9523, Sigma-Aldrich), tyramine (T2879, Sigma-Aldrich), nicotine (AC181420050, ACROS Organics), levamisole (AC187870100, ACROS Organics), choline (AC110295000, ACROS Organics), glycine (G46-1, Fisher Scientific), d-tubocurarine (T2397, Sigma-Aldrich), gabazine (S106, Sigma-Aldrich), and histamine (H7250, Sigma-Aldrich) were first dissolved in water to make frozen stock solutions (10 to 100 mM), which were diluted to final concentrations using extracellular solutions before use.

Data analyses

The duration and charge transfer of PSC bursts were quantified with Clampfit software (version 10, Molecular Devices) as previously described 16 . The frequency of PSC bursts was manually counted. Amplitudes of junctional current and membrane voltage were quantified based on the mean amplitude during the last 100 ms at each voltage step (250 ms) or current injection step (1,000 ms) using pClamp. Calcium imaging data were analysed as previously described 54 . Data graphing and statistical analyses were performed with Origin Pro 2015 (OriginLab Corporation, Northampton, MA). Data are shown as mean±s.e. Either ANOVA (with Tukey’s HSD test) or unpaired t-test was used for statistical comparisons as specified in figure legends. P<0.05 is considered to be statistically significant. The sample size (n) equals to the number of cells or cell pairs recorded, or the number of worms used in locomotion analyses.

Data availability

All data generated or analysed during this study are included in this published article and its Supplementary Information files.


Drugs that act in Synaptic Cleft

As said earlier, synaptic cleft acts as a site of
action of different drugs. These drugs include the following.

Curare drug

It is a drug that stops the action of acetylcholine at postsynaptic neuron. It is a non-depolarizing muscle relaxant that blocks the activation of acetylcholine receptors. It acts through the synaptic cleft and prevents the depolarization of post-synaptic neuron.

Strychnine

It is a poisonous drug acting mainly on motor neurons
in the spinal cord. It acts through the synaptic cleft and blocks the
activation of acetylcholine and glycine receptors causing uncontrolled muscle
spasm. It is used as a neurotoxin.

Morphine

It is a well-known pain killer and sedative drug. It acts through synaptic cleft and activates the mu-receptors on postsynaptic neurons.

Acetylcholine esterase inhibitors

These drugs decrease inhibit the acetylcholine enzyme present in the synaptic cleft. As a result, they prevent the degradation of acetylcholine. These drugs are classified as indirect-acting muscarinic agonists. They include physostigmine, pyridostigmine, neostigmine, etc.

Alcohol

Alcohol binds to GABAA receptors an
increase the inhibitory effects of GABA. It also acts through the synaptic
cleft.

Conclusion/Summary

Synaptic cleft is a space between two neurons, connecting them to one another forming a synapse.

It is bound on one side by pre-synaptic neuron and
have post-synaptic neuron on the other side. The presynaptic neuron is always
an axon terminal. Depending on the type of synapse, the post-synaptic neuron
may be

  • An axon, as in axo-axonic synapse
  • Dendrite in axo-dendritic synapse
    or
  • Cell body or soma, as in
    axo-somatic synapses

When a nerve impulse reaches the presynaptic terminal, it causes release of neurotransmitters into the synaptic cleft. These neurotransmitters diffuse through the synaptic cleft and bind to the receptors on post-synaptic neurons.

This causes the transmission of nerve impulses from pre-synaptic to post-synaptic neuron.

Functions performed by synaptic cleft include

  • Diffusion of neurotransmitters
  • Degradation of neurotransmitters
  • Regulation of nerve impulse
    transmission
  • Site of drug action

Alterations in nerve impulse transmission has been associated with a number of disorders including:


INTRODUCTION

Nervous system function is defined by connections between specific neurons. These links include chemical synapses that utilize neurotransmitters to evoke postsynaptic responses and gap junctions that regulate ion flow between coupled neurons. Although some progress has been made towards understanding the molecular basis of chemical synaptic specificity (Sanes and Yamagata, 2009 Shen and Scheiffele, 2010), little is known about how neurons choose partners for gap junction assembly (Bennett and Zukin, 2004 Hestrin and Galarreta, 2005). Both types of synapses are active in motor circuits that regulate body movements (Charlton and Gray, 1966 Westerfield and Frank, 1982 Van Der Giessen et al., 2008 Li et al., 2009). The key role of transcription factor codes in motor circuit neuron fate suggests that genetic programs define the specificity of these connections (Briscoe et al., 2000 Shirasaki and Pfaff, 2002). Downstream targets with roles in synaptic specificity are largely unknown but probably include a combination of diffusible cues and cell-surface proteins that regulate synaptogenic responses (Pecho-Vrieseling et al., 2009).

