Kainate receptor pore‐forming and auxiliary subunits regulate channel block by a novel mechanism

Key points

• Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion‐channel block by facilitating blocker permeation.
• Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α‐helical region.
• Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism.
• Our findings have broad implications for work on polyamine block of other cation‐selective ion channels.

Abstract

Channel block and permeation by cytoplasmic polyamines is a common feature of many cation‐selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α‐helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation‐selective ion channels.

Abbreviations

iGluR

ionotropic glutamate receptor

KAR

kainate receptor

l‐Glu

l‐glutamate

MD

molecular dynamics

POPC

1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine

Put

putrescine

RMSD

root mean square deviation

SMD

steered molecular dynamics

Spm

spermine

Spd

spermidine

     

Signalling beyond photon absorption: extracellular retinoids and growth factors modulate rod photoreceptor sensitivity

Key points

• We propose that the end product of chromophore bleaching in rod photoreceptors, all‐trans retinol, is part of a feedback loop that increases the sensitivity of the phototransduction cascade in rods.
• A previously described light‐induced hypersensitivity in rods, termed adaptive potentiation, is reduced by exogenously applied all‐trans retinol but not all‐trans retinal.
• This potentiation is produced by insulin‐like growth factor‐1, whose binding proteins are located in the extracellular matrix, even in our isolated retina preparation after removal of the retinal pigmented epithelium.
• Simple modelling suggests that the light stimuli used in the present study will produce sufficient all‐trans retinol within the interphotoreceptor matrix to explain the potentiation effect.

Abstract

Photoreceptors translate the absorption of photons into electrical signals for propagation through the visual system. Mammalian photoreceptor signalling has largely been studied in isolated cells, and such studies have necessarily avoided the complex environment of supportive proteins that surround the photoreceptors. The interphotoreceptor matrix (IPM) contains an array of proteins that aid in both structural maintenance and cellular homeostasis, including chromophore turnover. In signalling photon absorption, the chromophore 11‐cis retinal is first isomerized to all‐trans retinal, followed by conversion to all‐trans retinol (ROL) for removal from the photoreceptor. Interphotoreceptor retinoid‐binding protein (IRBP) is the most abundant protein in the IPM, and it promotes the removal of bleached chromophores and recycling in the nearby retinal pigment epithelium. By studying the light responses of isolated mouse retinas, we demonstrate that ROL can act as a feedback signal onto photoreceptors that influences the sensitivity of phototransduction. In addition to IRBP, the IPM also contains insulin‐like growth factor‐1 (IGF‐1) and its associated binding proteins, although their functions have not yet been described. We demonstrate that extracellular application of physiological concentrations of IGF‐1 can increase rod photoreceptor sensitivity in mammalian retinas. We also determine that chromophores and growth factors can limit the range of a newly described form of photoreceptor light adaptation. Finally, fluorescent antibodies demonstrate the presence of IRBP and IGFBP‐3 in isolated retinas. A simple model of the formation and release of ROL into the extracellular space quantitatively describes this novel feedback loop.

Abbreviations

AP

adaptive potentiation

AP‐4

(±)‐2‐amino‐4‐phosphonobutyric acid

CNG

cyclic nucleotide gated

DAPI

4′,6‐diamidino‐2‐phenylindole

ERG

electroretinography

FGF

fibroblast growth factor

IGF‐1

insulin‐like growth factor‐1

IGFBP

insulin‐like growth factor binding protein

IGF‐1R

insulin‐like growth factor‐1 receptor

IPM

interphotoreceptor matrix

IR

insulin receptor

IRBP

interphotoreceptor retinoid‐binding protein

RAL

all‐trans retinal

ROL

all‐trans retinol

ROS

rod outer segment

RPE

retinal pigmented epithelium

     

Acid‐sensing ion channel 1a drives AMPA receptor plasticity following ischaemia and acidosis in hippocampal CA1 neurons

Key points

• The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (AMPAR) plasticity – an anoxic form of long‐term potentiation and a delayed increase in Ca²⁺‐permeable (CP) AMPARs – contribute to this susceptibility by increasing excitotoxicity.
• In CA1, the acid‐sensing ion channel 1a (ASIC1a) is known to facilitate LTP and contribute to ischaemic acidotoxicity.
• We have examined the role of ASIC1a in AMPAR ischaemic plasticity in organotypic hippocampal slice cultures exposed to oxygen glucose deprivation (a model of ischaemic stroke), and in hippocampal pyramidal neuron cultures exposed to acidosis.
• We find that ASIC1a activation promotes both forms of AMPAR plasticity and that neuroprotection, by inhibiting ASIC1a, circumvents any further benefit of blocking CP‐AMPARs.
• Our observations establish a new interaction between acidotoxicity and excitotoxicity, and provide insight into the role of ASIC1a and CP‐AMPARs in neurodegeneration. Specifically, we propose that ASIC1a activation drives certain post‐ischaemic forms of CP‐AMPAR plasticity.

