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ABSTRACT Dysfunctional N-methyl-D-aspartate receptors (NMDARs) and cyclic adenosine monophosphate (cAMP) have been associated with deficits in synaptic plasticity and cognition found in

neurodegenerative and neuropsychiatric disorders such as Alzheimer’s disease (AD) and schizophrenia. Therapeutic approaches that indirectly enhance NMDAR function through increases in

glycine and/or D-serine levels as well as inhibition of phosphodiesterases that reduces degradation of cAMP, are expected to enhance synaptic strength, connectivity and to potentially impact

cognition processes. The present in vivo study investigated effects of subcutaneous administration of D-serine, the glycine transporter 1 (GlyT1) inhibitor SSR504734 and the PDE4 inhibitor

rolipram, on network oscillations, connectivity and long-term potentiation (LTP) at the hippocampi circuits in Sprague-Dawley rats. In conscious animals, multichannel EEG recordings assessed

network oscillations and connectivity at frontal and hippocampal CA1–CA3 circuits. Under urethane anaesthesia, field excitatory postsynaptic potentials (fEPSPs) were measured in the CA1

subfield of the hippocampus after high-frequency stimulation (HFS) of the Schaffer collateral-CA1 (SC) pathway. SSR504734 and rolipram significantly increased slow theta oscillations (4–6.5 

Hz) at the CA1–CA3, slow gamma oscillations (30–50 Hz) in the frontal areas and enhanced coherence in the CA1–CA3 network, which were dissociated from motor behaviour. SSR504734 enhanced

short-term potentiation (STP) and fEPSP responses were extended into LTP response, whereas the potentiation of EPSP slope was short-lived to STP with rolipram. Unlike glycine, increased

levels of D-serine had no effect on network oscillations and limits the LTP induction and expression. The present data support a facilitating role of glycine and cAMP on network oscillations

and synaptic efficacy at the CA3–CA1 circuit in rats, whereas raising endogenous D-serine levels had no such beneficial effects. SIMILAR CONTENT BEING VIEWED BY OTHERS HYDROGEN SULFIDE AND

POLYSULFIDES INDUCE GABA/GLUTAMATE/D-SERINE RELEASE, FACILITATE HIPPOCAMPAL LTP, AND REGULATE BEHAVIORAL HYPERACTIVITY Article Open access 31 October 2023 D-CYCLOSERINE ENHANCES THE

BIDIRECTIONAL RANGE OF NMDAR-DEPENDENT HIPPOCAMPAL SYNAPTIC PLASTICITY Article Open access 09 January 2024 D-SERINE RECONSTITUTES SYNAPTIC AND INTRINSIC INHIBITORY CONTROL OF PYRAMIDAL

NEURONS IN A NEURODEVELOPMENTAL MOUSE MODEL FOR SCHIZOPHRENIA Article Open access 12 December 2023 INTRODUCTION N-methyl-D-aspartate receptors (NMDARs) and cyclic adenosine monophosphate

(cAMP) play a pivotal role in plastic mechanisms of learning and memory1. Dysfunctional NMDARs and cAMP signalling have been associated with deficits in synaptic plasticity and cognitive

decline found in neuropsychiatric and neurodegenerative disorders such as schizophrenia and Alzheimer’s disease (AD)2,3,4. Therapeutic approaches that enhance NMDAR function through

increases in endogenous ligands of the NMDAR, as well as inhibition of phosphodiesterases, which reduces degradation of cAMP, are expected to enhance endogenous neurorepair and synaptic

strength to potentially impact cognition processes5,6,7. The strength of the glutamatergic neurotransmission is tightly controlled by the synaptic concentration of glycine and D-serine near

NMDA receptors. Glycine and D-serine are endogenous ligands at the glycine B site of the NMDA receptor, which act as a requisite co-agonist of glutamate for the activation of this receptor8.

Glycine, which generally acts as an inhibitory neurotransmitter, has an excitatory activity at the strychnine-insensitive coagonist site8. D-serine, which is released from astrocytes is

more potent at the strychnine-insensitive binding site than glycine9. On the one hand, levels of synaptic glycine are tightly controlled by the specific transporter GlyT1 localized on glial

cells and neurons closely associated with the NMDA receptor10. Several well tolerated, high affinity GlyT1 inhibitors have been developed and shown to increase central glycine levels for a

positive functional impact on central glutamatergic transmission and to possess the preclinical profile of putative antipsychotics properties in preclinical animal models11,12,13,14. On the

other hand, reducing D-serine levels impairs NMDAR-mediated processes in several structures, including the hippocampus, prefrontal cortex, nucleus accumbens or amygdala. Functional studies

using enzymatically, or genetically induced depletion of D-serine showed reduction of synaptic NMDARs currents and thereby alteration in synaptic plasticity at the level of the

hippocampus9,15,16, amygdala17, and nucleus accumbens18, the retina19 and the hypothalamus20. The role of D-serine at NMDARs is further illustrated by studies showing that synaptic and

cognitive impairments during aging is linked to a downregulation of D-serine synthesis21,22. Detailed analysis of the contribution of the two co-agonists in the regulation of NMDARs at the

hippocampus CA1 level revealed that D-serine would preferentially act on synaptic NMDARs whilst glycine would modulate extra-synaptic NMDARs15. The integrity of the hippocampal formation is