Wnt signaling functions as a key regulator of synaptic assembly in the brain and at the neuromuscular junction (Budnik and Salinas, 2011). For example, in cerebellar neurons, Wnt7a activates a cytoplasmic pathway that promotes local assembly of presynaptic components whereas Wnt-dependent synaptic assembly at the Drosophila neuromuscular junction can also depend on transcriptional regulation (Packard et al., 2002 Ahmad-Annuar et al., 2006 Ataman et al., 2006 Miech et al., 2008). Wnts might also function as antagonistic cues to limit synapse formation (Inaki et al., 2007 Klassen and Shen, 2007) and, in at least one case, adopt opposing roles that either promote or inhibit synaptogenesis (Davis et al., 2008). Although multiple members of the Wnt family are expressed in the developing spinal cord and have been shown to regulate axon trajectory and neuron fate, explicit roles in synaptogenesis have not been uncovered (Lyuksyutova et al., 2003 Liu et al., 2005 Agalliu et al., 2009). Here, we describe our finding that opposing Wnt signaling pathways regulate the specificity of synaptic inputs in a nematode motor circuit.

In C. elegans, backward movement depends on connections between AVA interneurons and VA class motor neurons, whereas forward locomotion requires AVB input to VB motor neurons ( Fig. 1 ) (Chalfie et al., 1985 Ben Arous et al., 2010 Haspel et al., 2010). The specificity of these connections is controlled by the UNC-4 homeodomain transcription factor, which functions in VA motor neurons (Miller et al., 1992). In unc-4 mutants, AVA inputs to VAs are replaced with gap junctions from AVB and backward locomotion is disrupted. The characteristic anterior polarity of VA motor neurons is not perturbed, however, which suggests that UNC-4 regulates the specificity of synaptic inputs but not other traits that distinguish VAs from sister VB motor neurons (White et al., 1992 Miller and Niemeyer, 1995). UNC-4 functions as a transcriptional repressor with the conserved Groucho-like protein UNC-37 to block expression of VB-specific genes (Pflugrad et al., 1997 Winnier et al., 1999) ( Fig. 3 ). We have shown that one of these VB proteins, the HB9 (MNX1) homolog CEH-12, is sufficient to rewire VA motor neurons with VB-type inputs (Von Stetina et al., 2007b). Thus, these findings revealed a regulatory switch in which differential expression of the transcription factors, UNC-4 versus CEH-12, in VAs results in alternate sets of presynaptic inputs. This mechanism, however, shows regional specificity along the length of the ventral nerve cord. Ectopic expression of ceh-12 in unc-4 mutants is limited to posterior VA motor neurons and VA input specificity in this location depends on ceh-12. These findings suggest that UNC-4 might regulate multiple targets that function in parallel to specify inputs to selected VA motor neurons in different ventral cord domains (Von Stetina et al., 2007b). Here, we report the discovery that ceh-12 expression in posterior VA motor neurons is activated by a specific Wnt protein, EGL-20, that is secreted from adjacent cells in this region. We propose that UNC-4 normally prevents VAs from responding to EGL-20 by antagonizing a canonical Wnt signaling pathway utilizing the Frizzled (Frz) receptors MOM-5 and MIG-1. We have also identified a separate Wnt pathway, involving the Frz receptor LIN-17 and the Wnt ligands LIN-44 and CWN-1, that preserves VA inputs by blocking CEH-12 expression in anterior VAs. Our results have uncovered a key role for the UNC-4 transcription factor in modulating the relative strengths of Wnt signaling pathways with opposing roles in synaptic choice. The widespread occurrence of regional Wnt signaling cues in the developing spinal cord could be indicative of similar functions for transcription factors in regulating synaptic specificity in the vertebrate motor circuit.

Diagram of the C. elegans motor neuron circuit. Interneurons from the head and tail extend axons into the ventral nerve cord to synapse with specific motor neurons. The forward circuit (red) includes AVB and PVC interneurons and DB (not shown) and VB motor neurons. The backward circuit (blue) includes AVA, AVD and AVE interneurons and DA (not shown) and VA motor neurons. VAs and VBs arise from a common progenitor but are connected to separate sets of interneurons (AVA and AVB shown for simplicity).