Abstract

The CA1 region of the hippocampus is particularly vulnerable to ischaemic damage. While NMDA receptors play a major role in excitotoxicity, it is thought to be exacerbated in this region by two forms of post‐ischaemic AMPA receptor (AMPAR) plasticity – namely, anoxic long‐term potentiation (a‐LTP), and a delayed increase in the prevalence of Ca²⁺‐permeable GluA2‐lacking AMPARs (CP‐AMPARs). The acid‐sensing ion channel 1a (ASIC1a), which is expressed in CA1 pyramidal neurons, is also known to contribute to post‐ischaemic neuronal death and to physiologically induced LTP. This raises the question does ASIC1a activation drive the post‐ischaemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD), and dissociated cultures of hippocampal pyramidal neurons (HPNs) exposed to low pH (acidosis). We find that both a‐LTP and the delayed increase in the prevalence of CP‐AMPARs are dependent on ASIC1a activation during ischaemia. Indeed, acidosis alone is sufficient to induce the increase in CP‐AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP‐AMPARs. By demonstrating that ASIC1a activation contributes to post‐ischaemic AMPAR plasticity, our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells, and provide insight into the role of ASIC1a and CP‐AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP‐AMPAR plasticity, and that inhibiting ASIC1a affords neuroprotection.

Abbreviations

a‐LTP

anoxic LTP

AMPAR

AMPA receptor

ASIC1a

acid‐sensing ion channel 1a

CP‐AMPAR

calcium‐permeable AMPAR

HPN

hippocampal pyramidal neuron

I–V

current–voltage

KO

knockout

LTP

long‐term potentiation

NASPM

1‐naphthyl acetyl spermine

NFATc

nuclear factor of activated T cells

NMDAR

NMDA receptor

OGD

oxygen glucose deprivation

OHSC

organotypic hippocampal slice culture

PcTx1

psalmotoxin 1

PI

propidium iodide

RI

rectification index

TBS

theta‐burst stimulation

WT

wild type

     

Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2

Key points

• Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability.
• KARs associate with the auxiliary proteins neuropilin‐ and tolloid‐like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date.
• M3–S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner.
• We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity.
• We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR‐specific modulators to alter receptor function.

Abstract

Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit‐ and Neto isoform‐specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3–S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3–S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand‐binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3–S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3–S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR‐specific modulators that alter the function of auxiliary proteins in native receptors.

Abbreviations

ATD

amino terminal domain

AMPAR

AMPA receptor

CUB

C1r/C1s‐Uegf‐BMP

eGFP

enhanced green florescent protein

iGluR

ionotropic glutamate receptor

KAR

kainate receptor

LBD

ligand binding domain

NMDAR

NMDA receptor

Neto

neuropilin‐ and tolloid‐like

TARP

transmembrane AMPA receptor regulatory protein

     

Large conductance Ca2+‐activated K+ (BK) channels promote secretagogue‐induced transition from spiking to bursting in murine anterior pituitary corticotrophs

Key points

• Corticotroph cells of the anterior pituitary are electrically excitable and are an integral component of the hypothalamic‐pituitary‐adrenal axis which governs the neuroendocrine response to stress.
• Corticotrophs display predominantly single spike activity under basal conditions that transition to complex bursting behaviours upon stimulation by the hypothalamic secretagogues corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP); however, the underlying mechanisms controlling bursting are unknown.
• In this study, we show that CRH and AVP induce different patterns of corticotroph electrical activity, and we use an electrophysiological approach combined with mathematical modelling to show the ionic mechanisms for these differential effects.
• The data reveal that secretagogue‐induced bursting is dependent on large conductance Ca²⁺‐activated K⁺ (BK) channels and is driven primarily by CRH whereas AVP promotes an increase in single‐spike frequency through BK‐independent pathways involving activation of non‐selective cation conductances.
• As corticotroph excitability is differentially regulated by CRH and AVP this may allow corticotrophs to respond appropriately to different stressors.

Abstract

Anterior pituitary corticotroph cells are a central component of the hypothalamic‐pituitary‐adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophic hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modelling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium‐ and voltage‐activated potassium (BK) channels in spontaneous and secretagogue‐induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non‐selective conductances. Co‐stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors.

