Luo 2020

Journal club 7/20

https://www.nature.com/articles/s41380-020-0793-6

Overview

Trying to learn more about signaling pathways, and my project involves chronic ketamine treatment

Molecular mechanism of cognitive impairment is unknown. 28 exposure to ketamine induves decreased expression og Glutamate receptor subiunits (GluA1, GluA2, GluN2A, GluN2B) [What is the normal structure of glutamate receptors, and what are these different kinds?]

NMDARs are tetraheteromeric channel pores formed by the obligatory GluN1 subunit, plus GluN2 or GluN3 subunits. These subunits contain several variants: GluN1 subunits with eight splice variants, four GluN2 subunits (GluN2A–D), and two GluN3 subunits. Each subunit has, structurally, an extracellular N-terminal, a reentrant loop that forms the channel pore, and an intracellular C-terminal. Functional NMDARs, in the forebrain of the CNS, are formed by two GluN1 subunits and two GluN2/3 subunits [6]. Glutamate binds the GluN2 subunit while D-serine and glycine, two coagonists, bind the GluN1 subunit in synaptic and extrasynaptic sites, respectively [7]. The domain for Mg2+ block and Ca2+ permeability is in the pore formed by the internal loop [6]. GluN2 subunits give specific and key biophysical and pharmacological properties, including sensitivity to glutamate, protons, polyamines, and Zn2+, modulation by glycine, Ca2+ permeability, and differential channel kinetics, including deactivation time and open probability [8]. GluN2 subunits control the trafficking and delivery of NMDARs to the plasma membrane and synaptic compartments through their intracellular domains [6].

decreased expression of synaptic proteins Syn and PSD-95

Decreased dendrite spine density

impairments in long term potentiation and synaptic transmission in ca1

deteriorated learning and memory in mice

All persists 28 days after the last injection.

Expression of CaMK11B, ERK1/2, CREB, NF-kB are inhibited by ketamine.

CaMKIIB overexpression rescues effects on glutamate subunit expression, sine density, LTP, synaptic transmission, and cognition.

Inhibiting the cAMkiib-erk1/2-creb-nfkb SIGNALING MAY MEDIATE KETAMINE-ASSOCIATED COGNITIVE IMPAIRMENTSBY RESTRAINING SYNAPTIC SIGNALING. I.E. HYPOFUNCTION OF THE GLUTAMATERGIC SYSTEM.

Methods

6-9 week old MALE mice

Ketamine: 30mg/kg (fairly high dose) used for most of the studies

lentiviral CaMKIIB-mcherry overexpression construcrt. mcherry as a control.

2 photon imaging: Spine densities were assessed by measuring the number of spines along 100–150 μm segments of CA1 neurons. The shape (stubby, mushroom, long thin, and filopodial), size, density, and volume of the spines were analyzed using Imaris 9.3 software

The spatial memory and learning of animals was assessed by Morris water maze test as previously described [45]. First, all mice received training in the Morris water maze for 5 days without any treatments. Then, starting on day 6, the mice were injected with 1 μl (108 TU/μl) of control vector or lentiviral vector carrying CaMKIIβ in the bilateral hippocampi. On day 7 after injection, mice were treated with vehicle or ketamine (30 mg/kg) once a day for 28 consecutive days. Tests were performed 24 h after the last injection of ketamine

Results

Chronic in vivo exposure to ketamine-induced dose- and time-dependent long-lasting reductions in glutamate receptor expression in the hippocampus.

FIG 1

After 1, 3, and 5 days of treatment with 30mg/kg ketamine, expression of Glutamate receptor sybunits decreases. (a, b)

The expression of glutamate receptors also decreases in a dose-wise manner, with 10mg/kg dose for 28 days decreaseing GluA1, GluA2, Glu2NA, expression only, and 30mg/kg dose also decerasing expression of glu2nb. none o fthe doses affected glun1.

D: In addition, using the 30mg/kg dose for 28 days, and then after 10, 14, or 28 days after the final injection, the effect on expression still lasts.

E, F, G: Surface protein is the functional form. Ttaol, intracellular, and surface protein, and phosphorylated protein are reduced.