UNC-4 regulates connectivity in the motor neuron circuit. (A) UNC-4 functions with co-repressor UNC-37 to block expression of the VB gene CEH-12 and to preserve VA-type inputs (blue). (B) De-repression of CEH-12 in posterior VAs in unc-4 and unc-37 mutants results in the mis-wiring of VAs with VB-type inputs (red) and disrupted backward locomotion. EGL-20 promotes CEH-12 expression. (C) Mutations in ceh-12 eliminate ectopic connections with AVB and partially suppress the Unc-4 phenotype. (D-F) Locomotion assays. ‘Unc’ animals cannot crawl backward. ‘Suppressed’ worms show detectable backward movement. ceh-12(gk391) and egl-20(n585) mutants strongly suppress the Unc-4 phenotype of hypomorphic alleles unc-4(e2323) and unc-37(e262) (D) and unc-4(e2322ts) (E). A mig-1(e1787) mutation partially suppresses Unc-4 movement (E). ceh-12 and egl-20 mutants partially suppress the null allele unc-4(e120) (F). * Pπ.05, ** Pπ.01, *** Pπ.0001. § Pπ.05 versus unc-4 egl-20. n�. N.S., not significant.


Types of Cell Signaling

The sequence of cell signaling remains is the same for most cell but depending on the distance separating two communicating cells, cell signaling can be classified into several different categories.

Cell signaling can be broadly classified into intracellular signaling and intercellular signaling. Intracellular signaling occurs within the cell in response to internal and external stimuli. In other words, it is simply when a cell is talking to itself and working independently. On the other hand, intercellular signaling is where a cell talks to the other cells of the body. In many cases, signaling might involve cells talking to themselves and to others in order to generate a response.

In the case of intercellular signaling, the type of signal can be further classified on the basis of the distance travelled.

Types of intracellular signaling (Photo Credit : CNX OpenStax/Wikimedia Commons)

Autocrine Signaling: Sometimes cells can produce signaling molecules that bind to receptors on their own membrane. In this way, it&rsquos possible for cells to send messages to themselves! Although it sounds strange, autocrine signaling is essential during development, as it ensures correct cell division and maintenance of cell identity. Think of it as setting reminders and writing notes to yourself it may seem weird, but it&rsquos necessary sometimes.

Direct-Contact Signaling: Some cells lie very close to each other and are in direct contact. Such cells have passages that connect them. For example, gap junctions in animal cells and plasmodesmata in plant cells are such passages that connect neighbouring cells. Signaling molecules can easily pass through these passages. This feature enables a group of cells to respond to a signal received by just one cell.

Paracrine Signaling: This form of communication takes place between cells that are near each other, but are not connected. In this case, the cells talk via the diffusion of chemical signaling molecules across short distances. Synaptic signaling between neurons (brain cells) is an example of paracrine signaling. Neurons release signaling molecules called neurotransmitters in the gap between themselves and the next neuron. This gap is known as a synapse. Hence, synaptic signaling allows our brain and central nervous system to work together by sending messages across multiple neurons.

Synaptic Signalling (Photo Credit : CNX OpenStax/Wikimedia Commons)

Endocrine Signaling: This is a method employed by cells that are far apart from each other . Like a package that is shipped internationally, signaling molecules may travel through the bloodstream to reach the target cell. Such molecules are called hormones . For example, the hormone adrenaline, released by the adrenal gland present atop the kidneys, is the fight or flight hormone. Adrenaline is released under stress and is responsible for increasing heart rate and blood pressure, redistributing blood to muscles, ramping up glucose production and much more. Hence, this hormone travels throughout the body from the adrenal gland via the bloodstream to the cardiac muscles to increase pumping, as well as to the liver for glucose production.