Abbreviations

ACTH

adrenocorticotrophic hormone

AVP

arginine vasopressin

BF

burstiness factor

BK channel

large conductance Ca²⁺‐ and voltage‐activated K⁺ channel

BK‐far

BK channels located distantly from voltage‐gated Ca²⁺ channels

BK‐near

BK channels in close proximity to voltage‐activated Ca²⁺ channels

CRH

corticotrophin‐releasing hormone

GFP

green fluorescent protein

HPA

hypothalamic‐pituitary‐adrenal

IK channel

intermediate conductance Ca²⁺‐activated K⁺ channel

IP₃

inositol trisphosphate

NMDG

N‐methyl‐d‐glucamine

PKA

protein kinase A

PKC

protein kinase C

POMC

proopiomelanocortin

STREX

stress regulated exon

ZERO

BK channels lacking STREX insert

     

High dendritic expression of I h in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons

Key points

• The hyperpolarization‐activated cation current I _h is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown.
• We used cell‐attached patch recordings to determine the localization profile of I _h along the somatodendritic axis of nigral dopamine neurons in slices from young rats.
• I _h density is higher in axon‐bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon‐lacking dendrites.
• Dual current‐clamp recordings revealed a similar contribution of I _h to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain.
• The I _h blocker ZD 7288 increased the temporal summation in all dendrites with a comparable effect in axon‐ and non‐axon dendrites.
• The strategic position of I _h in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine.

Abstract

Dendrites of most neurons express voltage‐gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization‐activated cation current I _h present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h‐channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of I _h can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating I _h current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell‐attached patch‐clamp recordings, we find a higher I _h current density in the axon‐bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. I _h is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of I _h in axon‐ and non‐axon‐bearing dendrites. The presence of I _h close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement.

Abbreviations

ABD

axon‐bearing dendrite

aEPSP

artificial excitatory postsynaptic potential

AP

action potential

DA neuron

dopamine neuron

FITC

fluorescein isothiocyanate

I_h

h‐current

IR–DGC

infrared−Dodt gradient contrast

nABD

axon‐bearing dendrite

PLP neuron

pyramidal‐like principal neuron

r

correlation coefficient

R_in

input resistance

TH

tyrosine hydroxylase

ZD 7288

4‐ethylphenylamino‐1,2‐dimethyl‐6‐methylaminopyrimidinium chloride

     

The extracellular matrix molecule brevican is an integral component of the machinery mediating fast synaptic transmission at the calyx of Held

Key points

• The proteoglycan brevican is a major component of the extracellular matrix of perineuronal nets and is highly enriched in the perisynaptic space suggesting a role for synaptic transmission.
• We have introduced the calyx of Held in the auditory brainstem as a model system to study the impact of brevican on dynamics and reliability of synaptic transmission.
• In vivo extracellular single‐unit recordings at the calyx of Held in brevican‐deficient mice yielded a significant increase in the action potential (AP) transmission delay and a prolongation of pre‐ and postsynaptic APs.
• The changes in dynamics of signal transmission were accompanied by the reduction of presynaptic vGlut1 and ultrastructural changes in the perisynaptic space.
• These data show that brevican is an important mediator of fast synaptic transmission at the calyx of Held.

Abstract

The extracellular matrix is an integral part of the neural tissue. Its most conspicuous manifestation in the brain are the perineuronal nets (PNs) which surround somata and proximal dendrites of distinct neuron types. The chondroitin sulfate proteoglycan brevican is a major component of PNs. In contrast to other PN‐comprising proteoglycans (e.g. aggrecan and neurocan), brevican is mainly expressed in the perisynaptic space closely associated with both the pre‐ and postsynaptic membrane. This specific localization prompted the hypothesis that brevican might play a role in synaptic transmission. In the present study we specifically investigated the role of brevican in synaptic transmission at a central synapse, the calyx of Held in the medial nucleus of the trapezoid body, by the use of in vivo electrophysiology, immunohistochemistry, biochemistry and electron microscopy. In vivo extracellular single‐unit recordings were acquired in brevican‐deficient mice and the dynamics and reliability of synaptic transmission were compared to wild‐type littermates. In knockout mice, the speed of pre‐to‐postsynaptic action potential (AP) transmission was reduced and the duration of the respective pre‐ and postsynaptic APs increased. The reliability of signal transmission, however, was not affected by the lack of brevican. The changes in dynamics of signal transmission were accompanied by the reduction of (i) presynaptic vGlut1 and (ii) the size of subsynaptic cavities. The present results suggest an essential role of brevican for the functionality of high‐speed synaptic transmission at the calyx of Held.