critical for normal memory function, hence much experimental interest focused to characterize structural and functional changes of the hippocampus throughout aging and in disease animal

models. Key mechanisms proposed to explain impaired cognitive processing are associated with deficits of network oscillations at the fronto-hippocampal circuit and impaired synaptic

plasticity related to long-term potentiation (LTP)23,24. Network oscillations represent fundamental mechanisms enabling coordinated activity between multiple association regions during

normal brain functioning. Hippocampal theta oscillations have been found to drive processing in the prefrontal cortex24,25. Increased gamma band (30–100 Hz) oscillations occur during the

transient brain states that are associated with attention and stimulus recognition26,27. More recently, several studies have suggested that gamma oscillations nested within theta (4–12 Hz)

oscillations play a role in working memory functions24. Also, substantial data suggest that corticothalamic28,29 and hippocampal networks30 make use of beta (12–30 Hz) and gamma (30–100 Hz)

frequency band activities for long-distance transmission of information among task-related brain sites, although a number of those studies were carried out in brain slices or animal model of

diseases31,32,33. LTP is most commonly induced by a combined activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and NMDA receptors. NMDAR-activity-dependent LTP is

suggested as a mechanism for short- and long-term memory acquisition34,35. Presynaptic depolarisation leads to exocytosis of glutamate into the synaptic cleft, activates many of the

postsynaptic proteins, including the cAMP. cAMP/PKA and cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathways involved in the LTP expression, maintenance and memory

enhancement36. To regulate the signalling of both pathways, the phosphodiesterase (PDE) enzyme family hydrolyses cAMP and cGMP preventing kinase activity3. There are 11 PDE subgroups found

in varying levels across the nervous system. There is a strong case for the regulation of synaptic plasticity by PDE4, which is the most widely studied PDE and is selective for cAMP over

cGMP. PDE4 hydrolyses cAMP and is found in the hippocampus and cortex among other areas in rodents37. Rolipram exhibits memory-enhancing effects in rodents. A decrease of PDE4 isoforms has

been shown in AD patients and the PDE4 inhibitor rolipram has demonstrated memory enhancements36 as well as displaying a good antidepressant effect but with unpleasant side effects38. PDE4

inhibition rescues impaired LTP and prevents object recognition memory deficits in an animal model of psychosis39. In the present work, we modulated D-serine, glycine and cAMP levels to

ascertain their functions in network oscillations and synaptic plasticity in healthy rats. By increasing synaptic concentration of glycine or D-serine in the vicinity of NMDA receptors,

blockade of GlyT1 and/or D-serine are expected to potentiate glutamatergic transmission. We demonstrate that increased levels of glycine and cAMP increased hippocampal network activity and

LTP in healthy rats. Under physiological conditions, synaptic plasticity in vivo at CA1 did not require high levels of D-Serine. MATERIALS AND METHODS ANIMALS All experimental procedures

were conducted in strict accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and with the European

Communities Council Directive 2010/63/UE of 22/09/2010 and were approved by local ethical committee. Experiments were carried out on Sprague-Dawley male rats 180–250 g (Harlan Nederland)

housed in individually ventilated cages located in a sound-attenuated chamber under the controlled light/dark cycle (light: 7.00–19.00) with standard food and water available ad libitum.

SURGERY AND ELECTRODE IMPLANTATION NETWORK OSCILLATIONS AND CONNECTIVITY Surgery was performed under Isoflurane anaesthesia as described earlier40,41. In brief, animals were equipped with

two stainless steel fixing screws (diameter 1 mm) for the recording of frontal electroencephalographic activities (EEG) inserted bilaterally in the left and right cortex (FL/FR: AP + 2 mm, L

 ± 2 mm from Bregma). Four stainless steel wires used for intra-hippocampal electrodes (CA1: AP −3.14 mm, L ± 1.8 mm, V + 2.8 mm; CA3: AP −4.5 mm, L ± 3.8 mm, V + 4 mm from Bregma,

respectively). In addition, stainless steel wires (7N51465T5TLT, 51/46 Teflon Bilaney, Germany) were placed in the muscle of the neck to record the electromyogram activity (EMG). Electrodes

were connected to a pin (Future Electronics: 0672-2-15-15-30-27-10-0) with a small insert (track pins; Dataflex: TRP-1558-0000) and were fit into a 10-hole connector, after which the whole

assembly was fixed with dental cement to the cranium. Animals were given at least 2 weeks as a recovery period. RECORDING, ANALYSIS OF SPECTRAL OSCILLATIONS AND NETWORK CONNECTIVITY EEG

recordings were derived from six brain regions under vigilance waking condition during the dark circadian phase40,41. Additionally, a general motion level was monitored in the home cage by

two passive infrared (PIR) detectors placed above each recording cage, and artifact-free waking epochs with low-voltage fast EEG activity, high to moderate EMG and body activities were

considered in the analysis. Epochs with high-voltage slow cortical waves in the absence of EMG and locomotor activities were discarded. A notch FIR filter at 50 Hz was applied to avoid

voltage related to power line interferences. Baseline EEG recordings of 30 min started 2 h after the light offset to avoid confounding circadian effect on EEG. Afterwards, EEG signals were

recorded for 3 h after vehicle or drug administration (_n_ = 8 animals for each condition). Continuous EEG and EMG field potentials were acquired at a sampling rate of 512 Hz with an input

range of ± 500 mV through a Biosemi ActiveTwo system (Biosemi, Amsterdam, Netherlands), which replaces the conventional ground electrodes by two separate electrodes: the common mode sense