Results

Loss of Bbs proteins affect principal neuron dendritic morphology

Given that primary cilia are required for the formation of neuronal dendrites [21], we investigated the effect of loss of ciliary Bbs proteins on the dendritic morphology of principal neurons of Bbs mouse models. We measured dendritic length, spine count, and spine density of dentate gyrus (DG), basolateral amygdala (BLA), and layer V pyramidal neurons of the frontal cortex using a Golgi-Cox impregnation method. We found that the total spine density was reduced by 55% in DG granule cells of P42 old Bbs4 −/− mice (Fig 1A and 1B and S1 Video). Total spine density on the basal and apical dendrites (further referred to as basal and apical spine density) of layer V neurons was reduced by 55% and 54% (Fig 1A and 1E), respectively, and total basal and apical spine density of BLA neurons was reduced by 23% and 22%, respectively (Fig 1A and 1J). Sholl analysis revealed a significant reduction in spine density of all branches and per 30-μm interval in DG with the exception of the most distal branch and a 300-μm circle in Bbs4 −/− mice (Fig 1C and 1D). Similar Sholl analysis results were found in apical and basal dendrites of Layer V neurons, where dendritic spine density per branch order and per 30-μm interval was affected (Fig 1F–1I). Apical and basal BLA dendrites of Bbs4 −/− mice revealed unequal patterns in spine reduction affecting only a few branches and concentric circles (Fig 1K–1N). A number of dendritic intersections in DG, Layer V, and BLA neurons were not affected when compared with control mice (S1A–S1E Fig). Total dendritic length was reduced by 48% in DG neurons and by 25% in basal dendrites of layer V cortex neurons in Bbs4 −/− mice. Change in the length of apical dendrites of layer V cortex neurons in Bbs4 −/− mice was not statistically significant. BLA apical and basal dendrites showed a statistically significant length reduction of 14% and 19%, respectively (S1F–S1J Fig). Overall, these data show significant aberrations in dendritic morphology in the Bbs mouse model.

(A) Representative images of Golgi-impregnated DG, BLA, and Layer V pyramidal neurons of P42 Bbs4 −/− and Bbs4 +/+ mice (100x scale bar, 5 μm). (B-D) Spine density of DG neurons. (B) Total spine density. (C) Spine density per branch order. (D) Spine density per 30-μm interval. (E-I) Spine density of layer V pyramidal neurons. (E) Total spine density. (F) Spine density in apical dendrites per branch order. (G) Spine density in basal dendrites per branch order. (H) Spine density in apical dendrites per 30-μm interval. (I) Spine density in basal dendrites per 30-μm interval. (J-N) Total spine density of BLA. (J) Total spine density. (K) Spine density in apical dendrites per branch order. (l) Spine density in basal dendrites per branch order. (M) Spine density in apical dendrites per 30-μm interval. (N) Spine density in basal dendrites per 30-μm interval (Nmice/WT = 5 Nmice/KO = 7, Ncells/WT = 25, Ncells /KO = 35, mean ± SD, ***P < 0.001 **P < 0.01 *P < 0.05). One-way ANOVA, Tukey post hoc test except for B, E, J, where unpaired t test was used. Underlying data are available in S1 Data. Bbs4, Bardet-Biedl syndrome 4 BLA, basolateral amygdala DG, dentate gyrus KO, knockout ns, not significant WT, wild type.

To determine when the dendritic architecture of Bbs4 −/− DG neurons begins to change, we analysed dendritic length and spine density per branch order and per 30-μm interval at E19.5 and P21. The results of P21 were similar to those obtained at P42: we observed a significant reduction in dendritic length and spine density and no significant changes in a number of dendritic intersections (S2A–S2F Fig). By contrast, at E19.5, the density of dendritic filopodia (dendritic protrusions on developing neurons) in Bbs4 −/− DG neurons were not affected. However, the dendritic length was significantly reduced at E19.5 (S2A and S2G–S2K Fig). Taken together, the Bbs4 −/− murine model shows a progressive decrease in dendritic spine density at P21 (38%) and P42 (55%), but not at late embryonic stages (S2L Fig).

To investigate whether similar dendritic abnormalities can be detected in other Bbs models, we analysed the DG dendrite morphology of P21 Bbs5 and Bbs1 M390R models. Notably, loss of the Bbs5 protein led to significant reduction in DG dendritic length (34%) and overall spine density (32%) in dentate granule cells of knockout mice (S3A–S3C Fig). Sholl analysis also revealed abnormal spine density in Bbs5 −/− mice, with a significant spine reduction from the second to fifth branch order and from the 60-μm to 150-μm interval, respectively (S3D–S3F Fig). Interestingly, Bbs1 M390R/M390R was associated with consistent but marginal abnormalities in spinogenesis of DG neurons showing only a 10% reduction in the total spine density. However, dendritic length was not affected (S3A and S3G–S3K Fig). This finding is in agreement with our clinical observations that BBS1 M390R patients have the mildest cognitive phenotype.

To determine whether specific subtypes of spines were overrepresented on DG neurons of Bbs4 −/− mice, we analysed spines based on their size and shape (S4A Fig) [22]. We observed that total spine count was reduced in all spine subtypes except ‘branched’ spines. However, when the reduction of dendritic length of Bbs4 −/− neurons was taken into account, we found that only the density of ‘thin’ spines was significantly reduced (22%) (S4B–S4E Fig).