Abbreviations

ANF

auditory nerve fibre

AP

action potential

AVCN

anterior ventral cochlear nucleus

CF

characteristic frequency

CNS

central nervous system

ECM

extracellular matrix

EPSP

excitatory postsynaptic potential

LSO

lateral superior olive

MNTB

medial nucleus of the trapezoid body

PN

perineuronal net

SNR

signal‐to‐noise ratio

VCN

ventral cochlear nucleus

vGlut

vesicular glutamate transporter

     

Hypoxic induction of T‐type Ca2+ channels in rat cardiac myocytes: role of HIF‐1α and RhoA/ROCK signalling

Key points

• T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are downregulated as development progresses. However, these channels are re‐expressed in adult cardiomyocytes under pathological conditions.
• Hypoxia induces the upregulation of the T‐type Ca²⁺ channel Ca_v3.2 mRNA in cardiac myocytes, whereas Ca_v3.1 mRNA is not significantly altered.
• The effect of hypoxia on Ca_v3.2 mRNA requires hypoxia inducible factor‐1α (HIF‐1α) stabilization and involves the small monomeric G‐protein RhoA and its effector ROCKI.
• Our results suggest that the hypoxic regulation of the Ca_v3.2 channels may be involved in the increased probability of developing arrhythmias observed in ischemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abstract

T‐type Ca²⁺ channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re‐expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage‐activated T‐type Ca²⁺ channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Ca_v3.2 mRNA, whereas Ca_v3.1 mRNA is not significantly altered. The effect of hypoxia on Ca_v3.2 mRNA was time‐ and dose‐dependent, and required hypoxia inducible factor‐1α (HIF‐1α) stabilization. Patch‐clamp recordings confirmed that T‐type Ca²⁺ channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl₂ (a T‐type channel blocker) demonstrated that the Ca_v3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Ca_v3.2 channel electrophysiological properties. The small monomeric G‐protein RhoA and its effector Rho‐associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Ca_v3.2 channel and the stabilization of HIF‐1α that occurred in response to hypoxia. These results suggest a possible role for Ca_v3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca²⁺ overload.

Abbreviations

Ca_V channels

voltage‐gated Ca²⁺ channels

DRB

5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole

DMOG

dimethyloxalylglycine

DPI

diphenyliodonium

HIF

hypoxia inducible factor

NMDG

N‐methyl‐d‐glucamine

NRVMs

neonatal rat ventricular myocytes

P_O2

partial pressure of oxygen

ROCK

Rho‐associated kinase

ROS

reactive oxygen species

siRNA

small interfering RNA

     

Primary visual cortex shows laminar‐specific and balanced circuit organization of excitatory and inhibitory synaptic connectivity

Key points

• Using functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory neurons in layers 2/3–6 of the mouse visual cortex (V1).
• Laminar‐specific synaptic wiring diagrams of excitatory neurons were constructed on the basis of circuit mapping.
• The present study reveals that that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks in V1.

Abstract

In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer‐by‐layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer‐specific organization of excitatory inputs. Remarkably, the spatial extent of inhibitory inputs of excitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer‐by‐layer synaptic wiring diagrams of excitatory neurons in the visual cortex.

Abbreviations

aCSF

artificial cerebrospinal fluid

DAPI

4′‐6‐diamidino‐2‐phenylindole

LSPS

laser scanning photostimulation

V1

primary visual cortex

     

Endogenous casein kinase‐1 modulates NMDA receptor activity of hypothalamic presympathetic neurons and sympathetic outflow in hypertension

Key points

• Increased NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus can increase sympathetic nerve discharges leading to hypertension.
• In this study, we determined how protein kinases and phosphatases are involved in regulating NMDA receptor activity and firing activity of presympathetic neurons in the hypothalamus in normotensive and hypertensive rats.
• We show that casein kinase‐1 inhibition increases NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus and augments sympathetic nerve discharges in normotensive, but not in hypertensive, rats.
• Our data indicate that casein kinase‐1 tonically regulates NMDA receptor activity by interacting with casein kinase‐2 and protein phosphatases in the hypothalamus and that imbalance of NMDA receptor phosphorylation can augment the excitability of hypothalamic presympathetic neurons and sympathetic nerve discharges in hypertension.
• These findings help us understand the neuronal mechanism of hypertension, and reducing the NMDA receptor phosphorylation level may be effective for treating neurogenic hypertension.

Abstract

Increased N‐methyl‐d‐aspartate receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus is involved in elevated sympathetic outflow in hypertension. However, the molecular mechanisms underlying augmented NMDAR activity in hypertension remain unclear. In this study, we determined the role of casein kinase‐1 (CK1) in regulating NMDAR activity in the PVN. NMDAR‐mediated excitatory postsynaptic currents (EPSCs) and puff NMDA‐elicited currents were recorded in spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats. The basal amplitudes of evoked NMDAR‐EPSCs and puff NMDA currents were significantly higher in SHRs than in WKY rats. The CK1 inhibitor PF4800567 or PF670462 significantly increased the amplitude of NMDAR‐EPSCs and puff NMDA currents in PVN neurons in WKY rats but not in SHRs. PF4800567 caused an NMDAR‐dependent increase in the excitability of PVN neurons only in WKY rats. Also, the CK1ε protein level in the PVN was significantly lower in SHRs than in WKY rats. Furthermore, intracerebroventricular infusion of PF4800567 increased blood pressure and lumbar sympathetic nerve activity in WKY rats, and this effect was eliminated by microinjection of the NMDAR antagonist into the PVN. In addition, PF4800567 failed to increase NMDAR activity in brain slices of WKY rats pretreated with the protein phosphatase 1/2A, calcineurin, or casein kinase‐2 inhibitor. Our findings suggest that CK1 tonically suppresses NMDAR activity in the PVN by reducing the NMDAR phosphorylation level. Diminished CK1 activity may contribute to potentiated glutamatergic synaptic input to PVN presympathetic neurons and elevated sympathetic vasomotor tone in neurogenic hypertension.