(CMS) active electrode and the driven right leg (DRL) passive electrode. This common mode reference for online data acquisition and impedance measures is a feedback loop driving the average

potential across the montage close to the amplifier zero. The signals were amplified and analogue band-pass filtered between 1 and 100 Hz and was digitized with 24-bit resolution. The

analysis was performed using a MATLAB toolbox described earlier41. Briefly, EEG spectral power density was calculated using a Welch’s method with a Hanning window function. Power was

expressed as relative power for each frequency over 1–256 Hz. Average relative power in each frequency bin of each location was averaged across animals to obtain the spectrum relative to

total power spectrum. For the sake of clarity in presenting this spectral data, graphs only shown the frequency range between 1 and 30 Hz and inset plots from 30 to 100 Hz. COHERENCE In

order to describe interconnectivity between pairs of EEG electrodes, coherence measure was used, which describe level of connectivity as a value in interval [0, 1] (where 1 corresponds to

complete perfect relation) for each frequency band f. Coherence is estimated as Coh(f) = |SAB(f)|2/(SAA(f)SBB(f)); where SAB is the cross-spectrum between the signals A and B from two

different electrodes; SAA, is the autospectrum of the signal A; SBB, is the autospectrum of the signal B. Coherence analysis allows assessment of pairwise synchronization of LFP/EEG signals

to shed more light onto the interaction between different brain networks. ELECTROPHYSIOLOGY IN-VIVO LTP Rats were anesthetized with an intraperitoneal injection of urethane (ethyl carbamate,

1.5 gm/kg, i.p.), and supplemented as necessary (0.2 mL/100 g) dependent upon the response to a paw pinch. Core body temperature was monitored and maintained at 37 °C through a heating pad

and rectal probe for the duration of the experiments. Once the skull was exposed, two bregma-referenced holes were drilled to insert stimulating twisted-wire bipolar and recording monopolar

electrodes constructed from Teflon-coated tungsten wires (75 µm external diameter). Recordings of field excitatory post synaptic potentials (fEPSPs) were made from the stratum radiatum in

the CA1 area of the right hippocampal hemisphere in response to stimulation of the ipsilateral Schaffer collateral–commissural pathway. The electrode implantation sites were identified using

stereotaxic coordinates, with the stimulating electrodes −3.4 mm posterior to bregma, −2.5 mm lateral to midline and 1.9–2.4 mm ventral, and the recording site located −4.2 mm posterior to

bregma, −4 mm lateral to the midline and 2.5–3.4 mm ventral. Before the experiment, the correct placement of SC-CA1 implants were finely adjusted by altering the depth of both stimulation

and recording electrodes in 10 µm increments for optimal field post synaptic potentials (fEPSP) evoking through the oscilloscope. Two epidural screws were inserted in the skull over the

cerebellum served, respectively, as the reference and the recording ground. During surgery, all efforts were made to minimize animal suffering. HFS FOR LTP INDUCTION Stimuli were delivered

using a constant current isolator unit (multichannel system MC STG4002). The induced field potential response of the SC1 was passed through the active two electrodes Biosemi amplifier

(Differential amplifier, Netherlands) and digitized at 3 kHz. At the beginning of each experiment, an input–output (I/O) curve with stimulus at a frequency of 0.033 Hz and intensities

ranging from 1 to 10 Volts was generated for each animal to determine the maximum fEPSP slope, and averaging five responses per intensity, then the intensity of test stimulus was set at a

level that evoked an fEPSP slope of 50% of the maximum was used for all subsequent stimulations. After the determination of I/O curves, the test stimulation was applied every 30 s before and

after tetanic stimulation. For each time point measured during the experiments, five records of evoked responses at the frequency of 0.033 Hz were averaged. Baseline activity was measured

every 2.5 min for at least 1 h to ensure stable baseline. The last 30 min of the baseline recording (12 time points), immediately after drug application was averaged and used as control for

LTP induction. Tetanisation was induced using a high-frequency stimulation (HFS) 200-Hz protocol consisting of square pulses (0.2 msec stimulus duration, 10 bursts of 20 stimuli, 2 s

inter-burst interval) at a stimulus intensity that evoked an fEPSP slope that was approximately 50% of the maximal response. fEPSP were recorded during 120 min after HFS to determine

possible changes in the synaptic response of SC1 neurons. LTP measurements were derived from field EPSP ratios of the normalized slope average obtained 120-min following HFS divided by the

normalized slope average collected 30 min prior to HFS. Slope of putative fEPSP were measured between the end of stimulus artefact and the trough of the negative peak. The slope of the fEPSP

was calculated using a linear fit least square analysis on the 80% interval between the artefact end and the negative peak. fEPSP slopes were obtained every 2.5 min as an average of 5

responses at 0.033 Hz and were then expressed as percentage change from baseline (defined as the last 30 min prior tetanisation). LTP was defined as an increase in fEPSP slope that is

maintained above 120% relative to baseline for 2 h following HFS administration. At the end of the electrophysiological study, three 30-s electrical pulses of 500 µA were delivered to

produce a lesion at the end tip of the stimulation and recording electrodes and brains were harvested for histological verification of electrodes placement. Brain sections (20 µm) were

examined using a light microscope. Animals with incorrect electrode placement were excluded from the study. DRUGS The GlyT1i (SSR504734), D-serine, and rolipram were purchased from Sigma