Reduced contextual and cued fear memory but no impairment in anxiety-like behaviour in Bbs4 −/− mice

Hippocampus, amygdala, and prefrontal cortex are structures involved in learning, memory, and social interaction. To investigate whether the loss of dendritic spines of DG, BLA, and prefrontal cortex neurons correlates with behavioural changes in Bbs4 −/− mice, we performed a set of behavioural tests. Bbs mice are known to develop a number of defects, including visual impairment and obesity [23]. To minimise the effect of these confounding factors, we performed the tests in younger mice (8 weeks). According to the majority of the literature and our own assessments, retinal degenerative changes in this Bbs4 model begin to develop at 7–8 weeks, making visual impairment unlikely to account for the differences in the test. Similarly, obesity should not confound our results, as at this age there are no weight differences in Bbs4 −/− and Bbs4 +/+ mice. To assess fear memory, we performed contextual and cued fear conditioning tests. In the conditioning session at Day 1, freezing behaviour and distance travelled during the first 150 seconds without introducing a conditioned stimulus (tone) and unconditioned stimulus (footshock) were used to evaluate baseline activity in the novel environment of the contextual fear experiment. The loss of Bbs4 did not affect the percentage of freezing or distance travelled in baseline activity of Bbs4 −/− male and female mice. However, after introduction of the paired tone-foot shock stimulus at Day 1, post hoc analysis revealed a significant decrease in the percentage of freezing and an increase in the distance travelled on Day 2 of Bbs4 −/− female mice compared with female control mice. Percentage of freezing and an increase in distance travelled were not statistically significant in male mice (Fig 2A, 2B and 2D).

(A) Schematic presentation of the contextual and cued fear conditioning test. At Day 1, mice were placed in the fear conditioning chamber for 616.6 seconds. After 150 seconds, a 5-second tone is played, followed by a 0.5-second, 0.5-mA shock. The tone and shock are repeated two more times at 150-second intervals. At Day 2 mice were placed in exactly the same chamber for 300 seconds without tones or shocks. After 4 hours (Day 2), mice are placed in the altered context and left for 180 seconds. At 180 seconds, a 5-second tone is played, which is repeated twice at 60-second intervals. The first 150 seconds of the conditioning trial were used as a baseline for the context data. The first 180 seconds in the altered context were used as the baseline for the cue data. (B) Freezing (%) in the contextual memory test. (C) Freezing (%) in the cue memory test. (D) Distance travelled (cm) in the conditioning test, context test, and cued test (females: NWT = 11, NKO = 11 males: NWT = 13, NKO = 12 mean ± SD, ***P < 0.001 **P < 0.01 *P < 0.05). One-way ANOVA, Tukey post hoc test. # It was noted that there was a significant level of reduction of percent time freezing and distance travelled in Bbs4 −/− mice when unpaired, two-tailed t test (P < 0.05) was used. Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4 KO, knockout WT, wild type.

Altered context at Day 2 before the introduction of the tone was used as a baseline for the cue data. The data revealed that there was no significant change in the percentage of freezing in altered context baseline activity (Fig 2C and 2D). During the cued conditioning session with the tone (Day 2, 180–360 seconds), the percentage of freezing was significantly reduced, and distance travelled increased in Bbs4 −/− male but not in female mice (Fig 2C and 2D). This set of results indicates that loss of Bbs4 protein affects contextual and cued fear memory in a gender-task–dependent manner.

Miniature excitatory postsynaptic currents amplitude is increased in Bbs4 −/− DG neurons

The morphology of dendritic spines is highly dynamic, and their formation and maintenance depend on synaptic function and neuronal activity [24]. To assess synaptic and neuronal function in a Bbs model, we measured intrinsic and synaptic properties of hippocampal granule cells of 3–4-week-old Bbs4 −/− and Bbs4 +/+ mice in acute hippocampal slices. We found that intrinsic properties of Bbs4 −/− neurons were unaffected compared with age-matched control littermates (Fig 3A–3C). To evaluate the synaptic properties of granule cells in these two groups, we measured miniature excitatory postsynaptic currents (mEPSCs). Notably, while the frequency of mEPSCs was not different between the two groups, mEPSC amplitudes were significantly larger in Bbs4 −/− neurons (Fig 3D–3F). These data make it unlikely that decreased neuronal activity underlies diminished spine density. On the contrary, the observed increase in mEPSC amplitudes suggests an activation of compensatory mechanisms at the presynaptic and/or postsynaptic sites in response to spine loss.