Abbreviations

ABP

arterial blood pressure

aCSF

artificial cerebrospinal fluid

AMPA

α‐amino‐3‐hydroxy‐4‐isoxazoleproprionic acid

AMPAR

AMPA receptor

CK1

casein kinase‐1

CK2

casein kinase‐2

CNQX

6‐cyano‐7‐nitroquinoxaline‐2,3‐dione

EPSC

excitatory postsynaptic current

GABA

γ‐aminobutyric acid

HR

heart rate

LSNA

lumbar sympathetic nerve activity

NMDA

N‐methyl‐d‐aspartate

NMDAR

NMDA receptor

PVN

paraventricular nucleus

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

PP2B

protein phosphatase 2B

PKC

protein kinase C

RVLM

rostral ventrolateral medulla

SHR

spontaneously hypertensive rat

WKY

Wistar–Kyoto

     

Ca2+ current facilitation determines short‐term facilitation at inhibitory synapses between cerebellar Purkinje cells

Key points

• Short‐term facilitation takes place at GABAergic synapses between cerebellar Purkinje cells (PCs).
• By directly patch clamp recording from a PC axon terminal, we studied the mechanism of short‐term facilitation.
• We show that the Ca²⁺ currents elicited by high‐frequency action potentials were augmented in a [Ca²⁺]_i‐dependent manner.
• The facilitation of synaptic transmission showed 4–5th power dependence on the Ca²⁺ current facilitation, and was abolished when the Ca²⁺ current amplitude was adjusted to be identical.
• Short‐term facilitation of Ca²⁺ currents predominantly mediates short‐term facilitation at synapses between PCs.

Abstract

Short‐term synaptic facilitation is critical for information processing of neuronal circuits. Several Ca²⁺‐dependent positive regulations of transmitter release have been suggested as candidate mechanisms underlying facilitation. However, the small sizes of presynaptic terminals have hindered the biophysical study of short‐term facilitation. In the present study, by directly recording from the axon terminal of a rat cerebellar Purkinje cell (PC) in culture, we demonstrate a crucial role of [Ca²⁺]_i‐dependent facilitation of Ca²⁺ currents in short‐term facilitation at inhibitory PC–PC synapses. Voltage clamp recording was performed from a PC axon terminal visualized by enhanced green fluorescent protein, and the Ca²⁺ currents elicited by the voltage command consisting of action potential waveforms were recorded. The amplitude of presynaptic Ca²⁺ current was augmented upon high‐frequency paired‐pulse stimulation in a [Ca²⁺]_i‐dependent manner, leading to paired‐pulse facilitation of Ca²⁺ currents. Paired recordings from a presynaptic PC axon terminal and a postsynaptic PC soma demonstrated that the paired‐pulse facilitation of inhibitory synaptic transmission between PCs showed 4–5th power dependence on that of Ca²⁺ currents, and was completely abolished when the Ca²⁺ current amplitude was adjusted to be identical. Thus, short‐term facilitation of Ca²⁺ currents predominantly mediates short‐term synaptic facilitation at synapses between PCs.

Abbreviations

AAV

adeno‐associated virus

AHP

afterhyperpolarization

AP

action potential

[Ca²⁺]_i

intracellular Ca²⁺ concentration

CaM

calmodulin

Cm

membrane capacitance

DCN

deep cerebellar nuclei

EGFP

enhanced green fluorescent protein

GC

granule cell

IN

inhibitory interneuron

PC

Purkinje cell

PPD

paired‐pulse depression

PPF

paired‐pulse facilitation

PPR

paired‐pulse ratio

PSC

postsynaptic current

     

μ opioid receptor activation hyperpolarizes respiratory‐controlling K?lliker–Fuse neurons and suppresses post‐inspiratory drive

Key points

• In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker–Fuse (KF) maintains upper airway patency and a normal respiratory pattern.
• In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats.
• Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post‐inspiration phase of the respiratory cycle.
• In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance.
• These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid‐induced respiratory disturbances, particularly the impairment of upper airways.

Abstract

Opioid‐induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker–Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi‐intact rats, injection of opioid agonists DAMGO or [Met⁵]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart–brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post‐inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid‐induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage‐clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl₂, characteristics of a G protein‐coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine‐mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by μ opioid agonists are likely mediators of the opioid‐induced loss of post‐inspiration and induction of apneusis.