1H shows that the mRNA levels of these subunits are decreased, suggesting a change in transcription.

Fig 1 thus shows that 28 days of 30mg/kg dosage decreases expression of subunits at the transcriptional level

'''Fig 2. Chronic in vivo exposure to ketamine reduced synaptic/extrasynaptic glutamate receptor expression, dendritic spine density, and synaptic protein expression in the hippocampus'''.

A. Mean intensity of subunit staining decreases after 28 days of ketamine treatment. colocalisation with Syn is used to show the synaptic expression. In B, the merge with psd-95 is used to validate synaptic expression

C red box is sampling for immunosaining.

Chronic exposure to ketamine decreased dendritic spine density and synaptic protein expressionD shows the density of dendritic spinces, with 2 photon imaging of segments from CA1 neurons expressing DGP. decreased dendritic spines in the ketamine treated mice. divided by morhology.

E. decreased expression of syn and psd-95 after 28 days of ketamine,

F. Continued decreased expression, 28 days after last injection.

These results indicated that repeated ketamine exposure decreased dendritic spine density and presynaptic/postsynaptic density. The expression of Syn and PSD-95 was also significantly decreased in female mice treated with ketamine daily for 28 days compared to vehicle controls (Supplementary Fig. 1b) and in mice injected with ketamine three times a week for a total of 28 times (Supplementary Fig. 2b).

3. Chronic exposure to ketamine reduced the expression and phosphorylation of CaMKIIβ, ERK1/2, CREB, and NF-κB

Shift focus to the molecular pathway that may account for the alterations in glutamate receptor expression

calcium influx through activated NMDAR can cause persistent Ca2+-independent activation of CaMKIIβ, a key molecule associated with the glutamate receptor signaling pathway

assessed the expression and phosphorylation of CaMKIIβ, ERK1/2, CREB, and NF-κB

A. chronic exposure to ketamine for 28 days reduced both the expression and phosphorylation of these signaling molecules

B. upon camKII overexpression, the decrease in the receptor molecules was mitigated.

Mice were injected with scrambled vectors or lentiviral vectors carrying CaMKIIβ in the bilateral hippocampi. Beginning on day 7 after injection, the mice were treated with vehicle or ketamine (30 mg/kg) once a day for 28 consecutive days

c. reductions in dendritic spine density were attenuated by overexpression of CaMKIIβ

cam KIIB overexpression has no effect on the vehicle-treated group

rescues the effect on dendrites in keamine-treated mice

[could have had a sham injection group?]

results further suggested that CaMKIIβ played critical roles in modulating glutamate receptor expression and dendritic spine density.

Fig. 4: Chronic in vivo exposure to ketamine-induced impairments in LTP and basal synaptic transmission in the hippocampal CA1 area, and these impairments were alleviated by overexpression of CaMKIIβ.

LTP is regarded as the cellular basis for learning and memory [3]. Previous studies have shown that acute ketamine administration alters LTP [3, 25, 26, 28]. However, no studies have investigated the effects of chronic ketamine administration on LTP or their potential mechanisms.

Methods:

''At least 20 min of stable baseline recordings were obtained prior to LTP stimulation. Hippocampal LTP was induced by theta-burst stimulation (TBS) consisting of a series of 10 bursts of 5 stimuli at 100 Hz (200 ms interburst interval, repeated three times) [26]. The input-output function was tested before recording LTP, and the baseline stimulation strength was set to provide fEPSPs with amplitudes of 30% of the maximum derived from the input-output function. ACSF was used as the bath solution. Bicuculline (10 μM) was included in the bath solution to block ionotropic gamma-aminobutyric acid (GABA) receptors.''