(A) Comparison of firing patterns in response to current injections during current clamp recordings from hippocampal granule cells. (B) Bar graphs summarising passive membrane properties. No significant differences were found between Bbs4 −/− and Bbs4 +/+ mice in input resistance (left), membrane time constant (middle), and resting membrane potential (right). (C) Firing frequency was plotted against current injection amplitudes. No significant differences were found between Bbs4 −/− and Bbs4 +/+ mice. (D) mEPSCs were recorded from hippocampal granule cells in Bbs4 −/− and Bbs4 +/+ mice (N = 6). (E) Cumulative probability plot comparing mEPSC amplitudes between Bbs4 −/− and Bbs4 +/+ mice. mEPSC amplitudes in granule cells of Bbs4 −/− mice are significantly larger (N = 6, P < 0.05, Kolmogorov-Smirnov test). (F) Cumulative probability plot comparing inter-event intervals (IEIs) of mEPSCs between Bbs4 −/− and Bbs4 +/+ mice (N = 6 P = 0.27, Kolmogorov-Smirnov test). Underlying data are available in S3 Data. Bbs4, Bardet-Biedl syndrome 4 IEI, inter-event interval KS, Kolmogorov-Smirnov mEPSC, miniature excitatory postsynaptic current.

IGF-1R downstream signalling is dysregulated in Bbs4 −/− synaptosomes

A number of tyrosine kinase receptors (RTKs), including IGF, RET, TrkB, PDGF, and EphB are known to enhance dendritic growth and promote the formation and maintenance of dendritic spines [7,25]. To assess the signalling of RTK in the synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice, we quantified the phosphorylation level of RTKs using a Phospho-RTK Array. Given that dendritic spine loss occurs between P1 and P21 and to capture the initial signalling changes before potential compensatory mechanisms may start taking place, we used synaptosomal fractions of P7 mice. Synaptosomal fractions from Bbs4 −/− and Bbs4 +/+ mice were incubated with the membrane containing immobilised RTK antibodies followed by detection of RTK phosphorylation by a pan anti-phospho-tyrosine antibody. Interestingly, phosphorylation levels of a number of RTKs were altered, including insulin and IGF1 receptors (Fig 4A and S5 Fig). We focused on IGF-1R/insulin receptor (IR) signalling, as it is known to have a profound effect on neuroplasticity in the CNS [7–9]. Pull-down experiments confirmed that phosphorylation of IGF-IR/InsulinR was decreased in the P7 enriched synaptosomal fraction of Bbs4 −/− mice (Fig 4B). Additionally, phosphorylation levels of Akt, a downstream target of canonical IGF signalling, were significantly reduced (Fig 4B). Next, we tested the phosphorylation level of insulin receptor substrate P53 (IRS p58), an adaptor protein that is phosphorylated by IR and IGF-1R [26]. Interestingly, IRS p58 protein has previously been shown to be highly enriched in the PSD of glutamatergic synapses, highlighting the role of this protein in neurons [27]. We found that phosphorylation of IRS p58 was significantly reduced in synaptosomal fractions of P7 Bbs4 −/− mice (Fig 4B). Furthermore, as the activities of IGF-1R and IRS p58 depend on interaction with Rho family GTPases [28, 29], we investigated the activities of Rac1 and RhoA GTPases. We observed that activity of RhoA was increased and, concurrently, Rac1 activity was decreased in the enriched synaptosomal fraction of P7 Bbs4 −/− mice (Fig 4C and 4D). We next assessed whether dysregulation of IGF-1 signalling in Bbs −/− mice affects the levels of N-methyl-D-aspartate (NMDA) and Alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid (AMPA) receptors in the total and synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice by western blotting. We observed a significant increase in the level of NMDA and AMPA receptors in the synaptosomal fraction of P7 Bbs4 −/− mice, whereas no changes in the receptors’ levels in the total brain fraction were detected (Fig 4E). These data are consistent with our previous findings of increased mEPSC amplitudes in Bbs4 −/− neurons (Fig 3E), suggesting a compensatory mechanism in response to spine loss.