Abbreviations

ACSF

artificial cerebrospinal fluid

ANOVA

analysis of variance

AP

action potential

CTAP

d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH₂

CV

coefficient of variation

cVN

central vagus nerve

DAMGO

([d‐Ala², N‐Me‐Phe⁴, Gly⁵‐ol]‐enkephalin

DNQX

6,7‐dinitroquinoxaline‐2,3‐dione

E2

stage 2 expiration

GIRK

G protein‐coupled inwardly rectifying potassium

KF

Kölliker–Fuse

LC

locus coeruleus

ME

[Met]⁵enkephalin

NLX

naloxone

PB

parabrachial

PN

phrenic nerve; post‐I, post‐inspiration; preBötC, preBötzinger complex; scp, superior cerebellar peduncle; T _E, expiratory time; T _I, inspiratory time

     

Functional characterization of spikelet activity in the primary visual cortex

Key points

• In vivo whole‐cell patch‐clamp recordings in cat visual cortex revealed small deflections in the membrane potential of neurons, termed spikelets.
• Spikelet statistics and functional properties suggest these deflections originate from a single, nearby cell.
• Spikelets shared a number sensory selectivities with the principal neuron including orientation selectivity, receptive field location and eye preference.
• Principal neurons and spikelets did not, however, generally share preferences for depth (binocular disparity).
• Cross‐correlation of spikelet activity and membrane potential revealed direct effects on the membrane potential of some principal neurons, suggesting that these cells were synaptically coupled or received common input from the cortical network.
• Other spikelet–neuron pairs revealed indirect effects, likely to be the result of correlated network events.

Abstract

Intracellular recordings in the neocortex reveal not only the membrane potential of neurons, but small unipolar or bipolar deflections that are termed spikelets. Spikelets have been proposed to originate from various sources, including active dendritic mechanisms, gap junctions and extracellular signals. Here we examined the functional characteristics of spikelets measured in neurons from cat primary visual cortex in vivo. Spiking statistics and our functional characterization of spikelet activity indicate that spikelets originate from a separate, nearby cell. Spikelet kinetics and lack of a direct effect on spikelet activity from hyperpolarizing current injection suggest they do not arise from electrical coupling to the principal neuron being recorded. Spikelets exhibited matched orientation tuning preference and ocular dominance to the principal neuron. In contrast, binocular disparity preferences of spikelets and the principal neuron were unrelated. Finally, we examined the impact of spikelets on the principal neuron''s membrane potential; we did observe some records for which spikelets were correlated with the membrane potential of the principal neuron, suggesting that these neurons were synaptically coupled or received common input from the cortical network.

Abbreviations

DSI

disparity selectivity index

F_o

mean Fourier amplitude

F₁

first Fourier harmonic amplitude

FWHM

full‐width half‐maximum

ISI

inter‐spikelet interval

ODI

ocular dominance index

RF

receptive field

STA

spike‐triggered average

V1

primary visual cortex

VTA

voltage‐triggered average

     

GABAB receptor‐mediated feed‐forward circuit dysfunction in the mouse model of fragile X syndrome

Key points

• Cortico‐hippocampal feed‐forward circuits formed by the temporoammonic (TA) pathway exhibit a marked increase in excitation/inhibition ratio and abnormal spike modulation functions in Fmr1 knock‐out (KO) mice.
• Inhibitory, but not excitatory, synapse dysfunction underlies cortico‐hippocampal feed‐forward circuit abnormalities in Fmr1 KO mice.
• GABA release is reduced in TA‐associated inhibitory synapses of Fmr1 KO mice in a GABA_B receptor‐dependent manner.
• Inhibitory synapse and feed‐forward circuit defects are mediated predominately by presynaptic GABA_B receptor signalling in the TA pathway of Fmr1 KO mice.
• GABA_B receptor‐mediated inhibitory synapse defects are circuit‐specific and are not observed in the Schaffer collateral pathway‐associated inhibitory synapses in stratum radiatum.

Abstract

Circuit hyperexcitability has been implicated in neuropathology of fragile X syndrome, the most common inheritable cause of intellectual disability. Yet, how canonical unitary circuits are affected in this disorder remains poorly understood. Here, we examined this question in the context of the canonical feed‐forward inhibitory circuit formed by the temporoammonic (TA) branch of the perforant path, the major cortical input to the hippocampus. TA feed‐forward circuits exhibited a marked increase in excitation/inhibition ratio and major functional defects in spike modulation tasks in Fmr1 knock‐out (KO) mice, a fragile X mouse model. Changes in feed‐forward circuits were caused specifically by inhibitory, but not excitatory, synapse defects. TA‐associated inhibitory synapses exhibited increase in paired‐pulse ratio and in the coefficient of variation of IPSPs, consistent with decreased GABA release probability. TA‐associated inhibitory synaptic transmission in Fmr1 KO mice was also more sensitive to inhibition of GABA_B receptors, suggesting an increase in presynaptic GABA_B receptor (GABA_BR) signalling. Indeed, the differences in inhibitory synaptic transmission between Fmr1 KO and wild‐type (WT) mice were eliminated by a GABA_BR antagonist. Inhibition of GABA_BRs or selective activation of presynaptic GABA_BRs also abolished the differences in the TA feed‐forward circuit properties between Fmr1 KO and WT mice. These GABA_BR‐mediated defects were circuit‐specific and were not observed in the Schaffer collateral pathway‐associated inhibitory synapses. Our results suggest that the inhibitory synapse dysfunction in the cortico‐hippocampal pathway of Fmr1 KO mice causes hyperexcitability and feed‐forward circuit defects, which are mediated in part by a presynaptic GABA_BR‐dependent reduction in GABA release.