Aldrich. SSR504734 (2.5, 10 and 40 mg/kg) was formulated in 10% CD + 1 HCl + NaCl. Rolipram (1, 3 and 10 mg/kg) was formulated in 10% CD + 1 HCl + NaCl. D-serine (20, 80 and 320 mg/kg) was

dissolved in NaCl + H2O + NaOH. Drugs were administered subcutaneously in a volume of 1 ml/100 g body weight. DATA ANALYSIS EEG Result for EEG spectrum metrics were calculated for each

frequency bin and were expressed as relative total power spectra during 3 h following the administration of test drugs. Animal numbers were chosen to ensure adequate statistical power

comparable to previously published papers. Data distribution was assumed to be normal and were presented as mean values with 95% confidence intervals (CI). One-way ANOVA followed by

Dunnett’s post hoc test were used to assess difference in means between vehicle and different drug doses for EEG power and coherence measures while considering the heterogeneity between

animals. ANOVA results are reported in figures’ captions, and mean data are visualised as box plots with significant differences based on post hoc analysis indicated by asterisks (*_p_-value

 < 0.05, **_p_-value < 0.01). LTP In vivo electrophysiology fEPSP responses recorded after application of drugs were expressed as percentage of change from baseline. The slope of the

fEPSP was calculated using a linear fit least square analysis on the 80% interval between the artefact end and the negative peak. fEPSP slopes were obtained every 2.5 min as an average of 5

responses at 0.033 Hz and were then expressed as mean percentage change from baseline (defined as the last 30 min prior tetanisation) ± SEM. Firstly, in order to assess longitudinal changes

after application of HFS, mixed-effect modelling was applied. Time after HFS was log-transformed as tnew = ln(1 + told/5), where told is a real time expressed in minutes. This transformation

allowed to linearize the data. As a next step, fEPSP relative to baseline (%) variable was modelled as tnew * condition + (1|animal), where condition variable is categorical variables

describing vehicle and different drug concentrations. Effect of condition variable on intercept (to assess initial differences in fEPSP responses) and slop (to assess differences in

attenuation of fEPSP responses in time) of the model were tested. Of primary interest there were differences between vehicle and drug. Secondly, in order to understand difference between

conditions at each time point after HFS, repeated measures analysis of variance (ANOVA) followed by a post-hoc test (Dunnett’s test) were used. _p_ < 0.05 was considered statistically

significant. RESULTS EFFECTS OF SSR504734, D-SERINE AND ROLIPRAM ON NETWORK OSCILLATION AND CONNECTIVITY Previous reports reveal the presence of two different theta (slow (4–6) and fast

(6.5–8 Hz) and gamma bands (slow (30–50 Hz) and fast (50–100 Hz)). Both rhythms are involved in the communication between CA1 and CA3 and between CA1 and entorhinal cortex40,42,43 and showed

sensitivity to pharmacological treatment40. Therefore, we evaluated LFP power in slow, and fast theta-gamma rhythms of the CA1–CA3-frontal circuit during treatment with D-serine, SSR504734

and rolipram. No major effect was observed on slow and high theta or gamma spectra after D-serine treatment. No frequency band showed consistent changes between 0 and 100 Hz across regions

from pre-injection levels and compared to vehicle (Fig. 1). In addition, there was no site-pair by frequency changes for peak coherence after the administration of different does of D-serine

(Fig. 4 only mean peak slow theta coherence was displayed over the 3 h period following the administration). In contrast, LFPs recorded after SSR504734 and rolipram were associated with

similar pattern of power and peak coherence changes. The EEG of rats shifted to continuous synchronized activity to slow theta oscillations at the CA1 network after the administration of

both drugs in the model (Figs. 2 and 3). Rolipram further synchronized slow theta at the CA3 level (Fig. 3), while no major changes were observed in the activity of other frequency bands in

CA1–CA3 network. SSR504734 at the higher dose yielded additional patterns of activity consisted with enhancing effect on frontal-CA1 network gamma power (Fig. 3). Coherence of both SSR504734

and rolipram peaked at the slow theta frequency in the CA1–CA3 network (Fig. 4 middle and right panel, respectively). To assess whether changes in EEG slow theta activity (4–6.5 Hz)

oscillations were associated with changes in motor behaviour following different treatments, the time course of motor activity at different time points revealed a reduction in activity

levels (Fig. 5). Next, we examined whether the observed differences in hippocampal oscillation are associated with changes in synaptic plasticity. EFFECTS OF EXOGENOUS D-SERINE ON LTP The

effect of exogenous D-serine (20, 80, 320 mg/kg) was investigated on LTP induction and maintenance. Baseline input/output (I/O) curves for fEPSP slopes were not significantly different

confirming that the SC fibres in all dose groups had similar basal synaptic transmission (Fig. 6). D-serine had no effect on basal synaptic activity. Mixed-effect model revealed significant

effect of D-serine after the HFS tetanisation on short term potentiation “STP” (model’s intercept: _p_ < 0.001, and each concentration of drug was significantly different from vehicle