(A) Phospho-RTK array reveals significant decrease in phosphorylation of insulin and IGF1 receptors in Bbs4 −/− (N = 2, mean ± SD). (B) Pull-down analysis shows aberrant IGF-1R downstream signalling. Bbs4 −/− and Bbs4 +/+ enriched synaptosomal fractions were incubated with mouse anti-phosphotyrosine antibody overnight, followed by incubation with Dynabeads M-280 for 2 hours. Immunoblotting analysis of the proteins eluted from the beads was performed using anti-IGFR/InsR, anti-Akt, and anti-IRS p58 antibodies. Input: the total brain protein fraction before the incubation with anti-phosphotyrosine antibody, which indicates the total level of IGFR/InsR, Akt, and IRS p58 in Bbs4 −/− and Bbs4 +/+ mice. (C, D) RhoA and Rac1 G-LISA Activation Assays. Levels of activated RhoA (c) and Rac1 (d) were measured in the total brain extracts and enriched synaptosomal fraction of Bbs4 −/− and Bbs4 +/+ mice (N = 3, mean ± SD unpaired t test). (E) Representative image of western blot analysis of NMDA and AMPA receptors levels in the total brain extract and enriched synaptosomal fraction of Bbs4 −/− and Bbs4 +/+ mice. (F) Representative western blots of autophagy markers LC3-II and p62 in the synaptosomal fractions of Bbs4 −/− and Bbs4 +/+ mice at P1, P7, P14, and P21 (N = 3, mean ± SD). LC3-I is a cytosolic form of LC3. LC3-II is a LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. LC3-II and p62 levels were quantified by measuring western blot band intensities using the Image J programme (N = 3, mean ± SD, unpaired t test). Housekeeping genes (actin, GAPDH, etc.) could not be used as normalisation controls due to the changes in their gene expression levels in Bbs4 −/− mice (our unpublished observations). (G) Measurement of oxidative phosphorylation (OXPHOS) complex activities in the whole brain homogenates of Bbs4 −/− and Bbs4 +/+ mice. Units: mU:U CS raw data were normalised to citrate synthase N = 4, mean ± SD ns, not significant unpaired t test. Underlying data are available in S2 Data. AMPAR, alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid receptor NMDAR, N-methyl-D-aspartate receptor Bbs4, Bardet-Biedl syndrome 4 CI, mitochondria complex I CII, mitochondria complex II CIII, mitochondria complex III CS, citrate synthase, GAPDH, Glyceraldehyde 3-phosphate dehydrogenase GluR, glutamate receptor IGF-1R, insulin-like growth factor receptor InsR, insulin receptor LC3, microtubule-associated protein 1A/1B-light chain 3 LC3-I, cytosolic form of LC3 LC3-II, LC3-phosphatidylethanolamine conjugate recruited to autophagosomal membranes OXPHOS, oxidative phosphorylation RTK, tyrosine kinase receptor SCC, succinate:cytochrome c oxidoreductase (= complex II + III combined) WB, western blot.

One of the possible mechanisms of dendritic spine pruning is macroautophagy [18], a process that is tightly regulated by IGF-1R signalling and small GTPases. To test whether autophagy is dysregulated in our Bbs model, we analysed the level of autophagy markers LC3-II and p62 in the enriched synaptosomal fractions isolated from Bbs4 −/− and Bbs4 +/+ mice brains at different postnatal stages. We observed a significant increase in LC3-II level at P1 and P7 of Bbs4 −/− mice (Fig 4F). Given the widely recognised notion that the level of p62 correlates inversely with autophagy, it was unexpected to see an increase in p62 in P1 synaptosomes in our experiment. However, it is in line with reports that p62 levels can be up-regulated during high autophagic flux due to a multifunctional role for p62 [30]. To exclude mitochondrial dysfunction and oxidative stress as triggers of autophagic induction [31], we assessed the activities of respiratory chain complexes I, II, III, IV and succinate:cytochrome c oxidoreductase (SCC complex II and III combined) in total brain homogenates of P7 Bbs4 −/− and Bbs4 +/+ mice. Oxidative phosphorylation (OXPHOS) complex activities were determined, and the results were normalised to the activity of citrate synthase (CS). We found no significant differences in the activities of OXPHOS between Bbs4 −/− and Bbs4 +/+ mice (Fig 4G), thus ruling out mitochondrial dysfunction as a cause of autophagy in BBS. Together, our findings suggest that aberrant IGF-1 signalling may lead to dysregulation of various cellular pathways that are known to control dendritic spine morphology and plasticity.