Abbreviations

AP

action potential

APV

2‐amino‐5‐phosphonopentanoic acid

CV

coefficient of variation

DNQX

6,7‐dinitroquinoxaline‐2,3‐dione

E/I

excitatory/inhibitory

EPSP

excitatory postsynaptic potential

FFI

feed‐forward inhibitory

FMRP

fragile X mental retardation protein

Fmr1

fragile X mental retardation 1

FWHM

full‐width half‐maximum

FXS

fragile X syndrome

GABA_AR

γ‐aminobutyric acid type A receptor

GABA_BR

γ‐aminobutyric acid type B receptor

IPSP

inhibitory postsynaptic potential

KO

knock‐out

PPR

paired‐pulse ratio

RMS

root mean square

SC

Schaffer collateral

SLM

stratum lacunosum moleculare

SR

stratum radiatum

TA

temporoammonic

WT

wild‐type

     

Functions of synapsins in corticothalamic facilitation: important roles of synapsin I

Key points

• The synaptic vesicle associated proteins synapsin I and synapsin II have important functions in synaptic short‐term plasticity.
• We investigated their functions in cortical facilitatory feedback to neurons in dorsal lateral geniculate nucleus (dLGN), feedback that has important functions in state‐dependent regulation of thalamic transmission of visual input to cortex.
• We compared results from normal wild‐type (WT) mice and synapsin knockout (KO) mice in several types of synaptic plasticity, and found clear differences between the responses of neurons in the synapsin I KO and the WT, but no significant differences between the synapsin II KO and the WT.
• These results are in contrast to the important role of synapsin II previously demonstrated in similar types of synaptic plasticity in other brain regions, indicating that the synapsins can have different roles in similar types of STP in different parts of the brain.

Abstract

The synaptic vesicle associated proteins synapsin I (SynI) and synapsin II (SynII) have important functions in several types of synaptic short‐term plasticity in the brain, but their separate functions in different types of synapses are not well known. We investigated possible distinct functions of the two synapsins in synaptic short‐term plasticity at corticothalamic synapses on relay neurons in the dorsal lateral geniculate nucleus. These synapses provide excitatory feedback from visual cortex to the relay cells, feedback that can facilitate transmission of signals from retina to cortex. We compared results from normal wild‐type (WT), SynI knockout (KO) and SynII KO mice, in three types of synaptic plasticity mainly linked to presynaptic mechanism. In SynI KO mice, paired‐pulse stimulation elicited increased facilitation at short interpulse intervals compared to the WT. Pulse‐train stimulation elicited weaker facilitation than in the WT, and also post‐tetanic potentiation was weaker in SynI KO than in the WT. Between SynII KO and the WT we found no significant differences. Thus, SynI has important functions in these types of synaptic plasticity at corticothalamic synapses. Interestingly, our data are in contrast to the important role of SynII previously shown for sustained synaptic transmission during intense stimulation in excitatory synapses in other parts of the brain, and our results suggest that SynI and SynII may have different roles in similar types of STP in different parts of the brain.

Abbreviations

DKO

double knock‐out

dLGN

dorsal lateral geniculate nucleus

EPSC

excitatory post‐synaptic current

EPSCctr

control EPSC

KO

knockout

PPF

paired‐pulse facilitation

PTP

post‐tetanic potentiation

RRP

readily releasable pool

STP

short‐term plasticity

SV

synaptic vesicle

SynI

synapsin I

SynII

synapsin II

TC

thalamo‐cortical

WT

wild‐type

     

Long‐term exercise‐specific neuroprotection in spinal muscular atrophy‐like mice

Key points

• The real impact of physical exercise parameters, i.e. intensity, type of contraction and solicited energetic metabolism, on neuroprotection in the specific context of neurodegeneration remains poorly explored.
• In this study behavioural, biochemical and cellular analyses were conducted to compare the effects of two different long‐term exercise protocols, high intensity swimming and low intensity running, on motor units of a type 3 spinal muscular atrophy (SMA)‐like mouse model.
• Our data revealed a preferential SMA‐induced death of intermediate and fast motor neurons which was limited by the swimming protocol only, suggesting a close relationship between neuron‐specific protection and their activation levels by specific exercise.
• The exercise‐induced neuroprotection was independent of SMN protein expression and associated with specific metabolic and behavioural adaptations with notably a swimming‐induced reduction of muscle fatigability.
• Our results provide new insight into the motor units’ adaptations to different physical exercise parameters and will contribute to the design of new active physiotherapy protocols for patient care.