[_p_ < 0.001]) and inhibition effect on LTP in time (model’s slope: _p_ < 0.001, and each concentration of drug, except for 40 mg/kg, was significantly different from vehicle [_p_ <

 0.001]). Absence of significant difference of the 40 mg/kg injection on model’s slope (_p_ = 0.77) characterizes, that LTP after vehicle and 40 mg/kg injections were inhibited at the same

rate. More precisely, after HFS tetanisation, D-serine impaired STP (−7.2%, −16.1% and −17%: _p_ = 0.02, respectively) and the expression to LTP (over 2 h). Further analysis into the last

period of the recording session revealed a significant inhibitory effect on LTP maintenance, particularly with the higher dose (90–120 min post-HFS, −18%: _p_ = 0.03) (Fig. 6). EFFECTS OF

SSR504734 ON LTP The effect of increased glycine levels via reuptake inhibition on LTP expression was investigated. I/O curves were not significantly different confirming that the SC fibres

in all dose groups were of a similar excitability (Fig. 7). SSR504734 had no influence on basal synaptic transmission. Mixed-effect model did not reveal significant effect of SSR504734 after

the HFS tetanisation on STP (model’s intercept: _p_ = 0.058), but the effect on enhancement of LTP in time was significant (model’s slope: _p_ < 0.001). More precisely, SSR504734 at 10 

mg/kg failed to enhance the slope of fEPSPs above vehicle levels during the entire recording session of 2 h post-treatment. However, there was a significant effect of SSR504734 at the dose

of 40 mg/kg on LTP maintenance as revealed during the last period of the recording session after HFS (90–120 min post-HFS, +41%: _p_ = 0.001). EFFECTS OF ROLIPRAM ON LTP RESPONSE The effect

of increasing cAMP levels with the selective blocker of PDE4 reuptake inhibition on LTP expression was studied. The results showed no significant difference between group I/O curves

confirming there was no difference in the excitability SC-CA1 synapses in the study groups (Fig. 8). Similarly, basal synaptic transmission was not affected by application of rolipram.

Mixed-effect model revealed significant effect of rolipram after the HFS tetanisation on STP (model’s intercept: _p_ < 0.001) and enhancement effect on LTP in time (model’s slope: _p_ 

< 0.001). Detailed analysis of difference driving above’s significance results showed, that the dose of 3 mg/kg, rolipram led to the increased STP (+25%, _p_ = 0.04, Fig. 8), however,

this transient potentiation did not express into LTP. At the lowest dose of 1 mg/kg, rolipram had no significant effect on STP and the expression into LTP as compared to vehicle (Fig. 8).

DISCUSSION In the present study, we sought to define patterns of network oscillatory activities and plasticity amongst anatomically and functionally connected brain region after modulation

of both NMDA and cAMP signalling. Both SSR504734 and rolipram enhanced network slow theta oscillations, connectivity in the CA1–CA3 circuit. SSR504734 enhanced LTP response, whereas the

plasticity was short-lived to STP with rolipram. Unlike glycine, increased levels of D-serine had no effect on network oscillations and limits the induction and expression of LTP. Neural

oscillations are critical mechanisms allowing dynamic coupling of intra- and inter-regional brain regions during information processing44,45. For instance, theta oscillatory rhythm derived

from concerted generators including cholinergic and GABAergic, as well as glutamatergic networks in different locations, plays a key role in the function of the hippocampus and associated

cortical networks46. Afferent cholinergic and glutamatergic input from the medial septum-diagonal band of Broca, as well as from CA3 and entorhinal cortex provide support for theta

oscillations at the CA1 circuit46. Modulation of the glutamatergic NMDA signalling through local modulation of septal circuit mediates the generation of hippocampal theta rhythm47. Theta

oscillations may facilitate synchrony between hippocampus and prefrontal cortex network required for learning and memory consolidation48. Gamma oscillations (30–100 Hz) are prominent in the

cortical-hippocampal network and have been shown to appear during a variety of memory tasks in rats, monkeys, and humans43,49. Gamma rhythms occur as two distinct variants that are thought

to route different streams of information entering hippocampal subfield CA142,50. Slow gamma (30–50 Hz) may promote direct inputs from CA3 to CA1, which is believed to be associated with

memory retrieval51,52. Fast gamma (50–100 Hz) may facilitate direct transmission from the medial entorhinal cortex that transmit ongoing spatial information51,53. Functional roles of theta

and gamma oscillations in mnemonic processes has been established54, and while disruption of this oscillatory rhythms has been associated with cognitive deficits described in psychiatric and

neurodegenerative disorders55,56. Pharmacological and several new therapeutic techniques, such as transcranial magnetic stimulation, transcranial direct current stimulation, and closed-loop

stimulation, have profound direct and indirect effects on ongoing oscillatory activity in the brain57, and restoration of theta and gamma like rhythmicity restores learning and memory

capabilities in rats58,59,60. SSR504734 AND ROLIPRAM BUT NOT D-SERINE ENHANCED NETWORK OSCILLATIONS AND CONNECTIVITY Abundant evidence points to the importance of NMDA receptors in