''Whole-cell patch-clamp recordings were obtained under voltage clamp using a MultiClamp700B amplifier to measure EPSCs in CA1 neurons in response to stimulation of the Schaffer collateral pathway. Stimuli were elicited via a concentric electrode placed in the stratum radiatum. The AMPA/NMDA ratio was calculated as the ratio of the peak EPSC recorded at −70 mV (AMPAR-mediated EPSC) to the EPSC recorded at +40 mV 50 ms after afferent stimulation (NMDAR-mediated EPSC)''

A. Via electrophysiological recording, we found that compared with vehicle exposure, chronic in vivo exposure to ketamine for 28 days significantly reduced LTP in the hippocampal CA1 area (Fig. 4a).

recorded LTP in the hippocampal CA1 area in mice in which CaMKIIβ was overexpressed via injection of a lentiviral vector carrying CaMKIIβ into the bilateral hippocampi before ketamine injection. We found that the LTP impairment induced by chronic ketamine exposure for 28 days was ameliorated by overexpression of CaMKIIβ

field EPSP: extracellular signal from synaptic excitation of neurons in the same direction. (striatum radiatum of CA1)

excitatory postsynaptic potential (EPSP) is the change in membrane voltage of a postsynaptic cell following the influx of positively charged ions into a cell

Don't know what the black line is

BLACK LINE: PRE- BURST STIMULUS. RED: POST (TO invoke LTP)

TOP: REPRESENTATIVE fEPSPs. bottom: time course of changes in fepsp slope. mean values of potentiation from 50-60m following stimulation. n=11-12, 4 animals per group. (2-3 recrdings per animal?)

B. still reduced 28 days after last injection

Overexpression of CaMKIIβ alleviated impairments in basal synaptic transmission and alterations in AMPA/NMDA current ratios in the hippocampus caused by chronic ketamine administration

basal synaptic transmission, we measured input-output function in the CA1 area of the hippocampus

C, reduction in input-output function was found in mice with ketamine, and the reduction was attenduated by camk2 overexpression. top: representative fepsp waveforms. Bottom: inut-output function curves. Normalized to maximum intensity.

D. PPRs (Paired-pulse ratios) of evoked EPSC (excitatory post synaptic currents) at different interstimulus intervals. Increased PPR in mice kiven chronic ketamine at short interpulse intervals, which indicated that the release of glutamate from presynaptic sites was decreased. Attenuated by overexpression of CAMK11B. left: representative traces at 30ms paired pulse intervales. Right: PPRs (EPSC2/EPSC1) in each group.

PPR is inversely proportional to the probability of presynaptic neurotransmitter release [41], this result suggested that presynaptic glutamate release was decreased by chronic ketamine exposure.

To determine effect of ketamine on spontaneous synaptic transmission, we recorded sEPSCs in the CA1 region

e. frequency and amplitude of sEPSCs (spontaneous EPSC) was sig decreased in ketamine-treated mice. Attenuated by OE. top: sample traces of sEPSCs. Bottom: changes in frequency and amplitude of sEPSCs.

SUGGESTS probability of presynaptic glutamate release and the function of postsynaptic glutamate receptors were decreased

To functionally characterize the AMPA and NMDA glutamate receptors in animals exposed to ketamine, we measured the ratio of NMDAR-mediated EPSCs to AMPAR-mediated EPSCs in the CA1 region

f. AMPA/NMDA current ratios were elevated in mice receiving ketamine, again reversed. Left. Representative current traces at +40 and -70 mV. mEASURED AT TIME MARKED BY RED DOT. r: ampar/nmdar-MEDIATED CURRENT RATIOS OF EACH GROUP.

total expression of both decreased (fig 1) so may be due to larger decrease in NMDAR function induced by chronic ket.

* =VS SCRAMBLE-VEHICLE. #: VS SCRAMBLE-KET

Impairments in learning and memory caused by chronic ketamine exposure were improved by overexpression of CaMKIIβ

Morris water maze test and mice injected in the bilateral hippocampi with a lentiviral vector carrying CaMKIIβ before ketamine injection

A: Schedule

B: Increased latency time to find the platform: impaired spatial learning. attenuated by camkii

d: no sig diff in time spent in the target quadrant {what does this mean: would expect to see decreased time in target quadrant if spatial memory is harmed]

e: locomotor behavior is unaltered

We found that chronic ketamine exposure increased the latency time to reach the platform and reduced the number of times crossed the former platform location in a Morris water maze test, which suggested impairments in spatial learning acquisition and maintenance, respectively.