Abstract

Spinal muscular atrophy (SMA) is a group of autosomal recessive neurodegenerative diseases differing in their clinical outcome, characterized by the specific loss of spinal motor neurons, caused by insufficient level of expression of the protein survival of motor neuron (SMN). No cure is at present available for SMA. While physical exercise might represent a promising approach for alleviating SMA symptoms, the lack of data dealing with the effects of different exercise types on diseased motor units still precludes the use of active physiotherapy in SMA patients. In the present study, we have evaluated the efficiency of two long‐term physical exercise paradigms, based on either high intensity swimming or low intensity running, in alleviating SMA symptoms in a mild type 3 SMA‐like mouse model. We found that 10 months’ physical training induced significant benefits in terms of resistance to muscle damage, energetic metabolism, muscle fatigue and motor behaviour. Both exercise types significantly enhanced motor neuron survival, independently of SMN expression, leading to the maintenance of neuromuscular junctions and skeletal muscle phenotypes, particularly in the soleus, plantaris and tibialis of trained mice. Most importantly, both exercises significantly improved neuromuscular excitability properties. Further, all these training‐induced benefits were quantitatively and qualitatively related to the specific characteristics of each exercise, suggesting that the related neuroprotection is strongly dependent on the specific activation of some motor neuron subpopulations. Taken together, the present data show significant long‐term exercise benefits in type 3 SMA‐like mice providing important clues for designing rehabilitation programmes in patients.

Abbreviations

ChAT

choline acetyltransferase

Chodl

chondrolectin

CK

creatine kinase

CMAP

compound muscle action potential

ERRβ

oestrogen‐related receptor β

MyHC

myosin heavy chain

NMJ

neuromuscular junction

SMA

spinal muscular atrophy

SMN

survival of motor neuron

TBS

Tris‐buffered solution

     

Heart failure induces changes in acid‐sensing ion channels in sensory neurons innervating skeletal muscle

Key points

• Heart failure is characterized by an elevated sympathetic state and exercise intolerance, which is partially driven by exaggerated autonomic reflexes triggered by skeletal muscle afferents.
• Acid‐sensing ion channels (ASICs) are highly expressed in skeletal muscle afferents and contribute to exercise mediated reflexes.
• Here we show that ASIC currents recorded from isolated skeletal muscle sensory neurons display diminished pH sensitivity, altered desensitization kinetics, and faster recovery from desensitization in a mouse model of heart failure.
• These results indicate ASICs in muscle afferents are altered in heart failure, and may contribute to the associated sympathoexcitation and exercise intolerance.

Abstract

Heart failure is associated with diminished exercise capacity, which is driven, in part, by alterations in exercise‐induced autonomic reflexes triggered by skeletal muscle sensory neurons (afferents). These overactive reflexes may also contribute to the chronic state of sympathetic excitation, which is a major contributor to the morbidity and mortality of heart failure. Acid‐sensing ion channels (ASICs) are highly expressed in muscle afferents where they sense metabolic changes associated with ischaemia and exercise, and contribute to the metabolic component of these reflexes. Therefore, we tested if ASICs within muscle afferents are altered in heart failure. We used whole‐cell patch clamp to study the electrophysiological properties of acid‐evoked currents in isolated, labelled muscle afferent neurons from control and heart failure (induced by myocardial infarction) mice. We found that the percentage of muscle afferents that displayed ASIC‐like currents, the current amplitudes, and the pH dose–response relationships were not altered in mice with heart failure. On the other hand, the biophysical properties of ASIC‐like currents were significantly different in a subpopulation of cells (40%) from heart failure mice. This population displayed diminished pH sensitivity, altered desensitization kinetics, and very fast recovery from desensitization. These unique properties define these channels within this subpopulation of muscle afferents as being heteromeric channels composed of ASIC2a and ‐3 subunits. Heart failure induced a shift in the subunit composition of ASICs within muscle afferents, which significantly altered their pH sensing characteristics. These results might, in part, contribute to the changes in exercise‐mediated reflexes that are associated with heart failure.

Abbreviations

ASICs

acid‐sensing ion channels

BP

blood pressure

DiI

1,1‐dioctadecyl‐3,3,3,3 tetramethylindocarbocyanine perchlorate

DRG

dorsal root ganglion

EPR

exercise pressor reflex

HF

heart failure

HR

heart rate