patterning neuronal networks and synaptic transmission. Positive modulation of the co-agonist binding site on the NMDA receptor has been proposed as a novel therapeutic approach to overcome

negative symptomatology and cognitive dysfunction seen in those diseases61. Glycine and D-serine are endogenous ligands to the NMDA modulatory site62, and ligands modulating NMDA receptor

transmission, have being developed such as GlyT-1 inhibitors to potentially elevate brain glycine or targeting enzymes, such as d-amino acid oxidase (DAAO) to slow the breakdown and increase

the brain level of D-serine63,64. In the present study we further evaluated effects of the glycine inhibitor, exogenous D-serine or modulating cAMP levels on synaptic network oscillations

and plasticity. The oscillatory activity across the CA1 network of structures studied in the present work was dominated by changes in the slow theta rhythm and related coherent activity.

Thus, indirect modulation of NMDA signalling through inhibition of the GYT1 may support the hypothesis that the release of endogenous D-serine from astrocytes did not saturate and,

therefore, enough to generate and entrain synchronized theta rhythm in CA1–CA3 network during information processing. However, exogenous supply of D-serine failed to promote similar changes

in network oscillation and connectivity, likely because D-serine levels might have reached saturation and therefore shunt down synchronized theta rhythm. In addition, SSR504734 enhanced slow

gamma at the frontal and CA1 structures, which of rhythm has been hypothesized to promote memory retrieval. Therefore, the evoked slow theta and gamma network oscillations at the CA1 and

frontal networks, respectively suggests a positive modulation effect of the glycine site of the NMDA receptor on attentional processing and memory operations phases. Alterations in cAMP

signalling are thought to contribute to neurocognitive and neuropsychiatric disorders. Phosphodiesterases play an essential role in orchestrating the compartmentalized degradation of cAMP,

leading to local changes in cAMP signalling in specific subcellular domains in the cell65. The cAMP signalling pathway is a second messenger that has a key role in several intracellular

cascades, including the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway which is critically involved in learning and memory5. Changes in cAMP levels has been

shown to regulate theta activity, and rolipram administered intravenously evoked an arousal EEG pattern period (low amplitude fast waves) in the cortical EEG and synchronization of the

hippocampal theta waves with increased voltages66. In the amygdalo-hippocampal pathways, an increase in intracellular cAMP concentration facilitates regular firing and oscillatory activity

at the theta frequency range67, supporting synaptic signal transfer between those functionally connected neuronal populations during retrieval of conditioned fear. Disruption of theta

activity results in spatial memory deficits, whereas the restoration of theta-like rhythmicity reverse learning deficits in rats59,60. In the present work, the enhanced slow theta activity

confirmed the potential positive modulatory effect of rolipram on neural networks. GLYCINE TRANSPORTER INHIBITOR AND ROLIPRAM, BUT NOT D-SERINE, ENHANCED IN VIVO PLASTICITY LTP AND GLYCINE

Activity-dependent synaptic plasticity, such as NMDAR-dependent LTP has been proposed as a cellular mechanism underlying learning and memory in the brain68,69. The GlyT1 antagonist NFPS

increased NMDAR channel opening in a dose-dependent manner in Sprague-Dawley prefrontal cortex slices70. Similarly, the antagonist CP-802,079 enhanced LTP induced by HFS in hippocampal

slices14. However, the effects of both these antagonists appear irreversible. Depoortère et al.71 first reported on the neurochemical, electrophysiological and pharmacological

characteristics of the selective, reversible GlyT1 inhibitor SSR504734. The compound enhanced in a concentration-dependent manner the NMDA component of CA1 excitatory postsynaptic currents

therefore further confirming the role of the GlyT1 in NMDA excitability. In the present study, SSR504734 enhanced the LTP expression at the highest dose of 40 mg/kg for the entire 2-h

post-tetanisation recording period. The significantly enhanced and enduring LTP is likely the result of increased NMDAR channel openings70 as this would cause a larger postsynaptic influx of

Ca2+ and activation of LTP maintenance mechanisms. The lower dose of SSR504734 given, 10 mg/kg had no effect on LTP as compared to the vehicle. Microdialysis studies in the prefrontal

cortex and nucleus accumbens revealed a significant increase in the extracellular glycine concentrations for 45–180 min after the application of SSR504734 at the dose of 10 mg/kg71,72.

According to this time frame, the HFS in this study fell 15 min before synaptic glycine concentrations were increased. It is, therefore, possible that SSR504734 (10 mg/kg) successfully

increased glycine concentrations in this study. However, as this would not have occurred prior to or during the tetanisation, the extra glycine at the synapse following GlyT1 inhibition

would not have been able to enhance NMDAR excitability before administration of the HFS. As a result, the NMDAR-dependent LTP of CA1 would not have been enhanced by the GlyT1 inhibition. The

40 mg/kg dose may also have a faster time frame for glycine concentration increase following injection due to the higher concentration of compound available. Extracellular increase in

glycine concentration has not been investigated at doses above 10 mg/kg of SSR50473471,72. A subsequent microdialysis study could confirm the hypothesis that HFS was administered before peak

glycine levels with the 10 mg/kg dose and that this effect was shifted rightwards following 40 mg/kg. SSR504734 has also been investigated in mice performing in an operant delayed

alternation cognitive task63. Success in the task, which relies on working memory, became more difficult with longer time intervals and delays above 8 s between trials proved to be

challenging. Animals treated with the dose of 30 mg/kg successfully completed tasks with intervals of 12–18 s, intervals at which control animals could no longer perform above chance levels.