Synaptic localisation of BBS proteins

The role of Bbs proteins in the regulation of primary cilia has been recently broadened by studies showing that Bbs proteins are involved in microtubular stabilisation, actin remodelling, transcriptional regulation, and endosomal trafficking [16,17,32]. Taking into account this broad spectrum of Bbs functions as well as our current results elaborating the role of Bbs, such as reduction in dendritic spine density along with aberrant synaptic IGF receptor signalling and altered neurotransmitter receptor levels (NMDA and AMPA), we hypothesised that Bbs proteins may play a vital role in neuronal synapses. Re-evaluation of our earlier mass spectrometric analyses of synaptosomal [33,34] and crude synaptosomal fractions [35] of the rat cortex, dorsal striatum, and DG revealed the presence of Bbs1, Bbs2, Bbs4, Bbs5, Bbs7, and Bbs10 proteins (S1 Table).

To elaborate on synaptic localisation of BBS proteins biochemically, we enriched the cytosolic, detergent-soluble synaptosomal (DSS pre-synapse enriched), and PSD fractions of synaptosomal preparations from adult rat hippocampi using a previously described method (S6 Fig) [36]. Label-free MS1 intensity-based LC-MS quantitation revealed a high abundance of Bbs1, Bbs2, Bbs5, and Bbs9 proteins in the PSD fraction, whereas Bbs7 was present mostly in the cytosolic fraction (Fig 5A and 5B). A low level of Bbs4 protein was also unambiguously identified in the PSD fractions (Fig 5A and 5B). Immunofluorescence analysis of Bbs4 and Bbs5 localisation confirmed the presence of Bbs punctae throughout the entire dendritic tree of mouse dissociated hippocampal neurons (Fig 5C and S7A–S7C Fig). Collectively, these data clearly indicate the presence of Bbs proteins in neuronal processes and PSDs.

(A) Proteomic profile of the BBS proteins in biochemical fractions using nano-LC-MS/MS analysis. Protein levels of Bbs proteins and synaptic markers were estimated by label-free LC-MS analyses from following biochemical fractions of the rat hippocampi: cytosolic, detergent-soluble synaptosomal preparation (DSS, pre-synapse enriched), and postsynaptic density preparation (PSD). Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (B) The protein abundances illustrated in the heat map are obtained from the total MS1 peptide intensities scaled to the mean of all the samples. Protein levels of the presynaptic (VGLU1, SYP) and postsynaptic (NMDAR1, PSD95) protein markers are enriched in DSS and PSD preparations, respectively. (C) Representative image of immunolabelling of Bbs proteins. Cultured mouse hippocampal neurons at low density were immunolabelled with Bbs4 and Bbs5 antibodies (red), phalloidin (green), and beta-III tubulin (green) after 6 days in vitro (DIV6). Scale bar, 20 μm (top panel) and 5 μm (bottom panels). Underlying data are available in S4 Data. BBS, Bardet-Biedl syndrome DIV6, six days in vitro hippocampal culture DSS, detergent-soluble synaptosomal LC-MS/MS, liquid chromatography- tandem mass spectrometry MS, mass spectrometry NMDAR1, N-methyl-D-aspartate receptor 1 PSD, postsynaptic density SYP, synaptophysin TUBB3, β-tubulin III encoded by TIBB3 gene VGLU1, vesicular glutamate transporter 1.


ELI5: Why do neurons connect as synapses instead of physically connecting to each other?

The synapses you're thinking of - as do most people - are called chemical synapses. As you know, between communicating neurons there is a small gap called a synaptic cleft. Neurotransmitters are small chemicals that leave one neuron (the PREsynaptic neuron) and move across the synaptic cleft to alter the state of the next neuron (the POSTsynaptic neuron). The cells do not touch for a few reasons.

All animal cells, including neurons, have a barrier surrounding them called a plasma membrane. Neurotransmitters in the presynaptic neuron leave in a complex process where balls with neurotransmitters inside (called vesicles) fuse with this plasma membrane and release the chemicals. These then move across the synapse and interact with the outside of the postsynaptic cell at specific reception points. If the postsynaptic cell was touching the presynaptic cell, neurotransmitters would not often be in the right place to interact with these receptors.

You may imagine that there would not be a membrane between the two neurons, but this would not allow differentiation of the kinds of signals neurons send. Some neurons have neurotransmitters that excite the postsynaptic neuron, while some stop it from activating or have a different effect. If two neurons were a continuous cell, when one cell activated, the electric charge would travel to the next. This maybe could work in some neurons, but not in neurons that stop the next neuron from activating.

tldr if neurons touched they wouldn't be able to send signals to other neurons.