At intervals of 12 s, SSR504734 (30 mg/kg) enhanced the percentage of correct choices as did 10 mg/kg, however, this was not a significant effect. Other studies that used SSR504734 showed

efficacy in behavioural studies with doses higher than 10 mg/kg73,74. Therefore, the present results support the earlier behavioural studies. SSR504734 1(0 mg/kg) may enhance glycine levels

but not sufficient to overcome the effect of other factors in an intact brain. Indeed, the other NMDAR coagonist D-serine exhibits higher affinity for the strychnine-insensitive binding

site9. This binding competition could prevent intermediately increased glycine levels from having a detectable effect on LTP or behavioural response. The results of this work suggest that

reuptake inhibition with SSR504734 and the two GlyT1 inhibitors NFPS12 and CP 802,07914 did indeed lead to increased synaptic levels of glycine, therefore increasing its role as a

facilitator of NMDAR excitability and the enhancement of LTP following HFS. The results suggest a beneficial effect of GlyT1 inhibitors on hippocampal-dependent forms of memory deficient,

and further provide a compelling rationale for using GlyT1 inhibitors to indirectly potentiate NMDA receptor functions. LTP AND D-SERINE Endogenous D-serine can be released in an

activity-dependent manner and, in turn, contributes to the induction of LTP and LTD19,75. D-serine is a more potent agonist of the NMDAR than glycine9, and D-serine is moderately better than

glycine in penetrating the blood–brain barrier when administered systemically, therefore, it was expected that the high doses of D-serine given in this experiment would potentiate the LTP

response. However, this was not the case with the middle dose (40 mg/kg) had a slightly depotentiation effect on LTP levels and the highest dose (320 mg/kg) decreasing LTP response. Whilst

some groups have reported enhancing LTP with D-serine application16,76, the consensus in the literature is that exogenous D-serine has no effect on LTP in wild-type animals even though its

increased NMDAR-mediated post-synaptic responses in hippocampal slices77,78,79. D-serine treatment alone has no effect on LTP induction, while it decreased the basal glutamatergic

neurotransmission, which weakens the efficacy of the agonist on synaptic plasticity and depresses basal AMPA receptor-mediated neurotransmission in young animals. This property may,

therefore, limit the potency of the agonist to increase the magnitude of synaptic plasticity, especially in aged rats as AMPAR-mediated neurotransmission was reduced in these animals80,81.

D-serine had no effect on spatial learning and memory per se82, whereas exogenous application of D-serine has however been shown to restore potentiation in aged animal models of decreased

D-serine concentrations77 or in pharmacologically induced glial metabolism disruption79. Another difference that may explain discrepancies of results may be related to stimulation protocols

used to induce LTP. In both groups that showed an enhancement of LTP under control conditions have used theta-burst protocol, which may activate different signalling pathways as compared to

HFS as well as induce a different pattern of Ca2+ signalling83. However, plasticity studies that used HFS protocols did not reveal significant increase of the LTP response following

D-serine, which may cause endogenous D-serine levels to saturate following tetanisation and shunts inhibition of afferent inputs which thus display a depression (an LTD-like effect) instead

of an LTP at the soma78. This may explain a negative trend in basal synaptic activity and the LTP responses that were seen in this study using young, wild type rats and HFS protocol. The

novel finding reported here is that the highest dose of exogenous D-serine reduced synaptic potentiation below control levels, which could lead to receptor internalization. Both D-serine and

glycine binding have been suggested to prime the NMDA receptor for clatherin-dependent endocytosis upon glutamate binding and receptor activation in hippocampal cells84. In this case, the

high dose of D-serine would increase the number of receptors primed for internalisation. Upon application of the HFS, which would cause a large glutamate release, postsynaptic depolarisation

would cause NMDA receptor endocytosis. If an enough NMDA receptor were internalised this would reduce the levels of signalling Ca2+ and therefore reduce the activation of LTP signalling

mechanisms. As the study by Nong et al.84 focussed more prominently on the role of glycine in NMDA receptor internalisation, further immunocytochemical assays using D-serine could strengthen

this hypothesis. Another possibility for the decreased LTP response after high dose of D-serine may result in synaptic excitotoxicity. Indeed, high concentrations of this amino acid have

been measured in pathological, neurotoxic states such as cerebral ischemia85. In AD, amyloid-β has been shown to induce D-serine release from microglia leading to neurodegeneration86. It has

also been reported that exogenous D-serine has a dose-dependent bell-shaped effect on LTD magnitude75, which could reflect changes in the LTD/LTP threshold as set out in the Bienenstock,

Cooper, and Munro (BCM) model. Accordingly, the high level of D-serine 320 mg/kg may be closer to the LTP threshold than lower or higher doses of exogenous D-serine and inducing a lower

level of LTP than under vehicle or 320 mg/kg conditions. However, to our knowledge it has not been investigated whether this is also the case for LTP as single concentrations of D-serine

were generally used in previous studies. Whilst both glycine and D-serine directly modulate the excitability of NMDA receptors and enhance NMDAR-mediated synaptic transmission, clinical

trials involving direct administration of both amino acids produced mixed results in improving cognitive impairments in schizophrenic patients87. The present study provides evidence that

D-serine controls NMDAR-dependent LTP, whilst glycine influence neurotransmission at a different level, by activating extrasynaptic glycine receptors distributed along the apical dendrite.

Future studies will evaluate mechanistic approaches targeting D-serine modulatory sites, for example by inhibition of the enzyme d-amino acid oxidase (DAAO), which slows the break-down of

D-serine, or by its transporter, the alanine-serine-cysteine-1 (Asc-1). LTP AND ROLIPRAM Alterations in the activity of PDE4 has been associated with cellular mechanisms underlying

structural and synaptic damage in experimental models of mood and neurological diseases6. Pharmacological inhibition of PDE4 activity promotes synaptic plasticity and memory88. Mice lacking

all PDE4D isoforms display either memory enhancements or impairments, depending on the task used17. The PDE4 selective inhibitor, rolipram, prevents memory deficits associated with sleep

loss89, aging90, muscarinic or NMDA receptor blockade39, and mouse models of Alzheimer’s disease91. LTP can be induced in the SC-CA1 synapse of the hippocampus by stimulation in the theta

frequency range (5–12 Hz), an effect that depends on activation of the cAMP pathway92. The amyloid beta-induced inhibition of LTP in slices was reversed following direct application of

rolipram93. In this study, an enhancement of early phase of LTP was observed following the administration of rolipram at 3 mg/kg. This would appear contradictory with the hypothesised role

of the cAMP/PKA pathway in late memory consolidation and late-LTP36. Late-LTP is a more persistent and robust form of LTP lasting for 8–10 h that requires PKA activation for protein

synthesis and to facilitate LTP maintenance94. Late-LTP can be induced using a strong tetanisation procedure such as the repetition of HFS trains94. Similar late-LTP levels have been

recorded in vitro and in vivo using application of rolipram to enhance cAMP levels39,88. Wiescholleck and Manahan-Vaughan39 reported that this dose in vivo caused a transient chemically

induced potentiation lasting an hour without tetanisation. However similar doses of rolipram in vitro show an effect on basal synaptic transmission and have been reported to have a negative

impact on LTP perhaps through a toxic effect88. The amplification of transient cAMP by rolipram and its impact on LTP levels is sensitive to the time at which the HFS protocol is

administered88. The enhancing effect of rolipram was only seen when the hippocampal slice was perfused with the compound during tetanisation, however, LTP was no different from saline if the

perfusion occurred after HFS88. Most likely the highest dose used in this study transiently amplified the cAMP to elicit a potentiation but not sufficiently to fully activate CREB

signalling or AMPA insertion at the synapse95. The enhancement of LTP by rolipram, although transient, shows that the HFS model is responsive to enhanced cAMP levels following the inhibition

of PDE4. The cAMP/PKA path is not the only signalling pathway regulated by the PDEs; cGMP and the related PKG also activate the transcription factor CREB, which may be involved in earlier

LTP and memory consolidation to the cAMP pathway36. Therefore, a combined enhancement of both cAMP and cGMP could activate mechanisms whilst also inducing the protein processes necessary for

facilitating late-LTP. The present study provides evidence that the glycine modulatory site was required for the induction of NMDAR-dependent LTP and connectivity whilst exogenous D-serine

negatively influenced neurotransmission. Unlike glycine, increased level of D-serine does limit the induction and expression of LTP in the rat CA1. We hypothesized that high levels of

D-serine might results in a shunt of synaptic inputs to downregulate NMDARs currents and eventually leading to synaptic depression after application of HFS at the somatic level (Fig. 9). Our

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Neuroscience, Janssen Research & Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, B-2340, Beerse, Belgium A. Ahnaou, T. Broadbelt, R. Biermans, H. Huysmans, N. V.

Manyakov & W. H. I. M. Drinkenburg Authors * A. Ahnaou View author publications You can also search for this author inPubMed Google Scholar * T. Broadbelt View author publications You

can also search for this author inPubMed Google Scholar * R. Biermans View author publications You can also search for this author inPubMed Google Scholar * H. Huysmans View author

publications You can also search for this author inPubMed Google Scholar * N. V. Manyakov View author publications You can also search for this author inPubMed Google Scholar * W. H. I. M.

Drinkenburg View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to A. Ahnaou. ETHICS DECLARATIONS CONFLICT OF INTEREST

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THIS ARTICLE CITE THIS ARTICLE Ahnaou, A., Broadbelt, T., Biermans, R. _et al._ The phosphodiesterase-4 and glycine transporter-1 inhibitors enhance in vivo hippocampal theta network

connectivity and synaptic plasticity, whereas D-serine does not. _Transl Psychiatry_ 10, 197 (2020). https://doi.org/10.1038/s41398-020-00875-6 Download citation * Received: 08 February 2020

* Revised: 21 May 2020 * Accepted: 26 May 2020 * Published: 18 June 2020 * DOI: https://doi.org/10.1038/s41398-020-00875-6 SHARE THIS ARTICLE Anyone you share the following link with will

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